ACTA » AGRICULTURAE SLOVENICA Biotehniška fakulteta Univerze v Ljubljani Biotechnical Faculty University of Ljubljana Acta agriculturae Slovenica • ISSN 1581-9175 • 109 - 2 • Ljubljana, september 2017 Acta agriculturae Slovenica Volume / Letnik 109 • Number / Številka 2 • 2017 VSEBINA / CONTENTS Izvirni znanstveni članki / Original research articles J. N. ODEDINA, S. O. ADIGBO, P. KULAKO, P. ILUEBBEY, T. O. FABUNMI, C. G. AFOLABI, A. A. OSIPITAN 165 Effect of cropping system and age of plant at harvest on tuber rot and performance of elite cassava varieties in derived savannah Učinki načina gojenja in starosti rastlin ob spravilu na pojavljanje gnilobe gomoljev pri elitnih sortah manioke v antropogeni savani Nigerije Akhtar MAHMOOD, Muhammad Saleem HAIDER, Qurban ALI, Idrees Ahmad NASIR 175 Multivariate analysis to assess abscisic acid content association with different physiological and plant growth related traits of Petunia Uporaba multivariatne analize za ocenitev povezave med vsebnostjo abscizinske kisline in različnimi z rastjo povezanimi fiziološkimi znaki pri petuniji R. M. GAAFAR, Mai ALLAM, Rasha SABRY2 and M. SAKER 187 Molecular genetic analysis of some North African barley germplasms Molekularna genetska analiza nekaterih severnoafriških genskih virov ječmena S. ALAGHEMAND, H. AYYUBI, E. HASANPOUR, J.A. OLFATI, M. SAADATIAN, Y. HAMIDOGHLI S. KAZEMI, H. KARIMI 197 Effects of organic fertilizers on growth and biochemical characteristics of Fenugreek Učinki organskih gnojil na rast in biokemijske lastnosti sabljastega triplata Sali ALIU, Imer RUSINOVCI, Shukri FETAHU, Skender KAQIU, Dukagjin ZEKA 205 Assessment of morphological variability and chemical composition of some local pepper (Capsicum annuum L.) populations on the area of Kosovo Ovrednotenje morfološke spremenljivosti in kemične sestave nekaterih populacij paprike (Capsicum annuum L.) na Kosovu Amin LAMIAN, Hassanali NAGHDI BADI, Ali MEHRAFARIN, Mehdi SEIF SAHANDI 215 Changes in essential oil and morpho-physiological traits of tarragon (Artemisia dracuncalus L.) in responses to arbuscular mycorrhizal fungus, AMF (Glomus intraradices N.C. Schenck & G.S. Sm.) inoculation under salinity Spremembe v vsebnosti eteričnih olj in morfološko-fizioloških lastnosti pehtrana (Artemisia dracuncalus L.) kot odziv na inokulacijo z arbuskularno mikorizno glivo (Glomus intraradices N.C. Schenck & G.S. Sm.) v razmerah slanosti Pooran GOLKAR, Ehsan SHAHBAZI, Mojtaba NOURAEIN 229 Combining ability x environment interaction and genetic analysis for agronomic traits in safflower (Carthamus tinctorius L.): biplot as a tool for diallel data Analiza interakcije okolja in kombinacijske sposobnosti in genetska analiza agronomskih lastnosti žafranike (Carthamus tinctorius L.): biplot kot orodje za anlizo dialelnih križanj Masoumeh ABEDINI, Behrokh DAIE-HASSANI, Shirwan MALAEI 241 UVA + B treatment affects antioxidant system and phytochemicals of parsley plant under different concentrations of Zn Obravnavanje z UVA + UVB vpliva na antioksidacijski sistem in kemizem petršilja pri različnih koncetracijah Zn Bashir O. BELLO, D. AMINU 251 Genetic relationships among okra (Abelmoschus esculentus (L.) Moench) cultivars in Nigeria Genetska razmerja med izbranimi sortami jedilnega osleza (Abelmoschus esculentus (L.) Moench) v Nigeriji Matjaž GLAVAN1, Aljaž MALEK, Marina PINTAR, Helena GRČMAN 261 Prostorska analiza kmetijskih zemljišč v zaraščanju v Sloveniji Spatial analysis of the abandonment of agricultural land in Slovenia Hamidreza MIRI, Maryam SADEGHI, Abdolreza JAFARI, Mohammad Mehdi RAHIMI 281 Effect of CO2 elevation and UV-A radiation on growth responses of Zinnia, Petunia, Coxcomb, and Marigold Učinek povečane koncentracije CO2 in povečanega UV-A sevanja na rastni odziv cinije, petunije, petelinjega grebena in žametnice Zahra MIRFATTAHI, Soheil KARIMI, Mahmoud Reza ROOZBAN 291 Salinity induced changes in water relations, oxidative damage and morpho-physiological adaptations of pistachio genotypes in soilless culture S slanostjo vzpodbujene spremembe v vodnem režimu, oksidativne poškodbe in morfološko-fiziološke prilagoditve genotipov pistacije v breztalnem gojenju 303 315 325 331 337 349 357 363 383 393 403 417 425 435 443 457 465 473 475 Muhammad Q. NAWAZ, Khalil AHMED, Syed S. HUSSAIN, Muhammad RIZWAN, Muhammad SARFRAZ, Ghulam M. WAINSE, Muhammad JAMIL Response of onion to different nitrogen levels and method of transplanting in moderately salt affected soil Odziv čebule na različna gnojenja z dušikom in tehnike sadnje v razmerah zmerno slanih tal John K. M. KUWORNU, Alfred B. NARH JNR, Irene S. EGYIR, Edward E. ONUMAH, Solomie GEBREZGABHER Willingness to pay for excreta pellet fertilizer: Empirical evidence from Ghana Pripravljenost za nakup gnojilnih pelet iz fekalij: empirični primer iz Gane Jože PODGORŠEK Unfair practices and illicit conduct in food supply chains in Slovenia Nepoštene prakse in nedovoljena ravnanja v verigi preskrbe s hrano v Sloveniji Masoumeh JALALI, Mina RASTGOU Natural incidence of bean viruses in the northwest of Iran Razširjenost virusov fižola v severozahodnem Iranu Rajko BERNIK, Peter KUHAR The analysis of technical suitability of the equipment for application of plant protection products in Southeastern Slovenia Analiza tehnične ustreznosti opreme za uporabo sredstev za zaščito rastlin v jogovzhodni Sloveniji Abdul MAJEED, Zahir MUHAMMAD, Manzoor HUSSAIN, Habib AHMAD In vitro allelopathic effect of aqueous extracts of sugarcane on germination parameters of wheat In vitro alelopatski učinki vodnih izvlečkov sladkornega trsa na parametre kalitve navadne pšenice Hassan NIKKHAHKOUCHAKSARAEI, Hamlet MARTIROSYAN Assessment of heritability and genetic advance for agronomic traits in durum wheat (Triticum durum Desf.) Ovrednotenje dednosti in genetske prednosti agronomskih lastnosti trde pšenice (Triticum durum Desf.) Antara SEAL, Ranjan BERA, Anupam DATTA, Susmita SAHA, Rupkatha R. CHOWDHURY, Kajal SENGUPTA, Arun K. BARIK, Ashis K. CHATTERJEE Evaluation of an organic package of practice towards integrated management of Solanum tuberosum and its comparison with conventional farming in terms of yield, quality, energy efficiency and economics Ovrednotenje nabora ekoloških postopkov v integrirani pridelavi krompirja (Solanum tuberosum L.) v primerjavi s konvencionalno glede na pridelek, kakovost, energetsko učinkovitost in ekonomičnost Abd El-Hafeez ZOHRY, Samiha OUDA, Wael HAMD-ALLA, El-Sayed SHALABY Evaluation of different crop sequences for wheat and maize in sandy soil Ovrednotenje različnih kolobarjev za koruzo in pšenico na peščenih tleh Ghader HABIBI, Atousa VAZIRI High salicylic acid concentration alters the electron flow associated with photosystem II in barley Velika koncentracija salicilne kisline spreminja pri ječmenu fotosintezni, s fotosistemom II povezan elektronski pretok Mohammad Reza NAGHAVI, Marouf KHALILI Evaluation of genetic diversity and traits relations in wheat cultivars under drought stress using advanced statistical methods Ovrednotenje genetske raznolikosti in razmerij med lastnostmi pri sortah krušne pšenice v razmerah sušnega stresa s statističnimi metodami Eskandari HAMDOLLAH, Alizadeh-Amraie ASHRAF Effect of partial root-zone irrigation system on seed quality changes of Persian clover (Trifolium resupinatum L.) during seed development and maturation Vpliv delnega namakanja korenin na spremembe v kakovosti semena perzijske detelje (Trifolium resupinatum L.) med njegovim razvojem in zorenjem Rajko BERNIK, Bojan GOSPODARIC, Jošt POTRPIN, Uroš BENEC The use of the model for determining potato (Solanum tuberosum L.) tuber distribution in the soil Uporaba modela za ugotavljanje razporeditve gomoljev krompirja (Solanum tuberosum L.) v tleh Borut BOHANEC, Tanja BOHINC, Filip VUČAJNK Vrednotenje pridelka koruze za sonarave oblike kmetovanja Evaluation of maize crops for sustainable farming Mohsen JANMOHAMMADI, Nasrin MOHAMMADI, Fariborz SHEKARI, Amin ABBASI, Mohammad ESMAILPOUR The effects of silicon and titanium on safflower (Carthamus tinctorius L.) growth under moisture deficit condition Učinki silicija in titana na rast žafranike (Carthamus tinctorius L.) v razmerah pomankanja vode Barbara GERIČ STARE, Saša ŠIRCA, Gregor UREK First report of nematodes Parasitylenchus bifurcatus Poinar & Steenberg, 2012 parasitizing multicolored Asian lady beetle Harmonía axyridis (Pallas, 1773) in Slovenia Prva najdba ogorčic Parasitylenchus bifurcatus Poinar & Steenberg, 2012, parazitov pisane polonice Harmonia axyridis (Pallas, 1773) v Sloveniji Ingrid ALLEMANN, Maria Elizabeth CAWOOD, James ALLEMANN Influence of altered temperatures on allelopatic properties of Amaranthus cruentus L. Vpliv spremenjenih temperatur na alelopatske lastnosti mehiškega zrnatega ščira Amaranthus cruentus L. Popravki Errata Navodila avtorjem Notes for authors doi: 10.14720/aas.2017.109.2.01 Original research article / izvirni znanstveni članek Effect of cropping system and age of plant at harvest on tuber rot and performance of elite cassava varieties in derived savannah ODEDINA1 J. N., ADIGBO2* S. O., KULAKO3 P., ILUEBBEY3 P., FABUNMI1 T. O., AFOLABI4 C. G., and OSIPITAN4 A. A. Received February 12, 2016; accepted August 03, 2017. Delo je prispelo 12. februarja 2016, sprejeto 03. avgusta 2017. ABSTRACT Devastated tuber rot disease among farmers prompted the evaluation of the elite improved varieties in the intercrop and the practice of delaying harvesting when there is glut in the market necessitated this study. Trial was carried out at the Federal University of Agriculture, Abeokuta between 2011 and 2014 to evaluate yield performance of 21 elite cassava varieties planted as sole crop verse intercropped and harvested at different age. The 2 x 21 x 3 factorial experiment was laid out in randomized complete block design and replicated three times. The tuber yield obtained from sole plot in 2011/2012 cropping season was significantly higher than intercrop whereas those of 2012/2014 cropping season were similar. Land Equivalent Ratio was above one in both cropping seasons indicating that the performance of the improved varieties in intercrop was efficient. The pooled mean tuber yield showed that TMS 30572, 92/0326, 95/0211, 01/1371, 00/0338, 01/0046, 00/0098, 01/1097, 01/0085, 98/0581 and 98/510 were among the top eight varieties. Harvesting could be delayed up to 15 months after planting to reduce tuber rot. Key words: cassava; intercrop; tuber rot; delay harvest; Nigeria IZVLEČEK UČINKI NAČINA GOJENJA IN STAROSTI RASTLIN OB SPRAVILU NA POJAVLJANJE GNILOBE GOMOLJEV PRI ELITNIH SORTAH MANIOKE V ANTROPOGENI SAVANI NIGERIJE Zaradi prakticiranja odloga spravila pridelka manioke, kadar se pojavlja njen višek na trgu in pojavljanja uničujoče gnilobe gomoljev, se je pojavila potreba po ovrednotenju elitnih sort te tropske gomoljevke, gojene v medsadnji. Poskus je bil izveden na Federal University of Agriculture, Abeokuta med 2011 in 2014 z namenom ovrednotenja pridelka 21 elitnih sort manioke, posajene v monokulturi ali v kombinaciji z drugimi kulturami in pospravljene v različnih časovnih obdobjih. Faktorski 2 * 21 * 3 poskus je bil izveden po sistemu naključnih blokov s tremi ponovitvami. Pridelek gomoljev na površinah z monokulturo je bil v rastni sezoni 2011/2012 značilno večji kot na površinah z medsadnjo, v rastni sezoni 2012/2014 pa sta bila pridelka podobna. Ekvivalent zemljišča je bil nad ena v obeh rastnih sezonah in kaže prednost izboljšanih sort, gojenih v medsadnji. Analiza povprečnih vrednosti pridelka gomoljev je pokazala, da so bile sorte TMS 30572, 92/0326, 95/0211, 01/1371, 00/0338, 01/0046, 00/0098, 01/1097, 01/0085, 98/0581 in 98/510 med osmimi najdonosnejšimi. Za zmanjšanje gnilobe je izkop gomoljev lahko zamaknjen do 15 mesecev po sadnji. Ključne besede: manioka; medkultura; gniloba gomoljev; poznejši izkop; Nigeria 1 INTRODUCTION Cassava (Manihot spp.) belongs to the family of Euphorbiaceae. Cassava is one of the most important food crops in Africa, South America and Asia. It derives its importance from the fact that its starchy, thickened, tuberous roots are a valuable source of cheap calories, especially in the developing countries where calorie deficiency and malnutrition are widely spread. Its usage as a source of ethanol for fuel, energy in animal feed, 1 Federal University of Agriculture, Abeokuta, Nigeria; Plant Physiology and Crop Production Department 2 Federal University of Agriculture, Abeokuta, Nigeria; Crop Research Programme, IFSERAR; Corresponding author: adigboso@funaab.edu.ng 3 International Institute of Tropical Agriculture, Ibadan, Nigeria 4 Federal University of Agriculture, Abeokuta, Nigeria; Crop Protection Department Acta agriculturae Slovenica, 109 - 2, september 2017 str. 165 - 173 J. N. ODEDINA et al. and starch for industries is increasing. Cassava contributes the largest share of daily per capita food consumption (1.6 kg) in Nigeria (FAOSTAT, 2003) and ranked number one among the top 20 commodities produced in Nigeria (www.fao.org/faostat/en/#rankings/commodities_by_co untries) for more than estimated 800 million people around the world (Akparobi et al., 1998; Lebot, 2009). Nigeria, Thailand, Indonesia and Brazil were ranked as first, second, third and fourth respectively, among the top 20 countries producing cassava in the world. The current estimated cassava production in 2013 for Nigeria, Thailand, Indonesia and Brazil were 47.4, 30.2, 23.0 and 21.5million tonnes, respectively (www.fao.org/faostat/en/#rankings/countries_by_comm odities). Total area harvested in 2009 was 3.13 million ha, with an average yield of 11.7 t ha-1 (FAO, 2010). It is produced predominantly (99 %) by small farmers with 1-5 ha of land intercropped with yams, maize, or legumes in the rainforest and savannah agro-ecologies of Southern, Central, and lately Northern Nigeria. The world production of cassava root was estimated to be 184 million tonnes in 2002. IFSERAR, (2009) conducted a diagnostic survey in South West Nigeria and reported that the local varieties grown among the farmers were not only low yielders but their ability to tolerate, or resist new strains of diseases and pests occasioned, perhaps, by climate change. Mwangi et al. (2004) similarly reported that the root rots are an important constraint to cassava production in humid forest and forest transition of Central and West Africa and can impact negatively on food security to several millions people inhabiting the regions. Rotting is known to increase significantly if mature plants are left in the soil for extended period of time (Oyeka, 2004). Yield loss was estimated at 20 to 100 % in Democratic Republic of Congo (Mwangi et al., 2004). These challenges necessitated the evaluation of 21 promising varieties collected from International Institute of Tropical Agriculture (IITA) in maize/cassava intercrop. IITA have released several high yielding varieties but there performances in the intercrop as well as their tolerant or resistant level to cassava root rot disease have not been documented. There is therefore the need to ascertain the performance of these elite cassava varieties under the predominant intercropping systems among the resource constraint farmers in the region. Besides, the highly perishable nature of cassava tubers has compelled the farmers to harvest only when there is availability of market or family need. This delay harvesting enables farmers to leave the mature plant in the soil as a form of storage. This storage period enable the farmers to keep the fresh tuber in good quality for an extended period. Growth and dry matter accumulated will continue since cassava is believed to mature 7-24 months. Most cassava varieties attain optimum weight at 18 months after planting when starch accumulation is highest (Ekanayake et al., 1997). Hammer et al. (1987), who evaluated sequential harvests to age 24 months, reported that root rot occurred in the second year. Sagrilo et al. (2006) quoted Sagrilo et al. (2002) that cassava harvested at 21 months could improve storage root yield compared to 12 months. Ebah-Djedji et al.(2012) who harvested cassava sequentially at 11, 13, 15 and 17 months after planting in Cote d' Ivoire recommended that tuberous root of improved cassava varieties should be harvested at 13 MAP to obtain optimum dry matter content. These inconsistencies in the appropriate time of harvesting to obtain optimum dry matter content and quality is further aggravated by the prevailing tuber rot. Consequently, there is the need to ascertain the appropriate time to harvest these elite cassava varieties. This will ensure maximum dry matter accumulation without losing much of the tuber to root rot, particularly when harvesting is delayed because of poor market arrangement. The objectives of this study therefore were to: evaluate the performance of the improved varieties, 2) investigate the effect of intercropping on the elite cassava varieties and 3) determine the effect of delay harvest on the cassava tuber rot. 2 MATERIALS AND METHODS The study was carried out at the Institute of Food Security, Environmental Resource and Agricultural Research (IFSERAR) farm, Federal University of Agriculture, Abeokuta in 2011/2012 and repeated in 2012-2014 cropping seasons. The experiment was laid out in a Randomized Complete Block Design in split plot and replicated three times in the 2011/2012. Cropping systems and variety factors were assigned to the main plot and sub plot, respectively. However, in 2012-2014 cropping season, harvesting date (12, 15 and 18 months) was varied as the third factor i.e. sub subplot (split split-plot) to gain additional information. The intercrop proportion mixture and population adopted was additive series. Table 1 shows the twenty one elite cassava varieties collected from International Institute of Tropical Agriculture (IITA), Ibadan. Benlate treated stem cuttings of 25 cm were planted into plot size of 9 m x 7 m (1.2 ha experimental field) at spacing of 1 m x 1 m in July 2011 and harvested in July, 2012. The cuttings obtained from the harvest were replanted in 166 Acta agriculturae Slovenica, 109 - 2, september 2017 Effect of cropping system and age of plant ... performance of elite cassava varieties in derived savannah July 2012 and harvested sequentially in July (12 months after planting MAP), October 2013 (15 MAP) and January 2014 (18 MAP). Three seeds per hole of treated maize 'SUWAN 1' variety was alternately planted in-between cassava stands (in the intercrop plots only) to evaluate the performance of cassava under intercropping (i.e. additive series). Maize was harvested at green stage. Weeding was carried out at 3, 9 12 WAP. Other weedings were done once in a month. Fertilizer 400 kg/ha N: P: K: Mg (12:12:17:2) was applied in the 2011/2012 cropping season whereas 2012/2014 trial did not receive fertilizer because of circumstance beyond our control. 2.1 Data collection on cassava 2.1.1 Plant height (cm): 5 randomly selected cassava plants within the plot were measured with aid of graduated meter rule from the ground level to the highest leaf. 2.1.2 Stem girth (mm): Vernier caliper was used to determine the stem girth (at 10 cm above the ground) of 5 randomly selected cassava stems within the plot. Table 1: Selected cassava varieties used for the experiment 2.1.3 Tuber girth (mm): Vernier caliper was used to determine the tuber girth of 5 randomly selected freshly harvested tubers from ten up rooted cassava stands samples 2.1.4 Number of tubers per plant: Determined by average number of freshly harvested tuber from the ten samples uprooted. 2.1.5 Rot incidence (%): This was done by dividing the rotted tubers by total tuber multiplied by 100. 2.1.6 Tuber yield (t/ha): The mass of uprooted tuber from the ten sampled cassava stand was converted to t/ha. (i.e. mass of sampled/sampled area*10000/1000} 2.2 Data analysis: Data collected were subjected to analysis of variance using GenStat Edition 12. Significant means were separated by using DMRT at 5 % probability. Variety Tuber color TMS98/0581 White TMS 01/1797 White TMS 95/0211 White TME 1 White TMEB 693 White TMS 01/0046 White TMS 01/0093 White TMS 00/0338 White TMS 01/1097 White TMS 01/1086 White TME B 419 White TMS 30572 White TMS 01/1371 Yellow TMS 01/0085 White TMS 98/0510 White TMS 01/0131 White TMS 98/0505 White TMS 92/0326 White TMS 01/0098 White TMS 01/1368 Yellow TMS 97/JW2 Yellow Acta agriculturae Slovenica, 109 - 2, september 2017 169 J. N. ODEDINA et al. 3 RESULTS AND DISCUSSION 3.1 Influence of intercropping on the plant height of elites cassava varieties at 12 MAP Plant height and stem girth are essential component to determine plant growth particularly when intercrop is involved. The plant height of the 21 varieties obtained at 12 months after planting varied significantly (P>0.05) from each other in the 2011/2012 cropping season (Table 2). TME B 419 had the tallest plants but comparable to TMEB 693, TMS 01/1097, TMS 01/1797, TME 1, TMS 01/1086, TMS 01/1371,TMS 97/JW2 and TMS 92/0326. Whereas, TMS 98/505 had the shortest plant which was similar to those of TMS 01/0098, TMS 01/0131, TMS 01/0046, TMS 01/0093 and TMS 00/0338. In 2012/2014 cropping season, TMS 97/JW2 and TMS01/0093 had the tallest plants at 12 MAP but were similar to those of TMS 95/0211, TME 1, TMS 00/00338, TME B 419, TMS 30572, TMS 01/1371, TMS 01/0085, TMS 98/0510, TMS 92/0326, TMS 01/0098 and TMS 01/1386 (Table 3). However, TMS 01/1797, TMEB 693, TMS 01/0046, TMS 01/1097, TMS 01/1086, TMS 01/0131 and TMS 98/0505 had the shortest plants in 2012/2014.The plant heights in the two seasons were at variance except those of TMS 97/JW2, TME B 419 and TMS 01/1371 which were consistently top on the list, whereas TMS 01/0131, TMS 01/0046 and TMS 98/0505 constantly had the shortest plants. This consistency in plant height implies that the varieties were stable in the different environment, whereas the others were influenced more by the environment. 3.2 Influence of intercropping on the tuber number of elites cassava varieties at 12 MAP The number of fresh tubers observed in 2011/2012 on cassava varieties TME 1, TMS 98/0505, TMS 97/JW2, TMS 98/0581, TMS 01/1097, TMS 01/1386, TMS 30572, TMS 01/1086, TMS 01/0085 and TMS 00/0338 were similar but significantly higher than those of TMS 01/1371 and TMS 01/0131 varieties in 2011/2012 cropping season (Table 2). The varieties TMS 30572, TME 1, TMS 01/0093, TMS 00/0338, TMS 1097, TMS 01/0046, TMEB 693, TMS 98/0510 and TMS 92/0326 in 2012/2014 were among the top varieties with high number of fresh tuber while TME B 419 had the least (Table 3). TME 1, TMS 01/1097, TMS 30572 and TMS 00/0338 were constantly ranked amongst the top varieties with high number of tubers in 12 MAP of the two seasons. The variance in tuber number could be genetically inherent and was considered as vital yield attribute that contributes immensely to the increase in tuber yield. It has been documented that the increase in yield were attributed to increase in number of tuber/stand and single root mass (Kogram et al., 2002). 3.3 Influence of intercropping on the tuber girth of elites cassava varieties at 12 MAP The cropping system and variety did not influence tuber girth in 2011/2012 (Table 2), however, in 2012/2014 the varieties varied significantly among each other in 2012/2014 at 12 MAP (Table 3). 'TMS 98/0510' had the highest tuber girth while 'TMS 97/JW2' had the least. The stem girths of the varieties were influenced by cropping systems in both seasons of the trial at 12 MAP (Tables 2 and 4). However, the varieties TMS 00/0338 and TMS 98/0505 consistently recorded the highest and the lowest, respectively in 12 MAP of both cropping season. 3.4 Influence of intercropping on the root rot of elites cassava varieties at 12 MAP The cropping systems did not influence tuber rot infection in both seasons but there were significant differences among the varieties in 12 MAP of 2011/2012 (Table 2), they were however similar in 2012/2014 (Table 4). The rot incidence observed in 2011/2012 was high and ranges between 9.8 and 22.5 % while that of 2012/2014 was low and range between 0.00 and 0.94 % at 12 MAP. The lost incurred during 2011/2012 cropping season is in consonant with the finding of Mwangi et al. (2004) who documented 20 to 100 % tuber lost. 3.5 Influence of intercropping on the LER and tuber fresh mass of elites cassava varieties at 12 MAP The Land Equivalent Ratio (LER) was similar in both cropping season but above one suggesting that intercrop plots was more productive. The fresh tuber mass of the varieties varied in the two cropping seasons at 12 MAP (Tables 2 and 4). Sole cassava plots had significantly higher tuber mass than intercrop in 2011/2012 (Table 2) but similar 2012/2014 cropping seasons (Table 4). In 2011/2012 cropping season, TMS 98/0505, TMS 97/JW2, TME 1, TMS 30572, TMS 95/0211, TMS 92/0326, TMS 01/0085, TMS 01/0098, and TMS 98/0581 varieties were the nine topmost in terms of fresh tuber mass at 12 MAP. Whereas the following varieties: TMS 01/1086, TMS 01/1368, TMS 98/0510, TMS 01/1097, TMS 01/1371, TMS 01/1797 and TMS 01/0046 closely followed. However, 'TMEB 693' had the lowest tuber yield. The fresh tuber yields range between 27.5 and 57.4 t ha-1 in 2011/2012 while those of 2012/2014 was 12 to 32.3 t ha-1. The yield range obtained in 2011/2012 was substantially higher than that of 2012/2014 at 12 MAP as expected. However, the results obtained in 2012/2014 was comparable to range of 9.9 to 30.1, 8.49 to 28.38, and 10.0 to 26.9 t ha-1 as 166 Acta agriculturae Slovenica, 109 - 2, september 2017 Effect of cropping system and age of plant ... performance of elite cassava varieties in derived savannah reported by IITA (1987), Maroya et al. (2010) and Ssemakula and Dixon (2007), respectively. The eleven topmost varieties in 2012/2014 cropping season at 12 MAP, were TMS 00/0338, TMS 30572, TMS 92/00326, TMS 01/1097, TMS 98/0581, TMS 01/0046, TME 1, TMS 98/0510, TMS 01/0085, TMS 01/1371,and TMS 95/0211 in that order. These were closely followed by TMS 01/1086, TMS 01/0093, TMS 01/1797, TMS 01/0098, TMS 01/1368, TME B 419 and TMS 01/0131 varieties. While TMS 97/JW2 variety had the lowest tuber yield. The variation in tuber yield of the varieties agreed with the finding of Howeler (2007); Mulualem and Ayenew (2012); Odedina et al. (2012) who reported that yields of cassava roots vary with cultivars, plant growth conditions (soil, climate, rainfall) and agronomic practices. It is pertinent to note that the variation in tuber yield of the 21 varieties was only observed in 12 MAP (Tables 2 and 4) while the harvest made at 15 and 18 MAP had similar tuber yield. This is, perhaps, an indication that maturity had not been attained and thus dry matter accumulation were at variance at 12 MAP. Although, number of tuber, stem girth and tuber girth varies among the varieties at 15 and 18 MAP (Table 3) but all the varieties had similar tuber yield (Table 4). This is an indicative of the fact that all the varieties tested in this trial attained maturity period after 12 MAP. Based on the definition of maturity period of cassava by Benesi et al. (2008) is the point where maximum or near maximum yield is obtained. The topmost 3 consistent varieties in the two cropping seasons were TMS 30572, TMS 92/0326 and TMS 98/0581. Although 'TMS 01/1371' and 'TMS 01/1386' were not listed among top yielder, but had beta carotene as an advantage and statistically comparative yield with the top varieties in the two cropping seasons. Table 2: Influence of intercropping on the tuber rot, agronomic parameters and tuber yield performance of elites cassava varieties in 2011/2012 cropping season Treatment Plant height Fresh Tuber girth Stem girth Rot incidence LER Fresh tuber (m) tuber (mm) (mm) (%) mass (t ha-1) no.plant-1 Cropping System (CS) Sole 2.74 7.2 63.66 28.93 14.3 - 44.2a Intercrop 2.72 6.9 62.22 28.52 14.3 - 39.4b LSD NS NS NS NS NS - 2.07 Variety (V) TMS 98/0581 2.78bcde 7.3a-e 69.13 32.75ab 22.5a 1.96 41.7abc TMS 01/1797 2.98abc 7.0b-f 59.97 27.94abc 18.3a-d 1.73 38.5bcd TMS 95/0211 2.61cdef 6.8b-f 67.04 28.75abc 11.7ef 1.40 46.5abc TME 1 2.93abcd 8.7a 64.40 28.26abc 10.2f 1.96 47.6abc TMEB 693 3.13ab 7.0b-f 57.22 27.12abc 13.4d-f 1.56 27.5d TMS 01/0046 2.49efg 6.7c-f 66.14 32.22abc 16.3b-f 1.86 38.1bcd TMS 01/0093 2.49efg 6.2d-f 60.44 25.76bc 18.2a-d 1.60 35.3cd TMS 00/0338 2.31fg 7.5a-e 58.90 34.24a 12.0ef 1.56 34.5cd TMS 01/1097 3.00abc 6.0ef 61.23 29.72abc 11.8ef 1.73 40.1bcd TMS 01/1086 2.69adef 8.0abc 60.02 26.88abc 13.5c-f 1.96 41.2bc TME B 419 3.23a 6.7c-f 61.85 27.96abc 12.9d-f 1.73 37.0cd TMS 30572 2.78bcde 8.0abc 65.23 28.68abc 13.6c-f 1.73 47.2abc TMS 01/1371 2.90abcd 5.7f 58.45 29.19abc 20.1ab 1.56 39.4bcd TMS 01/0085 2.75cde 7.3a-e 63.83 27.81abc 16.6a-e 1.50 45.8abc TMS 98/0510 2.66cdef 6.2d-f 73.30 31.51abc 19.5abc 2.03 40.6bcd TMS 01/0131 2.54defg 5.7f 60.41 27.23abc 16.6a-e 1.56 36.6cd TMS 98/0505 2.21g 8.2ab 66.08 24.52c 12.9d-f 1.70 57.4a TMS 92/0326 2.85abcde 7.0b-f 68.70 28.65abc 13.9c-f 1.50 45.9abc TMS 01/0098 2.40fg 6.7c-f 61.75 28.95abc 15.0b-f 1.90 45.1abc TMS 01/1368 2.82bcde 7.7abc 61.01 31.16abc 11.5ef 1.80 40.3bcd TMS 97/JW2 2.85a-e 8.2ab 56.58 25.97abc 9.8f 1.90 51.3ab SE (V) 0.201 0.75 12.53 4.147 3.07 NS 6.69 CS X V NS NS NS NS NS NS NS NS = not significant Acta agriculturae Slovenica, 109 - 2, september 2017 169 J. N. ODEDINA et al. Table 3: Influence of intercropping on agronomic performance of elite cassava varieties at different age of plant in 2012/2014 cropping season Plant height (m) Fresh tuber No. Tuber girth (mm) Treatment 12 MAP 15 MAP 18 MAP 12 MAP 15 MAP 18 MAP 12 MAP 15 MAP 18 MAP Cropping systems (CS) Sole 2.25 2.71 2.87 6.1 6.6 5.71 57.22 67.1 64.1 Intercrop 2.31 2.76 2.94 6.3 6.6 5.67 58.66 66.9 65.0 LSD NS NS NS NS NS NS NS NS NS Variety (V) TMS 98/0581 2.66ab 3.15abc 3.04abc 6.2b-f 6.7a-d 5.6a-e 64.1ab 66.1a-e 69.5abc TMS 01/1797 1.70d 2.12d 2.21bc 5.5c-f 4.8cd 4.5cde 55.0bc 64.9a-e 57.4cde TMS 95/0211 2.27a-d 2.41bcd 2.73abc 6.2b-f 6.2a-d 6.0a-d 62.0abc 66.2c-e 68.1a-d TME 1 2.27a-d 2.93a-d 3.13abc 7.2a-d 7.2abc 7.6ab 59.4abc 67.7a-e 56.9de TMEB 693 1.97cd 2.35bcd 2.53a 8.0a-c 8.5a 7.7ab 52.2bc 57.4e 57.7b-e TMS 01/0046 1.94cd 2.22cd 2.20bc 7.0a-e 6.8a-d 6.3abc 61.1abc 66.6a-e 69.3a-d TMS 01/0093 2.80a 3.78a 3.66a 8.2ab 8.8a 6.7abc 55.4bc 58.5e 66.2a-e TMS 00/0338 2.49abc 2.74bcd 2.85abc 7.3a-d 6.5a-d 5.7a-e 53.9bc 63.0b-e 62.6a-e TMS 01/1097 2.09bcd 2.43bcd 2.90abc 6.3a-f 6.3a-d 6.0a-d 56.2abc 74.0ab 63.8a-e TMS 01/1086 2.11bcd 2.79bcd 2.94abc 5.0def 6.3a-d 4.7cde 55.0bc 61.1c-e 61.3a-e TME B 419 2.41abc 2.54bcd 2.78abc 4.2f 5.1bcd 3.2e 56.9abc 74.1ab 72.6ab TMS 30572 2.26a-d 2.62bcd 3.07abc 8.7a 8.3ab 8.3a 60.2abc 71.2a-d 63.7a-e TMS 01/1371 2.43abc 3.10abc 3.16ab 5.8b-f 7.5abc 6.5abc 53.5bc 68.5a-e 68.4a-d TMS 01/0085 2.23a-d 2.77bcd 3.00abc 5.8b-f 7.7abc 5.5b-e 58.8abc 72.5abc 63.3a-e TMS 98/0510 2.25a-d 2.79bcd 2.98abc 7.0a-e 6.7a-d 5.7a-e 68.3a 74.9a 74.3a TMS 01/0131 1.94cd 2.34bcd 2.23bc 5.2def 5.0b-d 4.3cde 55.4bc 67.6a-e 60.7b-e TMS 98/0505 2.05bcd 2.32bcd 2.12c 4.7ef 3.7d 3.3de 61.1abc 71.4a-d 65.2a-e TMS 92/0326 2.41abc 3.02a-d 3.40a 7.3a-d 7.5a-c 6.0a-d 63.7abc 72.1a-d 71.6abc TMS 01/0098 2.48abc 3.26ab 3.43a 5.8b-f 6.8a-d 5.3b-e 56.8abc 62.0c-e 53.9e TMS 01/1368 2.35abc 2.53bcd 3.09abc 4.7ef 5.7a-d 5.3b-e 56.0abc 67.7a-e 68.8a-d TMS 97/JW2 2.77a 3.12ab 3.52a 4.7ef 6.5a-d 5.3b-e 51.6c 60.4de 60.9a-e SE 0.65 0.96 1.02 2.5 3.4 2.8 12.5 11.9 13.8 CS x V NS NS NS NS NS NS NS NS S NS = not significant, S = significant Table 4: Influence of intercropping on root rot and tuber yield of elite cassava varieties at different age of plant in 2012/2014 cropping season Stem girth (mm) Root rot incidence (%) Fresh tuber mass (t ha-1) Treatment 12 MAP 15 MAP 18 MAP 12 MAP 15 MAP 18 MAP LER 12 MAP 15 MAP 18 M Cropping systems (CS) Sole 23.9 23.1 23.3 0.29 0.48 11.61 23.7 31.0 27.0 Intercrop 23.52 23.7 23.0 0.31 0.73 11.06 - 24.6 31.5 26.6 LSD NS NS NS NS NS NS - NS NS NS Variety (V) TMS 98/0581 27.8ab 24.7abc 24.4a 0.39 1.60 35.14 1.46 29.3ab 30.0 23.6 TMS 01/1797 22.9c-g 21.2bcd 23.1ab 0.14 0.44 4.93 1.23 22.1a-d 29.2 26.7 TMS 95/0211 23.8b-f 24.9abc 24.1a 0.5 2.39 10.99 1.10 24.5abc 38.9 35.8 TME 1 23.3c-g 25.0abc 23.0ab 0.12 0.94 28.02 1.46 26.7abc 26.5 22.4 TMEB 693 22.1d-g 22.2a-d 20.7ab 0.0 0.50 14.40 1.06 19.7cde 25.4 27.2 TMS 01/0046 25.2a-e 20.7cd 21.0ab 0.94 1.40 11.20 1.36 27.4abc 37.3 25.5 TMS 01/0093 20.8f-g 22.2a-d 22.2ab 0.24 0.56 12.10 1.10 22.6a-d 30.7 26.2 TMS 00/0338 29.2a 22.7a-d 27.1a 0.73 0.73 7.57 1.06 32.3a 29.1 29.5 TMS 01/1097 24.7b-f 25.1abc 23.3ab 0.24 1.43 5.36 1.23 29.6ab 31.7 25.8 TMS 01/1086 21.9efg 21.6a-6 21.6ab 0.00 0.78 17.48 1.46 23.1a-d 29.2 24.4 TME B 419 23.0c-g 24.7abc 24.4a 0.27 0.80 4.74 1.23 21.3a-d 27.0 20.8 TMS 30572 23.7b-f 22.5a-d 25.3a 0.14 2.09 21.97 1.23 32.0a 39.0 35.4 TMS 01/1371 24.2b-f 26.3a 24.4a 0.00 1.78 20.02 1.06 25.0abc 37.5 32.7 TMS 01/0085 22.8c-g 23.1a-d 23.3ab 0.56 0.65 4.61 1.10 25.6abc 33.6 30.5 TMS 98/0510 26.5abc 23.5a-d 26.2a 0.27 1.24 18.34 1.53 26.6abc 30.4 24.7 TMS 01/0131 22.2d-g 23.7a-d 21.8ab 0.27 1.49 4.25 1.06 20.1a-d 24.2 20.2 TMS 98/0505 19.5g 22.0a-d 17.7b 0.14 1.01 11.33 1.20 15.2cd 23.1 17.7 TMS 92/0326 23.7b-f 26.2ab 23.7a 0.00 1.43 16.31 1.10 30.1ab 38.5 31.8 TMS 01/0098 24.0b-f 24.4a-d 23.0ab 0.71 1.01 2.85 1.40 22.0a-d 33.3 32.3 TMS 01/1368 26.2a-d 25.0abc 23.8a 0.27 0.56 2.92 1.30 21.6a-d 39.4 21.2 TMS 97/JW2 21.0fg 19.5d 21.4ab 0.94 0.27 8.80 1.50 12.0d 21.5 28.3 SE 4.2 5.0 5.8 NS NS NS NS 12.3 NS NS CS X V NS NS NS NS NS NS NS NS NS NS NS = not significant, S = significant 170 166 Acta agriculturae Slovenica, 109 - 2, september 2017 Effect of cropping system and age of plant ... performance of elite cassava varieties in derived savannah 3.6 Influence of age at harvest and intercropping on the plant height, tuber girth and tuber rot of elites cassava varieties at 12, 15 and 18 MAP The pooled mean plant height, tuber girth and tuber rot obtained from 2012/2014 showed significant difference among the ages of plant at harvest (Table 5). The similarity in height and tuber girth of plants harvested at 15 and 18 MAP buttressed the fact that maturity had been attained compared to 12 MAP. The higher incidence root rot recorded in 18 MAP accounted for the apparent decline in fresh tuber mass. This result contradicts the finding of Mulualem and Ayenew (2012) who recommended 18 months as the appropriate age to harvest cassava to get the desired yield. The pool mean of varieties TMS 01/0093, 97/JW2 and 01/0098 had similar but the tallest plants. However, varieties 97/JW2 and 01/0098 were not significantly taller than those of TMS 92/0326, TMS 98/0581 and TMS 01/1371. The following varieties TMS 01/0131, TMS 98/0505, TMS 01/1797, TMEB 693 and TMS 01/0046 had the shortest plants. The pooled mean fresh tuber number of varieties TMS 30572, TMEB 693, TMS 01/0093 and TME 1 were similar and highest among the others. But the tuber number of varieties TMS 01/0093 and TME 1 were not significantly higher than at TMS 92/0326. Variety TMS 98/0505 had the minimum number of fresh tubers but not significantly lower than TMS 01/1797, TME B 419 and TMS 01/0131.The tuber girth of varieties TMEB 693, TMS 01/0093, TMS 00/0338, TMS 01/1086, TMS 97/JW2, TMS 01/1797 and TMS 01/0098 were similar but significantly lower than those of TMS 98/0510, TMS 92/0326, TME B 419 and TMS 98/0581. The stem girth of varieties TMS 98/0510, TMS 00/0338 and TMS 98/0581 were significantly higher compared to others whereas TMS 97/JW2 and TMS 98/0505 had the lowest. 3.7 Influence of age at harvest and intercropping on the tuber yield of elites cassava varieties at 12, 15 and 18 MAP Although the tuber yield recorded for the three ages were similar but dropped at 18 MAP evidently due to rot damage. Ebah-Djedji et al. (2012) reported decline in cassava tuber at 17 months old, however, the decline was not linked to root rot. Hammer et al. (1987) reported that root rot occurred in the second year but was not specific on the number of months. The tuber fresh mass of varieties were significantly different from one another. The following varieties TMS 30572, TMS 92/0326, TMS 95/0211, TMS 01/1371, TMS 00/0338, TMS 01/0046, TMS 00/0098 and TMS 01/1097 were among the topmost eight varieties whereas TMEB693, TMEB 419, 01/0131, TMS 97/JW2 and TMS 98/0505 were the least. Generally, the consistence in plant heights values at 12 MAP of varieties TMS 97/JW2, TME B 419 and TMS 01/1371 (which ranked among the top) and those of TMS 01/0131, TMS 01/0046 and TMS 98/0505 (at the bottom of the list) in both cropping seasons are indication of their stability despite differences in crop management. Besides, the ability of TMS 30572, TMS 92/0326 and TMS 98/0581 to constantly rank among the first six varieties at 12 MAP in the two seasons makes them candidates to be recommended to farmers (Table 6). The wide gap in tuber yield between the two cropping seasons could be attributed to the fertilizer application. Although, farmers hardly use fertilizer for cassava production because of the notion that cassava can thrive on marginal soils that cannot sustain other crops. This trial connotes that the addition of fertilizer can substantially enhance tuber yield. Odedina et al. (2012) who worked on integrated nutrient management reported similar gap between control and other sources of nutrient. Ironically, appreciable quantity of root rot was observed in the first cropping season compared to the second, it was not quite clear if the addition of fertilizer was responsible for the tuber rot. Consequently, there is the need to validate whether or not fertilizer application to cassava influences root rot. 4 CONCLUSIONS The study has shown that the cassava varieties were not affected by intercropping but Land Equivalent Ratio was above one in both cropping seasons indicating that the performance of the improved varieties in intercrop was efficient. Plant height and tuber girth were higher in 15 and 18 MAP than 12. On the bases of their consistent performance at 12 MAP, in the two cropping seasons, TMS 30572, TMS 92/0326 and TMS 98/0581 are candidate varieties to be recommended to farmer with or without resource constraints. The pooled mean tuber yield showed that TMS 30572, TMS 92/0326, TMS 95/0211, TMS 01/1371, TMS 00/0338, TMS 01/0046, TMS 00/0098, TMS 01/1097, TMS 01/0085, TMS 98/0581 and TMS 98/510 are top eight varieties. The incidence of tuber rot was highest at 18 MAP hence; harvesting could be delayed up to 15 MAP to reduce Acta agriculturae Slovenica, 109 - 2, september 2017 169 J. N. ODEDINA et al. tuber rot. The three yellow flesh tuber varieties counterpart. identified had comparable performance with their white Table 5: Performance of elite cassava varieties as influenced by age of plant at harvest and intercropping in 2012/2014 cropping season Treatment Plant height Fresh tuber Tuber girth Stem girth Root rot incidence Fresh tuber mass (m) no.plant-1 (mm) (mm) (%) (t ha-1) Age at harvest (H) 12 MAP 2.28b 6.2 57.9b 23.73 1.95b 24.12 15 MAP 2.73a 6.6 67.0a 23.39 0.90c 31.23 18 MAP 2.90a 5.7 64.6a 23.17 3.43a 26.78 LSD 0.30 NS 5.23 NS 0.67 NS Cropping systems (CS) Sole 2.61a 6.1a 62.8a 23.44a 2.09a 27.21a Intercrop 2.67a 6.2a 63.5a 23.42a 2.09a 27.55a LSD NS NS NS NS NS NS H x CS NS NS NS NS NS NS Variety (V) TMS 98/0581 2.95bcd 6.1defg 66.6bc 25.6ab 2.89a 27.76bcdefg TMS 01/1797 2.01j 4.9hijk 59.1fgh 22.4fgh 1.52a 26.01defgh TMS 95/0211 2.47fghi 6.1defg 65.5bcd 24.2bcdef 2.33a 32.98abc TME 1 2.78cdef 7.3abc 61.4defg 23.8bcdef 2.49a 25.21defgh TMEB 693 2.28ghij 8.1a 55.8h 21.7gh 1.76a 24.10efghi TMS 01/0046 212ij 6.7cd 65.7bcd 22.6efg 2.71a 30.09abcde TMS 01/0093 3.41a 7.9ab 60.0efgh 21.7gh 1.99a 26.53cdefgh TMS 00/0338 2.69def 6.5cde 59.8efgh 26.4a 2.25a 30.31abcde TMS 01/1097 2.48fgh 6.2cdefg 64.3bcde 24.4bcde 1.61a 29.04abcdef TMS 01/1086 2.61defg 5.3fghi 59.2fgh 21.7gh 1.89a 25.57defgh TME B 419 2.58efg 4.1jk 67.9abc 24.0bcdef 1.77a 23.04fghi TMS 30572 2.65def 8.4a 65.1bcd 23.8bcdef 2.27a 35.48a TMS 01/1371 2.90b-e 6.6cde 63.5cdef 25.0abcd 1.98a 31.72abcd TMS 01/0085 2.67def 6.3cdefg 64.9bcd 23.1defg 2.09a 28.88bcdef TMS 98/0510 2.67def 6.4cdefg 72.5a 25.4ab 2.37a 27.21bcdefg TMS 01/0131 2.17hij 4.8ijk 61.2defg 22.6efg 1.64a 21.52ghi TMS 98/0505 2.16hij 3.9k 65.9bcd 19.7i 1.93a 18.64i TMS 92/0326 2.94bcd 6.9bcd 69.1ab 24.5abcde 2.02a 33.68ab TMS 01/0098 3.06abc 6.0defgh 57.5gh 23.8bcdef 2.00a 29.19abcdef TMS 01/1368 2.65def 5 2ghij 64.2cde 25.0abcd 1.66a 27.40bcdefg TMS 97/JW2 3.15ab 5.5efghi 57.6gh 20.6hi 2.74a 20.58hi SE (V) 0.36 1.18 4.9 2.0 NS 6.56 H X V NS NS NS NS NS NS CS x V NS NS NS NS S NS H x CS NS NS NS NS NS NS H X CS x V NS NS NS NS NS NS NS = not significant, S = significant 5 ACKNOWLEDGEMENTS The authors wish to thank the Institute of Food Security, Environmental Resources and Agricultural Research (IFSERAR), Federal University of Agriculture, Abeokuta for funding the project and Institute International of Tropical Agriculture (IITA), Ibadan for providing the bulky cassava planting materials and technical support in data collection. 6 REFERENCES Akparobi, S.O., Togun S.O. and Ekanayake, I.J. (1998). Assessment of cassava genotypes for resistance to cassava mosaic disease, cassava bacterial blight and cassava green mite at lowland and mid altitude site in Nigeria. African. Crop Science. Journal, 6, 385396. doi:10.4314/acsj.v6i4.27790 Benesi, I.R.M., Labuschagne, M.T., Herselman, L. Mahungu, N.M. and Saka, J.K. (2008).The effect of genotype, location and season on cassava starch extraction. Euphytica, 160, 59-74. doi:10.1007/s10681-007-9589-x 17? 2 Acta agriculturae Slovenica, 109 - 2, September 2017 Effect of cropping system and age of plant ... performance of elite cassava varieties in derived savannah Ebah-Djedji, B.C., Dje, K.M., N'Zue, B., Zohouri, G.P. and Amani, N.G. (2012). Effect of harvest period on starch and dry matter content from the tuberous roots of improved cassava (Manihot esculenta Crantz) varieties. Pakistan Journal of nutrition, 11 (5), 414-418 doi:10.3923/pjn.2012.414.418 Ekanayake, I.J., Osiru, D.S.O. and Porto, M.C.M. (1997). Agronomy of cassava IITA Research Guide, 60 pp1-20 Food and Agriculture Organization Statistics (FAOSTAT) (2003). www.faostat. Org. Accessed 1/04/2013 FAOSTAT. (2010). www.faostat. Org Accessed 10/03/2013 FAO (2005) FAO STATS. Food and Agriculture Organization of United Nations, Rome, Italy. http://www.fao.org. Accessed 07/05/2012 FAO http ://fao. org/faostat/en/#rankings/commodities_by _countries. Commodities by Countries search. Accessed by 1/08/2017 FAO http ://fao. org/faostat/en/#rankings/countries_by_co mmodities. Countries by Commodities search. Accessed by 1/08/2017 Hammer, G. L., Hobman, F. R. and Shepherd, R. K. (1987). Effects of Planting Time and Harvest Age on Cassava (Manihot esculenta) in Northern Australia. I. Crop Growth and Yield in Moist Environments. Experimental Agriculture, ,23, 401414. doi: 10.1017/S0014479700017361 Howeler, R.H. (2007). Agronomic practices for sustainable cassava production in Asia. In: Centro International de Agricultura Tropical (CIAT). Cassava research and development in Asia: Exploring new opportunities for an ancient crop. Proceedings of the 7th Regional Workshop, held in Bangkok, Thailand, Oct 28 - Nov 1. International Institute of Tropical Agriculture (IITA) (1987). Annual Report of Root, Tuber and Plantain Improvement Program. Ibadan, Nigeria. 90 pp. IITA Institute of Food Security, Environmental Resources and Agricultural Research, (IFSERAR) Federal University of Agriculture, Abeokuta FUNAAB. (2009). Diagnostic survey of Southwestern Agricultural zone of Nigeria. www.unaab.edu.ng. Accessed 10/01/2015. Kogram, C., Maneekao, S., Poosri Boonguer. (2002). Influence of chicken manure on cassava yield and soil properties. Paper presented at 17th WCSS 1421 August 2002, Bangkok, Thailand Lebot, V. (2009). Tropical roots and tuber crops: cassava, sweet potato, yams and aroids. Crop Production science in horticulture series. 17, CABI Publishing, Londres, pp. 50-103. Maroya, N. G., Asante, I. K. and Dixon, A. (2010). Genotype by environment interaction effect on beta-carotene of yellow root cassava (Manihot esculenta Crantz) genotypes in Ghana. Proc. 11th ISTRC-AB Symposium. Kinshasa, DR Congo. 4-8 October, 2010. Mulualem, T. and Ayenew, B. 2012. Cassava (Manihot esculentus Cranz) varieties and harvesting stages influenced yield and yield related component. Journal of Natural Science Research, 2, (10), 122128. Mwangi, M., Bandyopadhyay, R., Dixon, A.G.O. and Tatahangy, W. (2004).The status of fungal rot diseases as constraint to cassava production and utilization in eastern Democratic Republic of Congo. Proceedings of 9th Triennial Symposium of the International Society of Tropical Root Crops-Africa Branch, held from 30st October - 5th November, at Whitesands Hotel, Mombasa, Kenya. Odedina, J.N. Ojeniyi, S. and Odedina, S.A. (2012). Integrated nutrient management for sustainable cassava production in south Western Nigeria. Archives of Agronomy and Soil Science, 58 (1), 132 - 140. doi:10.1080/03650340.2012.695865 Odedina, J.N., Ojeniyi, S., Odedina, S.A., Fabunmi, T.O., Olowe, V. (2015). Growth and yield responses of cassava poultry manure and time of harvest in rainforest Agro-ecological zone of Nigeria. International Journal of Agricultural Sciences and Natural Resources, 2, (3), 67-72 Oyeka, T.J. (2004). Cassava root rot fungi in Nigeria; variability in Botryodiplodia theobromae isolates and evaluation of cassava germplasm for root rot resistance. PhD thesis University of Ibadan . Sagrilo, E. Filho, P.S.V. Pequeno, M.G.,Vidigal/ M.C.G., Scapim, C. A., Kvitschal, M.V., Maia, R.R. and Rimoldi, F. (2006). Effect of harvest period on foliage Production and dry matter distribution in five cassava cultivars during the second plant cycle. Brazilian Archives of Biology and Technology, 49,(6), 1007-1018. doi:10.1590/S1516-89132006000700019 Ssemakula Gorrettie and Alfred Dixon (2007). Genotype X environment interaction, stability and agronomic performance of carotenoid-rich cassava clones. Scientific Research and Essay, 2 (9), 390399. Acta agriculturae Slovenica, 109 - 2, september 2017 169 doi: 10.14720/aas.2017.109.2.02 Original research article / izvirni znanstveni članek Multivariate analysis to assess abscisic acid content association with different physiological and plant growth related traits of Petunia Akhtar MAHMOOD1, Muhammad Saleem HAIDER1, *Qurban ALI2, Idrees Ahmad NASIR2 Received February 13, 2016; accepted June 01, 2017. Delo je prispelo 13. februarja 2016, sprejeto 01. junija 2017. ABSTRACT Petunia is an important and beautiful ornamental flowering plant, grown throughout the world for its beauty and attraction. Different Petunia hybrids have been developed by petunia growing countries of the world. The prescribed study was conducted to investigate the association of abscisic acid with seed yield and its contributing traits of petunia line. Data for different physiological, morphological and petunia seed yield traits was recorded, analyzed and interpreted for final inferences. From results it was showed that the petunia lines IAGS-P8, IAGS-P9 and IAGS-P11 performed well for most of the studied traits. It was shown from multivariate analysis techniques that stomata conductance, chlorophyll b contents, seed area, chlorophyll a contents, flower fresh mass, flowers per plant, seed mass and abscisic acid contributed higher to seed yield per plant in petunia. The abscisic acid contents showed positive and significant association and contribution towards seed yield of petunia genotypes. It was suggested that selection on the basis of abscisic acid may be useful to develop good seed yield per plant and large number of flowers per plant in petunia under stressful environmental conditions. Key words: petunia; multivariate analysis; heritability; genetic advantage; abscisic acid content; seed yield IZVLEČEK UPORABA MULTIVARIATNE ANALIZE ZA OCENITEV POVEZAVE MED VSEBNOSTJO ABSCIZINSKE KISLINE IN RAZLIČNIMI Z RASTJO POVEZANIMI FIZIOLOŠKIMI ZNAKI PRI PETUNIJI Petunija je pomembna in lepa okrasna rastlina, ki se goji širom po svetu zaradi lepote in privlačnosti. V številnih državah, kjer jo gojijo, so bili vzgojeni različni križanci. Pričujoča raziskava je bila opravljena z namenom preučiti povezavo med vsebnostjo abscizinske kisline in lastnostmi, povezanimi s pridelkom semena preučevanih linij petunij. Izmerjene so bile različne fiziološke in morfološke lastnosti, ki vplivajo na pridelek semen, analizirana in pojasnjena je bila njihova povezava. Izsledki so pokazali, da so se linije petunij IAGS-P8, IAGS-P9 in IAGS-P11 izkazale kot primerne za večino analiziranih lastnosti. Multivariatna analiza je pokazala, da so parametri kot so stomatarna prevodnost, vsebnost klorofila b in a, površina semen, sveža masa cvetov, število cvetov na rastlino, masa semen in vsebnost abscizinske kisline prispevali največ k večjemu pridelku semena na rastlino. Vsebnost abscizinske kisline je imela značilen pozitiven vpliv na pridelek semena vseh genotipov petunij. Zaradi tega se priporoča, da je izbor genotipov petunije na osnovi večje vsebnosti abscizinske kisline primeren za vzgojo rastlin z velikim pridelkom semena na rastlino in velikim številom cvetov v stresnih okoljskih razmerah. Ključne besede: petunija; multivariatna anliza; dednost; genetska prednost; vsebnost abscizinske kisline; pridelek semena 1 INTRODUCTION The genus Petunia is an important ornamental plant of high economic imperativeness on the whole agriculture. It gives dominant qualities to serve as model plant for contemplating plant improvement. The history of Petunia development as a crop is accent with implications for an advanced array of added ornamental crops (Cantor et al., 2015; Gerats and Vandenbussche, 2005). This generally developed genus of flowering plants fits in with Solanaceae gang. It is an important ornamental plant in landscape because of colour diversity. A large number of hybrids and varieties have been developed with diverse color and patterns (Ganga, 1 Institute of Agricultural Sciences, University of the Punjab Lahore, Pakistan 2 Centre of Excellence in Molecular Biology, University of the Punjab Lahore, Pakistan; *Corresponding author: saim1692@gmail.com Acta agriculturae Slovenica, 109 - 2, september 2017 str. 165 - 173 Akhtar MAHMOOD et al. 2011). This plant is native to Brazil, Argentina or Uruguay and included over 35 species (Dole and Wilkins, 1999). Petunias are considered an annual ornamental plant but may be perennial in warmer climates (Berenschot et al., 2008). Petunia genus includes about 20 species of South American origin, mostly perennials but developed as annuals (Mallona et al., 2010), 14 annual species (Toma, 2009), but Petunia hybrida Hort. (P. axillaris Lam. x P. violacea Lindl.) is a species which presents the biggest decorative value (Berenschot et al., 2008; Vandenbussche et al., 2016). Presently the researches have also confirmed that the genus Petunia is consisted of 14 closely related species (Stuurman et al., 2004). The modern petunias have been developed through hybrid breeding like the violet petunia (P. violacea, and P. integrifolia (Hook.) Schinz & Thell.) and ambrosial agrarian or white petunia (Petunia axillaris). A large number of researchers are also working on finding the real ancestors because their ancestors are rarely cultivated today. Petunia is a small sprawling plant with large number of flowers, grown throughout the world for its beauty and interactive colors (Anderson, 2006; Bala, 2007). Petunias blossom abundantly even in hot summers and new varieties even in seasons with top clamminess (Bala, 2007; Florin et al., 2012). The petunia is long day plant due to which it is used for landscape proposes (Anderson, 2006; Currey and Lopez, 2013). The present study was conducted to develop inbred lines of petunia through selfing for growing seasons. The data of various morphological, physiological and seed yield traits was recorded to access the performance of inbred lines under development. The identification of promising inbred lines for the development of petunia hybrids. 2 MATERIAL AND METHODS Prescribed research work was conducted in the research area of Institute of Agricultural Sciences, University of the Punjab Lahore, Pakistan. Twelve petunia lines, IAGS-P1, IAGS-P2, IAGS-P3, IAGS-P4, IAGS-P5, IAGS-P6, IAGS-P7, IAGS-P8, IAGS-P9, IAGS-P10, IAGS-P11 and IAGS-P12 were selected and grown in the field during 2015. Selfing of all the lines was carried out for 4 successive growing seasons (2011-14) to develop inbred lines. The selfed seed was collected to develop next generation, grown in 2015 and for obtaining data for various traits, such as leaf temperature (LT), photosynthetic rate (A), stomata conductance (gs), water use efficiency (WUE), sub-stomata CO2 concentration (Ci) and transpiration rate (E) (by using IRGA-LI-6262 (Infrared Gas Analyzer, LI-COR Biosciences designs, USA), chlorophyll a content (Chl. a), chlorophyll b content (Chl. b) in fresh matter (measured through the dimethyl sulfoxide extraction method (Hiscox and Israelstam, 1979), plant height (PH), leaves per plant (LPP), flowers per plant (FPP), leaf area (LA), stem diameter (SD), leaf length (LL), fresh leaf mass (FLM), seeds per fruit (SPF), leaf width (LW), flower mass (FM), seed mass (SM), fresh stem mass (FSM), seed area (SA, measusered by using Digital Micrometer Screw Guage, Model: 1658DGT/25), 100-seed mass (HSM), abscisic acid (ABA) contnets (using HPLC method (Seo and Koshiba, 2002)), and seed yield per plant (SYP). The data were statistically analyzed by using analysis of variance technique (Steel et al., 1997). 3 RESULTS AND DISCUSSION The results from Table 1 persuaded that significant differences among all the studied traits were found. The highest heritability (h2bs) was found for photosynthetic rate, sub-stomata CO2 concentration, stomata conductance, water use efficiency, plant height, leaves per plant, flowers per plant, seeds per fruit, leaf area, seed yield per plant and abscisic acid contents. The genetic advantage was found higher for all studied traits except sub-stomata CO2 concentration, flowers per plant, seeds per fruit, seed mass while moderate for abscisic acid content and leaves per plant. The higher broad sense heritability referred the dominance type of gene action and suggested that the selection for such traits may be helpful to develop petunia hybrids with much of vigor and ability to tolerate harsh environmental conditions as higher value of h2bs was recorded for abscisic acid content. Higher concentration of abscisic acid contnets in plant body gives an extra advantage to grow in drought conditions with higher and well performance. Various researchers while working on different crop plants have described about higher heritability for these traits as reported in our study (Aaliya et al., 2016; Ali et al., 2015; Ali et al., 2013; Ali et al., 2014a; Mahmood and Haider, 2016). Genetic advance indicated the presence of additive type of gene action hence the traits with higher genetic advance suggested that the selection of lines may also be helpful to develop synthetic varieties. The similar findings for different crops have been reported by various researchers (Ali and Ahsan, 2015; Ali et al., 2014b; Ali et al., 2014c; Khorasani et al., 2011; Mahmood and Haider, 2016). The results 176 Acta agriculturae Slovenica, 109 - 2, september 2017 Multivariate analysis to assess ... with different physiological and plant growth related traits of Petunia (Supplementary Material Table S1) indicated that the lines IAGS-P8, IAGS-P9 and IAGS-P11 were better performing than all of the other lines also seen form figure 1a (principal component analysis) that the lines IAGS-P2, IAGS-P8, IAGS-P9 and IAGS-P11 fall in quadrant I which indicates the highest and best performance for respective traits. The lines which showed the best performance may be used for the development of good quality cultivars, with large number of flowers, stress tolerant and multicolor petunia hybrids and varieties (Ali et al., 2013; Florin et al., 2012; Mahmood and Haider, 2016). Table 1: Genetic components for morpho-physiology and yield traits of petunia Traits M.S G.M GV GCV % PV PCV % EV ECV % h2bs% GA% Photosynthetic rate (^g CO2 s ^ 123.533* 14.104 39.803 167.991 43.927 176.480 4.124 54.074 90.612 74.728 Leaf tTemperature (oC) 137.453* 21.194 38.440 134.674 60.574 169.058 22.134 102.194 63.459 40.898 Chlorphyll a (mg g"1 fr. mass.) 14.245* 3.199 4.238 115.095 5.770 134.298 1.532 69.203 73.447 96.786 Chlorphyll b (mg g"1 fr. mass) 17.345* 1.578 3.037 138.730 11.271 267.256 8.234 228.429 26.945 100.608 Stomata conductance (mmol m"2 s"1) 1.323* 0.031 0.417 366.764 0.489 397.167 0.072 152.400 85.276 337.958 Transpiration rate (mm day"1) 1.025* 0.884 0.258 54.013 0.509 75.873 0.251 53.286 50.678 71.773 sub-stomata CO2 concentration (^mol mol 1 CO2) 234.534* 148.889 77.000 71.914 80.533 73.546 3.533 15.404 95.613 10.114 Water use efficiency (%) 36.345* 6.792 11.700 131.248 12.945 138.055 1.245 42.814 90.382 84.026 Leaves per plant 233.342* 86.750 77.452 94.489 78.438 95.089 0.986 10.661 98.743 17.692 Plant height (cm) 219.245* 55.818 70.571 112.441 78.104 118.290 7.533 36.736 90.355 25.107 Stem diameter (cm) 1.026* 0.511 0.308 77.594 0.411 89.647 0.103 44.896 74.919 164.889 Flowers per plant 287.345* 141.000 92.791 81.123 101.764 84.955 8.973 25.227 91.183 11.449 Leaf length (cm) 36.124* 6.324 11.333 133.868 13.458 145.880 2.125 57.967 84.210 85.731 Leaf width (cm) 4.897* 1.358 1.017 86.539 2.863 145.198 1.846 116.591 35.522 77.676 Leaf area (cm2) 41.255* 6.353 12.934 142.685 15.387 155.628 2.453 62.138 84.058 91.087 Fresh leaf mass (g) 3.522* 0.654 0.629 98.096 2.263 186.031 1.634 158.066 27.806 112.255 Fresh stem mass (g) 214.255* 49.224 67.574 117.166 79.107 126.771 11.533 48.404 85.421 27.088 Flower mass (g) 2.148* 0.601 0.391 0.806 1.366 1.945 0.975 127.396 28.608 97.648 Seeds per fruit 996.357* 866.167 326.331 61.380 343.694 62.992 17.363 14.158 94.948 3.567 100"seed mass (mg) 64.235* 12.049 16.357 116.515 31.520 161.741 15.163 112.180 51.895 42.437 Seed area (mm) 2.087* 0.357 0.434 110.317 1.218 184.745 0.784 148.192 35.657 193.490 Seed yield per plant 1.024* 0.117 0.313 163.648 0.397 184.283 0.084 84.732 78.859 745.625 Seed mass (mg) 97.573* 50.140 24.671 70.146 48.231 98.078 23.560 68.548 51.152 12.434 Abscisic Acid contents (mg/100g fresh leaf mass) 524.156* 115.124 162.969 118.979 198.219 131.217 35.250 55.335 82.217 17.646 * = significant at 5 % probability level, mean sum of squares (M.S), grand mean (G.M), genotypic variance (GV), genotypic coefficient of variance (GCV %), phenotypic variance (PV), phenotypic coefficient of variance (PCV %), environmental variance (EV), environmental coefficient of variance (ECV %), broad sense heritability (h2bs %), genetic advance (GA) The correlation analysis provides best opportunity to the researchers for selecting genotypes of crop plant to improve crop plant growth and production (Ali et al., 2016; Ali et al., 2014c). The results from table 2 indicated that significant correlation was found for photosynthetic rate with chlorophyll a contents, plant height, sub-stomata CO2 concentration, leaf width, abscisic acid and seeds per fruit. Abscisic acid contents was found to be significantly correlated with most of the studied traits including photosynthetic rate, chlorophyll a contents, chlorophyll b contents, plant height, sub-stomata CO2 concentration, transpiration rate, water use efficiency, leaf temperature, leaf area, leaf width, fresh shoot mass, seeds per fruit, 100-seed mass, seed mass and seed yield per plant. Seed yield per plant was significantly correlated with photosynthetic rate, transpiration rate, leaf temperature, leaves per plant, fresh leaf mass, stem diameter, seed area, seed mass, seeds per fruit, flowers per plant and abscisic acid contents. The positive and significant correlation revealed that the selection of lines to develop hybrids and synthetic varieties may be helpful to improve the growth and development of petunia. The significant correlation of abscisic acid content with morphological traits, seed yield and physiological traits indicated that the selection of petunia lines on the basis of good abscisic acid production may be fruitful to improve drought tolerance in petunia (Aaliya et al., 2016; Abbas et al., 2016; Filipovic et al., 2014). The growth of petunia was adversely affected by changing the environmental optimum temperature of 25 °C with minimum circadian light intensity to be 13 Wm-2 (Kaczperski et al., 1991). It has been observed that temperature and light caused major effects on growth and development of petunia. Therefore, new petunia varities and hybrids should be developed which can tolerate varying environmental conditions to Acta agriculturae Slovenica, 109 - 2, September 2017 Akhtar MAHMOOD et al. continue optimal plant growth and development. However, the holdup of plant growth, development, the access of CO2 by stomata in an optimized environmental condition has shown not any extensive adverse effect on petunia plants (Blanchard and Runkle, 2009). Stepwise regression analysis was performed to predict the trait(s) that were highly contributing towards the petunia seed yield per plant. Stepwise regression analysis provides an opportunity to select crop plant genotypes with higher contribution traits to improve crop yield and production (Aaliya et al., 2016; Abbas et al., 2016). The results from Table 3 showed that stomata conductance, chlorophyll a contents, flowers per plant, leaf area, flower fresh mass, seed area, seeds per fruit, seed mass and abscisic acid contenta contributed more to seed yield per plant but it could be biased as preceding literature has also been reported the error effects of stepwise regression (El-Badawy and Mehasen, 2011) while handling a large number of independent variables. The Intercept = 145.754, R2 = 0.863, Adjust R2 = 0.336 and Standard Error = 0.812 was found with expected regression equation as fellow: Y = 145.754 + (7.144X0 + (-1.254 X2) + (3.898 X3) + (-6.651X4) + (121.14X5) + (-4.582X6) + (0.018X0 + (-0.042X8) + (1.145X9) + (0.163X10) + (-0.063X„) + (2.463X12) + (-0.125X13) + (-17.215X14) + (41.006X15) + (11.267X16) + (-11.175X17) + (2.825X18) + (8.982X19) + (0.256X20) + (6.902X21) + (21.267X22) + (25.926X23) The use of PCA (principal component analysis) to overcome the error effect of large number of independent variables in breeding experiments and find overall attributed variation in dependent structure (Ali et al., 2015; Filipovic et al., 2014; Goodarzi et al., 2015; Marjanovic-Jeromela et al., 2011). It has also been reported that the eigenvalues (in PCA) showed primary significance for numerical diagnostics to evaluate variation endorsed by a large number variables on the dependent structure and their data matrix in a graphical display (Greenacre, 2010). Therefore, we have also performed principle component analysis (PCA) to inspect the traits which were contributing higher towards petunia seed yield per plant. Our data generated four PCA as shown in Table 4 with diverse variation among all of the studied traits. It was found that the PC1, PC2, PC3 and PC4 contributed variation of 35.60 %, 24.60 %, 17.90 % and 11.3 % while their cumulative proportion was 25.2 %, 43.20 %, 57.20 % and 73.10 % respectively. PC1 and PC2 contributed higher variation for respective studied traits (Fig. 1a) the eigenvalues of these four PCs was higher than 1 (Fig. 1b). The Figure 1a also showed that the petunia lines IAGS-P2, IAGS-P8, IAGS-P9 and IAGS-P11 showed better performance for most of the studied traits. 17ft 176 Acta agriculturae Slovenica, 109 - 2, september 2017 Multivariate analysis to assess ... with different physiological and plant growth related traits of Petunia Table 2: Correlation among various morpho-physiological and yield traits of petunia Traits A LT Chl. a Chl. b gs E Ci WUE LPP PH SD FPP LL LW LA FLM FSM FM SPF HSW SA SM ABA LT 0.2024 Chl. a 0.8228* 0.0852 Chl. b 0.0197 -0.2476 0.0282 gs -0.2867 -0.2754 -0.2040 0.4668* E 0.0079 0.4140* -0.0727 0.8246* 0.4179* Ci 0.4950* -0.2424 0.4296* 0.0152 0.4287* 0.0697 WUE 0.1222 -0.2718 0.4619* 0.7206* 0.4996* 0.8442* 0.2587 LPP -0.2284 0.2949 -0.2064 -0.2864 -0.2048 0.6049* -0.2224 0.6517* PH 0.4889* 0.0884 0.1840 0.5719* 0.2407 0.4948* 0.1474 -0.0464 -0.2226 SD -0.0772 0.2017 0.2204 -0.2140 -0.2642 0.4087* 0.2221 -0.2726 0.0482 0.1820 FPP 0.1809 -0.4272* 0.4499* 0.4462* 0.6668* -0.2286 -0.2616 0.4426* -0.2204 0.2420 0.2846 LL -0.0172 0.1487 -0.0152 0.4146* 0.2267 0.5144* -0.0225 0.4184* 0.2298 0.6272* 0.7846* -0.4044* LW 0.4454* -0.2097 0.2184 0.2649 0.4011* 0.2166 0.1872 0.4426* 0.4649* 0.4250* 0.2819 0.4487* -0.0998 LA -0.0988 0.1709 0.0484 0.4282* 0.4186* 0.6729* 0.2268 0.4761* -0.0258 0.4480* 0.6828* -0.4292* 0.9210* 0.2848 FLM -0.2756 -0.0711 0.2048 0.4242* 0.2044 0.0602 0.2902 0.2690 0.4792* -0.0848 -0.2441 0.4787* 0.2478 -0.2162 0.2826 FSM 0.2586 0.0874 -0.0951 0.8227* 0.7284* 0.7286* -0.2976 0.6617* 0.2229 0.4228* 0.4016* 0.7602* 0.1291 0.4848* 0.6144* 0.4962* FM 0.4268* 0.4892* 0.2216 0.5122* -0.2800 0.8444* 0.0062 -0.0209 0.2824 -0.0691 -0.2471 -0.0049 0.4442* 0.2229 -0.4496* -0.2210 0.4468* SPF 0.4417* -0.2624 -0.2290 0.0262 0.6072* 0.2427 -0.4290* 0.0702 -0.1222 -0.0821 -0.2815 0.6472* -0.0104 -0.2489 -0.2488 -0.2559 0.4417* 0.0109 HSM 0.2220 0.0852 0.0289 0.2801 0.2698 0.2241 -0.2069 0.2428 0.2402 0.4799* -0.2092 -0.4772* 0.4224* 0.4472* 0.4400* -0.0049 0.1089 0.2227 -0.2910 SA 0.2224 -0.0276 0.4487* -0.4649* -0.4144* -0.4196* -0.2204 -0.2422 0.0090 0.4724* 0.1642 0.2487 0.4104* -0.2822 -0.4246* 0.4184* 0.1262 0.4474* -0.0584 -0.4251 SM -0.2780 0.4446* 0.2201 0.2084 0.2294 -0.0742 -0.0224 0.2144 -0.2874 0.2422 0.4928* -0.2649 0.4114* -0.4291 -0.1608 0.0275 -0.0047 -0.2452 -0.2056 -0.4778* -0.2148 ABA 0.4874* 0.5678* 0.4291* 0.6019* 0.6201* 0.5211* 0.2949 0.5404* 0.7011* 0.0150 -0.2548 0.5052* 0.2578 0.4029* 0.4122* -0.2109 0.4672* -0.0241 0.4402* 0.4122* -0.0252 0.5046* SYP 0.4686* 0.4744* -0.2714 -0.2221 0.4062* 0.4179* -0.2422 0.4978* 0.4494* -0.0664 0.4180* 0.4444* -0.2852 -0.2274 -0.2542 0.4222* 0.2217 0.0212 0.4284* 0.0642 0.4851* 0.4295* 0.5642* *= Significant at 5 % probability level, A = photosynthetic rate, LT = leaf temperature, Chl. a = chlorophyll a content, Chl. b = chlorophyll b content, E = transpiration rate, gs = stomata conductance, Ci = sub-stomata CO2 concentration, WUE = water use efficiency, LPP = leaves per plant, PH = plant height, SD = stem diameter, FPP = flowers per plant, LL = leaf length, LW = leaf width, LA = leaf area, FLM = fresh leaf mass, FSM = fresh stem mass, FM = flower mass, SPF = seeds per fruit, HSM = 100-seed mass, SA = seed area, SM = seed mass, ABA = abscisic acid content, SYP = seed yield per plant Acta agriculturae Slovenica, 109 - 2, september 2017 Akhtar MAHMOOD et al. Table 3: Stepwise regression analysis for various traits of petunia for seed yield Traits Coefficients B Standard Error t Stat Cumulative R Partial R2 % Xi Photosynthetic rate 7.144 0.553 2.013 0.1673 16.73 X2 Leaf temperature -1.254 0.127 -2.320 0.2151 21.51 X3 Chlorphyll a 3.898 2.531 1.114 0.2573 25.73 X4 Chlorphyll b -6.651 1.632 1.052 0.2661 26.61 X5 Stomata conductance 121.14 45.125 2.525 -0.235 23.50 X6 Transpiration rate -4.582 3.153 -1.172 0.266 26.60 X7 Sub-stomata CO2 concentration 0.018 0.053 -1.512 -0.263 26.30 X8 Water use efficiency -0.042 0.351 -0.153 0.5631 56.31 X9 Leaves per plant 1.145 0.121 1.522 0.2634 26/34 X10 Plant height 0.163 0.086 -1.315 0.2534 25.34 X11 Stem diameter -0.063 0.015 0.063 0.4743 47.43 X12 Flowers per plant 2.463 8.535 0.279 0.5386 53.86 X13 Leaf length 0.125 0.015 2.233 -0.2157 21.57 X14 Leaf width -17.215 16.815 -1.037 0.2353 23.53 X15 Leaf area 41.006 24.150 -0.137 -0.2327 23.27 X16 Fresh leaf mass 11.267 8.759 0.521 0.0333 3.33 X17 Fresh stem mass -11.175 5.115 -1.255 -0.0847 8.47 X18 Flower mass 2.825 0.131 0.522 0.3562 35.62 X19 Seeds per fruit 8.982 10.525 -1.248 -0.2237 22.37 X20 100-seed mass 0.256 0.052 -0.113 0.3644 36.44 X21 Seed area 6.902 4.315 1.522 -0.1245 12.45 X22 Seed mass 21.267 15.517 1.535 0.3252 32.52 X23 Abscisic acid contents 25.926 11.258 0.463 0.5437 54.37 Intercept = 145.754, R2 = 0.863, Adjust R2 ' = 0.336, Standard Error = 0.812 1 80 176 Acta agriculturae Slovenica, 109 - 2, september 2017 Multivariate analysis to assess ... with different physiological and plant growth related traits of Petunia Table 4: Principal component analysis Eigen value 6.147 3.9536 4.-0717 3.0313 Proportion 0.356 0.246 0.179 0.113 Cumulative 0.252 0.432 0.572 0.731 Traits PC2 PC2 PC3 PC4 Photosynthetic rate 0.689 0.425 0.026 0.055 Leaf temperature -0.25 0.083 -0.336 -0.093 Chlorphyll a 0.035 0.376 0.07 0.036 Chlorphyll b 0.286 -0.005 -0.025 -0.227 Stomata conductance 0.222 0.228 -0.297 -0.047 Transpiration rate 0.323 -0.037 0.252 -0.262 Sub-stomata CO2 concentration 0.087 0.163 0.029 0.002 Water use efficiency 0.291 0.183 0.223 -0.202 leaves per plant -0.204 -0.125 -0.34 0.257 Plant height -0.166 0.372 0.237 -0.033 Stem diameter -0.113 0.143 -0.044 -0.224 flowers per plant -0.413 -0.216 0.325 -0.208 Leaf length 0.114 -0.202 -0.02 0.258 Leaf width 0.214 0.323 0.076 0.248 Leaf area 0.301 -0.017 0.028 0.242 Fresh leaf mass 0.231 -0.007 -0.245 -0.042 Fresh stem mass -0.314 -0.064 0.216 0.002 Flower mass -0.291 0.216 0.018 0.337 Seeds per fruit -0.031 -0.216 0.414 -0.234 200-seed mass 0.262 0.109 -0.074 0.372 Seed area -0.291 0.212 0.012 -0.206 Seed yield per plant -0.231 -0.012 -0.126 -0.056 Seed mass -0.061 0.135 -0.105 -0.499 Abscisic acid contents 0.284 0.218 0.136 0.075 «-£3 2 «»o 0 -1 - -2 -3 - 1AGS-P2 Plant height Chl / stem diameter SS C / IAGS-P1 Flower weight f O seed araa Temp^a^^ seed yield per plant 1£GS-P11 orphyll a ^«v Leaf width °IAGS-P6 \ O2 Concentration N. Abscisic Acid Wateruseefficiency TA,-c- Da \ t Stomata Conductance 0AGS^pr-»rA 100-seed weight 0IAGS-p9\ Fresh leaf weightChlorPhy|1 b | ^ IAOS p3 leaves per plant \ flowers per plaffieds per Photosynthetic R - ^^^^ IAC Transpiration Rate Leaf Area 1 Genotypes fruit Leaf length J JAGS-P10 / ate3AGS-P5 ^^ ÎS-P7 ^^ 0IAGS-P4 OIAG^-- -5.0 -2.5 0.0 First Principle Component (29.6%) 2.5 5.0 a: Principle components 0 3 0 Acta agriculturae Slovenica, 109 - 2, september 2017 str. 165 - 173 Akhtar MAHMOOD et al. 8 7 6 5 > 4 c eg W 3 2 1 0 8 10 12 14 16 18 Principle Component Number b: Scree plot Figure 1: a. Principle component analysis of yield and its attributing traits, b. Scree plot and respective eigen values Principal factor analysis was performed by using principle component analysis values, to check that traits which were directly contributing and highly associated with petunia seed yield per plant. The factor 1 was found to be highly contributing factor trait which contributes 48.20 % in total variation were chlorophyll a, chlorophyll b, transpiration rate, stomata conductance, leaves per plant, water use efficiency, leaf area, leaf length, seed yield per plant stem diameter and abscisic acid content (Table 5). Abscisic acid content and seed yield per plant were found the most contributing traits of petunia. Various researchers have suggested that the selection of crop plant genotypes on the basis of the factor analysis (traits from factor 1) may be supportive to develop higher yield hybrids and synthetic varieties of crop plants. While the traits which fall in factor 2 (from factor loading table 5) indicated that the selection of crop plant genotypes on the basis of such traits will not be helpful as the segregation will take place in the next growing generations (Ali et al., 2016; Filipovic et al., 2014; Mahmood and Haider, 2016). The better performance of petunia lines for chlorophyll a, transpiration rate, chlorophyll b, leaves per plant, stomata conductance, leaf length, water use efficiency, leaf area, stem diameter, seed yield per plant, seed mass and abscisic acid content revealed that the accumulation or assimilation of organic matter/compounds will be higher in the plant body. It has been also found the accumulation or assimilation of organic biomass in plant body is very essential for the proper enhanced growth and development of petunia plant (Hladni et al., 2011; Huang, 2007; Huang and Yeh, 2009; Mahmood and Haider, 2016). The accumulation of organic compounds generally takes place in the leaves, stem and flowering parts of a plant body. The results from our study were well supported by results which demonstrated the role of factor analysis for effective selection criteria in maize breeding program (Filipovic et al., 2014). In order to understand about the genetic association among petunia lines, cluster analysis was performed (Khorasani et al., 2011; Mostafavi et al., 2011). The results from clustering showed that the petunia lines IAGS-P2 and IAGS-P12 followed by IAGS-P8 and IAGS-P9 were highly associated with each other as compared with other petunia lines (Fig. 2a) the association was verified through the development of minimum spanning tree (Fig. 2b) that showed smaller distance between petunia lines IAGS-P8 and IAGS-P9 while IAGS-P2 and IAGS-P12 were having IAGS-P1 in between them through the use of eigen values. So, from results it may be revealed that the petunia lines IAGS-P8 and IAGS-P9 were highly associated with each other and may be used as two separate male or female lines to develop petunia hybrids as also verified by mean performance and principal component analysis Figure 1a results of these lines. Also the petunia line IAGS-P11 showed better performance for almost all under studied traits, so it may also be used as male to develop good quality petunia hybrids (Mahmood and Haider, 2016). It was also suggested that in future breeding program of IAGS-P8, IAGS-P9 and IAGS-P11, these traits may be important for primary selection of synthetic petunia varieties and hybrids to increase seed yield per plant of petunia under various environmental regimes of Pakistan and other growing countries. Moreover, the hybrid seed production technology proved to be more efficient as it reduced the cost, time and increase 0 1 ft? 176 Acta agriculturae Slovenica, 109 - 2, september 2017 Multivariate analysis to assess ... with different physiological and plant growth related traits of Petunia efficacy for better selection in petunia improvement should cover different years and locations. programs. Still, further studies are required which Table 5: Factor loadings for different traits of petunia Factor1 Factor loadings % Communality Chlorophyll a 0.674 Chlorophyll b 0.735 Stomata conductance 0.643 Transpiration rate 0.743 Water use efficiency 0.879 Leaves per plant 0.568 Stem diameter 0.789 Leaf length 0.678 Leaf area 0.568 Seed yield per plant 0.567 Abscisic acid content 0.876 48.2 Factor2 Sub-stomata CO2 concentration -0.563 Plant height -0.577 Fresh leaf mass -0.636 22.1 Factor3 Leaves per plant 0.323 Leaf length 0.325 Leaf area 0.327 Fresh leaf mass 0.241 Seed yield per plant 0.263 11.1 Factor4 Photosynthetic rate 0.221 Leaves per plant 0.135 Plant height 0.119 Stem diameter 0.219 Leaf area 0.287 Flower mass 0.153 100-seed mass 0.206 8.62 Cumulative variance 90.02 Acta agriculturae Slovenica, 109 - 2, September 2017 Akhtar MAHMOOD et al. IAGS-P1 IAGS-P2 IAGS-P12 IAGS-P11 IAGS-P7 IAGS-P3 IAGS-P4 IAGS-P5 IAGS-P6 IAGS-P9 IAGS-P8 IAGS 10 a: Dendrogram J_l_I_I_I_I_I_I_I_L 1.00 0.98 0.96 0.94 0.92 0.90 0.88 0.86 0.84 Minimum Spanning Tree 0.4 ^IAG „IAGS-P3 ■T / \ N. i \ -i \ i \ i 0.2 \ i / V i * i x IAGS-P2 ^iAGS-P1 _0.0 .S-P4 X X IAGS-P5 x IAGS-P8 x IAGS-P9 x IAGS- -0.3 -0.2 -0.1 -0.0 0 \ \ x IAGS-P1 2 -0.2 \ \ \ ___ x IAGS-P 1 1 -0 4 1 0.2 0.3 0.4 0.5 x IAGS-P7 i i {IAGS-P10 P6 second dimension b: Minimum spanning tree Figure 2: a. Dendrogram analysis based on hierarchal clustering. Association of petunia lines on genetic basis of all studied traits, b. Minimum spanning tree using eigene values for petunia lines on the basis of all studied traits 184 176 Acta agriculturae Slovenica, 109 - 2, september 2017 Multivariate analysis to assess ... with different physiological and plant growth related traits of Petunia 4 CONCLUSION The present study concluded that abscisic acid contents showed positive and significant association and contribution towards seed yield of petunia genotypes. It was suggested that selection on the basis of abscisic acid content may be useful to develop good seed yield per plant and large number of flowers per plant in petunia under stressful environmental conditions. 5 CONFLICT OF INTEREST The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. 6 REFERENCES Aaliya, K., Qamar, Z., Nasir, I. A., Ali, Q., and Munim, A. F. (2016). Transformation, evaluation of gtgene and multivariate genetic analysis for morpho-physiological and yield attributing traits in Zea mays. Genetika, 48, 423-443. doi:10.2298/GENSR1601423A Abbas, H. G., Mahmood, A., and Ali, Q. (2016). Zero tillage: A potential technology to improve cotton yield. Genetika, 48,761-776. doi: 10.2298/GENSR160234761A. Ali, F., Kanwal, N., Ahsan, M., Ali, Q., Bibi, I., and Niazi, N. K. (2015). Multivariate Analysis of Grain Yield and Its Attributing Traits in Different Maize Hybrids Grown under Heat and Drought Stress. Scientifica, 2015. doi:10.1155/2015/563869 Ali, Q., and Ahsan, M. (2015). Correlation analysis for various grain contributing traits of Zea mays. African Journal of Agricultural Research, 10, 2350-2354. doi:10.5897/AJAR2013.7838 Ali, Q., Ahsan, M., Ali, F., Aslam, M., Khan, N. H., Munzoor, M., Mustafa, H. S. B., and Muhammad, S. (2013). Heritability, heterosis and heterobeltiosis studies for morphological traits of maize (Zea mays L.) seedlings. Advancements in Life Sciences 1,52-63. Ali, Q., Ahsan, M., Kanwal, N., Ali, F., Ali, A., Ahmed, W., Ishfaq, M., and Saleem, M. (2016). Screening for drought tolerance: comparison of maize hybrids under water deficit condition. Advancements in Life Sciences, 3, 51-58. Ali, Q., Ali, A., Ahsan, M., Nasir, I. A., Abbas, H. G., and Ashraf, M. A. (2014a). Linex Tester analysis for morpho-physiological traits of Zea mays L seedlings. Advancements in Life Sciences, 1, 242-253. Ali, Q., Ali, A., Awan, M. F., Tariq, M., Ali, S., Samiullah, T. R., Azam, S., Din, S., Ahmad, M., and Sharif, N. (2014b). Combining ability analysis for various physiological, grain yield and quality traits of Zea mays L. Life Science Journal, 11, 540-551. doi:10.7537/marslsj 1108s14.114 Ali, Q., Ali, A., Waseem, M., Muzaffar, A., Ahmad, S., Ali, S., Awan, M., Samiullah, T., Nasir, I., and Tayyab, H. (2014c). Correlation analysis for morpho-physiological traits of maize (Zea mays L.). Life Science Journal, 11, 9-13. doi:10.7537/marslsj 1112s14.02 Anderson, N. O. (2006). "Flower breeding and genetics: issues, challenges and opportunities for the 21st century" . Springer Science & Business Media. doi:10.1007/978-1-4020-4428-1 Bala, M. (2007). General and special floriculture. Ed. de Vest, Timi^oara, 105-106. Berenschot, A. S., Zucchi, M. I., Tulmann-Neto, A., and Quecini, V. (2008). Mutagenesis in Petunia x hybrida Vilm. and isolation of a novel morphological mutant. Brazilian Journal of Plant Physiology, 20, 95-103. doi:10.1590/S1677-04202008000200002 Blanchard, M. G., and Runkle, E. S. (2009). Use of a cyclic high-pressure sodium lamp to inhibit flowering of chrysanthemum and velvet sage. Scientia horticulturae, 122, 448-454. doi:10.1016/j.scienta.2009.06.016 Cantor, M., Kaizbai, E., and Erzsebet, B. (2015). The Behavior of Some Petunias Varieties for Improvement the Romanian Assortment. Bulletin of University of Agricultural Sciences and Veterinary Medicine Cluj-Napoca. Horticulture, 72, 39-44. doi:10.15835/buasvmcn-hort:10664. Currey, C. J., and Lopez, R. G. (2013). Cuttings of Impatiens, Pelargonium, and Petunia propagated under light-emitting diodes and high-pressure sodium lamps have comparable growth, morphology, gas exchange, and post-transplant performance. HortScience, 48, 428-434. Dole, J. M., and Wilkins, H. F. (1999). "Floriculture: principles and species," Prentice-Hall inc. Acta agriculturae Slovenica, 109 - 2, September 2017 Akhtar MAHMOOD et al. El-Badawy, M. E. M., and Mehasen, S. (2011). Multivariate analysis for yield and its components in maize under zinc and nitrogen fertilization levels. Australian journal of basic and applied sciences, 5, 3008-3015. Filipovic, M., Babic, M., Delic, N., Bekavac, G., and Babic, V. (2014). Determination relevant breeding criteria by the path and factor analysis in maize. Genetika, 46, 49-58. doi:10.2298/GENSR1401049F Florin, T., Petra, S., Zamfir-Vasca, D., and Vasilescu, T. (2012). Research on influence of top shoots pinching on plant growth and blossoming of some new varieties of petunia. Scientific Papers. Series B. Horticulture. Ganga, M. (2011). C. Kole (ed.), Wild Crop Relatives: Genomic and Breeding Resources, Plantation and Ornamental Crops. Wild Crop Relatives: Genomic and Breeding Resources: Plantation and Ornamental Crops, 209. ISBN 978-3-642-21201-7 Gerats, T., and Vandenbussche, M. (2005). A model system for comparative research: Petunia. Trends in plant science, 10, 251-256. doi: 10.1016/j.tplants.2005.03.005 Goodarzi, F., Hassani, A., Darvishzadeh, R., and HATAMI, H. (2015). Genetic variability and traits association in castor bean (Ricinus communis L.). Genetika, 47, 265-274. doi: 10.2298/GENSR1501265G Greenacre, M. J. (2010). "Biplots in practice". Fundacion BBVA. Hiscox, J. T., and Israelstam, G. (1979). A method for the extraction of chlorophyll from leaf tissue without maceration. Canadian Journal of Botany, 57, 13321334. doi:10.1139/b79-163 Hladni, N., Jocic, S., Miklic, V., Saftic-Pankovic, D., and Kraljevic-Balalic, M. (2011). Interdependence of yield and yield components of confectionary sunflower hybrids. Genetika, 43, 583-594. doi: 10.2298/GENSR1103583H Huang, L. C. (2007). Behavioral differences in prepurchase processes between purchasers of flowers for self use and for gift use. HortTechnology, 17, 183-190. Huang, L.-C., and Yeh, T.-F. (2009). Floral consumption values for consumer groups with different purchase choices for flowers. HortTechnology, 19, 563-571. Kaczperski, M., Carlson, W., and Karlsson, M. (1991). Growth and development of Petunia hybrids as a function of temperature and irradiance. Journal of the American Society for Horticultural Science, 116, 232237. Khorasani, S. K., Mostafavi, K., Zandipour, E., and Heidarian, A. (2011). Multivariate analysis of agronomic traits of new corn hybrids (Zea maize L.). International Journal of AgriScience, 1, 314-322. Mahmood, A., Haider, M. S. A., Qurban N., Idrees A. (2017). Estimation of genetic potential and association among morpho-physiological traits of Petunia. Journal of Research in Ecology, 5. EC0359. Mallona, I., Lischewski, S., Weiss, J., Hause, B., and Egea-Cortines, M. (2010). Validation of reference genes for quantitative real-time PCR during leaf and flower development in Petunia hybrida. BMC Plant Biology, 10, 1. doi:10.1186/1471-2229-10-4 Marjanovic-Jeromela, A., Marinkovic, R., Ivanovska, S., Jankuovska, M., Mijic, A., and Hristov, N. (2011). Variability of yield determining components in winter rapeseed (Brassica napus L.) and their correlation with seed yield. Genetika, 43, 51-66. doi:10.2298/GENSR1101051M Mostafavi, K., Shoahosseini, M., and Geive, H. (2011). Multivariate analysis of variation among traits of corn hybrids traits under drought stress. International Journal of AgriScience, 1, 416-422. Seo, M., and Koshiba, T. (2002). Complex regulation of ABA biosynthesis in plants. Trends in plant science, 7, 41-48. doi:10.1016/S1360-1385(01)02187-2 Stuurman, J., Hoballah, M. E., Broger, L., Moore, J., Basten, C., and Kuhlemeier, C. (2004). Dissection of floral pollination syndromes in Petunia. Genetics, 168, 1585-1599. doi:10.1534/genetics. 104.031138 Toma, F. (2009). Floriculture and floral art. Ed. Invel Multimedia, Bucharest. Vandenbussche, M., Chambrier, P., Bento, S. R., and Morel, P. (2016). Petunia, Your Next Supermodel? Frontiers in plant science, 7. doi:10.3389/fpls.2016.00072. 176 Acta agriculturae Slovenica, 109 - 2, september 2017 doi: 10.14720/aas.2017.109.2.03 Original research article / izvirni znanstveni članek Molecular genetic analysis of some North African barley germplasms *R. M. GAAFAR1, Mai ALLAM2, Rasha SABRY2 and M. SAKER2 Received February 22, 2016; accepted April 03, 2017. Delo je prispelo 22. februarja 2016, sprejeto 03. aprila 2017. ABSTRACT Isozyme and RAPD markers were used to characterize 29 barley accessions, which were collected from North Africa. In addition, resistance gene sequences were employed to develop molecular markers using RT-PCR approach. High level of polymorphism was found with both RAPD and isozyme markers, where RAPD showed that 60 % of amplified bands were polymorphic. Peroxidase showed three polymorphic loci (7 allelic bands). Isozymes cluster analysis successfully separated the barley accessions into three geographically distinct groups. RAPD investigation demonstrated that Egyptian accessions were grouped into two obvious groups. Moreover, the Tunisian accessions showed no distinct clustering, while high dissimilarities were revealed by the Algerian accessions. In the RT-PCR, from six primer pairs selected, primer pair AF092524P1P2 successfully amplified two specific amplicons of approximately (340 & 220 bp) and (360 & 270 bp), respectively in two Egyptian barley genotypes (El-Awamah and Awlad-Ali). One primer pair DN988165P1P2 gave only one specific amplicon in both barley genotypes of 250 and 270 bp, respectively. The markers developed could be used in improving barley crop by assisting in breeding selection of resistance genotypes. Key words: RT-PCR; resistance genes; barley; genetic diversity; RAPD IZVLEČEK MOLEKULARNA GENETSKA ANALIZA NEKATERIH SEVERNOAFRIŠKIH GENSKIH VIROV JEČMENA Z izoencimskimi in RAPD markerji je bilo ovrednoteno 29 akcesij ječmena, nabranih v Severni Afriki. Za razvoj molekularnih markerjev na osnovi RT-PCR so bila uporabljena nukleotidna zaporedja genov za odpornost. Ugotovljen je bil velik polimorfizem RAPD in izoencimskih markerjev, kjer je bilo 60 % namnoženih RAPD markerjev polimorfnih. Peroksidaza je pokazala tri polimorfne lokuse (7 alelelov). Z analizo izoencimskih skupin so bile akcesije ječmena uspešno razdeljene v tri razločne geografske skupine. Raziskava RAPD je pokazala, da se egiptovske akcesije razločno povežejo v dve skupini. Tunizijske akcesije niso pokazale razločnega povezovanja, pri alžirskih pa so bile ugotovljene velike razlike. V RT-PCR analizi, je od šestih izbranih začetnih oligonukeltodiov par AF092524P1P2 uspešno namnožil dva specifična amplikona s približno 340 in 220 in 360 in 270 baznih parov pri dveh egiptovskih genotipih ječmena (El-Awamah in Awlad-Ali). Par začetnih oligonukleotidov DN988165P1P2 je pomnožil le en specifični fragment pri obeh genotipih ječmena z 250 in 270 baznimi pari. Razviti markerji se bodo lahko uporabili pri izboru genotipov za izboljšanje ječmena v žlahtnjenju na odpornost. Ključne besede: RT-PCR; geni za odpornost; ječmen; genska raznolikost; RAPD markerji 1 INTRODUCTION Barley (Hordeum vulgare L.) is one of the most pivotal cereal crops in the world. It is cultivated in the temperate zones. The haploid genome of barley is about 5.1 Gbp (Mayer et al., 2012). Due to compatibility and inter-fertility of the cultivated and wild species (share a common genome, n =7), wild species of barley and primitive landraces provide precious sources of genetic variability in a number of beneficial traits (Nevo, 1992; Ceccarelli et al., 1995). Consequently, search for genetic variation that might be useful for plant breeding programs is very essential as well as collection and conservation of wild relatives of the cultivated species and the endemic varieties (Brown et al., 1990). PCR-based molecular markers (e.g. RAPD, SSR, STS, and ISSR) have been used in barley to uncover genetic variation, genotype identification and mapping of genes (Sánchez et al., 1996; Fernández et al., 2002; Matus and Hayes, 2002; Tanyolac, 2003). Particularly, RAPD markers are found to be valuable in case of self- 1 * Botany Department, Faculty of Science, Tanta University, Tanta, Egypt, corresponding author: redagaafar@science.tanta.edu.eg 2 Plant Biotechnology Department, National Research Centre, Dokki, Giza, Egypt Acta agriculturae Slovenica, 109 - 2, september 2017 str. 165 - 173 R. M. GAAFAR et al. pollinating species, which show a relatively low level of intraspecific polymorphism, as in hexaploid wheat (Devos and Gale, 1992; Joshi and Nguyen, 1993) and in cultivated barley (Barua et al., 1993; Chalmers et al., 1993). Recently, cDNA sequences are being used to develop markers, which are very useful since they are gene-based markers (Parchman et al., 2010). Moreover, the progress in sequencing and documentation in public database has facilitated sequence data mining and development of DNA markers without any difficulty (Bhattachryya et al., 2014). Resistance gene analogs (RGAs) were frequently found to be in close genetic distance to known resistance loci, hence suggesting their possible role in disease resistance responses in plants (Fourmann et al., 2001). The gene sequences, which encode proteins containing a nucleotide-binding site (NBS) and C-terminal leucine-rich repeats (LRRs) constitute the largest class of R genes in flowering plants (Dangl and Jones, 2001). Wild barley has been known to carry valuable sources of useful genes for barley breeding. For example, genes that are associated with resistance to diseases (Fetch et al., 2003), tolerance to abiotic stress (Ellis et al., 2000), other key agronomic traits (Vanhala and Stam, 2006), and quality traits (Shen et al., 2011). The development of new barley lines, tolerant to abiotic and biotic stresses is an essential part of the continued improvement of the crop. Moreover, it can assist in the amelioration of other relative crops. Wild barley would be a valuable source of novel genetic variation for environmental stress tolerance. However, it depends on identifying of suitable genetic variation and the development of marker-assisted selection, which allows effective cultivar development (Ellis et al., 2000). The aims of this study are to uncover the genetic variability in a barley germplasm collected from North Africa, compare peroxidase isozymes and RAPD diversity in the studied materials, and amplify disease resistance sequences from some Egyptian landraces, which could be used as molecular markers in assisted marker selection of the resistance lines of barley. 2 MATERIALS AND METHODS 2.1 Plant materials Twenty-nine cultivars, genotypes and landraces of barley were obtained from National Institute of Agricultural Research Tunisia (INRAT), National Institute of Agricultural Research Algeria (INRAA), National Research Centre, Egypt (NRC), and Agricultural Genetic Engineering Research Institute, Egypt (AGERI). For resistance genes amplification, two Egyptian barley genotypes El Awamah & Awlad Ali were selected. The barley cultivar names, places and country of origin are listed in Table 1. 2.2 Isozyme analysis To electrophoretically examine peroxidase (PER, E.C.1.11.1.7) isozymes, crude extraction of the twenty- nine genotypes from Algeria, Tunisia & Egypt was done using 0.1M Tris-HCl buffer in 4°C for two hours (Gottlieb, 1981). Then, the homogenates were centrifuged at 14.000 rpm for 20 minutes at 4°C using Centrion K3 centrifuge (Centrion, UK). Electrophoretic separation of the extracts was carried out in 10 % native PAGE (Laemmli, 1970). The peroxidase enzyme was stained as described by Soltis et al (1983) as follows: gels were incubated in 100 ml staining solution containing 0.05 M acetate buffer (pH = 5.0) and 65 mg benzidine. Two ml of 0.1 M CaCl2 were added as a catalyst. Finally, two ml of H2O2 were added as the substrate and the gels were kept in refrigerator until dark brown bands appeared. All isozyme bands were assessed according to their relative distances. 188 Acta agriculturae Slovenica, 109 - 2, september 2017 Molecular genetic analysis of some North African barley germplasms Table 1. Places, names and country of origin of the barley cultivars and landraces Serial Place of origin or cultivar name Country of origin 1 Sidimehdi Algeria 2 Temasine Algeria 3 Kasrmegarine Algeria 4 RasEllouche Algeria 5 Saida Algeria 6 Tichedielt Algeria 7 Nailia Algeria 8 Rihone-03 Algeria 9 Azrir Algeria 10 Tozeurt Tunisia 11 Tozeur2 Tunisia 12 Manel Tunisia 13 SidiBeozid Tunisia 14 Kibilliz Tunisia 15 Tomban Tunisia 16 Gabes Tunisia 17 KairooaA Tunisia 18 Rihan Tunisia 19 Jerba Tunisia 20 Arish (Sinai) Egypt 21 Giza123 Egypt 22 Giza 126 Egypt 23 Giza 129 Egypt 24 Giza 125 Egypt 25 Giza 2000 Egypt 26 Giza 127 Egypt 27 Matrooh (Awama) Egypt 28 Giza 131 Egypt 29 Giza 130 Egypt 2.3 DNA extraction and RAPD analysis DNA isolation of the different barley cultivars, genotypes and landraces was performed using the CTAB method of Doyle and Doyle (1990). For RAPD-PCR analysis, 2 random 10-mer primers OP A3 and OPG3 were used. The random primers used in our study were ordered from SNEF medical, Germany. PCR reactions were conducted in a total volume of 20 pl reaction mix containing 2 pl of 10* reaction buffer, containing 2 mM MgCl2, 2 pl of 0.2 mM dNTP, 0.1^l (0.5 U) of Taq DNA polymerase (Sigma, USA), 40 ng of genomic template DNA, and 10 pmol primer in a preheated thermocycler (Biometra, Germany) under the following conditions: 3 min at 95°C, followed by 44 cycles of 2 min at 92°C, 1 min at 37°C, and 2 min at 72°C. The reaction was finally incubated at 72°C for 10 min. For selecting the optimal conditions of the RAPD PCR, different optimization experiments were performed. Acta agriculturae Slovenica, 109 - 2, september 2017 str. 165 - 173 R. M. GAAFAR et al. The PCR products were separated by electrophoresis on a 1 % agarose gels using 1 x TAE buffer. Then, gels were stained with ethidium bromide (10 mg/ml) and visualized under UV light. A 100 bp DNA ladder (Axygen, USA ) was used as a molecular DNA standard. 2.4 Resistance sequences and primers selection Forty-six sequences showed resistance to different pathogens were retrieved from NCBI gene bank. These sequences were coded for different proteins included definsin (maize and wheat), superoxide dismutase (maize and wheat), catalase (barley and Cynodon). Six primer pairs were designed using Primer3 software. After primer selection with the Primer3 program, the complementarity of the primer pairs (primer dimmer and internal complementarity) was checked and the expected annealing temperatures were manually calculated. Table 2 contains the primer sequences, primers length and the expected PCR products in bp for the six primer pairs that were selected from resistance sequences, which were obtained from the NCBI gene bank. Table 2: Oligonucleotide primers used for RT-PCR amplification Primer name Sequence Primer length Sequence/plant Expected PCR fragments AB089942P1 GGTGTGAAGCGAGCAAGC AB089942P2 CAGTGGCATCGTTATTACATCA AF092524P1 CTACGTCGCCCACTACAACAAG AF092524P2 CCAACAGCGGGAAACTCAAG 18 bp 22 bp 22 bp 20 bp Definsin/wheat SOD/wheat 522 bp 565 bp AJ849917P1 GGCCACAACGCTAGTACAATCTT AJ849917P2 CATGCGTATCACTCAATCTGCC CV064086P1 CGGCCATGGATCCCTACAAG CV064086P2 CTCCTGCATGTTGGTCTTCGG DN988165P1 GTATCTTCATGTCATTGCTCGC DN988165P2 CTCCGGCTGGTTCCTTTC X17564P1 AGGGCACCATCTTTTTCACC X17564P2 GCGACGCTCTTATTTCACGA 23 bp 22 bp 20 bp 21 bp 22 bp 18 bp 20 bp 20 bp Definsin/Zea CatIso1/barley cDNA CatIso3/Cynodon SOD/Zea 433 bp 509 bp 153 bp 516 bp The primers were ordered from Metabion International AG 2.5 RNA isolation For RNA isolation from the two barley genotypes, leaves of the 7 day old seedlings of the barley genotypes were used. To avoid any contamination with RNase, all solutions were treated overnight with DEPC at a final concentration of 0.1 %. All non-disposable equipment, like glass and porcelain, was cleaned with 0.5 % SDS/0.5 M NaOH and rinsed with DEPC treated water and wiped with 70 % ethanol. Total RNA was isolated using TRIzol reagent of the TriFast (PeqGOLD) extraction kit. In this method, 0.2 -0.5 g of fresh leaves was ground to fine powder in liquid nitrogen. The powder was added directly to an Eppendorf tube containing 0.5 ml TRIzol reagent. The homogenate was incubated at room temperature (RT) for 5 min. After adding 0.2 ml chloroform, the tube was repeatedly inverted by hand for 15 sec. The mixture was incubated for 15 - 20 min at RT. Then, the upper aqueous phase was separated by centrifugation at 12000 rpm for 15 min at 4°C. The RNA was precipitated using 1 vol. isopropanol for 10 min at RT and afterwards by centrifugation at 12000 rpm for 10 min at 4°C. The pellet was washed with 70 % ethanol and resuspended in DEPC treated water (0.943 g/ml, BioBasic INC). To check the quality and quantity of the isolated RNA, RNA agarose gel electrophoresis was performed. Electrophoresis was carried out on 1.5 % agarose gel for 1h at 70 V. 2.6 RT-PCR analysis RT-PCR was carried out using Ready-To-Go RT-PCR Beads kit (Amersham Biosciences). Each bead is optimized to allow the first-strand cDNA synthesis and PCR reactions to proceed sequentially (One-step Protocol for RT-PCR). In the One-step protocol, 188 Acta agriculturae Slovenica, 109 - 2, september 2017 Molecular genetic analysis of some North African barley germplasms primers for the first-strand cDNA synthesis and PCR were added along with the template to an RT-PCR Bead. First, for each reaction tube, 39 ^l DEPC-treated water was added. The tube was taped to mix the water with the bead. To dissolve the bead, the tube was incubated on ice for 5 min. and the tube contents were gently pipetted up and down. After that, to each bead (contains 2 units of Taq DNA polymerase, 10 mM Tris-HCl, 60 mM KCl, 1.5 mM MgCl2 200 ^M dNTPs, MuLV reverse transcriptase, RNAguard™ Ribonuclease Inhibitor and stabilizer when brought to a final volume of 50 ^l) 1 ^l of the first-strand primer (0.5 ^g), 5 ^l of 10 pmol PCR primer pairs (resistance sequences based primers) and 5 ^l of the template RNA were added. For positive control reaction, 50 ^l of DEPC-treated water were added to control mix bead and then the entire contents of the control tube were transferred into a tube containing a RT-PCR Bead. The reaction tubes were incubated at 42°C for 15-30 min in heat block or thermal cycler. Then, the reaction tubes were incubated at 95°C for 5 min in order to inactivate the reverse transcriptase. The PCR analysis was carried out in MiniCycler™ (MJ Research) thermocycler for 30 cycles with a 1 min denaturation step at 94°C, 1 min annealing at 58°C and 1 min extension at 72°C. After RT-PCR amplification, the PCR products were resolved on 2 % agarose gel electrophoresis and the amplification profiles of the primer pairs were analyzed. 2.7 Data analysis Peroxidase loci were labeled sequentially with those migrating closest to the anodal end designated as number 1, while alleles at each locus were labeled alphabetically from the most anodal band. A tree illustrating the genetic diversity among barley landraces and varieties, using unweighted pair-group method with arithmetic average (UPGMA), was generated based on peroxidase isozymes data using NTSYS-pc version 2.1 (Rohlf, 2000). The RAPD amplicons (bands) were recorded as 0 (absence) or 1 (presence) and were also analyzed by cluster analysis using the UPGMA method using the NTSYS-pc version 2.1. The goodness of fit of the cluster analysis, for both of isozymes and RAPD data, was determined by calculating the cophenetic value matrix from the tree matrix in order to carry out the Mantel test (Mantel, 1967) to determine the agreement between the two matrices. 3 RESULTS 3.1 PER isozyme analysis Only three loci (PER 1, PER 2 and PER 3) were obtained from peroxidase enzyme analysis in the 29 accessions and varieties of barley. Out of these three loci, two loci (PER 2 and PER 3) were polymorphic in all accessions obtained from Algeria and Tunisia. All the three loci (PER 1 "one band", PER 2 "2 bands" and PER 3 "one band") were monomorphic in Egyptian varieties and landraces, where three accessions (Giza 129, Matrouh-Awama and Giza 131) showed one band with slower electrophoretic mobility. PER 1 and PER 3 were monomorphic with only one band shown in the Egyptian accessions and varieties. PER 3 displayed three polymorphic isozymes (bands) in the Algerian and Tunisian landraces. However, in case of PER 1 and PER 2, two polymorphic bands were observed in Algerian accessions. Nevertheless, in the Tunisian accessions, PER 1 (one band) was monomorphic and PER 2 (one band) was also monomorphic. Three Tunisian accessions (Tomban, Gabes and KairooaA) showed one unique allele in locus PER 2. Based on the profiles of peroxidase isozymes, cluster analysis grouped all Egyptian barley varieties into one main cluster group at a distance of 1.5 (Fig. 1). However, only one landrace (Matrouh) was contained into a separate subgroup with two accessions (Manel and Tozeurt) from Tunisia and one accession (Sidimehdi) from Algeria. Similarly, Algerian and Tunisia accessions were grouped into one main at the same distance (1.5). Two accessions (Azrir: Algeria and Gabes: Tunisia) were found in one of the subclusters with two Egyptian barley varieties. A unique main cluster contained only one Algerian accession (Temasine), see Fig. 1. Cophenetic correlation based Mantel t-test was r = 0.800, which indicated a good fit to the dendrogram obtained with the cluster analysis. Acta agriculturae Slovenica, 109 - 2, september 2017 193 R. M. GAAFAR et al. Sidimehdi i Toz eurt I Manel Matrooh 1 Kasrmegarine i Saida 1 ~~ Kibilliz-1_| _ Rihan ' Rasellouche | __ Tomban ' KairooaA 1 _ Nailia i Rihone03 | I SidiBeozid ' Tozeur2 | Jerba 1 Tichedielt ' Azrir i Gabes 1 Giza129-1_| I Giza131-1 Arish i Giza123' I Giza126 i___1 Giza125' I_I _ Giza130-' Giz a2000 i_| Giza1271 Temasine ' 0.00 0.43 0.86 1.29 1.72 Distance Figure 1: Cluster analysis of the isozymes-derived data of 29 barley accessions collected from three North African countries 3.2 RAPD analysis RAPD analysis based on two random primers (OPA3 and OPG3) showed that about 60 % of amplified bands were polymorphic. The number of bands (alleles) ranged from 3 to 12 per primer, with an average of 6 per primer. Cluster analysis based on RAPD data revealed that the two accessions (Sidimehdi and Temasine) from Algeria were clearly separated at a distance of 2.47 as two identical genomes from all other barley accessions. The Egyptian accessions were grouped into two obvious groups; one contained 4 accessions (Giza126, Giza2000, Giza127 and Giza125) and the second contained two accessions (Giza123 and Giza129). However, only one accession (Giza130) was out of these two groups. Moreover, it was obvious that the accessions of each group were genetic invariable (showed high similarity). The Tunisian accessions showed no distinct clustering. The most high dissimilarities were revealed by the Algerian accessions (Fig. 2). Cophenetic correlation based on Mantel t-test, used to measure the goodness of fit of RAPD cluster analysis, was r = 0.977, which showed a very good fit to the generated dendrogram. Sidimehdi i Temasine ' Kasrmegarine > Rihone03-' I__ Nailia I RasEllouche i Azrir i Tozeurt i — Tozeur2 Arish i I Matrooh Manel i i I SidiBeozid ' Giza126 -1 Giza2000 _| Giza127 -' —| _ Giza125 Gabes ' Kibilliz-1_| -1 Giza131-' Tomban i KairooaA I ' Rihan Giza123 i_| -1 Giza129' Jerba ' i Giza130-' _ Tichedielt 1 Saida 1_____ 0.00 0.62 1.24 1.85 2.47 Distance Figure 2: Cluster analysis of the RAPD-derived data of 29 barley accessions collected from three North African countries 3.3 RT-PCR and cDNA analysis The total RNA was isolated with TriFast method, which ensured high quality total RNA in sufficient amount. RNA was checked for quality and quantity using agarose gel electrophoresis. To amplify the selected resistance gene-sequences from the Egyptian barley genotypes, which may represent candidate sequences 19? 188 Acta agriculturae Slovenica, 109 - 2, september 2017 Molecular genetic analysis of some North African barley germplasms for the resistance gene analogs of barley; comparative RT-PCR amplifications with each primer pair (Table 2) were performed. The total RNA isolated from two Egyptian barley genotypes (El-Awamah and Awlad-Ali) was used as template for RT-PCR analysis. The genomic DNA of the barley genotypes was also used to check and compare the amplification profiles when using RNA and DNA as templates. In case of DNA template, ordinary PCR analysis was performed. Figure 3 showed that the primer combinations AB089942P1P2 and CV064086P1P2 amplified 3 weak amplicons in both barley genotypes (El-Awamah and Awlad-Ali). The primer pair AF092524P1P2 successfully amplified two PCR amplicons of approximately (340 & 220 bp) and (360 & 270 bp), respectively in both barley genotypes (Fig. 3). However, primer pair DN988165P1P2 gave only one amplicon in both barley genotypes (El-Awamah and Awlad-Ali) of 250 and 270 bp, respectively. The primer pairs AJ849917P1P2 and X17564P1P2 failed to amplify any fragments from both barley genotypes. Figure 3: PCR amplification of specific DNA markers based on primers derived from resistance gene ortholog sequences, cDNA was used as template. +ve: positive control Although, the primer combinations AB089942P1P2 and CV064086P1P2 have amplified three faint bands in the One-Step RT-PCR, they were successful to amplify only one specific PCR fragments when genomic barley DNA (El-Awamah) was used as template. The two PCR fragments were of approximately 230 bp and 200 bp, respectively (Fig. 4). Similarly, primer pair DN988165P1P2 amplified only one specific fragment of 700 bp. The X17564P1P2 primer pair failed to amplify any PCR product, while primer pairs AF092524P1P2 and AJ849917P1P2 amplified very faint fragment of 450 bp. Figure 4: PCR amplification of specific DNA markers based on primers derived from resistance gene ortholog sequences, genomic DNA extracted from El-Awamah landrace was used as template Acta agriculturae Slovenica, 109 - 2, september 2017 193 R. M. GAAFAR et al. 4 DISCUSSION In this study, high genetic variability was observed among the barley landrace accessions and varieties collected from the three North African countries (Algeria, Egypt and Tunisia). However, relatively low genetic diversity was shown among the landrace accessions and varieties from the same region. In addition, development of molecular markers based on pathogen-resistance gene sequences was successful and two gene-based primer pairs amplified specific DNA fragments, which could be used in barley crop improvement. The possible explanation of the low genetic diversity among varieties (collected from one country) is that they have a relatively narrow gene pool. Similar finding has been observed in the polish old oat cultivars, where low genetic variation of the old cultivars collection was noticed, which has been related to the entirely limited gene pool of such cultivars (Boczkowska et al., 2014). Evaluation of the degree of genetic diversity within cultivated barley and its related wild germplasm is necessary for barley crop improvement and for the conservation of barley genetic resources (Boczkowska et al., 2014; Gepts, 2006). Therefore, estimation of genetic diversity of the North African barley landrace accessions and varieties were examined using isozymes and RAPD molecular markers. RAPD markers provide a powerful tool for studying all aspects of genetic variability and genetic structure of the populations. Genetic data derived from isozymes are more robust this due to isozymes are codominant markers. However, isozyme analysis is restrained because fewer number of loci are generated. RAPDs are dominant markers. Therefore, there is less information per band. However, RAPD analysis has several advantages over other marker types: it has more loci that can be tested with RAPD, it is simple, has low cost, and it needs little amount of plant DNA (Garcia Mas et al., 2000). It is well known that genetic variability of the wild populations is essential for plants to be adapted to environment (Nevo et al., 1997). Canadian durum wheat cultivars, which were analyzed by AFLP markers, showed that the cultivars that had been bred from landraces were characterized by higher genetic heterogeneity compared to those that were derived from commercial cultivars (Soleimani et al., 2002). The current results indicated that it is possible to use specific primers based on the resistance gene sequences to amplify PCR products using cDNA or DNA as templates. Also, the amplified PCR products would help in identification of RGAs from barley. Wild barley and Middle Eastern landraces have already proven to be a very beneficial source of genes for modern crop improvement (Ellis et al., 2000). The most obvious example is the development of barley varieties that have mlo allele, which showed resistant to powdery mildew (Thomas et al., 1998). Similar approach was used to develop gene-based markers, which were used to construct a dense linkage map in yellow fever mosquito Aedes aegypti (Linnaeus in Hasselquist, 1762). In this approach, cDNA sequences were downloaded from GenBank and primers were set to amplify PCR fragments of about 500 bp (Fulton et al., 2001). The identification of QTL and in special cases, gene cloning, are steps in the process of building a program for the genetic manipulation of abiotic stress tolerance (Ellis et al., 2000) without using transformation. Fourmann et al. (2001) reported the development and mapping in Brassica napus L. of a series of resistancegene analogs based on existing sequences of nucleotide-binding resistance genes. Some of the sequences could be amplified in B. oleracea L. and B. rapa L. and were employed as helpful markers, which were linked to disease resistance in the three major cultivated Brassica species. Recently, barley genetic resources were mined for genes and alleles relevant for non-specific resistance (NR) to powdery mildew, which is caused by Blumeria graminis (DC.) Speer.f. hordei (Bgh). In that study, eleven candidate genes were identified, where they showed significant SNP or haplotype associations with the Bgh-phenotypes in a worldwide collection of spring barley (Spies et al., 2012). Marker assisted selection (MAS) signifies that DNA markers, which are tightly-linked to target loci are exploited as a substitute for assist population phenotyping (Collard et al., 2005). In our study, some of the sequences amplified specific DNA fragments (Fig. 4), which could also be used for marker assisted selection in the breeding program of barley crop improvement. Similarly, Giovanelli et al. (2002) developed gene-based markers, which were associated with cotyledon-stage downy mildew (Hyaloperonospora parasitica (Pers.) Constant.) resistance in broccoli. They proposed that such markers could be used for marker-assisted selection to generate downy mildew resistant varieties. 188 Acta agriculturae Slovenica, 109 - 2, september 2017 Molecular genetic analysis of some North African barley germplasms In conclusion, genetic diversity analysis revealed that relatively low genetic variability was found among the landraces and varieties from the same region. Using RT- PCR-based approach, it was successful to amplify gene-based DNA marker bands, which could be employed in the breeding of disease-resistant barley genotypes. 5 REFERENCES Barua U.M., Chalmers, K.J., Hackett, C.A., Thomas, W.T.B., Powell, W., Waugh, R. (1993). Identification of RAPD markers linked to a Phynchosporium secalis resistance locus in barley using near isogenic lines and bulked segregant analysis. Heredity, 71, 177-184. doi:10.1038/hdy.1993.122 Bhattacharyya, U., Pandey, S.K., Dasgupta, T. (2014). Identification of EST-SSRs and FDM in sesame (Sesamum indicum L) through data mining. Scholarly Journal of Agricultural Science, 4(2), 6069. Boczkowska, M., Harasimiuk, M., Onysk, A. (2014). Studies on genetic variation within old polish cultivars of common oat. Cereal Research Communications. doi:10.1556/CRC.2014.0025. Brown, A.D.H., Burdon, J.J., Grace, J.P. (1990). Genetic structure of Glycine canescens. A perennial relative of soybean. Theoretical and Applied Genetics, 79, 729-736. doi:10.1007/BF00224237 Ceccarelli, S., Grando S., van Leur, J.A.G. (1995). Barley landraces of the Fertile Crescent offer new breeding options for stress environments. Diversity, 112-113. Chalmers, K.J., Barua, U.M., Hackett, C.A., Thomas, W.T.B., Waugh, R., Powell, W. (1993). Identification of RAPD markers linked to genetic factors controlling the milling energy requirement of barley. Theoretical and Applied Genetics, 87, 314-320. doi:10.1007/BF01184917 Collard, B.C.Y., Jahufer, M.Z.Z., Brouwer, J.B., Pang, E.C.K. (2005). An introduction to markers, quantitative trait loci (QTL) mapping and marker-assisted selection for crop improvement: The basic concepts. Euphytica, 142, 169-196. doi:10.1007/s10681-005-1681-5 Dangl, J.L. & Jones, J.D. (2001). Plant pathogens and integrated defence responses to infection. Nature, 411, 826-833. doi:10.1038/35081161 Devos, K.M. & Gale M.D. (1992). The use of random amplified polymorphic DNA markers in wheat. Theoretical and Applied Genetics, 84, 567-572. doi:10.1007/bf00224153 Doyle, J.J. & Doyle, J.L. (1990). Isolation of plant DNA from fresh tissue. Focus, 12, 13-15. Ellis, R.P., Forster, B.P., Robinson, D., Handley, L.L., Gordon, D.C., Russell, J.R. et al. (2000). Wild barley: a source of genes for crop improvement in the 21st century?. Journal of Experimental Botany, 51, 9-17. doi:10.1093/jxb/51.342.9 Fernández, M.E., Figueiras, A.M., Benito, C. (2002). The use of ISSR and RAPD markers for detecting DNA polymorphism, genotype identification and genetic diversity among barley cultivars with known origin. Theoretical and Applied Genetics, 104, 845-851. doi:10.1007/s00122-001-0848-2 Fetch, T.G., Steffenson B., Nevo, E. (2003). Diversity and sources of multiple disease resistance in Hordeum spontaneum Plant Disease Journal, 87, 1439-1448. Fourmann, M., Chariot, F., Froger, N., Delourme, R., Brunel, D. (2001). Expression, mapping, and genetic variability of Brassica napus disease resistance gene analogues. Genome, 44, 1083-1099. doi:10.1139/g01-098 Fulton, R.E., Salasek, M.L., DuTeau, N.M., Black, W.C. (2001). SSCP analysis of cDNA markers provides a dense linkage map of the Aedes aegypti genome. Genetics, 158, 715-726. Garcia Mas, J., Oliver, M., Gomez Paniagua, H., de Vicente, M.C. (2000). Comparing AFLP, RAPD and RFLP markers for measuring genetic diversity in melon. Theoretical and Applied Genetics, 101, 860-864. doi:10.1007/s001220051553 Gepts, P. (2006). Plant genetic resources conservation and utilization: The accomplishments and future of a societal insurance policy. Crop Science, 46, 22782292. doi:10.2135/cropsci2006.03.0169gas Giovanelli, J.I., Farnham, M.W., Wang, M., Strand, A.E. (2002). Development of sequence characterized amplified region markers linked to downy mildew resistance in broccoli. American Society for Horticultural Science, 127, 597-602. Gottlieb, LD. (1981). Electrophoretic evidence and plant populations. Progress Photochemistry, 7, 146. Joshi, C.P. & Nguyen, H.T. (1993). RAPD (Random amplified polymorphic DNA) analysis based on intervarietal genetic relationships among hexaploid Acta agriculturae Slovenica, 109 - 2, september 201l R. M. GAAFAR et al. wheats. Plant Science, 93, 95-103. doi:10.1016/0168-9452(93)90038-2 Laemmli, U.K. (1970). Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature, 227, 680-685. doi:10.1038/227680a0 Mantel, N.A. (1967). The detection of disease clustering and a generalized regression approach. Cancer Research, 27, 209-220. Matus, I.A. & Hayes, P.M. (2002). Genetic diversity in three groups of barley germplasm assessed by simple sequence repeats. Genome, 45, 1095-1106. doi:10.1139/g02-071 Mayer, K.F.X., Waugh, R., Brown, J.W.S., Schulman, A., Langridge, P., Platzer, M., et al. (2012). A physical, genetic and functional sequence assembly of the barley genome. Nature, 491, 711-716. doi:10.1038/nature11543 Nevo, E. 1992. Origin, evolution, population genetic and resources for breeding of wild barley. Hordeum spontaneum in the fertile Crescent. In: Shewry PR (ed) Barley: Genetics, Biochemistry, Molecular Biology and Biotechnology (pp.19-143). CAB international: Wallingford. Nevo, E., Kirzhner V.M., Beiles, A., Korol, A.B. (1997). Selection versus random drift: long-term polymorphism persistence in small populations (evidence and modeling). Philosophical Transactions of the Royal Society : Biological Sciences, 352, 381-389. doi:10.1098/rstb.1997.0028 Parchman, T.L., Geist, K.S., Grahnen, J.A., Benkman, C.W., Buerkle, C.A. (2010). Transcriptome sequencing in an ecologically important tree species: Assembly, annotation, and marker discovery. BMC Genomics, 11, 180. doi:10.1186/1471-2164-11-180 Rohlf, F.J. (2000). NTSYS-pc: Numerical Taxonomy and Multivariate Analysis System. Version 2.1. Exeter Software, Setauket, NY. Sánchez de la Hoz, M.P., Dávila, J.A., Loarce, Y., Ferrer, E. (1996). Simple sequence repeat primers used in polymerase chain reaction amplifications to study genetic diversity in barley. Genome, 39, 112117. doi:10.1139/g96-015 Shen, Y., Lebold, K., Lansky, E.P., Traber, M.G., Nevo, E. (2011). "Tocol-omic" diversity in wild barley. Chemistry & Biodiversity, 8, 2322-2330. doi:10.1002/cbdv.201000363 Soleimani, V.D., Braum, B.R., Johnson, D.A. (2002). AFLP and pedigree-based genetic diversity estimates in modern cultivars of durum wheat [Triticum turgidum L. subsp. durum (Desf.) Husn.]. Theoretical and Applied Genetics, 104, 350-357. doi:10.1007/s001220100714 Soltis, D.E., Haufler, C.H., Darrow, D.C., Gastony G. J. (1983). Starch gel electrophoresis of ferns: a compilation of grinding buffers, gel and electrode buffers, and staining schedules. American Fern Journal, 73, 9-27. doi:10.2307/1546611 Spies, A., Korzun, V., Bayles, R., Rajaraman, J., Himmelbach, A., Hedley, P.E. et al. (2012). Allele mining in barley genetic resources reveals genes of race-non-specific powdery mildew resistance. Frontiers in Plant Science, 2, 113-135. doi:10.3389/fpls.2011.00113 Tanyolac, B. (2003). Inter-simple sequence repeats (ISSR) and RAPD variation among wild barley (Hordeum vulgare subsp. spontaneum) populations from west Turkey. Genetic Resources and Crop Evolution, 50, 611-614. doi:10.1023/A:1024412814757 Thomas, W.T.B., Baird, E., Fuller, J.D., Lawrence, P., Young, G.R., Russell J. et al. (1998). Identification of a QTL decreasing yield in barley linked to Mlo powdery mildew resistance. Molecular Breeding, 4, 381-393. doi:10.1023/A:1009646115967 Vanhala, T.K. & Stam, P. (2006). Quantitative trait loci for seed dormancy in wild barley (Hordeum spontaneum C. Koch). Genetic Resources and Crop Evolution, 53,1013-1019. doi:10.1007/s10722-004-7368-2 188 Acta agriculturae Slovenica, 109 - 2, september 2017 doi: 10.14720/aas.2017.109.2.04 Original research article / izvirni znanstveni članek Effects of organic fertilizers on growth and biochemical characteristics of Fenugreek 1S. ALAGHEMAND, 1H. AYYUBI, 1E. HASANPOUR, 1J.A. OLFATI, 2*M. SAADATIAN, 1Y. HAMIDOGHLI 1S. KAZEMI AND 1H. KARIMI Received April 10, 2016; accepted April 10, 2017. Delo je prispelo 10. aprila 2016, sprejeto 10. aprila 2017. ABSTRACT Fenugreek (Trigonella foenum-graecum L.) is an annual herb used as organic (green) manure and has medicinal applications. Organic fertilizers are used in sustainable agriculture of vegetables. Sources of organic manure and their effects on growth and yield characteristics of plants need to be determined. Effects of vermicompost and vermiwash were determined on qualitative and quantitative factors of chemical content, development and yield of fenugreek (Trigonella foenum-gaecum L.) from May to July 2012 at Agriculture College of University Guilan. A completely randomized block design with 3 replications was used. Treatments included 7 t/ha of cow manure, vermicompost, vermiwash (obtained from 7 t/ha vermicompost); 7 t/ha of leachate vermicompost + vermiwash and a control (no fertilization). Use of organic fertilizers beneficially affected plant height, pod length, pod fresh and dry mass, 1000-seed mass, plant fresh and dry mass, internode length and percents of leaves protein and nitrogen. Use of organic fertilizers may increase yield and yield components of fenugreek and its yield efficiency. Key words: Trigonella foenum-graecum L.; yield components and their chemical composition; organic fertilizer; sustainable agriculture; vermicompost IZVLEČEK UČINKI ORGANSKIH GNOJIL NA RAST IN BIOKEMIJSKE LASTNOSTI SABLJASTEGA TRIPLATA Sabljasti triplat (Trigonella foenum-graecum L.) je enoletno zelišče, ki se uporablja kot organsko (zeleno) gnojilo in je uporabno tudi v medicini. Organska gnojila se uporabljajo v trajnostni pridelavi zelenjave. Vire organskih gnojil in njihove učinke na rast in pridelek rastlin je potrebno še preučiti. Učinki komposta deževnikov (vermikomposta) in njihovih izcedkov (vermiwash) na kakovostne in količinske parametre kemijske sestave, razvoja in pridelka sabljastega triplata so bili preučevani od maja do julija 2012 na Agriculture College of University Guilan. Uporabljen je bil popoln naključni bločni poskus s tremi ponovitvami. Obravnavanja so obsegala: 7 t/ha kravjega gnoja, vermikompost, vermiwash (pridobljen iz 7 t/ha vermikomposta); 7 t/ha izcedka vermikomposta + vermiwash in kontrola (brez gnojenja ). Uporaba organskih gnojil je ugodno vplivala na višino rastlin, dolžino strokov, suho maso, maso 1000 semen, svežo in suho maso rastlin, dolžino internodijev in na odstotek beljakovin in dušika v rastlinah. Uporaba organskih gnojil lahko poveča učinkovitost pridelka in njegovih komponent pri sabljastem triplatu. Ključne besede: Trigonella foenum-gaecum L.; pridelek in njegova kemijska sestava; organska gnojila; trajnostno kmetijstvo; vermikompost 1 INTRODUCTION Fenugreek (Trigonella foenum-gaecum L.) is an annual herb used as organic (green) manure and has medicinal applications (Kaviarasan et al., 2007; Bukhari et al. 2008; Haouala et al., 2008). Seed of fenugreek contains lysine and L-tryptophan, proteins, mucilaginous fiber and saponins, coumarin, fenugreekine, nicotinic acid, sapogenins, phytic acid, scopoletin and trigonelline (Bukhari et al., 2008). Polyphenol content in vegetables can be affected by environmental factors, cultivation, and conditions at, and after, harvest (Suthar, 2008). Organic fertilizers improve soil pH and cation exchange capacity (CEC) and increase micro-organism diversity 1 Department of Horticultural Science, Faculty of Agricultural Sciences, University of Guilan, Rasht, Iran 2 Soran University, Faculty of Education, General Sciences Department, Soran, Kurdistan Regional Government, Iraq; Corresponding author: saadatian@hortilover.net Acta agriculturae Slovenica, 109 - 2, september 2017 str. 165 - 173 S. ALAGHEMAND et al. and activity (Renato et al., 2003). Excessive applications of chemical fertilizers reduce plant performance due to soil acidification, reduced soil biological activities, degradation of soil physical features, and lack of micronutrients (Adediran et al., 2004). Use of compost materials results in improved soil fertility (Kasthuri et al., 2011). The garbage earthworm (Eisenia fetida (Savigny, 1826)) digests organic waste and converts it to materials beneficial for plant growth; the final product of its activity is called vermicompost (Gunadi et al., 2002). Materials produced by earthworms contain several nutritional substances easily available to plants (Suthar, 2008; Taylor et al., 2003). Vermicompost has high porosity, high potential of uptake and storage of mineral elements, which are gradually released. It also has high water holding capacity, and is used in sustainable agriculture of vegetables (Arancon et al., 2004a; Atiyeh et al., 2002). This research was conducted to evaluate effects of sources of organic manure on growth and yield characteristics of fenugreek to identify which is the best for natural antioxidants, total phenol and protein content in seed. 2 MATERIAL AND METHODS For producing vermicompost, 25 g/kg or 2.5 kg/m2 earthworm were added to the cow manure bed and vermicomposted for 2 months (Peyvast et al., 2008). For producing vermiwash, 50 l of water was added to 100 kg vermicompost and after 48 hrs extracts were purified by passage through a textile filter. The experiment was conducted at the University of Guilan Campus, Agriculture Faculty, Rasht, Iran (altitude 7 m below mean sea level, 37°16' N, 51°3' E), from May to July 2012. Annual average temperature is 15.9 °C. Thermal amplitude is 35.3 °C in summer and 6.6 °C in winter. Average rainfall is about 1359 mm per year with most occurring during late summer. The loam soil was plowed, disked and clods broken. Raised seedbeds, 0.3 m wide and 15 cm high, were prepared. Seed were planted in rows 15 cm apart at a depth of 0.5-1.0 cm on 23 April 2012. The bed surface was covered with a 1 cm of vermicompost or soil. Before spreading fertilizers, 3 mixed samples of soil were selected from 0 to 30 cm depth with 3 replications. Samples were dried in the air, and contents determined (Table 1). Furrow irrigation was used at 2 day intervals. Table 1: Chemical and physical characteristics of soil (Greenhouse building at University of Guilan, 2012) Soil texture Clay % Silt % Sand % Available K (mg/l soil extract) Available P (mg/l soil extract) Total nitrogen % Organic carbon % Total saturation acidity % EC (exchange cation)x103 loam 19 30 51 219 65.6 0.168 1.08 7.44 1 A completely randomized block design with 3 replications was used. Plots were 4 m2. Treatments included 7 t/ha of vermicompost; vermiwash (obtained from 7 t/ha vermicompost); vermicompost leachate + vermiwash from 7 t/ha vermicompost and a no fertilizer control (levels of nutrition in the soil sufficient for the crop which the recommended nutrition levels may not be correct and we are testing the additional fertilizer to determine if it provides a benefit). Vermicompost and leachate vermicompost were spread over beds and vermiwash applied 3 times at 7 day intervals. At flowering plant height, plant fresh and dry mass, pod fresh and dry mass, number of nodes, number of lateral branches, internode length, number of pods, pod length, number of seed per pod, total phenol, percent of antioxidant activity, fresh yield, dry yield, and contents of nitrogen and protein in leaves were determined. The Bradford protocol (Bradford, 1976) was used to determine protein amount in seed. The DPPH ability for scavenging free radicals was determined based on the method of Du et al. (2009) with minor modifications. Briefly, 50 pi of fenugreek extracts were added to 950 pi of a 6.25x10-5 M solution of DPPH in methanol. A control sample containing the same amount of solvent in place of extract was used for measuring maximum DPPH absorbance. The reaction took place in the dark for 30 min; absorbance at 517 nm was measured to determine the concentration of remaining DPPH. The 198 198 Acta agriculturae Slovenica, 109 - 2, september 2017 Effects of organic fertilizers on growth and biochemical characteristics of Fenugreek percent DPPH, which was scavenged (% DPPHsc), was calculated using: % DPPHsc = Acont - Asamp/Acont x 100 where Acont is absorbance of the control, and Asamp is absorbance of the sample. Total phenols were determined spectro-photometricaly using Folin-Ciocalteau colorimetric method with modifications as described by Singleton et al. (1999). Data were subjected to ANOVA in SAS (ver. 9.1, SAS Institute, Inc., Cary, NC) and means separated using the Tukey test. 3 RESULTS AND DISSCUSSION The organic fertilizers did not affect numbers of pods/plant, number of seed/pod, number of nodes, number of lateral branches, antioxidant capacity, total phenols, and dry yield (Tables 2 - 4). Fertilizer affected plant height, pod length, pod fresh mass, 1000-seed mass, plant fresh mass and internode length; there were no differences between vermicompost types (Tables 2 -4). In no fertilizer control the soil nutrient content is sufficient for some, but not all, benefits to the crop. However the lowest value was obtained from the unfertilized control of many traits. Organic fertilizers gave the lowest 1000 seed mass, plant fresh mass, plant dry mass, protein and nitrogen content of seed were obtained from control plants; fertilization decreased pod dry mass (Table 2 - 4). Plants treated with vermicompost were the tallest, as reported by Yadav et al. (2003), Arguello et al. (2006) and Almulla et al. (2012) in other crops. Organic fertilizer enhanced plant height what could be due to the plant hormone auxin (Muscolo et al. 1999). Organic fertilizer can affect soil properties and suppress plant diseases and improve plant health. Pod length was increased by vermicompost treatment and pod length may be related to seed yield via indirect effects of vermicompost on seed yield via pod length. The highest pod fresh mass was due to the vermicompost treatment. The 1000-seed mass were different between vermicompost and vermivash treatments. Plants treated with vermicompost had the highest total yield/ha (Table 4). It may be that chemical and physical properties of humic acid in vermicompost increase uptake of hormones and nutrients resulting in improved growth and yield (Arancon et al., 2005), increased soil microorganism activity (Arancon et al., 2004b), improved nitrogen accumulation, and increased total yield. Plants treated with vermiwash + vermicompost had the longest internodes. Renato (2003) reported that organic fertilizers in the soil can increase element uptake. Organic manure increases soil aggregation, aeration, water holding capacity, and supply roots with an extended source of nutrients (Rani and Nishana, 2012). Vermiwash increased fresh and dry mass; it may be that organic fertilizers increased water absorption through distribution and development of roots. Mucus deposit of epidermal cells and coelomic fluid produced by worms contain plant hormones and chemical exudates (Rani and Nishana, 2012). Antioxidant capacity was not affected by organic fertilizer which contrasts to those of Haghighi (2011) using sewage sludge on leafy vegetable growth. High antioxidant capacity was recorded in the vermicompost treatment. Haghighi (2011) indicated that a deficit of nutrient availability and uptake in the control induced antioxidant activity. Pant et al. (2009) claimed that low plant growth and N concentration caused high level of antioxidant activity in leafy vegetables. Amounts of phenolic compounds in plants grown under organic conditions are higher than those grown under inorganic conditions (Asami et al., 2003; Dixon, 2001) which contrast with our results. Higher total phenolic compounds can cause the decrease in environmental stress to plants (Asami et al., 2003). Higher levels of total phenolic content was found in plants treated with vermicompost compared with those grown with Osmocote (a type of trade fertilizer) and this was attributed to a gradual release of available nutrients in plants from vermicompost (Asami et al., 2003; Pant et al., 2009; Wang and Lin, 2002). Protein and nitrogen contents were affected by fertilizers as also reported by Kasthuri et al. (2011) who found that Municipal Solid Waste Compost affected protein content of fenugreek. The protein and nitrogen contents increased with vermiwash + vermicompost leachate treatment. It may be that soil microorganism activity under high manure (Arancon et al., 2004b) results in nitrogen accumulation in plants due to increases in protein and organic manure affected soil amylase, invertase activity and cellulose and increased microbial biomass (Kasthuri et al., 2011). Vermicompost (Peyvast et al., 2008) and vermiwash (Suthar, 2010) were proposed as organic fertilizers but there is no information on effects of the combined application of vermiwash and vermicompost on vegetables. The main problem that can arise from excessive application of vermicompost is toxicity due to high salt content. With leaching, negative effects related to high salinity decreased and continuous application of this material may be possible. Vermiwash and leachate vermicompost can be used as fertilizers for cultivation of organic fenugreek. Application of organic fertilizers may help alleviate salinity and sodium problems that develop as a result of excessive chemical fertilizers and irrigation (Almulla et al., 2012). Using sustainable and environmentally friendly organic materials can increase Acta agriculturae Slovenica, 109 - 2, september 2017 S. ALAGHEMAND et al. fertility without negative effects on human health and were continued during next year and their beneficially the environment. Organic fertilizers effects especially effect should be examined. vermicompost, vermiwash and vermicompost leachate Table 2: Mean comparison of some measured characteristics in fenugreek (Greenhouse building at University of Guilan, 2012) Treatment Means Plant height (cm) Number of pods per plant Plant fresh mass (g) Pod length (cm) Pod fresh mass (g) Pod dry mass (g) ermicompost 51a 73.33aa 11.67aa 13.2a 8.41a 1.02b Vermiwash 48a 68.67a 11a 12.81a 7.57a 1.35ab Vermiwash+vermicompost 45.67a 74a 11.33a 12.53a 7.74a 1.15ab Control 35.67b 64.33a 10.33a 10.36b 5.78b 1.45a a values in columns followed by the same letter are not significantly different, P<0.05, Tukey test. Table 3: Mean comparison of some measured characteristics in fenugreek (Greenhouse building at University of Guilan, 2012) Treatment Means 1000-seed mass (g) Number of nodes Number of lateral branches Plant fresh mass (g) Plant dry mass (g) Internode length (cm) Vermicompost 1.49a 9.33aa 9.33aa 49.36b 23.69ab 6.18a Vermiwash 1.49a 9a 9.33a 57.12a 26.04a 5.89a Vermiwash+vermicompost 1.28b 8.33a 9.33a 52.27ab 22.57ab 6.22a Control 0.78c 8.33a 9.67a 43.86c 19.44b 4.24b a values in columns followed by the same letter are not significantly different, P<0.05, Tukey test. 198 Acta agriculturae Slovenica, 109 - 2, september 2017 Effects of organic fertilizers on growth and biochemical characteristics of Fenugreek Table 4: Mean comparison of some measured characteristics in fenugreek (Greenhouse building at university of guilan, 2012) Treatment Means Total yield (g) Dry yield (g) Total phenol (mg galic acid /100 g) Antioxidant capacity (% of inhibition) Seed protein (%) Nitrogen in seed (%) Vermicompost 2453.9aa 298.43a 4.9aa 39.02a 24.35ab 4.60ab Vermiwash 2076.9ab 370.35a 4.66a 38.31a 23.83ab 4.50ab Vermiwash+vermicompost 2280.6ab 331.76a 4.58a 38.22a 24.65a 4.65a Control 1490.4b 372.13a 4.34a 37.23a 20.83b 3.93b a values in columns followed by the same letter are not significantly different, P<0.05, Tukey test. 4 CONCLUSIONS According to the data the addition of the fertilizer provided improvement to some measured variables but a cost:benefit analysis will have to be done to determine if the cost of the fertilizer justifies its use. Further study is needed to confirm these results under field conditions. Use of organic fertilizers beneficially affected plant height, pod length, pod fresh and dry mass, 1000-seed mass, plant fresh and dry mass, internode length and percents of leaves protein and nitrogen. Use of organic fertilizers may increase yield and yield components of fenugreek and its yield efficiency. 5 REFERENCES Adediran JA., Taiwo LB., Akande MO., Sobulo RA., Idowu OJ. (2004). Application of organic and inorganic fertilizer for sustainable maize and cowpea yields in Nigeria. Journal of Plant Nutrition, 27, 1163-1181. doi:10.1081/PLN-120038542 Almulla L., Bhat NR., Lekha VS., Thomas B., Ali S., George P., Xavier M. (2012). Effect of three organic fertilizer formulations on growth and yield of cherry tomato (Lycopersicon esculentum cv. Sakura) under soilless organic greenhouse production system. European Journal of Scientific Research, 80(3), 281-288. Arguello JA., Ledesma A., Nunez SB., Rodriguez CH., Goldfarb MDD. (2006). Vermicompost effects on bulbing dynamics, nonstructural carbohydrate content, yield and quality of Rosado paraguayo garlic bulbs. Horticultural Sciences, 41(3), 589592. Arancon N., Edwards CA., Bierman PC., Metzger JD. (2004a). Influences of vermicomposts on field strawberries: 1. Effects on growth and yields. Bioresource Technology, 93, 145-153. doi:10.1016/j .biortech.2003.10.014 Arancon NQ., Edwards CA., Atieyh RM., Metzger JD. (2004b). Effect of vermicomposts produced from food waste on the growth and yields of greenhouse peppers. Bioresource Technology, 93, 139-143. doi:10.1016/j.biortech.2003.10.015 Arancon NQ., Galvis PA., Edwards A. (2005). Suppression of insect pest populations and damage to plants by vermicomposts. Bioresource Technology, 96(10), 1137-1142. doi:10.1016/j .biortech.2004.10.004 Acta agriculturae Slovenica, 109 - 2, september 2017 S. ALAGHEMAND et al. Asami D., Hang Y., Barnett D., Mitchelle A. (2003). Comparison of the total phenolic and ascorbic content of freeze-dried and air dried marionberry, strawberry, and corn grown using conventional, organic and sustainable agricultural practices. Journal of Agricultural and Food Chemistry, 51, 1237-1241. doi:10.1021/jf020635c Atiyeh RM., Arancon N., Edwards CA., Metzger JD. (2002). The influence of earthworm processed pigmanure on the growth and productivity of marigolds. Bioresource Technology, 81, 103-108. doi:10.1016/S0960-8524(01)00122-5 Bradford M. (1976). A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Analytical Biochemistry, 72, 248-254. doi:10.1016/0003-2697(76)90527-3 Bukhari SB., Bhanger MI., Memon S. (2008). Antioxidative activity of extracts from Fenugreek seeds (Trigonella foenum-graecum). Pakistan Journal of Analytical & Environmental Chemistry, 9(2), 78-83. Dixon RA. (2001). Natural products and plant disease resistance. Nature, 411, 843-847. doi: 10.1038/35081178 Du G., Li M., Ma F., Liang D. (2009). Antioxidant capacity and the relationship with polyphenol and vitamin C in Actinidia fruits. Journal of Agricultural and Food Chemistry, 113, 557-562. doi:10.1016/j.foodchem.2008.08.025 Gunadi B., Edwards CA., Blount C. (2002). The influence of different moisture levels on the growth, fecundity and survival of Eisenia foetida (Savigny) in cattle and pig manure solids. Soil Biology and Biochemistry, 39, 19-24. Haghighi M. (2001). Sewage sludge application in soil improved leafy vegetable growth. Journal of Biological & Environmental Sciences, 5(15), 165167. Haouala R., Hawala S., El-Ayeb A., Khanfir R., Boughanmi N. (2008). Aqueous and organic extracts of Trigonella foenum-graecum L. inhibit the mycelia growth of fungi. Journal of Environmental Sciences, 20, 1453-1457. doi: 10.1016/S1001 -0742(08)62548-6 Kasthuri H., Shanthi K., Sivakumar S., Rajakumar SH., Son K., Song YC. (2011). Influence of municipal solid waste compost (MSWC) on the growth and yield of green gram [Vigna radiate (L.) Wilczek], fenugreek (Trigonella foenum-graecum L.) and on soil quality. Iranian Journal of Environmental Health Science & Engineering, 8(3), 285-294. Kaviarasan S., Naik GH., Gangabhagirathi R., Anuradha CV., Priyadarsini KI. (2007). In vitro studies on antiradical and antioxidant activities of fenugreek (Trigonella foenumgraecum) seeds. Journal of Agricultural and Food Chemistry, 103, 31-37. doi:10.1016/j.foodchem.2006.05.064 Muscolo A., Bovalo F., Gionfriddo F., Nardi S. (1999). Earthworm humic matter produces auxin-like effects on Daucus carota cell growth and nitrate metabolism. Soil Biology and Biochemistry, 31, 1303-1311. doi:10.1016/S0038-0717(99)00049-8 Pant AP., Radovich TJK., Hue NV., Talcottb ST., Krenekb KA. (2009). Vermicompost extracts influence growth, mineral nutrients, phytonutrients and antioxidant activity in pakchoi (Brassica rapa cv. Bonsai, Chinensis group) grown under vermicompost and chemical fertilizer. Journal of the Science of Food and Agriculture, 89, 23832392. doi:10.1002/jsfa.3732 Peyvast Gh., Olfati JA., Madeni S., Forghani A. (2008). Effect of vermicompost on the growth and yield of spinach (Spinacia oleracea L.). Journal of Food Agriculture and Environment, 16(1), 110-113. Rani TS., Nishana H. (2012). Influence of different sources of organic manure, vermiwash and AMF on growth and yield of bhindi (Abelmoschus esculentus L. Moench). Plant Archives, 12(1), 7578. Renato Y., Ferreira ME., Cruz MC., Barbosa JC. (2003). Organic matter fractions and soil fertility underinfluence of liming, vermicopmpost and cattle manure. Bioresource Technology, 60, 59-63. Singleton VL., Orthofer R., LamuelaRaventos RM. (1999). Analysis of total phenols and other oxidation substrates and antioxidants by means of Folin-Ciocalteu reagent. Methods of Enzymology, 299, 152-178. doi:10.1016/S0076-6879(99)99017-1 Suthar S. (2008). Bioconversion of postharvest crop residues and cattle shed manure into value-added products using earthworms Eudrilus eugeniae Kinberg. Ecological Engineering, 32, 206-214. doi:10.1016/j. ecoleng.2007.11.002 Suthar S. (2010). Pilot-scale vermireactors for sewage sludge stabilization and metal remediation process: Comparison with small-scale vermireactors. Ecological Engineering, 2009.12.016. doi:10.1016/j.ecoleng.2009.12.016 Taylor M., Clarke WP., Greenfield PF. (2003). The treatment of domestic wastewater using small-scale vermicompost filter beds. Ecological Engineering. ?0? 198 Acta agriculturae Slovenica, 109 - 2, september 2017 Effects of organic fertilizers on growth and biochemical characteristics of Fenugreek 21(2-3), 197-203. doi:10.1016/j.ecoleng.2003.12.003 Wang SY., Lin S. (2002). Compost as soil supplement enhanced plant growth and fruit quality of strawberry. Journal of Plant Nutrition, 25, 11432259. doi:10.1081/pln-120014073 Yadav RL., Keshwa GL., Yadav SS. (2003). Effect of integrated use of FYM and sulphure on growth and yield of isabgol. Journal of Medicinal and Aromatic Plant Sciences, 25, 668-671. Acta agriculturae Slovenica, 109 - 2, september 2017 doi: 10.14720/aas.2017.109.2.05 Original research article / izvirni znanstveni članek Assessment of morphological variability and chemical composition of some local pepper (Capsicum annuum L.) populations on the area of Kosovo Sali ALIU, Imer RUSINOVCI, Shukri FETAHU, Skender KAÇIU and Dukagjin ZEKA Received May 12, 2016; accepted April 12, 2017. Delo je prispelo 12. maja 2016, sprejeto 12. aprila 2017. ABSTRACT Seven local pepper populations (Capsicum annuum L.) from different geographical regions of Kosovo, were evaluated for morphological traits, chemical composition, and antioxidant contents using standard analytical techniques. All local peppers populations were characterized for different morphological traits from seedling emergence to crop maturity. The total genetic variation for plant height (PH) was 11.72 cm or expressed in relative values was 27.94 %. Average of leaf area (LA) per plant was 2308.38 cm2, while the lowest value for LA was 1136 cm2. Yield per plant ranged from 265 to 691 g plant-1. The acidity level was ranging from 1.44 to 1.61 %, carbohydrates varied greatly from 4.21 to 6.07 %. Vitamin C (as ascorbic acid) content in fresh fruit ranged from 65.544 to 520.51 mg 100g-1 of fresh mass. Minerals were of reasonable levels with Fe (15.31 mg kg-1), Ca (216.71 mg kg-1), Na (406.01 mg kg-1), K (1851 mg kg-1), and Zn (5.74 mg kg-1). Key words: Capsicum annuum L.; genetic diversity; antioxidant content; mineral composition IZVLEČEK OVREDNOTENJE MORFOLOŠKE SPREMENLJIVOSTI IN KEMIČNE SESTAVE NEKATERIH POPULACIJ PAPRIKE (Capsicum annuum L.) NA KOSOVU S standardnimi analitskimi tehnikami so bile ovrednotene morfološke lastnosti, kemijska sestava in vsebnost antioksidantov sedmih lokalnih populacij paprike (Capsicum annuum L.) iz različnih geografskih območij Kosova. Za vse populacije so bile ovrednotene različne morfološke lastnosti od vznika do zrelosti plodov. Celokupna genetska spremenljivost za višino rastlin (PH) je bila 11.72 cm, izražena v relativnih vrednostih je bila 27.94 %. Povprečna listna površina na rastlino (LA) je bila 2308.38 cm2, najmanjša 1136 cm2. Pridelek na rastlino je bil med 265 in 691 g na rastlino. Vsebnost kislin je bila med 1.44 in 1.61 %, ogljikovih hidratov med 4.21 in 6.07 %. Vsebnost vitamina C v svežih plodovih je bila med 65.544 in 520.51 mg 100g-1 sveže mase. Vsebnosti mineralov so bile v običajnih območjih in sicer Fe, 15.31 mg kg-1, Ca, 216.71 mg kg-1, Na, 406.01 mg kg-1, K, 1851 mg kg-1, in Zn, 5.74 mg kg-1. Ključne besede: Capsicum annuum L.; genetska raznolikost; vsebnost antioksidantov; mineralna sestava 1 INTRODUCTION In the world, several hundred types of peppers are cultivated. Chili pepper (Capsicum spp.) is a solaneceous plant, whose centre of origin in Middle America and Mexico is centre of genetic diversity and domestication (Pickersgill, 1971). Csillery (2006) indicates that the first component description of Capsicum was given in Hungarian herbal by Dioszegi and Fazekas (1807) cited by Bozokalfa et al., (2009). Pepper (Capsicum annuum L.) is an important agricultural crop, not only because of its economic importance, but also for the nutritional value of its fruits, mainly due to the fact that they are an excellent source of natural colours and antioxidant compounds (Conforti et al., 2007; Deepa et al., 2007). Peppers are one of the main vegetables planted in Kosovo regarding production area and economic importance. The dominate type of pepper production is open field cultivation. Also, the local pepper genotypes in Kosova are rich in diversity with different populations where it has been cultivated for centuries and which is very useable for human food (Aliu et al., 2012; Kaciu et al., 2010). Almost indispensable food, salads, condiment of every Kosovar cuisine is pepper. In Kosovo from total area planted with vegetables, 4449 ha or expressed in 1 University of Prishtina, Faculty of Agriculture and Veterinary, Department of Crop Science, Kosovo; Corresponding author: sali.aliu@uni-pr.edu, dukagjinzeka@yahoo.com Acta agriculturae Slovenica, 109 - 2, september 2017 str. 165 - 173 Sali ALIU et al. percentage is 30.68 % are planted with pepper (MAFRD, 2014). Most pepper cultivars currently grown in Kosovo are open pollinated. Some local populations are still grown on many small farms due to consumer demand. For decades, these have been cultivated in different environments and growing techniques. Almost all the cultivars grown are of landraces types which are characterized by a wide range of observable variability. Since their introduction into the world, peppers are cultivated in various environments and a number of different populations were developed (Govindarajan, 1986). In general, they are genetically diverse and well adapted to the locations where they have been developed (Votava et al., 2005). Estimating genetic diversity and determining the relationships between germplasm collections helps ensure germplasm is efficiently collected and managed (Bozokalfa et al., 2009). Data on the level of genetic diversity of a germplasm collection may also increase the efficiency of efforts to improve this crop (Geleta et al., 2005). Pepper fruits are also source of vitamins A, complex B1 and B2, C and minerals such as dietary calcium, iron, magnesium etc. (Bosland,1992). A number of studies report that hot pepper seeds are rich in minerals content (Zou et al, 2015; Jarret et al., 2013; Park et al., 2006). The content of vitamin C in the pepper fruit is higher than in Citrus (Finger et al., 2010). The pepper fruit is a rich source of vitamin A, E, C and P in green chilli (Hosmani, 1993; Howard et al., 2000; Marin et al., 2004). They have a high level of vitamins C and E as well as the total of antioxidants is completed by phenolic compounds, which occur in peppers in connection with sugars (Materska et al., 2003; Shotorbani and Jamei, 2013). Antioxidants are beneficial because of their protective roles against multiple diseases such as cancer, anemia, diabetes and cardiovascular diseases. The compounds perform their function by counteracting the oxidizing effects on lipids by scavenging highly reactive oxygen free radicals, the major oxidizing factors for the oxidative modification of low density lipoprotein and nucleic acids (Perucka and Malgorzata, 2007). However, there is little information available about nutritional constituents of hot pepper seeds grown in Kosova. Therefore, the specific objective of this study was to evaluate morphological traits, and antioxidant content in local pepper populations. 2 MATERIALS AND METHODS 2.1 Plant material and collection of samples Local pepper populations were collected from different bio-climatic regions throughout Kosovo. Kosovo has a central geographic position on the Balkan Peninsula. It lies between latitude 41°50'58 "and 43°51'42" north and 20°01'30" and longitude 21°48'02" east. Seven local peppers populations (LPP) were collected from various agro-climatic regions. The localities of the sampling sites for the Kosova region were: Krusha, Shtime, Figure 1: Geographical areas of local pepper populations 206 Acta agriculturae Slovenica, 109 - 2, september 2017 Lipjan, Viti, Mitrovica, Istog and Decani. The altitudes of the sampling sites ranged from 306 to 649 m above sea level. The overall climate of Kosovo is a modified continental type, with some elements of a sub-mediterranean climate in the extreme south. Summers are hot with extreme temperatures of up to 37°C. The average annual rainfall is 720 mm (HMIK, 2008). These were selected to represent various geographical areas. (Figure 1). Assessment of morphological variability ... local pepper (Capsicum annuum L.) populations on the area of Kosovo A total of seven genotypes within 7 geographical was coded as; RKS-A, RKS-B, RKS-C, RKS-D, RKS-regions were studied under different field conditions E, RKS-F and RKS-G. over summer 2014. Plant material used in this research Table 1: Pepper populations (Capsicum spp.) analyzed within their geographical origin Code Longitude Latitude Elevation Geographical origin Local name RKS-A 020°39'19" 42°18'29" 310 Krusha Babure RKS-B 021°32'23" 42°27'27" 577 Shtime Somborka RKS-C 021°10'43" 42°29'04" 564 Lipjan Somborka RKS-D 021°24'02" 42°21'47" 494 Viti Dolma RKS-E 021°54'10" 42°51'47" 521 Mitrovica Somborka RKS-F 21°04'05" 42°36'39'' 479 Istog Somborka RKS-G 20°17'48" 42°31'42" 649 Decan Somborka All accessions were characterized for different agro-morphological traits from seedling emergence to crop maturity. All characteristics were measured in the field and laboratory at the normal harvest time. The methodology used to record qualitative values from seedling to harvest was obtained from the descriptor for Capsicum from International Plant Genetic Resources Institute (IPGRI 1995). The experimental design was a split plot with randomized complete block with three replications. Plants from local pepper populations were collected in farmer's fields during the period when full maturity of the plants were reached. Local paper populations were collected from different bio-climatic regions throughout Kosovo. To investigate the different traits which were including in our study we collected per location 30 plants x 7 locality = 210 plants (for each location = 3 repetitions x 10 plants/repetition = 30 plants/populations).The following characteristics were measured in Plant breeding laboratory, Faculty of Agriculture, Department of Crop science: plant height (PH), fruit diameter (FD), plant mass without fruits and root (PM), root mass (RM), number of flowers (FN), leaf number (LN), number of fruits (NF), fruit mass (FM), and yield per plant (YP). Leaf area (LA) per plant was measured by planimeter. Ascorbic acid extraction: The vitamin 'C' as ascorbic acid content (AAC) in fresh pepper fruits was determined by the titratability of 5 g of the blended pulp homogenized with 50 ml of oxalic acid at a concentration of 12 %. The titratable solution consisted of 2.6 sodium indophenol dichlorophenol. Results were expressed in mg of ascorbic acid per 100 g of pulp. pH: was measured by using a digital pH meter with the application of the electrode directly in to the blended pulp. The carbohydrates were determined by equipment of refractometer type R 200 (Reichert technologies, USA) precision placing a small sample of blended pulp on the reading prism. Results were expressed as percentage (%). Water content: 5 g of fruit were taken from each replication, cut into pieces dried in a temperature 105oC in equipment type AD-MF50 until constant mass (90 minutes). Results were expressed in percentage. Mineral composition: Mineral elements including Fe, Ca, Zn, Mg, Na, K and Cu in (mg kg1 ) were determined through burning and mineralizing of samples of fruit at 550 oC for 4 to 6 hours. Samples were digested in HCl and subsequently element concentrations were estimated using an atomic absorption spectrophotometer (1100 B Perkin-Elmer, Germany). Statistical analyses: all statistical analyses were performed with the SPSS software (version 15.0, SPSS Inc., 2006) to investigate the difference between the populations. Effects of the studied traits were evaluated by ANOVA. Mean separation within columns was done by Duncan's Multiple Range test. In order to assess the differentiation of local pepper populations (LPP's) based on all variables that were measured, the Canonical Discriminant Analyses (CDA) were applied. CDA is a technique for classifying set of observations into predefined classes. Acta agriculturae Slovenica, 109 - 2, september 2017 211 Sali ALIU et al. 3 RESULTS AND DISCUSSION The collection of local pepper populations investigated from Kosovo showed considerable variation in morphological and biometric fruit parameters. Plant height (PH) varied from 32.16 to 46.89 cm. The differences between them are 14.73 cm or expressed in percentage is 35.12 %. Results are presented in Table2. According to the analysis of variance (ANOVA) the differences for fruit diameter (FD) between populations were significant. The genetic variation for FD among them was with maximum of 4.73 cm and a minimum of 3.41 cm or differences among populations was 1.32 cm or 31.42 %. Similar range of FD in different pepper genotypes (2.74 to 4.57 cm) was reported by Bozokalfa et al. (2009) and Bassiony et al. (2010) (5.75 till 6.97 cm). The genetic variability for root mass (RM) ranges from 149.30 g plant-1 to 100.31 g plant-1 which was significant at level of probability of LSD (p = 0.01). Differences for these two populations were 48.99 g plant-1 or expressed in relative value was 47.79 %. The significantly higher value for number of flowers (NF) was characterized for population RKS-A with 73.55 flowers per plant, while lower number of flowers was recorded at RKS-C and RKS-E (38.01 flowers per plant). The differences between them were 35.54 flowers per plant or 70.45 %. The leaf number (LN) per plant also showed significant differences among populations. This trait segregated in a manner similar to plant yield. The variability varied from 217.01 to 125.88 leaves per plant, and had significant difference of 91.13 per plant or 52.45 %. Obtained results were in accordance with those obtained by Bassiony et al. (2010), obtaining LN from 158.5 to 191.67 leaves per plant. The leaf area is a one of the crucial factors in photosynthesis. The population RKS-B produced a higher (2308 cm2 plant-1) leaf area per plant (LA), while lower value was determined for population RKS-E, namely 1136 cm2 plant-1. The differences between two populations were 1171.56 cm2 plant-1 or expressed in percentage 64.92 %. The results concerning LA are given in Table 2. Also other characters varied substantially. The number of fruits (NF) across pepper populations ranged from 9.44 to 19.66. In our case this trait had an effect an on yield per plant. Plant high yields depend on many factors, the most important factors are the structure of genotypes and environments. Production capacity is a complex character, a result of few morphological components, number of flowers, fruit size, and fruit mass (Madosa et al., 2008). The highest significant contribution (691.44 g) in production (yield) per plant was determined at population RKS-D, followed by RKS -A, a value of 540.66 g plant-1. The major difference among populations is 425.96 g plant-1 or 91.34 % respectively. Results are presented in Table 2. 206 Acta agriculturae Slovenica, 109 - 2, september 2017 Assessment of morphological variability ... local pepper (Capsicum annuum L.) populations on the area of Kosovo Table 2: Morphological and agronomic traits recorded in different pepper populations PH FD RM PM FN LN LA NF FM YP Populations (cm) (mm) g plant-1 g plant-1 plant-1 plant-1 cm2 plant1 plant-1 g plant-1 g plant-1 RKS-A 46.89±0.16a 4.42±0.005a 138.21±0.005a 116.23±0.015a 73.55±0.015a 162.33±0.025bc 1392±0.473b 15.11±0.055ab 35.71±0.015ab 540.66±0.050b RKS-B 42.44±0.10a 3.96±0.010b 113.65±0.020b 86.71±0.010b 57.44±1.73b 217.01±0.026a 2308.±0.451a 10.66±0.020cd 38.01±0.040a 402.21±0.412c RKS-C 46.55±0.02a 4.47±0.010a 135.36±0.010b 111.76±0.020a 38.01±0.040c 178.33±0.015ab 2175±0.666a 13.33±0.015bc 32.83±0.055ab 431.91±0.196bc RKS-D 41.66±0.05a 4.73±0.18a 149.30±0.005a 120.14±0.011a 45.220.043bc 185.11±0.066ab 2009±0.100a 19.66±0.020a 35.09±0.035ab 691.44±0.294a RKS-E 32.16±0.02b 3.41±0.005b 100.31±0.015b 77.73±0.010b 38.01±0.040c 125.88±0.043c 1136±0.252b 9.44±0.015d 27.68±0.11b 265.48±0.247d RKS-F 33.26±0.02b 3.89±0.010b 101.21±0.010b 79.85±0.017b 37.21±0.015c 120.25±0.164c 1212±0.100b 9.78±0.035d 25.21±0.09b 425.96±0.230bc RKS-G 40.25±0.025a 4.41±0.010a 115.25±0.011b 78.56±0.020b 44.52±0.010bc 124.78±0.066c 1354±0.950bc 13.24±0.025bc 31.25±0.020ab 425.96±0.225bc Mean 40.45±0.025 4.2±0.005 121.89±0.010 95.854±0.017 47.708±0.016 159.09±0.035 1655.54±0.0.493 13.031±0.037 32.25±0.010 454.8±0.133 LSDp=0.05 8.58 1.03 20.86 12.23 10.86 85.05 1239.04 3.37 9.32 132.79 0.01 12.49 1.50 30.36 17.79 15.81 90.34 1802.69 4.90 13.56 193.20 * Values are given as means of three replicates ± SD. Means with different superscript letters within a column are significantly different (P < 0.05). Notes: PH - plant height; FD - fruit diameter; RM-root mass; PMW - plant mass ; FN - flower number; LN - leaf number; LA - leaf area; NF- number of fruits; FMW- fruit mass; YP - yield per plant Acta agriculturae Slovenica, 109 - 2, september 2017 209 Sali ALIU et al. The acidity level is from 1.44 to 1.61 % at populations RKS-E and RKS-B, respectively (Table3). Apart from this, these acids make up the energetic reserves and the metabolic reactions that involve the synthesis of pigments, enzymes and other materials and degradation of pectins and celluloses, which are essential in different processes (Antoniali et al., 2007). Table 3: Average values for some fruit parameters of local pepper populations Population Acidity mg 100 g-1 pH values Carbohydrates % *AAC content mg 100 g-1 Water content % RKS-A 1.52±0.005b 5.03±0.037b 4.21±0.010c 65.12±0.011a 91.33±0.147b RKS-B 1.61±0.006a 5.12±0.010b 6.07±0.068a 58.54±0.045d 90.56±0.168b RKS-C 1.52±0.010b 5.27±0.020a 4.21±0.015c 62.21±0.121b 93.26±0.0921a RKS-D 1.53±0.010b 5.24±0.020a 6.14±0.015a 52.51±0.010e 89.71±0.134b RKS-E 1.44±0.005bc 5.07±0.041b 5.59±0.016b 60.91±0.071c 90.76±0.157b RKS-F 1.54±0.005b 5.02±0.046b 5.51±0.032b 59.54±0.623d 87.85±0.735b RKS-G 1.51±0.004b 5.11±0.456b 4.38±0.274c 57.65±0.231d 92.35±0.257b Average 1.52±0.057 5.15±0.015 5.24±0.041 59.86±0.219 91.12±0.166 LSD p = 0.05 0.13 0.11 0.39 0.17 1.33 * AAC- ascorbic acid content. Values are given as means of three replicates ± SD. Means with different superscript letters within a column are significantly different (P < 0.05). The content of carbohydrates varied greatly within and among LPP's, with values ranging from 4.21 to 6.07 %. The difference among populations for dry fruit mass is 1.86 % or expressed in percentage values was 35.49% higher. AAC is the least complex vitamin found in plants and is synthesized from glucose or other carbohydrates (Kays, 1991). Vitamin C (as ascorbic acid) content in fresh fruit ranged from 65.544 to 520.51 mg 100 g-1 of fresh mass. The difference among pepper populations for AAC was 12.61 mg 100 g-1 or genetic variation was 21.06 % (Table 3). It was found, that hot pepper cultivars were richer in vitamin C, than the sweet ones. As other studies have shown, the highest or the lowest values of vitamin C in C. annuum are dependent on the variety and the maturity stage of the fruits (Khadi et al., 1987; Howard et al., 2000). For example, in the C. annuum pepper grown in Turkey, a variation that ranged from 15.2 to 64.9 mg 100 g-1 fresh fruit was reported (Topuz and Ozdemir, 2007). And another study conducted in India with the same species showed a variation that ranged from 48.23 to 192.63 mg 100 g-1 as reported by Deepa et al. (2006). One of the factors affecting the production of plant biomass is the concentration of mineral elements. The differences among local pepper populations (LPP) are presented in Table 4. According to the results the highest average values of zinc (Zn) (7.86 mg kg-1) and copper (Cu) (13.9 mg kg-1) was determined LPP coded for population RKS-A. While with the greatest accumulation of iron (Fe) was recorded for RKS-C population with the average value of 23.16 mg kg-1. In our study the other elements including; Ca, Na, K and Mg were on the higher significance results. Results are given in |Table 4. ? i o 206 Acta agriculturae Slovenica, 109 - 2, september 2017 Assessment of morphological variability ... local pepper (Capsicum annuum L.) populations on the area of Kosovo Table 4: The average mineral content (mg kg-) in pepper fruits Populations Zn1 Cu1 Fe1 Ca1 Na1 K1 Mg1 RKS-A 7.86±0.043a 13.9±0.17a 14.33±0.13b 236.76±0.59ab 225.76±0.10d 5337.06±4.1a 280.3±0.63b RKS-B 7.06±0.072ab 0.82±0.21e 14.66±0.13b 216.13±0.40b 238.86±0.24c 2360.83±2.81ab 271.9±0.35bc RKS-C 4.93±0.077bc 3.26±0.025b 23.16±0.05a 230.2±0.95ab 982.13±0.14a 572.16±1.15e 323.6±0.28a RKS-D 2.93±0.066c 1.04±0.141d 13.33±0.10c 319.01±0.69a 670.66±0.55b 898.16±0.35d 322.53±0.085a RKS-E 5.63±0.0921b 1.31±0.005c 13.01±0.095c 83.46±0.13c 247.2±0.17c 1998.76±0.27c 176.01 ±0.41c RKS-F 4.75±0.0945bc 3.11±0.015b 13.78±0.141c 210.11±0.011b 228.78±0.66cd 895.63±0.78d 275.56±0.092bc RKS-G 7.02±0.0461ab 0.98±0.016e 14.89±0.21b 221.3±0.10b 248.7±0.53c 901.23±0.51cd 285.23±0.15b Mean 5.74±0.034 3.490.055± 15.31±0.061 216.71±0.11 406.01±0.45 1851.98±1.24 276.45±1.03 * Values are given as means of three replicates ± SD. Means with different superscript letters within a column are significantly different (P < 0.05). Elemental nutrient composition of local pepper populations (mg kg-1) The canonical discriminant analysis (CDA) of the traits is presented in Figure 2. The first canonical functions described 93.4 % and a second canonical function is 5.6 % of the existing variance. The CDA analysis reported here differentiates LPP's on the basis of similarity. The first group is consisted by RKS-A and RKS-B, second group is consisted by RKS-C and RKS-G. The other populations are separated as individuals, RKS-D, RKS-E and RKS-F. The main elements that can affect the classification to the different groups were the genotype and environment which had influenced on the content of mineral composition and quality traits. The genotype and environmental interaction effects tend to be large when there is a wide variation among genotypes for different traits (Kanf, 1998). Figure 2: Canonical discriminant function at local pepper populations Acta agriculturae Slovenica, 109 - 2, september 2017 211 Sali ALIU et al. 4 CONCLUSIONS The study showed that there was a significant morphological variability among local pepper populations. Some populations are interesting for quantitative traits including leaf area and yield per plant. A high variability was also determined for vitamin C, carbohydrates and mineral composition. The cluster analysis shows that there is an obvious diversity among the populations collected in different geographical regions. The observed morphological diversity among pepper populations is helpful for breeding programs aimed in selecting superior genotypes. These local pepper populations should be included in broader genetic analyses and should be considered as a source of new genetic variability used for the development of inbred lines in the Kosovo breeding program. The evaluation of plant genetic resources has been considered of prime importance, especially in those species having economic importance. 5 ACKNOWLEDGEMENTS The first author would like to express his sincere appreciation to all of my co-authors for their continuous support and suggestions in preparing of this article. 6 REFERENCES Antoniali S., Paulo A., Maria M., Fuziki T., Sanches J. (2007). Physico-chemical characterization of Zarco HS yellow bell pepper for different ripeness stages. Scientia Agricola, 64, 1, 19-22. doi:10.1590/S0103-90162007000100003 Aliu S., Rusinovci I., Fetahu Sh, Salihu S., Zogaj R. (2012).Nutritive and Mineral Composition in a Collection of Cucurbita pepo L. Grown in Kosova. Food and Nutrition Sciences, 3, 634-638. doi:10.4236/fns.2012.35087 Bozokalfa K., Esiyok D., Turhan K. (2009). Patterens of phenotypic variation in a germplasm collection of pepper (Capsicum annuum L) from Turkey. Spanish Journal of Agricultural Research, 1, 83-95. doi:10.5424/sjar/2009071-401 Bassiony, A., Fawzy,F., Samad,H., Riad,G.( 2010). Growth, yield and fruit quality of sweet pepper plants as effected by potassium fertilization. Journal of American Science, 6, 722-729. Bosland P. (1992). Chiles; a diverse crop. Horticultural Technology,2, 6-10. Conforti, F., G.A. Statti, and F. Menichini. (2007). Chemical and biological variability of hot pepper fruits (Capsicum annuum var. acuminatum L.) in relation to maturity stage. Food Chemistry, 102, 1096 - 1104. doi: 10.1016/j .foodchem.2006.06.047 Csillery G.(2006). Pepper taxonomy and the botanical description of the species. Acta Agronomica Hungarica, 54, 151-166. doi: 10.1556/AAgr.54.2006.2.5 Deepa, N., C. Kaur, B. George, B. Singh, and H.C. Kapoor. (2007). Antioxidant constituents in some sweet pepper (Capsicum annuum L.) genotypes during maturity. Food Science and Technology, 40,121-129. doi:10.1016/j.lwt.2005.09.016 Deepa, N., C. Kaur, B. Singh, and H.C. Kapoor. (2006). Antioxidant activity in some red sweet pepper cultivars. Journal of Food Composition and Analysis, 19, 572-578. doi:10.1016/j.jfca.2005.03.005 Emilian Madosa , S. Ciulca , Giancarla Velicevici , C. Avadane , Lavinia Sasu , Adriana Cioroga , Ilijana Fri§kan.(2008).Study correlations between component characters of production capacity of sweet. Bulletin UASVM, Horticulture, 65(1). ISSN 1843-5254; e ISSN 1843-5394. p:90-94. Geleta L., Labuschagne M., Viljoen C. (2005). Genetic variability in pepper estimated by morphological data and amplified fragment length polymorphism markers. Biodiversity and Conservation, 14, 23612375. doi:10.1007/s10531 -004-1669-9 Govindaijan, V. (1986). Capsicum production technology, chemistry and quality part II-processed products, standards, world production. Critical Reviews in Food Science and Nutrition, 23, 207288. doi:10.1080/10408398609527426 Hosmani, M. M. (1993). Chilli Crop (Capsicum annuum), Bharat Photo Offset Works, Dharwad. Howard L.R., Talcott S.T., Brenes C.H., Villalon B. (2000). Changes in phytochemical and antioxidant activity of selected pepper cultivars (Capsicum species) as influenced by maturity. Journal of ? 1 ? 206 Acta agriculturae Slovenica, 109 - 2, september 2017 Assessment of morphological variability ... local pepper (Capsicum annuum L.) populations on the area of Kosovo Agricultural and Food Chemistry, 48, 1713-1720. doi:10.1021/jf990916t HMIK (Hydro-meteorological Institute of Kosovo).(2008). Database for sum of Temperatures and Rainfall. . URL http://. www.ammk-rks.net. IPGRI. (1995). International Plant Genetic Resources Institute. Rome. ISBN 92-9043-216-0. Josefa M. Navarro J., Pilar Flores P.,Garrido C, Vicente Martinez. (2005). Changes in the contents of antioxidant compounds in pepper fruits at different ripening stages, as affected by salinity. Food Chemistry, 96, (2006): 66-73. doi:10.1016/j.foodchem.2005.01.057 Jarret, R. L., Levy, I. J., Potter, T. L., & Cermak, S. C. (2013). Seed oil and fatty acid composition in Capsicum spp. Journal of Food Composition and Analysis, 30(2), 102-108. doi:10.1016/j.jfca.2013.02.005 Kanf, M. (1998). Using genotype by environment interaction for crop cultivar development. Advanced Agronomy, 62,199-252. Kays S.J. (1991). Postharvest physiology of perishable plant products. New York Van Nostrand Reinhold. 532 p. doi: 10.1007/978-1-4684-8255-3 Khadi, B.M., J.V. Goud, and V.B. Patil. (1987). Variation in ascorbic acid and mineral content in fruits of some varieties of chilli (Caspsicum annuum L.). Plant Foods for Human Nutrition, 37, 9- 15. doi: 10.1007/BF01092295 Kagiu S, Shala-Mayrhofer V, Mirecki N, Aliu S ,Jezik K. (2010). Influence of environment in electrochemical quality of tomato. International Journal of Horticultural Science, 17-20. MAFRD. (2014). Ministry of Agriculture, Forestry, Rural Development of Kosovo, Statistics Division. Marin A., Ferreres F., Tomas-Barberan F.A., Gil M.I. (2004). Characterization and quantitation of antioxidant constituents of sweet pepper (Capsicum annuum L). Journal of Agricultural and Food Chemistry, 52, 3861-3869. doi:10.1021/jf0497915 Materska M., Perucka I., Stochmal A., Piacente S., Oleszek W. (2003). Quantitative and qualitative determination of flavonoids and phenolic acid derivatives from pericarp of hot pepper fruit cv. Bronowicka Ostra. Polish Journal of Food and Nutrition Sciences, 12/53, SI 2, 72-76. Pickersgill, B.( 1971). Relationships between weedy and cultivated forms in some species of chilli peppers (genus Capsicum). Evaluation, 25, 683691. Irena Perucka, Malgorzata Materska. (2007). Antioxidant vitamin contents of capsicum annuum fruit extracts as affected by processing and varietal factors. ACTA Scientiarum Polonorum Technologia Alimentaria, 6(4):67-74. Park, H., Lee, S., Jeong, H., Cho, S., Chun, H., Back, O., Kim, D., & Lillehoj, H. S. (2006). The nutrient composition of the herbicide-tolerant green pepper is equivalent to that of the conventional green pepper. Nutrition Research, 26(10), 546-548. doi:10.1016/j.nutres.2006.09.001 SPSS-15.(2006). Statistical package program. Shotorbani N., Jamei R., R. (2013). Antioxidant activities of two sweet pepper Capsicum annuum L. varieties phenolic extracts and the effects of thermal treatment. Avicenna Journal of Phytomedicine, 3(1), 25-34. Topuz, A., and F. Ozdemir. (2007). Assessment of carotenoids, capsaicinoids and ascorbic acid composition of some selected pepper cultivars (Capsicum annuum L.) grown in Turkey. Journal of Food Composition and Analysis, 20,596-602. doi:10.1016/j.jfca.2007.03.007 Votava E., Baral B., Bosland P. (2005). Genetic diversity of chile (Capsicum annuum L.) landraces from northern new Mexico, Colorado and Mexico. Economic Botany, 59(1), 8-17. doi:10.1663/0013-0001(2005)059[0008:GDOCCA]2.0.CO;2 Zou Y, Ma K, Tian M. (2015). Chemical composition and nutritive value of hot pepper seed (Capsicum annuum) grown in Northeast Region of China. Food Science and Technology (Campinas), 35(4), 659-663. doi: 10.1590/1678-457X.6803 Acta agriculturae Slovenica, 109 - 2, september 2017 211 doi: 10.14720/aas.2017.109.2.06 Original research article / izvirni znanstveni članek Changes in essential oil and morpho-physiological traits of tarragon (Artemisia dracuncalus L.) in responses to arbuscular mycorrhizal fungus, AMF (Glomus intraradices N.C. Schenck & G.S. Sm.) inoculation under salinity Amin LAMIAN1, Hassanali NAGHDI BADI2, Ali MEHRAFARIN2*, Mehdi SEIF SAHANDI2 Received June 05, 2016; accepted July 06, 2017. Delo je prispelo 05. junija 2016, sprejeto 06. julija 2017. ABSTRACT This study aimed to evaluate the arbuscular mycorrhizal fungi (AMF) (Glomus intraradices N.C. Schenck & G.S. Sm.) inoculation and salinity effect on qualitative and quantitative changes in tarragon yield. Treatments included inoculation, and non-inoculation of AMF, and five salinity levels of irrigation water (with the electrical conductivity of 0, 2, 4, 6, and 8 dS m-1). The results showed the plant height, SPAD value, number of leaves, dry mass of leaves and shoot per plant were reduced under salinity condition. The various levels of salinity decreased the content of tarragon essential oil and some its components consist of a-pinene, limonene, Z-ocimene, E-ocimene, and methyl chavicol while, it increased the content of bornyl acetate, eugenol, methyl eugenol, caryophyllene, germacrene, and a-farnesene. AMF inoculation without salinity had the greatest positive effect on the evaluated traits of tarragon. Also, it improved the morpho-physiological traits under salinity due to alleviation of the harmful effects of salinity. Although the essential oil content was reduced with the AMF inoculation, the methyl chavicol amount was increased by the AMF inoculation under salinity condition. Key words: arbuscular mycorrhizal fungi; Artemisia dracunculus L.; electrical conductivity; essential oils; Glomus intraradices; methyl chavicol; morpho-physiological traits; salinity IZVLEČEK SPREMEMBE V VSEBNOSTI ETERIČNIH OLJ IN MORFOLOŠKO-FIZIOLOŠKIH LASTNOSTI PEHTRANA (Artemisia dracuncalus L.) KOT ODZIV NA INOKULACIJO Z ARBUSKULARNO MIKORIZNO GLIVO (Glomus intraradices N.C. Schenck & G.S. Sm.) V RAZMERAH SLANOSTI Namen te raziskave je bil oceniti vpliv inokulacije z arbuskulamo mikorizno glivo (Glomus intraradices N.C. Schenck & G.S. Sm.) in učinek slanosti na spremembe v kakovosti in količini pridelka pehtrana. Obravnavanja so obsegala inokulacijo in brez inokulacije z AMG ter pet stopenj slanosti vode za namakanje (z električno prevodnostjo 0, 2, 4, 6, in 8 dS m-1). Rezultati so pokazali, da so se vrednosti znakov kot so višina rastlin, SPAD vrednost, število listov, suha masa listov in število poganjkov na rastlino zmajšale v razmerah slanosti. Različne slanosti so zmanjšale vsebnosti eteričnih olj , med njimi a-pinena, limonena, Z-ocimena, E-ocimena in metil kavikola medtem, ko so se vsebnosti bornil acetata, eugenola, metil eugenola, kariofilena, germakrena in a-farnezena povečale. Inokulacija z AMG v razmerah brez slanosti je imela največje pozitivne učinke na vse ovrednotene lastnosti pehtrana. Izboljšala je tudi morfološke in fiziološke lastnosti v razmerah slanosti. Čeprav je bila vsebnost steričnih olj zmanjšana ob inokulaciji z AMG, se je vsebnost metil kavikola povečala v razmerah slanosti. Ključne besede: arbuskularne mikorizne glive; Artemisia dracunculus L.; električna prevodnost; eterična olja; Glomus intraradices; metil kavikol; morfološko-fiziološke lastnosti; slanost 1 Department of Horticulture Science, Science and Research Branch, Islamic Azad University (IAU), Tehran, Iran 2 Medicinal Plants Research Center, Institute of Medicinal Plants, ACECR, Karaj, Iran; "Corresponding author: A.Mehrafarin@gmail.com Acta agriculturae Slovenica, 109 - 2, september 2017 str. 165 - 173 Amin LAMIAN et. al 1 INTRODUCTION Tarragon (Artemisia dracunculus L.) is an herbaceous, perennial plant with alternate leaves of linear shape and light green color (Fernandez-Lizarazo et al., 2011). It is native to Russia and Siberia, alluvial valley areas, but today is spread also in the western areas of North America. However, tarragon is widespread also in parts of temperate Asia, as well as Central Asia and Eastern Europe (Fernandez-Lizarazo et al., 2011; Obolskiy et al., 2011). The tarragon leaves contain about 0.3 % essential oil of which methyl chavicol comprise approximately 70 % of its component. (Chopra et al., 1986; Verma et al., 2010). This plant possesses a wide range of health benefits, therefore, it widely used in traditional medicine & pharmaceutical industry. It's health benefits that can be pointed include appetizing, stomach tonic, diuretic, anti-scurvy, mild anti-worm and induce of menstruation. Tarragon is used in traditional medicine for the resolution of joint pain, hiccups and treatment of painful menstruation. Despite contradictory statements, it was recently reported the tarragon extract is anticancer agent for some cancer cells line such as human leukemic cancer cells, human endometrial cancer cells, human breast cancer cells, etc. (Obolskiy et al., 2011). Saline soil and saline irrigation are one of the most important environmental stresses, particularly in arid and semi-arid regions (Greenway and Munns, 1980; Kuznetsov and Shevyakova, 1997). Salt stress during vegetative and reproductive stage reduces crops biomass and yield (Aslam et al., 1993). Salinity affects plant growth and metabolism through disturbing several physiological processes of plants (Levitt, 1980; Hoshida et al., 2000). The first effect of salinity on plants is reducing water uptake even when the soil is completely wet, due to the decreased soil solution potential. Even more, cell membrane function may be damaged due to intracellular ion homeostasis disruption in plants exposed to salinity. Under these circumstances, some physiological disorders occur and programmed cell death imposed ultimately (Fan et al., 2013). Moreover, salt stress in plants leads to nutrients imbalance, reduction of the photosynthesis efficiency, increasing free radicals production such as superoxide anion (O2.-), hydrogen peroxide (H2O2), hydroxyl radical (OH.) and singlet oxygen (1O2), and metabolic toxicity which causes the destruction of the cell membrane (Greenway and Munns, 1980; Fan, et al., 2013; Sankar, et al., 2007; Agarwal and Shaheen: 2007). Plants responses to salinity stress are complex thus, salinity tolerance mechanisms are not clear yet. During the evolution and adaptation to salinity stress, various molecular mechanisms have been developed in plants for confronting the salinity. Some plants mechanisms cope with salinity by regulating ion homeostasis with accumulation of compatible solutes for osmotic adjustment, free radical scavenging, alterations in membrane structures, and phytohormone biosynthesis stimulation. It was also reported that the ion homeostasis was regulated via selective accumulation of ions, ion exclusion as well as limited ions uptake by root, ion transport to the leaves and that distribution at cellular and plant level (Fan et al., 2013; Hasegawa et al., 2000; Jaleel et al., 2007). Some microorganisms such as plant growth promoting rhizobacteria and fungi, especially arbuscular mycorrhizal fungi (AMF), can improve the plant growth and crop yield in saline soils via alleviating destructive effects of salinity stress (Cho et al., 2006). Arbuscular mycorrhizal fungi inhabit the rhizosphere and have a symbiotic association with the roots of most terrestrial plant species (Gini et al., 2003; Smith and Read 1997). The most common AMF in saline soil are species from the genus Glomus (Ho, 1987; Wang et al., 2004). Molecular studies revealed that about 80 % of fungi spores in saline soil belong to a single species, Glomus geosporum (T.H. Nicolson & Gerd.) C. Walker (Wilde et al., 2009). Wu et al. (2010) reported that root inoculation of Citrus tangerina Tanaka by G. geosporum and Paraglomus occultum (C. Walker) J.B. Morton & D. Redecker could improve growth, photosynthesis rate, root architecture, and ionic balance under 100 mM NaCl. Kapoor et al. (2002) reported AMF diminished the adverse effects of salinity and thereby increased coriander (Coriandrum sativum L.) growth. However, the root inoculation of sour orange (C. aurantium L.) and sweet orange (C. sinensis (L.) Osbeck) by G. intraradices under the 30, and 60 mM salt had no effect on salinity tolerance, but the Cl-concentration increased in root (Zou et al., 2013). AMF can significantly improve resistance of host plants to varied biotic and abiotic stresses. Arbuscular mycorrhiza leads to better nutrients mobility in nutrient poor soils (Marschner and Dell, 1994). Thus, the growth of plants coexisted with AMF is enhanced due to the increased nutrients uptake, especially N and P (Marschner and Dell, 1994). However, the role of AMF in stress conditions and salinity may not be limited only to this nutritional effect (Ruiz-Lozano and Azcon, 2000). The tolerance mechanisms to salinity in plant symbiosis with AMF is consisted with improved osmotic potential adjustment, enhanced water use efficiency, production of plant growth hormones, reduced detrimental effect of oxidative stress, mitigation of toxic ion effects or improved nutritional status (Auge, 2001). According to reports on the alleviating detrimental salinity effects via the AMF root OI f\ 216 Acta agriculturae Slovenica, 109 - 2, september 2017 Changes in essential oil and morpho-physiological ... inoculation under salinity inoculation, this study was aimed for evaluation the under AMF (Glomus intraradices) inoculation and qualitative and quantitative changes of tarragon yield salinity condition. 2 MATERIALS AND METHODS 2.1 Plant preparation and growth conditions This study was conducted in an experimental greenhouse of the Medicinal Plants Institute (MPI) affiliated with the Academic Center for Education, Culture and Research (ACECR) in Karaj as a factorial experiment in the randomized complete block design (RCBD) with 4 replications during 2015. The research station is located at 1472 m a. s. l., 35° 54' N and 50° 53' E. The same rooted transplants of tarragon (Artemisia dracunculus L. var. sativa) were provided from MPI seed bank (1096-MPISB), and were transferred into pots. Plants were grown in a plastic greenhouse for 3 months (from March 21 to June 20, 2015) with a photon flux density about 1300 ^mol m-2 s-1, 16 h light and 8 h dark period and the average temperature of 21/16 °C for day/night. The soil texture was loam-silt, its physio-chemical properties contains 0.08 % nitrogen, 36.2 ppm phosphorus, 49.8 ppm potassium, 7.9 pH, and electrical conductivity (EC) 1.2 dS m-1. 2.2 Treatments Treatments included inoculation and non-inoculation with AMF (Glomus intraradices N.C. Schenck & G.S. Sm.) as the first factor, and five salinity levels of irrigation water (with the EC of 0, 2, 4, 6, and 8 dS m-1) as the second factor. The leaching fraction equal 0.5 was used in irrigation practices in order to less salt accumulation. The electrical conductivity of the solutions was measured by EC meter (HI9811, Hanna, USA) and the salinity levels were kept constant throughout the experiment period for irrigation water. Same amount of irrigation water were applied for each treatment during the growing period. The salinity treatments began 30 days after transplanting via irrigation water with an interval every two days until the harvest time. The treatment solutions were made with saline water and distilled water depending on target salinity, while the control treatment of salinity was prepared with double distilled water. Natural saline water was obtained from Hoz-e-Soltan Lake in Qom, Iran. The major ions of the saline water were: 128 g l-1 Na+, 218.7 g l-1 Cl-, 1.23 g l-1 K+, 19.5 g l-1 Mg2+, 0.086 g l-1 Ca2+, and 48.8 g l-1 SO42-. For this experiment, 40 uniform plastic pots (20 cm upper diameter x 15 cm bottom diameter x 18 cm height) as experimental plots were divided into subgroups with or without AMF inoculation. Inocula consisted of soil possessing fungal spores, hyphae and mycelium. According to the method of Tommerup (1992), fungus identification was checked using light microscope (AXIO Imager; Carl Zeiss, Jena, Germany). AMF inoculum was multiplied in the open pots culture of sweet corn (Zea mays L. convar. saccharata var. rugosa Bonaf.) as a host and after six months of plant growth, the shoots were eliminated and the underground parts were stored for two months in polyethylene bags at 5 °C. Thirty grams of the AMF inoculum (root fragments with 85 % of colonized roots length) was added to 3.0 kg of autoclaved (121°C, 0.11 MPa, 1 h) soil for AMF inoculation (Carretero et al., 2008). Non-mycorrhizal treatments received the same amount of autoclaved AMF inoculum. 2.3 Essential oils analysis The harvested plant materials of tarragon were air-dried in a shaded place at a convenient temperature (24 ± 2 °C) during 6 days. Essential oils of the aerial parts were extracted by hydro-distillation method for 3 h using Clevenger-type apparatus. The essential oils were dried over anhydrous sodium sulfate and kept on 4 °C until further analysis (British Pharmacopoeia, 1988). The extracted essential oils were identified by gas chromatography (GC) and gas chromatography coupled with mass spectrometry (GC/MS) analysis. GC/MS analysis was carried out on an Agilent instrument coupled with a Agilent 5973N Mass system equipped with flame ionization detector (Hewlett-Packard Company, USA) and a SGE BPX5 capillary column (30 m x 0.25 mm; 0.25 ^m film thicknesses, Kinesis Ltd., UK). Temperature program included: an oven temperature held for 5 minutes at 50 °C and enhanced to 240 °C with 3 °C per min rate. Then, enhancement of temperature was programmed up to 300 °C with 15 °C per min rate and this temperature was held for 3 minutes. Other operating conditions include: carrier gas was He with a flow rate of 0.5 ml min-1; injector and detector temperatures were 290 °C, and split ratio, 1:25. Mass spectra were taken at 70 eV (Socaci et al., 2008). The components of the essential oils were identified by comparison of their mass spectra and retention indices with those published in the literature and presented in the MS computer library (Adams, 2001). 2.4 Measurements and statistical analysis The sampling was conducted in the onset of flowering stage as all plants were harvested 93 days after transplanting. For future accuracy and to reduce errors, samples were picked in four replicates randomly from the separate experimental plot. The analyzed morpho- Acta agriculturae Slovenica, 109 - 2, september 2017 221 Amin LAMIAN et. al physiological traits were plant height, the number of leaves, SPAD value, leaf dry mass, shoot dry mass. The SPAD values were recorded using a SPAD-502 meter (Konica-Minolta, Japan). All the data were subjected to statistical analysis (one-way ANOVA) using SAS software (Ver. 9.2). The difference between treatments means was compared by Duncan's multiple range test at 5 % confidence interval. 3 RESULTS AND DISCUSSION Results showed that the AMF inoculation had a significant effect on the plant height (p < 0.05), number of leaves, SPAD value, and leaf and shoot dry mass (p < 0.01). Also, analysis of variance showed that different levels of salinity had a significant (p < 0.01) effect on these traits. Between the salt stress and AMF inoculation was observed a significant interaction in the number of leaves (p < 0.05), leaf and shoot dry mass (p < 0.01) (Table 1). Table l: Analysis of variance for the effects of arbuscular mycorrhizal fungi (AMF), Glomus intraradices inoculation and salinity on morpho-physiological traits of tarragon S.O.V. Df. Mean Square Plant height Number leaves of Leaf dry mass Shoot dry mass SPAD value Replication 3 8.99 ns 91.6 ns 0.005 * 0.005 * 9.76 ns AMF inoculation 1 190.14 * 11262.4 ** 0.22 ** 0.51 ** 106.27 ** Salinity (S) 4 109.9 ** 1828.4 ** 0.03 ** 0.075 ** 48.92 ** AMF X (S) 4 47.68 ns 154.4 * 0.026 ** 0.139 ** 16.69 ns Error 27 26.07 51.37 0.0016 0.0078 7.22 CV ( %) 8.84 6.47 7.22 10.02 7.34 ns: non-significant differences; *: significant at p < 0.05; **: significant at p < 0.01. The plant height and SPAD value were increased for 7.8, and 9.3 percentage by AMF inoculation, respectively (Figure 1), but their amount were decreased under salinity condition. In comparison with the control treatment, the plant height and SPAD values were significantly reduced with increasing salinity to 4 and 6 dS m-1, what indicated that the leaf chlorophyll was more susceptible to raise of salinity (Figure 2, 3). These results also confirm the finding of other studies (Bernstein et al., 2010; Dolataadian et al., 2011; Amira and Qados, 2011; Mukhtar balal et al., 2011). Reduced osmotic potential in salinity condition is resulted in arrest of cell division and elongation (Jacoby, 1994). In addition, Na+ and Cl- accumulation have toxic effects on the cell division and photosynthetic system, a reason for reduced plants growth. Also, salinity stress reduced biosynthesis and transport of cytokinin and gibberellin, but ABA biosynthesis was increased. These factors are contributing to the reduction of plant height under salt stress compared to control (Jacoby, 1994). Destruction of chloroplasts, chlorophyll photo-oxidation and prevented chlorophyll biosynthesis are the main reasons for the decline in content of photosynthetic pigments under salinity conditions (Sultan, 2005). It has also been reported that reduced chlorophyll amount is a consequence of increased chlorophyllase activity under salt stress (Reddy and Vora, 2005). Glutamate is a precursor for proline and chlorophyll biosynthesis. Thus, increased proline production in salt stress decreases glutamate availability in the chlorophyll biosynthesis (Drazkiewicz, 2000). TIO 216 Acta agriculturae Slovenica, 109 - 2, september 2017 75 60 45 30 15 Changes in essential oil and morpho-physiological ... inoculation under salinity AMF inoculation AMF non-inoculation b I Plant height (cm) SPAD value (SPAD) Figure 1: Effect of arbuscular mycorrhizal fungi (AMF), Glomus intraradices inoculation on plant height (cm), and SPAD value (SPAD). The vertical bars represent standard errors of the means 75 60 S 45 30 53 M J 15 Ph 4 Salinity (dS m-1) Figure 2: Effect of irrigation water salinity on plant height (cm). The vertical bars represent standard errors of the means b a 0 0 0 2 6 8 Salinity (dS m-1) Figure 3: Effect of irrigation water salinity on SPAD value (SPAD). The vertical bars represent standard errors of the means Acta agriculturae Slovenica, 109 - 2, September 2017 Amin LAMIAN et. al The number of tarragon leaves was increased by AMF inoculation. However, the number of leaves was reduced through increasing salt concentration under either inoculation or non-inoculation of AMF. The highest number of leaves (with an average of 146.28) was observed in AMF inoculation without the salinity. However, the lowest of it (67.91) was obtained at the 6 dS m-1 salinity without using the AMF (Figure 4). These findings confirm the results of other studies (Bernstein et al., 2010; Amira and Qados, 2011; Mukhtar balal et al., 2011). During salinity stress, plant leaf area was reduced due to smaller leaves formation and leaf abscission. Thus, the photosynthetic capacity diminished and the supply of assimilates for optimal growth was reduced. In addition, the rapid leaves senescence under salt stress caused the reduction of the leaf area durability (Munns, 1993). Use of the AMF could ameliorate the tarragon leaf and shoot dry mass so that AMF inoculation in non-saline condition produced the greatest leaf dry mass (0.76 g) and its minimum (0.39 g) was observed in the 8 dS m-1 salinity without AMF inoculation (Figure 5). Similar to leaf dry mass, the highest shoot dry mass was obtained when the AMF inoculation was used in no-salinity stress (1.33 g) conditions and it's the least amounts was gained in the 8 dS m-1 salinity without AMF inoculation (0.62 g). Also, leaf and shoot dry mass were increased with increasing salinity to 4 dS m-1 withour AMF inoculation. In contrast, increasing salinity to more than 4 dS m-1 led to reduced leaf dry mass compared to the control (Figure 6). These findings are consistent with other studies (Kapoor et al., 2002; Ben Khaled et al., 2003; Rabie and Almadini, 2005; Gupta and Rutaray, 2005; Saleh and Al-Garni, 2006; Porras-Soriano et al., 2009). Plant dry mass reduction in saline conditions is a response to spent metabolic energy for coping with the salt stress (Parida and Das, 2005). Main factors that influenced the plant dry mass consisted of reduced leaf area, increased chlorophyll destruction, reduced photosynthesis rate, toxic effects of Na+ and Cl-accumulation, decreased water uptake, and imbalance in nutrients (Sankar et al., 2007; Agarwal and Shaheen, 2007; Verma and Mishra, 2005). However, some authors reported that phosphorous nutrition can reduce the detrimental effects of salinity stress on plant growth. Therefore, AMF with increased phosphorous uptake can ameliorate for the harmful effects of salinity stress. On the other hand, the potassium content was increased in AMF inoculated plants. Thus, it protected host plant against adverse effect to sodium through enhanced potassium to sodium ratio (Marschner and Dell, 1994; Ruiz-Lozano and Azcon, 2000; Jeffries et al., 2003). Also, it was reported that use of AMF in lettuce is the reason for the roots extension. In addition, the photosynthesis rate and water use efficiency were improved, while the evapotranspiration was reduced (Ruiz- Lozano et al., 1996). The tarragon essential oil content in this study was linearly reduced by the increased salinity. The AMF inoculation in various salinity levels reduced the essential oil content more than when AMF was not used, especially under the 8 dS m-1 salinity. Therefore, the greatest content (1.15 %) of essential oil was obtained in the AMF inoculation under no salinity condition, while its minimum (0.2 %) was acquired in the AMF inoculation along with the 8 dS m-(Figure 7). salinity Figure 4: Effect of irrigation water salinity and arbuscular mycorrhizal fungi (AMF), Glomus intraradices inoculation on number of leaves. The vertical bars represent standard errors of the means 216 Acta agriculturae Slovenica, 109 - 2, september 2017 Changes in essential oil and morpho-physiological ... inoculation under salinity 0,9 0,6 0,3 bc cd £ E 2CTGCAxEnv ^ CTSCAxEnv (5) The dominance ratio (Singh & Pawar, 2005) which explains the ratio of dominance to additive gene effects was calculated using following equation: \2a2 .(6) Xjk = m + gi + gj + Sij + eijk (1) in each location, where, Xijk is the observed value for a cross between the ith and jth parents in the kth replication, m is population mean; gi and gj are GCA values of the ith and jth parents, respectively; sij is the SCA value for the hybrid between the ith and jth parents and eijk is the residual. Combined analysis over locations for combining ability analysis, genetic parameters ($A and ¿2d) and heritability estimates (broad sense and narrow sense heritability) was conducted according to Perkins (1970) by the following formulae: 2.3 Biplot interpretations GGE biplot analysis is expressed as: Yj - Pj = ^£¡1%+ X2el2nj2+ £ij Where, Yij is genotypic values of the combination (F2 hybrids) between genotypes i and tester j for a given trait; pj average value of all combinations with tester j, X and X2 are singular values for PC1 and PC2. The £i1 and £i2 are PC1 and PC2 eigenvectors for genotypes i (entry), respectively; nj1 and nj2 are PC1 and PC2 eigenvectors for tester j, respectively; £ij is the residual of model for inbred i and tester j (Yan & Hunt 2002). In diallel crosses, a parent is both an entry and a tester (Yan & Hunt 2002). Biplot analysis for seed yield per plant done by GGE biplot software (Yan 2001). 3 RESULTS AND DISCUSSION s 2 s D 3.1 Combining ability analysis in individual locations The analysis of variance for the combining ability of evaluated traits for each individual location (Isfahan, Maragheh and Shahrekord) is presented in Table 1. Analysis of variance revealed significant genotypic differences for all studied traits in Isfahan (Table 1a). Combining ability analysis also showed that GCA and SCA mean squares were significant for all traits studied. The combining ability analysis in Maragheh revealed that the genotypes showed a significant difference for all studied traits (Table 1b). The mean squares of GCA were significant for all studied traits (Table 1b). The SCA mean squares were not significant for seed mass (Table 1b). The analysis of variance in Shahrekord revealed a significant difference between genotypes for all studied traits (Table 1C). The mean squares of GCA were significant for capitula per plant, seeds per capitulum and yield per plant (Table 1 C). The SCA mean squares were also significant for capitula per plant, seeds per capitulum and yield per plant (Table 1C). The total comparison of GCA and SCA mean squares presented different ratios of ¿2gca/ ¿2sca (Table 1). Genetic differences were more pronounced under high temperatures (Isfahan) than the low ones (Shahrekord and Maragheh). Also, such other factors as location altitude and relative rainfall might have played a role in the expression of genotypic differences in each location. According to the obtained results, under cold and dry climates (Shahrekord), genotype differences were less pronounced for traits; so, intercrossing of candidate genotypes at this location would result in flat breeding (Barten et al 1993). Acta agriculturae Slovenica, 109 - 2, September 2017 Pooran GOLKAR et al. Table 1: Analysis of variance for combining ability for agronomic traits in safflower under different environment Mean squares of studied traits Isfahan df CP SC SW YP Replication 2 9.39** 27.47** 1.5 45.52** Genotype 35 11.66** 194.73** 49.52** 172.33** GCA 7 37.99** 833.52** 194.32** 594.14** SCA 28 5.08* 35.04** 13.32** 66.88** Error 70 2.95 2.09 5.9 2.94 s2gca 1.10 26.62 6.03 17.58 5 SCA 0.71 10.98 2.47 21.31 52a 2.19 53.23 12.07 35.15 52d 0.71 10.98 2.47 21.31 Maragheh df CP SC SW YP Replication 2 6.52 42.14 66.61 148.34** Genotype 35 7.52** 39.4** 60.07** 77.38** GCA 7 19.14** 35.07 153.12** 237.39** SCA 28 4.59* 40.47** 36.78 37.35* Error 70 2.86 18.52 23.58 22.6 52GCA 0.49 0 3.88 6.67 52SCA 0.58 7.32 4.40 4.92 52a 0.97 0.00 7.76 13.34 52d 0.58 7.32 4.40 4.92 Shahrekord df CP SC SW YP Replication 2 23.06** 49.03* 99.59** 3.88 Genotype 35 14.41** 130.03** 60.92** 159.37** GCA 7 25.83** 532.35** 243.21** 514.26** SCA 28 11.55** 29.43** 15.33 70.35** Error 70 2.83 14.09 14.85 4.16 52GCA 0.48 16.76 7.60 14.80 52SCA 2.91 5.11 0.16 22.06 5S2CAA 0.95 33.53 15.19 29.59 52d 2.91 5.11 0.16 22.06 * and ** significant at P<0.05 and P<0.01, respectively; CP: capitula per plant, SC: seeds per capitulum, SW: 1000-seed weight, YP: yield per plant. 3.2 Combining ability across all locations Information on the relative importance of general and specific combining ability can be helpful in the analysis and interpretation of the genetic basis of important traits in safflower. Analysis of variance combined over environments showed that different environments had a significant effect on all studied traits (Table 2). The results obtained from the combined analysis of variance indicated a significant genotypic difference in F2 diallel experiment for all traits studies (Table 2). Combined analysis of variance for combining ability over three locations revealed that GCA mean squares were highly significant for all the traits (Table 2). The SCA mean square was highly significant, except for SCA of seeds per capitulum (Table 2). This result implied that selection for the improvement of seeds per capitulum could be delayed to later generations until the nonadditive portion would be mitigated to the additive portion as this trait showed the higher magnitude of SCA variance, as compared to GCA variance in the respective location. The proportion of 52gca to 52sca varied from trait to trait. On the other hand, the proportion of 52gca :52SCA showed that most total genetic variability for seeds per capitulum and seed mass was more associated with GCA (fixable genetic portion) than SCA (non- fixable genetic portion) effects (Table 7^7 232 Acta agriculturae Slovenica, 109 - 2, september 2017 Combining ability x environment interaction and ... safflower (Carthamus tinctorius L.): biplot as a tool for diallel data 2); thus simple selection would confer the rapid improvement of these traits. The genotypex environment interaction was significant for seeds per capitulum and yield per plant (Table 2), thereby suggesting significant variation among F2 genotypes for the mentioned traits in their sensitivity to the geographical location. The conditions in this experiment were favorable for detecting genotype x environment interactions, since different temperatures in three locations represented extreme environmental conditions under which high seed yield safflower genotypes could be commercially grown (Suresh et al 2013). Thus, for population improvement of F2 hybrid combinations, evaluation of capitula per plant and seed mass should be sufficient in one location. With considering to combining abilities (GCA and SCA) interaction with different locations, GCA x location interaction was significant for all studied traits, except for seed mass (Table 2), and the SCA x location interaction was significant for seeds per capsule and yield per plant (Table 2). These significant interactions suggested that the selection of different parental genotypes and specific hybrids was required for developing populations specific to each location. 3.3 Genetic components for Seed yield per plant and its components across different environments Genetic components for seed yield and its components (capitula per plant, seeds per capitulum and 1000- seed mass) are presented in Table 3. The higher proportion of 52d (1.66) to 5 a (11) and dominance ratio (1.73) implied the predominance role of dominance gene action, rather than the additive one, for the genetic control of capitula per plant (Table 2). This finding was similar to those obtained in the previous reports (Shahbazi and Saeidi 2007; Golkar et al., 2012; Deedawat et al 2015). According to the obtained results, the large proportion of 52A to 52D and dominance ratio (less than unity) indicated the predominance role of additive gene effects, rather than the dominance ones, in the genetic control of seeds per capitulum and seed mass in safflower. Our findings were consistent with previous reports (Mandal and Banerjee 1997; Shahbazi and Saeidi 2007; Nakhaei et al 2014).The comparison of the magnitude of 52gca (12.61) and 52sca (0.0017) and dominance ratio (1.01) for seed yield per plant indicated the additive - dominance genetic control of this trait (Table 2). The previous studies observed the predominant role of dominance gene action for seed yield per plant (Singh et al 2008; Deedawat et al 2015) and additive gene action in salinity stress (Nakhaei et al 2014). So, this new type of genetic control for the seed yield of safflower per plant could be utilized by both breeding methods based on selection and hybridization. Acta agriculturae Slovenica, 109 - 2, September 2017 Pooran GOLKAR et al. Table 2: Analysis of variance for combining ability for agronomic traits in safflower under different environment Mean squares of the studied traits Source of variation df CP SC SW YP Environment 2 120.9** 13515.1** 1017.4** 4213.7** Rep (Environment) 6 12.99 39.55 55.9 65.9 Genotype 35 27.84** 227.28** 137.54** 365.1** GCA 7 70.85** 931.97** 531.23** 1282.7** SCA 28 17.09** 51.1 39.11** 135.71** Gen* Environment 70 2.88 68.44** 16.48 21.9** GCA* Environment 14 6.07* 234.50** 29.72 31.5** SCA* Environment 56 2.08 26.93** 13.18 19.5** Error 210 2.88 11.57 14.78 9.9 Genetic parameters 52qca 0.55 7.48 5.28 12.6 s2sca 1.66 2.68 2.88 12.9 52qca:52sca 0.33 2.79 1.83 0.97 52a 1.1 14.96 10.56 25.2 52d 1.66 2.68 2.88 12.9 h2b (%) 0.87 0.70 0.86 0.93 h2n (%) 0.34 0.59 0.68 0.62 Dominance ratio 1.73 0.59 0.73 1.01 CP: Capitula per plant, SC: seeds per capitulum, SW: 1000-seed weight, YP: yield per plant. * and ** significant at P<0.05 and P<0.01, respectively 3.4 Heritability of the traits The efficiency of selection largely depends on the extent of genetic variability present in the population and the heritability of the concerned character (Mohammadi and Pourdad, 2009; Acquaah, 2012). The broad- sense heritability ranged from 73 (%) in seeds per capitulum to 93 (%) in seed yield per plant (Table 2). High estimates of broad-sense heritability for seed yield and its components in this study denoted that dominance or epistatic effects as other types of genetic effects might be contributed in their variation. Although, the higher value of broad - sense heritability proposed that selection would be more effective and improvement could be expected for the traits in future breeding programs (Singh and Pawar, 2005;Mohammadi and Pourdad, 2009). The high broad- sense heritability for the number of seeds per capitulum (about 70 %) was similar to that in the previous reports (Mohammadi and Pourdad, 2009). A low value for the broad - sense heritability (about 60 %) of seed yield per plant was reported (Shahbazi and Saeidi, 2007; Mohammadi and Pourdad, 2009) that was different from that found in this study. Different methods for heritability estimation resulted in different values of heritability for the same trait (Acquaah, 2012). In F2 generation, the narrow- sense heritability ranged from 34 % for capitula per plant to 68 % in seed mass (Table 2). The ranges of narrow-sense heritability in low and medium values (13 % < hn < 68 %) implied that a moderate progress would be achieved through selection for these traits. 3.5 Parental GCA and SCA of the crosses Selecting parents based on their genetic merit is a vital component in the utilization of genetic resources. The estimates on gca effects and the mean performance of the parents would help the breeder to understand the genetic architecture and the potentiality of the selected parents in F2 and later generations (Suresh et al., 2013). The GCA effects for different traits across three environments are presented in Table 3. In the case of capitula per plant, GE629i8, K2i and Mex-22-191 possessed high positive GCA effects that were suitable for breeding programs aimed at increasing the number of capitula per plant in safflower (Table 3). The GCA effects for CP varied from 1.21 (GE62918) to -1.03 (ISF14) (Table 3). The mean of parental genotypes for CP varied from 26.20 (GE62918) to 21.18 (A2) (Table 3). The seeds per capitula showed wide variation for GCA effects, from -5.94 (GE62918) to 3.07 (C4n0). The 234 Acta agriculturae Slovenica, 109 - 2, september 2017 Combining ability x environment interaction and ... safflower (Carthamus tinctorius L.): biplot as a tool for diallel data positive and significant GCA effects for Cm, C4n0, ISF14 and Mex.22-191 demonstrated the useful role of these genotypes in safflower breeding to improve the number of seeds per capitulum. The mean of seeds per capitulum for parental genotypes varied from 38.96 (C4110) to 20.81 (GE62918). The GCA effects for1000-seed mass ranged from -2.69 (C4110) to 4.54 (IL.111). The highest mean for seed mass (41.09) was attributed to IL.111 (Table 3). Table 3: General combining ability (GCA) effects and mean values for seed yield and different traits in safflower Trait CP SC SW GEfi C, C4 GCA Mean GCA Mean GCA Mean 1.21 26.20 -5.94 20.81 0.96 33.27 -0.36 23.54 1.62 35.43 -2 28.72 -0.28 22.20 3.07 38.96 -2.69 28.69 ISF14 A2 K21 IL.111 Mex.22-1 -1.01 -1.03 1.18 -0.25 0.53 22.41 21.18 26.17 24.04 24.20 1.43 -1.41 1.58 -3.21 2.84 35.91 28.40 30.07 26.27 35.53 -2.36 0.24 -0.31 4.54 1.62 27.32 30.42 25.59 41.09 30.77 (gi) 0.16 0.33 0.37 LSD (mean) 1.57 3.16 3.57 CP: capitula per plant, SC: seeds per capitulum (SC), SW: 1000-seed weight, YP: yield per plant. 3.6 Specific combining ability of the crosses Estimates of SCA effects of the 28 crosses for CP, SC and SW across the combined data are given in Table 4.The scope exploitation of hybrids for further breeding cycles in any crop largely depends on 1) mean performance of the hybrids over a range of environments, and 2) the specific combining ability effects of the parents (Suresh et al., 2013). So, the hybrids identified based on SCA effects could be exploited in heterosis breeding (Zhang et al., 2015). The SCA for capitula per plant varied from -4.32(CmxIL.111) to 3.06 (A2xIL.111). However, the crosses of low x low GCA with the negative sign led to a superior hybrid with a positive sign indicating epistatic gene actions in controlling capitula per plant (Singh and Pawar, 2005). The specific combining ability for seeds per capitulum ranged from -5.09 (ISF14xGE62918) to 4.18 (A2xGE62918) (Table 4). The specific combining ability effects for seed weight varied from 3.19 (A2xISF14) to A2xMex.22-191 (3.94) (Table 4). According to the obtained results, the hybrid genotype compromising A2 with GE629i8 and Mex.22-191 could be used in breeding projects for the improvement of seeds per capitulum and seed weight in safflower, respectively. Acta agriculturae Slovenica, 109 - 2, September 2017 Pooran GOLKAR et al. Table 4: Specific combining ability effects of different F2 generations for agronomic traits in safflower Crosses CP SC SW GE62918xCm 1.08 -1.65 -0.57 GE62918 X C4110 -0.61 0.56 1.16 GE62918XISFi4 1.25 -5.09 1.43 GE62918 x A2 -1.22 4.18 1.64 GE62918 xk21 0.09 -0.69 -1.78 GE62918XIL111 -0.24 0.57 -1.70 GE62918 xMex. 22-191 -0.23 2.08 1.29 C111 X C4110 1.48 1.47 -1.87 Cm X ISF14 -0.46 0.82 1.60 C111XA2 -0.99 -1.75 0.69 C111 xk21 1.32 -1.98 2.46 Cm xIL. 111 -4.32 3.3 -2.92 Cmx Mex.22-191 1.00 0.76 -0.67 C04110 X ISF14 -1.18 -1.35 -1.33 C4110 X A2 -0.18 -4.77 -2.15 c4110xk21 0.57 0.38 0.26 C4110XIL.111 1.04 -0.09 0.09 C4110xMex.22-191 0.96 3.56 -0.10 ISF14X A2 1.39 3.92 -3.19 ISF14XK21 -1.06 2.41 -1.35 ISF14XIL.111 -0.19 -1.78 0.27 ISF14xMex.22-191 -0.92 0.37 2.63 A2XK21 -1.47 0.54 1.88 A2xIL.111 3.06 1.54 1.50 A2xMex.22-191 0.58 -0.75 3.94 K21XIL.111 -0.12 -2.25 1.58 K21 xMex.22-191 0.68 1.27 0.68 IL.111xMex.22-191 -0.68 -1.26 1.36 (Sij)±SE 0.44 0.89 1.01 (Si,-Skl)±SE 0.71 1.43 1.62 (Si,-Slk)±SE 0.76 1.52 1.71 CP: capitula per plant, SC: seeds per capitulum, SM: 1000-seed weight. 3.7 The Biplot graphs for seed yield per plant Existing new superior inbred lines with enhanced combining abilities is one of the goals of safflower breeding programs. Seed yield is a complicated multigenic trait that its yield depends on the great influence of environmental situations (Bocanski et al., 2011). In this study, the GGE biplot technique allowed the effective and rapid view of GCA and SCA effects of the parental genotypes, best lines and tester and their performance in different crosses in this study. Even though GGE biplot analysis of diallel data is widely used to determine combining ability and heterotic responses in many crops for yields, such as oil crops [rape seed (Sincik et al., 2011), cotton (Hamoud, 2014), maize (Bocanski et al., 2011; Badu-Apraku et al., 2013)] and forages such as alfalfa (Milic et al. 2011). This method has rarely been reported for diallel data analysis for safflower at multi location trials. 3.7.1 GCA and SCA of the parents for seed yield per plant The GCA and SCA of parental genotypes are presented in Figure 1. Biplot accounted for 87.5 % (PCA1 = 66.1 %; PCA2 = 23.3 %) of the total variance, which was partitioned, according to conventional analysis by Griffing, to GCA of parental genotypes and SCA of its crosses. General combining abilities of parents (entries) were increased in the direction of the arrow on ATC (Average Tester Coordinate) abscissa, ranking the genotypes by their GCA, as shown: G8 (Mex.22-191) > G6 (K21) > G3 (C4110) > G2 (Cm) > G7 (IL.111) > G4 (Isf-14)> G5 (A2) > G1 (GE 62918) (Figure 1). The highest (40.37) and lowest (23.22) means for yield per plant were observed at K21 and GE62918, respectively. Therefore, it is necessary to select parents with high general GCA, so that it would benefit the offspring for seed yield and its components. 236 Acta agriculturae Slovenica, 109 - 2, september 2017 Combining ability x environment interaction and ... safflower (Carthamus tinctorius L.): biplot as a tool for diallel data The specific combining ability effects for seed yield per plant ranged from -9.41 (C4110 x ISF14) to 5.36 (K^x Mex.22-191) and A2 x Mex.22-191) (Figure 1). So, by considering the positive and significant GCA effects of parental genotypes of this cross, the K21 x Mex.22-191 cross with the highest positive SCA for yields per plant could the best cross combination for developing high yielding hybrid safflower varieties. PC1 -66.1%, PC2- 21 4%, Sift- 37 5% Transform -1}, Scaling - a, CeltVring - 2,5VP - 1 T4 T5 G4 \ T6 TT^j- n V7 \ \n V T8 n C3 T3 -3.2 -2.4 -1.6 -0.8 0.0 0.8 1.6 2.4 3.2 4.0 4.8 5.6 p c 1 Figure 1: The biplot based on diallel data for seed yield per plant with the average tester ordination view, for representation of GCA and SCA of eight parental genotypes in safflower. Codes of the genotypes are: Gi = GE62918, G2 = C111, G3 = C4110, G4 = ISF14, G5 = A2, G6 = K21, G7 = IL.111 and G8 = Mex.22.191; codes of the testers are: T1 = GE62918, T2 = C111, T3 = C4110, T4 = ISF14, T5 = A2, T6 = K21, T7 = IL.111 and T8 = Mex.22.191. 3.7.2 Heterotic groups detection for seed yield per plant Identifying, developing and retaining broad-range of heterotic germplasms should be considered as an important aim in safflower breeding. It is not be expected that heterosis could be occurred in highly recombinant germplasm either, since crossing populations that are very distant results to a loss of advantageous epistatic effects (complementary gene interactions) that exist within parental populations (Sakiroglu and Brummer, 2007; Milic et al., 2011). GGE biplot could discriminate the heterotic groups in this study. According to tester vector, the PCA1 scores were positive for all testers. According to Figure 2, the genotypes of 4 (ISF14), 5 (A2) and 6 (K21) were one group located above PC2 guideline, and the genotypes of G1 (GE62918, G2 (Cm), G3 (C4110) and G8 (Mex.22-191) were the other group located below the PC2 guideline. Moreover, testers of T4, T5 and T6 interacted positively with genotypes G4 (Isf14), G5 (A2), G6 (K21), G7 (IL.111) and G8 (Mex.22-191), but they interacted negatively with G1 (GE62918), G2 (Cm) and G3 (C4110). Similarly, testers T1, T2, T3 and T8 interacted positively with G1 (GE62918), G2 (C111) and G3 (C4110). This interaction pattern clearly indicated heterosis in twelve crosses, which was (4, 5, 6) x (1, 2, 3, 8) and defined as better than those in both parents. Heterosis could also occur between germplasms that are divergent and genetically distant, s something not necessarily dependent on geographic origin (Sakiroglu and Brummer 2007). Similar to this report, safflower genotypes with different geographical regions from Iran (Isfahan, Azarbayejan and Kordestan) and three genotypes with different country origins (Germany, Iran and Mexico) were grouped at the same heterotic groups. Tester 7 (IL.111), which was located near the ATC abscissa, did not seem to belong to any of the groups. An ideal tester should be discriminating of the entries and be highly representative of all testers (Yan & Hunt, 2002). So, the T6 was the best tester in this data set. Acta agriculturae Slovenica, 109 - 2, September 2017 Pooran GOLKAR et al. Figure 2: Determination of heterotic groupings by the average tester coordination for seed yield per plant in safflower. Codes of the genotypes are: Gi = GE629i8, G2 = Cm, G3 = C4n0, G4 = ISF14, G5 = A2, G6 = K2i, G7 = IL.Ill and G8 = Mex.22.i9i; Codes of the testers are: Ti = GE629i8, T2 = Cm, T3 = C4ii0, T4 = ISFi4, T5 = A2, T6 = K2i, T 7= IL. ill and T8 = Mex.22.191. 3.7.3 Polygon view of the biplot for seed yield per plant According to Figure 3, the biplot was divided into four sectors, with genotypes G8 (Mex.22-191), G4 (Isf14), G1 (GE62918) and G3 (C4110) as the corner genotypes. No testers fell in the G1 (GE62918) and G4 (Isf14) sectors, suggesting that genotypes of l and 4 were not the best mating parents with any of the genotypes. Moreover, this showed that these genotypes produced the poorest hybrids with some or all of the testers. As the single tester (T3) fell in the G3 (C4ll0) sector, G3 (C4ll0) was the best mating partner with T3. All of the testers, except T3, fell in the G8 (Mex.22-191)sector, indicating that the genotype 8 (Mex.22-191) was the best mating parent with the mentioned testers, but the crosses of G8 (Mex.22-191) by testers of 6 and 5 were better to some extent. According to the polygon view at Fig 3, the best crosses for seed yield were represented by G8 (Mex.22-191) x G5 (A2) and G8 (Mex.22-191) x G6 (K21). PC1 = 66.1 PC2 = 21.4%, Sum = 87.5 Transform = 0, Scaling = 0, Centering = l, SVP = 2 T4 - T5 / J G5 d / T6 i? / T8 / 1 / (y / / 73 -2.4 -1.6 -0.8 0.0 0.8 1.6 2.4 3.2 4.0 4.8 PC1 Figure 3: Polygon view of the biplot, showing the best crosses among all possible combinations for seed yield per plant in safflower. Codes of the genotypes are: G1 = GE62918, G2 = C111, G3 = C4110, G4 = ISF14, G5 = A2, G6 = K21, G7 = IL.111 and G8 = Mex.22.191; codes of the testers are: T1 = GE62918, T2 = C111, T3 = C4110, T4 = ISF14, T5 = A2, T6 = K21, T7 = IL.111 and T8 = Mex.22.191. 238 Acta agriculturae Slovenica, 109 - 2, september 2017 Combining ability x environment interaction and ... safflower (Carthamus tinctorius L.): biplot as a tool for diallel data 4 CONCLUSION This study was undertaken to study combining ability x environment interaction in safflower by diallel design. Combining ability x environment interaction was carried out to regulate testing over more than one environment for population improvement purposes and the early testing of superior safflower new hybrids. High-yielding cultivars could be especially necessary to ensure success for safflower cultivation in arid and semi arid regions of Iran. So, this study revealed the importance of gaining knowledge about combining ability and the identification of superior genotypes across different environments. Considering the contribution of different genetic components in genetic control of a trait showed that an appropriate strategy for improvement of each desirable trait could be achieved. It may also be worthwhile to attempt bi-parental mating in the segregating generation among some selected crosses such as A2 x Mex.22-191 and K2i x IL. 111 and A2 x IL. 111 to permit superior recombination. The greater negative GCA values of IL. 111 for seed mass, K21 for capitula per plant and yield per plant, and C4n0 for seeds per capitulum imply the capacity of these parents in producing superior progenies when combined with another parent for improving the noted traits, respectively. In this study, the superior genotypes across three environments could be used in recombination breeding programs to accumulate suitable genes responsible for improving seed yield in safflower. 5 REFERENCES Acquaah, G. (2012). Principles of plant genetics and breeding. 2nd Edition. Wiley-Blackwell, Oxford. doi:10.1002/9781118313718 Barten, J.H., Elkind, Y., Scott, J.W., Vidavski, S. & Kedar, N. (1993). Diallel analysis over two environments for blossom-end scar size in tomato. Euphytica, 65, 229-237. doi:10.1007/BF00023087 Bocanski, J., Nastasic, A., Stanisavljevic, D., Sreckov, Z., Mitrovic,B., Treskic , S., & Vukosaljev, M. (2011). Biplot analysis of diallel crosses of NS maize inbred lines. Genetica, 43(2), 277-284. doi: 10.2298/gensr1102277b Badu-Apraku, B., Oyekunle, M., Fakorede, M.A. B., Vroh,I., Akinwale, R.O., & Aderounmu, M. (2013). Combining ability, heterotic patterns and genetic diversity of extra-early yellow inbreds under contrasting environments. Euphytica, 192, 413-433. doi.:10.1007/s10681-013-0876-4 Dajue, L. & Mundel, H.H. (1996). Safflower (Carthamus tinctorius L.). IPGRI, Italy. Deedawat, S.K., Patel, S.R., Patil, S.S., & Patel, N.N. (2015). Combining ability studies on yield, oil content and other traits in safflower (Carthamus tinctorious L.). Green Farming International Journal, 6(5), 991-993. Golkar, P., Arzani, A. & Rezai, A.M.. 2012. Genetic Analysis of Agronomic Traits in Safflower (Carthamus tinctorious L.). Notulae Botanicae Horti Agrobotanici Cluj-Napoca, 40(1):276-281. Golkar, P. (2014). Breeding improvements in safflower (Carthamus tinctorius L.): A review. Australian Journal of Crop Science, 8(7),1079-1085. Gouda, R.K., Kage, U., Lohithaswa, H.C., Shekara, B.G., & Shobha, D. (2013). Combining ability studies in maize (Zea Mays L.). Molecular Plant Breeding, 3(14), 116-127. doi:10.5376/mpb.2013.04.0014 Griffing, B. (1956). Concept of general and specific combining ability in relation to diallel crossing systems. Australian Journal of Crop Science , 9, 463-493. doi: 10.1071/bi9560463 Gupta, R.K. & Singh, S.B. (1988). Diallel analysis for seed yield, oil content and other economic traits in safflower (Carthamus tinctorius L.). Genetika-Yugoslavia, 20,161-173. Hamoud, H.M.E. (2014). Use of biplot approach for genetic analysis of yield and related traits in cotton (Gossypium barbadense). Journal of Plant Breeding and Crop Science, 6, 41-47. doi:10.5897/JPBCS2013.0432 Kizil, S., Cakmak, O., Kirici, S., & Inan, M. (2008). A comprehensive study on safflower (Carthamus tinctorius L.) in semi-arid conditions. Biotechnology and Biotechnology Equipment, 22(4), 947-953. doi:10.1080/13102818.2008.10817585 Knowles, P.F. (1969). Centers of plant diversity and conservation of crop germplasm Safflower. Economic Botany, 23, 324-329. doi: 10.1007/BF02860678 Mandal, A.B. & Banerjee, S.P. (1997). Diallel analysis of yield and yield components in safflower (Carthamus tinctorius L.). Genetics and Breeding, 51, 211-215. Acta agriculturae Slovenica, 109 - 2, September 2017 Pooran GOLKAR et al. Milic, D., Katic,S., Karagic, D., Gvozdanovic-Varga, J., Petrovic and, S. & Bocanski, J. (2011). Genetic control of agronomic traits in alfalfa (M. sativa ssp.sativa L.). Euphytica, 182, 25-33. doi:10.1007/s10681-011-0434-x Mohammadi, R. & Pourdad, S.S. ( 2009). Estimation, interrelationships and repeatability of genetic variability parameters in spring safflower using multi-environment trial data. Euphytica, 165:313324. doi:10.1007/s10681-008-9789-z Nakhaei, M., Baghizadeh,A., Mohammadi-Nejad, G., & Golkar, P. (2014). Genetic analysis of salt tolerance in safflower (Carthamus tinctorius L.). Annual Research and Review in Biology, 4(1), 337-346. doi:10.9734/ARRB/2014/5909 Nassimi, A.W., Raziuddin, R., Ali, S., Hassan, G., & Ali, N. (2006). combining analysis for maturity and other traits in rapeseed (Brassica napus L.). Journal of Agronomy, 5(3), 523-526. doi:10.3923/ja.2006.523.526 Perkins, J.M. (1970). Environmental and genotype-environmental components of variability. VI. Diallel sets of crosses. Heredity, 25, 29-40. doi: 10.1038/hdy.1970.4 Sakiroglu, M. & Brummer, E.c. (2007). Little heterosis between alfalfa populations derived from the midwestern and southwestern United States. Crop Science, 47, 2364-2371. doi: 10.213 5/cropsci2006. 12.0803 SAS Institute. (2011). Base SAS 9.3 Procedures guide. SAS Inst., cary, Nc. Shahbazi, E. & Saeidi, Gh. 2007. Genetic analysis for yield components and other agronomic characters in safflower (Carthamus tinctorius L.). Genetics and Breeding, 36, 11-20. Sincik, M., Goksoy, A.T., Turan, Z.M. (2011). The heterosis and combining ability of diallel crosses of rapeseed inbred lines. Notulae Botanicae Horti Agrobotanici Cluj-Napoca, 39(2), 242-248. Singh, S. & Pawar, I.S. (2005). Theory and Application of Biometrical Genetics. CBS Press. Singh, V., Kolekar , N.M., & Nimbkar, N. 2005. Breeding strategy for improvement of flower and seed yield in safflower. The 7th International Safflower Conference. Wagga Wagga, Australia. Suresh, R., Chandra Babu R., S. Michael Gomez & Shanmugasundaram, P. (2013). Genetic analysis of yield traits in rice under irrigated and water stress environments. Indian Journal of Genetics, 73 (2), 162-168. doi:10.5958/j.0975-6906.73.2.023 Weiss, E.A. (2000). Oil seed Crops. 2nd ed. Blackwell, London. Yan, W. & Hunt, L.A. (2002). Biplot analysis of diallel data. Crop Science, 42, 21-30. doi:10.213 5/cropsci2002.0021 Yan, W. (2001). GGE biplot- A Windows application for graphical analysis of multi- environment trial data and other types of two-way- data. Agronomy Journal, 93, 1111-1118. doi: 10.2134/agronj2001.9351111x Zhang, Y., Kang, M.S., & Lamkey, K.R. (2005). Diallel-SAS 05: A comprehensive program for Griffing's and Gardner-Eberhart analysis. Agronomy Journal, 97, 1097-1106. doi:10.2134/agronj2004.0260 Zhang, X., Lv, CH., Guo, B., & Rugen, Xu. (2015). Combining ability of different agronomic traits and yield Components in hybrid barley. PLoS One, 10(6), e0126828. doi:10.1371/journal.pone.0126828 240 Acta agriculturae Slovenica, 109 - 2, september 2017 doi: 10.14720/aas.2017.109.2.08 Original research article / izvirni znanstveni članek UVA + B treatment affects antioxidant system and phytochemicals of parsley plant under different concentrations of Zn Masoumeh ABEDINI1, Behrokh DAIE-HASSANI1, Shirwan MALAEI2 Received September 14, 2016; accepted February 28, 2017. Delo je prispelo 14. septembra 2016, sprejeto 28. februarja 2017. ABSTRACT Decline in ozone layer that followed by enhanced solar UV radiation is a limiting factor for some plants. In this study the effect of UVA+UVB radiation on parsley plant was studied hydroponically at different concentrations of Zn (1.5 and 6.5 ^m). UV radiation at both concentrations of Zn, slightly decreased the plant growth and significantly increased the carotenoids, flavonoids, total phenols and H2O2 contents, but had no effect on chlorophylls content. At concentration of 1.5 ^m of Zn, UV radiation caused significant increases in the MDA and anthocyanin contents and the activities of POD and CAT enzymes, but decreased the soluble sugars and protein contents. At concentration of 6.5 ^m of Zn, UV radiation caused significant increases in the CAT activity, but had no significant effect on other parameters. Results suggest that parsley plant tolerates UVA+UVB radiation particularly at concentration of 6.5 ^mof Zn. Key words: antioxidant enzymes; parsley; photochemicals; UVA + B; Zn IZVLEČEK OBRAVNAVANJE Z UVA + UVB VPLIVA NA ANTIOKSIDACIJSKI SISTEM IN KEMIZEM PETRŠILJA PRI RAZLIČNIH KONCETRACIJAH Zn Tanjšanje ozonske plasti, ki mu sledi povečano UV sončevo sevanje sta omejujoči dejavnik za nekatere rastline. V tej raziskavi je bil preučevan učinek UVA+UVB sevanja na hidroponsko gojen peteršilj pri različnih koncentracijah Zn (1.5 in 6.5 ^m). UV sevanje je pri obeh koncentracijah cinka rahlo zmanjšalo rast in značilno povečalo vsebnost karotenoidov, flavonoidov, celokupnih fenolov in H2O2, a brez učinka na vsebnost klorofilov. Pri koncentraciji 1.5 |im Zn, je UV sevanje povzročilo značilno povečanje vsebnosti MDA in antocianinov, povečalo je aktivnosti encimov POD in CAT, a zmanšalo vsebnost topnih sladkorjev in beljakov. Pri koncentaraciji 6.5 ^m Zn je UV sevanje povzročilo značilno povečanje v aktivnosti CAT, a ni imelo značilnega učinka na ostale parametre. Rezultati nakazujejo, da peteršilj dobro prenaša UVA+UVB sevanje, še posebej pri koncentraciji 6.5 ^m Zn. Ključne besede: antioksidacijski encimi; peteršilj; kemizem rastlin; UVA + B; Zn 1 INTRODUCTION Interruption of ozone layer as an outcome of human activities has resulted to enhanced intensity of UV radiation on the Earth surface. Plants have obligate requirement for sun light are more susceptible to this radiance. UV radiation increases the production of reactive oxygen species (ROS) that are extremely cytotoxic (Mahdavian et al., 2008; Czegeny et al., 2016). The antioxidant system is one of the most important mechanisms responsible for detoxifying the free radicals. Non-enzymatic antioxidant system includes biochemicals such as carotenoids, flavonoids, ascorbic acid and glutathione. Flavonoids commonly absorb the light in the region of 280-320 nm and thus are capable to protect the plant from damage (Eichholz et al., 2011; Reshmi and Rajalakshmi, 2012). Carotenoids also have antioxidant properties and act as an internal filter against UV radiation (Nasibi and Kalantari, 2005). Enzymatic antioxidants such as catalase, peroxidase and superoxide dismutase can moderate the UV-induced injuries by protecting the photosynthetic pathway and cellular components (Wei et al., 2013). A wide range of morphological, 1 Assistant Professor of Payame Noor University, Faculty of Basic Sciences, Department of Biology, Iran, corresponding author: ms_abedini@pnu.ac.ir 2 M.Sc. student of Payame Noor University, Faculty of Basic Sciences, Department of Biology, Iran Acta agriculturae Slovenica, 109 - 2, september 2017 str. 241 - 240 Masoumeh ABEDINI et al. physiological and biochemical responses of plants have been reported to elevate the UV resistance. Some plants are more tolerant to UV radiation than others because they activate a variety of mechanisms against stress (Fedina et al., 2010; Wei et al., 2013). Zinc is an essential micronutrient and involves in the various metabolic pathways in plants (Alloway, 2008). The positive role of Zn in different environmental stresses such as salinity, drought and high irradiance was reported by several authors (Hassan et al., 2005; Weisany et al., 2012; Michael and Krishnaswamy, 2014). In this study the effect of UVA + B treatment on parsley plant antioxidant system and phytochemicals was investigated at two concentrations of Zn. 2 MATERIALS AND METHODS The seeds of parsely plant (Petroselinum crispum Mill. var. neapolitanum) was achieved from the Agricultural Research Center of Tabriz, Iran. 2.1 Plants growth condition Plants were grown hydroponically in a growth chamber with a temperature of 28/20 °C, 16 h photoperiod and relative humidity of 70 %. Seeds were germinated in petri-dishes and transferred to plastic containers with 2 l of Cooper nutrient solution (50 %) and pre-cultured for 7 days. After pre-culturing period plants were transferred to the full strength nutrient solution, containing two levels of Zn (1.5 and 6.5 pm) as zinc sulphate. Applied UV doses that were received by one-half of plants were 20.5 and 176 kJ m-2 day-1 for UVA and UVB respectively (supplied with 30 W, UV lamps, Philips; UVA 30 %, UVB 5 %). 20 days after treatments, the plants were harvested and stored in -80 °C for further analyses. 2.2 Photosynthetic pigments and phytochemicals assays Fresh leaf tissues were homogenized with 80 % aqueous acetone. The extracts were centrifuged for 10 min at 4000 g. Chlorophylls and carotenoids contents were determined spectrophotometrically at 470, 646.8 and 663.2 nm using equations described by Lichtenthaler (1987). Anthocyanins were extracted with acidified methanol (methanol: HCl, 99:1, v/v) solution on a shaker in the dark at 4 °C per 48 h. After filtering, the absorbance of samples were measured spectrophotometrically at 550 nm and calculated using an extinction coefficient of 33000 mol-1cm-1 (Wagner, 1979). Total phenolic of shoots was extracted by 80 % aqueous methanol for 20 min using ultrasonic bath. The mixture was centrifuged at 14000 g for 5 min.To 0.5 ml of supernatant, 1.5 ml (1:10 v/v diluted with distilled water) Folin-Ciocalteau reagent was added and allowed to stand for 5 min at 22 °C. After 5 min, 2 ml of 7.5 % of sodium carbonate was added. These mixtures were incubated for 90 min in the dark with intermittent shaking. After incubation, development of blue color was measured at 725 nm. The phenolic content was calculated on the basis of standard curve of gallic acid (Fletcher and Kott, 1999). The total flavonoid content of shoots was determined using the aluminum chloride assay through colorimetry. An aliquot (1 ml) of extracts were taken in different test tubes then 6 ml of distilled water was added followed by the addition of 0.3 ml of sodium nitrite (5 % NaNO2, w/v) and allowed to stand for 6 min. Later 0.3 ml of aluminum trichloride (10 % AlCl3) was added and incubated for 6 min, followed by the addition of 2 ml of sodium hydroxide (NaOH, 4 % w/v). After 15 min of incubation the mixture turns to pink and its absorbance was measured at 510 nm. The total flavonoid content was calculated on the basis of standard curve of quercitine (Toor and Savage, 2005). 2.3 Antioxidant enzymes assays To obtain the crude extract, 0.1 g parsley leaves were homogenized in 3 ml of 10 mmol l-1 potassium phosphate buffer (pH = 7), containing 0.2 % polyvinyl pyrrolidone. The homogenate was centrifuged at 21,000 g at 4 °C for 20 min. The resulting supernatant was used to measure the activities of superoxide dismutase (SOD), peroxidase (POD) and catalase (CAT) and protein content. The activity of SOD was measured according to its capacity to inhibit photochemical reduction of nitroblue tetrazolium. The reaction mixture contained 2.65 ml of 67 mmol l-potassium phosphate buffer (pH = 7.8), 0.2 ml of 0.1 mmol l-1 EDTA solution containing 0.3 mmol l-1 sodium cyanide, 0.1 ml of 1.5 mmol l-1 NBT, 50 ml of 0.12 mmol l-1 riboflavin and 50 ml enzyme extract. The amount of enzyme that catalyzed 50 % inhibition from photochemical reduction of NBT was defined as one unit (U) of SOD. Due to the possibility of auto-oxidation of the substrates, control assays were prepared in the absence of plant extract (Winterbourn et al., 1976). Guaiacol POD was assayed in plant shoots, following the method of Chance and Maehly (1955). The reaction 1XI 242 Acta agriculturae Slovenica, 109 - 2, september 2017 UVA + B treatment affects antioxidant system and phytochemicals of parsley plant under different concentrations of Zn mixture contained 1.50 ml of 100 mmol l-1 citrate-phosphate - borate buffer solution (pH = 7.5), 50 ^l of 15 mmol l-1 guaiacol, 25 ^l enzyme extract and 50 ^l of 3.3 mmol l-1 H2O2. The polymerization of guaiacol was initiated by adding H2O2 and an increase in absorbance at 470 nm was recorded for 3 min. POD activity was calculated using the extinction coefficient, 26.6 (mmol l-:)-1 cm-1, for guaiacol. The generation of 1 ^mol of tetra guaiacol per min was catalyzed by the amount of enzyme that was introduced as one unit of POD. The CAT activity was determined by monitoring the decrease in absorbance at 240 nm for 3 min due to dismutation of H2O2. The reaction mixture contained 1.50 ml of 100 mmol l-1 citrate-phosphate-borate buffer solution (pH = 7.5), 50 ^l enzyme extract and 13 ^l of 10 mmol l-1H2O2. The amount of enzyme for dismutation of 1 ^mol l-1 H2O2 per min was expressed as one unit. Extinction coefficient for H2O2 at 240 nm was considered 39.4 (mmol l-1)-1cm-1 (Obinger et al., 1997). 2.4 Total proteins and soluble sugars Total protein content was measured by the method of Bradford (1976) using bovine serum albumin as a standard. The soluble sugars content was measured by DuBois et al. (1956) method. 2.5 Hydrogen peroxide and malondialdehyde assays The hydrogen peroxide (H2O2) content was estimated according to the Harinasut et al. (2003). Samples were homogenized with 0.1 % (w/v) trichloroaceticacid (TCA). Mixture was centrifuged at 12000 g for 15 min. To 0.5 ml of the supernatant, 0.5 ml of 10 mM phosphate buffer (pH = 7.0) and 1 ml of 1 m potassium iodide (KI) was added. The mixture was incubated at 25°C for 15 min. The absorbance was measured at 390 nm. The H2O2 content was calculated from a standard curve prepared in a similar way. Lipid peroxidation was estimated from the amount of malondialdehyde (MDA) formed in a reaction mixture (Heath and Packer, 1968). Leaf tissues were homogenized in 0.1 % (w/v) (TCA). The homogenate was centrifuged at 10,000 g for 5 min. To 1 ml of the supernatant, 4 ml of 20 % TCA containing 0.5 % thiobarbituric acid was added. The mixture was incubated at 95 °C in a water bath for 30 min, and then quickly cooled on ice. The mixture was centrifuged at10,000 g for 15 min and the absorbance was measured at 532 nm. MDA levels were calculated from 1,1',3,3'-tetra ethoxy propan standard curve. 2.6 Statistical Analysis Experiment was conducted in complete randomized design with 3 replications. Analysis of variance was performed using InStat (3.0) software. The data were presented as the means ± SE for each treatment. Means were compared with Tukey's Multiple Range Test at the 5 % probability level. 3 RESULTS AND DISCUSSION 3.1 Growth parameters In this study, UV radiation non significantly decreased the fresh and dry mass and lengths of parsley plants shoots and roots at both levels of applied Zn. Application of Zn at concentration of 6.5 ^m significantly (p < 0.05) increased the dry and fresh mass of shoots (Fig 1), but non significantly increased the dry and fresh mass of roots (Fig 2) and plant length (Fig 3) in compared to plants received concentration of 1.5 ^m of Zn at normal and UV radiation conditions. The effect of UV radiation on plants growth is varying among different species. In the wide range of species, plant growth decreases in response to UV radiation, but in some cases the growth is not affected or it is even promoted by this radiation (Fedina et al., 2010, Ravindran et al., 2010; Zlatev et al., 2012). The induced changes in the plant's growth regulators biosynthesis and transport by UV radiation are responsible for the decreased growth of plants (Toosi et al., 2009). Similar to results obtained from this study, the biomass and production of potato, clover, oat and barley plants did not dramatically affect by 24-33 % increases in UV radiation during the growing season (Hakala et al., 2002). The ability of plant in the prevention of growth reduction under UV treatment is an indicator for plant tolerance (Smith et al., 2000). In this study, the application of Zn at high concentration could improve the growth of parsley under normal condition and UV radiation. The beneficial effects of Zn on plants growth are related to its necessity for carbohydrate and protein metabolism, membrane integrity, auxin synthesis and reproduction (Alloway, 2008). Acta agriculturae Slovenica, 109 - 2, september 2017 83 Masoumeh ABEDINI et al. Figure 1: Effect of UV treatment on the fresh and dry mass of parsley shoots under different concentrations of Zn Figure 2: Effect of UV treatment on the fresh and dry mass of parsley roots under different concentrations of Zn 1.45+UV Zn 2 Zn 2+UV Figure 3: Effect of UV treatment on the length of parsley shoots and roots under different concentrations of Zn 3.2 Photosynthetic pigments The concentrations of chlorophylls a, b and total were not significantly affected by UV treatment and Zn concentrations. But, carotenoids content and carotenoid/chlorophyll ratio of UV treated plants increased significantly (p < 0.05) in both concentrations of Zn. Application of Zn at concentration of 6.5 ^m slightly increased the carotenoid content and carotenoid/chlorophyll ratio of plants compared to the concentration of 1.5 ^m of Zn in both conditions (Table 244 Acta agriculturae Slovenica, 109 - 2, september 2017 UVA + B treatment affects antioxidant system and phytochemicals of parsley plant under different concentrations of Zn 1). According to results obtained from this study, parsley plant could effectively protects its own chlorophyll content against the enhanced UVA + B radiation by increasing the carotenoids content in both levels of Zn. Similar to this study, Salama et al. (2011) reported that in Rumex vesicarius L. the chlorophyll content of plant was not affected notably by UV treatment. The carotenoids are involved in the photosynthetic structures protection against the destructive effects of UV radiation (Nasibi and Kalantari, 2005). The efficacy of carotenoids in protecting the photosystems is likely due to their function as efficient quenchers of high energy of short wave radiation. The plant capacity in the protection of photosynthetic pigments content under enhanced UV conditions restores plant photosynthesis rate and tolerates plant against this stress (Levall and Bornman, 2006; Reshmi and Rajalakshmi, 2012). Zn application in this study did not affect the chlorophylls contents of parsley plant significantly. Similar to this result are those obtained for maize plants at different concentrations of Zn under the condition without stress (Saeidnejad and Kafi, 2013). Table 1: Effect of UV treatment on photosynthetic pigments of parsley plant under different concentrations of Zn treatment Chlorophyll a (mg g-1FM) Chlorophyll b (mg g-1FM) Total chlorophyll (mg g-1FM) Carotenoid (mg g-1FM) Carotenoid/ chlorophyll Zn 1.5 ^m 0.14 ± 2.53 a 0.48 ± 3.07 a 0.15 ± 5.93 a 0.11 ±0.31 b 0.01 ± 0.05 bc Zn 1.5 ^m + UV 0.04 ± 2.63 a 0.09 ± 2.34 a 0.34 ± 5.15 a 0.97 ± 3.027 a 0.07 ± 0.48 a Zn 6.5 ^m 0.031 ± 2.59 a 3.33 ± 0.12 a 0.6 ± 5.95 a 0.19 ±0.54 b 0.09 ± 0.07 b Zn 6.5 ^m + UV 0.07 ± 2.55 a 0.27 ± 3.1 a 0.41 ± 5.64 a 0.65 ± 3.44 a 0.08 ±0.6 a Each value represented as mean ± SE (n = 3); mean values followed by the same letter (s) are not significantly different (p < 0.05). 3.3 Phenolic compounds UV treatment induced a significant increase in the anthocyanin content of parsley plants at concentration of 1.5 ^m of Zn, but a slight increase was seen at concentration of 6.5 ^m of Zn. The flavonoids concentration of plants significantly (p < 0.05) increased by UV treatment in both levels of applied Zn. There were some increases in the total phenols contents of plants treated with UV at both levels of Zn wich they were not significant. In normal conditions without UV radiation, application of Zn at concentration of 6.5 ^m caused non-significant increase in these parameters compared to the concentration of 1.5 ^mof Zn (Table 2). The increased level of UV absorbing phenolics is the common protective response to enhanced UV radiation in plant species (Reshmi and Rajalakshmi, 2012; Wei et al., 2013). It has been demonstrated that UV-B photoreceptor, UV RESISTANCE LOCUS8 protein (gene name: UVR8), absorbs UV-B light through conserved tryptophan residues (Mach, 2016). Absorbing UV-B causes the apparent UVR8 homodimer to dissociate into monomers, which interact with constitutively photomorphogenic1 (COP1), an E3 ubiquitin ligase (Rizzini et al., 2011). This interaction induces genes encoding protective factors such as phenylpropanoid biosynthesis pathway, and damage-repair factors such as photolyases (Fasano et al., 2014). Furthermore, the role of phenolic compounds as a product of phenyl propanoid pathway in the free radicals scavenging was also proved (Nasibi and Kalantari, 2005). It was proposed that plants with low levels of phenolic compounds are sensitive to UV radiation (Kim and Rodrigo, 2001; Zlatev et al., 2012). In this study, application of concentration of 6.5 ^mof Zn increased the phenolic compounds compared to 1.5 ^m. Our results about the positive effect of Zn application on biosynthesis of phenolic compounds is parallel to that reported for Pistacia vera L. by Tavllali et al. (2010) under saline condition. 3.4 Total proteins and soluble sugars Total proteins and soluble sugars contents of UV treated plants decreased significantly (p < 0.05) in plants which received 1.5 ^m of Zn, but the induced decreases in total proteins and soluble sugars in plants received 6.5 ^m of Zn were not significant. At normal condition there was no main difference in these parameters between two levels of applied Zn (Table 2). The reductive effect of UV radiation on protein content is related to direct DNA injury, amino acid destruction and proteins and enzymes inactivation induced by UV radiation (Salama et al., 2011; Zlatev et al., 2012). Moreover, UV radiation causes the detrimental effects in the structure of RNA molecules and thus disrupts protein synthesis (Ulm and Nagy, 2005). According to this study, zinc application at high concentration in Acta agriculturae Slovenica, 109 - 2, september 2017 245 Masoumeh ABEDINI et al. UVA + B treated plants increased the protein content. The beneficial effect of Zn application on the protein content was reported for many species such as wheat plants under stress conditions (Morshedi and Farahbakhsh, 2010). Zn is necessary for the activity of the enzyme RNA polymerase and it protects the ribosomal RNA from attack by the enzyme ribonuclease. It is proposed that the most fundamental effect of zinc on protein metabolism is through its involvement in the stability and function of genetic material (Alloway, 2008). The results attained for soluble sugars in this study were parallel to that obtained for total protein content. It has been proposed that UV radiation by inactivation the photosynthetic enzymes such as rubisco and some other Calvin cycle enzymes and damaging the photosystem II proteins adversely affects the photosynthesis and decreases the sugar synthesis (Zu et al., 2004; Zlatev et al., 2012). In this study, the induced reduction in soluble sugars by UVA + B treatment was not significant at sufficient amounts of Zn.The positive effect of Zn application on soluble sugar content probably is related to its role in protection of photosynthetic enzymes from UV damages and contribution in the structure of enzyme ribulose bisphosphat carboxylase (Alloway, 2008). The enhanced amounts of soluble sugars by sufficient Zn application was reported for different plant species such as Cucurbita pepo L. under stressful and normal conditions (Sorkhi Lalelou et al., 2013). Table 2: Effect of UV treatment on anthocyanins, total phenolics, flavonoids, soluble sugars and total protein contents of parsley plant under different concentrations of Zn treatment Anthocyanins (mg g-1FM) Flavonoids (mg g-1FM) Total phenols (mg g-1FM) Soluble sugars (mg g-1DM) Total protein (mg g-1FM) Zn 1.5 ^m 1.4 ± 0.08 b 1.35 ± 0.082 b 8.61 ± 0.7 b 109.8 ± 3.63 a 27.6 ± 0.59 a Zn 1.5 ^m + UV 1.94 ± 0.07 a 2.53 ± 0.32 a 14.32 ± 0.12 ab 79.1 ± 2.7 b 25.4 ± 0.21 b Zn 6.5 ^m 1.47 ± 0.06 b 1.39 ± 0.09 b 10.43 ± 0.7 ab 111.8 ± 4.23 a 28.7 ± 0.44 a Zn 6.5 ^m + UV 1.51 ± 0.045 ab 2.62 ± 0.22 a 16.11 ± 0.1 a 96.8 ± 6.3 a 26.53 ± 0.38 a Each value represented as mean ± SE (n = 3); mean values followed by the same letter (s) are not significantly different (p < 0.05). 3.5 Antioxidant system In this study, UV treatment significantly (p < 0.05) increased the activities of POD and CAT enzymes, but slightly decreased the activity of SOD enzyme at concentration of 1.5 ^m of Zn. In plants received the concentration of 6.5 ^mof Zn, UV treatment had no significant effect on the activities of SOD and POD, but significantly increased the CAT activity. In the conditions with no UV radiation, application of 6.5 ^m of Zn could increase the SOD activity of plants compared to 1.5 ^m Zn, but did not affect the POD and CAT activities considerably (Table 3). H2O2 content of plants significantly (p < 0.05) increased in response to UV treatment in both levels of Zn. UV treated plants that received the concentration of 1.5 ^m of Zn had the highest amounts of H2O2. Under condition without UV radiation, plants received 6.5 ^m of Zn had slightly lower amount of H2O2 compared to plants which received 1.5 ^m of this element. The MDH content of plants increased significantly only in the UV treated plants which received 1.5 ^m of Zn. In the plants fed with concentration of 6.5 ^m of Zn, UV could not enhance this metabolite considerably (Table 3). UV radiation induces oxidative stress in plants (Hakala et al., 2002; Tossi et al., 2009). The increased levels of ROS in plants damage biomolecules such as lipids and result to MDA formation as the breakdown product of polyunsaturated fatty acids of membranes. The effect of sufficient Zn application on controlling the production of these detrimental components was reported by authors in different full stress conditions (Tavallali et al., 2010; Weisany et al., 2012), but there is no available reference concerning role of this element under UV condition. Zinc plays a key role in controlling the generation and detoxification of free oxygen radicals and subsequent lipid membrane oxidation (Alloway, 2008). It has been demonstrated that Zn ions have strong inhibitory effect on membrane bound NADPH oxidase (Cakmak and Marschner, 1988; Kawano et al., 2002). 246 Acta agriculturae Slovenica, 109 - 2, september 2017 UVA + B treatment affects antioxidant system and phytochemicals of parsley plant under different concentrations of Zn The activities of antioxidant enzymes POD and CAT in UV treated plants increased in this study. The enhancement in the activities of these enzymes in response to UV-B treatment were reported by several authors (Nasibi and Kalantari, 2005; Czégény et al., 2016). Antioxidant enzymes play a significant role in the dynamic equilibration between free oxygen radicals production and destruction. The responses of antioxidant enzymes to UV radiation vary among plants species (Tossi et al., 2009; Salama et al., 2011; Czégény et al., 2016). The increasing in the activities of antioxidant enzymes could be the indicator of build-up of a protective mechanism to reduce oxidative damages induced by stress (Harinasut et al., 2003; Chawla et al., 2013). According to results obtained from this study, the produced H2O2 effectively removed by POD and especially CAT in plants fed with concentration of 6.5 ^m Zn. There was a small decrease in the activity of SOD enzyme in response to UV treatment in parsley plant in low level of applied Zn that was improved by application of 6.5 ^m of Zn. Reduction in the activity of SOD enzyme as a result of UV-B radiation has been reported for sun flower plant (Costa et al., 2002). It has been proposed that a high amount of H2O2 is able to inhibit Cu-Zn-SOD via the reduction of Cu2+ to Cu+ (Casano et al. 1997). Zn is able to facilitate the biosynthesis of antioxidant enzymes (Cakmak, 2000) and its effects on improvement of antioxidant system of plants under various stresses have reported by several authors (Tavallali et al., 2010; Weiasany et al., 2012; Michael and Krishnaswamy, 2014). Table 3: Effect of UV treatment on antioxidant enzymes activities that defined as unit (U) and MDH and H2O2 contents of parsley plant under different concentrations of Zn treatment SOD activity (U mg-1 pro min-1) POD activity (U mg-1 pro min-1) CAT activity (U mg-1 pro min-1) H2O2 (^mol g-1FM) MDA (nmol g-1FM) Zn 1.5 ^m 0.4 ± 0.03 ab 0.12± 0.09 b 0.45 ± 0.007 b 0.096 ± 0.005 c 1.74 ± 0.097 b Zn 1.5 ^m + UV 0.3 ± 0.015 b 0.25±0.04 a 1.3 ± 0.045 a 0.23 ± 0.023 a 5.57 ± 1.11 a Zn 6.5 ^m 0.47 ± 0.07 a 0.17±0.06 b 0.42 ± 0.09 b 0.088 ± 0.003 c 1.64 ± 0.017 b Zn 6.5 ^m + UV 0.5 ± 0.04 a 0.19 ± 0.02 b 1.5 ± 0.029 a 0.18 ± 0.01 b 2.7 ± 0.5 b Each value represented as mean ± SE (n = 3); mean values followed by the same letter (s) are not significantly different (p < 0.05). 4 CONCLUSIONS The results of this study showed the relative tolerance of parsley plant against applied doses of UV radiation at both concentrations of Zn, but it was more obvious at concentration of 6.5 ^m. This plant could effectively increase UV absorbing phenolic compounds and carotenoids in response to UV radiation that are involved in photosynthetic apparatus protection. Moreover the induced increases in the activities of antioxidant enzymes in UV treated plants are responsible to moderate the ROS production in this plant. According to results obtained from this study, Zn application at concentration of 6.5 ^m had positive effects on parsley resistance to UV radiation. 5 ACKNOWLEDGEMENTS The authors would like to express their sincere Tabriz, Faculty of sciences, for supply of laboratory appreciation to Dr. Rogieh Haji-Boland, University of equipment. 6 REFERENCES Alloway, B. J. (2008). Zinc in Soils and Crop Nutrition. Brussels, Belgium and Paris, IZA & IFA. Bradford, M. (1976). A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Annual Review of Biochemistry, 72(1-2), 248-254. doi:10.1016/0003-2697(76)90527-3 Acta agriculturae Slovenica, 109 - 2, september 2017 87 Masoumeh ABEDINI et al. Cakmak, I. (2000). Tansley review no. 111. New Phytologist, 146(2), 185-205. doi:10.1046/j.1469-8137.2000.00630.x Cakmak, I., Marschner, H. (1988). Increase in membrane permeability and exudation in roots of zinc deficient plants. Journal of Plant Physiology, 132(3), 356-361. doi:10.1016/S0176-1617(88)80120-2 Casano, L., Gomez, L., Lascano, C., Trippi, V. (1997). Inactivation and degradation of Cu/Zn-SOD by active oxygen species in wheat chloroplasts exposed to photooxidative stress. Plant and Cell Physiology, 38(4), 433-440. doi:10.1093/oxfordjournals.pcp.a029186 Chance, B., Maehly, A. C. (1955). Assay of catalases and peroxidases, Methods in Enzymology. New York, NY: Academic Press. Chawla, S., Jain, S., Jain, V. (2013). Salinity induced oxidative stress and antioxidant system in salt-tolerant and salt-sensitive cultivars of rice (Oryza sativa L.). Journal of Plant Biochemistry and Biotechnology, 22(1), 27-34. doi:10.1007/s13562-012-0107-4 Cooper, A. (1988). 1. The system. 2. Operation of the system. In G. Books (ed), The ABC of Nutrient Film Technique (pp. 3-123). England, UK: Intl Specialized Book Service Inc. Costa, H., Gallego, S. M., Tomaro, M. A. L. (2002). Effect of UV-B radiation on antioxidant defense system in sunflower cotyledons.Plant Science, 162(6), 939-945. doi:10.1016/S0168-9452(02)00051-1 Czegeny, G., Matai,A., Hideg,E: (2016). UV-B effects on leaves-oxidative stress and acclimation in controlled environments. Plant Science, 248, 57-63. doi:10.1016/j.plantsci.2016.04.013 DuBois, M., Gilles, K. A., Hamilton, J. K., Rebers P. A., Smith, F. (1956). Colorometric method for determination of sugars and related substances. Anals of Chemistry, 28(3), 350-356. doi:10.1021/ac60111a017 Eichholz, I., Huyskens-Keil, S., Keller, A., Ulrich, D., Kroh, L. W., Rohn, S. ( 2011). UV-B-induced changes of volatile metabolites and phenolic compounds in blueberries (Vaccinium corymbosum L.). Food Chemistry, 126(1), 60-64. doi:10.1016/j.foodchem.2010.10.071 Fasano, R., Gonzalez, N., Tosco, A., Piaz, F. D., Docimo, T., Serrano, R., Grillo, S., Leone, A., Inze, D. (2014).Role of Arabidopsis UV RESISTANCE LOCUS 8 in plant growth reduction under osmotic stress and lowlevels of UV-B. Molecular Plant, 7(5), 773-791. doi:10.1093/mp/ssu002 Fedina, I., Hidema, J., Velitchkova, M., Georgieva, K., Nedeva, D. (2010). UV-B induced stress responses in three rice cultivars. Biologia Plantarum, 54(3), 571574. doi:10.1007/s10535-010-0102-3 Fletcher, R. S., Kott, L. S. (1999). Phenolics and cold tolerance of Brassica napus. Plant Agriculture, 1, 1-5. Hakala, K., Jauhiainen, L., Koskela, T., Kayhko, P., Vorne, V. (2002). Sensitivity of crops to increased ultraviolet radiation in northern growing conditions. Journal of Agronomy and Crop Science, 188(1), 8-18. doi:10.1046/j.1439-037x.2002.00536.x Harinasut, P., Poonsopa, D., Roengmongkol, K., Charoensataporn, R. (2003). Salinity effects on antioxidant enzymes in mulberry cultivar. ScienceAsia, 29, 109-113. doi:10.2306/scienceasia1513-1874.2003.29.109 Hassan, M. J., Zhang, G., Wu, F., Wei, K., Chen, Z. (2005). Zinc alleviates growth inhibition and oxidative stress caused by cadmium in rice. Journal of Plant Nutrition and Soil Science, 168(2), 255-261. doi:10.1002/jpln.200420403 Heath, R. L., Packer, L. (1968). Photoperoxidation in isolated chloroplasts, I. Kinetics and stoichiometry of fatty acid peroxidation. Archives of Biochemistry and Biophysics, 125(1), 189-198. doi:10.1016/0003-9861(68)90654-1 Kawano, T., Kawano, N., Muto, S., Lapeyrie, F. (2002). Retardation and inhibition of the cation-induced superoxide generation in BY-2 tobacco cell suspension culture by Zn2+ and Mn2+. Physiologia Plantarum, 114(3), 395-404. doi:10.1034/j.1399-3054.2002.1140309.x Kim, B., Rodrigo, L. (2001). Arabidopsis mutant tolerant to lethal ultraviolet-B levels shows constitutively elevated accumulation of flavonoids and other phenolics. Plant Physiology,126(i), 1105-1115. doi:10.1104/pp.126.3.1105 Levall, M., Bornman, J. F. (2006). Selection in vitro for UV-tolerant sugar beet (Beta vulgaris) somaclones. Physiologia Plantarum, 88(1), 37-43. doi:10.1111/j.1399-3054.1993.tb01757.x Lichtenthaler, H. K. (1987). Chlorophylls and carotenoids, Pigments of photosynthetic biomembranes. Methods in Enzymology, 148, 350-382. doi:10.1016/0076-6879(87)48036-1 Mach, J. (2016). How plants take the bad with the good, conserved UV-B perception and signaling in Chlamydomonas. Plant Cell, 28(4), 825. doi:10.1105/tpc.16.00279 Mahdavian, K., Ghorbanli, M., Kalantari, K. M. (2008). The Effects of ultraviolet radiation on the contents of chlorophyll, flavonoid, anthocyanin and proline in Capsicum annuum L. Turkish Journal of Botany, 32, 25-33. Michael, P. I., Krishnaswamy, M. (2014). Membrane damage and activity of antioxidant enzymes in response to zinc and high irradiance stress in cowpea 74-8 88 Acta agriculturae Slovenica, 109 - 2, september 2017 UVA + B treatment affects antioxidant system and phytochemicals of parsley plant under different concentrations of Zn plant. International Journal of Current Research and Academic Review, 2(10), 112-128. Morshedi, A., Farahbakhsh, H. (2010). Effects of potassium and zinc on grain protein contents and yield of two wheat genotypes under soil and water salinity and alkalinity stresses. Plant Ecophysiology, 2, 67-72. Nasibi, F., Kalantari, K. M. (2005). The effects of UV-A, UV-B and UV-C on protein and ascorbate content, lipid peroxidation and biosynthesis of screening compounds in brassica napus. Iranian journal of science and technology transaction a- science, 29(A1), 39-48. Obinger, C., Maj, M., Nicholls, P., Loewen, P. (1997). Activity, peroxide compound formation, and heme d synthesis in Escherichia coli HPII catalase. Archives of Biochemistry and Biophysics, 342(1), 58-67. doi:10.1006/abbi.1997.9988 Ravindran, K. C., Indrajith, A., Pratheesh, P. V., Sanjiviraja, K., Balakrishnan, V. (2010). Effect of ultraviolet-B radiation on biochemical and antioxidant defense system in Indigofera tinctoria L. seedlings.International Journal of Engineering, Science and Technology, 2(5), 226-232. doi:10.4314/ijest.v2i5.60154 Reshmi, G. R., Rajalakshmi, R. (2012). Drought and UV stress response in Spilanthes acmellaMurr. (toothache plant). Journal of Stress Physiology and Biochemistry, 8(4), 110-129. Rizzini, L., Favory, J. J., Cloix, C., Faggionato, D., O'Hara, A., Kaiserli, E., Baumeister, R., et al. (2011). Perception of UV-B by the arabidopsis UVR8 protein. Science, 332(6025), 103-106. doi:10.1126/science.1200660 Saeidnejad, A. H., Kafi, M. (2013). Alleviative effects of Zinc on physiological properties and antioxidants activity of maize plants under salinity stress. International Journal of Agriculture and Crop Sciences, 5(5), 529-537. Salama, H. M. H., Al-Watban, A. A., Al-Fughom, A. T. (2011). Effect of ultraviolet radiation on chlorophyll, carotenoid, protein and proline contents of some annual desert plants. Saudi Journal of Biological Sciences, 18(1), 79-86. doi:10.1016/j.sjbs.2010.10.002 Smith, J. L., Burritt, D. J., Bannister, P. (2000). Shoot dry weight, chlorophyll and UV-B- absorbing compounds as indicators of a plant's sensitivity to UV-B radiation. Annals of Botany, 86(6), 1057-1063. doi:10.1006/anbo.2000.1270 Tavallali, V., Rahemi, M., Eshghi, S., Kholdebarin, B., Ramezanian, A. (2010). Zinc alleviates salt stress and increases antioxidant enzyme activity in the leaves of pistachio (Pistacia vera L. Badami) seedlings. Turkish Journal of Agriculture and Forestry, 34(4), 349-359. Toor, R. K., Savage, G. P. (2005). Antioxidant activity in different fractions of tomatoes. Food Research International, 38(5), 487-494. doi:10.1016/j.foodres.2004.10.016 Tossi, V., Lamattina, L., Cassia, R. (2009). An increase in the concentration of abscisic acid is critical for nitric oxide-mediated plant adaptive responses to UV-B irradiation. New phytologist, 181(4), 871-879. doi:10.1111/j.1469-8137.2008.02722.x Ulm, R., Nagy, F. (2005). Signalling and gene regulation in response to ultraviolet light. Current Opinion in Plant Biology, 8(5), 477-482. doi:10.1016/j.pbi.2005.07.004 Wagner, G. J. (1979). Content and vacuole/extravacuole distribution of neutral sugars, free amino acids, and anthocyanin in protoplasts. Plant Physiology, 64(1), 88-93. doi:10.1104/pp.64.1.88 Wei, Z. F., Luo, M., Zhao, C. J., Li, C. Y., Gu, C. B., Wang, W., Zu, Y. G., Efferth, T., Fu, Y. J. (2013). UV-induced changes of active components and antioxidant activity in Postharvest Pigeon Pea [Cajanus cajan (L.) Millsp.] Leaves. Journal of Agriculture and Food Chemistry, 61(6), 1165-117. doi:10.1021/jf304973f Weisany, W., Sohrabi, Y., Heidari, G., Siosemardeh, A., Ghassemi-Golezani, K. (2012). Changes in antioxidant enzymes activity and plant performance by salinity stress and zinc application in soybean (Glycine max L.). Plant Omics, 5(2), 60-67. Winterbourn, C. C., McGrath, B. M., Carrell, R. W. (1976). Reactions involving superoxide and normal and unstable haemoglobins. The Biochemical Journal, 3(155), 493-50. doi:10.1042/bj1550493 Zlatev, S. Z., Lidon, F. J. C., Kaimakanova1, M. (2012). Plant physiological responses to UV-B radiation. Emirates Journal of Food Agriculture, 24(6), 481-501. doi:10.9755/ejfa.v24i6.481501 Zu, Y., Li, Y., Chen, J., Chen, H. (2004). Intraspecific responses in grain quality of 10wheat cultivars to enhanced UV-B radiation under field conditions. Journal of Photochemistry and Photobiology,74(2-3), 95-100. doi:10.1016/j.jphotobiol.2004.01.006 Acta agriculturae Slovenica, 109 - 2, september 2017 89 doi: 10.14720/aas.2017.109.2.09 Original research article / izvirni znanstveni članek Genetic relationships among okra (Abelmoschus esculentus (L.) Moench) cultivars in Nigeria Bashir O. BELLO1* and D. AMINU2 Received October 11, 2016; accepted September 04, 2017. Delo je prispelo 11. oktobra 2016, sprejeto 04. septembra 2017. ABSTRACT This study was conducted on okra (Abelmoschus esculentus (L.) Moench) cultivars at the Teaching and Research Farm, University of Maiduguri, Nigeria. The objective was to evaluate the level of genetic divergence and heritability of eight characters in 2015 and 2016 dry seasons using irrigation. The results showed highly significant (p<0.01) differences in the ten okra cultivars for days to anthesis, plant height, fresh capsule length, fresh mass per capsule and fresh capsule diameter across the two years. A high genotypic coefficient of variation, heritability, and genetic advance were detected in all the characters except for days to anthesis and fresh capsule diameter. This implied that different genetic constitution and preponderance of additive effects governed these characters, thus presenting a significant opportunity for selection. The Mahanalobis D2 analysis allotted the ten cultivars into four clusters. The highest was cluster I comprising four cultivars, followed by cluster II containing three cultivars, cluster III consisting two cultivars, and cluster IV with mono genotypic. The three most superior okra cultivars (Salkade, Y'ar gagure and Kwadag) for yield and related characters could be exploited directly or introgressed with other promising ones in future breeding programs. Key words: diversity; genetic advance; heritability; okra; fruit yield IZVLEČEK GENETSKA RAZMERJA MED IZBRANIMI SORTAMI JEDILNEGA OSLEZA (Abelmoschus esculentus (L.) Moench) V NIGERIJI Raziskava je bila opravljena na izbranih sortah jedilnega osleza oz. okre (Abelmoschus esculentus (L.) Moench) na Teaching and Research Farm, University of Maiduguri, Nigeria. Namen je bil ovrednotiti raven genetske pestrosti in dednost osmih lastnosti sort osleza ob namakanju v sušnem obdobju 2015 in 2016. Rezultati so pokazali značilne razlike (p<0.01) pri desetih sortah v dolžini obdobja (številu dni) do cvetenja, višini rastlin, dolžini svežih plodov ('strokov'), sveži masi plodov in v premeru svežih plodov v obeh vegetacijskih sezonah. Visoke vrednosti genotipskega koeficienta variabilnosti, dednosti in genetskega napredka, povezanega s selekcijo, so bile ugotovljene pri vseh preučevanih lastnostih, razen pri številu dni do cvetenja in premeru svežih plodov. To nakazuje veliko genetsko raznolikost in prevladovanje aditivnih učinkov pri genetski kontroli teh lastnosti, kar se lahko ugodno uporabi pri selekciji. Mahanalobisova D2 analiza je razdelila deset sort v štiri skupine (klastre). Največja skupina I je vsebovala 4 sorte, tej sledi skupina II s tremi sortami, nato skupina III z dvema sortama in mono-genotipska skupina IV. Tri najboljše sorte (Salkade, Y'ar gagure and Kwadag) glede na pridelek in z njim povezane lastnosti bi lahko bile uporabljene neposredno ali v križanjih z drugimi obetajočimi sortami v bodočih žlahtniteljskih programih. Ključne besede: diverziteta; genetska prednost; dednost; jedilni oslez (okra); pridelek plodov 1 "Department of Biological Sciences, Fountain University, Osogbo, and Department of Crop Production. University of Maiduguri, Nigeria, corresponding author: obbello2002@yahoo.com 2 Department of Crop Production, University of Maiduguri, Nigeria Acta agriculturae Slovenica, 109 - 2, september 2017 str. 251 - 240 Bashir O. BELLO and D. AMINU 1 INTRODUCTION Okra (Abelmoschus esculentus (L.) Moench) usually referred to as Lady's finger belongs to the Malvaceae family (Kishor et al., 2016). Okra is proposed to originate from the Tropical Africa from where it extensively spread to Asia, America, Southern Europe and other countries (Muhammad et al., 2013). In 2008, the five top most okra producing countries were Iraq, Nigeria, Togo, Sudan and India (FAOSTAT, 2010). However, Nigeria ranks third in okra amid fruit vegetables based on production and consumption, succeeding pepper and tomato (Ibeawuchi, 2007). The okra local cultivars diverged in growth habits including leaf arrangement and size, fruits branching, height and maturity period. During the vegetative stage, okra growth patterns are similar, but those that were highly vigorous produced improved leaf area and accumulated dry matter (Akanbi et al., 2010). The unripe green finger-like seed capsule of okra, usually called "pod" are processed and consumed as stews and salads, soups, sliced, boiled and fried vegetables (Akanbi et al., 2010; Daniela et al., 2012). The fruits contain effortlessly digestible fiber, fat-free contents and low calories (Kumar & Sreeparvathy, 2010; Reddy et al., 2013). Okra fruits are used for soups and stews thickening due to its mucilaginous and tender texture nature, (Ijoyah & Dzer, 2012; Das et al., 2013). The fruit contents comprises of 9.7 % carbohydrate, 86.1 % water, 1.0 % fiber, 0.8 %, 0.2 % fat and 2.2 % protein (Saifullah & Rabbani, 2009). Furthermore, the unripe pods are very rich sources of potassium, vitamins, calcium, and other minerals. Okra is tolerant to various climatic conditions and adaptable to the Nigeria agroecology. The collection of desirable plant germplasm relies on the proven accession features and genetic divergence, which are essential in genetic resources utilization (Olaoye et al., 2009; AdeOluwa & Kehinde, 2011). Genetic diversity denotes the variability in different crop species, and its links with accessions identification, which is important in gene bank curators (Bello et al., 2012ab; Bello et al., 2011; Osekitar & Akinyele, 2008). Morphological characterization of plants has been recommended as the first step to be adopted prior to far-reaching molecular research and biochemical analyses (Akash et al., 2013). Many researchers reported a substantial morphological degree of variance in the West African okra varieties (Adeniji, et al., 2007; Akanbi et al., 2010; AdeOluwa & Kehinde, 2011). The improvement in plant breeding scheme leans on high genetic differences in the population and the magnitude of inheritance of favorable attributes (Olawuyi et al., 2015, Bello et al., 2014ab). The variability obtained in a population are apportioned into non-heritable and heritable parameters utilizing genetically related components including heritability, the genotypic coefficient of variation and genetic advance, which are the core for selection (Muluken et al., 2016; Seth et al., 2016). The expected response to selection and methods of selection are assessed on high heritability values of the characters. Cluster analysis is one of the powerful tools in determining genetic divergence among varieties of crops. The objective of this study was to evaluate the level of genetic divergence and heritability of ten okra cultivars in the stress-free irrigation conditions of Nigeria Sudan savannah, with the view to devising a breeding strategy for okra selection for further improvement. 2 MATERIALS AND METHODS Field experiments were conducted on okra (Abelmoschus esculentus (L.) Moench.) cultivars at the Teaching and Research Farm, University of Maiduguri, Nigeria to evaluate the level of genetic divergence and heritability of eight characters in 2015 and 2016 dry seasons using irrigation. Ten okra cultivars were used; of which six cultivars (Salkade, Yar'gagure, Kwadam, Lai-lai, Yar'duwi and Y'ar kwami.) were obtained from Gagure, Gulani Local Government Area of Yobe State and four (Kwalpuku, Composite Kwadag and Mola Kwadag) were acquired from Borno State Agricultural programme, Maiduguri, Nigeria, and coded as described in Table 1. 252 Acta agriculturae Slovenica, 109 - 2, september 2017 Genetic relationships among okra (Abelmoschus esculentus (L.) Moench) cultivars in Nigeria Table 1: The morphological descriptions of ten okra cultivars studied Code Cultivars Morphological descriptions P1 Salkade This cultivar is tall containing broad leaves, a red stem and few flowers, long fruit with a small diameter. The fruit is long, white and smooth with a small diameter. P2 Y'ar duwi It has short pale green stem, few flowers, and small slim fruit with no spine P3 Composite It has a dark green fruit of medium size with medium diameter. It also has a green stem and broad leaves with many flowers. P4 Y'ar gagure It has a pale green spiny fruit, broad diameter, and long stem. It also has red and sparsely flowers. P5 Kwadag It has a long stem with few flowers, big capsules with spine and a red stem. P6 Kwalpuku It has a short stem, small leaves with many flowers and spiny fruits. P7 Y'ar kwami It has a dark green fruit with many flowers and big capsules with spines. P8 Mola Kwadag It has a short green stem with small finger-like leaves. It also has many flowers with big capsules. P9 Lai-lai It is runner-like, short, dark green with medium capsule diameter. It also has a white stem and small leaves with many flowers. P10 Kwadam This cultivar has a short and white stem, medium leaves and spiny capsules. The field experiment was based on Randomized Complete Block Design with three replications. The plot was 216 m2, divided into 33 plots of 2 x 2 m with 1 m spacing between replications, and 0.5 m between treatments. Weeding was carried out manually at 3, 6, and 9 weeks after sowing (WAS). A compound fertilizer, N.P.K. 15:15:15 was applied at the rate of 60 kg N/ha in twice, first at three weeks after planting and then at flowering. Two milliliters of Ultracide 40EC insecticide in 15 liters was applied fortnightly to control insect pests. Light watering was applied using a watering can at every morning and afternoon. This was continued for a week for rapid and well establishment of the germinated seedlings. For the evaluation of the eight studied characters; from each plot, ten (10) plants were randomly chosen. The eight studied characters include fresh capsule length, fresh capsule yield per plant (g), the number of primary branches per plant, days to anthesis, the number of capsules per plant, fresh capsule diameter (cm), fresh mass per capsule (g) and plant height (m). Individual year analysis of variance (AnOVA) was calculated, and then a combined ANOVA across the two years with the use of SAS PROC GLM model (Version 9.2, Volume 1), to determine the mean squares for every character (SAS Institute, 2011). A mixed model of the SAS PROC GLM model was utilized for the ANOVA. Cultivars were considered as fixed effects, while replication as a random effect. The degree of variation was estimated employing % coefficient of variation p < 0.05. Also, the variations in the character means were computed with the use of Least Significant Difference (LSD). However, genotypic coefficient of variation (GCV) and phenotypic coefficient of variation (PCV) were computed based on the formula proposed by Burton (1952) with the use of PROC GLM model of SAS (SAS Institute, 2011, Version 9.2, Volume 1). The broad sense heritability was determined as recommended by Johnson et al. (1955). The estimate of the expected genetic advance for each character was ascertained using the procedure of Allard (1960). The approach of Comstock & Robinson (1948) was followed for estimating the genetic advance in a percentage of the mean. To evaluate genetic divergences in the ten okra cultivars, the Mahalanobis' D2 model (Mahalanobis 1936) and its auxiliary analysis were applied following the method of Rao (1952). 3 RESULTS AND DISCUSSION The combined analysis of variance revealed exceedingly significant (p < 0.01) differences in the studied okra cultivars for days to anthesis, plant height, fresh capsule length, fresh mass per capsule and fresh capsule diameter across the two years (Table 2). It shows that the genetic parameters of the parental materials were quite dissimilar. This result corroborates with that of several earlier researchers (Akinyele & Osekita, 2006; Nwangburuka et al., 2011; Nwangburuka et al., 2012; AdeOluwa & Kehinde, 2013; Muluken et al., 2016). Again, the cultivars exhibited significant (P<0.05) differences for fresh capsule yield per plant and the number of capsules per plant. On the other hand, nonsignificant differences in the cultivars for the number of primary branches per plant revealed that the genetic parameters of the okra cultivars were very intact. The ANOVA also showed variations in the studied cultivars for almost all the characters. This variation could be used via selection to improve the okra studied characters. This result is supported by Acta agriculturae Slovenica, 109 - 2, september 2017 Bashir O. BELLO and D. AMINU several previous researchers (Duzyaman, 2005; Salesh et al., 2010; Nwangburuka et al., 2012; Hazem et al., 2013; Amoatey et al., 2015). The first-order cultivar x year interaction was significant for all the okra characters. It signified that environment condition affected the wide variation of these characters, and is considered as a key parameter for the yield of crops. The okra yield potential is essential in producing many capsules per plant which could be attained by means of timely harvesting the fresh fruits to enable the development of more branches. This effort will undeviatingly increase the crop yield. This finding is also in conformity with the previous researchers (Akinyele & Osekita 2006; Mehta et al., 2006; Alade et al., 2008). Table 2: Combined ANOVA for eight studied okra characters in Maiduguri, Nigeria across two years Sources of Days to Primary Fresh Capsules Plant Fresh Fresh Fresh variation anthesis branches capsule per plant height capsule capsule mass per plant yield per length diameter per plant capsule Year (Y) 8.22 22.88 9.67 11.54 6.61 20.92 6.25 6.63 Rep (Year) 19.27 18.63 12.63 4.86 4.44 6.54 14.74 19.22 Cultivars 862.29** 174.11** 2376.11** 39.19** 4286.82** 5.85** 67.66** 65.44** Cultivar x Year 63.82* 54.65* 45,53 41.53* 72.97* 34.74* 38.37* 38.45* Error 24.59 4.15 5.89 1.14 8.28 0.34 4.11 7.21 *,**, significant at P< 0.05 and P< 0.01 respectively The mean performance of the studied okra cultivars showed a significant difference in days to anthesis (Table 3). The maximum number of days to anthesis (50.69 days) was recorded for 'P4', while the minimum number of days (42 days) was obtained in 'P10'. The average number of days to anthesis was 46.82 days and five cultivars had higher days to anthesis than the average. It denoted that the assessed cultivars differed morphologically from one other especially on flower bearing habits, similar to the findings of Muluken et al. (2016). Besides, all the studied okra cultivars varied decidedly for plant height with 'P5' being the tallest while P3 cultivar was the shortest. The average plant height in 'P5' and 'P4' was 1.25m which was higher than the overall mean height. The number of primary branches per plant significant differed in the cultivars with 'P1' possessing the greatest number, whereas 'P10' exhibited the least. Subsequently, the greatest number of primary branches was obtained from 'P1', followed by 'P7', 'P5', and 'P4' which were more than the average. It is obvious that the number of primary branches varied significantly at the early growth of okra, as previously reported by Jagan et al. (2013). The average number of capsules per plant recorded was 28.03 with about half of the cultivars accomplishing a greater average. The 'P4' was outstanding with the greatest number of capsules per plant, followed by 'P1'', ''P5', and 'P9'. The highest number of fresh capsule length was attained in 'P1', while 'P6' had the lowest. This variation might be the differences in the number of bearing internodes and plant height in the cultivars. Half of the okra cultivars had an extended fresh capsule length than the average length. Fresh capsule length was reported to vary significantly from one accession to the other since it invariably articulates the distinctiveness (Nwangburuka et al., 2012; Hazem et al., 2013; Amoatey et al., 2015). The 'P2', 'P4', and 'P5' had fresh capsule diameter more than the average, while 40 % of the cultivars produced more than the average fresh capsule mass. The okra capsule length is at variance from one cultivar to the other, perhaps due to differences in days to anthesis and other morphological characters. However, 'P1', 'P4', and 'P5' also possessed capsule yield greater than the average. 254 Acta agriculturae Slovenica, 109 - 2, september 2017 Genetic relationships among okra (Abelmoschus esculentus (L.) Moench) cultivars in Nigeria Table 3: Mean performance for eight studied okra characters in Maiduguri, Nigeria across two years Cultivars Number of Days to Fresh Number of Fresh Plant height Fresh Fresh mass per primary anthesis capsule capsules per capsule at harvest capsule capsule (g) branches per yield per plant (no.) length (m) diameter plant (no.) plant (g) (cm) (cm) P1 4.23 50.33 598.65 33.75 14.88 1.44 1.22 16.23 P2 2.84 43.11 479.38 25.34 12.34 1.14 1.56 15.96 P3 1.89 44.23 428.62 22.54 12.52 1.11 1.48 14.73 P4 3.51 50.75 616.97 34.45 14.73 1.39 1.83 16.56 P5 3.75 49.31 622.67 34.35 14.37 1.49 1.84 16.83 P6 3.18 43.00 488.38 23.92 11.49 1.18 1.51 13.14 P7 3.33 45.34 431.63 25.18 13.37 1.18 1.52 14.34 P8 2.66 49.11 457.92 24.88 12.84 1.21 1.48 14.64 P9 2.12 47.93 532.85 28.64 11.59 1.16 1.52 14.98 P10 1.67 45.11 580.38 28.23 13.64 1.22 1.44 15.11 Mean 2.92 46.82 523.75 28.03 13.08 1.25 1.54 15.25 Range 2.56 7.33 194.05 11.91 3.39 0.38 0.62 3.68 SE± 2.759 11.849 11.73 6.654 6.149 6.111 11.234 10.171 LSD a 0.05 1.45 2.23 3.57 4.62 2.14 1.11 1.01 1.43* CV % 6.39 4.36 6.83 4.92 10.52 7.82 7.45 7.18 Variability parameters estimated viz. GCV, PCV, heritability and genetic advance studied characters are depicted in Table 4. The large magnitude of PCV and GCV with a small difference between the two heredity parameters indicated a smaller amount of environmental influence on the phenotypic expression. Muluken et al. (2016) earlier buttressed this extrapolation. The GCV and PCV ranged between 1.1-33.3 % and 2.4 %-48.5 %, respectively, for fresh capsule yield and fresh capsule diameter. Several researchers reported the consistent differences of okra cultivars due to cultivars and environmental interactions (Thirupathi et al., 2012; AdeOluwa & Kehinde, 2013; Ehab et al., 2013; Adekoya et al., 2014). This statement also showed the level of productivity in crops, as statistical groupings of the cultivars were believed to be distinguished. Several researchers corroborate these discoveries for West African okra germplasm (Nwangburuka et al., 2012; AdeOluwa & Kehinde, 2013; Adekoya et al., 2014). The number of primary branches and fresh capsule diameter, on the other hand, were having the lowest estimate of below 10 % for PCV and GCV. This expressed a slight range of difference and hindered possibility for selection of these characters. Furthermore, the least GCV and PCV estimate of characters, implied higher impacts environmental conditions on these characters therefore; selection based on phenotypic basis will not be useful for the genetic progress of the crop (Chaurasia et al., 2011; Bharathiveeramani et al., 2012; Das et al., 2012; Sankara & Pinaki, 2012; Thirupathi et al., 2012; Ehab et al., 2013; Kishor et al., 2016]. Contrariwise, days to anthesis possessed medium GCV and PCV values (Chaurasia et al., 2011). It indicated that genetic effects influenced these characters. Therefore, these characters are responsive to selection for onward improvement. The number of capsules per plant, fruit length, plant height, the number of primary branches, fresh capsule length, fresh capsule yield per plant and fresh mass per capsule exhibited high values more than 20 % for both PCV and GCV with a considerably low degree of variation between the two. This result substantiates with the findings of Ehab et al. (2013). Many researchers nevertheless, noticed that high magnitude of GCV and PCV inferred a low environmental manifestation effects on the characters, which probably increase greater improvement prospects through selection scheme (Salesh et al., 2010; Bharathiveeramani et al., 2012; Nwangburuka et al., 2012; Swati et al., 2014; Kishor et al., 2016). Thus, selections of favorable characters by utilizing high phenotypic and genotypic estimates could be exploited in enhancing the characters during the breeding program. Broad sense heritability estimates ranged from 25.84 % for the number of capsules per plant to 93.84 % for fresh capsule yield per plant (Table 4). As described by Robinson et al. (1955), heritability is categorized as least with a range of 0-30 %, fair (31-60 %) and best (> 60 %). In the present research, a broad sense heritability of greater than 60 % was obtained for capsule yield, plant height, days to anthesis, capsule length, capsule diameter, and capsule mass. These agronomic characters seemed to respond effectively to the pressure of selection. Whenever heritability is up to 80 % or more of a character, selection would be easy for such character. Thus, selection for all these characters might result in an increase in capsule yield of okra. A great heritability also showed a great genetic base. A close association between the phenotype and cultivar could be the cause of small environmental interplay conditions (Jagan et al., 2013; Muluken et al., 2016). Fairly broad sense heritability estimate was observed for the number of primary branches. The least heritability value, Acta agriculturae Slovenica, 109 - 2, september 2017 Bashir O. BELLO and D. AMINU however, was obtained for the number of capsules per plant. This alluded that these cultivars may not be improved via direct selection. Whenever a character is of a range between medium and high heritability, a selection due to specific performance can allow rapid progress. Fairly heritability implied improvement via selection. The least heritability also indicated ineffectual direct selection for the advancement of the characters owing to environmental masking effects (Nwangburuka et al., 2012; Bello & Olawuyi, 2015; Muluken et al., 2016). As described by Johnson et al. (1955), genetic advance as percent mean were categorized as high (>20 %), moderate (10 < 20 %) and low (0 < 10 %). Based on this ranking, the number of capsules per plant, the number of primary branches, capsule length, plant height, capsule yield per plant and capsule mass possessed the genetic advance of greater or equal to 20 % (Table 4). This revealed the predominance of additive genetic effects for these characters. Capsule diameter and days to anthesis showed low and moderate genetic advance, respectively. Appropriately, this result depicted that expected progress from the selection of the cultivars is between 16.1 % (days to anthesis) and 51.8 % (capsule yield). This substantiates with the findings of Olawuyi et al. (2015) and Hazem et al. (2013). The relative amount of heritable variability is not enough to determine the GCV only, except with the aid of heritability and genetic advance. The high estimates of heritability coupled with genetic advance offered sufficient information on each character and indicated a genotypic response to selection (Pradip et al., 2010; Sibsankar et al., 2012). High heritability and genetic advance were observed for all the characters studied except days to anthesis and fresh capsule diameter (Table 4). This showed that differences in the genetic background would enable great opportunity for selection. Furthermore, this demonstrated the preponderance of additive gene effects for these characters, instead of the environmental influences. Thus, selection can be made based on the phenotypic expressions of okra characters for the improvement of yield (Muluken et al., 2016). As moderate heritability and high genetic advance were noted for the number of primary branches of okra, low heritability and high genetic advance estimates were detected for the number of capsules per plant. This could also be on high environmental influences controlling the expression of the characters. This, therefore, possibly hinders the opportunity for selection for crop improvement due to the prevalence of non-additive (dominant and/or epistatic) or non-fixable effects. Jagan et al. (2013) earlier noticed these findings. In a condition of low heritability and genetic advance for the characters, unique approaches such as hybridization and recurrent selection should be adopted (Bozokalfa et al., 2013; Jagan et al., 2013). Table 4: Estimates of genotypic coefficient of variation (GCV), phenotypic coefficient of variation (PCV) and environmental coefficient of variation (ECV) for eight studied okra characters in Maiduguri, Nigeria across two years Characters GCV (%) PCV (%) Heritability Genetic Genetic H2(bs) (%) Advance as % Advance of mean (GA) Days to anthesis 17.7 18.1 86.7 16.1 8.7 Number of primary branches per 24.8 29.9 48.41 20.2 7.4 plant Plant height 18.9 24.2 72.54 24.3 20.8 Fresh capsule diameter 1.1 2.4 84.54 6.9 7.4 Number of capsules per plant 21.9 27.3 25.84 28.7 18.6 Fresh capsule length 20.7 22.5 79.98 37.5 6.8 Fresh capsule yield per plant 33.3 48.5 93.84 51.8 103.3 Fresh per capsule 20.9 22.5 71.30 41.6 16.1 The parameters of four different Clusters, respective cultivars, and their numbers are presented in Table 5. Cluster I possessed the highest with 4 cultivars followed by Cluster II with 3 cultivars and Cluster III with 2 cultivars, whereas Cluster IV had one cultivar (mono genotypic). The highly varied cultivars, P7, P8, P9, and P10 were obtained from Cluster I. These clusters outline showed that geographical variation had an indirect association with genetic diversity. Genetic diversity in okra germplasm with the use of cluster analysis had earlier been reported by several researchers (Akotkar et al., 2010; Das et al., 2012; Umrao et al., 2014; Seth et al., 2016). In general, the cultivars distribution patterns from diverse geographical regions into discrete clusters were at random. This result might be attributed to the free exchange and recurrent genetic constitution efforts 256 Acta agriculturae Slovenica, 109 - 2, september 2017 Genetic relationships among okra (Abelmoschus esculentus (L.) Moench) cultivars in Nigeria by the farmers and plant breeders of the diverse agroecological zones in Nigeria. Besides, the disparity of selection pressure owing to regional okra favorites could improve the resemblance of the cultivars. Lack of relationship between the genetic diversity and geographical distance indicated that forces like natural and artificial selection, the exchange of genetic material, genetic drift and spontaneous mutation could lead to genetic diversity instead of geographical origin (Pradip et al., 2010; Seth et al., 2016). Therefore, selection for outcrossing of okra cultivars ought to base on genetic diversity instead of geographic diversity. Table 5: Clustering form of 10 okra cultivars by Tocher's method Cluster_Number of cultivars_Okra cultivars I 4 P7, P8, P9, and P10 II 3 P1, P2, and P4 III 2 P5 and P6 IV 1 P3 Widely varied inter-cluster distances in the four clusters of okra cultivars are shown in Table 6. The inter-cluster distances were higher than the intra-cluster distances, signifying high genetic diversity among the cultivars of the varied groups. The intra-cluster distance in the ten studied cultivars indicated a low value between cluster IV and I (4.57), revealing similar relationships in the cultivars of these clusters (Pradip et al., 2010). The greatest intra-cluster estimate was observed between clusters III and I followed by between clusters IV and III. This depicts that the cultivars in these clusters diverged greatly. Therefore, outcrossing the cultivars featuring in these clusters could enhance transgressive segregations and generation progress. This has been as earlier opined by Umrao et al. (2014) and Seth et al. (2016). Conclusively, an involvement of highest yielding cultivars (Salkade, Y'ar gagure and Kwadag) could be exploited directly or hybridized to enhance novel recombinants and exploit transgressive segregates with high genetic yield potentials. Table 6: Mean intra (bold) and inter-cluster D2 values of 4 clusters for 10 okra cultivars formed by Torcher's method Cluster I II III IV I - II 8.54 - III 14.2 7.24 - IV 4.57 6.13 9.83 - 4 ACKNOWLEDGEMENT The authors appreciated the Farm manager and his of Maiduguri, Maiduguri, Nigeria for supporting this officials at the Teaching and Research Farm, University research. 5 REFERENCES Adekoya, M. A., O. J. Ariyo, O. B. Kehinde and A. E. Adegbite. (2014). Correlation and path analyses of seed yield in okra ( Abelmoschus esculentus (L.) Moench) grown under different cropping seasons. Pertanika Journal of Tropical Agricultural Sciences, 37(1), 39-49. Adeniji, O.T., O. B. Kehinde, M. O. Ajala and M. A. Adebisi. (2007). Genetic studies on seed yield of West African okra (Abelmoschus caillei) (A chev.) Stevels). Journal of Tropical Agriculture, 45(1-2), 36-41. AdeOluwa, O. O. & O. B. Kehinde. (2011). Genetic variability studies in West African Okra (Abelmoschus caillei). Agriculture and Biological Journal of North America, 2(10): 1326-1335. doi: 10.5251/abjna.2011.2.10.1326-1335 Akanbi, W.B., A. O. Togun, J. A. Adediran and E. A. O. Ilupeju. (2010). Growth, dry matter and fruit yield components of okra under organic and inorganic sources of nutrients. American-Eurasian Journal of Sustainainable Agriculture, 4(1), 1-13. Akash, M. W., S. M. Shiyab and M. I. Saleh. (2013). Yield and AFLP analyses of inter-landrace Acta agriculturae Slovenica, 109 - 2, september 2017 Bashir O. BELLO and D. AMINU variability in okra ( Abelmoschus esculentus L.). Life Science Journal, 10(2), 2771-2779. Akotkar, P.K., D. K. De and A. K. Pal. (2010). Genetic variability and divergence in okra (Abelmoschus esculentus L. Moench). Electronic Journal of Plant Breeding, 1(4), 393-398. Akinyele, B. O. & O. S. Osekita. (2006). Correlation and path coefficient analyses of seed yield attributes in okra (Abelmoschus esculentus (L.) Moench). African Journal of Biotechnology, 5(14), 1330-1336. Aladele, S. E., Ariyo, O. J., and de Lapena, R. (2008). Genetic relationships among West African okra (Abelmoschus caillei) and Asian cultivars (Abelmoschus esculentus) using RAPD. African Journal of Biotechnology, 7(10), 22-34. Allard, R. W. (1960). Principles of plant breeding. New York: John Willey and Sons. Pp. 485. Amoatey, H. M., G. Y. P. Klu, E. K. Quartey, H. A. Doku, F. L. Sossah, M. M. Segbefia and J. K. Ahiakpa. (2015). Genetic divergence studies in 29 accessions of okra (Abelmoschus spp (L.) using 13 quantitative traits. American Journal of Experimental Agriculture, 5(3), 217-225. doi:10.9734/AJEA/2015/12306 Bello, O. B., M. S. Afolabi, S. A. Ige, S. Y. Abdulmaliq, M. A. Azeez, J. Mahmood. (2011). Yield response of diallelic crossed maize (Zea mays L.) cultivars to varying nitrogen regimes in Nigeria. Journal of Bio-Sciences, 19, 43-52. Bello, O. B., S. Y. Abdulmaliq, S. A. Ige, J. Mahmood, F. Oluleye, M. A. Azeez, M. S. Afolabi. (2012a). Evaluation of early and late/intermediate maize varieties for grain yield potential and adaptation to a southern guinea savanna agro-ecology of Nigeria. International Journal of Plant Research, 2(2), 1421. doi:10.5923/j.plant.20120202.03 Bello, O. B., M. S. Afolabi, S. A. Ige, S. Y. Abdulmaliq, M. A. Azeez and J. Mahmood. (2012b). Nitrogen use efficiency and grain yield in a diallelic cross of maize populations. International Journal of Plant Research, 2(3), 94-102. doi:10.5923/j.plant.20120203.08 Bello, O. B., O. J. Olawuyi, Ige SA, J. Mahsmood, M. S. Afolabi, M. A. Azeez and S. Y. Abdulmaliq. (2014a). Agro-nutritional variations of quality protein maize (Zea mays L.) in Nigeria. Journal of Agricultural Sciences, 59(2), 101-116. doi:10.2298/JAS1402101B Bello, O. B., O. J. Olawuyi, S. Y. Abdulmaliq, S. A. Ige, J. Mahmood, M. A. Azeez and M. S. Afolabi. (2014b). Yield performance and adaptation of early and intermediate drought-tolerant maize cultivars in Guinea savanna of Nigeria. Sarhad Journal of Agriculture, 30(1), 53-66. Bello, O. B., & O. J. Olawuyi. (2015). Gene action, heterosis, correlation and regression estimates in developing hybrid cultivars in maize. Tropical Agriculture, 92(2), 102-117. Bharathiveeramani, B, M. Prakash and S. A. Prakash. (2012). Variability studies of quantitative characters in Maize (Zea mays L.). Electronic Journal of Plant Breeding, 3(4), 995-997. Bozokalfa, K. M., D. I. Esiyokhulya and T. A. Kaygisiz. (2010). Estimates of genetic variability and association studies in quantitative plant traits of Eruca spp. Landraces. Genetika, 42(3), 501-512. doi:10.2298/GENSR1003501B Burton, G. W. (1952). Quantitative inheritance in grasses. In Proceeding of the 6th GrasslandConference, 7, 227-283. Chaurasia, P.C., K. C. Rajhans and M. Yadav. (2011). Correlation coefficient and path analysis in okra [ Abelmoschus esculentus (L.) Moench]. Indian Horticultural Journal, 1, 32-43. Comstock, R. E. & H. F. Robinson (1948). The components of genetic variance in population of biparental progenies and their use in estimating the average degree of dominance. Biometrics, 4, 254266. doi:10.2307/3001412 Daniela, F. O., M. Alicia, R. C. Alicia, H. Rodolfo and Z. V. Sonia. (2012). Quality attributes of okra (Abelmoschus esculentus L. Moench) pods as affected by cultivar and fruit size. Journal of Food Research, 1(4), 224-235. doi:10.5539/jfr.v1n4p224 Das, S., A. Chattopadhyay, S. B. Chattopadhyay, S. Dutta and P. Hazra. (2012). Genetic parameters and path analysis of yield and its components in okra at different sowing dates in the Gangetic plains of eastern India. African Journal of Biotechnology, 11, 16132-16141. doi: 10.5897/AJB 12.545 Das, S, A, Chattopadhyay, S. B. Chattopadhyay, S. Dutta and P. Hazra. (2013). Breeding okra for higher productivity and yellow vein mosaic tolerance. International Journal of Vegetable Science, 19, 58-77. doi: 10.1080/19315260.2012.675024 Duzyaman, E. (2005). Phenotypic divergence within a collection of distinct okra (Abelmoschus esculentus) cultivars derived from Turkish land races. Genetic Resources and Crop Evolution, 52(8), 1019-1030. doi: 10.1007/s10722-004-6118-9 258 Acta agriculturae Slovenica, 109 - 2, september 2017 Genetic relationships among okra (Abelmoschus esculentus (L.) Moench) cultivars in Nigeria Ehab, A. A. I., Y. A. Mohamed and M. M. Ali. (2013). Genetic behavior of families selected from some local okra [ Abelmoschus esculentus (L.) Moench] populations in Egypt. Plant Breeding and Biotechnology, 1(4), 396 -405. doi:10.9787/PBB.2013.1.4.396 FAOSTAT. (2010). Economic and Social Department. The Statistics Division. Major Food and Agricultural Commodities and Producers. Retrieved from http://faostat.fao.org/site/339/default.aspx Hazem, A, S. Obiadalla-Ali, M. H. Z. Eldekashy and A. A. Helaly (2013). Combining ability and heterosis studies for yield and its components in some cultivars of okra [Abelmoschus esculentus (L.) Moench]. American-Eurasian Journal of Agriculture and Environmental Sciences, 13(2), 162-167. Ibeawuchi, I. K. (2007). Intercropping a food production strategy for resource poor farmers. Nature Science, 5(1), 46-49. Ijoyah, M. O. & D. M. Dzer. (2012). Yield Performance of okra ( Abelmoschus esculentus L. Moench) and maize (Zea mays L.) as affected by time of planting maize in Makurdi, Nigeria. Agriculture and Biological Journal of North America, 1(6), 13281332. doi:10.5251/abjna.2010.1.6.1328.1332 Jagan, K. K., K. R. Ravinder, M. Sujatha, V. Sravanthi and V.R. Madhusudhan. (2013). Studies on genetic variability, heritability and genetic advance in okra [Abelmoschus esculentus (L.) Monech]. IOSR Journal of Agriculture and Veterinary Sciences, 5(1), 59-61. doi:10.9790/2380-0515961 Johnson, H. W., H. F. Robinson and R. E. Comstock. (1955). Estimation of genetic and environmental variability in soybeans. Agronomy Journal, 47, 314-318. doi:10.2134/agronj1955.00021962004700070009x Kishor, D. S., K. Aiya, K. J. Yogeesh, K. Y. Vinod and K. Hee-Jong. (2016). Genotypic variation among okra ( Abelmoschus esculentus (L.) Moench) germplasms in South Indian Plant Breeding and Biotechnology, 4(2), 234-241. doi:10.9787/PBB.2016.4.2.234 Kumar, P. S. & S. Sreeparvathy (2010). Studies on heterosis in okra (Abelmoschus esculentus (L.) Moench). Electronic Journal of Plant Breeding 1(6), 1431-1433. Mahalanobis, P. C. (1936). On the generalized distance in statistics. Proceedings of the National Institute of Science of India, 2, 49-55. Mehta, D. R., Dhaduk, L. K. and K. D. Patel (2006). Genetic variability, correlation and path analysis studies in okra {Abelmoschus esculentus (L.) Moench}. Agricultural Science Digest, 26(1), 1518. Muhammad, R. S., A. Muhammad, Z. Khurram, M. J. Muhammad, A. Saeed, I. Qumer and N. Aamir (2013). Growth, yield and seed production of okra as influenced by different growth regulators. Pakistan Journal of Agricultural Science, 50(3), 387-392. Muluken, D., M. Wassu and G. Endale (2016). Variability, heritability and genetic advance in Ethiopian okra [ Abelmoschus esculentus (L.) Monech] collections for tender fruit yield and other agro-morphological traits. Journal of Applied Life Sciences International, 4(1), 1-12. doi:10.9734/JALSI/2016/19483 Nwangburuka, C. C., Kehinde, O. B., Ojo, D. K., Denton, O. A., & Popoola, A. R. (2011). Morphological classification of genetic divergence in cultivated okra, Abelmoschus esculentus (L) Moench using principal component analysis (PCA) and single linkage Cluster analysis (SLCA). African Journal of Biotechnology, 70(54), 11165-11172. doi:10.5897/AJB11.285 Nwangburuka, C.C., O. A. Denton, O. B. Kehinde, D. K. Ojo and A. R. Popoola. (2012). Genetic variability and heritability in cultivated okra [Abelmoschus esculentus (L.) Moench]. Spanish Journal of Agricultural Research, 10(1), 123-129. doi:10.5424/sjar/2012101-021-11 Olaoye, G., O. B. Bello, L. S. Olayiwola and A. Y. Abubakar. (2009). Analyses of moisture deficit grain yield loss in drought tolerant maize (Zea mays L.) germplasm accessions and its relationship with field performance. African Journal of Biotechnology, 8(14): 3229-3238. Olawuyi, O.J., O. B. Bello, C. V. Ntube and A. O. Akanmu (2015). Progress from selection of some maize cultivars' response to drought in the derived savanna of Nigeria. AGRIVITA, 37(1), 8-17. doi:10.17503/Agrivita-2015-37-1-p008-017 Osekita, O. S. & B. O. Akinyele (2008). Genetic analysis of quantitative traits in ten cultivars of okra - Abelmoschus esculentus (Linn.) Moench. Asian Journal of Plant Sciences, 7, 510-513. doi:10.3923/ajps.2008.510.513 Pradip, K., D. K. Akotkar and A. K. De Pal. (2010). Genetic variability and divergence in okra [ Abelmoschus esculentus (L).Moench]. Electronic Journal of Plant Breeding, 1(4), 393-398. Acta agriculturae Slovenica, 109 - 2, september 2017 Bashir O. BELLO and D. AMINU Purushothama J. & G. Narshimulu. (2012). Genetic variability analysis for the selection of elite cultivars based on pod yield and quality from the germplasm of okra [Abelmoschus esculentus (L.) Moench]. Journal of Agricultural Technology, 8, 639-655. Rao, T. S. (1972). Note on natural variability for some qualitative and quantitative characters in okra. Indian Journal of Agricultural Sciences, 42, 437438. Reddy, M. T., K. H. Babu, M. Ganesh, H. Begum, R. S. K. Reddy and J. D. Babu (2013). Exploitation of hybrid vigour for yield and its components in okra [Abelmoschus esculentus (L.) Moench]. American Journal of Agricultural Science and Technology, 1, 1-17. doi:10.7726/aj ast.2013.1001 Robinson, H. F., R. E. Comstock and P. H. Harvey (1955). Estimates of heritability and the degree of dominance in maize. Agronomy Journal, 41, 353359. doi: 10.2134/agronj1949.00021962004100080005x Saifullah M & M. G. Rabbani (2009). Evaluation and characterization of Okra (Abelmoschus esculentus L. Moench.) cultivars. SAARC Journal of Agriculture, 7(1), 92-99. Salesh K. J., A. Deepak and T. R. Ghai (2010). Variability studies for yield and its contributing traits in okra. Electronic Journal of Plant Breeding, 1(6), 1495-1499. Sankara, R. K. & A. Pinaki. (2012). Performance of okra [Abelmoschus esculentus (L.) Moench] cultivars under summer and rainy environments. International Journal of Advance Life Sciences, 2, 17-26. SAS (2011). Statistical analysis system (SAS) software for windows version 9.2. Vol.1, SAS Institute. Cary, N.C. USA. Seth, T., A. Chattopadhyay, S. Chatterjee, S. Dutta and B. Singh (2016). Selecting parental lines among cultivated and wild species of okra for hybridization aiming at YVMV disease resistance. Journal of Agricultural Science and Technology. 18, 751-762. Sibsankar, D., C. Arup, B. C. Sankhendu, D. Subrata and H. Pranab (2012). Genetic parameters and path analysis of yield and its components in okra at different sowing dates in the Gangetic plains of eastern India. African Journal of Biotechnology, 11(95), 16132-16141. doi:10.5897/AJB12.545 Swati, B., N. Reena, R. Meenakshi and P. K. Jain (2014). Genetic variability in okra [Abelmoschus esculentus (L.). Moench]. An International Quarterly Journal of Environmental Sciences, 6, 153-56. Thirupathi, R. M., B. K. Hari, M. Ganesh, R. K. Chandrasekhar, H. Begum, R. B. Purushothama and G. Narshimulu. 2012. Genetic variability analysis for the selection of elite cultivars based on pod yield and quality from the germplasm of okra [Abelmoschus esculentus (L.) Moench]. Journal of Agricultural Technology, 8, 639-655. Umrao, V., S. K. Sharma, R. Kumar, V. Kumar and A. Sharma (2014). Genetic variability and divergence anal ysis in okra [ Abelmoschus esculentus (L.) Moench]. HortFlora Research Spectrum, 3(2), 127-132. 100 Acta agriculturae Slovenica, 109 - 2, september 2017 doi: 10.1472G/aas.2G17.109.2.10 Original research article / izvirni znanstveni članek Prostorska analiza kmetijskih zemljišč v zaraščanju v Sloveniji Matjaž GLAVAN1*, Aljaž MALEK1, Marina PINTAR1, Helena GRČMAN1 Received October 11, 2016; accepted Janury 30, 2017. Delo je prispelo 11. oktobra 2016, sprejeto 30. januarja 2017. IZVLEČEK V prispevku je predstavljena prostorska analiza razredov dejanske rabe kmetijskih zemljišč v zaraščanju (razred rabe 1410) in kmetijskih zemljišč, poraslih z gozdnim drevjem (razred rabe 1800), z namenom ugotoviti vpliv izbranih naravnih dejavnikov (delež gozda, nadmorska višina, naklon površja, ekspozicija, poplavnost) in statusa upravljanja območja na pojavljanje in razporeditev teh zemljišč v Sloveniji. Vsi ti dejavniki vplivajo na upravljavske odločitve lastnikov. Ugotovili smo, da so zemljišča razreda rabe 1410 razporejena po celotni Sloveniji z glavnino vzdolž osi od jugozahoda proti severovzhodu, zemljišč razreda rabe 1800 je največ v jugozahodnem delu države. Na občinski ravni nismo našli izrazite povezave med deležem gozda in deležem raziskovanih razredov rab. Z večanjem nadmorske višine in naklona se odstotek rabe 1410 in 1800 poveča. Ugotovili smo večjo pojavnost razreda rabe 1400 na pogosteje poplavljenih območjih. Zaraščanje je večje znotraj območij z različnimi statusi upravljanja (npr. Natura 2000). Za izboljšanje stanja in preprečevanje nadaljnjega zaraščanja bi bilo v statističnih regijah, kjer sta razreda rabe 1410 in 1800 najbolj prisotna, potrebno pospešeno izvajati zakon o kmetijskih zemljiščih, ki določa odpravljanje zaraščanja na območjih z večjo boniteto zemljišč. Ključne besede: kmetijska zemljišča; prostorska analiza; zaraščanje; raba tal ABSTRACT SPATIAL ANALYSIS OF THE ABANDONMENT OF AGRICULTURAL LAND IN SLOVENIA In this study we performed a spatial analysis to determine the influence of selected environmental factors (forests share, elevation a.s.l., slope, aspect, flooding) and the management status of an area on the spatial distribution of the abandoned agricultural land (land use class 1410), and agricultural land with forest trees (land use class 1800) in Slovenia. All these natural factors influence the management decisions of landowners. We found out that land use class 1410 is distributed over the entire country, along the south-west to the north-east axis. The majority of the land use class 1800 can be found in the south-western part of the country. On municipality level, the link between the forest and studied land uses was weak. The percentage of land use classes 1410 and 1800 grows with increasing altitude a.s.l. and slope. Land use class 1400 is more frequent in flooded areas. The percentage of the abandoned agricultural land is greater in areas with special management status (e.g. Natura 2000 areas). A strict, professional and fast implementation of the Agricultural Land Act is needed to improve the situation and prevent further agricultural land losses. Key words: agricultural land; spatial analysis; abandonment; land use 1 UVOD Na svetovni ravni delež kmetijskih površin narašča in površin gozda upada (FAOSTAT, 2013). Razlog je predvsem v krčenju gozdov za potrebe kmetovanja. Rahla rast gozdnih površin je opazna v Evropi, Aziji in tudi ponekod v Severni Ameriki. Izguba svetovnih gozdnih površin v obdobju od leta 1990 do 2000 je bila 8,3 milijonov ha/leto in v obdobju od leta 2000 do 2010 5,2 milijona ha/leto (FAO, 2010). Gozd obsega preko 4 milijarde ha oziroma 31 % površine kopnega, na drugi strani kmetijska zemljišča skoraj 5 milijard ha oziroma 38 %. Primarni gozdovi (brez vpliva človeka) predstavljajo 36 % površine, sekundarni (antropogeno preoblikovani) 57 % in gozdne plantaže 7 % (FAO, 2010). Največ gozda, glede na površino, ima Južna Amerika (49 %) in najmanj Azija (19 %). Največ kmetijskih zemljišč po površini ima Azija (53 %) in najmanj Evropa (21 %) (FAOSTAT, 2013). Enako kot v Severni Ameriki in Evropi je tudi v Sloveniji opazen trend povečanja površin gozda in upadanja kmetijskih površin (Keenleyside in Tucker, 2010; FAO, 2010; ZGS, 2011; SURS-STAT, 2012; EU, 2013). Razlogi za zmanjšanje obsega kmetijskih površin so opuščanje in zaraščanje za kmetijstvo manj ugodnih 1 Univerza v Ljubljani, Biotehniška fakulteta, Jamnikarjeva 101, 1000 Ljubljana; Corresponding author: matjaž.glavan@bfum-lj.si Acta agriculturae Slovenica, 109 - 2, september 2017 str. 261 - 240 Matjaž GLAVAN in sod. zemljišč ter urbanizacija za kmetijstvo ugodnejših zemljišč (Cunder, 1998; Jankovič, 2003; Hladnik, 2005). V obdobju od leta 2002 do 2007 je bilo v povprečju urbaniziranih 11 ha kmetijskih zemljišč dnevno (Vrščaj, 2008). Stopnja prehranske samooskrbe se manjša, saj za zadovoljevanje potreb prebivalcev vedno več hrane uvažamo (Plut, 2011). Na državni ravni se s preoblikovanjem krajine in vse večjim uvažanjem hrane povečujeta izguba pridelovalnega potenciala tal in prebivalstva ter prehranska odvisnost od tujine. Zaraščanje je proces, ki nastane po prenehanju kmetijske obdelave (npr. paše, košnje, oranja) in poteka na opuščenih kmetijskih zemljiščih (FAO, 2006). Zemljišča so prepuščena naravnemu razvoju - sukcesiji. Sukcesija je spreminjanje združb organizmov v času kot posledica vpliva rastlinstva na določeno okolje. Sukcesijski proces je usmerjen, sprva v združbo z malo vrst, z medvrstnimi povezavami se razvije kompleksna (klimaksna, pedoklimaksna) združba, ki je značilna za neko okolje. Primarna sukcesija se odvija na območjih, kot so npr. ozemlja po umiku ledenika, ozemlja, prekrita z magmo, novonastali ognjeniški otoki. Sekundarna sukcesija se odvija na območjih po določeni motnji (požar, poplava), ki le delno odstrani rastlinstvo s prizadetega območja, ali na območjih, kjer je bila opuščena določena dejanska raba (kmetijska zemljišča, urbana zemljišča) (Wilfing, 1993; Krebs, 2001). Vzroke oziroma dejavnike, ki otežujejo in dražijo kmetijsko pridelavo ter privedejo do opuščanja kmetijskih zemljišč, delimo na naravno pridelovalne, strukturne, socio-ekonomske in agrarno politične in so običajno medsebojno prepleteni (Cunder, 1998). Naravno pridelovalni dejavniki, ki vplivajo na kmetijsko pridelavo, so naklon, nadmorska višina, usmerjenost zemljišč (ekspozicija površja), lastnosti tal in prisotnost drugih razredov rabe na območju. Strukturni vzroki izhajajo iz neugodne lastniške in posestne strukture, kot so velikost in oblika parcel, velikost kmetijskega gospodarstva, razdrobljenost parcel, njihova oddaljenost od lastnikov in dostopnost (Cunder, 1998; Gellrich in Zimmermann, 2007; Ilc, 2008; Corbelle Rico in sod., 2012; Zaragozi in sod., 2012). Socio-ekonomski vzroki so prepleteni in vezani na lastnosti kmetovalcev in družbene razmere, kamor prištevamo spremembe tržnih razmer, splošno nezanimanje za kmetovanje, možnost zaposlitve ob kmetovanju v neposredni bližini, selitev v urbane centre in staranje prebivalstva (MacDoland in sod., 2000; Khanal in Watanabe, 2006; Ilc, 2008, Diaz in sod., 2011). Med vzroke za opuščanje kmetovanja in spodbujanje zaraščanja sodijo tudi agrarnopolitične odločitve (plačilne pravice) ter finančna (neposredna plačila) in davčna politika (katastrski dohodek), ki vplivajo na stroške pridelave (Cunder, 1998; Khanal in Watanabe, 2006). Renwick in sod. (2013) ugotavljajo, da lahko v primeru opustitve neposrednih plačil in ukrepov za podporo trgom v okviru skupne kmetijske politike (SKP) odstotek opuščenih površin znotraj Evropske unije naraste za 8 %. Nezanemarljiv je tudi vpliv naravovarstvene in okoljevarstvene zakonodaje, ki lahko spodbuja kmetijsko pridelavo (npr. ohranjanje tradicionalne kmetijske krajine na območjih Natura 2000, košnja strmih travnikov) ali opuščanje (npr. vzpostavljanje naravne vegetacije za krepitev biodiverzitete) (MacDonald in sod., 2000; Diaz in sod., 2011). Zaraščanje opuščenih kmetijskih površin ima lahko mnogo posledic. Skupna biotska raznovrstnost se sprva zaradi naselitve pionirskih vrst poveča, a se ob vzpostavitvi kompleksnejših združb (gozd) ponovno zmanjša in ustali. Izjema je vzpostavitev monokultur, kot so nekatere invazivne vrste pri spontanem zaraščanju ali iglavci pri tvorbi plantaž (EU, 2013). Mozaičnost in heterogenost kulturne krajine se s povečevanjem površine gozda manjša, kar vpliva na okolje, življenje ljudi, turizem, dostopnost za rekreacijo ipd. (Pogačnik in sod., 1995; Jankovič, 2003; Pogačnik in sod., 2011). Večje homogene enote enega razreda dejanske rabe lahko vodijo v poenostavljeno gospodarjenje (MacDonald in sod., 2000). Opuščena in zaraščena zemljišča predstavljajo vir nadaljnjega širjenja gozdov in divjadi, a hkrati prispevajo tudi k povečanju biodiverzitete (EC, 1980; Keenleyside in Tucker, 2010; Renwick in sod., 2013), stabilizaciji terena, zmanjšanju erozije tal (EU, 2013) in povečanju sposobnosti zadrževanja vode, kot je podaljšana retenzija vode v času obilnih padavin (Poyatos in sod., 2003; Garcia Ruiz in Lana Renault, 2011). Preden se določi potrebne ukrepe za preprečevanje ali odpravljanje zaraščanja, je treba celostno analizirati vzroke opuščanja in zaraščanja kmetijskih zemljišč in na podlagi tega določiti ustrezne ukrepe za preprečevanje ali odpravljanje zaraščanja na nekem območju, če so tla primerna za kmetijsko obdelavo (Hočevar in sod., 2004; Pogačnik in sod., 2011). Če preprečevanje ali odpravljanje zaraščanja ni smiselno, se določi alternativno sedanji dejanski rabi zemljišča, ali se jih prepusti sukcesiji (Cunder, 1998; MacDonald in sod., 2000). Zaraščajoče se površine lahko vzpostavimo nazaj v kmetijske s pomočjo rekultivacije (Pogačnik in sod., 1995). Rekultivacija je odvisna od oddaljenosti, dostopnosti in oblike parcele ter pedogeoklimatskih dejavnikov (Corbelle Rico in sod., 2012). Poznamo več načinov rekultivacije, kot so požig zaraščene površine, ročno ali mehanično odstranjevanje lesne biomase in rekultiviranje s pomočjo živali (Vidrih in sod., 1996; Vodlan, 2006). Če preprečevanje in odpravljanje zaraščanja ni možno ali smiselno, je možno alternativno 262 Acta agriculturae Slovenica, 109 - 2, september 2017 Prostorska analiza kmetijskih zemljišč v zaraščanju v Sloveniji ekstenzivno kmetovanje v obliki manj intenzivne pridelave zelišč, dišavnic (npr. pridelava sivke na Krasu), različnih vrst jagodičja (npr. pridelava brinovih jagod na Krasu) (Cunder, 1998) ali drevesno-pašne dejanske rabe (Pogačnik in sod., 1995; Vidrih, 2010). V oddaljenih, težje dostopnih območjih lahko pogozdovanje služi za pridobivanje lesa, lov, rekreacijo ali kot naravovarstveno območje (Cunder, 1998; Cojzer, 2011; Corbelle Rico in sod., 2012). Pri oblikovanju kmetijske politike, ki bi preprečevala opuščanje kmetovanja, je potreben teritorialen in prostorski pristop (Renwick in sod., 2013). Zakonodaja se običajno razvija v smeri spodbujanja lokalnega gospodarstva (npr. združevanje kmetov, razvoj izdelkov z dodano vrednostjo), poenostavljanja spremembe lastništva, pravice do koriščenja zemljišča, milejše davčne politike, neposrednih plačil in pomoči pri odkupu pridelkov. V prispevku nameravamo s pomočjo javno dostopnih prostorskih podatkov ugotoviti prostorsko razporeditev kmetijskih zemljišč v zaraščanju in kmetijskih zemljišč, poraslih z gozdnim drevjem po slovenskih občinah in statističnih regijah. Ugotoviti smo želeli tudi, kakšen je vpliv izbranih naravnih dejavnikov (naklon, nadmorska višina, ekspozicija, poplavnost, delež gozda) ter statusa upravljanja območja (NATURA 2000, parki in rezervati, območje z omejeni dejavniki, vodovarstveno območje, območje življenjskega prostora rjavega medveda) na razporeditev izbranih razredov dejanskih rab. V analizo smo vključili podatke o dejanski rabi zemljišč, kot je bila zaznana v času nastajanj tega prispevka. 2 MATERIALI IN METODE 2.1 Prostorski sloji in opis območja raziskovanja Za potrebe prostorske analize smo v državnih ustanovah pridobili ustrezne prosto dostopne georeferencirane digitalne prostorske sloje (Preglednica 1, Slika 1). Preglednica 1: Nabor in vir georeferenciranih digitalnih podatkovnih slojev, uporabljenih v prostorskih analizah kmetijskih zemljišč v zaraščanju v Sloveniji Table 1: Dataset and source of georeferenced data used in spatial analysis of the abandonment of agricultural land in Slovenia Podatkovni prostorski sloj Vir podatka Dejanska raba zemljišč (RABA) Ministrstvo za kmetijstvo, gozdarstvo in prehrano (MKGP, 2011) Območja z omejenimi dejavniki (OMD) Državna meja Geodetska uprava Republike Slovenije (GURS, 2011) Meje občin Digitalni model višin 25mx25m (DMV) Ekološko pomembna območja (EPO) Območja Nature 2000 (NATURA) Zavarovana območja narave (ZO) Življenjski prostor rjavega medveda (ŽPRM) Poplavna območja (PO) Vodovarstvena območja (VVO) Agencija Republike Slovenije za okolje (ARSO, 2011) Meje statističnih regij Statistični urad Republike Slovenije (SURS, 2011) Po definiciji iz določanja razredov dejanske rabe je kmetijsko zemljišče v zaraščanju (v nadaljevanju: raba 1410) zemljišče, ki se zarašča zaradi opustitve kmetovanja ali preredke obdelave zemljišč, kar je lahko posledica neugodnih naravnih razmer za kmetovanje (naklon, osončenost, nadmorska višina, lastnosti tal, itd.) ali socio-ekonomskih razlogov (starost in izobraženost lastnikov, velikost kmetije, itd.) (Slika 1c) (MKGP, 2012). Na njem se pojavlja invazivno in trnasto rastlinje ter drevesa in grmičevje različnih starosti, katerih pokrovnost je 20-75 %. Drevesa so majhna, mlada in posamično razporejena. Neobdelanih vinogradov in drugih trajnih nasadov ne uvrščamo v ta razred, dokler so na letalskih posnetkih (ortofoto) vidne vrste (MKGP, 2012). Kmetijsko zemljišče, poraslo z gozdnim drevjem (v nadaljevanju: raba 1800) je površina, porasla s travinjem, na kateri rastejo posamična gozdna drevesa oziroma grmi in se redno, Acta agriculturae Slovenica, 109 - 2, september 2017 Matjaž GLAVAN in sod. vsaj enkrat letno, popase oziroma pokosi (Slika 1c). Rabo 1800 smo vključili v analizo, ker v primeru opuščanja kmetijske pridelave na teh zemljiščih predstavljajo izhodišče za zaraščanje. V primeru uveljavljanja neposrednih plačil so ta zemljišča uvrščena med kmetijska zemljišča. Pokrovnost travinja je vsaj 80 %, pokrovnost drevesnih krošenj oziroma grmov je manjša od 75 % (MKGP, 2012). V Sloveniji je bilo v letu 2011 25.278 ha rabe 1410, kar predstavlja 1,25 % celotne površine države. Zemljišč rabe 1800 je bilo 9.496 ha, kar predstavlja 0,47 % celotne površine države (MKGP, 2011). Zemljišča rabe 1410 so razporejena po celotni Sloveniji z glavnino v osi od jugozahoda proti severovzhodu, medtem ko je zemljišč rabe 1800 največ v jugozahodnem delu države. V razred dejanske rabe gozd (v nadaljevanju: raba 2000) spadajo zemljišča, porasla z gozdnim drevjem v obliki sestoja ali drugim gozdnim rastjem, ki zagotavlja katero koli funkcijo gozda. Med gozd se uvršča tudi zemljišča, poraščena z ruševjem (če je pokrovnost večja kot 75 %), površine, kjer je bila izvedena pomladitvena sečnja in so zato začasno brez drevja, poseke na trasah daljnovodov, cevovodov in smučarskih vlečnic, sanacije pogorišč, površine z mladimi drevesi, plantaže gozdnega drevja in gozdne drevesnice, ki so del gozda in jih na podlagi letalskih posnetkov (ortofoto) ne moremo posebej izločiti (MKGP, 2012). V razred gozd ne spadajo površine, poraščene z drevjem, kot so npr.: mestni parki in vrtovi (te se uvrščajo pod razred rabe 3000 -pozidano in sorodno zemljišče), posamične skupine dreves, ki imajo manjšo površino kot 2.500 m2 in rekreacijska območja. Jase v gozdu se izločijo kot kmetijska zemljišča, če so večje od 5.000 m2. Izločajo se tudi manjše površine, če so ta zemljišča v register kmetijskih gospodarstev vpisana kot GERK. Iz gozda so izločene ceste, ki so širše od 2 m in vodijo do naselij ali posameznih hiš (MKGP, 2012). Po evidenci MKGP je bilo v Sloveniji v času raziskave 1.210.010 ha gozdov, kar predstavlja 59,89 % površine države. Slika 1: Prostorska razporeditev: (a) občin in statističnih regij, (b) združenih razredov kmetijske dejanske rabe od 1100 do 1800, (c) kmetijskih zemljišč v zaraščanju (1410) in kmetijskih zemljišč poraslih z gozdnim drevjem (1800) - meje poligonov so odebeljene za boljšo vidljivost, (d) gozda (razred rabe 2000) v Sloveniji Figure 1: Spatial distribution of: (a) municipalities and statistical regions, (b) joined agricultural land use classes from1100 to 1800, (c) abandoned agricultural land use class (1410) and agricultural land use with forest trees class (1800) - polygon borders are made bold for better visibility, (d) forest (land use class 2000) in Slovenia 104 Acta agriculturae Slovenica, 109 - 2, september 2017 Prostorska analiza kmetijskih zemljišč v zaraščanju v Sloveniji Digitalni model višin (DMV) smo za potrebe analize razvrstili v 10 razredov nadmorskih višin oz. v 200 metrske višinske pasove (Slika 2a). Največji odstotek od celotne površine države predstavljajo višinski pas od 200,01 do 400,00 m (33,07 % površine države). Najmanjši odstotek od celotne površine države predstavljajo višinski pas od 1600,01 do 1800,00 m (0,88 % površine države). Razrede naklona smo razdelili po merilih za bonitiranje zemljišč na 8 razredov, kot jih določa Pravilnik o določanju in vodenju bonitete zemljišč (RS, 2008) (Slika 2b). Največji odstotek od celotne površine države predstavljajo območja z naklonom od 0 do 6 % (21,12 %). Najmanjši odstotek predstavljajo območja z naklonom, večjim od 65 % (7,29 %). Površino Slovenije smo razdelili na pet razredov ekspozicije površja (Slika 2c). V analizi smo uporabili prostorske podatke poplavnih območij (Slika 2d) redkih poplav z dogodki s povratno dobo deset (Q10) do dvajset (Q20) let in pogostih poplav z dogodki s povratno dobo dve (Q2) do pet (Q5) let. Območij redkih poplav je v Sloveniji 29.344 ha (1,45 % Slovenije), območij pogostih poplav je 6.422 ha (0,32 % Slovenije). Slika 2: Prostorska razporeditev razredov (a) nadmorske višine, (b) naklon, (c) ekspozicija površja in (d) pogostost poplavljanja v Sloveniji Figure 2: Spatial distribution of (a) altitude above sea level classes (m), (b) slope classes (%), (c) surface exposition (°), and (d) flood areas in Slovenia V analizo smo vključili območja petih izbranih upravljavskih statusov (Slika 3). Izbor je temeljil na: (1) območja so določena v zakonodaji; (2) območja so umeščena v prostor z jasnimi mejami; (3) režim upravljanja območij vpliva na kmetijsko dejavnost in odločanje lastnikov o vrsti dejanske rabe zemljišč. Ta območja so: (a) NATURA 2000; (b) zavarovana območja; (c) območja z omejenimi dejavniki za kmetijsko dejavnost; (d) vodovarstvena območja in (e) območje življenjskega prostora rjavega medveda. Območja NATURA 2000 so bila potrjena z Uredbo o posebnih varstvenih območjih (RS, 2004a) in jih delimo na območja, določena na podlagi direktive o habitatih -SCI (79/409/EGS) in območja, določena na podlagi direktive o pticah - SPA (92/43/EGS) (Slika 3a). Prostorski podatkovni sloj zavarovanih območij narave prikazuje območja, ki so zavarovana po različnih predpisih o ohranjanju narave (Slika 3b). Zavarovana območja se po Zakonu o ohranjanju narave delijo na ožja zavarovana območja (naravni spomenik, strogi naravni rezervat in naravni rezervat) in širša zavarovana območja (narodni, regijski in krajinski park) (RS, 2004b). Acta agriculturae Slovenica, 109 - 2, september 2017 Matjaž GLAVAN in sod. Slika 3: Prostorska razporeditev: (a) območij NATURA 2000, (b) območij nacionalnih, regijskih ali krajinskih parkov, (c) območij z omejenimi dejavniki za kmetijsko dejavnost, (d) vodovarstvenih območij in (e) območij življenjskega prostora rjavega medveda v Sloveniji Figure 3: Spatial distribution of (a) NATURA 2000 areas, (b) national, regional or landscape parks areas, (c) naturally less favoured areas for agricultural activities, (d) water protection areas, and (e) areas of life environment of brown bear in Sloveni Leta 2011 je bilo 286 območij Natura 2000 s skupno površino 720.286 ha, kar je predstavljalo 35,53 % površine države (Slika 3a). Prostorski podatkovni sloj zavarovanih območij narave obsega 252.929 ha (12,48 % Slovenije) in prikazuje območja, ki so zavarovana po različnih predpisih o ohranjanju narave (Slika 3b). Območja z omejenimi dejavniki za kmetijsko dejavnost (OMD) obsegajo 1.751.251 ha (86,38 %) in se v prostorskem sloju delijo na hribovska gorska območja v obsegu 1.467.573 ha (72,39 %), druga območja v obsegu 81.196 ha (4,00 %) in območja s posebnimi omejitvami v obsegu 202.482 ha (9,99 %) (RS, 2015) (Slika 3c). Vodovarstvena območja (VVO) v Sloveniji obsegajo 350.202 ha (17,27 %) in se glede na režim varovanja za zaščito vodnih virov delijo na državni nivo v obsegu 129.261 ha (6,38 %), občinski nivo v obsegu 216.160 ha (10,66 %) in vrelčni nivo v obsegu 4.781 ha (0,24 %) (Slika 3d). Območje življenjskega prostora rjavega medveda se deli na štiri dele (Slika 3e). Osrednje območje obsega 347.625 ha 266 Acta agriculturae Slovenica, 109 - 2, september 2017 Prostorska analiza kmetijskih zemljišč v zaraščanju v Sloveniji (17,32 %), od tega predstavlja 305.972 ha (15,25 %) območje manjše naseljenosti ljudi in 41.653 ha (2,08 %) območje gostejše naseljenosti ljudi. Robno območje obsega 255.662 ha (12,74 %), prehodno 308.523 ha (15,37 %), območje izjemne (redke) prisotnosti medveda pa obsega 1.095.003 ha (54,56 %) (Slika 3e). Pravilnik o razvrstitvi kmetijskih gospodarstev v območja z omejenimi možnostmi za kmetijsko dejavnost (OMD) določa tri vrste območij (RS, 2015) (Slika 3c). V hribovska gorska območja (HGO) uvrščamo območja, kjer je pridelava na zemljiščih omejena zaradi nadmorske višine in/ali naklona zemljišč na manjši nadmorski višini. Za druga območja z omejenimi dejavniki (DO) so značilne tako neugodne naravne razmere (geologija, tla, konfiguracija terena, parcelna struktura) kot tudi socio-ekonomska in demografska struktura (odseljevanje, negativni prirast, brezposelnost, velikost kmetij). V območja s posebnimi omejitvami (PO) prištevamo območja, kjer se kmetovanje nadaljuje, da bi zagotovili ohranjanje ali izboljšanje okolja, ohranjanje življenjskega prostora na podeželju in varovanje turističnega potenciala območja. VVO območja se glede na režim varovanja za zaščito vodnih virov delijo na državni, občinski in vrelčni nivo (Slika 3e). Nivoji se delijo še na kategorije varstvenih režimov (od 0 do 5). Najstrožji režim varovanja je v kategorijah 0, 1 in 2. V 3. in 4. kategoriji je režim blažji. Kategorija 5 zajema le vrelčni nivo vodovarstvenih območij. Vodovarstvena območja državnega nivoja se določajo glede na Pravilnik o kriterijih za določitev vodovarstvenih območij (RS, 2004c), vodovarstvena območja občinskega in vrelčnega nivoja se določajo z odloki in medobčinskimi uradnimi vestniki s pripadajočimi strokovnimi podlagami. Kategorija varstvenega režima vpliva predvsem na način gnojenja kmetijskih zemljišč in uporabe fitofarmacevtskih sredstev (strožji režim - manjša uporaba gnojil in sredstev za varstvo rastlin), kar se odraža v obsegu pridelka in prihodkov. Območje življenjskega prostora rjavega medveda se deli na pet delov: (a) osrednje območje - manjša naseljenost ljudi; (b) osrednje območje - območje gostejše naseljenosti ljudi; (c) robno območje; (d) prehodno in (e) območje izjemne (redke) prisotnosti medveda. Območja oz. cone opredeljuje Strategija upravljanja z rjavim medvedom v Sloveniji iz leta 2002, ki je vodilo za načrtovanje akcijskega načrta, za upravljanje z rjavim medvedom (MOP, 2002, 2007). 2.2 Metodologija prostorske analize Prostorska analiza je bila opravljena z geografskim informacijskim sistemom Esri ArcGis 10.0. Iz podatkovnega sloja RABA smo za nadaljnjo uporabo pripravili sloje rabe 1410, rabe 1800 in rabe 2000. Urejanju površin novih slojev v atributnih tabelah je sledil njihov uvoz v MS Office Excel, kjer je sledila statistična analiza. Za ugotavljanje odvisnosti pojavljanja rabe 1410 in rabe 1800 od izbranih naravnih danosti (nadmorska višina in naklon) sta bila uporabljena regresijski model in Pearsonov koeficient korelacije (povezanost odstotka rabe 1410 in rabe 1800 z rabo 2000 na območju posameznih občin). Pri preučevanju razširjenosti rabe 1410 in rabe 1800 so bili uporabljeni odstotki določene rabe glede na celotno površino kmetijskih zemljišč v izbranem razredu nadmorske višine oz. naklona. Za ugotavljanje povezave med rabo 1410 in rabo 1800 ter rabo 2000 so bili uporabljeni odstotki rabe od površin občin. 3 REZULTATI IN DISKUSIJA 3.1 Občine in statistične regije Zemljišča z rabo 1410 so razporejena po celi Sloveniji z glavnino v osi od jugozahoda proti severovzhodu. Glede na delež površine regije je raba 1410 najbolj razširjena v obalno-kraški statistični regiji in pomurski statistični regiji. Glavnina kmetijskih zemljišč rabe 1800 je v jugozahodnem delu države (obalno-kraška, goriška in notranjsko-kraška statistična regija) (Preglednica 2). Acta agriculturae Slovenica, 109 - 2, september 2017 Matjaž GLAVAN in sod. Preglednica 2: Površina (ha) in odstotek (%) zemljišč rabe 1410 (kmetijsko zemljišče v zaraščanju) in 1800 (kmetijsko zemljišče poraslo z gozdnim drevjem) glede na celotno površino in površino kmetijskih zemljišč (KZ) po statističnih regijah Slovenije Table 2: Area (ha) and share (%) of abandoned agricultural land class (1410) and agricultural land use with forest trees class (1800) in comparison to total and agricultural land area (KZ) in statistical regions in Slovenia Statistična regija Površina območje KZ Raba 1410 Raba 1800 ha ha ha % regije % KZ ha % regije % KZ Gorenjska 213.660 43.312 1.360 0,64 3,14 704 0,33 1,63 Goriška 232.550 52.000 3.613 1,55 6,95 1.734 0,75 3,33 Jugovzhodna Slovenija 267.509 69.891 2.842 1,06 4,07 679 0,25 0,97 Koroška 104.080 24.351 737 0,71 3,03 205 0,20 0,84 Notranj sko -kraška 145.634 37.685 1.275 0,88 3,38 1.789 1,23 4,75 Obalno-kraška 104.445 36.083 3.072 2,94 8,51 3.270 3,13 9,06 Osrednjeslovenska 255.496 80.372 2.570 1,01 3,20 513 0,20 0,64 Podravska 216.967 107.897 3.550 1,64 3,29 162 0,07 0,15 Pomurska 133.753 81.425 3.126 2,34 3,84 34 0,03 0,04 Savinjska 238.398 86.021 1.800 0,76 2,09 294 0,12 0,34 Spodnjeposavska 88.514 39.145 1.112 1,26 2,84 54 0,06 0,14 Zasavska 26.375 7.602 222 0,84 2,91 57 0,22 0,75 Skupaj 2.027.380 665.782 25.278 1,25 3,80 9.496 0,47 1,43 Največ rabe 1410 je v delih regij (Kras, Brkini, Barje, Haloze, Slovenske gorice, Goričko), kjer ob sočasnih spremembah razmer na trgu dela in na kmetijskem trgu prihaja do neugodnih socio-ekonomskih razmer. Tudi v analizi ukrepov upravljanja progama Natura 2000 za sektor kmetijstva so kot enega pomembnih razlogov za zaraščanje in zmanjševanje biotske pestrosti navedli socio-ekonomske vidike (starost, bolezen, ni naslednika) (Žvikart in sod., 2013). Ob tem se kmetijstvo ni zmožno dovolj hitro prilagoditi npr. z lastniško strukturo ali velikostjo parcel. Pogosto je omejujoč faktor naravna danost (naklon, tla), zato je v takih razmerah edina možnost ekstenzifikacija pridelave. Obalno-kraška statistična regija predstavlja izredno priložnost za krepitev drevesno-pašnega sistema na že obstoječih kraških gmajnah in za njegovo uvedbo na širšem območju, po vzoru savan in dehes. O možnosti ekstenzivnega koriščenja manj zanimivih kmetijskih zemljišč ter njegovih prednostih ob omejenih naravnih danostih za intenzivno kmetijsko pridelavo piše več avtorjev (Knap, 2008; Eler in sod., 2008; Vidrih, 2010). V regijah in občinah, kjer je zaraščanja manj, bi bilo na podlagi sosednje vrste dejanske rabe in lastnosti zemljišč treba razmisliti, v katero smer razvijati nadaljnjo dejansko rabo teh zemljišč. Ena od možnosti je sečnja lesne biomase iz zaraščenih območij in predelava v sekance ali pelete (Vodlan, 2006; Humar, 2008). 3.2 Naravni dejavniki Statistična analiza je pokazala, da imajo glede na površino največji odstotek rabe 2000 občine Osilnica (3.191 ha, 88,11 %), Črna na Koroškem (13.605 ha, 87,23 %), Dolenjske Toplice (9.459 ha, 85,83 %), Kostel (4.769 ha, 85,03 %) in Lovrenc na Pohorju (7.160 ha, 84,80 %). Zemljišča rabe 1410 in 1800 v teh občinah predstavljajo manj kot 1 % površine občine. S pomočjo Pearsonovega koeficienta korelacije smo ugotavljali povezanost deležev rabe 1400 in rabe 1800 z rabo 2000 (Slika 4). Med rabama 1410 in 2000 (Pearsonov koeficient korelacije: - 0,3) in med rabama 1800 in 2000 (Pearsonov koeficient korelacije: 0,26) ni opaznih izrazitih vzorcev (Slika 4). Občine na JZ države z večjim odstotkom gozda imajo večji odstotek rabe 1800. Z izvajanjem kmetijske dejavnosti (paša, občasna sečnja in mulčenje) se slednja počasneje ali sploh ne zaraščajo, že obstoječa starejša drevesa rastejo naprej, medtem ko se podrast stalno odstranjuje. Vendar vzdrževanja rabe 1800 ni možno izvajati strojno, zato so pašne živali nujno potrebne. Vzreja pašnih živali zahteva stalno fizično prisotnost pastirja ali dobro varovano ogrado, ki preprečuje dostop divjim zverem, ki na območje rabe 1800 dostopajo iz sosednjih gozdov (Černe in sod., 2010). Prav škodni dogodki kot posledica napada divjih živali (volkovi, medvedi) so glavni razlog opuščanja pašništva na območju obalno-kraške in notranjsko-kraške regije (Pogačnik in sod., 2006). 268 Acta agriculturae Slovenica, 109 - 2, september 2017 Prostorska analiza kmetijskih zemljišč v zaraščanju v Sloveniji 8 - 8 b) 7 - ♦ 7 6 - 6 E o ♦ S 35 - ♦ o OO 5 -O ♦ ♦ # i> 0 ♦ !S>4 ♦ ♦ "i ♦ * ♦ f ■b - ♦♦ ♦ * * ♦ >N 3 ♦ o 2 - ♦ V ♦ ♦ * . * ♦ * \: ♦ % 9» O 2 1 - 1 ♦ ♦ ♦ . % ♦ t % * ♦ * : v* ❖ 0 10 20 30 40 SO 60 70 80 90 100 10 20 30 40 50 60 70 80 90 100 Delež zemljišč rabe 2000 (%) Delež zemljišč rabe 2000 (%) Slika 4: Razsevni grafikon predstavlja povezanost razredov dejanske rabe (a) kmetijska zemljišča v zaraščanju (1410) in (b) kmetijska zemljišča porasla z gozdnim drevjem (1800) z odstotkom gozdne rabe (2000) po občinah Figure 4: Scattered chart presenting connections between classes of actual land use (a) abandoned agricultural land (1410) and (b) agricultural land with forest trees class (1800) and forest (2000) by municipality Slika 5: Primerjava deležev razredov dejanske rabe kmetijsko zemljišče v zaraščanju (1410) in kmetijsko zemljišče poraslo z gozdnim drevjem (1800) v odvisnosti od (a) nadmorske višine in (b) naklon po regresijskem modelu Figure 5: Comparison between shares of actual land use classes of abandoned agricultural land (1410) and agricultural land with forest trees (1800) in dependence from (a) altitude above sea level and (b) slope by regression model Ugotavljali smo, kako se z nadmorsko višino in naklonom spreminja delež posameznih razredov rabe 1410 in rabe 1800. Uporabili smo regresijski model (Slika 5). Opazovan je bil odstotek rabe 1410 in 1800 od vseh in samo od kmetijskih zemljišč. V primeru upoštevanja celotne površine območja posameznega višinskega pasu se z večanjem nadmorske višine in naklona odstotek kmetijskih površin in s tem tudi rabe 1410 in 1800 zmanjšuje (Preglednica 3). V povprečju se odstotek zemljišč rabe 1410 in 1800, glede na odstotek kmetijskih zemljišč, na vsakih 100 m nadmorske višine poveča za 0,29 % in 0,59 %. V povprečju se odstotek zemljišč rabe 1410 in 1800, s povečanjem naklona za 10 %, poveča za 1,5 % in 0,8 %. Rezultati naše analize kažejo, da imata nadmorska višina in naklona vpliv na zaraščanje in se ujema z ugotovitvami drugih raziskav (Poyatos in sod., 2003; Hočevar in sod., 2004; Gellrich in Zimmermann, 2007, Ketiš in sod., 2014). Rezultat analize vpliva nadmorske višine in naklona nakazuje na povečanje površin rabe 1410 in 1800 v primeru opuščanja kmetijske obdelave v obliki paše ali košnje strmih travnikov v hribovitih predelih Slovenije. Še posebej izrazit je pojav v hribovsko-gorskem okolju, kjer opažajo upad populacije gozdnih kur zaradi zaraščanja gozdih jas, gorskih pašnikov in gozdnih robov, ki zaradi oddaljenosti niso več zanimivi za obdelavo (Kus Veenvliet, 2012). Kmetijska politika se zaveda vpliva opuščanja kmetijskih zemljišč, zato v okviru Programa razvoja podeželja Republika Slovenija Acta agriculturae Slovenica, 109 - 2, september 2017 Matjaž GLAVAN in sod. (2014-2020) namenja neposredna plačila za košnjo habitatnih travnikov na naklonih, večjih od 50 %. Iz podatkov (Preglednica 3) je razvidno, da so severne in zahodne osojne lege manj ugodne za kmetovanje, kar privede do opuščanja in vodi v zaraščanje. Delno na večji odstotek zaraščanja vpliva tudi relief in smer poteka gorskih grebenov v Sloveniji, ki marsikje potekajo od severozahoda protu jugovzhodu in so na severnih in zahodnih legah bolj strmi ter s tem manj primerni za kmetijstvo. Zemljišča zahodnih in južnih leg na manjših nadmorskih višinah do 400 metrov imajo dober potencial za nadaljnjo, tudi bolj intenzivno kmetijsko pridelavo, kot so vinogradništvo in sadjarstvo ter oljkarstvo v Slovenski Istri in Goriških Brdih. Rabe 1800 se večinoma pojavljajo v jugozahodni Sloveniji, na območjih apnenčastih, dolomitnih in flišnih kamnin. Brez prisotnosti človekovega delovanja bi jih prekrival gozd s ekonomsko manj zanimivimi termofilnimi vrstami, ki so prilagojene tudi sušnim razmeram. Kmetovalci so jih zato izkoristili kot površine s kombiniranim gozdno-pašnim sistemov. Če teh površin ne vzdržujemo z redno pašo, se lahko hitro zarastejo z grmovno zarastjo in postanejo neprehodne. Večina teh površin je z obstoječo pašno rabo dosega svoj maksimalen izkoristek (Vidrih, 2005). Površin rabe 1400 je glede na odstotek razreda in odstotek kmetijskih zemljišč več na območjih pogostih kot redkih poplav (Preglednica 3). Analiza je pokazala večji vpliv poplavnih območij na rabo 1410 kot na rabo 1800. Manjši vpliv na rabo 1800 izhaja iz dejstva, da je večina te rabe na specifičnih lokacijah, kjer se zaradi matične podlage (apnenec) in topografije (naklon) poplave ne pojavljajo. To potrjuje tudi majhen obseg (48 ha) teh zemljišč na poplavnih območjih. Poplavljanje posredno vpliva na zaraščanje kmetijskih zemljišč, saj lahko redne poplave otežujejo proces kmetovanja in zmanjšujejo pridelek. To lahko na mokrotnih travnikih in mokriščih (Cerkniško polje, Ljubljansko barje), privede do zmanjšanja rastlinske in živalske pestrosti, ki se najbolj očitno odrazi v številu ptic vlažnih travnikov (Žgavec in sod., 2013; Bordjan in Bordjan, 2014). ?70 270 Acta agriculturae Slovenica, 109 - 2, september 2017 Prostorska analiza kmetijskih zemljišč v zaraščanju v Sloveniji Preglednica 3: Površine (ha) in odstotek (%) rab kmetijsko zemljišče v zaraščanju (1410) in kmetijsko zemljišče poraslo z gozdnim drevjem (1800) v različnih razredih (a) nadmorskih višin (m), (b) naklon (%), (c) ekspozicije površja (°) in (d) pogostosti poplav glede na površino kmetijskih zemljišč (KZ) Table 3: Area (ha) and share (%) of abandoned agricultural land use class (1410) and agricultural land use with forest trees class (1800) in different classes of (a) altitude above sea level classes (m), (b) slope classes (%), (c) solar radiation exposition (°), and (d) flood areas in comparison to total area of agricultural land use (KZ) Površina območje KZ raba 1410 raba 1800 ha ha ha % razreda % KZ ha % razreda % KZ (a) Razredi nadmorske višine (m) 0 - 200 180.280 103.525 3.803 2,11 3,67 347 0,19 0,33 200,01 - 400 670.932 319.051 11.127 1,66 3,49 1.923 0,29 0,60 400,01 - 600 472.631 134.435 4.841 1,02 3,60 2.620 0,55 1,95 600,01 - 800 308.479 62.029 2.554 0,83 4,12 1.767 0,57 2,85 800,01 - 1000 168.112 24.142 1.312 0,78 5,43 1.169 0,70 4,84 1000,01 - 1200 95.741 7.621 659 0,69 8,64 472 0,49 6,20 1200,01 - 1400 61.972 4.298 444 0,72 10,34 325 0,52 7,56 1400,01 - 1600 31.449 5.047 337 1,07 6,69 531 1,69 10,52 1600,01 - 1800 17.768 3.235 179 1,01 5,53 221 1,24 6,83 1800,01 > 19.927 2.302 59 0,30 2,58 120 0,60 5,22 Skupaj 2.027.293 665.685 25.316 1,25 3,80 9.495 0,47 1,43 (b) Naklon (%) 0 - 6 428.089 265.174 6.947 1,62 2,62 1.665 0,39 0,63 6,01 - 11 191.912 83.824 2.660 1,39 3,17 1.448 0,75 1,73 11,01 - 17 228.292 89.362 2.989 1,31 3,35 1.432 0,63 1,60 17,01 - 24 247.958 82.288 3.254 1,31 3,95 1.333 0,54 1,62 24,01 - 34 300.696 77.089 3.770 1,25 4,89 1.374 0,46 1,78 34,01 - 50 315.478 49.780 3.501 1,11 7,03 1.218 0,39 2,45 50,01 - 65 167.137 11.315 1.299 0,78 11,48 526 0,31 4,65 65,01 > 147.731 6.852 896 0,61 13,07 498 0,34 7,27 Skupaj 2.027.293 665.685 25.316 1,25 3,80 9.495 0,47 1,43 (c) Razredi ekspozicije površja (°) Sever 436.290 96.624 4.116 0,94 4,26 1.382 0,32 1,43 Vzhod 483.991 153.663 5.800 1,20 3,77 1.925 0,40 1,25 Jug 532.092 198.361 7.918 1,49 3,99 3.657 0,69 1,84 Zahod 445.154 126.822 5.445 1,22 4,29 2.368 0,53 1,87 Ravnina 129.766 90.214 2.037 1,57 2,26 163 0,13 0,18 Skupaj 2.027.293 665.685 25.316 1,25 3,80 9.495 0,47 1,43 (d) Razredi pogostosti poplav Redke poplave 29.344 20.496 803 2,74 3,92 44 0,15 0,21 Pogoste poplave 6.422 4.984 209 3,25 4,19 4 0,06 0,07 Skupaj 35.766 25.480 1.012 2,83 3,97 48 0,13 0,19 Opomba: Zaradi uporabe rastrskih slojev v izračunu je skupna površina Slovenije nekoliko manjša. 3.3 Dejavniki upravljanja območij Poudariti je treba, da je vpliv upravljavskega statusa območja na zaraščanje težje ovrednotiti, saj se različni statusi prostorsko prekrivajo. Iz analize je razvidno, da sta odstotka površin rabe 1410 in rabe 1800 glede na površino območja različno zastopana znotraj in zunaj območij. Glede na odstotek kmetijskih zemljišč na območjih je površina rab 1410 in 1800 večja znotraj kot zunaj obravnavanih območij (Preglednica 4). Upravljavski status območja lahko vpliva tako na zaraščanje kot na preprečevanje slednjega. Nekatera območja Natura 2000 so opredeljena kot kmetijske krajine. Različne raziskave potrjujejo, da opuščanje kmetijske dejavnosti negativno vpliva na populacije Acta agriculturae Slovenica, 109 - 2, september 2017 Matjaž GLAVAN in sod. vrst, ki so od nje odvisne (MacDonald in sod., 2000; Kraj in Koren, 2011; Miličic in sod., 2011; Žgavec in sod., 2013; Žvikart in sod., 2013; Bordjan in Bordjan, 2014). Vpliv upravljavskega statusa območja ni omejen le na območje izvajanja, temveč tudi na njegovo neposredno okolico. Diaz in sodelavci (2011) ugotavljajo, da je zaraščanja več v bližini narodnih parkov, kar je posledica vzpostavljanja prvotne vegetacije. Območja Nature 2000 in različne oblike parkov lahko z režimi upravljanja ali kmetijsko-okoljsko podnebnih plačil vplivajo na intenzivnost obdelave (npr. čas ali število košenj) in način kmetovanja (konvencionalno, integrirano, ekološko) (Žgavec in sod., 2013; Žvikart in sod., 2013). Zemljišča so lahko zaradi omejitev in okoljskih ukrepov manj produktivna in stroški vloženega dela in materiala večji, kar lahko privede do opuščanja kmetijske dejavnosti in posledično do zaraščanja (Glavan in Pintar, 2013). Območja Nature 2000 in parkov lahko ob primernem upravljanju pripomorejo k varovanju zaščitenega tipa kraj ine in tako vplivajo na preprečevanje zaraščanja kmetijskih zemljišč, a je za to potrebna kmetijska obdelava in pridelava (Knap, 2008; Gutman, 2011; Pogačnik, 2011; Miličic, 2011; Kus Veenvliet, 2012; Žgavec in sod., 2013). Na varovanih območjih, ki ležijo na izjemno dobrih tleh in legah, je opaziti trend intenziviranja obdelave in odpravljanja grmovne in drevesne zarasti, kar ima lahko kvarne posledice za biodiverziteto (Žgavec in sod., 2013; Žvikart in sod., 2013; Glavan in sod., 2015). Le vključevanje in izobraževanje kmetov ter sprememba odnosa nacionalne okoljske politike do kmetov bosta omogočila varovanje habitatov kulturne krajine. Preglednica 4: Površine (ha) in delež (%) razredov dejanske rabe (a) kmetijsko zemljišče v zaraščanju (1410) in (b) kmetijsko zemljišče poraslo z gozdnim drevjem (1800) na območjih z in brez izbranega upravljavskega statusa ter glede na površino območja in kmetijskih zemljišč (KZ) Table 4: Area (ha) and share (%) of actual land use classes (a) abandoned agricultural land (1410) and (b) agricultural land with forest trees (1800) in the areas with and without selected management status in comparison to total area and area of agricultural land (KZ) Površina Območje območje KZ Z upravljavskim statusom Brez upravljavskega statusa ha ha ha % območja % KZ ha % območja % KZ (a) Raba 1410 Natura 2000 720.286 158.011 9.478 1,32 6,00 15.800 1,21 3,11 Območja z omejenimi dejavniki 1.751.251 506.082 21.387 1,22 4,23 3.891 1,41 2,44 Življenjski prostor medveda* 2.006.813 665.588 24.884 1,24 3,74 - - - Narodni park 83.808 8.913 731 0,88 8,25 24.543 1,26 3,74 Regijski parki 43.442 16.113 509 1,17 3,16 24.770 1,25 3,81 Krajinski parki 117.004 47.708 2.534 2,17 5,31 22.744 1,19 3,68 Naravni rezervati 8.675 1.132 95 1,1 8,42 25.183 1,25 3,79 Vodovarstvena območja 350.202 106.954 4.776 1,36 4,47 20.502 1,22 3,67 (b) Raba 1S00 Natura 2000 720.286 158.011 6.065 0,84 3,84 3.430 0,26 0,68 Območja z omejenimi dejavniki 1.751.251 506.082 9.380 0,54 1,85 116 0,04 0,07 Življenjski prostor medveda* 2.006.813 665.588 9.403 0,47 1,41 - - - Narodni park 83.808 8.913 428 0,51 4,81 9.067 0,47 1,38 Regijski parki 43.442 16.113 143 0,33 0,89 9.352 0,47 1,44 Krajinski parki 117.004 47.708 236 0,20 0,49 9.260 0,48 1,50 Naravni rezervati 8.675 1.132 22 0,25 1,92 9.474 0,47 1,43 Vodovarstvena območja 350.202 106.954 3.462 0,99 3,24 6.033 0,36 1,08 * Življenjski prostor rjavega medveda je določen na celotnem ozemlju Republike Slovenije. 979 272 Acta agriculturae Slovenica, 109 - 2, september 2017 Prostorska analiza kmetijskih zemljišč v zaraščanju v Sloveniji Prisotnost rjavega medveda in njegovo povzročanje gospodarske škode lahko otežujeta rejo živali na prostem, predvsem rejo drobnice. Rjavi medved je zelo domač v zaraščajočih in opuščenih kmetijskih površinah, ki se marsikje zaradi intenzivnega zaraščanja širijo do urbanih naselij in kmetijskih površin v okolici (Jarni, 2011). Zato je akcijski načrt za upravljanje z rjavim medvedom predvidel intenzivno čiščenje zaraščajočih površin v okolici naselij (MOP, 2007). Prav gotovo bo morala kmetijska politika slediti zavezam, zapisanim v zakonu o kmetijskih zemljiščih, in nameniti finančna sredstva za odpravljanje zaraščanja. Največji delež zemljišč rabe 1410 je glede na celotno površino OMD na območjih PO in glede na površino kmetijskih zemljišč na območjih DO (Preglednica 5). Odstotek zemljišč rabe 1800 je glede na površino območja največji na območjih s posebnimi omejitvami in glede na površino kmetijskih zemljišč na hribovskih gorskih območjih (Preglednica 5). Največje površine in velik odstotek zaraščanja na HGO so posledica tega, da ta območja obsegajo skoraj 75% države in zemljišča na večjih nadmorskih višinah ter večjih naklonih. Slednja naravna dejavnika imata izrazit vpliv na odločanje lastnikov zemljišč o obdelavi (Slika 5). Tipi OMD so glede na odstotke med seboj dokaj izenačeni. Neposredna plačila za OMD so bila uvedena z namenom blaženja vplivov neugodnih razmer za kmetovanje (nadmorska višina, naklon, kraška skalovitost in razgibanost terena ter poplavnih območij), s čimer se preprečuje opuščanje kmetovanja. S sistemom točkovanja, ki ga je pripravil Kmetijski inštitut Slovenije, je to prostorsko in analitsko en od najnatančneje dodelanih ukrepov skupne kmetijske politike v Sloveniji. Pri določanju zunanjih mej območij se lahko poleg naravnih danosti upošteva še demografske razmere (odseljevanje, negativni prirast, brezposelnost, velikost kmetij). Kljub temu, da se ob predstavitvi ukrepa OMD pogosto navaja ugodne vplive na preprečevanje zaraščanja, so v strokovni literaturi analize vpliva ukrepa na nivoju kmetij redke. Obstoječi študiji, ki obravnavata kmetijsko-okoljske ukrepe v okviru Programa razvoja podeželja (PRP) navajata, da na intenziteto obdelave in sestavo travne ruše močno vplivajo socio-ekonomski dejavniki, kot sta starost in izobrazba kmeta ter premajhna finančna vrednost neposrednih plačil, ki ne spodbujajo k vključitvi v ukrepe (Žgavec in sod., 2013; Ketiš in sod., 2016). Lastniki oz. obdelovalci zemljišč se za vključitev v OMD-ukrepe odločajo prostovoljno in za vsako parcelo pred uveljavljanjem ukrepa pretehtajo ekonomsko upravičenost obdelave. Razlogi za opuščanje ali nadaljevanje obdelave ne vključujejo le neposrednih plačil, temveč tudi pridelek na parceli, stroške, nastale z obdelavo, čas, potreben za vzdrževanje, dostopnost, možnost strojne obdelave. Če le en od teh dejavnikov ne izkazuje pozitivnega izida, lahko to vodi v opustitev obdelave in zaraščanje parcele. Acta agriculturae Slovenica, 109 - 2, september 2017 Matjaž GLAVAN in sod. Preglednica 5: Površine (ha) in odstotek (%) zemljišč dejanske rabe kmetijsko zemljišče v zaraščanju (1410) in kmetijsko zemljišče poraslo z gozdnim drevjem (1800) znotraj območij (a) Natura 2000, (b) varovanih narodnih, regijskih in krajinskih parkov ter naravnih rezervatov, (c) življenjskega prostora rjavega medveda, (d) z omejenimi dejavniki in (e) vodovarstvenih območij glede na površino območja in kmetijskih zemljišč (KZ) Table 5: Area (ha) and share (%) of actual land use abandoned agricultural land (1410) and agricultural land with forest trees (1800) within the areas of (a) Natura 2000, (b) national, regional or landscape parks, (c) life environment of brown bear, (d) naturally less favoured areas for agricultural activities and (e) water protection areas in comparison to total area and area of agricultural land (KZ) Površina Območja območja KZ raba 1400 raba 1800 % območja % območja ha ha ha % KZ ha % KZ (a) Natura 2000 Območje, pomembno za skupnost (SCI) 257.082 51.265 2.788 1,08 5,44 1.000 0,39 1,95 Posebno območje varstva (SPA) 82.178 25.440 883 1,07 3,47 318 0,39 1,25 Kombinacija SCI - SPA 381.026 81.306 5.807 1,52 7,14 4.747 1,25 5,84 Skupaj 720.286 158.011 9.478 1,32 6 6.065 0,84 3,84 (b) Varovana območja narodnih, regijskih in krajinskih parkov ter naravnih rezervatov Narodni park 83.808 8.913 736 0,88 8,25 428 0,51 4,81 Regijski parki 43.442 16.113 509 1,17 3,16 143 0,33 0,89 Krajinski parki 117.004 47.708 2.534 2,17 5,31 236 0,20 0,49 Naravni rezervati 8.675 1.132 95 1,10 8,42 23 0,25 1,92 Skupaj 252.929 73.866 3.874 1,53 5,24 830 0,33 1,12 (c) Življenjski prostor rjavega medveda Območje izjemne/redke prisotnosti 1.095.003 465.454 15.759 1,44 3,39 3.891 0,36 0,84 Osrednje območje 347.625 65.635 3.030 0,87 4,62 2.439 0,70 3,72 - Območje gostejše naseljenosti ljudi 41.653 18.931 550 1,32 2,90 129 0,31 0,68 - Območje manjše naseljenosti ljudi 305.972 46.704 2.481 0,81 5,31 2.310 0,76 4,95 Prehodno območje 308.523 55.082 2.431 0,79 4,41 1.304 0,42 2,37 Robno območje 255.662 79.418 3.664 1,43 4,61 1.770 0,69 2,23 Skupaj 2.006.813 665.588 24.884 1,24 3,74 9.403 0,47 1,41 (d) Območja z omejenimi dejavniki (OMD) DO - druga območja 81.196 24.387 1.144 1,41 4,69 114 0,14 0,47 HGO - hribovska gorska območja 1.467.573 375.745 16.305 1,11 4,34 7.599 0,52 2,02 PO - območja s posebnimi omejitvami 202.482 105.950 3.938 1,94 3,72 1.668 0,82 1,57 Skupaj 1.751.251 506.082 21.387 1,22 4,23 9.380 0,54 1,85 (e) Vodovarstvena območja (VVO) - državni, občinski, vrelčni nivo 0 97 20 0 0,14 0,71 0 0,01 0,05 1 7.778 2.591 126 11,65 22,57 39 0,85 2,17 2 67.512 17.637 995 5,19 15,48 618 1,02 3,95 3 238.749 75.481 2.964 1,24 3,93 1.942 0,81 2,57 4 31.303 8.281 610 1,95 7,37 861 2,75 10,40 5 4.781 2.944 80 1,68 2,72 2 0,04 0,06 Skupaj 350.202 106.954 4.776 1,36 4,47 3.462 0,99 3,24 774. 274 Acta agriculturae Slovenica, 109 - 2, september 2017 Prostorska analiza kmetijskih zemljišč v zaraščanju v Sloveniji Rezultati za VVO potrjujejo opozorila kmetov, da se bodo območja najstrožjih vodovarstvenih režimov začela zaraščati, saj so pogoji za izvajanje običajne ekonomsko upravičene kmetijske dejavnosti zelo strogi in kmete odvračajo od obdelovanja teh zemljišč (Preglednica 5). Tako je omejena uporaba mineralnih gnojil v ravninskih območjih ter izvajanje paše na kraških in visokogorskih pašnikih. Z vidika varovanja vodnih virov je tak trend zaželen, a po drugi strani kvari krajinsko podobo območij in socio-ekonomski položaj kmeta. Povečanje zarasti in prehajanje v gozdno vegetacijo ima tudi negativen vpliv, saj se poveča evapotranspiracija, kar zmanjšuje količino vode, ki napaja vodni vir (Ulaga in sod., 2008). Predlagamo, da se v bodoče pred uvajanjem režima predhodno preveriti kratko-, srednje- in dolgoročne vplive na kmetijsko dejavnost, vrsto dejanske rabe in možnost pojava grmovne in drevesne zarasti. 4 SKLEPI S to raziskavo smo prvič po sprejetju prenovljenega zakona o kmetijskih zemljiščih (RS, 2011), ki v sedmem členu lastnikom, zakupnikom in drugim uporabnikom nalaga preprečevanje in odpravljanje zaraščanja, opravili prostorsko in statistično analizo z namenom identifikacije območij, ki so najbolj izpostavljena zaraščanju. Raziskava je pokazala, da je zaraščanje proces, ki se pojavlja po celotni Sloveniji z glavnino v osi od jugozahoda proti severovzhodu. Analiza povezave zaraščanja s prisotnostjo gozda na območju občine je pokazala, da ni izrazitih vzorcev, ki bi potrjevali domnevo, da večji odstotek gozda pomeni večji odstotek zaraščanja. Povezave med preučevanimi razredi dejanske rabe bi bilo treba podrobno preučiti na ravni posameznega območja zaraščanja, kjer bi opravili analizo učinka sosedstva rabe 2000 na razširjenost rabe 1410 in analizo opuščanja kmetijske obdelave na zemljiščih rabe 1800 (npr. vpliv napadov zveri na pašno živino). Površine rabe 1410 in rabe 1800 so povezane z naklonom in nadmorsko višino, saj se z večanjem vrednosti obeh spremenljivk odstotek obeh rab glede na celotno površino kmetijskih zemljišč v občini poveča. Analiza je pokazala, da je raba 1410 na poplavnih območjih bolj razširjena od rabe 1800. Manjši vpliv na rabo 1800 izhaja iz dejstva, da je razširjena na specifičnih lokacijah, kjer se zaradi prepustnih apnenčastih kamnin in topografije (naklon) poplave ne pojavljajo. To potrjuje tudi majhen obseg rabe 1800 (48 ha) na poplavnih območjih. Vpliv posameznega upravljavskega statusa območja na zaraščanje je težje ovrednotiti, ker se jih ponekod izvaja več vzporedno. Analiza je pokazala, da je zaraščanje večje na območjih z upravljavskim statusom. Še posebno izstopa dejstvo, da bolj kot je striktno okoljsko varovanje (kombinacija Nature 2000 območij SCI - SPA, narodni park, naravni rezervat, osrednje območje življenjskega prostora rjavega medveda, 1. in 2. kategorija VVO), večji je odstotek rabe 1410 od vseh kmetijskih zemljišč. V prostoru bodo z nadaljnjim razvojem kmetijske tehnologije in pridelovalnih procesov vedno obstajala zemljišča, ki bodo podvržena procesom zaraščanja. Dinamiko zaraščanja ali krčitve zarasti narekuje ekonomski interes lastnikov in zakupnikov zemljišč. Raziskava je pokazala, da se zaraščajo tudi kmetijska zemljišča v ravnini, saj do naklona 6 % obsegajo skoraj 7.000 ha rabe 1410 in 1.700 ha rabe 1800. Skupno te površine presegajo obseg vseh delujočih (6.077 ha) in delno delujočih (647 ha) namakalnih sistemov Sloveniji. Na območjih, kjer je zaraščanje najbolj razširjeno, bi bilo treba pospešeno izvajati zakon o kmetijskih zemljiščih, ki določa odpravljanje zaraščanja na območjih z večjo boniteto zemljišč. Opraviti bi bilo treba analizo vzrokov za opuščanje kmetijske obdelave na posameznih območjih in predlagati ukrepe za preprečevanje (komasacije, izboljšanje dostopnosti, finančna spodbuda ali kazni) in odpravljanje zaraščanja (agromelioracije za izboljšanje fizikalnih, kemijskih in bioloških lastnosti tal, rekultivacija s pomočjo drobnice) ter nadaljnjo obdelavo zemljišč, ki bi ustrezala naravno pridelovalnim razmeram območja (reja avtohtonih pasem, izdelki z dodano vrednostjo, npr. sivka s Krasa), načrtno pogozdovanje (biomasa, plantaže gozdnega drevja). Podatki o dejanski rabi zemljišč kažejo, da se proces zaraščanja nadaljuje, zato bi morali odločevalci nadaljevati aktivnosti, ki spodbujajo preprečevanje in odpravljanje zaraščanja (ARSO, 2016). Vendarle je pomembno, da vlogo pri tem prevzamejo tudi lokalne skupnosti, ki bolje poznajo razmere na terenu in možnosti izkoriščanja prostora. Dober ukrep za zmanjševanje zaraščanja je, v januarju 2017 sprejeta, uredba o izvajanju ukrepa odpravljanja zaraščanja na kmetijskih zemljiščih, katere izvajanje se financira s sredstvi, zbranimi iz naslova odškodnine zaradi spremembe namembnosti zemljišč (RS, 2017). Uredbo je pripravilo Ministrstvo za kmetijstvo, gozdarstvo in prehrano z namenom financiranja izvedbe agromelioracij na kmetijskih zemljišči v zaraščanju, s čimer bi jih ponovno usposobili za kmetijsko pridelavo. A uredba ureja le odpravljanje zaraščanja in ne deluje preventivno. Zato bo v prihodnje treba določiti tudi ukrepe za preprečevanje zaraščanja, kot so preprečevanja plazenja, odvodnjavanje talne vode, Acta agriculturae Slovenica, 109 - 2, september 2017 Matjaž GLAVAN in sod. izgradnja teras, odpravljanje skalovitosti in izravnave terena, izgradnja poljskih in gozdnih poti, vzdrževanje osuševalnih sistemov in komasacije preko povečevanja površine kmetijskih parcel. Preventivni ukrepi preprečujejo zaraščanje, kar je dolgoročno finančno in energijsko učinkoviteje. Pomembno je, da s preventivnimi ukrepi omogočimo, da je zemljišče ves čas v pridelovalni funkciji in da z vlaganji izboljšujemo dostopnost in pridelovalni potencial zemljišč. 5 ZAHVALA Delo je bilo financirano strani Javne agencija za raziskovalno dejavnost Republike Slovenije (ARRS) in Ministrstva za kmetijstvo, gozdarstvo in prehrano (MKGP) v okviru Ciljnega raziskovalnega programa. Naslov projekta je Strokovne podlage za določitev območij primernih za odpravljanje zaraščanja (V4-1149). 6 VIRI ARSO. 2011. Geoportal ARSO. Ljubljana. Ministrstvo za okolje in prostor. Agencija Republike Slovenije za okolje. http://gis.arso.gov.si/geoportal/catalog/main/home. page (23. nov. 2011) ARSO. 2016. [KM10] Sprememba rabe zemljišč in kmetijstvo. Kazalci okolja Slovenije. Agencija Republike Slovenije za okolje. http://kazalci. arso. gov. si/?data=indicator&ind_id=4 60 (30. dec. 2016) Bordjan D., Bordjan A. 2014. Effects of overgrowing at Cerknica Polje (southern Slovenia) on breeding farmland birds/ Vpliv zaraščanja Cerkniškega polja (južna Slovenija) na gnezdilke kmetijske krajine. Acrocephalus, 35: 153-163. doi:10.1515/acro-2014-0009 Cojzer M. 2011. Značilnosti zaraščanja in možnosti usmerjanja sukcesijskega razvoja sestojev pionirskih drevesnih in grmovnih vrst na novonastalih gozdnih površinah. Doktorska disertacija. Ljubljana, Univerza v Ljubljani, Biotehniška Fakulteta, Oddelek za gozdarstvo in obnovljive vire: 195 str. Corbelle Rico E., Crecente Maseda R., Sante Riveira I. 2012. Multi-scale assesment and spatial modelling of agricultural land abandonment in a European peripheral region: Galicia (Spain), 1956 - 2004. Land Use Policy, 29: 493-501. doi:10.1016/j.landusepol.2011.08.008 Černe R., Jerina K., Jonozovič M., Kavčič I., Stergar M., Krofel M., Marenče M. 2010. Škode od volkov v Sloveniji - analiza v okviru projekta Life+ SloWolf Akcija A4. Ljubljana, Zavod za Gozdove; Univerza v Ljubljani, Biotehniška fakulteta, Oddelek za gozdarstvo in obnovljive gozdne vire, Ljubljana: 12 str. Cunder T. 1998. Zaraščanje kmetijskih zemljišč v slovenskem alpskem svetu. Sonaravni razvoj v slovenskih Alpah in sosedstvu. V: 1. Melikovi geografski dnevi, Kranjska Gora, 5-7 nov. 1998. Ljubljana, Univerze v Ljubljani, Filozofske fakultete, Oddelek za geografijo: 165-175 Diaz G.I., Nahuelhual L., Echeverria C., Marin S. 2011. Drivers of land abandonment in Southern Chile and implications for landscape planning. Landscape and Urban Planning, 99: 201-217. doi: 10.1016/j. landurbplan.2010.11.005 EC. 1980. Effects on environment of the abandonment of agricultural land. Brussels, Commission of European Communities: 193 str. http://aei.pitt.edu/36390/1M.2517.pdf (31. avg. 2012) Eler K., Čop J., Vidrih M. 2008. Trajnostna raba pašnikov Primorskega krasa v prihodnosti. V: Novi izzivi v poljedelstvu, Rogaška Slatina, 4-5 december 2008: 358-364 EU. 2013. Assessing the risk of farmland abandonment in the EU - Final report. Luxembourg, European Commission, Joint Research Centre, Institute for Environment and Sustainability: 132 str. http://ec.europa.eu/agriculture/external-studies/2013/farmland-abandonment/fulltext_en.pdf (12. apr. 2013) FAO. 2006. The role of agriculture and rural development in revitalizing abandoned/depopulated areas. Latvia, Riga, Comission on Agriculture, 34th Session of the European Comission: 24 str. http://www.fao.org/fileadmin/user_upload/Europe/docu ments/Publications/Abandoned_en.pdf (1. jun. 2013) FAO. 2010. Global Forest Resources Assessment. Rim, Food and Agriculture Organization of the United 276 Acta agriculturae Slovenica, 109 - 2, september 2017 Prostorska analiza kmetijskih zemljišč v zaraščanju v Sloveniji Nations (FAO): 378 str. http://www.fao.org/forestry/fra/fra2010/en/ (9. jun. 2013) FAOSTAT. 2013. Food and Agricuture Organization of the United Nations. http://faostat3.fao.org/home/E (2. jul. 2013) Garcia Ruiz J.M., Lana Renault N. 2011. Hydrological and erosive consequences of farmland abandonment in Europe, with special reference to the Mediterranean region - A review. Agriculture, Ecosystems and Environment, 140: 317-338. doi:10.1016/j.agee.2011.01.003 Gellrich M., Zimmermann N.E. 2007. Investigating the regional - scale pattern of agricultural land abandonment in the Swiss mountains: A spatial statistical modelling approach. Landscape and Urban Planning, 79: 65-76. doi:10.1016/j.landurbplan.2006.03.004 Glavan M., Kozinc Z., Pintar M. 2015. Kmetijstvo in upravljanje s poplavnimi območji ob reki Dravi -projekt LiveDrava. Zbornik referatov - Mišičev vodarski dan 2014. Maribor, Vodnogospodarski biro Maribor: 8-15. http ://www.mvd20.com/zbornik.php?page=letnik (9. dec. 2015) GURS. 2011. Zbirke prostorskih podatkov. 2011. Ljubljana, Geodetska uprava RS. http://www.e-prostor.gov.si/si/zbirke_prostorskih_podatkov/ (23. nov. 2011) Gutman L. 2011. Rekultivacija zaraščajočih kmetijskih zemljišč s pašo domačih živali na območju krajinskega parka Goričko. Diplomsko delo. Ljubljana, Univerza v Ljubljani, Biotehniška Fakulteta, Oddelek za agronomijo: 40 str. Hladnik D. 2005. Spatial structure of disturbed landscapes in Slovenia. Ecological Engineering, 24: 17-27. doi:10.1016/j.ecoleng.2004.12.004 Hočevar M., Kušar G., Cunder T. 2004. Monitoring in analiza zaraščanja kraške krajine v GIS okolju. Zbornik gozdarstva in lesarstva, 75: 21-52 http://rkg.gov.si/GERK/documents/RABA_IntKlju c_20110101.pdf (5. jan. 2012) Ilc R. 2008. Zaraščanje kmetijskih zemljišč na območju Ribniško - Kočevske doline. Diplomsko delo. Ljubljana, Univerza v Ljubljani, Biotehniška Fakulteta, Oddelek za zootehniko: 52 str. Jankovič J. 2003. Nekdaj goli Kras spet gozdnat. Sodobno kmetijstvo, 36, 6: 12-18 Jarni K. 2011. Vpliv antropogenih virov hrane na prostorsko razporeditev rjavega medveda (Ursus arctos). Diplomsko delo. Ljubljana, Univerza v Ljubljani, Biotehniška Fakulteta, Oddelek za biologijo: 65 str. Keenleyside C., Tucker G. M. 2010. Farmland Abandonment in the EU: an Assessment of Trends and Prospects. Report prepared for WWF. London, Institute for European Environmental Policy: 93 str. http://www.ieep.eu/assets/733/Farmland_abandonm ent_in_the_EU_- _assessment_of_trends_and_prospects_-_FINAL_15-11-2010_.pdf (18. jan. 2011) Ketiš, K., Eler, K., Udovč, A., Batič, F. (2014). Dependence of the conservation status of acid grasslands at the Pohorje and Kozjak on socioeconomic parameters. Acta agriculturae Slovenica, 103 (2), 233 - 244. doi:10.14720/aas.2014.103.2.8 Khanal N.R., Watanabe T. 2006. Abandonment of agricultural land and its consequences - a case study in the Sikles area, Gandaki basin, Nepal Himalaya. Mountain Research and Development, 26, 1: 32-40. doi: 10.1659/0276-4741(2006)026[0032:A0ALAI]2.0.C0;2 Knap M. 2008. Rekultiviranje zaraščenih površin s kozami na območju Notranjskega regijskega parka. Diplomsko delo. Ljubljana, Univerza v Ljubljani, Biotehniška Fakulteta, Oddelek za zootehniko: 42 str. Kralj T., Koren D. 2011. Analiza stanja kmetijstva. V: Izhodišča za Načrt upravljanja Triglavskega narodnega parka 2012-2022. Kus Veenvliet J. (Ur.). Bled, Javni zavod Triglavski narodni park: 25 str. http://www.tnp.si/images/uploads/analiza_kmetijstv a.pdf (18. nov. 2015) Krebs C.J. 2001. Ecology: The experimental analysis of distribution and abundance (5th Edition). San Francisco, Benjamin Cummings: 608 str. Kus Veenvliet J. 2012. Analiza doseganja ciljev Strategije ohranjanja biotske raznovrstnosti v Sloveniji. Končno poročilo. Naročnik: Ministrstvo za okolje in prostor. Nova vas, Zavod Symbiosis: 235 str. MacDonald D., Carbtree J.T., Wiesinger G., Dax T., Stamou N., Fleury P., Lazpita Guiterrez J., Gibon A. 2000. Agricultural abandonment in mountain areas in Europe: Environmental consequences and policy response. Journal of Environmental Management, 59: 47-69. doi:10.1006/jema.1999.0335 Miličic V., Perpar A., Kramarič F., Udovč A. 2011. Analiza stanja kmetijstva na območju krajinskega parka Ljubljansko barje - končno poročilo -revidirano in dopolnjeno besedilo. Naročnik: Javni zavod Krajinski park Ljubljansko barje. Ljubljana, Acta agriculturae Slovenica, 109 - 2, september 2017 Matjaž GLAVAN in sod. Univerza v Ljubljani, Biotehniška fakulteta, Oddelek za agronomijo, Katedra za agrometeorologijo, urejanje kmetijskega prostora, ekonomiko ter razvoj podeželja: 90 str. MKGP. 2011. MKGP portal. Ministrstvo za kmetijstvo, gozdarstvo in prehrano. http://rkg.gov.si/GERK/ (23. nov. 2011) MKGP. 2012. Interpretacijski ključ - podroben opis metodologije zajema dejanske rabe kmetijskih gozdnih zemljišč. Ljubljana, Ministrstvo za kmetijstvo, gozdarstvo in prehrano. MOP. 2002. Strategija upravljanja z rjavim medvedom (Ursus arctos) v Sloveniji. Ljubljana, Ministrstvo za okolje in prostor: 31 str. http://www.mop.gov.si/fileadmin/mop.gov.si/pageu ploads/podrocj a/velike_zveri/strategij a_rj avi_medv ed_2002.pdf (15. dec. 2011) MOP. 2007. Akcijski načrt upravljanja z rjavim medvedom (Ursus arctos L.) v Sloveniji. Ljubljana, Ministrstvo za okolje in prostor: 23 str. http://www.mop.gov.si/fileadmin/mop.gov.si/pageu ploads/podrocj a/velike_zveri/akcij ski_nacrt_medve d_predlog_apr07.pdf (15. dec. 2011) Plut D. 2011. Prehranska varnost planeta in Slovenije. Ljubljana, Svet za varstvo okolja Republike Slovenije: 4 str. http://www.mko.gov.si/fileadmin/mko.gov.si/pageu ploads/svo/53seja_Plut.pdf (5. maj 2013) Pogačnik M., Kompan D., Vidrih T., Kotar M., Matičič B. 1995. Rekultiviranje opuščenih hribovitih kraških zemljišč z drobnico. Sodobno kmetijstvo, 28, 5: 225-229 Pogačnik M, Batič F. 2006. Sistemi sobivanja domačih in divjih živali - zaključno poročilo - CRP "Konkurenčnost Slovenije 2001-2006". Ljubljana, Univerza v Ljubljani, Veterinarska fakulteta: 13 str. Pogačnik A., Foški M., Drobne S., Konjar M., Soss K., Lipar P., Brilly M., Vidmar A., Žura M., Juvanc A., Maher T., Marsetič R., Detellbach S., Strnad I., Šemrov D., Fatur M. 2011. Analiza stanja, razvojnih teženj ter usmeritev za strateški prostorski razvoj Slovenije - zaključno poročilo -CRP "Konkurenčnost Slovenije 2006-2013". Univerza v Ljubljani, Fakulteta za gradbeništvo in geodezijo, Ljubljana: 249 str. Poyatos R., Latron J., Llorens P. 2003. Land use and land cover change after agricultural abandonment -the case of a mediterranean mountain area (Catalan pre-pyrenees). Mountain Research and Development, 23, 4: 362-368. doi:10.1659/0276-4741(2003)023[0362:LUALCC]2.0.C0;2 Renwick A., Jansson T., Verburg H.P., Revordero Giha C., Britz W., Gocht A., McCracken D. 2013. Policy reform and agricultural land abandonment in the EU. Land Use Policy, 30: 446-457. doi:10.1016/j.landusepol.2012.04.005 RS. 2004a. Uredba o posebnih varstvenih območjih. Ur. l. RS, št.49/04, 110/04, 59/07, 43/08, 8/12, 33/13, 35/13 - popr., 39/13 - odl. US in 3/14 RS. 2004b. Zakon o ohranjanju narave. Uradni list RS, št. 96/04, 61/06 - ZDru-1, 8/10 - ZSKZ-B in 46/14 RS. 2004c. Pravilnik o kriterijih za določitev vodovarstvenega območja. Uradni list RS, št. 64/04, 5/06, 58/11 in 15/16 RS. 2008. Pravilnik o določanju in vodenju bonitete zemljišč. Ur. l. RS, št. 47/08 RS. 2011. Zakon o kmetijskih zemljiščih. Ur.l. RS, št. 71/11 in 58/12 RS. 2015. Pravilnik o razvrstitvi kmetijskih gospodarstev v območja z omejenimi možnostmi za kmetijsko dejavnost. Ur.l. RS, št. 12/15 in 84/16 RS. 2017. Uredba o izvajanju ukrepa odpravljanje zaraščanja na kmetijskih zemljiščih. Ur.l. RS, št. 3/17 SURS-GIS. 2011. Baza geostatistični podatkov Statističnega urada Republike Slovenije. http://www.stat.si/gis/ (23. nov.2011) SURS-STAT. 2012. Statistični letopis 2012. Ljubljana, Statistični urad Republike Slovenije http://www.stat.si/StatWeb/glavnanavigacija/podat ki/publikacije/statisti%C4%8Dni-letopis?leto=2012 (2. jul. 2013) Ulaga F., Kobold M., Frantar P. 2008. Analiza časovnih sprememb vodnih količin slovenskih rek. Zbornik referatov - Mišičev vodarski dan 2008. Maribor, Vodnogospodarski biro Maribor: 9-17 http://www.mvd20.com/zbornik.php?page=letnik (17. nov. 2015) Vidrih T. 2005. Drevesno-pašna raba. Naše travinje, 1, 1: 6-8. Vidrih T. 2010. Ciljna paša na posekah kraškega pašnika. Naše travinje, 6, 1: 4-6 Vidrih T., Pogačnik M., Kotar M., Kompan D., Juntes P., Matičič B. 1996. Cilji in naloge razvojno raziskovalnega centra za rekultiviranje opuščenih kraških in hribovitih površin Vremščica. V: Možnosti razvoja reje drobnice v Sloveniji, Postojna, Hotel Jama, 27-29 november 1996. Slovenj Gradec, Kmetijska založba: 73-78 Vodlan M. 2006. Poraba energije pri čiščenju zaraščenih kmetijskih zemljišč na dveh lokacijah v ?78 118 Acta agriculturae Slovenica, 109 - 2, september 2017 Prostorska analiza kmetijskih zemljišč v zaraščanju v Sloveniji Sloveniji. Diplomsko delo. Ljubljana, Univerza v Ljubljani, Biotehniška Fakulteta, Oddelek za agronomijo: 43 str. Vrščaj B. 2008. Strukturne spremembe kmetijskih zemljišč. Njihova urbanizacija in kakovost v obdobju 2002 - 2007. Hmeljarski bilten, 15: 73-84 Wilfing H. 1993. Ekologija. Celovec: Mohorjeva založba: 120 str. www.fao.org/world/regional/reu/eca/eca_en/eca34_3.pd f (18. jul. 2013) Zaragozi B., Rabasa A., Rodriguez Sala J.J., Navarro J.T., Belda A., Ramon A. 2012. Modelling farmland abandonment: A study combining GIS and data mining techniques. Agriculture, Ecosystems and Environment, 155: 124-132. doi:10.1016/j.agee.2012.03.019 ZGS. 2011. Poročilo Zavoda za gozdove Slovenije o gozdovih za leto 2011. Ljubljana, Zavod za gozdove Slovenije: 133 str. http ://www. zgs.si/slo/gozdovi_slovenij e/o_gozdovi h_slovenije/letna_porocila/index.html (15. apr. 2015) Žvikart M. 2013. Analiza ciljev in ukrepov programa upravljanja območij Natura 2000 - Operativni program upravljanja z območij Natura 2000 v Sloveniji 2014-2020 - SI Natura 2000 Management - akcija A1, A2 - Sektor kmetijstvo. Ljubljana, Zavod RS za varstvo narave: 38. Žgavec, D., Eler, K., Udovč, A.,Batič; F. (2013). Contribution of agricultural policy measures to maintain grassland areas (the case of Radensko Polje Landscape Park). Acta agriculturae Slovenica, letnik 101(1), 77-86 Acta agriculturae Slovenica, 109 - 2, september 2017 doi: 10.14720/aas.2017.109.2.11 Original research article / izvirni znanstveni članek Effect of CO2 elevation and UV-A radiation on growth responses of Zinnia, Petunia, Coxcomb, and Marigold Hamidreza MIRI1, Maryam SADEGHI2, Abdolreza JAFARI3, Mohammad Mehdi RAHIMI4 Received October 25, 2016; accepted July 20, 2017. Delo je prispelo 25. oktobra 2016, sprejeto 20. julija 2017. ABSTRACT In order to evaluate the effect of CO2 elevation and UV radiation on growth responses of zinnia, petunia, coxcomb, and marigold, a study was conducted in 2015 at Arsanjan Islamic Azad University, Iran. The experimental design was factorial arranged in completely randomized design with three replications. Treatments were included four ornamental species (zinnia, petunia, coxcomb, and marigold), CO2 concentration at two levels (350 and 700 ppm), and UV radiation at two levels (with and without UV radiation). Results showed that elevating of CO2 concentration from 350 ppm to 700 ppm increased morphological and physiological characters of C3 plants, especially marigold. Meanwhile, increasing CO2 concentration from 350 ppm to 700 ppm, decreased effects of UV damage on plants' morphological and physiological characters. The highest leaf number, shoot dry mass, plant height and water use efficiency of C4 plant (coxcomb flower) were observed at 350 ppm of CO2 concentration without UV radiation while, the highest leaf number, shoot dry mass and leaf pigments of C3 plants (zinnia, petunia, and marigold flower) were obtained at 700 ppm of CO2 concentration without UV radiation. The results showed that the activity of catalase and peroxidase enzymes under UV radiation was increased in all of plants. Overall, it is concluded that, the recognition of plants resistant to UV radiation and high levels of CO2 concentration in the future may be better for environmental production and distribution as ornamental plants in town landscapes, where ecophysiological traits should be considered. Key words: ornamental plants; climate change; morphological and physiological traits; UV radiation IZVLEČEK UČINEK POVEČANE KONCENTRACIJE CO2 IN POVEČANEGA UV-A SEVANJA NA RASTNI ODZIV CINIJE, PETUNIJE, PETELINJEGA GREBENA IN ŽAMETNICE Z namenom ovrednotenja učinka povečanega CO2 in UV sevanja na rastni odziv cinije, petunije, petelinjega grebena in žametnice je bil v letu 2015 izveden poskus na Arsanjan Islamic Azad University, Iran. Poskus je bil popoln naključni faktorski poskus s tremi ponovitvami. Obravnavanja so obsegala štiri vrste okrasnih rastlin (cinija, petunija, petelinov greben in žametnica), dve koncentraciji CO2 (350 in 700 ppm), in dve jakosti UV sevanja (brez in z UV sevanjem). Rezultati so pokazali, da je povečana koncentracija CO2 iz 350 ppm na 700 ppm povečala vrednosti morfoloških in fizioloških znakov C3 rastlin, še posebej žametnice. Povečanje koncentracije CO2 iz 350 ppm na 700 ppm je zmanjšalo učinke poškodb po UV v morfoloških in fizioloških znakih. Največje število listov, največja masa suhe snovi in največja učinkovitost izrabe vode so bili pri C4 rastlinah (petelinji greben) zabeleženi pri 350 ppm CO2 brez UV sevanja, pri C3 rastlinah (cinija, petunija in žametnica) je bila največja vrednost znakov kot so število listov, suha masa poganjkov in vsebnost listnih pigmetov ugotovljena pri 700 ppm CO2 brez UV sevanja. Aktivnosti katalaze in peroksidaze sta se v razmerah UV sevanja povečali pri vseh rastlinah. V splošnem lahko zaključimo, da je pri izbiri okrasnih rastlin za učinkovitejše ozelenjevanje urbanih površin potrebno upoštevati tudi njihove morfološko fiziološke lastnosti. Ključne besede: okrasne rastline; morfološko fiziološke lastnosti; klimatske spremembe; UV sevanje 1 Department of Agronomy and Crop Breeding, College of Agriculture, Arsanjan Branch, Islamic Azad University, Arsanjan, Iran, corresponding author: hmiri6@gmail.com 2 Department of Agronomy and Crop Breeding, College of Agriculture, Arsanjan Branch, Islamic Azad University, Arsanjan, Iran 3 Department of Biology, Arsanjan Branch, Islamic Azad University, Arsanjan, Iran 4 Department of Agronomy and Crop Breeding, College of Agriculture, Arsanjan Branch, Islamic Azad University, Arsanjan, Iran Acta agriculturae Slovenica, 109 - 2, september 2017 str. 281 - 240 Hamidreza MIRI et al. 1 INTRODUCTION Nowadays, unstable symptoms on the Earth such as environmental pollution and species extinction caused by increased emissions of greenhouse gases, in combination with changes in solar radiations intensity appeared to be unavoidable (Xing, 2009; Ziska and Blumenthal, 2010). Morphological traits and physiological processes of plants are affected by different climate change aspects such as CO2 elevation, high temperature, ultra violet (UV) radiation, and quantity and dispersal of rainfall (Fuhrer, 2003). It is reported that climate changes has affected flowering initiation, physiology, water relations, ions absorption, photosynthesis and respiration of plants (Mortensen, 1987). CO2 plays an important role in process of global warming and climate changes (Holden and Hoyer, 2005). The level of CO2 in the atmosphere is rising at an unprecedented rate, has increased from 280 ppm at the beginning of the industrial revolution to 380 ppm today, and is expected to double pre-industrial levels sometime during this century (Hennessy et al., 2008; Karl et al., 2009). Generally, CO2 elevation could increase net photosynthesis of potted plants, cut flowers, and vegetables (Croonenborghs et al., 2009). Significant increase of water use efficiency (WUE) and decrease of stomatal conductance were observed in plants treated by CO2 elevation (Lincoln and Couvet, 1989; Prior et al., 2011). Kamali et al. (2011) reported that CO2 elevation from 380 to 1050 ppm could increase shoot and root dry mass, height, number of leaves and leaf area of coxcomb (Celosia argentea L.). Shoor et al. (2010) showed that increasing CO2 level to 700 ppm could accelerate marigold (Tagetespatula L.) flowering time. Plants, as sessile organisms that require sunlight for growth and development, are inevitably exposed to UV wavelengths (200-400 nm), which represent almost 7 % of the electromagnetic radiation emitted from the sun. Plants responses to ozone layer destruction and high UV radiation include widespread range of bio-chemical, physiological, morphological, and anatomical changes (Zhang et al., 2003). High doses of UV radiation may damage macromolecules, including DNA and proteins, and induce the production of reactive oxygen species (ROS), affecting photosynthetic pigments, cell membrane integrity and viability (Horii et al., 2007). Rahimzadeh et al. (2011) reported that UV-A radiation decreased shoot dry mass, protein, and chlorophyll content of savory (Satureja hortensis L.). Kazemi Ghale et al. (2011) showed that in radish plants treated with UV radiation photosynthesis rate decreases due to leaf area reduction, leaf thickness increases, and observes bio-chemical changes of chlorophyll pigments. Golbazhagh et al. (2010) reported the reduction of sunflower growth such as shoot dry mass, root length, and leaf area caused by increasing exposure time of different doses of UV-A radiation. Sarikhani (2013) showed that the UV-A radiation reduced peppermint yield. Also, antioxidant and secondary metabolite activity increased when peppermint was treated by UVA radiation. By considering the climatic changes the objective of this research was to determine the influence of CO2 elevation and UV-A radiation on morphological and physiological properties of zinnia, petunia, coxcomb, and marigold . Study of climate changes effect on zinnia, petunia, coxcomb, and marigold production, quality, and marketable properties are very important because these plants are widespread ornamental species. 2 MATERIALS AND METHODS In order to evaluate the effect of CO2 elevation and UVA radiation on morphological and physiological parameters of zinnia (Zinnia elegans Jacq.), petunia (Petunia x hybrida 'Grandifloras'), coxcomb (Celosia cristata L.), and Mexican marigold (Tagetes erecta L.) a study was conducted in 2015 at Islamic Azad University, Arsanjan branch, Iran (53° 19' E, 29° 55' N and 1690 m). The Experimental design was factorial arranged in completely randomized design with three replications. Treatments included plant species (zinnia, petunia, coxcomb, and marigold), CO2 concentration at two levels (ambient CO2 (350 ppm) and elevated CO2 (700 ppm)), and UV radiation at two levels (with and without UV-A radiation). The experiment was conducted in two environmentally controlled growth chambers with four compartments to apply CO2 and UV-A treatments, with the mean air temperature of 25/14 °C (day/night) and relative humidity of 75 (day) and 60 % (night). At the beginning of the experiment, five seeds were planted in 5 cm deep of each of the 48 pots (18 cm height and 14 cm diameter) filled with a silty-loam soil with 1.16 % organic matter, 15 to 18 % sand, 50 to 56 % silt, 10 to 15 % clay and pH of 7.5. After seed germination, seedlings were thinned to one per pot at the four-leaf stage. Then, half of the pots were moved into the ambient CO2 chamber and the other half, into the elevated CO2 chamber. Pots were uniformly irrigated (EC = 0.78 ds m-1 of water) every 3 days. Half of the 282 Acta agriculturae Slovenica, 109 - 2, september 2017 Effect of CO2 elevation and UV-A radiation on growth responses of Zinnia, Petunia, Coxcomb, and Marigold pots in the chambers was exposed by UV-A radiation (5 min per days) from fluorescent tubes (T9 Black light blue fluorescent- Schwan Company), used to produce UV-A radiation.They were installed at 50 height from each pot. Measured traits were leaf number, shoot dry mass, plant height, water use efficiency, chlorophyll-a and carotenoid content, and activity of catalase and peroxidase enzymes. In order to the assessment leaf pigments content, 20 ml aceton (80 %) was mixed with 0.5 g of leaf fresh mass, then, the mixture was centrifuged (with 3000 rpm for 10 min). Then, leaf pigments were determined by spectrophotometer (UV2100 Plus model - USA) device (wavelength 470 and 663 nm) (Arnon, 1967). In order to measure activity of catalase and peroxidase, 5 ml of potassium phosphate buffer (100 m mol and pH 7.5) was blend with 0.5 g of leaf fresh mass, then, the mixture was centrifuged (with 12000 rpm for 60 min). Afterwards, activity of catalase and peroxidase were determined using method described by Pereia et al. (2002) and Fielding and Hall (1978) , respectively. All data were submitted to an analysis of variance (ANOVA) and Duncan test was used to verify the significant differences among treatment means at the 5 % probability level (Little and Jackson, 1978). 3 RESULTS AND DISCUSSION Effect of CO2 and species interaction was significant on leaves' number, shoot dry mass, plant height, water use efficiency, chlorophyll a, activity of catalase and peroxidase (Table1). Results showed that the highest leaf number and shoot dry mass were observed in coxcomb flower + ambient CO2 (Figure1 (a) and (b)). The highest WUE was obtained in coxcomb flower + ambient CO2 (Figure1 (d)) and the highest catalase activity was achieved in coxcomb flower + elevated CO2 (Figure1 (g)). Results showed that the highest plant height was observed in Zinnia + elevated CO2 (Figure1 (c)) also, the highest chlorophyll a was achieved in marigold + ambient CO2 (Figure1 (e)) while, the highest peroxidase activity were achieved in petunia + ambient CO2 (Figure1 (h)). Generally, the highest leaf number and shoot dry mass were observed in coxcomb in comparison to the C3 plants due to quickly establishment and better use of soil nutrients by coxcomb seedlings (Hammer et al., 2005; Wortman et al., 2011). Shoor et al. (2010) reported that elevating CO2 concentration to 700 ppm could be increased marigold height (approximately 50 %) as a result improved plant photosynthesis capacity and allocated more assimilates to vegetative growth. It seems that elevating CO2 concentration improved WUE due to high CO2 concentration into inter-cellular space and transpiration reduction by stomatal closer (Fuhrer, 2003). Furthermore, increasing chlorophylla and carotenoid pigments by elevated CO2 had significant role in photosynthesis rate and photosystem II protection for improving radiation absorption and increasing photosynthesis capacity (Mavrogianopoulos et al., 1999; Joseph et al., 2008). Miri and Rastegar (2012) showed that elevated CO2 could increase chlorophyll index (approximately 6 to 30 %) in soybean, lambsquarters, panicum, and pigweed. In addition, shoot dry mass, WUE, chlorophyll a, catalase and peroxidase activity were significantly influenced by UV-A radiation and species interaction (Table 1). According to results, morphological and physiological parameters showed a reduction as affected by UV-A radiation. The highest leaf number, shoot dry mass, and WUE, and chlorophyll a were observed in coxcomb and marigold , respectively, without UV-A radiation (Figure 2 (a), (b), (d), and (e)). Meanwhile, the catalase activity in all plants increased by UV-A radiation (Figure 2 (g)). It seems that in plant subjected to UV-A radiation less transfer of photosynthetic assimilate occurred due to reduction of photosynthesis capacity (Balouchi et al., 2008). Reduction in growth and development and reduction of cell division also was observed by UV-A radiation (Smirnoff and Wheelev, 2000; Gao et al., 2003). But, plants response differently to UV-A radiation as affected by species and environmental factors such as plant water status, photosynthetically active radiation (PAR), and nutrients availability (Mark and Tevini, 1996; Balouchi et al., 2009). In general, results showed that shoot dry mass, plant height, WUE, chlorophyll a and carotenoid pigments, and catalase activity were significantly influenced by CO2 concentration, UV-A radiation, and plant species (Table 1). The highest leaf number and shoot dry mass were obtained in coxcomb + ambient CO2 + without UV-A radiation (Figure 3 (a) and (b)). Also, the highest WUE was observed in coxcomb + at elevated CO2 + without UV-A radiation (Figure 3 (d)). Meanwhile, the highest plant height was achieved in Zinnia + elevated CO2 + without UV-A radiation (Figure3 (c)). According to our research, results showed that UV-A radiation could decrease growth parameters of plant species due to its impact on photosynthesis capacity, but, elevating CO2 could lead to improve photosynthesis capacity and high assimilate transfer to vegetative growth (Ziska and McClung, 2008; Croonenborghs et al., 2008; He et al., 2013). Acta agriculturae Slovenica, 109 - 2, September 2017 283 Hamidreza MIRI et al. Table 1: The summary of the source of variation and the mean square of shoot dry mass (g per plant), leaf number (per plant), water use efficiency (g l-1), chlorophyll a and carotenoid (mg g-1), catalase and peroxidase activity (u mol g-1 min-1) Source of variation df Shoot dry mass Leaf number Plant height Water use efficiency Chlorophyll a Carotenoid Catalase activity Peroxidase activity Plant species 3 0.521** 154.750** 1689.607** 3.81** 46.192** 21.175** 3.644** 1207.464** CO2 level 1 0.000ns 4.083ns 552.028** 1.31** 1.602ns 1.522ns 1.367** 54.957** PlantxCO2 3 0.013** 26.750** 30.184* 1.10** 3.323* 0.102ns 2.719** 41.788** UV radiation 1 0.003* 24.083* 0.445ns 1.97ns 6.237* 0.0471ns 4.025** 145.718** PlantxUV 3 0.003* 0.528ns 8.196ns 7.68** 9.105** 0.622ns 0.533** 39.719** CO2xUV 1 0.001ns 0.083ns 80.808** 1.11ns 4.466* 0.218ns 0.035ns 11.903™ PlantxCO2xUV 3 0.009** 9.417ns 95.731** 1.35** 6.633** 1.828* 4.572** 7.987ns Error 32 0.001 4.042 10.386 1.72 0.945 0.460 0.055 4.438 CV (%) 12.70 14.10 18.00 13.60 11.50 13.07 18.20 16.90 Note: * and ** significant at the 0.05 and 0.01 level, respectively; ns, not significant Acta agriculturae Slovenica, 109 - 2, september 2017 Effect of CO2 elevation and UV-A radiation on growth responses of Zinnia, Petunia, Coxcomb, and Marigold Figure 1: The effect of flower species and CO2 concentration interaction on leaf number (a), shoot dry mass (b), plant height (c), water use efficiency (d), chlorophyll a content (e), carotenoid content (f), catalase activity (g), and peroxidase activity (h). (According to standard error, the means with same overlap not significant) Acta agriculturae Slovenica, 109 - 2, September 2017 285 Hamidreza MIRI et al. Figure 2: The effect of plant species and UV-A radiation interaction on leaf number (a), shoot dry mass (b), plant height (c), water use efficiency (d), chlorophyll a content (e), carotenoid content (f), catalase activity (g), and peroxidase activity (h). (According to standard error, the means with same overlap not significant) 286 Acta agriculturae Slovenica, 109 - 2, september 2017 Effect of CO2 elevation and UV-A radiation on growth responses of Zinnia, Petunia, Coxcomb, and Marigold Pnuli Figure 3: The effect of plant species, CO2 concentration, and UV-A radiation interaction on leaf number (a), shoot dry mass (b), plant height (c), water use efficiency (d), chlorophyll a content (e), carotenoid content (f), catalase activity (g), and peroxidase activity (h). (According to standard error, the means with same overlap not significant). It seems that elevating CO2 concentration could increase growth of C3 plants (zinnia, petunia, and marigold) in comparison to the C4 ones (coxcomb ) due to a better use of environmental sources for growth and development. However, UV-A radiation reduced plant growth parameters, but, increasing CO2 concentration could reduce destructive effects of UV-A radiation on analysed plants species. It is concluded that, the highest growth parameters of zinnia, petunia, and marigold were achieved under elevated CO2 without UV-A radiation, but, the highest growth parameters of coxcomb were obtained in ambient CO2 without UV-A radiation. Acta agriculturae Slovenica, 109 - 2, September 2017 127 Hamidreza MIRI et al. 4 ACKNOWLEDGMENT This project was funded by a grant from the Research Council and Graduate Center of Islamic Azad University, Arsanjan branch, Iran. 5 REFERENCES Arnon, A. N. (1967). Method of extraction of chlorophyll in the plants. Agronomy Journal, 23, 112-121. Balouchi, H. R. Modres Sanvi, A.M., Emam, Y. & Barzegar, M. (2008). The effect of water stress, CO2 elevation and UV radiation on quality parameters of durum wheat. Agriculture and Natural Research Journal, 45, 167-181. Balouchi, H. R. Sanavy, S.A.M. Emam, Y. & Dolatabadian, A. (2009). UV radiation, elevated CO2 and water stress effect on growth and photosynthetic characteristics in durum wheat. Plant Soil and Environment, 55, 443-453. Croonenborghs, S. Ceusters, J. Londers, E. & De Proft, M.P. (2009). Effects of elevated CO2 on growth and morphological characteristics of ornamental bromeliads. Horticultural Science, 121, 192-198. doi:10.1016/j.scienta.2009.01.018 Fielding, J.L. & Hall, J. (1978). Abiochemical and cytochemical study of peroxidase activity in root Pea. Journal of Experimental Botany, 29, 98-112. Fuhrer, J. (2003). Agro-ecosystem responses to combinations of elevated CO2, ozone, and global climate change. Agriculture, Ecosystem and Environment, 97, 1-20. doi:10.1016/S0167-8809(03)00125-7 Gao, W. Zhena, Y. Slusser, J.R. & Gordon, M. (2003). Impact of enhanced Ultraviolet-B irradiance on cotton growth, development, yield, and qualities under field conditions. Agricultural and Forestry Meteorology, 120(5), 241-248. Golbaz Hagh, A., Khara, H. & Darvishzadeh, J.R. (2010). The effect of UV-A radiation on growth of four genotype sunflowers. Biodiversity symposium, 1064-1066 pp. Hammer, G.L., Chapman, S., Oosterom, E.V., Podlich, D.W. (2005) Trait physiology and crop modeling as a framework to link phenotypic complexity to underlying genetic system. Australian Journal of Agricultural Research, 56, 947-960. doi: 10.1071/AR05157 He, J. M. Ma, X. G. Zhang, Y. Sun, T.F. Xu, F.F. Chen, Y.P. Liu, X. & Yue, M. (2013). Role and interrelationship of Ga protein, hydrogen peroxide, and nitric oxide in ultraviolet B-induced stomatal closure in Arabidopsis leaves. Plant Physiology, 161, 1570-1583. doi: 10.1104/pp. 112.211623 Hennessy, K. Fawcett, R. Kirono, D. Mpelasoka, F. Jones, D. Bathols, B. J. Whetton, P. Stafford, M. S. Howden, M. Mitchell, C. & Plummer, N. (2008). An assessment of the impact of climate change on the nature and frequency of exceptional climatic events. CSIRO, Bureau of Meteorology, Canberra, Australia, 33 pp. Holden, E. & Hoyer, K.G. (2005). The ecological Foot Prints of Fuels, Transportation Research Part D, N.10, 395 pp. Horii, A. Mccup, P. & Shetty, K. (2007). Enhancement of seed vigour following insecticide and phenolic elicitor treatment. Bio-resource Technology, 98, 623-632. doi: 10.1016/j.biortech.2006.02.028 Joseph, C. V. Leon, H.V. & Allen, J.R. (2008). Growth at elevated CO2 delays the adverse effects of drought stress on leaf photosynthesis of the C4 sugarcane. Journal of Plant Physiology.25, 45-51. Kamali, M. Shoor, M. Goldani, M. Selahvarzi, Y. & Tehranifar, A. (2011). The effect of CO2 elevation on morphological parameters of Coxcomb. First symposium of meteorological and irrigation management. 1-2 October. Karl, T. R. Melillo, J. M. & Peterson, T. C. (2009). Global climate change impacts in the United States, Cambridge University Press. 196 p. Kazemi Ghaleh, R., Shekari, F., Enaeti, V. (2011). The effect of UV radiation on morphological and germination of radish. First symposium of agricultural and sciences. Zanjan University. 19-21 September. Lincoln, D.E. & Couvet, D. (1989). The effect of carbon supply on allocation to allelochemicals and caterpillar consumption of peppermint. Oecologia, 78, 112-11. doi: 10.1007/BF00377205 Mark, U. & Tevini, M. (1996). Combination effect of UV-B radiation and temperature on sunflower and maize seedlings. Journal of Plant Physiology, 148, 49-56. doi:10.1016/S0176-1617(96)80293-8 288 Acta agriculturae Slovenica, 109 - 2, september 2017 Effect of CO2 elevation and UV-A radiation on growth responses of Zinnia, Petunia, Coxcomb, and Marigold Mavrogianopoulos, G.N. Spanakis, J. & Tsikalas, P. (1999). Effect of CO2 enrichment and salinity on photosynthesis and yield in melon. Science Horticulture, 79, 51-63. doi:10.1016/S0304-4238(98)00178-2 Miri, H.R. & Rastegar, A. (2012). The effect of CO2 elevation on growth and competitiveness between soybean, panicum, lambsquarters, and pigweed (In Persian). Crop Production Journal, 1, 1-18. Mortensen, L.M. (1987). CO2 enrichment in greenhouses. Crop responses. Science Horticulture, 33, 1-25. doi:10.1016/0304-4238(87)90028-8 Pereia, G.J. Molina, G. & Zeved, R.A. (2002). Activity of antioxidant enzymes in responses to cadmium in Crotalaria juncea. Plant and Soil, 239, 123-132. doi:10.1023/A:1014951524286 Prior, S.A. Runion, G.B. Marble, S.C. Rogers, H.H. Gilliam, C.H. & Torbert, H.A. (2011). A review of elevated atmospheric co2 effects on plant growth and water relations: implications for horticulture. Horticultural Science, 46, 158-162. Rahimzadeh, P. Hosseini, S. & Dilmaghani, K. (2011). Effects of UV-A and UV-C radiation on some morphological and physiological parameters in Savory (Satureja hortensis L. ). Annals of Biological Research, 2, 164-171. Sarikhani, H. (2013). The effect of UV-A radiation on growth and physiological of pepper mint (In Persian). Plant Production Journal, 2, 35-44. Shoor, M. M. Zargarian, & S. Bostani. (2010). Evaluation of the effect of CO2 elevation on Tagetes morphological and anatomical under green house. Horticulture Journal, 2, 128-135. Smirnoff, N. & Wheelev, G.L. (2000). Ascorbic acid in plants: biosynthesis and function critical. Journal of Plant Science, 19, 267-290. Wortman, S.E. Adam, S.D. Brian, J. & Lindquist, J.L. (2011). Integrating management of soil nitrogen and weeds. Weed Science, 59, 162-170. doi:10.1614/WS-D-10-00089.1 Xing, Y. (2009). A framework model for assessing sustainability impacts of urban development, Accounting Forum, Volume 33, Issue 3, September 2009, 209-224 pp. Zhang, M. An, L. Feng, H. Chen, T. Chen, K. Liu, Y. Tang, H. Chang, J. & Wang, X. (2003). The cascade mechanisms of nitric oxide as second messenger of ultraviolet-B in inhibiting mesocotyl elongations. Photochemistry and Photobiology, 77, 219-225. doi:10.1562/0031- 8655(2003)077<0219:TCM0N0>2.0.C0;2 Ziska, L.H. Blumenthal, D.M. Runion, G.B. Hunt, E.R. & Diaz-Soltero, H. (2010). Invasive species and climate change: an agronomic perspective. Climatic Change. DOI 10.1007/s10584-010-9879-5. doi:10.1007/s10584-010-9879-5 Ziska, L.H. & McClung, A. (2008). Differential response of cultivated and weedy (red) rice to recent and projected increases in atmospheric carbon dioxide. Agronomy Journal, 100, 12591263. doi:10.2134/agronj2007.0324 Acta agriculturae Slovenica, 109 - 2, September 2017 289 doi: 10.14720/aas.2017.109.2.12 Original research article / izvirni znanstveni članek Salinity induced changes in water relations, oxidative damage and morpho-physiological adaptations of pistachio genotypes in soilless culture Zahra MIRFATTAHI1, Soheil KARIMI1*, Mahmoud Reza ROOZBAN1 Received November 19, 2016; accepted August 03, 2017. Delo je prispelo 19. novembra 2016, sprejeto 03. avgusta 2017. ABSTRACT Selecting salt tolerant rootstocks is a sustainable approach for developing fruit trees in salinity prone areas. 60-day-old seedlings of Pistacia vera 'Akbari' and 'Ghazvini', and P. vera 'Ghazvini' x P. atlantica (GxA) were subjected to 0, 50, 100 and 150 mM NaCl in half strength Hoagland's nutrient solution. After 45 days, the growth, water relations, and oxidative damage parameters were investigated. Salt stress reduced plant biomass, height, crown diameter and leaf number, but increased specific leaf area (SLA) of the seedlings. Under salt stress, the growth of 'Akbari' seedlings was higher than the other genotypes. Accumulation of malondialdehyde (MDA) and proline was observed in the leaves of salt affected seedlings. 'Ghazvini' seedlings had the highest MDA concentration and the lowest cell membrane stability in their leaves. Degredation of photosynthetic pigments under salt stress was lower in the leaves of 'Akbari' seedlings than that in other genotypes. Increase in leaf succulence was observed in 'Akbari' and GxA seedlings in response to salt stress. Relative water content and concentration of anthocyanins in the leaves of pistachio genotypes remained unchanged under salt stress. The results revealed that monitoring leaf abscission, SLA, leaf succulence, MDA concentration, and photosynthetic pigments provide suitable contrast for screening salt tolerance in pistachio. Furthuremore, 'Akbari' was found to be the most salt tolerant genotype. Key words: interspecific hybrid; leaf pigments; morphophysiological adaptation; salt stress; oxidative stress; Pistacia vera IZVLEČEK S SLANOSTJO VZPODBUJENE SPREMEMBE V VODNEM REŽIMU, OKSIDATIVNE POŠKODBE IN MORFOLOŠKO-FIZIOLOŠKE PRILAGODITVE GENOTIPOV PISTACIJE V BREZTALNEM GOJENJU Izbor na slanost tolerantnih podlag je primeren pristop pri razvoju sadnih dreves na območjih podvrženih zasoljevanju. 60-dni stare sejanke pistacije (Pistacia vera 'Akbari' in 'Ghazvini', ter P. vera 'Ghazvini' x P. atlantica (GxA)) so bile izpostavljene 0, 50, 100 in 150 mM NaCl v polovični Hoaglandovi hranilni raztopini. Parametri rasti, vodnih razmer in oksidativnih poškodb so bili preučeni po 45 dneh. Solni stres je zmanjšal biomaso rastlin, višino, premer krošnje in število listov, a povečal specifično listno površino (SLA) sejank. Pod solnim stresom je bila rast sejank 'Akbari' večja kot drugih genotipov. Akumulacija malondialdehida (MDA) in prolina je bila opažena v listih od soli prizadetih sejank. Sejanke 'Ghazvini' so imele največjo koncentracijo MDA in najmanjšo stabilnost celičnih membran listov. Razgradnja fotosinteznih pigmentov v listih sejank 'Akbari'je bila v solnem stresu manjša kot pri drugih genotipih. Povečanje sukulence listov kot odziv na solni stres je bilo opaženo pri sejankah 'Akbari' and GxA. Relativna vsebnost vode in vsebnost antocianinov v listih sta pri vseh genotipov pistacije ostali nespremenjeni v solnem stresu. Izsledki so odkrili, da daje spremljanje odpadanja listov, SLA, listne sukulence, koncentracije MDA in fotosinteznih pigmentov primernen nabor znakov za odkrivanje tolerance na sol pri pistaciji. 'Akbari' je bil prepoznan kot na sol najbolj tolranten genotip. Ključne besede: medvrstni križanci; listni pigmenti; morfološko-fiziološke adaptacije; solni stres; oksidativni stres; Pistacia vera 1 INTRODUCTION Salinity is one of the most serious constraints to agricultural crop production in arid and semi-arid areas. More than 1.5 Mha of irrigated lands are taken out of production each year due to high salt accumulation in the soil (Munns and Tester, 2008). Salinity reduces the growth and development of plant by affecting water availability to plant, absorption of mineral nutrients, and ion homeostasis (Parida and Dos, 2005). Furthermore, 1 Department of Horticultural Science, College of Aburaihan, University of Tehran, Tehran, Iran; Corresponding author: skarimi@ut.ac.ir Acta agriculturae Slovenica, 109 - 2, september 2017 str. 291 - 302 Zahra MIRFATTAHI et al. burst of regeneration of reactive oxygen species (ROS) under this situation damages proteins, lipids and the genetic material (Gill and Tuteja, 2010). Morphological, physiological, and biochemical adaptations to salt stress, such as stomatal closure, osmotic adjustment, ion exclusion and compartmentation, and increase in the antioxidative activity have been widely discussed in the literature (Hishida et al., 2014). Pistachio (Pistacia vera L.) is originally native to arid and semi-arid regions of the middle east and Persia. Although the plant is relatively salt tolerant (Ferguson et al., 2002), intensified salt built up in the soil of these regions due to using poor quality water for irrigation in association with frequent drought periods has reduced its production over recent decades (Karimi and Rahemi, 2012). Selecting salt tolerant rootstocks is an effective approach for sustainable development of pistachio in such salt prone areas. In this regard, understanding the effects of salinity on pistachio is of a crucial importance for establishing a successful rootstock breeding program. Although there are many reports on evaluating salt tolerance of pistachio genotypes and related species, some controversies in results of these researches can be found. The issue is mainly due to physico-chemical differences in growing media, and using convenient criteria for evaluating salt tolerance of pistachio which are mainly developed for herbaceous annual crop species. However, performing such experiments in soilless culture may reduce experimental errors by increasing uniformity of growing medium. Absence of complex interactions between soil, the stressor, and plant in soilless condition enhances repeatability of results and provide a clear understanding of the plant responses to salinity. Therefore, in this study the effects of salt stress were evaluated on the growth, morpho-physiological adaptations, and biochemical characteristics of pistachio seedlings in soilless culture in order to screen salt tolerance. Moreover, effectiveness of different criteria for screening salt tolerance in pistachio seedlings was investigated. 2 MATERIAL AND METHODS 2.1 Plant material and experimental conditions This study was conducted at the Department of Horticultural Science of University of Tehran in 201415. Seeds of pistachio 'Ghazvini' and 'Akbari' were obtained from Pistachio Research Station of Damghan, Iran. Seeds of interspecific hybrid P. vera 'Ghazvini' x P. atlantica Desf. (GxA) were obtained by controlled hybridization (Morovati, 2013). The seeds were soaked for 24 hrs and then treated with 0.2 % captain fungicide for 4 hrs. The seeds were transferred to clean containers with moistened filter paper and allowed to germinate for 5 days at room temperature. Three germinated seeds were sown in 4.5 liter plastic pots containing 3.0 kg of coco-peat and perlite mixture (2:1 volume ratio) in a greenhouse with average day/night temperatures of 30/24 °C and air humidity of 10/22 %. The pots were irrigated with tap water for about 30 days. At four leaved stage, the seedlings started to receive half-strength Hoagland's nutrient solution (Hogland and Arnon, 1950) for a 60-day period and then subjected to four NaCl concentrations (0, 50, 100 and 150 mM) in the nutrient solution for 45 days. The seedlings were irrigated with the nutrient solutions every 48 hrs. To avoid salt build-up in the pots, the plants were irrigated enough to ensure drainage of 30 % of the solutions. 2.2 Growth parameters After 45 days of exposure to the salt stress plant height, leaf number and crown diameter of the seedlings were measured. Then, the plants were harvested and their fresh mass and dry mass were determined. Specific leaf area (SLA) was measured by determining dry mass of fifteen leaf discs (0.90 cm diameter) from the 4-5th fully expanded leaves from shoot top according to Eq. 1: Eq. 1 2.3 Water relations Leaf water potential (TLeaf) was measured at midday (11:00-12:00) using a portable pressure chamber device (Soil Moisture Equipment Corp., USA). TLeaf was measured immediately after excising the 4-5th fully expanded leaves from the top of the stem. Leaf relative water content (RWC) was measured by punching fifteen discs (0.90 cm diameter) from the developed leaves. The discs were weighted (FM), floated on distillated water for 24 hrs (at 4 C in dark) to obtain turgid mass (TM), and finally their dry mass was recorded 72 hrs after placing at 70 °C (DM). RWC was calculated according to Eq. 2: RWC = Eq. 2 Leaf water content was expressed as the percentage equivalent of the ratio of the mass of water (FM - DM) to the leaf dry matter (Md) (Eq. 3). fm-DM Leaf water content = ——— X 1UU Eq. 3 Md Area basis leaf water content (succulence index) was expressed as the ratio of the mass of water to the area of 292 Acta agriculturae Slovenica, 109 - 2, september 2017 Salinity induced changes in water relations, ... adaptations of pistachio genotypes in soilless culture the leaf sample (LAS). In this order, fifteen foliar discs (0.90 cm diameter) from the developed leaves of shoot top were used (Eq. 4). FMDM Succulence = , . _— Eq. 4 LAS 2.4 Leaf proline concentration Leaf proline concentration was measured according to the method described by Bates et al. (1973). Leaf tissue (0.1 g) was extracted in 10 ml of 3 % sulphosalicylic acid and then, 2 ml of ninehydrin reagent and 2 ml acetic acid were added to 2 ml of the extract. Then, the samples were heated in boiling water for 60 min. Four milliliters of toluene was added to each sample and vortexed for 15-20 seconds. The absorbance of toluene phase was measured at 532 nm using a spectrophotometer (Perkin Elmer, Lambada 25, USA). The concentration of proline was determined according to an external standard curve. 2.5 Oxidative damage parameters Cell membrane stability index (CMS) was determined by measurement of electrolyte leakage from leaf samples. Fifteen leaf discs (0.90 cm diameter) were excised from fully expanded young leaves. The leaf discs were washed three times in deionized water and incubated in 15 ml deionized water (40°C) for 30 min. The initial conductance (CO of the incubation solution was measured using an electrical conductance meter. Leaf tissue in the incubation solution was killed by placing the samples in boiling water for 10 min. The conductance of the solution (Cmax) was determined at room temperature. CMS was calculated by using the following formula (Eq. 5): CMS=l-(C7CinflJxl00 Eq.5 Lipid peroxidation was assessed by measurement of malondialdehyde (MDA) concentration in fully expanded young leaves, according to the method described by Heath and Parker, (1986). Leaf tissue (200 mg) was homogenized in 10 ml of 0.1 % trichloroacetic acid (TCA) and centrifuged at 1000 g for 5 min. Then 4 ml TCA acid 20 % and thiobarbituric acid 0.15 % were added to 1 ml of the supernatant. The absorbance was measured at 532 nm by spectrophotometry (PerkinElmer, Lambda 25, USA). 2.6 Leaf pigments In addition to evaluation of leaf greenness by using a SPAD 502 chlorophyll meter (Minolta Co., Japan), concentrations of chlorophylls and carotenoids were measured in the 4-5th developed leaves according to the method described by Lichtenthaler (1987). Fresh tissue (15x0.64 cm2 leaf discs) was extracted in 80 % acetone and after centrifuging at 4800 rpm for 20 min, the absorption of supernatant was read at 470, 647 and 664 nm using a spectrophotometer (PerkinElmer, Lambda 25, USA). The concentrations of chlorophyll a (Chl a) and b (Chl b), and carotenoids were calculated according to the following formulas (Eq. 6-8): Chl a = 12.25A664 - 2.79A647 Eq. 6 Chl b = 21.51A647 - 5.10A664 Eq. 7 Carotenoids = (1000A470 - 1.8Ca - 85.02Cj)/198 Eq. 8 For measuring anthocyanins, 500 mg fresh leaf tissue was extracted in 10 ml of methanol acidified with 1 % HCl at 4 oC for 24 hrs. The absorbance of the extract was determined at 550 nm using a spectrophotometer (PerkinElmer, Lambda 25, USA). Anthocyanin concentration was calculated using an extinction coefficient of 33000 mol-1 cm-1 (Wagner, 1979). 2.7 Statistical analyses The experiment was conducted as factorial (3 genotypes x 4 NaCl concentrations) based on a completely randomized design with three replications. Three pots were considered in each replication and mean of these pots were considered as a replication for each treatment. In sum, 108 pots were used in the experiment. The data were subjected to ANOVA and the means were compared using Duncan's multiple range test (DMRT) at P < 0.05. The statistical analyses were performed using the SPSS software (v. 21.0). 3 RESULTS The effects of salt stress on growth of pistachio seedlings are represented in Table 1. Shoot height, crown diameter, leaf number, and dry mass of the seedlings were significantly reduced under 100 and 150 mM NaCl treatments. Shoot height and leaf number of 'Akbari' seedlings were significantly higher than the other genotypes. Trunk diameter of 'Akbari' seedlings was significantly higher than the other genotypes and the lowest value was found in GxA. Fresh mass of the seedlings significantly decreased by increasing NaCl concentration in the nutrient solution. Among the genotypes, seedlings of 'Akbari' had the highest fresh and dry mass at the end of the experiment; no significant differences were observed between fresh and dry mass of 'Ghazvini' and GxA seedlings. SLA significantly increased under 100 and 150 mM NaCl treatments. The highest SLA was found in GxA and 'Akbari' seedlings had the lowest SLA. Acta agriculturae Slovenica, 109 - 2, september 2017 Zahra MIRFATTAHI et al. Table 1: The effects of salt stress and plant genotype on growth of pistachio seedlings Plant height Crown diameter Leaf number Fresh mass Dry mass SLA (cm) (mm) (g) (g) (cm2 g-1) Genotype 40.3at 0.010b Akbari 5.79a 22.9a 81.0a 36.1a Ghazvini 35.1b 4.78b 19.2b 56.6b 24.7b 0.011ab GxA 32.9b 4.44c 18.5b 47.8b 20.7b 0.012a NaCl (mM) 0.010b 0 39.9a 5.43a 24.6a 77.2a 35.0a 50 35.8ab 5.12ab 21.7ab 65.5b 28.4ab 0.010b 100 33.8b 4.87bc 18.8bc 53.1c 23.6b 0.012a 150 34.2b 4.63c 16.2c 48.5c 21.5c 0.012a ANOVA Genotype ** ** * ** ** * NaCl Level * ** ** ** ** ** NaClxGenotype ns ns ns ns ns ns Difference among the treatments was analyzed by 3 genotypes x 4 salt stress ANOVA; ns, *, ** indicate nonsignificant, and significant differences at 0.05 and 0.01, respectively. The data are means of 3 replicates; Mean separation was performed according to DMRT (P < 0.05) and similar letters indicate no significant difference between mean values. Relative water content (RWC) and water potential (^Leaf) of the leaves were not affected by the NaCl treatments. However, TLeaf was significantly higher in the leaves of 'Akbari' seedlings (-0.68 MPa) and the lowest value (-0.892 MPa) was found in 'Ghazvini' leaves (Table 2). Leaf water content and succulence were significantly affected by the interactive effects of salt stress and plant genotype (P < 0.01). Water content significantly increased in the leaves of 'Akbari' and GxA under 100 and 150 mM NaCl stress up to 21 and 41 percent, respectively. However, no significant changes were observed in leaf water content of 'Ghazvini' under salt stress (Figure 1). Unlike the other genotypes, area basis leaf water content was significantly reduced in the leaves of 'Ghazvini' under 150 mM NaCl by 16.2 percent (Figure 1). Figure 1: The interactive effects of salt stress and genotype on water content and succulence of the leaves of pistachio seedlings. Mean separation according to DMRT at P < 0.05 (n = 3) Significant increase in leaf proline concentration was observed under severe salt stress. Among the genotypes, 'Ghazvini' and 'Akbari' had more leaf proline content than GxA (Table 2). Cell membrane stability index (CMS) remained unchanged under salt stress, too (Table 2). However, CMS in the leaves of 'Ghazvini' was significantly lower than the other genotypes. Significant accumulation of malondialdehyde (MDA) concentration was found in the leaves of the seedlings under salt stress and the highest increase in MDA concentration (45.9 %) was observed under 150 mM NaCl treatment. GxA had the highest leaf MDA concentration (15.5 mmol g-1), and the lowest concentration (10.56 mmol g-1) was found in 'Akbari' (Table 2). 294 Acta agriculturae Slovenica, 109 - 2, september 2017 Salinity induced changes in water relations, ... adaptations of pistachio genotypes in soilless culture Table 2: The effects of salt stress and plant genotype on relative water content (RWC), water potential (¥Leaf), proline, cell membrane stability index (CMS), and concentration of malondialdehyde (MDA) in the leaves of pistachio seedlings RWC ^Leaf Proline CMS MDA (%) (MPa) (pmol g -1) (%) (mmol g-1) Genotype -0.680at 267.8ab 10.65b Akbari 83.3 90.7a Ghazvini 84.0 -0.892b 296.7a 83.5b 12.26ab GxA 85.3 -0.744ab 218.1b 92.1a 15.50a NaCl (mM) 231.2b 10.72b 0 84.2 -0.851 88.8 50 84.0 -0.683 258.2b 90.7 11.94ab 100 85.3 -0.755 271.0ab 88.2 13.25ab 150 83.6 -0.851 283.3a 87.0 15.65a ANOVA Genotype ns ** * ** * NaCl Level ns ns * ns * NaClxGenotype ns ns ns ns ns Difference among the treatments was analyzed by 3 genotypes x 4 salt stress ANOVA; ns, *, ** indicate nonsignificant, and significant differences at 0.05 and 0.01, respectively. The data are means of 3 replicates; Mean separation was performed according to DMRT (P < 0.05) and similar letters indicate no significant difference between mean values. Table 3 represents the effects of salt stress and plant genotype on concentration of leaf pigments. Chlorophyll a:b ratio (Chl a:b) significantly increased in the leaves of pistachio seedlings in response to salt stress. Chl a:b in the leaves of 'Akbari' was significantly lower than the other genotypes. Leaf greenness, which was measured by SPAD, significantly reduced under salt stress. However, no significant difference was observed in leaf color of the genotypes. Salt stress significantly reduced total chlorophyll concentration in the leaves. 'Akbari' had the highest leaf chlorophyll concentration than the other genotypes. Concentration of anthocyanins and carotenoids in the leaves remained unchanged under salt stress, however, concentration of carotenoids in the leaves of 'Akbari' was higher than 'Ghazvini' and GxA. Table 3: The effects of salt stress and plant genotype on concentration of pigments in the leaves of pistachio seedlings SPAD Chl a:b Total Chls Carotenoids Anthocyanins (mg cm-2) (mg cm-2) (mmol g-1) Genotype 0.395bt Akbari 55.0 29.2a 2.23a 280.9 Ghazvini 55.9 0.420a 25.4b 2.03b 282.1 GxA 56.1 0.423a 23.6b 1.90b 282.3 NaCl (mM) 0.396b 0 57.8a 27.9a 2.13 289.5 50 55.4b 0.422a 23.8b 1.90 279.3 100 54.6b 0.421a 26.8ab 2.08 283.3 150 54.9b 0.416a 25.2b 2.06 275.3 ANOVA Genotype ns ** ** ** ns NaCl Level * ** * ns ns NaClxGenotype ns ns ns ns ns Difference among the treatments was analyzed by 3 genotypes x 4 salt stress ANOVA; ns, *, ** indicate nonsignificant, and significant differences at 0.05 and 0.01, respectively. The data are means of 3 replicates; mean separation was performed according to DMRT (P < 0.05) and similar letters indicate no significant difference between mean values. Acta agriculturae Slovenica, 109 - 2, september 2017 Zahra MIRFATTAHI et al. Chlorophylls:carotenoids ratio (Chl:Crt) was 'Akbari' and 'Ghazvini' seedlings. A significant significantly affected by the interactive effect of salt decrease in Chl:Crt ratio was found in the leaves of stress and plant genotype (P < 0.01). Chl:Crt ratio G*A under salt stress (Figure 2). remained unchanged in the leaves of salt stressed □ 0 Q50 H100 ■ 150 mM NaCl 14 - Akbari Ghazvini G> 0 (2) MWTP = 0 if MWTP* < 0 (3) where MWTPi* is the end user's (farmer's) unobserved 'maximum willingness to pay' amount for the excreta pellets; MWTPi is an end user's (farmer's) actual 'maximum willingness to pay amount' for the excreta pellets; X' is vector of explanatory variables; ft is vector of unknown true coefficients; a is the intercept; and £i is disturbance term, which is assumed to be normally independently distributed, i.e., NID (0, c2) and independent of xi. Assuming that the variable is zero, then the dependent variable in the Tobit regression model is a continuous variable (i.e. the maximum amount quoted by end users to pay for 50 kg excreta pellets as fertilizer). The amount end users were willing to pay was hypothesized to be influenced by the following factors: Age of the Respondent (AGE): This is a continuous variable indicating the age of the respondent in years. The age of respondent may have either negative or positive effect on WTP decision. Aged farmers may not see the need for long term sustainability of soil fertility and may be conservative to new ideas hence may show negative attitude towards the faecal sludge fertilizer. On the other hand, as they grow much older, they may become more experienced thereby willing to conserve the soil through the use of excreta pellets and hence positively influencing their WTP decision (Tessema & Holden 2006; Asgary et al., 2004; Dong et al., 2003). Experience (EXP): Farming experience leads to increased willingness to improve the soil for better productivity (Tessema & Holden, 2006). Hence experience is expected to positively influence WTP decision. Education Level of the Respondent (EDUCATION): This parameter refers to the number of years that the respondent has spent in a formal school. Following Paulos (2002) and Yitayal (2004), the study assumed that respondents who had high level of education better understood the problems of soil erosion or degradation and its consequences and therefore would be willing to invest in a product that increases fertility and conservation of the soil. Household Head (HHD): This is a dummy variable which takes a value of 1 if the respondent is the head of the household; and 0 otherwise. It is hypothesized that being a household head is expected to influence willingness to pay positively. Household Size (HSIZE): This is a continuous variable which refers to the number of family members in the household. This explanatory variable is included because it affects the labor supply at the household level. Also, households with more people to feed will be willing to pay for a new input that would increase yield (Agyekum et al., 2014). Gender of the Respondent (Gender): dummy variable (1= male, 0 = otherwise) and is included in the model to find out the influence of gender on WTP. Males have a probability of getting better access to information than females. Therefore, a positive influence is hypothesized for male respondent. Female farmers tend to adopt new technologies at a lower rate than males due to seemingly limited access to information and resource (Doss & Morris, 2001). Farm Size (Farmsize): It is a continuous variable expressed in terms of hectares of cultivated land and expected to have a positive effect on the willingness to pay. This is expected because farmers (end users) with larger land size tend to be commercially oriented and hence would invest in an input that will positively affect production (Oladele, 2008; Cofie, 2010). Farm Income (FARMINC): This is a continuous variable. It is measured as the annual farm income from sale of produce. It is expected to have a positive relationship with WTP. As income increases it positively affects the WTP for waste as fertilizer. This can be linked to farmers having more money to afford such a product. Numerous empirical studies have reported positive relationships between income and adoption of agricultural technologies and input (Faye & Deininger, 2005; Holden & Shiferaw, 2002; Mbata, 2008). Aware of Faecal Fertilizer (Aware): This is a dummy variable which assumes 1 for "yes I am aware of faecal fertilizers", and 0 otherwise. It is hypothesized that respondents who have prior knowledge of faecal sludge-based fertilizer would be more willing to use excreta pellets since they are less bulky and the unpleasant smell is removed. Also, farmers who were aware of 318 318 Acta agriculturae Slovenica, 109 - 2, september 2017 Willingness to pay for excreta pellet fertilizer: Empirical evidence from Ghana other available agricultural inputs were more receptive to paying for these inputs (Asrat et al., 2004). Quantity of Fertilizer Previously Used (Number of 50 kg bags): A continuous variable that measures the amount of fertilizer used in the previous year. A unit was pegged at 50 kg bag based on the fact that the majority of the respondents bought fertilizers packaged in 50 kg bags. It is hypothesized that an increase in the quantity used will increase the probability of end users WTP. Unit Cost (unit cost): This is a continuous variable that measures the average cost of a unit (50 kg bag) of fertilizer used in the previous year. It is hypothesized that an increase in price of the cost of fertilizer would lead to an increase in the probability of WTP. Previous Use of Organic Fertilizer (Used Organic): This is a dummy variable that assumes 1 for "yes I have ever used organic fertilizer" and 0 otherwise. It was hypothesized that previous use of organic fertilizer would positively influence the end users WTP decision. Membership of Farmer Based Organization (member of FBO): This is a dummy variable which assumes 1 for "yes I am a member" and 0 for otherwise. It is hypothesized that being a member of a farmer-based organization (FBO) will positively affect the WTP amount. Own Current Land (own land): This is a dummy variable which assumes 1 for "yes I own the land that am currently working on" and 0 for otherwise. Following Obuobie et al., (2006), a positive relation with maximum WTP amount is hypothesized. The previous study revealed that most farmers on hired land were not allowed by the land owners to use faecal sludge based fertilizer in the city of Tamale in the northern region of Ghana. 3 RESULTS AND DISCUSSION 3.1 Preliminary results The study found that about 69 % of the farmers interviewed were aware of excreta pellets as fertilizer whilst 31 % of the total respondents were not aware. This indicates that the use of faecal sludge as fertilizer is not an alien concept to farmers. The farmers agreed with the assertion that fertilizer derived from human excreta should be certified by a relevant body such as the Environmental Protection Agency (EPA) before its introduction onto the market, and that certification by a relevant authority would lead to increased willingness to pay for excreta pellet fertilizer. Farmers also agreed that excreta pellets can be used as a replacement for conventional chemical fertilizers. Farmers in northern Ghana disclosed that human excreta when used in farming produce better and safer yields (i.e. enhance agricultural productivity) than conventional fertilizers, hence it should be used in place of the conventional fertilizers (Kranjac-Berisavljevic, 2009). Farmers perceived that such a fertilizer has a positive effect on long term fertility of soils. With regard to health, farmers did not agree that using faecal-based fertilizer would expose them to several major health risks. However, they did indicate that use of raw or untreated human excreta on farmland is associated with some skin infections, diarrhea, foot rot and vomiting. The majority of respondents interviewed (91.1 %) claimed that they would not buy excreta pellets if it is sold at the same price as conventional fertilizer. However, they would buy excreta pellets if cheaper than conventional 4 Please note that 342 respondents out of the total sample of 461 were chemical fertilizers; in other words, they expect the price of the product to be lower than the price of conventional chemical fertilizers (Cofie et al, 2009). Majority of respondents that indicated their WTP for excreta pellets, only 29 % stated they were not willing to pay. The reasons stated were: those excreta pellets have not been certified for use by farmers, especially under global GAP; and that some farmers also claimed that excreta pellets need to be tested on their produce before they would be willing to buy. One of the reasons for not willing to pay was that the product was not certified and not tested in their locality to ascertain its quality. Some were also of the view that since the product is made from human excreta which are waste coupled with disposal being a major problem to local authorities, it should not be sold. In general, over 70 % of respondents were willing to pay for excreta pellets as fertilizer and this level of approval is consistent with previous studies (Danso et al. 2006, and Agyekum et al. 2014). Table 1 shows the distribution of the willingness to pay amounts by the respondents.4 Nine respondents (representing 2.6 %) were willing to pay less than GHS 10.00 [$ 4.22] for a 50kg bag of the excreta pellets. Forty eight respondents (representing 14 %) were willing to pay between GHS 10.00 and GHS 19.99 [$ 4.22 - $ 8.43]. Also, one hundred and ninety seven respondents (representing 57.6 %) were willing to pay between GHS 20.00 and GHS 29.99 [$ 8.44 - $ 12.65]. ng to pay for the excreta pellets. Acta agriculturae Slovenica, 109 - 2, september 2017 John K. M. KUWORNU et al. Seventy seven respondents (representing about 22.5 %) (representing about 3.2 %) were willing to pay GHS were willing to pay between GHS 30.00 and GHS 39.99 40.00 and GHS 50.00 [$ 16.88 - $ 21.10]. [$ 12.66 - $ 16.87], and eleven respondents Table 1: Maximum willingness to pay amount WTPA Frequency Percentage <10 9 2.6 10-19.99 48 14.0 20-29.99 197 57.6 30-39.99 77 22.5 40-50 11 3.2 Total 342 100 3.2 Tobit regression model results of the factors influencing willingness to pay maximum amount The minimum price per 50 kg bag quoted was GHS 2.00 [$ 0.84] and a maximum of GHS 50.00 [$ 21.10]. The mean price quoted was GHS 22.83 [$ 9.63]. This mean amount was quite higher than that quoted by respondents in Agyekum et al. (2014) for 50 kg of faecal compost. The Tobit regression model results of the factors that influenced willingness to pay amounts quoted by the farmers are presented in Table 2. Being a household head was significant and had a positive relation with the willingness to pay. A household head was willing to pay GHS 5.12 [$ 2.16] more for a bag of excreta pellets compared to an end user who is not the head of the household. This willingness to pay more for such an input may be due to the responsibilities associated with such a position that would make the farmer want to invest in soil conservation (Gebremedin, 2012). Farm size was also significant and positively influenced WTP amount, as farm size increased by a hectare, the WTP amount increased by GHS 0.48 [$ 0.20]. This could be due to high cost of use of inorganic fertilizer, as farm size increases end users would be more willing to use faecal sludge-based fertilizers. End users with larger farm size would also be willing to pay more for excreta pellets because as stated by Oladele (2008) they are commercially oriented and hence would like to invest in an input that will impact positively on production. The results also revealed a negative and significant relation between the amounts an end user was willing to pay and if he had ever used an organic fertilizer. Thus, if the end user has ever used organic fertilizer, he was willing to pay GHS 2.41 [$ 1.02] less for the 50 kg of the excreta pellets compared to a respondent who has never used organic fertilizer. This relation could be attributed to the cheaper prices of organic fertilizer available as some farmers acquired organic fertilizers for free and are only required to pay for the transportation cost. Hence, this category of farmers will not be willing to pay a high price for another organic product. This assertion is consistent with previous studies in which farmers with compost or manure experience were willing to pay less for faecal compost (Drechsel et al. 2004, Danso et al. 2006). Also, if organic fertilizer were available, use by farmers may be very low due to limited knowledge and management among farmers (Obour et al., 2015). As expected, there is a positive relation between willingness to pay amount and the average unit cost of fertilizer currently being used by the end user. A GHS 1 increase in the unit cost of the current fertilizer being used will lead to paying GHS 0.06 [$ 0.03] more for excreta pellets (though negligible). This can be attributed to an end user looking to invest in cheaper alternatives. 320 Acta agriculturae Slovenica, 109 - 2, september 2017 Willingness to pay for excreta pellet fertilizer: Empirical evidence from Ghana Table 2: Tobit regression results of the factors influencing maximum willingness to pay amount Variable Coefficient P>t Gender -2.160 0.147 Age 0.030 0.608 Household Head 5.119*** 0.001 Household Size 0.050 0.813 Own Land -0.146 0.903 Used Organic -2.406** 0.030 No Of 50 kg -0.006 0.271 Unit Cost 0.040** 0.022 Farm Size 0.480** 0.024 Income 0.000 0.376 Education -0.058 0.660 FBO Member 1.731 0.147 Experience -0.050 0.405 Aware Of Faecal Fertilizer 1.183 0.309 Constant 11.969 0.000 Number of obs 459 F(14, 445) 2.800 Prob > F 0.001 Pseudo R2 0.001 Log pseudolikelihood -1770.3 *, ** and *** denotes 10 %, 5 % and 1 % significant level 4 CONCLUSIONS AND IMPLICATIONS The results of this study revealed that the perceptions about fertilizer derived from human excreta are positive. Majority of respondents did not have religious, health and cultural hindrances to the use of excreta product. They described excreta pellet as a potentially good replacement for conventional fertilizer. Respondents also indicated that they would use a faecal sludge-based fertilizer provided it was certified by the Environmental Protection Agency since this certification and labeling would lead to trust in the product. Interestingly, almost all respondents indicated that they would not purchase excreta pellet if it is sold at the same price as conventional chemical fertilizer. However, these respondents mentioned that they would purchase the excreta pellet if it is sold cheaper than the conventional chemical fertilizers on the market. There is a potential for the marketing of excreta pellet as fertilizer in Ghana. Producers of different backgrounds were interested in using the product for both crop and non-crop agricultural enterprises as they expressed positive perception about the product. End users were willing to pay a minimum of GHS 2.00 [$ 0.84], a maximum of GHS 50.00 [$ 21.10] and a mean of GHS 22.83 [$ 9.63]. to obtain a 50 kg bag of excreta pellet fertilizer. The results of the Tobit regression model revealed that being a household head, unit cost of current fertilizer used, and farm size positively influenced the willingness to pay amount whereas previous use of organic fertilizer influenced the willingness to pay amount negatively. Thus, the marketing of excreta pellet as fertilizer has prospects in Ghana. End users are interested in reducing cost hence would welcome any input that is cheaper and effective. Based on these findings, this paper recommends that marketing of faecal sludge based fertilizer should target people in decision making positions such as household heads with large farm sizes and businesses in the manufacturing sector of Ghana should find innovative ways of reducing the cost of production of excreta pellet fertilizer so as to reduce the price when offered on the market. A cheaper product will be wholly accepted by end users. The Ghana Standards Authority should support manufacturing companies to satisfy certification requirements of the product before introduction onto the market. Demonstration plots should be identified at the district level so as to give first hand education on the use of the product. For florists, the product should be tested on their flowers to dispel the notion that it might be too strong for the plants and may cause wilting. Also a protocol or brochure should be added to the product (when sold), indicating how product should be applied to meet specific crop or plant nutrients requirements. Marketing of the product should target farmer-based organizations, people in social positions such as household heads and people with larger land holdings. Information about the efficacy of product will flow to other end users. Government institutions such as Environmental Protection Agency (EPA), Council for Acta agriculturae Slovenica, 109 - 2, september 2017 John K. M. KUWORNU et al. Industrial and Scientific Research (CSIR) and the Ministry of Food and Agriculture (MoFA) should establish clear guidelines and standards on the use of faecal sludge-based fertilizers. When establishing quality guidelines, the institutional capacities for controlling and enforcing them should also be taken into account. More importantly, political will and legal tools should be adequate enough to enforce quality standards. This will go a long way to help maintain end users' confidence in the product for the long term since the raw material is inexhaustible. MoFA must intensify its education on the use of protective clothing especially gloves when applying the excreta pellet fertilizer. 5 ACKNOWLEDGEMENTS The authors gratefully thank International Water funding for this study. The authors also thank the Management Institute, Accra, Ghana for providing respondents for their answers and time. 6 REFERENCES Agyekum, E. O., Ohene-yankyera, K., Keraita, B., Fialor, S. C. & Abaidoo, R. C. (2014). Willingness to Pay for Faecal Compost by Farmers in Southern Ghana. Journal of Economics and Sustainable Development 5(2), 18-25. Asgary, A., K.Willis, A. A. Taghvaei, and M. Rafeian (2004). 'Estimating rural households' willingness to pay for health insurance', European Journal of Health Economics. 5.209-215. Asrat, P., K. Belay, and D. Hamito. 2004. Determinants of farmers' willingness to pay for Soil Conservation Practices in the Southeastern Highlands of Ethiopia. Land Degradation and Development, 15, 423-438. doi: 10.1002/ldr.623 Cofie O. & Adamtey N., (2009). Nutrient Recovery from Human Excreta for urban and peri-urban Agriculture. Contribution to SuSanA Food Security Working Group meeting during the WEDC international conference in Addis Ababa, Ethiopia. April 15 2009. Cofie, O., Adeoti, A., Nkansah-boadu, F., & Awuah, E. (2010). Resources , Conservation and Recycling Farmers perception and economic benefits of excreta use in southern Ghana. Resources,Conservation & Recycling, 55(2), 161166. doi:10.1016/j.resconrec.2010.09.002 Danso, G., Drechsel, P. Fialor, S. and Giodano, M. (2006). Estimating the demand for municipal waste compost via farmers' willingness-to-pay in Ghana. Waste Management Journal, 26(12), 1400-9. doi:10.1016/j.wasman.2005.09.021 Dong, H., B. Kouyate, J. Cairns, F. Mugisha and R. Sauerborn (2003). 'Willingnessto pay for community based insurance in Burkina Faso. Health Economics, 12(10), 849-862. doi:10.1002/hec.771 Doss, C. R., & Morris, M. L. (2001). How does gender affect the adoption of agricultural innovations: The case of improved maize technology in Ghana. Agricultural Economics, 25, 27-39. doi:10.1016/S0169-5150(00)00096-7 Drechsel P, Cofie O, Fink M, Danso G, Zakari F, & Vasquez R. (2004). Closing the rural-urban nutrient cycle. Options for municipal waste composting in Ghana. Final scientific report on IDRC project 100376, IWMI-West Africa, Accra. Escherich, T. (1885). Ueber die bacterien des milchkothes, Artz Intelligenz-Blatt, Mün Med Wcsht, 32, pg. 243. Escherich, T. (1885). Die darmbakterien des neugeborenen und säuglings, Fortsch der Med, 3, (pg. 515-22) 547-54. Foti R, Nyakudya I, Moyo M, Chikuvire J, & Mlambo N. (2007). Determinants of Farmer Demand for "Fee-for-Service Extension in Zimbabwe: The Case of Mashonaland Central Province. Journal of International Agricultural and Extension Education, 14 (1), 95-104. doi:10.5191/jiaee.2007.14108 Faye, I., & Deininger K. (2005). Do New Delivery Systems Improve Extension Access? Evidence from Rural Uganda. Paper presented at the American Agricultural Economics Association Annual Meeting, July 24-27, Providence, RI, US. Gebremedin, G. (2012). Households' willingness to pay for soil conservation practices in Adwa Woreda, Ethiopia : A Contingent Valuation Study. A Thesis Submitted To The Faculty Of Development Studies In Partial Fulfilment For The Requirements Of Masters of Science Degree In Agricultural and Applied Economics, University Of Malawi. 322 Acta agriculturae Slovenica, 109 - 2, september 2017 Willingness to pay for excreta pellet fertilizer: Empirical evidence from Ghana Harris, P.J.C., Allison, M., Smith, G., Kindness, H.M. & Kelly, J. (2001). 'The Potential Use of Waste Streams. Products for Soil Amelioration in Periurban Interface Agricultural Production Systems', in Pay Drechsel and Agmar Kunze (eds), Waste Composting for Urban and Peri-irban Agriculture: Closing the Rural-Urban Nutrient Cycle in Sub-Saharan Africa, CABI Publishing, Wallingford, Oxon, UK, pp. 1-28. doi:10.1079/9780851995489.0001 Hofny-Collins A. (2006) The Potential for Using Composted Municipal Waste in Agriculture: The case of Accra, Ghana. Doctoral thesis Swedish University of Agricultural Sciences Uppsala 2006. Acta Universitatis agriculturae Sueciae, 65, 16526880. Holden, S. T., and B. Shiferaw. (2002). "Poverty and Land Degradation: Peasants' Willingness to Pay to Sustain Land Productivity." In The Adoption of Natural Resource Management Practices: Improving Sustainable Agricultural Production in Sub-Saharan Africa, edited by. C. B. Barrett, F. M. Place, and A. A. Aboud, 91-102. New York: CABI Publishing. doi:10.1079/9780851995847.0091 Kranjac-Berisavljevic G., (2009). Use of Faecal Sludge for Agriculture in Tamale Metropolis: perception of Farmers, Consumers and Relevant Agencies. Presentation from the 2009 World Water Week. Mariwah, S., & Drangert, J.-O. (2011). Community perceptions of human excreta as fertilizer in periurban agriculture in Ghana. Waste Management & Research, 0(0), 1 -8. doi:10.1177/0734242x10390073 Mbata, G. I. O. (2008). Determinants of profitability and willingness to pay for metropolitan waste-use in urban agriculture of The Federal Capital Territory , Abuja , Nigeria Federal Capital Territory Agricultural Development Programme , Gwagwalada , Abuja. Journal of Tropical Agriculture, Food, Environment and Extension, 7(1), 41-46. Nikiema J., Cofie O., Impraim R., & Drechsel P. (2012). Fortified Excreta Pellets for Agriculture. International Water Management Institute - West Africa Office, www.iwmi.org. Obour, P. B., Dadzie, F. A., Kristensen, H.L., Rubœk, G. H., Kjeldsen, C., & Saba, C. K. S. (2015). Assessment of farmers' knowledge on fertilizer usage for peri-urban vegetable production in the Sunyani Municipality, Ghana. Resources, Conservation & Recycling, 103, 77-84. doi:10.1016/j.resconrec.2015.07.018 Obuobie, E., Keraita, B., Danso, G., Amoah, P., Cofie, O., Rachid-Sally, L. & Drechsel, P. (2006). Irrigated Urban Vegetable Production in Ghana. Characteristics, Benefits and Risks. CSIR-INST Accra, Ghana. Pp.70-98. Oladele, O. I. (2008). Factors Determining Farmers' Willingness To Pay For Extension Services In Oyo State, Nigeria. Agricultura Tropica Et Subtropica, 41(4), 165-170. Paulos, A. (2002). Determinants of Farmers" Willingness to Participate in Soil Conservation Practices in the Highlands of Bale: The case of Dinsho farming system area. MSc. Thesis, Alemaya University. Haramaya, Ethiopia. Phuc, P. D., Konradsen, F., Phuong, P. T., Cam, P. D., & Dalsgaard, a. (2006). Practice of using human excreta as fertilizer and implications for health in Nghean Province, Vietnam. The Southeast Asian journal of tropical medicine and public health, 37(1), 222-9. Retrieved from http://www.ncbi.nlm.nih.gov/pubmed/16771238. Tessema, W., & Holden, S. (2006). Soil Degradation, Poverty, and Farmers" Willingness to Invest in Soil Conservation: A case from a Highland in Southern Ethiopia. Ethiopian Economic Association, Proceedings of the Third International Conference on the Ethiopian Economy, Vol. 2, pp 147- 164. Tobin, J. (1958). Estimation of relationships for limited dependent variables. Econometrica, 26 (1), 24-36. doi:10.2307/1907382 Yitayal, A. (2004). Determinant of Use of Soil Conservation Measures by Small Holder Farmers; in Gimma Zone, Dedo District. An MSc Thesis Presented to School of Graduate Studies of Alemaya Universtity. Zurbrugg, C., Drescher, S., Rytz, I., Sinha, A. H. M. M., & Enayetullah, I. (2005). Decentralised composting in Bangladesh, a win-win situation for all stakeholders. Resources, Conservation and Recycling, 43(3), 281-292. doi:10.1016/j.resconrec.2004.06.005 Acta agriculturae Slovenica, 109 - 2, september 2017 doi: 10.14720/aas.2017.109.2.15 Original research article / izvirni znanstveni članek Unfair practices and illicit conduct in food supply chains in Slovenia Jože PODGORŠEK1 Received January 19, 2017; accepted May 05, 2017. Delo je prispelo 19. januarja 2017, sprejeto 05. maja 2017. ABSTRACT The food supply chain in Slovenia is highly developed, but it involves unequally developed stakeholders with different bargaining power. Upon reviewing all stakeholders through participatory research, it is made clear that in the whole food supply chain, retail chains generally have the greatest and primary producers the smallest bargaining power. For this reason, in the process of regulating mutual relations in contractual commitments and mutual operations, unfair practices and illicit conduct often emerge, through which the parties with significant market power impose additional discounts, rebates and other contributions on the parties with smaller market power in order to improve their own financial management. Unfair practices and illicit conduct lead to the weakening of the entire food supply chain, so it is important to recognise such tendencies in the food chain. In this article, the autor would like to draw attention to the illicit conduct and unfair practices in Slovenia which are used by retail chains in their interpersonal relationships and their dealings with suppliers. Key words: unfair practices; illicit conduct; food supply chain; significant market power; imposition of conditions IZVLEČEK NEPOŠTENE PRAKSE IN NEDOVOLJENA RAVNANJA V VERIGI PRESKRBE S HRANO V SLOVENIJI Veriga preskrbe s hrano v Sloveniji je izredno razvita, vendar v njej sodelujejo različno razviti deležniki z različno pogajalsko močjo. Ob pregledu vseh deležnikov preko participativnega raziskovanja se izlušči, da imajo največjo pogajalsko moč v celotni verigi preskrbe s hrano trgovske verige in praviloma najmanjšo primarni pridelovalci. Zaradi tega so pri urejanju medsebojnih odnosov pogosto v pogodbenih zavezah in medsebojnem poslovanju prisotne nepoštene prakse in nedovoljena ravnanja, s katerimi stranke z znatno tržno močjo vsiljujejo dodatne popuste, rabate in druge prispevke strankam z manjšo tržno močjo z namenom izboljševanja svojega finančnega poslovanja. Zaradi nepoštenih praks in nedovoljenih ravnanj prihaja do slabitve celotne verige preskrbe s hrano, zato je pomembno, da jih v verigi preskrbe s hrano prepoznamo. V tem prispevku želim opozoriti na nepoštene prakse in nedovoljena ravnanja, ki so jih trgovske verige v Sloveniji vključile v medsebojne odnose pri poslovanju z dobavitelji. Ključne besede: nepoštene prakse; nedovoljena ravnanja; veriga preskrbe s hrano; znatna tržna moč; vsiljevanje pogojev 1 INTRODUCTION The food supply chain is a major employer in Europe and the Slovenian area. On the basis of the data in the report presented by the European Parliament (Jackiewicz, 2015), more than 47 million people in the EU are employed by the food sector. Production, processing, logistics and food sales in Slovenia employ all together about 87.000 people (ReSURSKZ, 2011), and additional new jobs will be opened up, which is due to the increasing self-sufficiency in Slovenia. Because of the quality jobs in the operating agri-food chain, the needs of the state for various social transfers are being consequently reduced. Increased production and food processing have lead to the growing consumption of raw materials for agricultural production and thus the revenues to the state budget have increased. A well-functioning agri-food chain promotes economic growth equally well and increases the purchasing power of rural areas. Increasing demand for food of Slovenian origin generates other aspects of preserving the Slovenian countryside as well, enabling the development of other 1 Food supply chain relationships ombudsman, The Ministry of agriculture, forestry and food, Dunajska 22, SI-1000 Ljubljana, Slovenia, e-mail address: joze.podgorsek@gov.si Translator: dr. Alenka Divjak, Visoka šola za upravljanje podeželja Grm Novo mesto, Sevno 13, 8000 Novo mesto, e-mail address: alenka.divjak@guest.arnes.si Acta agriculturae Slovenica, 109 - 2, september 2017 str. 325 - 302 Jože PODGORŠEK industries, such as tourism, sports, recreation, and more. In view of all these aspects, it is extremely important that the food supply chain is solid and sustainable. The strength of the food supply chain is undoubtedly influenced by the relations between its various partners as well. Because of the uneven market position of various partners involved in the food supply chain, smaller food producers in particular are affected by unfair practices which in the long run weaken the partners on whom these practices are imposed. In general, unfair practices can be defined as the practices that significantly deviate from sound business conduct, being contrary to good faith and honest conduct, and which are unilaterally imposed on the others by one partner or a group of partners. Illicit conduct can be classified as non-compliance with payment deadlines and the imposition of conditions (additional payments, discounts, promotions, unfair delivery conditions, counter trade by non-competitive conditions, transfer of business risk to trade suppliers, etc.) (ZKme1B, 2014). Such improper practices evolve there where is no balance in the financial and consequently in the bargaining power of business partners, impairing in this way the entire EU economy, because as a result of such actions, businesses (especially small and medium ones) are losing the ability to invest and foster innovations and they consequently do not decide to expand their business in the single market. Attention should be paid to the factor of fear, when the weaker partners decide not to initiate legal action in spite of the exisisting possibility to do so, and irrespective of the damage, they simply accept unfair practices because they are afraid that a stronger partner may otherwise terminate a business relationship. Although the imbalances in bargaining power are a completely legitimate component of the functioning of the market, the abuse of a stronger position can distort the relationship between the companies, which often leads to unfair trading practices. This question concerning the transactions between enterprises has been increasingly coming to the force in recent years, and although it is difficult to evaluate all its dimensions, there are concrete statistics and market evidence revealing that unfair practices in the food chain are fairly widespread, especially in certain parts of the agri-food chain. It is being recognized by many Member States that such practices can cause a lot of damage, so they are taking action against them, while the other members are planning to do the same. The rules in this area as well as the extent of this problem vary greatly among the Member States. At the same time, market participants are trying to confront the problem by developing principles of good practice in vertical relationships and by designing self-regulatory frameworks for the implementation of these principles (Evropska komisija, 2014; Kocsis and Nedeczky, 2013). However, as unfair practices are widespread and they are becoming more and more problematic, the question is being raised in this article: to what extent can self-regulatory mechanisms actually help to restore market equilibrium? Voluntary mechanisms that encourage companies to refrain from unfair practices should supposedly mitigate this problem to some degree, but they certainly cannot solve it (Jackiewicz, 2015). For the effective prevention of unfair practices and illicit conduct, the identification of such unfair practices is of the utmost importance. For that reason, in the continuation of this article, its author will present the identified suspicions of unfair practices and illicit conduct that occur in the Slovenian food supply chain, with the emphasis on the retail chains recognized as the parties with significant market power. 2 MATERIAL AND METHODS In Slovenia, the food supply chain is extremely dynamic and, due to the proverbial incoherence of individual actors, it is fairly different from similar chains in the neighbouring countries. So in Slovenia, there are the following types of food supply chains (Podgoršek, 2016): 1. agricultural holding — retail chain (typical for the sector of fresh vegetables, potatoes), 2. agricultural holding - cooperative - retail chain (some examples in the sector of vegetables and fruit), 3. agricultural holding — food processing company - retail chain (typical for the sector of meat and grain), 4. agricultural holding - cooperative - food processing company - retail chain (typical for the milk sector), 5. food processing company - retail chain (typically for the sector of drinks). Due to the highly fragmented structure of different stakeholders, a decision has been taken by the author to analyze the relationships in the food supply chain at the point where all retail chains and their suppliers join in. To establish the suspicion of unfair practices and illicit conduct, the method of participatory active research has been chosen. This is one of the research tools connecting participants in this research with the purpose of finding a common definition and a solution to the problem. For this reason, one of the important 326 Acta agriculturae Slovenica, 109 - 2, september 2017 Unfair practices and illicit conduct in food supply chains in Slovenia objectives of this research is also its active contribution to the solution to certain social problems. It is oriented to promoting skills, community development, social justice, wider accessibility and the participation of different stakeholders (Podmenik and Bembic, 2015). In accordance with the selected method, the author personally interviewed a variety of Slovenian suppliers of retail chains in Slovenia, maintaining in this way the anonymity of the participants in the research, as all the time throughout the present research, it was possible to recognize the participants' fear that sanctions might be imposed on them by retail chains in the case of their identity being revealed. For this reason, the obtained information was combined and merged in such a form that a clear source of information can no longer be identified. The research has been conducted in all the main groups of suppliers who have been divided into the following five groups: - meat and meat products, - milk and dairy products, - fruits and vegetables (fresh and processed), - the manufacture of grain mill and bakery-manufacture, - the manufacture of other food products. The research was conducted among different types of suppliers, such as farms, agricultural cooperatives and agro-processing companies. 3 RESULTS AND DISCUSSION In the Slovenian area, unfair practices and illicit conduct have been actively dealt with for many years due to the increasing concern of both stakeholders and agricultural policy who recognized the seriousness of the existing anomalies. The first serious attempt to restrict the development of unfair practices was the signing of the Code of good business practices among stakeholders in the agri-food chain at the Agra Fair 2011. The Code signatories (the Slovenian Chamber of Commerce, the Slovenian Chamber of Commerce and Industry, The Chamber of Agriculture and Forestry, the Slovenian Chamber of Craft and Small Business and the Cooperative Association of Slovenia) then agreed to develop positive relationships and promote the joint development of the food supply chain. The Code also envisaged the introduction of the net purchase prices in retail chains, planning to implement the agreement by early 2013 (Kodeks, 2011). Due to non-compliance with the commitments contained in the Code, the Law on amending the Law on agriculture (Zkme-1B, 2014) was accepted. The amendment to the Law on agriculture set a 45-day maximum payment period for perishable foodstuffs and 90 days for other foods. It also identified illicit conduct and provided a legal basis for the appointment and functioning of a food supply chain relationships ombudsman. The first ombudsman was appointed on 3 January, 2015, for a period of five years. The ombudsman's task is to monitor the behaviour of stakeholders in the food supply chain, to publish examples of good business practices and to notify the Public Agency for the Competition Protection of any prohibited practices, whereby the Public Agency has to protect personal information and business secrets of the parties. On the basis of the analysis of the results obtained by the method of participatory active research, the author of the article divided the allegations of unfair practices and illicit conduct, made by individual participants, into five groups, without making any direct references to the participants in order to prevent the revelation of the trade secrets of individual stakeholders. biomass of wheat. Results revealed that germination percentage was neither affected by different plant parts nor by extract concentrations. Interaction between plant parts and concentrations were also non-significant. Germination (%) was maximum (91.8) in control conditions. Slighter decrease in germination was observed in petri-dishes treated with different extract concentrations of different plant parts; however, the differences among means of concentrations and plant parts for germination percentage were insignificant and they ranged between 89.7 - 90.9 % which did not differ significantly from 91.8 % in control (Table 1). 3.1 Retail chain 1: In this chain, the following allegations of unfair practices have been found: - the imposition of additional discounts and rebates amounting to over 20 % according to the value of delivered food products. 3.2 Retail chain 2: In this chain, the following allegations of unfair practices have been found: - the imposition of additional discounts in the form of charging superrabates (different rates depending on the supplier), additional payments for marketing up to 2 % of current output, - the requirement for agreed action prices of items for products in the weekly specials for the period from 16 days before the special (also for food products with a shorter shelf life) and Acta agriculturae Slovenica, 109 - 2, september 2017 Jože PODGORŠEK up to 7 days after the completion of the special, which means that they require the action price for the article in a weekly special for the period of 1 month, - the return of already delivered and absorbed goods - the transfer of risk to suppliers, - the signing of a contract with the company for transferring money, recommended by the retail chain (the company for transferring money is through the founders associated with the retail chain), and billing the service of transfer up to 1 % in relation to payments made, - the dictation of action prices that are lower than the prices in comparable neighboring countries, with the possibility of immediate loss of business in the case of the supplier's disagreement with the proposed price, - the application of fines and penalties on the delayed or failed delivery of a certain item, in no proportion to the damage caused, - the increase of various discounts in the case of the exclusion of the supplier of this trading system and his re-integration into the network of suppliers. 3.3 Retail chain 3: In this chain, the following allegations of unfair practices have been found: - the imposition of individual contracts on individual suppliers to pay up to 5 % of the total turnover of the previous year, - contractual restriction of the right to charge default interest on the late payment of invoices and transfer of commercial risk to the supplier in the case of sales actions, - bound trade at non-competitive prices in the case of franchises, - opaque rejection of goods due to possible defects in food products or crops, - exclusion of one's own-brand products which are in other retail chains marketed by the same suppliers under the commercial brand of a competitive retail chain. 3.4 Retail chain 4: In this chain, the following allegations of unfair practices have been found: - the transfer of the business risk to the supplier in the case of action sales, - payment defaults and late payments for nutritional products, the lack of consent to the sale of receivables overdue to the purchasers of receivables, - contractual restriction of the right to charge default interest on the untimely paid accounts, - late payments (in some cases more than 90 days after currency), - bound trade at non-competitive prices in the case of franchises. 3.5 Retail chain 5: In this chain, the following allegations of unfair practices have been found: - the coercion of small suppliers into the exclusive sale of their products only through a particular retail chain. The list of suspected unfair practices and illicit conduct is unfortunately being constantly updated. During the action, taken by the food supply chain relationships ombudsman in one of the retail chains, compulsory payments for early payment of invoices (paid within 20 days instead of envisaged 45 days) were replaced with compulsory promotional rebate, which is probably due to the alertness to a usurious interest rate in the case of pre-payment of bills. It should be noted that suppliers continue to pay special promotions according to the price list of the retail chain. 4 CONCLUSIONS Unfair trade practices are recognized throughout Europe and they are quite common. In the pan-European survey, conducted among suppliers in the food chain, 96 % of suppliers stated that they met with at least one form of unfair trade practices (Evropska komisija, 2014). However, there are considerable differences among individual EU countries. Thus, some national studies have shown different shares of the existence of unfair trade practices. In Spain, the national survey has found that 56 % of suppliers experienced retroactive changes in the contract terms. In Italy, a survey has shown that 57 % of producers often or always accept unilateral retroactive changes, from fear of commercial retaliation in the case of the rejection of changes. The overall impact of unfair trade practices is difficult to assess and quantify in quantitative terms, however, as a result of these practices, those parties are directly and negatively affected. Because of the unfair trade practices, the income of suppliers is undoubtedly reduced. The unfair practices and illicit conduct in the food supply chains are dealt with by various European countries in different ways. The Czech Republic applies the Act on abuse of a dominant market position in the market of agricultural and food products (Official 328 Acta agriculturae Slovenica, 109 - 2, september 2017 Unfair practices and illicit conduct in food supply chains in Slovenia Journal of the EU, 2013). The law in question among others prohibits the sale of a product at a price lower than its purchase price and only the net price is to be considered, without any additional discounts and rebates. In Italy, they use the Regulation on economic relations in the sale of food and agricultural products (Oggetto, 2016), which specifies mandatory and prohibited elements in contracts. The control over the implementation of the regulation is kept by the Authority for the competition and the market, which can also rely in special cases on the operational support of the Financial Guard (Guardia di Finanza). In neighboring Hungary, it is the Trade act (RS Government, 2013) which governs the relations in the food supply chain and which defines the abuses committed by traders with the dominant market power. The law should protect the suppliers and it is based on the protection of free trade and entrepreneurship. From 2012 onwards, the law no longer applies to food products. Therefore, the chain of food products and the prevention of unfair practices are today regulated by the Trade act and the Law on unfair distribution practices. The supervision of the implementation of the legislation is in the domain of the State Office for the safety of the food chain. To sum up, the food supply chain in different EU countries is regulated in different ways. All the countries, however, share a common interest in regulating the relations existing in this chain and endeavour to prevent the exploitation of negotiating superiority achieved by individual stakeholders. Therefore, this problem, in addition to the food supply chain relationships ombudsman, is dealt with by the Public Agency of the Republic of Slovenia for Protection of competition, which was notified of the suspicions of unfair practices and illicit conduct by the ombudsman in January 2016. Nevertheless, it is likely that in Slovenia it will also be necessary to adopt a legislative framework through which the relations in the food supply chain will be regulated and unfair practices prevented, especially the pressure exerted on weaker negotiators by the parties with significant market power. 5 REFERENCES Evropska komisija. (2014). Sporočilo komisije evropskemu parlamentu, svetu, evropskemu ekonomsko socialnemu odboru in odboru regij, Evropska komisija, Strasbourg, COM(2014) 472 final, 15.7.2014. Jackiewicz, D. B. (2015). Osnutek poročila o nepoštenih trgovinskih praksah v verigi preskrbe s hrano, Evropski parlament, Odbor za notranji trg in varstvo potrošnikov, Bruselj, 9 str. Kodeks. (2011). Kodeks dobrih poslovnih praks med deležniki v agroživilski verigi. Objavljeno na spletni strani: https://www.gzs.si/pripone/K0DEKS%20-%2023%208%202011.pdf. 1.12.2016. Kocsis, M., Nedeczky, A. (2013). Your RFI concerning the assesment of harmful effects in the food supply chain, Budapest, 4 str. Oggetto. (2016). Oggetto: DL 24.1 2012 n. 1, art. 62 recante »Disciplina delle relazioni commerciali in materia di cessione di prodotti agricoli e agroalimentari«. Podgoršek, J. (2016). Letno poročilo varuha odnosov v verigi preskrbe s hrano, Ljubljana, 22 str. Podmenik, D., Bembič, M. (2015). Novi raziskovalni pristopi v družbeni geografiji: participativno akcijsko raziskovanje. Geografski vestnik, 87,2, 105 - 113. doi:10.3986/GV87207 ReSURSKŽ. (2011). Resolucija o strateških usmeritvah razvoja slovenskega kmetijstva in živilstva do leta 2020 - »Zagotovimo.si hrano za jutri« (ReSURSKŽ), Uradni list RS, št. 25/11, 4.4.2011. Uradni list EU. (2013). Mnenje Evropsko ekonomsko-socialnega odbora o zeleni knjigi o nepoštenih trgovinskih praksah v oskrbni verigi z živili in neživili med podjetji v Evropi. Uradni list Evropske unije. COM(2013) 37final, 7 str. Vlada RS. (2013). Predlog Zakona o spremembah in dopolnitvah Zakona o kmetijstvu. Objavljeno na spletni strani: http://imss.dz- rs. si/imis/316beffb1414f838cf7e.pdf 1.12.2016 ZKme-1B. (2016). Zakon o spremembah in dopolnitvah Zakona o kmetijstvu, Uradni list RS, št. 26/14, 14.4.2014. Acta agriculturae Slovenica, 109 - 2, september 2017 doi: 10.14720/aas.2017.109.2.16 Original research article / izvirni znanstveni članek Natural incidence of bean viruses in the northwest of Iran Masoumeh JALALI1, Mina RASTGOU* Received January 23, 2017; accepted June 01, 2017. Delo je prispelo 23. januarja 2017, sprejeto 01. junija 2017. ABSTRACT Bean is considered as one of the most important legumes around the world. Viral diseases are a major yield reducing factor in bean production. Bean samples with virus-like symptoms like severe or mild mosaic, vein banding, leaf curling, blistering and necrosis were collected from different bean fields in Urmia (Northwest of Iran) during the growing seasons of 2013 and 2014. Bean common mosaic virus (BCMV), Bean common mosaic necrosis virus (BCMNV), Bean yellow mosaic virus (BYMV), Cucumber mosaic virus (CMV), Tomato spotted wilt virus (TSWV), Tomato mosaic virus (ToMV) and Tomato yellow leaf curl virus (TYLCV) were detected by double antibody sandwich enzyme-linked-immunosorbent assay. Mixed infection of BCMV and BCMNV were found. BCMNV was the most frequent virus in this region whereas BYMV and TYLCV were each detected just in one sample. This is the first report of BCMNV, BCMV, BYMV, TSWV, TMV and TYLCV incidence on bean in Urmia, Iran. Key words: BCMV; BYMV; BCMNV; TYCLV; TMV; TSWV; Iran; ELISA IZVLEČEK RAZŠIRJENOST VIRUSOV FIŽOLA V SEVEROZAHODNEM IRANU Fižol je ena izmed najpomembnejših stročnic širom po svetu. Virusne bolezni so najpomembnejši dejavnik zmanjševanja njegovega pridelka. Vzorci fižola z znamenji virusnih ukužb kot so blagi ali izraziti mozaik, obžilna razbarvanja, zvijanje, mehurjavost in nekroza listov so bili nabrani z različnih fižolovih polj v okolici Urmie (severozahodni Iran) v rastnih sezonah 2013 in 2014. Virus navadnega mozaika fižola (BCMV), virus navadnega mozaika in nekroze fižola (BCMNV), virus rumenega mozaika fižola (BYMV), virus mozaika kumar (CMV), virus pegavosti in uvelosti paradižnika (TSWV), virus mozaika paradižnika (ToMV) in virus rumenenja in kodravosti paradižnika (TYLCV) so bili ugotovljeni z ELISA testom. Najdena je bila mešana okužba z BCMV in BCMNV. BCMNV je bil najpogostejši virus na tem območju, medtem, ko sta bila BYMV in TYLCV ugotovljena samo v po enem vzorcu. To je prvo poročanje o pojavljanju BCMNV, BCMV, BYMV, TSWV, TMV in TYLCV na fižolu v Urmiji, Iran. Ključne besede: BCMV; BYMV; BCMNV; TYCLV; TMV; TSWV; Iran; ELISA 1 INTRODUCTION The common bean (Phaseolus vulgaris L.) is one of the major food legumes produced on over 26 million hectares worldwide (Mavric and Šustar-Vozlič, 2004; Loebenstein et al., 2009). Virus diseases are a major yield reducing factor in bean production. Thirty four different virus species have been reported to infect common bean naturally (Arli-Sokman et al., 2016). Economically the most important viruses are potyviruses including Bean common mosaic virus (BCMV), Bean common mosaic necrosis virus (BCMNV) and Bean yellow mosaic virus (BYMV). Other important viruses on bean are Cucumber mosaic virus (CMV), Southern bean mosaic virus (SBMV), Tobacco streak virus (TSV) and Tomato aspermy virus (TAV) (Brunt et al., 1996; Kumar et al., 1994). These viruses are transmitted by insect vectors, mainly aphids with the exception of TSV that is transmitted by thripses and SBMV that is transmitted by been leaf beetle. Some of them can be transmitted by seed (Loebenstein et al., 2009). Insect transmission is very important for virus spread on short distances while seed transmission is the most important factor in the spread of viruses around the world. Because of the high seed transmission rate (up to 83 %), BCMV and BCMNV are economically the 1 Department of Plant Protection, College of Agriculture, Urmia University, Urmia, Iran * Department of Plant Protection, College of Agriculture, Urmia University, Urmia, Iran, Po. Box: 165, Iran; email: m.rastgou@urmia.ac.ir Acta agriculturae Slovenica, 109 - 2, september 2017 str. 331 - 302 Masoumeh JALALI, Mina RASTGOU most important viruses of common bean (Mavric and Sustar-Vozlic, 2004). For example, BCMV can reduce yield up to 24 % (Kumar et al., 1994). BCMV, BYMV and BCMNV have been reported from bean-growing regions of Iran (Kaiser and Mossahebi, 1974; Shahraeen et al., 2002; Shahraeen et al., 2005; Peyambari et al., 2011; Ghasemzadeh et al., 2012; Ghobakhloo et al., 2012; Salari et al., 2013). Other viruses reported on bean from Iran include Alfalfa mosaic virus (AMV), Tobacco mosaic virus (TMV), Tomato mosaic virus (ToMV), Bean leaf roll virus (BLRV), Soybean mosaic virus (SMV), Bean pod mottle virus (BPMV), Bean curly top virus (BCTV), Broad bean wilt virus (BBWV), Broad bean stain virus (BBSV) and Faba bean necrotic yellows virus (FBNYV) (Kaiser and Mossahebi, 1974; Shahraeen et al., 2005; Alavi and Massumi, 2014). BCMV has been recognized as a major constraint on bean production in Iran. Bean is one of the most important cultivated crop in West Azarbaijan province covering around 1346 ha with the mean yield of 2 t ha-1, but until now there was no data on viral diseases of this crop in this region. The occurrence, frequency and distribution of seven viruses including BCMV, BCMNV, BYMV, CMV, Tomato spotted wilt virus (TSWV), Tomato mosaic virus (ToMV) and Tomato yellow leaf curl virus (TYLCV) in bean growing areas of Urmia, the largest bean growing region in West Azarbaijan province were studied in 2013 and 2014 and are presented in the article. 2 MATERIALS AND METHODS 2.1 Plant material A set of 195 symptomatic samples of bean showing severe or mild mosaic, vein banding, leaf curling, blistering and necrosis on leaves was collected from bean growing areas of Emamzadeh-Gharachelar, Ghafar Behi, Jabal, Balo, Nazloo (Urmia University campus) and Aliabad regions around the city of Urmia during the 2013 and 2014 growing seasons. Each sample was put in a plastic bag, labelled and stored at 4 °C. 2.2 Virus detection Double antibody sandwich enzyme linked immunosorbent assay (DAS-ELISA) was used for detection of BYMV, BCMV, BCMNV, TSWV, CMV, ToMV, TYLCV according to Clark and Adams (1977) method with minor changes using commercial polyclonal antisera obtained from DSMZ (Braunschweig, Germany). ELISA plates were coated with coating buffer containing anti-BCMV, BYMV, BCMNV, TSWV, CMV, ToMV and TYLCV polyclonal antibodies (1:1000) and the plates were incubated at 37 °C for 3 h. Each sample was diluted 1:5 with extraction buffer (8 g NaCl, 0.2 g KH2PO4, 0.2 g NaN3, 0.5 ml Tween 20 and 2 % PVP in 1 l of distilled H2O, pH 7.4). The diluted plant sap extracts were added to the wells (100 ^l). Two replicates were used for each sample. Plates were kept at 4 °C overnight, then rinsed three times with washing buffer. Conjugated polyclonal antibody was diluted (1:1000) in conjugate buffer and was loaded into each well. The plate was incubated at 37 °C for 3 h. Finally, 10 mg of p-nitrophenyl phosphate (Sigma) in 10 ml of substrate buffer was added to the wells and incubated at room temperature for 30-60 min. Absorbance values were read at 405 nm (A405) using a microplate reader (BioTek ELX-808, USA). Healthy plants were used as negative controls and samples were considered to be positive when the absorbance values at 405 nm values exceeded at least tree time the mean of the negative controls. 2.3 Maintenance of the viruses Bean plants were planted in pots in a greenhouse and inoculated with virus isolate prepared from systemically infected leaves macerated in a chilled sterilized 0.01 M cold phosphate buffer (K2HPO4 + KH2PO4), pH 7.0 (1 : 6 [w/v] tissue : buffer). Inoculation on plants at the three-leaf stage was done using the rub method on carborundum-dusted leaves. The plants were observed weekly for symptom development for 5 weeks and samples for DAS-ELISA were taken at each observation time. 3 RESULTS AND DISCUSSION During summer of 2013 and 2014, 195 bean samples with virus-like symptoms such as mosaic on leaves, leaf distortion, downward curling, mottling, vein necrosis and local lesions (Figure 1) were collected from bean farms in different areas and villages around Urmia and subjected to DAS-ELISA that revealed the presence of all tested viruses as shown in Table 1. 96 samples were collected in 2013 and 99 samples in 2014. 12 samples collected in 2013 and 17 collected in 2014 were infected with BCMNV, 8 and 2 with BCMV, 1 and 0 with 1.1. J 332 Acta agriculturae Slovenica, 109 - 2, september 2017 Natural incidence of bean viruses in the northwest of Iran BYMV and TYLCV, 2 and 1 with ToMV and CMV and 2 and 0 with TSWV (Table 1). DAS-ELISA results showed the presence of BCMNV and BCMV in all bean-producing parts around Urmia where samples were taken. BYMV, TYLCV and TSWV were only detected in Aliabad, the largest bean-growing area in Urmia. ToMV was detected in Emamzadeh-Gharachelar and Nazloo and CMV was detected in Emamzadeh-Gharachelar and Ghafar Behi regions. Mixed infection of BCMNV and BCMV were also detected in two samples from Aliabad region. According to DAS-ELISA results BCMNV was the dominant virus in Urmia, so one of the isolates was inoculated and monitored on Phaseolus vulgaris L. in the greenhouse. The necrotic symptoms were seen 5 to 6 days after inoculation (Figure 2) and the presence of the virus was confirmed by DAS-ELISA. Table 1: Incidence of seven viruses in common bean plants collected in Urmia Place of Year of No. of BCMNV BCMV BYMV TYLCV ToMV CMV TSWV sampling sampling samples Emamzadeh- 2013 15 4 2 - - 1 1 - Gharachelar 2014 16 5 1 - - - - Ghafar-Behi 2013 12 2 1 - - - 1 - 2014 15 3 1 - - - - - Jabal 2013 14 2 1 - - - - - 2014 16 3 - - - - - - Balo 2013 12 1 1 - - 1 - - 2014 13 - - - - - - - Nazloo 2013 12 1 1 - - - - - 2014 10 1 - - - 1 - - Aliabad 2013 31 2 2 1 1 - - 2 2014 29 5 - - - - 1 - total 2013 96 12 8 1 1 2 2 2 2014 99 17 2 - - 1 1 - Rate of 2013 - 12.5 8.3 1.04 1.04 2.1 2.1 2.1 infection (%) 2014 - 17.2 2.1 - - 1.04 1.04 - Figure 1: Symptoms of A, vein banding, B, blistering, C, mottling and D, necrosis on virus-infected bean plants -3-3-3 Acta agriculturae Slovenica, 109 - 2, september 2017 3 Masoumeh JALALI, Mina RASTGOU m Figure 2: Necrosis induced by BCMNV after inoculation on bean The incidence and distribution of BCMV, BCMNV, BYMV, TYLCV, TMV and TSWV in bean growing areas of Urmia were not similar to each other. BCMNV infection was the highest. As in Urmia, BCMV and BCMNV are the most prevalent viruses in common bean also in other areas of the world. Since both viruses were found in the same areas and even in the same plant, recombinations between them are possible and can lead to creation of new strains or even new pathotypes (Vallejoes et al., 2006) in Urmia. Previous studies reported that the incidences of BCMNV strains is lower than those of BCMV strains in most bean production areas of the world (Berger et al., 1997; Kostova et al., 2003; Petrovic et al., 2010), but our two year survey in Urmia bean-producing regions indicated that BCMNV is prevalent in most regions. In most visited areas, especially in Aliabad region, high rate of viral infection was observed. DAS-ELISA test showed different infection rates in various places. The highest infection rate of BCMNV was found in Emamzadeh-Gharachelar and Aliabad probably due to virulence of this virus, high population of the vectors in these areas and suitable weather condition for infection. TYLCV was found on common bean with a disease incidence of 50 to 70 % showing thickening and crumpling of leaves and stunting in northern Anhui Province, China (Ji et al., 2012). Hosseini and Eini Gandomani (2014) detected gemini viruses on bean in Zanjan province, Iran. We detected TYLCV in only one symptomatic sample in Aliabad region. BCMNV was reported for the first time and in high abundance in Urmia indicating the spread of viral diseases with climate change and global warming. There has not been any data on occurrence and importance of viral diseases on bean and their spread in nature in the province since now. Investigation on occurrence, spread and determination of dominant viruses of bean are very important especially for implementation of proper diagnostic methods and management techniques, especially breeding for resistance. According to the local observations, symptoms of viral diseases on bean farms of this province are increasing considerably in recent years. Our results also showed high rate of virus infection in the area and confirmed these observations. -3-3/1 334 Acta agriculturae Slovenica, 109 - 2, september 2017 Natural incidence of bean viruses in the northwest of Iran Figure 3: Percentage of incidence of BCMNV, BCMV, BYMV, TYLCV, ToMV, CMV and TSWV in bean fields of Urmia during 2013-2014 growing seasons 4 CONCLUSION Viruses are known to greatly reduce bean yield. BCMV and BCMNV, two economically very important bean viruses transmitted by several aphid species and by seed, were found in high incidence in West Azarbaijan province, Urmia region using DAS-ELISA. Most of the mosaic and systemic necrosis symptoms observed in bean plants in the field could be attributed to these two viruses. Additionally, the presence of BYMV, TYLCV, ToMV, CMV and TSWV was confirmed. To our knowledge this is the first report of TSWV incidence on bean in Iran and the first report of the presence of BCMNV, BCMV, BYMV, TYLCV, ToMV, CMV and TSWV in West Azarbaijan province, Urmia (Iran). Results of this study are the base for further work on ecology, epidemiology, diversity and breeding for resistance to bean viruses in Urmia and also in Iran. 5 REFERENCES Alavi, S., and Massumi, H. (2014). Distribution, biological properties and genetic diversity of Iranian tomato mosaic virus isolates. Iranian Journal of Plant Pathology, 50 (1), 37-52. Arli-Sokmen, M., Deligoz, I., and Kutluk-Yilmaz, N. (2016). Characterization of Bean common mosaic virus and Bean common mosaic necrosis virus isolates in common bean growing areas in Turkey. European Journal of Plant Pathology, 146, 1-16. doi:10.1007/s10658-016-0886-x Berger, P.H., Wyatt, S. D., Shiel, P. J., Silbernagel, M. J., Druffel, K., and Mink, M. I. (1997). Phylogenetic analysis of the Potyviridae with emphasis on legume-infecting potyviruses. Archives of Virology, 142, 1979-1999. doi: 10.1007/s007050050216 Brunt, A.A, Crabtree, K., Dallwitz, M.J., Gibbs, A.J., Watson, L., and Zurcher, E.J. (1996). Plant viruses online: descriptions and lists from the VIDE database. Version: 20th August 1996. URL http://biology.anu.edu.au/Groups/MES/vide/. Clark, M.F., and Adams, A.N. (1977). Characteristics of the micro plate method of enzyme-linked immunosorbent assay for the detection of plant viruses. Journal of General Virology, 34, 475-483. doi:10.1099/0022-1317-34-3-475 Ghobakhloo, A., Pourrahim, R., Elahinia, S.A., and Farzadfar, SH. (2012). Molecular RT-PCR detection of Bean common mosaic virus--BCMV in Melilotus sp. Proc. 12th Iranian Genetics Congress, Tehran, Iran, pp. 70-74. Ghasemzadeh, A., Sokhandan Bashir, N., and Khakvar, R. (2012). Identification of important viruses for Legominosae family with using of universal primers from East-Azarbaijan province. Proc. 12th Iranian Genetics Congress, Tehran, Iran, pp. 92-97. Hosseini, V., and Eini Gandomani, A. (2014). Molecular Determination of geminiviruses of bean in Zanjan province. Proc. Iran's 21th Congress of Plant Protection. Urmia, Iran, 405. Ji, Y.H., Cai, Z.D., Zhou, X.W., Liu, Y.M., Xiong, R.Y., Zhao, T.M., Yu, W.G., Tao, X.R., and Zhou, Y.J. (2012). First report of Tomato yellow leaf curl virus infecting common bean in China. Plant Disease, 96, 1229. doi: 10.1094/PDIS-03-12-0258-PDN Kaiser, W.J., and Mosahebi, GH. (1974). Natural infection of mungbean by Bean common mosaic virus. Acta agriculturae Slovenica, 109 - 2, september 2017 3 Masoumeh JALALI, Mina RASTGOU Phytopathology, 64, 1209-1214. doi:10.1094/Phyto-64-1209 Kostova, D., Lisa, V., Milne, R. G., Vaira, A. M., Dellavalle, G., and Tsorlianis, S. (2003). Virus diseases of vegetable crops in Southern Bulgaria. PhytopathologiaMediterranea, 42, 3-8. Kumar, C.A., Khetarpal, R.K., Parakh, D.B., Singh, S., and Nath, R. (1994). Check list on seed transmitted viruses: Leguminous hosts. National Bureau of Plant Genetic Resources, New Delhi-110012, 14 p. Loebenstein, G., Tottappilly, G., Fuentes, S., and Cohen, J. (2009). Virus and phytoplasma diseases. In: Loebenstein, G. and G. Thottappilly (eds.). The Sweet Potato. Springer, Berlin, Germany. doi:10.1007/978-1-4020-9475-0_8 Mavric, I., and Sustar-Vozlic, J. (2004). Virus diseases and resistance to bean common mosaic and bean common mosaic necrosis Potyvirus in common bean (Phaseolus vulgaris L.). Acta Agriculturae Slovenica, 85(1), 181-190. Petrovic, D., Ignjatov, M., Nikolic, Z., Vujakovic, M., Vasic, M., Milosevic., et al. (2010). Occurrence and distribution of viruses infecting the bean in Serbia. Archives of Biological Science, 62(3), 595-601. doi: 10.2298/ABS1003595P Peyambari, M., Koohi Habibi, M., Mosahebi, GH, and Izadpanah, K. (2011). Study of bean common mosaic virus (BCMV) in several provinces and reaction of three bean genotypes to BCMV infection. Journal of Plant Protection, 25(3), 250-257. Salari, N., Seyyed Musavi, M., Shahraeen, N., Ghorbani, Sh., and Maleki, M. (2013). Identification and diagnosis of isolates of bean common mosaic virus and bean common mosaic necrosis virus by immunocapture RT-PCR. New Cellular and Molecular Biotecnology Journal, 5(11), 21-28. Shahraeen, N., Hassani Mehrban, A., Mostaed, M., and Ghotbi, T. (2002). Bean common mosaic necrosis virus (BCMNV) incidence report in bean farms in Arak and Lorestan provinces. Proc, Iran 15th Plant Protection Congress, Kermanshah, Iran, p. 287. Shahraeen, N., Ghotbi, T., Dezaje, A and Sahandi, A. (2005). A survey of viruses affecting French bean (Phaseolus vulgaris L.) in Iran includes a first report of Southern bean mosaic virus and Bean pod mottle virus. Plant Disease, 89, 1012. doi:10.1094/PD-89-1012B Vallejos, C. E., Astua-Monge, G., Jones, V., Plyler, T. R., Sakiyama, N. S., and Mackenzie, S. A. (2006). Genetic and molecular characterization of the I locus of Phaseolus vulgaris. Genetics, 172, 1229-1242. doi:10.1534/genetics. 105.050815 336 Acta agriculturae Slovenica, 109 - 2, september 2017 doi: 10.14720/aas.2017.109.2.17 Original research article / izvirni znanstveni članek The analysis of technical suitability of the equipment for application of plant protection products in Southeastern Slovenia Rajko BERNIK1, Peter KUHAR2 Received January 23, 2017; accepted July 26, 2017. Delo je prispelo 23. januarja 2017, sprejeto 26. julija 2017. ABSTRACT Technical testing of the equipment for the application of plant protection products (PPP) was performed in Southeastern Slovenia in the period from 2004 to 2013. The technical conditions of boom or orchard sprayers was examined in details and the following parts were checked: drive, anti-drip valves, nozzles, filters, pipes and tubes, manometers, all valves, pressure regulators, agitator, pump, liquid discharge, spray solution reservoir and spray boom or fan system. The analysis revealed a poor condition of most devices in the initial years. However, technical suitability improved drastically until 2013. Technical condition was not directly related to the region of inspection although the lowest number of defective sprayers was recorded in the Posavje region. This can be linked to higher frequency of use and maintenance of the sprayers in this region as it stands out as the area with larger farms. Occasional technical disorders of the equipment can be recorded each year. It is therefore essential to continue with regular technical inspection to ensure optimal and accurate functioning of the sprayers. Key words: boom sprayers; orchard sprayers; technical conditions; cross application IZVLEČEK ANALIZA TEHNIČNE USTREZNOSTI OPREME ZA UPORABO SREDSTEV ZA ZAŠČITO RASTLIN V JOGOVZHODNI SLOVENIJI V letih od 2004 do 2013 smo opravljali tehnične preglede za nanos FFS na območju jugovzhodne Slovenije. Pregledovali smo dve vrsti naprav in sicer škropilnice in pršilnike. Ugotavljali smo njihovo tehnično brezhibnost. Na vsaki napravi so bili pregledani sklopi kot so pogon, protikapni ventili, šobe, filtri, cevi, manometri, pipe in zasuni, regulatorji tlaka, mešalo, črpalka, praznjenje, rezervoar ter škropilne letve oziroma puhala pri pršilnikih. Analizirali smo podatke o napakah in ugotovili, da je bilo tehnično stanje naprav v začetnem obdobju, zelo slabo. Tehnično stanje naprav se je do leta 2013 zelo izboljšalo. Ugotovili smo tudi, da tehnično stanje naprav ni odvisno od območja njenega nahajanja. Je pa območje Posavja izstopalo po najmanjšem deležu okvarjenih naprav. Razlog za to vidimo predvsem v tem, da se naprave nahajajo na velikih kmetijah, kjer jih redno uporabljajo in vzdržujejo. Ker pa se okvare in napake, sicer v manjši meri, pojavljajo vsakoletno sklepamo, da je s pregledi potrebno nadaljevati, če hočemo obdržati doseženo stanje tehnične brezhibnosti, kajti naprave se ob uporabi prej ali slej obrabijo. Ključne besede: škropilnice; pršilniki; fitofarmacevtska sredstva; tehnično stanje; prečni nanos 1 prof. dr., univ. dipl. ing. str, Jamnikarjeva 101, SI-1000 Ljubljana, Slovenija, e-mail: rajko.bernik@bf.uni-lj.si 2 mag., univ. dipl. ing. agr, Grm Novo mesto, center biotehnike in turizma, Sevno 13, 8000 Novo mesto, Slovenija, e-mail: peter.kuhar@amis.net Acta agriculturae Slovenica, 109 - 2, september 2017 str. 337 - 302 Rajko BERNIK, Peter KUHAR 1 INTRODUCTION Devices used for the application of plant protection products (PPP) are machines, which enable uniform application of PPP to the plant surface. Devices usually utilize water to dissolve and apply PPP and can be categorized as sprayers and air assisted sprayers (Mrhar, 1997). Phytopharmaceutical products, which are applied via sprayers, destroy, suppress, control or deter harmful organisms and prevent their negative effect on plant growth and development or storage of plant products (Blazic, 2009). Technical flawless of the spraying equipment is crucial for optimal distribution of PPP. The quality of the sprayer is important but it does not guarantee good results if the device is not properly managed and maintained. Up-to-date development of spraying equipment and its technical assistance and inspection are generally focused to ensure accurate PPP application (exact dosage, uniform distribution) and ameliorate other factors (such as working speed) (Roettele et al., 2011). Sprayers must provide exact application and uniform dosage of PPP during the entire lifespan of the device. To ensure their proper functioning, sprayers must be regularly tested and potential technical faults eliminated (Ganzelmeier, 2004a). SIST EN 13790-1 (2004) and SIST EN 13790-2 (2004) standards were implemented in EU member states in order to unify the technical demands for testing of the devices for PPP application. The purpose of the standards is to ensure comparable testing conditions throughout the EU. Their contents can be summarized into the following significant points (Ganzelmeier, 2004b): (1) unification of different procedures, findings and technical demands, previously implemented in specific EU member states; (2) methodology and technical requirements are based on successful practices, previously implemented in specific EU member states; (3) high technical level is ensured with minimum time and funding; (4) EU member states are obliged to accept and implement new standards and withdraw old standards; (5) standards represent the basis for the unification of technical testing in the EU and serve as potential interactive tool in the future; (6) standards set technical requirements but do not regulate the decisions of specific EU member states. In case of Poland, technical testing of the devices for the application of PPP started in 1995 and became mandatory in 1999. Holownicki et al. (2004) reported similar national standards to that of SIST EN 13790-1 (2004) and SIST EN 13790-2 (2004) prior to their implementation. However, several parts of the spraying equipment were tested according to a less strict methodology, which frequently only included visual assessment. Norway began technical testing of spraying equipment in 1990 (boom sprayers) and in 1995 (air assisted sprayers) on a voluntary level. The testing became mandatory for all devices in 2000. Although national technical tests were very sophisticated, slight changes were made after the implementation of SIST EN 173901 (2004) and SIST EN 13790-2 (2004) (Bjugstad et al., 2004) standards. Italy started voluntary testing of air assisted sprayers in the Bolzano region as far back as in 1980. Oddly, technical testing of spraying devices has not been performed in several other regions to this day. In 2004, only 9 of 20 Italian regions had developed a system for technical testing. Some technical testing stations were established in 1996 - 1999 but most were founded in 2002 - 2004 (Balsari et al., 2004). Up to now, 120 testing stations are active in Italy but the testing is only mandatory in Tuscany region. Most technical tests are performed by private institutions. Public testing stations are mostly focused on voluntary testing and experimental testing of the spraying equipment (Balsari et al., 2004). Germany implemented technical testing practices of spraying devices in late 1960 (boom sprayers) and in the middle of 1980 (air assisted sprayers) on a voluntary level. About 1000 testing stations are located in Germany today, which cover 2000 different locations. Testing became mandatory in 1993 and until then approximately 30.000 devices were tested each year. Today, as many as 63.000 sprayers are tested yearly (Osteroth, 2004). In Slovenia, technical testing of spraying equipment was already defined in 1994 Plant Health Act (Zakon o zdravstvenem ..., 1994). Tests have been performed for the last two decades and the aim of the present paper is to present the testing results to a broader scientific community. The effects and results of the testing are discussed and strategies for their improvement suggested. 338 Acta agriculturae Slovenica, 109 - 2, september 2017 The analysis of technical suitability of the equipment for application of plant protection products in Southeastern Slovenia 2 MATERIALS AND METHODS Experimental data include the records of technical testing from 2004 until 2013. Inspections were performed at 86 different locations in Southeastern Slovenia, encompassing Agricultural advisory services Trebnje, Novo Mesto, Metlika, Črnomelj, Krško, Sevnica and Brežice. The research covers the area from Trebnje, Bela krajina, Novo Mesto, Škocjan, Šentjernej, Kostanjevica na Krki, Krško, Brežice to Bizeljsko. Micro locations (testing stations) varied each year (according to the suggestions of local communities) but this did not affect the groups of spraying devices subjected to inspection. Technical testing was performed according to Rules on terms and procedures, which must be met by all authorized supervisory companies for regular inspection of PPP application devices (Pravilnik o pogojih ..., 2000, Pravilnik o spremembi pravilnika ..., 2002, Pravilnik o spremembah in dopolnitvi ..., 2005). The details on technical inspection are specified in aforementioned Rules on terms and procedures. 2.1 Measuring devices and other equipment - Measuring set Herbst ROT-650/60/40/10 - Measuring of pump flow - Measuring of the working pressure and the pressure gauge - Measuring burettes Herbst ED 16 ECO - Mobile Electronic Sprayer Test Equipment SprayerTest 1000 - Wireless rectifier Linksys - Computer IBM Lenovo R60 - Drip tray - Aluminum tracks for the trolley - Inspection protocol (printed) Boom sprayers and air assisted sprayers were inspected separately. The following sections of the boom sprayer were inspected: spray solution reservoir, liquid discharge, agitator, pressure gauge, gate valves and other valves, manometer, pipes and tubes, filters, nozzles, spray boom, anti-drip valves and drive. Several parts of the air assisted sprayer are identical to the parts of a boom sprayer but some segments are different. The following segments of air assisted sprayers were inspected: spray solution reservoir, liquid discharge, agitator, pressure gauge, gate valves and other valves, manometer, pipes and tubes, filters, nozzles, anti-drip valves, drive and blower. 2.2 Data analysis Data were statistically analyzed in program R, version 3.0.2. Regression models- linear mixed models were used to interpret the correlation between the year and rate of faults. The level of risk was 5 %. 3 RESULTS AND DISCUSSION 3.1 Technical condition of all spraying devices Technical condition of spraying devices varied according to the year of inspection. Data is presented in Table 1. Table 1: Data on inspected devices (data on boom sprayers and air assisted sprayers merged) in a particular year Preglednica 1: Podatki o pregledanih napravah (škropilnice in pršilniki skupaj) v posameznem letu Year 2004 2005 2006 2007 2008 2009 2010 2011 2012 2013 Inspected devices 1440 2037 799 1419 1012 2006 1013 1939 1056 1823 Defected devices 708 851 374 517 188 150 56 63 39 29 Share of defected devices 49.2% 41.8% 46.8% 36.4% 18.6% 7.5% 5.5% 3.2% 3.7% 1.6% Acta agriculturae Slovenica, 109 - 2, september 2017 Rajko BERNIK, Peter KUHAR Figure 1: Share of defected devices in a particular year (data on boom sprayers and air assisted sprayers merged) Slika 1: Delež okvarjenih naprav za obe vrsti naprav v posameznem letu The share of defected spraying devices declined consistently from 2004 until 2013, with a slight increase detected in 2006. The greatest decline in the share of defected sprayers was observed between the years 2004 and 2009, when the share dropped to less than 10 %. The share of defected devices gradually declined until 2013 but in a smaller proportion compared to the initial experimental period. 3.2 Technical condition of boom sprayers Data on boom sprayer technical testing is presented in Table 2. Table 2: Data on inspected boom sprayers in a particular year Preglednica 2: Podatki o pregledanih škropilnicah v posameznem letu Year 2004 2005 2006 2007 2008 2009 2010 2011 2012 2013 Inspected devices 1080 1504 594 1085 713 1461 719 1381 722 1287 Defected devices 594 731 316 472 170 132 48 56 28 23 Share of defected devices 55.0% 48.6% 53.2% 43.5% 23.8% 9.0% 6.7% 4.1% 3.9% 1.8% 340 Acta agriculturae Slovenica, 109 - 2, september 2017 The analysis of technical suitability of the equipment for application of plant protection products in Southeastern Slovenia Figure 2: Share of defected boom sprayers in a particular year Slika 2: Delež okvarjenih škropilnic v posameznem letu We assumed that the share of defected boom sprayers decreased during the 10-year period. Data analysis was performed on several segments of the boom sprayer. Table 3: Results of data analysis using the linear mixed model for several boom sprayer segments Preglednica 3: Rezultati linearnega mešanega modela na podatkih za posamezne sklope - škropilnice Coefficient Standard error df t p-value Intersect 0.079 0.016 116 4.863 0.0000 Year -0.010 0.002 116 -4.895 0.0000 The negative value of the coefficient suggests that the defects on boom sprayers decreased in the examined period. Acta agriculturae Slovenica, 109 - 2, september 2017 Rajko BERNIK, Peter KUHAR Year Figure 3: Share of defects on individual segments of a boom sprayer depending on the year - 0 marks year 2004 Slika 3: Delež napak za posamezen sklop v odvisnosti od leta - število 0 na abscisni osi ponazarja začetno leto 2004 Figure 3 depicts the decrease of share of devices with defects in all segments of boom sprayers in the period from 2004 to 2013. Some differences were minor but the condition of several segments was significantly improved. 3.3 Technical condition of air assisted sprayers Differences in technical conditions of air assisted sprayers are reported in Table 4. Table 4: Data on inspected air assisted sprayers in a particular year Preglednica 4: Podatki o pregledanih napravah (pršilniki) v posameznem letu Year 2004 2005 2006 2007 2008 2009 2010 2011 2012 2013 Inspected devices 356 531 204 332 299 541 294 555 334 534 Defected devices 114 119 58 45 18 18 8 7 11 6 Share of defected devices 32.0% 22.4% 28.4% 13.6% 6.0% 3.3% 2.7% 1.3% 3.3% 1.1% 149 342 Acta agriculturae Slovenica, 109 - 2, september 2017 The analysis of technical suitability of the equipment for application of plant protection products in Southeastern Slovenia Figure 4: Share of defected air assisted sprayers in a particular year Slika 4: Delež okvarjenih pršilnikov v posameznem letu The share of defected air assisted sprayers ranged between 20 and 30 % in the period of 2004- 2006 and dropped to 13.6 % in 2007. The share was less than 10 % after 2008. Data were statistically analyzed and a decrease of defected air assisted sprayers was expected in the 10-year period. Analysis was performed on several segments of air assisted sprayers. Table 5: Results of the linear mixed model analysing data of several air assisted sprayer segments Preglednica 5: Rezultati linearnega mešanega modela na podatkih za posamezne sklope - pršilniki Coefficient Standard error df t p-value Intersect 0.023 0.008 116 3.313 0.0012 Year -0.004 0.001 116 -3.239 0.0016 The negative value of the coefficient denotes a decrease of defects in air assisted sprayers during the examined period. Acta agriculturae Slovenica, 109 - 2, september 2017 Rajko BERNIK, Peter KUHAR fan system. anti-drip valves drive gate valves and otliei valves manometers pipes and tirbes filtri nozzles •-_; p ^ reservoir liquid discharge pump agitator pressure regulators 1 ! 1 I 1 ! 1 1 1 ! l l l l l l l l l l 02468 02468 02468 Year Figure 5: Share of defects on individual segments of an air assisted sprayer depending on the year - 0 marks year 2004 Slika 5: Delež napak za posamezen sklop v odvisnosti od leta - številka 0 na abscisni osi ponazarja začetno leto 2004 The share of defects on all segments of air assisted sprayers decreased in the period from 2004 to 2013. The only exceptions were defects on the air blower, which increased slightly from 2009 to 2012. 3.4 Technical condition linked to the region of the technical testing The locations for technical testing were not the same each year and therefore, the area was divided into seven units, each corresponding to the jurisdiction of a single Agricultural advisory service (Trebnje, Novo Mesto, Metlika, Črnomelj, Krško, Sevnica and Brežice). Three main regions were formed based on geographical similarities for easier data analysis: (1) Osrednja Dolenjska (combining KSS Novo Mesto and Trebnje; NMTr), (2) Bela Krajina (combining KSS Črnomelj and Metlika; ČrMe) and (3) Posavje (combining KSS Brežice, Krško and Sevnica; BKKSe). Results report combined data on defects of both types of sprayers for each region. 344 Acta agriculturae Slovenica, 109 - 2, september 2017 The analysis of technical suitability of the equipment for application of plant protection products in Southeastern Slovenia Figure 6: Share of defected sprayers in a specific region during the period of 2004-2013 Slika 6: Delež okvarjenih naprav v posameznem območju v letih 2004-2013 The share of defect sprayers was somewhat higher in Bela Krajina region in 2006 compared to 2004 and decreased significantly after 2006. A similar pattern was detected in Osrednja Dolenjska region, where the share of defected sprayers increased slightly in 2006 and decreased dramatically after that period. In Posavje region the pattern shows a significant decrease of defected sprayers from 2004 until 2007. In that year the share increased prior to a significant decrease of defected sprayers after 2008. 4 CONCLUSIONS 4.1 Technical condition of all spraying devices As anticipated, technical condition of all spraying devices (boom sprayers and air assisted sprayers) increased during the examined time period. Although technical inspection of PPP spraying devices in Slovenia began two decades ago, it only became mandatory in 2002. The initial technical condition of spraying devices was therefore poor, which can be ascribed to the general non-attendance at testing prior to 2002. Only environmentally conscious individuals and larger farmers tested their sprayers on a regular basis as they were aware of the importance of proper functioning of the devices. The latter are only effective in distribution of PPP if their technical condition is optimal. Frequently, leakage, poor distribution of the liquid solution and inadequate dosing (output) were recorded at testing, which resulted in reduced quality of application and economic loss. Every deviation from standard functioning of the sprayer inevitably leads to inefficient use of PPP. Sadly, it seems that many farmers were not concerned with inferior performance of their sprayers as they only Acta agriculturae Slovenica, 109 - 2, september 2017 Rajko BERNIK, Peter KUHAR cultivate small areas of land and are thus satisfied with reduced performance - functioning of the devices. Small-scale farmers only require minor quantities of PPP and do not regard plant protection as a major economic cost of farming. This can be linked to a great number of defects on sprayers in the initial years of technical testing, i.e. in 2004 and 2005. Later, farmers were obliged to test their spraying equipment by law and a qualified mechanic serviced their devices on site. Many minor defects were corrected and users were simultaneously educated on a proper use and maintenance of the spraying equipment. The results of this practice came to sight in the following years as the number of defects on sprayers greatly reduced. This proves the importance of regular technical testing for improved condition of spraying equipment in Slovenia. However, defects can be recorded each year and it is therefore essential to proceed with technical testing of PPP sprayers in the future. Spraying devices have a limited lifespan and technical performance of every sprayer is reduced in time. For example, defected sprayers from the period of 2004-2008 were once in immaculate condition but because of their use and/or poor maintenance flaws occurred. That is why sprayers must be tested on a yearly basis to limit the use of faulty equipment on Slovenian farms and uncover hidden defects on the equipment such as inferior transversal distribution of spraying solution. Technical condition of boom sprayers As expected, technical condition of boom sprayers greatly improved in the experimental period. Many defects were recorded on different segments of boom sprayers in 2004 and 2005 and their number was reduced in later years. Technical condition of air assisted sprayers As in boom sprayers, technical condition of air assisted sprayers improved dramatically from 2004 to 2013. Most defects were similarly recorded in 2004 and 2005 and a superior condition of these devices was recorded in later years. 4.2 Technical condition linked to the region of the technical testing Locations were grouped according to the jurisdiction of Agricultural advisory services and three main regions were formed based on geographical similarities of the area. We assumed that the technical condition of spraying devices is not defined by region and the hypothesis was confirmed. No significant differences were detected among the three regions. Nevertheless, smaller share of defected sprayers was recorded in the Posavje region which can be ascribed to several factors. Many large farms are active in this region and consequently, the farmers possess newer and better equipment for PPP application. The spraying devices are less prone to develop any defects and are also regularly serviced. Experiences show, that small-scale farmers frequently use defected sprayers on their land. The devices are old and poorly maintained but the farmers cannot afford new mechanization due to non-favorable economic calculation based on limited land use. The other reason for superior results of the Posavje region may be linked to better technical support of Agricultural advisory services and education on the importance of proper PPP use in this area. These practices should be inspected in detail and implemented in other areas. 5 REFERENCES Balsari P., Marucco P., Oggero G., Tamagnone M. (2004). Inspection of sprayers in Italy with special regard to te Piemonte region. V: First European workhop on standardised procedure for the inspection of sprayer in europe. Mitteilungen aus der Biologischen Bundesanstalt. Braunschweig, Germany, 27. - 29. apr. 2004. Ganzelmeier H., Wehmann H. J. (ed.). Berlin, Biologischen Bundesanstalt für Land- und Forstwirtschaft Berlin und Braunschweig: 43-50 Bjugstad N., Hermansen P., Fridheim D. F. (2004). Testing of sprayers in Norway. V: First European workhop on standardised procedure for the inspection of sprayer in europe. Mitteilungen aus der Biologischen Bundesanstalt. Braunschweig, Germany, 27. - 29. apr. 2004. Ganzelmeier H., Wehmann H. J. (ed.). Berlin, Biologischen Bundesanstalt für Land- und Forstwirtschaft Berlin und Braunschweig: 57-62 Blažič M., Bolčič Tavčar M., Bukovec P., Drofenik J., Fatur T., Jukič Soršak L., Koprivnikar B. M., Lešnik M., Malovrh M., Šarc L., Vranac S., van der GEEST B. (2009). Gradivo za usposabljanje prodajalcev FFS in izvajalcev varstva rastlin. Ljubljana, Ministrstvo za kmetijstvo, gozdarstvo in prehrano, Fitosanitarna uprava Republike Slovenije: 94 str. 346 Acta agriculturae Slovenica, 109 - 2, september 2017 The analysis of technical suitability of the equipment for application of plant protection products in Southeastern Slovenia Ganzelmeier H. (2004a). Preface. V: First European workhop on standardised procedure for the inspection of sprayer in europe. Mitteilungen aus der Biologischen Bundesanstalt. Braunschweig, Germany, 27. - 29. apr. 2004. Ganzelmeier H., Wehmann H. J. (ed.). Berlin, Biologischen Bundesanstalt für Land- und Forstwirtschaft Berlin und Braunschweig: 4 Ganzelmeier H. (2004b). European standard EN 13790 -the basis for sprayer inspections in Europe. V: First European workhop on standardised procedure for the inspection of sprayer in europe. Mitteilungen aus der Biologischen Bundesanstalt. Braunschweig, Germany, 27. - 29. apr. 2004. Ganzelmeier H., Wehmann H. J. (ed.). Berlin, Biologischen Bundesanstalt für Land- und Forstwirtschaft Berlin und Braunschweig: 24 - 42 Holownicki R., Doruchowski G., Godyn A., Swiwchowski W. (2004). Obligatory inspection of sprayers in Poland - organisation and first experiences. V: First European workhop on standardised procedure for the inspection of sprayer in europe. Mitteilungen aus der Biologischen Bundesanstalt. Braunschweig, Germany, 27. - 29. apr. 2004. Ganzelmeier H., Wehmann H. J. (ed.). Berlin, Biologischen Bundesanstalt für Land- und Forstwirtschaft Berlin und Braunschweig: 51-56 Mrhar M. (1997). Kmetijski stroji in naprave. Ljubljana, Kmečki glas: 226 str. Osteroth H. J. (2004). Inspection of sprayers in Germany - results and experience over past decades. V: First European workhop on standardised procedure for the inspection of sprayer in europe. Mitteilungen aus der Biologischen Bundesanstalt. Braunschweig, Germany, 27.-29. apr. 2004. Ganzelmeier H., Wehmann H. J. (ed.). Berlin, Biologischen Bundesanstalt für Land- und Forstwirtschaft Berlin und Braunschweig: 68 -73 Pravilnik o pogojih in postopkih, ki jih morajo izpolnjevati in izvajati pooblaščeni nadzorni organi za redno pregledovanje naprav za nanašanje fitofarmacevtskih sredstev. (2000). Uradni list RS št. 72/00 Pravilnik o spremembah in dopolnitvi Pravilnika o pogojih in postopkih, ki jih morajo izpolnjevati in izvajati pooblaščeni nadzorni organi za redno pregledovanje naprav za nanašanje fitofarmacevtskih sredstev. (2005). Ur. l. RS št. 97/05 Pravilnik o spremembi pravilnika o pogojih in postopkih, ki jih morajo izpolnjevati in izvajati pooblaščeni nadzorni organi za redno pregledovanje naprav za nanašanje fitofarmacevtskih sredstev.( 2002). Uradni. list RS št. 18/02 Roettele M., Balsari P., Doruchowski G., Marucco P., Wehman H. J. (2011). Environmentally Optimized Sprayer Background and documentation. http://www.topps-eos.org/documents/EOS-Handbook_fin15_3-2011.pdf (23. 12. 2015) SIST EN 13790-1. Agricultural machinery - Sprayers -Inspection of sprayers in use - Part 1: Field crop sprayers. 2004: 20 str. SIST EN 13790-2. Agricultural machinery - Sprayers -Inspection of sprayers in use - Part 2: Air-assisted sprayers for bush and tree crops. 2004: 18 str. Zakon o zdravstvenem varstvu rastlin. (1994). Uradni list RS, št. 82/94 Acta agriculturae Slovenica, 109 - 2, september 2017 doi: 10.14720/aas.2017.109.2.18 Original research article / izvirni znanstveni članek In vitro allelopathic effect of aqueous extracts of sugarcane on germination parameters of wheat Abdul MAJEED1*, Zahir MUHAMMAD2, Manzoor HUSSAIN3 and Habib AHMAD4 Received January 26, 2017; accepted March 27, 2017. Delo je prispelo 26. januarja 2017, sprejeto 27. marca 2017. ABSTRACT Allelopathy - interactions among plants for resources along with competition - is a composite phenomenon which has spacious potentials of application in agriculture. Understanding of interactions among plants, particularly cultivated crops, may be helpful in modifying crop cultivation pattern with consequent yields increments. In this study, we investigated the allelopathic effects of aqueous extracts of root, stem peels and leaves of sugarcane (Saccharum officinale L.) cultivar 51 at concentrations 0, 2.5, 5.0, 7.5 and 10.0 g/l on germination indices and seedling biomass of wheat (Triticum aestivum L.) cultivar Pirsabak-2005. Results demonstrated that higher concentration (10.0 g/l) of extracts of root, stem peels and leaves significantly decreased mean germination time (MGT) but increased shoot and seminal root growth and seedling dry biomass; however, germination percentage was affected neither by extract concentration nor by plant parts used in the study. Extract concentrations up to 7.5 g/l had no effect on the studied parameters of wheat. Our result suggests that sugarcane's allelopathy demonstrates healthy effects on wheat growth and that wheat could be cultivated in sequential rotation in field conditions. Key words: allelopathy; sugar cane; common wheat; germination parameters; biomass; crop rotation IZVLEČEK In vitro ALELOPATSKI UČINKI VODNIH IZVLEČKOV SLADKORNEGA TRSA NA PARAMETRE KALITVE NAVADNE PŠENICE Alelopatija - interakcije med rastlinami za vire preko tekmovanja - je kompleksen fenomen, ki ima za uporabo v kmetijstvu velik pomen. Razumevanje teh interakcij med rastlinami, še posebej med gojenimi, lahko pomaga pri spreminjanju načinov pridelave z znatnim povečanjem pridelka. V raziskavi so bili preučevani alelopatski učinki vodnih izvlečkov korenin, stebel in listov sladkornega trsa (Saccharum officinale L.), kultivarja 51, v koncentracijah 0, 2.5, 5.0, 7.5 in 10.0 g/l na kalitvene parametere in biomaso kalic krušne pšenice (Triticum aestivum L.), sorte Pirsabak-2005. Rezultati so pokazali, da so večje koncentracije (10.0 g/l) izvlečkov korenin, stebla in listov značilno zmanjšale povprečni čas kalitve (MGT), a povečale rast semenskih korenin in poganjkov ter biomaso kalic, na odstotek kalitve pa niti koncentracija izvlečkov niti rastlinski organ nista vplivala. Izvlečki v koncentracijah do 7.5 g/l niso imeli učinka na preučevane parametre pšenice. Rezultati nakazujejo, da ima sladkorni trs pozitivne alelopatske učinke na rast pšenice in da lahko poljščino gojimo v kolobarju neposredno za sladkornim trsom. Ključne besede: aleopatija; sladkorni trs; krušna pšenica; biomasa; parametri kalitve; kolobar 1 INTRODUCTION Allelopathy is a composite process occurring in natural habitats as well as in cultivated fields and is generally perceived as a mechanism of plants and microbes' capacity to maintain their dominance over others or at least to coexist in a given environment. Through allelopathy alone or in combination with competition, plants can influence survival capability of in-range plants and other microorganisms in a manner that they are constrained to either migrate to somewhere else or to remain in the habitat in a defensive mode. The 1 Department of Botany, Government Degree College Naguman Peshawar, Peshawar, Khyber Pakhtunkhwa, Pakistan; *Corresponding author: majeedpsh@gmail.com 2 Department of Botany, University of Peshawar, Peshawar, Khyber Pakhtunkhwa, Pakistan 3 Department of Botany, Hazara University Mansehra, Khyber Pakhtunkhwa, Pakistan 4 Islamia College University Peshawar, Peshawar, Khyber Pakhtunkhwa, Pakistan Acta agriculturae Slovenica, 109 - 2, september 2017 str. 349 - 356 Abdul MAJEED et al. influential capacity of one plant over the other is because of the metabolic compounds (known as allelochemicals) they release into the surrounding environments either as volatile substances, rain leachates, decomposed products or direct secretion to rhizosphere with potential negative or positive interactions with other plants and microbes (Barkosky et al., 2000; Barto et al., 2010; Rice, 2012). Allelochemicals are generally secondary metabolites present in different concentrations in different plant parts (leaves, stem, barks, flowers, seeds etc.) which upon release into the rhizosphere tend to modify the resource consumption capacity by several mechanisms i.e., alteration of cell membrane permeability, changing enzymatic activity, triggering genetic defects and disturbing photosynthesis of the competitor plants (Gonzalez and Estevez-Braun, 1997; Wu et al., 2000; Barto et al., 2010; Majeed et al., 2012). Interactions among plants for resources through allelopathy lead to physiological and biochemical modifications which may result in the establishment of a successful plant community by eliminating or restricting the susceptible species, although many plants exhibit positive allelopathic effects on the surrounding plants (Elijarrat and Barcelo, 2001; Maharjan et al., 2007; Hussain et al., 2010). Thus allelopathy may be successfully employed in agriculture for enhancing crop productivity and weed management (Fang et al., 2013). Common wheat (Triticum aestivum L.) in the family Poaceae is an important agronomic crop widely cultivated for grains which are used in food and several other processed products. After maize and rice, wheat is ranked the third most widely produced grain crops in the world which has significant impact on meeting global food demands and dietary needs (Hou et al., 2014). The crop in Pakistan is generally cultivated in rotation with several other crops such as maize, rice, tobacco and sugarcane. Knowledge about allelopathic nature of preceding crops may be helpful in modifying cultivation pattern of wheat with other crops. During the last few years, allelopathic activities of several plants and agricultural crops including wheat have been rigorously worked out (Khaliq et al., 2013; Muhammad and Majeed, 2014). Sampietro and Vattuone (2006) observed root elongation of some selected weeds and crops at lower concentrations while declined root growth at higher concentration of aqueous extracts of sugarcane straw. Nikneshan et al. (2011) investigated aqueous extracts of air-dried leaf powder of eight cultivars of sunflower for allelopathic activities on wheat with decrease in germination indices at higher extract concentrations. Majeed et al. (2012) observed drastic effects of higher but stimulatory effects of lower concentrations of aqueous extracts of Chenopodium album L. on plant height, tillers, spike length and grain yield of wheat. Variable results were obtained for germination, plant height; fresh and dry mass of shoot and root of wheat under the allelopathic influence of three weeds species viz: Asphodelus tenuifolius Cav., Euphorbia hirta L., and Fumaria indica (Hausskn.) Pugsley (Jabeen et al., 2013). Ullah et al. (2013) recorded suppressive effects on germination of 20 varieties of wheat treated with aqueous leaf extracts of Eucalyptus camaldulensis Dehnh., Acacia nilotica (L.) Willd. ex Delile, Helianthus annuus L. and Parthenium hysterophorus L. Although sugarcane's allelopathy has been worked out against weeds and some crops; however, reports on its allelopathic investigation against wheat are scarce in literature. The aim of this work was to study the allelopathic activity of root, stem peels and leaves of sugarcane on germination and seedling growth of wheat in laboratory conditions. 2 MATERIALS AND METHODS 2.1 Plant materials Mature plants of Sugarcane (Saccharum officinale L.) cultivar 51 were collected at harvesting stage from cultivated fields in Naguman, Peshawar during 2013. Different plants parts i.e., leaves, stem peels and root were separated and dried under shade conditions. Dried parts were ground to powder with an electric grinder for further use. In order to get aqueous extracts of different concentration, 2.5, 5.0, 7.5 and 10.0 g of dried powder of each part i.e., leaves, stem peels and roots were soaked for 24 h in 1litre distilled water each at room temperature. The soaked materials were filtered through muslin cloth after 24 hours. Filtrate was again filtered through filter paper (Whatman No. 1) in sterilized flasks to get extracts of different concentrations. Aqueous extracts were stored at 4 °C in a refrigerator. 2.2 Bioassay Seeds of wheat (Triticum aestivum L.) cultivar Pirsabak-2005 were obtained from Agricultural Research Institute, Tarnab, Peshawar. Seeds were placed on twice folded filter paper as seed beds in petri-dishes. Each petri-dish was provided with 10 ml of respective concentrated aqueous extracts. Control seeds were provided with the same volume of distilled water. Each Petri dish had 10 seeds; each treatment was further replicated 4 times. The experiment was arranged in a 350 Acta agriculturae Slovenica, 109 - 2, september 2017 In vitro allelopathic effect of aqueous extracts of sugarcane on germination parameters of wheat Completely Randomized Manner at room temperature (20-25 °C) with 12 h photo period. The experiment was performed at Botany Department, Government Degree College Naguman Peshawar during 2013. After 72 hours, data on germination, seminal root and shoot length of seedling was recorded. Germination (%) was calculated as number of germinated seeds in each replicate after 72 hours till final reading. Mean germination time was determined as: MGT = E (di x ni) /Eni ; where n represents number of germinated seeds at ith day and d is the number of days counted from the beginning till the completion of germination (Basra et al., 2005). Dry biomass of seedling was determined as previously described by Muhammad and Hussain (2012). 2.3 Statistical analysis Results were statistically analyzed by the analysis of variance (ANOVA). Least significant difference (LSD) was used to determine significant mean values of the studied parameters at p<0.05. 3 RESULTS 3.1 Germination (%) In this experiment, the allelopathic activity of root, stem peels and leaves of sugarcane which were prepared as aqueous extracts in different concentrations were studied on germination parameters and dry biomass of wheat. Results revealed that germination percentage was neither affected by different plant parts nor by extract concentrations. Interaction between plant parts and concentrations were also non-significant. Germination (%) was maximum (91.8) in control conditions. Slighter decrease in germination was observed in petri-dishes treated with different extract concentrations of different plant parts; however, the differences among means of concentrations and plant parts for germination percentage were insignificant and they ranged between 89.7 - 90.9 % which did not differ significantly from 91.8 % in control (Table 1). Table 1: Effect of different concentrations of aqueous extracts of plant parts (root, stem peel and leaves) of sugarcane on germination (%) of wheat Plant parts Concentration (g/l) Plant parts means Control 0 2.5 5.0 7.5 10.0 Roots 91.8a 90.9a 90.7a 90.9a 91.4a 90.98a Stem peel 91.8a 90.7a 91.1a 90.9a 91.0a 91.10a Leaves 91.8a 90.8a 90.9a 90.7a 89.7a 90.64a Concentration means 91.8a 90.8a 90.9a 90.83a 90.7a 3.2 Mean Germination Time MGT was calculated to assess the average number of days taken from the beginning of germination of seeds till completion of germination. MGT was significantly affected by plant parts as well as concentration of extracts. Interaction between plant parts and concentration was also recorded as significant. It was observed that lower extract concentrations up to 7.5 g/l of each plant part had no effect on this parameter; however, 10.0 g/l extract significantly minimized germination time particularly when roots and peel extracts were applied (Table 2). Leaf extract at the highest dose (10 g/l) significantly prolonged meant time of germination which was recorded as 5.8 days. In each plant part at highest extract concentration, MGT was lowest than control where it was 4.7 days except in leaf extract which revealed maximum value for this parameter. 3.3 Shoot growth Analysis of variance (ANOVA) determined significant differences for values of shoot length of wheat seedlings at 10g/l concentrated extracts but insignificant effect at lower concentrations. Interactions were also significant. Shoot length was 15.9 mm in control plates which were treated with distilled water. There were no significant differences between values in extract concentrations 2.5, 5 and 7.5 g/l of respective plant parts which revealed results almost consistent with those of control; however, 10 g/l extract of root and stem peels significantly increased shoot length (16.8 and 16.7 mm respectively). Unexpectedly, the same concentrated extract of leaves had adverse effect on shoot growth which was significantly reduced (14.2 mm) as compared to control and other plant part extracts (Table 3). Acta agriculturae Slovenica, 109 - 2, september 2017 351 Abdul MAJEED et al. Table 2: Effect of different concentrations of aqueous extracts of plant parts (root, stem peel and leaves) of sugarcane on mean germination time (MGT) (days) of wheat Plant parts Concentration (g/l) Plant parts means Control 0 2.5 5.0 7.5 10.0 Roots 4.7a 4.3a 4.6a 4.8a 3.4c 4.36a Stem peel 4.7a 4.4a 4.7a 4.9a 3.7b 4.48a Leaves 4.7a 4.8a 4.8a 4.9a 5.8bc 4.4a Concentration means 4.7a 4.5a 4.7a 4.86a 3.3c LSD (p<0.05) for plant parts = 3.245; concentration = 2.769; interaction 3.120 Values in columns and rows with different alphabets are significantly different Table 3: Effect of different concentrations of aqueous extracts of plant parts (root, stem peel and leaves) of sugarcane on shoot length (mm) of wheat Plant parts Concentration (g/l) Plant parts means Control 0 2.5 5.0 7.5 10.0 Roots 15.9a 16.0a 15.9a 15.3a 16.8b 90.98a Stem peel 15.9a 15.8a 15.7a 15.5a 16.7b 91.1a Leaves 15.9a 15.4 15.6a 15.7a 14.2bc 90.64a Concentration means 15.9a 15.8a 15.7a 15.5a 15.9a LSD (p<0.05) for plant parts = 1.971; concentration = 3.963; interaction 4.7612 Values in columns and rows with different alphabets are significantly different 3.4 Seminal root length Like other indices, seminal root length was significantly influenced by concentrations of the extracts as well as different plant parts. Lower concentration had no effect on RL whose values were similar to control with slight variations. Interaction between plant parts and concentrations at highest dose were significant. Control condition and extract concentrations up to 7.5 g/l recorded almost similar values of seminal root length which ranged between 27.3-27.9 mm. Maximum length (30.1 mm) of seminal root was observed in root extract at 10g/l concentration followed by the same extract concentration of stem peels and leaves which yielded consistent values 29.4 and 29.3 mm respectively (Table 4). 3.5 Seedling dry biomass Dry biomass of seedling was calculated in each replicate of each treatment and then averaged to determine individual seedling's biomass. Results demonstrated significantly different values for different plant parts, 10g/l extract concentration and interactions but insignificant differences were observed for lower concentrations (2.5 - 7.5 g/l). Values of dry biomass slightly varied under lower extract concentrations of each plant part; however, they were statistically similar to control values. Root and stem peels extract at 10 g/l resulted in highest dry biomass of seedlings which were recorded as 40.1 and 39.2 mg respectively when compared to control (36.1 mg). The lowest value for dry biomass was observed in 10 g/l leaves extract which was 34.2 mg, significantly different from control as well as root and peel extracts at the same concentration (Table 5). 350 Acta agriculturae Slovenica, 109 - 2, september 2017 In vitro allelopathic effect of aqueous extracts of sugarcane on germination parameters of wheat Table 4: Effect of different concentrations of aqueous extracts of plant parts (root, stem peel and leaves) of sugarcane on seminal root length (mm) of wheat Plant parts Concentration (g/l) Control 0 2.5 5.0 7.5 10.0 Plant parts means Roots Stem peel Leaves Concentration means 27.8a 27.8a 27.8a 27.8a 27.7a 27.5a 27.3a 27.5a 27.6a 27.7a 27.4a 27.5a 28.0a 27.9a 27.8a 27.9a 301b 28.4a 29.4b 28.06a 29.3b 27.9a 29.6b LSD (p<0.05) for plant parts = 11.09; concentration = 7.939; interaction 1.890 Values in columns and rows with different alphabets are significantly different Table 5: Effect of different concentrations of aqueous extracts of plant parts (root, stem peel and leaves) of sugarcane on dry biomass seedling-1 (g) of wheat Plant parts Concentration (g/l) Plant parts means Control 0 2.5 5.0 7.5 10.0 Roots 36.1a 35.8a 36.9a 36.5a 40.1b 37.08c Stem peel 36.1a 36.4a 36.3a 36.9a 39.2b 36.98a Leaves 36.1a 36.7a 36.4a 36.7a 34.2c 36.02a Concentration means 36.1a 36.3a 36.5a 36.7a 37.8ab LSD (p<0.05) for plant parts = 5.213; concentration = 1.7821; interaction 6.379 Values in columns and rows with different alphabets are significantly different 4 DISCUSSION Germination is an important indicator which depicts the plants' response to changes in the environment, resources or any allelopathic stress induced as a result of allelochemicals released from donor plants (Hussain et al., 2010). Germination indices are generally used to detect potential stimulatory or inhibitory allelopathic activity of the test plant (Hussain and Reigosa, 2014). In our study, germination percentage was not affected by aqueous extract of different plant parts of S. officinale. Moreover, concentrations of the extracts were also unable to stimulate or inhibit the germination percentage of wheat. This is in contradiction with previous studies on the allelopathy of Hypericum myrianthum Cham. & Schltdl. (Fritz et al., 2007), Eucalyptus camaldulensis (Ahmed et al., 2008), Prosopis juliflora (Sw.) DC. (Siddiqui et al., 2009), Dodonaea viscosa Jacq. (Barkatullah et al., 2010) and Halianthus annus L. (Muhammad and Majeed, 2014) on germination of wheat and other target crops which revealed significant retardation of germination under the allelopathy of the respective plants at different extract concentrations. We used the highest concentration as 10 g l-1 which is relatively low as compared to extract concentrations used in other studies. Thus, non-responsiveness of seed germination to alleopathic stress in this study might be due to relatively low concentration or possible resistance exhibited by wheat seeds to potential allelopathic activity of sugarcane. Mean germination time (MGT) is another important parameter which determines the energy of germination capacity of seeds in a stressed environment (Bonciarelli, 1995). MGT of target plants in a particular allelopathic assay may either be increased or decreased, depending on the concentration and type of allelopathic compounds. Phenolic compounds, in general, have been sought to prolong germination time as they may possibly interfere with seed dormancy and enzymatic activity necessary for rapid germinability. In this study, MGT was significantly reduced by root and peel extracts at 10 g/l concentration but prolonged under leaves extract. This suggest that root and peels extracts of sugarcane have some potent alleochemicals with stimulatory effect on germination time. On the other hand, leaves extract may possibly possess phenolics which had detrimental effect on this parameter. Acta agriculturae Slovenica, 109 - 2, september 2017 351 Abdul MAJEED et al. Previously, Sampietro & Vattuone (2006) isolated phenolic compounds from straw of sugarcane which had deteriorating effects on germination and general growth of different weeds and crops. Shoot and seminal root growth were significantly increased by root and stem peels extract at higher concentration which demonstrated stimulatory allelopathy of sugarcane. We assume that root and peel extracts of sugarcane may contain carbohydrates complexes and unknown diffusible allelochemicals which induced stimulatory response in wheat seedling with consequent increase in shoot and seminal root length. However, leaf extract showed detrimental effects on the lengths of shoot and seminal root. Previously stimulatory effect of rice hull extracts (Seyyednejad et al., 2010) but inhibitory effect of Jatropha curcas L. (Aburge and Sam, 2010), Dodonaea viscosa (Barkatullah et al., 2010) and oleander and walnut leaf extracts (Unal, 2013) on shoot length of different plants including wheat have been reported. Similarly, our results regarding reduced shoot and seminal root length under leaf extracts are in line with findings of Batlang and Shusho (2007), Singh et al. (2009), Sadehgi et al. (2010) and Unal (2013) who reported shoot and seminal root suppression of wheat in response to aqueous extracts of different allelopathic plants. Primary effect of allelochemicals may probably be alteration in cell membrane permeability of the target plant which can cause secondary effects such as changes in water and mineral absorptions potentials, changes in pH, enzymatic alterations etc. thus causing either stimulatory or inhibitory effects (Barkosky et al. 2000; Gatti et al., 2010; Majeed et al., 2012). Allelopathic stress may either elevate the level or induce the inhibition of carbohydrates and protein contents of target plant which build up more proline content as stress indicator; consequently plant growth is either increased or reduced (Batish et al., 2007; Al-Johani et al., 2012). Increase in dry biomass was observed under allelopathic effects of 10 g/l root and peel extract which are supported by the findings of Mubeen et al. (2012) on barley and Han et al. (2013) on lettuce who documented increase in dry biomass in response to alleopathic aqueous extracts of diverse plants. Decrease in biomass of seedlings treated with leaf extracts in this study are in agreement with Singh et al. (2005), Jamil et al. (2009), Ullah et al. (2013) and Jabeen et al. (2013) who reported suppression in fresh and dry biomass of seedling in Brassica sp., wild oat and wheat under the influence of allelopathy of some weeds and crops. Differences in results of different studies may be due to different experimental conditions, extract concentrations and test plants they used. The increase in dry biomass of wheat seedlings treated with root and stem peel extract in the current study may probably be due to increase in photosynthetic rate and greater accumulation of photo-assimilate in the shoot, increase in thickness and length of seminal root, decrease in proline content and greater potassium ion uptake induced by allelopathic extracts of S. officinale; as in earlier studies some authors argued that botanical extracts could have positive influence on these phenomena thus stimulating biomass of the receptor plant (Rizvi and Rizvi, 1992; Gatti et al., 2010; Ibrahim et al., 2013). Similarly, reduced biomass, seminal root and shoot length under leaf extract suggests the presence of phenolic and growth inhibitory compounds in leaves of sugarcane and these reductions may be regarded as a result of induced oxidative damage and activation of cellular anti- oxidant systems which in turn changed permeability patterns of cell membrane, irregularities in mineral and ion uptake and the activation of stress enzymes (Oracz et al., 2007; Khaliq et al., 2012). 5 CONCLUSION The present study suggests that different plant parts of sugarcane exhibit both stimulatory and inhibitory allelopathy against wheat in germination bioassay. Roots and stem peel extracts at higher concentration promoted germination time, shoot and seminal root growth and dry biomass of seedling while leaf extract had negative effects on these parameters. Pot culture study is underway to confirm our lab bioassay findings. 6 REFERENCES Abugre, S. & Sam, S.J. (2010). Evaluating the allelopathic effect of Jatropha curcas aqueous extract on germination, radicle and plumule length of crops. International Journal of Agriculture and Biology, 12, 769-772. Ahmed, R., Hoque, A.T.R., & Hossain, M.K. (2008). Allelopathic effects of leaf litters of Eucalyptus camaldulensis on some forest and agricultural crops. Journal of Forestry Research, 19(1), 19-24. doi:10.1007/s11676-008-0003-x 350 Acta agriculturae Slovenica, 109 - 2, september 2017 In vitro allelopathic effect of aqueous extracts of sugarcane on germination parameters of wheat Al-Johani, N.S., Aytah, A.A., & Boutraa, T. (2012). Allelopathic impact of two weeds on growth and photosynthesis of barley (Hordeum vulgare L.). Pakistan Journal of Botany, 44(6), 1865-1872. Barkatullah, Hussain, F., & Ibrar. M. (2010). Allelopathic potential of Dodonaea viscosa (L.) Jacq. Pakistan Journal of Botany, 42(4), 23832390. Barkosky, R.R., Einhellig, F.A., & Butler, J.L. (2000). Caffeic acid-induced changes in plant-water relationships and photosynthesis in leafy spurge Euphorbia esula. Journal of Chemical Ecology, 26(9), 2095-2109. doi:10.1023/A:1005564315131 Barto, K., Friese, C., & Dipollini, D. (2010). Arbuscular mycorrhizal fungi protect a native plant from allelopathic effects of an invader. Journal of Chemical Ecology, 36(4), 351-360. doi:10.1007/s10886-010-9768-4 Basra, S.M.A., Farooq, M., Tabassam, R., & Ahmad, N. (2005). Physiological and biochemical aspects of pre-sowing seed treatments in fine rice (Oryza sativa L.). Seed Science and Technology, 33(3), 623-628. doi:10.15258/sst.2005.33.3.09 Batish, D.R., Lavanya, K., Singh, H.P., & Kohli, R.K. (2007). Phenolic allelochemicals released by Chenopodium murale affect the growth, nodulation and macromolecule content in chickpea and pea. Plant Growth Regulation, 51(2), 119-128. doi:10.1007/s10725-006-9153-z Batlang, U., & Shushu, D.D. (2007). Allelopathic activity of sunflower (Helianthus annuus L.) on growth and nodulation of bambara groundnut (Vigna subterranea (L.) Verdc.). Journal of Agronomy, 6 (4), 541-547. doi:10.3923/ja.2007.541.547 Bonciarelli, F. (1995). Fondamenti di Agronomia Generale. Ed. Agricole, Bologna, Italy, pp. 372. Eljarrat, E., & Barcelo, D. (2001). Sample handling and analysis of allelochemical compounds in plants. Trends in Analytical Chemistry, 20(10), 584-590. doi:10.1016/S0165-9936(01)00104-2 Fang, C., Zhuang, Y., Xu, T., Li, Y., & Lin, W. (2013). Changes in rice allelopathy and rhizosphere microflora by inhibiting rice phenylalanine ammonia-lyase gene expression. Journal of Chemical Ecology, 39(2), 204-212. doi:10.1007/s10886-013-0249-4 Fritz, D., Bernardi, A.P., Haas, J.S., Ascoli, B.M., Bordignon, S.A.D.L., & Von Poser, G. (2007). Germination and growth inhibitory effects of Hypericum myrianthum and H. polyanthemum extracts on Lactuca sativa L. Revista Brasileira de Farmacognosia, 77(1), 44-48. doi:10.1590/S0102-695X2007000100010 Gatti, A.B., Ferreira, A.G., Arduin, M., & Perez, S.C. (2010). Allelopathic effects of aqueous extracts of Artistolochia esperanzae O. Kuntze on development of Sesamum indicum L. seedlings. Acta Botanica Brasilica, 24(2), 454-461. doi:10.1590/S0102-33062010000200016 Gonzalez, J.A., & Estevez-Braun, A. (1997). Phytonematicidal activity of aromatic compounds related to shikimate pathway. Pesticide Biochemistry and Physiology, 58(3): 193-197. doi:10.1006/pest.1997.2294 Han, X., Cheng, Z., Meng, H.U., Yang, Y., & Ahmad, I. (2013). Allelopathic effect of decomposed garlic (Allium sativum L.) stalk on lettuce (L. sativa var. crispa). Pakistan Journal of Botany, 45(1), 225233. Hou, J., Jiang, Q., Hao, C., Wang, Y., Zhang, H., & Zhang, X. (2014). Global selection on sucrose synthase haplotypes during a century of wheat breeding. Plant Physiology, 164(4), 1918-1929. doi:10.1104/pp. 113.232454 Hussain, F., Ahmad, B., & Ilahi, I. (2010). Allelopathic effects of Cenchrus ciliaris L. and Bothriochloa pertusa (L.) A. Camus. Pakistan Journal of Botany, 42, 3587-3604. Hussain, M.I., & Reigosa, M.J. (2014). Evaluation of herbicide potential of sesquiterpene lactone and flavonoid: impact on germination, seedling growth indices and root length in Arabidopsis thaliana. Pakistan Journal of Botany, 46, 995-1000. Ibrahim, M., Ahmad, N., Shinwari, Z.K., Bano, A., & Ullah, F. (2013). Allelopathic assessment of genetically modified and non-modified maize (Zea mays L.) on physiology of wheat (Triticum aestivum L.). Pakistan Journal of Botany, 45(1), 235-240. Jabeen, N., Ahmed, M., Shaukat, S.S., & Iram-us-Slam. (2013). Allelopathic effects of weeds on wheat (Triticum aestivum L.) germination and growth. Pakistan Journal of Botany, 45(3), 807-811. Jamil, M., Cheema, Z.A., Mushtaq, M.N., Farooq, M., & Cheema, M.A. (2009). Alternative control of wild oat and canary grass in wheat fields by allelopathic plant water extracts. Agronomy for Sustainable Development, 29(3), 475-482. doi:10.1051/agro/2009007 Khaliq, A., Matloob, A., Aslam, F., Mushtaq, M.N., & Khan, M.B. (2012). Toxic action of aqueous wheat straw extract on horse purslane. Planta Daninha, Acta agriculturae Slovenica, 109 - 2, september 2017 351 Abdul MAJEED et al. 30(2), 269-278. doi:10.1590/S0100- 83582012000200005 Khaliq, A., Matloob, A., Khan, M.B., & Tanveer, A. (2013). Differential suppression of rice weeds by allelopathic plant aqueous extracts. Planta Daninha, 31(1), 21-28. doi:10.1590/S0100-83582013000100003 Maharjan, S., Shrestha, B.B., & Jha, P.K. (2007). Allelopathic effects of aqueous extract of leaves of Parthenium hysterophorus L. on seed germination and seedling growth of some cultivated and wild herbaceous species. Scientific World, 5(5): 33-39. Majeed, A., Chaudhry, Z., & Muhammad, Z. (2012). Aallelopathic assessment of fresh aqueuous extracts of Chenopodium album L. for growth and yield of wheat (Triticum aestivum L.). Pakistan Journal of Botany, 44(1), 165-167. Mubeen, K., Nadeem, M.A., Tanveer, A., & Zahir, Z.A. (2012). Allelopathic effects of sorghum and sunflower water extracts on germination and seedling growth of rice (Oryza sativa L.) and three weed species. Journal of Animal and Plant Science, 22(3), 738-746. Muhammad, Z., & Majeed, A. (2014). Allelopathic effects of aqueous extracts of sunflower on wheat (Triticum aestivum L.) and maize (Zea maize L.). Pakistan Journal of Botany, 46(5), 1715-1718. Muhammad, Z., & Hussain, F. (2012). Effect of NaCl salinity on the germination and seedling growth of seven wheat genotypes. Pakistan Journal of Botany, 44(6), 1845-1850. Nikneshan, P., Karimmojeni, H., Moghanibashi, M., & Hosseini, N. (2011). Allelopathic potential of sunflower on weed management in safflower and wheat. Australian Journal of Crop Science, 5(11), 1434-1440. Oracz, K., Bailly, C., Gniazdowska, A., Come, D., Corbineau, F., & Bogatek, R. (2007). Induction of oxidative stress by sunflower phytotoxins in germinating mustard seeds. Journal of Chemical Ecology, 3(2), 251-264. doi:10.1007/s10886-006-9222-9 Rice, E.L. (2012). Allelopathy: Physiological Ecology. Academic Press, Inc. USA. Pp.1 Rizvi, S.J.H., & Rizvi, V. (1992). Allelopathy: Basic and Applied Aspects. London, Chapman & Hall. Pp. 480. doi:10.1007/978-94-011-2376-1 Sadeghi, S., Rahnavard, A., & Ashrafi, Z.Y. (2010). Response of wheat (Triticum aestivum) germination and growth of seedling to allelopathic potential of sunflower (Helianthus annuus) and barley (Hordeum vulgare L.) extracts. Journal of Agricultural Technology, 6, 573-577. Sampietro, D.A., & Vattuone, M.A. (2006). Sugarcane straw and its phytochemicals as growth regulators of weed and crop plants. Plant Growth Regulation, 48(1), 21-27. doi:10.1007/s10725-005-5135-9 Seyyednejad, S.M., Koochak, H., Najafabade, F.P., & Kolahi, M. (2010). Allelopathic effect of aquatic hull extract of rice (Oryza sativa L.) on growth of Silybum marianum and Echinochloa crus-galli. African Journal of Agricultural Research, 5(16), 2222-2226. Siddiqui, S., Bhardwaj, S., Khan, S.S., & Meghvanshi, M.K. (2009). Allelopathic effect of different concentration of water extract of Prosopis juliflora leaf on seed germination and radicle length of wheat (Triticum aestivum Var-Lok-1). American-Eurasian Journal of Scientific Research, 4(2), 8184. Singh, B., Vikaspal, S., & Kumar, M. (2009). Effect of Tinospora cordifolia aqueous extract on traditional food crops of Garhwal Himalaya. International Journal of Sustainable Agriculture, 1(2), 36-40. Singh, H.P., Batish, D.R., Pandher, J.K., & Kohli, R.K. (2005). Phytotoxic effects of Parthenium hysterophorus residues on three Brassica species. Weed Biology and Management, 5(3), 105-109. doi:10.1111/j. 1445-6664.2005.00172.x Ullah, A., Khan, E.A., Baloch, M.S., Nadim, M.A., Sadiq, M., & Noor, K. (2013). Allelopathic effect of herbaceous and woody plants species on seed germination and seedling growth of wheat. Pakistan Journal of Weed Science Research, 19(3), 357-375. Unal, B.T. (2013). Effects of growth regulators on seed germination, seedling growth and some aspects of metabolism of wheat under allelochemical stress. Bangladesh Journal of Botany, 42(1), 65-72. doi:10.3329/bjb.v42i1.15865 Wu, H., Pratley, J., Lemerle, D., & Haig, T. (2000). Evaluation of seedling allelopathy in 453 wheat (Triticum aestivum) accessions against annual ryegrass (Lolium rigidum) by the equal-compartment-agar method. Crop and Pasture Science, 51(7), 937-94. doi:10.1071/AR00017 O C/T 350 Acta agriculturae Slovenica, 109 - 2, september 2017 doi: 10.14720/aas.2017.109.2.19 Original research article / izvirni znanstveni članek Assessment of heritability and genetic advance for agronomic traits in durum wheat (Triticum durum Desf.) Hassan NIKKHAHKOUCHAKSARAEI1, Hamlet MARTIROSYAN2 Received January 29, 2017; accepted July 08, 2017. Delo je prispelo 29. januarja 2017, sprejeto 08. julija 2017. ABSTRACT In order to evaluate the amount of heritability for desirable agronomic characteristics and the genetic progress associated with grain yield of durum wheat (Triticum durum Desf.), a split plot experiment was carried out with four replications during three cropping seasons (2009-2012). Three sowing dates (as environmental factor) and six durum wheat varieties (as genotypic factor) were considered as main and sub factors respectively. Analysis of variance showed interaction effects between genotypes and environments in days to ripening, plant height, spike length, number of grains per spike, number of spikes per unit area, grain mass and grain yield. The grain yield showed the highest positive correlation with number of grains per spike also grain mass (91 % and 85 %, respectively). A relatively high heritability of these traits (82.1 % and 82.2 %, respectively) suggests that their genetic improvement is possible. The maximum genetic gain (19.6 %) was observed for grain mass, indicating this trait should be a very important indicator for durum wheat breeders, although the climatic effects should not be ignored. Key words: durum wheat; grain yield; plant genetics; yield components IZVLEČEK OVREDNOTENJE DEDNOSTI IN GENETSKE PREDNOSTI AGRONOMSKIH LASTNOSTI TRDE PŠENICE (Triticum durum Desf.) Z namenom ovrednotenja dednosti željenih agrononmskih lastnosti in genetskih procesov povezanih s pridelkom zrnja trde pšenice (Triticum durum Desf.) je bil izveden poskus z deljenkami s štirimi ponovitvami v rastnih sezonah 20092012. Tri datumi setve kot okoljski dejavniki in šest sort trde pšenice kot genetski dejavnik so bili uporabljeni kot glavni in podrejeni dejavniki. Analiza variance je pokazala interakcijske učinke med genotipi in okoljem v dnevih do zrelosti, višini rastlin, dolžini klasov, številu zrna na klas, številu klasov na enoto površine, masi zrn in pridelku zrnja. Pridelek zrnja je pokazal največjo pozitivno korelacijo s številom zrn na klas in maso zrnja, 91 % in 85 %. Relativno velika dednost teh lastnosti, 82.1 % in 82.2 % nakazuje, da je možno njuno genetsko izboljšanje. Največja genetska pridobitev (19.6 %) je bila opažena pri masi zrnja, kar nakazuje, da bi morala biti ta lastnost zelo pomemben kazalnik za žlahtnitelje trde pšenice, čeprav tudi podnebni dejavniki ne bi smeli biti zanemarjeni. Ključne besede: trda pšenica; pridelek zrnja; rastlinska genetika; komponente pridelka 1 INTRODUCTION Durum wheat (Triticum durum Desf.) is cultivated on 21 million hectares, about 10 % of all cultivated areas in the world. Durum wheat is an important and popular crop in the Mediterranean region and is used for food products as couscous, bulgur, and pasta (Gisslen, 2001). Durum wheat genotypes had shown better adaptation to varying environments than common wheat (T. aestivum L.) (Khazaei et al., 2013). So, selection of the stable durum wheat genotypes for achieving both high grain yield and good quality is very important. It is important to use appropriate selection method and selection intensity for traits of interest, proper statistical assessment of genetic variation, the magnitude of heritability (usually represented by h2), genetic coefficient of variation, and response to selection. 1 Department of Agronomy and Plant Breeding, Qaemshahr Branch, Islamic Azad University, Qaemshahr, Iran; * Corresponding author: hsnnikkhah@yahoo.com 2 Department of Plant Cultivation, Armenian National Agrarian University, Yerevan, Armenia This article is part of the Ph.D. thesis entitled « Influence of some agro technical measures on the productivity of durum wheat (Triticum durum Desf.) varieties (under the conditions of Mazandaran province of Iran) », issued by Hassan Nikkhahkouchaksaraei, supervisor Assist. Prof. Hamlet Martirosyan, Ph. D. Acta agriculturae Slovenica, 109 - 2, september 2017 str. 349 - 356 Hassan NIKKHAHKOUCHAKSARAEI, Hamlet MARTIROSYAN Genetic variability is an important factor in hybridization program for producing high yielding progenies. The effective selection depends on the amount of genetic variability and amount of heritability indices (Heidari, 2010). For having a good response to selection, high genetic variation and high heritability are needed (Shukla et al., 2006). There is a direct relationship between heritability and response to selection which is referred to as genetic progress. The expectation of a response to selection is called genetic advance (G.A.). High genetic advance coupled with high heritability estimate offers the best effective condition for selection (Larik et al., 2000). Therefore, genetic advance is an important indicator associated with selection that aids plant breeder in his work (Shukla et al., 2006; Memon et al., 2005). High genetic variation for traits under selection as well as high heritability, are crucial for having good response to selection (Shukla et al., 2006). Manal (2009) reported high heritability accompanied by high genetic advance for spike length and 1000 grain-mass in his study of heritability and genetic advance of yield traits in common wheat (T. aestivum) under drought condition. This fact suggests that selection should lead to a fast genetic improvement of trait. The purpose of this study was to identify the traits which can be used as selection markers and can also help to predict the grain yield of durum wheat. 2 MATERIALS AND METHODS The experiments were conducted during three crop seasons (2009-2012), at the experimental field of the Islamic Azad University, Qaemshahr Branch, Mazandaran Province of Iran (36 ° 30 ' N, 52 ° 48 ' E, 28 m above sea level). The experiments were designed as split-plots based on randomized complete block design with four replicates. Three sowing dates (as environmental conditions) were 25 October, 25 November and 25 December and were randomized as main plots. Six durum wheat genotypes ('Yavaros', 'Tarro-3', 'Shwa/Mald', 'Stork', 'Behrang' and 'Syrian-4') from different origin (CYMMIT and ICARDA) were used as subplots. Each plot included fifteen rows 5 m long and 0.18 m apart. The seed rate was 500 viable seeds per one square meter. Based on soil test, urea fertilizer (46, 0, 0; N. P. K.) as source of nitrogen, and triple superphosphate fertilizer (0, 46, 0; N. P. K.) as source of phosphorus were used. Herbicide, fungicide and insecticide were used as usual. The area of 3 square meters was harvested to estimate grain yield and related traits (including the number of days to ripening, plant height, spike length, number of grains per spike, number of spikes per square meter and grain mass) at when plants were mature. Analysis of variance and combined analysis of variance (Steel and Torrie, 1980) were conducted on data by using the statistical SAS program (SAS Institute, 2008), in according to following statistical model. Years with random effect as well as treatments and sowing dates with fixed effects had considered in this model (Yazdi Samadi et al., 1997). Mean comparisons by using Duncan's Multiple Range Test. ijkl n + G, + Bjk + Dk+ Yi + GDk + GYll + DYa + GDYm + E, ijkl ijk (Steel and Torrie, 1980) Where, XijU is the amount of each trait in a plot. The mean of trait, the effect of genotype, the effect of repetition, the effect of sowing date, the effect of binary interaction (respectively, including the effect of genotype x sowing date, genotype x year, planting date x year), the effect of triplet interaction (genotype x sowing date x year) and residual effect or experimental error, have been shown from left to right, respectively. Phenotypic coefficient of variation (S2p) depends on genetic variation (S2g), environmental variation (S2e) and their interactions. / à2 = à2 + à2 + à2 + à2 + à2 à2 à2 g + à gy + à gd + à gyd + gd = (M5 - M6 - M? + MS) / rdy 'gy = (M? - MS + M9) / rd 'gd = (M? - MS + M9) / ry Ogyd = (M9 - M8) / r d2e = M9 Also the following formulas are used for calculating the coefficients of variation (Burton, 1952). G.CV. = (^S2g/Y) x 100 P.CV. = (<82p / Y) x 100 E.CV. = ( 45 mm). As per Venkatasalam et al. (2012), addition of organic manure significantly influenced the beneficial microorganisms to colonize in rhizosphere and stimulate plant growth by providing necessary nutrients besides synthesizing some plant hormones; which may be the reason for increase in plant height, number of leaves and tuber mass, irrespective of potato variety. 370 3 7 8 Acta agriculturae Slovenica, 109 - 2, september 2017 Evaluation of an organic package of practice ... farming in terms of yield, quality, energy efficiency and economics Table 4: Growth attributes of potato under studied farming systems (as average of both seasons) Treatment Potato Variety Emergence (%) Plant height (cm) No. of compound leaves /plant No. of tuber (thousand/ha) Processing Total Grade (> 45 mm) Pilot Area I: Mathurapur village (district: North 24 Paraganas), West Bengal, India Conventional Crop KufriJyoti 93.3 - 96.8 (95.1c)[±4.91] 43.3 - 49.8 (45.5)[±2.53] 44.2 - 46.8 (44.6c)[±2.13] 294.69b 539.7b Management (CCM) KufriChandramu khi 93.7 - 97.1 (95.6c)[±5.03] 46.7 - 54.1 (49.6)[±2.11] 48.3 - 54.4 (50.2b)[±2.37] 275.67c 501.45c Green Farming utilizing IRF KufriJyoti 97.5 - 99.8 (98.6a)[±4.13] 43.7 - 52.8 (48.1)[±2.05] 44.8 - 53.7 (50.3b)[±2.76] 325.53a 586.74a KufriChandramu khi 97.1 - 99.4 (97.7ab)[±4.11] 47.1 - 59.3 (56.2)[±2.09] 49.6 - 59.8 (56.6a)[±2.14] 287.25bc 529.23b Pilot Area II: Bhabanipur village, (district: Nadia), West Bengal, India Conventional Crop KufriJyoti 94.3 - 98.8 (96.8ns)[±5.88] 44.3 - 51.8 (48.8d)[±2.09] 43.7 - 54.8 (47.5c)[±1.71] 263.27d 494.44d Management (CCM) Super 6 95.2 - 98.4 (96.5ns)[±5.44] 49.3 - 53.8 (50.7c)[±2.01] 48.7 - 63.4 (57.5ab)[±2.23] 302.13b 543.24b Green Farming utilizing IRF KufriJyoti 96.5 - 99.2 (97.4ns)[±4.79] 46.2 - 59.5 (54.4b)[±2.12] 43.3 - 53.5 (46.5c)[±2.19] 285.46c 530.13bc Super 6 96.3 - 98.3 (96.3ns)[±4.75] 48.3 - 59.8 (57.1a)[±2.13] 50.4 - 65.3 (59.3a)[±2.12] 365.34a 631.13a Note: Figure in parenthesis () represents mean value & Standard Error [± ]. The means marked with different letters in the same column were significantly different at P< 0.05 under Duncan's New Multiple Range Test. Nutrient Use Efficiency (NUE) in terms of 'Partial Factor Productivity' of applied nutrients (PFPnpk); were evaluated for both CCM and green farming plots. The advantage of PFPNPK is that it quantifies total economic output from any particular factor/nutrient, relative to its utilization from all resources in the system, including indigenous soil nutrients and nutrients from applied inputs (Cassman et al., 1996). Decline in PFPnpk may be attributed to nutrient imbalance, decline in indigenous soil-NPK supply, subsoil compaction, reduced root volume and increased incidence of pests and diseases (Karim & Ramasamy, 2000). Adoption of efficient NPK management practices is responsible for higher partial factor productivity (Yadav, 2003). The value was found to be significantly higher in green farming plots as compared to CCM (Table 5). 20.61 to 23.03 kg and 63.01 to 74.93 kg potato per kg NPK, were recorded under CCM and green farming plots respectively. Higher value in case of the later treatment may be attributed to adoption of organic plant management (using IRF). Evaluation of grade wise tuber distribution revealed higher production of large (>75 g) and medium sized (50-75 g) tubers under green farming (13.6 and 25.5 % respectively). This may be due to cumulative effect of organic and inorganic fertilizers sources (Mandal et al., 2005) as well as organic plant management (Figure 4). Higher bulking of tubers under integrated soil management was corroborated with the finding of Sharma et al. (1996). Acta agriculturae Slovenica, 109 - 2, september 2017 str. 349 - 356 Antara SEAL et al. Figure 4: Grade wise distribution of potato tubers (number and mass) under CCM and Green Farming Production of large sized tubers vis-à-vis higher yield would reflect efficient photosynthesis, phloem loading and translocation as well as synthesis of large molecular weight substances within storage organs (Singh, 1999). Hence, the observed phenomenon under green farming may indicate the positive influence of organic plant management (utilizing IRF) towards rapid bulking of tubers. Maximum tuber yield was obtained from variety Super-6 (33.0 t/ha) followed by 'Kufri Jyoti' (30.00 t/ha) in green farming plots, at Pilot Area II and I respectively. In both the Pilot Areas minimum tuber yield was obtained in CCM plots irrespective of potato variety (Table 5). Mohapatra et al. (2008) and Kumar et al. (2011) also reported improvement in tuber yield of potato with application of organic manure. Hence better yield performance in the green farming plots may be attributed to the release of organic acids following compost application which led to proper nutrient mineralization (Kumar et al., 2011) vis-à-vis organic plant management, which enabled adequate nutrient uptake (Kumar et al., 2008). 3 7 8 Acta agriculturae Slovenica, 109 - 2, september 2017 Evaluation of an organic package of practice ... farming in terms of yield, quality, energy efficiency and economics Table 5: Nutrient Use Efficiency (NUE) and yield attributes under studied farming systems (as average of both seasons) NUE* Grade wise number Treatment PotatoVariety (kg/kg [ mass (kg) of the tubers/ha] _NPK) 0 - 25 g 25 - 50 g 50 -75 g > 75 g Pilot Area I: Mathurapur village (district: North 24 Paraganas), West Bengal, India Conventional Crop Management (CCM) Green Farming utilizing IRF KufriJyoti KufriChandramu khi KufriJyoti KufriChandramu khi 22.42c 21.21c 68.12a 63.01 ab 184577bc 141283b 113914b 99925b [3402] [5041] [7452] [11855] 184478bc 135447c 105881bc 75644d [3300] [5043] [6668] [11239] 189001a 151320a 135021a 111398a [3457] [6549] [8543] [11451] 182950d 138214bc 118256b 89810c [3396] [5511] [7532] [11312] Total tuber yield (t/ha) 27.75b 26.25b 30.00a 27.75b Pilot Area II: Bhabanipur village, (district: Nadia), West Bengal, India Conventional Crop Management (CCM) Kufri Jyoti Super 6 Green Farming utilizing IRF g Kufn Jyoti Super 6 20.61d 23.03c 63.01b 74.93a 181900bc 134240cd 111017d 67283d [3211] [5221] [7219] [9849] 174445d 142003b 123804b 102987b [2927] [5352] [7718] [12503] 183006b 138438c 117206c 91479c [3338] [5600] [7868] [10944] 190115a 153617a 145917a 141480a [3401] [6331] [8918] [14350] 25.50d 28.50b 27.75b 33.00a *NUE was evaluated in terms of'Partial Factor Productivity' of Applied Nutrient (PFPnpk) Note: The means marked with different letters in the same column were significantly different at P< 0.05 under Duncan's New Multiple Range Test. Year wise comparison indicated an interesting pattern of crop yield under the studied farming systems (Figure 5). Under CCM, yield did not follow any consistent pattern during the two years study period. During 2nd year slightly lower yield was obtained under variety 'Jyoti', while just the opposite trend was recorded under varieties Chandramukhi and Super 6. In case of green farming however, yield increased in the 2nd year as compared to 1st year's performance; irrespective of experimental site and potato variety. During 2nd year while slightly lower (-1.79 percent) yield (on an average) was recorded under CCM, the same increased by about 7.16 percent, in case of green farming plots. Acta agriculturae Slovenica, 109 - 2, september 2017 365 Antara SEAL et al. Figure 5: Comparative yield performance of potato under CCM and green farming In case of green farming adoption of organic plant management (utilizing IRF) at different stages of plant growth; perhaps activated the plant metabolic functions leading to better nutrient uptake, assimilation, translocation and storage; finally culminating in higher tuber yield. Similar improvement in plant functioning under organic (IRF) plant management was documented in various other crops viz. tomato, black gram, green gram (Sengupta et al., 2011; Bera et al., 2014b; Barik et al., 2014b;) okra, cabbage (Barik et al., 2014a; 2014c) and rain-fed/ winter paddy (Mukhopadhyay et al., 2015). 3.2 Soil quality development Variation in soil properties was studied before initiation of experiment and after harvest of potato tubers (Table 6). Analysis revealed that soil of all the experimental plots were slightly acidic in reaction, pH varying from 6.46 to 6.49. Electrical conductivity (EC), which reflects soil fertility (except in salt effected problematic soil) was found to increase in case of compost treated plots indicating steady nutrient mineralization. Increasing trend of organic carbon was noticed in green farming plots, where Novcom compost was applied during land preparation. Potato produces more dry matter than any other food crop and therefore, requires higher amount of nutrient inputs (Kushwah and Singh, 2011).The imbalanced and indiscriminate use of fertilizers in intensive cropping system without adequate restorative practices may pose threat towards sustainability of the system (Jatav et al., 2013). Soil fertility in terms of available - N, P2O5, K2O and SO4-showed an increasing trend under both the studied farming systems. -3-7/1 3 7 8 Acta agriculturae Slovenica, 109 - 2, september 2017 Evaluation of an organic package of practice ... farming in terms of yield, quality, energy efficiency and economics Table 6: Change in soil quality under studied farming systems (as average of both seasons) Soil Physicochemical Properties Treatments pH EC Org.- C Av.- N Av.- P2O5 Av.- K2O Av.- SO4 (1: 2.5) (dSm-1) (%) <-------------kgha1---------------> Tt: Conventional Crop 6.46* 0.034 0.75 354.21 68.43 241.06 34.53 Management (CCM) (6.31) (0.035) (0.74) (394.32**) (73.45**) (245.62*) (35.67) T2: Green Farming 6.49 0.031 0.74 357.69 65.21 237.51 36.32 utilizing IRF (6.54) (0.038**) (0.79**) (386.21**) (68.79*) (235.21) (38.84*) Soil Biological Properties MBC SR FDA qMBC qCO2 qFDA QR T1: Conventional Crop 279.42 0.586 26.31 3.73* 2.10 0.351 0.08 Management (CCM) (271.31) (0.623**) (28.42) (3.67) (2.30**) (0.384**) (0.09) T2: Green Farming 278.16 0.584 25.21 3.76 2.10** 0.341 0.08 utilizing IRF (312.24**) (0.591) (39.23**) (3.95**) (1.89) (0.497**) (0.08) Note: MBC- Microbial Biomass Carbon (^g CO2-C/g dry soil), SR- Soil Respiration, FDA- Fluorescein Di-acetate Hydrolysis (^g/g dry soil), qMBC- Microbial Quotient (%), qCO2 - Microbial Metabolic Quotient, qFDA: Specific hydrolytic activity (%),QR- Soil Microbial Respiration quotient. Figure in parenthesis indicate analytical values ofpost harvest soil samples taking average of the two seasons. T - test for 2 dependent means (* significant at P<0.05 and * * significant at P<0.01). Microbial activity is probably the most important factor that controls nutrient re-cycling in soil. Microorganisms participate in disintegration and decomposition processes leading to the release of nutrients trapped in plant, animal debris, rock and minerals; as well as synthesize and release hormones that are essential for plant growth (Gogoi et al., 2003). Anderson (2003) pointed out that the MBC/OC ratio and metabolic quotient (qCO2) could be used as more sensitive indicators of soil microbial response to land use, soil management, and environmental variables. Microbial biomass carbon was found to increase under green farming, whereas slight reduction in value was noticed under CCM. Increase in soil respiration in green farming plots, might be due to increased microbial activity for utilization of added organic source. This was corroborated by enhanced FDA value, that represents overall enzymatic activity by soil microbes. Microbial quotient (qMBC) i.e., the ratio of Cmic/Corg, increased in green farming plots indicating increase in microbial activity. qCO2 value was found to increase under CCM. In general, conventional agro-systems present higher values in comparison to organic or natural ecosystems; indicating comparatively more stressed conditions under the former (Dilly & Munch, 1998). Simultaneously qFDA (FDA per unit organic carbon) value of soil decreased in CCM plots. Decrease in qFDA under conventional practice, perhaps indicated stress on microbial community, thereby requiring higher energy for maintenance. 3.2.1 Soil Development Index Soil Development Index (SDI) is a concept to quantify the extent of positive changes in different soil quality parameters for expression of overall soil development; to enable easy understanding of the end-users. (Bera et al., 2014a). Soil Development Index was noticeably higher under green farming plots (where Novcom compost was integrated with chemical fertilizer) as compared to CCM plots, receiving only chemical fertilizer (Fig. 6). The variation was especially significant in terms of soil biological properties, which was recordably higher in green farming plots as compared to plots receiving chemical fertilizers. Similar enhancement of soil quality under organic manure application was documented by Golabi et al. (2004), Gulshan et al. (2013), Das et al. (2013) and Bera et al. (2014). Acta agriculturae Slovenica, 109 - 2, september 2017 str. 349 - 356 Antara SEAL et al. Soil Development Index (SDI) under different management practices Conventional Crop Management Green Farming utilizing IRF Figure 6: Soil quality development under CCM and green farming 3.3 Potato quality under studied farming systems Analysis of different quality parameters of potato was done to assess qualitative differences in potato (if any) under varying management practice (Table 7). Mean specific gravity of potato was the highest (1.084) under green farming as compared to CCM (1.071). Specific gravity of potato positively correlated with dry matter and starch content, as was also noted by Marwaha (1997), Abbas et al. (2011) and Kaur & Agarwal (2014); but presented negative correlation with reducing sugar content (Iritani & Weller, 1974; Salamoni et al., 2000). Starch, comprising 65-80 % of dry matter content, is considered to be the main constituent of potato (Kadam et al., 1991a). Starch content (mean value 13.73 g / 100g tissue) was about 15.6 percent higher in case of potato grown under green farming as compared to those produced under CCM. Since starch comprises the largest part of dry matter, it has direct influence on technological quality, especially with regard to the texture of the processed products (Kadam et al., 1991b). Table 7: Quality of potato grown under studied farming systems Quality parameter Conventional Crop Management (CCM) Green Farming utilizing IRF Specific gravity 1.045 - 1.112 (1.071 ± 0.012) 1.049 - 1.124 1.084** ± 0.011 Starch (g/ 100g tissue) 6.81 - 19.91 (11.87 ± 2.034) 7.66 - 22.56 (13.73** ± 2.061) pH 5.99 - 6.10 6.11 - 6.38 (6.02 ± 0.016) (6.17** ± 0.019) Titratable acidity (%) 0.30 - 1.02 (0.51** ±0.115) 0.16 - 0.27 (0.23 ± 0.019) Vitamin C (mg/ 100 mg tissue) 10.80 - 12.00 (11.40 ± 0.364) 12.00 - 14.40 (13.20** ± 0.318) Figure in parenthesis indicate mean value and standard error, T - test for 2 independent means (* significant at P<0.05 and ** significant at P<0.01). 3 7 8 Acta agriculturae Slovenica, 109 - 2, september 2017 Evaluation of an organic package of practice ... farming in terms of yield, quality, energy efficiency and economics Factors that may interfere in a negative and/ or indirect way on the technological quality of tubers are pulp pH and total titratable acidity. The pH index determines deterioration potential by fermentation and the activity of enzymes (Cecchi, 1999).The phosphorylase enzyme acts predominantly on starch breakdown (Jadhav et al., 1991), with maximum activity at pH 5.5 (Iritani & Weller, 1973). In addition, pulp pH is variable and presents a negative correlation (-0.86) with reducing sugars accumulation (Iritani & Weller, 1973). Total acidity on the other hand quantifies organic acids present in foods and, in general, there is a tendency of reduction in their contents because of respiration and/or due to conversion into sugars (Chitarra & Chitarra, 1990), which could contribute toward browning of the fried product. Lower pH (6.02) value and higher titratable acidity (0.51 percent) in case of conventionally grown potato (as against those grown under green farming) indicated its inferior quality in terms of faster degradability and higher browning potential, when fried. Potatoes are a steady reliable source of vitamin C (ascorbic acid) in human diet (FAO 2008).Although vitamin C is sensitive to heat, and breaks down to some degree on cooking, enough is still retained to make cooked potatoes a rich source of this nutrient. Vitamin C works as antioxidant which helps protect cells from free radical damage apart from its requirement for healthy skin, teeth, gums, muscles and bones. It also helps with the absorption of iron from plant foods, which is poorly available to the body otherwise. Vitamin C (13.20 mg/ 100 mg tissue) was about 15.7 percent higher in potatoes grown under green farming as compared to their conventional counterparts, which corroborated the findings of Hamouz et al. (1999), Hamouz et al. (2005) and Zarzynska (2013). As per their observation ascorbic acid content in potato tubers was influenced by the method of cultivation; with higher levels in potatoes cultivated following ecological pathway i.e., without using any chemical protection as compared to those produced under conventional methods. According to Widmann et al. (2008) fertilizers used to increase potato growth represent an ecological risk because they penetrate potato's tissues, affect their metabolism and change the chemical composition of the tubers, reducing the content of their active principles. Therefore, potato grown under organic environment, tend to be qualitatively superior in comparison to its conventional counterpart. 3.4 Comparative energy use efficiency under studied farming systems Conventional agriculture's energy inefficiency is directly tied to the high energy consumption of producing and transporting synthetic pesticides and fertilizers used to grow these crops. Whereas organic agriculture utilizes manure, legumes, and other natural sources of nitrogen, which replace the fossil fuels used for manufacturing synthetic nitrogen fertilizer; with natural biological process (Ziesemer, 2007). Total energy input in the form of direct, indirect, renewable and non-renewable; are presented in Table 9. In case of green farming about 49 % of total energy input was in the form of direct energy, as compared to only 22 % in case of CCM plots. Energy productivity or the potato output per unit energy application (Fig. 7) was more under green farming (1.26 kg MJ-1) as compared to CCM (0.56 kg MJ-1). This can be attributed to the differences in the level of technology and other characteristics of each treatment (Zangeneh et al., 2010). Specific energy was 55.6 percent less under green farming vs. CCM; indicating higher sustainability under the former. The result was well corroborated with 34410.09 MJ ha-1 excess gain in net energy under green farming as compared to CCM. Energy intensiveness was computed as energy consumed in crop production per unit market value of crop produced (Lockeretz et al., 1977). Lower energy intensiveness under green farming (0.09 MJRs-1 vs. 0.21 MJRs-1 under CCM) indicated towards the sustainable agriculture practice. Acta agriculturae Slovenica, 109 - 2, september 2017 365 Antara SEAL et al. Figure 7: Comparative energy usage for potato cultivation under green farming and conventional crop management 3.5 Economics of potato cultivation under studied farming systems Total cost of potato cultivation (Table 8) was found to be almost the same (Rs. 1,57,012 under green farming and Rs. 1,57,462 under CCM) under both the farming systems. Net income under green farming was 30.98 percent higher as compared to CCM due to better tuber yields under the former. Green farming also led to higher economic gains as compared to CCM in terms of other financial components like net return, return per rupees invested and benefit cost ratio. The findings pointed towards the scope for financial security under this crop production system. Table 8: Economics of potato cultivation under studied farming systems Economics T1: Conventional Crop Management (CCM) T2: Green Farming utilizing IRF Total cost (Rs./ha) 157462 157012 Gross income (Rs./ha) 229125 250875 Net income (Rs./ha) 71663 93863 Returns per rupees invested 0.49 0.60 Benefit cost ratio 1.49 1.60 Note: 100 Indian Rupee = 1.487US$ or 1.325€or 1.050 British £ (as on 2016 June 14). 4 CONCLUSION Green farming practice utilizing IRF Technology delivered positive results encompassing all the critical aspects of crop (in this case potato) cultivation i.e., yield, quality, energy investments and soil health; besides ensuring economic sustenance. Higher tuber yield and rapid bulking of potato tubers under green farming indicated the positive influence of organic plant management (utilizing IRF) towards efficient photosynthesis, translocation and synthesis of large molecular weight substances within storage organs; or in other words better plant metabolic functioning. The finding was corroborated by the better technological quality of potato tubers under green farming versus conventional crop management. The criteria of economic sustenance under changing climatic pattern was also better ensured under this farming, which showed higher withstanding capacity against production loss or fall in market price; as compared to conventional -3-7Q 3 7 8 Acta agriculturae Slovenica, 109 - 2, september 2017 Evaluation of an organic package of practice ... farming in terms of yield, quality, energy efficiency and economics crop management. The study also revealed that complete cessation of chemical pesticides and other agrochemicals was possible even for an input intensive crop like potato, by undertaking initiatives towards activation of plant biochemical functions. Energy efficiency is an integral part of sustainable agriculture. As compared to conventional farming, green farming ensured higher use of renewable energy and significantly lower energy intensiveness for potato cultivation; thereby presenting the prototype for sustainable agriculture. 5 REFERENCES Abbas, G., Farooq, K., Hafiz, I.A., Hussain, A., Abbasi, N.A., Shabbir, G. (2011). Assessment of processing and nutritional quality of potato genotypes in Pakistan. Pakistan Journal Agricultural Sciences, 48(3), 169-175. Anderson, T.H. (2003). Microbial eco-physiological indicators to assess soil quality. Agriculture, Ecosystems and Environment, 98, 285-293. doi:10.1016/S0167-8809(03)00088-4 Banaeian, N., Omid, M., Ahmadi, H. (2011). Energy and economic analysis of greenhouse strawberry production in Tehran province of Iran. Energy Conversion and Management, 52(2), 1020-1025. doi:10.1016/j.enconman.2010.08.030 Barik, A.K., Chatterjee, A.K., Datta, A., Saha, S., Bera, R., Seal, A. (2014b). Evaluation of Inhana Rational Farming (IRF) Technology as an Effective Organic Option for Large Scale Paddy Cultivation in Farmer's Field - A Case Study from Kowgachi-II Gram Panchayat, North 24 Parganas, West Bengal. The International Journal of Science &Technology, 2 (5), 183-197. Barik, A.K., Chatterjee, A.K., Datta, A., Bera, R., Seal, A. (2014c). Evaluation of Inhana Rational Farming (IRF) Technology as an effective organic package of practice- a case study of state horticultural research & development station, Krishnanagar, Nadia, West Bengal. Centra European Journal of Experimental Biology, 3(3), 1-15. Barik, A.K., Chatterjee, A.K., Mondal, B., Datta, A., Saha, S., Nath, R., Bera, R., Seal, A. (2014a). Adoption of Rational Farming Technology for Development of a Model for Exploring Sustainable Farming Practice in Farmer's Field. The International Journal of Science & Technology, 2(4), 147-155. Bera, R., Datta, A., Bose, S., Dolui, A.K., Chatterjee, A.K., Dey, G.C., Barik, A.K., Seal, A. (2013b). Comparative Evaluation of Compost Quality, Process Convenience and Cost under Different Composting Method to assess their Large Scale Adoptability Potential as also Complemented by Compost Quality Index. International Journal of Scientific and Research Publications, 3(6), 406417. Bera, R., Khan, M., Mazumdar, D. (2013a). Soil Development Index (SDI) to evaluate effectivity of different organic inputs towards soil quality development under FAO-CFC-TBI Project at Maud T. E., Assam. Oral presentation G36 at: 100th Indian Science Congress; 2013 Jan 3-7; Kolkata, p.272. Bera, R., Seal, A., Datta, A., Saha, S., Dolui, A.K., Khan, M., Mazumdar, D. (2014a). Formulation of a Soil Development Index (SDI) to Evaluate the Effectivity of Organic Soil Management under FAO-CFC-TBI Project at Maud Tea Estate, Assam, India. International Journal of Advance Agricultural Research, 2(12), 318-329. Bera, R., Seal, A., Datta, A., Sengupta, K. (2014b). Evaluation of Inhana Rational Farming Technology as an Organic Package of Practice for Effective and Economic Vegetable Cultivation in Farmers' Field. Journal of Natural Product and Plant Resources, 4(33), 82-91. Bhatia, A.K., Pandita, M.L., Khurana, S.C. (1992). Plant growth substances and sprouting conditions. 1. Effect on haulm growth of plant raised from nursery rooted sprouts. Journal of the Indian Potato Association, 19, 21-24. Biswas, R.K., Majumder, D., Sinha, A. (2011). Impacts and Constraints Evaluation of Organic Farming in West Bengal. Study No. - 167. Visva - Bharati, Santiniketan, India: Agro-Economic Research Centre. Retrieved from: http://www.visva-bharati.ac.in/InstitutionsCentresSchools/Contents/A ERC-DETAIL/Summary-167.pdf Black, C.A. (1965). Method of soil analysis, Part-1. Madison, USA: Am. Soc. Agron. Inc. Cassman, K.G., Gines, G.C., Dizon, M.A., Simon, M.I., Alcatara, J.M. (1996). Nitrogen use efficiency in tropical lowland rice systems: contributions from indigenous and applied nitrogen. Field Crops Research, 47(1), 1-12. doi:10.1016/0378-4290(95)00101-8 Acta agriculturae Slovenica, 109 - 2, september 2017 365 Antara SEAL et al. Cecchi, H.M. (1999). Theoretical and practical fundamentals in food analysis (p. 211-212). Campinas: Unicamp Publishing House. Chatterjee, A.K., Bank, A. K., De, G.C., Dolui, A.K., .Seal, A. (2014). Adoption of Inhana Rational Farming (IRF) Technology as an Organic Package of Practice towards Improvement of Nutrient Use Efficiency of Camellia sinensis through Energization of Plant Physiological Functioning. The International Journal of Science & Technology, 2(6), 377-395. Chaboussou, F. (2004). Healthy crops: A new agricultural revolution (p. 244). Oxford, England: Jon Carpenter Publishing. Chitarra, M.I.F., Chitarra, A.B. (1990). Pos-colheita de frutos e hortaligas:fisiologia e manuseio. [Post -harvest of Fruits and Vegetables:Physiology and Handling] (p. 320). Lavras: FAEPE. Dias, M.C. (2012). Phytotoxicity: An Overview of the Physiological Responses of Plants Exposed to Fungicides. Journal of Botany, 2012, 1-4. doi:10.1155/2012/135479 Dilly O, Munch JC. (1998). Ratios between estimates of microbial biomass content and microbial activity in soils. Biology and Fertility of Soils, 27, 374-379. doi: 10.1007/s003740050446 Epstein, E. (1997). The science of composting (p. 383 -415). Lancaster, PA: Technomic Publishing . Evanylo, G. (2006). Compost Maturity and Indicators of Quality: Laboratory Analyses and On-Farm Tests. Retrieved from: http://www.mawaterquality.org /industry_change / compost_school / Compost % 20 quality_Evanylo.pdf Feltran, J.S., Lemos, L.B., Vieites, R.L. (2004). Technological quality and utilization of potato tubers. Scientia Agricola (Piracicaba, Braz.), 61(6), 598-603. doi: 10.1590/S0103-90162004000600006 FAI [Fertilizer Association of India]. (2007). The Fertilizer (control) Order 1985. New Delhi, India: FAI. FAO [Food and Agriculture Organisation]. (2008). Potatoes, nutrition and diet. Fact sheet. International Year of the Potato 2008. Retrieved from: http://www.fao.org/potato- 2008/en/potato/IYP-6en.pdf Garg, V.N. (2014). Training Workshop of Potato farmers of UP at Kannauj by Experts from Netherlands ; October 27, 2014; India. Retrieved from: http://vngarg.blogspot.in/2014/10/training-workshop-of-potato-farmers-of.html Ghorbani, R., Mondani, F., Amirmoradi, S., Feizi, H., Khorramdel, S., Teimouri, M., Sanjani, S., Aghel H. (2011). A case study of energy use and economical analysis of irrigated and dryland wheat production systems. Applied Energy, 88, 283-288. doi:10.1016/j.apenergy.2010.04.028 Gogoi, S., Bhuyan, M.K., Karmakar, R.M. (2003). Dynamics of microbial population in tea ecosystem. Journal of the Indian Society of Soil Science, 51 (3), 252-257. Golabi, M.H., Denney, M.J., Iyekar, C. (2004). Use of composted organic wastes as alternative to synthetic fertilizers for enhancing crop productivity and agricultural sustainability on the Tropical Island of Guam. In: Conserving Soil and Water for Society: Sharing Solutions. Paper No. 234. Proceedings of 13th International Soil Conservation Organisation Conference ; 2004 July 4-8; Brisbane, p.1-6. Gomez, K.A., Gomez, A.A . (1984). Statistical Procedures for Agricultural Research (p. 684). New York: John Wiley and sons. Gulshan, A.B., Saeed, H.M., Javid, S., Meryem, T., Atta, M.I., Amin-ud-Din, M. (2013). Effects of animal manure on the growth and development of okra (Abelmoschusesculentus L.). ARPN Journal of Agricultural and Biological Science, 8(3), 213-218. Hamouz, K., Lachman, J., Doak, P., Pivec, V. (2005).The effect of ecological growing on the potatoes yield and quality. Plant, Soil and Environment, 51 (9), 397-402. Hamouz, K., Lachman, J., Vokal, B., Pivec, V. (1999). Influence of environmental conditions and way of cultivation on the polyphenol and ascorbic acid content in potato tubers. Rostlinna Vyroba, 45(7), 293-298. Harwood, J., Heifner, R., Coble, K., Perry, J., Somwaru, A. (1999). Managing Risk in Farming: Concepts, Research, and Analysis. Agricultural Economic Report No. 774. US: Market and Trade Economics Division and Resource Economics Division, Economic Research Service, Department of Agriculture, p.1-70. Iritani, W.M., Weller, L.D. (1973).The development of translucent end tubers. The American Journal of Potato Research, 50, 223-233. doi: 10.1007/BF02849356 Iritani, W.M., Weller, L.D. (1974). Some factors influencing the pH of apical and basal portions of Russet Burbank and Norgold potatoes. The American Journal of Potato Research, 51, 119-125. doi: 10.1007/BF02851344 'j on 3 7 8 Acta agriculturae Slovenica, 109 - 2, september 2017 Evaluation of an organic package of practice ... farming in terms of yield, quality, energy efficiency and economics Jackson, M.L. (1973). Soil Chemical Analysis. New Delhi: Prentice Hall of India Pvt. Ltd. Jadhav, S.J., Mazza, G., Desai, U.T. (1991). Postharvest handling and storage. In: D.K. Salunkhe, S.S. Kadam, S.J. Jadhav. (Eds.) Potato production, processing and products (p.69-109). Boca Raton: CRC Press. Jatav, M.K., Dua, V.K., Kumar, M., Trehan, S.P., Kumar, S. (2013). Spatial distribution of soil available Nutrients in the potato growing pockets of Hoshiarpur district of Punjab. Potato Journal, 40 (2), 128-134. Kadam, S.S., Dhumal, S.S., Jambhale, N.D. (1991a). Structure, nutritional composition, and quality. In: D.K. Salunkhe, S.S. Kadam, S.J. Jadhav. (Eds.) Potato production, processing and products (p.9-35). Boca Raton: CRC Press. Kadam, S.S., Wankier, B.N., Adsule, N.R. (1991b). Processing. In: D.K. Salunkhe, S.S. Kadam, S.J. Jadhav. (Eds.) Potato production, processing and products (p.111-154). Boca Raton: CRC Press. Karim, A.A., Ramasamy, C. (2000). Expanding Frontiers of Agriculture: contemporary issues. Ludhiana, India: Kalyani Publishers. Kaur, S.,Aggarwal, P. (2014). Studies on Indian Potato Genotypes for their Processing and Nutritional Quality Attributes. International Journal of Current Microbiology and Applied Sciences, 3(8), 172-177. Khan, M.A., Hossain, S.M.A. (2007). Study on Energy Input, Output and Energy Use Efficiency of Major Jute Based Cropping Pattern. Bangladesh Journal of Scientific and Industrial Research, 42(2), 195202. doi: 10.3329/bjsir.v42i2.472 Kumar, M., Baishya, L.K., Ghosh, D.C., Gupta, V.K. (2011). Yield and quality of potato (Solanum tuberosum ) tubers as influenced by nutrients sources under rained conditions of Meghalaya. Indian Journal of Agronomy, 56(3), 260-66. Kumar, M., Jatav, M.K., Trehan, S.P. (2008).Contributing of organic sources to potato nutrition at varying nitrogen levels. In: Proceedings of Global Potato Conference; 2008 Dec 9-12. New Delhi: Indian Potato Association. Kushwah, V.S., Singh, S.P. (2011). Relative performance of low input and high input technology for potato production in India. Potato Journal, 38(1), 56-60. Lockeretz, W., Klepper, R., Commoner, B., Gertler, M,, Fast, S,, O'leary, D., Blobaum, R. (1977). Economic and energy comparison of crop production on organic and conventional corn belt farms. In W. Lockeretz (Eds.) Agriculture and Energy (p. 94). NY: Academic Press. Marwaha, R.S. (1997). Processing of potatoes: Current status, need, future potential and suitability of Indian varieties: A critical appraisal. Journal of Food Science and Technology, 3, 457-471. Mohapatra, B.K., Maiti, S., Satapathy, M.R. (2008). Integrated nutrient management in potato (Solanum tuberosum )-Jute (Corchorus olitorius) sequence. Indian Journal of Agronomy, 53 (3), 205-09. Mollah, M.R.A., Islam, N., Sarkar, M.A.R. (2011). Integrated nutrient management for potato mungbean-T.aman rice cropping pattern under level Barind agroecological zone. Bangladesh Journal of Scientific and Industrial Research, 36(4), 711-722. Mondal, S.S., Acharya, D., Ghosh, A., Bug, A. (2005). Integrated nutrient management on the Growth, productivity and quality of Potato in indo-gangetic plains of West Bengal. Potato Journal, 32 (1 - 2), 75-78. Mukhopadhyay, K., Mukherjee, S., Seal, A., Bera, R., Dolui, A.K., Rahman, F.H. (2015). Evaluation of new technology with fully organic inputs toward production of paddy with special reference to soil quality development- A case study of Howrah Krishi Vigyan Kendra. Article 65. In: Souvenir of National Seminar on Soil Health Management and food Security: Role of Soil Science Research and Education; 2015 Oct 8-10; Kolkata, India, p. 101102. Nambiar, K.K.M. (1998). Integrated use of organic manures and chemical fertilizers in red soils for sustainable agriculture. In J. Sehgal, W.E. Blum, K.S. Gajbhiye (Eds. ) Red and Lateritic Soils; Vol-1, Managing red and lateritic soils for sustainable agriculture (p.367-376). Netherlands: Balkema Publishers. Ojha, M.D., Saha, B. (2014). Organic Potato in Nalanda (Bihar): Using Eco-Friendly Agri. Bios Inputs. Indian Research Journal of Extension Education, 14 (3), 119 -121. Pandey, N.K. (2013). Economics of seed potato production in India. Summer School on Advances in Quality Potato Production and Post-harvest Management (2013 July 16 -Aug 5). Shimla, India: Central Potato Research Institute. Retrieved from: http://14.139.61.86/E-Book-SummerSchool/.html Peer, Q.J.A., Ahmad, N., Kaur, J., Chesti, M.H., Ahmad, H.S., Bhat, A., Bhat, B.A. (2013). Study on economics of potato growing towards livelihood security. African Journal of Agricultural Research, 8(45), 5639-5644. Acta agriculturae Slovenica, 109 - 2, September 2017 Antara SEAL et al. Raj, D., Joshi, V.K., Lal, B.B. (2011).Yield, quality and storability of the potato flour of different Indian cultivars. International Journal of Food and Fermentation Technology, 7(1), 111-117. Sadasivam, S., Manickam, A. (2011). Biochemical Methods (3rd edn.); Reprinted. New Delhi: New Age International (p) limited. Salamoni, A.I., Pereira, A da S., Viegas, J., Campos, A.D., Chala, C.S. de A. (2000). Genetic variance of reducing sugars and dry matter and their correlations with agronomic traits in potatoes. Agricultural Research Brazilian, 35, 1441-1445. Seal, A., Bera, R., Chatterjee, A.K., Dolui, A.K. (2012). Evaluation of a new composting method in terms of its biodegradation pathway and assessment of compost quality, maturity and stability. Archives of Agronomy and Soil Science, 58(9), 995-1012. doi: 10.1080/03650340.2011.565410 Seal, A., Bera, R., Mukhopadhyay, K., Mukherjee, S. (2015). Recycling of poultry litter through Novcom composting method: A case study from Howrah KrishiVigyan Kendra, West Bengal, India. Journal of Pharmaceutical and Scientific Innovation, 4(3), 176-179. doi: 10.7897/2277-4572.04339 Sen, H.S. (2011). Agriculture and Horticulture in West Bengal: Present Status Holds Promise while its Future Demands Long Term Planning with Focus on Commercialization. Presented at: Orientation Program for IAS Probationers 2010 batch at ATI; 2011 June 20; Kolkata, India. Retrieved from: http://hssen-hssen- coastalmanagement.blogspot.in/2011/06/agriculture -in-west-bengal. html Sengupta, K., Bhui, S., Mondal, M. (2011). Novcom-An Effective compost for blackgram production. Extended summary: International Symposium on System intensification towards Food and Environmental Security; 2011 Feb 24-27; Kalyani, India BCKV, p. 94. Sharma, R.C., V. Sunaina, N.C. Upadhyay., M. Kumar. (1996). Effect of green manuring, organics and biofertilizer on tuber production in potato on a typicUstochrept. In: Proceeding of the XIV National Symposium on Agronomy, Environment and Food Security for 21st century, CCSHAU, Hisar, India. 10-14 Dec 1996, p. 183-84. Singh, J.P. (1999). Potassium fertilization of potatoes in north India. In: Proceeding of IPI workshop on "essential role of potassium in diverse cropping system", held at the 16th world congress of soil science, Montpellier, France 20-26 August,1998. Basel, Switzerland: International Potash Institute, p. 123-27 Trautmann, N.M., Krasny, M.E. (1997). The science of composting. In: Composting in the classroom (p. 15). Ithaca, NY: Cornell University. Venkatasalam, E.P., Singh, S., Sharma, S. (2012). Effect of organic manures on yield and yield attributing characters of potato. Potato Journal, 39 (1), 84-87. Wheeler, B.E.J. (1969). An Introduction to Plant Diseases (p.301). London: John Wiley and Sons Ltd. Widmann, N., Goian, M., Ianculov, I., Dumbrava, D., Moldovan, C. (2008). Method to Starch Content determination from plants by Specific Weight. Scientific Papers, Animal Science and Biotechnologies, 41 (1), 814-818. Yadav, R.L. (2003). Assessing on-farm efficiency and economics of fertilizer N, P and K in rice wheat systems of India. Field Crops Research, 18, 39-51. doi:10.1016/S0378-4290(02)00198-3 Zahedi M, Eshghizadeh H R, Mondani F. 2014. Energy Use Efficiency and Economical Analysis in Cotton Production System in an Arid Region: A Case Study for Isfahan Province, Iran. International Journal of Energy Economics and Policy, 4(1), 4352. Zangeneh, M., Omid, M., Akram, A. (2010). A comparative study on energy use and cost analysis of potato production under different farming technologies in Hamadan province of Iran. Energy, 35(7), 2927-33. doi:10.1016/j.energy.2010.03.024 Zarzynska, K. (2013). Chemical Composition of Potato Tubers in Relation to Crop Production System and Environmental Conditions. Journal of Agricultural Science and Technology B, 3, 689-695. Ziesemer, J. (2007). Energy use in organic food systems. Report of Natural Resources Management and Environment Department Food and Agriculture Organization of the United Nations. August 2007, Rome. Retrieved from: http://www.fao.org/docs/eims/upload/233069/energ y-use-oa.pdf lOT 3 7 8 Acta agriculturae Slovenica, 109 - 2, september 2017 doi:10.14720/aas.2017.109.2.21 Original research article / izvirni znanstveni članek Evaluation of different crop sequences for wheat and maize in sandy soil Abd El-Hafeez ZOHRY1, Samiha OUDA2*, Wael HAMD-ALLA1 and El-Sayed SHALABY3 Received February 11, 2017; accepted February 26, 2017. Delo je prispelo 11. februarja 2017, sprejeto 26. februarja 2017. ABSTRACT The objective of this paper was to assess four crop sequence system including wheat and maize grown in sandy soil of Upper Egypt with respect to the applied irrigation amount for each crop sequence, total production and water productivity. Two field experiments were conducted in Egypt during 2013/14 and 2014/15 growing seasons. Each experiment included four crop sequences: maize then wheat (CS1); maize, short season clover (SSC) then wheat (CS2); cowpea, SSC then wheat (CS3); cowpea intercropped with maize, SSC then wheat (CS4). The lowest amount of applied water was added to CS1 which resulted with low value of wheat and maize yield and the lowest water productivity. The highest amount of applied water was applied to CS2 and CS4 (similar values). The highest wheat yield and water productivity were obtained in CS3. The highest maize yield and water productivity was obtained from CS4. The highest total production (170.88 and 213.43 CU ha-1 in the 1st and 2nd season, respectively) and water productivity (0.093 and 0.114 CU m-3 in the 1st and 2nd season, respectively) for the studied crop sequences was obtained from CS3. In conclusion, higher water productivity for wheat in sandy soil can be attain by cultivating two legume crops before it (CS3); and for maize, it should be intercropped with a legume crop (CS4). Key words: maize; wheat; short season clover; cowpea; cowpea intercropped with maize; cereal units; Assiut Governorate IZVLEČEK OVREDNOTENJE RAZLIČNIH KOLOBARJEV ZA KORUZO IN PŠENICO NA PEŠČENIH TLEH Predmet te raziskave je bilo ovrednotenje kolobarja za pšenico in koruzo na peščenih tleh v Zgornejm Egiptu glede na obseg namakanja v posameznem kolobarju, celokupno produkcijo in učinkovitost izrabe vode. Izvedena sta bila dva poljska poskusa v rastnih sezonah 2013/14 in 2014/15. Vsak poskus je obsegal štiri sosledja poljščin v kolobarju in sicer : 1 - koruza nato pšenica (CS1); 2 - koruza, kratkosezonska detelja (SSC), potem pšenica (CS2); 3 - čičerka, SSC, potem pšenica (CS3); 4 - čičerka v medsetvi s koruzo, SSC, potem pšenica (CS4). Najmanjša količina dodane vode je bila v sistemu CS1, kar je rezultiralo v majhnem pridelku pšenice in koruze in najmanjši učinkovitosti izrabe vode. Največ vode je bilo dodano v kolobarjih CS2 in CS4 (enake količine). Največji pridelek in največja učinkovitost izrabe vode sta bila dosežena v kolobarju CS3. Največji pridelek koruze in največja učinkovitost izrabe vode sta bila dosežena v kolobarju CS4. Največja celokupna produktivnost (170.88 in 213.43 CU ha-1 v prvi in drugi rastni sezoni) in največja učinkovitost izrabe vode (0.093 in 0.114 CU m-3 v prvi in drugi rastni sezoni) sta bili za preučevane kolobarje deseženi v kolobarju CS3. Zaključimo lahko, da je večja učinkovitost izrabe vode za pšenico na peščenih tleh dosežena v kolobarju z dvema metuljnicama pred njo (CS3) in za koruzo z medsetvijo metuljnice (CS4). Ključne besede: koruza; pšenica; kratkosezonska detelja; čičerka; medsetev čičerke v koruzo; žitne enote; Assiut upravna enota 1 INTRODUCTION Maize and wheat are very important cereal crops all over the world (Valipour 2012a). The cultivated area of these important crops is under competition with other crops with higher economic values (Valipour 2016). In Egypt, there is a large gap between the production of these two crops and its consumption. Therefore, it is important to increase its cultivated area by cultivating low fertile soil on the edges of the Nile Delta and 1 Crop Intensification Research Department; Field Crops Research Institute; Agricultural Research Center, Giza, Egypt 2 Water Requirements and Field Irrigation Research Department, Soils, Water and Environment Research Institute, Agricultural Research Center, Egypt; * Corresponding author: samihaouda@yahoo.com 3 AgronomyDepartment, Faculty of Agriculture, Assuit University, Egypt Acta agriculturae Slovenica, 109 - 2, september 2017 str. 349 - 356 Abd El-Hafeez ZOHRY et al. Valley. These soils are characterized by low fertility level and high water infiltration rate. Increasing productivity of unit land and water of these soils is a challenge under the prevailing situation of water deficiency and food insecurity in Egypt. For that reason, proper water management for the cultivated crops in such areas is essential. Accurate estimation of reference evapotranspiration is the main factor to attain that. Earlier studies compared different ETo equations for their accuracy revealed that Penman-Monteith equation is the most accurate because of its detailed theoretical base and its accommodation of small time periods (Valipour, 2014). It was found that air temperature and solar radiation contributed most to the temporal variation of ETo in the upper reaches, as well as solar radiation and wind speed were the determining factors for the temporal variation of ETo in the middle-lower reaches (Zhao et al., 2015). Furthermore, comparison between FAO Penman-Monteith with other methods that calculating reference evapotranspiration was done by several authors (Valipour, 2012b and 2014). The prevailing crop sequence in these areas is two crops per year (a winter then a summer crop). One of the management that could be done to increase productivity of unit land and water in these soils is changing crop sequence from two crops per year to three crops per year, with inclusion of legume crop (early winter, winter then summer crops). The major benefit resulted from this practice is improvement in soil fertility and increased farmers' income (Sheha et al., 2014). In addition, implementing intercropping in one season can play a similar role in increasing productivity of unit land and water (Kamel et al., 2010). A very common crop sequence in Egypt, either on fertile or low fertile soils, is the cultivation of wheat then maize, where both are cereals and its cultivation in a year on the same piece of land leads to imbalance in soil nutrients and decline in the yield of both crops (Hamd-Alla et al., 2015). Previous research on the effect of crop sequence on wheat yield indicated that it was significantly improved when cowpea preceded it. The opposite occurred when maize preceded wheat, where wheat yield was reduced (Hamd-Alla et al., 2015). Under these circumstances, wheat benefited from the residual effect of legume, which positively affected wheat yield (Kumpawat and Rathore, 2003). Furthermore, cultivating short season clover in September before wheat cultivation in November proved to increase wheat yield (Sheha et al., 2014). Another avenue to increase the productivity of unit land and water is intercropping, where one crop share its life cycle or part of it with another crop (Eskandari et al., 2010). This practice can be used as a way to improve soil fertility, increase land productivity and save on the applied irrigation water (Kamel et al., 2010). Furthermore, it increases water productivity as a result of using less water to irrigate two crops (Andersen, 2005). One example of intercropping systems is cowpea intercropped with maize, which has many advantages, such as increasing maize yield by 10% and reducing associated weeds (Hamd-Alla et al., 2014). Furthermore, no additional water will be applied to cowpea under this system (Kamel et al., 2016). In low fertile soil, such as in Egypt, legume/cereal intercropping system can increase soil fertility via raising its organic content and available nitrogen fixed by legume (Singh et al, 1986), which reduces fertilizer requirements for cereal crops, reduces costly inputs and ensure agricultural sustainability (Megawer et al., 2010). Furthermore, Banik et al. (2006) reported that intercropping can offer opportunity for stable agricultural production in low fertile soil, whereas mono cropping cannot ensure its stability. Although many studies were done to determine the effect of crop sequence and intercropping systems on maize and wheat productivity in old fertile soil in Egypt (Sheha et al., 2014; Nofal 2012; Zohry 2005a; Zohry, 2005b), there were no previous studies on different crop sequences including wheat and maize in low fertile soil existed on the edges of the Nile value. Such a study can enhance our knowledge about the impact of different crop sequences on the applied irrigation water for these two crops in the whole crop sequence and its consequent total production. Application of such a study in Egypt is important for policy makers and can allow reduction of food gap of these two crops. Thus, the objective of this paper was to assess four crop sequences systems including wheat and maize grown in sandy soil of Upper Egypt with respect to the applied irrigation amount for each crop sequence, total production and water productivity. These crop sequences were: maize then spring wheat; maize, short season clover then spring wheat; cowpea, short season clover then spring wheat; and cowpea intercropped with maize, short season clover then spring wheat. 2 MATERIALS AND METHODS Two field experiments were carried out at Arab El- growing seasons of 2013/14 and 2014/15. Each Awammer Research Station; Agricultural Research experiment included four crop sequences as follows: Center; Assiut Governorate; Upper Egypt during two 1. Maize then spring wheat (CS1); 384 Acta agriculturae Slovenica, 109 - 2, september 2017 Evaluation of different crop sequences for wheat and maize in sandy soil 2. Maize, short season clover then spring wheat (CS2); 3. Cowpea, short season clover then spring wheat (CS3); 4. Cowpea intercropped with maize, short season clover then spring wheat (CS4). The soil of the experiment was sandy (sand, 89.9 %, silt, 7.1 % and clay, 3.0 %), with soil pH equal to 8.4, EC was 0.33 dS m-1 and CaCO3 was 30.9 %. Furthermore, total N % was 0.008 and available P and K values were 8.31 and 64.00 mg kg-1, respectively. The soil was cultivated for the first time with this experiment. Soil chemical analysis was determined according to Jackson (1958). Irrigation water was applied each fourth day by using a solid-set sprinkler system. The rotary sprinkler (type Rc160) has 0.87 to 1.23 m3h-1 discharge at 2.10 to 2.5 bars nozzle pressure, with spacing of 9 meters between laterals and 7 meters between sprinklers. A differential pressure tank was connected to the sprinkler irrigation system to inject fertilizer via irrigation water. The soil moisture constants (% per mass) in the depth of 0 - 60 cm were measured. Field capacity was 12.5 - 11.8 %, wilting point was 4.9 - 4.9 % and bulk density was 1.57 -1.55 g cm-3. Reference evapotranspiration (ETo), crop evapotranspiration and irrigation schedule were determined using BISm model (Snyder et al., 2004) for weather data of 2013/14 and 2014/15 growing seasons. The model uses Penman-Monteith equation, as presented in Allen et al., (1989) to calculate ETo. Table 1 presents weather data and ETo values in both growing seasons in the studied site. There is no rain occurrence in Assuit governorate because it is located in Upper Egypt region. Table 1. Monthly weather data and ETo in 2013/14 and 2014/15 growing seasons in Assuit Agricultural Research Station 2013/14 growing season 2014/15 growing season SR TX TN WS ETo SR TX TN WS ETo Nov13 16.3 27.5 14.0 2.6 4.1 Nov14 16.5 25.6 12.2 2.9 4.1 Dec13 14.1 20.3 7.8 3.1 3.3 Dec14 14.3 22.5 9.1 2.5 3.3 Jan14 15.4 21.7 7.6 2.5 3.3 Jan15 15.2 19.0 5.7 2.7 2.9 Feb14 18.8 23.4 8.0 3.0 4.2 Feb15 17.5 21.8 7.8 2.9 3.8 Mar14 21.8 26.8 11.6 3.0 5.1 Mar15 18.1 26.9 11.9 3.2 5.0 Apr14 25.0 32.2 16.2 3.2 6.7 Apr15 25.9 29.2 12.8 3.6 6.6 May14 26.7 34.9 19.7 3.6 7.7 May15 27.8 34.7 18.9 3.5 7.8 Jun14 29.9 37.2 21.7 3.8 8.8 Jun15 26.8 35.7 21.0 4.0 8.7 Jul14 29.4 37.9 22.7 3.9 8.9 Jul15 29.4 37.7 22.6 3.5 8.6 Aug14 27.6 38.0 23.0 3.5 8.4 Aug15 25.5 40.2 25.4 3.9 9.3 Sep14 24.4 35.7 20.8 3.8 7.6 Sep15 24.2 35.5 20.6 3.6 7.8 Oct14 20.2 30.7 16.9 3.0 5.5 Oct15 20.0 30.5 16.7 2.8 5.8 Average 22.5 30.5 15.8 3.2 6.1 Average 20.9 29.9 15.4 3.3 6.0 SR = solar radiation (MJ/m /day), TX and TN = maximum and minimum temperature, respectively (°C), WS = wind speed (m s-1), ETo = reference evapotranspiration (mm day-1). Land preparation was done by ploughing the land twice and then the land was leveled. The experimental design was spilt plot design, where the year was considered to be in the main plot and crop sequences were in the subplots. The size of single experimental plot was 21 m2. Regarding to maize, 'SC130' hybrid was sown on 12/5/2013 and 5/5/2014 in the first and second season, respectively using 27 kg of maize grains. Sole maize or intercropped with cowpea, Vigna sinensis 'Cream'), was planted with 100 % of its recommended planting density on one side of narrow furrows (70 cm width), 25 cm apart between plants. Nitrogen fertilizer was added at the rate of 360 kg N ha-1 of ammonium nitrate (33.5 % N). It was applied in five equal doses, after 15, 25, 35, 45 and 55 days from planting. Maize was also fertilized with 74.4 kg P2O5 ha-1 of calcium super phosphate, (15.5 % P2O5) and potassium sulphate (48.8 % K2O) at the rate of 58.6 kg K2O ha-1, both were applied during land preparation, as recommended by Ministry of Agriculture and Land Reclamation in Egypt. Maize plants were harvested on 2/9/2013 and 25/8/2014 in the Acta agriculturae Slovenica, 109 - 2, september 2017 385 Abd El-Hafeez ZOHRY et al. first and second season, respectively and maize grain yield was measured. Cowpea seeds ('Cream') were planted in 12/5/2013 and 5/5/2014 in the first and second season, respectively using 15 kg of cowpea seeds. Sole cowpea was sown on one side of the narrow furrow (70 cm width), 36 cm apart between plants. Nitrogen fertilizer was added at the rate of 96 kg N/ha of ammonium nitrate (33.5 % N) with the second irrigation. The high applied rate of N fertilizer for cowpea is recommended by the Ministry of Agriculture and Land Reclamation in Egypt because it is known that, in these areas, the activity of soil bacteria could be limited. In addition, 74.4 kg P2O5 ha-1 of calcium super phosphate (15.5 % P2O5) was added during land preparation as recommended by Ministry of Agriculture and Land Reclamation in Egypt. First cut of cowpea was done on 12/7/2013 and 2/7/2014 in the first and second season, respectively. Second cut of cowpea was done on 22/8/2013 and 15/8/2014 in the first and second season, respectively. Regarding to cowpea intercropped with maize, cowpea was sown on one side of the narrow furrow (70 cm width) and maize was planted on the other side of the narrow furrow with (50 % and 100 % of the recommended rate for cowpea and maize, respectively). No fertilizes was applied to cowpea under this intercropping system. Short season clover seeds (Trifolium alexandrinum'Fahl') were planted with its recommended planting density in 15/9/2013 and 10/9/2014 in the first and second season, respectively using 60 kg of seeds. Nitrogen fertilizer was added at the rate of 72 kg N ha-1 of ammonium nitrate (33.5 % N), 20 days after planting as a result of low activity of the symbiosis bacteria in the soil. It was also fertilized with calcium super phosphate (15.5 % P2O5) as 37.2 kg P2O5 ha-1 during land preparation as recommended by Ministry of Agriculture and Land Reclamation in Egypt. Harvest was done in 20/11/2013 and 15/11/2014 in the first and second season, respectively. With respect to wheat, Triticum aestivum ' Sids 1', which is a common wheat cultivar was sown in 1/12/2013 and 25/11/2014 in the first and second season, respectively using 100 % of its recommended planting density (144 kg of grain yield). As recommended by Ministry of Agriculture and Land Reclamation in Egypt, nitrogen fertilizer was added as 288 kg N ha-1 in the form of ammonium nitrate (33.5 % N) in five equal doses, after 20, 40, 55, 70 and 85 days after planting. Phosphorus fertilizer was applied in the form of single super phosphate (15.5 % P2O5) as 74.4 kg P2O5 ha-1 and was incorporated into the soil during land preparation. Potassium in the form of potassium sulphate (48.8 % K2O) as 58.6 kg K2O ha-1 was applied during land preparation. Wheat was harvested on 20/4/2014 and 15/4/2015 in the first and second season, respectively, where wheat grain yields were measured. For all the studied crops, seeds yield was recorded on the basis of experimental plot area by harvesting all plants, weighted, and then all the plots were combined together . The biomass of all studied crops was removed from the field after harvest. Dry mass of cowpea and short season clover were measured. In the second year experiment, the experiment was implemented on the same area used for the first year experiment.pART27 2mgfp5-ER 103 explants. All the obtained data from the experiment of each season were subjected to the statistical analysis of complete randomized blocks design with four replications according to Gomez and Gomez (1984). Revised Least Significant Differences (LSD') at 5 % levels of probability was used for comparing means according to Waller and Duncan (1969). Crop water productivity Water productivity was calculated for each crop in the sequence, as well as for each crop sequence as a whole. Crop water productivity was calculated by dividing the obtained yield by applied water for each crop. To calculate water productivity for the whole crop sequence, calculation of Cereal Units (CU) (Brockhaus, 1962) for each crop in the sequence was done, then it was added together to obtain one value to represent the total yield from each crop sequence. The CU has been used as a common denominator in German agricultural statistics for decades and is mainly based on the nutritional value for livestock. It is also an appropriate unit for the description of agricultural products (Brankatschk and Finkbeiner, 2014). Furthermore, Macak et al., (2015) used CU to evaluate productivity of different crop rotations. This methodology is widely used in Egypt to evaluate the production of different intercropping systems. Abou-Keriasha et al., (2013) reported that according to Brockhaus (1962) 100 kg of either wheat or maize is equal to 1.0 CU. Furthermore, 100 kg of short season clover or cowpea equal to 1.14 and 1.12 CU, respectively. Thus, water productivity (CU mm-1) was calculated using the accumulated values of cereal units as numerator and the applied water in millimeters as dominator. 384 Acta agriculturae Slovenica, 109 - 2, september 2017 Evaluation of different crop sequences for wheat and maize in sandy soil 3 RESULTS 3.1 Applied water for crops and crop sequences Table 2 indicates that water requirements for all the studied crops were higher in the second year compared to the first growing season, except for wheat. With respect to the four crop sequences, the lowest amount of applied water was added to CS1, where only two crops were cultivated. The value of the applied water to CS2 and CS4 were similar in each growing season and different in both growing season. Furthermore, this amount was the highest, compared to what was applied to the other crop sequences. Table 2: Water requirements (WR, m3 ha-1) for the studied crops and crop sequences in both growing seasons WR WR 2013/14 2014/15 Wheat 6267 6200 Maize 8440 8947 Short season clover 5400 5507 Cowpea 6853 6933 Cowpea intercropped with maize 8440 8947 Maize then wheat (CS1) 14707 15147 Maize, short season clover then wheat (CS2) 20107 20653 Cowpea, short season clover then wheat (CS3) 18520 18640 Cowpea intercropped with maize, short season clover then wheat (CS4) 20107 20653 3.2 Effect of crop sequence on wheat productivity Table 3 shows that in all the studied crop sequences, there were significant differences between wheat yields (P < 0.05) in both growing seasons, where the lowest wheat yield was obtained when maize/wheat system was cultivated (CS1). In the second growing season, wheat yield was insignificantly higher. Furthermore, wheat cultivation after short season clover in CS2 increased wheat yield by 16 and 47 % in the first and second season, respectively, compared to maize cultivation before wheat system (CS1). The highest wheat yield was obtained when cowpea and short season clover were cultivated before it in both growing seasons (CS3), which increased its yield by 23 and 87 % in the first and second growing seasons, respectively. It can be also noticed, in all crop sequences, that wheat yield value was higher in the second growing season, compared to the first growing season. Table 3 also reveales that the highest water productivity for wheat was obtained when cowpea and short season clover were cultivated before it in CS3. This result was true in both growing seasons. Furthermore, the lowest water productivity was found when maize preceded wheat in CS1. Table 3: Spring wheat yield as affected by different crop sequences, percentage of yield increase (PI%) and water productivity (WP) in both growing seasons Crop sequence 2013/14 growing season 2014/15 growing season Wheat yield (ton ha-1)* PI (%) WP (kg m-3) Wheat yield (ton ha-1)* PI (%) WP (kg m-3) CS1 3.70d --- 0.59 3.73d --- 0.60 CS2 4.29c 16 0.68 5.49c 47 0.89 CS3 4.55a 23 0.73 6.98a 87 1.13 CS4 4.48b 21 0.72 6.05b 62 0.98 *Means with different letters indicated that it was significantly different 3.3 Effect of crop sequences on maize productivity Maize yield was insignificantly lower in the CS2 compared to the CS1 in the first growing season. In CS2, maize yield increased by 7 % in the second growing season as a result of the residual effect of the legume crops from the first growing season (Table 4). The highest yield was obtained when cowpea was intercropped with maize in both growing seasons (CS4). Acta agriculturae Slovenica, 109 - 2, september 2017 385 Abd El-Hafeez ZOHRY et al. Intercropping cowpea with maize resulted in 3 and 13 % increase in maize yield in the first and second growing season, respectively. The results in Table 4 also revealed that in both growing seasons, there were insignificant differences between maize yield values in the studied crop sequences (P <0.05). The highest water productivity for maize in both growing seasons were obtained when cowpea intercropped with maize (CS4), as a result of higher yield without any increase in the applied irrigation water for the intercropped system (Table 4). Table 4: Maize yield as affected by different crop sequences in both growing seasons, percentage of yield increase (PI%) and water productivity (WP) Crop sequence 2013/14 growing season 2014/15 growing season Maize yield (t ha-1)* PI (%) WP (kg m-3) Maize yield (t ha-1)* PI (%) WP (kg m-3) CS1 5.43a -- 0.64 5.40b -- 0.60 CS2 5.39a -1 0.64 5.79ab +7 0.65 CS4 5.62a +3 0.67 6.12a +13 0.60 *Means with different letters indicated that it was significantly different 3.4 Effect of crop sequence on productivity of short season clover Table 5 indicates that there were significant differences between short season clover productivity in the studied crop sequences (P < 0.05) in both growing seasons. The highest yield of short season clover was obtained when cowpea preceded it in both growing seasons (CS3), namely 35 and 37 % in the first and second season, respectively. However, when intercropping cowpea with maize preceded by short season clover (CS4), its yield was increased by 12 and 10 % only in the first and second growing season, respectively. Furthermore, water productivity for short season clover increased when cowpea preceded it in CS3, compared to the other two crop sequences (Table 5). Table 5: Short season clover dry yield as affected by different crop sequences in both growing seasons, percentage of yield increase (PI %) and water productivity (WP) Crop sequence 2013/14 growing season 2014/15 growing season Clover yield (t ha-1)* PI (%) WP (kg m-3) Clover yield (t ha-1)* PI (%) WP (kg m-3) CS2 4.30b -- 0.70 5.10b -- 0.93 CS3 5.82b 35 1.06 6.97a 37 1.27 CS4 4.79a 12 0.07 5.62b 10 1.02 *Means with different letters indicated that it was significantly different 3.5 Effect of crop sequence on cowpea productivity Table 6 revealed that cowpea yield was significantly different in the studied crop sequences in both growing seasons (P < 0.05). Thus, cowpea yield was reduced by 45 and 35 % in the first and second season, respectively. Accordingly, water productivity followed the same trend as cowpea yield did in both crop sequences, where it was lower under intercropping with maize in both growing seasons (Table 6). Table 6: Cowpea dry yield as affected by different crop sequences in both growing seasons, percentage of yield increase in its (PI %) and water productivity (WP) in both growing seasons Crop sequence 2013/14 growing season 2014/15 growing season Clover yield (t ha-1)* PI (%) WP (kg m-3) Clover yield (tha-1)* PI (%) WP (kg m-3) CS3 5.27a -- 0.76 5.72a -- 0.03 CS4 2.92b 45 0.42 3.70b 35 0.53 *Means with different letters indicated that it was significantly different -3 0Q 384 Acta agriculturae Slovenica, 109 - 2, september 2017 Evaluation of different crop sequences for wheat and maize in sandy soil 3.6 Total production of each crop sequence and its water productivity The accumulated cereal units for each crop sequence are presented in Table 7. The results showed that the lowest value of accumulated cereal units were found for wheat followed by maize and it was higher in the second growing season. On the contrary, the highest values were obtained when cowpea preceded wheat and followed by short season clover in CS3 in both growing seasons. Furthermore, the highest percentage of increase in total yield of cereal units was found in CS3, namely 88 and 124 %, in the first and second season, respectively. Table 7: Yield of crop sequences in cereal units (CU) in both growing seasons and percentage of increase (PI%) Crop Total yield in 2013/14 (CU ha-1) PI (%) Total yield in 2014/15 (CU ha-1) PI sequence (%) CS1 90.91 -- 95.17 -- CS2 145.72 60 170.90 80 CS3 170.88 88 213.43 124 CS4 132.12 45 166.12 75 Table 8 reveals that CS3 attained the highest water productivity, compared to rest of crop sequences in both growing seasons. Table 8: Water productivity (CU mm-1) for each crop sequence in both growing seasons 2013/14 season 2014/15 season Maize/wheat (CS1) 0.062 0.063 Maize/clover/wheat (CS2) 0.073 0.082 Cowpea/ clover/wheat (CS3) 0.093 0.114 Cowpea with maize/clover then wheat (CS4) 0.066 0.080 4 DISCUSSION AND CONCLUSION In this paper, four crop sequences included two major and important crops in Egypt were evaluated. The evaluation was done on the basis of its applied amount of irrigation water, on its total production calculated using cereal units method and on its water productivity. Our results indicated that the applied amount of irrigation water for each crop in the four sequences was higher in the second year compared to the first year, except for wheat. Table 1 indicated that monthly ETo values in the first growing season of wheat (NovemberApril) were lower than its counterpart in second season from January to April, which resulted in lower water requirements for wheat in the second growing season. Although the value of monthly ETo was lower in June and July in the second growing season of maize (May-September), it has a negligible effect of the applied water to maize and the amount was higher in the second season. In CS4, cowpea was intercropped maize, thus it obtained its water requirements from the applied amount to maize, which resulted in similar applied water to what was applied for CS4 (Table 2). Kamel et al., (2016) indicated that intercropping cowpea with maize did not require applying extra water to cowpea because it shared the applied water to maize. Wheat cultivated in CS1 achieved the lowest productivity (Table 3), where it was planted after maize and both crops are exhausted to the soil, especially when its fertility is low. Hamd-Allah et al. (2015) indicated that low productivity of wheat was obtained when maize preceded it. Consequently, the lowest total production as expressed by cereal units was obtained for CS1. The increase in wheat yield in the second growing season can be explain by lower temperature in January and February in the second growing season, which could increase wheat tillering and positively affected grain yield. Porter and Gawith (1999) indicted that the optimum temperature for wheat shoot growth is 20.3 °C. Whereas, Hakim et al., (2012) stated that 20-25 °C is consider optimum for growth and development of spring wheat. Furthermore, wheat water productivity was the lowest in CS1in both growing seasons. Furthermore, wheat yield was increased by inclusion of short season clover in CS2, or cultivation of cowpea and short season clover before it in CS3 in the first growing season. The second highest wheat yield value resulted from CS4 in the first growing season. In the second growing season, wheat yield was increased by higher values in CS2, CS3 and CS4 (Table 3). Accordingly, the Acta agriculturae Slovenica, 109 - 2, september 2017 385 Abd El-Hafeez ZOHRY et al. highest value of wheat water productivity was obtained in CS3. This result attributed to the residual effect of the two legume crops (cowpea and short season clover) cultivated before wheat on increasing available nitrogen, which benefited wheat yield in the second growing season. This result is supported by the findings of Espinoza et al., (2015). The inclusion of legumes in a cropping sequence can also improve soil quality, porosity, and structure (McCallum et al., 2004) and influence specific microorganism populations in the rhizosphere (Osborne et al., 2010) for the benefit of following crops. Regarding to maize in CS1, its yield was decreased in the second growing season, as a result of wheat cultivation before it in the first growing season (Table 4). Consequently, the lowest water productivity for maize existed in CS1 (Table 4), as well as the lowest total production (Table 7) and the lowest water productivity (Table 8) existed in the studied four crop sequences. The existence of legume crop (s) in the crop sequences CS2 and CS4 resulted in increasing maize yield and its water productivity (Table 4). This result can be attributed to the ability of legume crops to facilitate the absorption of P and K in the soil by cereal crops, in addition to its role in providing N through N-fixing rhizobium. Bado et al., (2006) stated that N2 -fixing legumes supply N to the subsequent crops through fallen senescent leaves and below ground parts, leading to an increase in succeeding crop yield. Hassan et al., (2010) indicated that legumes mobilize P in the soil during its growth, which increase P uptake of the following cereals. Ferguson et al., (2013) indicated that legumes have the ability to remove calcium and magnesium in the soil more than cereals and replace it with hydrogen, which results in removing OH- ions and increases H+ thus lowering the soil pH. Regarding to maize, intercropping cowpea with it in CS4 increased its yield in the first growing season. Moreover, higher increase in maize yield was noticed in the second growing season, as a result of the residual effect of short season clover in CS4 (Table 4). Furthermore, the highest maize water productivity was attained in CS4 (Table 4). Previous research on intercropping cowpea with maize in clay soil under surface irrigation indicated that maize yield was increased by 10 %, as a result of increased nitrogen content in the soil, reduction in the associated weeds competing with maize plants (Zohry, 2005a) and reduction in biological enemies that attack maize plants (Hamd-Alla, 2015) and it was also observed in our experiment. Inclusion of pure stand of cowpea in the crop sequence resulted in more positive effect on soil fertility, compared to its effect when it is intercropped with a soil exhausted crop like maize (Zohry, 2005a). The pure stand of cowpea produced higher yield compared to cowpea intercropped with maize as a result of lower plant density for cowpea, as well as interspecific competition between cowpea and maize, where maize is the main crop in this system and cowpea is the secondary crop (Dahr et al., 2013). Gharnbari et al., (2010) indicated that cowpea intercropped with maize increased absorbed photosynthetically active radiation. This effect is shown in our experiment, where maize yield was increased, compared to sole maize planting. Furthermore, this intercropping system reduces water evaporation and improves conservation of soil moisture (Gharnbari et al., 2010). For that reason, in our experiment, the applied amount to sole maize was similar to what was applied to cowpea intercropped with maize. Kariaga (2004) concluded that this intercropping system reduced runoff through maintaining ground cover and also it reduced soil erosion. Our results showed that there was superiority in water productivity for CS3 (only wheat was included), compared to CS1 (both maize and wheat were included) due to the high used amounts of water, which resulted in the highest yield values in both growing seasons. The two legume crops preceded wheat in this crop sequence resulted in higher wheat yield value. CS2 recorded the second with respect to the value of water productivity, where both maize and wheat were included, in addition to short season clover preceded wheat. However, the applied amount of water was the highest, with lower total yield in CU than what was obtained by CS3. In general, Najibnia et al., (2014) indicated that intercropping system was superior in water productivity, compared to sole planting. Thus, the best crop sequence for maize with respect to water productivity was CS4. Thus, it can be concluded that to attain higher yield and water productivity for wheat in new reclaimed soil in Upper Egypt, two legume crops should be cultivated before it. Similarly, to achieve higher yield and water productivity for maize in these types of soils, maize should be intercropped with legume crop, and another legume crop should follow it to benefit from its residual effect in the following growing season. The results of this experiment can be with great benefits to other countries with similar weather and soil conditions in the arid and semiarid regions. 384 Acta agriculturae Slovenica, 109 - 2, september 2017 Evaluation of different crop sequences for wheat and maize in sandy soil 5 REFERENCES Abou-Keriasha M.A., Eisa N. M.A. and Lamlom M.M. (2013). Benefits of legume crops in rotation and intercropping for increased production and land use. Egyptian Journal of Agronomy, 35(2), 183197. Allen R.G., Jensen M.E., Wright J.L. and Burman R.D, (1989). Operational estimate of reference evapotranspiration. Agronomy Journal, 81, 650662. doi:10.2134/agronj1989.00021962008100040019x Andersen M.K., (2005). Competition and complementarily in annual intercrops: the role of plant available nutrients. PhD thesis, Department of Soil Science, Royal Veterinary and Agricultural University, Copenhagen, Denmark. Bado B.V., Bationo A., and Cescas M.P., (2006). Assessment of cowpea and groundnut contributions to soil fertility and succeeding sorghum yields in the Guinean savannah zone of Burkina Faso (West Africa). Biology and Fertility of Soils, 43, 171-176. doi:10.1007/s00374-006-0076-7 Banik P.,Midya A., Sarkar B. K. and Ghose S. S., (2006). Wheat and chickpea intercropping systems in an additive series experiment: Advantages and weed smothering. European Journal of Agronomy, 24, 325-332. doi:10.1016/j.eja.2005.10.010 Brankatschk G. and Finkbeiner M. 2014. Application of the Cereal Unit in a new allocation procedure for agricultural life cycle assessments. Journal of Cleaner Production, 73, 72-79. doi:10.1016/j.jclepro.2014.02.005 Brockhaus, J. 1962. ABC der Landwirtschaft, Band (i). A-K p. 488-489. VEB, Brock HausVerlag, Leipzig. Dhar P.C., Awal M.A., Sultan M.S., Rana M.M., Sarker A., 2013.Interspecific competition, growth and productivity of maize and pea in intercropping mixture. Journal of Crop Science, 2(10), 136-143. Eskandari H., Ghanbari A., and Javanmard A. 2009. Intercropping of cereals and legumes for forage production. Notulae Scientia Biologicae, 1 (1), 7 -13. Espinoza S., Ovalle C., Zagal E., Matus I., Pozo A., 2015. Contribution of legumes to the availability of soil nitrogen and its uptake by wheat in Mediterranean environments of central Chile. Chilean Journal of Agricultural Research, 75(1), 111-121. doi:10.4067/S0718-58392015000100016 FAO. 2003. Unlocking the water potential of agriculture. FAO Corporate Document Repository. Rome, FAO. Ferguson B.J., Lin M.H., Gresshoff P.M, 2013. Regulation of legume nodulation by acidic growth conditions. Plant Signal Behavior, 8(3), e23426. doi:10.4161/psb.23426 Ghanbari A., M. Dahmardeh, B. A. Siahsar and M. Ramroudi. 2010. Effect of maize (Zea mays L.) -cowpea (Vigna unguiculata L.) intercropping on light distribution, soil temperature and soil moisture in arid environment. Journal of Food, Agriculture and Environment, 8(1):102-108. Gomez K.A., and Gomez A.A., 1984. Statistical procedures for agriculture research 2nd Edition. John Wiley and Sons. New York, pp. 317-333. Hakim M.A., Hossain A., Teixeira da Silva J.A., Zvolinsky V.P., Khan M.M. 2012. Yield, protein and starch content of 20 wheat (Triticum aestivum L.) genotypes exposed to high temperature under late sowing conditions. Journal of Science Research, 4(2):477-489. doi:10.3329/j sr.v4i2.8679 Hamd-Alla W.A., Shalaby E.M., Dawood R.A., and Zohry A.A., 2015. Effect of crop sequence and nitrogen fertilization on productivity of wheat. Elixir International Journal of Agriculture, 88, 36215-36222. Hassan H.M., Marschner P., McNeill A., 2010. Growth, P uptake in grain legumes and changes in soil P pools in the rhizosphere. 19th World Congress of Soil Science, Soil Solutions for a Changing World, 1 - 6 August 2010, Brisbane, Australia. Jackson M.L., 1958. Soil Chemical Analysis. Prentice Hall. Englewood Cliffs. New Jersey. USA. Kamel, A.S., El-Masry M.E. and Khalil H.E., 2010. Productive sustainable rice based rotations in saline-sodic soils in Egypt. Egyptian Journal of Agronomy, 32(1):73-88. Kamel A.S., Zohry A.A.and Ouda, S., 2016. Unconventional Solution to Increase Crop Production under Water Scarcity. In: Major Crops and Water Scarcity in Egypt. Springer Publishing House. pp 99-114. Kariaga, B.M. 2004, Intercropping maize with cowpeas and beans for soil and water management in western Kenya. 13th International Soil Conservation Organization Conference - Brisbane, July 2004. Paper No. 993. Kirkegaard J., Christen O., Krupinsky J., Layzell D., 2008. Review: Break crop benefits in temperate wheat production. Field Crops Research, 107, 185195. doi:10.1016/j.fcr.2008.02.010 Acta agriculturae Slovenica, 109 - 2, september 2017 385 Abd El-Hafeez ZOHRY et al. Kumpawat, B.S. and Rathore, S.S., 2003. Effect of preceding grain legumes on growths, yield, nutrient content and uptake by wheat under different nitrogen levels. Field Crops Research, 25(2), 209214. Kwari J.D., 2005. Soil fertility status in some communities of southern Borno. Final report to PROSAB Project, Maiduguri, Nigeria. p. 21. Macák M., Zák S., Andrejcíková M. 2015. Productivity and macro elements content of cereal and legume crops. Acta fytotechn zootechn, 18, 160-162. McCallum M.H., Kirkegaard J.A., Green T., Cresswell H.P., Davies S.L., and Angus J.F., 2004. Improved subsoil macro-porosity following perennial pastures. Australian Journal of Experimental Agriculture, 44, 299-307. doi:10.1071/EA03076 Megawer E.A, Sharaan A.N., and El-Sherif A.M., 2010.Effect of intercropping patterns on yield and its components of barley, lupine or chickpea grown in newly reclaimed soil. Egyptian Journal of Applied Science, 25(9), 437-452. Najibnia, S., Koocheki, A., N. Mahallati , and M. Porsa. 2014. Water capture efficiency, use efficiency and productivity in sole cropping and intercropping of rapeseed, bean and corn. European Journal of Sustainable Development. 3(4): 347-358. doi:10.14207/ej sd.2014.v3 n4p3 47 Nofal, N., 2012. Effect of intercropping faba bean on sugar beet under different nitrogen fertilization. MSc thesis, El-Minia University. Egypt. Osborne C.A., Peoples M.B., and Janssen P.H., 2010. Detection of a reproducible, single-member shift in soil bacterial communities exposed to low levels of hydrogen. Applied Environmental Microbiology, 76, 1471-1479. doi:10.1128/AEM.02072-09 Rochester, I.J., M.B. Peoples, N.R. Hulugalle, R.R. Gault, and G.A. Constable, 2001. Using legumes to enhance nitrogen fertility and improve soil conditions in cotton cropping systems. Field Crops Research, 70, 27-41. doi:10.1016/S0378-4290(00)00151-9 Porter, J. R., M. Gawith. 1999. Temperatures and the growth and development of wheat: a review. European Journal of Agronomy, 10:23-36. doi:10.1016/S1161-0301(98)00047-1 Sheha A.M., Nagwa R. Ahmed and A.M. Abou-Elela. 2014. Effect of crop sequence and nitrogen levels on rice productivity. Annals of Agricultural Science, 52 (4): 451 - 460. Singh, N.B., Singh, P.P., and Nair, K.P., 1986. Effect of legume intercropping on enrichment of soil nitrogen, bacterial activity and productivity of associated maize crops. Experimental Agriculture, 22, 339-344. doi:10.1017/S0014479700014587 Snyder R.L., Orang M., Bali K. and Eching S., 2004. Basic irrigation scheduling BIS. http://www.waterplan.water.ca.goV/landwateruse/w ateruse/Ag/CUP/Californi/Climate_Data_010804.xl s. Valipour M. 2012a. HYDRO-MODULE determination for Vanaei village in Eslam Abad Gharb, Iran. ARPN Journal of Agricultural and Biological Science, 7(12):968-976. Valipour, M. 2012b. Ability of Box-Jenkins Models to Estimate of Reference Potential Evapotranspiration (A Case Study: Mehrabad Synoptic Station, Tehran, Iran). IOSR Journal of Agriculture and Veterinary Science, 1(5): 1-11. doi:10.9790/2380-0150111 Valipour, M. 2014. Analysis of potential evapotranspiration using limited weather data. Applied Water Science. doi:10.1007/s13201-014-0234-2. doi:10.1007/s13201-014-0234-2 Valipour M. 2016. Variations of land use and irrigation for next decades under different scenarios. Irriga, Botucatu, Ediçao Especial, Irrigaçao, p. 262-288. Zhao, J., Xu, Z., Zuo, D. and Wang, X. 2015. Temporal variations of reference evapotranspiration and its sensitivity to meteorological factors in Heihe River Basin, China. Water Science and Engineering, 8(1): 1-8. doi:10.1016/j.wse.2015.01.004 Zohry A.A., 2005a. Effect of preceding winter crops and intercropping on yield, yield components and associated weeds in maize. Annals of Agricultural Science,43(1), 139-148. Zohry, A.A., 2005b. Effect of relaying cotton on some crops under bio-mineral N fertilization rates on yield and yield components. Annals of Agricultural Science,43(1), 89-103. Yan F., Schubert S., Mengel K., 1996. Soil pH changes during legume growth and application of plant material. Biology and Fertility of Soils, 23(3), 236242. doi:/10.1007/BF00335950 384 Acta agriculturae Slovenica, 109 - 2, september 2017 doi: 10.14720/aas.2017.109.2.22 Original research article / izvirni znanstveni članek High salicylic acid concentration alters the electron flow associated with photosystem II in barley Ghader HABIBI1*, Atousa VAZIRI1 Received February 09, 2017; accepted May 08, 2017. Delo je prispelo 09. februarja 2017, sprejeto 08. maja 2017. ABSTRACT In this study, the effects of exogenously applied salicylic acid (0.5 and 5 mM SA) on the rates of photosystem II (PSII) activity was analysed in 4-week-old barley (Hordeum vulgarelBahman') seedlings using chlorophyll (Chl) a fluorescence transient (OJIP) measurements. No evident changes in Chl and carotenoid contents as well as chlorophyll fluorescence transient curves were observed in either of the studied concentrations after 24 h of SA application. After 5 d, low SA concentration (0.5 mM) increased PSII activity, Chl b and carotenoid contents in barley seedlings. In contrary, 5 days after 5 mM SA treatment, the maximal quantum efficiency of PSII (Fv/Fm) and the Performance Index (PIABS), as an indicator of PSII structure and functioning, were significantly decreased. This lower Fv/Fm and PIABS coupled with lower levels of Chl b and carotenoids, and lower values of photosynthetic electron transport chain components including the electron transport flux (pEo) and the inferred oxygen evolving complex activity (Fv/Fo). By monitoring the chlorophyll a fluorescence rise kinetics, from the initial "O" level to the "P" (the peak) level, a dramatic increase in "Of phase was detected, which coincides with an increased photo-reduction of QA as a result of blockage of electron flow. This study provided the evidence that the high concentration of SA induced damage to different sites of the PSII. Key words: photosynthetic pigments; photosynthetic electron flow; Hordeum vulgare 'Bahman'; OJIP transient flourescence; salicylic acid IZVLEČEK VELIKA KONCENTRACIJA SALICILNE KISLINE SPREMINJA PRI JEČMENU FOTOSINTEZNI, S FOTOSISTEMOM II POVEZAN ELEKTRONSKI PRETOK V raziskavi so bili preučevani učinki dodajanja salicilne kisline (0.5 in 5 mM) na aktivnost fotosistema II (PSII) pri 4-tedne starih kalicah ječmena (Hordeum vulgare'Bahman') z meritvami fluorescence (OJIP) klorofila a (Chla). Nobenih sprememb v vsebnosti klorofila in karotenoidov kot tudi ne sprememb v fluorescenci ni bilo opaznih po 24 urah dodajanja obeh koncentracij salicilne kisline. Po petih dneh so se v kalicah ječmena pri dodani manjši koncentraciji salicilne kisline (0.5 mM) povečali aktivnost PSII, vsebnost Chl b in karotenoidov. Nasprotno sta se pet dni po obravnavanju s 5 mM salicilno kislino značilno zmanjšala učinkovitost PSII (Fv/Fm) in PIABS indeks kot indikatorja zgradbe in delovanja PSII. Zmanjšanje Fv/Fm in PIABS je bilo povezano z zmanjšanjem vsebnosti klorofila b in karotenoidov ter z manjšimi vrednostimi komponent fotosintezne elektronske verige, vključno s elektronskim pretokom (pEo) in z njim povezano aktivnostjo kompleksa, ki sprošča kisik (Fv/Fo). Pri spremljanju povečanja fluorescence klorofila a od začetne "O" na največjo vrednost "P" je bilo opazno njeno dramatično povečanje v fazi "OJ", kar je soupadalo s povečano fotoredukcijo QA kot posledica blokade fotosintezenega elektronskega pretoka. Raziskava dokazuje, da večja koncentracija salicilne kisline povzroči poškodbe na večih mestih PSII. Ključne besede: fotosintezna barvila; fotosintezni elektronski pretok; Hordeum vulgare 'Bahman', OJIP prehodna fluorescenca; salicilna kislina Corresponding author: Associate Prof. Dr. Ghader Habibi, Biology Department, Payame Noor University, PO BOX 19395-3697 Tehran, Iran, Telephone: +98 482 322 2702, Fax: +98 422 822 8055, Email: gader.habibi@gmail.com 1 Department of Biology, Payame Noor University (PNU), Iran Acta agriculturae Slovenica, 109 - 2, september 2017 str. 349 - 356 Ghader HABIBI, Atousa VAZIRI 1 INTRODUCTION Salicylic acid (SA), as a common plant-produced phenolic compound, plays an important role in plant growth and development as well as in tolerance to biotic and abiotic stresses (Li et al., 2014; Khan et al., 2014; Janda and Ruelland, 2015). In recent years the involvement of SA in the plant growth and yield (Javaheri et al., 2012), and the regulation of some photosynthetic reactions (Arfan et al., 2007; Li et al., 2014) has widely been studied. It has been suggested that the effects of SA on plant physiological and biochemical processes depends on the concentration of the applied SA (Miura and Tada, 2014). At low concentrations (0.1-0.5 mM for most plants), it enhances the efficiency of the antioxidant system and the efficiency of PSII photochemistry (Chen et al., 2016), whereas at higher concentrations (1-10 mM for most plants) it increases oxidative damage (Hara et al., 2012; Miura and Tada, 2014). Although negative effect of SA is probably correlated with an imbalance in antioxidant metabolism (Hasanuzzaman et al., 2013), the specific mechanisms of SA-mediated damages remain elusive. It is assumed that, plants respond to high SA depend on PSII response to this stress (Chen et al. 2016). To address this issue, the chlorophyll (Chl) a fluorescence transient (OJIP) measurements were used to study photosynthetic apparatus functioning in response to various SA concentrations and incubation times in this study. Chl a fluorescence induction (OJIP, where O (or Fo) is the minimum fluorescence when all QA (the primary quinone acceptor of PSII) are in the oxidized state, P (or Fmax) is the maximal fluorescence when all QA is in the reduced state (QA-)) has been studied extensively in photosynthesis physiology research (Jee, 1995; Kalaji et al., 2011; Hamdani et al., 2015). The reduction of QA by PSII causes chlorophyll a fluorescence to rise from its minimal fluorescence level "O" to a "J" level (or Fj). Fluorescence rise from "J" level to the "I" level (or Fi) is related to the filling up of the plastoquinone pool. Finally, a traffic jam of electrons on the electron acceptor side photosystem I generates a fluorescence rise from the "I" level to the "P" level. The analysis of chlorophyll a fluorescence signals using 'JIP-test', explores the information about the structure and function of the photosynthetic apparatus mostly related to PSII (Strasser et al., 2000; Bussotti et al., 2007) as well as some parameters due to energy fluxes for light absorption (ABS), trapping (TR) of excitation energy and electron transport (ET) per reaction center (RC) or per sample area called cross-section (CS) (Strasser et al., 2000). As a noted above, the exact mechanisms by which SA affects photochemistry remain obscure. The present paper is the first report on the SA-mediated changes in specific chlorophyll fluorescence parameters. In order to improve our knowledge of barley photosynthetic apparatus in response to SA treatment, the OJIP fluorescence transient was measured in barley plants in responses to different concentrations of SA. 2 MATERIALS AND METHODS 2.1 Plant material and harvest The randomly selected healthy seeds of barley (Hordeum vulgare 'Bahman') were sterilized with 5 % sodium hypo-chlorite solution for five minutes prior to sowing. Seeds were then sown on filter paper moistened with distilled water. Ten-day-old seedlings were transferred to modified Hoagland nutrient solution (Johnson et al. 1957) containing 6 mM KNO3, 4 mM Ca(NO3)2, 2 mM NH4H2PO4, 1 mM MgSO4, 50 ^M H3BO3, 2 ^M MnSO4, 2 ^M ZnSO4, 0.5 ^M CUSO4, 0.5 ^M H2MoO4 and 0.02 mM FeSO4-EDTA for 15 days prior to the start of treatments. The pH of the nutrient medium was adjusted to 5.5-5.7. The seedlings were grown in a controlled growth room under a 16/8 light/dark cycle and a photosynthetically active radiation (PAR) of 200 ± 30 ^mol m-2 s-1 and an average day/night temperature of 25 ± 1/18 ± 1 °C. Salicylic acid (SA) was dissolved in absolute ethanol then added drop wise to water (ethanol/water: 1/1000 v/v, pH was adjusted to 5.7) (Williams et al. 2003). At 25 days after germination, the foliar application of SA was carried out in the morning (between 08:00 and 10:00) with a compression sprayer of 1 L capacity. Non-SA applied plants were sprayed with ethanol/water (1/1000 v/v). At 1 and 5 days after treatment, the plants were harvested and the recent fully expanded and mature leaves were used for measurement of chlorophyll fluorescence and other analysis. 2.2 Chlorophyll a fluorescence measurements Chlorophyll a fluorescence transients (OJIP transients) were measured with a Packet-PEA chlorophyll fluorimeter (Plant Efficiency Analyser, Hansatech Instruments Ltd., King's Lynn, Norfolk, PE 32 1JL, England) in dark-adapted (for at least 20 min) leaves of barley. We used the JIP-test (Strasser and Strasser, 394 Acta agriculturae Slovenica, 109 - 2, september 2017 High salicylic acid concentration alters the electron flow associated with photosystem II in barley 1995; Strasser et al., 2004) to analyse chlorophyll a (ABS), trapping (TR) of excitation energy and electron fluorescence rises. The measured and calculated transport (ETR) per reaction center (RC) or per sample parameters are described in Tab 1. Specific parameters area called cross-section (CS). were calculated from energy fluxes for light absorption Table 1: Some groups of measured and calculated parameters using the JIP-test (Yusuf et al., 2010) Nomenclature_Explanation_ Data extracted from the recorded fluorescence transient OJIP Area Total complementary area between Fo and Fm (reflecting the size of the plastoquinone pool) FJ Fluorescence intensity at the J-step (2 ms) of OJIP F[ Fluorescence intensity at the I-step (30 ms) of OJIP Fluorescence parameters derived from the extracted data Fm or Fmax Maximal chlorophyll fluorescence intensity measured when all photosystem II (PSII) reaction centers are closed Fo Minimal fluorescence (all PSII RCs are assumed to be open) Fv Variable chlorophyll fluorescence (Fm-Fo) Vj Relative variable fluorescence at time J (relative variable fluorescence at phase J of the fluorescence induction curve The specific energy fluxes (per reaction center, RC) AB S/RC Light absorption flux (for PSII antenna chlorophylls) per RC DI/RC Dissipation energy flux per RC ET/RC Maximum electron transport flux (further than QA-) per RC TR/RC Trapped (maximum) energy flux (leading to QA reduction) per RC The phenomenological energy fluxes (per excited cross-section of leaf, CS) ABS/CS Absorbed photon flux per cross section TR/CS Maximum trapped excitation flux per cross section ET/CS Electron transport flux from QA to QB per cross section DI/CS Dissipation energy flux per cross section Quantum yields and efficiencies ^Eo or phi (Eo)=ETo/AB S Quantum yield for electron transport (ET) De-excitation rate constants of PSII antenna kN Non-photochemical de-excitation rate constant kP Photochemical de-excitation rate constant Performance index PIABS The performance index that is calculated as: (RC/ABS)x(^Po/(1-^Po))x(^o/(1- ^o)), where, RC is for reaction center; ABS is for absorption flux; g & 2 -ü o ¡X "8 S s >o 0 1 o. 1,5 0 X B iS v a V ^ B B T T r J? j? •«p 4? E A L^F-1 S S Akcesija 5 4 1 Slika 2: Povprečni indeks okužbe s koruzno progavostjo (Setosphaeria turcica (Luttr.) K.J. Leonard & Suggs) (±SE) v Krasincu (2016) Figure 2: Average index of infection by northern leaf blight (Setosphaeria turcica (Luttr.) K.J. Leonard & Suggs) (±SE) at Krasinec (2016) * različne črke pomenijo statistično značilno razliko po Duncanu (P<0,05) 4 RAZPRAVA Pridelki koruze so bili na obeh lokacijah in v obeh letih močno odvisni od genotipa. 'Rdeča bohinjka' in delno 'Rumena bohinjka', podobno pa tudi 'LJ 180' so imele pretežno majhne pridelke, so pa bile tudi med najbolj ranimi, torej je bil manjši pridelek pričakovan. Sodobni hibridni sorti 'P 9074' in 'Ronaldinio KWS' sta imeli pričakovano največji pridelek. Pridelek populacije 'P 9074' je bil na Laboratorijskem polju Biotehniške fakultete skoraj dvakrat večji od populacije 'Metliška Plut'. Prav slednja je pokazala zadovoljive rezultate na vseh poskusih in je zato lahko primerna za lokalno pridelavo tudi v ekološkem načinu pridelave. 'Metliška Plutje raznolika glede na dolžino in obarvanost storžev (Slika 3), kar pa bi lahko z nadaljnjo selekcijo izboljšali. 440 Acta agriculturae Slovenica, 109 - 2, september 2017 Vrednotenje pridelka koruze za sonarave oblike kmetovanja Slika 3: Variabilnost akcesije 'Metliška Plut' pri vzdrževalcu (Aleš Plut, Cerovec 17, Semič) (pridelek 2016). Figure 3: Variability of accession 'Metliška Plut' at maintainers site (Aleš Plut, Cerovec 17, Semič) (yield 2016) Rezultati naše raziskave kažejo, da se lokalne populacije različno odzivajo na abiotične in biotične dejavnike. Andjelkovic in sod. (2014) denimo navajajo, da so bile različne akcesije koruze različno odporne na sušo. Ugotovili smo, da obstajajo razlike med posameznimi populacijami koruze glede na pridelovalne sisteme. V Osrednji Sloveniji smo koruzo gojili na konvencionalen način, medtem ko smo v jugovzhodni Sloveniji poskus z različnimi genotipi koruze zasnovali na ekološki kmetiji. V obeh letih poskusa je po pridelku zrnja med populacijami izstopala 'Metliška Plut'. V drugem letu poskusa je bil njen pridelek primerljiv s hibridnimi sortami v obeh letih poskusa. V 2016 smo raziskavo nadgradili, saj smo poleg pridelka spremljali tudi indeks okuženosti s koruzno progavostjo. Hibridni sorti sta bili na obeh lokacijah najmanj dovzetni na širjenje koruzne progavosti. Da so hibridne sorte koruze manj dovzetne za napad škodljivih organizmov, v svojemu delu navajajo Beaver in sod. (2011). Indeks okuženosti s koruzno progavostjo je bil manjši na poskusu v osrednji Sloveniji, kar med drugim lahko pripišemo dobri kmetijski praksi. 5 ZAKLJUČEK Poskusi uporabne vrednosti slovenskih populacij in sort koruze v dveh različnih regijah so pokazali, da lokalne populacije po pridelku in odpornostjo proti boleznim niso neposredno konkurenčne sodobnim hibridnim sortam. Vsekakor pa je potrebno poudariti, da zlasti populacija poltrdinke 'Metliška Plut' odstopa od ostalih in je primerna za pridelavo na izbranih lokacijah. Akcesijo že razmnožujemo in pripravljamo za vpis na sortno listo. Acta agriculturae Slovenica, 109 - 2, september 2017 Borut BOHANEC, Tanja BOHINC, Filip VUČAJNK 6 ZAHVALA Raziskava je bila izvedena v sklopu projekta V4-1313 'Vzpostavitev sistema vzdrževalne selekcije in pridelave semenskega materiala kmetijskih rastlin za sonaravne oblike kmetovanja'. Zahvaljujemo se izr. prof. dr. Ludviku Rozmanu za aktivnost pri štartu projekta in dolgoletnemu delu na genskih virih koruze. 7 VIRI Andjelkovic, V., Kravic, N., Ignjatovic-Micic, D., Dumanovic, Z., Vancetovic, J. (2014). Estimation of drought tolerance among maize landraces from mini-core collection. Genetika-Belgrade, 46(3), 775-788. doi:10.2298/GENSR1403775A Beaver, J.S., Brunner, B.R., Armstrong, A. (2011). Release of sweet corn (Zea mays L.) open-pollinated cultivar 'Suresweet 2011'. Journal of Agriculture of the University of Puerto Rico, 9J(1/2):105-110. Fullerton, R.A. (1982). Assessment of leaf damage caused by northern leaf blight in maize, New Zealand Journal of Experimental Agriculture, 10, 313-316. doi:10.1080/03015521.1982.10427890 Kutka, F. (2011). Open-pollinated vs. hybrid maize cultivars. Sustainability, 3, 1531-1554. doi:10.3390/su3091531 Muntean, L., Has, I., Has, V., Gulea, A. (2012). Comparison between some corn synthetic populations obtained from Transylvanian local populations and a corn synthetic population based on elite inbred lines. Bulletin of University of Agricultural Sciences and Veterinary Medicine Cluj-Napoca. Agriculture, 69(1),133-142. Rozman, L. (2012). Genska banka koruze v Sloveniji. Acta agriculturae Slovenica, 99(3), 317-328. Shelton, A.C., Tracy, W.F. (2015). Recurrent selection and participatory plant breeding for improvement of two organic open-pollinated sweet corn (Zea mays L.) populations. Sustainability, 7,5139-5152. doi:10.3390/su7055139 442 Acta agriculturae Slovenica, 109 - 2, september 2017 doi:10.14720/aas.2017.109.2.27 Original research article / izvirni znanstveni članek The effects of silicon and titanium on safflower (Carthamus tinctorius L.) growth under moisture deficit condition Mohsen JANMOHAMMADI1*, Nasrin MOHAMMADI1, Fariborz SHEKARI1, Amin ABBASI1, Mohammad ESMAILPOUR2 Received June 01, 2017; accepted July 26, 2017. Delo je prispelo 01. junija 2017, sprejeto 26. julija 2017. ABSTRACT Safflower is one of important crop in semi-arid regions of the world, where the precipitations are limited. In order to investigate the effect of foliar spray of nano-silicon dioxide (10 and 20 mM) and nano titanium dioxide (25 and 50 mM) and water-deficit stress (irrigation after 110 mm evaporation) on growth parameters and yield components of spring safflower a field experiment was carried out at the highland semi-arid region, in, North West of Iran. Water deficit stress significantly reduced morpho-physiological traits such as ground cover, canopy width, leaf fresh mass, leaf are and plant height) as well as yield components (e.g. capitulum diameter, seed mass and seed number per capitulum). However, the plants grown under water deficit condition showed the higher harvest index than well irrigated plants. Comparison of the foliar treatments showed that the both nano-particles (silicon and titanium) improved the plant growth and yield components over the control. However, the effect of nano-silicon was more prominent than titanium. The highest amount of seed oil was recorded under well irrigated condition (irrigation after 60 mm evaporation) with foliar application of nano-titanium. The percentage of palmitic acid, arachidic acid and myristic acid in seed increased by nano-titanium application. Altogether, principal component analysis indicated that spray of 10 mM nano silicon dioxide was best foliar treatments under all moisture regimes. Key words: safflower; agronomic traits; foliar spraying; nano-particles; principal component analysis; semi-arid region IZVLEČEK UČINKI SILICIJA IN TITANA NA RAST ŽAFRANIKE (Carthamus tinctorius L.) V RAZMERAH POMANKANJA VODE Žafranika je pomembna poljščina v semiaridnih območjih, kjer so padavine omejene. Z namenom ugotavljanja učinkov pršenja listov z nano-silicijevim (10 in 20 mM) in nano titanovim dioksidom (25 in 50 mM) ter stresa zaradi vodnega deficita (namakanje po 110 mm evaporacije) na rastne parametre in komponente pridelka pomladanske žafranike je bil izveden poljski poskus na višinskem semiaridnem območju severno zahodnega Irana. Vodni deficit je značilno zmanjšal morfološko-fiziološke lastnosti (pokritost tal, širino krošnje, svežo maso listov, listno površino in višino rastlin) kot tudi komponente pridelka (premer koška, maso semen in število semen na košek). Kljub temu so imele rastline, ki so rastle v razmerah pomanjkanja vode večji žetveni indeks kot dobro namakane. Primerjava foliarnih obravnavanj je pokazala, da so oboji nano delci (silicijevi in titanovi) izboljšali rast rastlin in komponente pridelka v primerjavi s kontrolo. Učinek silicijevih nano delcev je bil bolj izražen kot titanovih. Največja vsebnost olja v semenih je bila v dobro namakanih rastlinah (namakanje po 60 mm evaporacije) s foliarno dodajanimi nano delci titana. Odstotki palmitinske, arahidonske in miristične kisline v semenih so se povečali po uporabi nano titanovih delcev. Analiza glavnih komponent je pokazala, da je bilo pršenje z 10 mM nano silicijeva dioksida najboljše foliarno obravnavanje pri vseh vodnih režimih. Ključne besede: žafranika; agronomske lastnosti; foliarno pršenje; nano-delci; analiza glavnih komponent; semiaridna območja 1 Department of Genetic and Plant Production, Faculty of Agriculture University of Maragheh, P.O. Box 55181-83111, Maragheh, Iran; *Corresponding author: jmohamad@alumni.ut.ac.ir 2 Department of Medicinal and Aromatic Plants, Jahrom university, Jahrom, Iran Acta agriculturae Slovenica, 109 - 2, september 2017 str. 349 - 356 Mohsen JANMOHAMMADI et al. 1 INTRODUCTION The world's population is growing rapidly, so that by 2050, it is predicted to reach 9.1 billion, 34 %more than today (UN, 2013). Indeed, majority of this population growth is expected to occur in developing countries. These trends refer to the imperative note that, the food security is (and will increasingly continue to be) faced with a major challenge, necessitating anincrease in food production in these areas. However, limited remaining arable lands and restricted water resources bolds the significance of improvingcrop management and implementing strategies to increase drought toleranceto achieve the required massive increase in global food production. Additionally, climate change is one of the main factors restricting the yield, performance and stability of crop production. However, in terms of climate, most of developing countries are located in semi-arid areas which are characterized by highly variable and unpredictable rainfall and total precipitations below potential evapotranspiration, making them frequently engaged with drought stress. However, even at mild intensity, drought stress can inhibit photosynthesis and stomatal conductance significantly, resulting in considerable yield reductions (Shahrokhnia & Sepaskhah, 2017). Safflower (Carthamus tinctorius L.) is one of the oldest cultivated crops which is partially adapted to water-deficit zones. Being a highly branched, herbaceous, thistle-like annual plant, it is commercially cultivated for extracting vegetable oil from its seeds. Besides, safflower is grown for flowers used for coloring, flavoring foods, dyes, medicinal properties, and forage. Safflower can be considered as a promising substitute crop in dryland agro-ecosystems due to its unique agronomic characteristics (Hussain et al., 2016). Safflower is aboriginal to semi-arid environments, and its adaptation to water-deficit conditions is largely resulted from its extended roots which can penetrate to a depth of down to 2 meters to absorb nutrients (HaghighatiMalek & Ferri, 2014). Nutrient requirements of safflower are similar to those of winter cereal such as wheat and barley, but compared to other annual plants, safflower has a longer growth period. Although the deep rootsystem of safflower allows the plant to utilize nutrients and water from lower depths, its long, dry spell without effective precipitation can negatively affect the seed yield. High evapotranspiration, limited water resources and other parameters in semi-arid regions has invoked the interests for studying the effect of water-deficit stress. Indeed, it is necessary to find or develop appropriate techniques for producing crop under water limited condition (Asadzade et al., 2015). This can be partially reached through growing drought-tolerant varieties while setting forth more rational strategies for exploiting landscape. The use of fertilizers and improving the soil water holding capacity represent other options (Murungweni et al., 2016). Additionally, application of innovative nanotechnologies in agriculture (including the advantageous nano-particles) has been shown to be a promising approach to improve crop production considerably (Lal, 2008; Liu & Lal, 2015). Ingredients with particle sizes smaller than 100 nm in at least one dimension are generally classified as nano-materials. A number of engineered nano-materials have been investigated for use in agricultural scope to increase crop productivity and enhance crop protection (Khot et al., 2012). In this regard, foliar application of beneficial nano-particles has opened a new avenue for makingnanotechnology a feasible option in the field. Among the wide range of inorganic nano-materials introduced recently, silicon (Si) and titanium (Ti) nanoparticles have gained the largest deals of focus. Although silicon is the second most abundant element in the earth's crust (following oxygen), biological role of Si in plants is yet to be thoroughly studied for specific plants. Being absorbed by plants in the form of silicic acid, siliconis the only nutrient element that is not toxic at high concentrations (Ma et al., 2001; Ma, 2004). Beneficial roles of Si in inducing defense mechanisms under various biotic and abiotic stresses have been reported (Van Bockhaven et al., 2013; Zhu & Gong, 2014). More interestingly, alleviative effects of Si under water-deficit stress have been observed (Shiet et al., 2016). Likewise, titanium is the ninth most abundant element in the earth's crust; it is introduced as a beneficial element for plant growth. Nano-titanium dioxide (nTiO2) has been reported as an effective photo-catalyst under ultraviolet radiation (Gupta & Tripathi, 2011). Therefore, leaf-spray of nano titanium dioxide may improve the efficiency of plant photosynthesis and related physiological activities, thereby improvingthe plant growth. Foliar spray of nTiO2solution on barley under supplemental irrigation conditions may positively affect some morphophysiological characteristics like days to anthesis, chlorophyll content and straw yield (Janmohammadi et al., 2016b). However, there have been reportson possible adverse effects of nTiO2 suspension on corn leaf development and transpiration (Asli & Neumann, 2009). Also, these authors reposted that, titanium has an inhibitory effect on hydraulic conductivity of roots. Jaberzadeh et al. (2013) showed that, foliar application of low concentrations of nTiO2on wheat seedling increased the plant biomass and seed yields under water-deficit stress. These studies supported several earlier studies showing that the response to a nano-material is significantly dependent 444 Acta agriculturae Slovenica, 109 - 2, september 2017 The effects of silicon and titanium on safflower (Carthamus tinctorius L.) growth under moisture deficit condition on environmental conditions and plant species (Choi et al., 2003; Zheng et al., 2005; Choi et al., 2010; Mastronardi et al., 2015). Although comparative studies have been carried out on the effects of nano-particles, most studies have been done on cultured samples underartificial conditions, with their applicability to plant responses under natural water-deficit condition remained unclear. The objectives of the present research were: (1) to evaluate foliar application of nTiO2 and nano-silicon dioxide (nSiO2) on agronomic traits of safflower in a semi-arid, high-elevation Mediterranean area, (2) to assesschanges in fatty acids under water-deficit stress, and (3) to identify optimum nano-material and its concentration for safflower. 2 MATERIAL AND METHODS Field experiments were conducted during 2015/16 growing season at the Maragheh Agricultural Research Institute located in a semi-arid area in northwestern Iran. Geographically, the study area was located at 46°16'Eand 37°23'N (altitude = 1485 m from mean sea level). Based on Koppen's classification, this area has a semi-arid and cold temperate climate with annual precipitation of 375 mm, consisting of 73 % rain and 27 % snow. Total rainfall during the growing season was measured at 97.8 mm. The area is located within Sahand Mountain highs in northwestern Iran, providing itwith very cold winters with minimum air temperatures falling below -15 °C and more than 100 days with freezing temperatures. Average maximum and minimum temperatures during the growing season were 21°C and 8°C, respectively.Local soil was clay loam in typeand low in organic carbon (0.43 %) with a pH value of 6.85 and total nitrogen and CaCO3 contents of 0.17 % and 19 %, respectively. Electrical conductivity (EC) and iron, manganese, copper, zinc, and potassium contents of the soil were measured at 0.84 ds m-1, 1.62 ppm, 6.37 ppm, 0.49 ppm, 0.73 ppm, and 627 ppm, respectively. The field was left as fallow for a year before the cultivation. The previous cultivated crop in experimental site was bread wheat. The experimental field was ploughed once in early fall and harrowed twice to bring the soil to fine tilth one week before planting. The recommended dose of fertilizer (100 kg of N and 70 kg of P2O5 per hectare) was applied in the form of urea and triple superphosphate at the time of seed bed preparation. The experimental design was factorial on the basis of randomized complete block in three replicates. The experiments were arranged as split-plot, based on the randomized complete block design with three replications. Two irrigation treatments, namelywell irrigated (W: irrigation after 60 mm evaporation from class A pan), and water-deficit conditions (S: irrigation after 110 mm evaporation from class A pan) were assigned to the main plots and suspensions of nanoparticlesat different concentrations were allocated to sub plots. Under water-deficit condition,irrigation gravimetric water content (0g) of soil was measured before the irrigation, indicating16 % to 27 % water content at a depth of 30 cm.The treatments were subjected to foliar application at 5 levels including control (spray of distilled water), nSiO2(at 10 and 20 mM) and nTiO2 (at 25 and 50 mM). Nanoparticles were purchased from the Pishgaman Nano Co., Iran. According to the manufacturer, particle sizes of the purchased SiO2 ranged within 20-100 nm. Synthesized nanoparticles were characterized morphologically by transmission electron microscopy (Figure 1). According to the results, specific surface area of the nano-sized particles was 180-600 m2 g-1at 99.7 % purity. Figure 1: Transmission electron microscopy (TEM) micrograph of synthesized nano-silicon dioxide (a) nano-titanium dioxide (b) Acta agriculturae Slovenica, 109 - 2, september 2017 445 Mohsen JANMOHAMMADI et al. A productive and thorn-less variant of safflower, locally referred to as 'Golestan', was used in the present study. Before the start of the experiment seed were propagated in isolated fields under full irrigated condition, according to Sabaghnia et al. (2015), in northwest of Iran. Seeds were treated with a mixture of carboxin (5,6-dihydro-2-methyl-1,4-oxathiin-3 -carboxamide) and actellic (a.i. pirimiphos methyl) at (2:1) to minimize the probability of seed- and soil-borne diseases. The seedswere sown at 30-cm row spacing in 2.5 x 6 m plots (15 m2) using an experimental seed drill, on March 28th. All plots were irrigated twice after sowing and subsequent irrigations were applied according to the treatments (W and S) by drip irrigation system. During the irrigations, the plots were irrigated to up to 70 % of field capacity. All necessary cultural practices and plant protection measures were taken uniformly for all plots during the entire period of experimentation. Weeds were controlled by hand weeding in spring. Nanoparticlesuspensions were applied using an atomizer sprayer. Foliar spray treatments were initiated 40 days after planting and repeated once each 10 days until grain filling stage. Relative water contents were evaluated at the beginning of capitulum formation stage (BBCH scale: 50). Five fresh leaves of the same size and age were collected from five plants from each treatment, and then weighted (FM). Leaf segments were kept immersed in distilled water for 24 h at room temperature in the dark. The turgid mass (TM) of the leaves was measured before having the leaves oven-dried at 80 °C for 72 h until constant mass and then reweighted (DM). The fresh mass, turgidity, and dry weights of the leaf segments were used to determine hydration and relative water content according to Sangakkara et al. (1996).Accordingly, hydration was determined as H (%) = 100 - 100 (Dm / Fm) and relative water content (RWC) was determined as RWC (%) = [(FM- DM) / (Tm- Dm)] x 100. Leaf area was estimated at the end of flowering (BBCH scale: 69). Non-contact surface temperature of the leaves was measured at early flowering stage (BBCH scale: 61) using an IR thermometer device (Testo830-T2). Measurements were done on 10 plants per plot. Chlorophyll index was measured on 10 fully expanded leafs of a plant at each plot using a portable chlorophyll meter (SPAD) at capitulum and fruit development stage (BBCH scale: 71). The ground cover was evaluated in terms of the amount of dead plant material covering the soil surface. Average canopy spread was measured as the average horizontal width of the plant canopy, taken from right to left as one moves around the plant. Ground cover and canopy spread were measured during the flowering stage (BBCH = 65; 50 % of florets open in flowers on the main shoot). For eliminating the border effects, lateral rows at both ends of each plot were excluded from the measurements. At maturity stage, the plants were cut at ground level from two middle rows and then oven-dried at 80 C until a constant mass was reached. Seeds were separated from straw by crushing. The seed and straw (stem plus leaves) were weighted by a balance and yields were determined per unit area for different treatments. Total biomass was also calculated by summing upsafflower seed and straw. Evaluated agronomic traits were number of branches per plant, stem diameter (mm), capitulum diameter (mm), number of capitula per plant, number of achenes per capitulum, achene mass per capitulum, and thousand-achene mass. Harvest index was calculated as the ratio of achene yield to aboveground dry matter at maturity. Protein and oil contents (%) of the seeds were measured using a near-infrared seed analyzer (Zeltex). The oil contents of the samples were determined according to Darinkaboud and Gharibi (2016) using soxhlet extraction technique. For this purpose, the seeds were ground in a mill to a particle size of 0.5 mm. Then 500 mg of the seed meal was transferred into a weighed cellulose extraction thimble which was then sealed with cotton wool. The samples were dried in the thimbles (at 60 °C for 15 h) before reweighting the thimbles. The oil was extracted with petroleum ether in a 500-ml soxhlet instrument at 70 °C for 10 h. Oil contents of the samples were determined after drying and weighing the extracted samples with the thimbles. Contents of palmiticacid, arachidic acid and myristic acid were evaluated by gas chromatography according to Rudolphi et al. (2012). For this purpose, 200 mg of the seed meal was mixed with 0.5 ml of sodium-methylate. The mixes were then incubated twice in a water bath at 20 °C for 10 min and mixed in between the incubation courses. After adding 300 ^l of NaHSO4 (5 %) and 300 ^l of iso-octane to the mix, the sample was centrifuged at 2,000 U/min for 10 min. Subsequently, 200 ^l of the upper liquid phase was analyzed by gas chromatographically (Chrompack CP 9001, equipped with a flame ionizing detector).The data was subjected to analysis of variance (ANOVA) using MSTATC statistical package. Differences were compared by Least Significant Difference Test (LSD) at alpha 0.05. 446 Acta agriculturae Slovenica, 109 - 2, september 2017 The effects of silicon and titanium on safflower (Carthamus tinctorius L.) growth under moisture deficit condition 3 RESULTS Results of ANOVA showed that, most of the morphophysiological traits were significantly affected by moisture regimes and foliar treatments (Table 1). The main effects of moisture regime and foliar treatment as well as their interaction effects (moisture regime x foliar treatment) were found to be statistically significant (p< 0.01) for the ground cover percentages. Water-deficit conditions reduced this trait considerably. Assessments of ground cover between different combined treatments revealed that, the highest values were recorded for the plants grown under well irrigated condition and those subjected to foliar application with 25 mM Ti, 10 mM Si and 50 mM Ti, respectively (Figure 2). A parallel trend was also observed for canopy width, wherethe plant treated with the nano-particles at low concentration showed the widest canopy. The smallest canopy width was, however, recorded for the plants treated with distilled water (control) and 20 mM Si under water-deficit stress. Figure 2: The effect of foliar application of nano-particles on ground cover percentage of safflower plants under different moisture condition. Ti: nano titanium dioxide, Si: nano silicon dioxide, W: well irrigated, S: water deficit condition. The numbers beside the nano-particles refers to the concentration of nanoparticles suspension. Number of zero refers to control conditions (spraying of distilled water). Vertical bars in each column are standard error. Between the columns with different names there are statistically significant differences. Likewise, assessment of fresh leaf mass showed that this trait has been significantly affected by both moisture regimes and foliar treatments (Table 1). Water-deficit stress reduced the fresh leaf mass by 33 % in comparison with that under well irrigated condition. A comparison on average fresh leaf mass between the foliar treatments revealed that, the highest mass were those of the plants treated with nano-particle suspensions at low concentration (10 mM Si and 25 mM Ti). Water-deficit stress was seen to significantly increase the canopy temperature (by up to 2 °C), while the plants treated with titanium nano-particles had lower canopy temperatures (Table 1). Acta agriculturae Slovenica, 109 - 2, september 2017 447 Mohsen JANMOHAMMADI et al. Table 1: Effect of different moisture deficit stress and foliar application of nano-practices on morpho-physiological traits of safflower (Carthamus tinctorius L.) plants Treatment GC CW FWL CT PH RWC CHL LA DCE Well irrigated 81.46a 34.61a 14.56a 26.72b 58.96a 75.00a 49.11a 666.30a 72.13a moisture deficit 67.93b 24.10b 10.88b 28.71a 52.42b 64.13b 49.53a 465.78b 64.06b control 67.00c 23.38c 8.97d 28.01a 49.10b 65.33c 42.75b 464.91c 63.33c Nano-TiO2 25 mM 80.00a 34.51a 13.85ab 27.31ab 60.29a 72.00a 53.65a 557.82b 70.83ab Nano-TiO2 50 mM 75.50ab 27.73b 11.37b 26.97bc 54.35ab 70.50ab 48.14ab 537.09bc 68.00ab Nano-SiO2 10 mM 81.33a 34.67a 16.40a 27.55ab 60.06a 73.33a 52.56a 674.93a 72.16a Nano-SiO2 20 mM 69.66bc 26.47bc 13.02bc 28.75a 54.66ab 66.66bc 49.81ab 573.10b 66.16bc Level of significance S ** ** ** ** ** ** NS ** ** N ** ** ** * * ** * ** * SxN ** ** NS NS NS * NS * NS GC: ground cover percentage, CW: canopy width (cm), FWL: leaves fresh mass (g), CT: canopy temperature (°C), PH: plant height (cm), RWC: relative water content (%), CHL: chlorophyll content (SPAD unit), LA: leaf area (cm2), DCE: number of day from sowing to capitulum emergence. Different letters within columns indicate statistically significant differences at the 5 % level of significance. S: moisture stress, N: nano-particles, SxN: the interaction effect of nano-particles and stress. NS = Not significant, * = Significant at 5 % level of probability, ** = Significant at 1 % level of probability. Investigation of plant height indicated that, the water-deficit regime reduced this trait by 12 % compared with that of well irrigated plants. On the other hand, most of the plants treated with the nano-particles were longer than control plants (treated with distilled water). Investigation of relative water content (RWC) showed that, application of nTiO2 and nSiO2 at low concentration could improve leaf water status under both moisture regimes. However, the positive impact of 10 mM Si was more prominent than those of other treatments considered. Also, application of 20 mM nSiO2 under water-deficit regimes failed to rectify leaf water statue significantly (Figure 3). Evaluation of chlorophyll content (SPAD unit) revealed that, the application of nano-particles affected this trait (p< 0.05) significantly. Foliar spray of 25 mM Ti, 50 mM Ti, 10 mM Si and 20 mM Si could increase the chlorophyll content by 25 %, 12 %, 23 % and 16 %, respectively, in comparison with control plants (Table 1). Leaf area was seen to be significantly affected by both factors (p< 0.01). The largest leaf area was recorded for the plant grown under well irrigated condition with foliar application of 10 mM Si, while the lowest value was recorded for the plants grown under water-deficit condition without nano-particles application or those treated with 25 mM Ti. T T W-0 W Ti25 W Tiso W-Siio W-Si,„ S-0 S-Ti25 S-Tis0 S-Si10 S-Si2( Figure 3: Mean comparison of relative water content of safflower leaves under different foliar treatment with nano-particles and moisture regimes 448 Acta agriculturae Slovenica, 109 - 2, september 2017 The effects of silicon and titanium on safflower (Carthamus tinctorius L.) growth under moisture deficit condition Figure 4: The impact of foliar spray of different nano-particles on leaf area of safflower plants under different moisture regimes A glancing comparison between the two moisture regimes showed superior positive effects of n-TiO2 under well irrigated condition (Figure 4). Phenological development was also seen to be affected by moisture regimes, so that the plant grown under water-deficit condition reached capitulum emergence stage way earlier. On the other hand, foliar application of the nano-particles delayed the developmental stage considerably, as compared to control plants (Table 1). The effects of moisture regimes and foliar treatments on yield componentsare shown in Table 2. Investigation of mean capitulum diameter showed that, the water-deficit stress reduced this trait by 18 % in comparison with that under well irrigated condition. The largest capitulum was recorded for plants treated with 25 mM Ti and 10 mM Si, while the smallest one was of control plants and those treated with 20 mM Si. Number of the capitula per plant exhibited a significant decrease under water-deficit stress. Accordingly, this parameter responded to foliar spray positively, so that the application of Si and Ti suspensions at low concentration could increase this yield component slightly (p< 0.01). Table 2: Mean comparison of yield and yield components of Safflower (Carthamus tinctorius L.) as affected by irrigation levels and nano-particles Treatment MCD NCP TSW SNC SY BY HI PRO OIL Well irrigated 29.66a 7.46a 39.20a 29.53a 1268.53a 5011.00a 25.44b 13.90b 30.74a moisture deficit 25.26b 6.88b 32.33b 23.17b 958.17b 3521.66b 27.26a 19.29a 26.31b control 23.47c 6.13bc 34.05b 24.49c 1058.33c 4204.33ab 25.36b 18.82a 25.44c Nano-TiO2 25 mM 31.70a 8.00a 38.20a 28.99a 1156.66ab 4547.00a 25.72b 16.42b 32.32a Nano -T1O2 50 mM 25.98bc 6.80b 33.67b 25.76bc 1055.00c 4068.33a 26.39ab 14.38c 30.07a Nano -S1O2 10 mM 30.38ab 8.13a 38.04a 27.25ab 1224.06a 4289.33ab 28.66a 17.54a 26.81c Nano -S1O2 20 mM 25.78bc 6.82b 34.85b 25.26bc 1071.33c 4225.66ab 25.62b 15.82bc 28.01b Level of significance S * * ** ** ** ** * ** ** N * * ** * ** ** * ** * SxN NS NS * NS NS ** NS NS * MCD: mean capitulum diameter (mm), NCP: number of capitula per plant, TSW: thousand seeds mass (g), SNC: number of seed per capitulum, SY: seed yield (kg ha-1), BY: biological yield (kg ha-1), HI: harvest index (%), PRO: seed protein content (%), OIL: seed oil content (%; Soxhlet) .Different letters within columns indicate statistically significant differences at the 5 % level of significance. S: moisture stress, N: nano-particles, SxN: the interaction effect of nano-particles and stress. NS = Not significant, * = Significant at 5 % level of probability, ** = Significant at 1 % level of probability. Evaluation of thousand-seed mass revealed that moisture regime-foliar treatmentinteraction effect was statistically significant at 95 % confidence level. Significantly lower seed mass (by 21 %) were observed under water-deficit stress conditions. The heaviest seeds were those of the plants grown under well irrigated Acta agriculturae Slovenica, 109 - 2, september 2017 449 Mohsen JANMOHAMMADI et al. condition and treated with Si and Ti suspensions at low for the plants grown under water-deficit stress and concentration, while the smallest seeds were recorded sprayed with 50 mM Ti and 20 mM Si (Figure 5). LSD- 3.25 a W C W-Tii5 W-Ti;o W-Si10 \V-Si20 S-C S Ti 25 S-TI50 S-Siio S-Si20 Figure 5: Mean comparison of thousand seed mass in safflower plants under different foliar treatment with nano-particles and moisture regimes As one of the most important yield indicators, number of seeds per capitulum decreased noticeably under water shortage (by 27%). Furthermore, foliar treatment affected the number of seeds per capitulum at 95% confidence level (p< 0.05), so that the largest number of seeds was recorded for the plants treated with 25 mM Ti suspension. Results showed that, water-deficit stress could reduce the seed yield by 32 %, as compared to that under well irrigated condition. Also, seed yield responded to foliar treatments significantly, so that the application of 10 mM Si increased the seed yield by 15 % over the control plants. All by all, the highest seed yield was obtained by applyingSi and Ti suspensions at low concentration. Furthermore, the results showed that, water scarcity tends to reduce the biological yield considerably (by 42 %). A comparison on average values for combined treatments showed that, the highest biological yield was related to the plant grown under well irrigated condition with foliar application of 25 mM Ti, while the lowest yield was recorded for the plant grown under water-deficit condition with foliar application of 50 mM Ti (Figure 6). Figure 6: The impact of foliar treatment with different nano-particles on biological yield of safflower plants under different moisture regimes 450 Acta agriculturae Slovenica, 109 - 2, september 2017 The effects of silicon and titanium on safflower (Carthamus tinctorius L.) growth under moisture deficit condition Interestingly, water-deficit stress increased the harvest index by 7 % in comparison to that under well irrigated condition. Also foliar application of 10 mM Si increased the harvest index by 13 % over the control plants. The first component in principal component analysis (PCA) clearly separated moisture regimes, with the second component revealingthe statistical distinction of foliar application of 10 mM Si from other spray treatments under well irrigated condition (Figure 7). Similarly, PCA showed that, although there is no significant difference between foliar treatments under water-deficit stress, application of nano-particles at low concentration (10 mM Si and 25 mM Ti) tends to outperform other treatments. Evaluation of quantitative traits also revealed that, even though most of these parameters were affected by both factors, the responseswere somewhat different for quantitative traits. Water scarcity resulted in a considerable increase in seed protein percentage (Table 2). On the other hand, evaluation of the effect of foliar treatments on seed protein content reveled that, all treatments, except for 10 mM Si, reduced this parameter significantly. S-Si10 • • S-Si20 • • S-Ti„ W-Si,„ S-T'so • • • WTi25 s-c W-SiM • W-Ti50 # W-C l- & 0 E o -l- -2 0.0 First Component Figure 7: Principal component analysis (PCA) of combined treatments (moisture regimes and nano-particles foliar application) in safflower plants. Ti: nano titanium dioxide, Si: nano silicon dioxide, W: well irrigated, S: water deficit condition. The numbers beside the nano-particles refers to the concentration of nano-particles suspension. Number of zero refers to control conditions (spraying of distilled water). However, water-deficit stress reduced the seed oil content most intensively (Table 2). Although foliar spray improved the oil contentin general terms, the greatest increase was seen to be related to the application of Ti. Moreover, fatty acid profiles of safflower seed oils revealed that these component were significantly affected by moisture regimes and foliar treatments (Figure 8). Noticeably lower palmitic acid content was seen in water shortage. On the other hand, foliar application of nTiO2, regardless of the concentration, under well irrigated condition resulted in the highest level of palmitic acid. Adverse effects of water shortage on arachidic acid and myristic acid contents were more prominent than that on palmitic acid content (Figure 8). However, the response of arachidic acid to foliar treatments was largely similar to that of palmitic acid, where the highest fat acid content was obtained by foliar application of 50 mM Ti. All by all, the effect of foliar treatment under well irrigated condition was more distinguished on arachidic acid and palmitic acid contents, rather than meristic acid content. Acta agriculturae Slovenica, 109 - 2, september 2017 291 Mohsen JANMOHAMMADI et al. Figure 8: A stacked bar chart which display the change of fatty acids between the combined treatments. Ti: nano titanium dioxide, Si: nano silicon dioxide, W: well irrigated, S: water deficit condition. The numbers beside the nano-particles refers to the concentration of nano-particles suspension. Number of zero refers to control conditions (spraying of distilled water) 4 DISCUSSION It was found that water-deficit stress in the semi-arid region reduced plant growth and seed yield considerably. On the other hand, foliar application of TiO2 and SiO2nano-particles could significantly affect the evaluated traits. Although the effects of nanoparticles were more obvious under well irrigated condition, low concentrations of SiO2nano-particles could significantly improve some of crucial agronomic traits such as ground cover percentage. As a very essential attribute of spring crop in a semi-arid region, fast canopy closure may affect the economic yield extensively. In Mediterranean semi-arid areas, precipitation regime is anticipated to be dominated by low, erratic and unpredictable rainfalls. The moisture supplied to the soil from rain is mostly lost by evaporation. Fast canopy closure and a high percentage of ground cover under the Mediterranean conditions may provide numerous benefits. Extended canopy can improve the capturing and use of solar radiation for photo-assimilate synthesis throughout rainy spring months (Soleimanzadeh & Gooshchi, 2012). Also, quick ground covering can reduce the moisture loss through evaporation and increase competitive power of plant against weeds. This also accords with our earlier observations which showed that, canopy width and ground cover percentage are significantly affected by foliar application of SiO2nano-particles (Janmohammadi et al., 2016b). On the contrary, if the nanoparticles show a good translocation through the phloem, the application should be done via foliar spraying. In addition, the nano-materials moving along the phloem are likely to be accumulated in the plant organs which may act as sink, such as seeds. The beneficial effects of Si have been thought to be due to the precipitation of amorphous silica in plants, which acts as a protectant. As a physico-mechanical barrier, nano-silicon can protect plants against biotic and abiotic stresses (Ma, 2004). Nano-silicon can boost plant's defense mechanismsincluding the accumulation of lignin, phenolic compounds, and phytoalexins (Ma & Yamaji, 2006). Nano-silicon can also deposit on the walls of epidermis and vascular tissues of the stem and leaf surface in most plants; it further controls physiological properties of plants. Also during the pathogenic attack, Si can induce rapid and extensive defense mechanisms (Fauteux et al., 2005). From a physiological standing point, silicon is able to increase the plasma membrane integrity by providing more stable lipids involved in cell membrane (Sahebi et al., 2015). Previous studies have suggested that, application of nano-silicon may alleviate the adverse effects of environmental stress on plants, increasingtheir water-use efficiency and photosynthesis rate (Ma, 2004). It has been suggested that, exogenous silicon may improve plant growth by enhancing antioxidant defense (Karimi and Mohsenzadeh, 2016). Nano-Si can also reduce grazing-resulted damages, such as that by insects, through changes in the tissue level to reduce palatability. Compared to control, treated plants with Si maintained higher stomatal conductance, relative water content, and higher water potential. Also, their leaves 452 Acta agriculturae Slovenica, 109 - 2, september 2017 The effects of silicon and titanium on safflower (Carthamus tinctorius L.) growth under moisture deficit condition were larger and thicker, thereby limiting the loss of water through transpiration (Hattori et al., 2005) and reducing water consumption (Eneji et al., 2008). Si further affects the root structure and improves root resistance in dry soils (Hattori et al., 2005). It has been observed that, Si increased antioxidant defense capabilities and therefore maintained physiological processes such as photosynthesis (Pei et al., 2010). The results demonstrated that, the low concentration of nano-particles could simultaneously increase the leaf area, chlorophyll concentration, and seed yield components. Within a plant, the source is the photosynthesizing tissue or the organ exportingcarbon skeletons, the sink is the one requiring carbon feed, the sink strength is the ability of a tissue or an organ to mobilize photo-assimilates, the sink size is the capacity of a tissue or organ to import and store further compounds from the source(s), and the sink activity is measured by the rate of respiration. Our findings revealed that, both of the moisture regimes and foliar treatments affected the source-sink relation considerably. Water scarcity was seen to reduce source and sink sizes significantly, while increasing the sink strength. Water stress increased the seed protein content noticeably. Seed protein was resulted from protein degradation in source tissue and amino acid remobilization for the seed. Available soil, water, temperature, nutrients, light, and CO2 are indubitably important drivers of plant growth (Pessarakli, 2014). The former three (water, temperature, and nutrients) are, however, fundamentally different from the latter two (light and CO2) because they can affect both sink and source activities, while light and CO2 can only affect the source activity (Fatichi et al., 2014). It seems that, foliar application of nano-particles increases the plant growth and seed yield through improving the function of photosynthetic apparatus (source activities) and photo-assimilate translocation.These results are consistent with those of other studies and suggest thatthe application of Si may represent an approach to improve the growth of this crop and increase its production in arid or semi-arid areas where water is at a premium;this technique, however, would not fully substitute for an adequate water supply (Kaya et al., 2006). However, in some of the evaluated traits, especially qualitative aspects and canopy temperature, the application of nTiO2ended up with better results. The main reason for the improvements of these traits could have been the photo-sterilization and photo-generation of "active oxygen (e.g. superoxide) and hydroxide anions" by n-TiO2 that could increase the plant stress resistance and promote efficiency of stomatal conductivity (Zheng et al., 2005). nTiO2 increases plant growth by enhancing nitrogen metabolism (Yang et al., 2006) which improves the absorption of nitrate in spinach, and also by accelerating the conversion of inorganic nitrogen into organic nitrogen which increases the fresh and dry masses. Morteza et al. (2013) suggested that foliar utilization of nTiO2 can improve plant growth and grain yield by improving the biosynthesis of pigments and conversionof light energy to chemical energy, thereby increasing photosynthetic efficiency. Effects of nTiO2 on the content of light harvesting complex II (LHC II) on thylakoid membranes of spinach increases LHC II content (Hong et al., 2005; Lei et al., 2007). These promote energy transfer and oxygen evolution in photosystem II (PS II) of spinach (Lei et al., 2007). It was found that, nano-anatase TiO2 promotes antioxidant stress by decreasing the accumulation of superoxide radicals, hydrogen peroxide, and malonyl dialdehyde content while enhancing the activities of superoxide dismutase, catalase (ascorbate peroxidase), and guaiacol oxidase, thereby increasing the evolution oxygen rate in spinach chloroplasts under UV-B radiation (Lei et al., 2008).. Khot et al. (2012) concluded that, the nano size of TiO2 might have increased the absorption of inorganic nutrients, accelerated the breakdown of organic substances, and also caused quenching of oxygen free radicals formed during the photosynthetic process, hence increasing the photosynthetic rate. This finding supports our previous research which showed that foliar application of nTiO2 on barley plants in a semi-arid region positively affected growth rate and seed yield (Janmohammadi et al., 2016a). It seems that, nTiO2 plays a significant role in activatingdefense mechanism and modulating biosynthesis of phytohormones such as cytokinins and gibberellin (Mandeh et al., 2012). However, the accumulation of the nano-particles in plant tissue can be very important point from food safety perspective, therefore; their measurement in future experiments can be very valuable and provide precious information. 5 CONCLUSION Our examination of the effects of SiO2 andTiO2 nano-particles on safflower found that these nano-particles had differing effects on plant growth and quantitative and qualitative aspects of seed yield, and that the concentration of the nano-particles played largely contributed to these differences. Results revealed that, water-deficit stress drastically reduced the seed yield and seed oil content, while increasing the seed protein percentage. For almost all evaluated traits, the best performance was recorded under well irrigated Acta agriculturae Slovenica, 109 - 2, september 2017 293 Mohsen JANMOHAMMADI et al. condition with spraying low concentrations of nano-particles. Under water-deficit stress, however, the differences between foliar treatments were not significant.Our study suggests that, foliar application of SiO2 suspension at low concentration can be a suitable agronomic monument for improving safflower performance. 6 REFERENCES Asadzade, N., Moosavi, S. G., Seghatoleslami, M. J. (2015). Effect of low irrigation and Zn and SiO2 nano-fertilizers and conventional fertilizers on morphophysiological traits and seed yield of sunflower. Biological Forum, 7(1), 357-364. Asli, S., Neumann, P.M. (2009). Colloidal suspensions of clay or titanium dioxide nanoparticles can inhibit leaf growth and transpiration via physical effects on root water transport. Plant, Cell & Environment, 32, 577-584. doi:10.1111/j.1365- 3040.2009.01952.x Darinkaboud, B. A., GharibiAsl, S. (2016). The oil and protein content of Isfahahn's safflower seed in different periods of irrigation, levels of humic acid and superabsorbent. International Journal of Life Sciences and Pharma Research, Special Issue, 5663. Eneji, A. E., Inanaga, S., Muranaka, S., Li, J., Hattori, T., An, P., Tsuji, W. (2008). Growth and nutrient use in four grasses under drought stress as mediated by silicon fertilisers. Journal of Plant Nutrition, 31, 355-365. doi:10.1080/01904160801894913 Fatichi, S., Leuzinger, S., Körner, C. (2014). Moving beyond photosynthesis: from carbon source to sink-driven vegetation modeling. New Phytologist, 201(4), 1086-1095. doi:10.1111/nph.12614 Fauteux, F., Remus-Borel, W., Menzies, J. G., Bélanger, R. R. (2005). Silicon and plant disease resistance against pathogenic fungi. FEMS Microbiology Letter, 249, 1-6. doi:10.1016/j.femsle.2005.06.034 Frazier, T. P., Burklew, C. E., Zhang, B. (2013). Titanium dioxide nanoparticles affect the growth and microRNA expression of tobacco (Nicotiana tabacum). Functional & Integrative Genomics, Available online, doi:10.1007/s10142-013-0341-4 Haghighati-Malek, A., Ferri, F. (2014). Effects of nitrogen and phosphorus fertilizers on safflower yield in dry lands condition. International Journal of Research in Agricultural Sciences, 1, 2348-3997. Hattori, T., Inanaga, H., Araki, H., An, P., Morita, S., Luxova, M., Lux A. (2005). Application of silicon enhanced drought tolerance in Sorghum bicolor. Physiologia Plantarum, 123, 459-466. doi:10.1111/j.1399-3054.2005.00481.x Hong, F., Zhou, J., Liu, C., Yang, F., Wu, C., Zheng, L., Yang, P. (2005). Effect of nano-TiO2 on photochemical reaction of chloroplasts of spinach. Biological Trace Element Research, 105 (1-3), 269279. doi:10.1385/BTER:105:1-3:269 Hussain, M. I., Lyra, D. A., Farooq, M., Nikoloudakis, N., Khalid, N. (2016). Salt and drought stresses in safflower: a review. Agronomy for Sustainable Development, 36 (1), 4-13. doi:10.1007/s13593-015-0344-8 Janmohammadi, M., Amanzadeh, T., Sabaghnia, N., Dashti, S. (2016a). Impact of foliar application of nano micronutrient fertilizers and titanium dioxide nanoparticles on the growth and yield components of barley under supplemental irrigation. Acta Agriculturae Slovenica, 107(2), 265-276. doi: 10.14720/aas.2016.107.2.23 Janmohammadi, M., Amanzadeh, T., Sabaghnia, N., Ion, V. (2016b). Effect of nano-silicon foliar application on safflower growth under organic and inorganic fertilizer regimes. Botanica Lithuanica, 22(1), 53-64. doi: doi:10.1515/botlit-2016-0005 Karimi, J., Mohsenzadeh, S. (2016). Effects of silicon oxide nanoparticles on growth and physiology of wheat seedlings. Russian Journal of Plant Physiology, 63(1), 119-123. doi:10.1134/S1021443716010106 Kaya, C., Tuna, L., Higgs, D. (2006). Effect of silicon on plant growth and mineral nutrition of maize grown under water-stress conditions. Journal of Plant Nutrition, 29(8), 1469-1480. doi:10.1080/01904160600837238 Khot, L. R., Sankaran, S., Maja, J. M., Ehsani, R., Schuster, E. W. (2012). Applications of nanomaterials in agricultural production and crop protection: a review. Crop Protection, 35, 64-70. doi:10.1016/j.cropro.2012.01.007 Lei, Z., Mingyu, S., Xiao, W., Chao, L., Chunxiang, Q., Liang, C., Fashui, H. (2007). Effects of nano-anatase on spectral characteristics and distribution of LHCII on the thylakoid membranes of spinach. Biological Trace Element Research, 120 (1-3), 273283. doi: 10.1007/s12011 -007-8025-3 Lei Z., Mingyu S., Xiao W., Chao L., Chunxiang Q., Liang C., Hao H, Xiaoqing L, Fashui, H. 2008. 454 Acta agriculturae Slovenica, 109 - 2, september 2017 The effects of silicon and titanium on safflower (Carthamus tinctorius L.) growth under moisture deficit condition Antioxidant stress is promoted by nano-anatase in spinach chloroplasts under UV-B radiation. Biological Trace Element Research, 121(1), 69-79. doi:10.1007/s12011-007-8028-0 Liu, R., Lal, R. (2015). Potentials of engineered nanoparticles as fertilizers for increasing agronomic productions. Science of the Total Environment, 514, 131-139. doi:10.1016/j.scitotenv.2015.01.104 Ma, J. F., Miyake, Y., Takahashi, E. 2001. Silicon as a beneficial element for crop plants, in Silicon in Agriculture, (Eds.) New York, NY: Elsevier Science Publishing, 17-39. doi:10.1016/S0928-3420(01)80006-9 Ma, J. F., Yamaji, N. (2006). Silicon uptake and accumulation in higher plants. Trends in Plant Science, 11(8), 392-397. doi:10.1016/j.tplants.2006.06.007 Ma, J.F. (2004). Role of silicon in enhancing the resistance of plants to biotic and abiotic stresses. Soil Science and Plant Nutrition, 50, 11-18. doi: 10.1080/00380768.2004. Mandeh, M., Omidi, M., Rahaie, M. (2012). In vitro influences of TiO2 nanoparticles on barley (Hordeum vulgareL.) tissue culture. Biological trace element research, 150(1-3), 376-380. doi:10.1007/s12011-012-9480-z Mastronardi, E., Tsae, P., Zhang, X., Monreal, C., DeRosa, M. C. (2015). Strategic role of nanotechnology in fertilizers: potential and limitations. In Nanotechnologies in Food and Agriculture (pp. 25-67). Springer International Publishing. Switzerland, Cham. doi:10.1007/978-3-319-14024-7_2 Morteza, E., Moaveni, P., Farahani, H. A., Kiyani, M. (2013). Study of photosynthetic pigments changes of maize (Zea mays L.) under nano Tio2 spraying at various growth stages. SpringerPlus, 2(1), 1-5. doi.10.1186/2193-1801-2-247 Murungweni, C., Wijk, M. T., Smaling, E. M. A., Giller, K. E. (2016). Climate-smart crop production in semi-arid areas through increased knowledge of varieties, environment and management factors. Nutrient Cycling in Agroecosystems, 105(3), 183197. doi: 10.1007/s10705-015-9695-4 Pei, Z.F., Ming, D. F., Liu, D., Wan, G. L., Geng, X. X., Gong H. J., Zhou, W. J. (2010). Silicon improves the tolerance of water-deficit stress induced by polyethylene glycol in wheat (Triticum aestivum L.) seedlings. Journal of Plant Growth Regulation, 29, 106-115. doi: 10.1007/s00344-009-9120-9 Pessarakli, M. (2014). Handbook of plant and crop physiology. CRC Press. United State, Florida. Rudolphi, S., Becker, H. C., Schierholt, A., von Witzke-Ehbrecht, S. (2012). Improved estimation of oil, linoleic and oleic acid and seed hull fractions in safflower by NIRS. Journal of the American Oil Chemists' Society, 89(3), 363-369. doi:10.1007/s11746-011-1920-y Sahebi, M., Hanafi, M. M., Siti Nor Akmar, A., Rafii, M. Y., Azizi, P., Tengoua, F., Mayzaitul Azwa, F., Shabanimofrad, M. (2015). Importance of silicon and mechanisms of bio-silica formation in plants. BioMed research international, 1-16. doi:10.1155/2015/396010 Sabaghnia, N., Ahadnezhad, A., Janmohammdi, M. (2015). Genetic variation in garden cress (Lepidium sativum L.) germplasm as assessed by some morphological traits. Genetic Resources and Crop Evolution, 5(62): 733-745. doi:10.1007/s10722-014-0192-4 Sangakkara, H. R., Hartwig, U. A., Nosberger, J. (1996). Response of root branching and shoot water potential of Phaeseolus valgaris L. to soil moisture and fertilizer potassium. Journal of Agronomy and Crop Science, 177, 165-173. doi:10.1111/j.1439-037X. 1996.tb00234.x Shahrokhnia, M. H., Sepaskhah, A. R. (2017). Physiologic and agronomic traits in safflower under various irrigation strategies, planting methods and nitrogen fertilization. Industrial Crops and Products, 95, 126-139. doi:10.1016/j.indcrop.2016.10.021 Shi, Y., Zhang, Y., Han, W., Feng, R., Hu, Y., Guo, J., Gong, H. (2016). Silicon Enhances Water Stress Tolerance by Improving Root Hydraulic Conductance in Solanum lycopersicum L. Frontiers in plant science, 7. doi:10.3389/fpls.2016.00196 UN (United Nations Department of Economic and Social Affairs, Population Division), (2013). World Population Prospects: the 2012 Revision. Yang, F., Hong, F., You, W., Liu C., Gao, F., Wu, C., Yang, P. (2006). Influence of nano-anatase TiO2 on the nitrogen metabolism of growing spinach. Biological Trace Element Research, 110 (2), 179190. doi: 10.1385/BTER:110:2:179 Zheng, L., Hong, F., Lu, S., Liu, C. (2005). Effect of nano-TiO2 on strength of naturally aged seeds and growth of spinach. Biological Trace Element Research. 104, 83e91. doi:10.1385/BTER:104:1:083 Acta agriculturae Slovenica, 109 - 2, september 2017 295 doi: 1G. 1472G/aas.2G17.1G9.2.28 Original research article I izvirni znanstveni članek First report of nematodes Parasitylenchus bifurcatus Poinar & Steenberg, 2012 parasitizing multicolored Asian lady beetle Harmonia axyridis (Pallas, 1773) in Slovenia Barbara GERIČ STARE1*, Saša ŠIRCA1, Gregor UREK1 Received June 15, 2017; accepted September 11, 2017. Delo je prispelo 15. junija 2017, sprejeto 11. septembra 2017. ABSTRACT In years 2015 and 2016 around 200 specimens of ladybird species Harmonia axyridis were collected in Slovenia. Ladybirds were dissected and checked under stereomicroscope for presence of nematodes. Nematodes were discovered in samples of ladybirds H. axyridis collected at Brdo pri Lukovici and Trboje. The incidence of parasitized ladybirds was around 30 %. Nematodes were analysed morphometrically and the characteristic features of Parasitylenchus bifurcatus nematodes were observed: straight stylet lacking basal thickenings, a bursa and a forked tail tip in the vermiform females and juvenile males. The identity was confirmed with 18S rDNA region sequence (acc. no. LT629306 and LT629307) which showed high similarity (>99.9 % nucleotide identity) to the P. bifurcatus sequences in the public domain. This is a first report of P. bifurcatus nematode species found parasitizing ladybird Harmonia axyridis in Slovenia. The species compromises fecundity of its host and has therefore a potential to be used as a biological control agent to control high abundance of invasive H. axyridis ladybirds. Key words: parasitic nematodes; ladybirds beetles; Parasitylenchus bifurcatus; Harmonia axyridis; harlequin ladybird; biological control IZVLEČEK PRVA NAJDBA OGORČIC PARASITYLENCHUS BIFURCATUS POINAR & STEENBERG, 2012, PARAZITOV PISANE POLONICE HARMONIA AXYRIDIS (PALLAS, 1773) V SLOVENIJI V letih 2015 in 2016 smo v Sloveniji nabrali okrog 200 osebkov pisane polonice Harmonia axyridis. Pikapolonice smo secirali in pod lupo preverili prisotnost parazitskih ogorčic. Ogorčice so bile prisotne v pikapolonicah nabranih na Brdu pri Lukovici in v Trbojah. Stopnja parazitiranih pikapolonic je bila okrog 30 %. Morfometrična analiza je razkrila karakteristične znake ogorčic vrste Parasitylenchus bifurcatus: ravno bodalo brez zadebelitev pri osnovi, burso in razcepljeno konico repa pri črvastih samicah in ličinkah. Identiteto najdenih ogorčic smo potrdili z določitvijo nukleotidnega zaporedja odseka 18S rDNA (št. v javni bazi: LT629306 in LT629307), ki kaže veliko podobnost z zaporedji vrste P. bifurcatus dostopnimi v javnih bazah (več kot 99,9 odstotna enakost). Gre za prvo najdbo vrste P. bifurcatus, parazita pisane polonice Harmonia axyridis, v Sloveniji. Ogorčice vrste P. bifurcatus zmanjšajo rodnost svojega gostitelja in so kot take potencialni biotični agens za nadzor invazivne vrste pikapolonice H. axyridis. Ključne besede: parazitske ogorčice; Parasitylenchus bifurcatus; pikapolonice; pisana polonica; Harmonia axyridis; biotično varstvo 1 INTRODUCTION The multicolored Asian lady beetle, Harmonia axyridis (Pallas, 1773) (Coleoptera: Coccinellidae) originating from Asia has been applied as a biocontrol agent to control aphids and other harmful insects in orchards, vineyards, greenhouses, crop fields, and gardens (Koch 2003, Poinar and Steenberg 2012). It has been introduced to North America several times in the beginning of 20th century, to Eastern Europe in 1960s and become commercially available as a biological control agent in 1980s in the Western Europe. While its' ravenous appetite resulted in efficient biological control of harmful organisms, no one predicted that 1 Ph.D, Agricultural Institute of Slovenia, Plant Protection Department, Hacquetova ulica 17, SI-1000 Ljubljana, Slovenia, corresponding author: barbara.geric@kis.si Acta agriculturae Slovenica, 109 - 2, september 2017 str. 349 - 356 Barbara GERIČ STARE, Saša ŠIRCA, Gregor UREK establishment of this introduced species in the natural environment will result in rapid spread and build-up of large populations endangering biodiversity of native insects as it feeds on numerous species of insects. It is also a fruit pest since it feeds and aggregates on apples, pears and grapes. It endangers wine production as wine produced from grape clusters containing adult beetles has an unpleasant flavour and odour. Further, it is a nuisance for humans since they try to overwinter in homes, release haemolymph from their legs when agitated (reflex bleed) leading to unpleasant odours and stains, and even occasionally bites humans and cause allergic reactions (Kovach, 2004). Because of the above mentioned reasons the European and Mediterranean Plant Protection Organization (EPPO) has removed Harmonia axyridis from the list of recommended biological control agents in 2009 (EPPO, 2016). Multicolored Asian lady beetle has rapidly spread even in the countries where it has not been purposely introduced (e.g. Austria, Denmark, Great Britain) (Brown et al., 2008). The first observation of H. axyridis in natural environment in Slovenia dates back to 2007 (Polak, 2013). There is no known evidence that H. axyridis was used for biological control purposes in Slovenia. Its spread in Slovenia may result from (illegally) imported specimens escaped from greenhouses or gardens or it has spread naturally from the neighbouring countries, possibly from Austria. Since then, multicolored Asian lady beetle has become widely spread in Slovenia (Laznik et al., 2012, Polak, 2013). The multicolored Asian lady beetle has a great reproductive and expansion potential (Polak, 2013). Furthermore, the successful invasion of this species can be attributed to extreme resistance of this species to diseases and parasites that attack other ladybirds. In Slovenia chemical control of multicolored Asian lady beetle is limited as there is no registered pesticide for controlling this species in agricultural production. However biocides can be used for the species control in household environments which are not intended for crop production. Therefore researchers have allocated their efforts in the search of natural enemies of multicolored Asian lady beetle that could reduce their numbers (Raak-van den Berg et al., 2014). Several natural enemies attacking adult multicolored Asian lady beetle have been reported, among them the nematode Parasitylenchus bifurcatus Poinar and Steenberg, 2012 (Tylenchida, Hexatylina: Iontonchioidea, Parasitylenchidae), entomopathogenic fungus Hesperomyces virescens Thaxt. (Ascomycota: Laboulbeniomycetes: Laboulbeniales), ectoparasitic mite Coccipolipus hippodamiae (McDaniel and Morrill, 1969) (Acarina: Podapolipidae), insect parasitoid Dinocampus coccinellae (Schrank, 1802) (Hymenoptera: Braconidae) and bacteria of the genus Spiroplasma (Majerus et al., 1999, Raak-van den Berg et al., 2014, Haelewaters et al., 2017). Adult multicolored Asian lady beetles have been found parasitized by nematodes in Denmark in 2009 and the parasitic nematode was subsequently described as a new nematode species Parasitylenchus bifurcatus (Harding et al., 2011, Poinar and Steenberg, 2012). Diagnostic morphological characters of Parasitylenchus bifurcatus are a straight stylet lacking basal thickenings, an excretory pore opening at the level of or somewhat posterior to the nerve ring and a gubernaculum, a narrow bursa in the males and a forked tail tip in the vermiform (infective) females and juvenile males. The characteristic cleft tail tip giving rise also to the name of this new species separates it clearly from a similar species Parasitylenchus coccinellinae Iperti and van Waerebeke. Molecular marker often used as a character for nematode identification, the sequence of nearly full length SSU rDNA is available in public domain (Raak-van den Berg et al., 2014). Different developmental stages of P. bifurcatus, first generation parasitic females, subsequent generation parasitic females, vermiform (infective) females, males and juvenile nematodes, occurred together in the body cavity of both female and male H. axyridis (Harding et al., 2011, Poinar and Steenberg, 2012, Raak-van den Berg et al., 2014). The prevalence of infected adult ladybirds collected in nature ranged from 2 - 47 % and increased up to 60 % when field-collected ladybirds were incubated in the laboratory for 30 days (Poinar and Steenberg, 2012, Haelewaters et al., 2017). These high rates of parasitism imply that P. bifurcatus is a potential biological control agent of H. axyridis as parasitism results in depletion of the fat body and partial or complete atrophy of the reproductive organs of the host. Further, P. bifurcatus parasitism has been shown as significant mortality factor of H. axyridis in the laboratory conditions (Poinar and Steenberg, 2012) and strong association between female failure to reproduce and infection with P. bifurcatus has been documented (Raak-van den Berg et al., 2014). Interestingly, P. bifurcatus parasitizes only adult host, while larvae and pupae are not included in its life cycle. With several subsequent generations of nematodes within the host, the numbers of nematodes can reach several hundred or even thousand juveniles. It is not documented how P. bifurcatus finds and enters uninfected adult beetles, but it is proposed that infective vermiform females with thick cuticle leave infected ladybirds while these are aggregated and enter other uninfected ones (Poinar and Steenberg, 2012). The origin of P. bifurcatus infection in H. axyridis is unknown. One possibility is that it could have arrived with H. axyridis from Asia or North America, while the 458 Acta agriculturae Slovenica, 109 - 2, september 2017 First report of nematodes Parasitylenchus bifurcatus ... Asian lady beetle Harmonia axyridis (Pallas, 1773) in Slovenia other possibility is that infection was acquired from endemic European ladybirds (Poinar and Steenberg, 2012, Raak-van den Berg et al., 2014). So far, P. bifurcatus infecting H. axyridis has been documented from Denmark (Poinar and Steenberg, 2012), the Netherlands (Raak-van den Berg et al., 2014), the Czech Republic and Poland (Haelewaters et al., 2017), Kentucky and West Virginia in USA (Tove Steenberg, personal communication, May 28, 2015). The objective of this study was to examine H. axyridis in Slovenia and to check whether it is parasitized by nematodes. 2 MATERIALS AND METHODS 2.1 Samples In years 2015 and 2016 around 200 samples of adult ladybird species Harmonia axyridis from six different locations in Slovenia were collected and identified using morphological keys and species descriptions (Reitter, 1911; Freude et al., 1967; Chapin and Brou 1991; Riedel and Bastian, 2005; Polak, 2013). Ladybirds were dissected one to four days after sampling and individual nematodes were collected under the binocular in 0.9 % NaCl solution. The nematodes for morphometrical analysis were heat killed (at 65 °C), fixed in 4 % formalin and transferred on slides for further examination and measurements. Nematodes for molecular analysis were placed into 1 ^l of dH20 in 1.5 ml tubes and stored at -20 oC until isolation of DNA. 2.2 Morphomerics Morphometric analysis encompassing measurements of common nematode body features was performed on 10 fixed nematode specimens of each nematode life stage using Nikon TiE microscope. 2.3 Molecular identification 2.3.1 DNA extraction DNA was extracted from twenty nematodes. The Promega Genomic DNA Wizard purification kit (Madison, WI, USA) was used after a slight modification of the manufacturer's instructions. Extracted DNA was diluted in 20 ^l of dH20. 2.3.2 PCR amplification For the amplification of 18S rDNA fragment, two set of primers, 1096F and 1912R, and 1813F and 2646R described by Holterman et al. (2006) were used. Both primers sets give overlapping sequences and together produce an 18 rDNA gene sequence of approximately 1.600 bp. PCR reactions contained 1 |l of isolated DNA, 1x GoTaq buffer (Promega), 1.5 mM MgCl2, 2.5 mM of each of the dNTPs, 1 |M of each of the primers, 1 U GoTaq Flexi DNA Polymerase (Promega) and distilled water up to 25 | l. The amplification was carried out in a thermal cycler Veriti (Applied Biosystems) using the amplification program as described by Holterman et al. (2006). Electrophoresis was performed on a 1 % TBE agarose gel to detect and inspect the amplified DNA product. 2.3.3 Cloning PCR products were cloned using a pGEM®-T Easy vector kit (Promega) and transformed into competent cells of E. coli JM109 (Promega) according to the manufacturer's instructions. White colonies were selected in the blue/white colour screening for further analysis. 2.3.4 Isolation of plasmid DNA and sequencing Selected clones were grown in 5 ml of LB medium with ampicillin (150 ^g/ml) incubated overnight at 37 °C on a rotation shaker at 300 rpm. Overnight cultures were centrifuged at 2.700 rcf for 10 min, and the pellet used for isolation of plasmid DNA by GeneJet Plasmid Miniprep (Thermo Scientific) according to the manufacturer's instructions. The isolated plasmids were sequenced by Macrogen Inc. (Korea) using universal primers SP6 and T7. 2.3.5 Sequence analysis DNA sequences were assembled using the computer software BioEdit v. 7.0.5.2 (Hall 1999). NCBI blastn suite was used to determine sequence similarity to other sequences in the public domain. Acta agriculturae Slovenica, 109 - 2, september 2017 Barbara GERIČ STARE, Saša ŠIRCA, Gregor UREK 3 RESULTS AND DISCUSSION In year 2015 none of the dissected ladybirds' specimens exhibited nematode parasites. In 2016 parasitic nematodes were recovered from H. axyridis collected at two locations, Brdo pri Lukovici and Trboje. The incidence of parasitized ladybirds from the samples was around 30%, which is in accordance with previous studies finding up to 35 % incidence in Denmark (Poinar and Steenberg, 2012). Even higher incidence up to 47 % was reported from the Czech Republic, (Haelewaters et al., 2017), while lower incidence was reported for the Netherlands (Raak-van den Berg et al., 2014). Three different nematode life/developmental stages within the individual host were found, including subsequent generation parasitic females, vermiform (infective) females and males (Figure 1). First generation parasitic females were not encountered in analysed samples. Numbers of nematodes found in ladybirds varied, but up to several hundreds of nematodes could be found in some specimens. Contemporaneity of different developmental stages within a host and large nematode populations within a single beetle have been observed before (Poinar and Steenberg, 2012). Common nematode body features were measured in morphometric analysis. The nematodes extracted from ladybirds were identified as P. bifurcatus (Table 1, Figure 1). The characteristic observed features of P. bifurcatus nematodes were straight stylet lacking basal thickenings, a narrow bursa and a forked tail tip in the vermiform females and juvenile males. Subsequent generation parasitic females from Trboje had smaller body length of 886.4 ^m (782.0-1098.0) and body width of 72.3 ^m (59.0-81.0) compared to 1300 ^m (920 -1600) and 195 ^m (158-271) of P. bifurcatus nematodes from Denmark, respectively. All the other morphometrical characters were in the range of P. bifurcatus species description (Poinar and Steenberg, 2012). It can therefore be concluded that P. bifurcatus species morphometric characters of subsequent generation parasitic females may have somewhat higher variability than previously reported. Figure 1: Different developmental stages of Parasitylenchus bifurcatus nematodes, parasites of Harmonia axyridis ladybirds. Arrows indicating straight stylet lacking basal thickenings (St), spicule (Sp) and a forked tail tip (T). Scale bars = 20 ^m 460 Acta agriculturae Slovenica, 109 - 2, september 2017 First report of nematodes Parasitylenchus bifurcatus ... Asian lady beetle Harmonia axyridis (Pallas, 1773) in Slovenia Table 1: Morphometric characters of subsequent generation parasitic females, vermiform (infective) females and males of P. bifurcatus nematodes isolated from H. axyridis from Trboje. All measurements are in ^m, presented as mean ± standard deviation, with the range in parentheses Character Subsequent generation parasitic females (n = 10) Vermiform (infective) females (n = 10) Males (n=10) Body length 886.4 ± 124.2 646.9 ± 60.5 605.6 ± 58.8 (782.0-1098.0) (573.0-752.0) (530.0-689.0) Body width 72.3 ± 8.1 (59.0-81.0) 16.8 ± 1.6 (14.4-18.6) 21.2 ± 3.3 (17.9-25.8) Stylet length 13.3 ± 0.5 (12.7-13.9) 12.6 ± 1.4 (10.6-14.2) 9.4 ± 0.3 (9.1-9.7) Tail length 32.3 ± 1.3 (30.9-34.0) 37.8 ± 2.9 (35.2-41.0) 38.3 ± 4.6 (33.0-44.2) Tail width 37.1 ± 3.3 10.7 ± 1.1 15.7 ± 1.6 (34.2-41.0) (9.5-11.8) (13.7-17.6) Distance from head to 219.3 ± 13.0 108.5 ± 3.5 103.6 ± 9.5 excretory pore (206.5-234.0) (101.5-114.0) (90.9-117.0) Vulva position % 93.9 ± 0.8 (93.0-94.8) 87.5 ± 0.8 (86.2-88.0) - Spicule length - - 11.8 ± 0.5 (11.3-12.2) Spicule width at base 3.7 ± 0.3 (3.4-4.0) Gubernaculum length 4.1 ± 0.4 (3.8-4.8) Bursa length - - 8.6 ± 0.9 (7.3-9.2) Two sequences were determined from nematodes extracted from the sample of Harmonia axyridis from Brdo, Slovenia. Two determined sequences of 18S rDNA region are 1.623 bp long and differ among them in 1 bp position. Both determined sequences belong to P. bifurcatus based on a high similarity to the P. bifurcatus sequences in the public domain determined with NCBI BlastN tool (99.94 % identity to P. bifurcatus sequence with acc. no. KC875397.1). Sequences obtained in this study were deposited at the European Nucleotide Archive, EMBL Nucleotide Sequence Database with the accession numbers LT629306 and LT629307. While no sequences are available for the species P. coccinellinae Iperti and Waerebeke to assess the phylogenetic relationship of these two related species, the bifurcated tail has not been reported for P. coccinellinae (Iperti and van Waerebeke, 1968, Poinar and Steenberg, 2012). Therefore, based on morphological and molecular characters this is a first report of nematodes P. bifurcatus parasitizing multicolored Asian lady beetles H. axyridis in Slovenia. 4 CONCLUSIONS Multicolored Asian lady beetles collected in Slovenia in 2016 were found to be parasitized by nematode P. bifurcatus. Identification was confirmed with both, morphological and molecular characters. Nematode species P. bifurcatus compromises fecundity of its host and has therefore a potential to be used as a biological control agent to control high abundance of invasive ladybirds of H. axyridis species. However, future studies on biology, possible transmission and effect of this parasitic nematode for native species of ladybirds are needed before any practical application. It is critical that the introduced biological control agent does not become pest themselves as has happened in the past with the intentional introduction of H. axyridis into North America and Europe. To our knowlede P. bifurcatus is at the moment not used anywhere to manage H. axyridis, but studies needed for justification and approval of such use are in progress (T. Steenberg, personal communication, December 1, 2015). In EU there is no unified federal legislation regulating use of biological control agents (BCA) (Loomans, 2015). Some EU member states have national regulations, which are based on international standards, but are implemented in different ways (Hunt et al., 2011 Acta agriculturae Slovenica, 109 - 2, september 2017 Barbara GERIČ STARE, Saša ŠIRCA, Gregor UREK cited in Loomans, 2015). Introduction of exotic BCA is covered in the following international acts: the International Plant Protection Convention, the Plant Protection Product Acts and the Convention on Biodiversity (Loomans, 2015). In Slovenia the use of biological control agent is regulated with the Plant Health Act (Zakon o zdravstvenem varstvu rastlin, Uradni list RS št. 62/07- uradno prečiščeno besedilo, 36/10 in 40/14 - ZIN-B). Detailed procedures for introduction, cultivation, trade and use of invertebrate BCA are subject to the Rules on biological control of plant pests (Pravilnik o biotičnem varstvu rastlin, Uradni list RS, št. 45/06, 28.4.2006). BCA are classified in two lists: the List of indigenous and the List of non-indigenous species of invertebrate organisms for biological control. The lists are maintained by the Administration based on the EPPO list (PM6/3) and on the basis of the results of researches, professional or scientific articles. Provisions of these regulations do not apply for the introduction and use of microorganisms for biological control. They are regulated by legislation in the field of plant protection products and are subject to different risk assessment in the registration procedure. Microorganisms are regulated by EU Regulation (EC) No. 1107/2009 of the European Parliament and of the Council of 21 October 2009 concerning the placing of plant protection products on the market or equivalent regulations. P. bifurcatus is not included on the lists of approved BCA in Slovenia. Further studies on safety and efficacy would be needed to include P. bifurcatus on this list. Use of parasitic nematodes in plant protection is an accepted practice. Entomopathogenic nematodes are widely used as biocontrol agents to control several harmuful insect species in integrated pest management programs and slug-parasitic nematodes are used to control certain slugs and snails. Both groups are available as comercial biocontrol products and have been used for decades. Further, there are several studies exploring potential of predatory nematodes to control plant parasitic nematodes and potential of fungalfeeding nematodes for the control of soilborne plant pathogens (Grewal et al., 2005). Although P. bifurcatus is a parasite of an insect species it is not an entomopathogenic nematode. Entomopathogenic nematodes kill their hosts relatively quickly (typically within 24-48 h of infection) with the help of their associated bacteria. On the other hand, P. bifurcatus is a typical parasite which can co-exist with its host for an extended time without killing it, but it does cause depletion of fat body as well as partial or complete atrophy of the insect's reproductive organs (Poinar and Steenberg, 2012, Haelewaters et al., 2017). As discussed above, P. bifurcatus may have potential as a biocontrol agent of H. axyridis. 5 ACKNOWLEDGEMENTS This work was financially supported by the Slovenian Research Agency in the frame of Research Programme Agrobiodiversity (P4-0072). Link with Dr. Tove Steenberg has been established within the frame of FP7 Project CropSustaIn, grant agreement FP7-REGPOT- CT2012-316205. The authors are grateful to Ana Kerin for technical support with molecular analysis, to Dr. Matej Stopar and Dr. Tinka Bacic for collecting ladybirds and Dr. Jaka Raziger for identification of ladybirds. 6 REFERENCES Brown, P. M. J., Adriaens, T, Bathon, H., Cuppen, J., Goldarazena, Hagg, T., Kenis M., ... Roy, D. B. (2008). Harmonia axyridis in Europe: spread and distribution of a non-native coccinellid. BioControl, 55(1), 5-21. doi:10.1007/s10526-007-9132-y Chapin, J. B., Brou, V. A. (1991). Harmonia axyridis (Pallas), the third species of the genus to be found in the United States (Coleoptera: Coccinellidae). Proceedings of the Entomological Society of Washington, 95(3), 630-635. EPPO. (2016). EPPO standard PM 6/3(4): List of biological control agents widely used in the EPPO region (web pages only), Version October 2016, Retrieved from http://archives.eppo.int/EPPOStandards/biocontrol_we b/deletions.htm Freude, H., Harde, K. W., Lohse, G. A., (1967). Die Käfer Mitteleuropas. Band 7. Krefeld: Goecke & Evers Verlag. Grewal, P. S., Ehlers, R. U., Shapiro-Ilan, D. I. (2005). Nematodes as biocontrol agents. Wallingford, CAB Publishing. doi:10.1079/9780851990170.0000 Hall, T. A. (1999). BioEdit: a user-friendly biological sequence alignment editor and analysis program for Windows 95/98/NT. Nucleic Acids Symposium Series, 41, 95-98. 302 Acta agriculturae Slovenica, 109 - 2, september 2017 First report of nematodes Parasitylenchus bifurcatus ... Asian lady beetle Harmonia axyridis (Pallas, 1773) in Slovenia Harding, S., Poinar, G. O. Jr, Dimitrova, D. V., Steenberg, T. (2011). Parasitylenchus sp. (Tylenchomorpha: Allantonematidae) parasitizing field populations of Harmonia axyridis (Coleoptera: Coccinellidae). European Journal of Entomology, 108, 487-488. doi:10.14411/eje.2011.062 Haelewaters, D., Zhao, S. Y., Clusella-Trullas, S. Cottrell, T. E., De Kesel, A., Fiedler, L., Herz, A., ... Roy. H. E. (2017). Parasites of Harmonia axyridis: current research and perspectives. BioControl, 62, 355-371. doi:10.1007/s10526-016-9766-8 Holtermanm M., van der Wurff, A., van den Elsen, S., van Megen, H., Bongers, T., Holovachov, O., Bakker J., Helder, J. (2006). Phylum-wide analysis of SSU rDNA reveals deep phylogenetic relationships among nematodes and accelerated evolution toward crown clades. Molecular Biology and Evolution, 23(9), 17921800. doi: 10.1093/molbev/msl044 Hunt, E. J., Loomans, A. J. M., Kuhlmann, U. (2011). An international comparison of Invertebrate Biological Control Agent Regulation: What can we learn? In: R. U. Ehlers (Ed.), Regulation of Biological Control Agents, 1st Edition, XII (pp. 79-112). Springer. doi:10.1007/978-90-481 -3664-3_3 Iperti, G, van Waerebeke, D. (1968). Description, biologie et importance d'une nouvelle espèce d'Allantonematidae (Nématoda), parasite des coccinelles aphidiphages: Parasitylenchus coccinellinae, n. sp. Entomophaga, 13, 107-119. doi:10.1007/BF02371781 Koch, R. L. (2003) The multicolored Asian lady beetle, Harmonia axyridis: a review of its biology, uses in biological control, and non-targeted impacts. Journal of Insect Science, 3, 32. doi: 10.1093/jis/3.1.32 Kovach, J. (2004). Impact of the multicolored Asian lady beetle as a pest of fruit and people. American Entomologist, 50, 165-167. Laznik, Z., Milevoj, L., Trdan, S. (2012). Multicoloured Asian lady beetle (Harmonia axyridis [Pallas], Coleoptera, Coccinellidae) - invasive beneficial species. Acta agriculturae Slovenica, 99(2), 225-234. Loomans A. J. M. (2015). Environmental benefits and risks of biological control: evaluation of natural enemies as a basis for releasing BCAS in The Netherlands, Zbornik predavanj in referatov 12. Slovenskega posvetovanje o varstvu rastlin z mednarodno udeležbo, 7-13. Majerus, T. M. O., Graf von der Schulenberg J. H., Majerus, M. E. N., Hurst, G. D. D. (1999) Molecular identification of a male-killing agent in the ladybird Harmonia axyridis (Pallas) (Coleoptera: Coccinellidae) Insect Molecular Biology, 8, 551-555. doi:10.1046/j .1365-2583.1999.00151 .x Mizell, R. F. III. (2007). Impact of Harmonia axyridis (Coleoptera: Coccinellidae) on native arthropod predators in pecan and crape myrtle. Florida Entomologist, 90, 524-553. doi:10.1653/0015-4040(2007)90[524:IOHACC]2.0.CO;2 Poinar, G. O. Jr, Steenberg, T. (2012). Parasitylenchus bifurcatus n. sp. (Tylenchida: Allantonematidae) parasitizing Harmonia axyridis (Coleoptera: Coccinellidae). Parasites & Vectors, 5, 218 doi: 10.1186/1756-3305-5-218 Raak-van den Berg, C. L., van Wielink, P. S., de Jong, P. W., Gort, G., Haelewaters, D., Helder, J., van Lenteren J. C. (2014). Invasive alien species under attack: natural enemies of Harmonia axyridis in the Netherlands. BioControl, 59, 229-240. doi:10.1007/s10526-014-9561-3 Polak, S. (2013). Harlekinska polonica (Harmonia axyridis) - nevarnost z vzhoda. Trdoživ, 2, 11-14. Reitter, E. (1911). Fauna Germanica. Die Käfer des Deutschen Reiches. III. Band. Stuttgart: K. G. Lutz' Verlag. Riedel, A., Bastian, J. (2005). Der Asiatische Marienkäfer Harmonia axyridis (Pallas, 1773) (Col., Coccinellidae) - über den Stand seiner Ausbreitung in Mitteleuropa und Hinweise zu seiner Erkennung. Mitteilungen des Entomol Vereins Stuttgart, 40, 117122. Acta agriculturae Slovenica, 109 - 2, september 2017 doi: 10.14720/aas.2017.109.2.29 Original research article / izvirni znanstveni članek Influence of altered temperatures on allelopatic properties of Amaranthus cruentus L. Ingrid ALLEMANN1, Maria Elizabeth CAWOOD1*, James ALLEMANN2 Received July 04, 2017; accepted July 22, 2017. Delo je prispelo 04. julija 2017, sprejeto 22. julija 2017. ABSTRACT The relationships between allelochemicals and environmental factors are a key factor for the growth of plants under rotation. We investigated the allelopathic potential of Amaranthus cruentus L, grown under different temperature conditions in in vitro bioassays. An inhibitory effect on germination and growth of lettuce (Lactuca sativa L.), tomato (Solanum lycopersicum L.), pepper (Capsicum annuum L.) and cucumber (Cucumis sativus L.) was observed when seeds were subjected to the leaf litter of Amaranthus cruentus. Analysis from our study indicated that germination percentage was significantly affected by growth temperatures (T) of the amaranth (P < 0.0001), litter concentration (C) (P < 0.0001), vegetable type (V) (P < 0.0001), the T x V interaction (P = 0.0041) and V x C interaction (P < 0.0001). Pepper was the most sensitive with a decline in germination percentage at increasing concentrations (0, 1 and 5 mg ml-1) of litter. Hypocotyl and seminal root lengths were adversely influenced by the plant litter for all the temperature treatments, although effects were most severe when exposed to the leaf litter of the hot temperature treatment. The inhibition caused by the litter was dependent on growth temperature and concentration, while each vegetable species showed different levels of sensitivity. Key words: Amaranthus cruentus; allelopathy; vegetables; germination; hypocotyl; seminal root; crop rotation IZVLEČEK VPLIV SPREMENJENIH TEMPERATUR NA ALELOPATSKE LASTNOSTI MEHIŠKEGA ZRNATEGA ŠČIRA Amaranthus cruentus L. Odnosi med alelokemikalijami in okoljskimi dejavniki so odločilni za rast rastlin v kolobarju. V razmerah in vitro poskusa je bil preučevan alelopatski potencial mehiškega zrnatega ščira (Amaranthus cruentus L.) v različnih temperaturnih razmerah. Inhibitorni učinek na kalitev in rast vrtne solate (Lactuca sativa L.), paradižnika (Solanum lycopersicum L.), paprike (Capsicum annuum L.) in kumar (Cucumis sativus L.) je bil ugotovljen, kadar so bila semena teh vrtnin izpostavljena listnim ostankom mehiškega zrnatega ščira. Analize so pokazale, da so na odstotek kalitve značilno vplivali rastna temperatura (T) ščira (T) (P < 0.0001), količina listnih ostankov (C) (P < 0.0001) in vrsta vrtnine (v) (P < 0.0001) kot tudi interakciji T x V (P = 0.0041) in V x C (P < 0.0001). Najbolj občutljiva je bila paprika z upadom odstotka kalitve pri naraščanju koncentracije ščira (0, 1 in 5 mg ml-1). Na dolžino hipokotila in semenske korenine so ostanki ščira vplivali negativno pri vseh temperaturah, vendar so bili učinki izrazitejši pri visokih temperaturah. Inhibicija, ki so jo povzročili ostanki ščira, je bila odvisna od njihovih količin in rastnih temperatur, vendar je vsaka od vrtnin pokazala različno občutljivost. Ključne besede: Amaranthus cruentus; alelopatija; vrtnine; kalitev; hipokotil, semenska korenina; setveni kolobar 1 INTORODUCTION Amaranthus cruentus L., also known as "Mexican grain amaranth" belongs to the family Amaranthaceae and is characterised by being an annual, pseudo-cereal with broad leaves which are used as a leafy vegetable and a forage crop (Saunders & Becker, 1984; Steckel, 2004; Yaacob et al., 2004.). Amaranthus spp. have been traditionally used in medicine for the treatment of various diseases and their antioxidant properties have been recorded by various authors (Olumayokun et al., 2004; Dhellot et al., 2006; Mensah et al., 2008). The habitat where amaranth is found differs dramatically and the temperatures that they can grow ranges from 20- 1 Department of Plant Sciences, University of the Free State, Bloemfontein, South Africa *Corresponding author: cawoodme@ufs.ac.za 2 Department of Soil, Crop and Climate Sciences, University of the Free State, Bloemfontein, South Africa This article is part of a Master thesis entitled » Influence of abiotic stress on allelopathic properties of Amaranthus cruentus L. «, issued by Ingrid Allemann, Supervisor Dr Maria Elizabeth Cawood, Ph. D., Co-Supervisor Dr James Allemann, Ph.D. Acta agriculturae Slovenica, 109 - 2, september 2017 str. 349 - 356 Ingrid ALLEMANN et al. 35 °C (Grosz-Heilman et al., 1990; Guo & Al-Khatib, 2003). Given its several advantages as a crop, its unique nutritional properties, and its use as food and feed (Ayo, 2001; Bavec & Bavec, 2006; Grobelnik et al., 2009), grain amaranth is receiving increasing attention as an alternative crop worldwide. Amaranthus cruentus is one of three amaranthus species cultivated as vegetable and grain source. The other two are Amaranthus caudatus L. and Amaranthus hypochondriacus L. (Olaniyi, 2007; Olaniyi et al., 2008). Recently, Mlakar et al. (2012) compared the allelopathic activities of extracts prepared from fresh roots, stems, leaves, and inflorescence with seeds of weedy and grain amaranths. It was found that at varying concentrations, the aqueous extracts of the grain amaranth, A. cruentus, exert allelopathic activity. Compared to the pigweed amaranth, the grain species displayed a stronger inhibitory effect on the germination process, and root elongation of garden cress. Allelopathy can affect a whole range of aspects in agroecosystems namely, weed management, plant reproduction, species associations, the mulching effect on crops and the succession and rotation of cultivated species (Chon et al., 2006; Rawat et al., 2017). These results point out the problematic consequence when amaranth and more specific, A. cruentus, is cultivated in crop rotation systems. Understanding this biological occurrence could help to improve applications in both natural and agricultural systems and is helpful for planning and managing cropping systems (Gronle et al., 2015). Furthermore, climate change influences a plant's chemical response and the ecological function of plant allelochemicals (Harvey & Malcicka, 2015). Sudden changes in temperature may influence the production of chemical compounds and the allelopathic properties of a plant (Maqbool et al., 2013). The allelopathic potential of Amaranthus retroflexus increased when grown at high latitudes compared to plants grown at low latitudes, indicating the role of temperature on the expression of allelochemicals (Wang et al., 2017). Therefore, the aim of this study was to investigate the relationship between temperature variation and allelopathic effects of different concentrations of plant litter of A. cruentus on the germination and growth of some vegetable species in order to determine the crops' feasibility for intercropping and in rotation systems. 2 MATERIALS AND METHODS 2.1 Plant material Amaranthus cruentus 'Anna' seeds were planted in pots containing a soil-compost (80:20 v/v) mixture and grown at 28/21 °C; day/night temperatures in climate controlled chambers at The Department of Agriculture, University of the Free State. Fifty pots per chamber with an average of three plants per pot were kept in three different chambers and daylight was set at 12 hours in order to prevent flowering. The plants were watered every second day and fertilised with Nutrifeed solution (Starke Ayres). After three months plants were subjected to cool (14/7 °C) and hot (33/40 °C) temperatures. Stress continued for 14 days where after aerial parts of the plants were harvested and the plant material lyophilised, ground into a fine powder and kept at 4 °C until further analyses. Optimum (28/21 °C) temperature grown plants serve as temperature controls. 2.2 Vegetable seeds Seeds were obtained from Starke Ayres: Tomato 'Money Maker', Sweet Pepper 'California Wonder', Cucumber v 'Ashley' and Lettuce 'Great Lakes'. 2.3 Allelopathy bioassay The 'sandwich method' of Fujii et al. (2003) was used for determining the in vitro phytotoxicity of the leaf litter from the different temperature treatments of A. cruentus on the various vegetable seeds. For this method a 5 ml layer of 0.5 % (w/v) sterile water agar was poured into each well of a sterile multi-dish plate and allowed to set. The A. cruentus leaf litter was placed on this bottom layer and a second layer of 5 ml sterile water agar was added on top. This made a sandwich of powdered leaf litter between the two layers of agar (10 ml in total). This method was used in order to physically separate the A. cruentus samples from the tested seeds, however allows for diffusion of any active component from the sample through the barrier agar layer. The phytotoxicity of 10 and 50 mg of the powdered leaf litter was measured (1 and 5 mg ml-1 litter per well) against the different vegetable seeds. Controls contain no leaf material. Vegetable seeds were surface sterilised by washing in 96 % ethanol for 1 minute followed by 1.30 min in 3.5 % NaCl and finally back into ethanol for 30 seconds. After sterilization the seeds were placed on sterile filter paper and left to dry within a laminar flow cabinet (Labotec, airflow from above). The seeds were then positioned vertically on the top layer of agar in each well, plates were closed and incubated at 25 °C in black bags (full darkness) for 3-7 days depending on the vegetable used. On the 3rd to 7th day the germination percentage and length of the seminal root and hypocotyl was measured. Each of the experiments was done in triplicate and presented as the mean of the replicates. 466 Acta agriculturae Slovenica, 109 - 2, september 2017 Influence of altered temperatures on allelopatic properties of Amaranthus cruentus L. 2.4 Statistical analysis The results were expressed as means with least significant difference (LSD). Analysis of variance (ANOVA) was performed using SAS 9.3 (Institute Inc., Cary, NC, USA, 2008) statistical programme for data and Tukey-Kramer's LSD procedure for comparison of means. Significance of differences compared to the control groups was determined using the t-test (Steel & Torrie, 1980). 3 RESULTS AND DISCUSSION A. cruentus is used as a food crop, and both its grain and leaves being consumed. This plant is now being considered as a new agricultural crop; it is therefore important to understand how this plant will interact within a changing environment and how this will influence other plants. In addition to the increase in average temperatures, global warming is also characterized by an increase in the frequency of the occurrence of extremely high and low temperatures (Wagner, 1996; IPCC 2007). This study showed that allelopathic activity of A. cruentus could be affected by environmental temperatures, which can affect other crops negatively in rotation systems. Highly significant (P < 0.01) differences in germination percentage of the various vegetables exposed to the litter of amaranth plants grown under stress or optimal temperature conditions can be seen in Figure 1. Vegetables exposed to plant litter of amaranth grown under hot temperature conditions showed the most significant inhibition in germination (Figure 1). Peppers were the most sensitive with 60 % germination inhibition. Lettuce was the least affected with only 22 % inhibition and affected more negatively by amaranth litter from plants cultivated at optimal temperatures than by that of plants grown at stress temperatures (Figure 1). It appeared as though germination of cucumber, pepper and tomato seeds were generally more sensitive to amaranth litter than lettuce. Steinsiek et al. (1982) reported a more severe allelopathic effect of an aqueous extract of common wheat (Triticum aestivum L) straw on the inhibition of germination and growth of selected weed species at 35 °C than at 25 °C or 30 °C. Einhellig & Echrich (1984) claimed that grain sorghum (Sorghum bicolor (L.) Moench.) and soybean (Glycine max (L.) Merr.) were more susceptible to ferulic acid when they were grown at higher temperatures. It was also observed that at 26/22 °C (day/night) temperature, the allelopathic inhibition of chlorogenic acid or tomatine was greater on the growth of an insect herbivore, Manduca sexta (Linnaeus, 1763) than at 26/14 °C (Stamp & Osier, 1997). 90 80 70 60 50 40 30 20 10 0 Heat Cold Optimal Cucumber Heat Cold Optimal Heat Cold Optimal Lettuce Pepper Treatment and vegetable type Heat Cold Optimal Tomato Figure 1: Response of the germination percentages of various vegetables to amaranth litter from plants grown at different temperatures. The data are expressed as average values. P < 0.01 (LSD(T:0.05) = 2.71). n = 96. Acta agriculturae Slovenica, 109 - 2, september 2017 Ingrid ALLEMANN et al. 120 100 80 60 5 40 20 0 1 5 Cucumber 01 Lettuce 01 Pepper 01 Tomato Litter [mg ml-1] Figure 2: Influence of increasing litter concentration on the germination percentage of four vegetables (LSD(T:0.05) 2.71). n = 96 Our results emphasise the importance of A. cruentus litter concentration on germination and growth of vegetables. The significant interaction between amaranth litter concentration and the effect on the four vegetable species is illustrated in Figure 2 and Table 1. Here it is clear that germination percentage and growth of all four vegetables decreased significantly with increased concentration of amaranth litter that was added to the growth medium, however, each vegetable species showed different levels of sensitivity. Peppers were the most sensitive to amaranth litter with the germination percentage falling from 100 % through 54 % to 22 % at the 0, 1 and 5 mg ml-1 litter concentrations respectively. Tomatoes were the least affected, with germination inhibition of only 17 % and 43 %, followed by 19 % and 33 % for lettuce and 36 and 66 % for cucumber at 1 and 5 mg ml-1. From Table 1, an inhibition of 34 %, 76 % and 78 % in hypocotyl growth and 35 %, 68 % and 85 % in seminal root growth occurred in cucumber, pepper and tomato respectively at 1 mg ml-1. An increase in concentration to 5 mg ml-1 resulted in growth inhibition of 65 % for cucumber and more than 90 % for pepper and tomato (Table 1). Table 1: Effect of different concentrations of amaranth litter on the average hypocotyl and seminal root lengths of various vegetables Leaf litter Hypocotyl length (mm) [mg ml-1] Cucumber Pepper Tomato 0 33.15 ± 23.82 18.49 ± 13.99 39.56 ± 13.82 1 21.74 ± 24.62 4.39 ± 5.45 8.62 ± 9.93 5 11.87 ± 15.19 1.67 ± 2.64 2.05 ± 4.77 AVG 22.25 8.18 16.74 LSD(T < 0.05) 11.06 4.44 4.67 Leaf litter Seminal root length (mm) [mg ml-1] Cucumber Pepper Tomato 0 23.59 ± 10.32 13.38 ± 5.08 26.69 ± 6.67 1 15.36 ± 15.94 4.33 ± 5.76 4.08 ± 3.09 5 8.82 ± 10.58 1.23 ± 2.06 2.54 ± 2.89 AVG 15.92 6.31 33.31 LSD(T < 0.05) 6.45 2.33 2.28 n = 13. 0 5 5 5 468 Acta agriculturae Slovenica, 109 - 2, september 2017 Influence of altered temperatures on allelopatic properties of Amaranthus cruentus L. According to Qasem (2010), allelopathy plays a significant role in seed dormancy, seed germination and seedling emergence. The influence of allelochemicals on the balance of plant populations and species stability, microorganisms, natural enemies and insect populations, and the spread of pathogens is another important role of allelopathy (Qasem, 2010). From the results it is clear that germination of all vegetables tested, was adversely affected by both concentrations of amaranth litter irrespective of the temperature treatment to which the plants were exposed (Figure 3). Analysis from our study therefore indicated that germination percentage was significantly affected by growth temperatures (T) (P < 0.0001) of the amaranth, litter concentration (C) (P < 0.0001), vegetable type (V) (P < 0.0001), the T x V interaction (P = 0.0041) and V x C interaction (P < 0.0001). The T x V x C (P = 0.0540) interaction showed no statistical differences. 80 - 60 - Ü 40 20 0 1 5 Litter [mg ml-1] H Hot ElCold □ Optimal Figure 3: Two way interaction (ANOVA) between litter concentration and temperature on germination percentage of vegetables. P < 0.01 (LSD(T:0.05) = 2.73). n = 96 0 Lettuce growth exhibited a two way interaction between concentration of plant litter and temperature treatment (Figure 4). Litter concentration and temperature treatments were highly significant in reducing hypocotyl (LSD(t < 005) = 3.43) and seminal root (LSD(T < 005) = 3.41) lengths respectively. From Figure 4 it is clear that the hot and optimal temperatures at both concentrations of litter, had a more severe effect on growth than the cool temperature treatment. Concentrations of 5 mg ml-1 for all the treatments inhibited hypocotyl and seminal root development almost completely. Acta agriculturae Slovenica, 109 - 2, september 2017 Ingrid ALLEMANN et al. Litter [mg/mL] Litter [mg/mL] Figure 4: Results of two-way ANOVA of the effect of litter concentration and temperature and their interaction on hypocotyl (LSD(T < 005) = 3.43) and seminal root lengths (LSD(T < 005) = 3.41) of lettuce seedlings. n = 13. It is demonstrated in this study that the environment for the cultivation of A. cruentus is important and that compounds responsible for allelopathy, are present even at optimal growth conditions. Furthermore, with increased concentrations of litter a decrease in germination and seedling development occurred. Therefore, it is possible that more leftover plant material in a field can have a more severe allelopathic effect on the following harvest. It was also clear that vegetables displayed diversity in reaction towards the temperature treatments and type of extract. In crop rotation systems it is important, because if germination is affected by compounds of A. cruentus produced under stress conditions, farmers can incur financial losses. 4 CONCLUSION The in vitro bioassay results of the plant litter from A. cruentus indicated that the allelopathic compounds inhibiting germination and growth of vegetables are produced by amaranth irrespective of cultivation temperature, although the litter of plants grown under stressful conditions have a greater allelopathic effect than those at optimal environments. A difference in the response of vegetables and sensitivity to increasing concentration may have a negative impact in crop rotation systems. Results showed that it is vital to understand under which conditions amaranth was cultivated. Field trials will contribute to the confirmation of the phytotoxicity of the leaf litter and will be conducted in future studies. This will add to the understanding of allelopathic properties of A. cruentus in crop rotating systems. 5 REFERENCES Ayo, J.A. (2001). The effect of amaranth grain flour on the quality of bread. International Journal of Food Properties, 4(2), 341-351. doi:10.1081/JFP-100105198 Bavec, F., & Bavec, M. (2006). Grain amaranths. In: Organic Production and Use of Alternative Crops Pp 88-98. CRC Press/Taylor and Francis, Florida. doi:10.1201/9781420017427 Chon, S.U., Jennings, J.A., Nelson, C.J. (2006). Alfalfa (Medicago sativa L.) autotoxicity: Current status. Allelopathy Journal, 18, 57-80. Dhellot, R.J., Matouba, E., Maloumbi, M.G., Nzikou, J.M., Safou, D.G., Linder, M. Parmentier, M. (2006). Extraction, chemical composition and nutritional characterization of vegetable oils: Case of Amaranthus hybridus (var 1 and 2) of Congo Brazzaville. African Journal of Biotechnology, 5, 1095-1102. Einhellig, F.A., & Echrich, P.C. (1984). Interaction of temperature and ferulic acid stress on grain sorghum and soybean. Journal of Chemical Ecology, 10, 161-70. doi:10.1007/BF00987653 Fujii, Y., Parvez, S.S., Parvez, M.M., Ohmae, Y., Iida, O. (2003). Screening of 239 medical plant species for allelopathic activity using the sandwich method. Weed Biology and Management, 3, 233-241. doi:10.1046/j.1444-6162.2003.00111.x 470 Acta agriculturae Slovenica, 109 - 2, september 2017 Influence of altered temperatures on allelopatic properties of Amaranthus cruentus L. Grobelnik, S.G., Turinek, M., Jakop, M., Bavec, M., Bavec, F. (2009). Nutrition value and use of grain amaranth: potential future application in bread making. Agricultura, 6, 43-53. Gronle, A., Heb, J., Böhm, H. (2015). Weed suppressive ability in sole and intercrops of pea and oat and it is interaction with ploughing and crop interference in organic farming. Organic Agriculture, 5, 39-51. doi:10.1007/s13165-014-0095-x Grosz-Heilmann, R., Golz, J.T., Helgeson, D.L. (1990). Amaranth: A Food Crop From the Past for the Future (No. 121422). North Dakota State University, Department of Agribusiness and Applied Economics. Guo, P., & Al-Khatib, K. (2003). Temperature effects on germination and growth of redroot pigweed (Amaranthus retroflexus), Palmer amaranth (A. plameri), and common waterhemp (A. rudis). Weed Science, 51, 869-875. doi:10.1614/P2002-127 Harvey, J.A., & Malcicka, M. (2015). Climate Change, Range Shifts and Multitrophic Interactions. In: Biodiversity in Ecosystems- Linking Structure and Function Pp 85-109. Publisher: InTechOpen. IPCC (Contribution of Working Group II to the Fourth Assessment Report of the Intergovernmental Panel on Climate Change). (2007). Cambridge University Press, Cambridge, United Kingdom and New York, NY, USA. Maqbool, N., Wahid, A., Farooq, M., Cheema, Z.A., Siddique, K.H.M. (2013). Allelopathy and Abiotic Stress Interaction in Crop Plants. In: Allelopathy: Current Trends and Future Applications Pp 451468. Springer-Verlag, Germany. doi:10.1007/978-3-642-30595-5_19 Mensah, J.K., Okoli, R.I., Ohaju-Obodo, J.O., Eifediyi, K. (2008). Phytochemical, nutritional and medical properties of some leafy vegetables consumed by Edo people of Nigeria. African Journal of Biotechnology, 7(14), 2304-2309. Mlakar, S.G., Jakop, M., Bavec, M., Bavec, F. (2012). Allelopathic effects of Amaranthus retroflexus and Amaranthus cruentus extracts on germination of garden cress. African Journal of Agricultural Research, 7(10), 1492-1497. Olaniyi, J. O. (2007). Evaluation of yield and quality performance of grain amaranth varieties in the Southwestern Nigeria. Research Journal of Agriculture, 1(2), 42-45. Olaniyi, J. O., Adelasoye, K. A., Jegede, C. O. (2008). Influence of nitrogen fertilizer on the growth, yield and quality of grain amaranth varieties. World Journal of Agricultural Science, 4(4), 506-513. Olumayokun, A., Olajid, B.R., Ogunleya, T., Erinle, O. (2004). Anti-inflammatory properties of Amaranthus spinosus. Pharmaceutical Biology, 521-525. Qasem, J.R. (2010). Allelopathy importance, field application and potential role in pest management: a review. Journal of Agricultural Sciences and Technology, USA 4 (6), 104-120. Rawat, L.S., Maikhuri, R.K., Bahuguna, Y.M., Jha, N.K., Phondani, P.C. (2017). Sunflower Allelopathy for Weed Control in Agriculture Systems. Journal of Crop Science and Biotechnology, 20(1), 45-60. doi:10.1007/s12892-016-0093-0 Saunders, R., & Becker, R. (1984). Amaranthus: a potential food and feed resource. Advances in Cereal Science and Technology, 6, 357-396. Stamp, N.E., & Osier, T.L. (1997). Combined effects of night-time temperature and allelochemicals on performance of a solanaceae specialist herbivore. Ecoscience, 4, 286-95. doi: 10.1080/11956860.1997.11682407 Steckel, J. (2004). The dioecious Amaranth spp: Here to stay. Weed Technology, 21, 567-570. doi: 10.1614/WT-06-045.1 Steel, R. G. D., & Torrie, J. H. (1980). Principles and Procedures of Statistics Pp 633. Second Edition, New York: McGraw-Hill. Steinsiek, J.W., Oliver, L.R., Collins, F.C. (1982). Allelopathic Potential of Wheat (Triticum aestivum) Straw on Selected Weed Species. Weed Science, 30, 495-497. Wagner, D. (1996). Scenarios of extreme temperature events. Climatic Change, 33, 385-407. doi: 10.1007/BF00142585 Wang, C., Liu, J., Zhou, J. (2017). N deposition affects allelopathic potential of Amaranthus retroflexus with different distribution regions. Annals of the Brazilian Academy of Sciences, 89(2), 919-926. doi:10.1590/0001-3765201720160513 Yaacob, J.S., Taha, R.M., Mat Nor, N.A., Aziz, N. (2004). Pigment analysis and tissue culture of Amaranthus cruentus L. In: De Neve, S., Boehme, M., Everaarts, A., Neeteson (Eds.), First International Symposium on Sustainable Vegetable Production in South East Asia, Acta horticulturae, 958, ISHS 2012. Acta agriculturae Slovenica, 109 - 2, september 2017 POPRAVKI / ERRATA Spremembe v avtorstvu člankov / changes in authorship Avtorji / Authors: Radojko Pelengic, Barbara Pipan, Vladimir Meglič, Denis Rusjan Tiskano / Printed: Ovrednotenje genskih virov belih sort žlahtne vinske trte (Vitis vinifera L.) = Evalution of genetic resources of white grapevine varieties (Vitis vinifera L.) / Radojko Pelengic ... [et al.].- Ilustr. - Dostopno tudi na: http://aas.bf.uni-lj.si/december2012/19Pelengic.pdf, URN:NBN:SI:doc-GTI6AHLW, http://www.dlib.si/details/URN:NBN:SI:doc-GTI6AHLW. - Besedilo v slov. - Bibliografija: str. 437-438. - Abstract; Izvleček Objavljen v: Acta agriculturae Slovenica. - ISSN 1581-9175. - Letn. 99, št. 3 (2012), str. 433-438. 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