AActa agriculturae Slovenica • eISSN 1854-1941 • 118 – 2 • Ljubljana, junij 2022 118•2 2022ACTA AGRICULTURAE SLOVENICA Acta agriculturae Slovenica Letnik / Volume 118 · Številka / Number 2 · 2022 eISSN 1854-1941 Glavna in odgovorna urednika / Editors-in-Chief Franc BATIČ, rastlinska pridelava / plant production Jernej OGOREVC, živalska prireja / animal production Področni uredniki / Section Editors Franc BATIČ (botanika in ekologija rastlin / botany and plant ecology), Majda ČERNIČ-ISTENIČ (agrarna ekonomika in razvoj podeželja / agricultural economics and rural development), Jure ČOP (pridelovanje krme / fodder production), Zalika ČREPINŠEK (agrometeorolologija / agrometeorology), Marko FLAJŠMAN (poljedelstvo / field crops), Matjaž GLAVAN (urejanje kmetijskih zemljišč / agricultural land management), Helena GRČMAN (pedologija / soil science), Andrej GREGORI (gojenje gob / mushrooms growing), Metka HUDINA (hortikultura / horticulture), Anton IVANČIČ (genetika in biotehnologija / genetics and biotechnology), Jernej JAKŠE (genetika in biotehnologija / genetics and biotechnology), Damjana KASTELEC (statistika / statistics), Aleš KOLMANIČ (poljedelstvo / field crops), Zlata LUTHAR (genetika in biotehnologija / genetics and biotechnology), Andrej LAVRENČIČ (pridelovanje krme / fodder production), Marina PINTAR (urejanje kmeti- jskih zemljišč / agricultural land management), Andrej SIMONČIČ (varstvo rastlin / plant protection), Stanislav TRDAN (var- stvo rastlin / plant protection), Andrej UDOVČ (agrarna ekonomika in razvoj podeželja / agricultural economics and rural de- velopment), Andreja URBANEK-KRANJC (fiziologija rastlin / plant physiology), Rajko VIDRIH (živilstvo / food technology), Dominik VODNIK (fiziologija rastlin / plant physiology), Filip VUČANJK (kmetijsko strojništvo / agricultural machinery) Peter DOVČ (živalska biotehnologija / animal biotechnology, populacijske študije / population studies, genomika / genomics), Milena KOVAČ (selekcija in biometrija / selection and biometry), Janez SALOBIR (prehrana / nutrition) Mednarodni uredniški odbor / International Editorial Board Dunja BANDELJ (Koper, Slovenia), Iryna BANDURA (Melitopol, Ukraine), Michael BLANKE (Bonn, Germany), Marko FLAJŠMAN (Ljubljana, Slovenia), Jürg FUHRER (Liebefeld-Bern, Switzerland), Helena GRČMAN (Ljubljana, Slovenia), Metka HUDINA (Ljubljana, Slovenia), Anton IVANČIČ (Maribor, Slovenia), Lučka KAJFEŽ BOGATAJ (Ljubljana, Slovenia), Damijana KASTELEC (Ljubljana, Slovenia), Iztok KOŠIR (Žalec, Slovenija), Chetan KESWANI (Varanasi, India), Ivan KREFT (Ljubljana, Slovenia), Jaromír LACHMAN (Prague, Czech Republic), Salim LEBBAL (Khenchela, Algeria), Mario LEŠNIK (Maribor, Slove- nia), Zlata LUTHAR (Ljubljana, Slovenia), Senad MURTIĆ (Sarajevo, Bosnia and Herzegovina), Alessandro PERESSOTTI (Udine, Italy), Hardy PFANZ (Essen, Germany), Slaven PRODANOVIĆ (Belgrade, Serbia), Naser SABAGHNIA (Maragheg, Iran), Ola- lekan Suleiman SAKARIYAWO (Abeokuta, Nigeria), Andrej SIMONČIČ (Ljubljana, Slovenia), Giuseppe SORTINO (Palermo, Italy), Bojan STIPEŠEVIĆ (Osijek, Croatia), Massimo TAGLIAVINI (Bolzano, Italy), Željko TOMANOVIĆ (Beograd, Serbia), Stanislav TRDAN (Ljubljana, Slovenia), Andrej UDOVČ (Ljubljana, Slovenia), Rajko VIDRIH (Ljubljana, Slovenia), Dominik VODNIK (Ljubljana, Slovenia), Alena VOLLMANNOVA (Nitra, Slovak Republic) Drago BABNIK (Ljubljana, Slovenia), Tomaž BARTOL (Ljubljana, Slovenia), Michel BONNEAU (Saint Gilles, Belgium), Milena KOVAČ (Ljubljana, Slovenia), Amarendra Narayan MISRA (Balasore, Orissa, India), Zdenko PUHAN (Zürich, Switzerland), Dejan ŠKORJANC (Maribor, Slovenia), Jernej TURK (Maribor, Slovenia) Tehnični uredniki / Technical Editors Karmen STOPAR, Jure FERLIN, Jože STOPAR Oblikovanje / Graphic art and design Milojka ŽALIK HUZJAN Jezikovni pregled / Proofreading Avtorji v celoti odgovarjajo za vsebino in jezik prispevkov / The authors are responsible for the content and for the language of their contributions. Založnik in izdajatelj / Publisher and Issuer Založila / Published by: Založba Univerze v Ljubljani / University of Ljubljana Press Za založbo / For the Publisher: Gregor MAJDIČ, rektor Univerze v Ljubljani / the Rector of the University of Ljubljana Izdala / Issued by: Univerza v Ljubljani, Biotehniška fakulteta / University of Ljubljana, Biotehnical Faculty Za izdajatelja / For the Issuer: Nataša POKLAR ULRIH, dekanja Biotehniške fakultete UL / the Dean of the Biotehnical Faculty UL Naslov Uredništva / Editorial Office Address Univerza v Ljubljani, Biotehniška fakulteta, Acta agriculturae Slovenica Jamnikarjeva ulica 101, SI-1000 Ljubljana E-naslov / E-mail Acta.Agriculturae.Slovenica@bf.uni-lj.si Spletni naslov / Web address http://ojs.aas.bf.uni-lj.si Avtorske pravice / Copyright Avtorji / By the authors Članki so odprtodostopni v skladu z določili in pogoji licence Creative Commons CC BY 4.0 – Priznanje avtorstva (https://creativecommons.org/licenses/by/4.0/deed.sl) Open access articles are distributed under the terms and conditions of the Creative Commons Attribution (CC BY 4.0) licence (https://creativecommons.org/licenses/by/4.0/) Acta agriculturae Slovenica izhaja samo kot spletna revija, skupni letnik pa praviloma obsega štiri številke. / Acta agriculturae Slovenica is published only as an online journal with four issues per year in one common volume. Trenutno revija ne zaračunava stroškov za predložitev in obdelavo vključenih prispevkov. / The journal does not charge APCs or submission charges. Acta agriculturae Slovenica izhaja s finančno pomočjo / is published with the financial support: Javne agencije za raziskovalno dejavnost Republike Slovenije / Slovenian Research Agency. Acta agriculturae Slovenica je vključena v / is included into: Scopus (SJR, SNIP), DOAJ, WOS Zoological Records, CrossRef, CAB Abstracts, FSTA, Google Scholar, dLib, COBISS. Ovitek: Noduli na koreninah soje po inokulaciji semen z aktivnim sevom bakterije Bradyrhizobium japonicum (Kirchner, 1896), Jordan, 1982 D87 v razvojni stopnji začetka cvetenja: А – inoku- lacija semen na dan setve, B – inokulacija semen 7 dni pred setvijo (Foto: Petro Pukhtaievych, 1–11) Cover: Nodules on soybean roots at seeds inoculation by the active strain Bradyrhizobium japonicum (Kirchner, 1896), Jordan, 1982 D87 at the budding-beginning of flowering stage: A – inoculation at the sowing day, B – inoculation in 7 days before sowing (Photo: Petro Pukhtaievych, 1–11) Acta agriculturae Slovenica Volume / Letnik 118 · Number / Številka 2 · 2022 Table of Contents / Kazalo Original Scientific Article / Izvirni znanstveni članek Open vertical farms: a plausible system in increasing tomato yield and encouraging natural suppression of whiteflies Navpični način gojenja: racionalen sistem za povečanje pridelka paradižnika in vzpodbuda za sonaravno zatiranje tobačnega ščitkarja Suleiman MUSTAPHA, Abdulrasak Kannike MUSA, Oluropo Ayotunde APALOWO, Abdrahaman Adebowale LAWAL, Olaniyi Israel OLAYIWOLA, Helen Olaide BAMIDELE, Robert Omotayoman UDDIN II 1–9 Comprehensive seed priming assessment of Hibiscus sabdariffa L. in germination and seedling growth stage under salt stress Ocena predobravnavanja semen vrste osleza Hibiscus sabdariffa L. v razvojnih stopnjah kalitve in kalice v razmerah solnega stresa Mostafa AHMADIZADEH, Ashkan ASGARI, Hossein PASALARI 1–17 Comparative study between fungicides and some chemical inducers for controlling root rot incidence of green bean (Phaseolus vulgaris L.) under field conditions Primerjalna raziskava fungicidov in kemičnih vzpodbujevalcev za nadzor koreninske gnilobe pri fižolu (Phaseolus vulgaris L.) v razmerah na prostem Nehal Samy EL-MOUGY, Nadia Gamel EL-GAMAL, Mohamed Saied Ali KALIL, Mokhtar Mohamed ABDEL-KADER 1–9 Influence of different sources of nitrogen fertilizer and weed control on yield, yield components and some qualitative traits of chickpea (Cicer arietinum L.) cultivars under dryland conditions of Khorramabad Vpliv različnih dušikovih gnojil in uravnavanja plevelov na pridelek, komponente pridelka in kakovostne lastnosti sort čičerke (Cicer arietinum L.) v sušnih razmerah Khorramabada Sajad KORDI, Tayebeh DANAYE-TOUS, Soheila DASTBORHAN 1–13 The effect of some additives on the rheology of dough and quality of bread Učinki nekaterih dodatkov na reološke lastnosti testa in kakovost kruha Xhabir ABDULLAHI, Gafur XHABIRI, Erhan SULEJMANI, Faton SELIMI 1–7 Sublethal effects of some insecticides on the functional response of Aenasius bambawalei Hayat, 2009 (Hymenoptera: Encyrtidae) Subletalni učinki nekaterih insekticidov na funkcionalen odziv vrste Aenasius bambawalei Hayat, 2009 (Hymenoptera: Encyrtidae) Zeinab RAFATIAN, Nooshin ZANDI-SOHANI, Fatemeh YARAHMADI 1–8 Morphological, biochemical, and nutritional value of prickly and smooth fruit spinach Morfološka, biokemična in hranilna vrednost špinače z gladkimi in bodečimi plodovi Reza ABOLGHASEMI, Maryam HAGHIGHI, Nematollah ETEMADI 1–13 Studies of the impact of environmental conditions and varietal features of sweet cherry on the accumulation of vitamin C in fruits by using the regression analysis method Preučevanje vpliva vremenskih dejavnikov in lastnosti sort na vsebnost vitamin C v plodovih češenj z metodo regresijske analize Іryna IVANOVA, Мarina SERDYUK, Vira MALKINA, Tetiana TYMOSHCHUK, Marharyta VOROVKA, Ivan MRYNSKYI, Anastasiia ADAMOVYCH 1–12 Genotypic variation in response to drought stress is associated with biochemical and transcriptional regulation of ureides metabolism in common bean (Phaseolus vulgaris L.) Genetska spremenljivost odziva navadnega fižola (Phaseolus vulgaris L.) na sušni stres je povezana z biokemičnim in transkripcijskim uravnavanjem presnove ureidov Motlalepula PHOLO-TAIT, Thuto KGETSE, Gaone Nthabeleng TSHEKO, Olerato Tshotlhe THEDI, Katso LETHOLA, Ebenezer Oteng MOTLAMME, Moagisi Innocent ITHUTENG, Samodimo NGWAKO 1–9 Enhancement of shoot proliferation and evaluation of biotic elicitation effects on anatomical changes of pseudo stem and anti-lipid peroxidation activity of Curcuma mangga Val. Pospeševanje tvorbe poganjkov in ovrednotenje elicitacijskih učinkov na anatomske spremembe navideznih stebel in proti maščobne peroksidacijske aktivnosti kurkume (Curcuma mangga Val.) Fariz ABRAHAM, Lai-Keng CHAN, Gunawan INDRAYANTO, Peng Lim BOEY 1–11 Effects of γ-radiation on chickpea (Cicer arietinum) varieties and their tolerance to salinity stress Učinki γ-sevanja na sorte čičerke (Cicer arietinum L.) in njihova toleranca na slanostni stres Amal Abdel-Nasser ABDOUN, Laila MEKKI, Aladdin HAMWIEH, Abdelfattah BADR 1–16 Symbiotic and physiological indicators of soybean inoculated of Bradyrhizobium japonicum single-strain in 7 days before sowing Simbiontski in fiziološki indikatorji soje, inokulirane sedem dni pred setvijo s sevom bakterije Bradyrhizobium japonicum Nadiya VOROBEY, Kateryna KUKOL, Petro PUKHTAIEVYCH, Tetyana KOTS 1–11 Review Article / Pregledni znanstveni članek The usage of beneficial insects as a biological control measure in large-scale farming - a case study review on Trichogramma spp. Uporaba koristnih žuželk kot merilo biotičnega varstva pri kmetovanju na velikih zemljiščih - pregledna raziskava na primeru parazitoidnih os iz rodu Trichogramma Aleksandar IVEZIĆ, Branislav TRUDIĆ, Gordon DRAŠKIĆ 1–13 Acta agriculturae Slovenica, 118/2, 1–9, Ljubljana 2022 doi:10.14720/aas.2022.118.2.2272 Original research article / izvirni znanstveni članek Open vertical farms: a plausible system in increasing tomato yield and encouraging natural suppression of whiteflies Suleiman MUSTAPHA 1, 2, 3, Abdulrasak Kannike MUSA 1, Oluropo Ayotunde APALOWO 4, Abdraha- man Adebowale LAWAL 1, Olaniyi Israel OLAYIWOLA 5, Helen Olaide BAMIDELE 1, Robert Omotayo UDDIN II 1 Received July 07, 2021; accepted March 21, 2022. Delo je prispelo 7. julija 2021, sprejeto 21. marca 2022 1 Department of Crop Protection, University of Ilorin, Nigeria 2 Division of Crop Protection, ICAR-Indian Institute of Horticultural Research, Bengaluru, India 3 Corresponding author, e-mail: juniorsuleiman78@gmail.com 4 Department of Crop Science and Horticulture, Nnamdi Azikiwe University, Awka, Nigeria 5 Institute of Global Food Security, School of Biological Sciences, Queen’s University Belfast, UK Open vertical farms: a plausible system in increasing toma- to yield and encouraging natural suppression of whiteflies Abstract: This study evaluated the effectiveness of open vertical farming in increasing tomato yield and also recruit- ing the presence of ecological service providers in the control of whiteflies. The experiment compared the horizontal farm- ing approach to novel outdoor vertical farm design. Using both raised and flatbeds to represent horizontal farm, tomato plants were grown in a spacing of 3.6 and 2.4 m2 respectively while the vertical farm covered a land space of 1.8 m2 hav- ing three arrays with array 1 at ground level, array 2 and 3 were elevated at 110 and 220 cm high respectively. Data col- lected included the numbers of Bemisia tabaci (Gennadius, 1889) and predatory spiders and; tomato fruit yield (g). Re- sults indicated that the mean number of predatory spiders in the vertical farm from 6 – 10 weeks after transplanting were able to supress B. tabaci populations when compared to the horizontal farm. The total fruit yield harvested indicated that the vertical farm produced more tomato fruit yield compared to the horizontal farm. It is plausible that the practice of out- door vertical farming may be a step approach solution to land shortages and also a sustainable system for integrated pest management. Key words: Vertical farm; Bemisia tabaci; predator-prey interaction; biological control; tomato; insect pest Navpični način gojenja: racionalen sistem za povečanje pri- delka paradižnika in vzpodbuda za sonaravno zatiranje to- bačnega ščitkarja Izvleček: V raziskavi je bilo ovrednoteno gojenje para- dižnika v navpičnem sistemu z namenom povečanja pridelka in kot način ekološkega uravnavanja tobačnega ščitkarja. V poskusu sta bila primerjana dva načina gojenja in sicer obi- čajen vodoraven in navpičen sistem gojenja na prostem. Pri vodoravnem načinu gojenja so bile uporabljene visoke in na- vadne grede, kjer je posamezna rastlina paradižnika pokrivala 3,6, oziroma 2,4 m2. Pri navpičnem načinu gojenja je posa- mezna rastlina zavzemala 1,8 m2 v treh višinah in sicer na tleh (1), na višini 110 cm (2) in 220 cm (3). Parametri, ki so bili merjeni so obsegali število osebkov škodljivca (Bemisia tabaci (Gennadius, 1889) in predatorskih pajkov ter pridelek paradižnika (g). Rezultati so pokazali, da je številu predator- skih pajkov v navpičnem sistemu gojenja v 6 do 10 tednih po sadnji bolje uspelo zatreti populacijo škodljivca v primerjavi z vodoravnim načinom gojenja. Tudi celokupen pridelek pa- radižnika je bil pri navpičnem načinu gojenja večji kot pri vodoravnem. Iz izsledkov lahko zaključimo, da je gojenje pa- radižnika v navpičnem načinu gojenja na prostem racionalen korak pri reševanju pomanjkanja zemljišč kot pri trajnostnem uravnavanju škodljivcev. Ključne besede: navpišni način gojenja; Bemisia ta- baci; interakcija plenilec-plen; biološki nadzor; paradižnik; škodljive žuželke Acta agriculturae Slovenica, 118/2 – 20222 S. MUSTAPHA et al. 1 INTRODUCTION Food insecurity is fast becoming an increasingly vital matter worldwide (Al-Kodmany, 2018). It has been predicted that the urban population will constantly rise in the coming decades. At the same time, land experts (i.e., ecologists, agronomists, and geologists) warn of in- tensifying shortages of farmland (Corvalan et al., 2005; Healy and Rosenberg, 2013; Thomaier et al., 2015). With a rapidly expanding population and changing climate, pressures on food production systems are expected to increase in the coming years (FAO, 2018). Traditional farming methods cannot produce enough food to feed the world’s growing population and may fail in future (Despommier, 2013, Touliatos et al., 2016; Muller et al., 2017). Therefore, there is urgently the need for trans- formative solutions in food production. Vertical farm- ing has been proposed as a way out in addressing the problem of farmland shortages because of its promises in maximizing small spaces to grow more crops and its sustainability to the environment (Corvalan et al., 2005; Despommier, 2014; Healy and Rosenberg, 2013; Thomaier et al., 2015) although, the effects it has on dif- ferent insect pest complex is still not fully studied. Herein, we examined how outdoor vertical farm- ing might be used to increase yields and also in the sus- tainable management of insect pests by investigating the fruit crop tomato which easily adapts to this tech- nique. Tomato, having lots of culinary and nutritional benefits is attacked by different pests a major of which are the whiteflies (Varela et al., 2003; Waiganjo et al., 2006). Whiteflies are highly polyphagous and are also known vectors of the tomato yellow leaf curl viruses (TYLCV) (Scholthof et al., 2011). The whitefly Bemi- sia tabaci (Hemiptera: Aleyrodidae) larvae produces honeydew on which sooty moulds grows, reducing the photosynthetic capabilities of the plant and result- ing in defoliation and stunting (European and Medi- terranean Plant Protection Organization, 2004). The primary method used to control the insect pest is by the application of insecticides which unfortunately be- ing practiced mainly in traditional horizontal farming has led to the development of resistance to numerous types of insecticides, reduction of beneficial arthropods and causing negative impacts on human health and the environment at large (Denholm et al., 1998; Matthews, 2008). Natural enemies are helpful in curtailing the de- structiveness of the insect pest with previous reports on predators such as wasps, lacewings, mites and also spiders effective in bringing whitefly population down (Gerling, 2001). Although, with limited information on its application and implication in vertical farming ap- proach (Roberts et al., 2020). It is also not clear if the technique of vertically growing crops outdoors could be useful in checking the population of B. tabaci by natural intervention of predators as observed in other farming practices. Therefore, the current study is an at- tempt to answer the following questions: Can outdoor vertical farms increase the yields of crops?, and, would it be sustainable enough to support natural suppression of key insect pests? Fig. 1: Schematic diagram of the open-air vertical structure used for this experiment Acta agriculturae Slovenica, 118/2 – 2022 3 Open vertical farms: a plausible system in increasing tomato yield and encouraging natural suppression of whiteflies 2 MATERIALS AND METHODS 2.1 STUDY SITE The study was conducted at the Department of Agronomy, Faculty of Agriculture, University of Ilorin (latitude 8o29’9N and longitude 4o35’38E), Kwara State. This area is located in the Southern Guinea Savannah Ecological Zone of Nigeria. The area was characterized by a bimodal rainfall with peaks in June and Septem- ber and an annual rainfall between 1000-1240 mm. The study was carried out from December 2016 to March 2017. 2.2 ARRANGEMENTS FOR THE HORIZONTAL AND VERTICAL FARMS The flatbeds were made in a size of 150 cm × 160 cm (2.4 m2) each with an inter-bed spacing of one me- tre (1 m). The plot spacing for each raised bed was 240 × 150 cm (3.6 m2) with an inter-bed spacing of one me- tre (1 m). The vertical farm was built using open-air vertical structures (Fig. 1) 3.2 m high using 8 cm wide and 5 cm thick wood (Garg and Balodi, 2014). It was built in a spacing of 120 × 150 cm (1.8 m2) with an inter vertical farm spacing of 1 m apart. 2.3 TREATMENTS AND EXPERIMENTAL DE- SIGN The experiment was laid out in a randomized com- plete block design with four replicates. The horizontal farm was made into two types of beds; raised beds and flatbeds, these served as the treatments in the horizon- tal farm. While the vertical farm was made having three arrays; with Array 1 at ground level (GL), Array 2 at 110 cm high and Array 3 at 220 cm high and these served as treatments in the vertical farm. 2.4 LAND PREPARATION AND PLANTING ON THE HORIZONTAL AND VERTICAL FARMS The raised and flatbeds in the horizontal farm were prepared on farmland previously cultivated for tomato having sandy loam soil that was well drained. The to- mato variety used for this study was UC82B packaged and supplied by the trademark company Technisem. Tomato seedlings were grown in the nursery in a screen house and were transplanted 4 weeks after sowing on the horizontal farm at a spacing of 50 × 80 cm on both beds according to the manufacturer’s spacing instruc- tion, with each bed containing a total of twelve tomato seedlings and 36 seedlings for the three replicates per bed type. In the vertical farm, tomato seedlings were transplanted from the nursery into 7 litre buckets with a diameter of 25 cm and a height of 23 cm filled with sandy loam soil with also history of tomato cultivation. Each vertical array contained twelve (12) buckets with a total of thirty-six (36) buckets per vertical farm and 108 buckets for the three replicates. Tomato seedlings that were introduced to the third array of a growing height of 220 cm high were gradually introduced to this height from ground level to 110 cm high and finally to 220 cm high within four days interval. At 2 weeks after transplanting (WAT), tomato seed- lings were lightly pruned by cutting off a few branches to encourage its growth and acclimation in both the vertical and horizontal farm. N.P.K (15-15-15) fertilizer was applied at the rate of 120 kg ha-1 3WAT to boost the growth of the crops in both farms. The fertilizer was applied by ring placement into drills 5 cm deep and 7 cm away from the plant and covered with soil (Olaniyi et al., 2010). Watering of tomato was done daily in both the vertical and the horizontal farm at 8:00 am using a watering can. 2.5 COLLECTION AND IDENTIFICATION OF ASSOCIATED ARTHROPODS Adult whiteflies were collected using aspirator and yellow sticky traps while a x10 magnifying lens was used for the observation of the presence of puparia or pupal cases underneath tomato leaves before taking leaf samples for viewing under a stereo microscope. All col- lected whitefly samples were identified to the species level on the basis of morphological characters of adults, puparium and/or pupal case (Simala et al., 2009). The observed spiders in this experiment were not identified to species, genus, nor family level due to the lack of taxonomist specialized in arachnology in the country hence difficulties were experienced in speciat- ing spiders. 2.5.1 Data collection Nine (9) tomato plants were selected at random from each replicate from both the horizontal and ver- tical farm and data was collected for the number of adult whiteflies, predatory spiders and total fruit yield (g). The numbers of whiteflies were estimated by divid- ing the crop canopy into three layers: upper (> 40 cm), Acta agriculturae Slovenica, 118/2 – 20224 S. MUSTAPHA et al. intermediate (20-40 cm) and lower (0-20 cm) and se- lecting five leaves from each layer per plant which were gently turned over to the abaxial side to count the total number of adult whiteflies (Sequeira and Naranjo, 2008; López et al. 2010). This was performed from 7:30-9:30 am. Numbers of spiders were estimated by counting the total number of spiders seen per crop. This was carried out from 7-9:30 pm when spiders were observed to be very active and could be easily spotted with a flashlight. The number of B. tabaci on the tomato plants in both the vertical and horizontal farm were determined to have reached action threshold when above 5 of the in- sect were counted per leaf according to Ellsworth and Martinez-Carrillo (2001). Tomato fruits harvested from both farms at the end of the experiment were measured on a weighing scale calibrated in grams. 2.5.2 Data Analysis Data was presented in mean and standard error of mean (SEM) and significant differences between means were separated according to Kruskal-Wallis one- way analysis of variance by allocating ranks to means. Spearman correlation analysis was done to determine the association between population of spiders, B. tabaci and fruit yield using SPSS 20th Edition. 3 RESULTS 3.1 Bemisia tabaci ON BOTH HORIZONTAL AND VERTICAL FARMS Table 1 shows that the infestation of B. tabaci started at 1 week after transplanting (WAT) in both the horizontal and vertical farms with the flatbed having a significantly (H (4) = 11.500, p = 0.021) higher mean number of 12.81. There were no significant differences between the mean rank of B. tabaci in both the horizon- tal and vertical farms from 2 to 4 WAT. The horizontal farm, on the other hand, reached its peak population of B. tabaci at 5 WAT in the flat bed with a mean number of 14.69 (H (4) = 11.360, p = 0.023) and 9 WAT in the raised bed with a mean population of 13.35 (H (4) = 13.745, p = 0.008) significantly higher than the vertical farm (Table 1). Throughout the experiment, the hori- zontal farm experienced the most numbers of B. tabaci adults affecting tomato plants when compared to the vertical farm as seen in Table 1. 3.2 SUPPRESSION OF Bemisia tabaci BY SPIDERS Table 2 shows that there were no significant differ- ences between the mean rank numbers of the predatory spiders observed at 3 (H (4) = 6.222, p = 0.183) and 4 (H (4) = 8.038, p = 0.090) WAT in both the vertical and horizontal farms. Even though a significant population of predatory spiders was observed at 5 WAT (H (4) = 10.678, p = 0.030) in the vertical farm, it was not able to bring the population of B. tabaci below the action threshold (Tables 1 and 2). Tables 1 and 2 also revealed that biological sup- pression by natural intervention of spiders (Fig. 2) was initially achieved only in the vertical farm at 6 WAT and started with the mean spider number of 13.00 in Array 3 significantly (H (4) = 12.616, p = 0.013) able to bring down the population of B. tabaci to the mean number of 0.00 (Table 1). This continued further as the number of spiders gradually increased in the vertical farm from 7 to 10 WAT and reached its peak at 10 WAT in array 3 with the mean number of 35.00 which was highly significantly (H (4) = 12.994, p = 0.011) effec- tive in suppressing the population of B. tabaci below the action threshold when compared to the horizontal farm which was above it throughout the period of the experiment (Table 1 and 2), this was as a result of the Farm Type Population of B. tabaci in horizontal and vertical farms (WAT) 1 2 3 4 5 6 7 8 9 10 Horizontal Raised bed 9.15d 8.61c 7.26b 11.75e 13.22d 9.54d 8.28b 9.51c 13.35c 8.84b Flat bed 12.81e 9.75d 10.64d 10.51c 14.69e 11.84e 9.12c 7.04b 9.38b 10.80c Vertical Array 1 8.72c 7.75b 13.79e 6.54a 7.64c 4.99c 0.00a 0.00a 0.00a 0.00a Array 2 6.52b 7.68a 6.96a 8.87d 7.38b 3.73b 0.00a 0.00a 0.00a 0.00a Array 3 5.30a 11.00e 8.08c 6.33b 5.80a 0.00a 0.00a 0.00a 0.00a 0.00a SEM 0.82 0.50 1.44 1.35 1.24 1.05 1.28 0.98 0.60 0.61 Table 1: Number of Bemisia tabaci on the vertical and horizontal farm Superscripts within column indicates mean rank number according to Kruskal-Wallis Test, with a = rank 1, b = rank 2, c = rank 3, d = rank 4 and e = rank 5; 1 being the lowest to 5 the highest rank, SEM = Standard error of mean Acta agriculturae Slovenica, 118/2 – 2022 5 Open vertical farms: a plausible system in increasing tomato yield and encouraging natural suppression of whiteflies significantly low numbers of spiders recorded in the horizontal farm which were less than that of the verti- cal farm as shown in Table 2. 3.3 MASS OF TOMATO FRUIT YIELD IN HORI- ZONTAL AND VERTICAL FARM The total tomato fruit yield (g) harvested from both the horizontal and vertical farms are shown in Table 3. There was no significant (H (4) = 7.767, p = 0.101) differences between the vertical and the horizon- tal farm. Further observations on the mean rank of the fruit mass indicated that array 3, array 2 and array 1 of the vertical farm had the highest tomato fruit mean mass of 67.10, 61.20 and 55.10 respectively when com- pared to the horizontal farm (Table 3). 3.4 CORRELATION BETWEEN NUMBER OF B. tabaci, NUMBER OF SPIDERS AND FRUIT YIELD Significantly negative correlation (Rs = -0.999) was observed between the number of spiders and the num- ber of B. tabaci in the vertical farm as shown in Table 4. The horizontal farm, on the other hand, the numbers of spiders observed did not have a significantly negative correlation effectively enough to reduce the population of B. tabaci (Rs = -0.318) compared to the vertical farm. Table 4 also showed that the fruit yield in both the horizontal and vertical farm was affected by the population of B. tabaci having a negative correlation of -0.28 and -0.813 respectively. However, the correlation of the number of spiders in the vertical farm with the fruit yield (Rs = 0.806) showed that the presence of the spiders positively influenced the fruit yield in the verti- cal farm compared to the horizontal farm which had a negative correlation of -0.256 in its fruit yield as seen in Table 4. 4 DISCUSSION The current study is a novel approach in indicating the usefulness of outdoor vertical farming technique in sustainable crop production. Despommier as described by Corvalan et al. (2005) and Al-Kodmany (2018) hinted that vertical farming system will succeed only if they function by imitating natural ecological processes. The outdoor vertical farming system could be useful in supporting the practice of growing crops organically. The design allows for the natural use of sunlight and also encourages the natural interactions with ecologi- cal service providers as observed in this research. The experiment indicated that spiders acted as positive ser- vice providers in terms of natural suppression against whiteflies affecting tomato grown using open-air verti- cal farms. The technique of outdoor vertical farm un- like the indoor ultra-modernized versions allows for the interaction of plants with beneficial arthropods. Plants’ evolutionary response to pest damage is to emit Farm Type Population of predatory spiders in horizontal and vertical farms (WAT) 3 4 5 6 7 8 9 10 Horizontal Raised bed 0.00a 0.33a 0.00a 0.67a 1.00b 1.00b 6.30b 0.00a Flat bed 0.00a 0.33a 0.33b 0.67b 1.70a 0.30a 4.30a 0.00a Vertical Array 1 0.00a 0.33a 9.33e 5.00c 15.00c 21.00c 23.70e 16.30b Array 2 0.67c 1.00b 7.67c 10.33d 20.30d 22.30d 21.70d 20.00c Array 3 0.33b 3.33c 8.67d 13.00e 34.00e 27.00e 21.00c 35.00d SEM 0.19 0.73 1.25 1.10 4.02 2.49 3.61 2.31 Table 2: Number of predatory spiders on the vertical and horizontal farm Superscripts within column indicates mean rank number according to Kruskal-Wallis Test, with a = rank 1, b = rank 2, c = rank 3, d = rank 4 and e = rank 5; 1 being the lowest to 5 the highest rank, SEM = Standard error of mean Farm Type Mass of tomato fruit (g) Horizontal Flat bed 16.90a Raised bed 30.50a Vertical Array 1 55.10a Array 2 61.20a Array 3 67.10a SEM 14.03 Table 3: Mass of tomato fruits (g) in both horizontal and vertical farms Acta agriculturae Slovenica, 118/2 – 20226 S. MUSTAPHA et al. a unique chemical signal known as herbivore induce plant volatiles- a distress signal to recruit the services of predatory arthropods who feed off these pest (Karban and Baldwin, 1997; Thaler, 1999; Kessler and Baldwin, 2001; Lou et al., 2006; Pickett et al., 2006). This study positively indicated that the ability of crops to emit this substance is not restricted using open vertical farming techniques. The vertical farm created a favourable niche Figure 2: Showing presence of spiders in the vertical farm. Pictures a, b: green spiders observed using camouflage to hunt for preys; pictures c, d: two different species of spider on their web to capture prey Horizontal farm Vertical Farm BTH SPH BTV SPV Horizontal farm BTH - - - - SPH -0.318 - -0.788 - FYH -0.28 -0.256 0.693 -0.699 Vertical farm BTV 0.363 - - - SPV -0.333 0.802 -0.999** - FYV -0.083 0.303 -0.813 0.806 Table 4: Correlation between no. of B. tabaci, no. of spiders and fruit yield in the vertical and horizontal farm *Correlation is significant at the 0.05 level (2 tailed), ** Correlation is significant at the 0.01 level (2-tailed) BTH=B. tabaci horizontal farm, BTV= B. tabaci vertical farm, SPH= Spiders horizontal farm, SPV= Spiders vertical farm, FYH= Fruit yield horizontal farm, FYV= Fruit yield vertical farm, - = no correlation Acta agriculturae Slovenica, 118/2 – 2022 7 Open vertical farms: a plausible system in increasing tomato yield and encouraging natural suppression of whiteflies for the increase in the population of spiders by having adequate platforms where spiders could set up webs and could easily move around to reach and capture their prey within the varying heights (Jayakumar and Sankari 2010). Spiders, being a carnivorous arthropod, typically preying on insects, positively provided an im- portant service in keeping the population of B. tabaci below the action threshold by natural intervention in the vertical farm in all three arrays (Marshall, 2006; Oy- eniyi and Oyeseyi, 2014). Sahu et al. (1996) and Jayakumar and Sankari (2010) studied the predatory efficiency of spiders in the suppression of pests in some field crops. In their study, there was no comparison to different growing heights to the predatory potential of spiders as shown in this research that growing heights using vertical farm could also be resourceful in influencing the increase of spi- ders to control pests. Correlation analysis also revealed that the rise in the population of spiders has a strong effect in suppressing the population of B. tabaci in the vertical farm when compared to the horizontal farm. The increased population of spiders in the vertical farm limited the population of whiteflies (Jayakumar and Sankari, 2010). The practice of vertical farming is considered to promote sustainable agricultural prac- tices more than that adopted by conventional farming method (horizontal farm), which refers to large scale, outdoor agriculture that embraces techniques that en- gage heavy irrigation, intensive tillage and excessive use of fertilizers, and pesticides (Despommier, 2007; Healy and Rosenberg, 2013). The fruit yield data collected from both the ver- tical and horizontal farm indicated that even though the horizontal farm produced fruit yield that was not significantly different from the different arrays of the vertical farm, the same was not enough yield in com- parison to the vertical farm that gave better fruit yield. The increased fruit yield harvested from the vertical farm may be due to the ability of the farm to grow crops in arrays in limited space by stacking crops above each other which is an advantage over the horizontal farm- ing method that makes use of huge expanse of land (Garg and Balodi, 2014; Hossain et al., 2015). With a lit- tle utilized space of about 1.8 square metre in the verti- cal farm, more fruit yield was gotten when compared to the horizontal farm space of 3.6 square metre and this is indicative of the facts that this technique could be used to increase food yield where land is fast becoming a limited resource. Also, the different heights of the ver- tical arrays in an outdoor situation would ensure that elevated plants have greater and better access to ambi- ent amount of sunlight which will positively affect the performance of the crop to produce more fruit (Garg and Balodi, 2014). By vertically growing crops, it would not only mitigate the need for more land, it would also produce available growing space in the air where crops could be grown in arrays to get more yield as shown in this study (Sarkar and Majumder, 2015; Hossain et al., 2015; Despommier, 2009; Garg and Balodi, 2014). This meth- od also ensured the maximum use of land for tomato production without wastage and could address the loss of cultivable land by utilizing the spaces around house- holds by suspending crops vertically and may eliminate the need to create additional farmland and also help create a cleaner environment with the use of less crop protection products such as pesticides that contami- nates the environment and by encouraging the activi- ties of natural enemies like spiders against the activities of insect pests (Despommier, 2009; Hossain et al., 2015). We would like to put some caution on the inter- pretation of our result. While we did not identify the species of spiders and also report direct consumption of whiteflies by them through molecular analysis of their gut contents, the increased presence of spiders in the vertical farm may have had a threatening effect on the whiteflies and as such reduced their numbers sig- nificantly. Similarly, Southon et al. (2019) in an experi- ment they conducted by studying biological control of predatory wasps against the insect pest fall armyworm, observed that the presence of wasps negatively affected the feeding habit of fall armyworm, reduced their body mass and also kept them in hiding. It is plausible to in- fer here that whiteflies may have noticed the increasing population of spiders in the vertical farm and as such felt threatened and would rather derived nutrition else- where far from the presence of predators. 5 CONCLUSION This study observed the use of open vertical farm- ing in increasing the presence of predatory arthropods such as spiders in the natural suppression of B. taba- ci, a major pest of tomato and also to increase yield. Since vertical farming is fast becoming an acceptable trend worldwide due to the overwhelming population increase, the technique could be practiced to produce crops in tight spaces to boost yield to feed the grow- ing populace. Horizontal farming, on the other hand, is just not sufficient enough to meet the needs of this ever-increasing population due to the rapid rate of ur- banization. Outdoor vertical farming in comparison to the traditional horizontal technique indicated that natural suppression by ecological service providers Acta agriculturae Slovenica, 118/2 – 20228 S. MUSTAPHA et al. could be plausible on crops grown using the technique. Although this is just a pilot trial, further investigations are necessary to ascertain the level of effectiveness open vertical farms will pose in the future to ensure continu- ous sustainable production of food as an alternative to the dwindling agricultural land resources. The practice could be encouraged to minimizing the dependency on chemical pesticides which have been studied to have deleterious effects. Also, the presence of predatory ar- thropods could be further influenced for future inte- grated pest management in open vertical farms. There is, therefore, the need to begin considering this tech- nique as an urban approach to the lack of cultivable lands for food production. 6 CONFLICT OF INTEREST The authors declare that the research was conduct- ed in the absence of any commercial or financial rela- tionships that could be construed as a potential conflict of interest. 7 REFERENCES Al-Kodmany, K. (2018). The vertical farm: a review of devel- opments and implications for the vertical city. Buildings, 8(24), 2-36. https://doi.org/10.3390/buildings8020024 Corvalan, C., Hales, S. & McMichael, A. J., (2005). Ecosystems and human well-being: Health synthesis; World Health Or- ganization: Geneva, Switzerland. Denholm, L., Cahill, M., Byrne, F. J. & Devonshire, A.L. (1996). Progress with documenting and combating insecticide résistance in Bemisia. In: Gerling, D. and Mayer, R.T., (eds.), Bemisia 1995: Taxonomy, Biology, Damage, Control and Management, Intercept Ltd. Andover, Hants. UK, pp. 577-603. Denholm, L., Cahill, M., Dennehy, T. J. & Horowitz, A. R. (1998). Challenges with managing insecticide résistance in agri- cultural pests, exemplified by the whitefly Bemisia tabaci. Philosophical Transactions of the Royal Society (Lond. B), 353:1757-1767. https://doi.org/10.1098/rstb.1998.0328 Despommier, D. (2007). The vertical farm essay 1: Reduc- ing the impact of agriculture on ecosystem function and services. Retrieved November 8, 2011, from http://www. verticalfarm.com/more?essay1 Despommier, D. (2009). The rise of vertical farms. Scientific American, 301, 80-87. https://doi.org/10.1038/scientifi- camerican1109-80 Despommier, D. (2010). The Vertical Farm: Feeding the World in the 21st Century; Thomas Dunne Books: New York, NY, USA. Despommier, D. (2013). Farming up the city: The rise of ur- ban vertical farms. Trends in Biotechnology, 31, 388–389. https://doi.org/10.1016/j.tibtech.2013.03.008 Despommier, D. (2014). Encyclopedia of Food and Agricul- tural Ethics (Vertical Farms in Horticulture); Springer: Dordrecht, The Netherlands. https://doi.org/10.1007/978- 94-007-0929-4_88 Ellsworth, P. C. & Martinez-Carrillo, J. L. (2001). IPM for Bemisia tabaci: a case study from North America. Crop Protection, 20, 853-869. https://doi.org/10.1016/S0261- 2194(01)00116-8 European and Mediterranean Plant Protection Organiza- tion (2004). Diagnostic protocols for regulated pests. Bulletin, 34, 281–288. https://doi.org/10.1111/j.1365- 2338.2004.00729.x FAO, 2018. Food and Agriculture Organization of the United Nations: FAOSTAT. http://www.fao.org/faostat/en/#data/ EL (last access: 9 March 2018). Garg, A. & Balodi, R. (2014). Recent Trends in Agriculture: Vertical Farming and Organic Farming. Advances in Plants & Agriculture Research, 1(4), 142-144. https://doi. org/10.15406/apar.2014.01.00023 Gerling, D., Alomar, O. & Arnô, J., (2001). Biological control of Bemisia tabaci using predators and parasitoids. In Naranjo, S. E. and Ellsworth, P. C. (eds.), Spécial Issue: Challenges and Opportunities for Pest Management of Bemisia tabaci in the New Century. Crop Protection, 20, 779-799. https:// doi.org/10.1016/S0261-2194(01)00111-9 Healy, R. G. & Rosenberg, J. S. (2013). Land Use and the States; Routledge: New York, NY, USA. https://doi. org/10.4324/9781315064406 Hossain, E., Nabi, S. N. & Kaminski, A. (2015). Vertical ag- riculture: Homestead horticulture suspended in sacks. Penang, Malaysia: WorldFish, Program Brief Jayakumar, S. & Sankari, A. (2010). Spider population and their predatory efficiency in different rice establishment techniques in Aduthurai, Tamil Nadu. Journal of Biopes- ticides, 3, 020-027. Karban, R. & Baldwin, I. T. (1997). Induced Responses to Her- bivory. Chicago University Press, Chicago, Illinois, USA. https://doi.org/10.7208/chicago/9780226424972.001.0001 Kessler, A. & Baldwin, I. T. (2001). Defensive function of herbivore-induced plant volatile emissions in nature. Science, 291, 2141–2144. https://doi.org/10.1126/sci- ence.291.5511.2141 López, R., Carmona, D., Vincini, A. M., Monterubbianesi, G. & Caldiz, D. (2010). Population dynamics and damage caused by the leafminer Liriomyza huidobrensis Blanchard (Diptera: Agromyzidae) on seven potato processing va- rieties grown in temperate environment. Neotropical En- tomology, 39(1), 108-114. https://doi.org/10.1590/S1519- 566X2010000100015 Lou, Y., Xiaoyan, H., Turlings, T. C. J., Cheng, J., Xuexin, C. & Gongyin, Y. (2006). Differences in induced volatile emis- sions among rice varieties result in differential attraction and parasitism of Nilaparvata lugens eggs by the parasi- toid, Anagrus nilaparvatae in the field. Journal of Chemical Ecology, 32, 2375–2387. https://doi.org/10.1007/s10886- 006-9151-7 Marshall, S. A. (2006). Insect: Their Natural History and Diver- sity. Firefly Books Matthews, G. A. (2008). Attitudes and behaviours regard- Acta agriculturae Slovenica, 118/2 – 2022 9 Open vertical farms: a plausible system in increasing tomato yield and encouraging natural suppression of whiteflies ing use of crop protection products—A survey of more than 8500 smallholders in 26 countries. Journal of Crop Protection, 27, 834-846. https://doi.org/10.1016/j.cro- pro.2007.10.013 Muller, A., Ferré, M., Engel, S., Gattinger, A., Holzkämper, A., Huber, R., Müller, M. & Six, J. (2017). Can soil-less crop production be a sustainable option for soil conservation and future agriculture? Land Use Policy, 69, 102–105. htt- ps://doi.org/10.1016/j.landusepol.2017.09.014 Olaniyi, J. O., Akanbi, W. B., Adejumo, T. A. & Akande, O. G., (2010). Growth, fruit yield and nutritional quality of tomato varieties. African Journal of Food Science, 4(6), 398–402. Oyeniyi, A. O. & Oyeseyi, J. O. (2014). Diversity and distribu- tion of spiders in southwestern Nigeria. Natural Resources, 5, 926-935. https://doi.org/10.4236/nr.2014.515079 Pickett, J. A., Bruce, T. J. A., Chamberlain, K., Hassanali, A., Khan, Z. R., Matthes, M. C., Napier, J. A., Smart, L. E., Wadhams, L. J. & Woodcock, C. M. (2006). Plant volatiles yielding new ways to exploit plant defence. In: Dicke, M., Takken, W. (Eds.), Chemical Ecology: From Gene to Eco- system. Springer, Netherlands, pp. 161–173. https://doi. org/10.1007/978-1-4020-5369-6_11 Roberts, J. M., Bruce, T. J. A., Monaghan, J. M., Pope, T. W., Leather, S. R. & Beacham, A. M. (2020). Vertical farming systems bring new consideration for pest and disease management. Annals of Applied biology, 176, 226-232. https://doi.org/10.1111/aab.12587 Sahu S., Sing, R. & Kumar, P. (1996). Host preference and feed- ing potential of spiders predaceous in insect pests of rice. Journal of Entomological Research, 20(2), 145-150. Sarkar, A. & Majumder, M. (2015). Opportunities and chal- lenges in sustainability of vertical eco farming: A review. Journal of Advanced Agricultural Technologies, 2, 98-105. https://doi.org/10.12720/joaat.2.2.98-105 Scholthof, K. B. G., Adkins, S., Czosnek, H., Palukaitis, P. & Jacquot, E. (2011). Top 10 plant viruses in molecular plant pathology. Molecular Plant Pathology, 12, 938–954. https://doi.org/10.1111/j.1364-3703.2011.00752.x Sequeira, R. V., Naranjo & S. E. (2008). Sampling and man- agement of Bemisia tabaci (Genn.) biotype B in Australian cotton. Crop Protection, 27, 1262–1268. https:// doi.org/10.1016/j.cropro.2008.04.002 Šimala, M., Milek, T. M. & Korić, B. (2009). Whitefly species (Hemiptera: Aleyrodidae) recorded on imported orna- mental plants in croatia from 2005–2008. Institute for plant protection in agriculture and forestry of Re- public of Croatia, 389-396. Southon, R. J, Fernandes, O. A., Nascimento, F. S. & Sumner, S. (2019). Social wasps are effective biocontrol agents of key lepidopteran crop pests. Proceedings of the Royal So- ciety B. Soc. B., 286, 20191676. https://doi.org/10.1098/ rspb.2019.1676 Thaler, J. (1999). Jasmonic acid mediated interactions between plants, herbivores, parasitoids and pathogens: a review of field experiments in tomato. In: Agrawal, A. A., Tuzun, S., Bent, E. (Eds.), Induced Plant Defenses Against Pathogens and Herbivores (pp.319-334). APS Press, St. Paul, Minne- sota. Thomaier, S, Specht, K, Henckel, D, Dierich, A, Siebert, R, Freisinger, U. B & Sawicka, M. (2015) Farming in and on urban buildings: Present practice and specific novelties of zero-acreage farming (Z Farming). Renewable Agricul- ture and Food Systems, 30, 43–54. https://doi.org/10.1017/ S1742170514000143 Touliatos, D., Dodd, I. C. & McAinsh, M. (2016). Vertical farm- ing increases lettuce yield per unit area compared to con- ventional horizontal hydroponics. Food and Energy Secu- rity, 5, 184–191. https://doi.org/10.1002/fes3.83 Varela, A. M., Serf, A. & Lohr, B. (2003). A guide to IPM in tomato production in eastern and southern Africa. Interna- tional Centre of Insect Physiology and Ecology Waiganjo, M. M., Wabule, N. M., Nyongesa, D., Kibaki, J. M., Onyango, I., Wepukhulu, S. B. & Muthoka, N. M. (2006). Tomato production in Kirinyaga district, Kenya. A baseline Survey report. 3-4. Acta agriculturae Slovenica, 118/2, 1–17, Ljubljana 2022 doi:10.14720/aas.2022.118.2.2417 Original research article / izvirni znanstveni članek Comprehensive seed priming assessment of Hibiscus sabdariffa L. in germination and seedling growth stage under salt stress Mostafa AHMADIZADEH 1, 2, Ashkan ASGARI 1, 3, Hossein PASALARI 1 Received November 10, 2021; accepted March 22, 2022. Delo je prispelo 10. novembra 2021, sprejeto 22. marca 2022 1 Areas, Minab Higher Education Center, University of Hormozgan, Bandar Abbas, Iran; Research Group of Agroecology in Dryland 2 Corresponding author, e-mail: ahmadizadeh.mostafa@gmail.com 3 Corresponding author, e-mail: asgariashkan6@gmail.com Comprehensive seed priming assessment of Hibiscus sab- dariffa L. in germination and seedling growth stage under salt stress Abstract: This study was performed to appraise the ef- fects of several seed pretreatment solutions and priming time on seed germination indices and growth characteristics of Hi- biscus sabdariffa L. in various salt stress levels. Seed priming was accomplished by KCl (1 and 2 %), Na2SO3 (0.5 and 1 %), KNO3 (0.5 and 1 %), and Ca2CO3 (1 and 2 %) as halopriming and distilled water as hydropriming at 12 and 24 h priming durations and control (non-primed), then primed seeds ex- posed to four levels (0, 50, 100, 200 mM) of NaCl solutions. The highest germination percentage was observed in 12 and 24 h hydropriming (63.3 and 53.3 %) and non-primed (56.6 %) under normal condition, respectively. Besides, there was no germinated seed at 24 h priming by 0.5 and 1 percent- age of KNO3. Under saline condition, 24 h 2 % Ca2CO3 had the highest germination percentage (43.3 %) in 50 mM, while 12 h treatment with 0.5 % Na2SO3 (33.3 %) had high germina- tion percentage in 100 mM levels of saline conditions. Also, the highest germination rate index was observed in 0.5 % Na2SO3 with 12 h treatment time (4.05 and 3.95 respectively) in 50 and 100 mM levels of saline conditions. Overall, salt stress considerably reduced germination and growth traits of Hibiscus sabdariffa L. seedlings. Considering the effect of vari- ous seeds priming of Hibiscus sabdariffa L. on germination indices like germination percentage and mean germination time, the importance of priming duration and type of prim- ing solutions could be concluded. Key words: abiotic stress; medicinal plant; roselle; seed treatment Ocena predobravnavanja semen vrste osleza Hibiscus sab- dariffa L. v razvojnih stopnjah kalitve in kalice v razmerah solnega stresa Izvleček: V raziskavi so bili ocenjeni učinki predobrav- navanja semen z različnimi raztopinami in časi obravnavanja na kalitveni indeks in rastne lastnosti vrste Hibiscus sabdarif- fa L. v razmerah različnega solnega stresa. Predobravnavanje semen je bilo izvedeno z raztopinami KCl (1 in 2 %), Na2SO3 (0,5 in 1 %), KNO3 (0,5 in 1 %), in Ca2CO3 (1 in 2 %) kot obravnavanje s solmi in z destilirano vodo kot vodno obrav- navanje za 12 in 24 h ter kontrolo (brez predobravnavanja). Po tem so bila ta semena izpostavljena raztopinam štirih koncentracij natrijevega klorida (0, 50, 100, 200 mM NaCl). Največji odstotek kalitve je bil ugotovljen pri semenih, ki so bila predobravnavana z vodo za 12 in 24 ur (63,3 in 53,3 %) in pri netretiranih semenih (56,6 %) v normalnih razmerah. Pri predobravanavanju semen za 24 ur z 0,5 in 1 % raztopino KNO3 ni vzklilo nobeno seme. V razmerah slanosti je imelo 24 urno obravnavanje z 2 % raztopino Ca2CO3 največji odsto- tek kalitve (43,3 %) pri 50 mM med tem, ko je imelo12 urno obravnavanje z 0,5 % raztopino Na2SO3 (33,3 %) še vedno velik odstotek kalitve v razmerah 100 mM slanosti. Največja vrednost indeksa kalitve je bila ugotovljena pri obravnavanju z 0,5 % raztopino Na2SO3, z 12 urnim časom obravnavanja (4,05 in 3,95) v razmerah 50 in 100 mM slanosti. Nasplošno je solni stres znatno zmanjšal kalitev in rastne parameter se- jank osleza Hibiscus sabdariffa L.. Upoštevaje učinke različnih predobravnavanj semen osleza Hibiscus sabdariffa L. na kali- tvena indeksa kot sta odstotek kalitve in poprečni čas kalitve je potrebno pri tem posebej upoštevati pomen časa obravna- vanja in vrsto raztopine za obravnavanje. Ključne besede: abiotski stres; zdravilna rastlina; Hibi- scus sabdariffa L.; obravnava semen Acta agriculturae Slovenica, 118/2 – 20222 M. AHMADIZADEH et al. 1 INTRODUCTION Roselle (Hibiscus sabdariffa L.) is a member of the Malvaceae family (Shruthi et al., 2018), which origi- nally belonged to Malaysia and India (Mahadevan et al., 2009), and cultivated in tropical and subtropical climates (Da-Costa-Rocha et al., 2014). It is perennial or annual sub-shrub or woody-based herb, and widely grown in subtropical and tropical zones (Ibrahim et al., 2013). These plant species have played a key role in people’s living because they provide humanity’s needs that are food, clothes, shelter, and medicines (Riaz & Chopra, 2018)polysaccharides and organic acids thus having enormous prospective in modern therapeutic uses. The study aimed to review and document all the available evidence and information about the calyces of Hibiscus sabdariffa (roselle. Roselle is used in tradition- al medicine, due to overfill in phytochemicals like poly- phenols, particularly anthocyanins, polysaccharides, and organic acids; hence, it has significant potential in modern medicinal applications (Riaz & Chopra, 2018; Sukkhaeng et al., 2018). It is traditionally cultivated ow- ing to the usage of calyces, stems, leaves, and seeds as all organs have pharmacological and other uses (Wright et al., 2007). Calyx products are applied in indigenous medicine to treat high blood pressure, liver diseases and fever (Ali et al., 2005). Roselle extracts are increasingly developed for medications, food, and cosmetics (Farns- worth & Bunyapraphatsara, 1992). The presence of salt in the water or soil is consid- erable challenge for plant production in the world. It is most prevalent in dryland and coastal areas. Due to un- suitable irrigation and drainage management, limited rain, high evaporation, and saline irrigation water, salt concentration in the soil and water is increasing inland (Ibrahim, 2016). This problem takes about 3.7 million acres of the area of food production every year (Munns & Tester, 2008). Therefore, half of the cultivation area will be lost by the Mid-21st century (Wang et al., 2003). Salt stress became a limitation factor to the production of the crops, and the majority of crops are extremely sensitive to saline soil and water (Lin et al., 2017; Ah- madizadeh et al., 2016). Seed germination and seedling growth are the susceptible stages to abiotic stress, and abiotic stress can be slowed or stopped the germination of seeds (Ahmadizadeh et al., 2011; Galal, 2017). Rouhi et al. (2011), Ahmadizadeh et al. (2011), Ansari et al. (2013), and Ebrahimi et al. (2014) stated that raising the stress had a negative impact on the germination rate. In the past decade, several strategies have been ap- plied to improve abiotic stress tolerance in crops. There are various methods to enhance crop growth and devel- opment in salt-affected conditions (Hussain et al., 2016; Feghhenabi et al., 2020). One of the appropriate meth- ods is pretreatment, like prime the seeds with various materials before sowing (Ali et al., 2017; Subramanyam et al., 2019). Seed pretreatment as a practical, cost-effec- tive, and low-risk enhancing germination of seed and seedling growth through pre-germinating metabolic processes improvement (Jime’nez-Arias et al., 2015; Mi- gahid et al., 2019). Priming of seed is moderate stress, which activates a stress-reaction mechanism (Bhanu- prakash & Yogeesha, 2016). Priming of seed is a physi- ological method of seed hydration and drying to ame- liorate the pre-germinate metabolism under stressed conditions. The primed seeds exhibit quicker and nor- mal seed germination (Hasanuzzaman & Fotopoulos, 2019), and seed priming adjusts the biochemical and physiological of the embryo. Priming also decreases the seeds sensitivity to unfavorable conditions (Afzal et al., 2016). Several researchers have indicated that seed prim- ing enhances the well establishment and growth of plants (Farooq et al., 2010; Kerchev et al., 2020; Fegh- henabi et al., 2020). The beneficial impacts of seed priming in saline conditions have been shown in sev- eral crops for instance, pepper (Khan et al., 2009), okra (Dkhil et al., 2014), tomato (Ebrahimi et al., 2014), ro- sella (Galal, 2017), and Silybum marianum (L.) Gaertn. (Migahid et al., 2019). Latef et al. (2020) studied the im- pact of priming with Al2O3 nanoparticles on the growth of roselle, and the results showed that Al2O3 nanoparti- cles influenced growth traits, like dry mass, fresh mass, root, and shoot length. Shruthi et al. (2018) concluded pretreatment with GA3, KNO3, and hot water to study the influence of seed priming on germination of Ro- selle (Hibiscus sabdariffa L.), they indicated the posi- tive impact of seed pretreatment on the properties of germination speed and germination percentage. Nas- sar (2010) reported the positive results of seed priming and organic fertilizer on the yield and quality of ro- selle. Sheyhakinia et al. (2020) showed ameliorate of salt stress tolerance by jasmonic acid in roselle. Their results showed that jasmonic acid protected roselle seedlings against salinity damage. Germination indices and seed- ling traits of two tomato cultivars are influenced by the great potential value of seed treatment with CaCl2 and KNO3 solution under salinity conditions. In contrast, enhanced salinity concentrations led to a significant reduction in germination indices and seedlings growth (Ebrahimi et al., 2014). Soil salinity is one of the principal widespread abiotic stresses, which has adverse effects on crop pro- duction (Ismail et al., 2007; Ahmadizadeh et al., 2021). Appropriate seed germination is a prerequisite for the successful stand establishment of plants in unfavorable Acta agriculturae Slovenica, 118/2 – 2022 3 Comprehensive seed priming assessment of Hibiscus sabdariffa L. in germination and seedling growth stage under salt stress environments such as low moisture, saline water, and soil, which are limiting germination factors (Ahmadi- zadeh, 2013). The fast and uniform germination and seedling establishment are influential factors for plant performance. There are several priming techniques, which are helpful for successful stand establishment of plants in abiotic stress conditions. Therefore, the study aimed to investigate the effects of various priming in- cluding KCl, Na2SO3, KNO3, and Ca2CO3 and hydro- priming of seeds on germination at different priming durations under normal and various levels of salinity conditions. 2 MATERIAL AND METHODS In order to assess the effects of various seed prim- ing compounds and priming durations on seed germi- nation indices and growth characteristics of Hibiscus sabdariffa L. seedlings in various levels of salt stress conditions were studied at the agricultural laboratory of Minab higher education center, university of Hor- mozgan. An experiment was conducted in a factorial experiment based on a completely randomized design with three replications. Priming treatments consisted of halopriming with KNO3 (0.5 and 1 %), Na2SO3 (0.5 and 1 %), KCl (1 and 2 %) and Ca2CO3 (1 and 2 %), and hydropriming with distilled water and control (non- primed), priming durations was 12 and 24 h, then seeds exposed to four levels (0, 50, 100, 200 mM) of NaCl so- lution. For any treatments, disinfected seeds were im- mersed in 50 ml of the prepared solution for 12 and 24 h in covered glass containers to preserve evaporation loss. The seeds were then rinsed with distilled water several times, afterward dried back at room tempera- ture (25 °C) for 24 h to be dried (Ebrahimi et al., 2014; Ibrahim, 2016; Aghdaei et al., 2019). Fifteen healthy primed seeds of roselle were placed in petri dishes on two layers of filter paper, then 8 ml of the salinity solutions (0, 50, 100, and 200 mM NaCl). To germinate the seeds, the petri dishes were put in an incubator at 26 ± 1◦C. Germination of Hibiscus sab- dariffa L. the seeds were counted as a germinated seed once they displayed extension of radicle almost 2 mm. The germination count was recorded every 24 h up to 7 days. At the end of the first week, and germination percentage (GP), germination rate index (GRI), mean germination time (MGT), germination index (GI), and vigor index (VI) were calculated based on the following equations (Al-Mudaris, 1998): GP = (N/M) * 100 (1) where GP is germination percentage, N is the total number of germinated seeds at the end of seven days, and M is the total number of cultivated seeds. GRI = (G1/1) + (G2/2) + …+ (Gx/x) (2) G1 = germination percent in first day. G2 = germination percent in second day to final experiment, MGT = (fx) / f (3) where f is the number of newly germinated seeds on each day and x is the day of counting, GI = (7 * N1) + (6 * N2) + (5 * N3) + … (4) N1, N2, . . . = the number of germinated seeds in first day, second day and . . ., VI = GP × Seedling length (SL) (El- ouaer & Hannachi, 2012), (5) Also, fresh shoot mass, fresh root mass, shoot length, root length, dry shoot mass and dry root mass were measured. Mass of root and shoot were measured from the sample mass before and after drying at 70 °C for 12 h. the data were analyzed using SAS software, and means comparisons were done by the least significant difference test (LSD) at p < 0.05 level of confidence. The Excel software was used to draw figures. 3 RESULT AND DISCUSSION Seed germination and early establishment of seed- ling are the crucial stages for the crops, and these two stages are the delicate growth stages in unfavorable en- vironments (Begcy et al., 2018). Seed germination is sometimes prevented or delayed under different abiotic stresses (Fazlali et al., 2013; Muhie et al., 2020a,b). Ro- selle is sensitive to germination and early seedling de- velopment in saline conditions (Bahaeldeen et al., 2012; Al-Tohafi et al., 2015; Kadamanda, 2019). Considering to the value and privilege of the Hibiscus sabdariffa products, particularly medicinal value, this study was conducted to evaluate the influence of various priming approaches in different NaCl concentrations on germi- nation indices and seedling traits of roselle plants in petri dish at a controlled experimental environment. The analysis of variance showed a highly significant (p < 0.01) difference between various priming treatments in terms of germination indices and seedling growth traits (Table 1). Also, the result revealed a significant difference among the salinity levels. The priming × sa- linity interaction effect was significant for all studied traits (Table 1). High concentrations of salinity pre- vent and reduce the performance of most plants, but seed emergence is the utmost momentous process for well seed germination in medicinal plants (Nadjafi et al., 2006; Reed et al., 2022). The highest germination Acta agriculturae Slovenica, 118/2 – 20224 M. AHMADIZADEH et al. rate and percentage of T. polium seeds were obtained at concentrations of 500-2500 ppm GA3. Washing and chilling (5°C. The highest germination percentage (GP) (36.93 %) was observed in normal condition, while un- der the salinity conditions the highest GP (23.24 %) was observed under 50 mM salinity condition (Table 2). The decreasing in the percentage of germination may be associated with the increase of external osmotic pressure that has an impact on the water absorption of the seed, as well as, owing to the accumulation of some ions in the embryo, which may result in stimulation of the metabolic processes of germination and ultimate- ly leading to cells death in the embryo (Maher et al., 2013; Feghhenabi et al., 2020). Afkari Bajehbaj (2010) and Shereiwy et al. (2021) demonstrated that enhanc- ing salinity levels decreased final germination in seeds, but, the adverse impact of salinity on primed seeds was less than unprimed seeds. The highest germination rate index (GRI) (5.06) was observed in normal condition, while under the salinity conditions the highest GRI was observed under 50 mM salinity condition that had sig- nificantly different from normal condition (Table 2). The highest GI was observed in normal condition, under the low level of salinity conditions the GI reduc- tion was 46 percentage, and the highest GI in stress condition was observed under 50 mM salinity condi- tion (Table 2). The highest VI was observed in nor- mal condition, and there was significantly difference between the various levels of salinity stress (Table 2). The means comparison under different levels of salin- ity stress revealed the highest MGT under 50, and 100 mM salinity conditions (Table 2). In this respect, Kaveh et al. (2011), Thiam et al. (2013), and Ibrahim (2016) indicated that enhancing the concentration of salinity improved germination time and reduced the germina- tion percentage. In general, low levels of salinity cause a dormancy and low impact on the germination rate, but ascending concentration of salt prevents the seed germination and reduces the percentage of germina- tion (Khan & Weber, 2006; Shannon & Grieve, 1998). There was a significant difference between control and salinity conditions in terms of shoot and root mass. The highest of shoot fresh mass (0.19 g), root fresh mass (0.023 g) and root dry mass (0.002 g) were observed at non-salinity condition (control) but the highest shoot dry mass (0.019) was achieved at salinity condition (Ta- ble 2), suggesting that root growth is more sensitive to salinity than shoot growth. Amiri et al. (2010) with the study of germination characteristics of Cynara scolymus L. and Echinacea purpurea (L.) Moench under salinity stress demonstrated that shoot dry mass was reduced by enhancing salt concentration in studied medicinal plants. The similar finding was reported by research- M ea n Sq ua re S. O .V D F G P G RI M G T G I V I Sh L RL FS hM FR M D Sh M D RM Sh /R Pr im 18 86 1. 39 ** 15 .2 5* * 4. 78 ** 90 7. 27 ** 49 42 5. 72 ** 14 .9 3* * 5. 62 ** 0. 02 ** 0. 00 04 4* * 0. 00 03 3* * 0. 00 00 03 5* * 20 2. 13 ** Sa lin ity 3 12 15 6. 73 ** 23 5. 69 ** 33 .8 2* * 88 4. 45 ** 10 01 02 3* * 21 3. 34 ** 57 .5 6* * 0. 34 ** 0. 00 48 ** 0. 00 34 ** 0. 00 00 37 2* * 13 94 .1 1* * P* S 54 19 7. 69 ** 4. 51 ** 1. 19 ** 13 32 3. 17 ** 21 36 7. 41 ** 2. 73 ** 1. 33 ** 0. 00 4* * 0. 00 01 4* * 0. 00 00 68 ** 0. 00 00 00 77 ** 88 .4 4* * Er ro r 15 2 35 .4 1 0. 98 0. 69 43 .1 4 31 52 .8 6 0. 50 0. 18 0. 00 1 0. 00 00 1 0. 00 00 08 9 0. 00 00 00 13 25 .1 2 Ta bl e 1: A na ly si s o f v ar ia nc e fo r sa lin ity a nd p ri m in g eff ec ts o n so m e ge rm in at io n ch ar ac te ri st ic s o f H ib is cu s s ab da ri ffa ** : i s s ig ni fic an t a t 1 % , G P: G er m in at io n Pe rc en ta ge , G RI : G er m in at io n Ra te In de x, M G T: M ea n G er m in at io n Ti m e, G I: G er m in at io n In de x, V I: V ig or In de x, S hL : S ho ot le ng th , R L: R oo t L en gt h, F Sh M : F re sh S ho ot M as s, FR M : Fr es h R oo t M as s, D Sh M : D ry S ho ot M as s, D RM : D ry R oo t M as s, Sh /R : S ho ot /R oo t r at io Acta agriculturae Slovenica, 118/2 – 2022 5 Comprehensive seed priming assessment of Hibiscus sabdariffa L. in germination and seedling growth stage under salt stress Sa lin ity m M G P (% ) G RI M G T (d ay ) G I V I Sh L (c m ) RL (c m ) FS hM (g ) FR M (g ) D Sh M (g ) D RM (g ) Sh /R 0 36 .9 3 a 5. 06 7 a 1. 66 6 b 38 .6 3 a 31 4. 5 a 4. 85 9 a 2. 62 8 a 0. 19 00 a 0. 02 36 a 0. 01 67 b 0. 00 20 2a 8. 35 0 c 50 23 .2 4 b 2. 29 7 b 2. 36 7 a 20 .8 4 b 13 6. 7 b 3. 36 3 b 1. 60 1 b 0. 16 73 b 0. 01 61 b 0. 01 81 a 0. 00 15 7b 11 .7 11 b 10 0 19 .3 8 c 1. 86 9 c 2. 27 4 a 17 .4 9 c 80 .3 c 2. 39 4 c 1. 21 7 c 0. 12 73 c 0. 01 14 c 0. 01 90 a 0. 00 13 7c 14 .6 11 a 20 0 1. 49 d 0. 16 1 d 0. 69 3 c 1. 35 d 2. 84 d 0. 24 3 d 0. 19 6 d 0. 01 56 d 0. 00 15 d 0. 00 26 c 0. 00 01 3d 3. 09 2 d LS D 2. 20 24 0. 36 65 0. 30 92 2. 43 09 20 .7 8 0. 26 42 0. 16 1 0. 01 22 0. 00 12 0. 00 11 0. 00 01 1. 85 49 Ta bl e 2: M ea n co m pa ri so n of s al in ity le ve ls o n ge rm in at io n in di ce s a nd s ee dl in g gr ow th tr ai ts in H ib is cu s s ab da ri ffa G P: G er m in at io n Pe rc en ta ge , G RI : G er m in at io n R at e In de x, M G T: M ea n G er m in at io n Ti m e, G I: G er m in at io n In de x, V I: V ig or In de x, S hL : S ho ot le ng th , R L: R oo t L en gt h, F Sh M W : F re sh S ho ot M as s, FR M : F re sh R oo t M as s, D Sh M : D ry S ho ot M as s, D RM : D ry R oo t M as s, Sh /R : S ho ot /R oo t r at io ers in other species consisting of roselle (Galal, 2017), safflower (Kaya et al., 2003; Khodadad, 2011), triticale (Atak et al., 2006), and tomato (Ebrahimi et al., 2014). There are multiple pretreatment approaches ap- plied and classification based on the priming com- pounds. These comprise halo-priming, hydro-priming, hormone priming, osmo-priming, solid matrix, hard- ening, stratification, and thermal shock and humidifi- cation. The hydropriming, osmo-priming, haloprim- ing, and hormone priming techniques commonly had been used for seed treatment (Ashraf & Foolad, 2005; Eskandari, 2013; Paparella et al., 2015). Water potential, temperature, seed vigor, priming duration, seed primed storage condition, and plant species are the factors that influence the response of the seed to priming. There- fore, the optimization and fine-tuning of the priming approach is substantial to obtain the best outcome (Ra- tikanta & Kalipada, 2013). Hydropriming affect some of the required meta- bolic processes for germination to happen without ger- mination be accomplished, faster imbibition, further, softening of seed coat led to lesser mechanical preven- tion as a result of priming (Askari-Nejad & Farahmand, 2012). Seed pretreatment by inorganic salts enhances the enzymes activity engaged in the germination of seed and changes the mobilization of organic substanc- es’ to various embryo parts (Aghdaei et al., 2019). Prim- ing for 12 h with 0.5 % Na2SO3, 12 and 24 h with 2 % Ca2CO3, 12 h with 1 % KCl, 12 h with 1 % Na2SO3 and various hydro-prim showed the highest GP (Table 3). The highest GRI was in 12 h priming with 0.5 % Na2SO3, 1 % Na2So3 and hydro-prim (Table 3). The GRI illus- trate the percentage of germination on every day of the germination period. Higher GRI values display prompt and high germination (Fuller et al., 2012). Priming with 0.5 % Na2SO3 and 1 % Ca2CO3 in 12 h revealed the lowest median germination time (MGT) (Table 3). The lower MGT showed the faster germination of a seeds population (Fuller et al., 2012). The highest GI was in 12 h priming with 0.5 % Na2SO3, hydro-prim, and 24 h with 2 % Ca2CO3 (Table 3). Priming with 12 h 0.5 % Na2SO3, hydro-prim, and 24 h with 2 % Ca2CO3 showed the highest VI (Table 3). In contrast, 12 and 24 h prim- ing with two percentage KCl had the lowest GRI, GI, VI, and GP (Table 3). The highest shoot length was in 24 h priming with 2 % Ca2CO3 (4.19 cm), 1 % Ca2CO3 (3.91 cm), 1 % Na- 2SO3 (3.89 cm), and 12 h priming with 1 % KCl (3.74 cm), there were no statistically significant difference among these treatments. Priming with 24 h with 2 % Ca2CO3 showed the highest root length (Table 3). In terms of shoot mass at 24 h priming with 2 % Ca2CO3 (0.19 g), 1 % Ca2CO3 (0.18 g), and 1 % Na2So3 (0.17 Acta agriculturae Slovenica, 118/2 – 20226 M. AHMADIZADEH et al. es have been utilized for better germination of seeds in both normal and unfavorable environments (Jisha et al., 2012). The positive and affirmative effects of priming were observed in adverse condition than optimal con- ditions (Ashraf & Foolad, 2005; Chen & Arora, 2011; Ibrahim, 2016). Suggested priming mechanisms were consisting of the incidence of epigenetic alterations, also the transcription factors accumulation and inactive and inhibition of signaling proteins. These mechanisms are induced against the stress, hence improved resulting in a well and effective defense mechanism (Tanou et al., 2012). Some treatments and techniques were able to de- velop well establishment of crops in stressful conditions (Soeda et al., 2005; Patade et al., 2009). The highest GP was in 0, 12, 24 hours hydroprim- ing under normal condition. Also, 2 % Ca2CO3, 0.5 % Na2So3, and 1 % KCl in 12 hours treatment showed high GP under normal condition (Figure 1). enhancing the growth characteristics resulting from seed priming by water soaking could be owing to the impact of seed hydropriming on the fast and sound establishment of plants (Ashraf & Foolad, 2005). The lowest GP was ob- served in 0.5 % KNO3 in 24 hours pretreatment in all studied conditions, 0.5 % KNO3 in 12 hours pretreat- ment under 200 mM salinity condition, as well as, the same results were observed by 24 pretreatments of 1 % g) had the highest shoot mass that there were no sta- tistically significant difference among these treatments. Also, 24 h priming with 2 % Ca2CO3 root mass (Table 3). The highest dry shoot mass was in 24 h priming with 1 % Ca2CO3, and 2 % Ca2CO3, priming with 24 h 2 % Ca2CO3, hydro-prim, and 12 h with 2 % Ca2CO3 showed the highest dry root mass (Table 3). The positive effects of various priming approaches like priming the tomato seed by potassium nitrate on germination (Lara et al., 2014), hydropriming on sorghum and rice germina- tion percentage (Farooq et al., 2006; Moradi & Younesi, 2009), and salicylic acid on Solanum melongena L. seed germination percentage (Mahesh et al., 2017), as well as, enhance of the shoot and root length of cotton (Gos- sypium hirsutum L.) in hydropriming (Shaheen et al., 2015) have been reported. Plant cell turgor reduction and decrease of shoot and root length caused by salinity stress (Werner & Finkelstein, 1995). Also, it was suggested that salinity stress acts firstly on water uptake. Moreover, Na+ and Cl- accumulation prevent the metabolism of cells divid- ing and expanding (Neumann, 1997), less germination, and even resulting in seed or embryo death. In addi- tion, salt stress leads prevent and decrease the enzymes activities that may be significantly associated with seed germination (Katembe et al., 1998). Priming approach- Figure 1: Effect of various priming under four level of salt stress on germination percentage of Hibiscus sabdariffa L. 1: non-primed, 2: Hydro 12h , 3: Hydro 24 h, 4: KNO3_0.5 % 12 h , 5: KNO3_0.5 % 24 h , 6: KNO3_1 % 12 h, 7: KNO3_1 % 24 h, 8: Na2SO3_0.5 % 12 h, 9: Na2SO3_0.5 % 24 h , 10: Na2SO3_1 % 12 h, 11: Na2SO3_1 % 24 h, 12: KCl_1 % 12 h, 13: KCl_1 % 24 h, 14: KCl_2 % 12 h , 15: KCl_2 % 24 h, 16: Ca2CO3_1 % 12 h, 17: Ca2CO3_1 % 24 h, 18: Ca2CO3_2 % 12 h , 19: Ca2CO3_2 % 24 h Acta agriculturae Slovenica, 118/2 – 2022 7 Comprehensive seed priming assessment of Hibiscus sabdariffa L. in germination and seedling growth stage under salt stress Na2SO3, and 2 % KCl under 200 mM salinity condition, and 1 % Ca2CO3 in 12 hours pretreatment under 200 mM salinity condition (Figure 1). In saline conditions, 2 % Ca2CO3, 0.1 % Na2SO3 under 50 mM salinity, and 0.1 % Na2SO3 in 100 mM salinity showed the high GP. However, there was significantly different with the con- trol condition (Figure 1). The result implied that the best treatments in terms of GP were 12 h Hydro, 12 h Na2SO3_0.5 %, 24 h Ca2CO3_1 %, 24 h Ca2CO3_2 % in control, 100 mM salinity, 200 mM salinity, and 50 mM salinity conditions, respectively (Figure 1). Hydropriming (12 and 24 h), 0.5 % Na2SO3, and 1 % Na2SO3 in 12 h had the highest GRI in the normal condition. In saline conditions, 12 h seed priming by 0.5 % Na2SO3 under 50 mM salt stress condition and 12 h priming with 1 % KCl in 200 mM salt stress condi- tion, while there were zero GRI in 24 h pretreatment of seeds with 0.5 % KNO3 and 1 % KNO3 under salinity conditions, as well as, 1 % Na2SO3, 1 % Ca2CO3 and 2 % KCl under 200 mM salinity condition (Figure 2). The highest MGT was in 1 % Ca2CO3 and 2 % KCl with 12 and 24 priming hours under 200 and 100 mM salinity conditions, respectively. Also, there were no significant differences in 24 h priming with 2 % Ca2CO3 under 50 mM salinity conditions, 0.5 % Na2SO3, hydro-prim and 1 % Na2SO3 under 100 mM salinity conditions, and 12 h priming with 1 % Na2SO3 and 1 % Ca2CO3 in 100 mM Figure 2: Effect of various priming under four level of salt stress on germination rate index of Hibiscus sabdariffa L. 1: non-primed, 2: Hydro 12h , 3: Hydro 24 h, 4: KNO3_0.5 % 12 h , 5: KNO3_0.5 % 24 h , 6: KNO3_1 % 12 h, 7: KNO3_1 % 24 h , 8: Na2SO3_0.5 % 12 h, 9: Na2SO3_0.5 % 24 h , 10: Na2SO3_1 % 12 h, 11: Na2SO3_1 % 24 h, 12: KCl_1 % 12 h, 13: KCl_1 % 24 h , 14: KCl_2 % 12 h , 15: KCl_2 % 24 h, 16: Ca2CO3_1 % 12 h, 17: Ca2CO3_1 % 24 h, 18: Ca2CO3_2 % 12 h , 19: Ca2CO3_2 % 24 h salinity condition (Table 4). Similar results were report- ed by (Farooq et al., 2006) and (Qadir et al., 2011), who reported reducing MGT using CaCl2 primed seeds. Hydropriming (12 and 24 h) had the highest GI in normal conditions, and there were no significant differ- ences with hydropriming (control), 0.5 % Na2So3, and 1 % Na2SO3 in 12 h in normal condition (Table 4). The seeds with twelve-hour priming of 1 % KCl in 100 mM salinity, 0.5 % Na2So3, and 0.1 % Na2SO3 under 50 mM salinity showed high GI, but there were significant dif- ferences with the control condition (Table 4). Enhanc- ing the germination rate in treatment seeds can be il- lustrated through the enhanced synthesis of protein, the less term of metabolism in the germination stage, the influence on cell membrane phospholipids (Ansa- ri et al., 2013), enhance of cell division rate (Taylor & Harman, 1990), and faster absorption of water, better development in these seeds, which all eventually lead to enhancing of seed germination duration. Shahverdi et al. (2017), with priming the stevia seeds, indicated a considerable correlation between the germination per- centage enhancement and seed germination improve- ment factors. It seems that the efficiency of pretreat- ment of seed is affiliated with the elements like type and concentration of priming compound, duration of seed treatment by compounds (duration of priming). The 12 h treatment with hydro-prim, 1 % Na2So3, Acta agriculturae Slovenica, 118/2 – 20228 M. AHMADIZADEH et al. Pr im % Ti m e G P (% ) G RI M G T (d ay )G I V I Sh L (c m ) RL (c m ) FS hM (g ) FR M (g ) D Sh M (g ) D RW (g ) Sh /R H yd ro - C on tr ol 27 .9 17 2. 73 19 1. 91 04 25 .7 5 14 3. 33 1. 94 17 1. 20 58 0. 13 33 3 0. 01 53 33 0. 01 63 75 0. 00 16 33 8. 83 3 12 28 .3 33 3. 69 03 1. 94 92 28 .1 67 20 0. 46 2. 68 33 1. 80 83 0. 13 58 3 0. 01 54 17 0. 01 63 67 0. 00 16 42 7. 60 8 24 24 .5 83 3. 25 69 1. 74 05 25 .0 83 15 5. 67 2. 39 17 1. 62 5 0. 13 75 0. 01 78 33 0. 01 17 17 0. 00 18 83 4. 77 2 K N O 3 0. 5 12 19 .5 8 2. 01 39 1. 82 5 18 .3 33 88 .2 9 2. 46 67 0. 77 5 0. 13 16 7 0. 01 33 33 0. 01 56 67 0. 00 14 58 8. 27 7 24 0 0 0 0 0 0 0 0 0 0 0 0 1 12 22 .0 8 2. 68 75 1. 76 81 22 .0 83 12 6. 83 2. 82 5 1. 07 5 0. 12 5 0. 01 49 92 0. 01 51 25 0. 00 13 33 12 .9 93 24 0 0 0 0 0 0 0 0 0 0 0 0 N a 2 SO 3 0. 5 12 29 .5 83 4. 21 11 1. 36 82 31 .5 83 18 4. 92 3. 30 83 0. 1 0. 13 5 0. 01 51 58 0. 01 82 92 0. 00 13 67 11 .8 86 24 23 .3 33 2. 63 19 2. 01 41 22 .4 17 16 0. 33 3. 50 83 1. 74 17 0. 14 25 0. 01 27 92 0. 01 69 33 0. 00 13 67 13 .1 81 1 12 25 .8 33 3. 55 14 2. 02 31 24 .7 5 15 8. 67 2. 92 5 1. 47 5 0. 12 16 7 0. 01 13 42 0. 01 51 67 0. 00 12 17 10 .3 6 24 16 .2 5 1. 67 64 2. 39 15 13 .9 17 15 5. 67 3. 89 17 1. 95 83 0. 17 41 7 0. 01 43 5 0. 01 35 75 0. 00 10 5 11 .2 25 K C l 1 12 26 .2 5 3. 16 25 1. 96 7 25 .9 17 19 5. 5 3. 74 17 1. 52 5 0. 14 16 7 0. 01 33 75 0. 01 48 75 0. 00 11 75 10 .4 14 24 20 .8 33 2. 12 08 2. 29 43 19 .6 67 13 9 2. 72 5 1. 42 5 0. 13 16 7 0. 01 15 5 0. 01 65 5 0. 00 13 25 11 .5 48 2 12 14 .1 67 1. 31 25 1. 94 94 12 .7 5 63 .8 8 2. 28 33 0. 90 83 0. 10 58 3 0. 00 70 83 0. 01 52 58 0. 00 08 98 13 .8 17 24 14 .1 67 1. 25 2. 04 31 12 .2 5 67 .1 7 2. 29 17 1. 10 83 0. 10 83 3 0. 00 84 08 0. 01 29 75 0. 00 07 33 14 .1 43 C a 2 C O 3 1 12 17 .5 2. 10 42 1. 62 96 17 .3 33 13 3. 24 3. 17 5 1. 55 75 0. 12 91 7 0. 01 68 33 0. 01 24 75 0. 00 14 92 7. 63 3 24 21 .2 5 2. 54 86 1. 97 56 21 .1 67 14 5. 25 3. 91 67 2. 19 17 0. 18 08 3 0. 01 78 33 0. 01 94 33 0. 00 17 33 12 .9 76 2 12 25 .4 17 2. 63 75 2. 01 92 23 .8 33 18 9. 04 3. 14 17 1. 87 5 0. 13 75 0. 01 94 17 0. 01 59 67 0. 00 18 6. 90 2 24 27 .5 2. 99 86 2. 21 99 26 .5 83 23 9. 38 4. 19 17 2. 94 17 0. 19 0. 02 52 5 0. 02 08 67 0. 00 20 75 12 .0 33 LS D 5% 4. 80 01 0. 79 87 0. 67 38 5. 29 8 45 .2 89 0. 57 58 0. 35 08 0. 02 65 0. 00 27 0. 00 24 0. 00 03 4. 04 27 Ta bl e 3: M ea n co m pa ri so n of v ar io us p ri m in g on g er m in at io n in di ce s a nd s ee dl in g gr ow th tr ai ts in H ib is cu s s ab da ri ffa L . G P: G er m in at io n Pe rc en ta ge , G RI : G er m in at io n R at e In de x, M G T: M ea n G er m in at io n Ti m e, G I: G er m in at io n In de x, V I:V ig or In de x, S hL : S ho ot le ng th , R L: R oo t L en gt h, F Sh M : F re sh S ho ot M as s, FR M : F re sh R oo t M as s, D Sh M : D ry S ho ot M as s, D RM : D ry R oo t M as s, Sh /R : S ho ot /R oo t r at io Acta agriculturae Slovenica, 118/2 – 2022 9 Comprehensive seed priming assessment of Hibiscus sabdariffa L. in germination and seedling growth stage under salt stress and 1 % KCl had the highest VI in normal condition, and there were no significant differences with 0.5 % Na2SO3, 1 % Ca2CO3, 2 % Ca2CO3, and 1 % KNO3 in 12 h treatments under normal condition, and 0.5 % Na- 2SO3 (12 h priming) in 50 mM salinity condition, 1 % Na2SO3, and hydro-prim in 24 h treatment at normal condition (Figure 3). The 12 h treatment with 0.5 % Na- 2SO3 in 50 mM salinity condition, 1 % KCl, 1 % Ca2CO3, 1 % Na2SO3, and the 24 h treatment with 1 % Na2SO3 had the highest shoot length in normal condition. Also, there were no significant differences with 12 h treat- ment by 1 % KNO3 under 100 mM salinity, 1 % KNO3, Figure 3: Effect of various priming under four level of salt stress on vigor index of Hibiscus sabdariffa L. 1: non-primed, 2: Hydro 12h , 3: Hydro 24 h, 4: KNO3_0.5 % 12 h , 5: KNO3_0.5 % 24 h , 6: KNO3_1 % 12 h, 7: KNO3_1 % 24 h , 8: Na2SO3_0.5 % 12 h, 9: Na2SO3_0.5 % 24 h , 10: Na2SO3_1 % 12 h, 11: Na2SO3_1 % 24 h, 12: KCl_1 % 12 h, 13: KCl_1 % 24 h , 14: KCl_2 % 12 h , 15: KCl_2 % 24 h, 16: Ca2CO3_1 % 12 h, 17: Ca2CO3_1 % 24 h, 18: Ca2CO3_2 % 12 h , 19: Ca2CO3_2 % 24 h Figure 4: Effect of various priming under four level of salt stress on shoot length of Hibiscus sabdariffa L. 1: non-primed, 2: Hydro 12h , 3: Hydro 24 h, 4: KNO3_0.5 % 12 h , 5: KNO3_0.5 % 24 h , 6: KNO3_1 % 12 h, 7: KNO3_1 % 24 h , 8: Na2SO3_0.5 % 12 h, 9: Na2SO3_0.5 % 24 h , 10: Na2SO3_1 % 12 h, 11: Na2SO3_1 % 24 h, 12: KCl_1 % 12 h, 13: KCl_1 % 24 h , 14: KCl_2 % 12 h , 15: KCl_2 % 24 h, 16: Ca2CO3_1 % 12 h, 17: Ca2CO3_1 % 24 h, 18: Ca2CO3_2 % 12 h , 19: Ca2CO3_2 % 24 h Acta agriculturae Slovenica, 118/2 – 202210 M. AHMADIZADEH et al. 2 % Ca2CO3 in normal condition, 24 h treatment by 1 % KCl, and 0.5 % Na2SO3 in normal condition (Figure 4). The highest root length was in hydro-prim, 1 % Ca2CO3 at 12 h in normal condition, and 0.5 % Na2SO3 at 12 h in 50 mM salinity condition, and there were no significant differences with 2 % Ca2CO3 at 12 h, 1 % Na2SO3, and hydroprim at 12 h in normal condition (Figure 5). Neumann (1995) also reported that salinity could quickly prevent root growth and thus the ability to uptake water and essential mineral nutrition. The 12 h treatment with 1 % Na2SO3, in 50 mM salinity condi- tion, 1 % Ca2CO3 and 1 % KCl had the highest shoot mass in normal condition. Also, there were no signifi- cant differences with 1 % Na2SO3 at 24 h, 2 % Ca2CO3 in 12 h treatment in normal condition and 1 % KNO3 in 12 treatment under 100 mM salinity (Table 4). The 24 h treatment with 1 % Na2SO3, 1 % KCl, and the 12 h treatment with 2 % Ca2CO3 had the high- est root mass in normal condition. Also, there were no significant different with 12 treatment of 1 % Na2SO3, 0.5% Na2SO3 in normal condition and 1 % Ca2CO3 un- der 50 mM salinity (Table 4). The results showed several priming had high dry shoot mass under various salin- ity conditions, for instance 2 % Ca2CO3, 1 % Ca2CO3, 1 % Na2SO3, 0.5 % Na2SO3, 0.5 % KNO3, and 1 % KCl illustrated the highest dry shoot mass under 100 mM salinity condition. Besides, 0.5 % Na2SO3, 1 % Ca2CO3, 0.5 % KNO3, 2 % Ca2CO3, 1 % KCl, 1 % KNO3 and 2 % KCl had the highest dry shoot mass under 50 mM salin- ity condition (Table 4). Seed priming by CaCl2, KCl, and NaCl were figured out to be effective in diminishing the negative impact of salinity on wheat via their effects on changing the levels of various plant phytohormones (Iqbal et al., 2006). The highest dry root length was in hydro-prim, 1 % Na2SO3, and 1 % KCl at 24 h, and 1 % Ca2CO3 at 12 h in normal condition, also, there were no significant differences with hydro-prim (12 h), 2 % Ca2CO3 (12 h), 0.5 % Na2SO3 (12 and 24 h), and 1 % KCl (24 h) in 50 mM salinity condition, 0.5 % Na2SO3 (24 h), 1 % Na2SO3 (24 h), and 1 % KNO3 (12 h) in 100 mM salinity condi- tion (Table 4). Abdollahi & Jafari (2012) demonstrated that KNO3 3 % treatment enhanced root length to primary shoot ratio more than NaCl 1 % under saline condition. This enhances the water uptake by the plant that may help the growth development of seedlings in saline conditions. In addition, application of the four potassium nitrate concentrations (0, 0.5, 1, and 2 %) on time to 50 percentage germination, and germination percentage of amaranth seeds revealed that using 0.5 percent of potassium nitrate decreased time to 50 % seed germination (Musa & Lawal, 2015). The 24 h treatment with 1 % KCl, 1 % Ca2CO3, and 2 % Ca2CO3 had the highest shoot and root ratio in 100 mM salinity condition. Also, there were no significant differences with 12 treatments of 1 % Ca2CO3, 2 % KCl, Figure 5: Effect of various priming under four level of salt stress on root length of Hibiscus sabdariffa L. 1: non-primed, 2: Hydro 12h , 3: Hydro 24 h, 4: KNO3_0.5 % 12 h , 5: KNO3_0.5 % 24 h , 6: KNO3_1 % 12 h, 7: KNO3_1 % 24 h , 8: Na2SO3_0.5 % 12 h, 9: Na2SO3_0.5 % 24 h , 10: Na2SO3_1 % 12 h, 11: Na2SO3_1 % 24 h, 12: KCl_1 % 12 h, 13: KCl_1 % 24 h , 14: KCl_2 % 12 h , 15: KCl_2 % 24 h, 16: Ca2CO3_1 % 12 h, 17: Ca2CO3_1 % 24 h, 18: Ca2CO3_2 % 12 h , 19: Ca2CO3_2 % 24 h. Acta agriculturae Slovenica, 118/2 – 2022 11 Comprehensive seed priming assessment of Hibiscus sabdariffa L. in germination and seedling growth stage under salt stress Prim (%) Time salt MGT (day) GI FShM (g) FRM (g) DShM (g) DRM (g) Sh/R Hydro Control 0 2.05 56.33 0.19 0.020 0.0192 0.0025 7.79 50 2.85 18 0.19 0.021 0.0209 0.0017 16.36 100 2.73 28.66 0.14 0.02 0.0253 0.0023 11.17 200 0 0 0 0 0 0 0 12 h 0 1.58 68.66 0.15 0.025 0.0227 0.0023 10.07 50 2.30 23.33 0.21 0.023 0.0213 0.0020 10.52 100 2.9 19.33 0.17 0.013 0.0213 0.0022 9.82 200 1 1.33 0 0 0 0 0 24 h 0 1.49 59 0.19 0.03 0.0196 0.0033 5.90 50 2.19 28.66 0.21 0.021 0.0155 0.0023 6.71 100 2.94 10.66 0.14 0.016 0.0116 0.0018 6.46 200 0.33 2 0 0 0 0 0 KNO3_0.5 % 12 h 0 1.72 33.66 0.18 0.016 0.0201 0.00196 10.35 50 2.72 20.33 0.18 0.023 0.0221 0.0022 10.28 100 2.85 19.33 0.16 0.013 0.0203 0.0016 12.47 200 0 0 0 0 0 0 0 24 h 0 0 0 0 0 0 0 0 50 0 0 0 0 0 0 0 100 0 0 0 0 0 0 0 200 0 0 0 0 0 0 0 KNO3_1 % 12 h 0 2.02 40.33 0.21 0.043 0.0189 0.003 6.34 50 1.94 22 0.19 0.011 0.0216 0.0013 17.68 100 2.10 24.66 0.096 0.005 0.0198 0.0010 27.94 200 1 1.33 0 0 0 0 0 24 h 0 0.66 1.66 0.06 0.001 0.005 0.00053 3.12 50 0 0 0 0 0 0 0 100 0 0 0 0 0 0 0 200 0 0 0 0 0 0 0 Na2SO3_0.5 % 12 h 0 1.26 55.66 0.20 0.023 0.0189 0.00163 11.57 50 1.97 35 0.17 0.018 0.0239 0.00206 11.61 100 1.89 33.66 0.11 0.015 0.0228 0.00126 19.35 200 0.33 2 0.04 0.003 0.0075 0.0005 5 24 h 0 1.64 44.66 0.18 0.017 0.0202 0.0018 12.81 50 2.85 22.66 0.2 0.016 0.0211 0.00203 10.45 100 2.55 21 0.15 0.015 0.0236 0.00156 15.62 200 1 1.33 0.03 0.002 0.0027 0.0002 13.83 Continued on the next page Table 4: Mean comparison of priming and salinity interaction effect on germination indices and seedling growth traits in Hibiscus sabdariffa L. Acta agriculturae Slovenica, 118/2 – 202212 M. AHMADIZADEH et al. Na2SO3_1 % 12 h 0 1.36 45.33 0.14 0.016 0.0136 0.002 6.78 50 3.09 30 0.19 0.018 0.0217 0.00163 13.32 100 2.63 22.33 0.15 0.009 0.0252 0.00123 21.32 200 1 1.33 0 0 0 0 0 24 h 0 2.12 33.33 0.31 0.034 0.0188 0.0021 9.12 50 4.77 6.66 0.20 0.009 0.0116 0.00073 16.92 100 2.66 15.66 0.17 0.013 0.0238 0.0013 18.85 200 0 0 0 0 0 0 0 KCl_1 % 12 h 0 1.66 48 0.27 0.031 0.016 0.0018 8.91 50 2.70 28.66 0.14 0.010 0.0207 0.00173 12.33 100 1.83 26 0.15 0.011 0.0228 0.00117 20.40 200 1.66 1 0 0 0 0 0 24 h 0 1.89 42.66 0.21 0.025 0.0202 0.00243 8.64 50 2.66 23.66 0.19 0.015 0.0251 0.00203 12.32 100 2.61 12 0.12 0.005 0.0207 0.00083 25.22 200 2 0.333 0 0 0 0 0 KCl_2 % 12 h 0 2.21 25.66 0.15 0.007 0.0162 0.001267 13.56 50 2.58 15 0.18 0.009 0.0223 0.001467 15.48 100 3 10.33 0.09 0.0116 0.0224 0.00086 26.21 200 0 0 0 0 0 0 0 24 h 0 2.36 19.33 0.15 0.0103 0.0172 0.0012 15.12 50 3.22 9 0.12 0.0143 0.0173 0.00073 24.05 100 2.58 20.66 0.16 0.009 0.0174 0.001 17.4 200 0 0 0 0 0 0 0 Ca2CO3_1 % 12 h 0 1.96 38.66 0.23 0.041 0.0172 0.0032 5.33 50 2.05 20.33 0.14 0.0143 0.0137 0.001 13.67 100 2.5 10.33 0.13 0.012 0.0189 0.001767 11.51 200 0 0 0 0 0 0 0 24 h 0 1.81 31 0.24 0.032 0.0171 0.00213 8.66 50 2.11 24 0.20 0.009 0.0198 0.0015 13.54 100 2.13 22.66 0.14 0.019 0.0206 0.00233 8.88 200 1.83 7 0.13 0.010 0.0201 0.000967 20.81 Ca2CO3_2% 12 h 0 1.91 50.66 0.21 0.034 0.0169 0.0026 6.62 50 2.46 28.66 0.21 0.030 0.0253 0.00246 10.54 100 3.03 14.33 0.12 0.012 0.0215 0.00213 10.44 200 0.66 1.66 0 0 0 0 0 24 h 0 1.879 39.33 0.28 0.034 0.0199 0.0025 7.88 50 2.45 40 0.22 0.039 0.0196 0.003 6.65 100 2.21 20.66 0.16 0.014 0.0239 0.0017 14.47 200 2.33 6.33 0.09 0.013 0.0203 0.0010 19.11 LSD 5 % - - 1.34 10.59 0.05 0.005 0.0048 0.0006 8.0855 MGT: Mean Germination Time, GI: Germination Index, FShM: Fresh Shoot Mass, FRM: Fresh Root Mass, DShMW: Dry Shoot Mass, DRM: Dry Root Mass, Sh/R: Shoot/Root ratio Acta agriculturae Slovenica, 118/2 – 2022 13 Comprehensive seed priming assessment of Hibiscus sabdariffa L. in germination and seedling growth stage under salt stress and hydro-prime in 50 mM salinity condition (Table 4). Seed priming is one of the simple, low risk and cost ap- proaches used to cope with the adverse effect of salin- ity in agricultural lands. The privilege of seed priming or pretreatment in unfavorable conditions have been studied in several crops, instance hot pepper (Khan et al., 2009), tomato (Ebrahimi et al., 2014), pepper (Aloui et al., 2014), lettuce (Nasri et al., 2011), pea (Pisum sa- tivum L.) (Naz et al., 2014), and maize (Abraha & Yo- hannes, 2013). Soil salinity has adverse effects on agri- culture productivity. Therefore, agronomic and genetic solutions to enhancing salt tolerance are urgently re- quired. We concluded that applying easy and low-cost techniques such as priming can remarkably increase the germination seed in salinity condition. 4 CONCLUSIONS Priming is a technique that is capable to improve the performance of seeds in salinity stress conditions. Under saline condition, 24 h 2 % Ca2CO3 had the high- est germination percentage (43.3 %) in 50 mM, while 12 h treatment with 0.5 % Na2SO3 (33.3 %) had high ger- mination percentage in 100 mM levels of saline condi- tions. Also, the highest germination rate index was ob- served in 0.5 % Na2SO3 with 12 h treatment time (4.05 and 3.95 respectively) in 50 and 100 mM levels of saline conditions. There was no geminated seed at 24 h prim- ing by 0.5 and 1 percentage of KNO3, while priming with 1 % of KNO3 at 12 h showed good performance in terms of shoot mass trait under saline condition. The result of various priming on studied traits revealed the importance of the type of priming compound and priming duration. The result implied that the best treat- ments in terms of GP were 12 h hydropriming, 24 h Ca2CO3_2 %, 12 h Na2SO3_0.5 %, 24 h Ca2CO3_1 %, in control, 50 mM salinity, 100 mM salinity, and 200 mM salinity conditions, respectively. We suggested perform- ing the same studies with the suitable material at the precise concentration on similar species to determine and understand the reliability and efficiency of the ap- proaches. Also, supplementary research should concen- trate on molecular, metabolic, and physiological stimu- late with priming agents in salt stress. Moreover, future studies need to assess germination and early seedling growth at the field condition. 5 ACKNOWLEDGMENTS This research was conducted in a research project (No. 99.3821) at University of Hormozgan. The authors are thankful to Minab Higher Education Center, Uni- versity of Hormozgan to provide laboratory facilities and equipment. 6 REFERENCES Abdollahi, F., & Jafari, L. (2012). Effect of NaCl and KNO3 priming on seed germination of canola (Brassica Napus L.) under salinity conditions. International Journal of Ag- riculture, Research and Review, 2, 573–579. Abraha, B., & Yohannes, G. (2013). The role of seed priming in improving seedlinggrowth of maize (Zea mays L.) under salt stress at field conditions. Agricultural Sciences, 4(12), 666–672. https://doi.org/10.4236/as.2013.412089 Afkari Bajehbaj, A. (2010). The effects of NaCl priming on salt tolerance in sunflower germination and seedling grown under salinity conditions. African Journal of Biotechnolo- gy, 9(12), 1764–1770. https://doi.org/10.5897/AJB10.1019 Afzal, I., Rehman, H. U., Naveed, M., & Basra, S. M. A. (2016). Recent advances in seed enhancements. In S. . Araujo & A. . Balestrazzi (Eds.), New Challenges in Seed Biology - Basic and Translational Research Driving Seed Technology. IntechOpen. https://doi.org/10.5772/64791 Aghdaei, M., Nemati, S. H., Samiei, L., & Sharifi, A. (2019). Ef- fect of some priming methods on germination and veg- etative parameters of pepino (Solanum muricatum Aiton). EurAsian Journal of BioSciences, 13, 1919–1929. Ahmadizadeh, M. (2013). Physiological and agro-morpho- logical response to drought stress. Middle East Journal of Scientific Research, 13(8). Ahmadizadeh, M., Valizadeh, M., Zaefizadeh, M., & Shahbazi, H. (2011). Evaluation of interaction between genotype and environments in term of germination and seedling growth in durum wheat landraces. Advances in Environ- mental Biology, 5(4). Ahmadizadeh, M., Babaeian-Jelodar, N., Mohammadi-Nejad, G., Bagheri, N., & Singh, R. K. (2021). High-density link- age mapping for agronomic and physiological traits of rice (Oryza sativa L.) under reproductive-stage salt stress. Journal of Genetics, 100(2), 1–12. https://doi.org/10.1007/ S12041-021-01301-6 Ahmadizadeh, M., Vispo, N. A., Calapit-Palao, C. D. O., Pan- gaan, I. D., Viña, C. Dela, & Singh, R. K. (2016). Reproduc- tive stage salinity tolerance in rice: a complex trait to phe- notype. Indian Journal of Plant Physiology, 21(4), 528–536. https://doi.org/10.1007/s40502-016-0268-6 Ali, B., Al Wabel, N., & Blunden, G. (2005). Phytochemical, pharmacological and toxicological aspects of Hibiscus sabdariffa L.: a review. Phytotherapy Research, PTR, 19(5), 369–375. https://doi.org/10.1002/PTR.1628 Ali, Q., Daud, M. K., Haider, M. Z., Ali, S., Rizwan, M., Aslam, N., … Zhu, S. J. (2017). Seed priming by sodium nitro- prusside improves salt tolerance in wheat (Triticum aes- tivum L.) by enhancing physiological and biochemical parameters. Plant Physiology and Biochemistry PPB, 119, 50–58. https://doi.org/10.1016/J.PLAPHY.2017.08.010 Al-Mudaris, M. A. (1998). Notes on various parameters re- Acta agriculturae Slovenica, 118/2 – 202214 M. AHMADIZADEH et al. cording the speed of seed germination. Der Tropenland- wirt - Journal of Agriculture in the Tropics and Subtropics, 99(2), 147–154. Aloui, H., Souguir, M., Latique, S., & Hannachi, C. (2014). Germination and growth in control and primed seeds of pepper as affected by salt stress. Cercetari Agro- nomice in Moldova, 47(3), 83–95. https://doi.org/10.2478/ CERCE-2014-0029 Al-Tohafi, S.A., Hussain, A.S., Habeeb, H.A., & Azab, N.H. (2015). Response of growth and yield of roselle plant (Hi- biscuss abdariffa L.) for adding (clean salt) and spraying with organic fertilizer (humic aljohara) in high salinity soil. Kufa Journal for Agricultural Sciences, 7(1). Amiri, M. B., Rezvani Moghaddam, P., Ehyai, H. R., Fallahi, J., & Aghhavani Shajari, A. (2010). Effect of osmotic and sa- linity stresses on germination and seedling growth indices of Cynara scolymus and Echinacea purpurea. Environmen- tal Stresses in Crop Sciences, 3(2), 165–176. Ansari, O., Azadi, M. S., Sharif-Zadeh, F., & Younesi, E. (2013). Effect of hormone priming on germination characteristics and enzyme activity of mountain rye (Secale montanum) seeds under drought stress conditions. Journal of Stress Physiology & Biochemistry, 9(3), 61–71. Ashraf, M., & Foolad, M. R. (2005). Pre‐sowing seed treat- ment—a shotgun approach to improve germination, plant growth, and crop yield under saline and non‐saline con- ditions. Advances in Agronomy, 88, 223–271. https://doi. org/10.1016/S0065-2113(05)88006-X Askari Nejad, H. & Farahmand, S. (2012). Evaluating the po- tential of seed priming techniquesin improving germina- tion and early seedling growth of Aeluropus macrostachys under salinity stress condition. Annals of Biological Re- search, 3, 5099-5105. Atak, M., Kaya, M. D., Kaya, G., Cikili, Y., & Ciftci, C. Y. (2006). Effects of NaCl on the germination, seedling growth and water uptake of triticale. Turkish Journal of Agriculture and Forestry, 30, 39–47. Bahaeldeen, M. B., Sulaiman, A. A., & Adam, A. (2012). Roselle (Hibiscus sabdariffa L.) in Sudan, cultivation and their uses. Bulletin of Environment, Pharmacology and Life Sci- ences, 1(6), 48–54. Begcy, K., Sandhu, J. & Walia, H. (2018) Transient heat stress during early seed development primes germination and seedling establishment in rice. Frontiers in Plant Science, 9, 1768. https://doi.org/10.3389/fpls.2018.01768 Bhanuprakash, K., & Yogeesha, H. S. (2016). Seed priming for abiotic stress tolerance: An overview. In R. N. K. . Srini- vasa, K. S. . Shivashankara, & R. H. Laxman (Eds.), Abi- otic Stress Physiology of Horticultural Crops (pp. 103–117). Springer, New Delhi. https://doi.org/10.1007/978-81-322- 2725-0_6 Bruce, T. J. A., Matthes, M. C., Napier, J. A., & Pickett, J. A. (2007). Stressful “memories” of plants: Evidence and pos- sible mechanisms. Plant Science, 173(6), 603–608. https:// doi.org/10.1016/J.PLANTSCI.2007.09.002 Chen, K., & Arora, R. (2011). Dynamics of the antioxidant system during seed osmopriming, post-priming germi- nation, and seedling establishment in Spinach (Spinacia oleracea). Plant Science : An International Journal of Ex- perimental Plant Biology, 180(2), 212–220. https://doi. org/10.1016/J.PLANTSCI.2010.08.007 Da-Costa-Rocha, I., Bonnlaender, B., Sievers, H., Pischel, I., & Heinrich, M. (2014). Hibiscus sabdariffa L. a phytochemi- cal and pharmacological review. Food Chemistry, 165, 424- 443. https://doi.org/10.1016/j.foodchem.2014.05.00 Dkhil, B. Ben, Issa, A., & Denden, M. (2014). Germination and seedling emergence of primed okra (Abelmoschus es- culentus L.) seeds under salt stress and low temperature. American Journal of Plant Physiology, 9(2), 38–45. https:// doi.org/10.3923/AJPP.2014.38.45 Ebrahimi, R., Ahmadizadeh, M., & Rahbarian, P. (2014). En- hancing stand establishment of tomato cultivars under salt stress condition. South Western Journal of Horticulture, Biology and Environment, 5(1), 19–42. Elouaer, Mohamed Aymen, Hannachi, C. (2012). Seed prim- ing to improve germination and seedling growth of saf- flower (Carthamus tinctorius) under salt stress. EurAsian Journal of Biosciences, 6, 76–84. https://doi.org/10.5053/ ejobios.2012.6.0.9 Eskandari, H. (2013). Effects of priming technique on seed germination properties, emergence and field performance of crops: a review. International Journal of Agronomy and Plant Production , 4(3), 454–458. Farnsworth, N. R., & Bunyapraphatsara, N. (1992). Thai me- dicinal plants: recommended for primary health care system. Medicinal Plant Information Center: Mahidol University, 409. Farooq, M., Basra, S. M. A., Afzal, I., & Khaliq, A. (2006). Op- timization of hydropriming techniques for rice seed in- vigoration. Seed Science and Technology, 34(2), 507–512. https://doi.org/10.15258/SST.2006.34.2.25 Farooq, Muhammad, Barsa, S. M. A., & Wahid, A. (2006). Priming of field-sown rice seed enhances germination, seedling establishment, allometry and yield. Plant Growth Regulation, 49(2), 285–294. https://doi.org/10.1007/ S10725-006-9138-Y Farooq, Muhammad, Basra, S. M., Wahid, A., & Ahmad, N. (2010). Changes in nutrient-homeostasis and reserves metabolism during rice seed priming: Consequences for seedling emergence and growth. Agricultural Sciences in China, 9(2), 191–198. https://doi.org/10.1016/S1671- 2927(09)60083-3 Fazlali, R., Eradatmand Asli, D., & Moradi, P. (2013). The effect of seed priming by ascorbic acid on bioactive compounds of naked seed pumpkin (Cucurbita pepo var. styriaca) un- der salinity stress. International Journal of Farming and Al- lied Sciences, 2(17), 587–590. Feghhenabi, F., Hadi, H., Khodaverdiloo, H., & van Genuchten, M. T. (2020). Seed priming alleviated salinity stress during germination and emergence of wheat (Triticum aestivum L.). Agricultural Water Management, 231, 106022. https:// doi.org/10.1016/J.AGWAT.2020.106022 Fuller, M. P., Hamza, J. H., Rihan, H. Z., & Al-Issawi, M. (2012). Germination of primed seed under NaCl stress in wheat. International Scholarly Research Network (ISRN) Botany, 1–5. https://doi.org/10.5402/2012/167804 Galal, A. (2017). Physico-chemical changes in karkade (Hi- biscus sabdariffal L.) seedlings responding to salt stress. Acta agriculturae Slovenica, 118/2 – 2022 15 Comprehensive seed priming assessment of Hibiscus sabdariffa L. in germination and seedling growth stage under salt stress Acta Biologica Hungarica, 68(1), 73-87. https://doi. org/10.1556/018.68.2017.1.7 Hasanuzzaman, M., & Fotopoulos, V. (2019). Priming and pre- treatment of seeds and seedlings. (M. Hasanuzzaman & V. . Fotopoulos, Eds.), Priming and Pretreatment of Seeds and Seedlings (1st ed.). Springer Singapore. https://doi. org/10.1007/978-981-13-8625-1 Hussain, S., Khan, F., Hussain, H. A., & Nie, L. (2016). Physi- ological and biochemical mechanisms of seed priming- induced chilling tolerance in rice cultivars. Frontiers in Plant Science, 0(FEB2016), 116. https://doi.org/10.3389/ FPLS.2016.00116 Ibrahim, E. A. (2016). Seed priming to alleviate salinity stress in germinating seeds. Journal of Plant Physiology, 192, 38– 46. https://doi.org/10.1016/J.JPLPH.2015.12.011 Ibrahim, E. B., Abdalla, A. W. H., Ibrahim, E. A., & Naim, A. M. E. (2013). Variability in some Roselle (Hibiscus sabdar- iffa L.) genotypes for yield and its attributes. International Journal of Agriculture and Forestry, 3(7), 261–266. Iqbal, M., Ashraf, M., Jamil, A., & Ur-Rehman, S. (2006). Does seed priming induce changes in the levels of some endog- enous plant hormones in hexaploid wheat plants under salt stress? Journal of Integrative Plant Biology, 48(2), 181– 189. https://doi.org/10.1111/J.1744-7909.2006.00181.X Ismail, A. M., Heuer, S., Thomson, M. J., & Wissuwa, M. (2007). Genetic and genomic approaches to develop rice germ- plasm for problem soils. Plant Molecular Biology, 65(4), 547–570. https://doi.org/10.1007/s11103-007-9215-2 Jime’nez-Arias, D., Borges, A. A., Luis, J. C., Valdés, F., Sandal- io, L. M., & Pe’rez, J. A. (2015). Priming effect of mena- dione sodium bisulphite against salinity stress in Arabi- dopsis involves epigenetic changes in genes controlling proline metabolism. Environmental and Experimental Botany, 120, 23–30. https://doi.org/10.1016/J.ENVEXP- BOT.2015.07.003 Jisha, K. C., Vijayakumari, K., & Puthur, J. T. (2012). Seed prim- ing for abiotic stress tolerance: an overview. Acta Physiolo- giae Plantarum, 35(5), 1381–1396. https://doi.org/10.1007/ S11738-012-1186-5 Kadamanda, R. (2019). The salinity effect on leaf pigment content of roselle (Hibiscus sabdariffa L.) at different growth stages. Journal of Food, Agriculture & Environment, 17(1), 36-39. Katembe, W. J., Ungar, I. A., & Mitchel, J. P. (1998). Effect of salinity on germination and seedling growth of two Atri- plex species (Chenopodiaceae). Annals of Botany, 82(2), 167–175. https://doi.org/10.1006/ANBO.1998.0663 Kaveh, H., Nemati, H., Farsi, M., & Vatandoost Jartoodeh, S. (2111). How salinity affect germination and emergence of tomato lines. Journal of Biological & Environmental Sci- ences, 5(15), 159–163. Kaya, C., Ak, B. E., & Higgs, D. (2003). Response of salt‐ stressed strawberry plants to supplementary calcium ni- trate and/or potassium nitrate. Journal of Plant Nutrition, 26(3), 543–560. https://doi.org/10.1081/PLN-120017664 Kaya, M. D., Ipek, A., & Ozturk, A. (2003). Effects of different soil salinity levels on germination and seedling growth of safflower (Carthamus tinctorius L.). Turkish Journal of Ag- riculture and Forestry, 27, 221–227. Kerchev, P., van der Meer, T., Sujeeth, N., Verlee, A., Stevens, C.V., Van Breusegem, F., & Gechev, T. (2020). Molecu- lar priming as an approach to induce tolerance against abiotic and oxidative stresses in crop plants. Biotechnol- ogy Advances, 40, 107503. https://doi.org/10.1016/j.bio- techadv.2019.107503 Khan, H. A., Ayub, C. M., Pervez, M. A., Bilal, R. M., Shahid, M. A., & Ziaf, K. (2009). Effect of seed priming with NaCl on salinity tolerance of hot pepper (Capsicum annuum L.) at seedling stage. Soil & Environmental, 28(1), 81–87. Khan, H. A., Pervez, M. A., Ayub, C. M., Ziaf, K., Bilal, R. M., Shahid, M. A., & Akhtar, M. A. (2009). Hormonal priming alleviates salt stress in hot Pepper (Capsicum annuum L.). Soil and Environmental, 28(2), 130–135. Khan, M. A., & Weber, D. J. (2006). Ecophysiology of high sa- linity tolerant plants (Tasks for Vegetation Science). (M. A. Khan & D. J. Weber, Eds.), Ecophysiology of High Salinity Tolerant Plants (1st ed.). Springer Netherlands. https://doi. org/10.1007/1-4020-4018-0 Khodadad, M. (2011). An evaluation of safflower genotypes (Carthamus tinctorius L.), seed germination and seedling characters in salt stress conditions. African Journal of Ag- ricultural Research, 6(7), 1667–1672. Lara, T. S., Marcel, J., Lira, S., Rodrigues, A. C., Rakocevic, M., Alvarenga, A. A., & Lira, J. M. S. (2014). potassium nitrate priming affects the activity of nitrate reductase and anti- oxidant enzymes in tomato germination. Journal of Ag- ricultural Science, 6(2), 72–80. https://doi.org/10.5539/jas. v6n2p72 Latef, A. A. H. A., Zaid, A., Alhmad, M. F. A., & Abdelfattah, K. E. (2020). The impact of priming with Al2O3 nanoparti- cles on growth, pigments, osmolytes, and antioxidant en- zymes of egyptian Roselle (Hibiscus sabdariffa L.) cultivar. Agronomy, 10(5), 681. https://doi.org/10.3390/AGRONO- MY10050681 Lin, J., Wang, Y., Sun, S., Mu, C., & Yan, X. (2017). Effects of arbuscular mycorrhizal fungi on the growth, photosyn- thesis and photosynthetic pigments of Leymus chinensis seedlings under salt-alkali stress and nitrogen deposition. Science of the Total Environment, 576, 234–241. https://doi. org/10.1016/j.scitotenv.2016.10.091 Mahadevan, N., Shivali, P., & Kamboj, J. (2009). Hibiscus sab- dariffa L. -an overview. Natural Product Radiance, 8(1), 77–83. Maher, S., Fraj, H., & Cherif, H. (2013). Effect of NaCl prim- ing on seed germination of Tunisian fenugreek (Trigonella foenum-graecum L.) under salinity conditions. Journal of Stress Physiology & Biochemistry, 9(2), 86–96. Mahesh, H., Murali, M., Anup Chandra Pal, M., Melvin, P., & Sharada, M. (2017). Salicylic acid seed priming instigates defense mechanism by inducing PR-Proteins in Solanum melongena L. upon infection with Verticillium dahliae Kleb. Plant Physiology and Biochemistry : PPB, 117, 12–23. https://doi.org/10.1016/J.PLAPHY.2017.05.012 Migahid, M. M., Elghobashy, R. M., Bidak, L. M., & Amin, A. W. (2019). Priming of Silybum marianum L. Gaertn seeds with H2O2 and magnetic field ameliorates seawater stress. Heliyon, 5(6), e01886. https://doi.org/10.1016/J.HELI- YON.2019.E01886 Acta agriculturae Slovenica, 118/2 – 202216 M. AHMADIZADEH et al. Moradi, A., & Younesi, O. (2009). Effects of osmo- and hydro- priming on seed parameters of grain sorghum (Sorghum bicolor L.). Australian Journal of Basic and Applied Sciences, 3(3), 1696–1700. Muhie, S.H., Yildirim, E., Memis, N., & Demir, I. (2020a). Ver- micompost priming stimulated germination and seed- ling emergence of onion seeds against abiotic stresses. Seed Science and Technology, 48(2), 153-157. https://doi. org/10.15258/sst.2020.48.2.02 Muhie, S., Özdamar, C., Gökdaş, Z., Njie, E.S., Memiş, N., & Demir, İ. (2020b). Effect of solid matrix priming with sea- weed extract on germination and seedling performance of onion seeds under abiotic stress conditions. Black Sea Journal of Agriculture, 3(4), 233-238. Munns, R., & Tester, M. (2008). Mechanisms of salinity toler- ance. Annual Review of Plant Biology, 59(1), 651–681. htt- ps://doi.org/10.1146/annurev.arplant.59.032607.092911 Musa, M., & Lawal, A. A. (2015). Influence of priming concen- tration on the growth and yield of amaranth (Amaranthus cruentus L.) in sokoto semi-arid zone of Nigeria. Jour- nal of Plant Sciences, 3(1), 30. https://doi.org/10.11648/J. JPS.20150301.15 Nadjafi, F., Bannayan, M., Tabrizi, L., & Rastgoo, M. (2006). Seed germination and dormancy breaking techniques for Ferula gummosa and Teucrium polium. Journal of Arid Environments, 64(3), 542–547. https://doi.org/10.1016/J. JARIDENV.2005.06.009 Nasri, N., Kaddour, R., Mahmoudi, H., Baatour, O., Bouraoui, N., & Lachaâl, M. (2011). The effect of osmopriming on germination, seedling growth and phosphatase activities of lettuce under saline condition. African Journal of Bio- technology, 10(65), 14366–14372. https://doi.org/10.4314/ ajb.v10i65 Nassar, K. H. (2010). Effect of seed priming and microdose of organic fertilizer on yield and quality of Roselle. University of Khartoum. Naz, F., Gul, H., Hamayun, M., Sayyed, A., Khan, H., & Sher- wani, S. (2014). Effect of NaCl stress on P. sativum ger- mination and seedling growth with the influence ofseed priming with potassium (KCL and KOH). American- Eurasian Journal of Agricultural & Environmental, 14(11), 1304–1311. Neumann, P. (1997). Salinity resistance and plant growth re- visited. Plant, Cell & Environment, 20(9), 1193–1198. htt- ps://doi.org/10.1046/J.1365-3040.1997.D01-139.X Neumann, P. M. (1995). Inhibition of root growth by salin- ity stress: Toxicity or an adaptive biophysical response? Structure and Function of Roots, 299–304. https://doi. org/10.1007/978-94-017-3101-0_39 Paparella, S., Araújo, S. S., Rossi, G., Wijayasinghe, M., Car- bonera, D., & Balestrazzi, A. (2015). Seed priming: state of the art and new perspectives. Plant Cell Reports, 34(8), 1281–1293. https://doi.org/10.1007/S00299-015-1784-Y Patade, V. Y., Bhargava, S., & Suprasanna, P. (2009). Haloprim- ing imparts tolerance to salt and PEG induced drought stress in sugarcane. Agriculture, Ecosystems & Envi- ronment, 134(1–2), 24–28. https://doi.org/10.1016/J. AGEE.2009.07.003 Qadir, I., Khan, Z. H., Khan, R. A., & Afzal, I. (2011). Evaluation the potential of seed priming techniques in improving germination and early seedling growth of various range- land grasses. Pakistan Journal of Botany, 43, 2797–2800. Ratikanta, M. ., & Kalipada, P. (2013). vegetable seed priming: a low cost, simple and powerful techniques for farmers’ livelihood. International Journal of Bio-Resource and Stress Managemen, 4(4), 475–481. Reed, R.C., Bradford, K.J. & Khanday, I. (2022). Seed germina- tion and vigor: ensuring crop sustainability in a changing climate. Heredity, 1-10. https://doi.org/10.1038/s41437- 022-00497-2 Riaz, G., & Chopra, R. (2018). A review on phytochemistry and therapeutic uses of Hibiscus sabdariffa L. Biomedicine & Pharmacotherapy = Biomedecine & Pharmacotherapie, 102, 575–586. https://doi.org/10.1016/J.BIOPHA.2018.03.023 Rouhi, H. R., Aboutalebian, M. A., & Sharif-zadeh, F. (2011). Effects of hydro and osmopriming on drought stress tol- erance during germination in four grass species. Inten- tional Journal of AgriScience , 1(2), 107–114. Shaheen, H. L., Iqbal, M., Azeem, M., Shahbaz, M., & Shehzadi, M. (2015). K-priming positively modulates growth and nutrient status of salt-stressed cotton (Gossypium hir- sutum) seedlings. Archives of Agronomy and Soil Science, 62(6), 759–768. http://doi.org/10.1080/03650340.2015.10 95292 Shahverdi, M., Omidi, H., & Tabatabaei, S. (2017). Determi- nation of optimum duration and concentration of stevia (Stevia rebaudiana Bert.) seed priming with boric acid (H3BO3). Türkiye Tarımsal Araştırmalar Dergisi, 4(1), 24– 30. https://doi.org/10.19159/tutad.300701 Shannon, M. C., & Grieve, C. M. (1998). Tolerance of vegeta- ble crops to salinity. Scientia Horticulturae, 78(1–4), 5–38. https://doi.org/10.1016/S0304-4238(98)00189-7 Sheteiwy, M.S., Shao, H., Qi, W., Daly, P., Sharma, A., Shagha- leh, H., Hamoud, Y.A., El-Esawi, M.A., Pan, R., Wan, Q., & Lu, H. (2021). Seed priming and foliar application with jasmonic acid enhance salinity stress tolerance of soy- bean (Glycine max L.) seedlings. Journal of the Science of Food and Agriculture, 101(5), 2027-2041. https://doi. org/10.1002/jsfa.10822 Sheyhakinia, S., Bamary, Z., Einali, A., & Valizadeh, J. (2020). The induction of salt stress tolerance by jasmonic acid treatment in roselle (Hibiscus sabdariffa L.) seedlings through enhancing antioxidant enzymes activity and metabolic changes. Biologia, 75(5), 681–692. https://doi. org/10.2478/S11756-020-00444-8 Shruthi, K., Balakrishna, P., & Sreeramu, B. S. (2018). the ef- fects of seed treatments on germination and other seed quality attributes of rosella (Hibiscus sabdariffa Var. sab- dariffa L.). International Journal of Science, Environment, 7(1), 201–206. Sivritepe, N., Sivritepe, H. O., & Eris, A. (2003). The effects of NaCl priming on salt tolerance in melon seedlings grown under saline conditions. Scientia Horticulturae, 97(3–4), 229–237. https://doi.org/10.1016/S0304-4238(02)00198-X Soeda, Y., Konings, M., Vorst, O., Houwelingen, A., Stoo- pen, G., Maliepaard, C., Geest, A. (2005). Gene expres- sion programs during Brassica oleracea seed maturation, osmopriming, and germination are indicators of pro- Acta agriculturae Slovenica, 118/2 – 2022 17 Comprehensive seed priming assessment of Hibiscus sabdariffa L. in germination and seedling growth stage under salt stress gression of the germination process and the stress toler- ance level. Plant Physiology, 137(1), 354–368. https://doi. org/10.1104/PP.104.051664 Subramanyam, K., Du Laing, G., & Van Damme, E. J. M. (2019). Sodium selenate treatment using a combination of seed priming and foliar spray alleviates salinity stress in Rice. Frontiers in Plant Science, 0, 116. https://doi.org/10.3389/ FPLS.2019.00116 Sukkhaeng, S., Promdang, S., & Doung-ngern, U. (2018). Fruit characters and physico-chemical properties of roselle (Hi- biscus sabdariffa L.) in Thailand—A screening of 13 new genotypes. Journal of Applied Research on Medicinal and Aromatic Plants, 11, 47–53. https://doi.org/10.1016/J.JAR- MAP.2018.10.001 Shruthi, K., Balakrishna, P., & Sreeramu, B.S. (2018). The ef- fects of seed treatments on germination and other seed quality attributes of rosella. International Journal of Sci- ence, Environment and Technology, 7, 201-206. Tanou, G., Fotopoulos, V., & Molassiotis, A. (2012). Prim- ing against environmental challenges and pro- teomics in plants: Update and agricultural per- spectives. Frontiers in Plant Science, 0(SEP), 216. https://doi.org/10.3389/FPLS.2012.00216 Taylor, A. G., & Harman, G. E. (1990). Concepts and tech- nologies of selected seed treatments. Annual Review of Phytopathology, 28, 321–339. https://doi.org/10.1146/AN- NUREV.PY.28.090190.001541 Thiam, M., Champion, A., Diouf, D., & Ourèye SY, M. (2013). NaCl effects on in vitro germination and growth of some senegalese cowpea (Vigna unguiculata (L.) Walp cultivars. International Scholarly Research Notices (ISRN) Biotechnol- ogy, 1–11. https://doi.org/10.5402/2013/382417 Wang, W., Vinocur, B., & Altman, A. (2003). Plant responses to drought, salinity and extreme temperatures: towards genetic engineering for stress tolerance. Planta, 218(1), 1–14. https://doi.org/10.1007/S00425-003-1105-5 Werner, J. E., & Finkelstein, R. R. (1995). Arabidopsis mu- tants with reduced response to NaCl and osmotic stress. Physiologia Plantarum, 93(4), 659–666. https://doi. org/10.1111/J.1399-3054.1995.TB05114.X Wright, C., Van-Buren, L., Kroner, C., & Koning, M. M. (2007). Herbal medicines as diuretics: a review of the scientific evidence. Journal of Ethnopharmacology, 114(1), 1–31. https://doi.org/10.1016/J.JEP.2007.07.023 Acta agriculturae Slovenica, 118/2, 1–9, Ljubljana 2022 doi:10.14720/aas.2022.118.2.1744 Original research article / izvirni znanstveni članek Comparative study between fungicides and some chemical inducers for controlling root rot incidence of green bean (Phaseolus vulgaris L.) under field conditions Nehal Samy EL-MOUGY 1, Nadia Gamel EL-GAMAL 1, Mohamed Saied Ali KALIL 1, Mokhtar Moham- ed ABDEL-KADER 1, 2 Received June 20, 2020; accepted March 24, 2022. Delo je prispelo 20. junija 2020, sprejeto 24. marca 2022 1 Plant Pathology Department, National Research Centre, Giza, Egypt 2 Corresponding author, e-mail: mokh_nrc@yahoo.com Comparative study between fungicides and some chemical inducers for controlling root rot incidence of green bean (Phaseolus vulgaris L.) under field conditions Abstract: Root rot disease caused by Rhizoctonia solani J.G. Kuhn and Fusarium solani (Mart.) Sacc. is a major prob- lem restricting profitable farming of green bean (Phaseolus vulgaris L.). Under field conditions, some chemical inducers compared with chemical fungicides were evaluated for con- trolling bean root rot disease. Significant effect was observed for all applied treatments against disease incidence compared with control. Applied treatments of seed dressing plus foliar spray showed the highest reduction of root rot incidence fol- lowed by seed dressing then foliar spray. Salicylic acid as seed followed by foliar spray showed the highest suppressive effect against disease incidence followed by glutathione treatments. Furthermore, application of calcium silicate revealed higher effect against disease incidence compared with potassium and sodium silicate at both pre-, and post-emergence plant growth stages. Fungicidal treatments showed affect disease incidence in a lower extent compared with Plant Resistance Inducers (PRI). Treatment of Rizolex T50 followed by Topsin M70 was more effectively in controlling root rot than each fungicide alone. Such applied treatments could be useful for controlling root rot disease under field conditions Key words: bean; fungicide alternatives; root rot; Rizolex T50; Topsin M70 Primerjalna raziskava fungicidov in kemičnih vzpodbujeval- cev za nadzor koreninske gnilobe pri fižolu (Phaseolus vulga- ris L.) v razmerah na prostem Izvleček: Koreninska gniloba, ki jo povzročata glivi Rhi- zoctonia solani J.G. Kühn in Fusarium solani (Mart.) Sacc. je glavni omejujoči dejavnik za donosno pridelavo stročjega fižola(Phaseolus vulgaris L.). V razmerah poljskega poskusa so bili primerjani učinki kemičnih vzpodbujevalcev in kemičnih fungicidov v njihovi sposobnosti nadzora koreninske gnilobe. V primerjavi s kontrolo so bili opaženi značilni učinki vseh obravnavanj proti bolezni. Obravnavanja s kapsuliranimi se- meni in foliarnimi pršili so pokazala največje zmanjšanje gnilo- be, tem so sledila obravnavanja s kapsuliranimi semeni in nato obravnavanja s foliarnimi pršili. Salicilna kislina kot sredstvo obdelave semen in kot naknadno foliarno pršilo je imela naj- večji učinek na zaviranje bolezni, temu je sledilo obravnavanje z glutationom. Nadalje je bila uporaba kalcijevega silikata bolj učinkovita pri zatiranju bolezni v primerjavi s kalijevim in na- trijevim silikatom v obravnavanjih pred in po vzniku rastlin. Obravnavanja s fungicidi so pojav bolezni bolj zmanjšala kot tista z alternativnimi sredstvi. Obravnavanje s fungicidom Ri- zolex T50 in nato s fungicidom Topsinn M70 je bilo bolj učin- kovito pri nadzoru gnilobe kot uporabi posameznih fungicidov. Našteta obravnavanja bi torej lahko bila koristna za nadzor fižo- love gnilobe v poljskih razmerah. Ključne besede: fižol; alternativni fungicidi; koreninska gniloba; Rizolex T50; Topsin M70 Acta agriculturae Slovenica, 118/2 – 20222 1 INTRODUCTION Soilborne plant pathogens are considered the main problems in agricultural production all-over the world that they affected seriously on plant stand caus- ing great losses in produced yield. Therefore, growing plants are exposed to invasion by various soilborne pathogens during their different growth stages starts from seed sowing up to maturity. Bean (Phaseolus vulgaris L.) is one of food legume species that widely cultivated for domestic use, exportation, and it has considerable importance for human food especially in developing countries (Baudoin et al., 2001). Certain fungi could attack bean plants causing root rot, wilt and leaf spot diseases which greatly influenced plant stand and subsequently yield production. Root rot disease caused by particular soilborne pathogens have an effect on emerged seedlings and may be occurs earlier when seeds are attacked during their emergence causing pre- emergence infection leading to the need of re-sowing the missed hills or dead plants. The most harmful soilborne fungi causing root rot disease of bean are Fusarium solani Sacc., Sclerotium rolfsii Sacc. and Rhizoctonia solani J.G. Kühn (Abdel- Kader, 1997; El-Mougy et al., 2007). A high buildup of root rot pathogen inoculums due to successive culti- vation on the same land could leads to causes serious yield losses. Therefore, control of this disease is consid- ered important especially in new reclaimed soil where green bean is wide prevalence crop in Egypt. The present research focuses on comparing the use of fungicides and natural compounds that are capable to control root rot incidence. The objective of this study was evaluate some chemical fungicides and chemical inducers as alternatives to fungicide application to achieve effective management against the incidence of green bean root rot disease when used as seed dressing and/or foliar spray under natural field conditions. 2 MATERIALS AND METHODS 2.1 TESTED MATERIALS: Green bean seeds (Phaseolus vulgaris ‘Giza 3’) kindly obtained from Vegetables Crop Research De- partment, Agricultural Research Centre, Giza, Egypt. The tested chemicals potassium silicate, calcium sili- cate, glutathione, salicylic acid and sodium silicate pur- chased from Al-Gamhoria Company Ltd. for chemicals and medicinal instruments, Cairo, Egypt. Meanwhile, the fungicides Rizolex-T 50 WP (20 % Tolclophos- methyl and 30 % Thiram) Sumi Agro Co. and Topsin M70 WSB (thiophanate-methyl 70 %) Martin’s Co. pur- chased from local market. 2.2 FIELD EXPERIMENTS: A field located at Al-Nubaria region, Beheira governorate, Egypt was chosen for this experiment. This field is well known by the authors to be charac- terized with semi homogeneous distribution with root rot pathogens mainly Fusarium solani, Sclerotium rolf- sii and Rhizoctonia solani as naturally heavily infested soil. In addition, at prior growing season to the present study, samples of growing bean plants showing root rot disease symptoms were collected and subjected to iso- lation trails for the causal organisms. The present field experiment was carried out for two successive growing seasons (March and September, 2019) to evaluate the efficacy of some chemical induc- ers and fungicides applied as seed dressing and/or foliar spray against root rot incidence. The experimental field contains plots (6 × 7 m), each included 12 rows with 35 holes. In all plots green bean seeds of ´Giza 3` were sown at the rate of three seed/hole. Seed dressing was carried out before sowing, meanwhile foliar spray was applied twice, the first was at the two true leaves age of emerged seedlings and the second after 15-day interval. All treated plants were sprayed using 20 l sprayer for each plot. 2.3 APPLICATIONS TO THE EXPERIMENTAL FIELD For the two cultivation seasons the same proposed treatments were designed as follows: a) Seed treatment at the rate of 3 g kg-1 T1- potassium silicate T2- calcium silicate T3- glutathione T4- salicylic acid T5- sodium silicate T6- Rizolex T50 b) Seed treatment at the rate of 3g l-1 + foliar spray at the rate of 3 g l-1 T7- potassium silicate + potassium silicate T8- calcium silicate + calcium silicate T9- glutathione + glutathione T10- salicylic acid + salicylic acid T11- sodium silicate + sodium silicate T12- Rizolex T50 (3 g l-1) + Topsin M70 (3 g l-1) N. S. EL-MOUGY et al. Acta agriculturae Slovenica, 118/2, –2022 c) Foliar spray at the rate of 3 g l-1 T13- potassium silicate T14- calcium silicate T15- glutathione T16- salicylic acid T17- sodium silicate T18- Topsin M70 T19- untreated control 2.4 DISEASE AND YIELD ASSESSMENT Three plots as replicates were used for every spe- cific treatment as well as untreated control. All plots were conducted in completely randomized block de- sign. The traditional agricultural practices, that is, soil plowing, fertilization, irrigation, etc., were followed at all experimental plots. Monitoring and scouting for dis- eases incidence in all cultivated plots were preformed weekly (El-Mougy and Abdel-Kader et al., 2018). At all applied treatments and control as well the percent of pre-emergence root rot disease incidence was recorded after 15 days from sowing date as numbers of emerged seedlings referring to the numbers of sown seeds. Meanwhile, the percentage of post-emergence disease infection were recorded after 15, 30 and 45 days as numbers of diseases plants referring to the numbers of emerged seedlings. Percentage of healthy survivals was calculated as the numbers of survival plants referring to the numbers of sown seeds. Accumulated yield was determined as fresh pods (kg/plot) for each particular treatment at the end of growing season. The increase of obtained yield in relative to comparison treatment was also calculated. 2.5 STATISTICAL ANALYSIS The obtained data subjected to analysis of variance using IBM SPSS software version 14.0. Mean separation performed using Duncan’s Multiple Range Test at p ≤ 0.05 by the MSTAT-C software 3 RESULTS The fungi isolated from the bean plants showing root rot symptoms were identified as Fusarium solani and Rhizoctonia solani. The results showed that all ap- plied treatments had announced effects against root rot incidence compared with the control (Table 1). Applied treatments of a seed dressing + foliar spray showed lower root rot disease incidence followed by seed dressing alone, and then foliar spray alone. Moreover, the effects of the fungicide applications were lower than those of the chemical inducers and followed similar trend as the applied methods mentioned above. The results also indicate that the application of salicylic acid as a seed and/or foliar spray showed the highest reduction in root rot incidence followed by the glu- tathione treatments. The percentage of pre- and post- emergence root rot was recorded as 5.3 %, 8.0 % [T4]; 5.5 %, 6.5 % [T10] and 15.6 %, 21.0 % [T16] followed by 7.3 %, 12.6 % [T3]; 7.5 %, 9.6 % [T9] and 14.3 %, 27.0 % [T15] when compared with the 30.0 %, 57.3 % of the untreated control treatment [T19]. Moreover, the pre- and post-emergence potassium silicate [T1], [T7] applications showed greater disease reduction when compared with the calcium [T2[, [T8] and sodium sili- cate [T5], [11]. For the seed dressing, seed dressing + foliar spray, and foliar spray treatments disease reduction was cal- culated as 70.0 %, 73.8 % [T2]; 72.0 %, 80.8 % [T8] and 38.0 %, 46.5 % [14] in relation to the control treatment [T19], respectively. Meanwhile for the same applied treatments disease reductions of 61.3 %, 74.6 % [T1]; 60.0 %, 82.0 % [T7] and 21.6 %, 38.9 % [T13] were iden- tified. Likewise, for treatments [T5], [T11], and [T17] the percentages of root rot disease reduction were 60.0 %, 69.4 %; 56.6 %, 75.0 %, 30.0, and 41.8 % at pre-, and post-emergence growth stages, in respective order. The fungicidal treatment data showed that the combination of two fungicides applied as a seed dress- ing + foliar spray resulted in lower disease incidence when compared with the seed dressing and foliar spray, in descending order (Table 1). The percentage of root rot incidence for [T12] was 9.5 % and 10.6 % with dis- ease reduction of 68.3 % and 81.5 % at the pre-, and post-emergence growth plant stages, respectively. Fur- thermore, for the [T6] treatment, disease incidence and reduction were recorded as 9.6 %, 14.6 %, and 68.0 %, 74.5 % for both plant growth stages in parallel, respec- tively. Meanwhile a higher root rot incidence of 27.3 %, 40.0 % and its lower reduction of 9.0 %, 30.1 % were recorded when Topsin M70 [18] was applied only as a foliar spray, although it significantly differed when compared with the untreated control [T19]. The results also revealed that applied treatments increased plant survival when compared with the un- treated control (Table 1). The highest percentage of plant survival recorded with the seed dressing + foliar spray was followed by seed dressing then foliar spray treatments, respectively. The highest percentages of plant survival were 88.0 %, 81.9 %, and 81.5 % with the [T10], [T9], and [T8] treatments, followed by 86.6 %, 78.6 %, and 77.3 % with the [T4], [T3], and [T2] treat- Comparative study between fungicides and some chemical inducers for controlling root rot incidence of green bean (Phaseolus vulgaris L.) ... Acta agriculturae Slovenica, 118/2 – 20224 N. S. EL-MOUGY et al. ments, and 63.3 %, 58.6 %, and 50.6 % with the [T16], [T15], and [T14] treatments. The other applied chemi- cal inducer treatments ranged between 37.5 % with the [T13] treatment to 77.6 % with the [T7] treatment for plant survival. For the fungicide applications there was 79.8 %, 75.6 %, and 32.6 % survival with the [T12] Ri- zolex T50 WP + Topsin M70 WSB followed by [T6] Ri- zolex-T50 WP and [T18] Topsin M70 WSB, respectively which was significant when compared with 12.6% for the [T19] control treatment. In contrast, the data indicated that the obtained decreases in root rot disease incidence due to the cur- rent applied treatments resulted in an increase in plant survival which was subsequently reflected in the accu- mulated product yield. The green pod yields from the beans showed similar tendencies to the reduction in disease incidence identified in Table 1 and Fig. 1. The highest obtained yields recorded were 27.8, 26.2, and 24.6 kg/plot with an increase over the control calculated as 78.2 %, 67.9 %, and 57.6 % for the [T10], [T9], and [T8] treat- ments which included a seed dressing + foliar spray treatments, respectively. There were moderate yield in- creases of 70.5 %, 55.1 %, and 42.9 % for the [T4], [T3], and [T2] treatments which involved seed dressing. Per- centages of 71.7 %, 50.0 %, and 36.5 % indicated lower increases in the produced yields and were recorded for the [T16], [T15], and [T14] treatments which in- volved foliar sprays. The rest of the applied chemical treatments revealed a yield increase between 12.8 % for [T13] with a foliar spray and 44.8 % for [T7] with a seed dressing + foliar spray. Root rot diseases incidence (%) Pre- emergence** Red.* (%) Post- emergence** Red.* (%) Plant survivals (%) Seed dressing [T1] Potassium silicate 11.6 ± 0.8 cd 61.3 14.5 ± 1.9 de 74.6 73.8 ± 1.0 f [T2] Calcium silicate 9.0 ± 0.5 e 70.0 15.0 ± 1.0 de 73.8 77.3 ± 1.1f [T3] Glutathione 7.3 ± 0.5 f 75.6 12.6 ± 0.5 e 78.0 78.6 ± 2.0 f [T4] Salicylic acid 5.3 ± 0.7 g 82.3 8.0 ± 1.5 fg 86.0 86.6 ± 1.2 g [T5] Sodium silicate 12.0 ± 2.4 cd 60.0 17.5 ± 0.8 d 69.4 70.5 ± 1.7 f [T6] Rizolex-T50 9.6 ± 0.8 e 68.0 14.6 ± 1.4 de 74.5 75.6 ± 1.9 f Seed dressing + Foliar spray [T7] Potassium silicate 12.0 ± 1.1 cd 60.0 10.3 ± 1.6 f 82.0 77.6 ± 1.1f [T8] Calcium silicate 8.4 ± 0.8 ef 72.0 11.0 ± 1.7 e 80.8 81.5 ± 1.7 g [T9] Glutathione 7.5 ± 0.8 f 75.0 9.6 ± 2.8 f 83.2 81.9 ± 1.7 g [T10] Salicylic acid 5.5 ± 0.8 g 81.6 6.5 ± 2.6 g 88.6 88.0 ± 0.4 g [T11] Sodium silicate 13.0 ± 1.1 cd 56.6 14.3 ± 2.3 de 75.0 72.6 ± 0.8 f [T12] Rizolex-T50 + Topsin M70 9.5 ± 2.9 e 68.3 10.6 ± 1.0 f 81.5 79.8 ± 1.5 f Foliar spray [T13] Potassium silicate 23.5 ± 2.4 ab 21.6 35.0 ± 1.5 ab 38.9 37.5 ± 0.8 b [T14] Calcium silicate 18.6 ± 2.3 b 38.0 30.6 ± 2.0 b 46.5 50.6 ± 2.0 d [T15] Glutathione 14.3 ± 2.7 c 52.3 27.0 ± 1.3 bc 52.8 58.6 ± 1.0 d [T16] Salicylic acid 15.6 ± 1.2 c 48.0 21.0 ± 0.6 c 63.3 63.3 ± 0.5 e [T17] Sodium silicate 21.0 ± 0.6 ab 30.0 33.3 ± 3.3 ab 41.8 45.6 ± 1.2 c [T18] Topsin M70 27.3 ± 0.4 a 9.0 40.0 ± 0.3 ab 30.1 32.6 ± 0.8 b [T19] Control 30.0 ± 1.5 a - 57.3 ± 1.3 a - 12.6 ± 1.8 a Table 1: Average efficacy of some chemical inducers and fungicides against bean root rot incidence during two growing sea- sons under field conditions * Red. = Reduction ** Pre-emergence calculated after 15 days from seed sowing *** post-emergence calculated after 45 days from seedlings emergence Means ± standard deviations within a column followed by the same letter are not significantly different by Duncan multiple range test at p < 0.05 5 Comparative study between fungicides and some chemical inducers for controlling root rot incidence of green bean (Phaseolus vulgaris L.) ... Acta agriculturae Slovenica, 118/2 – 2022 For the fungicidal applications, yield was increased by 26.9 % for [T12] in response to the application of Rizolex T50 WP as a seed dressing followed by a foliar spray with Topsin M70 WSB. Meanwhile yield increases of 19.2 % and 17.3 % were recorded for [T6] and [T18] with the Rizolex T50 WP seed dressing and Topsin M70 WSB foliar spray, respectively. 4 DISCUSSION In the current study the fungi isolated from the collected bean plants that showed root rot symptoms were identified as Fusarium sp. and Rhizoctonia solani Root rot disease of green caused by F. solani and R. sola- ni has been previously reported (Abdel-Kader, 1997, El- Mougy et al., 2007, El-Mougy and Abdel-Kader, 2018). Additionally, common bean, cowpea, and faba bean, which are suitable pathogen hosts, are regularly grown in the study field, thus, it is assumed that there is an in- creasing population of the soilborne root rot pathogen as these crops are considered suitable hosts. This inves- tigation aimed to evaluate the use of chemical inducers as fungicide alternatives when compared with chemical fungicides applied as seed dressing and/or foliar sprays to control the incidence of green bean root rot disease under field conditions. The obtained results revealed that the applied chemical inducers as well as the fungi- Fig. 1: Average accumulated yield of bean plants and its increase (%) in response to application of some chemical inducers and fungicides at two growing seasons under field conditions cidal treatments were all highly effective at reducing disease incidence and increasing yield. In this regard, salicylic acid treatments as a seed dressing and/or foliar spray had high efficacy against root rot incidence and yield increase. These results are in accordance with those of previous studies (El-Mougy et al., 2019). El- Mohamady et al. (2017) reported on the use of chemi- cal inducers, such as chitosan (CH), salicylic acid (SA), and humic acid (HA) for the control of bean root rot caused by Fusarium solani and Rhizoctonia solani under both greenhouse and field conditions. They found that soaking bean seeds in CH 1.0 g/l-1 + SA 5% followed by foliar applications at half of this concentration, caused a superior reduction in both damping-off and root rot incidence when compared with their other applied treatments. In addition, Anderson (1988) reported that salicylic acid as a phenolic compound acts as a regula- tor key of the internal coding network in plants under either abiotic or biotic stress. It plays a major role in the plant resistance functions against pathogens as it pro- motes the production of pathogenesis-related proteins (PRPs). Furthermore, salicylic acid was accountable for the aggregation of phytoalexins in viable plant tissues. Mandel et al. (2009) stated that external or internal op- erators might ultimately affect the host plant physiolo- gy, leading to the fast and harmonic activation of de- fense-genes in plants which were susceptible to parasite infections. However, Jabnoun et al. (2015) reported that Acta agriculturae Slovenica, 118/2 – 20226 N. S. EL-MOUGY et al. systemic acquired resistance for controlling tomato fungal diseases could be induced by using salicylic acid and chitosan as chemical inducers. Likewise, several workers reported yield increases when chemical induc- ers or fungicides were applied (El-Mougy et al. 2007, El-Mohamady and Abd-El-Baky, 2008, Abd-El-Kareem et al., 2013, Abdel-Kader et. al., 2014, El-Mohamady et al., 2017, El-Mougy et al., 2019). In the present study the used chemical inducers, salicylic acid and potassium, calcium, sodium silicate had high activity against root rot incidence and yield increase as well as when applied as a seed dressing and/or foliar spray. Recently, the ap- plication of several chemical inducers received a large amount of attention due to various investigations into the control of plant diseases. Glutathione (GSH) as an antioxidant has a role in regulating plant tolerance to biotic stresses by repressing localized necrotic symp- toms following viral infections. Utilizing the pharmaco- logical and transgenic approaches confirmed the role of GSH for reducing disease symptoms in plants which were induced by pathogen infections. Furthermore, re- cent studies have shown that GSH also has a key role in restricting pathogen levels. In fact, it seems that GSH is a vital agent responsible for the elicitors involved in dif- ferent types of plant disease resistance (Gullner et al., 2017). Furthermore, glutathione (GSH) was reported to be involved in the activation and regulation of the bio- synthetic processes involved in plant defense (Bolter et al., 1993). Moreover, glutathione functions include sev- eral roles in biosynthetic pathways, detoxification, anti- oxidant biochemistry, and redox homeostasis (Noctor et al., 2012). In contrast, applications of silicon salts proved their activity against pathogenic fungal growth in vitro as well as plant disease incidence. Silicates were reported to have efficacy for reducing plant diseases in rice (Datnoff et al., 1997), strawberry (Kanto et al., 2006), wheat (Belanger et al., 2003), and cucumber (Menzies et al., 1992). Biggs et al. (1997) stated that PDA medium supplemented with calcium silicate in- hibited 65% of the growth of Monilinia fructicola (G.Winter) Honey the causal agent of peach brown rot. Furthermore, it also inhibited mycelial growth for sev- eral phytopathogenic fungi grown on potassium sili- cate amended media in vitro (Bekker et al., 2006, 2009). Li et al. (2009) also reported an inhibitor effect for so- dium silicate against the growth of Fusarium sulphu- ureum Schltdi. in vitro. Moreover, an in vivo foliar spray of potato plants with 100 and 200 mM sodium silicate was found to control tuber dry rot disease effectively. They concluded that sodium silicate has direct fungi- toxic effects against the fungal pathogen. Furthermore, it was reported that soluble potassium silicate applied as a root and foliar spray caused reductions in disease incidence as well as an increase in growth and fruit quality for capsicum plants (Jayawardana et al., 2014). Ultimately, Shen et al. (2010) stated that it is probable that a reduction in disease incidence in plants treated with silicon sources under field conditions is not likely to be attributed to the fungistatic effects of silicon, but could act as a physical block which directly prevents pathogen penetration to plant tissues or indirectly by enhancing plant defense responses. It is suggested that silicon may act as the first protecting block in treated plants and could inhibit pathogen colonization and subsequent infection. Therefore, potassium silicate could be used as a fungicide since it has direct inhibitor effects on fungal growth and its ability to increase plant self-defense systems (Menzies et al., 1992) and strength- en plant cell walls, inhibiting disease infection (Epstein, 1999). The current investigation showed the lesser ef- fects of contact and systemic fungicides for controlling bean root rot incidence when compared with chemical inducers, despite their superior activity over control treatments against disease occurrence. The fungicide Rizolex (Tolclofos - methyl) is an organophosphate es- ter chemical compound which has protective, curative and slightly systemic action and has high fungitoxicity to Rhizoctonia solani and Sclerotium rolfsii (Ohtsuki and Fujinami, 1982). Rizolex was reported to actively re- duce the incidence of southern stem rot caused by R. solani and S. rolfsii and increase peanut yield (Csinos, 1985). Rizolex T50 is a seed treatment fungicide that delivers protection against a broad spectrum of soil borne and seed/seedling diseases, including Rhizoctonia damping-off and Fusarium (Hopkins 2013). Hamed (2008) reported that Rizolex completely inhibited the growth of Rhzictonia solani (100 %) at all concentra- tions (0.025, 0.05, 0.25, and 0.5 ppm) in vitro. Recently, El-Mohamady et al. (2017) concluded that using chem- ical inducers in comparison with the fungicide Rizolex had superior effects to RIS alone against bean damping- off, root rot diseases, and increasing the produced yields, and therefore may be considered as an eco- friendly applied method for the control of soil-borne plant pathogens. Topsin M70 (thiophanate-methyl) is a fungicidal substance which belongs to the agent group of benzimidazoles. It is a wide range systemic fungicide controlling a wide variety of plant pathogens (Hirschfeld et al. 2010). The primary effect of thiophanate-methyl is caused by the transformation product methyl-benzimi- dazole-2yl-carbamate, which binds to the fungal tubu- lin and disturbs the formation of the spindle apparatus during mitosis so that homologous chromosomes can- not divide, and cell growth will be inhibited. It is ab- sorbed by the treated plant roots, leaves, and has a pro- tective and curative action (Saber, et. al, 2011). A Acta agriculturae Slovenica, 118/2 – 2022 7 Comparative study between fungicides and some chemical inducers for controlling root rot incidence of green bean (Phaseolus vulgaris L.) ... combine use of the fungicides Topsin-M and Dimecron showed significant increase in phenolic content (Sid- diqui et. al., 1999). Increase in phenolic and phenolic content produced as a result of stress may act as a pro- tective compound against pathogenic fungi and insects (Friend, 1979). In vitro tests with Rizolex-T and Topsin- M at 200 ppm have completely inhibited the growth of R. solani and F. oxysporum Schlecht. emend. Snyder & Hansen the causal pathogens of root rot and wilt com- plex disease, which used soil culture medium in some surveyed nurseries (Abdel-Kader et. al., 2004). Moreo- ver, the use of Topsin M70 as a foliar spray was effective against various plant diseases, such as citrus mold caused by Penicillium italicum Wehmer (Kanan and Al- Najar, 2009), Fusarium mangiferae Kanan and Al-Najar, 2009), Fusarium mangiferae Britz, Wingfield & Marasas on mango (Iqbal et al., 2010), Phytophthora infestans (Mont.) de Bary the causal agent of tomato late blight (Meya et al., 2014), faba bean chocolate spot caused by Botrytis fabae Sardina (Moustafa et al., 2015), Lasiodip- lodia theobromae (Pat.) Griffon & Maubl. the causal of die-back of grapevine (El-Habbaa et al., 2016) and Fusarium oxysporum f. sp. capsici the causal agent of wilt disease on chili pepper (Bashir et. al., 2018). The present investigation demonstrates that the applied chemical inducers and fungicides were efficient at controlling green bean root rot disease incidence and increased the accumulated yield. Treatments of seed dressing followed by foliar spray showed higher effec- tivity on disease incidence and produced greater yields than with the seed dressing or foliar spray individually. 5 CONCLUSIONS The results of the current study suggest that the combined application method of chemical inducers such as seed and/or foliar sprays was superior to sin- gle treatments for reducing root rot incidence on green bean plants and increasing accumulated pod yields. The commercial fungicides Rizolex T50 WP and Topsin M70 WSB showed similar trends for controlling disease incidence. Such treatments may be used commercially and could be said to have characteristics such as being eco-friendly, safe, cheap, and an easily applied alterna- tive fungicide methods for use in natural field condi- tions. 6 REFERENCES Abdel-Kader, M.M. (1997). Field application of Trichoderma harzianum as biocide for control of bean root rot disease. Egyptian Journal of Phtopathology, 25, 19-25. Abdel-Kader, M.M., El-Bahr, M.K. & El-Mougy, N.S. (2004). Pathogenic Fungi and Soil Conditions Causing Root Rot and Wilt Disease Complex during Acclimatization of Tis- sue Culture-Derived Banana Plantlets. Egyptian Journal of Phytopathology, 32(1-2), 37-48. https://www.research- gate.net/publication/237702728 Abdel-Kader, M.M., El-Mougy, N.S., & Nadia Gad. (2014). Some Control Measures against Lupine Wilt Disease Incidence under Field Conditions. International Jour- nal of Engineering and Innovative Technology, 4(2), 239-243. http://www.ijeit.com/Vol%204/Issue%202/ IJEIT1412201408_41.pdf Abd-El-Karem, F. Abdel-Kader, M.M., Fotouh, Y.O., Abd-Alla, A.M. El-Mougy, N.S., El- Mohamedy, R.S. & El-Gamal, N.G. (2013). Induction of Systemic Resistance in faba bean Against chocolate spot Diseases Severity Using Chemical Inducers under Field Conditions. Journal of Applied Sciences Research, 9(6), 4006-4014. http://www. aensiweb.com/old/jasr/jasr/2013/4006-4014.pdf Anderson, A.J. (1988). Elicitors the hypersensitive response and phytoalexins. Pages 103-110 in: Physiology and Bio- chemistry of Plant-Microbial Interactions. American Soci- ety of Plant Physiology, Rockville, MD. Bashir, M.R., Atiq, M., Sajid, M., Mohsan, M., Abbas, W., Alam, M.W. & Bashir, M. (2018). Antifungal exploitation of fun- gicides against Fusarium oxysporum f. sp. capsici causing Fusarium wilt of chilli pepper in Pakistan. Environmental Science and Pollution Research, 25(7), 6797‐6801. https:// doi.org/10.1007/s11356-017-1032-9 Baudoin, J.P., Camarena, F., Lobo, M., & Mergeai, G. (2001). Breeding Phaseolus for intercrop combinations in Andean highlands. In: Cooper, H.D., Spillane, C. and Hodgkin, T. eds. Broadening the genetic bases of crop. Oxford, UK: CABI Publishing, pp. 373-384. https://doi. org/10.1079/9780851994116.0373 Bekker, T.F., Kaiser, C., & Labuschagne, N. (2009). The anti- fungal activity of potassium silicate and the role of pH against selected plant pathogenic fungi in vitro. S South African Journal of Plant and Soil 26(1), 55-57. https://doi. org/10.1080/02571862.2009.10639934 Bekker, T.F., Kaiser, C., Van Der Merwe, R., & Labuschagne, N. (2006). In vitro inhibition of mycelial growth of several phytopathogenic fungi by soluble silicon. South African Journal of Plant and Soil, 23(3), 169-172. https://doi.org/1 0.1080/02571862.2006.10634750 Belanger, R.R., Benhamou, N., & Menzies, J.G. (2003). Cyto- logical evidence of an active role of silicon in wheat re- sistance to powdery mildew (Blumeria graminis f. sp. trit- ici). Phytopathology, 93, 402–12. https://doi.org/10.1094/ PHYTO.2003.93.4.402 Bolter, C., Brammall, R.A., Cohen, R., & Lazarovits, G. (1993). Glutathione alterations in melon and tomato roots fol- lowing treatment with chemicals which induce disease resistance to Fusarium wilt. Physiological and Molecular Plant Pathology, 42(5), 321-336. https://doi.org/10.1016/ S0885-5765(05)80009-8 Csinos, A.S. (1985). Activity of Tolclofos - Methyl (Rizolex) on Acta agriculturae Slovenica, 118/2 – 20228 N. S. EL-MOUGY et al. Sclerotium rolfiii and Rhizoctonia solani in Peanut. Peanut Science, 12, 32-35. https://www.peanutscience.com/doi/ pdf/10.3146/pnut.12.1.0008 Datnoff, L.E., Deren, C.W., & Snyder, G.H. (1997). Silicon fer- tilization for disease management of rice in Florida. Crop Protection, 16, 525–531. https://doi.org/10.1016/S0261- 2194(97)00033-1 El-Habbaa, G.M., Mahdy, A.M.M., Mohamed, F.G., & El- Shaery, S.A. (2016). Biological and chemical control of grapevine die-back disease and their effect on defense related enzymes. International Journal of Scientific & Engineering Research, 7(3), 345-351. https://www.ijser. org/researchpaper/Biological-and-chemical-control-of- grapevine-die-back-disease-and-their-effect-on-defense- related-enzymes.pdf El-Mohamedy, R.S.R., & Abd–El-Baky, M.M.H. (2008). Ef- fect of seed treatment on control of root rots disease and improvement of growth and yield of pea plants. Middle Eastern and Russian Journal of Plant Science and Biotech- nology, 2(2), 84-90. http://www.globalsciencebooks.info/ Online/GSBOnline/images/0812/MERJPSB_2(1&2)/ MERJPSB_2(2)84-90o.pdf El-Mohamedy, R.S.R., Shafeek, M.R., Abd El-Samad, E.H., Salama, D.M., & Rizk, F. A. (2017). Field application of plant resistance inducers (PRIs) to control important root rot diseases and improvement growth and yield of green bean (Phaseolus vulgaris L.). Australin Journal of Crop Science, 11(5), 496-505. https://doi.org/10.21475/ ajcs.17.11.05.p260 El-Mougy, N.S., & Abdel-Kader, M.M. (2018). Control of green bean root rot incidence using bioagent and anti- oxidants as seed dressing under natural field conditions. BioScience Research, 15(2), 1401-1408. https://www.isisn. org/BR15(2)2018/1401-1408-15(2)2018BR18-190.pdf El-Mougy, N.S., El-Gamal, N.G., & Abdel-Kader, M.M. (2007). Control of wilt and root rot incidence in Phaseolus vul- garis L. by some volatile compounds. Journal of Plant Protection Research, 47(3), 255–265. http://www.plant- protection.pl/Control-of-wilt-and-root-rot-incidence-in- Phaseolus-vulgaris-L-by-some-plant-volatile,91109,0,2. html El-Mougy, N.S., El-Mohamady, R.S., El-Gamal, N.G., & Ab- del-Kader, MM (2019). Efficacy of some chemical re- sistance inducers agents and nitrogen-fixing rhizobium for suppressing root rot and wilt diseases incidence of phaseolus vulgaris L. under natural field conditions. Bio- Science Research, 16(1), 834-842. https://www.isisn.org/ BR16(1)2019/834-842-16(1)2019BR18-566.pdf Epstein, E. (1999). Silicon. Annual Review of Plant Physiol- ogy and Plant Molecular Biology, 50, 641-664. https://doi. org/10.1146/annurev.arplant.50.1.641 Friend, J. (1979). Phenolic substances and plant disease. Re- cent Advances in Phytochemistry, 12, 557-588. https://doi. org/10.1007/978-1-4684-3372-2_17 Gullner G., Zechmann B., Künstler A., & Király L. (2017) The Signaling Roles of Glutathione in Plant Disease Re- sistance. In: Hossain M., Mostofa M., Diaz-Vivancos P., Burritt D., Fujita M., Tran LS. (eds) Glutathione in Plant Growth, Development, and Stress Tolerance. Springer, Cham. https://doi.org/10.1007/978-3-319-66682-2_15 Hamed, F.R. (2008). Effect of Rizolex on Rhizoctonia solani Kuhn isolates and biocontrol fungus Trichoderma harzi- anum Rifai in vitro. Journal of Kerbala University, 6(1), 218- 223. https://www.iasj.net/iasj?func=fulltext&aId=50236 Hirschfeld, T., Ellner, F., Buschhaus, H., & Gossmann, M. (2010). New insight in the mode of action of thiophanate- methyl. Modern fungicides and antifungal compounds VI. 16th International Reinhardsbrunn Symposium, Frie- drichroda, Germany, April 25-29, 2010 pp.417-421. https:// www.cabdirect.org/cabdirect/abstract/20153047734 Hopkins, M. (2013). Valent’s Rizolex Fungicide Seed Treat- ment Approved. https://www.croplife.com/crop-inputs/ fungicides/valents-rizolex-fungicide-seed-treatment-ap- proved/ Jayawardana, H.A.R.K., Weerahewa, H.L.D. & Saparamadu, M.D.J.S. (2014). Effect of root or foliar application of solu- ble silicon on plant growth, fruit quality and anthracnose development of capsicum. Tropical Agricultural Research, 26(1), 74–81. https://doi.org/10.4038/tar.v26i1.8073 Iqbal, Z., Pervez, M.A., Ahmed, S., Iftikhar, Y., Yasin, M., Nawaz, A., Ghazanfar, M.u., Dasti, A.A., & saleem, A. (2010). De- termination of minimum inhibitory concentrations of fungicides against fungus Fusarium mangiferae. Pakistan Journal of Botany, 42(5), 3525-3532. http://www.pakbs. org/pjbot/PDFs/42(5)/PJB42(5)3525.pdf Jabnoun, K.H., El-Mohamedy, R.S.R., Abdel-Kareem, F.I., Ben Abdallah, R.A., Gueddes-Chahed, M., & Daami- Remadi, M. (2015). Variation in chitosan and salicylic acid efficacy towards soil-borne and air-borne fungi and their suppressive effect of tomato wilt severity. Journal of Plant Pathology and Microbiology, 6(11), 1-10. https://doi. org/10.4172/2157-7471.1000325 Kanan, G.J.M., & Al-Najar, R.A.K. (2009). In vitro deferential sensitivity of Pencillium italicum causing postharvest rot to citrus fruits in Jordan to chemical fungicides and their combinations. Journal of Plant Protection Research, 49(1), 77-86. https://doi.org/10.2478/v10045-009-0010-8 Kanto, T., Miyoshi, A., Ogawa, T., Maekawa, K., & Aino, M. (2006). Suppressive effect of liquid potassium silicate on powdery mildew of strawberry in soil. Journal of Gen- eral Plant Pathology, 72, 137–42. https://doi.org/10.1007/ s10327-005-0270-8 Li, Y.C., Bi, Y., Ge, Y.H., Sun, X.J., & Wang, Y. (2009). Antifun- gal activity of sodium silicate on Fusarium sulphureum and its effect on dry rot of potato tubers. Journal of food Science, 74(5), 213-218. https://doi.org/10.1111/j.1750- 3841.2009.01154.x Mandel, M.J., Wollenberg, M.S., Stabb, E.V., Visick, K.L., & Ruby, E.G. (2009). A single regulatory gene is sufficient al- ter bacterial host range. Nature, 458, 215-218. https://doi. org/10.1038/nature07660 Menzies, J., Bowen, P., Ehret, D., & Glass, A.D.M. (1992). Fo- liar applications of potassium silicate reduce severity of powdery mildew on cucumber, muskmelon and zucchini squash. Journal of American Society for Horticultural Sci- ence, 117, 902-905. https://journals.ashs.org/jashs/view/ journals/jashs/117/6/article-p902.xml Menzies, J., Bowen, P., Ehret, D., & Glass, A.D.M. (1992). Fo- Acta agriculturae Slovenica, 118/2 – 2022 9 Comparative study between fungicides and some chemical inducers for controlling root rot incidence of green bean (Phaseolus vulgaris L.) ... liar applications of potassium silicate reduce severity of powdery mildew on cucumber, muskmelon, and zucchini squash. Journal of American Society for Horticultural Sci- ence, 117, 902–905. https://journals.ashs.org/jashs/view/ journals/jashs/117/6/article-p902.xml Meya, A.I., Mamiro, D.P., Kusolwa, P.M., Maerere, A.P., Sibu- ga, K.P., Erbaugh, M., Miller, S.A., & Mtui, H.D. (2014). Management of tomato late blight disease using reduced fungicide spray regimes in Morogoro, Tanzania. Tanzania Journal of Agricultural Sciences, 13(2), 8-17. http://www. suaire.suanet.ac.tz:8080/xmlui/handle/123456789/1303 Moustafa, M.S.H., El-Dakar, H.A.M., & Ismail, A.A. (2015). Adverse effect of rainfall on the efficacy of some fungi- cides used to control chocolate spot of faba bean (Vicia faba). International Journal of Scientific & Engineering Research, 6(12), 915-925. https://www.ijser.org/research- paper/Adverse-Effect-of-Rainfall-on-the-Efficacy-of- -some-Fungicides-Used-to-Control-Chocolate-Spot-of- Faba-Bean-Vicia-faba.pdf Noctor, G., Mhamdi, A., Chaouch, S., Han, Y., Neukermans, J., Marquez, B., Garcia, A., Guillaume, G., & Foyer, C.H. (2012). Glutathione in plants: an integrated overview. Plant, Cell and Environment, 35, 454–484. https://doi. org/10.1111/j.1365-3040.2011.02400.x Ohtsuki, S. and Fujinami, A. (1982). Rizolex (tolclofos-me- thyl). Japan Pesticide Information, 41, 21-25. http://pascal- francis.inist.fr/vibad/index.php?action=getRecordDetail &idt=PASCALAGROLINEINRA83X0220933 Saber, A.S., Mahran, A.H., & Abdel-Maksoud, M.A. (2011). Suppressive effect of Ginkgo biloba extract (EGb 761) on topsin induced ovarian toxicity and oxidative stress in al- bino rats. Journal of Applied Pharmaceutical Science, 1(4), 46-54. https://www.japsonline.com/abstract.php?article_ id=59&sts=2 Shen, X., Zhou, Y., Duan, L., Li, Z., Eneji, A.E. & Li, J. (2010). Silicon effects on photosynthesis and antioxidant parame- ters of soybean seedlings under drought and ultraviolet-B radiation. Journal of Plant Physiology 167(15), 1248-1252. https://pubmed.ncbi.nlm.nih.gov/20713250/ Acta agriculturae Slovenica, 118/2, 1–13, Ljubljana 2022 doi:10.14720/aas.2022.118.2.1803 Original research article / izvirni znanstveni članek Influence of different sources of nitrogen fertilizer and weed control on yield, yield components and some qualitative traits of chickpea (Cicer arietinum L.) cultivars under dryland conditions of Khorramabad Sajad KORDI 1, 2, Tayebeh DANAYE-TOUS 3, Soheila DASTBORHAN 1 Received July 30, 2020; accepted April 03, 2022. Delo je prispelo 30. julija 2020, sprejeto 3. aprila 2022 1 Department of Plant Ecophysiology, Faculty of Agriculture, University of Tabriz, Tabriz, Iran 2 Corresponding author, e-mail: sajad.kordi@gmail.com 3 Department of Agronomy and Plant Breeding, Faculty of Agriculture, Islamic Azad University, Khorramabad Branch, Iran Influence of different sources of nitrogen fertilizer and weed control on yield, yield components and some qualita- tive traits of chickpea (Cicer arietinum L.) cultivars under dryland conditions of Khorramabad Abstract: A field experiment was conducted to evalu- ate yield, yield components, and some qualitative traits of chickpea (Cicer arietinum L.) cultivars under nitrogen fertil- izers and weed control in dryland conditions of Khorram- abad during the 2017 - 2018 growing season. Treatments were arranged in split-split-plot based on a randomized complete block design with three replications. The main factor included F1: control (without fertilizer); F2: bio-fertilizer (Rhizobium); F3: 100 % chemical fertilizer and F4: integration of bio-fertil- izer + 50 % chemical fertilizer; sub-factor consisted of three cultivars of chickpea (Adel, Mansour, and Arman) and sub- sub-factor included weeds control (weeding) and weed infest- ed (non-weeding). The results indicated that nitrogen fertiliz- ers, especially the integration of bio-fertilizer + 50 % chemical fertilizer, had a positive effect on all studied traits. The highest number of pods per plant, grain yield, and biological yield were obtained from the Arman cultivar with the application of bio-fertilizer + 50 % chemical fertilizer and for the same cultivar under weed control conditions. The maximum num- ber of pods per plant (28.2) and amount of grain protein con- tent (25.3 %) were obtained by integrating of bio-fertilizer + 50% nitrogen chemical fertilizer and weeds control. In gener- al, the Arman cultivar has priority over other cultivars for the grain yield under Khorramabad climate conditions, and in- tegration of bio-fertilizer + 50 % chemical fertilizer could be considered as a means to reduce the consumption of chemical fertilizers for sustainable agriculture. Key words: chickpea; grain protein; grain yield; hectoli- ter mass; Rhizobium; weed control Vpliv različnih dušikovih gnojil in uravnavanja plevelov na pridelek, komponente pridelka in kakovostne lastnosti sort čičerke (Cicer arietinum L.) v sušnih razmerah Khorrama- bada Izvleček: Za ovrednotenje pridelka, njegovih kompo- nent in nekaterih kakovostnih lastnosti sort čičerke (Cicer ari- etinum L.) je bil izveden poljski poskus z gnojenjem z različ- nimi dušikovimi gnojili in načini zatiranja plevelov v sušnih razmerah Khorramabada v rastnih sezonah 2017 in 2018. Obravnavanja so bila izvedena kot popolen naključni bločni poskus z deljenkami s tremi ponovitvami. Glavna obravnava- nja so bila: F1: kontrola (brez gnojil); F2: biognojila (Rhizobi- um); F3: 100 % mineralna gnojila in F4: integracija biognojil + 50 % mineralnih gnojil. Podobravnavanja so obsegala tri sorte čičerke (Adel, Mansour in Arman) in dva načina uravnava- nja plevelov (zatiranje, kontrola). Rezultati so pokazali, da je imelo gnojenje z dušikovimi gnojili, še posebej hkratna upo- raba biognojil z dodatkom 50 % mineralnih gnojil, pozitiven učinek na vse preučevane lastnosti. Največje število strokov na rastlino, največji pridelek zrnja in biološki pridelek so bili doseženi pri sorti Arman pri uporabi biognojil z dodatkom 50 % mineralnih gnojil in zatiranju plevelov. Podobno sta bila največje število strokov na rastlino (28,2) in največja vsebnost beljakovin v zrnju (25,3 %) dosežena pri hratni uporabi bio- gnojil in 50 % mineralnih dušikovih gnojil in zatiranju ple- velov. V splošnem se je sorta Arman izkazala v pridelku zrnja boljše kot ostale v podnebnih razmerah Khorramabada in hkratno uporabo biognojil z dodatkom 50 % mineralnih gno- jil lahko smatramo kot primeren način gnojenja za zmanj- ševanje porabe mineralnih gnojil v trajnostnem kmetijstvu. Ključne besede: čičerka; beljakovine v zrnju; pridelek zrnja, hektoliterska masa; Rhizobium, pletev Acta agriculturae Slovenica, 118/2 – 20222 S. KORDI et al. 1 INTRODUCTION Chickpea (Cicer arietinum L.) is the third main grain legume in the world, with an annual global pro- duction of 14.24 million tons from an area of 14.79 million ha (FAO, 2018). It is an essential component of the agricultural system in all over Iran, because this crop fits well in rotation patterns and can grow under low fertility and different soil and climate conditions. The main provinces producing chickpeas in Iran are Lorestan and Kermanshah (Mekuanint et al., 2018). The potential yield of chickpea cultivars is approximately 4 t ha−1, while the average national yield is about 533 kg ha-1 (Khorsandi et al., 2016). The gap between actual and potential yields is mainly due to poor crop man- agement such as imbalanced use of fertilizer, the lack of effective rhizobial strain, unavailability of high-quality seeds, and also damages caused by pests and diseases (Togay et al., 2008; Mekuanint et al., 2018). Moreover, Iran has nitrogen deficient soil and therefore, plants use a low amount of nitrogen which affects physiological processes and decreases photosynthesis activities, pro- duction of assimilate and biomass, and eventually yield (Ghilavizadeh et al., 2013). Application of chemical fertilizers, especially ma- cronutrients, can generally increase biomass produc- tion by 2-3 times (Elliott and Abbott, 2003), that is the reason why farmers are applying high amounts of chemical fertilizers, which are very costly and hazard- ous to the environment. Therefore, alternative sources of chemical fertilizers and the application of organic fertilizers (e.g., bio-fertilizers) are considered as op- tions for sustainable agriculture to improve soil qual- ity in modern agriculture (Chen et al., 2014; Meena et al., 2015). The utilization of bio-fertilizer (e.g., Rhizo- bium species) has become of paramount importance in the agriculture for their potential role in food safety, improving crop yield, and decreasing greenhouse gas emissions (Ghilavizadeh et al., 2013; Raei et al., 2015). The absence of compatible strains and low popula- tion of Rhizobium in the soil are essential limitations for nodule formation in chickpea (Kantar et al., 2010; Wolde-Meskel et al., 2018). Inoculation with effective strains at planting time is recommended if the popula- tion density of compatible rhizobia is less than 50 cells per gram of soil (Thies et al., 1991a, b; Wolde-Meskel et al., 2018). Previous studies showed that inocula- tion of chickpea seeds with Rhizobium could increase plant growth, grain yield, and biomass yield (Funga et al., 2016; Khaitov et al., 2016; Tena et al., 2016; Wolde- Meskel et al., 2018). The weak ability of chickpea crops to compete with weeds is a vital issue in low input and organic farming systems (Melander, 1993). The critical period of weed interference in chickpea is 15 to 60 days after sowing in Iran, and the presence of weed at this time can cause severe loss of the yield (Mohammadi et al., 2005; Gupta et al., 2016). Hence, weed control needs to be undertaken during the initial periods of chick- pea growth. Hand weeding is a well-proven effective method of weed control in chickpea fields in Iran. But implementation of this method is costly for farmers and can be used on small farms (Mohammadi et al., 2005). Mousavi (2010) reported that by twice weeding, the grain yield of chickpea was significantly increased (by 174 %) compared to weed infested treatment. The objective of this study was to determine the effects of bio-fertilizer and weed control and their interactions on yield, yield components, and some qualitative traits of chickpea in Khorramabad condition. 2 MATERIALS AND METHODS 2.1 LOCATION AND PLANT MATERIALS This study was conducted in the Experimental Farm of the Pole Baba Hossein, Khorramabad, Iran (33°25′N, 48°19′E, and altitude 1,171 m), during the 2017 - 2018 growing season. The meteorological data during the experimental period are presented in Table 1. Physical and chemical characteristics of soil at the depth of 0-40 cm are shown in Table 2. Chickpea seeds were planted in early January 2018 in plots, consisting Month Precipitation (mm) Maximum temperature (°C) Minimum temperature (°C) Average temperature (°C) Jan 50.1 23.5 - 4.3 7.6 Feb 68.7 21.6 - 4.3 8.3 Mar 62.7 23.5 1.1 11.7 Apr 103.7 30.3 3.3 15.2 May 151.7 30.0 5.6 17.2 Jun 12.1 37.0 11.8 24.6 Table 1: Khorramabad meteorological station monthly statistics in the experiment period Acta agriculturae Slovenica, 118/2 – 2022 3 Influence of different sources of nitrogen fertilizer and weed control on yield, yield components and some qualitative traits ... of six 2-meter rows spaced 30 cm apart. The intra-row plant spacing was 10 cm. Hand weeding was done in weed control treatments during the growing season. 2.2 TREATMENTS AND EXPERIMENTAL DE- SIGN The experiment was conducted as split-split- plot based on Randomized Complete Blocks Design (RCBD) with three replications. The main factor in- cluded F1: control (without application of fertilizer); F2: bio-fertilizer (Rhizobium); F3: 100 % nitrogen chemical fertilizer and F4: integration of bio-fertilizer + 50 % nitrogen chemical fertilizer. Sub-factor consisted of chickpea cultivars (Adel, Mansour, and Arman), and sub-sub-factor included weed control (weeding) and weed infestation (non-weeding). 2.3 FERTILIZER AND MICROBIAL INOCULA Before cultivation, 100 kg ha-1 triple superphos- phate was added to all plots according to the soil test. With the last plowing before planting, 50 and 25 kg N ha-1 as urea were added to 100 % chemical fertilizer and integration of bio-fertilizer + 50 % chemical fertilizer, respectively. The strain of the used Rhizobium bio-ferti- lizer was Mesorhizobium ciceri SWRI-3 which consisted of 108 colony forming units/ml (CFU ml-1) inoculant and was purchased from Soil and Water Research Insti- tute, Karaj, Iran. Before planting, the seeds were mixed entirely with bio-fertilizer and kept for half an hour in the shade to dry. The liquid bio-fertilizer (Rhizobium) was applied at the amount of 2 l ha-1. The dried seeds were planted in early January. 2.4 TRAITS MEASUREMENT The traits measured in this study included plant height, number of pods per plant, 100-grain mass, grain yield, biological yield, harvest index, hectoliter mass, and grain protein content. Five plants from each plot were selected randomly to determine the plant height and number of pods per plant. To measure the 100-grain mass, five samples containing 100 grains were randomly collected from each plot, and their mass was recorded. To measure the hectoliter mass, a container with known mass and volume was completely filled with the chickpea seeds (Singh and Goswami, 1996; Kordi and Ghanbari, 2019). After filling the container, excess seeds were removed by passing a flat stick across the top surface. The seeds were not compacted in any way. The container was weighed on a digital balance (Model GT2100, Germany) reading to 0.01 g. Hectoliter mass (ρb) was calculated by the ratio of seeds mass in the container (Mb) to its volume (Vb): The ρb was recorded from the average of 10 sam- ples for each treatment. To measure the grain yield, all plants in the one meter-length center of two rows located in the middle of each plot were taken, and grain yield was recorded with a portable balance and calculated based on 12 % seed moisture. To measure the dry biological yield, in- cluding aerial parts and roots, the samples were dried in an oven at 75 °C for 72 h and then weighed. The harvest index (HI) was accounted as follows: HI = (Grain yield / Biological yield) × 100 For determination of crude protein content, the nitrogen content of grains was obtained by the Kjel- dahl method (digestion of organic matter with sulfuric acid in the presence of a catalyst; rendering the reaction product alkaline; distillation and titration of the liber- ated ammonia; and calculation of the nitrogen content) (Jensen, 1996). Crude protein content (Cp) of grain was determined as: Cp = 6.25 × C2 where C2 is the total grain nitrogen concentration on a dry matter. 2.5 DATA ANALYSIS SAS (version 9.1) and MSTAT-C statistical soft- wares were used for the analysis of variance (ANOVA) Soil texture Clay (%) Silt (%) Sand (%) pH EC (dS m-1) Total N (%) Available P (ppm) Available K (ppm) Clay loam 31.2 42.0 26.8 7.97 1.04 0.11 6.1 430 Table 2: Physical and chemical analysis of soil before the experiment Acta agriculturae Slovenica, 118/2 – 20224 S. KORDI et al. and comparisons of means, respectively. Duncan’s mul- tiple range test, at p ≤ 0.05, was used to rank the differ- ences among means. The graphs were drawn by Excel, and error bars were assigned based on standard error (SE). 3 RESULTS AND DISCUSSION 3.1 PLANT HEIGHT The result of variance analysis showed that plant height was affected by fertilizer, cultivar, and weeding. The interaction effect of cultivar × weeding was sig- nificant on the mentioned trait (Table 3). In all studied cultivars, the plant height under weed infested treat- ment was lower than that under weed control condi- tions. This decrement was 12.8, 8.7, and 17 % in Adel, Mansour, and Arman cultivars, respectively. The high- est plant height (66.0 cm) was obtained by the Arman cultivar under weed control conditions (Figure 1). It has been reported that weed competition has a negative effect on plant height in chickpea (Ratnam et al., 2011). Weeds compete with crops for essential nutrients, avail- able water, and light used for photosynthesis (Merga and Alemu, 2019), and reduce crop yield. The results of previous experiments also indicated that hand weeding increased the plant height of chickpea (Rathod et al., 2017). Among fertilizer treatments, the highest (59.4 cm) and the lowest (54.3 cm) plant height were related to the integration of bio-fertilizer + 50 % nitrogen chemi- cal fertilizer and control (without fertilizer) treatments, respectively (Figure 2). Application of Rhizobium (F2), 100 % chemical fertilizer (F3), and integration of Rhizo- bium + 50 % nitrogen chemical fertilizer (F4) increased the plant height by 5.5, 5.9, and 9.2 %, respectively, compared to the control treatment (without fertilizer). These results are in line with the findings of Amany (2007) and Caliskan et al. (2008), who reported that plant height increased with the application of nitrogen fertilizer. Khan et al. (2017) stated that the application of Rhizobium increases the plant height of chickpea. The solubilizing ability of Rhizobium species may in- crease nitrogen availability in the soil, and the plants can uptake the required amount of nutrients (Khaitov and Abdiev, 2018). 3.2 NUMBER OF PODS PER PLANT According to the results of variance analysis (Table 3), the number of pods per plant was affected by sim- ple effects of fertilizer, cultivar, and weeding as well as the interaction effects of fertilizer × cultivar, fertilizer × weeding, and cultivar × weeding (Table 3). In all stud- ied cultivars, maximum pods per plant were observed by applying of Rhizobium + 50 % nitrogen chemical fertilizer. On the other hand, the Arman cultivar had the highest pods per plant under all fertilization treat- Mean squares df Source of variation Grain protein content Hectoliter mass Harvest index Biological yield Grain yield 100-grain mass Pods per plant Plant height 0.5 ns0.00002 ns0.5 ns15477.4 ns691.8 ns0.6 ns13.8 ns9.5 ns2Replication 33.3**0.0006*35.85**2911530.8**602344.3**46.0 **85.8**77.6**3Fertilizer (F) 0.30.000073.156240.84618.30.61.54.36Error 1 6.6**0.0008**16.8**5485802.1**419913.9**218.6**86.3**798.7**2Cultivar (C) 0.4 ns0.00003 ns15.6**61900.3*23171.4**3.7**5.0 *9.2 ns6F × C 0.20.000031.419679.52488.90.7 1.74.416Error 2 10.7**0.002**531.4**11947331.1**4262226.7**67.9**1106.5**1111.9**1Weeding (W) 0.5*0.00003 ns3.1 ns32900.8 ns2078.9 ns0.1 ns30.5**6.4 ns3F × W 0.7*0.00002 ns15.3**120062.7*47801.8**6.9*15.7 **53.2**2C × W 0.1 ns0.000006 ns0.9 ns9939.7 ns3261.5 ns0.5 ns2.8 ns7.2 ns6F × C × W 0.20.000022.6273614502.61.51.93.024Error 3 1.70.64.94.04.93.76.53.0---C.V (%) Table 3: Analysis of variance of grain yield, yield components and some qualitative traits of chickpea cultivars *,** and ns show significant difference at probability of 5 %, 1 % and no significant difference, respectively Acta agriculturae Slovenica, 118/2 – 2022 5 Influence of different sources of nitrogen fertilizer and weed control on yield, yield components and some qualitative traits ... ments. Adel cultivar without fertilization had the lowest number of pods per plant (16.3), whereas the highest pods number per plant (26.6) was obtained by the Ar- man cultivar with the application of Rhizobium + 50 % nitrogen fertilizer (Figure 3). The number of pods per plant generally depends on the cultivar (Ayaz et al., 2004). It is also affected by environmental factors and management practices (Knott, 1987). Yadav et al. (2011) reported that seed inoculation with Rhizobium enhanced nodulation, growth, and yield of legumes. In- creasing the number of pods per plant under inocula- tion treatment can be due to the effect of Rhizobium on N, P, and K uptake, some enzyme activities, and root development (Wu, 2000). Many studies found positive effects of Rhizobium inoculation on the number of pods per plant in chickpea (Meena et al., 2013; Khaitov et al., 2016). The result of mean comparisons of fertilizer × weeding showed that in all fertilizer treatments, espe- cially the application of 100 % chemical fertilizer, weed control increased the pods number per plant compared to weed infested treatment. This increment was 43.2, 28.6, 69.9, and 40.8 % under F0, F1, F2, and F3 treat- Figure 1: Plant height of chickpea cultivars under weed control and weed infested conditions. Different letters indicate a significant difference at p ≤ 0.05 (Duncan test) Figure 2: Plant height of chickpea under different nitrogen sources. F0, F1, F2, F3: Control, bio-fertilizer, 100 % chemical fertiliz- er, and bio-fertilizer + 50 % chemical fertilizer, respectively. Different letters indicate a significant difference at p ≤ 0.05 (Duncan test) Figure 3: Number of pods per plant of chickpea cultivars under different nitrogen sources. F0, F1, F2, F3: Control, bio-fertilizer, 100 % chemical fertiliz- er, and bio-fertilizer + 50 % chemical fertilizer, respectively. Different letters indicate a significant difference at p ≤ 0.05 (Duncan test) Figure 4: Number of pods per plant of chickpea under dif- ferent nitrogen sources and weed control and weed infested conditions. F0, F1, F2, F3: Control, bio-fertilizer, 100 % chemical fertiliz- er, and bio-fertilizer + 50 % chemical fertilizer, respectively. Different letters indicate a significant difference at p ≤ 0.05 (Duncan test) Acta agriculturae Slovenica, 118/2 – 20226 S. KORDI et al. ments, respectively. Integration of Rhizobium + 50 % ni- trogen chemical fertilizer combined with weed control produced the highest number of pods per plant (28.2), while the lowest number was related to weed infested treatment without fertilizer (15.5) (Figure 4). The num- ber of pods per plant is one of the most important fac- tors affecting the yield of pulse crops such as chickpea. The availability of nitrogen may reduce the weed com- petition pressure in the crops (Shafiq et al., 1994). Togay et al. (2008) reported that the plants from inoculated seeds with Rhizobium had a higher number of pods per plant compared to the control. In all cultivars, weeding improved the number of pods per plant. However, the positive effect of weeds control on pods per plant in the Adel cultivar was high- er than the other cultivars. The highest number of pods per plant (26.8) was obtained in the Arman cultivar under weed control conditions (Figure 5). The higher number of pods per plant in weed control conditions could be due to the lack of competition of weeds with chickpea plants in the field. Chickpea is sensitive to weed interference due to its slow growth rate and lim- ited leaf development at the early stage of crop growth and establishment (Kaushik et al., 2014). 3.3 100-GRAIN MASS Based on the results of variance analysis (Table 3), simple effects of fertilizer, cultivar, and weeding were significant on 100-grain mass. Also, the interaction ef- fects of fertilizer × cultivar and cultivar × weeding were significant for this trait (Table 3). The mean compari- sons of fertilizer × cultivar showed that in all fertilizer treatments, the maximum 100-grain mass belonged to the Mansour cultivar. In all three cultivars, the mini- mum 100-grain mass was related to control treatment (without fertilizer) (Figure 6). Increasing 100-grain mass under inoculation treatment can be due to the improved traits such as leaf area and photosynthetic pigments, which finally causes an increase in photosyn- thetic products (Nyoki and Nakidemi, 2016). The 100-grain mass of chickpea under weed con- trol conditions was higher than under weed infested treatment in all studied cultivars, especially the Adel cul- tivar. The highest (37.4 g) and lowest (29.2 g) 100-grain mass were achieved from the Mansour cultivar under Figure 5: Number of pods per plant of chickpea cultivars under weed control and weed infested conditions. Different letters indicate a significant difference at p ≤ 0.05 (Duncan test) Figure 6: 100-grain mass of chickpea cultivars under differ- ent nitrogen sources. F0, F1, F2, F3: Control, bio-fertilizer, 100 % chemical fertiliz- er, and bio-fertilizer + 50 % chemical fertilizer, respectively. Different letters indicate a significant difference at p ≤ 0.05 (Duncan test) Figure 7: 100-grain mass of chickpea cultivars under weed control and weed infested conditions. Different letters indicate a significant difference at p ≤ 0.05 (Duncan test) Acta agriculturae Slovenica, 118/2 – 2022 7 Influence of different sources of nitrogen fertilizer and weed control on yield, yield components and some qualitative traits ... weed control conditions and the Adel cultivar under weed infested treatment, respectively (Figure 7). Most weeds exhibit faster initial growth than crops such as chickpea, thereby inhibiting crop growth, which might affect photosynthesis and crop yield (Tepe et al., 2011). It appears that some factors like nutrient deficiency and the level of plants competition over light and nutrient resources under weed infested treatment could be con- sidered as reduction factors for production. 3.4 GRAIN YIELD Fertilizer, cultivar, and weeding had significant effects on the grain yield of chickpea. The interaction effects of fertilizer × cultivar and cultivar × weeding were also significant for grain yield (Table 3). The result of mean comparisons showed that the application of nitrogen fertilizer, especially Rhizobium + 50 % nitro- gen chemical fertilizer, increased grain yield in all three cultivars. The highest grain yield (1662.5 kg ha-1) was related to the Arman cultivar with applying Rhizobium + 50 % nitrogen chemical fertilizer (Figure 8). Inocula- tion of chickpea seeds with Mesorhizobium ciceri strain resulted in a 23 % increase in grain yield compared to the control treatment (without fertilizer). In the present research, increment of the grain yield resulted from the application of different nitrogen sources in studied cul- tivars, especially the Arman cultivar, may be due to the more plant height and number of pods per plant in this condition (Figures 1, 2, 3). It seems that the positive ef- fects of Rhizobium inoculation on chickpea can be a re- sult of nitrogen supply for the crop (Togay et al., 2008). The effect of Rhizobium bacteria on plant growth is not only through nitrogen fixation, but it is also associated with the ability of the Rhizobium bacteria to synthesize phytohormones like auxin. Some phytohormones, in- cluding auxin, enhance root growth and development as well as promoting water and nutrients uptake (Wer- ner and Newton, 2005). It has been reported that in- oculation of chickpea seeds with Rhizobium improves grain yield by 9.6 - 27.9 % (Gupta and Namdeo, 1996). Increasing the nitrogen rate from 0 to 50 kg N ha−1 significantly improved the number of pods per plant, 1000-grain mass, grain yield, biological yield, and har- vest index in chickpea (McKenzie and Hill, 1995). There is a negative correlation between soil mineral nitrogen content and the number or mass of rhizobia nodes, meaning that high mineral nitrogen reduces rhizobia activity (Flajšman et al., 2020). Mean comparisons indicated that in all studied cultivars, especially the Adel cultivar, weed control in- creased chickpea grain yield compared to weed infested treatment. The highest (1726.5 kg ha-1) and the lowest (948 kg ha-1) grain yields were achieved from the Ar- man cultivar with weed control and Adel cultivar under weed infested treatment, respectively. Weed control led to a 62.2, 37.4, and 34 % increase in grain yield of the Adel, Mansour and Arman cultivars, compared to weed infested treatment, respectively (Figure 9). This result indicates that poor weed management is one of the ma- jor grain yield limiting factors in chickpea. It has been reported that weed interference can decrease chickpea yield by more than 85 % (Ratnam et al., 2011). Figure 8: Grain yield of chickpea cultivars under different nitrogen sources. F0, F1, F2, F3: Control, bio-fertilizer, 100 % chemical fertiliz- er, and bio-fertilizer + 50 % chemical fertilizer, respectively. Different letters indicate a significant difference at p ≤ 0.05 (Duncan test) Figure 9: Grain yield of chickpea cultivars under weed con- trol and weed infested conditions. Different letters indicate a significant difference at p ≤ 0.05 (Duncan test) Acta agriculturae Slovenica, 118/2 – 20228 S. KORDI et al. 3.5 BIOLOGICAL YIELD The biological yield was significantly affected by simple effects of fertilizer, cultivar and weeding as well as interaction effects of fertilizer × cultivar and cultivar × weeding (Table 3). Similar to grain yield (Figure 8), the biological yield of chickpea cultivars increased with the application of nitrogen fertilizers. In all studied cultivars, applying nitrogen fertilizer (especially Rhizo- bium + 50 % nitrogen chemical fertilizer) enhanced biological yield compared to the control (Figure 10). The maximum (5104.1 kg ha-1) and minimum (3139.2 kg ha-1) biological yields were recorded for the Arman cultivar with the application of Rhizobium + 50 % ni- trogen chemical fertilizer and the Adel cultivar without fertilizer (control), respectively (Figure 10). Nitrogen is one of the most essential nutrients with a considerable effect on plant growth and productivity (Tripathi et al., 2015). The production of phytohormones by Rhizo- bium species enhances root growth and development through improved water and nutrients uptake (Spaepen et al., 2009). According to Khaitov and Abdiev (2018), the combined application of bio-fertilizer and nitrogen fertilizer leads to a positive impact on basic metabo- lism, grain yield, and biomass. These results show that the integration of bio-fertilizer and nitrogen chemical fertilizer can be useful for crops production. Togay et al. (2008) found that inoculation of chickpea seeds with Rhizobium has significantly increased the plant height, number of branches per plant, and biological yield. Namvar et al. (2011) reported that the application of nitrogen increases the production of total dry matter in plants, which can be caused by increasing the plant height (Figure 2), number of branches per plant, and number of pods per plant (Figure 3) that eventually re- sults in high grain and biological yields (Figures 8, 10). Weeds control increased biological yield com- pared to weed infested treatment in all three cultivars of chickpea. This increment was 31, 20.2, and 16.6 % in the Adel, Mansour, and Arman cultivars, respectively. The highest biological yield (4908.5 kg ha-1) was ob- tained by the Arman cultivar under weed control con- ditions (Figure 11). Chickpea is highly susceptible to weed competition due to its slow growth rate and short stature at the early stage of crop growth and establish- ment (Singh et al., 2017). Therefore, under weed control conditions, soil moisture and nutrients are provided for the crop to increase biological yield (Khan et al., 2002). 3.6 HARVEST INDEX (HI) The data presented in Table 3 showed that simple effects of fertilizer, cultivar, and weeding were significant on the harvest index. The interaction effects of fertilizer × cultivar and cultivar × weeding were also significant for the mentioned trait (Table 3). The application of different nitrogen sources improved the harvest index. The highest harvest index (35.8 and 35.4) was achieved by the Adel cultivar with the application of 100 % nitro- gen chemical fertilizer and the Mansour cultivar with the application of Rhizobium + 50 % chemical nitrogen fertilizer, respectively (Figure 12). Malik et al. (2006) reported that inoculation of soybean seeds with Rhizo- bium has significantly increased the harvest index. On Figure 10: Biological yield of chickpea cultivars under differ- ent nitrogen sources. F0, F1, F2, F3: Control, bio-fertilizer, 100 % chemical ferti- lizer, and bio-fertilizer + 50 % chemical fertilizer, respec- tively.Different letters indicate a significant difference at p ≤ 0.05 (Duncan test) Figure 11: Biological yield of chickpea cultivars under weed control and weed infested conditions. Different letters indicate a significant difference at p ≤ 0.05 (Duncan test) Acta agriculturae Slovenica, 118/2 – 2022 9 Influence of different sources of nitrogen fertilizer and weed control on yield, yield components and some qualitative traits ... the other hand, Flajšman et al. (2019) showed that the soybean harvest index was not influenced by bacteria seed inoculation. Weed control led to a 24.2, 14.7, and 15.4 % increase in harvest index in the Adel, Mansour, and Arman cultivars, respectively, compared to weed infested treatment. The highest harvest index (37.4) was obtained by the Adel cultivar under weed control conditions (Figure 13). Pooniya et al. (2009) found that weed management played an important role in improv- ing the harvest index in the chickpea. According to the results of this research, it seems that weed control had a greater effect on grain yield than biological yield. 3.7 HECTOLITER MASS According to the results of variance analysis (Table 3), the hectoliter mass was affected by fertilization, cul- tivar, and weeding. The interaction effects of the treat- ments were not significant for this trait (Table 3). The application of different sources of nitrogen improved hectoliter mass in the chickpea. Among the various fer- tilizer treatments, the highest (0.770 g cm-3) and low- est (0.757 g cm-3) hectoliter mass were achieved in the integration of Rhizobium + 50 % chemical fertilizer and without fertilization (control), respectively (Figure 14). These results are in agreement with the findings of Kordi and Ghanbari (2019). They reported that differ- Figure 12: Harvest index response of chickpea cultivars to different nitrogen sources. F0, F1, F2, F3: Control, bio-fertilizer, 100 % chemical ferti- lizer, and bio-fertilizer + 50 % chemical fertilizer, respec- tively.Different letters indicate a significant difference at p ≤ 0.05 (Duncan test) Figure 13: Harvest index of chickpea cultivars under weed control and weed infested conditions. Different letters indicate a significant difference at p ≤ 0.05 (Duncan test) Figure 14: Hectoliter mass of chickpea under different nitro- gen sources. F0, F1, F2, F3: Control, bio-fertilizer, 100 % chemical ferti- lizer, and bio-fertilizer + 50 % chemical fertilizer, respec- tively.Different letters indicate a significant difference at p ≤ 0.05 (Duncan test) Figure 15: Hectoliter mass of different chickpea cultivars. Different letters indicate a significant difference at p ≤ 0.05 (Duncan test) Acta agriculturae Slovenica, 118/2 – 202210 S. KORDI et al. ent sources of nitrogen resulted in changes in the hec- toliter mass of maize, and the highest and the lowest hectoliter mass appeared in the integration of bio-ferti- lizer + 75 % chemical fertilizer and without fertilization (control), respectively. Evaluation of different chickpea cultivars in terms of hectoliter mass showed that the maximum (0.770 g cm-3) and minimum (0.758 g cm-3) hectoliter mass were related to the Arman and Adel cul- tivars, respectively (Figure 15). The mean comparisons indicated that under weed control conditions, the hec- toliter mass of chickpea was higher than that in weed infested treatment (Figure 16). The reduction of hecto- liter mass under weed infested treatment can be due to the decreased traits such as 100-grain mass, leaf area, and photosynthetic pigments, which finally reduces as- similates production. 3.8 GRAIN PROTEIN CONTENT The results showed that the simple effects of fer- tilizer, cultivar, and weeding were significant on grain protein content. The interaction effects of fertilizer × weeding and cultivar × weeding were also significant for this trait (Table 3). Integration of Rhizobium + 50 % chemical fertilizer had the highest grain protein con- tent under weed control and weed infested conditions. However, maximum grain protein content was obtained by applying Rhizobium + 50 % chemical fertilizer under weed control conditions (Figure 17). The grain protein content is used as one of the most important parame- ters for measuring grain quality. Nitrogen is an integral part of the protein and has a vital role in the quality of crops due to its involvement in the synthesis of amino acids and proteins (Caliskan et al., 2008). Nitrogen defi- ciency is one of the limiting factors of yield in most of the crops (Liu et al., 2015). Adding nitrogen in any form (as a chemical fertilizer or bio-fertilizer) increases the grain protein content of crops. It has been reported that inoculation and nitrogen fertilization has resulted in a significant increase in grain protein content of chick- pea (El-Hadi and Elsheikh, 1999). Kordi and Ghanbari (2019) reported that the highest and the lowest protein contents in maize grain were achieved from the inte- gration of bio-fertilizer + 75 % chemical fertilizer and Figure 16: Hectoliter mass of chickpea under weed control and weed infested conditions. Different letters indicate a significant difference at p ≤ 0.05 (Duncan test) Figure 17: Grain protein content of chickpea under different nitrogen sources and weed control and weed infested condi- tions. F0, F1, F2, F3: Control, bio-fertilizer, 100 % chemical fertiliz- er, and bio-fertilizer + 50 % chemical fertilizer, respectively. Different letters indicate a significant difference at p ≤ 0.05 (Duncan test) Figure 18: Grain protein content of chickpea cultivars under weed control and weed infested conditions. Different letters indicate a significant difference at p ≤ 0.05 (Duncan test) Acta agriculturae Slovenica, 118/2 – 2022 11 Influence of different sources of nitrogen fertilizer and weed control on yield, yield components and some qualitative traits ... without fertilization (control), respectively. Nitrogen- fixing bacteria activity increases the nitrogen fertilizer recovery by providing a part of the required nitrogen during the growing season and reducing the nitrogen loss within the soil (Jalilian et al., 2012). It is reported that Rhizobium has facilitated the uptake of nutrients in chickpea through the development of the root system (Rudresh et al., 2005). The result of mean comparisons indicated that the Adel cultivar had higher grain protein content com- pared to the other studied cultivars in both weed con- trol and weed infested conditions. The highest (24.6 %) and the lowest (22.7 %) grain proteins were achieved from the Adel cultivar under weed control conditions and the Mansour cultivar under weed infested treat- ment, respectively (Figure 18). The higher competitive ability of weeds compared to chickpea under weed in- fested treatment led to a significant reduction of avail- able nitrogen and finally decreased the amount of pro- tein in plants. Tanveer et al. (2015) reported that the grain protein content of chickpea decreased with in- creasing weed density. 4 CONCLUSIONS The results obtained from this research clearly in- dicated that the application of nitrogen fertilizer, espe- cially Rhizobium + 50 % chemical fertilizer, improved all the investigated parameters compared to the control treatment (without fertilizer). Thus, the integration of Rhizobium + 50% chemical fertilizer can be used as the most appropriate treatment for reducing the extensive use of chemical fertilizers in agriculture and paving the way for sustainable agricultures. Hand weeding had a positive and significant effect on yield, yield compo- nents, and some qualitative traits of chickpea cultivars. According to the results of this research, the Arman cultivar has priority over other cultivars for the grain yield under the climate conditions of Khorramabad. 5 ACKNOWLEDGEMENTS This research was conducted as an internal re- search project in Islamic Azad University, Khorrama- bad Branch. All material and spiritual rights of the paper are preserved for Islamic Azad University, Khor- ramabad Branch. 6 REFERENCES Amany, A.B. (2007). Effect of plant density and urea foliar application on yield and yield components of chickpea (Cicer arietinum L.). Research Journal of Agriculture and Biological Sciences, 3(4), 220-223. Ayaz, S., McKenzie, B.A., Hill, G.D., McNeil, D.L. (2004). Vari- ability in yield of four grain legume species in a subhu- mid temperate environment. II. Yield components. The Journal of Agricultural Science, 142(1), 21-28. https://doi. org/10.1017/S0021859604004113 Caliskan, S., Ozkaya, I., Caliskan, M.E., Arslan, M. (2008). The effect of nitrogen and iron fertilization on growth, yield, and fertilizer use efficiency of soybean in Mediterranean- type soil. Field Crops Research, 108(2), 126-132. https:// doi.org/10.1016/j.fcr.2008.04.005 Chen, X., Cui, Z., Fan, M., Vitousek, P., Zhao, M., Ma, W., … Zhang, F. (2014). Producing more grain with lower en- vironmental costs. Nature, 514, 486-489. https://doi. org/10.1038/nature13609 El Hadi, E.A., Elsheikh, E.A.E. (1999). Effect of Rhizo- bium inoculation and nitrogen fertilization on yield and protein content of six chickpea (Cicer arietinum L.) cultivars in marginal soils under irrigation. Nutri- ent Cycling in Agroecosystems, 54(1), 57-63. https://doi. org/10.1023/A:1009778727102 Elliott, D.E., Abbott, R.J. (2003). Nitrogen fertilizer use on rain-fed pasture in the Mt. Lofty Ranges, South Australia. 1. Pasture mass, composition and nutritive characteristics. Australian Journal of Experimental Agriculture, 43(6), 553- 577. https://doi.org/10.1071/EA01131 FAO STAT. (2018). Crops. http://faostat3.fao.org/download/Q/ QC/E. Accessed 7 February, 2018. Flajšman, M., Šantavec, I., Kolmanič, A., Kocjan Ačko, D. (2019). Bacterial seed inoculation and row spacing affect the nutritional composition and agronomic performance of soybean. International Journal of Plant Production,13, 183-192. https://doi.org/10.1007/s42106-019-00046-8 Flajšman, M., Mihelič, R., Kolmanič, A., Kocjan Ačko, D. (2020). Influence of soil amended with zeolite and/or mineral N on agronomic performance and soil mineral N dynamics in a soybean–winter triticale crop rotation field experiment. Cereal Research Communications, 48, 239- 246. https://doi.org/10.1007/s42976-020-00030-3 Funga, A., Ojiewo, C.O., Turoop, L., Mwangi, G.S. (2016). Sym- biotic effectiveness of elite rhizobia strains nodulating Desi type chickpea (Cicer arietinum L.) varieties. Journal of Plant Sciences, 4(4), 88-94. Ghilavizadeh, A., Darzi, M.T., Haj Seyed-Hadi, M. (2013). Ef- fects of biofertilizer and plant density on essential oil con- tent and yield traits of ajowan (Carum copticum). Middle East Journal of Scientific Research, 14(11), 1508-1512. Gupta, S.C., Namdeo, S.L. (1996). Effect of Rhizobium strains on symbiotic traits and grain yield of chickpea. Indian Journal of Pulses Research, 9(1): 94-95. Gupta, K.C., Gupta, A.K., Saxena, R. (2016). Weed manage- Acta agriculturae Slovenica, 118/2 – 202212 S. KORDI et al. ment in cowpea [Vigna unguiculata (L.) Wasp.] under rainfed conditions. International Journal of Agricultural Science, 12(2), 238-240. https://doi.org/10.15740/HAS/ IJAS/12.2/238-240 Jalilian, J., Modarres-Sanavy, S.A.M., Saberali, S.F., Sadat- Asilan, K. (2012). Effects of the combination of benefi- cial microbes and nitrogen on sunflower seed yields and seed quality traits under different irrigation regimes. Field Crops Research, 127, 26-34. https://doi.org/10.1016/j. fcr.2011.11.001 Jensen, E.S. (1996). Grain yield, symbiotic N2 fixation and interspecific competition for inorganic N in pea – bar- ley intercrops. Plant and Soil, 182, 25-38. https://doi. org/10.1007/BF00010992 Kantar, F., Shivakumar, B.G., Arrese-Igor, C., Hafeez, F.Y., González, E.M., Imran, A., Larrainzar, E. (2010). Efficient biological nitrogen fixation under warming climates. In: S.S. Yadav, D.L. McNeil, R. Redden, S.A. Patil (Eds.), Cli- mate Change and Management of Cool Season Grain Leg- ume Crops (pp. 283-306). Springer, New York. https://doi. org/10.1007/978-90-481-3709-1_15 Kaushik, S.S., Rai, A.K., Sirothia, P., Sharma, A.K., Shukla, A.K. (2014). Growth, yield and economics of rain fed chick- pea (Cicer arietinum L.) as influenced by integrated weed management. Indian Journal of Natural Products and Re- sources, 5(3), 282-285. Khaitov, B., Kurbonov, A., Abdiev, A., Adilov, M. (2016). Effect of chickpea in association with Rhizobium to crop pro- ductivity and soil fertility. Eurasian Journal of Soil Science, 5(2), 105-112. https://doi.org/10.18393/ejss.2016.2.105- 112 Khaitov, B., Abdiev, A. (2018). Performance of chickpea (Cicer arietinum L.) to biofertilizer and nitrogen application in arid condition. Journal of Plant Nutrition, 41(15), 1980- 1987. https://doi.org/10.1080/01904167.2018.1484134 Khan, M.A., Gul, B., Weber, D.J. (2002). Improving seed ger- mination of Salicornia rubra (Chenopodiaceae) under sa- line conditions using germination regulating chemicals. Western North American Naturalist, 62(1), 101-105. Khan, N., Nawaz, F., Khan, A., Ul Haq, N., Ullah, S., Rehman Khalil, A.U., … Ali, M. (2017). Effect of farmyard ma- nure and Rhizobium inoculation on growth of chickpea (Cicer arietinum L.) variety ’Karak-03’. Pure and Applied Biology, 6(1), 378-384. http://dx.doi.org/10.19045/bsp- ab.2017.60037 Khorsandi, H., Valizadeh-Osalo, G., Sadeghzadeh-Ahari, D., Farayedi, Y. (2016). Study on effects of nitrogen starter and spray fertilizer application differences on chickpea geno- type and variety yields and yield components in dryland condition. Iranian Journal of Dryland Agriculture, 4(2), 211-228. Knott, C.M. (1987). A key for stages of development of the pea (Pisum sativum L.). Annals of Applied Biology, 111(1), 233- 245. https://doi.org/10.1111/j.1744-7348.1987.tb01450.x Kordi, S., Ghanbari, F. (2019). Evaluation of yield, yield com- ponents and some physiological and qualitative traits of corn affected by chemical and biological nitrogen fertiliz- ers. Acta Scientiarum Polonorum: Hortorum Cultus, 18(1), 3-12. https://doi.org/10.24326/asphc.2019.1.1 Liu, K., Li, Y., Hu, H., Zhou, L., Xiaom, X., Yu, P. (2015). Esti- mating rice yield based on normalized difference vegeta- tion index at heading stage of different nitrogen applica- tion rates in southeast of China. Journal of Environmental and Agricultural Sciences, 2, 13. Malik, M.A., Cheema, M.A., Khan, H.Z., Wahid, M.A. (2006). Growth and yield response of soybean (Glycine max L.) to seed inoculation and varying phosphorus levels. Journal of Agricultural Research, 44(1), 47-53. McKenzie, B.A., Hill, G.D. (1995). Growth and yield of two chickpea (Cicer arietinum L.) varieties in Canterbury, New Zealand. New Zealand Journal of Crop and Horticultural Science, 23(4), 467-474. https://doi.org/10.1080/01140671 .1995.9513925 Meena, M.R., Dawson, J., Prasad, M. (2013). Effect of biofer- tilizers and phosphorus on growth and yield of chickpea (Cicer arietinum L.). Bioinfolet, 10, 235-237. Meena, R.S., Meena, V.S., Meena, S.K., Verma, J.P. (2015). The needs of healthy soils for a healthy world. Journal of Clean- er Production, 102, 560-561. https://doi.org/10.1016/j. jclepro.2015.04.045 Mekuanint, Y., Tsehaye, Y., Egziabher, Y.G. (2018). Response of two chickpea (Cicer arietinum L.) varieties to rates of blended fertilizer and row spacing at Tselemti district, Northern Ethiopia. Advances in Agriculture, 2018, 1-8. https://doi.org/10.1155/2018/5085163 Melander, B. (1993). Modelling the effects of Elysmus re- pens L. (Gould). Competition on yield of cereals, peas and oilseed rape. Weed Research, 33, 99-108. https://doi. org/10.1111/j.1365-3180.1994.tb01977.x Merga, B., Alemu, N. (2019). Integrated weed management in chickpea (Cicer arietinum L.). Cogent Food & Agriculture, 5(1), 1620152. https://doi.org/10.1080/23311932.2019.162 0152 Mohammadi, G., Javanshir, A., Rahimzadeh-Khooie, F., Mo- hammadi, S.A., Zehtab-Salmasi, S. (2005). Critical period of weed interference in chickpea. Weed Research, 45(1), 57-63. https://doi.org/10.1111/j.1365-3180.2004.00431.x Mousavi, S.K. (2010). Chemical weed control in autumn sow- ing of chickpea (Cicer aretinum L.) at Lorestan province. Iranian Journal of Pulses Research, 1(2), 131-142. Namvar, A., Seyed-Sharifi, R., Sedghi, M., Asghari-Zakaria, R., Khandan, T., Eskandarpour, B. (2011). Study on the ef- fects of organic and inorganic nitrogen fertilizer on yield, yield components, and nodulation state of chickpea (Cicer arietinum L.). Communications in Soil Science and Plant Analysis, 42(9), 1097-1109. https://doi.org/10.1080/00103 624.2011.562587 Nyoki, D., Ndakidemi, P.A. (2016). Effects of rhizobia inocu- lation, phosphorus and potassium on chlorophyll con- centration of soybean grown under maize intercropping system. International Journal of Plant & Soil Science, 13(6), 1-10. https://doi.org/10.9734/IJPSS/2016/30710 Pooniya, V., Rai, B., Jat, R.K. (2009). Yield and yield attributes of chickpea (Cicer arietinum L.) as influenced by various row spacings and weed control. Indian Journal of Weed Science, 41(3 & 4), 222-223. Raei, Y., Kordi, S., Ghanbari, F., Shayan, A.A., Shahkarami, G., Fatahi, S. (2015). The effect of Azospirilium bacteria Acta agriculturae Slovenica, 118/2 – 2022 13 Influence of different sources of nitrogen fertilizer and weed control on yield, yield components and some qualitative traits ... and salicylic acid effects on drought stress tolerance in Ocimum basilicum L. medicinal plant. Advances in Biore- search, 6, 44-53. Rathod, P.S., Patil, D.H., Dodamani, B.M. (2017). Integrated weed management in chickpea (Cicer arietinum L.) under rainfed conditions of Karnataka, India. Legume Research, 40(3), 580-585. https://doi.org/10.18805/lr.v0iOF.9611 Ratnam, M., Rao, A.S., Reddy, T.Y. (2011). Integrated weed management in chickpea (Cicer arietinum L.). Indian Jour- nal of Weed Science, 43(1 & 2), 70-72. Rudresh, D.L., Shivaprakash, M.K., Prasad, R.D. (2005). Effect of combined application of Rhizobium, phosphate solubi- lizing bacterium and Trichoerma spp. on growth, nutrient uptake and yield of chickpea (Cicer arietinum L.). Applied Soil Ecology, 28(2), 139-146. https://doi.org/10.1016/j.ap- soil.2004.07.005 Shafiq, M., Hassan, A., Ahmad, N., Rashid, A. (1994). Crop yields and nutrient uptake by rain-fed wheat and mung- bean as affected by tillage, fertilization, and weeding. Journal of Plant Nutrition, 17(4), 561-577. https://doi. org/10.1080/01904169409364750 Singh, K.K., Goswami, T.K. (1996). Physical properties of cumin seed. Journal of Agricultural Engineering Research, 64(2), 93-98. https://doi.org/10.1006/jaer.1996.0049 Singh, B., Somanagouda, G., Das, R.C., Lal, G. (2017). Effects of integrated weed management practices on nutrient uptake by weeds and chickpea (Cicer arietinum L.). International Journal of Current Microbiology and Applied Sciences, 6(3), 2338-2343. https://doi.org/10.20546/ijcmas.2017.603.267 Spaepen, S., Vanderleyden, J., Okon, Y. (2009). Plant growth- promoting actions of rhizobacteria. Advances in Botani- cal Research, 51, 283-320. https://doi.org/10.1016/S0065- 2296(09)51007-5 Tanveer, A., Javaid, M.M., Irfan, M., Khaliq, A., Yaseen, M. (2015). Yield losses in chickpea with varying densities of dragon spurge (Euphorbia dracunculoides). Weed Science, 63(2), 522-528. https://doi.org/10.1614/WS-D-13-00049.1 Tena, W., Wolde-Meskel, E., Walley, F. (2016). Response of chickpea (Cicer arietinum L.) to inoculation with native and exotic Mesorhizobium strains in Southern Ethiopia. African Journal of Biotechnology, 15(35), 1920-1929. htt- ps://doi.org/10.5897/AJB2015.15060 Tepe, I., Erman, M. Yergin, R. Bükün, B. (2011). Critical period of weed control in chickpea under non-irrigated condi- tions. Turkish J of Agriculture and Forestry, 35(5), 525-534. Thies, J.E., Singleton, P.W., Bohlool, B.B. (1991a). Influence of the size of indigenous rhizobial populations on es- tablishment and symbiotic performance of introduced rhizobia on field-grown legumes. Applied and Environ- mental Microbiology, 57(1), 19-28. https://doi.org/10.1128/ aem.57.1.19-28.1991 Thies, J.E., Singleton, P.W., Bohlool, B.B. (1991b). Modelling symbiotic performance of introduced rhizobia in the field by use of indices of indigenous population size and nitro- gen status of the soil. Applied and Environmental Micro- biology, 57(1): 29-37. https://doi.org/10.1128/aem.57.1.29- 37.1991 Togay, N., Togay, Y., Cimrin, K.M., Turan, M. (2008). Effect of Rhizobium inoculation, sulfur and phosphorus applica- tions on yield, yield components and nutrient uptake in chickpea (Cicer aretinum L.). African Journal of Biotech- nology, 7(6), 776-782. Tripathi, L.K., Thomas, T., Singh, V.J., Gampala, S., Kumar, R. (2015). Effect of nitrogen and phosphorus application on soil nutrient balance in chickpea (Cicer arietinum L.) cul- tivation. Green Farming, 6(2), 319-322. Werner, D., Newton, W.E. (2005). Nitrogen fixation in agricul- ture, forestry, ecology, and environment. New York, Spring- er. https://doi.org/10.1007/1-4020-3544-6 Wolde-Meskel, E., Heerwaarden, J.V., Abdulkadir, B., Kassa, S., Aliyi, I., Degefu, T., … Giller, K.E. (2018). Additive yield response of chickpea (Cicer arietinum L.) to rhizobium inoculation and phosphorus fertilizer across smallholder farms in Ethiopia. Agriculture, Ecosystems & Environment, 261, 144-152. https://doi.org/10.1016/j.agee.2018.01.035 Wu Fei-Bo. (2000). Effects of inoculation with nitrogen-fixing organisms on N, P and K Uptake, some enzyme activi- ties and lint yield in Sea Island Cotton (Gossypium bar- badense). Acta Phytophysiologica Sinica, 26(4): 273-279. Yadav, J., Verma, J.P., Rajak, V.K., Tiwari, K.N. (2011). Selec- tion of effective indigenous Rhizobium strain for seed inoculation of chickpea (Cicer aritenium L.) production. Bacteriology Journal, 1(1), 24-30. https://doi.org/10.3923/ bj.2011.24.30 Acta agriculturae Slovenica, 118/2, 1–7, Ljubljana 2022 doi:10.14720/aas.2022.118.2.2601 Original research article / izvirni znanstveni članek The effect of some additives on the rheology of dough and quality of bread Xhabir ABDULLAHI 1, 2, Gafur XHABIRI 1, Erhan SULEJMANI 1, Faton SELIMI 1 Received March 09, 2022; accepted April 22, 2022. Delo je prispelo 9. marca 2022, sprejeto 22. aprila 2022 1 Department of Food Technology, University of Tetova, Tetovo, North Macedonia 2 Corresponding author, e-mail: xhabir.abdullahi@unite.edu.mk The effect of some additives on the rheology of dough and quality of bread Abstract: The technology of production of baking prod- ucts today can not be imagined without the use of food ad- ditives. In this research it was aimed to investigate the use of some additives in wheat flour type 500 for bread production. The formulations and additives used in this study are: without additives for M0, emulsifiers (E 472e) for M1, calcium phos- phate (E341 ii) for M2, L-ascorbic acid (E300) for M3 and Damil additive complex (antifouling E170 - 0.06 %; emulsifier E472e -0.08 %; antioxidant E300 -0.01 %; fungal a-amylase - 0.01 %) for M4 formulation. The results showed that the use of additives positively affects some rheological qualities such as water absorption capacity, stability and energy of the dough. M4 bread had a higher specific volume than all breads with 5.14 cm 3 g-1, while M1 and M3 breads were similar. From the total points accumulated for the sensory qualities the M4 bread with a total of 88.8 points accumulated had the best qualities with volume, external appearance and very good crust and crumb taste. It is therefore recommended to use the Damil additive complex in bread production. Key words: additives; wheat flour; rheological charac- teristics; specific volume of bread Učinki nekaterih dodatkov na reološke lastnosti testa in ka- kovost kruha Izvleček: Tehnologije proizvodnje pekovskih izdelkov si ne moremo predstavljati brez uporabe aditivov za živila. V članku so predstavljeni rezultati raziskave, kakšna je uporab- nost dodatkov pšenični moki tip 500 za proizvodnjo kruha. V tej študiji smo uporabili naslednje recepture in dodatke: M0 brez dodatkov, M1 emulgator (E 472e), M2 kalcijev fos- fat (E341 ii), M3 L-askorbinsko kislino (E300) in M4 dodatek Damil. Rezultati so pokazali, da uporaba dodatkov pozitivno vpliva na nekatere reološke lastnosti, kot so sposobnost vpi- janja vode, stabilnost in energija testa. Kruh M4 je imel večji specifični volumen od vseh kruhov s 5,14 cm3 g-1, kruha M1 in M3 pa sta bila podobna. Od skupnih točk za senzorične la- stnosti je imel kruh M4 s skupno 88,8 zbranimi točkami naj- boljše lastnosti za volumen, zunanji videz, zelo dobro skorjo in prijeten okus. Zato je pri proizvodnji kruha priporočljiva uporaba kompleksa aditivov Damil. Ključne besede: aditivi; pšenična moka; reološke la- stnosti; specifična prostornina kruha Acta agriculturae Slovenica, 118/2 – 20222 X. ABDULLAHI et al. 1 INTRODUCTION Modern technology of production of baking prod- ucts has enabled the use of ingredients of suitable qual- ity and food additives with different functional proper- ties. The use of food additives in the baking industry has facilitated the control of the technological process, has enabled the extension of shelf life and the mainte- nance of freshness for a longer period of time (Grujić, 2005). Baking technology plays an important role in the food industry and has increased interest in the use of these products by consumers (Eddy et al., 2007). How- ever today it is impossible to imagine the production of baking products without additives. The addition of additives aims to give bakers the tolerance and flex- ibility required during the stages of the baking process (Hrušková & Novotná, 2003). It is common practice to use various additives such as emulsifiers, oxidants and enzymes to improve the quality of bread (Nanditha & Prabhasankar, 2009) However, the technological quality of the flour is one of the main factors when considering which im- prover should be used. In order for dough improvers to work better, they are in most cases composed of oxi- dizing agents (Biebaut, 1991; Morita et al., 1960). It has been proven that oxidizing agents such as L-ascorbic acid (E300) will increase the level of water absorption in flour and the strength of the dough from the oxidized sulfhydryl groups (-SH) to the disulfide bond (SS). Various empirical methods based on classical ex- tensograph instruments, alveograph, farinograph and mixograph are currently used to obtain data on the rheological properties and baking properties of flour (Uthayakumaran et al., 2002; Dobraszczyk & Morgen- stern, 2003; Tronsmo et al., 2003; Chiotelli et al., 2004). Oxidation generally affects the strength and exten- sibility of the dough. Its effect can be clearly demon- strated by extension tests measured with extensographs (Šimurina et al., 2002). During the baking process to achieve a high quality bread, local producers used as usual additives in order to increase the energy of the dough, to make the dough more elastic, to increase the volume of the bread, to improve the sensory values and to other purposes. The purpose of this research was to study the rheological and sensory qualities of bread produced from mixtures of type 500 flour with various additives such as: emulsifiers (E472e), calcium phosphate (E341), L-ascorbic acid (E300) and Damil additive complex (wheat flour, emulsifier E472e -0.08%; antifouling E170 - 0.06%; antioxidant E300 -0.01%; fungal a-amylase - 0.01%). 2 MATERIALS AND METHODS 2.1 MATERIALS Wheat flour type 500 was used for the production of bread, which was taken from the flour factory “Kokra e Art”- Tetovo, where physico-chemical properties were analyzed. The flour is supplemented with the following mixture of additives: emulsifier (E472e), calcium phos- phate (E341 ii), L-ascorbic acid and additive complex “Damil” (wheat flour, antifouling E170 - 0.06 %; emul- sifier E472e -0.08 %; antioxidant E300 -0.01 %; fungal a-amylase - 0.01 %) in quantities depending on the use of the additive as shown in Table 1. Since our country does not meet the demands of consumers for food or bread, we are dependent on wheat imports. With the mixture of imported and local wheat, the technological value of the flour also chang- es, which also affects the rheological properties of the dough and the quality of the bread. Therefore, due to the variable technological quality of flour, the use of ad- ditives has become important to standardize the flour in terms of rheological properties, to increase the vol- ume of bread to improve sensory values and for other purposes. This experimental design is based on our knowl- edge from previous research of various authors such as Baratto et al. (2015), Sana & Sinani (2017) and (Hor- Samples Flour T-500 (%) Emulsifier (E472e) (%) Calcium phosphate (E341 ii) (%) L-ascorbic acid (%) Damil additive mixture (%) M0 100 - - - - M1 100 0.3 - - - M2 100 - 0.01 - - M3 100 - - 0.02 - M4 100 - - - 0.02 Table 1: Flour samples design with flour type 500 and additives Acta agriculturae Slovenica, 118/2 – 2022 3 The effect of some additives on the rheology of dough and quality of bread vat et al., 2007). Considering that previous studies are mainly based on the use of additives separately, the novelty in our study is the use of the complex additive “Damil”. Bread samples are prepared and baked in the bread production company “Deni” -Skopje, based on the amount of flour mixtures of 300 g, additives from 0.02 to 0.30 % according to the table above. The amount of water is according to the absorption of water in the farinograph, while the amount of salt and yeast is 1.80% and 2.80%, respectively. The production of bread is car- ried out in a standardized way with the direct method where all the ingredients are added to the mixer. The kneading lasted 5 min at medium speed and then sep- arated and given their shape. The dough divided into pieces was placed in the fermentation chamber for 90 minutes at a temperature of 30 °C with 75 % relative humidity and was baked for 25 min at 180 °C in an elec- tric oven. After the breads come out of the oven, they are cooled for 2 hours at room temperature and sent for further evaluation of the quality of the bread. 2.2 METHODS Evaluation of physico-chemical properties of flour such as: protein content, moisture, ash and wet gluten of flour were performed with the Infratec 1241- FOSS. The device is based on NIR (Near Infra Red) technol- ogy and is designed to determine the basic chemical parameters of cereals and flour. The analysis of the rheological properties of the formulated mixtures of flour and additives was per- formed in the laboratory of the enterprise “Kokrra e Art” -Tetovo. To determine the rheological characteris- tics of the mixtures were used 300 g of flour, salt, yeast and additive. The rheological properties of the dough are determined with Farinograph Brabender accord- ing to the standard methods of ISO 5530-1 were the instrument measures the dough stability and degree of softening (Dapčević Hadnađev et al., 2011). Exten- sograph standard methods ISO 5530-2 where used for determination of the physical properties of the dough. The extensibility, resistance and energy of the dough were determined from the curve of the extensogram (Xhabiri & Sinani, 2011; Freund et al., 2006). Determination of moisture, ash, and energy value of bread was performed by standard methods ISO6492: 1999 (E), ISO5984: 2002, SOP628, SOP200). The sen- sory qualities of bread such as volume, appearance, aroma and taste of crust and crumb were also analyzed by a 15 member experienced sensory assessors. All the features of the analyzed breads were evaluated with 1-5 points, then the points obtained were multiplied by the coefficient of importance for each feature and the total points were obtained. The specific volume, Vsp (cm3 g-1) of bread was defined as the ratio of volume and mass of bread, where the mass was determined two hours after baking and cooling, while the volume of bread was de- termined by the method of removal of grains of millet (Kaluđerski & Filipović, 1998). Statistical analysis was performed using SPSS 16 software. The multiple comparison test and the level of significance of the differences between the treatments were taken into account (p < 0.05). All experiments were performed in three replications and the mean val- ues were given together with the standard deviations. Datas were also subjected to statistical analysis (Dun- can test - multivariate analysis, at significance level p <0.05). 3 RESULTS AND DISCUSSION 3.1 CHEMICAL COMPOSITION OF FLOUR The results of physico-chemical composition of flour used in this study are given in Table 2. Based on the analysis, the moisture content in type 500 flour is 14.00 ± 0.50% which indicates that the moisture content in type 500 flour is within the maxi- mum allowable limit (Official Gazette of Republic of Macedonia, 2014). The protein content in type 500 flour was 11.80 ± 0.10% and is approximate to the results of (Abdullahi et.al., 2016). Gluten has an important role in the quality of flour and affects water absorption, viscosity, elongation, elasticity, resistance to deforma- tion, gas holding capacity and hardening properties of dough (Lazarido et al., 2007, Wieser, 2007). The content of wet gluten in t 500 flour was 28.90 ± 0.20 %, indicat- ing that the flour is suitable for bread production. The ash content in type 500 flour was 0.55 ± 0.08 %, which indicates that only the endosperm part was obtained during processing. Physico-chemical parameters (%) (Mean ± SD) Moisture 1 4.00 ± 0.50 Protein 11.80 ± 0.10 Ash 0.60 ± 0.08 Wet gluten 28.90 ± 0.2 0 Table 2: Composition of flour Acta agriculturae Slovenica, 118/2 – 20224 X. ABDULLAHI et al. 3.2 RHEOLOGICAL PARAMETERS OF THE DOUGH Farinograph data of type 500 wheat flour with mixtures of additives are presented in Table 3. The re- sults showed the dependence of the mixing of additives with type 500 wheat flour however they were also influ- enced by the additives that were used. As an important parameter that has the greatest practical value is the ab- sorption of water, which is important in the evaluation of flour (Dapčević Hadnađev et al., 2011). Water ab- sorption was highest in sample M3 with 57.20 ± 1.04 %, while the lowest in sample M1 with 53.90 ± 0.80 % and these differences are significant. From the data of table 3 it can be seen that the control dough M0 has no sig- nificant differences in (p <0.05) with M1 and M4, but expresses significant differences with M2 and M3. The dough development time was much longer in the control dough M0, compared to the formulations with mixture of additives, therefore we have a signifi- cant difference (p < 0.05). This indicates that the dough development time in type 500 wheat flour without ad- ditive increases with increasing proteolytic degradation of proteins (Dua et al., 2009). Regarding the stability of the dough, there is a positive effect of additives in improving the stability of the dough, where samples M3 and M2 had a significant (p < 0.05) higher dough stability than the control dough. A positive effect of the addition of additives on the M2 sample was observed at the degree of softening, which had a better rate of 43 ± 1.06 FU. The degree was the same as that of the control M0, and both had a significant difference (p < 0.05) from M1, M3 and M4 samples. Although many authors emphasize that gluten is the main ingredient of the dough that affects the rheological qualities (Torbica et al., 2007) as well as increases the volume of baked products (Rakita, 2017). The addition of additives to the flour did not change the qualitative number observed from all samples. Data from extensograph analyzes are presented in Table 4 which shows that the use of some additives has influenced extensographic parameters. The low- est dough extensibility had M3 dough, while the other doughs had higher extensibility than M0 control dough. Martin et al. (2003) investigated the effect of pentosan- ase and oxidases on glutenin dough and macropolymer characteristics and reported higher dough extensibil- ity. M1 and M2 dough had lower resistance than M0 control dough, while M3 and M4 dough had higher resistance. The results obtained correspond to those of Ghanbari & Farmani (2013). The data show that the dough M1 had the same energy as the control dough M0, while in other doughs M2, M3, M4 the energy increased. The results obtained are comparable to those of Horvat et al (2007). The ideal ratio for bread production should be 1.5-2.5 and in most dough mixtures it is within the al- lowed limits while the M3 sample had a higher ratio that reaches up to 3.46. Extensograph parameters M0 M 1 M 2 M 3 M 4 Extensibility (mm) 153.00 ± 2.88b 165.00 ± 8.96bc 170.00 ± 8.88c 135.00 ± 9.64a 156.00 ± 5.51bc Resistance (EU) 307.00 ± 18.61a 272.00 ± 16.37a 269.00 ± 8.51a 546.00 ± 47.62b 344.00 ± 39.51a Energy (cm 2 ) 85.60 ± 3.05a 85.30 ± 12.58a 88.30 ± 7.64a 116.00 ± 15.51b 102.00 ± 3.78ab Relation R / E 2.03 ± 0.23a 1.63 ± 0.15a 1.56 ± 0.15a 3.46 ± 0.71b 2.23 ± 0.31a Table 4: Rheological qualities of doughs with exstensograf Different letters in the same order differ significantly, Duncan p <0.05 Farinograph parameters M0 M 1 M 2 M 3 M 4 Water absorption (%) 54.50 ± 0.32a 53.90 ± 0.80a 56.80 ± 0.24b 57.20 ± 1.04b 54.80 ± 0.73a Dough development (min) 3.10 ± 0.16b 1.30 ± 0.04a 1.25 ± 0.02a 1.70 ± 0.04a 1.28 ± 0.02a Dough stability (min) 0.90 ± 0.16a 1.05 ± 0.01a 4.56 ± 0.07b 5.10 ± 0.08b 1.09 ± 0.07a Degree of softening (FU) 43.00 ± 1.41a 52.00 ± 2.16b 43.00 ± 1.06a 77.00 ± 1.41c 54.00 ± 1.34b Quality number 71.00 ± 1.01c 67.00 ± 1.63b 75.00 ± 0.25c 80.00 ± 1.63d 64.00 ± 0.75a Table 3: Rheological properties of farinograph doughs Different letters in the same order differ significantly, Duncan p < 0.05 Acta agriculturae Slovenica, 118/2 – 2022 5 The effect of some additives on the rheology of dough and quality of bread Based on the organoleptic analyzes performed on the quality of bread mixes with wheat flour type 500 and some additives, it was observed significant improve- ments in bread mixes compared to control bread M0. Khan et al (2011) had similar results. Improvements are particularly noticeable in the appearance, taste of the crust and crumb. Pomeranz (1988) confirms that all breads made with type-500 flour, with or without ad- ditives, have their own characteristic taste and aroma. Better volume was in M4 and M3 bread rated with 4.8, while other breads have similar points to the control bread M0. This is also confirmed by the results obtained from the extensogram for the energy of the dough. M4 bread has better appearance while others have similar points to M0 control bread. Considering 3.3 SPECIFIC VOLUME OF BREAD The specific volume of M4 bread was higher than M0, while the specific volume of M2 bread was lower than the specific volume of M0 (p < 0.05). Similar re- sults have been found by Ribotta et al (2010). 3.4 SENSEORY PROPERTIES OF THE BREAD TYPES The quality of bread depends on the quality of the protein in the flour (Lasztity, 2002) therefore high pro- tein content has good effect on bread volume and per- formance (Pomeranz, 1988). Bread samples Volume (k = 4) Exterior (k = 3) Appearance of the crumb (k = 5) Aroma of the crust and crumb (k = 3) Taste of the crust and crumb (k = 5) Total M0 4.3 4. 3 4.6 4.1 4. 3 86.9 M1 4.2 4.4 4.5 4.0 4. 5 87.0 M2 4.3 4.3 4.5 4.1 4.5 87.3 M3 4.7 4.4 4.2 4.3 4.3 87.4 M4 4.7 4.5 4.4 4.0 4.5 88.8 Table 5: Sensory properties of the bread samples k-coefficient of importance Figure 1: Specific volume (cm3 g-1) of bread types Acta agriculturae Slovenica, 118/2 – 20226 X. ABDULLAHI et al. the appearance of the pores created and their size, most breads including control bread M0 have similar points, while bread M3 has fewer points. The aroma of crust and crumb was generally al- most identical to M0 control bread, but M3 bread had a slightly higher. The taste was very similar to all breads, including the control bread. All bread with additives had more accumulated points than control especially the M4 bread had highest points, which corresponds to the findings of Grujić et al (2009). 4 CONCLUSION The use of additives in wheat flour t-500 for bread production improved some rheological properties such as: water absorption capacity, dough stability and dough energy. M4 bread had shown much higher specific vol- ume than M0 control bread, while M1 and M3 bread had similar specific volume. According to the sensory profile (volume, external appearance as well as better aroma and taste of crust and crumb) the additive con- taining bread had higher points. Therefore, the use of Damil additive complex (wheat flour, antifouling E170 - 0.06 %; emulsifier E472e -0.08 %; antioxidant E300 -0.01 %; fungal a-amylase - 0.01 %) for the production of bread with type 500 flour may be recomended. 5 ETHICS The research does not involve human or animal subjects. 6 CONFLICT OF INTEREST The authors declare there is no conflict of interest. 7 ACKNOWLEDGMENT We thank the Flour Industry “Kokra e Art” in Tetovo for providing the flour and rheological measur- ments. 8 REFERENCES Abdullahi, XH., Idrizi, Xh., Sulejmani, E., Jusufi, E., Abdulahi, G. (2016). Qualitative indicators of some wheat cultivars that affect the flour quality in Pollog region. Albanian Journal of Agriculture Science, Special edition, 9-14. Baratto, C. M., Becker, N. B., Gelinski, J., and Silveira, S. M. (2015). Influence of enzymes and ascorbic acid on dough rheology and wheat bread quality. African Journal of Bio- technology, 14(46), 3124-3130. https://doi.org/10.5897/ AJB2015.14931 Biebaut, D. (1991). Flour improvers and raising agents. In: Food Additive User’s, page 242-256. Handbook Blackie Academic & Professional (Ed.), London. https://doi. org/10.1007/978-1-4757-5247-2_13 Chiotelli, E., Rolee, A., Le Meste, M. (2004). Rheological prop- erties of soft wheat flour doughs: effect of salt and triglic- erides. Cereal Chemistry, 81,459. https://doi.org/10.1094/ CCHEM.2004.81.4.459 Dapčević-Hadnađev, T., Pojić, M., Hadnađev, M., Torbica, T. (2011). The Role of Empirical Rheology in Flour Quality Control, Part of the book: Wide Spectra of Quality Control, Intech Open. https://doi.org/10.5772/24148 Dobraszczyk, B. J., Morgenstern, M. P. (2003). Rheology and the breadmaking process. Journal of Cereal Science, 38, 229. https://doi.org/10.1016/S0733-5210(03)00059-6 Dua, S., Lukow O.M., Humphreys, G., Adams, K. (2009). Ef- fect of extraction methods and wheat cultivars on gluten functionality. Open Food Science Journal, 3, 84-92. https:// doi.org/10.2174/1874256400903010084 Eddy, O.N., Udofia, G.P., Eyo, D. (2007). Sensory evaluation of wheat/cassava composite bread and effect of label infor- mation on acceptance and preference. African Journal of Biotechnology, 6(20), 2415-2418. https://doi.org/10.5897/ AJB2007.000-2379 Freund, W., & Kim, M.Y. (2006). In: Future of Flour–A Com- pendium of Flour Improvement, L. Popper, W. Schäfer&W. Freund, (Ed.), Verlag Agrimedia, Clenze, Germany, p. 101. Grujić, S. (2005). Prehrambeni aditivi – funkcionalna svojstva i primjena (Food additives - functional properties and appli- cation), Faculty of Technology, Banja Luka, p. 207. Ghanbari, M. & Farmani, J. (2013). Influence of hydrocolloids on dough properties and quality of Barbari:An Iranian leavened flat bread. Journal of Agricultural Science and Technology, 15, 545-555. Grujić, S., Savanović, D., Odžaković, B., Šavija, O., Popara, D. (2009). Uticaj odabranih sastojaka i prehrambenih aditiva na poboljšanje senzornih svojstava peciva (Influence of selected ingredients and food additives on the improve- ment of sensory properties of pastries) Glasnik hemičara, tehnologa i ekologa, 1, 159-165. Horvat, D., Magdić, D., Drezner, G., Šimiae, G., Dvojkoviae, K., Bročanac, M., & Lukinac, J. (2007). The effect of improver on dough rheology and bread properties. In 4th Interna- tional Congress FLOUR–BREAD, 07, 37–42. Hrušková, M. & Novotná, D. (2003). Effect of ascorbic acid on the rheological properties of wheat fermented dough. Czech Journal Food Science, 21, 137–144. https://doi. org/10.17221/3490-CJFS Kaluđerski, G. & Filipović, N. (1998). Metode ispitivanja kvaliteta žita, brašna i gotovih proizvoda (Methods of testing the quality of cereals, flour and finished products) Matica srpska, Novi Sad. p, 267-274. Khan, A. N., Anwaar, A. A., Bhatti, M.S., Randhawa, M. A., Ah- mad, A., and Yousaf, A. A. (2011). Effect of additives on Acta agriculturae Slovenica, 118/2 – 2022 7 The effect of some additives on the rheology of dough and quality of bread the shelf life extension of chapatti. Food Sciense and Tech- nology Researsh, 17(3), 203–208. https://doi.org/10.3136/ fstr.17.203 Lasztity, R. (2002). Prediction of wheat quality-succes and doubts. Periodica politechnica, Serbian Chemical Engi- neering, 46, p. 39-49. Lazarido, A., Duta, D., Papageorgiou, M., Belc, N., Biliaderis, C. G. (2007). Effects of hydrocolloids on dough rheology and bread quality parameters in gluten-free formulations. Journal of Food Engineering, 79, 1033–1047. https://doi. org/10.1016/j.jfoodeng.2006.03.032 Martin, C.P., Valera, R., Martinez-Anaya, M.A. (2003). Effectof pentosanase and oxidases on the characteristics of doughs and theglutenin macropolymer (GMP). Journal of Agri- cultural and Food Chemistry, 51(16), 4673–4679. https:// doi: 10.1021/jf0257695 Morita, N.K., Nakata, Z.H. and Toyosawa, L. (1996). Effect of á–glucosyl rutin as improvers for wheat dough and bread- making. Cereal Chemistry, 73, 99-104. Official Gazette of Republic of Macedonia, Regulation on the minimum conditions for trading, the quality and types of flour, the manner and methods of sampling, as well as the methods for analysis of the quality of the flour, 24(2014). Pomeranz, Y. (1988). Composition and functionality of wheat Flour components. In Wheat:Chemisty and Technology. 2, ed. Pomeranz Y. American Association of Cereals Chem- ists, St Paul, MN, USA, 219-370. Rakita, S. (2017). Ph.D. Thesis, Faculty of Tehnology, University of Novi Sad, Serbia, 49(1), 1-27. Ribotta, D. P., Pérez, T. G., Añón, C. M., León, E. A. (2010). Optimization of additive combination for improved soy– wheat bread quality. Food Bioprocess Technology, 3, 395 – 405. https://doi.org/10.1007/s11947-008-0080-z Sana, M. & Sinani, A. (2017). The Usage of Edible Additives with Different Gluten Quality in Baking Products. UBT International Conference, p 160. https://doi.org/10.33107/ ubt-ic.2017.160 Šimurina, O., Mastilivć, J., Psodorov, Ð., Filipčev, B. (2002). Uloga aditiva i mešavina aditiva u pekarskoj proizvodnji, Žito-Hleb, 29,105-116. Torbica, A., Antov, M., Mastilović, J., Knežević, D. (2007). The influence of changes in gluten complex structure on technological quality of wheat (Triticum aestivum L). Food Research International, 40(8), 1038-1045. https://doi. org/10.1016/j.foodres.2007.05.009 Tronsmo, K. M., Magnus, E.M., Baardseth, P., Schofield, J. D., Aamondt, A., Faergestad, E. M. (2003). Comparison of small and large deformation rheological properties of wheat dough and gluten. Cereal Chemistry, 80, 587. https:// doi.org/10.1094/CCHEM.2003.80.5.587 Uthayakumaran, S., Newberry, M., Phan-Tien, N., Tanner, R. (2002). Small and large strain rheology of wheat glu- ten. Rheologica Acta, 41, 162. https://doi.org/10.1007/ s003970200015 Wieser, H. (2007). Chemistry of gluten proteins. Food Mi- crobiology, 24(2), 115-119. https://doi.org/10.1016/j. fm.2006.07.004 Acta agriculturae Slovenica, 118/2, 1–8, Ljubljana 2022 doi:10.14720/aas.2022.118.2.2246 Original research article / izvirni znanstveni članek Sublethal effects of some insecticides on the functional response of Ae- nasius bambawalei Hayat, 2009 (Hymenoptera: Encyrtidae) Zeinab RAFATIAN 1, Nooshin ZANDI-SOHANI 1, 2, Fatemeh YARAHMADI 1 Received June 17, 2021; accepted April 23, 2022. Delo je prispelo 17. junija 2021, sprejeto 23. aprila 2022 1 Department of Plant Protection, Faculty of Agriculture, Agricultural Sciences and Natural Resources University of Khuzestan, Mollasani, Ahvaz, Iran 2 Corresponding author, e-mail: nzandisohani@yahoo.com; zandi@asnrukh.ac.ir Sublethal effects of some insecticides on the functional re- sponse of Aenasius bambawalei Hayat, 2009 (Hymenoptera: Encyrtidae) Abstract: Aenasius bambawalei Hayat, 2009 is one of the most effective natural enemies of Phenacoccus solenopsis Tin- sley, 1898. The sublethal effects of dimethoate, imidacloprid, and thiodicarb on the functional response of A. bambawalei to different densities of third instar nymphs of P. solenopsis were evaluated under laboratory conditions. Young females were exposed to the insecticides and then introduced to the host densities of 2, 4, 8, 16, 32, and 64 for 24 h. The results revealed a type III functional response in control and insec- ticide treatments. The handling time and maximum attack rates of A. bambawalei females were adversely affected by in- secticides. The longest handling time and the lowest value of maximum attack rate were observed in thiodicarb treatment, 5.03 h and 4.76, respectively. Therefore, for the simultaneous application of biological and chemical control of P. solenopsis, the influence of insecticides on the functional response be- havior of natural enemies must be evaluated. Key words: parasitoid; biological control; chemical con- trol; IPM Subletalni učinki nekaterih insekticidov na funkcionalen odziv vrste Aenasius bambawalei Hayat, 2009 (Hymenopte- ra: Encyrtidae) Izvleček: Vrsta Aenasius bambawalei Hayat, 2009 je naj- učinkovitejši naravni sovražnik škodljivca Phenacoccus sole- nopsis Tinsley, 1898. Subletalni učinki dimetoata, imidaklopri- da in tiodikarba na funkcionalni odziv vrste A. bambawalei na različne gostote nimf tretjega štadija škodljivca P. solenopsis so bili ovrednoteni v laboratorijskih razmerah. Mlade samice so bile izpostavljene insekticidom in nato prinešene gostitelju v gostotah 2, 4, 8, 16, 32, in 64 za 24 h. Rezultati so pokazali funkcionalni odziv tipa III pri kontroli in obravnavanjih z in- sekticidi. Na čas obravnavanja in največji napad samic vrste A. bambawalei so negativno vplivala obravnavanja z insektici- di. Najdaljši čas obravnavanja (5,03 h) in najmanjša vrednost maksimalnega napada (4,76) sta bila opažena pri obravnava- nju s tiodikarbom. Zaradi tega moramo pri hkratnem biolo- škem in kemijskem uravnavanju škodljivca P. solenopsis pred- hodno ovrednostiti vpliv insekticidov na funkcionalni odziv naravnega sovražnika. Ključne besede: parazitoid; biološki nadzor; kemijski nadzor; IPM Acta agriculturae Slovenica, 118/2 – 20222 Z. RAFATIAN et al. 1 INTRODUCTION Extensive application of synthetic insecticides has caused unsuitable effects on ecosystems such as insect resistance, pest outbreaks, pesticide residues in soil and products, and undesirable effects on non-target organ- isms (Ambrose et al., 2010). On the other hand, in an integrated pest management (IPM) program, success mainly depends on the simultaneous use of chemical compounds and biological control agents and insec- ticides are essential elements for pest population sup- pression (Abedi et al., 2012). Thus, in such systems, the compatibility of insecticides with biocontrol agents is the main concern for IPM managers (Martinou & Stavrinides, 2015). Parasitoid wasps can regulate the pest popula- tion and prevent the pest outbreak (Hentz et al., 1998). One of the major interactions between parasitoids and pests that can influence pest suppression is functional response (Holling, 1959). In parasitoids, functional re- sponse refers to the relationship between the number of hosts attack by a female parasitoid during a given time interval as a function of host density (Solomon, 1949). According to Holling (1959, 1966), there are three types of functional responses. In type I, the parasitoid has a constant search rate overall densities and the result is a linear response until parasitoid satiation (Hassell, 1978). In type II, the parasitoid response to the pest density is curvilinear and in higher densities changes to plateau. Type II response integrates parasitoid handling time, which defines as the time parasitoids spend for over- coming and parasitizing a host. Moreover, parasitoid cleaning and resting behavior before starting to search for a new host are included. Type III shows a sigmoid curve that rises to a plateau when the parasitoid feels satiation. The sigmoid form is due to a slow increase in the attack rate of the parasitoid in higher densities of the host (Holling, 1959, Hassell, 1978). Cotton mealybug, Phenacoccus soleopsis Tinsley (Hemiptera: Pseudococcidae), has been reported as a serious pest on cotton in the United States for the first time (Fuchs et al., 1991) and then from India and Pa- kistan (Hodgson et al., 2008, Sahito et al., 2011). It was also described as a pest of Hibiscus rosa-sinensis L. in Nigeria and Iran (Akintola & Ande, 2008, Joodaki et al., 2018). P. soleopsis is highly polyphagous and more than 202 plant species are attacked across the world includ- ing ornamentals, fruits, weeds, and field crops (Kumar et al., 2009, Fand & Suroshe, 2015). Several chemical in- secticides are used for managing mealybug infestation; however, due to the wax covers the whole body and the cryptic habit of the pest, the efficiency of the method is limited (Fand & Suroshe, 2015). The pest is attacked by 23 species of predators and 7 species of parasitoids in Iran and all of the reported parasitoids belong to the family Encyrtidae (Mossadegh et al., 2015). Among them, only the solitary parasitoid, Aenasius bambawa- lei Hayat is effective in suppressing the pest population and has a key role in its natural parasitism (Fand & Suroshe, 2015). Pesticide exposure is one of the several factors which can influence the functional response of Natural enemies (Martinou et al., 2015). The effect of sublethal concentrations of insecticides on different parasitoids such as Diaeretiella rapae (McIntoch, 1855) (Hym.: Bra- conidae) (Rezaei et al., 2014), Habrobraon hebetor Say, 1836 (Hym.: Braconidae) (Abedi et al., 2012, Mahdavi et al., 2013, Rashidi et al., 2018), Dolichogenidea tasman- ica (Cameron, 1912) (Hym.: Braconidae) (Paull et al., 2014), and Eretmocerus mundus Mercet, 1931 (Sohrabi et al., 2014) have been reported in previous studies. Nevertheless, there is no available information on the sublethal effects of insecticides on A. bambawalei. In the current study, the sublethal effects of dimethoate, imi- dacloprid, and thiodicarb on the functional response of the parasitoid wasp, A. bamabawalei, were investigated on P. solenopsis. 2 MATERIALS AND METHODS 2.1 INSECT REARING Different life stages of P. solenopsis were collected from twigs of Hibiscus rosa-sinensis L. available at the campus of Agricultural Sciences and Natural Resources University of Khuzestan. Young potato, Solanum tu- berosum L., sprouts (0.5-1.5 cm length) were used as a laboratory host of the mealybugs and the transfer was carried out using a fine brush. Then, the potato sprouts were kept in a container (24 × 10 × 16 cm) covered with fine mesh. The newly established colony was used in the experiments. To create the colony of A. bambawalei, the para- sitized nymphs of P. solenopsis were collected from the same H. rosa-sinensis twigs. A separate container was used to keep the mummies until the adults’ emergence. While the adult parasitoids appeared in the contain- ers they were moved by an aspirator to new containers containing 3rd instar nymphs of P. solenopsis. Both colo- nies were maintained in the incubators at 27 ± 2 ᵒC, 65 ± 5 % R. H., and 14 l: 10 D h, and all the experiments were carried out in the mentioned conditions. Acta agriculturae Slovenica, 118/2 – 2022 3 Sublethal effects of some insecticides on the functional response of Aenasius bambawalei Hayat, 2009 (Hymenoptera: Encyrtidae) 2.2 INSECTICIDES Dimethoate, imidacloprid, and thiodicarb were tested in the experiments. Table1 shows more informa- tion about insecticides. 2.3 FUNCTIONAL RESPONSE BIOASSAY For these experiments, the sublethal concentra- tions of 1, 0.5, and 25 ppm of dimethoate, imidacloprid, and thiodicarb were used, respectively. The cylindrical plastic containers (15 cm high and 7 cm diameter) were considered as exposure cages and impregnated to the insecticidal solution for 30 seconds. Distilled water was used as control. Then, they were allowed to dry for 24 h. After this time, 50 newly emerged mated female wasps (less than one day old) were released in each exposure cage for 24 h. Then, randomly selected females were transferred to the new containers consist of different densities of 2, 4, 8, 16, 32, and 64 third instar nymphs of P. solenpsis. The containers were transferred to the incubators with the above-mentioned conditions for 24 h. Then the females were removed and the contain- ers were kept in the incubator until the appearance of mummies. The number of parasitized nymphs was recorded. Ten replications were considered for this ex- periment. 2.4 STATISTICAL ANALYSIS Logistic regression analysis of the proportion of host-parasitoid (Na/N0) as a function of host density (N0) was used to determine the functional response type of A. bamabawalei as recommended by Juliano (2001).This was done by fitting the below polynomial function: (1) Where Na is the number of parasitized hosts, N0 is the initial number of hosts offered, and P0, P1, P2, and P3 are intercept, linear, quadratic, and cubic parameters, respectively. These parameters were calculated using the method of maximum likelihood (PROC CATMOD, SAS Institute, 2001). If the linear parameter (P1) is neg- ative, the type of functional response is II, whereas a positive linear parameter reveals a type III functional response (Juliano, 2001). In the second step, a nonlinear least-squares re- gression (PROC NLIN; SAS Institute Inc., 2001) was used to calculate the functional response parameters (Th and either a for type II functional response or b, c, and d for type III functional response) using Rog- er’s random parasitoid equation (Rogers, 1972; Juliano, 2001). The parameters were estimated according to the following equations: (2) where Na is the number of parasitized hosts, N0, the initial number of hosts, is the instantaneous search- ing efficiency (attack rate), T is the total amount of time available for searching (24h), and is the handling time. For modeling the type III functional response, the searching efficiency (a) in equation (2) was substituted with equation (3) with a function of host density. (3) Where a, b, c, and d are constants and must be es- timated. In cases where both d and c were not signifi- cantly different from 0; the case observed in this study, led to a = bN0 which was inserted into Equation (2). This yielded the following formula (Hassell, 1978): (4) Since thedata from the above experiment fitted the type III functional response, the functional response parameters were obtained using (3) and (4). The maximum attack rate (T/Th) which indicates the maximum number of hosts that can be parasitized by an individual parasitoid during 24 h, was estimated using calculated Th (Hassell, 2000). DosageFormulationCompanyActive ingredientInsecticide 0.5 ppm35 % SCAria ChimiImidaclopridConfidor® 1 ppm40 % ECKimiyaGohareKhakDimethoateRoxion® 25 ppm80 % DFMoshkfam Fars ThiodicarbLarvin® Table 1: Information about experimental insecticides Acta agriculturae Slovenica, 118/2 – 20224 Z. RAFATIAN et al. plained the functional response of A. bambawalei in all treatments (Table 3). The maximum number of 3rd instar nymphs that could be parasitized by A. bambawalei (T/Th) decreased from 8.35 in control to 6.49, 4.88, and 4.76 in imidaclo- prid, dimethoate, and thidicarb, respectively (Table 3). 4 DISCUSSION Pesticides are prevalent elements in integrated pest management programs and the study of their be- havioral effects such as functional response on natural enemies can be helpful for the success of IPM. Accord- ing to the results of the current study, the functional response of A. babmawalei in control and insecticide treatments was of type III. Parasitoids with type III functional response are density-dependent and can in- crease their search rate on higher densities of the host. Although a previous overview on functional response indicated that type II is more common in parasitoids (Fernandez-arhex & Corley, 2003), several studies in- dicate type III in parasitoid wasps such as Eretmocerus mundus Mercet on Bemisia tabaci Gennadius, 1889 (Sohrabi et al., 2014), Aphidius colemani Viereck, 1912 on Aphis gossypii Glover, 1877 (Van Steenis & El-Wha- wass, 1995) and Praon volucre (Haliday, 1833) on Myzus persicae (Sulzer, 1776) (Tazerouni et al., 2016). Accord- ing to an earlier study, type II functional response was 3 RESULTS The logistic regression analysis of the proportion of P. solenopsis parasitized by A. babmawalei indicated Type III functional response for this parasitoid in con- trol and all insecticidal treatments. The linear coeffi- cient, P1, was positive and the quadratic coefficient, P2, was negative in all treatments (Table 2). Therefore, the proportion of host parasitized was density-dependent, which shows a type III functional response (Figure 1). Roger’s random parasitoid equation was used for data analysis in control and other treatments. Accord- ing to the results of nonlinear least square regression parameters, c and d were not significantly different from zero; therefore, they were removed from the mod- el and a reduced model was used (Equation 4). Estimat- ed b values for the control, dimethoate, imidacloprid, and thiodicarb were 0.00620, 0.00304, 0.00344, and 0.00267, and estimated handling times in these treat- ments were 2.87, 4.92, 3.70, and 5.03 h, respectively (Ta- ble 3). Comparing the asymptotic 95 % confidence of b values in Table 3 indicated that imidacloprid had no significant effect on the value of the b of A. bambawa- lei; however, it was significantly reduced in dimethoate and thiodicarb treatments. Nevertheless, handling time (Th) significantly increased in all insecticide treatments. The values of r2, coefficient of determination, revealed that Rogers’s random parasitoid equation properly ex- P valueSEEstimateParametersTreatment 0.21050.3778-0.4730P0 (Constant)Control 0.36140.05780.0528P1 (Linear) 0.11890.00220-0.00344P2 (Quadratic) 0.10550.000022-0.000035P3 (Cubic) 0.00040.4598-1.6313P0 (Constant)Dimetoat 0.21600.06890.0853P1 (Linear) 0.13660.00261-0.00388P2 (Quadratic) 0.16190.0000260.000036P3 (Cubic) 0.00750.4118-1.1003P0 (Constant)Imidacloprid 0.29190.06250.0659P1 (Linear) 0.14080.00238-0.00350P2 (Quadratic) 0.14580.0000240.000034P3 (Cubic) 0.00140.4536-1.4500P0 (Constant)Thiodicarb 0.47870.06890.0488P1 (Linear) 0.34210.00262-0.00249P2 (Quadratic) 0.38940.0000260.000022P3 (Cubic) Table 2: Maximum likelihood estimates from logisitic regression analysis of the proportion of third instar nymphs of Pheno- coccus solenopsis parasitized by Aenasius bambawalei as a function of initial host density Acta agriculturae Slovenica, 118/2 – 2022 5 Sublethal effects of some insecticides on the functional response of Aenasius bambawalei Hayat, 2009 (Hymenoptera: Encyrtidae) Figure 1: Functional response of A. bambawalei to different densities of 3rd instar nymphs of P. solenopsis in different insecti- cide treatments. From the top: dimethoate, thiodicarb, imidacloprid, and control treatments. Symbols are observed data and lines were predicted by the model (Equation 2) Acta agriculturae Slovenica, 118/2 – 20226 Z. RAFATIAN et al. recorded for A. bambawalei to different densities of P. solenopsis at selected temperatures which is varied from the results of our study (Joodaki et al., 2018). Several factors including difference in experimental conditions, parasitoid strain, host plant cultivar, host species, the age of parasitoid and host, physiological state of the host may be responsible for the change of functional response type in parasitoid wasp (Sagarra et al., 2001, Ambrose et al., 2010, Asadi et al., 2012, Pasandideh et al., 2015, Tazerouni et al., 2016, Joodaki et al., 2018). In this study, the type of functional response did not change depending on different insecticide treatments. There is no available information about other studies reporting the effects of insecticides on the functional response of A. bambawalei. On the other hand, Habrobracon hebetor Say, 1836 showed type III functional response to dif- ferent densities of Anagasta kuheniella Zeller, 1879 in control and all insecticide treatments (Mahdavi et al., 2013). In another study, the type of functional response of Diaretiella rapae (McIntosh) did not change when it was exposed to primicarb and thiamethoxam (Rezaei et al., 2014). However, Sohrabi et al. (2014) reported an al- teration in type III functional response of Eretmocerus mundus Mercet in control treatment as well as imida- cloprid to type II in buprofezin treatment. Moreover, the sublethal concentrations of primicarb, cyperme- thrin, and dimethoate changed the functional response of D. rapae from type II to type III (De-Jiu et al., 1991). Handling time (the time needed to subdue and consume the prey item) and attack rate are important parameters used to assess the parasitoid functional response (Juliano, 2001). In this study, all insecticides had a significant negative effect on the handling time of parasitoids; however, the influence of thiodicarb was r2T/Th Th bTreatments 0.968.352.87 ± 0.0980 (2.6781-3.0704) 0.00620 ± 0.00103 (0.00484- 0.00825) Control 0.964.884.92 ± 0.3197 (4.2774-5.7773) 0.00304 ± 0.000881 (0.00128-0.00480) Dimethoate 0.866.493.70 ± 0.2602 (3.1747-4.2163) 0.00344 ± 0.00106 (0.00131-0.00556) Imidacloprid 0.884.765.03 ± 0.3203 (4.3940-5.6765) 0.00267 ± 0.000740 (0.00119-0.00415) Thiodicarb Table 3: Parameters (± SE) estimated by random parasitoid equation indicating functional response of A. bambawalei to different treatments The values in parentheses are 95 % confidence intervals; b: constant; Th: handling time; T/Th maximum attack rate even higher than the other insecticides and had the most adverse effect on the host-finding of the para- sitoid. The difference in handling time in insecticide treatments may be due to their various mode of action, which influence the neural system of parasitoids. The longer handling time in parasitoids exposed to insecti- cides could be related to the commotion of their neu- ral system (Rezaei et al., 2014). An increase in handling time under insecticide treatments has been reported in different parasitoids such as H. hebetor when exposed to spinosad (Dastgersi, 2009), and cypermethrin (Abedi et al., 2012). Among all insecticide treatments, dimethoate and thiodicarb had the most, and imidacloprid had the least negative effect on the value of maximum attack rate of the wasp. The parasitism rate of A. bambawalei in imida- cloprid treatment was 1.3 times more than dimethoate and thiodicarb; however, in control, it was distinctly higher than insecticide treatments. Insecticides can cause a repellent effect on parasitoids or decrease their host finding ability due to boosting disturbance and re- ducing the olfactometric abilities, which may result in a reduction in parasitism rate (Decourtye et al., 2004). 5 CONCLUSIONS The results confirmed the negative impact of in- secticides on the functional response of parasitoids and probably the success of biological control programs. Hence, for the simultaneous application of biological and chemical control in integrated pest management programs, the influence of insecticides on the differ- ent behavior of natural enemies such as functional re- sponse has to be evaluated. Such information is essen- tial to estimate the appropriate time to release natural enemies in the case of using insecticides. 6 ACKNOWLEDEMENTS The authors would like to thank Agricultural Sci- ences and Natural Resources University of Khuzestan, Iran, for the financial support of this research project. 7 REFERENCES Abedi, Z., Saber, M., Gharekhani, G., Mehrvar, A., Mahdavi, V. (2012). Effects of azadirachtin, cypermethrin, methoxyfe- nozide and pyridalil on functional response of Habrobra- con hebetor Say (Hym.:Braconidae). Journal of Plant Pro- tection Research, 52(3), 353-358. https://doi.org/10.2478/ v10045-012-0058-8 Acta agriculturae Slovenica, 118/2 – 2022 7 Sublethal effects of some insecticides on the functional response of Aenasius bambawalei Hayat, 2009 (Hymenoptera: Encyrtidae) Asadi, R., Talebi, A.A., Khalghani, J., Fathipour, Y., Moharrami- pour, S., AskariSiahooei, M. (2012). Age-specific function- al response of Psyllaephagus zdeneki (Hymenoptera: En- cyrtidae), parasitoid of Euphyllurapakistanica (Hemiptera: Psyllidae). Journal of Crop Protection, 1(1), 1-15. Akintola, A. J. & Ande, A. T. (2008). First record of Phena- coccus solenopsis Tinsley (Hemiptera: Pseudococcidae) on Hibiscus rosa-sinensis in Nigeria. Agricultural Journal (Medwell Journals, Pakistan) 3(1), 1-3. Ambrose, D. P., Rajan, S. J., Raja, J. M. (2010). Impacts of Syn- ergy-505 on the functional response and behavior of the reduviid bug, Rhynocoris marginatus. Journal of Insect Sci- ence, 10(1), 187. https://doi.org/10.1673/031.010.18701 Dastjerdi, H. R., Hejazi, M. J., Ganbalani, G. N., Saber, M. (2009). Sublethal effects of some conventional and biora- tional insecticides on ectoparasitoid, Habrobracon hebetor Say (Hymenoptera: Braconidae). Journal of Entomology, 6(2), 82-89. https://doi.org/10.3923/je.2009.82.89 De-Jiu, G., Ming-En, Y., Ren-Huan, H., Zhehuai, L. (1991). The effects of sublethal doses of insecticides on the foraging behavior of parasitoid, Diaeretiella rapae (Hym.: Braconi- dae). Acta EcologicaSinica, 4, 1-4. https://doi.org/10.1007/ BF02374300 Decourtye, A., Armengaud, C., Renou, M., Devillers, J., Cluzeau, S., Gauthier, M., Pham-Delègue, M.H. )2004(. Imidacloprid impairs memory and brain metabolism in the honeybee (Apismellifera L.). PesticideBiochemistry and Physiology, 78(2), 83-92. https://doi.org/10.1016/j. pestbp.2003.10.001 Fand, B. B. & Suroshe, S. S. (2015). The invasive mealybug Phenacoccus solenopsis Tinsley, a threat to tropical and subtropical agricultural and horticultural production systems–a review. Crop Protection, 69, 34-43. https://doi. org/10.1016/j.cropro.2014.12.001 Fuchs, T. W., Stewart, J. W., Minzenmayer, R., Rose, M. (1991). First record of Phenacoccus solenopsis Tinsley in cultivat- ed cotton in the United States. Southwestern Entomologist, 16(3), 215-221. Hassell, M. P. (1978). The dynamics of arthropod predator-prey systems. Princeton University Press. Hassel, M. (2000). The spatial and temporal dynamics of host- parasitoid interactions. OUP Oxford. Hentz, M. G., Ellsworth, P. C., Naranjo, S. E., Watson, T. F. (1998). Development, longevity, and fecundity of Che- lonussp. nr. curvimaculatus (Hymenoptera: Braconidae), an egg–larval parasitoid of pink bollworm (Lepidoptera: Gelechiidae). Environmenal Entomology, 27(2), 443-449. https://doi.org/10.1093/ee/27.2.443 Holling, C. S. (1959). Some characteristics of simple types of predation and parasitism. The Canadian Entomologist, 91(7), 385-398. https://doi.org/10.4039/Ent91385-7 Holling, C. S. (1966). The functional response of invertebrate predators to prey density. The Memoris of Entomological Society of Canada, 98(S48), 5-86. https://doi.org/10.4039/ entm9848fv Hodgson, C., Abbas, G., Arif, M. J., Saeed, S., Karar, H. (2008). Phenacoccus solenopsis Tinsley (Sternorrhyncha: Coc- coidea: Pseudococcidae), an invasive mealybug damaging cotton in Pakistan and India, with a discussion on sea- sonal morphological variation. Zootaxa, 1913(1), 1-35. https://doi.org/10.11646/zootaxa.1913.1.1 Joodaki, R., Zandi-Sohani, N., Zarghami, S., Yarahmadi, F. (2018). Temperature-dependent functional response of Aenasius bambawalei (Hymenoptera: Encyrtidae) to dif- ferent population densities of the cotton mealybug Phe- nacoccus solenopsis (Hemiptera: Pseudococcidae). Eu- ropean Journal of Entomology, 115, 326-331. https://doi. org/10.14411/eje.2018.032 Juliano, S. A. (2001). Nonlinear curve fitting: Predation and functional response curves. In S. M. Cheiner & J. Gurven (Eds.), Design and Analysis of Ecological Experiments, 2nd Edition, (pp.178-196), Chapman and Hall, London. Kumar, R., Kranthi, K. R., Monga, D., Jat, S. L. (2009). Natural parasitization of Phenacoccus solenopsis Tinsley (Hemip- tera: Pseudococcidae) on cotton by Aenasius bambawalei Hayat (Hymenoptera: Encyrtidae). Journal of Biological Control, 23(4), 457-460. Mahdavi, V., Saber, M., Rafiee-Dastjerdi, H., Mehrvar, A., Has- sanpour, M. (2013). Efficacy of pesticides on the function- al response on larval ectoparasitoid, Habrobracon hebetor Say (Hymenoptera: Braconidae). Archives of Phytopathol- ogy and Plant Protection, 46(7), 841-848. https://doi.org/1 0.1080/03235408.2012.753182 Martinou, A. F., Stavrinides, M. C. (2015). Effects of sublethal concentrations of insecticides on the functional response of two mirid generalist predators. PloS one, 10(12). https:// doi.org/10.1371/journal.pone.0144413 Mossadegh, M. S., Vafaei, S., Farsi, A., Zarghami, S., Esfandi- ari, M., Dehkordi, F.S… Seyfollahi, F. (2015).Phenacoccus solenopsis Tinsley (Sternorrhyncha: Pseudococcidae), its natural enemies and host plants in Iran. In Sh. Manzari (Ed.), Proceedings of the 1st Iranian International Congress of Entomology (pp. 159-167). Tehran, Iran. Paull, C. A., Schellhorn, N. A., Austin, A. D. (2014). Response to host density by the parasitoid Dolichogenidea tasmanica (Hymenoptera: Braconidae) and the influence of grape- vine variety. Bulletin of Entomological Research, 104(1), 79- 87. https://doi.org/10.1017/S0007485313000527 Pasandideh,A., Talebi, A. A., Hajiqanbar, H., Tazerouni, Z. (2015). Host stage preference and age-specific functional response of Praon volucre (Hymenoptera: Braconidae, Aphidiinae) a parasitoid of Acyrthosiphon pisum (Hemip- tera: Aphididae). Journal of Crop Protection, 4(4), 563-575. Rashidi, F., Nouri-Ganbalani, G., Imani, S. (2018). Sublethal effects of some insecticides on functional response of Habrobracon hebetor (Hymneoptera: Braconidae) when reared on two lepidopteran hosts. Journal of Economic En- tomology, 111(3), 1104-1111. https://doi.org/10.1093/jee/ toy069 Rezaei, N., Kocheyli, F., Mossadegh, M. S., Talebi Jahromi, K., Kavousi, A. (2014). Effect of sublethal doses of thiameth- oxam and pirimicarb on functional response of Diaere- tiella rapae (Hymenoptera: Braconidae), parasitoid of Lipaphis erysimi (Hemiptera: Aphididae). Journal of Crop Protection, 3(4), 467-477. Rogers, D. (1972). Random search and insect population models. Journal of Animal Ecology, 369-383. Sagarra, L.A., Vincent, C., Stewart, R.K. (2001). Body size as Acta agriculturae Slovenica, 118/2 – 20228 Z. RAFATIAN et al. an indicator of parasitoid quality in male and female Anagyrus kamali (Hymenoptera: Encyrtidae). Bulletin of Entomological Research, 91(5), 363-367. https://doi. org/10.1079/BER2001121 Sahito, H.A., Abro, G.H., Syed, T.S., Lanjar, A.G., Mal, B., Khaj- jak, A.S. (2011). Seasonal population abundance of cot- ton mealybug, Phenacoccus solenopsis Tinsley (Hemiptera: Pseudococcidae) and its natural enemies on various hosts. Pakistan Journal of Entomology Karachi, 26(1), 25-40. Sohrabi, F., Shishehbor, P., Saber, M., Mosaddegh, M. S. (2014). Effects of buprofezin and imidacloprid on the functional response of Eretmocerus mundus Mercet. Plant Protection Science, 50(3), 145-150. https://doi.org/10.17221/64/2012- PPS Tazerouni, Z., Talebi, A. A., Fathipour, Y., Soufbaf, M. (2016). Age-specific functional response of Aphidius matricariae and Praon volucre (Hymenoptera: Braconidae) on Myzus persicae (Hemiptera: Aphididae). Neotropical Entomol- ogy, 45(6), 642-651. https://doi.org/10.1007/s13744-016- 0413-y Van Steenis, M. J. & El-Khawass, K. A. M. H. (1995). Behaviour of Aphidius colemani searching for Aphis gossypii: func- tional response and reaction to previously searched aphid colonies. Biocontrol Science and Technology, 5(3), 339-348. https://doi.org/10.1080/09583159550039783 Acta agriculturae Slovenica, 118/2, 1–13, Ljubljana 2022 doi:10.14720/aas.2022.118.2.1557 Original research article / izvirni znanstveni članek Morphological, biochemical, and nutritional value of prickly and smooth fruit spinach Reza ABOLGHASEMI 1, 2, Maryam HAGHIGHI 3, Nematollah ETEMADI 3 Received March 11, 2020; accepted May 10, 2022. Delo je prispelo 11. marca 2020, sprejeto 10. maja 2022 1 Ph.D. student, Department of Horticulture, College of Agriculture, Isfahan University of Technology, Isfahan, Iran 2 Corresponding author, e-mail: r.abolghasemi@ag.iut.ac.ir 3 Associate Professor, Department of Horticulture, College of Agriculture, Isfahan University of Technology, Isfahan, Iran Morphological, biochemical, and nutritional value of prick- ly and smooth fruit spinach Abstract: This study aimed to investigate the morpho- logical (qualitative and quantitative traits) and biochemical characteristics (such as leaf pigments and total antioxidant capacity, vitamin E and C content, total soluble carbohydrate, total amino acid content, nitrate concentration, nitrate reduc- tase assay, oxalic acid content, Ca and Fe content) in spinach. The selected accessions in this study were prickly (’Varamin Prickly’) and smooth (’Monatol’) fruits of spinach selected among 44 accessions. This experiment was carried out in spring, arranged as a complete randomized block with three replicates and 18 observations. Results showed no significant differences between the two accessions for most qualitative and quantitative morphological traits. In contrast, biochemi- cal characteristics showed significant differences between the two accessions. Both accessions had high yields, but the dry biomass of ’Varamin Prickly’ accession was more than ’Monatol’ (smooth fruit). The results indicated that the fruit type does not appear to cause variations in morphological traits, and differences in accessions could be due to genetic sources and environmental distribution. The prickly fruit accession showed a significant superiority for most qualita- tive nutraceutical traits, including DPPH, flavonoid, phenol, carbohydrate, amino acid, fiber, and Fe content compared to smooth fruit accession. Finally, it was found that prickly fruit accession is very suitable for mechanized harvesting and human diet due to its appropriate plant, leaf, petiole, and qualitative nutraceutical traits and can be used for breeding purposes and cultivation fields. Key words: Iranian spinach accession; prickly fruit; smooth fruit; nutraceutical traits; Spinacia oleracea Morfološka, biokemična in hranilna vrednost špinače z gladkimi in bodečimi plodovi Izvleček: Namen raziskave je bil preučiti morfološke (kakovostne in količinske) in biokemijske lastnosti (listna barvila, celokupno antioksidacijsko sposobnost, vsebnost vitaminov E in C, celokupnih topnih ogljikovih hidratov, ce- lokupnih amino kislin, nitrata, oksalne kisline, Ca in Fe, prei- skus nitrate reduktaze) v špinači. Iz 44 akcesij so bile izbrane sorte z bodečimi (’Varamin Prickly’) im gladkimi (’Monatol’) plodovi. Poskus je bil izveden spomladi kot popolni naključni bločni poskus s tremi ponovitvami in 18 opazovanji. Rezultati so pokazali, da v večini kakovostnih in količinskih morfolo- ških lastnostih obeh tipov ni bilo značilnih razlik. Nasprotno so se vbiokemičnih lastnostih obeh tipov pokazale značilne razlike. Oba tipa akcesij sta imela velike pridelke, a je bila bio- masa ’Varamin Prickly’ večja kot pri akcesijah sorte Monatol. Rezultati so pokazali, da tip plodov ne povzroča raznolikosti v morfoloških lastnostih in, da bi razlike med akcesijami lah- ko bile genetskega ali okoljskega izvora. Akcesije z bodečimi plodovi so bile značilno superiorne v večini kakovostnih hranilnih lastnosti kot so DPPH, vsebnost flavonoidov, feno- lov, ogljikovih hidratov, amino kislin, vlaknin in Fe v primer- javi z akcesijami z gladkimi plodovi. Povzamemo lahko, da bi bile akcesije z bodečimi plodovi zelo primerne v bodočih pro- gramih žlahtnenja in pridelave na polju zaradi primernosti za strojno spravilo in prehrano ljudi zaradi primernih lastnosti habitusa, listov, listnih pecljev rastlin in ugodnih hranilnih lastnosti. Ključne besede: akcesije iranske špinače; bodeči plodo- vi; gladki plodovi; hranilne lastnosti; Spinacia oleracea Acta agriculturae Slovenica, 118/2 – 20222 R. ABOLGHASEMI et al. 1 INTRODUCTION Spinach (Spinacia oleracea L.) is one of the criti- cal and commercial vegetables planted worldwide for fresh and processed consumption (Morelock & Correll, 2008). As one of the origins of spinach, Iran has ranked the seventh producer globally (Shi et al., 2016). This vegetable is a fast-growing, cold season, and nutritious that is highly recommended in the human diet. Spinach has high levels of iron, dietary fiber, vitamins, antioxi- dants, and several phytochemical components. On the other hand, some compounds, such as nitrate and oxa- late, can be high accumulated in leaves that have poten- tial health hazards to humans (Koh et al., 2012). There- fore, selecting and introducing low-capacity nitrate and oxalate varieties is an essential aim in spinach breeding programs (Koh et al., 2012). Pprevious studies were devoted to the genetic structure associated with the morphological traits of spinach (Arif et al., 2013; Sabaghnia et al., 2014). High variation yield was reported among 54 Iranian spinach accessions (Sabaghnia et al., 2014). Nevertheless, vari- ation in the phenotypic characteristics of plants such as leaf type, fruit type (seed type), and even leaf color can affect plant growth and biochemical traits. Like, leaf color affects morphological and biochemical char- acteristics of Sassafras tsumu (Hemsl.) Hemsl. Jiang et al., 2016). Phenotypic differences were also reported in Vachellia nilotica subsp. indica (Benth.) Kyal. & Boatwr. due to having two types of smooth and prickly seeds. (Gorain et al., 2014). Sunflowers from different seeds differ in biochemical properties such as oil and protein content (Balalic et al., 2012). Fruit types, leaf morphology, leaf color (green vs. purple), and day to flowering are spinach’s most critical phenotypic characteristics for breeding classification and commercial purposes (Shi et al., 2016). Wrinkled leaves are marketable in the USA, whereas flat leaves are favorable in Iran (Avsar, 2011). Moreover, fruit forms used in mechanization and handy cultivation are differ- ent (Wu et al., 2015). There are two types of taxonomic varieties of spinach (S. oleracea var. spinosa Moench.) with prickly and (S. oleracea var. inermis Peterm.) with smooth fruits (Mei et al., 2010). Spinach cultivars, with prickly fruits are not suitable for mechanized cultiva- tion. So, it has been suggested that round and smooth fruits, is suited for mechanized spinach cultivation (Morelock & Correll, 2008). Some countries have solved the prickly problem of spinach fruit by using fruit coat- ing (Shi et al., 2016). It is reported that most European varieties of spinach fruits are smooth (Meng et al., 2017). Conversely, most prickly spinach fruits belong to the Asian region consisting of Korea, Japan, Iraq, and Iran. There is a remarkable diversity among the fruits of Asian spinach cultivars due to the vast territory and different climates (Meng et al., 2017; Shi et al., 2016). The researchers reported that prickly fruit spinach varieties had narrow and small leaves with long peti- oles that are generally resistant to low temperatures but sensitive to high temperatures (Meng et al., 2017; Mei et al., 2010). On the other hand, thick and wrinkled leaves with short petioles are generally observed in smooth fruit varieties (Mei et al., 2010). It is reported that smooth fruit varieties of spinach are tolerant to high temperatures and are sensitive to low temperatures (Mei et al., 2010). So, the appearance of spinach fruit is also an essential feature for spinach classification (Wu et al., 2015; Liu et al., 2004). Besides, the type of fruit may be affected by spin- ach quality. Some relationships were observed between the morphological and biochemical characteristics of spinach (Mei et al., 2010). Wu et al. (2015) investigated a wide variety of morphological traits of two smooth and prickly fruits spinach varieties. He reported signifi- cant differences in the growth habits of two fruits types, and morphological and biochemical classification of spinach fruits are critical to distinguish among them (Wu et al., 2015). Therefore, the qualitative assessment of prickly and smooth fruits of spinach, such as antioxi- dants, fiber, iron content, and accumulation of nitrate and oxalate, provided helpful information on cultivar selection (Wu et al., 2015). Recently, due to the extensive cultivation of spin- ach, there has been a vast demand for high-quality seed material for farmers (Jafari & Jalali, 2015). There is a lack of information on the differences between smooth and prickly fruit spinach for the future breeding program. Therefore, this study seems necessary as primitive in- formation for broadcasting future research. The present experiment was conducted to investigate and compare the morphological and biochemical characteristics of the prickly and smooth fruit accessions and the relation of fruit morphology with spinach’s biochemical and nutraceutical characteristics. 2 MATERIALS AND METHODS 2.1 PLANT MATERIAL AND FIELD EXPERI- MENT DESIGN Iranian spinach accession ’Varamin Prickly’ (prickly fruit) and ’Monatol’ accession (smooth fruit), in text later indicated as seeds, were selected from 44 spinach accessions provided by the Seed and Plant Im- provement Institute and the Gene Bank of Iran (SPII) Acta agriculturae Slovenica, 118/2 – 2022 3 Morphological, biochemical, and nutritional value of prickly and smooth fruit spinach and the Leibniz Institute of Plant Genetics and Crop Plant Research (IPK) gene bank (IPK, 2018) based on the previous author’s study (Abolghasemi et al., 2019). The selection was based on spinach’s yield and mecha- nized planting characteristics, such as leaf features, plant height, petiole length, fresh mass and yield, dry mass, days to flowering, and percentage of the female plant. In addition, these two accessions had different seed type that was selected for this study. The experiment was designed based on a randomized complete block design (RCBD) with 3 replications and 18 observations. The seeds were sown in a field located at the Isfahan University of Technology, Isfahan, Iran, in March 2018 (spring sowing). The conditions of daylight and tem- perature during spring are presented in Fig 1. The soil was sandy-clay, and manure fertilizer mixed with soil 40 t ha-1 pre-planting. The plot size was 2 m2. After seed germination and plant growth, 18 observations (plant bush) in each plot were selected. It should be noted that this study was carried out in the growth chamber for a more precise evaluation and better comparison of mor- phological and biochemical characteristics of these two accessions (smooth and prickly seed). Finally, the results of our research in controlled conditions confirmed our field cultivation results (data was not presented). There- fore, due to the similarity of the two studies, the field study results are presented in this manuscript. 2.2 MORPHOLOGICAL CHARACTERIZATION To investigate morphological features, 35-50 days after planting (due to non-homogeneous growth of spinach), 18 plants were selected from each plot. Then four leaves were selected from four directions of plants to measure the desired parameters. In the first step, the qualitative and visual characteristics of spinach acces- sions were evaluated based on the descriptor of the In- ternational Plant Genetic Resources Institute (IPGRI) presented in Table 1. (Arif et al., 2013; Jafari & Jalali 2017). Quantitative and morphological characteristics studied in this study included: 1000-grain mass, ger- mination percentage was calculated using the formula when 50 percent of the seeds were germinated (Ikic et al., 2012): Germination percentage = (number of germinated seeds / total number of seeds) × 100 when 50 percent of the seeds were germinated, leaf length (cm), leaf width (cm), petiole length (cm), petiole diameter (mm), leaf area (mm2), plant height (cm), fresh and dry mass (g), yield (kg ha-1), leaf numbers, male and female plants percent, days to flowering. The dry mass of the shoot was measured after putting the bush in an oven at 70 °C for 48 hours (Arif et al., 2013; Jafari & Jalali, 2017). 2.3 BIOCHEMICAL ANALYSIS Leaf pigments: Some fresh leaf tissue (5 g) was mixed with 80 % acetone, then filtered and balanced to 10 ml, and its absorption was measured in 663, 647, and 470 nm with a spectrophotometer (U-2100, JASCO, Japan) (Lichtenthaler, 1987). Chlorophyll a = (19.3 × A663 ‐ 0.86 × A647) Vol- ume / 100 Mass Chlorophyll b = (19.3 × A647 ‐ 3.6 × A663) Vol- ume /100 Mass Total chlorophyll = Chlorophyll a + Chlorophyll b Carotenoids = 100(A470) ‐ 3.27(mg g-1 Chl. a) ‐ 104 (mg g-1 Chl. b)/227 Total antioxidant capacity: The total antioxidant capacity of spinach was measured by the 2, 2-diphenyl- 1-picrylhydrazyl hydrate (DPPH) method (Prasad et al., 2008). Fresh tissue (0.1 g) was mixed in 1.0 ml MeOH and shacked for 2 hours, then centrifuged at 12000 g for 30 min. The supernatant (0.5 ml) was added to the 2.8 ml DPPH solution (0.1 mM) and incubated for 30 mat room temperature. Absorbance was read at 517 nm. The control sample was 2.8 ml DPPH, with the addition of 0.5 ml MeOH. The scavenging activity was determined by the following equation (Stojichevich et al., 2008): Inhibition % = (A control – A sample / A control) × 100 A: Absorption at 517 nm Total flavonoid content: 0.30 ml of the extract was mixed with 0.50 ml of NaNO2 (5 %). After that, Fig 1: Changes in temperature and day length in spinach spring cultivation Acta agriculturae Slovenica, 118/2 – 20224 R. ABOLGHASEMI et al. AlCl3.6H2O (10 %), 0.4 ml NaOH, and 0.20 ml were added. Then, its absorption was performed at 510 nm with a spectrophotometer (U-2100, JASCO, Japan) (Krizek et al., 1998). Total phenolic content: The Folin–Ciocalteu meth- od measured total phenolic content. We were using a spectrophotometer (U-2100, JASCO, Japan) at 765 nm. The standard curve was plotted with the Gallic acid so- lution (Raven, 2003). Vitamin E content: 0.2 ml of alcoholic extract mixed with 5.0 ml toluene, 1.0 ml of ferric chloride, and 3.5 ml of 2, 2-Bipyridine. The mixture was diluted with 10 ml ethanol (95 %), and after 2 min, absorbance was recorded at 525 nm. The standard curve was plotted with vitamin E (Wang & Galletta, 2002). Vitamin C content: Fresh leaf (1.0 g) mixed with 50 ml meta-phosphoric acid (6 %), keeps in the dark for 45 min then centrifuged at 6000 g for 15 min, then 1.0 ml of extract was added to 9.0 ml dichlorophenol indophenol solution (DCPIP) (0.025 %). The next ab- sorbance was read at 515 nm. The standard curve was plotted with different vitamin C concentrations (Djioua et al., 2009). Total soluble carbohydrate content: Total soluble carbohydrate content is measured by the anthrone method (Mc-Cready et al., 1950). For this purpose, 2.0 ml of anthrone was added to the alcoholic extract of the samples. Then, samples were boiled for 2 minutes in the water bath. After cooling and creating the color phase, the absorbance was read at 625 nm. The standard curve was plotted with the glucose standard (Mc-Cready et al., 1950). Total amino acid content: The ninhydrin method was used to measure free amino acids. The ethanol ex- tract of powdered samples was mixed with 1.0 ml of ninhydrin; after boiling and cooling, absorption was read in 575 nm wavelength by spectrophotometer (U- 2100, JASCO, Japan). Calibrated curves were plotted with alanine: y = 0.0198x - 0.0025(r2 = 0.997) (Shih- wen et al., 2006). Crude fiber content: Dried leaves (1.0 g) boiled with 200 ml of H2SO4 for 30 min. The extract was fil- tered, washed with boiling water, the residue boiled with 200 ml NaOH for 30 min then filtered, the solids solid part removed from the filter and placed in a tarred crucible for drying and ashing. It has placed tarred cru- cible with residue in an oven set at 100 ˚C for 8 hours. Then weighted the crucible with fiber residue and cal- culated the residue mass by subtracting the empty cru- cible mass from the crucible and sample mass. Then, the crucible was placed with the dried residue in the oven (550 ˚C) for 4 hours for ashing. Finally, we weighted the crucible with the ash residue in the analytical balance and calculated the ash mass by subtracting the empty crucible mass from the crucible and ash mass. The per- centage of crude fiber was calculated using the follow- ing equation: % Crude fiber content = ((dried residue mass – ash mass) / sample mass) × 100 Nitrate concentration: Nitrate concentration was evaluated by Narayana & Sunil (2009). Briefly, water ex- traction (0.1 g fresh leaf mixed in deionized water) was placed in a boiling water bath for 30 min and centri- fuged at 4000 g for 30 min. Then, extraction was mixed with 0.8 ml salicylic acid (5 %) in concentrated sulfuric acid (95 %) and cooled at room temperature. Then, 19 ml of sodium hydroxide (NaOH, 2N) was added, and the absorbance at 410 nm was determined with a spec- trophotometer (U-2100, JASCO, Japan). The standard curve was plotted with different KNO3 concentration using following standard formula get from standard curve; y = 0.053x + 0.035 (r2 = 0.997). Nitrate reductase assay: The leaf sample (100 mg) was suspended in 5.0 ml of phosphate buffer (0.1 M), KNO3 (0.02M), and propanol (5 %). The solution was kept in the dark water bath (37 °C) for 30 min. The solution was treated with 1.0 ml of sulfanilamide (1 %) and N-1-naphthyl-ethylenediamine (0.02 %). After 15 min, the absorbance was measured at 540 nm with a spectrophotometer (U-2100, JASCO, Japan). The stand- ard curve was plotted with the KNO2 solution: y = 0.0049x + 0.0092 (r2 = 0.988) (Narayana & Sunil, 2009). Oxalic acid content: The dry sample (0.5 g) was mixed with 30 ml HCl (0.25N) and put in a water bath for 15 min. Then, the supernatant was filtered and mixed with 5.0 ml sulfuric acid (2 N) and 2.0 ml potassium permanganate (0.003 M). After 10 minutes, the absorb- ance was detected at 528 nm. y = 0.9126x - 0.0705 (r2 = 0.9327) and standard oxalic acid solution (1 mg ml-1) was prepared with distilled water. (AOAC, 1970). Ca and Fe content: The concentration of Ca and Fe shoot was measured by atomic absorption (Perkin Elmer, 3030, Netherland) after digesting for 12 h with 2.5 ml HCl (36 %) (Nolte, 2003). 2.4 ANALYSIS OF DATA The experiment was arranged in RCBD with three replicates. The morphological and biochemical ob- tained data were analyzed using analysis of variance (ANOVA) by SAS 9.4 comparison of means was per- formed using the least significant difference (LSD) test at a 0.05 level of probability. The correlation between morphological and biochemical traits was also tested Acta agriculturae Slovenica, 118/2 – 2022 5 Morphological, biochemical, and nutritional value of prickly and smooth fruit spinach using the least significant difference (LSD) test at p ≤ 0.05. 3 RESULTS AND DISCUSSION 3.1 QUALITATIVE TRAITS The qualitative characteristics of two accessions based on the spinach descriptor are shown in Table 1. Usually, smooth seeds of spinach are generally more favorable in the world. The ’Monatol’ accession has a smooth seed (presented in Fig 2, C). On the other hand, prickly seed accession (Fig 2, A) is less desirable; they are challenging to plant (Asadi & Hasandokht, 2007). It has been reported that the best seed type for mecha- nized planting of spinach was a smooth and round seed (Morelock & Correll, 2008). In this study, both acces- sions had a gray background in seed color (Table 1). It has been reported that accessions with petiole standing, leaf sheath standing, wrinkled, or slightly wrinkled, are suitable for mechanical harvesting (Mei et al., 2010). Accordingly, ’Varamin Prickly’ (prickly seed) accession is more suitable for mechanical har- vesting than ’Monatol’ (smooth seed) accession because of petiole and leaf form (Table 1). Prickly seed acces- sion has a feature of leaf standing, petiole standing, and low wrinkle, green leaf color appropriate, which can be used in breeding programs, mechanical harvesting, and genetic modification (Table 1). In the USA and other western countries, wrinkled leaves of spinach are more marketable than flat leaves (Kuwahara et al., 2014). In confirmation of this report, smooth seed accession had wrinkle leaves (Fig 2, C, and D). The smooth seed accession has wrinkled dark green leaves and is more suitable for storage because the ventilation is better but hardly washable (Fig 2, D). Usually, spinach cultivars with lower leaf wrinkles have less nitrate content, and there is a direct correlation between leaf wrinkle and nitrate content (Arshi, 2000). So, no-wrinkle leaves as a desirable attribute are inter- ested in researchers. While in Iran, the most favorable spinach has a large, fleshy, thick, low leaf wrinkle and juicy leaf (Fig 2, B), and according to Table 1, the en- demic accession with prickly seed has these features. In Iran, the optimal form of spinach leaf is round, and overseas desirable triangular shapes have been reported (Kunicki et al., 2010). The seed type did not affect leaf shape (Table 1). Leaf color is critical in leafy vegeta- bles since green pigments are desirable and marketable for fresh and frozen spinach (Eftekhari et al., 2010). In this study, both accessions had a complimentary green color, although the foreign accession with smooth seed Se ed ty pe Se ed co lo r Pe tio le at tit ud e W ri nk le s of le af Le af th ic kn es s Le af Sh ea th at tit ud e Le af sh ap e Le af co lo r Le af Sh ea th Lo ba tio n of le af ti p Th e sh ap e of le af ti p W av e m ar gi n of le af Pr ic kl y se ed G ra y- ye llo w Er ec t Ve ry Lo w Th ic k Er ec t Br oa d ov al D ar k gr ee n C on ca ve Be nd C ir cu la r Ye s Sm oo th se ed G ra y- ye llo w H or iz on ta l H ig h Ve ry th ic k H or iz on ta l Br oa d ov al Ve ry da rk g re en C on ca ve U pw ar d C ir cu la r N o Ta bl e 1: Q ua lit at iv e m or ph ol og ic al fe at ur es o f p ri ck ly a nd sm oo th sp in ac h ac ce ss io ns (V ar am in P ri ck ly a nd M on at ol ) a cc or di ng to sp in ac h de sc ri pt or (A ri f e t a l., 2 01 3; Ja fa ri an d Ja la li 20 17 ) Acta agriculturae Slovenica, 118/2 – 20226 R. ABOLGHASEMI et al. was darker in color (Table 1, Fig 2). The leaf shape, leaf sheath, and leaf tip shape were similar in both acces- sions. In general, although the type of seed did not af- fect most of the quality characteristics of spinach in this study, the prickly seed type of spinach was much more desirable for mechanical harvesting than the smooth seed due to the attitude of the petiole and leaf sheath (Table 1). Researchers have reported that prickly seed spinach has better growth in appearance characteristics (Wu et al., 2015; Amoli, 2012) that can be observed in mechanical harvesting for the prickly seed (Table 1). 3.2 QUANTITATIVE TRAITS Analysis of variance showed a significant dif- ference between the two accessions in dry mass, 1000-grain mass, germination percentage, male and fe- male plants at 1 % probability level, and 5 % for petiole length (data was not presented). Mean comparison in Table 2 showed no significant difference in leaf num- ber, plant height, leaf length and width, petiole diam- eter, yield, fresh mass, leaf area, and day to flowering between prickly and smooth seed accessions. There is not much similarity between the investigations that can be reported in each region according to different spin- ach weather and growth conditions. In this study, peti- ole length was more than 100 % higher in the prickly seed accession (Iranian accession) than in the smooth accession, indicating that the plant’s form was more sta- ble in the prickly seed than the smooth seed accession and more suitable for mechanical harvesting (Table 2). Confirming this, it has been reported that plant shape and petiole length are crucial for mechanical harvesting (Shi et al., 2016). Dry mass was the highest in prickly seed accession (Table 2). So, the highest amount of dry mass (14.77 g) was observed in ’Varamin Prickly’ ac- cession (Table 2). yield, fresh and dry mass of ’Varamin Prickly’ was more suitable (Asadi & Hasandokht, 2007; Eftekhari et al., 2010; Jafari & Jalali, 2015). One of the desirable traits for spinach processing and packaging is dry mass, which directly relates to the smoothness of the leaf spinach (Arshi, 2000; Eftekhari et al., 2010). It can also be stated that the different water content in the tissues of these two accessions leads to the dif- ference in dry mass. In Asadi & Hasandokht’s (2007) study, the highest dry mass in ’Qom’ accession was re- ported with prickly seed. The mass of 1000-grains of prickly seed accession was 16.81 g, which had bigger seeds than the smooth seed accession 48.3 % more than the 1000-grain mass of smooth seed accession (Table 2). According to our results, Eftekhari et al. (2010) re- ported that the 1000-grain mass of prickly seed acces- sions was higher than that of smooth seed accession. Prickly seeds appear larger in appearance than smooth ones. The percentage of seed germination of smooth seed accession was higher than the prickly seed acces- sion (showed a 52.3 % increase in germination). There was a significant difference between the two accessions (Table 2), which may be due to more water absorption of smaller seeds during germination. Although prickly seeds are high in mass, it is hard to say that prickly seeds have a much larger surface area than smooth seeds. It is believed that the small seed varieties have higher wa- ter absorption capacity and better establishment due to the higher surface area to volume ratio (Zaferaniye, 2015). Observations on the germination process of spinach seeds have shown that genotypes with better germination had better vegetative growth and yield. (Jaliliyan, 2009; Zaferaniye, 2015). However, this study’s performance was not statistically significant (Table 2). Spinach is a leafy vegetable, so longer vegetative growth is desirable. It is reported that the late-flowering varie- ties are a priority to increase the spinach production period (Jaliliyan, 2009). In this regard, this study classi- fied prickly and smooth seed accessions as spring late flowering (near to 70 days) (Table 2). The researchers reported that the late-flowering accessions were mostly economically desirable (Asadi & Hasandokht, 2007). Therefore, spinach with better vegetative growth, yield, and appearance is more favorable to farmers. It should Fig 2: Comparison of ’Varamin Prickly’ spinach seed (A); leaf (B) and ’Monatol’ spinach seed (C); leaf (D) Acta agriculturae Slovenica, 118/2 – 2022 7 Morphological, biochemical, and nutritional value of prickly and smooth fruit spinach be noted that the result of two traits of yield and day to the flowering of spinach in two different seasons (spring and autumn) can be different because these two traits are affected by the type of growing season (Asadi & Hasandokht, 2007). In this study, both Iranian and foreign accessions (the prickly and smooth seed) were similar in day to flowering and yield (Table 2)). In con- firmation of various reports of spinach being dioecious , both of the accessions were male or female in this study as well (Morelock & Corell, 2008). As can be seen in table 2, the smooth seed accession had significantly more female plants than the Iranian prickly seed acces- sion. So smooth seed accession showed 93.5 % more female plants (Table 2). According to studies, spring spinach cultivation is valuable if the plant can flower later with female flowers (Zaferaniye, 2015). Jaliliyan (2009) recommends planting female varieties for eco- nomic yield. As a result, the smooth seed accession in this study confirms this statement. It has been reported that prickly seeds in winter and spring conditions usu- ally have better growth than smooth seeds (Wu et al., 2015). It can be concluded that prickly seed accession was similar to the smooth seed accession in many traits in spring cultivation. According to the spinach descriptor coding (Table 5), prickly seed accession showed higher valuable traits (a score of 33) in yield. Smooth seed accession has more scores in the mechanical harvesting traits (rated 31), indicating the superiority of mechanical harvesting. According to qualitative characteristics (Table 1), these results are related to more standing leaves and petiole in the Iranian accession, than in the foreign accession. Our results confirm previous reports on favorable veg- etative and mechanical harvesting characteristics for prickly seed accessions (Asadi & Hasandokht, 2007; Eftekhari et al., 2010; Jafari & Jalali, 2015). In the cat- egory of breeding traits, smooth seed accession (with a score of 13) was better than the Iranian accession (with a score of 12), which indicates that the foreign accession is better for breeding traits (Table 5). However, it seems that prickly seed accession should also be considered in terms of breeding characteristics due to near score according to the descriptor scale to smooth seed acces- sion (Table 5). 3.3 BIOCHEMICAL TRAITS Currently, many domestic and wild spinach acces- sions in the country may have more favorable biochem- ical characteristics than foreign accessions. Identifying these genotypes and crossing them improved cultivars with good biochemical characteristics (Sabaghnia et Fe m al e pl an t (% ) M al e pl an t (% ) D ay s t o flo w er in g G er m i- na tio n pe rc en t- ag e (% ) 10 00 -g ra in m as s (g ) Le af a re a (m m 2 ) D ry m as s (g ) Fr es h m as s (g ) Yi el d (k g h- 1 ) Pe tio le di am et er -( m m ) Pe tio le le ng th (c m ) Le af w id th (c m ) Le af le ng th (c m ) Pl an t he ig ht (c m ) Le af nu m be r Se ed ty pe 49 .5 9b 51 .8 6a 69 .6 6a 64 .7 1b 16 .8 1a 48 01 5a 14 .7 7a 60 .0 2a 30 86 7a 4. 35 a 12 .6 6a 7. 33 a 13 .7 3a 25 .0 0a 15 .6 6a Pr ic kl y se ed 95 .9 6a 3. 82 b 69 .0 0a 98 .5 8a 11 .3 3b 60 08 5a 8. 63 b 81 .7 7a 42 05 6a 4. 75 a 6. 20 b 9. 06 a 12 .0 0a 19 .6 6a 20 .2 0a Sm oo th s ee d 11 .2 0. 44 3. 16 13 .2 0. 04 57 66 8 1. 14 25 9. 2 68 59 5 0. 26 2. 84 1. 28 5. 24 8. 48 28 .4 0 St an da rd E rr or Ta bl e 2: M or ph ol og ic al tr ai ts o f p ri ck ly a nd sm oo th s ee d sp in ac h ac ce ss io ns D iff er en t l et te rs w ith in th e sa m e co lu m n in di ca te si gn ifi ca nt d iff er en ce s o f e ac h ty pe a t p ≤ 0 .0 5 by th e LS D te st Acta agriculturae Slovenica, 118/2 – 20228 R. ABOLGHASEMI et al. al., 2014). Results of the analysis of variance showed that there were differences between DPPH, flavonoids, amino acid, and iron content in a 1 % probability level and 5 % probability level in phenol, carbohydrate, and fiber content. No significant differences were observed in some biochemical traits such as photosynthetic pigments, vitamins E, C, nitrate and nitrate reductase activity, and the amount of oxalic acid. (data was not presented). There were significant differences between DPPH, flavonoids, and phenolic compounds they are increased in prickly Iranian accession (69.38 %, 0.76 %, and 79.55 mg 100 g-1 fresh mass, respectively, Table 3). Phenol and antioxidant properties of every region depend on many parameters such as climate, soil, altitude, and different species of plants (Mirzaei et al., 2010). Phenolic and an- tioxidant compounds of Iranian accessions ’Saleh-abad’ and ’Langrood’ had the highest amount of antioxidant compounds, and both of them had prickly seeds (Yosefi et al., 2010). Accordingly, the antioxidant capacity of prickly seed accession was 6.5 % higher than smooth seed accession (Table 3). Plants are one of the im- portant sources of antioxidants compounds that can protect cells from oxidative damage. Secondary plant metabolites such as total phenols and flavonoids de- rived from plants have strong potential for free radical sweeping in different parts of the plant, such as spinach leaves. Iranian spinach is rich in phenolic compounds with antioxidant properties (Chan et al., 2009). Spinach leaves contain active components of fla- vonoids, high antioxidants, and wide pharmacological and biochemical applications, including anti-allergic, anti-viral, and anti-cancer (Lamnitski et al., 2003; Berg- man et al., 2001). In the present study, the content of flavonoids in prickly seed accession was 67.1 % higher than in smooth seed varieties (Table 3). Flavonoids are one of the most widespread and diverse natural com- pounds that can absorb free radicals like other phenolic compounds. In oxidative stresses, phenolic compounds, especially flavonoids, can interact with membrane phospholipids through hydrogen bonding to the polar heads of phospholipids, thereby contributing to mem- brane integrity (Mirzaei et al., 2010). Under non-stress conditions, there were differences in antioxidant, fla- vonoid, and total phenolic activity in different spinach varieties (Barbarin et al., 2005). Soluble carbohydrates are nutrient chemicals valu- able to humans and a source of plant energy. Carbohy- drates of spinach are very important (Bavec et al., 2010). The highest amount of carbohydrate was observed in prickly seed accession with 0.64 mg g-1 fresh mass of spinach (Table 3). Hagen et al. (2009) reported that the carbohydrate content of leafy vegetables varied among the different accessions under the same growth condi- tions. The content of soluble sugars has been affected by pre-harvest growth temperature (Steindal et al., 2015). Amino acids are involved in the structure of spin- ach protein, and amino acids make up about 30 % of all spinach dry matter (Lisiewska et al., 2011). Few arti- cles have been published investigating factors affecting spinach amino acid content (Trejo-Tellez et al., 2005). The level of amino acids in spinach has been correlated with the amount of nitrogen content (Trejo-Tellez et al., 2005). Although nitrogen was not used as fertilizer in the present study, amino acid content was higher in Iranian prickly seed accession (Table 3) (66.6 %) than in other cultivars (Table 3). The studied accessions had significant differences in total fiber content. Prickly Iranian accession showed the highest fiber content (2.03 %) (Table 3). One of the causes of vegetable consumption is fiber. In this opin- ion, the studied Iranian spinach has superiority over the foreign accession in fiber content was more suggested for consumption as a diet food (Erfani et al., 2006). In confirming Iranian spinach accession showed 27.6 % more crude fiber content (Table 3). Spinach is one of the most significant nitrate accumulators because it has a very efficient absorp- tion system and an inefficient nitrate recovery system (Cantliffe, 2005). According to the results of the analysis of variance, nitrate accumulation and nitrate reductase activity in prickly and smooth seed accessions were not significant (data was not presented). It has been report- ed that there is a difference in nitrate accumulation only in smooth and wrinkled leaves (Arshi, 2000). Although the difference in leaf wrinkling between the two acces- sions was obvious, their leaves were not sufficiently dif- ferent in this trait. Probably, nitrate accumulation was not statistically significant (Table 3). Nitrate accumu- lation in vegetables is also reported to be affected by cultivars and even specific genotypic differences such as ploidy levels (Alamian et al., 2014). The studied accessions had significant differences in Fe content (Fig 3). Zaferaniye’s (2015) study reported that the Iranian accessions have higher iron content than the foreign accessions. In the present study, the Iranian spinach accession had higher Fe content, which was 62.1 % more than the foreign accession (Fig 3). Also, the concentration of iron in the Iranian and for- eign accessions of spinach varied from 30 to 50 mg g-1, which was consistent with the amount of iron in this study (Fig 3). Prickly accession was superior in mor- phological traits in most studies (Asadi & Hasandokht, 2007), and the present study also showed the superi- ority of nutraceutical traits in prickly seed accession. In the nutraceutical category, the superiority of the Acta agriculturae Slovenica, 118/2 – 2022 9 Morphological, biochemical, and nutritional value of prickly and smooth fruit spinach prickly seed accession over the smooth seed accession was quite evident (Table 5). Therefore, it is concluded that prickly seed accession had significant antioxidants, flavonoids, total phenols, carbohydrates, amino acids, fibers, and iron content, which was higher than the for- eign accession of smooth seed. Following the results of other researchers, Iranian accessions, including ’Vara- min Prickly’ have a significant superiority in terms of nutritional and functional traits compared to imported foreign accessions, which can be emphasized in the se- lection and improvement of Iranian spinach accessions (Erfani et al., 2006; Zaferaniye, 2015). 3.4 CORRELATION AMONG THE MORPHOLOG- ICAL AND BIOCHEMICAL TRAITS Morphological characteristics of leaves in spring conditions were impressed, as a positive correlation was observed between leaf length and leaf width (r = 0.79*) (Table 4). Similar to our results, Eftekhari et al. (2010) reported a significant correlation between leaf length and width in spinach accessions. Plant height was positively but not significantly correlated with petiole length (r = 0.74), indicating that increasing petiole length was desirable for mechanized harvesting (Table 4). Leaf number is one of the most important yield components in spinach (Jafari & Jalali, 2015), and as shown in table 4, this trait had a significant correla- tion with yield (r = 0.85*). Also, a significant positive relationship was observed between fresh and dry mass (r = 0.86*). There was also a positive relationship be- tween iron content, leaf area (r = 0.79*) and dry mass (r = 0.89**). A good correlation was observed between female plants and the amount of antioxidants’ activity (r = 0.81*), flavonoids (r = 0.96**) and total phenols con- tent (r = 0.61) (Table 4). Also, there was a significant correlation between the percentage of the female plant Se ed ty pe C hl or op hy ll (m g g F M -1 ) C ar ot en oi d (m g g F M -1 ) D PP H (% ) Fl av on oi d (% ) Ph en ol (m g/ 10 0 g F M -1 ) V it E (m g 10 0 g F M -1 ) V it C (m g 10 0 g F M -1 ) C ar bo hy dr at e (m g g F M -1 ) A m in o ac id (μ g g F M -1 ) Fi be r (% ) N itr at e (m g N O 3 g- 1 F M ) N itr at e R ed uc ta se (μ gr N O 2- g F M -1 h ) O xa lic A ci d (m g g- 1 F M ) Pr ic kl y se ed 7. 27 a 1. 65 a 69 .3 8a 0. 76 a 79 .5 5a 1. 04 a 20 .5 4a 0. 64 a 0. 05 a 2. 03 a 10 6. 2a 29 .1 1a 2. 09 a Sm oo th s ee d 6. 1a 1. 43 a 65 .1 1b 0. 25 b 78 .7 5b 0. 76 a 19 .4 9a 0. 36 b 0. 03 b 1. 59 b 10 7. 2a 38 .0 3a 3. 02 a St an da rd E rr or 0. 63 0. 02 5 2. 25 0. 00 3 29 .8 0. 02 5 1. 52 0. 00 7 0. 00 5 0. 04 1 46 5. 3 24 0. 8 0. 31 Ta bl e 3: B io ch em ic al tr ai ts o f p ri ck ly a nd sm oo th s ee d sp in ac h ac ce ss io ns D iff er en t l et te rs w ith in th e sa m e co lu m n in di ca te si gn ifi ca nt d iff er en ce s o f e ac h ty pe a t p ≤ 0 .0 5 by th e LS D te st Fig 3: The mineral concentration of prickly and smooth seed spinach accessions Acta agriculturae Slovenica, 118/2 – 202210 R. ABOLGHASEMI et al. Ta bl e 4: C or re la tio n co effi ci en ts 3 0 m or ph ol og ic al a nd b io ch em ic al tr ai ts st ud ie d on p ri ck ly a nd sm oo th s ee d sp in ac h ac ce ss io ns ** in di ca te s s ig ni fic an t c or re la tio ns a t t he le ve l o f p < 0 .0 1; * in di ca te s s ig ni fic an t c or re la tio n at th e le ve l o f p < 0. 05 . L .N : L ea f n um be r; P. H : P la nt h ei gh t;L .L : L ea f l en gt h; L .W : L ea f w id th : P .L : P et io le le ng th ; P .D : P et io le d ia m et er , Y : Y ie ld ; F .M : F re sh m as s; D .M : D ry m as s; L. A : L ea f a re a; S .W : 1 00 0- gr ai n m as s; G .P : G er m in at io n pe rc en ta ge ; D .F : D ay to fl ow er in g; M : M al e; F : F em al e; C h: C hl or o- ph yl l; C ar : C ar ot en oi ds ; F la v: F la vo no id s; Ph : P he no ls ; C ar b: C ar bo hy dr at es ; A .A : A m in o ac id s; Fi : F ib er ; N it: N itr at e; N itR D : N itr at e re du ct as e; O A : O xa lic a ci d Se ed ty pe N ut ra ce ut ic al a tt ri bu te s To ta l gr ad e C hl or op hy lls C ar ot en oi ds D PP H Fl av on oi ds Ph en ol s V it E V it C C ar bo hy dr at es A m in o ac id s Fi be r N itr at e N itr at e re du ct as e O xa lic ac id C a Fe Pr ic kl y se ed 3 2 4 4 3 3 4 4 4 5 2 1 3 3 4 48 Sm oo th s ee d 2 1 3 2 3 2 3 3 2 2 1 2 2 4 2 34 Yi el d an d m ec ha ni ca l h ar ve st in g at tr ib ut es Le af nu m be r Pl an t he ig ht Le af le ng th Le af w id th Pe tio le le ng th Pe tio le d ia m et er Yi el d Fr es h m as s D ry m as s Le af a re a G er m in at io n Pr ic kl y se ed 3 4 4 1 3 2 3 3 4 3 3 33 Sm oo th s ee d 4 2 2 2 1 2 4 4 1 4 5 31 Br ee di ng fe at ur es Se ed ty pe Se ed w ei gh t D ay to fl ow er in g M al e Fe m al e Pr ic kl y se ed 2 4 2 2 2 12 Sm oo th s ee d 1 2 2 4 4 13 Ta bl e 5: D es cr ip tiv e st at is tic s o f t he m ea su re d tr ai ts in sm oo th a nd p ri ck ly s ee d of sp in ac h ac ce ss io ns (S ab ag hn ia e t a l., 2 01 4; Ja fa ri a nd Ja la li, 2 01 5) † (1 = L ow p oi nt ; 2 = M ed iu m p oi nt ; 3 = H ig h po in t; 4 = ve ry h ig h po in t) Acta agriculturae Slovenica, 118/2 – 2022 11 Morphological, biochemical, and nutritional value of prickly and smooth fruit spinach with antioxidant properties and leaf iron content (Table 4). In confirmation of our results, Asadi & Hasandokht (2007) reported that female plants of spinach had more leafy, yield, and antioxidant properties. A significant correlation was observed between chlorophyll and carotenoid (r = 0.90*). Carotenoid has been reported to have a protective effect on chlorophyll (Macfarland & Burchett, 2001). A positive correlation was observed between ni- trate and petiole length (r = 0.63). However, this rela- tionship was not significant but consistent with other researchers’ reports about the direct relationship be- tween nitrate accumulation and petiole length (Asadi & Hasandokht, 2007; Jafari & Jalali, 2015). The correlation in Table 4 shows that vitamin C has a negative relationship with nitrate (r = -0.41) and a positive relationship with oxalic acid (r = 0.85*). In- creasing nitrate reduces vitamin C in spinach leaves. No significant negative relationship was observed between oxalic acid and nitrate (r = -0.50). According to studies, different results have been reported on nitrate and oxa- late relation; (Kaminishi & Kita, 2006) reported a nega- tive relationship between nitrate and oxalate in spin- ach. Koh et al. (2012) and Zhang et al. (2005) reported a positive correlation between these traits. These reports may indicate a complex pathway involved in nitrate and oxalate metabolism. Fiber content was positively correlated with female plant percentage (r = 0.90*) and negatively with male plant percentage (r = -0.78). It seems that in addition to photosynthetic pigments (Asadi & Hasandokht, 2007), the fiber content will also increase in the female bushes. 4 CONCLUSION It can be concluded that although the foreign smooth seed accession was better in breeding charac- teristics such as 1000-grain mass, germination percent- age, leaf thickness, and female plant %, ’Varamin Prick- ly’ (prickly seed) accession was significantly superior to nutraceutical traits such as antioxidant activity, total phenols, fibers, and iron content. Therefore, to enhance the quality traits in new spinach varieties, ’Varamin Prickly’ (prickly seed accession) is suitable for transfer- ring these traits and breeding purposes. These features may help obtain more information on the qualitative characteristics of Iranian spinach accessions or clarify the factors that influence the nutraceutical properties of Iranian accessions. Therefore, desirable Iranian ac- cessions for nutraceutical and morphological traits can be used in breeding programs for spinach cultivar pro- duction. 5 AUTHOR CONTRIBUTION STATE- MENTS Each named author has contributed to conducting the underlying research and drafting this manuscropt. 6 ACKNOWLEDGMENTS We thank Isfahan University of technology and the Iran National Science Foundation for supporting this work. 7 COMPLIANCE WITH ETHICAL STAND- ARDS (E.G., CONFLICT OF INTEREST) To the best of our knowledge, the named authors have no conflict of interest, financial or otherwise. 8 REFERENCE Abolghasemi, R., Haghighi, M., Etemadi, N., Soorni, A., & Jafari, P. (2019). Screening of some native and foreign ac- cessions of spinach for spring culture in Isfahan. Iran Ag- ricultural Research, 38(1), 87-99. Alamian, M., Eftekhari, S. A., Heidari, M., & Alamzadeh An- sari, N. (2014). Evaluation of nitrate accumulation and nitrate reductase activity in different vegetative growth of selected Iranian land races of Spinach (Spinacia oleracea L.). Journal of Crop Production, 3, 25-36. (In Persian). AOAC. (1970). Official methods of analysis. In W. Horwitz (Eds.), Association of official analythical chemists, 12th edn. Washington DC, USA. Arif, M., Jatoi, S. A., Rafique, T., & Ghafoor, A. (2013). Genetic divergence in indigenous spinach genetic resources for agronomic performance and implication of multivariate analyses for future selection criteria science. Development and Technology, 32, 7-15. Arshi, Y. (2000). Genetic improvement of vegetable crops. Mash- had Jahad Daneshgahi, pp. 725. Asadi, H., & Hasandokht, M. J. (2007). Study of genetic diver- sity on Iranian spinach genotypes. Journal of Agricultural Science, 38, 257-265. (In persian). Avsar, B. (2011). Genetic diversity of Turkish spinach cultivars. A master dissertation graduate school of engineering and sciences, Izmir Institute of Technology, Turkey, pp. 256. Balalić, I., Miklić, V., Jocić, S., Marinković, R., Cvejić, S., Hlad- ni, N., & Miladinović, D. (2012). Evaluation of NS sun- flower hybrids in small-plot trials via hybrid location in- teraction. Ratarstvo I Povrtarstvo, 49, 270-281. https://doi. org/10.5937/ratpov49-1772 Barbarin, A. M., Williams, J., Bettman, G., Hauber, D., & Rat- nayaka, H. (2005). Gas exchange and antioxvdant prop- erties of two spinach cultivars of contrasting morphology. Acta agriculturae Slovenica, 118/2 – 202212 R. ABOLGHASEMI et al. 102nd Annual International Conference of the American Society for Horticultural Science, Las Vegas, Vendal. Bavec, M., Turinek, M., Grobelnik-Mlakar, S., Slatnar, A., & Bavec, F. (2010). Influence of industrial and alternative farming systems on contents of sugars, organic acids, total phenolic content, and the antioxidant activity of red beet (Beta vulgaris L. ssp. vulgaris ‘Rote Kugel’). Journal of Agri- cultural and Food Chemistry, 58(22), 11825-11831. https:// doi.org/10.1021/jf103085p Bergquist, S. A., Gertsson, U. E., Nordmark, L. Y., & Olsson, M. E. (2007). Ascorbic acid, carotenoids, and visual quality of baby spinach as affected by shade netting and posthar- vest storage. Journal of Agricultural and Food Chemistry, 55(21), 8444-8451. https://doi.org/10.1021/jf070396z Cantliffe, D. J. (2005). Nitrate accumulation in table beets and spinach as affected by nitrogen, phosphorous and potas- sium nutrition and light intensity. Agronomy Journal, 65, 563-565. https://doi.org/10.2134/agronj1973.0002196200 6500040012x Chan, S. W., Lee, C. Y., Yap, C. F., Wan Aida, W. M., & Ho, C. W. (2009). Optimization of extraction conditions for phe- nolic compounds from Limaupurut (Citrus hystrix) peels. International Food Research Journal, 16, 203-213. Djioua, T., Charles, F., Lopez-Lauri, F., Filgueiras, H., Coudret, A., Freire, Jr. M., Ducamp-Collin, M. N., & Sallanon, H. (2009). Improving the storage of minimally processed mangoes (Mangifera indica L.) by hot water treatments. Postharvest Biology and Technology, 52, 221-226. https:// doi.org/10.1016/j.postharvbio.2008.10.006 Eftekhari, S. A. A., Hasandokht, M. R., Fatahi-Moghadam, M. R., & Kashi, A. (2010). Genetic diversity of some Iranian spinach (Spinacia oleracea L.) landraces using morpho- logical traits. Iranian Journal of Horticultural Sciences, 41, 83-93. (In persian). Erfani, F., Hasandokht, M. R., Barzegar, M., & Jabari, A. (2006). Determination and comparison of some nutrient in seven Iranian spinach cultivar. Iranian Journal of Food Science, 3(2), 27-33. Gorain, M., Sharma, N., Sharma, A., Kumar Meena, A., Sin- gh, S., Srivastava, K., & and Ahmed, S. I. (2014). Spatial prototype and phenotypes of variation in populations of prickly acacia in semi-arid regions in India. International Journal of Agricultural Science Research, 3(7), 112-120. Hagen, S. F., Borge, G. I. A., Solhaug, K. A., & Bengtsson, G. B. (2009). Effect of cold storage and harvest date on bio- active compounds in curly kale (Brassica oleracea L. var. acephala). Postharvest Biology and Technology, 51(1), 36- 42. https://doi.org/10.1016/j.postharvbio.2008.04.001 Ikic, I., Maričević, M., Tomasović, S., Gunjača, J., Satović, Z., Sarčević, H. (2012). The effect of germination temperature on seed dormancy in Croatian-grown winter wheats. Eu- phytica, 3(1), 25-34. https://doi.org/10.1007/s10681-012- 0735-8 IPK. (2018). Institute of Plant Genetics and Crop Plant Re- search. GBIS. http://gbis.ipk-gatersleben.de/gbis_i/home. jsf. Jafari, P., & Jalali, A. H. (2017). The effect of planting date on yield and nitrate accumulation in spinach (Spinacia olera- cea L.) In Isfahan weather conditions. Journal of Horticul- tural Science, 31(3), 533-542. (In persian). Jafari, P., & Jalali, A. H. (2015). Yield and nitrate accumulation comparison in five Iranian spinach landraces in Isfahan province. Journal of Crop Production, 13, 217-225. (In per- sian). Jaliliyan, J. (2009). Effect of plant density and supplemental ir- rigation on yield, yield components and protein content of four spinach cultivars in dry land conditions. Master of Sci- ence Degree in Agriculture, West Islamabad University. Jiang, A. P., Liu, J., & Jiang, J. M. (2016). Relationships of leaf color and pigment and nutrient elements in senescing leaves of Sassafras tsumu. For Research, 29, 362-368. (In Chinese). Kaminishi, A., & Kita, N. (2006). Seasonal change of nitrate and oxalate concentration in relation to the growth rate of spinach cultivars. Horticultural Science, 41, 1589-1595. https://doi.org/10.21273/HORTSCI.41.7.1589 Koh, E., Charoenprasert, S., & Mitchell, A. E. (2012). Effect of organic and conventional cropping systems on ascorbic acid, vitamin C, flavonoids, nitrate, and oxalate in 27 va- rieties of spinach (Spinacia oleracea L.). Journal of Agri- cultural and Food Chemistry, 60, 3144-3150. https://doi. org/10.1021/jf300051f Krizek, D. T., Britz, S. J., & Mirecki, R. M. (1998). Inhibitory ef- fect of ambient levels of solar UV-A and UV-B radiation on growth of CV. New red fire lettuce. Physiology Plant, 103, 1-7. https://doi.org/10.1034/j.1399-3054.1998.1030101.x Kunicki, E., Grabowska, A., Sękara, A., & Wojciechowska, R. J. F. H. (2010). The effect of cultivar type, time of cultivation, and biostimulant treatment on the yield of spinach (Spi- nacia oleracea L.). Folia Horticulturae, 22(2), 9-13. https:// doi.org/10.2478/fhort-2013-0153 Kuwahara, K., Suzuki, R., Ito, Y., Mikami, T., & Onodera, Y. (2014). An analysis of genetic differentiation and geo- graphical variation of spinach germplasm using SSR markers. Plant Genetic Resources, 12(2), 185-190. https:// doi.org/10.1017/S1479262113000464 Lamnitski, L., Bergman, M., Nyska, A., Ben-Shaul, V., & Gross- man, S. (2003). Composition, efficacy and safety of spin- ach extracts. Nutrition and Cancer, 46, 222-31. https://doi. org/10.1207/S15327914NC4602_16 Lichtenthaler, H. K. (1987). Chlorophylls and carotenoids: Pigments of photosynthetic biomembranes. Methods in Enzymology, 1(44), 350-382. https://doi.org/10.1016/0076- 6879(87)48036-1 Lisiewska, Z., Kmiecik, W., Gebczynski, P., & Sobczynska, L. (2011). Amino acid profile of raw and as eaten products of spinach (Spinacia oleracea L.). Food Chemistry, 126(2), 460-465. https://doi.org/10.1016/j.foodchem.2010.11.015 Liu, C. J., Lin, Q., & He, J. X. (2004). Methods and terminology of study on seed morphology from China. Acta Botanica Sinica, 24, 178-188. Mc-Cready, R. M., Silviera, V., & Owens, H. S. (1950). De- termination of starch and amylase in vegetables. Ana- lytical Chemistry, 22, 1156-1158. https://doi.org/10.1021/ ac60045a016 Mei, Y., Hou, L. P., Cui, Y. L., Chen, H. L., & Meng, S. C. (2010). Acta agriculturae Slovenica, 118/2 – 2022 13 Morphological, biochemical, and nutritional value of prickly and smooth fruit spinach Optimization and establishment of AFLP analyzing sys- tem in spinach (Spinacia oleracea L.). Acta Agriculturae Boreali Sinica, 25, 111-115. Meng, S., Liu, C., Xu, X. I., Song, S., Song, SH., Zhang, Z. H., & Liu, L. (2017). Comparison of morphological features of fruits and seeds for identifying two taxonomic varieties of Spinacia oleracea L. Canadian Journal of Plant Science, 98(2), 318-331. https://doi.org/10.1139/CJPS-2017-0119 Mirzaei, A., Akbartabar, M., Sadeghi, H., & Sharifi, B. (2010). The antioxidant activities and total phenolic of Artemi- sia martima, Achillea millefoliumand, Matricaria recutica. Journal Armaghan Danesh, 15(1), 243-252. Morelock, T. E., & Correll, J. C. (2008). Spinach. In J. Prohens & F. Nuez (Eds.), Vegetables I: Asteraceae, Brassicaceae, Che- nopodiaceae, and Cucurbitaceae (pp. 189-218). Springer, New York. Narayana, B., & Sunil, K. (2009). A spectrophotometric meth- od for the determination of nitrite and nitrate. Eurasian Journal of Analytical Chemistry, 4(2), 204-214. Nolte, J. (2003). ICP emission spectroscopy. Weinheim: Wiley- VCH Verlag GmbH and Co. KgaA, pp. 206-207. Prasad, P. V. V., Pisipati, S. R., Mutava, R. N., & Tuinstra, M. R. (2008). Sensitivity of grain sorghum to high temperature stress during reproductive development. Crop Science, 48, 1911-1917. https://doi.org/10.2135/cropsci2008.01.0036 Raven, J. A. (2003). Cyceing silicon: the role of accumula- tion in plants. New Phytologist, 3, 419-421. https://doi. org/10.1046/j.1469-8137.2003.00778.x Sabaghnia, N., Asadi-Gharneh, H. A., & Janmohammadi, M. (2014). Genetic diversity of spinach (Spinacia oleracea L.) landraces collected in Iran using some morphological traits. Acta Agriculturae Slovenica, 103(1), 101-111. https:// doi.org/10.14720/aas.2014.103.1.11 Shi, A., Mou, B., Evans, M., Clark, J. R., Motes, D., Correll, J., Xiong, H., Qin, J., Chitwood, J., & Weng, Y. (2016). Asso- ciation mapping of leaf traits in spinach (Spinacia oleracea L.). Plant Breeding, 135, 399-404. https://doi.org/10.1111/ pbr.12369 Shih-wen, S., Yi-cheng, L., Yih-ming, W., & Min-jane, C. (2006). Efficiency improvements on ninhydrin method for amino acid quantification. Journal of Food Composi- tion and Analysis, 19, 112-117. https://doi.org/10.1016/j. jfca.2005.04.006 Steindal, A. L. H., Rodven, R., Hansen, E., & Molmann, J. (2015). Effects of photoperiod, growth temperature and cold acclimatization on glucosinolates, sugars and fatty acids in kale. Food Chemistry, 174, 44-51. https://doi. org/10.1016/j.foodchem.2014.10.129 Stojičevič, S. S., Stanisavljević, I. V., Veličković, D. T., Veljković, V. B., & Lazić, M. L. (2008). Comparative of the antioxidant and antimicrobial activities of Sempervivum marmoreum L. extracts obtained by various extraction techniques. Journal of the Serbian Chemical Society, 73(6), 597-607. https://doi.org/10.2298/JSC0806597S Trejo-Tellez, L. I., Gomez-Merino, F. C., Rodriguea-Mendoza, M. N., & Alcantar-Gonzalez, G. (2005). Foliar fertilization with urea on the portioning of nitrogen in spinach. Jour- nal of Agricultural Science and Technology, 23, 495-503. Wang, S. Y., & Galletta, G. J. (2002). Compositional change in Colletotrichum (Anthracnose) infected strawberry fruit. Acta Horticulturae, 567, 815-819. https://doi.org/10.17660/ ActaHortic.1997.439.135 Wu, Y. N., Mei, Y., Cui, Y. L., Chen, H. L., & Meng, S. C. (2015). Morphological traits analysis of spinach (Spinacia olera- cea L.) germplasm resources. Journal of Henan Agricul- tural Science, 44, 119-124. Yosefi, Z., Tabaraki, R. H., Asadi Gharneh, A., & Mehrabi, A. A. (2010). Variation in antioxidant activity, total phenolic, and nitrate in spinach. International Jour- nal of Vegetable Science, 16(3), 233-242. https://doi. org/10.1080/19315260903577278 Zaferaniye, M. (2015). Evaluate vegetative and reproductive characteristics of indigenous and foreign rarities. Inter- national conference on applied research in agriculture, Mashhad, Iran, pp. 1-17. Zhang, Y., Lin, X., Zhang, Y., Zheng, S. J., & Du, S. (2005). Ef- fects of nitrogen levels and nitrate/ ammonium ratios on oxalate concentrations of different forms in edible parts of spinach. Journal of Plant Nutrition, 28, 2011-2025. https:// doi.org/10.1080/01904160500311086 Acta agriculturae Slovenica, 118/2, 1–12, Ljubljana 2022 doi:10.14720/aas.2022.118.2.2404 Original research article / izvirni znanstveni članek Studies of the impact of environmental conditions and varietal features of sweet cherry on the accumulation of vitamin C in fruits by using the regression analysis method Іryna IVANOVA 1, Мarina SERDYUK 2, Vira MALKINA 3, Tetiana TYMOSHCHUK 4, 5, Marharyta VOROVKA 6, Ivan MRYNSKYI 7, Anastasiia ADAMOVYCH 8 Received November 02, 2021; accepted April 03, 2022. Delo je prispelo 2. novembra 2021, sprejeto 3. aprila 2022 1 Department of Horticulture, Viticulture and Biochemistry, Faculty of Agrotechnology and Ecology, Dmytro Motornyi Tavria State Agrotechnological University, Melitopol, Ukraine 2 Department of Food Technology and Hotel and Restaurant Business, Faculty of Agrotechnology and Ecology, Dmytro Motornyi Tavria State Agrotechnological University, Melitopol, Ukraine 3 Department of Higher Mathematics and Physics, Faculty of Energy and Computer Technology, Dmytro Motornyi Tavria State Agrotechnological University, Melitopol, Ukraine 4 Department of Plant Protection, Faculty of Agronomy, Polissia National University, Zhytomyr, Ukraine 5 Corresponding author, e-mail: tat-niktim@ukr.net 6 Department of Pedagogy and Pedagogical Mastership, Bogdan Khmelnitsky Melitopol State Pedagogical University, Melitopol, Ukraine 7 Department of Botany and Plant Protection, Faculty of Agronomy, Kherson State Agrarian and Economic University, Kherson, Ukraine 8 Department of Social Sciences and Humanities, Dmytro Motornyi Tavria State Agrotechnological University, Melitopol, Ukraine Studies of the impact of environmental conditions and vari- etal features of sweet cherry on the accumulation of vitamin C in fruits by using the regression analysis method Abstract: The accumulation of vitamin C in sweet cher- ry fruits depends on the variety and environmental condi- tions. The aim of our research was to substantiate the rate of impact of weather factors as well as of varietal features on vitamin C accumulation in sweet cherry fruits. The variet- ies ʹKazkaʹ and ʹZabutaʹ, ʹKordiaʹ and ʹMirazhʹ were chosen as the best ones from among 33 varieties of early, medium and late term of ripening (7.31–10.67 mg 100 g-1) according to the average content of vitamin C in sweet cherry fruits. The stud- ies found that the environmental conditions of the research years had the largest impact on the vitamin C content in the fruits of late and early ripening varieties, and in the fruits of medium ripening variety the vitamin C amount depended on the varietal features. The practicability of forecasting of vitamin C content in sweet cherry fruits on the average in- dices for a group of early and late maturity varieties, but not separately for every pomological variety, has been proven. For the medium ripening variety this index can be forecasted within each pomological variety. The models of dependence of vitamins C accumulation on the impact of meteorological parameters were evaluated on the basis of the principle com- ponents analysis and the least square method. Key words: antioxidants; variety; terms of fruit ripening; vitamin C; weather conditions; principle components analysis Preučevanje vpliva vremenskih dejavnikov in lastnosti sort na vsebnost vitamin C v plodovih češenj z metodo regresij- ske analize Izvleček: Količina vitamin C v plodovih češenj je od- visna od sorte in okoljskih razmer. Namen te raziskave je bil ovrednotiti vpliv vremenskih dejavnikov in lastnosti sort na količino vitamina C v plodovih češenj. Izmed 33 zgodnjih, srednjih in poznih sort so bile izbrane najboljše sorte kot so ʹKazkaʹ,ʹZabutaʹ, ʹKordiaʹ in ʹMirazhʹ glede na poprečno vseb- nost vitamin C (7,31–10,67 mg 100 g-1) v plodovih. Raziskava je odkrila, da so imele na vsebnost vitamina C v plodovih zgo- dnjih in poznih sort češenj največji vpliv vremenske razmere posamezne rastne sezone, pri srednje dozorevajočih sortah pa so imele največji vpliv na vsebnost vitamina lastnosti sort. Dokazana je bila možnost napovedovanja vsebnosti vitamina C v plodovih zgodnjih in poznih sort na osnovi povprečnih indeksov, vendar ne za vsako sorto posebej. Za srednje do- zorevajoče sorte bi lahko uporabili ta indeks za vsako sorto posebej. Model za ugotavljanje odvisnosti kopičenja vitamina C v odvisnosti od vremenskih dejavnikov je bil ovrednoten na osnovi analize glavnih komponent in metode najmanjšega kvadrata. Ključne besede: antioksidanti; sorte; čas dozorevanja; vitamin C; vremenske razmere; analiza glavnih komponent Acta agriculturae Slovenica, 118/2 – 20222 I. IVANOVA et al. 1 INTRODUCTION Sensible nutrition of people is one of the main pri- orities of the state policy of most countries. The fruits are considered as an essential element of good nutri- tion, as they are the source of a vitamin complex. Vi- tamins belong to a group of indispensable nutrients of organic nature, which facilitate metabolism. They are scarcely synthesized in an organism, so they should be in taken with food. The amount of vitamin C in fruits (Bastos et al., 2015; Hayaloglu & Demir, 2015) is an im- portant characteristic of fruits for their consumption in raw condition, for processing and storing (Vasylyshy- na, 2018). One of the directions of solving the defined problem is to provide the population with fruits with a high amount of biologically active substances which are essential for people (Dhandevi & Jeewon, 2015). The sweet cherry fruits (Prunus avium L.) belong to the food stuff which contains digestible sugars, phe- nolic acids with a predominant amount of anthocya- nins, mineral substances and vitamins (Leong & Oey, 2012; Pissard et al., 2016; Ivanova et al., 2021a, 2021b, 2021c). Biologically active substances represented by phyto-nutrients and antioxidants provide antioxidant, anti-cancerogenic and anti-phlogogenic action on human organism (Ballistreri et al., 2013; Popescu et al., 2014; Legua et al., 2017). Due to this, sweet cherry fruits reveal some preventive effect against cardiovascular diseases, di- abetes, cancer, which is connected with an oxidative stress (He et al., 2007; Faniadis et al., 2008; Schmitz-Eiberger & Blanke, 2012). Sweet cherry fruits are characterized by a high amount of dietary medicative substances, which promote to organism functioning. Sweet cherry fruits contain water soluble (C, B) and fat soluble (A, E, K) vi- tamins (Antognoni et al., 2020). Vitamins take part in oxidation-reductions, respiration, nucleic acids forma- tion and amino-acids exchange, protein secretion as well as improve carbohydrate digestion. Besides, they control cholesterol metabolism, prevent the accumulation of harmful free radicals in body tissues, improve the resist- ance to infectious diseases as well as to unfavorable en- vironmental factors which cause overheating, excessive heat loss, hypoxia and improve person’s performance (Bastos et al., 2015). Vitamin C or L-ascorbic acid is one of the most important phyto-nutrients, which determines the biological value of sweet cherry fruits. Vitamin C be- longs to a group of water soluble vitamins (Antognoni et al., 2020). Vitamin C amount in sweet cherry fruits equals 7.26–10.78 mg 100 g-1 on the average. In living organ- isms, the ascorbic acid is an antioxidant, as it protects the organism against the oxidative stress and is a co-factor in essential vital enzyme reactions (Nowak et al., 2018; An- tognoni et al., 2020). The immunity protection and the maintaining of psychical processes in a proper condition are the most important functions of vitamin C. Vitamin C is one of the regulators of reductive-oxidative processes in living cells. Vitamin C scarcity results in metabolic disor- der in the whole organism (Prior, 2003). For a human an every-day need for vitamin C equals 50–100 mg. Vitamin C scarcity in a human diet can cause hypoavitaminosis and avitaminosis C, as this vitamin is not synthesized in an organism (Acero et al., 2019). Fresh fruits are the main source of vitamin C. This fact testifies to the expediency of prolonging the term of fruits consuming. Thus, it is im- portant to retain this valuable nutritive constituent under sweet cherry fruits keeping and processing (Correia et al., 2017). Palatability traits and biochemical composition of sweet cherry fruits depend on the genetic traits of the variety (Serrano et al., 2005; Faniadis et al., 2008; Cor- reia et al., 2020). The researchers have produced the va- rieties of stone fruits which meet modern requirements, but there are still some problems which are very topical (Correia et al., 2017; Grandi et al., 2017). The chemical composition of the fruits of any harvest, except varietal features, depend on the meteorological conditions of the growing period as well as of the zone of fruit growing (Hayaloglu & Demir, 2015; Luna-Vázquez et al., 2016). The researchers of the subtropical regions of Brazil estimated the chemical composition, identificated the biologically active compounds and estimated the antioxi- dant activity of berries and fruits including sweet cherries. It was established that the amount of ascorbic acid in the fruits of the subtropical regions of Brazil is much higher than in the zone with moderate climate (Rios de Souza et al., 2014). The formation of the biochemical composition of sweet cherry fruits as well as of their consumption value depend on the temperature, light intensity, fruit ripeness (Martini et al., 2017; Acero et al., 2019). The dependence of vitamin C amount on the meteorological indicators within the period from blooming to stone fruit ripening was studied by many scientists. A higher concentration of vitamin C in fruits was registered in the years with suf- ficient water availability (Lakatos et al., 2010, 2014). The level of ascorbic acid content in fruits is formed geneti- cally. But the weather factors during the growing period have great impact on the accumulation of vitamin C fund (Bieniek et al., 2011). The greatest amount of vitamin C is formed during the years with moderately warm and wet growing period. A sufficient fruits lightning is a very im- portant factor which affects the process of ascorbic acid synthesis (Kevers et al., 2011). On the basis of literature sources it can be stated that there is a strong correlation between vitamin C amount and the environmental con- ditions of the region of fruit growing. Under conditions of climate changes, stress abiotic factors have negative im- pact on the formation of vitamin C fund in sweet cherry Acta agriculturae Slovenica, 118/2 – 2022 3 Studies of the impact of environmental conditions and varietal features of sweet cherry on the accumulation of vitamin C in fruits ... fruits under conditions of a Southern Steppe sub-zone of Ukraine. Therefore, there is a necessity to study the pecu- liarities of vitamin C accumulation in sweet cherry fruits of different terms of ripening under the influence of stress weather factors in order to single out the most suitable varieties for fruit storing and processing. The aim of the study was to develop a model for vitamin C content forecasting in the fruits of early, me- dium and late terms of ripening depending on the envi- ronmental conditions. The received mathematic model is the basis for the forecasting of a test parameter of fruits quality in the regions with similar environmental conditions. The task of the research was to recommend the fruits of early, medium and late maturity varieties with a high amount of vitamin C for consuming fresh as well for fruit storing and processing. To achieve the aim, it is necessary to: - analyze the environmental conditions during the period of phenological stages of sweet cherry fruits growing and developing; - estimate the vitamin C amount in fruits on the stage of their economic maturity and choose the best varieties; - study the correlations between the accumulation of vitamin C and weather factors and to develop the mathematical models of their dependence; - estimate the rate of impact of each weather factor on the formation of vitamin C fund in test varieties of three groups. 2 MATERIALS AND METHODS The research was conducted during 2008–2019 in sweet cherry fruiteries in the Southern Steppe sub-zone of Ukraine. The region is characterized by insufficient wa- ter availability as to the amount of rainfalls. The climate is Atlantic-continental, dry with a high temperature regime. The dry winds are of Northeastern direction. According to the complex of climatic parameters, the test region is favorable for sweet cherry fruits growing (Table 1). The data of Melitopol meteorological station of the South of Ukraine (46° 49’N, 35° 22’E) were used for the calculation of the model of forecasting of vitamin C amount in sweet cherry fruits The research areas have black southern loamy soil, which was formed on the loesses. The agrochemical characteristic of soil is given in Table 2. The technology of sweet cherry growing in the ex- periment was standard for a given region. The scheme of trees planting in 2001 was – 5 × 3 m. The space between rows on the fruit plantation was kept under autumn fallow. All sweet cherry varieties on ‚Magaleb- ska‘ cherry rootstock were divided into three groups according to the term of ripening (early, medium and late): early term of ripening 7 varieties, medium term of ripening 13 varieties, late term of ripening 13 variet- ies. The fruits of an early ripening term were harvested in the third decade of June and in the first decade of July. The fruits of a medium ripening term were har- vested in the second decade of July. The fruits of a late ripening term were harvested in the third decade of July. To study the vitamin C amount, 100 fruits were chosen from 6 trees of each sweet cherry variety of the same age on the stage of heavy bearing with average intensity. There was a threefold frequency of variants in the experiment. The fruits of each variety were picked by hand from four different sides of the tree crown on Depth of arable layer, cm Humus content, % pH kCl Nutrient content, mg g-1 100 of soil N P2O5 K2O 40.0 1.38 6.9 27.0 90.0 154.0 Table 2: Agrochemical characteristics of topsoil of tested soil Readings Value Average annual air temperatures, °С 9.1–9.9 Average monthly air temperatures in the warmest months, С 20.5–23.1 Sum of active temperatures higher than 10 C from April to October, С 3316 Average amount of rainfalls per year, mm 475 Average annual relative air humidity, % 73 Average annual air velocity, m s–1 3 Hydrothermic coefficient 0.22–0.77 Table 1: Meteorological conditions of Southern Steppe sub-zone of Ukraine Acta agriculturae Slovenica, 118/2 – 20224 I. IVANOVA et al. the stage of economic maturity. After harvesting the fruits, they were weighed and counted (Serdyuk et al., 2020). The sweet cherry fruits were transported to the laboratory for 2–3 hours after their harvesting to esti- mate the tested parameter. During the period of sweet cherry fruits harvesting, their economic maturity was estimated by visual and organoleptic examination. The fruit pulp was rather firm, color and flavor were typical for each pomological variety. The sweet cherry fruits were harvested with a fruit-stalk. The fruits transpor- tation and storing were conducted under condition of preserving of the external appearance and flavor typi- cal for a variety. The restoring of Tilman’s reagent (2.6-dichlorphe- nol-indophenol) was taken as the basis of the technique on the evaluation of a mass fraction of vitamin C. The amount of ascorbic acid in the extracts (Serdyuk et al., 2020) was evaluated in terms of the amount of the reagent which was used for titration. The model of dependence of vitamin C amount in sweet cherry fruits on the weather factors was developed according to the schedule (Ivanova et al., 2021a, 2021b): 1. Evaluation of a mass fraction of ascorbic acid (AA). 2. Analysis of the weather factor during the years of the research. 3. Choosing the weather factor which show the cor- relation with vitamin C amount in fruits. 4. Developing the regression model of dependence of vitamin C amount in the fruits of sweet cherry varieties on the weather factor. 5. Evaluation and ranging of the rate of impact of each weather factor on the tested parameter of fruits quality. The statistical analysis was made using modern computer technologies DataMining – software environ- ment RStudio. 3 RESULTS AND DISCUSSION For the sweet cherry fruits of three groups of ripen- ing the average content of vitamin C equaled 8.17 mg 100 g-1 (Tables 3–5). The accumulation of vitamin C in the fruits of seven cultivars grown in Turkey was analyzed by Demir T. The average content of vitamin C in sweet cherry fruits varied between 4 and 7 g kg-1 of fresh mass (Demir, 2013). The ascorbate levels for 22 sweet cherry cultivars grown in southern of Italy was considerably lower – 0.034–0.260 g kg-1 of fresh mass (Matteo et al., 2016). The rate of accumulation of vitamin C fund in sweet cherry fruits of an early term of ripening equaled 7.10 mg 100 g-1, which is by 13.1 % lower as compared with the average index for three groups. In a group of an early term of ripening the varieties, ʹKazkaʹ (7.36 ± 1.40 %) and ́ Zabutaʹ (7.31 ± 1.49 %) had the largest vitamin C content on the average during the years of studies. In the fruits of ʹBigaro Burlatʹ variety vitamin C content was the lowest (6.84 ± 1.22 %). The fruits of ʹBigaro Burlatʹ (the early term ripening group) had the lowest content of vitamin C in 2018 – 5.02 mg 100 g–1, which is for 26.6 % lower than the average va- rietal parameters. The fruits of ʹMerchantʹ variety had a maximal accumulation of vitamin C (11.29 mg 100 g–1) in 2019, which is for 63.6 % higher as compared with the average varietal parameters. The highest content of vitamin C during the years of research was in ʹKazkaʹ and ʹZabutaʹ varieties, and the lowest – in ʹBigaro Burlatʹ variety (Table 3). The average content of vitamin C in sweet cherry Variety Vitamin C content, % Variation according to years, Vр, %average min max ʹBigaro Burlatʹ 6.84 ± 1.22 5.02 9.83 17.9 ʹZabutaʹ 7.31 ± 1.49 5.16 10.27 20.4 ʹKazkaʹ 7.36 ± 1.40 6.08 10.12 19.1 ʹMerchantʹ 6.90 ± 1.84 5.18 11.29 26.7 ʹRubinova Ranniaʹ 6.92 ± 1.28 5.12 9.03 18.5 ʹSweet Erlizʹ 7.26 ± 1.68 5.17 11.00 23.1 ʹValeriy Chkalovʹ 7.13 ± 1.54 5.19 10.12 21.5 Average value 7.10 ± 1.46 5.26 10.23 21.02 LSD05 0.579 – – – Таble 3: The accumulation of vitamin C in sweet cherry fruits of early term ripening varieties, mg 100 g-1 (2008–2019), x sx± , n = 5 Acta agriculturae Slovenica, 118/2 – 2022 5 Studies of the impact of environmental conditions and varietal features of sweet cherry on the accumulation of vitamin C in fruits ... fruits of two varieties (medium and late terms of ripen- ing) equals 8.93 ± 2.27 % and 8.48 ± 1.74 % respectively (Table 4, 5). The rate of formation of vitamin C fund in sweet cherry fruits of medium term of ripening ex- ceeds the average varietal parameters by 3.8 % and in the fruits of late term of ripening by 9.3 %. It has been established that the fruits of a medium term of ripening had the maximal content of vitamin C. In a medium term ripening group a minimal amount of vitamin C had the fruits of ʹDachnytsiaʹ, Variety Vitamin C content, % Variation according to years, Vр, %average min max ʹChervneva Ranniaʹ 5.95 ± 1.06 4.12 7.67 17.9 ʹDachnytsiaʹ 6.32 ± 1.11 5.01 7.88 17.5 ʹDilemaʹ 10.94 ± 2.20 8.19 14.51 20.1 ʹKordiaʹ 10.63 ± 1.81 8.01 13.85 17.1 ʹOktaviaʹ 10.11 ± 1.74 7.40 12.08 17.2 ʹOktaviaʹ 9.25 ± 2.25 5.12 12.47 24.3 ʹOrionʹ 10.46 ±1.83 7.51 12.82 17.8 ʹPervystokʹ 9.05 ± 1.59 6.34 11.18 17.5 ʹProstirʹ 7.73 ± 1.19 5.19 9.27 15.4 ʹTalismanʹ 10.48 ± 2.46 7.23 14.11 23.4 ʹTempʹ 8.07 ± 1.49 5.89 9.85 18.5 ʹUliublenytsia Turovtsevaʹ 9.02 ± 1.60 6.11 11.91 17.7 ʹVynkaʹ 8.08 ± 1.51 6.23 10.14 18.7 Average value 8.93 ± 2.27 6.33 11.36 18.7 LSD05 0.645 – – – Таble 4: Vitamin C content in sweet cherry fruits of medium term ripening varieties, mg 100 g–1 (2008–2019), x sx± , n=5 Variety Vitamin C content, % Variation according to years, Vр, %average min max ʹАnonsʹ 8.20 ± 1.59 5.71 11.81 19.3 ʹКarinaʹ 8.33 ± 1.48 5.78 10.28 17.7 ʹKolhoznytsiaʹ 7.85 ± 1.24 5.79 10.92 15.8 ʹКоsmichnaʹ 8.95 ± 1.60 6.69 12.03 17.9 ʹKrupnoplidnaʹ 7.74 ± 1.16 5.79 10.23 14.9 ʹMeotydaʹ 8.03 ± 1.45 5.61 10.72 18.1 ʹMirazhʹ 10.67 ± 1.49 8.28 14.14 14.0 ʹPrazdnichnaʹ 10.25 ± 2.02 7.61 13.08 19.7 ʹReginaʹ 7.29 ± 1.01 6.03 10.54 13.8 ʹSurpryzʹ 8.10 ± 1.47 5.65 11.01 18.2 ʹТemporionʹ 7.72 ± 1.44 5.01 9.76 18.7 ʹUdivitelnaʹ 7.58 ± 1.31 5.41 9.23 17.3 ʹZodiakʹ 9.60 ± 1.46 7.79 11.19 15.2 Average value 8.93 ± 2.27 6.33 11.36 18.7 LSD05 0.645 – – – Таble 5: Vitamin C content in sweet cherry fruits of late ripening varieties, mg 100 g-1 (2008–2019), x sx± , n = 5 Acta agriculturae Slovenica, 118/2 – 20226 I. IVANOVA et al. ʹTempʹ and ʹChervneva Ranniaʹ varieties of 2008. Vi- tamin C content was lower than the average varietal parameter by 20.7 %, 27.0 % and 30.7 % respectively. A maximal amount of vitamin C was registered in the fruits of ʹTalismanʹ (14.11 %) and ʹDilemaʹ (14.51 %) of 2010 that is by 34.6 and 32.6 % higher than the average varietal parameter. The highest accumulation of an av- erage content of vitamin C was registered in the fruits of ʹDilemaʹ (10.94 ± 2.20 %) and ʹKordiaʹ (10.63 ± 1.81 %) varieties. In a late term ripening group a minimal amount of vitamin C had the fruits of ʹTemporionʹ variety (5.01 %) of 2008 (Table 5). The content of vitamin C was by 9.8 % lower than the average varietal param- eter. The fruits of ʹMirazhʹ (14.14 %) and ʹPrazdnichnaʹ (13.08 %) of 2014 had a maximal amount of vitamin C that is by 34.9 and 27.6 % higher as compared with an average varietal parameter. A maximal accumulation of an average vitamin C content was registered in the fruits of ʹMirazhʹ (10.67 ± 1.49 %) and ʹPrazdnichnaʹ (10.25 ± 2.02 %). Especially valuable are the varieties whose fruits are characterized by a high and stable amount of vitamin C content (Leong & Oey, 2012). Their variation parameter Vр can be used as an indicator of stability – of a variety in reference to the meteorological conditions of different years of fruits growing. The variation of sampling can be considered insignificant or low under the Vp lower than 10 %, aver- age – under 10 – 20 % and high under 20 % and more. Therefore, the varieties whose fruits have a high and stable vitamin C content are of special value. The variability of vitamin C content during the years of research in the sweet cherry fruits of an early and medium terms of ripening was in the range from 15,4 tо 26,7 %. For a group of early ripening varieties (ʹValeriy Chkalovʹ, ́ Svit Earlizʹ, ́ Merchantʹ and ́ Zabutaʹ) the variation parameter equaled 20.4–26.7 %, for a group of medium terms ripening varieties (ʹTalismanʹ, ʹDilemaʹ, ʹOktaviaʹ) Vp equaled 20.1–24.3 %. It testifies to a maximal impact of weather factors on vitamin C content in the sweet cherry fruits of these groups. The variation parameter for ʹProstirʹ and ʹBigaro Burlatʹ va- rieties equals 15.4 % and 17.9 % respectively that testi- fies to their resistance to stress factors. The Vp range for the sweet cherry fruits of the late term of ripening varied within the average values 13.8–19.7 %. The lowest values of the variation param- eter were registered in ʹReginaʹ and ʹMirazhʹ varieties (Vр = 13.8–14.0 %). The highest vitamin C stability in a group of an early term of ripening is in ʹBigaro Burlatʹ variety (Vр = 17.9 %). The varieties ʹKazkaʹ and ʹZabutaʹ have the highest rate of vitamin C accumula- tion (7.36 ± 1.40 %) and (7.31 ± 1.49 %) respectively. The most perspective from the point of view of fruits storing and processing are ʹKordiaʹ (Vр = 17.1 %) of the medium term and ʹMirazhʹ (Vр = 14.0 %) of the late term of ripening. These varieties are characterized by a high and nearly high content of vitamin C as well as by minimal variability as compared with other va- rieties. The received results of the research have been confirmed by literature data (Bieniek et al., 2011). The chemical composition analysis of fruit revealed sig- nificant differences both between the cultivars and be- tween the years of the research. Environmental conditions, Factor A (Table 6) had the greatest impact on vitamin C content in the fruits of Source of variation Sum of squares Degree of freedom Dispersion Ffact Ftable095 Impact, % Group 1, early ripening sweet cherry varieties Factor A (year) 448.5 11 40.7 321.5 1.8 80.2 Factor В (cultivar) 9.9 6 1.6 13.0 2.2 1.7 Interaction АВ 79.6 66 1.2 9.5 1.4 1.2 Group 2, medium term sweet cherry varieties Factor A (year) 998.05 11 90.7 577.7 1.8 39.1 Factor В (cultivar) 1266.13 12 105.5 671.9 1.8 49.6 Interaction АВ 237.68 132 1.8 11.4 1.3 9.3 Group 3, late ripening sweet cherry varieties Factor A (year) 640.38 11 58.2 244.3 1.8 43.5 Factor В (cultivar) 513.89 12 42.8 179.7 1.8 34.9 Interaction АВ 241.40 132 1.8 7.67 1.3 16.4 Таble 6: The results of two factors dispersion analysis under the accumulation of vitamin C in sweet cherry fruits of different terms of ripening Acta agriculturae Slovenica, 118/2 – 2022 7 Studies of the impact of environmental conditions and varietal features of sweet cherry on the accumulation of vitamin C in fruits ... early and late terms of ripening. The rate of impact of the environmental conditions of the years of research (Factor A) for early ripening varieties equaled 80.2 %, for late ripening varieties only 43.5 %. The rate of im- pact on varietal features (Factor B) was significantly lower for these two groups of varieties in terms of ripe- ness rate. For early ripening varieties, it equaled 1.7 %, for late ripening varieties 34.9 %. It was established that the formation of vitamin C fund in the sweet cherry fruits of medium term of ripening depended on the varietal features (Factor B, 49.6 %). The impact of envi- ronmental conditions of the research years (Factor A) for this group of varieties equaled 39.1 %. The research shows the practicability of forecast- ing of vitamin C accumulation in fruits of early and late ripening sweet cherry varieties in terms of aver- age variety values but not for each pomological variety. The forecasting of accumulation of vitamin C fund in the fruits of medium ripening varieties is expedient to make in terms of average varietal parameters and rate of content for each pomological variety. To establish the correlation relationships between vitamin C content in fruits of early (Y1), medium (Y2), late (Y3) terms of ripening and the climatic factors, the following analysis was made. On the basis of the es- tablished matching coefficients of correlation rY1Xi, rY2Xi , rY3Xi were chosen the most significant factors. The signif- icance of these correlation coefficients was established by checking a statistical hypothesis H0: p = 0 (where p - is a correlation coefficient of general population) under the alternative hypothesis H1: p ≠ 0 under the reliability level α = 0,05. Student’s criteria was used to check the statistical hypothesis. It was established that significant correlation coefficients, under the significance level of 0.05 and the number of degrees of freedom k = 10, were within an interval [–0.55; 0.55]. Further experiments were conducted according to the schedule: First step. On the basis of the principle com- ponents analysis we build a set of the princi- pal components iPC ( 1 )i n= … represent- ed by a liniar combination of weather factors: 1 m i ij j j PC p X = =∑ , 1i n= … (1) where jX – weather factors parameters, 1..j m= iPC - principal components, 1i n= … ijp - coefficients, 1i n= … , 1..j m= . We choose first five principal components ( ), 1 ..5iPC i = , which provide more than 93,5 % of cumu- lative proportion of variance. Second step. The development of the regres- sion models of dependence of vitamin C value on the weather factors for each group of varie- ties from the principal components was as follows: 5 0 1 ˆ i i i Y b b PC = = + ⋅∑ (2) where Ŷ – vitamin C content, mg 100 g-1 iPC - principal components, 1 5i = … ib - regression coefficients, 1 5i = … . The regression equation for early ripening varie- ties is as follows: 1 1 2 3 4 5 ˆ 7.10762 0.41631 0.13542 0.61543 0.13960 0.25401Y PC PC PC PC PC= − + + + − The regression equation for medium ripening varieties is as follows: 2 1 2 3 4 58.9078 0.4252 0.1872 0.0064 0.7698 0.ˆ 1628Y PC PC PC PC PC= − + − + + The regression equation for late ripening varieties is as follows: 3 1 2 3 4 58.4881 0.3533 0.3044 0.0150 0.1113 0.ˆ 0869Y PC PC PC PC PC= − + − − − The value of the determination coefficient (R- squared) for early ripening varieties equals 0.9239, for medium –ripening, 0.7273, for late ripening varieties, 0.7069, it indicates that there is a strong impact of inde- pendent variables on the dependent variables. The p-value is < 0.05 for F-statistic value for all regression models, it testifies to the adequacy of the models according to Fisher’s criterion under the level of significance -0.05. Third step. We proceed from the principal com- ponents to the initial factors by applying formula 1. We carry on the standardization process of variable mod- els. We receive a regression model which characterizes the dependence of vitamin C value (for 1 2 3 ˆ ˆ, ˆ ,Y Y Y ) from weather factors. 1 ˆ n j j j Y a X = = ⋅∑  (3) where j j j j X X X X σ − = – values of weather Acta agriculturae Slovenica, 118/2 – 20228 I. IVANOVA et al. and environmental factors in a standardized form 1j n= … jX - arithmetic mean of factors jX , 1j n= … jX σ - standard deviation of factors jX , 1j n= … ja - model coefficients, 1j n= … Ŷ - vitamin C content, mg 100 g-1. Table 7 presents the coefficients of model (3) for vitamin C content in sweet cherry fruits of early 1( ˆ )Y , medium ( 2̂Y ) and late ( 3̂ )Y terms of ripening. The coefficients ( ) , 1 ..12i i∆ = were estimated by formula for each factor on the basis of the developed models: 2 ii yx i a r R ⋅ ∆ =  (4) where ia - coefficients of a regression model (3) iyx r - matching coefficients correlation 2R − determination coefficients. Twelve parameters of the climatic factors ( iX ) which in a particular growing period could have a significant impact on the ascorbic acid content in the sweet cherry fruits of early (Y1), medium (Y2) and late (Y3) ripening varieties were chosen (Table 8). These parameters are the following: air humid- ity; average monthly air humidity in May (Х2); aver- age monthly precipitation amount in May (Х1) and in June (Х11); average number of days with precipitation amount more than 1 mm in May (Х5), June (Х6) and July (Х7); average minimal relative air humidity in May (Х3) and in June (Х4); the amount of precipitation dur- ing the period after blooming (Х10); hydrothermal co- efficient (Х12). The parameters (Х8), the difference be- tween the average maximal and minimal temperatures in May, and in June (Х9) were chosen from among the temperature air indicators (°С). A complex of weather factors which have average and strong linear correlation dependence in terms of vitamin C content has been established for each group of varieties. The factors of impact which are expedient to study from the point of view of the importance and the logi- cality of the experiment, despite the insignificance of their correlation coefficients, have been found. The coefficients i∆ estimate the rate of each fac- tor in a total dispersion of vitamin C amount in sweet cherry fruits. On the basis of the estimated indices , 1 ..12i i∆ = we rank all the factors in terms of their impact from the most significant (rank 1) to the factor which has the lowest impact (rank 12). Table 7 repre- sents the values of index ,%i∆ and the rank of factors. For the experimental groups of sweet cherry varie- ties of three terms of ripening, i∆ varies in the range of 0.16 – 30.64 % (Table 7). According to the estimated in- dices ( ) , 1 ..12i i∆ = all the factors were divided into the ranks. An average monthly amount of precipitation in May (Х1) had a maximal impact on vitamin C content in sweet cherry fruits of early and late ripening varie- ties. An average precipitation amount in June (Х11) had impact on the formation of vitamin C fund in sweet cherry fruits of late term of ripening. This parameter got the first rank and varied in the range of i∆ indices from 14.92 % to 30.64 %. Air humidity indices had a significant impact on vitamin C content in sweet cherry fruits. The parameters of the second rank are average monthly relative air humidity in May (Х2), late term of ripening, total number of days with precipitation amount more than 1 mm in June (Х6), medium term of ripening, total number of days with precipitation amount more than 1 mm in July (Х7), early term of rip- ening. The significance of the rate of impact of these 1a 2a 3a 4a 5a 6a 1̂Y -0,529 -0,131 -0,207 -0,067 -0,234 -0,278 2̂Y -0,420 -0,164 -0,225 -0,082 -0,167 -0,263 3̂Y -0,360 -0,444 -0,478 0,070 -0,309 0,120 7a 8a 9a 10a 11a 12a 1̂Y -0,382 0,061 -0,021 -0,227 -0,183 0,101 2̂Y -0,320 0,129 0,056 -0,184 -0,090 0,056 3̂Y -0,166 0,477 0,147 0,004 0,380 0,064 Тable 7: Coefficients of a regression model in standardized factors Acta agriculturae Slovenica, 118/2 – 2022 9 Studies of the impact of environmental conditions and varietal features of sweet cherry on the accumulation of vitamin C in fruits ... factors, 6X∆ , 7X∆ , 2 ,X∆ was in the range of 11.57– 18.57 %. Weather factors which have a significant impact on vitamin C formation in sweet cherry fruits of three groups belong to the parameters of the third rank, they are: average minimal air humidity in May, % (Х3), a total number of days with precipitation amount more than 1mm in June (Х6). The rate of impact equaled 7 X∆ = 18.57 % for the varieties of an early term of ripening, and for the varieties of medium and late terms of ripening the rate of impact equaled 3X∆ 11.38 % and 14.81 % respectively. The factors of the 1–7th ranks with the rates of impact i∆ (6.09–30.64 %) had a maxi- mal impact on vitamin C content for early ripening va- rieties of sweet cherry. Relative factors term ( iX ) Factors Matching coefficients of correlation ( j iY X r ), indices of the impact rate of factors ( )i∆ and indices of the factors ranks for the varieties groups early ripening medium ripening late ripening 1 iy x r i∆ rank 2 iy x r i∆ rank 3 iy x r i∆ rank X1 Average monthly amount of precipitation in May, mm 0.889 30.64 1 0.651 23.86 1 0.535 13.45 4 X2 Average monthly relative air humidity in May, % 0.712 6.09 7 0.575 8.22 7 0.479* 14.87 2 X3 Average minimal relative air humidity in May, % 0.733 9.90 4 0.579 11.38 3 0.443* 14.81 3 X4 Average minimal relative air humidity in June, % 0.201* 0.88 11 0.348* 2.49 10 0.476* 2.34 11 X5 Total number of days with precipitation amount more than 1 mm in May, per day 0.531* 8.09 5 0.702 10.25 5 0.431* 9.29 5 X6 Total number of days with precipitation amount more than 1 mm in June, per day 0.604 10.97 3 0.503* 11.57 2 0.719 6.03 8 X7 Total number of days with precipitation amount more than 1 mm in July, per day 0.745 18.57 2 0.368* 10.28 4 0.634 7.37 7 X8 Difference between average maximal and minimal tem- peratures in May, °С –0.52* 2.07 9 –0.29* 3.35 9 –0.24* 8.24 6 X9 Difference between average maximal and minimal tem- peratures in June, °С –0.27* 0.36 12 –0.27* 1.35 12 –0.541 5.56 9 X10 Amount of precipitation in blooming period, mm 0.498* 7.37 6 0.631 10.13 6 0.528 0.16 12 X11 Average monthly amount of precipitation in June, mm 0.159* 1.90 10 0.683 5.38 8 0.563 14.92 1 X12 Hydrothermical coefficient 0.480* 3.15 8 0.452 1.74 11 0.661 2.97 10 Table 8: The table of the coefficients of matching correlation ( ) j iY X r , indices of the impact rate ( i∆ ) and their ranks, which characterize the impact of factors (Xі,) on vitamin C content in the sweet cherry fruits of three terms of ripening *some important factors which must be studied in the experiment as expedient and logical ones, though their correlation coefficients are not significant Acta agriculturae Slovenica, 118/2 – 202210 I. IVANOVA et al. Some additional impact on vitamin C content had the following parameters: average minimal relative air humidity in June (X4); the difference between average maximal and minimal temperatures in May (X8); the difference between average maximal and minimal tem- peratures in June (X9); average monthly precipitation amount in June (X11); hydrothermical coefficient (X12). The overall value of index i∆ for factors X12, X8 and X11, Х4, Х9 equaled 8.36 %. Factors of 1–8 ranks had a maximal impact on vitamin C content for medium ripening varieties. The range of i∆ for them was in the range of 5.38–23.86 %. A less significant impact on the formation of vitamin C content in sweet cherry fruits of a medium term of ripening had the climatic parameters like: average min- imal relative air humidity in June (X4), the difference between average maximal and minimal temperatures in May (X8); the difference between average maximal and minimal temperatures in June (X9); hydrothermical co- efficient (X11). The overall value of index i∆ for factors Х4, Х8, Х9, Х11, which belong to ranks 9–12 within the given group of sweet cherry varieties, equaled 8.93 %. All factors in a group of late ripening sweet cher- ry varieties ( i∆ – 5.56–14.92 %) had a largest impact on the formation of vitamin C content. The factors of 10–12 ranks (Х10, Х11, Х12) had a less significant impact on vitamin C content in sweet cherry fruits of a given group. The overall value of index i∆ for them equaled 5.47 %. The analysis of the rate of impact of weather fac- tors on vitamin C content in sweet cherry fruits of three terms of ripening testifies to the fact that the most sig- nificant climatic parameters are: humidity indices in May and June (the last months of fruits formation), the average monthly precipitation amount in May (X1) for early ripening and medium ripening varieties, and the precipitation depth in June (X11) for late ripening varie- ties respectively. 4 CONCLUSIONS 1. The varieties ʹKazkaʹ (7.36 mg 100 g–1), ʹZabutaʹ (7.31 mg 100 g–1) have been chosen in terms of vitamin C content for the group of an early term of ripening. ʹBugaro Burlatʹ variety (with a minimal variability pa- rameter Vр = 17.9 %) has been chosen in terms of min- imal variability parameter during the years of research. 2. A medium ripening variety ʹKordiaʹ (10.63 mg 100 g–1 under Vр = 17.1 %) and late ripening variety ʹMirazhʹ (10.67 mg 100 g–1 under Vр = 14.0 %) were chosen as the most perspective from the point of view of fruit storing and processing in terms of vitamin C content. The variation parameters of vitamin C content in sweet cherry fruits of three terms of ripening ranged from 13,8% to 26,7%. 3. The environmental conditions of the years of re- search (Factor A) – 43.5 % and 80.2 % respectively, had a dominating impact on vitamin C content in the fruits of late and early ripening varieties. 4. The impact of varietal features (Factor B) was dominating (49.6 %) in terms of vitamin C content in the sweet cherry fruits of medium term of ripening. 5. The models of dependence of vitamin C content on the impact of climatic conditions for three groups of varieties were developed by using the method of the principle components and the method of least squares. 6. The analysis of the rate of impact of each me- teorological parameter on vitamin C content in sweet cherry fruits was made by using the developed regres- sion models. The range of impact of the meteorological parameters in the formation of vitamin C fund has been established and their maximal values ( i ∆ 14.92 % to 30.6 %) for the groups of three-term ripening varieties have been estimated. 5 REFERENCES Acero, N., Gradillas, A., Beltran, M., García, A., Muñoz Min- garro, D. (2019). Comparison of phenolic compounds profile and antioxidant properties of different sweet cherry (Prunus avium L.) varieties. Food Chemistry, 279, 260–271. https://doi.org/10.1016/j.foodchem.2018.12.008 Antognoni, F., Potente, G., Mandrioli, R., Angeloni, C., Freschi, M., Malaguti, M., Hrelia, S., Lugli, S., Gennari, F., Muzzi, E., & Tartarini, S. (2020). Fruit quality characterization of new sweet cherry cultivars as a good source of bioactive phenolic compounds with antioxidant and neuroprotec- tive potential. Antioxidants (Basel, Switzerland), 9(8), 677. https://doi.org/10.3390/antiox9080677 Ballistreri, G., Continella, A., Gentile, A., Amenta, M., Fabroni, S., Rapisarda, P. (2013). Fruit quality and bioactive com- pounds relevant to human health of sweet cherry (Prunus avium L.) cultivars grown in Italy. Food Chemistry, 140, 630–638. https://doi.org/10.1016/j.foodchem.2012.11.024 Bastos, C.A.A., Barros, L., Dueñas, M., Calhelha, R. C., Quei- roz, M.J.R.P., Santos-Buelga, C., Ferreira, I.C.F.R. (2015). Chemical characterization and bioactive properties of Prunus avium L.: the widely studied fruits and the unex- plored stems. Food Chemistry, 173, 1045–1053. https://doi. org/10.1016/j.foodchem.2014.10.145 Bieniek, A., Kawecki, Z., Kopytowski, J., Zielenkiewicz, J. (2011). Yielding and fruit quality of Lithuanian sweet cherry cultivars grown under the climatic and soil con- Acta agriculturae Slovenica, 118/2 – 2022 11 Studies of the impact of environmental conditions and varietal features of sweet cherry on the accumulation of vitamin C in fruits ... ditions of Warmia. Folia Horticulturae, 23(2), 101–106. https://doi.org/10.2478/v10245-011-0015-4 Correia, S., Schouten, R., Silva, A. P., Gonçalves, B. (2017). Fac- tors affecting quality and health promoting compounds during growth and postharvest life of sweet cherry (Pru- nus avium L.) Frontiers in Plant Science, 8, 2166. https:// doi.org/10.3389/fpls.2017.02166 Correia, S., Aires, A., Queirós, F., Carvalho, R., Schouten, R., Silva, A.P., Gonçalves, B. (2020). Climate conditions and spray treatments induce shifts in health promoting com- pounds in cherry (Prunus avium L.) fruits. Scientia Hor- ticulturae, 263, 109–147. https://doi.org/10.1016/j.scien- ta.2019.109147 Demir, T. (2013). Determination of carotenoid, organic acid and sugar content in some sweet cherry cultivars grown in Sakarya, Turkey. Journal of Food, Agriculture and Envi- ronment, 11, 73–75. Dhandevi, P. E. M., & Jeewon, R. (2015). Fruit and vegetable intake: Benefits and progress of nutrition education inter- ventions-narrative review article. Iranian Journal of Public Health, 44, 1309. Faniadis, D. Drogoudi, P. D., Vasilakakis, M. (2008). Effects of cultivar, orchard elevation, and storage on fruit qua- lity characters of sweet cherry (Prunus avium L.). Scien- tia Horticulturae, 125, 301–304. https://doi.org/10.1016/j. scienta.2010.04.013 Grandi, M., Lugli, S., Correale, R. (2017). Fruit quality changes in postponed picking of new cherry cultivars. Acta Hor- ticulturae, 1161, 599–602. https://doi.org/10.17660/Acta- Hortic.2017.1161.95 Hayaloglu, A. A., & Demir, N. (2015). Physicochemical cha- racteristics, antioxidant activity, organic acid and sugar contents of 12 sweet cherry (Prunus avium L.) cultivars grown in Turkey. Journal of Food Science, 80(3), 564–570. https://doi.org/10.1111/1750-3841.12781 He, F. J., Nowson, C. A., Lucas, M., MacGregor, G. A. (2007). Increased consumption of fruit and vegetables is related to a reduced risk of coronary heart disease: meta-analy- sis of cohort studies. Journal of Human Hypertension, 21, 717–728. https://doi.org/10.1038/sj.jhh.1002212 Ivanova, I., Serdyuk, M., Malkina, V., Priss, T., Herasko, Т., Тymoshchuk, Т. (2021a). Investigation into sugars ac- cumulation in sweet cherry fruits under abiotic factors effects. Agronomy Research, 19(2), 444–457. https://doi. org/10.15159/ar.21.004 Ivanova, I., Serdiuk, M., Malkina, V., Bandura, I., Kovalenko, I., Tymoshchuk, T., Tonkha, O., Tsyz, O., Mushtruk, M., Ome- lian, A. (2021b). The study of soluble solids content accu- mulation dynamics under the influence of weather factors in the fruits of cherries. Potravinarstvo Slovak Journal of Food Sciences, 15, 350–359. https://doi.org/10.5219/1554 Ivanova, I., Serdyuk, M., Malkina, V., Tymoshchuk, T., Ko- telnytska, A., Moisiienko, V. (2021c). The forecasting of polyphenolic substances in sweet cherry fruits under the impact of weather factors. Agraarteadus, 32(2), 239–250. https://doi.org/10.15159/jas.21.27 Kevers, C., Pincemail, J., Tabat, J., Defrasgne, J. O., Dommes, J. (2011). Influence of cultsvator. Harvest time, storage con- ditions, and reelsng on the antioxidant capacity and phe- nolsc and ascorbic acid contents of apples and pears Jour- nal of agricultural and food chemistry, 59(11), 6165–6171. https://doi.org/10.1021/jf201013k Lakatos, L., Dussi, M. C., Szabо, Z. (2014). The influence of meteorological variables on sour cherry quality para- meters. Acta horticulturae, 1020, 287–292. https://doi. org/10.17660/ActaHortic.2014.1020.41 Lakatos, L., Szab, T., Sun, Z., Soltész, M. (2010). The role of meteorological variables of blossoming and ripening within the tendency of qualitative indexes of sour cherry. International Journal of Horticultural Science, 16(1), 7–10. https://doi.org/10.31421/IJHS/16/1/854 Legua, P., Domenech, A., Martínez, J.J., Sánchez-Rodríguez, L., Hernández, F., Carbonell-Barrachina, A.A., Melgarejo, P. (2017). Bioactive and volatile compounds in sweet cherry. Journal of Food and Nutrition Research, 5(11), 844–851. ht- tps://doi.org/10.12691/jfnr-5-11-8 Leong, S. Y., & Oey, I. (2012). Effects of processing on antho- cyanins, carotenoids and vitamin C in summer fruits and vegetables. Food Chemistry, 133, 1577–1587. https://doi. org/10.1016/j.foodchem.2012.02.052 Luna-Vázquez F. J., Ibarra-Alvarado, C., Rojas-Molina, A., Rojas-Molina, J. I., Yahia E. M., Rivera-Pastrana, D. M., Rojas-Molina, A., & Zavala-Sánchez, M. Á. (2016). Nutra- ceutical value of black cherry Prunus serotina Ehrh. fruits: antioxidant and antihypertensive properties. Molecules, 18(12), 14597–14612. https://doi.org/10.3390/molecu- les181214597 Martini, S., Conte, A., & Tagliazucchi D. (2017). Phenolic compounds profile and antioxidant properties of six sweet cherry (Prunus avium) cultivars. Food Research International, 97, 15–26. https://doi.org/10.1016/j.food- res.2017.03.030 Matteo, A. D., Russo, R., Graziani, G., Ritieni, A., Vaio, C. D. (2016). Characterization of autochthonous sweet cherry cultivars (Prunus avium L.) of southern Italy for fruit quality, bioactive compounds and antioxidant activity. Journal of the Science of Food and Agriculture. 97(9), 2782– 2794. https://doi.org/10.1002/jsfa.8106 Nowak, D., Gośliński, M., Wojtowicz, E., Przygoński, K. (2018). Antioxidant properties and phenolic compounds of vita- min C-rich juices. Journal of Food Science, 83(8), 2237– 2246. https://doi.org/10.1111/1750-3841.14284 Pissard, A., Lateur, M., Baeten, V., Magein, H., Dupont, P., Ta- bart, J., Pincemail J., Kevers, C. (2016). Determination of total phenolic compound content and antioxidant activity in cherry species and cultivars. Journal of Berry Research, 6, 81–91. https://doi.org/10.3233/JBR-150109 Popescu, S., Velciov, A.B., Riviș, A., Costescu, C., Petolescu, C. (2014). Antioxidant activity and phenolic content of sweet cherries (Prunus avium L.) from West and South-West of Romania. Bulletin UASVM Food Science and Technology, 71(2), 215–216. https://doi.org/10.15835/ buasvmcn-fst:10762 Prior, R. L. (2003). Fruits and vegetables in the prevention of cellular oxidative damage. American Journal of Clinical Nutrition, 78(3), 570–578. https://doi.org/10.1093/ajc- n/78.3.570S Rios de Souza, V., Pereira P. A. P., Teodoro da Silva, T.L., Carlos Acta agriculturae Slovenica, 118/2 – 202212 I. IVANOVA et al. de Oliveira Lima, L., Pio, R., Queiroz, F. (2014). Determi- nation of the bioactive compounds, antioxidant activity and chemical composition of Brazilian blackberry, red raspberry, strawberry, blueberry and sweet cherry fruits. Food Chemistry, 156, 362–368. http://dx.doi.org/10.1016/j. foodchem.2014.01.125 Schmitz-Eiberger, M. A. & Blanke, M. M. (2012). Bioactive components in forced sweet cherry fruit (Prunus avium L.), antioxidative capacity and allergenic potential as dependent on cultivation under cover. Food Science and Technology, 46, 388–392. https://doi.org/10.1016/j. lwt.2011.12.015 Serdуuk, M. E., Priss O. P., Haprindashvili, N. A., Ivanova, I. Ye. (2020). Research methods of fruit, vegetable and berry products. (Ch. 1). Melitopol : Liuks. (In Ukraine). Serrano, M., Guillén F., Martínez-Romero, D., Castillo S., Va- lero, D. (2005). Chemical constituents and antioxidant ac- tivity of sweet cherry at different ripening stages. Journal Agricultural Food Chemistry, 53(7), 2741–2745. https:// doi.org/10.1021/jf0479160 Vasylyshyna, О. (2018). The quality of sour cherry fruits (Pru- nus cerasus L.), treated with chitosan solution before stor- age. Acta Agriculturae Slovenica, 111(3), 633–637. https:// doi.org/10.14720/aas.2018.111.3.11 Acta agriculturae Slovenica, 118/2, 1–9, Ljubljana 2022 doi:10.14720/aas.2022.118.2.2541 Original research article / izvirni znanstveni članek Genotypic variation in response to drought stress is associated with biochemical and transcriptional regulation of ureides metabolism in common bean (Phaseolus vulgaris L.) Motlalepula PHOLO-TAIT 1, 2, Thuto KGETSE 3, Gaone Nthabeleng TSHEKO 3, Olerato Tsotlhe THEDI 3, Katso LETHOLA 1, Ebenezer Oteng MOTLAMME 1, Moagisi Innocent ITHUTENG 1 and Samodimo NGWAKO 4 Received February 23, 2022; accepted May 29, 2022. Delo je prispelo 23. februarja 2022, sprejeto 29. maja 2022 1 Department of Agricultural Research, Ministry of Agricultural Development and Food Security, Gaborone, Botswana 2 Corresponding author, e-mail: pholom@webmail.co.za 3 Department of Chemistry & Forensic Sciences, Botswana International University of Science and Technology, Palapye, Botswana 4 Faculty of Research and Graduate Studies, Botswana University of Agriculture and Natural Resources, Gaborone, Botswana Genotypic variation in response to drought stress is asso- ciated with biochemical and transcriptional regulation of ureides metabolism in common bean (Phaseolus vulgaris L.) Abstract: Ureidic legumes such as common bean (Phaseoulus valgaris L.) plants export nitrogen from the nod- ules to shoots and leaves as ureides during symbiotic biological nitrogen fixation. Common bean gene encoding allantoinase (allantoin amidohydrolase, EC 3.5.2.5), is a key enzyme that catalyses the hydrolysis of allantoin to allantoic acid. It plays a role in ureide generation for export and ureide catabolism to generate a nitrogen source in sinks tissues. As such, one of the adaptive mechanisms of plants to drought stress, is associ- ated with ureides accumulation. To identify genetic variation of common bean in response to drought stress, changes in the expression of ALLANTONAISE (PvALN) gene and ure- ides content were examined in the leaf tissues of the three common bean genotypes (CAL96, DAB514 and DAB541) and one tepary bean genotype (Phaseolus acutifolius A.Gray). Amongst all the genotypes, the suggested drought susceptibil- ity in DAB514 common bean genotype, was probably attrib- uted to a repressed PvALN expression rate which were cor- roborated by an impaired ureides levels, and reduced plant growth. On contrary, drought stress induced an upregulated relative expression of PvALN coupled with an increase in al- lantoin and allantoate in DAB541 common bean genotype. In addition, the sustained plant growth in CAL96 was probably attributed to a steady amount of allantoin synthesized under drought stress. Taken together, DAB541 and CAL96 common bean genotypes are the promising genotypes with an induced upregulated transcriptional control of catabolism and/or bio- synthesis of ureides, hence potential genotypes for selection and introduction under Botswana semi-arid conditions. Key words: common bean; drought stress; ureides: al- lantonaise; allantoin; allantoate Genetska spremenljivost odziva navadnega fižola (Phaseo- lus vulgaris L.) na sušni stres je povezana z biokemičnim in transkripcijskim uravnavanjem presnove ureidov Izvleček: Ureidne stročnice kot je navadni fižol (Phaseo- ulus valgaris L.) transportirajo med simbiontsko vezavo dušik iz nodulov v liste kot ureide. Pri navadnem fižolu je pomem- ben gen, ki kodira alantoinazo (alantoin amidohidrolaza, EC 3.5.2.5), ključni encim, ki katalizira hidrolizo alantoina v alan- toinsko kislino. Ta ima pomembno vlogo pri tvorbi ureidov za njihov eksport in razgradnjo kot vir dušika v tkivih ponora. Pri rastlinah je eden izmed prilagoditvenih mehanizmov na sušni stres povezan s kopičenjem ureidov. Za določitev ge- netske variabilnosti navadnega fižola na sušni stres so bile analizirane spremembe v izražanju gena za alantoinazo, AL- LANTONAISE (PvALN) in vsebnosti ureidov v listnih tkivih pri treh genotipih navadnega (CAL96, DAB514 and DAB541) in enem genotipu ostrega fižola, Phaseolus acutifolius A.Gray. Med vsemi genotipi bi občutljivost genotipa DAB514 nava- dnega fižola verejtno lahko pripisali zavrtju izražanja gena PvALN, kar je bilo povezano z zmanjšano tvorbo ureidov in slabšo rastjo. V nasprotju je sušni stres vzpodbudil povečano izražanje tega gena, kar je bilo povezano s povečanjem vseb- nosti alantoina in alantoata pri genotipu DAB541. Dodatno bi ohranjeno rast genotipa CAL96 lahko pripisali stalni količini allantoina, ki se sintetizira med sušnim stresom. Zaključimo lahko, da sta genotipa navadnega fižola DAB541 in CAL96 obetajoča, z vzpodbujeno povečano transkripcijsko kontrolo katabolizma in/ali biosinteze ureidov, ki bi lahko služila kot potencial za izbor in uvajanje ustreznih genotipov v sušnih razmerah Botswane. Ključne besede: navadni fižol; sušni stres; ureidi; alan- tonaza; allantoin; alantoat Acta agriculturae Slovenica, 118/2 – 20222 M. PHOLO-TAIT et al. 1 INTRODUCTION Common bean (Phaseolus vulgaris L.) is an im- portant grain legume in the human diet due to its high nutritional properties, such as proteins, vitamins and minerals (Broughton et al., 2003). One of the major benefits of common beans in agriculture is their capac- ity to symbiotically fix atmospheric nitrogen through associations with soil nitrogen-fixing rhizobia, thus re- ducing the need to use nitrogen fertilizers (Coleto et al., 2014). As such, common bean plants are not dependent on nitrogen fertilization for growth due to their abil- ity to form symbioses with atmospheric di-nitrogen fixing bacteroid located in root nodules. Plant growth and productivity is dependent on the accessibility of the newly available fixed nitrogen from the root to the vegetative and reproductive plant tissues. Ureide allantoin and its degradation derivate al- lantoate are a group of soil heterocyclic nitrogen com- pounds that play an essential role in the assimilation, metabolism, transport, and storage of nitrogen in high- er plants (Smith & Atkins, 2002). They serve as the ve- hicle for storage and xylem transport of symbiotically fixed nitrogen from root to the shoot, and as such play a key role in nitrogen utilization in ureide-type legumes (Kohl et al., 1990; Smith & Atkins, 2002; Zrenner et al., 2006). Once delivered to sink tissues, allantoin is con- verted to allantoate, which in-turn can be broken down completely to glyoxylate, releasing four molecules of ammonia and two molecules of CO2. Genes encoding allantoinase (allantoin amidohydrolase, EC 3.5.2.5), ca- talysis the first step in the degradation of the ureide al- lantoin and the synthesis of allantoate, the second most prominent ureide. It is therefore unique in this pathway such that it plays a role in ureide generation for export from the nodules as well as ureide catabolism to gener- ate a nitrogen source in leaves and other nitrogen sinks (Muñoz et al., 2001; Watanabe et al., 2014; Werner et al., 2013). Adaptive mechanisms of plants to abiotic stresses such as drought, include changes in the expression of genes involved, biosynthesis of compatible osmolytes and scavenging systems for reactive oxygen species (Han et al., 2014; Hasegawa et al., 2000). The inhibi- tion of nitrogen utilization under drought stress, is proposed to be attributed to N-feedback regulation, in which ureides would be among the signaling molecules triggering the inhibition (Charlson et al., 2009; King & Purcell, 2005; Rachid Serraj, Vadez et al., 1999). The in- duction and activation of enzymes with a subsequent increased levels of intermediary metabolites, particu- larly ureides allantoin and allantoate play a vital role in plant responses and adaptation to abiotic stresses (Alamillo et al., 2010; Smith & Atkins, 2002) . In soy- bean, high ureides levels in shoots and leaves correlated with nitrogen fixation inhibition (Rachid Serraj, Vadez, et al., 1999). In Arabidopsis thaliana (L.) Heynh. mu- tant lacking ALLANTONAISE (ALN), high levels of al- lantoin metabolites were reported due to an activated allantoin biosynthetic genes and/or repression of ALN expression rate. The response suggested that ureide me- tabolism and accumulation contribute to the abiotic stress response, which is regulated, at least in part, at the transcriptional level. In addition, this implied a possible elevated drought stress tolerance, possibly by reducing oxidative damage. (Irani & Todd, 2016). The symbiotic nitrogen fixation showed to be ex- tremely sensitive to drought stress and this effect could result in decreasing N accumulation and yield of leg- ume crops (Serraj, 1999; Rachid Serraj, 2003). However, ureide-exporting legumes, such as common beans are more sensitive to drought stress due to rapid decline in nitrogen fixation compared to amidic ones (Purcell et al., 2004; R Serraj, 1999; Rachid Serraj, Vadez et al., 1999). On contrary, a variable degree of nitrogen fixa- tion inhibition due to drought stress was found among the bean genotypes. An increase in both mRNA levels and ALN activity with a concomitant increase in roots, shoots and leaves ureide levels in common bean in re- sponse to drought was attributed to an elevated syn- thesis of allantoate (Alamillo et al., 2010). Remarkably, other studies demonstrated a positive correlation be- tween suppressed nitrogen fixation and accumulation of ureides in stems and leaves of both sensitive and tol- erant genotypes. Further variability was associated with the rise in allantoate level coupled with an increase in ALLANTOINASE gene expression and enzyme activity in the most sensitive genotype, which increased after inhibition of nitrogen fixation, suggesting that ureides originate in vegetative tissues as a response to water stress, probably mediated by the induction of allantoin- ase (Coleto et al., 2014). The overreliance on erratic rain coupled with rela- tively poor soil quality has resulted in poor productiv- ity of crops in Botswana, making the agricultural sector most vulnerable to climate change (FANRPAN, 2017). Crop diversification such as the use of drought-tolerant legumes with enhanced nitrogen fixation ability and improved utilization of the newly fixed nitrogen to en- hance crop productivity crops has been hailed as one of the potential adaptive measures to mitigate climate change. As such, Botswana has considered the introduc- tion of common bean into the cropping system as one of the climate smart agriculture approaches, combating poverty, environmental degradation, and improving soil health. This was further justified by its high nutri- Acta agriculturae Slovenica, 118/2 – 2022 3 Genotypic variation in response to drought stress ... in common bean (Phaseolus vulgaris L.) tive value and commercial benefits such as source of income for many rural household (Beebe et al., 2013; Molosiwa et al., 2019). However, information on the performance of the potential common bean genotypes for introduction, particularly nitrogen fixation and uti- lization capability and crop productivity under Botswa- na conditions remain elusive. Therefore, this study was conducted to identify the growth and genetic response of common bean genotypes under drought stress. Bio- chemical analysis of ureides-derived metabolites and transcriptional analysis of Phaseoulus vulguris ALLAN- TOINASE relative gene expression was conducted for the identification and selection of the best and promis- ing common bean genotypes in terms of nitrogen fixa- tion and utilization under drought stress. 2 MATERIALS AND METHODS 2.1 PLANT MATERIALS AND GROWTH CONDI- TIONS Common bean genotypes were selected based on their superior stability, adaptability and yield per- formance in the previous studies conducted at Sebele and Pandamatenga respectively (Molosiwa et al., 2019). These includes three common bean (Phaseolus vulgaris L.) genotypes (DAB541; DAB514; CAL96) and GK011 tepary bean (Phaseolus acutifolius A.Gray.; GK011), the latter being reported in previous studies as a drought tolerant bean. The experiment was conducted in a growth cabinet, in a randomized block design, with six replications, under a 16 h light/8 h dark photoperiod at 25 °C and a light intensity of 100-150 μmol photons m-2s-1. Plants were exposed to water holding treatment three (3) weeks after emergence. Drought stress ex- periment consisted of two treatments, namely drought stress treatment by withholding water application with a serious drought stress (35-45 % water holding capac- ity) and the control by ensuring maximum water hold- ing capacity by watering (70-80 %). 2.2 UREIDES ACCUMULATION: ALLANTOIN AND ALLANTOATE The determination of ureides allantoin and allan- toate were performed by differential analyses of glyoxy- late derivative according to published protocol (Lesca- no, 2020). Ureides were extracted from leaf 15 mg liquid nitrogen grounded leaf tissue samples by boiling it in 50 mM potassium phosphate buffer (pH 7.0). Homoge- nates were centrifuged at 18,000 xg for 25 min at 4 °C to ensure the absence of debris and a clear supernatant containing ureides. Six biological replicates of 100 μl aliquots of each sample were collected in three separate tubes for the measurement of endogenous glyoxylate, allantoic acid-derived glyoxylate and allantoin-derived glyoxylate. Glyoxylate is converted into glycolic acid- phenylhydrazone and then oxidized by ferricyanide in the presence of concentrated acid and phenylhydrazine to give red-colored 1,5-diphenylformazan. The absorb- ance of supernatants was measured using a spectropho- tometer at 535 nm. 2.3 TRANSCRIPTOMIC ANALYSIS OF DIFFER- ENTIALLY EXPRESSED GENES Total RNA was extracted using a Quick-RNA Miniprep Kit (Zymo Research Corporation, Irvine, CA, United States) as per the manufacturer’s protocol and treated with DNase1 (Zymo Research Corporation, Ir- vine, CA, United States). The quantity and quality of the isolated RNA were evaluated, respectively, using a Nan- oDrop ND-1000 UV–Vis Spectrophotometer (Thermo Fisher Scientific) and by 1 % electrophoresis agarose gels according to manufacturer’s instructions. The qual- ity of each cDNA and the RT-qPCR were checked per by using standard PCR reaction and the housekeeping gene PvACTIN-2 primers (Díaz-Leal et al., 2012) and PvALN (Table 1). These primer pairs were designed using GeneScript qPCR primer design (https://www. genscript.com/tools/pcr-primers-designer/advanced). Luna Universal qPCR Master Mix (New England Bio- lab Inc., MA, USA) and primers were used to determine RNA expression. The qPCR reactions were performed using triple replicates of cDNA samples in 96-well plates and performed on the LineGene 9600 (Hangzhou Bioer Techonology), following SYBR Green/FAM detection. Reactions were prepared in a total volume of 20 μl ac- cording to Luna® Universal qPCR Master Mix Protocol (M3003; New England Biolab Inc., MA, USA) contain- ing: 1x Luna Universal qPCR Master Mix, 10 μM of for- ward and reverse primer, 100 ng cDNA template and nuclease-free water. The PCR cycles consisted of 1 cycle of initial denaturing at 95 °C for 1 min, followed by 40 cycles of 95 °C for 15 s and 60 °C for 40 s. The melting curve was obtained by applying increasing temperature from 60 to 90 °C. The relative fold change for Phaseou- lus vulgaris ALLANTOINASE (PvALN) was calculated using the 2−ΔΔCt method (Livak & Schmittgen, 2001), and normalized against the housekeeping PvACTIN-2 gene (Díaz-Leal et al., 2012). Acta agriculturae Slovenica, 118/2 – 20224 M. PHOLO-TAIT et al. 2.4 DATA ANALYSIS The data collected were subjected to analysis of variance (ANOVA) using MINITAB computer software program, significant means were separated using pair- wise Tukey comparison at p < 0.05. 3 RESULTS 3.1 PHYSIOLOGICAL BIOMASS RESPONSE To determine the response of common bean to drought stress, fresh biomass was determined from above ground plant tissues after watering was withheld for 10 consecutive days. Biomass for the three geno- types including GK011, CAL96 and DAB541 did not differ under normal water growth conditions. However, a significant reduced biomass was observed in DAB514 common bean genotypes compared to GK011 tepary bean as well as compared to the two common bean genotypes, namely, CAL96 and DAB541. Comparing drought stressed plants to their relative control plants demonstrated no significant variation in biomass in GK011 tepary bean and DAB541 common bean gen- otype. However, drought stress induced a 38.6 % and 38.7 % significant reduction in biomass in CAL96 and DAB514 respectively. Further biomass comparisons were made between drought stressed plants in all the genotypes. The result indicated a 42.6 % reduction in Gene Forward Primers Reverse Primer ACTIN-2 (PvActin-2) 5ˈ-TTGCTTTCAAGGAGGGGGTATGC-3ˈ 5ˈ-GGAGCTTGGAACCTTTCGGTGC-3ˈ ALLANOTONAISE (PvALN) 5ˈ-ACAAGCATGATGCAGGTGCTGTGA-3ˈ 5ˈ-TGCCTCCACGACATCGCACA-3ˈ Table 1: Primer pairs used to determine expression of genes Figure 1: Biomass of common bean genotypes (Phaseolus vulgaris L.) in response to drought stress. Biomass was measured unbve ground plat tissues of three common bean genotypes (CAL97; DAB514; DAB541) and GK011 tepary bean. The standard error of mean of three independent flow cells is indicated by the error bars (n = 6). Bars with different lowercase letters indicate significant differences (p < 0.05) Acta agriculturae Slovenica, 118/2 – 2022 5 Genotypic variation in response to drought stress ... in common bean (Phaseolus vulgaris L.) biomass in drought stressed DAB514 plants relative to drought stressed GK011 tepary bean. In addition, a 35.6 % and a 50.6 % reduction in biomass in drought stressed DAB514 plants were observed in comparison to drought stressed CAL96 and DAB541 common bean plants respectively. Taken together, all comparisons demonstrated the existence of clear variation between DAB514 and DAB541 common bean genotypes in terms of biomass (Figure 1). 3.2 UREIDES ACCUMULATION IN RESPONSE TO DROUGHT STRESS To investigate the production of ureides-derived metabolites in response to drought stress, levels of al- lantoin and allantoate were measured in the leaves of drought stressed plants and control plants (Figure 2). The results were visualized using heat map (Figure 3), generated with MINITAB analytical software (version 21.1). The result demonstrated a significant increase in allantoin metabolite in CAL96 (50.1 %), DAB514 (45.5 %) and DAB541 (47.1 %) common bean genotypes compared to the GK011 tepary bean under normal wa- ter conditions. A significant 60.0 % and 23.8 % increase in allantoin accumulation between the plants under normal condition and drought stress was detected for GK011 tepary bean and DAB541common bean geno- types respectively. In contrast, allantoin content was not significantly affected between the control plants and the drought stressed DAB514 and CAL96 common bean genotypes. In respect to allantoate metabolite, the levels of allantoate were not significantly affected in CAL96 and DAB514 common bean genotypes compared to GK011 tepary bean under normal water growth condi- tions. The study further compared variation in allan- toate levels for the drought stressed plants compared to their relative control plants. The result exhibited 24.6 %, 26.5 % and 47.8 % of reduced allantoate levels for GK011 tepary bean and two common bean genotypes, namely, CAL96 and DAB514 in drought stressed plants compared to the control plants. In contrast, DAB541 common bean genotype elicited a 31.0 % significantly increased levels of allantoate in the drought stressed plants relative to their control plants. Taken together, the response of DAB541 common bean genotype under water stress showed a similar trend for both allantoin and allantoate. Thus, water stress induced a significant increase in both allantoin and allantoate metabolite lev- els (Figure 2). 3.3 RELATIVE GENE EXPRESSION OF UREIDE METABOLISM To assess the correlation between allantonaise ureide and changes in ALLANTONAISE (PvALN) rela- tive gene expression, both metabolic accumulation of allantonaise and relative PvALN gene expression was performed on the leaves of genotypes. To assess wheth- er the accumulation of ureides results from changes in the transcription of genes related to ureide metabolism, quantitative real time PCR was performed to determine the mRNA levels of genes coding for key enzymes in the synthesis of ureides, ALLANTONAISE (ALN). Expres- sion level of PvALN gene in the three replicates samples were normalized against the expression of ACTIN-2 as the internal control. According to the pairwise Tukey comparison, the relative expression of PvALN gene in water-deficit plants compared to the control plants was significantly depressed for all the common beans geno- types, except for DAB541. GK011 tepary bean showed the highest 7.7-folds reduction in the relative expression of PvALN. This decrease was however insignificantly different from DAB514 and CAL96 common bean gen- otypes, which also showed a decreased PvALN expres- sion rate by 3.2 and 5.4-folds respectively. Intriguingly, only DAB541 common bean genotype, showed an in- crease in the expression rate PvALN mRNA (1.2-folds) in the leaves of drought stressed plants relative to the control plants (Figure 4). 4 DISCUSSIONS Legumes are agronomically and economically im- portant in many cropping systems because of their abil- ity to assimilate atmospheric nitrogen and maintaining soil fertility. These are highly desirable traits to consider in the improvement of legume productivity for sustain- able agricultural practices (Serraj, 1999; Rachid Serraj, 2003, 2003; Rachid Serraj et al., 1999). Drought stress is one of the most important environmental factors that regulate plant growth and development and limit its production. Legumes exhibit reduction in nodulation and biological nitrogen fixation in response to drought stress (Pimratch et al., 2008). Accumulation of ureide compounds has been reported in several plant species under stress conditions, and a considerable number of research articles argue for a hindered rather than active ureide catabolism as the survival trait for plants sub- jected to periods of mild drought or salinity due to the Acta agriculturae Slovenica, 118/2 – 20226 M. PHOLO-TAIT et al. alternative prime stress signaling function of uric acid and allantoin. Figure 2: Ureides accumulation in response to drought stress. Ureides measurement consisted of allantoin (A) and allantoate (B) accumulation for pants under control (T0) and drought stress (T1). Ureides accumulation was measured on leaf tissues of three common bean genotypes (CAL97; DAB514; DAB541) and GK011 tepary bean. Bars with different letters are statistically different according to p < 0.05. The standard of mean of three independent flow cells is indicated by the error bars error (n = 6) Acta agriculturae Slovenica, 118/2 – 2022 7 Genotypic variation in response to drought stress ... in common bean (Phaseolus vulgaris L.) The current study evaluated the response of three common bean genotypes to drought stress at both bio- chemical and transcriptional level. Firstly, the response of common bean genotypes under normal growth conditions were tested against tepary bean (Phaseolus acutifolius A. Gray), a relatively higher drought-tol- erant crop than common bean (Phaseoulus vulgaris) and serving as genetic resource for food and genetic enhancement of related legumes (Mwale et al., 2020). The insignificant growth rate in terms of biomass un- der normal growth condition was accompanied by a significant increase in ureide allantoin levels in CAL96, DAB514 and DAB541 common bean genotypes rela- tive to GK011 tepary bean. On contrary, the allantoate content was not affected in CAL96 and DAB514 when compared to GK011 tepary bean. Taken together, the normal growth rate of common bean genotypes com- pared to GK011 tepary bean might have been sustained by an enhanced assimilation and metabolism of nitro- gen, which is attributed increased levels of allantoin and a sustained level of allantoate. Taking into consid- eration the 16 hours day growth period in the current study, this results are consistent with Arabidopsis thali- ana studies, which indicated that allantoin ureide deg- radation is important for the growth and development during vegetative growth under long-day conditions (Takagi et al., 2018). The response of bean genotypes was further evalu- ated under drought stress by comparing plants under drought stress against their relative control ones. In- triguingly, all the common bean genotypes, including tepary bean revealed a similar trend of induced inhib- ited plant growth under drought stress. However, only DAB514 common bean genotype showed a significant reduced plant growth in drought stressed plants com- pared to their relative control plants. The impaired plant growth rate in DAB514 was positively associat- ed with the reduction in both allantoin and allantoate levels, with a concomitant induced down-regulated PvALN relative gene expression. This response pro- posed an impaired ureides degradation at transcrip- tional level, which inevitably negatively affected assimi- lation and use of fixed N and eventually plant growth in DAB514 common bean genotype under drought stress. This finding is contrary to reports that indicated that DAB514 common bean genotype as a stable and high Figure 4: Relative Phaseolus vulgaris ALLANTONAISE (PvALN) gene expression in common beans in response to drought stress. Relative gene expression was measured on leaf tissues of three common bean genotypes (CAL97; DAB514; DAB541) and GK011 tepary bean. Bars with different letters are statistically different according to p < 0.05. The standard of mean of three independent flow cells is indicated by the error bars error (n = 3) Acta agriculturae Slovenica, 118/2 – 20228 M. PHOLO-TAIT et al. yielding genotype under drought stress (Molosiwa et al., 2019). Our results implies that DAB514 common bean genotype is a drought-sensitive genotype pos- sibly due to an impaired ureides metabolism at both chemical and transcriptional level with a substantial reduced plant growth. Though similar results were ob- served in terms of a suppressed expression of PvALN coupled with low levels of allantoate, the plant growth rate was not affected in water stressed CAL96 common bean genotype. The suppressed expression of PvALN in CAL96 common bean genotype might be responsible for an impaired rate of degradation of allantoin and the synthesis of allantoate, subsequently owing to a steady amount of allantoin synthesized under drought stress. Water deficit also resulted in another notable in- crease in ureides allantoin and allantoate levels cou- pled with an induced upregulated relative expression of PvALN in DAB541 common bean genotype. This is in concert with studies on Arabidopsis, Phaseolus vul- garis, and Soybean which demonstrated an increase in shoot ureides under drought stress (Alamillo et al., 2010; Ladrera et al., 2007; Rachid Serraj, 2003; Vadez & Sinclair, 2001). This advocated for an increased tran- scriptional regulation of purine metabolism by PvALN, which in turn resulted in enhancing both the degra- dation of the ureide allantoin and the synthesis of al- lantoate (Alamillo et al., 2010; Coleto et al., 2014). This response suggested that ureide accumulation is a gen- eral response to drought stress and is regulated at the transcriptional level mainly through the induction of allantonaise degradation and the subsequent allantoate synthesis in DAB541 common bean genotype leaf tis- sues. 5 CONCLUSIONS The current study evaluated the response of com- mon bean genotypes to drought stress by assessing ureides metabolism at biochemical and transcriptional level coupled with the ultimate plant growth in terms of biomass. Overall results suggested a degree of ge- netical variation among common bean genotypes. The enhanced plant growth or maintained growth rate un- der drought stress in DAB541 and CAL96 common bean genotypes was probably due to an enhanced degradation of the ureide allantoin and the synthesis of allantoate metabolites. These findings suggested an enhanced ureide generation for export and ureide ca- tabolism to generate a nitrogen source in leaves under drought. Therefore, the study concludes that DAB541 and CAL96 common bean genotype are potential gen- otypes for selection and introduction under Botswana semi-arid condition. Molecular reverse genetic stud- ies can further be conducted to confirm ureides me- tabolism and crop performance of DAB541 and CAL96 common bean genotypes under drought stress. 6 ACKNOWLEDGEMENTS This research was conducted at the Biotechnology and Genetics laboratory, Department of Agricultural Research for providing, who provided both infrastruc- ture and financial capital for consumables and molec- ular reagents. Many thanks to Legumes and Oil Seed Unit for providing us with the seed materials. 7 REFERENCES Alamillo, J. M., Luís Díaz-Leal, J., Victoria Sánchez-Moran, M. A., & Pineda, M. (2010). Molecular analysis of ureide ac- cumulation under drought stress in Phaseolus vulgaris L. Plant, Cell and Environment, 33, 1828–1837. https://doi. org/10.1111/j.1365-3040.2010.02187.x Beebe, S. E., Rao, I. M., Blair, M. W., & Acosta-Gallegos, J. A. (2013). Phenotyping common beans for adaptation to drought. Frontiers in Physiology, 4 MAR. https://doi. org/10.3389/fphys.2013.00035 Broughton, W. J., Hernández, G., Blair, M., Beebe, S., Gepts, P., & Vanderleyden, J. (2003). Beans (Phaseolus spp.) - Model food legumes. Plant and Soil, 252(1), 55–128. https://doi. org/10.1023/A:1024146710611 Charlson, D. V., Korth, K. L., & Purcell, L. C. (2009). Allantoate amidohydrolase transcript expression is independent of drought tolerance in soybean. Journal of Experimen- tal Botany, 60(3), 847–851. https://doi.org/10.1093/jxb/ ern332 Coleto, I., Pineda, M., Rodiñ, O. A. P., De Ron, A. ., & Alamillo, J. . (2014). Comparison of inhibition of N2 fixation and ureide accumulation under water deficit in four common bean genotypes of contrasting drought tolerance. Annals of Botany, 113, 1071–1082. https://doi.org/10.1093/aob/ mcu029 Díaz-Leal, J. L., Gálvez-Valdivieso, G., Fernández, J., Pineda, M., & Alamillo, J. M. (2012). Developmental effects on ureide levels are mediated by tissue-specific regulation of allantoinase in Phaseolus vulgaris L. Journal of Experimen- tal Botany, 63(11), 4095–4106. https://doi.org/10.1093/ jxb/ers090 FANRPAN. (2017). FANRPAN Policy Brief CLIMATE- SMART AGRICULTURE IN BOTSWANA. In Promoting a Conducive Policy Environment for a Food and Nutrition Secure Africa (Issue 2017). Irani, S., & Todd, C. D. (2016). Ureide metabolism under abiot- ic stress in Arabidopsis thaliana. Journal of Plant Physiolo- gy, 199, 87–95. https://doi.org/10.1016/j.jplph.2016.05.011 King, C. ., & Purcell, L. C. (2005). Inhibition of N2 fixation in soybean is associated with elevated ureides and ami- Acta agriculturae Slovenica, 118/2 – 2022 9 Genotypic variation in response to drought stress ... in common bean (Phaseolus vulgaris L.) no acids. Plant Physiology, 137, 1389–1396. https://doi. org/10.1104/pp.104.056317 Kohl, D. H., Lin, J. J., Shearer, G., & Schubert, K. R. (1990). Activities of the pentose phosphate pathway and enzymes of proline metabolism in legume root nodules. Plant Physiology, 94(3), 1258–1264. https://doi.org/10.1104/ pp.94.3.1258 Ladrera, R., Marino, D., Larrainzar, E., González, E. M., & Ar- rese-Igor, C. (2007). Reduced carbon availability to bacte- roids and elevated ureides in nodules, but not in shoots, are involved in the nitrogen fixation response to early drought in soybean 1[OA]. Plant Physiology, 145, 539–546. https://doi.org/10.1104/pp.107.102491 Lescano, I. (2020). Determination of ureides content in plant tissues. Bio-Protocol, 10(11), 1–9. https://doi.org/10.21769/ bioprotoc.3642 Livak, K. J., & Schmittgen, T. D. (2001). Analysis of relative gene expression data using real-time quantitative PCR and the 2−ΔΔCT method. Methods, 25(4), 402–408. htt- ps://doi.org/10.1006/METH.2001.1262 Molosiwa, O. O., Pharudi, J., Seketeme, S., Mashiqa, P., & Chirwa, R. (2019). Assessing yield stability and adapt- ability of Andean common bean genotypes in the semi- arid environment of Botswana. African Journal of Agri- cultural Research, 14, 1593–1600. https://doi.org/10.5897/ ajar2019.13988 Muñoz, A., Piedras, P., Aguilar, M., & Pineda, M. (2001). Urea is a product of ureidoglycolate degradation in chickpea. Purification and characterization of the ureidoglycolate urea-lyase. Plant Physiology, 125(2), 828–834. https://doi. org/10.1104/pp.125.2.828 Mwale, S. E., Shimelis, H., Mafongoya, P., & Mashilo, J. (2020). Breeding tepary bean (Phaseolus acutifolius) for drought adaptation: A review. Plant Breeding, 139, 821–833. https:// doi.org/10.1111/pbr.12806 Pimratch, S., Jogloy, S., Vorasoot, N., Toomsan, B., Patanothai, A., & Holbrook, C. C. (2008). Relationship between bio- mass production and nitrogen fixation under drought- stress conditions in peanut genotypes with different lev- els of drought resistance. Journal of Agronomy and Crop Science, 194(1), 15–25. https://doi.org/10.1111/J.1439- 037X.2007.00286.X Purcell, L. C., Serraj, R., SinclairT.R., & DeA. (2004). Soybean N2 fixation estimates, ureide concentration, and yield re- sponses to drought. Crop Science, 44, 484–492. https://doi. org/10.2135/cropsci2004.4840 Serraj, R. (1999). Review article. Symbiotic N2 fixation response to drought. Journal of Experimental Botany, 50(331), 143– 155. https://doi.org/10.1093/jexbot/50.331.143 Serraj, Rachid. (2003). Effects of drought stress on legume symbiotic nitrogen fixation: Physiological mechanisms. Indian Journal of Experimental Biology, 41(10), 1136–1141. Serraj, Rachid, Sinclair, T. R., & Purcell, L. C. (1999). Symbi- otic N2 fixation response to drought. Journal of Experi- mental Botany, 50(331), 143–155. https://doi.org/10.1093/ jxb/50.331.143 Serraj, Rachid, Vadez, V., Denison, R. F., & Sinclair, T. R. (1999). Involvement of ureides in nitrogen fixation inhibition in soybean. Plant Physiology, 119(1), 289–296. https://doi. org/10.1104/pp.119.1.289 Smith, P. M. C., & Atkins, C. A. (2002). Purine biosynthesis. Big in cell division, even bigger in nitrogen assimilation. Plant Physiology, 128(3), 793–802. https://doi.org/10.1104/ pp.010912 Takagi, H., Watanabe, S., Tanaka, S., Matsuura, T., Mori, I. C., Hirayama, T., Shimada, H., & Sakamoto, A. (2018). Dis- ruption of ureide degradation affects plant growth and development during and after transition from vegetative to reproductive stages. BMC Plant Biology, 18(1), 1–16. https://doi.org/10.1186/s12870-018-1491-2 Vadez, V., & Sinclair, T. R. (2001). Leaf ureide degradation and N2 fixation tolerance to water deficit in soybean. Journal of Experimental Botany, 52(354), 153–159. https://doi. org/10.1093/jxb/52.354.153 Watanabe, S., Matsumoto, M., Hakomori, Y., Takagi, H., Shi- mada, H., & Sakamoto, A. (2013). The purine metabolite allantoin enhances abiotic stress tolerance through syner- gistic activation of abscisic acid metabolism. https://doi. org/10.1111/pce.12218 Watanabe, S., Matsumoto, M., Hakomori, Y., Takagi, H., Shi- mada, H., & Sakamoto, A. (2014). The purine metabo- lite allantoin enhances abiotic stress tolerance through synergistic activation of abscisic acid metabolism. Plant, Cell and Environment, 37(4), 1022–1036. https://doi. org/10.1111/pce.12218 Werner, A. K., Medina-Escobar, N., Zulawski, M., Sparkes, I. A., Cao, F. Q., & Witte, C. P. (2013). The ureide-degrading reactions of purine ring catabolism employ three amido- hydrolases and one aminohydrolase in arabidopsis, soy- bean, and rice. Plant Physiology, 163(2), 672–681. https:// doi.org/10.1104/pp.113.224261 Zrenner, R., Stitt, M., Sonnewald, U., & Boldt, R. (2006). Py- rimidine and purine biosynthesis and degradation in plants. Annual Review of Plant Biology, 57, 805–836. htt- ps://doi.org/10.1146/annurev.arplant.57.032905.105421 Acta agriculturae Slovenica, 118/2, 1–11, Ljubljana 2022 doi:10.14720/aas.2022.118.2.2416 Original research article / izvirni znanstveni članek Enhancement of shoot proliferation and evaluation of biotic elicitation effects on anatomical changes of pseudostem and anti-lipid peroxida- tion activity of Curcuma mangga Val. Fariz ABRAHAM 1, Lai Keng CHAN 1, 2, 3, Gunawan INDRAYANTO 4, 5, Peng Lim BOEY 6, 7 Received November 10, 2021; accepted June 06, 2022. Delo je prispelo 10. novembra 2021, sprejeto 6. junija 2022 1 Universiti Sains Malaysia, School of Biological Sciences, Penang, Malaysia 2 D’Arboretum, Bukit Rambai, Melaka, Malaysia 3 Corresponding author, e-mail: merrilynchan@gmail.com 4 Airlangga University, Faculty of Pharmacy, Surabaya, Indonesia 5 University of Surabaya, Faculty of Pharmacy, Surabaya, Indonesia 6 Universiti Sains Malaysia, School of Chemical Sciences, Penang, Malaysia 7 Sunrich Biotech Sdn. Bhd., Kuala Ketil, Kedah, Malaysia Enhancement of shoot proliferation and evaluation of biot- ic elicitation effects on anatomical changes of pseudo stem and anti-lipid peroxidation activity of Curcuma mangga Val. Abstract: Mango turmeric (Curcuma mangga Val.) con- tains many bioactive compounds that are used for traditional treatment of various health problems and ailments. Slow propagation nature of C. mangga have resulted in short sup- ply to meet the market demand. The longitudinally incised half shoot explants promote 100 % increased of shoot num- ber compared with non-incised shoots with the formation of average 6.6 shoots/explant when they were cultured either vertically or horizontally on MS medium supplemented with 2.0 mg l-1 BA and 0.5 mg l-1 NAA. Biotic elicitation with 3.5 mg l-1 or 5.0 mg l-1 yeast extract or combination of 150 mg l-1 chitosan and 3.5 mg l-1 yeast extract did not promote shoot proliferation but exhibited anti-lipid peroxidation activ- ity slightly lower than quercetin, a potent plant antioxidant flavonoid and butyl hydroxyl toluene (BHT), a commercial preservative agent which is used as a positive control. While absolute ethanol which served as a negative control did not show any anti-lipid peroxidation activity. Biotic elicitation of C. mangga plantlets using similar elicitors resulted in anatom- ical changes of its pseudostem with reduced number of thin lignified xylem cells and the presence of druse suspected to be oxalate crystals inside the cortex cells with delicate cell wall. Key words: anti-lipid peroxidation activity; chitosan; mango turmeric; pseudostem; shoot proliferation; yeast ex- tract Pospeševanje tvorbe poganjkov in ovrednotenje elicitacij- skih učinkov na anatomske spremembe navideznih stebel in proti maščobne peroksidacijske aktivnosti kurkume (Cur- cuma mangga Val.) Izvleček: Kurkuma (Curcuma mangga Val.) vsebuje šte- vilne bioaktivne snovi, ki se uporabljajo pri tradicionalnem obravnavanju številnih zdravstvenih težav in obolenj. Poča- sen način njenega razmnoževanja povzroča njeno pomanj- kanje glede na veliko povpraševanje na trgu. Do polovice vzdolžno zarezani stebelni izsečki so stoodstotno povečali število poganjkov v primerjavi z nezarezanimi s tvorbo pov- prečno 6,6 poganjkov na izseček, če so bili gojeni navpično ali vodoravno v MS gojišču, obogatenim z 2,0 mg l-1 BA in 0,5 mg l-1 NAA. Biotično vzpodbujanje s 3,5 mg l-1 ali 5,0 mg l-1 izvlečka kvasa v kombinaciji z 150 mg l-1 hitozana in 3,5 mg l-1 izvlečka kvasa ni pospešilo tvorbe poganjkov ampak je pokazalo malo manjšo antiperoksidacijsko aktivnost v pri- merjavi s kvercetinom, močnim rastlinskim flavonoidnim an- tioksidatom in butil hidroksi toluenom (BHT), komercialnim zaščitnim sredstvom, ki sta bila uporabljena kot pozitivna kontrola. Uporaba absolutnega etanola kot negativne kon- trole ni pokazala nobene antiperoksidacijske aktivnosti. Bio- tično vzpodbujanje nastanka rastlinic korkume s podobnimi eliciatorji je povzročilo anatomske spremembe v nastajajočih navideznih steblih z zmanjšanjem števila tankih lignificiranih celic ksilema in prisotnostjo kritalnih kopuč, domnevno iz kalcijevega oksalata, v celicah primarne skorje, ki so imele zelo tanko celično steno. Ključne besede: antiperoksidacijska aktivnost za ma- ščobe; hitozan; kurkuma; navidezna stebla; tvorba poganjkov; izvlečki kvasa Acta agriculturae Slovenica, 118/2 – 20222 F. ABRAHAM et al. 1 INTRODUCTION Mango turmeric (Curcuma mangga Val.), a mem- ber of Zingiberaceae family, is commonly used in Ayurvedic, traditional Chinese medicine (TCM) and alternative Malay medicines (Ramadanil et al., 2019). It has been used as food spices, food preservatives and treatment of various health problems and minor ail- ments such as fever, stomach-ache, general debility, and postpartum care (Subositi and Wahyono, 2019; Furmuly and Azemi, 2020). It has been proven to have many health beneficial activities due to some bioactive compounds or secondary metabolites such as the alka- loids, flavonoids, tannins, terpenoid and curcuminoid that are present mainly in the rhizomes (Muchtaromah et al., 2018; Yuandani et al., 2019; Awin et al., 2020; Fi- triastuti et al., 2020; Maryam and Martiningsih, 2021). Conventionally, C. mangga is propagated via its rhizome during the rainy season. However, C. mangga and many other Curcuma species have slow propaga- tion rate and very prone to soil borne diseases and rhizome dormancy (Škorničková, 2007; Pikulthong et al., 2016; Soonthornkalump et al., 2020; Leong-Škornič- ková et al. 2021). This has resulted in insufficient sup- ply of C. mangga to meet the market demand. In vitro culture techniques have been employed as alternative measures for improving the production of plantlets for some Curcuma species such as C. longa (El-Hawaz et al., 2015; Marchant et al., 2021), C. aromatica (Sharmin et al., 2013; Mohanty et al., 2015), C. alismatifolia (Li et al., 2021) and C. zedoaria (Sudipta et al., 2020). Addition of elicitors into culture medium had been reported to enhance the production of useful secondary metabo- lites but high amount of elicitation was found to cause cell or tissue damage of the cultured materials (Espi- nosa-Leal et al., 2018). Our previous study (Abraham et al., 2011) had reported that biotic elicitation using yeast extract and chitosan did not enhance shoot pro- liferation but increased the production of total phenolic compounds which resulted in severe abnormality of the C. mangga in vitro plantlets especially the pseudo stems. Hence, the present study was carried out with three main objectives. Firstly, to determine whether a shoot incision technique could enhance in vitro shoot proliferation and used it as an alternative means for mass production of C. mangga plantlets. Secondly, to determine whether yeast extract and chitosan that have been found to promote production of total phenolic compounds with good free radical scavenging activity could also enhance the anti-lipid peroxidation activity of C. mangga. Thirdly, to study the effect of biotic elici- tation of yeast extract and chitosan on the anatomical structures of C. mangga pseudostem. 2 MATERIALS AND METHODS 2.1 ESTABLISHMENT OF IN VITRO PLANTLETS Young buds of C. mangga of approximately 1.5 cm3 in size, excised from the actively growing rhizomes dur- ing the raining season. They were washed with com- mercial detergent solution (Sunlight®, Unilever, Selan- gor, Malaysia) to remove all soil and organic matters and rinsed under running tap water for 40 min. The cleansed bud explants (1.5 cm3) were then immersed in 70 % ethanol (Chemical Industries (M) Sdn. Bhd., Selangor, Malaysia) for 10 min followed by surface ster- ilization with 20 % Clorox®, a commercial bleach solu- tion containing 5.3 % sodium hypochlorite (The Clorox Company, Oakland, CA), for 20 min. The surface-ster- ilized buds were then rinsed three times with sterile distilled water before being inoculated onto gelled MS (Murashige and Skoog, 1962) medium without any plant growth regulator (PGR). The cultures were incu- bated in a culture room regulated at 25 ± 2 ºC with con- tinuous illumination at average light intensity of 32.5 µmol m-2 s-1 for 6 weeks (Abraham, 2010). 2.2 EFFECT OF SHOOT INCISION ON SHOOT PROLIFERATION The shoot explants of 1.0 cm length were obtained from the 6 weeks old in vitro plantlets. Each shoot ex- plant was vertically cut into half or quarter while the non-incised whole shoot explants were used as control. These shoot explants were then cultured onto shoot proliferation medium, MS supplemented with 2 mg l-1 6-benzylaminopurine (BAP) and 0.5 mg l-1 1-naphtha- lene acetic acid (NAA) (Sigma-Aldrich (M) Sdn. Bhd. Subang Jaya, Malaysia) (Abraham, 2010). Three shoot explants were used for each experimental unit and ten experimental units were used for each explant type. The explants were placed vertically and horizontally on the proliferation medium. The number of shoots produced from each explant with different mode of placement was determined after 6 weeks of culture. 2.3 ANTI-LIPID PEROXIDATION ACTIVITY OF CURCUMA MANGGA 2.3.1 Sample extraction Our previous study (Abraham et al., 2011) have indicated that plantlets cultured in proliferation me- dium added with 3.5 mg l-1 yeast extract, 5.0 mg l-1 yeast Acta agriculturae Slovenica, 118/2 – 2022 3 Enhancement of shoot proliferation and evaluation of biotic elicitation effects on anatomical changes of ... Curcuma mangga Val. extract, and plantlets cultured in proliferation medium supplemented with150 mg l-1 chitosan plus 3.5 mg l-1 yeast extract produced high total phenolic compounds with high free radical scavenging activity (RSA). Hence, these plantlets together with plantlets cultured in shoot proliferation medium (Control) were selected for their anti-lipid peroxidation activity. Two grams (g) of dried sample derived from each treatment condition was grounded into powder form using blender (Philips, Se- langor, Malaysia). Each sample was placed into 250 ml conical flask and soaked with 100 ml methanol (Chemi- cal Industries Sdn. Bhd., Selangor, Malaysia) at 40 ºC for two hours and the soaking process was repeated three times. The methanol extracts from each sample were collected, combined, filtered, and evaporated using ro- tary evaporator machine (EYELA, N-N Series, Japan). 2.3.2 Anti-lipid peroxidation activity Anti-lipid peroxidation activity was determined using modified Ferric thiocyannate (FTC) method (Os- awa and Namiki, 1981). C. mangga sample extract (4 mg) was dissolved in 4 ml absolute ethanol (99.5 %) followed by addition of 8 ml 0.05M phosphate buffer (pH 7.0), 4.1 ml 2.5 % linoleic acid solution (Sigma, Ronkonkoma, NY) and 3.9 ml distilled water. The ex- tract solutions were kept in aluminium foil wrapped vessels and incubated at 40 °C. Butyl hydroxyl toluene (BTH) (Sigma, Ronkonkoma, NY) was used as a posi- tive control and 4 ml absolute ethanol served as a nega- tive control. A volume of 0.1 ml extract solution was added to 9.7 ml 7.5 % ethanol followed by 0.1 ml 30 % ammonium thiocyannate solution and 0.1 ml 0.02 M ferric chloride (Sigma, Ronkonkoma, NY) in 3.5 % HCl (v/v). The reaction was incubated for three minutes un- der dark condition. Three replicates were prepared for each sample and three repetition of spectrophotometer reading were applied for each sample. The absorbance of sample was measured at 500 nm wavelength using UV-Vis spectrophotometer (Mettler Toledo, Colum- bus, Ohio). This procedure was repeated every 24 hours until both the positive and negative controls gave the maximum absorbance. The degree of lipid peroxidation was represented by percentage of oxidized lipid in test- ed samples at the day before the absorbance decreased. 2.4 HISTOLOGY STUDY OF BASAL PSEU- DOSTEM The basal pseudostems of the morphological ab- normal C. mangga plantlets were selected for anatomi- cal study. They were trimmed into approximately 0.5 cm3 in size and were then immersed in FAA solution (40 % formaldehyde: acetic acid glacial: 95 % ethanol = 5: 5: 90) for fixation. During preparation of sections, the fixed tissues were passed through a series of alco- hol solutions, starting with 50 % ethanol/tetra butyl alcohol (TBA) (ethanol 95 %: absolute TBA: water = 4: 1: 5) and finally treated with absolute ethanol/TBA (ethanol 95 %: absolute TBA = 2.5: 7.5). After which the tissues were immersed in mixture solution of TBA and liquid wax with ratio 1:1 at 60-62 ºC for 24 hours. At the embedding stage, the tissues were immersed in liquid wax at 60-62 ºC for 12 hours. After the wax solidified, the wax blocks containing the tissue samples were sliced into thin slices with 15 µm thickness using rotary microtome (Leica, Germany) for slide prepara- tion. Double stained standard technique was used for the preparation of permanent slides. Each sliced section treated with a few drops of safranin after it was placed on the glass slide. This was followed by a few drops of 95 % ethanol to remove the excess safranin. After this, a few drops of Fast Green and 95 % ethanol were added respectively. Finally, one drop of xylene was added on the section. The sliced section was then covered with a glass cover slip and sealed with Shandon Mount (Shan- don, USA) as a mounting agent. The prepared slides were observed under light microscope (Olympus BX- 50, Japan) fitted with coloured video camera (JVC KF- 55B, Japan) and image analysing system (analySIS docu version 3.1, Germany) for determination of cell size and cell morphology. 2.5 STATISTICAL ANALYSIS For the effect of shoot incision and mode of place- ment on shoot proliferation, the experiment was con- ducted in complete randomized block design (CRBD). The data was analysed using two-way ANOVA and the best explant type was determined using Tukey’s HSD test at p ≤ 0.05. The anti-lipid peroxidation activity for all the selected samples was analysed using one-way ANOVA and the comparison of means was determined using to Tukey’s HSD test at p ≤ 0.05 3 RESULTS AND DISCUSSION 3.1 EFFECTS OF SHOOT INCISION ON SHOOT PROLIFERATION Shoot proliferation is an essential step for mass production of in vitro plantlets which is normally car- Acta agriculturae Slovenica, 118/2 – 20224 F. ABRAHAM et al. ried out by inducing multiple shoots formation using plant growth regulators. Many researchers have been using 6-benzylaminopurine (BAP) or benzyl adenine (BA) combination with naphthalene acetic acid (NAA) for multiple shoot induction in Zingiber species (Ab- bas et al., 2011; Zahid et al., 2021) and Curcuma species (Bejoy et al., 2012; Jala, 2012; Ferrari et al., 2016) with the formation of an average of 3 to 5 shoots per ex- plants. Similar result was obtained in the present study whereby an average of 3.3 and 3.7 shoots per shoot ex- plant were formed when the shoot explants were cul- tured horizontally and vertically respectively on MS medium supplemented with 2.0 mg l-1 BA and 0.5 mg l-1 NAA for induction of multiple shoot formation. When the shoot explants were cut longitudinally into half, the number of shoots formed per shoot explant was greatly enhanced with the formation of 6.6 shoots per shoot explant and the number of shots formed were not sig- nificantly different when the explants were placed hori- zontally or vertically. The quarterly cut shoot explants further enhanced the formation of multiple shoots with 7.6 and 8.4 shoots per shoot explant formed when the shoot explants were cultured horizontally and vertically respectively (Table 1). However, the multiple shoots de- rived from the quarterly cut shoot explants were small and became necrotic with copious release of phenolic compounds in the culture medium and eventually re- sulted in death of plantlets after two subculture cycles (6 weeks/cycle). Mode of inoculation either vertically or horizontally was statistically found to have no effect on promoting shoot proliferation in C. mangga. The half shoot explants grow into healthy multiple shoots when cultured on the shoot proliferation medium. Hence, half shoot explants were used for the subsequent stud- ies. Results obtained in the present study clearly dem- onstrated that shoot incision longitudinally could en- hance 100 % increment of shoot production. The en- hancement of shoot proliferation in longitudinally incised shoots was due to increased cut surface area that exposed to the culture medium for better absorp- tion of nutrients and inhibit apical dominance and hence induce more lateral shoot formation (Mok and Ho, 2019). Longitudinally dissecting of shoot explants to promote high multiple shoot formation has become a common practice in propagation of banana (Ahmed Hasan et al., 2020). Hence, shoot incision can be used as an alternative means to promote in vitro shoot multi- plication of C. mangga that resulted in more and faster production of plantlets. 3.2 ANTI-LIPID PEROXIDATION ACTIVITY OF BIOTIC ELICITED CURCUMA MANGGA Our previous study (Abraham et al., 2011) had shown that C. mangga plantlets, cultured in prolifera- tion medium supplemented with 3.5 mg l-1 yeast extract; 5.0 mg l-1 yeast extract or combination of 150 mg l-1 chitosan and 3.5 mg l-1 yeast extract, exhibited high free radical scavenging activity (RSA). These crude extracts together with the positive and negative controls were tested for their anti-lipid peroxidation activity using the ferric thiocyannate (FTC) assay. This method was used to measure the antioxidant activity of the stud- ied samples toward auto peroxidation of the linoleic acid. BHT (Butyl Hydroxyl Toluene) (Sigma, USA) was used as a positive control and 99.5 % ethanol served as a negative control. Positive control is essential for comparing the biotic elicited C. mangga extracts with BHT, a most used antioxidant as preservative in foods containing fats, pharmaceuticals, petroleum products and it inhibits autoxidation of unsaturated organic compounds. The C. mannga extracts used in this study were dissolved in 99.5 % ethanol, hence 99.5 % ethanol was used as the negative control. The negative control is used to show that any positive effects of the tested sam- ples are not due to the ethanol effect. The selected C. mangga extracts together with BHT (positive control) and 99.5 % ethanol (negative control) and quercetin, a potent plant antioxidants flavonoid, exhibited similar pattern of lipid peroxidation activity from day 0 to day Explants Vertical Horizontal No. of shoots/explant ± Se No. of shoots/shoot No. of shoots/explant ± Se No. of shoots/shoot Whole 3.7 ± 0.2 3.7c c 3.3 ± 0.3 3.3 c c Half 3.3 ± 0.3 6.6b b 3.3 ± 0.3 6.6 b b Quarter 1.9 ± 0.2 7.6a a 2.1 ± 0.2 8.4 a a Table 1: Effect of shoot explant incision and inoculation mode on enhancing mult iple shoot formation of C. mangga Mean values within the row followed by same superscript letter indicate not significantly different when the different explant types were vertically or horizontally placed on the culture medium. Mean values within the same column (for parameter No. of shoots/shoot explant) followed by different subscript alphabet indicate significantly different of shoot numbers for different explant types (Tukey, HSD, p < 0.05) Acta agriculturae Slovenica, 118/2 – 2022 5 Enhancement of shoot proliferation and evaluation of biotic elicitation effects on anatomical changes of ... Curcuma mangga Val. six. However, the oxidation of lipid (linoleic acid) of 99.5 % ethanol (negative control) drastically increased started from day seven until day ten while others re- main constant until day 10 (Figure 1). Quercetin is a potent antioxidant flavonoid found in many plant species such as onions, grapes, berries, broccoli, and citrus (David et al., 2016). Even though the three extracts obtained from biotic elicited plantlets of C. mangga showed slightly lower anti-lipid peroxida- tion activity when compared with quercetin, it could be assumed that C. mangga could also a potent antioxidant. Their anti-lipid peroxidation activity was also found to be lower when compared to BHT, a commercial pre- servative agent and a synthetic antioxidant. BHT and quercetin showed similar lipid-peroxidation activity with percentage of oxidized lipid as 29.5 ± 0.1 % and 30.0 ± 0.1 % respectively. Crude extract derived from C. mangga in vitro plantlets cultured in proliferation medium supplemented with 3.5 or 5.0 mg l-1 yeast ex- tract exhibited no difference in anti-lipid peroxidation activity. Plantlets cultured in proliferation medium sup- plemented with 3.5 mg l-1 yeast extract showed better anti-lipid peroxidation activity as compared to extract derived from C. mangga plantlets cultured in prolifera- tion medium supplemented with 150 mg l-1 chitosan plus 3.5 mg l-1 yeast extract. Plantlets cultured in pro- liferation medium without elicitation exhibit the least inhibition of lipid peroxidation (Table 2). It opened an interesting possibility to use C. mangga extracts as food preservative agents or topical medication for treatment of cold sore such as BHT. Besides using as food pre- servative agent, BHT has been used as medicine for the treatment of cold sore (Freeman et al., 1985). 3.3 EFFECT ON ANATOMY OF PSEUDOSTEM Our previous study (Abraham et al., 2011) had shown that the morphology of the plantlets cultured in the proliferation medium without elicitation (Control) were normal. While those cultured in proliferation me- dium with the addition of yeast extracts (3.5 mg l-1 or 5.0 mg l-1 were slightly abnormal with retarded growth. Those plantlets cultured in proliferation medium with combination of 150 mg l-1 chitosan and 3.5 mg l-1 yeast extract were grossly abnormal with chlorosis and glob- ular shaped shoots, fragile leaf petiole and experience severe growth retardation. Results obtained from the histological study of the pseudostem in the present study (Figure 2, Figure 3 & Table 3) clearly showed that these elicited plantlets did affect the anatomical struc- tures of the basal pseudostem of C. mangga and could be linked directly with the morphological character- istics of C. mangga plantlets as reported in Abraham et al. (2011). The plantlets cultured in the shoot pro- liferation medium without elicitation (Control) were consisted of smooth spherical to oval shape cortex cells Sample Oxidized lipid in sample (%) ± se Absolute ethanol (Negative Control) 100 a Quercetine 30.0 ± 0.1 e BHT (Butyl Hydroxyl Toluene) (positive control) 29.5 ± 0.1 e Extract from plantlets cultured without biotic elicitors 33.0 ± 0.2 b Extract of 3.5 mg l-1 yeast-extract treated plantlets 31.6 ± 0.2 d Extract of 5.0 mg l-1 yeast-extract treated plantlets 32.1 ± 0.1 cd Extract of 150 mg l-1 chitosan and 3.5 mg l-1 yeast-extract treated plantlets 32.5 ± 0.1 c Table 2: Percentage of oxidized lipid in tested samples at day 10 using ferric thiocyanate (FTC) assay Mean values within the column followed by same alphabets represent non-significantly different mean values based on Tukey, HSD at p ≤ 0.05 Figure 1: The kinetic of anti-lipid peroxidation of biotic elic- ited C. mangga extracts and controls [BHT (positive control); 95.5 % ethanol (negative control)] Acta agriculturae Slovenica, 118/2 – 20226 F. ABRAHAM et al. and normal xylem cells. The cortex cells did not con- tain any druse, a crystal substance present in plant such as calcium oxalate crystal. The well distributed cortex cells without druse and well-formed lignified xylems (Figure 2 a & b) were able to supply sufficient nutrients to support healthy and normal plantlets. For the plant- lets cultured in medium supplemented with 3.5 mg l-1 yeast extract, the xylems were also well-formed except the lignified layer of these xylem cells were not as thick as the ones observed in the control. Their cortex cells were found to contain druse, which could be seen as black dots inside the cells (Figure 2 c & d). The accu- mulation of druses was more obvious in plantlets that were cultured in proliferation medium supplemented with 5.0 mg l-1 yeast extract (Figure 2 e & f). The size of xylem cells of the control plantlets and that cultured in proliferation added with 3.5 mg l-1 yeast extract was not significantly different, with an average diameter of 32.9 ± 4.0 µm and 40.8 ± 5.1 µm respectively. They were double the size of the xylem cells present in the basal pseudostem of plantlets cultured in proliferation me- dium supplemented with 5.0 mg l-1 yeast extract (15.4 ± 0.8 µm). However, the number of xylem cells found in plantlets cultured with the presence of 3.5 mg l-1 yeast extract was only 22 when compared to that of control with 41 xylem cells. The plantlets cultured in medium supplemented with 5.0 mg l-1 yeast extract produced even much lesser number of not well-lignified xylems, with an average of only 9 xylem cells in each 4x optical magnification field (Table 3). The control plantlets de- veloped bigger cortex cells (43.0 ± 3.0 µm) compared to plantlets cultured with the proliferation medium sup- plemented with 3.5 mg l-1 and 5.0 mg l-1 yeast extract with diameter of cortex cells as 29.9 ± 1.8 µm and 30.8 ± 1.8 µm respectively, which were not significantly dif- ferent in size (Table 3). The smaller size of cortex cells with the presence of druse, and a smaller number of poor lignified xylem cells might not be able to provide sufficient nutrients derived from the culture medium to all parts of plantlets. Hence, it explained the retarded growth with abnormal characteristic of the plantlets that were cultured in proliferation medium supple- mented with 3.5 mg l-1 and 5.0 mg l-1 yeast extract as reported in our previous study. Most of the tissues of the plantlets cultured in proliferation medium supplemented with 150 mg l-1 chitosan and 3.5 mg l-1 yeast extract were fragile, and parts of the section were damaged after dehydration process (Figure 3 a & b). The size of some of the cortex could be measured and determined but not that of xy- lem cells because of cell damage. The number of the xy- lem cells (7 in each 4x optical magnification field) was roughly estimated from the location of the ruptured xylem cells. The size of the cortex cells found in the ba- sal pseudostem of C. mangga plantlets cultured in the shoot proliferation medium supplemented with 150 mg l-1 chitosan and 3.5 mg l-1 yeast extract was found to be not significantly different with those plantlets cultured in proliferation medium added with yeast extract (Ta- ble 3). The small number and fragile xylem cells could greatly reduce the absorption of nutrients, and this was linked directly with the severe growth retardation and gross morphological abnormality of the plantlets cultured in proliferation medium supplemented with yeast extract and chitosan as reported in Abraham et al. (2011). Yeast extract was reported to induce a com- plex stress response resulted in activation of secondary metabolites production (Farjaminezhad and Garoo- si, 2021; Kochan et al., 2017) but resulted in slow cell growth (Hedayati et al., 2021) and cell damage at high concentration (Sánchez-Sampedro et al., 2005). In the present study, the formation of druse (crys- tal inclusion inside cortex cells) was detected in C. mangga plantlets cultured in proliferation medium elic- ited with yeast extract. The druse inside the cells of C. mangga plantlets were suspected to be oxalate crystal because oxalate crystals are the most common crys- tal inclusion of higher plants (Franceschi and Horner, Elicitor treated peudostems Diameter ± s.e. (µm) Estimated No. of xylems/ 4x optical magnification fieldCortex Xylem Control 43.0 ± 3.0 a 32.9 ± 4.0 a 41 5.0 mg l-1 yeast extract 30.8 ± 1.8 b 15.4 ± 0.8 b 9 3.5 mg l-1 yeast extract 29.9 ± 1.8 b 40.8 ± 5.1 a 22 150 mg l-1 chitosan + 3.5 mg l-1 yeast extract 35.2 ± 3.9 b ND 7 Table 3: Summary of cortex and xylem diameter and estimated number of xylem cells in Curcuma mangga plantlets cul- tured in proliferation medium supplemented with biotic elicitors Mean values within the same column followed by different alphabet indicate significantly different values (Tukey, HSD, p ≤ 0.05). ND = Not determine Acta agriculturae Slovenica, 118/2 – 2022 7 Enhancement of shoot proliferation and evaluation of biotic elicitation effects on anatomical changes of ... Curcuma mangga Val. Figure 2: Histology images of basal pseudostem of C. mangga plantlets cultured in proliferation medium (Control) at 4 x opti- cal magnification (a) and 10 x optical magnification (b); Proliferation medium supplemented with 3.5 mgL-1 YE at 4 x optical magnification (c) and 10x optical magnification (d); Proliferation medium supplemented with 5.0 mg l-1 YE at 4 x optical magnification (e) and 10x optical magnification (f) Acta agriculturae Slovenica, 118/2 – 20228 F. ABRAHAM et al. Figure 3: Histology images of basal pseudostem of C. mangga plantlets cultured in proliferation medium supplemented with 150 mg l-1 chitosan and 3.5 mg l-1 yeast extract at 4x optical magnification (a) and 10x optical magnification (b) Acta agriculturae Slovenica, 118/2 – 2022 9 Enhancement of shoot proliferation and evaluation of biotic elicitation effects on anatomical changes of ... Curcuma mangga Val. 1980; Webb, 1999; Nakata, 2012). Even though oxalate crystals were often found in higher plant, the formation and function of oxalate crystals in plants were still un- clear (Webb, 1999). It was proposed that oxalate crystal might be involved in ion balance, plant defence, tissue rigidity and support, detoxification, light accumula- tor, and reflector (Franceschi and Homer, 1980; Doege, 2003). The possible function of oxalate crystal as part of plant defence might be relevant in C. mangga study. The oxalate crystal could not be detected in plantlets cultured in medium without elicitor (control) (Figure 2 a & b). Most part of the tissue of C. mangga plantlets cultured in medium supplemented with 150 mg l-1 chi- tosan and 3.5 mg l-1 yeast extract were damaged dur- ing dehydration process of histology preparation. The damaged tissue indirectly indicate that the cell walls of those plantlets were more delicate than the cell walls of plantlets cultured in medium supplemented with yeast extract or the control. This finding was also supported the observation on the visual morphological character- istic of those plantlets which exhibited brittleness on the abnormal formed shoots cultured in proliferation medium supplemented with 150 mg l-1 chitosan plus 3.5 mg l-1 yeast extract. Chitosan has been used to study the defence mech- anism of plants towards their fungal pathogens. Unlike yeast extract, chitosan is a compound with defined mo- lecular structure (polycationic b-1,4-linked-d-glucosa- mine polymers) which resemble to cell wall component of fungi (Walker–Simmons et al., 1983). The interaction between oligosaccharins and receptors located in the plant membranes results in the production of many plants defence secondary metabolites and many phe- nolic and terpenoid compounds (Kim and Lee 2011; Pang et al 2021). It was found to induce the synthesis of pathogenesis-related (PR) proteins and several de- fence enzymes (such as phenylalanine ammonia lyase and peroxidase) (Riaz et al., 2014). The plant defence compounds, synthesized after chitosan elicitation, have relevant physiological activity, mainly as antioxidants (the polyphenols), might have resulted in cell damage as well. 4 CONCLUSIONS Longitudinally half incised shoot explants and cultured vertically on shoot proliferation medium, MS supplemented with 2.0 mg l-1 benzyl adenine (BA) and 0.5 mg l-1 NAA, promote shoot proliferation by 100 %. This method can be used as an alternative technique for multiplication of the in vitro plantlets of C. mangga which normally propagate at a slow rate. Supplementa- tion of 3.5 or 5.0 mg l-1 yeast extract, or combination of 3.5 mg l-1 yeast extract and 150 mg l-1 chitosan, into the shoot proliferation medium could be used as an al- ternative mode for enhancing anti-lipid peroxidation activity. However, biotic elicitation with 3.5 or 5.0 mg l-1 yeast extract, or combination of 3.5 mg l-1 yeast extract and 150 mg l-1 chitosan did not affect the cortex cell size but a reduction in xylem cell numbers of C. mangga pseudostem. Supplementation of 5.0 mg l-1 yeast extract reduced the size of the xylem cell by more than half as compared to the xylem cell of those cultured in prolif- eration supplemented with 3.5 mg l-1 yeast extract and the control. The addition of 3.5 mg l-1 yeast extract and 150 mg l-1 chitosan into the proliferation medium re- sulted in abnormal anatomy of their pseudostems with damage of the xylem cells. 5 REFERENCES Abbas, M. S., Taha, H. S., Aly, U. I., El-Shabrawi, H. M., Gaber, E. S. I. (2011). In vitro propagation of ginger (Zingiber officinale Rosco). Journal of Genetic Engineering and Biotechnology, 9(2), 165–172. https://doi.org/10.1016/j. jgeb.2011.11.002 Abraham, F. (2010). Elicitation effect on the growth and bio- chemical activities of Curcuma mangga Val. (Zingiberace- ae) in vitro plantlets. Master of Science Thesis, Universiti Sains Malaysia, Penang, Malaysia. Abraham, F., Bhatt, A., Chan, L.K., Indrayanto, G., Sulaiman, S. F. (2011). Effect of yeast extract and chitosan on shoot proliferation, morphology and antioxidant activity of Curcuma mangga in vitro plantlets. African Journal of Biotechnology, 10(40), 7787–7795. https://doi.org/10.5897/ ajb10.1261 Ahmed Hasan, S., Khasim, S.M., Ramudu, J. (2020). Develop- ment of Standard Protocols for In Vitro Regeneration of Some Selected Banana Cultivars (Musa spp.) from India. In: Khasim, S.M., Long, C., Thammasiri, K., Lutken, H. (eds) Medicinal Plants: Biodiversity, Sustainable Utiliza- tion and Conservation. Springer, Singapore. https://doi. org/10.1007/978-981-15-1636-8_45 Awin, T., Mediani, A., Mohd Faudzi, S. M., Maulidiani, Leong, S. W., Shaari, K., Abas, F. (2020). Identification of α-glucosidase inhibitory compounds from Curcuma mangga fractions. International Journal of Food Properties, 23(1), 154–166. https://doi.org/10.1080/10942912.2020.17 16792 Bejoy, M., Dan, M., Anish, N. P., Anjana, R. G., Radhika, B.J., Manesh, K. (2012). Micropropagation of Indian ginger (Curcuma vamana Sabu & Mangaly): A wild relative of tumeric. Biotechnology, 11(6), 333–338. https://doi. org/10.3923/biotech.2012.333.338 David, A.V.A., Arulmoli, R., Parasuraman, S. (2016). Over- views of biological importance of quercetin: a bioactive flavonoid. Pharmacognosy Reviews, 10, 84-89. https://doi. org/10.4103/0973-7847.194044 Acta agriculturae Slovenica, 118/2 – 202210 F. ABRAHAM et al. Doege, S. J. (2003). The role of natural calcium oxalate crys- tals in plant defense against chewing insects. Inquiry: The University of Arkansas Undergraduate Research Journal, 4, 88–94. El-Hawaz, R.F., Bridges, W.C., Adelberg, J.W. (2015). In vitro growth of Curcuma longa L. in response to five min- eral elements and plant density in fed-batch culture sys- tems. PLOS One, 10(4), 1-13. http://doi10.1371/journal- pone.0118912 Espinosa-Leal, C.A., Puente-Garza, C.A., Garcia-Lara, S. (2018). In vitro plant tissue culture: means for production of biological active compounds. Planta, 248, 1-18. https:// doi.org/10.1007/s00425-018-2910-1 Farjaminezhad, R. and Garoosi, G. (2021). Improvement and prediction of secondary metabolites production under yeast extract elicitation of Azadirachta indica cell suspen- sion culture using response surface methodology. AMB Express, 11, 1-16. https://doi.org/10.1186/s13568-021- 01203-x Ferrari, M. P. de S., Antoniazzi, D., Nascimento, A. B., Franz, L. F., Bezerra, C. S., Magalhães, H. M. (2016). Evaluation of new protocols to Curcuma longa micropropagation: A medicinal and ornamental specie. Journal of Medicinal Plants Research, 10(25), 367–376. https://doi.org/10.5897/ jmpr2016.6109 Fitriastuti, D., Julianto, T.S., Iman, A. W. N. (2020). Identifica- tion and heme polymerization inhibition activity (HPIA) assay of ethanolic extract and fraction of Temu Man- gga (Curcuma mangga Val.) rhizome. EKSAKTA: Journal of Sciences and Data Analysis, 1(1), 64–72. https://doi. org/10.20885/eksakta.vol1.iss1.art10 Franceschi, V.R. and Horner, H.T. (1980). Calcium oxalate crystals in plants. The Botanical Review, 46, 361–427. htt- ps://doi.org/10.1007/BF02860532 Freeman DJ, Wenerstrom G, Spruance SL. (1985).Treatment of recurrent herpes simplex labialis with topical butylated hydroxytoluene. Clinical Pharmacology & Therapeutics, 38(1), 56-9. https://doi.org/10.1038/clpt.1985.134 Furmuly, A. M., Azemi, N. (2020). A review on golden spe- cies of Zingiberaceae family: Genus Curcuma. Journal of Critical Reviews, 7(16), 882–895. https://doi.org/10.31838/ jcr.07.16.114 Hedayati, A., Hemmaty, S., Nourozi, E. , Amirsadeghi, A. (2021) Effect of yeast extract on the gene expression and tropane alkaloids production in Atropa belladonna L. hairy roots. Russian Journal of Plant Physiology, 68, 102–109 (2021). https://doi.org/10.1134/S1021443721010088 Jala, A. (2012). Effects of NAA BA and sucrose on shoot in- duction and rapid micropropagation by trimming shoot of Curcuma longa L. International Transaction Journal of Engineering, Management, & Applied Sciences & Technolo- gies, 3(2), 101–109. http://TuEngr.com/V03/101-109.pdf. Leong-Škorničková, J., Soonthornkalump, S., Thongbai, W. (2021). Four new Curcuma species (Zingiberaceae) from Thailand. Blumea, 65, 244-253. https://doi.org/10.3767/ blumea.2021.65.03.09 Kim, Y.O. and Lee, E.J. (2011). Comparison of phenolic com- pounds and the effects of invasive and native species in East Asia: support for the novel weapons hypothesis. Ecological Research, 26, 87–94. https://doi.org/10.1007/ s11284-010-0762-7 Kochan, E., Szymczyk, P., Kuzma, L., Lipert, A., Szymanska, G. (2017). Yeast extract stimulates ginsenoside production in hairy root cultures of American ginseng cultivated in shake flasks and nutrient sprinkle bioreactors. Molecules, 22, 1-15. https://doi.org/10.3390/molecules22060880 Li, Y.Y., Tian, Q.L., Yu, H.W., Lu, L.M. (2021). Progress towards a molecular-level understanding of Curcuma alismatifolia. European Journal of Horticultural Science, 86(3), 328-334. https://doi.org/10.17660/eJHS.2021/86.3.12 Marchant, M.J., Molina, P., Montecinos, M., Guzman, L., Bal- ada, C., Fassio., C., Castro, M. (2021). In vitro propagation of Easter Islaand Curcuma longa from rhizome explants using temporary immersion system. Agronomy, 2121, 1-11. http://doi.org/10.3390/agronomy11112121 Maryam, S. and Martiningsih (2021). Antioxidant activity and total fenol content white saffron (Curcuma mangga Val). IOP Conference Series: Materials Science and Engi- neering, 1115(1), 012081. https://doi.org/10.1088/1757- 899x/1115/1/012081 Mohanty, S., Parida, R., Sandeep,I., Sahoo, S., Nayak, S. (2015). Evaluation of drug yielding potential of micropropagated Curcuma aromatica. International Journal of Pharmacy and Pharmaceutical Sciences, 7(4), 71-76. Mok., P.K. and Ho., W.S. (2019). Rapid in vitro propagation and efficient acclimatization of Neolamarckia cadamba. Asian Journal of Plant Science, 18, 153-163. https://doi. org/10.3923/ajps.2019.153.163 Muchtaromah, B., Ahmad, M., Romaidi, Nazilah, L. A., Naja, N. A. (2018). Antibacterial activity of water and ethanol extract of Allium sativum, Curcuma mangga, and Acorus calamus combination. Journal of Biological Researches, 24(1), 8–15. https://doi.org/10.23869/bphjbr.24.1.20182 Murashige, T. and Skoog, F. (1962). A revised medium for rapid growth and bio assays with tobacco tissue cul- tures. Physiologia Plantarum, 15, 473–497. https://doi. org/10.1111/j.1399-3054.1962.tb08052.x Nakata, P.A. (2012). Plant calcium oxalate crystal formation, function, and its impact on human health. Frontiers of Biology, 7, 254–266. https://doi.org/10.1007/s11515-012- 1224-0 Osawa, T. and Namiki, M. (1981). A novel type of antioxidant isolated from leaf wax of Eucalyptus leaves. Agricultural and Biological Chemistry, 45(3), 735–739. https://doi.org/1 0.1080/00021369.1981.10864583 Pang, Z., Chen, J., Wang, T., Gao, C., Li, Z. Guo, L., Xu, J., Cheng, Yi. (2021). Linking plant secondary metabolites and plant microbiomes: a review. Frontiers in Plant Science, 22, 1-22. https://doi.org/10.3389/fpls.2021.621276 Pikulthong, V., Teerakathiti, T., Thamchaipenet, A., Peya- choknagul, S. (2016). Development of somatic embryos for genetic transformation in Curcuma longa L. and Cur- cuma mangga Valeton & Zijp. Agriculture and Natural Resources, 50(4), 276-285. https://doi.org/10.1016/j.an- res.2015.08.004 Ramadanil, Damry, Rusdi, Hamzah, B., Zubair, M. S. (2019). Traditional usages and phytochemical screenings of se- lected Zingiberaceae from central Sulawesi, Indonesia. Acta agriculturae Slovenica, 118/2 – 2022 11 Enhancement of shoot proliferation and evaluation of biotic elicitation effects on anatomical changes of ... Curcuma mangga Val. Pharmacognosy Journal, 11(3), 505–510. https://doi. org/10.5530/pj.2019.11.80 Riaz, A., Riaz, A., Rattu, A.U.R., Tahir, M.I., Azeem, M. (2014). Phenylalanine ammonia-lyse (PAL) and peroxidase activ- ity in brown rust infected tissues of Pakistani wheat culti- vars. Pakistan Journal of Botany, 46(3), 1101-1107. https:// www.researchgate.net/publication/262896801 Sánchez-Sampedro, M. A., Fernández-Tárrago, J., Corchete, P. (2005). Yeast extract and methyl jasmonate-induced sily- marin production in cell cultures of Silybum marianum (L.) Gaertn. Journal of Biotechnology, 119(1), 60–69. htt- ps://doi.org/10.1016/j.jbiotec.2005.06.012 Sharmin, S.A., Alam, M.J., Sheikh, M.M.I., Zaman, R., Khale- kuzzaman, M., Mondal, S.C., Haque, M.A., Alam, M.F., Alam, I. (2013). Micropropagation and antimicrobial ac- tivity of Curcuma aromatica Salisb., a threatened aromatic medicinal plant. Turkish Journal of Biology, 37, 698-708. http://doi:10.3906/biy-1212-11 Škorničková, J. (2007). Taxonomic Studies in Indian Curcuma L. Ph.D Thesis, Charles University, Prague, Czech Repub- lic. Soonthornkalump, S., Ongsakul, A., Dolaji, A., Leong- Škorničková, J. (2020). Curcuma papilionacea (Zingiber- aceae), an unusual new species from southern Thailand. Phytotaxa, 432(1), 011-016. https://doi.org/10.11646/phy- totaxa.432.1.2 Subositi, D. and Wahyono, S. (2019). Study of the genus cur- cuma in Indonesia used as traditional herbal medicines. Biodiversitas, 20(5), 1356–1361. https://doi.org/10.13057/ biodiv/d200527 Sudipta, J., Asit, R., Ambika, S., Suprava, S., Biswabhusan, D., Basudeba, K., Sanghamitra, N. (2020). Rapid plant re- generation in industrially important Curcuma zedoaria revealing genetic and biochemical fidelity of the regener- ants. 3 Biotech, 10, 17. https://doi.org/10.1007/s13205-019- 2009-9 Walker-Simmons, M., Hadwiger, L., Ryan, C.A. (1983). Chi- tosans and pectic polysaccharides both induce the accu- mulation of the antifungal phytoalexin pisatin in pea pods and antinutrient proteinase inhibitors in tomato leaves. Biochemical and Biophysical Research Communications, 110(1), 194-199. http://10.1016/0006-291x(83)91279-2 Webb, M. A. (1999). Cell-mediated crystallization of calcium oxalate in plants. Plant Cell, 11(4), 751–761. https://doi. org/10.1105/tpc.11.4.751 Yuandani, Yuliasmi, S., Satria, D., Dongoran, R. F., Sinaga, M. S., Marpaung, N. H. A. (2019). Correlation between the phytochemical constituents of Curcuma mangga and its immunomodulatory effect. Rasayan Journal of Chemistry, 12(1), 1–6. https://doi.org/10.31788/RJC.2019.1215050 Zahid, N. A., Jaafar, H. Z., Hakiman, M. (2021). Micropropaga- tion of ginger (Zingiber officinale ‘Bentong’) and evalua- tion of its secondary metabolites and antioxidant activi- ties compared with the conventionally propagated plant. Plants, 10, 1–17. https://doi.org/10.3390/plants10040630 Acta agriculturae Slovenica, 118/2, 1–16, Ljubljana 2022 doi:10.14720/aas.2022.118.2.2538 Original research article / izvirni znanstveni članek Effects of γ-radiation on chickpea (Cicer arietinum) varieties and their tolerance to salinity stress Amal ABDOUN 1, 2, Laila MEKKI 1, Aladdin HAMWIEH 3 and Abdelfattah BADR 4 Received February 01, 2022; accepted June 07, 2022. Delo je prispelo 1. februarja 2022, sprejeto 7. junija 2022 1 Botany and Microbiology Department, Faculty of Science, Suez Canal University, Ismailia, Egypt 2 Corresponding author, e-mail: amal_abdoun@science.suez.edu.eg 3 International Center for Agriculture Research in Dry Land (ICARDA), Aleppo, Syria 4 Botany and Microbiology Department, Faculty of Science, Helwan University, Cairo, Egypt Effects of γ-radiation on chickpea (Cicer arietinum) variet- ies and their tolerance to salinity stress Abstract: Chickpea (Cicer arietinum L.) is a bisexual and self-pollinated legume. It improves the soil fertility through its natural ability to fix atmospheric nitrogen with its symbiotic bacteria. Salinity is one of the most important abiotic stress factors affecting plant growth. γ-radiation is a very effective tool for inducing mutations in many plants. This study evalu- ated the γ-radiation effect on germination, cell division and plant growth of first-generation plants. Seeds of seven chick- pea varieties were irradiated with γ-radiation doses ranging between 50 Gy and 600 Gy. Non-significant differences in germination percentage were recorded for seeds exposed to 50 Gy, 100 Gy, and 200 Gy of γ-radiation in comparison to the corresponding controls except ILC 484. The mitotic index (MI) of root cells increased at the low doses of 50 Gy, 100 Gy and 200 Gy comparing and reduced at the higher doses in all chickpea varieties to the control. All doses of γ-radiation induced a variable range of chromosomal abnormalities; the most common were bridges, laggard chromosomes, stickiness at metaphase, chromosome breaks, micronuclei and binucle- ate cells. The 300 Gy to 600 Gy doses induced degradation of nuclear membranes. The salinity treatments at 25 mM NaCl and 60 mM NaCl reduced seedling’s growth of all cul- tivars. The dose of 100 Gy alleviated the impact of salinity at a concentration of 25 mM NaCl for all varieties, except FLIP 84-188 and FLIP 97-263. The 60 mM NaCl treatment signifi- cantly reduced early growth of all cultivars and its effect was not alleviated by the γ-radiation. Key words: chickpea; γ-radiation; germination; mitotic index; chromosomal aberrations Učinki γ-sevanja na sorte čičerke (Cicer arietinum L.) in nji- hova toleranca na slanostni stres Izvleček: Čičerka (Cicer arietinum L.) je obojespolna samoprašna stročnica. Zaradi sposobnosti vezave atmosfer- skega dušika s simbiontskimi bakterijami izboljšuje rodovi- tnost tal. Slanost je eden izmed najpomembnejših abiotskih stresnih dejavnikov, ki vpliva na rast rastlin. V raziskavi je bil ovrednoten vpliv γ-sevanja na kalitev, celične delitve in rast rastlin F1 generacije čičerke. Semena sedmih sort čičerke so bila obsevana z γ-žarki v jakosti od 50 Gy do 600 Gy. V pri- merjavi s kontrolo so bile zabeležene neznačilne razlike v od- stotku kalitve pri semenih, ki so bila izpostavljena 50 Gy, 100 Gy in 200 Gy γ-sevanja, razen pri sorti ILC 484. V primerjavi s kontrolo se je pri vseh sortah povečal mitotski indeks (MI) celic rastnega vršička korenine, ki so bile obsevane z majhni- mi dozami 50 Gy, 100 Gy in 200 Gy ter zmanjšal pri obsevanju z večjimi dozami. Vse uporabljene doze γ-sevanja so vzpod- budile različen obseg kromosomskih aberacij. Najbolj pogoste so bile mostički, zaostali kromosomi in zlepljeni kromosomi v metafazah ter zlomljeni kromosomi, mikronukleusi in dvoje- drene celice po delitvi. Doze sevanja z jakostjo od 300 Gy do 600 Gy so vzpodbudile razpad jedrnih membran. Slanostna obravnavanja s 25 mM NaCl in 60 mM NaCl so zmanjšala rast sejank vseh sort. Doza obsevanja s 100 Gy je zmajšala učinek slanostnega stresa 25 mM NaCl pri večini sort, razen pri sor- tah FLIP 84-188 in FLIP 97-263. Obravnavanje z dozo 60 mM NaCl je značilno zmanjšalo zgodnjo rast pri vseh sortah in ne- gativnega učinka ni bilo mogoče zmanjašati z γ-obsevanjem. Ključne besede: čičerka; γ-sevanje; kalitev; mitotski in- deks; kromosomske aberacije Acta agriculturae Slovenica, 118/2 – 20222 A. ABDOUN et al. 1 INTRODUCTION Chickpea (Cicer arietinum L.) is an annual her- baceous self-pollinated legume (Ladizinsky & Adler, 1976). Its seeds are a significant source of proteins, car- bohydrates, vitamins, minerals and unsaturated fatty acids (Jimenez-Lopez et al., 2020; Jukanti, Gaur, Gowda, & Chibbar, 2012). It is also important for sustainable agriculture since fixing atmospheric nitrogen via sym- biotic bacteria provides rotational value to subsequent crops and improve the growth and yield of chickpea (Marques et al., 2020). The domesticated chickpea is di- vided into two major distinct chickpea types. One is the “microsperma” or ‘desi’ with small and dark colored seeds with reticulated surface and anthocyanin pig- mented aerial parts and pink or purple flowers (More- no & Cubero, 1978; Van der Maesen, 1972). The other is “macrosperma” or ‘kabuli’ with large seeds with beige seed coat and green aerial parts that lack anthocyanin pigmentation and with white flowers (Upadhyaya et al., 2008). Soil salinity is considered to be one of the most common abiotic stresses controlling agricultural pro- duction around the world by threatening crop yield, and agricultural sustainability (Munns & Gilliham, 2015). It will become progressively more severe over time due to climatic changes, unsuitable irrigation and excessive fertilization (Sun et al., 2018). Salinity affects crops in two ways; by osmotic stress caused by high concentrations of salts in the soil, which make it harder for roots to absorb water, and by ion stress caused by an increased levels of soluble salts within the plant cells caused by exchangeable sodium (Na+) during salinity stress (Munns et al., 2020). The impacts of salt stress on plants vary greatly depending on the type and dose of salt used, environmental factors, plant species, cultivars within a species, and plant development stages (Tabur, Avci, & Özmen, 2021). The osmotic stress induces for- mation of harmful free radicals, including reactive oxy- gen species (ROS) which causes oxidative damages and induces negative effects on the cell functional integrity (Gaafar, Hamouda, & Badr, 2016; Sharma, Jha, Dubey, & PessarakliM, 2012). Tolerance to salinity may conse- quently include variations in responses to these factors (Munns & Tester, 2008). Gamma rays have been used frequently in muta- tion breeding of grain legumes (Abdelfattah Badr, El- Shazly, & Halawa, 2014; Chopra, 2005; El-Azab, Ahmed Soliman, Soliman, & Badr, 2018; Soliman, Elkelish, Souad, Alhaithloul, & Farooq, 2020). Many mutant crop varieties resistant to diseases, cold, salt and with de- sired qualities have been developed using γ-radiation (Chopra, 2005; Gnanamurthy, Mariyammal, Dhana- vel, & Bharathi, 2012; Tshilenge-Lukanda, Kalonji- Mbuyi, Nkongolo, & Kizungu, 2013). Low frequency of γ-radiation may be beneficial, while the treatments with high doses can be harmful to germination, growth rate, vigor, pollen and ovule fertility (Singh, 2005). The γ-radiation has been used for mutation induction in chickpea (Amri-Tiliouine et al., 2018; Joshi-Saha, Red- dy, Petwal, & Dwivedi, 2015; Wani, 2009). Assessment of LD50, lethality, injury, mitotic, and meiotic aberra- tion frequency is required for determining sublethal doses for successful mutation breeding experiments (Bhat & Wani, 2017). At high doses, γ-radiation inter- act with several metabolites and cell components and can induce many cytogenetic mutations such as chro- mosomal rearrangements: chromatid and chromosome bridges, single and double fragments, micronuclei, and delayed chromosomes segregation (Abdelfattah Badr et al., 2014; El-Azab et al., 2018; Nazarenko & Izhbol- din, 2017). Kamble and Patil (2014) reported the rate of cell division (as mitotic index) and induced qualitative and quantitative chromosomal aberration comprising chromosomes, clumping, polyploidy, ring formation, stickiness, chromatin bridges, laggards, multipolarity at anaphase in chickpea. Shah, Mirza, Haq, and Atta (2008) tested the effect of γ-radiation doses ranging from 100 Gy to 1200 Gy in the first generation (M1) of four chickpea genotypes. The germination percentage (GP) reduced gradually with increasing γ-radiation doses from 400 Gy 1200 Gy. Brahmi et al. (2014) reported that the 150 Gy dose was determined as the optimum causing 50 % reduction in seed survival of local chickpea variety, but higher, more than 250 Gy doses caused a slow decline in germination rate; reaching values lower than 10 % for treatments of over 650 Gy. The shoot lengths of nine Cicer species, including three kabuli and four desi types as well as two annual wild species were inhibited with a 200 Gy of γ-radiation and growth curves gradually decreased at the 300 Gy and 400 Gy doses (Toker, Uzun, Canci, & Ceylan, 2005). Melki, Mhamdi, and Achouri (2011) in- vestigated the impact of low doses of γ-radiation from radioactive cobalt on chickpea growth, protein content in leaves and grains harvested from irradiated seeds. The dose of 20 Gy γ-radiation enhanced plant growth by 146.35 % compared to plants grown from non-irra- diated seeds. Sohrabi, Heidari, and Esmailpoor (2008) evaluated the effect of NaCl salinity at different levels (0, 3, 6 and 9 dS m-1) on chickpea and reported reduc- tion of plant growth, pod number, flowers, seed mass and seed number. The objective of this study is to evaluate the poten- tial of induced mutation with γ-radiation in chickpea varieties to alleviate the effects of salinity stress treat- Acta agriculturae Slovenica, 118/2 – 2022 3 Effects of γ-radiation on chickpea (Cicer arietinum) varieties and their tolerance to salinity stress ments on germination, seedling’s growth and cell divi- sion and chromosomes in the M1 chickpea. 2 MATERIALS AND METHODS 2.1 PLANT MATERIAL Seven varieties of chickpea (FLIP 81-71, FLIP 84- 188, FLIP 97-263, ILC 72, ILC 464, ILC 484 and ILC 2555) were obtained from International Center for Ag- ricultural Research in Dry Areas (ICARDA), current- ly hosted by the Agricultural Research Center (Giza, Egypt), and used in this study. 2.2 EXPERIMENTAL SET-UP Air-dried seeds of the chickpea varieties were ir- radiated with 50, 100, 200, 300, 400, 500 and 600 Gy of γ-radiation (dose rate 1.249 kGy h-1). The irradiation was done at the Atomic Energy Center, Nasr City, Cairo, Egypt using irradiation device GSR D1 (Germany). The 50 % lethal irradiation dose (LD50) was determined by calculating the germination and survival percentage. Germination percentage (GP) of irradiated seeds for all doses and their controls was determined on the 7th day of germination. 2.3 CYTOLOGICAL ANALYSIS The chickpea seeds were germinated in Petri dish- es and germinating roots (7 days) were fixed in freshly prepared Carnoy’s fixative for 24 h and kept at 4 °C until used. The fixed roots were washed briefly with distilled water, hydrolyzed with 1N HCl for 8-10 min at 60 °C or for 20-25 min at room temperature. The hydrolyzed roots were washed briefly again with distilled water and stained with the basic fuchsin stain (Germany) for 15 min at 23 °C. The stained root tips were cut off and squashed in a drop of 45 % acetic acid, using coverslip. The slides were examined using the 40 × magnification of the light microscope (KRÜSS, Germany) and five slides were examined for each treatment. Photomicro- graphs of abnormal and control cells were taken with digital camera (Fujifilm FinePix JV100 12 MP Digital Camera, China). The following data were measured and calculated for each treatment using the following equations MI (%) = (Number of cells in mitosis / Number of all examined cells) × 100. Abnormality type (%) = (Number of cells show- ing the specific abnormality type / Total number cells showing all abnormalities) × 100. Total abnormalities (%) = (Total number of cells showing all abnormalities / Total number of all exam- ined cells) × 100. 2.4 MORPHOLOGICAL ANALYSIS For studying the effects of NaCl salinity and of γ-radiation doses and their combination, the treated and control seeds were sown in 30 cm wide plastic boxes containing 30 kg soil (EC = 0.6 ds cm-1) with five replicates during the early winter season of 2018 -2019. Three treatments were applied, first: control plants were not treated by neither γ-radiation nor NaCl; second, the plants were treated with two concentrations of NaCl (25mM and 60mM NaCl); third, the plants were treated with γ-radiation doses (50, 100 and 200 Gy) and NaCl (25 mM NaCl or 60 mM NaCl) as combination treat- ments. Seven vegetative growth parameters were meas- ured after eight weeks from sowing: shoot length (cm), root length (cm), number of leaves per plant, shoot fresh biomass (g), root fresh biomass (g), shoot dry bio- mass (g) and root dry biomass (g). 2.5 STATISTICAL ANALYSIS The data were statistically analyzed using the ANOVA by IBM SPSS Statistics 25 software. The sig- nificant difference between the treatments comparing to the control in the same variety was recorded at an alpha level of 0.05 according to the Least Significant Difference (LSD) test. 3 RESULTS The seed GP calculations revealed no significant variations among varieties under normal condition. The highest GP of 100 % was recorded for ‘ILC 484’ and the lowest GP of (86.7 % (was recorded for ‹ FLIP 84-188’. In general, non-significant differences were recorded for seeds exposed to 50 Gy, 100 Gy, and 200 Gy doses of γ-radiation in comparison with the corresponding controls except ‘ILC 484’. The GP values for the studied varieties slightly decreased at 300 Gy and decreased sig- nificantly at 400 Gy, 500 Gy and 600 Gy of γ-radiation (Figure 2). The maximum inhibitory effect on germina- tion was recorded at 500 Gy for ‘FLIP 81-71’, ‘ILC 72’ (50 %)) and ‘ILC 2555’ (53.3 %) but the lowest value of Acta agriculturae Slovenica, 118/2 – 20224 A. ABDOUN et al. GP for the other four varieties was recorded at the maxi- mum dose of 600 Gy of γ-radiation. Cytological analysis of root meristematic cells, for all chickpea varieties, following seeds exposure to γ-radiation doses from 300 Gy to 600 Gy showed deg- radation of most nuclear membranes. Consequently, the cytological analyses were only made on the roots ex- posed to the γ-radiation doses of 50 Gy, 100 Gy and 200 Gy and the control (Figure 3). The MI and the chromo- somal abnormalities of the treatments are presented in Table 1. The MI showed significant variation between the different doses of gamma irradiation compared to the control in ‘FLIP 81-71’, ‘ILC 464’ and ‘ILC 484’. The lowest MI value (3.4) was scored in ‘FLIP 97-263’ at 50 Gy and 200 Gy while the highest MI value (7.3) was recorded in ‘ILC 72’ at 200 Gy. The results showed an increase in the MI with the increasing doses from 50 Gy to 200 Gy in all the studied chickpea varieties. All the applied doses of γ-radiation induced a variable range of mitotic chromosomal abnormali- ties; bridges, laggard chromosomes, sticky metaphase, chromosome breaks, micronuclei and binucleated cells (Table 1). Five of the studied chickpea varieties (FLIP 84-188, FLIP97-263, ILC 464, ILC 484 and ILC 2555) showed significant difference in bridge percentage be- tween the treatments. The appearance of laggard chro- mosome was more frequent in all treatments of all varieties except the 50 Gy of ‘ILC 2555’. The only sig- nificant difference of laggard chromosome percentage was recorded in ‘ILC 2555’. The highest value of laggard Figure 1: Photos illustrating the germinating seeds of the three chickpea varieties FLIP 81-71. FLIP 84-188, and FLIP 97-263 under control conditions and after exposure to 50, 100, 200, and 300Gy of γ-radiation Acta agriculturae Slovenica, 118/2 – 2022 5 Effects of γ-radiation on chickpea (Cicer arietinum) varieties and their tolerance to salinity stress Figure 2: Germination of seven chickpea varieties under control conditions and after exposure to γ-radiation. The mean val- ues ± standard errors are presented (n = 5) chromosomes (74.5 %) was induced by 200 Gy in ILC 72, while the highest value of sticky metaphase (81.5 %) was induced by 200 Gy in ILC 464. In three varieties, ILC 72, ILC 484 and ILC 2555, a highly significant dif- ference (p = 0.010, 0.000 and 0.001, respectively) in breaks was recorded. While ‘FLIP 81-71’ and ‘ILC 464’ showed significant difference (0.010, 0.002, respective- ly) in the presence of micronuclei, the significant differ- ence of binucleated cells was recorded in ‘FLIP 84-188’ and ‘FLIP 97-263’. The maximum values of the percent- age of micronuclei and binucleated cells were 45.0 % and 27.3 %, respectively, induced by 50 Gy γ-radiation in ‘ILC 464’. The total abnormalities increased with in- creased γ-radiation doses. The highest value of the total abnormalities induced by γ-radiation was 5.4%, which was recorded at 200 Gy dose in ‘ILC 72’ while the mini- mum value (2.0 %) was recorded at the 50 Gy dose in ‘FLIP 81-71’ and ‘FLIP 97-263’. Highly significant dif- ference appeared at the total abnormalities between all chickpea varieties (Table 1). The shoots of all chickpea varieties grew above ground after 12 days of sowing. The seedlings treated with 600 Gy and 500 Gy died after three weeks of sow- ing while seedlings exposed to 400 Gy and 300 Gy died after five weeks of sowing. The effect of the 60 mM NaCl treatment was sig- Acta agriculturae Slovenica, 118/2 – 20226 A. ABDOUN et al. nificantly higher than the 25 mM NaCl treatment in all studied varieties. In ‘FLIP 81-71’, measurements un- der the two salt treatments showed significant reduc- tions in shoot and root traits. Combining low doses of γ-radiation with the low concentration of NaCl treat- ments alleviated the effect of salinity treatments par- ticularly the shoot length, which scored the highest length in plants exposed to combination of 100 Gy of γ-radiation and 25 mM NaCl compared to the control value (Table 2). The data of ‘ILC 72’ illustrated that the combination of 100 Gy of γ-radiation and 25 mM NaCl treatment showed significant increase in shoot length (24.0 ± 0.58 cm) as compared with 25 mM salt treat- ment alone. On the other hand, the γ-radiation doses (50, 100 and 200 Gy) combined with 60 mM NaCl treat- ments showed non-significant increase in shoot length comparing with 60 mM salt treatment alone. The com- bination of γ-radiation doses of 50 Gy, 100 Gy and 200 Gy with the 25 mM NaCl, significantly increased root length (approx. 11 cm) compared to the control plants and plants exposed to 25 mM NaCl only of ‘ILC 464’. The data of ‘ILC 2555’ showed root length reductions Figure 3: Photographs illustrating types of chromosomal abnormalities induced in the root meristems of seedlings of seven chickpea varieties exposed to three doses of γ-radiation (50 Gy,100 Gy and 200 Gy): a) micronucleus induced by 50 Gy; b) bi- nucleated cell induced by 50 Gy; c) severe stickiness and disturbance at metaphase induced by 100 Gy; d) binucleated cell with sticky metaphase induced by 100 Gy;, e, f) stickiness at metaphase induced by 100 Gy; g) micronucleus, lag-chromosome and sticky metaphase induced by 200 Gy; h) multi-bridges with vagrant chromosome induced 50Gy; i) anaphase bridge induced by 200 Gy; j) anaphase bridge and lagging chromosomes induced by 50 Gy; k) anaphase multi-bridges induced by 50 Gy; l) telophase bridge break induced by 200 Gy. Acta agriculturae Slovenica, 118/2 – 2022 7 Effects of γ-radiation on chickpea (Cicer arietinum) varieties and their tolerance to salinity stress Va ri et y Tr ea tm en t To ta l ce lls ex am in ed M I (% ) LS D D iff er en t a bn or m al iti es (% ) To ta l To ta l ab no rm al iti es (% ) O ne -w ay A N O VA Br id ge LS D La g. C hr om . LS D St ic ky m et ap ha se LS D Br ea k LS D M ic ro nu cl ue s LS D Bi nu cl ei LS D LS D F- va lu e p- va lu e FL IP 81 -7 1 C on tr ol 23 55 3. 4 ± 0. 8 - 25 .0 ± 1 2. 6 - 31 .7 ± 1 5. 9 - 43 .3 ± 2 8. 5 - 0. 0 ± 0. 0 - 0. 0 ± 0. 0 - 0. 0 ± 0. 0 - 1. 5 ± 0. 1 - 10 .5 0. 00 4 50 G y 29 63 3. 6 ± 0. 3 ns 18 .8 ± 1 0. 5 ns 17 .4 ± 9 .0 ns 28 .2 ± 1 7. 4 ns 3. 7 ± 3. 7 ns 20 .8 ± 7 .0 * 11 .1 ± 1 1. 1 ns 2. 0 ± 0. 2 ns 10 0 G y 36 55 6. 0 ± 0. 8 * 24 .1 ± 3 .5 ns 36 .2 ± 5 .1 ns 35 .7 ± 2 .5 ns 0. 7 ± 0. 7 ns 2. 0 ± 1. 1 ns 1. 3 ± 1. 3 ns 2. 4 ± 0. 3 * 20 0 G y 39 67 5. 5 ± 0. 3 * 2. 9 ± 2. 9 ns 31 .8 ± 1 4. 1 ns 62 .9 ± 1 9. 4 ns 0. 8 ± 0. 8 ns 1. 2 ± 1. 2 ns 0. 4 ± 0. 4 ns 3. 3 ± 0. 3 * O ne -w ay A N O VA 0. 03 4 0. 29 7 0. 70 4 0. 63 6 0. 56 6 0. 01 0 0. 48 3 FL IP 84 -1 88 C on tr ol 53 85 5. 0 ± 1. 3 - 43 .3 ± 5 .1 - 43 .3 ± 5 .1 - 13 .3 ± 1 0. 2 - 0. 0 ± 0. 0 - 0. 0 ± 0. 0 - 0. 0 ± 0. 0 - 0. 7 ± 0. 1 - 30 .7 0. 00 0 50 G y 30 50 5. 2 ± 0. 3 ns 25 .9 ± 2 .0 * 49 .9 ± 1 .3 ns 7. 3 ± 3. 7 ns 0. 0 ± 0. 0a ns 10 .7 ± 4 .3 ns 6. 2 ± 1. 7 * 2. 1 ± 0. 4 * 10 0 G y 29 00 5. 3 ± 0. 6 ns 7. 8 ± 3. 9 * 30 .9 ± 1 5. 5 ns 46 .7 ± 2 6. 7 ns 1. 8 ± 0. 9 ns 12 .8 ± 6 .4 * 0. 0 ± 0. 0 ns 2. 9 ± 0. 3 * 20 0 G y 41 00 6. 8 ± 0. 8 ns 10 .9 ± 0 .8 * 53 .5 ± 3 .5 ns 28 .5 ± 2 .3 ns 5. 0 ± 2. 5 * 1. 0 ± 1. 0 ns 1. 0 ± 1. 0 ns 4. 8 ± 0. 3 * O ne -w ay A N O VA 0. 48 4 0. 00 0 0. 30 8 0. 29 2 0. 08 7 0. 10 6 0. 00 7 FL IP 97 -2 63 C on tr ol 61 55 3. 6 ± 0. 5 - 18 .8 ± 1 0. 5 - 69 .9 ± 1 0. 5 - 6. 4 ± 3. 2 - 5. 0 ± 5. 0 - 0. 0 ± 0. 0 - 0. 0 ± 0. 0 - 0. 6 ± 0. 1 - 7. 3 0. 01 1 50 G y 35 66 3. 4 ± 0. 4 ns 29 .2 ± 3 .8 ns 46 .9 ± 7 .6 ns 6. 0 ± 3. 0 ns 0. 0 ± 0. 0 ns 10 .6 ± 5 .3 ns 7. 4 ± 2. 2 * 2. 0 ± 0. 2 * 10 0 G y 36 88 3. 7 ± 0. 4 ns 0. 0 ± 0. 0 * 56 .0 ± 5 .4 ns 30 .9 ± 1 .2 * 0. 0 ± 0. 0 ns 13 .2 ± 6 .6 ns 0. 0 ± 0. 0 ns 2. 3 ± 0. 5 * 20 0 G y 22 10 3. 4 ± 0. 1 ns 14 .9 ± 1 .2 ns 62 .0 ± 6 .0 ns 9. 5 ± 1. 5 ns 0. 0 ± 0. 0 ns 0. 0 ± 0. 0 ns 9. 5 ± 1. 5 * 2. 5 ± 0. 4 * O ne -w ay A N O VA 0. 92 3 0. 03 7 0. 26 2 0. 00 0 0. 44 1 0. 11 7 0. 00 1 C on tin ue d on th e ne xt p ag e Ta bl e 1: C yt ol og ic al a na ly si s o f c hi ck pe a ro ot m er is te m sh ow in g m ito tic in de x (M I) , s pe ci fic a nd to ta l c hr om os om al a bn or m al iti es a fte r γ- ra di at io n Acta agriculturae Slovenica, 118/2 – 20228 A. ABDOUN et al. IL C 7 2 C on tr ol 58 90 4. 0 ± 0. 9 - 16 .7 ± 1 6. 7 - 66 .7 ± 1 6. 7 - 0. 0 ± 0. 0 - 0. 0 ± 0. 0 - 0. 0 ± 0. 0 - 16 .7 ± 1 6. 7 - 0. 4 ± 0. 1 - 36 .9 0 50 G y 30 00 5. 0 ± 1. 2 ns 17 .7 ± 9 .2 ns 53 .3 ± 3 .3 ns 11 .8 ± 8 .3 ns 0. 0 ± 0. 0 ns 13 .3 ± 1 3. 3 ns 3. 8 ± 3. 8 ns 3. 2 ± 0. 4 * 10 0 G y 28 00 6. 7 ± 0. 5 ns 19 .8 ± 1 .1 ns 47 .2 ± 2 .8 ns 13 .0 ± 1 .5 ns 7. 6 ± 2. 7 * 11 .0 ± 3 .1 ns 0. 0 ± 0. 0 ns 3. 4 ± 0. 3 * 20 0 G y 46 00 7. 3 ± 1. 5 ns 7. 1 ± 0. 8 ns 74 .5 ± 1 .9 ns 10 .1 ± 1 .7 ns 4. 4 ± 0. 2 ns 4. 0 ± 0. 1 ns 0. 0 ± 0. 0 ns 5. 4 ± 0. 5 * O ne -w ay A N O VA 0. 18 4 0. 78 9 0. 18 5 0. 20 6 0. 01 0. 52 3 0. 50 3 IL C 46 4 C on tr ol 34 41 3. 7 ± 0. 4 - 26 .1 ± 3 .9 - 21 .1 ± 1 0. 6 - 52 .8 ± 1 2. 1 - 0. 0 ± 0. 0 - 0. 0 ± 0. 0 - 0. 0 ± 0. 0 - 0. 3 ± 0. 2 - 42 .4 0 50 G y 30 65 4. 5 ± 0. 3 ns 0. 0 ± 0. 0 * 18 .5 ± 1 8. 5 ns 9. 2 ± 4. 9 * 0. 0 ± 0. 0 ns 45 .0 ± 1 1. 7 * 27 .3 ± 1 1. 7 * 2. 2 ± 0. 3 * 10 0 G y 33 50 7. 0 ± 0. 3 * 17 .7 ± 1 .5 * 52 .9 ± 1 0. 6 ns 30 .3 ± 8 .5 ns 1. 0 ±1 .0 ns 3. 2 ± 3. 2 ns 10 .5 ± 9 .1 ns 4. 4 ± 0. 3 * 20 0 G y 39 10 4. 9 ± 0. 3b * 0. 0 ± 0. 0 * 18 .5 ± 1 .0 ns 81 .5 ± 1 .0 * 0. 0 ± 0. 0 ns 0. 0 ± 0. 0 ns 0. 0 0. 0 ns 2. 2 ± 0. 2 * O ne -w ay A N O VA 0. 00 1 0. 00 0 0. 19 5 0. 00 1 0. 44 1 0. 00 2 0. 09 4 IL C 48 4 C on tr ol 35 29 3. 9 ± 0. 3 - 38 .1 ± 1 4. 3 - 38 .9 ± 2 0. 0 - 14 .7 ± 9 .8 - 0. 0 ± 0. 0 - 8. 3 ± 8. 3 - 0. 0 ± 0. 0 - 0. 5 ± 0. 1 - 20 .8 0 50 G y 42 00 6. 4 ± 0. 3 * 2. 8 ± 2. 8 * 40 .4 ± 2 9. 9 ns 5. 7 ± 3. 0 ns 0. 0 ± 0. 0 ns 25 .6 ± 1 4. 4 ns 25 .6 ± 1 7. 9 ns 2. 8 ± 0. 4 * 10 0 G y 28 00 5. 4 ± 0. 4 * 1. 5 ± 1. 5 * 55 .7 ± 1 .6 ns 13 .0 ± 2 .1 ns 11 .7 ± 1 .0 * 1. 2 ± 1. 2 ns 16 .9 ± 3 .5 ns 2. 9 ± 0. 4 * 20 0 G y 31 00 5. 4 ± 0. 2 * 20 .3 ± 0 .6 ns 40 .8 ± 0 .6 ns 14 .5 ± 0 .1 ns 10 .6 ± 0 .5 * 5. 0 ± 0. 2 ns 8. 9 ± 0. 4 ns 5. 1 ± 0. 6 * O ne -w ay A N O VA 0. 00 4 0. 02 4 0. 89 9 0. 60 2 0. 00 0 0. 25 2 0. 30 1 IL C 25 55 C on tr ol 63 45 4. 5 ± 0. 2 - 38 .8 ± 3 .6 - 61 .2 ± 3 .6 - 0. 0 ± 0. 0 - 0. 0 ± 0. 0 - 0. 0 ± 0. 0 - 0. 0 ± 0. 0 - 0. 5 ± 0. 1 - 16 .8 0. 00 1 50 G y 35 40 4. 6 ± 0. 3 ns 20 .8 ± 1 1. 0 ns 0. 0 ± 0. 0 * 26 .7 ± 1 8. 7 ns 0. 0 ± 0. 0 ns 41 .7 ± 2 1. 3 * 10 .8 ± 5 .8 * 2. 6 ± 0. 3 * 10 0 G y 30 50 4. 5 ± 0. 0 ns 9. 6 ± 0. 8 * 51 .2 ± 1 .2 ns 39 .2 ± 1 .8 * 0. 0 ± 0. 0 ns 0. 0 ± 0. 0 ns 0. 0 ± 0. 0 ns 2. 8 ± 0. 2 * 20 0 G y 41 50 4. 7 ± 0. 3 ns 9. 4 ± 4. 8 * 36 .5 ± 9 .6 * 41 .7 ± 4 .4 * 7. 5 ± 1. 8 * 0. 0 ± 0. 0 ns 4. 9 ± 2. 4 ns 3. 9 ± 0. 5 * O ne -w ay A N O VA 0. 82 5 0. 03 3 0. 00 0. 05 4 0. 00 1 0. 05 7 0. 12 M ea n va lu es ± S E (n = 5 ), an d p- va lu es (A N O VA ) a re p re se nt ed . ns = n on -s ig ni fic an t d iff er en ce , * d en ot es si gn ifi ca nt d iff er en ce a ga in st th e co nt ro l i n th e sa m e va ri et y at p < 0 .0 5 ac co rd in g to L SD te st . A bb re vi at io ns : M I, M ito tic In de x, L ag . C hr om ., La gg ar d ch ro m os om e, St ic ky m et ap ha se , M ic ro nu cl eu Acta agriculturae Slovenica, 118/2 – 2022 9 Effects of γ-radiation on chickpea (Cicer arietinum) varieties and their tolerance to salinity stress Va ri et y Tr ea tm en t Sh oo t l en gt h (c m ) R oo t l en gt h (c m ) N o. o f l ea ve s / p la nt Fr es h bi om as s ( g) D ry b io m as s ( g) LS D LS D LS D Sh oo t LS D R oo t LS D Sh oo t LS D R oo t LS D FL IP 81 -7 1 C on tr ol 24 .3 3 ± 0. 92 - 8. 77 ± 0 .1 5 - 25 .0 0 ± 3 .2 2 - 1. 33 ± 0 .0 4 - 0. 45 ± 0 .0 3 - 0. 31 ± 0 .0 2 - 0. 07 ± 0 .0 1 - 25 m M N aC l s al t 19 .3 3 ± 1. 86 * 8. 07 ± 0 .8 7 ns 20 .3 3 ± 3. 84 ns 0. 9 7 ± 0 .1 1 * 0. 49 ± 0 .0 9 ns 0. 28 ± 0 .0 03 ns 0. 06 ± 0 .0 06 ns 60 m M N aC l s al t 13 .8 3 ± 1. 59 * 4. 67 ± 0 .5 8 * 6. 00 ± 0 .5 8 * 0. 53 ± 0 .0 88 * 0. 30 ± 0 .0 58 ns 0. 21 ± 0 .0 09 * 0. 03 ± 0 .0 03 * 50 G y+ 25 m M N aC l 23 .0 0 ± 1. 53 ns 8. 50 ± 0 .3 5 ns 16 .6 7 ± 0. 88 * 1. 43 ± 0 .0 75 ns 0. 55 ± 0 .0 29 ns 0. 33 ± 0 .0 12 ns 0. 08 ± 0 .0 03 ns 10 0 G y+ 25 m M N aC l 25 .0 0 ± 1. 53 ns 7. 03 ± 0 .6 1 * 23 .0 0 ± 1. 16 ns 1. 30 ± 0 .0 58 ns 0. 38 ± 0 .1 03 ns 0. 36 ± 0 .0 73 ns 0. 03 ± 0 .0 03 * 20 0 G y+ 25 m M N aC l 20 .3 3 ± 0. 88 ns 7. 70 ± 0 .4 9 ns 23 .3 3 ± 0. 88 ns 1. 10 ± 0 .0 58 * 0. 26 ± 0 .0 06 * 0. 31 ± 0 .0 39 ns 0. 03 ± 0 .0 00 * 50 G y+ 60 m M N aC l 13 .3 3 ± 2. 40 * 6. 10 ± 0 .3 1 * 7. 67 ± 1 .2 0 * 0. 78 ± 0 .0 12 * 0. 36 ± 0 .0 24 ns 0. 28 ± 0 .0 07 ns 0. 03 ± 0 .0 02 * 10 0 G y+ 60 m M N aC l 17 .3 3 ± 1. 20 * 5. 50 ± 0 .6 8 * 14 .0 0 ± 1. 16 * 1. 05 ± 0 .0 53 * 0. 24 ± 0 .0 23 * 0. 33 ± 0 .0 12 ns 0. 03 ± 0 .0 00 * 20 0 G y+ 60 m M N aC l 17 .6 7 ± 0. 33 * 4. 93 ± 0 .3 0 * 12 .0 0 ± 11 .1 6 * 0. 92 ± 0 .0 09 * 0. 25 ± 0 .0 06 * 0. 28 ± 0 .0 38 ns 0. 03 ± 0 .0 00 * FL IP 84 -1 88 C on tr ol 21 .1 7± 0 .1 7 - 10 .8 3 ± 1. 09 - 22 .0 0 ± 3. 06 - 1. 38 ± 0 .0 34 - 0. 50 ± 0 .0 24 - 0. 22 ± 0 .0 19 - 0. 05 ± 0 .0 03 - 25 m M N aC l s al t 19 .1 7 ± 2. 40 ns 6. 50 ± 1 .5 3 * 20 .0 0 ± 5. 03 ns 0. 62 ± 0 .1 00 * 0. 32 ± 0 .1 07 * 0. 18 ± 0 .0 15 ns 0. 03 ± 0 .0 13 ns 60 m M N aC l s al t 20 .1 7 ± 0. 93 ns 8. 67 ± 0 .4 4 * 9. 67 ± 1 .2 0 * 1. 04 ± 0 .0 28 ns 0. 32 ± 0 .0 23 * 0. 21 ± 0 .0 18 ns 0. 03 ± 0 .0 00 * 50 G y+ 25 m M N aC l 9. 83 ± 0 .9 3 * 4. 87 ± 0 .3 2 * 17 .3 3 ± 1. 45 ns 0. 52 ± 0 .1 08 * 0. 14 ± 0 .0 09 * 0. 16 ± 0 .0 22 * 0. 01 ± 0 .0 01 * 10 0 G y+ 25 m M N aC l 15 .0 0 ± 2. 89 * 6. 50 ± 0 .5 8 * 19 .6 7 ± 1. 20 ns 0. 97 ± 0 .2 74 * 0. 31 ± 0 .0 22 * 0. 16 ± 0 .0 23 * 0. 02 ± 0 .0 05 * 20 0 G y+ 25 m M N aC l 11 .0 0 ± 1. 16 * 5. 20 ± 0 .2 5 * 11 .6 7 ± 0. 88 * 0. 62 ± 0 .0 79 * 0. 24 ± 0 .0 07 * 0. 12 ± 0 .0 06 * 0. 02 ± 0 .0 01 * 50 G y+ 60 m M N aC l 12 .0 0 ± 1. 16 * 4. 83 ± 0 .1 5 * 7. 00 ± 1 .1 6 * 0. 67 ± 0 .0 64 * 0. 19 ± 0 .0 26 * 0. 19 ± 0 .0 24 ns 0. 02 ± 0 .0 01 * 10 0 G y+ 60 m M N aC l 12 .6 7 ± 0. 88 * 6. 43 ± 0 .4 1 * 8. 33 ± 0 .3 3 * 0. 72 ± 0 .0 51 * 0. 29 ± 0 .0 15 * 0. 13 ± 0 .0 09 * 0. 03 ± 0 .0 01 * 20 0 G y+ 60 m M N aC l 16 .3 3 ± 0. 88 * 9. 40 ± 0 .2 1 ns 9. 33 ± 0 .6 7 * 0. 62 ± 0 .1 29 * 0. 35 ± 0 .0 18 * 0. 23 ± 0 .0 34 ns 0. 04 ± 0 .0 03 ns Ta bl e 2: M or ph ol og ic al m ea su re m en ts o f c hi ck pe a pl an ts fo llo w in g se ed e xp os ur e to N aC l t re at m en ts a lo ne a nd in c om bi na tio n w ith γ -r ad ia tio n FL IP 97 -2 63 C on tr ol 26 .8 3 ± 1. 92 - 5. 90 ± 0 .8 6 - 21 .6 7 ± 3. 28 3 - 2. 10 ± 0 .0 48 - 0. 42 ± 0 .0 52 - 0. 64 ± 0 .0 18 - 0. 07 ± 0 .0 09 - 25 m M N aC l s al t 24 .6 7 ± 0. 93 ns 4. 93 ± 0 .9 8 ns 18 .6 7 ± 2. 72 8 ns 0. 65 ± 0 .0 36 * 0. 21 ± 0 .0 64 * 0. 33 ± 0 .0 24 * 0. 04 ± 0 .0 07 * 60 m M N aC l s al t 19 .6 7 ± 2. 60 * 3. 90 ± 0 .3 1 * 12 .3 3 ± 0. 88 2 * 0. 75 ± 0 .1 24 * 0. 24 ± 0 .0 45 * 0. 30 ± 0 .0 27 * 0. 04 ± 0 .0 07 * 50 G y+ 25 m M N aC l 18 .8 3 ± 0. 60 * 4. 27 ± 0 .5 0 * 18 .3 3 ± 3. 75 6 ns 1. 02 ± 0 .0 89 * 0. 30 ± 0 .0 72 ns 0. 45 ± 0 .0 57 * 0. 05 ± 0 .0 03 ns 10 0 G y+ 25 m M N aC l 18 .3 3 ± 1. 67 * 8. 07 ± 0 .1 2 * 14 .6 7 ± 1. 76 4 * 1. 24 ± 0 .1 21 * 0. 44 ± 0 .0 35 ns 0. 51 ± 0. 03 5 * 0. 07 ± 0 .0 09 ns 20 0 G y+ 25 m M N aC l 16 .6 7 ± 2. 40 * 3. 87 ± 0 .3 4 * 13 .3 3 ± 1. 76 4 * 1. 05 ± 0 .1 87 * 0. 28 33 ± 0. 02 2 * 0. 48 ± 0 .0 55 * 0. 05 ± 0 .0 02 ns 50 G y+ 60 m M N aC l 16 .0 0 ± 1. 53 * 3. 60 ± 0 .2 3 * 18 .6 7 ± 0. 88 2 ns 0. 76 ± 0 .0 67 * 0. 26 ± 0 .0 21 * 0. 33 ± 0 .0 13 * 0. 05 ± 0 .0 01 ns 10 0 G y+ 60 m M N aC l 12 .3 3 ± 1. 45 * 7. 97 ± 0 .2 4 * 13 .0 0 ± 1. 15 5 * 0. 66 ± 0 .0 23 * 0. 43 ± 0 .0 32 ns 0. 33 ± 0 .0 21 * 0. 06 ± 0 .0 06 ns 20 0 G y+ 60 m M N aC l 14 .0 0 ± 1. 53 * 5. 17 ± 0 .5 24 ns 7. 33 ± 1 .2 02 * 0. 88 ± 0 .2 09 * 0. 38 67 ± 0. 03 9 ns 0. 40 ± 0 .0 73 * 0. 06 ± 0 .0 07 ns Acta agriculturae Slovenica, 118/2 – 202210 A. ABDOUN et al. Ta bl e 2 co nt in ue d IL C 7 2 C on tr ol 24 .3 0 ± 2. 00 - 3. 67 ± 0 .4 4 - 17 .0 0 ± 1. 00 - 0. 68 ± 0 .0 9 - 0. 06 ± 0 .0 03 - 0. 30 ± 0 .0 12 a - 0. 01 1 ± 0. 00 1- 25 m M N aC l s al t 18 .5 0 ± 2. 08 * 4. 50 ± 1 .5 3 ns 15 .3 3 ± 0. 88 ns 0. 77 ± 0 .0 9 ns 0. 17 ± 0 .0 64 * 0. 3± 0 .0 15 ns 0. 02 9 ± 0. 01 1 * 60 m M N aC l s al t 14 .3 3 ± 0. 88 * 3. 43 ± 0 .3 5 ns 7. 67 ± 0 .8 8 * 0. 74 ± 0 .0 7 ns 0. 10 ± 0 .0 08 ns 0. 32 ± 0 .0 15 ns 0. 01 7 ± 0. 00 1n s 50 G y+ 25 m M N aC l 18 .3 3 ± 1. 20 * 4. 90 ± 0 .2 1 ns 20 .3 3 ± 1. 45 ns 0. 78 ± 0 .0 4 ns 0. 14 ± 0 .0 19 * 0. 30 ± 0 .0 18 ns 0. 02 3 ± 0. 00 3* 10 0 G y+ 25 m M N aC l 24 .0 0 ± 0. 58 ns 5. 37 ± 0 .5 9 ns 19 .0 0 ± 3. 61 ns 1. 15 ± 0 .1 1 * 0. 07 ± 0 .0 12 ns 0. 46 ± 0 .0 44 * 0. 01 1 ± 0. 00 2n s 20 0 G y+ 25 m M N aC l 20 .6 7 ± 1. 20 ns 5. 30 ± 0 .4 7 ns 18 .6 7 ± 0. 88 ns 0. 96 ± 0 .0 9 * 0. 10 ± 0 .0 07 ns 0. 39 ± 0 .0 35 * 0. 01 6 ± 0. 00 1n s 50 G y+ 60 m M N aC l 14 .8 7 ± 0. 59 * 4. 07 ± 0 .5 8 ns 12 .0 0 ± 0. 58 * 0. 41 ± 0. 01 ns 0. 08 ± 0 .0 09 ns 0. 16 ± 0 .0 06 * 0. 01 4 ± 0. 00 2n s 10 0 G y+ 60 m M N aC l 13 .5 7 ± 0. 96 * 6. 20 ± 0 .4 6 * 10 .3 3 ± 0. 88 * 0. 36 ± 0 .0 3 ns 0. 10 ± 0 .0 09 ns 0. 14 ± 0 .0 15 * 0. 01 7 ± 0. 00 1n s 20 0 G y+ 60 m M N aC l 13 .4 3 ± 0. 74 * 4. 57 ± 0 .5 2 ns 11 .6 7 ± 0. 33 * 0. 38 ± 0 .0 4 ns 0. 09 ± 0 .0 03 ns 0. 15 ± 0 .0 18 * 0. 01 4 ± 0. 00 1n s IL C 4 64 C on tr ol 28 .7 3 ± 1. 47 - 9. 8 3 ± 0. 73 - 22 .6 7 ± 2. 67 - 1. 79 ± 0 .2 43 - 0. 43 ± 0 .1 23 - 0. 48 ± 0 .0 66 - 0. 06 ± 0 .0 12 - 25 m M N aC l s al t 24 .1 7 ± 0. 44 * 9. 00 ± 0 .2 9 ns 16 .3 3 ± 2. 03 * 1. 06 ± 0 .0 45 * 0. 31 ± 0 .0 09 ns 0. 29 ± 0 .0 12 * 0. 05 ± 0 .0 01 ns 60 m M N aC l s al t 20 .0 0 ± 1. 16 * 6. 33 ± 0 .6 0 * 10 .3 3 ± 1. 45 * 0. 98 ± 0 .0 15 * 0. 26 ± 0 .0 15 * 0. 26 ± 0 .0 04 * 0. 04 ± 0 .0 03 * 50 G y+ 25 m M N aC l 26 .0 0 ± 0. 58 ns 11 .1 7 ± 0. 44 ns 18 .0 0 ± 1. 16 ns 1. 35 ± 0. 03 5 * 0. 59 ± 0 .0 09 ns 0. 37 ± 0 .0 09 * 0. 09 ± 0 .0 01 * 10 0 G y+ 25 m M N aC l 28 .6 7 ± 0. 88 ns 11 .5 0 ± 0. 76 ns 22 .0 0 ± 0. 56 ns 1. 98 ± 0 .0 79 ns 0. 60 ± 0 .0 13 * 0. 53 ± 0 .0 21 ns 0. 09 ± 0 .0 02 * 20 0 G y+ 25 m M N aC l 18 .0 0 ± 1. 16 * 10 .9 7 ± 0. 26 ns 19 .6 7 ± 0. 88 ns 1. 03 ± 0 .0 91 * 0. 60 ± 0 .0 03 * 0. 28 ± 0 .0 25 * 0. 09 ± 0 .0 00 * 50 G y+ 60 m M N aC l 17 .5 0 ± 2. 02 * 7. 50 ± 1 .2 6 * 15 .0 0 ± 4. 73 * 0. 92 ± 0 .0 92 * 0. 36 ± 0 .1 02 ns 0. 25 ± 0 .0 25 * 0. 05 ± 0 .0 13 ns 10 0 G y+ 60 m M N aC l 18 .8 3 ± 0. 73 * 6. 50 ± 0 .2 9 * 10 .6 7 ± 0. 88 * 0. 95 ± 0 .0 37 * 0. 26 ± 0 .0 09 ns 0. 26 ± 0 .0 10 * 0. 04 ± 0 .0 03 * 20 0 G y+ 60 m M N aC l 17 .0 0 ± 1. 16 * 6. 83 ± 0 .4 4 * 10 .6 7 ± 0. 88 * 0. 91 ± 0 .0 39 * 0. 28 ± 0 .0 09 ns 0. 25 ± 0 .0 11 * 0. 05 ± 0 .0 02 * IL C 4 84 C on tr ol 19 .6 7 ± 1. 01 - 4. 27 ± 0 .5 6 - 21 .0 0 ± 2. 52 - 1. 44 ± 0 .2 3 - 0. 26 ± 0 .0 4 - 0. 49 ± 0 .0 8 - 0. 06 7 ± 0. 00 9 - 25 m M N aC l s al t 17 .0 0 ± 0. 58 ns 6. 93 ± 0 .1 2 * 33 .0 0 ± 2. 03 ns 1. 79 ± 0 .1 0 ns 0. 42 ± 0 .0 1 * 0. 6 ± 0. 03 ns 0. 10 3 ± 0. 00 3 * 60 m M N aC l s al t 14 .0 0 ± 0. 58 * 9. 57 ± 0 .5 4 * 8. 33 ± 0 .8 8 * 1. 3 ± 0. 12 ns 0. 47 ± 0 .0 5 * 0. 35 ± 0 .0 8 ns 0. 02 8 ± 0. 00 2 * 50 G y+ 25 m M N aC l 25 .5 0 ± 1. 32 * 6. 47 ± 0 .3 5 * 25 .0 0 ± 1. 73 ns 1. 89 ± 0 .1 6 ns 0. 14 ± 0 .0 1 * 0. 64 ± 0 .0 6 ns 0. 02 2 ± 0. 00 1 * 10 0 G y+ 25 m M N aC l 21 .3 3 ± 1. 20 ns 8. 57 ± 0 .3 5 * 24 .3 3 ± 1. 76 ns 1. 53 ± 0 .2 4 ns 0. 26 ± 0 .0 2 ns 0. 45 ± 0 .0 8 ns 0. 03 4 ± 0. 00 3 * 20 0 G y+ 25 m M N aC l 24 .0 0 ± 1. 15 * 8. 50 ± 0 .3 8 * 22 .0 0 ± 1. 15 ns 1. 72 ± 0 .0 5 ns 0. 28 ± 0 .0 2 ns 0. 58 ± 0 .0 2 ns 0. 03 6 ± 0. 00 3 * 50 G y+ 60 m M N aC l 15 .3 3 ± 1. 45 * 6. 17 ± 0 .6 7 * 13 .0 0 ± 1. 15 * 1. 18 ± 0 .0 4 ns 0. 28 ± 0 .0 2 ns 0. 4 ± 0. 02 ns 0. 02 7 ± 0. 00 2 * 10 0 G y+ 60 m M N aC l 14 .3 3 ± 1. 45 * 7. 73 ± 0 .2 4 * 10 .6 7 ± 1. 20 * 0. 81 ± 0 .3 6 ns 0. 21 ± 0 .0 1 ns 0. 27 ± 0 .1 2 ns 0. 02 7 ± 0. 00 1 * 20 0 G y+ 60 m M N aC l 16 .0 0 ± 1. 15 * 6. 13 ± 0 .4 9 * 12 .6 7 ± 1. 45 * 0. 83 ± 0 .3 7 ns 0. 24 ± 0 .0 2 ns 0. 28 ± 0 .1 3 ns 0. 03 1 ± 0. 00 3 * C on tin ue d on th e ne xt p ag e Acta agriculturae Slovenica, 118/2 – 2022 11 Effects of γ-radiation on chickpea (Cicer arietinum) varieties and their tolerance to salinity stress IL C 25 55 C on tr ol 22 .8 3 ± 0. 73 - 10 .1 7 ± 1. 01 - 24 .6 7 ± 0. 88 - 1. 26 ± 0 .1 30 - 0. 33 ± 0 .1 10 - 0. 24 ± 0 .0 45 - 0. 03 ± 0 .0 06 - 25 m M N aC l s al t 23 .8 3 ± 1. 59 ns 8. 67 ± 0. 44 ns 20 .3 3 ± 1. 45 * 1. 35 ± 0 .2 31 ns 0. 22 ± 0 .0 92 ns 0. 25 ± 0 .0 66 ns 0. 01 ± 0 .0 03 * 60 m M N aC l s al t 21 .1 7 ± 0. 60 ns 7. 33 ± 0. 60 * 10 .3 3 ± 0. 88 * 0. 97 ± 0 .0 72 ns 0. 25 ± 0 .0 65 ns 0. 19 ± 0 .0 12 ns 0. 03 ± 0 .0 06 ns 50 G y+ 25 m M N aC l 23 .1 7 ± 0. 60 ns 10 .7 3 ± 0. 19 ns 25 .6 7 ± 1. 76 ns 1. 04 ± 0 .0 22 ns 0. 37 ± 0 .0 72 ns 0. 24 ± 0 .0 12 ns 0. 04 ± 0 .0 03 * 10 0 G y+ 25 m M N aC l 25 .3 3± 2 .1 9 ns 7. 90 ± 0. 70 * 24 .6 7 ± 0. 88 ns 1. 48 ± 0 .2 31 ns 0. 49 ± 0 .1 98 ns 0. 34 ± 0 .0 23 * 0. 05 ± 0 .0 06 * 20 0 G y+ 25 m M N aC l 18 .5 0 ± 1. 26 * 5. 83 ± 0. 52 * 11 .6 7 ± 0. 88 * 1. 04 ± 0 .1 34 ns 0. 35 ± 0 .0 45 ns 0. 21 ± 0 .0 21 ns 0. 03 ± 0 .0 03 ns 50 G y+ 60 m M N aC l 20 .0 0 ± 2. 08 ns 9. 07 ± 0. 23 ns 11 .6 7 ± 0. 88 * 1. 24 ± 0 .0 84 ns 0. 26 ± 0 .0 95 ns 0. 26 ± 0 .0 18 ns 0. 03 ± 0 .0 06 ns 10 0 G y+ 60 m M N aC l 20 .0 0 ± 1. 15 ns 6. 60 ± 0. 47 ns 10 .6 7 ± 0. 67 * 1. 06 ± 0 .0 54 ns 0. 39 ± 0 .0 42 ns 0. 26 ± 0 .0 09 ns 0. 04 ± 0 .0 03 ns 20 0 G y+ 60 m M N aC l 16 .3 3 ± 1. 20 * 3. 97 ± 0. 29 ns 10 .0 0 ± 1. 00 * 0. 67 ± 0 .0 21 * 0. 28 ± 0 .0 25 ns 0. 18 ± 0 .0 06 ns 0. 03 ± 0 .0 00 ns M ea n va lu es ± S E ar e pr es en te d (n = 5 ). ns = n on -s ig ni fic an t d iff er en ce , * d en ot es si gn ifi ca nt d iff er en ce a ga in st th e co nt ro l i n th e sa m e va ri et y at p < 0 .0 5 ac co rd in g to L SD te st Acta agriculturae Slovenica, 118/2 – 202212 A. ABDOUN et al. under all treatments except combination of 50 Gy and 25 mM NaCl treatments. The number of leaves per plant increased by the treatments with the 25 mM NaCl and its contribution with all doses of γ-radiation in ‘ILC 484’, while the γ-radiation doses in combination with the 25 mM and 60 mM NaCl induced significant re- duction in shoot length, number of leaves and shoot and root biomass comparing to the control and salt treatment in ‘FLIP 97-263’. The combination of 100 Gy γ-radiation and 25 mM salt treatment induced signifi- cant increase in shoot fresh biomass comparing to the salt treatment only in ‘FLIP 84-188’ (Table 2). 4 DISCUSSION All varieties used in the current study, except ‘ILC 484’, germinated in the control range when irradiated low doses of γ-radiation (50 Gy, 100, Gy 200 and 300 Gy) , which in agreement with Shah et al. (2008), who reported that germination was not affected in the desi variety Pb2000 at γ-radiation doses of 100 Gy, 200 Gy and 300 Gy. High doses of γ-radiation (400 Gy, 500 Gy and 600 Gy) on the other hand decreased the GP sig- nificantly compared with the low doses and the control. The inhibition of germination, seedling growth, and other biological responses were frequently observed (Abdelfattah Badr et al., 2014; Kim, Lee, Back, Kim, & Lee, 2000; Toker et al., 2005). The reduction of GP at high doses of γ-radiation, has been reported in many plants including chickpea (Joshi-Saha et al., 2015; Mel- ki & Sallami, 2008; Shah et al., 2008). Low doses of ir- radiation, like low levels of other abiotic stresses, may increase the anti-oxidative capacity of the cells by pro- ducing ROS that mediate the acceleration of cell cycle entry to G0/G1 leading to a positive effect on the plant cell cycle machinery (Feher, Ötvös, Pasternak, & Pettkó- Szandtner, 2008; Sharma et al., 2012). On the contrary, high doses of γ-radiation may result in cell cycle ar- rest at G2/M phase during somatic cell division and/or damage in the genome (El-Azab et al., 2018; Preuss & Britt, 2003). However, the cytogenetics during germi- nation under abiotic stress is not well understood and requires attention. The retarded germination of seeds exposed to high doses of NaCl stress and the slow growth of seedlings under these treatments may be associated with slow cell division at the early emergence of seminal root and shot. It is widely accepted that the first action of abiotic stress on germination is moisture deficit resulting in poor plant stand at the early seedling phase and ham- pers early crop establishment (Kaydan & Yagmur, 2008; Shao, Chu, Jaleel, & Zhao, 2008). Mitotic index was ap- proved as an efficient short-term genetic bioassay via the United States Environmental Agency through the Gene-Tox Program in 1981 (Waters & Auletta, 1981) and was used as an indicator to characterize the cell activity and proliferation (Scofield, Jones, & Murray, 2014). Low doses of γ-radiation induced an increase in the proportion of dividing cells, whereas higher doses resulted in reduction in mitotic activity. A dose- dependent increase in mitotic indices was observed in cowpea following exposure to γ-radiation ranging from 10 to 300 Gy (Girija, Gnanamurthy, & Dhanavel, 2013). Similar findings were also found in cowpea cul- tivars (Abdelfattah Badr et al., 2014) and in soybean cultivars (El-Azab et al., 2018). In plant root tips, arrest in cell cycle progression is caused by check points that mediate the entry of cells into S-phase and mitosis (De Veylder, Joubès, & Inzé, 2003). The cell often sponta- neously continues cycle progression, but this is often followed by genome instability allowing cell survival at the cost of tolerating mutation including chromosomal abnormalities (Hartig & Beck, 2006). As explained in the results section, the cytological effects of γ-radiation on cell division in the root tip mi- tosis was made on plants following exposure to the low doses (50, 100 and 200 Gy). Higher γ-radiation doses from (300 to 600 Gy) caused degradation of most nu- clear membranes in the root meristematic cells of all varieties. This result is in agreement with Arian and Maqbool (2011) who reported that doses of 150 to 300 Gy induced oxidative damages and inhibition of cell di- vision in chickpea root tip cells. The γ-radiation also af- fected the cell division phases forming different abnor- mality types. The total abnormalities percent showed a highly significant difference at all the studied varieties. The total number of abnormal cells increased with the increase of γ-radiation doses of all the studied varieties. Similar result was reported by (Wani, 2009) in chickpea following γ-radiation and ethyl methane sulphonate and their combination treatments. Chromosomal abnormalities induced by γ-radiation include stickiness of chromosomes (Dha- navel, Gnanamurthy, & Girija, 2012). The highest value of sticky metaphase was recorded in ‘ILC 464’ at 200 Gy. Chromosome stickiness might be formed due to changes in specific non-histone proteins, histone pro- teins and DNA breaks induced during chromosome condensation (Piskadlo, Tavares, & Oliveira, 2017). The appearance of free and the lagging chromosomes was more frequent in all the treatments in the studied chick pea varieties except at 50 Gy in ‘ILC 2555’. The lag- ging chromosomes at ana-telophase might be formed due to the failure of spindle fibers to push the respec- tive chromosomes to the poles because of exposure to Acta agriculturae Slovenica, 118/2 – 2022 13 Effects of γ-radiation on chickpea (Cicer arietinum) varieties and their tolerance to salinity stress γ-irradiations. The ataxia telangiectasia and Rad3-relat- ed (ATR) plays an essential role in suppressing replica- tion stress from DNA damage. A mitosis-specific and R loop–driven ATR pathway supports faithful chro- mosome segregation, preventing formation of lag- ging chromosomes (Kabeche, Nguyen, Buisson, & Zou, 2018). Chromosomal bridges are commonly attributed to dicentric chromosomes originating from chromo- some exchange after chromosome double strand breaks (Cornforth & Goodwin, 1991). Chromosome breakage is usually considered to involve the DNA molecule re- sponsible for the linear stability of the chromosome. This aberration is the result of unfinished repair of DNA (Grant, 1978). Micronuclei usually arise from lag- ging chromosomes and fragments, which fail to reach the pole region in time and are included in the daughter cells as micronuclei (A Badr, 1986; Kumar, 1998). The micronuclei were more frequency observed in cells ex- posed to γ-radiation at low dose of 50 Gy, in all the va- rieties. The number of micronuclei could illustrate the individual sensitivity level to mutagens (Koteles, 1996; Köteles, Bojtor, Szirmai, Berces, & Otos, 1993). All plants exposed to 60 mM NaCl treatment died before reaching maturity. This result is in agreement with Khan, Siddique, Munir, and Colmer (2015) who stated that salinity severely inhibited plant growth, and led to some tissue death resulting in plant deaths. Hal- eem (2012) reported that high concentrations of NaCl treatments at 50, 100 and 200 mM caused depression in plant growth, total soluble protein content, photosyn- thetic pigments content, nucleic acids contents and all yield characteristics, and concluded that seed irradia- tion with γ-rays moderates the adverse effect of salin- ity stress compared to non-irradiated seeds. Khan et al. (2015) stated that the 60 mM NaCl treatment also re- duced stem and root dry mass of all chickpea genotypes when compared to their controls. Even at low (20 mM and 25 mM) salt concentration, chickpea growth was reduced significantly (Sadiki & Rabih, 2001). Salinity of 3 dS m−1 in field soils was reported to be the threshold for reduced shoot growth and yield in chickpea (Kater- ji, Van Hoorn, Hamdy, Mastrorilli, & Oweis, 2005; Rao, Giller, Yeo, & Flowers, 2002). 5 CONCLUSION The γ-radiation doses above 300 Gy induced deg- radation of nuclear membranes, whereas lower doses did not affect or slightly enhanced mitotic activities but induced different types of chromosomal abnormalities. The total number of abnormal cells increased with the increase of γ-radiation doses in all the studied varie- ties. Gamma-rays induced various types of qualitative and quantitative chromosomal aberration including chromosome bridges, laggard chromosomes, stickiness, chromosome breakage and micronuclei. The salinity treatments at 25 mM NaCl and 60 mM NaCl reduced seedling’s growth of all cultivars estimated as root and shoot length and biomass production. The application of γ-rays can moderate the adverse effect of low lev- els of salinity stress compared to non-irradiated seeds. The γ-radiation dose of 100 Gy alleviated the impact of NaCl salinity in chickpea plants at a concentration of 25 mM NaCl for all varieties, except ‘FLIP 84-188’and ‘FLIP 97-263’. On the other hand, the 60 mM NaCl treatment significantly reduced early growth of all cul- tivars and its effect was not alleviated by the γ-radiation application. 6 STATEMENTS AND DECLARATIONS Conflict of interest: The authors declare no com- peting interests. 7 REFERENCES Amri-Tiliouine, W., Laouar, M., Abdelguerfi, A., Jankowicz- Cieslak, J., Jankuloski, L., & Till, B. J. (2018). Genetic vari- ability induced by gamma rays and preliminary results of low-cost TILLING on M2 generation of Chickpea (Cicer arietinum L.). Frontiers in Plant Science, 9, 1568. Retrieved from https://www.frontiersin.org/articles/10.3389/ fpls.2018.01568/full Arian, A., & Maqbool, A. F. (2011). Gross mutations and oxi- dative damages induced by high doses of gamma rays in chickpea (Cicer arietinum L.) root tip cells. Institute of Plant Sciences, University of Sindh, Jamshoro, 16-20 Badr, A. (1986). Effects of the s-triazine herbicide tur- butryn on mitosis, chromosomes and nucleic acids in root tips of Vicia faba. Cytologia, 51(3), 571-577. Re- trieved from https://www.jstage.jst.go.jp/article/cytolo- gia1929/51/3/51_3_571/_article/-char/ja/ Badr, A., El-Shazly, H. H., & Halawa, M. (2014). Cytological effects of gamma radiation and its impact on growth and yield of M1 and M2 Plants of Cowpea Cultivars. Cytolo- gia, 79(2), 195-206. Retrieved from https://www.jstage.jst. go.jp/article/cytologia/79/2/79_195/_article/-char/ja/ Bhat, T. A., & Wani, A. A. (2017). Chromosome structure and aberrations: Springer. https://doi.org/10.1007/978-81-322- 3673-3 Brahmi, I., Mabrouk, Y., Charaabi, K., Delavault, P., Simier, P., & Belhadj, O. (2014). Induced mutagenesis through gam- ma radiation in chickpea (Cicer arietinum L.): develop- mental changes and improved resistance to the parasitic weed Orobanche foetida Poir. International Journal, 2(11), Acta agriculturae Slovenica, 118/2 – 202214 A. ABDOUN et al. 670-684. Retrieved from https://www.researchgate.net/ profile/Yassine-Mabrouk-2/publication/271705368_ Chopra, V. (2005). Mutagenesis: Investigating the process and processing the outcome for crop improvement. Current Science, 353-359. Retrieved from https://www.jstor.org/ stable/24110583 Cornforth, M., & Goodwin, E. (1991). Transmission of radiation-induced acentric chromosomal fragments to micronuclei in normal human fibroblasts. Radia- tion Research, 126(2), 210-217. Retrieved from https:// meridian.allenpress.com/radiation-research/article-ab- stract/126/2/210/39242/Transmission-of-Radiation-In- duced-Acentric De Veylder, L., Joubès, J., & Inzé, D. (2003). Plant cell cycle transitions. Current Opinion in Plant Biology, 6(6), 536- 543. Retrieved from De Veylder, L., Joubès, J., & Inzé, D. (2003). Plant cell cycle transitions. Current opinion in plant biology, 6(6), 536-543. https://doi.org/10.1016/j. pbi.2003.09.001 Dhanavel, D., Gnanamurthy, S., & Girija, M. (2012). Effect of gamma rays on induced chromosomal variation in cow- pea Vigna unguiculata (L.) Walp. International Journal of Current Science, 2012, 245-250. El-Azab, E. M., Ahmed Soliman, M., Soliman, E., & Badr, A. (2018). Cytogenetic impact of gamma irradiation and its effects on growth and yield of three soybean cultivars. Egyptian Journal of Botany, 58(3), 411-422. Retrieved from https://journals.ekb.eg/article_7900.html Feher, A., Ötvös, K., Pasternak, T. P., & Pettkó-Szandtner, A. (2008). The involvement of reactive oxygen species (ROS) in the cell cycle activation (G0-to-G1 transition) of plant cells. Plant Signaling & Behavior, 3(10), 823-826. https:// doi.org/10.4161/psb.3.10.5908 Gaafar, R. M., Hamouda, M., & Badr, A. (2016). Seed coat color, weight and eye pattern inheritance in gamma- rays induced cowpea M2-mutant line. Journal of Genetic Engineering and Biotechnology, 14(1), 61-68. Retrieved from https://www.sciencedirect.com/science/article/pii/ S1687157X15000669 Girija, M., Gnanamurthy, S., & Dhanavel, D. (2013). Cy- togenetics effect of gamma rays on root meristem cells of Vigna unguiculata (L.). European Journal of Experi- mental Biology, 3(2), 38-41. Retrieved from https:// www.researchgate.net/profile/D-Dhanavel/publica- tion/333260847_Cytogenetics_effect_of_gamma_rays_ on_root_meristem_cells_of_Vigna_unguiculata_L/ links/5ce4dd0d458515712eba7214/Cytogenetics-effect- of-gamma-rays-on-root-meristem-cells-of-Vigna-un- guiculata-L.pdf Gnanamurthy, S., Mariyammal, S., Dhanavel, D., & Bhar- athi, T. (2012). Effect of gamma rays on yield and yield components characters R3 generation in cowpea (Vigna unguiculata (L.). Walp.). International Journal of Plant Sci- ence, 2(2), 39-42. Grant, W. F. (1978). Chromosome aberrations in plants as a monitoring system. Environmental Health Perspectives, 27, 37-43. Retrieved from https://ehp.niehs.nih.gov/doi/ abs/10.1289/ehp.782737 Haleem, M. A. (2012). Pre-exposure to gamma rays alleviates the harmful effect of salinity on cowpea plants. Journal of Stress Physiology & Biochemistry, 8(4). Retrieved from https://cyberleninka.ru/article/n/pre-exposure-to-gam- ma-rays-alleviates-the-harmful-effect-of-salinity-on- cowpea-plants Hartig, K., & Beck, E. (2006). Crosstalk between auxin, cyto- kinins, and sugars in the plant cell cycle. Plant Biology, 8(03), 389-396. Retrieved from https://www.thieme-connect. com/products/ejournals/abstract/10.1055/s-2006-923797 Jimenez-Lopez, J. C., Singh, K. B., Clemente, A., Nelson, M. N., Ochatt, S., & Smith, P. (2020). Legumes for global food security. Frontiers in Plant Science, 11, 926. Retrieved from https://www.frontiersin.org/articles/10.3389/ fpls.2020.00926/full Joshi-Saha, A., Reddy, K. S., Petwal, V., & Dwivedi, J. (2015). Identification of novel mutants through electron beam and gamma irradiation in chickpea (Cicer arietinum L.). Journal of Food Legumes, 28(2), 1-6. Jukanti, A. K., Gaur, P. M., Gowda, C., & Chibbar, R. N. (2012). Nutritional quality and health benefits of chickpea (Cic- er arietinum L.): a review. British Journal of Nutrition, 108(S1), S11-S26. Retrieved from https://www.cambridge. org/core/journals/british-journal-of-nutrition/article/ nutritional-quality-and-health-benefits-of-chickpea-cic- er-arietinum-la-review/BCD8920297E987AAABBC12B- FF90EB0CF Kabeche, L., Nguyen, H. D., Buisson, R., & Zou, L. (2018). A mitosis-specific and R loop–driven ATR pathway promotes faithful chromosome segregation. Science, 359(6371), 108-114. Retrieved from https://www.science. org/doi/abs/10.1126/science.aan6490 Kamble, G., & Patil, A. (2014). Comparative mutagenicity of EMS and gamma radiation in wild chickpea. International Journal of Science and Technology, 3, 166-180. Retrieved from https://citeseerx.ist.psu.edu/viewdoc/download?doi =10.1.1.677.576&rep=rep1&type=pdf Katerji, N., Van Hoorn, J., Hamdy, A., Mastrorilli, M., & Oweis, T. (2005). Salt tolerance analysis of chickpea, faba bean and durum wheat varieties: I. Chickpea and faba bean. Agricultural Water Management, 72(3), 177-194. Retrieved from https://www.sciencedirect. com/science/article/pii/S037837740400229X?casa_ token=oeBq20KUQHMAAAAA:j0l_JkGs5I8-sr6ALn- R6e4YCB3y6jhNtNkK_eBgJFftC4JM8Jk4BtDwP2sSrS- DuPuAKdijgsg Kaydan, D., & Yagmur, M. (2008). Germination, seedling growth and relative water content of shoot in different seed sizes of triticale under osmotic stress of water and NaCI. African Journal of Biotechnology, 7. Khan, H. A., Siddique, K. H., Munir, R., & Colmer, T. D. (2015). Salt sensitivity in chickpea: growth, photosynthesis, seed yield components and tissue ion regulation in contrasting genotypes. Journal of Plant Physiology, 182, 1-12. https:// doi.org/10.1016/j.jplph.2015.05.002 Kim, J.-S., Lee, E.-K., Back, M.-H., Kim, D.-H., & Lee, Y.-B. (2000). Influence of low dose ${\gamma} $ radiation on the physiology of germinative seed of vegetable crops. Acta agriculturae Slovenica, 118/2 – 2022 15 Effects of γ-radiation on chickpea (Cicer arietinum) varieties and their tolerance to salinity stress Korean Journal of Environmental Agriculture, 19(1), 58-61. Retrieved from https://www.koreascience.or.kr/article/ JAKO200019756103350.page Koteles, G. (1996). The human lymphocyte micronucleus as- say. A review on its applicabilities in occupational and environmental medicine. Central European Journal of Oc- cupational and Environmenta Medicine, 2, 12-30. Köteles, G., Bojtor, I., Szirmai, S., Berces, J., & Otos, M. (1993). Micronucleus frequency in cultured lymphocytes of an urban population. Mutation Research/Genetic Toxicology, 319(4), 267-271. Retrieved from https://www.sciencedi- rect.com/science/article/abs/pii/0165121893900145 Kumar, S. (1998). Effect of gamma rays, EMS, DES on meiosis in Lathyrus sativus. Journal of. Cytology, 33, 139-147. Re- trieved from https://ci.nii.ac.jp/naid/10019329359/ Ladizinsky, G., & Adler, A. (1976). The origin of chickpea Cic- er arietinum L. Euphytica, 25(1), 211-217. Retrieved from https://link.springer.com/article/10.1007/BF00041547 Marques, E., Krieg, C. P., Dacosta-Calheiros, E., Bueno, E., Ses- sa, E., Penmetsa, R. V., & von Wettberg, E. (2020). The im- pact of domestication on aboveground and belowground trait responses to nitrogen fertilization in wild and culti- vated genotypes of Chickpea (Cicer sp.). Frontiers in Ge- netics, 11. Retrieved from https://www.ncbi.nlm.nih.gov/ pmc/articles/PMC7738563/ Melki, M., Mhamdi, M., & Achouri, A. (2011). Chickpea re- sponse to low doses of gamma radiation. Russian Agricul- tural Sciences, 37(4), 318-321. Retrieved from https://link. springer.com/article/10.3103/S1068367411040136 Melki, M., & Sallami, D. (2008). Studies the effects of low dose of gamma rays on the behaviour of chickpea under various conditions. Pakistan Journal of Biological Sciences: PJBS, 11(19), 2326-2330. Retrieved from https://europep- mc.org/article/med/19137865 Moreno, M.-T., & Cubero, J. (1978). Variation in Cicer arieti- num L. Euphytica, 27(2), 465-485. Retrieved from https:// link.springer.com/article/10.1007/BF00043173 Munns, R., Day, D. A., Fricke, W., Watt, M., Arsova, B., Bark- la, B. J., . . . Foster, K. J. (2020). Energy costs of salt toler- ance in crop plants. New Phytologist, 225(3), 1072-1090. Retrieved from https://nph.onlinelibrary.wiley.com/doi/ full/10.1111/nph.15864 Munns, R., & Gilliham, M. (2015). Salinity tolerance of crops– what is the cost? New Phytologist, 208(3), 668-673. Re- trieved from https://nph.onlinelibrary.wiley.com/doi/ full/10.1111/nph.13519 Munns, R., & Tester, M. (2008). Mechanisms of salinity tol- erance. Annual Review of Plant Biology, 59, 651-681. Retrieved from https://www.annualreviews.org/doi/ abs/10.1146/annurev.arplant.59.032607.092911 Nazarenko, M., & Izhboldin, O. (2017). Chromosomal rear- rangements caused by gamma-irradiation in winter wheat cells. Biosystems Diversity, 25(1). Retrieved from https:// cyberleninka.ru/article/n/chromosomal-rearrangements- caused-by-gamma-irradiation-in-winter-wheat-cells Piskadlo, E., Tavares, A., & Oliveira, R. A. (2017). Metaphase chromosome structure is dynamically maintained by con- densin I-directed DNA (de) catenation. Elife, 6, e26120. Retrieved from https://elifesciences.org/articles/26120 Preuss, S. B., & Britt, A. B. (2003). A DNA-damage-induced cell cycle checkpoint in Arabidopsis. Genetics, 164(1), 323- 334. https://doi.org/10.1093/genetics/164.1.323 Rao, D., Giller, K., Yeo, A., & Flowers, T. (2002). The effects of salinity and sodicity upon nodulation and nitrogen fixa- tion in chickpea (Cicer arietinum). Annals of Botany, 89(5), 563-570. Retrieved from https://academic.oup.com/aob/ article/89/5/563/205764?login=true Sadiki, M., & Rabih, K. (2001). Selection of chickpea (Cicer ar- ietinum) for yield and symbiotic nitrogen fixation ability under salt stress. Agronomie, 21(6-7), 659-666. Retrieved from https://hal.archives-ouvertes.fr/hal-00886149/ Scofield, S., Jones, A., & Murray, J. A. (2014). The plant cell cycle in context. Journal of Experimental Botany, 65(10), 2557-2562. https://doi.org/10.1093/jxb/eru188 Shah, T. M., Mirza, J. I., Haq, M. A., & Atta, B. M. (2008). Ra- dio sensitivity of various chickpea genotypes in M1 gen- eration I-Laboratory studies. Pakistan Journal of Botany, 40(2), 649-665. Retrieved from https://d1wqtxts1xzle7. cloudfront.net/34204731/PJB402649-with-cover-page- v2.pdf?Expires=1643712109&Signature=f5dIrLXLvrm C1FqJRo6dTeC2-ZmygN6JXGkcojuRo~dpFt7zlbRhX7 Z9SsozJPKgbdWosm2taOcu-qOE-mkspI8m43bgeg7d- Ly2lJa8iR10-WXnTWGl2Ybgrop6-hFvb69ApyTNjRU7s- DXISUaYDB680jDHwveI0Vkwpg3DQJYBrtFnl9mw-3a jxo1th0WX8L8b6RRWivH70gMbbpQeUVjX~hrwvAJ1i akk4QkO4Hdad02gfxNGNEXs6mEaRMD-tNnMg50ad- dU8xRJ-K8GJjXyFgbxdesLuOO22eLcaze2oQkl-yZyBfu- 1nae4IPj96T1Zoiy041pH7sMxCdPqLz3Q__&Key-Pair- Id=APKAJLOHF5GGSLRBV4ZA Shao, H.-B., Chu, L.-Y., Jaleel, C. A., & Zhao, C.-X. (2008). Wa- ter-deficit stress-induced anatomical changes in higher plants. Comptes Rendus Biologies, 331(3), 215-225. https:// doi.org/10.1016/j.crvi.2008.01.002 Sharma, P., Jha, A. á., Dubey, R. S., & PessarakliM, R. O. S. (2012). Oxidative damage and antioxidative defense mechanism in plants under stressful conditions. Journal of Botany, 2012, 1-26. Retrieved from https://downloads. hindawi.com/archive/2012/217037.pdf Singh, B. D. (2005). Mutations in crop improvement. In: Singh, B. D. (ed). Plant Breeding, Principles and Methods. Kalyani Publishers, Ludhiana, , pp. 698–731. Sohrabi, Y., Heidari, G., & Esmailpoor, B. (2008). Effect of sa- linity on growth and yield of Desi and Kabuli chickpea cultivars. Pakistan Journal of Biological Sciences: PJBS, 11(4), 664-667. Retrieved from https://europepmc.org/ article/med/18817146 Soliman, M., Elkelish, A., Souad, T., Alhaithloul, H., & Farooq, M. (2020). Brassinosteroid seed priming with nitrogen supplementation improves salt tolerance in soybean. Physiology and Molecular Biology of Plants, 26(3), 501-511. Retrieved from https://link.springer.com/article/10.1007/ s12298-020-00765-7 Sun, Z., Li, H., Zhang, Y., Li, Z., Ke, H., Wu, L., . . . Ma, Z. (2018). Identification of SNPs and candidate genes as- sociated with salt tolerance at the seedling stage in cot- ton (Gossypium hirsutum L.). Frontiers in Plant Science, 9, 1011. Retrieved from https://www.frontiersin.org/arti- cles/10.3389/fpls.2018.01011/full Acta agriculturae Slovenica, 118/2 – 202216 A. ABDOUN et al. Tabur, S., Avci, Z. D., & Özmen, S. (2021). Exogenous salicylic acid application against mitodepressive and clastogenic effects induced by salt stress in barley apical meristems. Biologia, 76(1), 341-350. Retrieved from https://link. springer.com/article/10.2478/s11756-020-00573-0 Toker, C., Uzun, B., Canci, H., & Ceylan, F. O. (2005). Ef- fects of gamma irradiation on the shoot length of Cicer seeds. Radiation Physics and Chemistry, 73(6), 365-367. Retrieved from https://www.sciencedirect.com/science/ article/pii/S0969806X05000721?casa_token=yH7l1OTA- dUAAAAA:kR_9_MCIoPSbud0WjplZ2Wz6ovsPE- jNNS7NSt9CYiBjUVAd8iujrB92hNmZ8zfzSnxzZshRzcA Tshilenge-Lukanda, L., Kalonji-Mbuyi, A., Nkongolo, K., & Kizungu, R. (2013). Effect of gamma irradiation on mor- pho-agronomic characteristics of groundnut (Arachis hy- pogaea L.). American Journal of Plant Sciences, 4(11), 2186. Retrieved from https://www.scirp.org/html/39582.html Upadhyaya, H. D., Dwivedi, S. L., Baum, M., Varshney, R. K., Udupa, S. M., Gowda, C. L., . . . Singh, S. (2008). Ge- netic structure, diversity, and allelic richness in com- posite collection and reference set in chickpea (Cicer arietinum L.). BMC Plant Biology, 8(1), 1-12. Retrieved from https://bmcplantbiol.biomedcentral.com/arti- cles/10.1186/1471-2229-8-106 Van der Maesen, L. J. G. (1972). Cicer L., A Monograph of the Genus, with Special References to Chickpea (Cicer arietinum L.). Its ecology and cultivation. Mededlingen landbouw ho- geschool (Communication Agricultural University), Wa- geningen. Wani, A. A. (2009). Mutagenic effectiveness and efficiency of gamma rays, ethyl methane sulphonate and their com- bination treatments in chickpea (Cicer arietinum L.). Asian Journal of Plant Sciences, 8(4), 318-321. https://doi. org/10.3923/ajps.2009.318.321 Waters, M. D., & Auletta, A. (1981). The GENE-TOX pro- gram: genetic activity evaluation. Jornal of Chemical In- formation and Computer Science, 21(1), 35-38. https://doi. org/10.1021/ci00029a007 Acta agriculturae Slovenica, 118/2, 1–11, Ljubljana 2022 doi:10.14720/aas.2022.118.2.1867 Original research article / izvirni znanstveni članek Symbiotic and physiological indicators of soybean inoculated of Bradyrhizobium japonicum single-strain in 7 days before sowing Nadiya VOROBEY 1, Kateryna KUKOL 1, Petro PUKHTAIEVYCH 1, 2, Tetyana KOTS 1,3 Received September 09, 2020; accepted June 13, 2022. Delo je prispelo 9. septembra 2020, sprejeto 13. junija 2022 1 Institute of Plant Physiology and Genetics, National Academy of Sciences of Ukraine, Kyiv, Ukraine 2 Corresponding author, e-mail: azotfixation@gmail.com 3 Institute of the Ukrainian Language, National Academy of Sciences of Ukraine, Kyiv, Ukraine Symbiotic and physiological indicators of soybean inocu- lated of Bradyrhizobium japonicum single-strain in 7 days before sowing Abstract: Results of investigation of soybean of the Almaz variety in inoculation with preparations based on nodule bacteria Bradyrhizobium japonicum (Kirchner, 1896), Jordan, 1982 B78, B157, D37, D87 are presented. Different periods of the soybean seeds inoculation were used - on the sowing day (control) and in 7 days before sowing (ex- perimental variants). The differences between control and experimental plants in the formation and functioning of the symbiotic apparatus and its functional activity, depending on the period between from seed inoculation to sowing were analysed. It was determined that the number of root nodules in the control plants was higher. The mass of nodules at the stage of 3 true leaves exceeded the control by 1.5–2.0 times in plants inoculated in 7 days before sowing, and the intensity of nitrogen fixation by 1.7–6.6 times. At the budding-beginning of flowering stage, the mass and intensity of N2 fixation by the nodules of control plants increased. As a result, the difference between the nitrogen fixing activity of control and experi- mental plants decreased significantly. Stimulating effect on aboveground mass of Bradyrhizobium japonicum strains with increased nitrogen fixing activity was noted. Optimal condi- tions for the formation and functioning of bean-rhizobial symbiosis were provided at the use of both of these terms of soybean inoculation. This reveals the possibility of effective application of early inoculation of soybean seeds with prepa- rations based on nodule bacteria Bradyrhizobium japonicum active strains. Key words: rhizobia; Bradyrhizobium japonicum; bacte- rial preparations; pre-sowing inoculation; nitrogen fixing ac- tivity; soybean Simbiontski in fiziološki indikatorji soje, inokulirane sedem dni pred setvijo s sevom bakterije Bradyrhizobium japoni- cum Izvleček: Predstavljeni so rezultati raziskave inokulaci- je soje, sorte Almaz, s simbiontsko bakterijo Bradyrhizobium japonicum (Kirchner, 1896), Jordan; sevi 1982 B78, B157, D37, D87). Inokulacija semen soje je bila izvedena na dan setve (kontrola) in sedem dni pred setvijo (različna obravnavanja v poskusu). Ugotovljene so bile razlike med kontrolo in različ- nimi obravnavanji v tvorbi in delovanju simbiontskega apa- rata glede na čas inokulacije. Ugotovljeno je bilo, da je bilo število koreninskih nodulov pri kontrolnih rastlinah večje. Masa nodulov je na razvojni stopnji soje tretjega pravega lista presegala kontrolo pri rastlinah inokuliranih sedem dni pred setvijo za 1,5–2,0 krat , vezava zračnega dušika pa za 1,7–6,6 krat. Na razvojni stopnji začetka cvetenja sta se masa nodulov in jakost vezave N2 pri kontroli povečali, s čemer se je značil- no zmanjšala razlika med kontrolo in obravnavanji. Opazen je bil tudi stimulacijski učinek inokulacije s sevi Bradyrhizo- bium japonicum na nadzemno biomaso soje zaradi povečane vezave dušika. Optimalne razmere za tvorbo in delovanje te rizobijske simbioze s sojo so se pojavile pri obeh načinih ino- kulacije. To nakazuje možnost učinkovite uporabe zgodnje inokulacije semen soje s pripravki aktivnih sevov bakterije Bradyrhizobium japonicum. Ključne besede: rizobiji; Bradyrhizobium japonicum; bakterijski pripravki; predsetvena inokulacija; aktivnost veza- ve zračnega dušika; soja Acta agriculturae Slovenica, 118/2 – 20222 N. VOROBEY et al. 1 INTRODUCTION The nodule bacteria Bradyrhizobium japonicum (Kirchner, 1896), Jordan, 1982 are the basis of biological microbial preparations for inoculation of soybean, that are characterized by multifunctional effect on plants. Their application increases resistance of plant to abiotic and biotic factors, the number and mass of root nod- ules, the intensity of symbiotic nitrogen fixation, the chlorophyll content in leaves, improves crop productiv- ity and grain quality. This reduces the use of expensive nitrogen fertilizers, and, as a consequence, reduces the negative impact on the environment (Patyka & Petry- chenko, 2004; Morgun & Kots, 2008; Kots, 2011; Kots et al., 2016; Kramarev & Artemenko, 2016). Increasing the sown areas of soybean testifies its important role in the agricultural complex (Berbenets, 2019). Usually pre-sowing seeds inoculation is carried out on the sowing day or in the day before, which pro- vides a period of 24 hours from preparation application on the seeds to getting inoculated seeds into the soil. It is difficult for growers with large sown areas to treat and sow seeds in the soil in one day (because it takes a long time, related consumables, equipment prepara- tion, and human resources, etc.). Pesticides with which are treated seeds to kill fungal and bacterial infections can also have a negative effect on nodulating bacteria Bradyrhizobium (soybean inoculants). All this compli- cates the pre-sowing treatment of seeds with biologi- cals in industrial conditions. Therefore, in recent years, the soybean cultivation technology has begun to use seeds with pre-treatment by plant protection products and preparations of nodule bacteria Bradyrhizobium japonicum, which are compatible with fungicides and insecticides. Thus, today there is a problem of provid- ing highly effective inoculants for early inoculation of pulses seeds, in particular soybean. They got the name pre-inoculants. In the segment of soybean inoculants on the Ukrainian market there is a wide range of trade names of preparations of domestic and foreign production (Kokorina & Kozhemyakov, 2010; Kots & Mamenko, 2015). According to the main criteria (type of micro- organisms, their titer), the domestic inoculants do not differ from the foreign ones. However, the presence of protectors (preservative, adhesive) in some foreign preparations, increases the treatment manufacturabil- ity, simplifies the process of soybean sowing (Slobody- anyuk, 2017). Pre-inoculants are used as a special element of soybean growing technology, which is in demand to- day. Pre-inoculant has advantages over conventional inoculants, even with small seed volumes. It contains an additional component that is able to form a protective film and protect bacteria on the surface of seeds from harmful environmental factors. It promotes additional seeds nutrition, provides better germination, increases germination energy and allows pre-treatment of seeds with inoculant. Currently known pre-inoculants, compatible with the original soybean seed treatment and with the pos- sibility of early application of the preparation before sowing seed in soil in 24 hours – RhizoFlo 5 (Saatbau Linz, Austria), in 1–2 days – Rhizobophyte (Ukraine), in 7 days – Biobacter (Lallemand, Uruguay), in 5–15 days – Rizoform + Static (Schelkovo Agrohim, Russia), in 21 days – Bioboost Plus (Liquid, Canada), in 45 days – HiCoat Super Extender (Agrocenter BASF, Germany) and in 90 days – HiCoat Super (Веcker Underwood, USA), Agribacter, Agribacter + Rise (Lallemand, Uru- guay) and other (Agritema, 2017). Improving the nodule bacteria biologicals for soybean in the direction of increase the time between seeds inoculation and their sowing will significantly in- crease their effectiveness and attractiveness as a tool for obtaining biological nitrogen (Laktionov et al., 2018), because biological nitrogen fixation is an unalterable way to provide plants with nitrogen and does not vio- late the natural environment ecology (Kots et al., 2016). Domestic preparations are also known on the Ukrainian market, including Rizoline + Rizosave inocu- lants with recommendation of early treatment of seeds (7–10 days before sowing). When using a tank mix with Rizoline 2 l t-1 + Rizosave 2 l t-1 and a fungicide Fever 0.4 l t-1, there were obtained soybean yields higher than control on 2.8–3.3 c ha-1 (Slobodyanyuk, 2017). Microbial preparations with natural film-forming compounds are known, whereby nodule bacteria on seeds are protected from negative external influences and remain viable for a long time. For example film- former for seed inlay, created on the basis of waste plant and animal origin, opens the possibility of seed inoculation in 25–30 days before sowing, maintain- ing the nodulating activity of rhizobia and increased yields depending on the variety by 12 % (Grishechkin & Golovina, 2014). Many Ukrainian farmers recognized the advantag- es of modern inoculation processing technologies. The additional harvests gave them the opportunity to re- coup costs, and to make significant profits. The most ef- fective will be the preparation that provides the highest concentration of live bacteria on the seeds at the time of their entry into the soil. Bacterial titers of preparations for soybean inoculation, which are widely available on the domestic market, range from 1 × 109 to 5 × 109 at the time of production and 2 × 108 – (2–4) × 109 at the Acta agriculturae Slovenica, 118/2 – 2022 3 Symbiotic and physiological indicators of soybean inoculated of Bradyrhizobium japonicum single-strain in 7 days before sowing end of the shelf life of the preparation. High-quality two-component preparation for soybean HiCoat®Super of the American company Becker Underwood has the highest of the Bradyrhizobium genus bacteria concen- tration (1 × 1010 cells per 1 g of preparation), and the longest among all known inoculants bacterial life on seeds. This allows seeds inoculation in 90 days before sowing and initiates nodules formation already in the initial stages of plant development. In the development modern preparations of nod- ule bacteria for legumes and pulses at the first stage it is important to study the period of viable bacteria on seeds to sowing, virulence and nitrogen fixing activity (NFA) due to pre-inoculation of seeds. Regarding the research of this problem, there are few publications, and the results obtained are debatable. Martyniuk S. et аl. (2002) observed a sharp drop in the number of bacteria Rhizobium lupini (Schroeter, 1886) Eckhardt et al., 1931 363a and 367a on inoculated lupine seeds Oligarch va- riety (creator – Leningrad Scientific Research Institute of Agriculture ‘Belogorka’, Russia), within 24–48 hours after inoculation. However, after added to inoculant of polymeric protector polyvinylpyrrolidone (PVP) at a concentration of 5 % even after 168 hours, sufficient bacteria quantity remained to form an effective sym- biosis, possibly due to the protective properties of this polymer and its action as an “adhesive”. That is, the use of PVP is potentially able to increase the allowable time between seeds inoculation and sowing up to six days. The scientists point to several factors that affect the effectiveness of early seeds treatment by inoculants. So, the most important of them are the ability of bac- teria to survive on seeds, seeds storage conditions, as well as the influence of other products (compounds) used in inoculation (Anghinoni et al., 2017). There are data in the literature on the treatment of soybean seeds with bulk peat preparations based on active strains of Bradyrhizobium japonicum SEMIA 5079 and SEMIA 5080 in 5 days before sowing and on the sowing day. The authors found that both methods of bacterization at the absence of the fungicides use provided the for- mation of nodules number at the level of control plants (Zilli et al., 2010). This indicated the ability of rhizobia to survive on the seeds of Glycine max L. (Merrill) for five days. Regarding the fixation of molecular nitrogen, these researchers did not find significant differences between the variants depending on the duration from inoculation of seeds to sowing. Bacterial preparations based on active strains of nodule bacteria can be effective for pre-sowing treat- ment of soybean seeds without the use of extenders. Thus, the aim of our investigation was to study the effectiveness formations and functioning of the sym- biotic apparatus, growth and development of soybean plants depending on the duration of the period from seeds inoculation with nodule bacteria Bradyrhizobium japonicum to sowing without excipients in microbial preparations. 2 MATERIALS AND METHODS The experiments were performed with the soy- bean (Glycine max L. (Merrill) seeds of Almaz variety (originator – Poltava State Agrarian Academy, Ukraine), included in the Register of plant varieties of Ukraine since 2007 and recommended for cultivation in the for- est-steppe of Ukraine (early ripening, high plasticity to climatic conditions). Inoculation of seeds was carried out with a liquid- phase preparation made on the basis different in sym- biotic activity of nodule bacteria В78, В157, D37, D87 strains (obtained as result of intergeneric conjugation between Escherichia coli S17-1 (pSUP5011::Tn5mob) and strains 646, 634b from the collection of N2-fixing microorganisms of the Institute of Plant Physiology and Genetics NAS of Ukraine. Restoration of physi- ological activity of Bradyrhizobium japonicum nodule bacteria after storage in the museum collection was car- ried out by standard microbiological methods (Netru- sov et al., 2005). The nodule bacteria were grown in biological tubes on a nutrient medium yeast mannitol agar (YMA) (Ne- trusov, 2005) for 7 days at +28 °C for preparing a liquid- phase preparation. Thereafter, the biomass of bacteria were washed off from agar, and transfered in glass flasks with liquid YM environment (10 ml of suspension per 350 ml of YM) and cultured during 7 days at a tempera- ture of 28 °C and constant aeration. The bacterial titer of the preparation, which were used for inoculation of soybean seeds was 4.1–4.5‧109 CFU (colony forming units) per g of the preparation. Soybean seeds were externally sterilized for 15 min with 70 % ethanol, washed with running water, inocu- lated for 1 h by prepared liquid microbial preparations and sowed in the substrate. Variants with inoculation of soybean seeds in 7 days before the day of sowing (experimental variants) and with inoculation of seeds in 1 hour before sowing (control) were included in the experimental scheme. Soybeans were grown on a sandy substrate (10 kg washed river sand, 8 plants in each pot on) with the introduction of Hellriegel nutrient mixture with 0.25 of nitrogen norm (1 norm was 708 mg Са (NO3)2 . 4 Н2О per kg of sand) (Grodzinsky & Grodzinsky, 1964). River sand is the sand extracted from riverbeds, which Acta agriculturae Slovenica, 118/2 – 20224 N. VOROBEY et al. is characterized by a high degree of purification and the absence of foreign inclusions. The pots with plants were placed on a specially equipped site of the Insti- tute of Plant Physiology and Genetics NAS of Ukraine under conditions at natural light, temperature and ar- tificial controlled irrigation (Figure 1). Sowing seeds – 18.05.2018, the first seedling – 23.05.2018. Repeatability of the experimental variants was 5 times. Selection of plants for the analysis was carried out in the stages: 3 true leaves (on the 28th day after ger- mination), budding-beginning of flowering (on the 35 days after germination), full flowering soybeans (on the 48 days after germination). The nodulating ability of Bradyrhizobium japoni- cum was determined by counting the number and mass of root nodules in 10 plants of each variant of the exper- iment. Biometric indices – the mass of the aboveground part of plants and roots – in 15 plants of each variant of the experiment. N2-fixation activity was determined by acetylene method in terms of acetylene regenera- tion activity by root nodules of soybean (Hardy et. al., 1968) and expressed in µmol of ethylene, produced by nodules of 1 plant for 1 hour. The roots with nodules were placed in hermetically sealed glass vials with a ca- pacity of 75 cm3, 10 % of acetylene of the total volume was injected through the rubber membrane. Incubation period with acetylene - 1 hour. A gas mixture contain- ing ethylene, formed as a result of acetylene reduction by nitrogenase, was analyzed on Agilent Technologics 6850 Network GC System (USA) gas chromatograph with flame ionization detector. Separation of gases was performed on a column Supelco Porapak N at thermo- stat temperature +55 °С and detector +150 °С. The gas carrier was nitrogen (50 ml per 1 min). Sampling capac- ity for analysis was 1 cm3. Pure ethylene was used as the standard. The amount of ethylene formed from acety- lene for 1 h under the action of nitrogenase incubated sample (the nitrogen fixing activity) was represented in molar units of ethylene formed per 1 plant for 1 hour – µmol C2H4 (plant h)-1. Experiment with determine of N2-fixation activity was repeated five times. The statistical processing of the obtained data was conducted using ANOVA and the Tukey HSD Test with the average values. The results were presented in the form of mean values and standard error (m ± SE). The difference between the data was considered significant, if p ≤ 0.05. 3 RESULTS AND DISCUSSION The ability to penetrate in the legume root through Figure 1: The pots with plants on a specially equipped site of the Institute of Plant Physiology and Genetics NAS of Ukraine Acta agriculturae Slovenica, 118/2 – 2022 5 Symbiotic and physiological indicators of soybean inoculated of Bradyrhizobium japonicum single-strain in 7 days before sowing root hairs, and in the process of complex morphophysi- ological changes to cause the formation of nodules in- dicates the virulence of nodule bacteria. Root nodules are the complex constructed organs of plants, the main structures of which are bacteria-infected tissue, where nitrogen is fixed, and the meristem. We established that plants depending on the du- ration of the period from seeds inoculation to sowing and the inoculants on basis of Bradyrhizobium japoni- cum differed for number of root nodules formed. In the control plants (inoculated on the sowing day) formed more root nodules compared to the plants of the exper- imental variants (inoculation in 7 days before sowing). In particular, at stage of the 3 true leaves formation on soybean roots 22.6–39.0 nodules were counted. At the same time, in plants whose seeds were inoculation in 7 days before sowing, nodule number ranged from 14.0 to 29.0 per root (Figure 2). More intensive nodules for- mation was fixed in the budding-beginning of flower- ing period of soybean: 30.0–45.0 pieces per 1 root in variants with seeds inoculation in 1 hour before sowing and 28.0–36.5 pieces per 1 root in variants with seeds inoculation in 7 days before sowing. Depending on the different time intervals between seed inoculation and sowing used in this study, no sig- nificant differences in the nodules location on the roots were observed. Symbiotic organs (nodules) formed mainly on the main root of soybean and branches of the first order at a depth of 1–17 cm and had a light pink color, indicating on the synthesis of leghemo- globin and nitrogen-fixing ability. It should be noted the inoculating strains showed different virulence. When roots were infected by strain В78 the number of formed nodules was the lowest among the studied variants for both terms of prepa- rations use. Probably due to the functional features of these rhizobia (reduced ability to survive on the seeds surface, less mobility and speed of penetration into the root meristem, etc.). Inoculation of soybeans with the preparation of nodule bacteria strain B157 was pro- vided the largest number of root nodules in plants in- oculated on the sowing day. In 7 days before sowing the largest nodules number was formed on the soybeans roots inoculated with nodule bacteria strain D37 (Fig- ure 2). It is known that after invasion of nodule bacteria Figure 2: Root nodules number (pcs. plant-1) in soybean Almaz variety depending on the period duration from seed treatment by strains of Bradyrhizobium japonicum (Kirchner, 1896), Jordan, 1982 D37, D87, В78, В157 to sowing (n = 10). m ± SE. An asterisk (*) indicates statistically significant difference between treatments (paired columns) at p ≤0.05; ns – no significant difference Acta agriculturae Slovenica, 118/2 – 20226 N. VOROBEY et al. in the roots of plants is realized by the formation of bacteroids and the growth of meristem due to which the mass of the nodules increases (Spaink et al., 2002). At the stage of 3 true leaves, in plants which seeds were inoculated in 7 days before sowing mass of nodules was 1.2–2.0 times higher than that of the variants at the sowing day. At the budding-beginning of flowering stage, the difference in this index between the variants has been decreasing due to a more intensive increase in the mass of root nodules in variants with seed inocula- tion on the sowing day (Table 1). Thus, in the variants with a prolonged period of 7 days from seeds inoculation to sowing, the nodule bacteria strains retained their functional activity, which Inoculant strain Development stage of plants: 3 true leaves budding-beginning of flowering 7 days before sowing on the sowing day 7 days before sowing on the sowing day D37 0.152 ± 0.006b 0.122 ± 0.010c * 0.235 ± 0.012a 0.214 ± 0.013b ns D87 0.203 ± 0.005c 0.101 ± 0.006b * 0.311 ± 0.016b 0.241 ± 0.020c * В78 0.097 ± 0.003a 0.063 ± 0.002a * 0.220 ± 0.011a 0.143 ± 0.010a * В157 0.198 ± 0.016c 0.113 ± 0.008bc * 0.305 ± 0.012b 0.292 ± 0.016d ns Table 1: Root nodules mass (mg plant-1) in soybean Almaz variety depending on the period duration from seed treatment to sowing (n = 10) m ± SE, * – significant difference at p ≤ 0.05; ns – no significant difference; interaction: a – not significant. For each strain and each variable, different letters a, b, c, d indicate significant differences Figure 3: Nodules on soybean roots at seeds inoculation by the active strain Bradyrhizobium japonicum (Kirchner, 1896), Jor- dan, 1982 D87 at the budding-beginning of flowering stage, А – inoculation at the sowing day, B – inoculation in 7 days before sowing Acta agriculturae Slovenica, 118/2 – 2022 7 Symbiotic and physiological indicators of soybean inoculated of Bradyrhizobium japonicum single-strain in 7 days before sowing was realized in the initial stages by active formation of root nodules (Figure 3). Brazilian researchers, studying the effectiveness of early seeds inoculation of Glycine max L. (Merrill) in 5 and 10 days before sowing, using of chemical plant protection products, have established, that seeds treated with pesticides based on fludioxonil and thiameth- oxane can be treated with bacterial preparations and stored for 10 days before sowing without negative im- pact on grain yield (Anghinoni et al., 2017). The same authors noted the influence of the duration from in- oculation of soybean seeds to sowing in the soil on cer- tain factors related to the nodulation. The issue of joint use of fungicides and inoculation requires the detailed study to ensure the effective formation and functioning of legume-rhizobial symbiosis and protection against phytopathogens of various etiologies. In model pot ex- periments in the Institute of Plant Physiology and Ge- netics NAS of Ukraine the effect of pesticides on the formation and functioning of the symbiotic apparatus of soybean plants was studied. A negative effect of a number of fungicides on the photosynthetic rate and nitrogen-fixing activity of soybean plants was studied. The strength of this effect depended on the preparation and the term of its use before sowing (Pavlyshche et al., 2017). The time interval of 7 days between seeds inocula- tion and sowing is permissible in the case of prepara- tions use based on mentioned active strains of nodule bacteria Bradyrhizobium japonicum with a high level of exopolysaccharides production. The latter can serve as natural substitutes for synthetic adhesive extenders, which are used in modern pre-inoculants. It is known that bacterial exopolysaccharides, forming a biofilm around rhizobial cells, provide their adsorption on the seed surface and protective function (Melnykova, 2019), thereby contributing to their preservation on seeds for a certain period of time and the restoration of physiological and symbiotic characteristics. It is also actual studying the practical application of a wide spectrum water-soluble synthetic polymers as adhesive and film-formers as a part of biologicals for improvement of bacteria adhesion on a seed surface (by type of multicomponent formulations in the produc- tion of modern chemical treaters). Russian scientists have tested for this purpose low and high molecular mass sodium alginate (FMC polymer), hydroxypropyl methylcellulose (HPMC) (Colorcon, “Colorcon, Inc.”, USA), polyethylene glycol (PEG), carbomer, polyvinyl alcohol (PVA) and polyvinylpyrrolidone (PVP). Poly- mers are also capable prolonging the expiration date of microbial preparations, increase their compatibility with chemical plant protectors, resistance to ultraviolet radiation, temperature differences, drying, increase the survival of rhizobia on the seeds surface, which allows of pre-treatment. In the study of survival of 634b strain on the ‘Belgorodska 7’ (creator – Federal State Budg- etary Educational Institution of Higher Education “V. Gorin Belgorod State Agricultural University”, Russia) soybean variety seeds under the influence of polymers of different origin and composition it was shown that polyvinylpyrrolidone 10 % solution is the most effec- tive of these compounds. Its use ensures the preserva- tion of 10 times more number of viable rhizobia on the seeds in 10 days after inoculation compared to control (Laktionov et al., 2019). The most important criterion for evaluating the effectiveness of symbiotic systems Glycine max – Bradyrhizobium japonicum is their molecular nitrogen fixation rate, which is based on the functioning of nod- ule bacteria enzyme nitrogenase and interrelated meta- bolic processes of symbiosis partners. At the stage of 3 true leaves the nitrogen fixation of soybean root nodules was 1.7, 6.6, 4.5 and 1.8 times more intensive in plants which seeds were inoculated by D87, В78 and В157 strains in 7 days before sowing, compared with control plants (inoculation in 1 hour before sowing with similar preparations). The high level NFA of symbiotic systems when treating with rhizobia 7 days before sowing seems to be the result of a sufficient number of microbial cells for become in- fected after this period. Researchers have found that the viability of nodule bacteria on seeds without the use of pesticides depends on the plant species and the biologi- cal qualities of microorganisms (Gemell et al., 2005), as well as the duration and storage conditions of inocu- lated seeds (Deaker et al., 2012). Pre-inoculation of soybean seeds (in 7 days before sowing) by preparation based on D87 strain provided the highest level of N2 assimilation due to the forma- tion of the largest nodules mass on the roots of plants (Figure 4). At the budding-beginning of flowering stage the nitrogen fixation activity of soybean root nodules has increased in variants with seeds inoculation on the sowing day. As a result, the difference between the NFA indicators of control (inoculation at the sowing day) and experimental plants (inoculation in 7 days before sowing) has decreased and gradually to equalize. The identified features of the formation and functioning of the symbiotic apparatus of soybeans, formed due to the use of different terms between inoculation and sowing, indicate a significant role of adaptive proper- ties of the microsymbiont during prolonged stay on the seeds before sowing and in the process of the formation of bean-rhizobial symbiosis. Therefore, the produce of Acta agriculturae Slovenica, 118/2 – 20228 N. VOROBEY et al. microbial preparations for pre-inoculation of seeds requires proper selection of strains of Bradyrhizobium japonicum taking into account their adaptive and physi- ological characteristics. Thus, as a result of the application of mentioned time periods from seed inoculation to sowing, a sym- biotic apparatus was formed on soybean roots, the ni- trogen fixation rate of which changed during the grow- ing season, that affected the plant supply by biological nitrogen. Intense assimilation of N2 by root nodules pro- vokes in plants a growing demand for photoassimilates and causes their redistribution (Kirizii et al., 2007). The regulatory role of nitrogen fixation in plant metabo- lism can stimulate or slow the growth of aboveground mass and rhizogenesis. At the stage of 3 true leaves, the aboveground and the root mass of inoculated plants (on the sowing day) outweighed the corresponding in- dices of plants bacterized in 7 days before sowing. Dur- ing the soybean growing season, the difference in root mass between plants of control and experimental vari- ants decreased and was not significant at the budding- beginning of flowering stage (Table 2). The root system of plants in all variants was well developed, with a large number of lateral roots, which provided an increase in the soybean nutrition area surface. Plant mass is one of the indices that characterizes the conditions of growth and development in different stages of the growing season. An actively functioning symbiotic apparatus is a more powerful sink of assimi- lates compared to vegetative growth. Therefore the pho- tosynthetic apparatus is not always fully able to provide the needs of all growth meristems in assimilates when the balance between growth, photosynthesis and nitro- gen fixation is disturbed. In the early period of functioning of the bean- rhizobial symbiotic system of soybean (3 true leaves stage) in control and experimental plants there was a positive relationship between the intensity of nitrogen- fixing activity and aboveground mass growth (Figure 4; Table 3). Figure 4: The nitrogen fixing activity, µmol C2H4 (plant h) -1, of root nodules of the Almaz soybean variety plants depending on the period duration from seed treatment by Bradyrhizobium japonicum (Kirchner, 1896), Jordan, 1982 D37, D87, В78, В157 strains to sowing (n = 5). m ± SE. An asterisk (*) indicates statistically significant difference between treatments (paired columns) at p ≤ 0.05; ns – no significant difference Acta agriculturae Slovenica, 118/2 – 2022 9 Symbiotic and physiological indicators of soybean inoculated of Bradyrhizobium japonicum single-strain in 7 days before sowing Then, before the budding-beginning of flowering stage in control plants, the growth in aboveground mass accelerated and outpaced the growth of plants bacte- rized in 7 days before sowing with strains D37, D87, B78 and B157 by 9.6, 13.3, 8, 2 and 14.5 % respectively. There was an intensive increase in the vegetative mass of plants in all variants of the experiment from the bud- ding stage to the full flowering stage. During the full flowering stage, which is related to the redistribution of assimilates and the formation of generative organs, the indicators of aboveground mass of control and ex- perimental plants, taking into account the error of the experiment, also did not differ significantly. Thus, when applying seed bacterization on the sowing day, this in- dicator was in the range of 6.82–7.23 g plant-1 and with seed inoculation in 7 days before sowing – 6.75–7.58 g plant-1. The dynamics of the aboveground mass for- mation of control and experimental plants was similar during the growing season. Optimal conditions for the formation and functioning of legume-rhizobial sym- biosis for plants were provided using both of period of soybean inoculation. In the vegetation experiment soybean were grown on a river sandy substrate with the introduction of Hellriegel nutrient mixture with 0.25 of nitrogen norm. It is probable that mineral nitrogen was used in the ear- lier stages of plant growth and development, and during the soybean flowering period the nutrition was mainly due to biologically N2 (due to the functioning of the symbiotic apparatus of plants). Therefore, the stimu- lation of vegetative growth was more active in plants inoculated with preparations of nodule bacteria D87, B78 and B157 strains with increased nitrogen fixation intensity. 4 CONCLUSIONS Thus, our studies have shown that inoculation of soybean seeds with microbial preparations based on Inoculant strain Development stage of plants: 3 true leaves budding-beginning of flowering 7 days before sowing on the sowing day 7 days before sowing on the sowing day D37 2.09 ± 0.08a 2.30 ± 0.09a * 3.34 ± 0.16a 3.08 ± 0.17a ns D87 2.04 ± 0.08a 2.28 ± 0.07a * 3.33 ± 0.17a 3.45 ± 0.11a ns В78 2.35 ± 0.09b 2.50 ± 0.07ab ns 3.19 ± 0.15a 2.95 ± 0.11a ns В157 2.08 ± 0.08a 2.31 ± 0.08a * 3.14 ± 0.12a 2.86 ± 0.12a ns Table 2: The root mass (g plant-1) of the Almaz soybean variety, under inoculation with nodule bacteria Bradyrhizobium japonicum (Kirchner, 1896), Jordan, 1982 (n = 15) m ± SE, * – significant difference at p ≤ 0.05; ns – no significant difference; interaction: a – not significant. For each strain and each variable, different letters a, b indicate significant differences Inoculant strain Development stage of plants: 3 true leaves budding-beginning of flowering full flowering 7 days before sowing on the sowing day 7 days before sowing on the sowing day 7 days before sowing on the sowing day D37 2.57a ± 0.12 2.50a ± 0.11 ns 3.86a ± 0.23 4.23a ± 0.13 ns 6.75a ± 0.28 6.82a ± 0.32 ns D87 2.84a ± 0.18 2.73ab ± 0.11 ns 4.06a ± 0.28 4.60a ± 0.32 ns 7.62a ± 0.38 7.21a ± 0.30 ns В78 2.87a ± 0.13 2.64ab ± 0.13 ns 3.77a ± 0.12 4.08a ± 0.30 ns 6.97a ± 0.32 7.18a ± 0.31 ns В157 2.75a ± 0.13 2.93b ± 0.14 ns 3.70a ± 0.24 4.24a ± 0.32 ns 7.58a ± 0.23 7.23a ± 0.27 ns Table 3: The aboveground mass (g plant-1) of the Almaz soybean variety, under inoculation with nodule bacteria Bradyrhizo- bium japonicum (Kirchner, 1896), Jordan, 1982 (n = 15)) m ± SE, * – significant difference at p ≤ 0.05; ns – no significant difference; interaction: a – not significant. For each strain and each variable, different letters a, b indicate significant differences Acta agriculturae Slovenica, 118/2 – 202210 N. VOROBEY et al. Bradyrhizobium japonicum on the sowing day caused the formation of more nodules on the plant roots. However, the mass of the formed nodules and the in- tensity of nitrogen fixation significantly dominated in plants inoculated in 7 days before sowing the seeds in the stage of the 3 true leaves only. The increase in the intensity of nitrogen fixation in control plants in the budding-beginning stage of flowering caused to the equalization of the difference of nitrogen fixation activ- ity between the variants with different terms between inoculation and sowing. This allows the effective use of bacterial preparations based on active strains D37, В78, D87, В157 for pre-sowing treatment of soybean seeds (in 7 days before sowing) without the use of extend- ers. In the future, it is advisable to study the ability to nodulate and assimilate N2 active strains of nodule bac- teria (and preparations based on them without the use of extender) under conditions of longer delay of seeds sowing from their inoculation. The results obtained are important for elucidation of the possibility of introduc- tion of nodule bacteria active strains obtained by bio- technological methods as a bacterial basis of prepara- tions for pre-inoculation of soybean. 5 REFERENCES Agritema. Catalog of products. (2017). Retrieved from https:// agritema.com/wp-content/uploads/2017/09/AGRITE- MA-Catalog_UA.pdf Anghinoni, F. B. G., Braccini, A. L., Scapim, C. A., Anghinoni, G., Ferri, G. C., Suzukawa, A. K. & Tonin, T. A. (2017). Pre-inoculation with Bradyrhizobium spp. in industrially treated soybean seeds. Agricultural Science, 8(7), 582–590. https://doi.org/10.4236/as.2017.87044 Berbenets, О. V. (2019). World-wide production of soya as an inexhaustible source of vegetable proteins and Ukraine’s place in the global trading market. Agrosvit, 10, 41–45. https://www.doi.org/10.32702/2306-6792.2019.10.41 Deaker, R., Hartley, E., & Gemell, G. (2012). Conditions affect- ing shelf-life of inoculated legume seed. Agriculture, 2(1), 38–51. https://doi.org/10.3390/agriculture2010038 Gemell, L. G., Hartley, E. J., & Herridge, D. F. (2005). Point-of- sale evaluation of preinoculated and custom-inoculated pasture legume seed. Australian Journal of Experimen- tal Agriculture, 45(3), 161–169. https://doi.org/10.1071/ EA03151 Grishechkin, V. V. & Golovina, E. V. (2014). Use of new or- ganic film-former (PPO) for conservation of viability of rhizobia at inoculation of seeds of soya and their influ- ence on formation of nodules and productivity. Legumes and Groat Crops, 1(9), 41–44. Grodzinsky, A. M. & Grodzinsky, D. M. (1964). Short reference book on plant physiology, Kyiv: Nauk. Dumka. Hardy, R. W. F., Holsten, R. D., Jackson, E. K. & Burns, R. C. (1968). The acetylene – ethylene assay for N2 fixation: laboratory and field evaluation. Plant Physiology, 42(8), 1185–1207. https://doi.org/10.1104/pp.43.8.1185 Kirizii, D. A., Vorobei, N. A. & Kots, S. Ya. (2007). Relation- ships between nitrogen fixation and photosynthesis as the main components of the productivity in alfalfa. Russian Journal of Plant Physiology. 54(5), 666–671. https://doi. org/10.1134/S1021443707050032 Kokorina, A. L. & Kozhemyakov, A. P. (2010). The Rhizobium- legume symbiosis and the use of microbiological preparations of complex action are an important reserve for increasing the productivity of arable land. Saint Petersburg: All-Russia Research Institute for Agricultural Microbiology. Kots, S & Mamenko, P. (2015). Soybean inoculation and in- crustation: a review of application technology and mar- ket of prerarats. Proposition. Special issue. Modern agro- technologies for the use of biological products and growth regulators, 24-28. Kots, S. Ya. (2011). Сurrent state of biological nitrogen fixation studies. Physiology and Biochemistry of Cultivated Plants, 43(3), 212–225. Kots, S. Ya., Vorobey, N. A., Kyrychenko, O. V., Melnykova, N. N., Mykhalkiv, L. M. & Pukhtaievych, P. P. (2016). Micro- biological Preparations for Agriculture, Kyiv: Logos. Kramarev, S. M. & Artemenko, S. F. (2016). The productivity of maize in crop short rotations with soybeans in the condi- tions of northern Steppe of Ukraine. News of Dnipropetro- vsk State Agrarian and Economic University, 42(4), 68–71. Laktionov, Yu. V., Kosulnikov, Y. V., Dudnicova, D. V., Yahno, V. V. & Kojemyakov, A. P. (2019). Pre-sowing protection of inoculated soybean Glycine max (L.) Merr. seeds by wa- ter-soluble polymer compositions and their solid-phase modification. Agricultural Biology, 54(5), 1052–1059 htt- ps://doi.org/10.15389/agrobiology.2019.5.1052eng Laktionov, Yu. V., Kosulnikov, Yu. V. & Dudnikova, D. V. (2018). The effect of water-soluble polymers on the survival of nodule lupine bacteria (Rhizobium lupini). Grain Economy of Russia, 3(57), 22–26. https://doi.org/10.31367/2079- 8725-2018-57-3-22-26 Martyniuk, S., Oron, J., Martyniuk, M. & Wozniakowska, A. (2002). Effects of interactions between chemical seed dressings and Bradyrhizobium japonicum on soybean seeds. Archives of Agronomy and Soil Science, 48(4), 305– 310. https://doi.org/10.1080/03650340214202 Melnykova, N. M. (2019). Effect of rhizobial exopolysaccha- rides on soybean seed germination and nodule develop- ment in the soybean-rhizobia symbiosis. Plant Physiology and Genetics, 51(5), 436–446. https://doi.org/10.15407/ frg2019.05.436 Morgun, V. V. & Kots, S. Ya. (2008). Symbiotic nitrogen fixa- tion and its significance in nitrogen plant nutrition: re- search status and prospects. Physiology and Biochemistry of Cultivated Plants, 40(3), 187–205. Netrusov, A. I., Yegorova, M. A., Zakharchuk, L. M., Kolotilova, N. N., Kotova, I. B., Semenova, E. V., … Judina, T. G. (2005). Workshop on Microbiology. Moscow: Akademiya Publ. Patyka, V. P. & Petrychenko, V. F. (2004). Microbial nitrogen fixation in modern fodder production. Feeds and Feed Production, 53, 3–6. Pavlyshche, A. V., Kiriziy, D. A. & Kots, S. Ya. (2017). The re- Acta agriculturae Slovenica, 118/2 – 2022 11 Symbiotic and physiological indicators of soybean inoculated of Bradyrhizobium japonicum single-strain in 7 days before sowing action of symbiotic soybean systems to the action of fungicides under various treatment. Plant Physiology and Genetics, 49(3), 237‒247. https://doi.org/10.15407/ frg2017.03.237 Slobodyanyuk, O. (2017). The nodules for soybean. Rizoline is a new answer for an old question. Agri Business Today, 347(5), 64–65. Spaink, H., Kondorosi, A & Hooykaas, P. (2002). The Rhizobi- aceae Molecular Biology of Model Plant – Assosiated Bacte- ria. Translated in rus. by Tikhonovich, I. A. & Provorov, N. A. St. Petersburg: Biont. Zilli, J. E., Campo, R. J. & Hungria, M. (2010). Effectiveness of Bradyrhizobium inoculation at pre-sowing of soybean. Pesquisa Agropecuaria Brasileira, 45(3), 335–337. https:// doi.org/10.1590/S0100-204X2010000300015 Acta agriculturae Slovenica, 118/2, 1–13, Ljubljana 2022 doi:10.14720/aas.2022.118.2.2402 Review article / pregledni znanstveni članek The usage of beneficial insects as a biological control measure in large- scale farming - a case study review on Trichogramma spp. Aleksandar IVEZIĆ 1, 2, Branislav TRUDIĆ 3, Gordon DRAŠKIĆ 4 Received November 02, 2021; accepted May 27, 2022. Delo je prispelo 2. novembra 2021, sprejeto 27. maja 2022 1 Department of Forecasting and Warning Service in Plant Protection, Agricultural extension service of Kikinda, Serbia 2 Corresponding author, e-mail: aleksandarivezic@yahoo.com 3 Forest Biodiversity and Restoration Team, Forestry Division, Food and Agriculture Organization of the United Nations, Viale delle Terme di Caracalla, Rome, Italy 4 Persida Inc., A Specialized Software Solutions Provider, Brooklyn, NY, United States The usage of beneficial insects as a biological control mea- sure in large-scale farming - a case study review on Tricho- gramma spp. Abstract: Large scale crops like maize, soybean, wheat and rice have changed the ecosystems worldwide, causing a major impact on global agricultural diversity. Intensive farming includes wide range of synthetic substances which are very often applied irrationally and excessively. Given the prevalence of large-scale farming in world agriculture, it is necessary to begin the transition from conventional crop pro- tection to integrated pest management (IPM) in these agro- ecosystems. One of the most important components of IPM are biological control measures with augmentative release of commercially available species of the genus Trichogramma Westwood, 1833 (Hymentoptera: Trichogrammatidae) as potentially successful and environmentally friendly methods. Besides Trichogramma, many other beneficial organisms are constantly being tested as potential biocontrol agents such as Chrysopa spp. (Neuroptera: Chrysopidae) and Orius spp. (Hemiptera: Anthocoridae). Minimizing the use of chemicals and replacing them with biological plant protection is fully in line with the agriculture development strategy and confirmed to be achievable in practice. It is especially important to apply such tactical decisions in the production of large-scale crops, which, at the same time, represent the biggest polluters of the environment as well. Key words: beneficial insects; biological control; Tricho- gramma spp.; large-scale crops; IPM Uporaba koristnih žuželk kot merilo biotičnega varstva pri kmetovanju na velikih zemljiščih - pregledna raziskava na primeru parazitoidnih os iz rodu Trichogramma Izvleček: Poljščine, kot so koruza, soja, pšenica in riž, ki se gojijo na velikih obdelovalnih zemljiščih, so globalno spre- menile ekosisteme in imajo globalno največji vpliv na raznoli- kost v kmetijstvu. Intenzivno kmetijstvo uporablja širok spek- ter sintetičnih snovi, ki so pogosto uporabljene neracionalno in v prevelikem obsegu. Zaradi prevladovanja kmetovanja na velikih zemljiščih v svetovnem merilu je potrebno začeti s prehodom iz konvencionalnega varstva kmetijskih rastlin na integrirano zatiranje škodljivih organizmov (IPM) v agroeko- sistemih. Med najpomembnejšimi komponentami integrira- nega varstva rastlin so ukrepi biotičnega zatiranja škodljivcev s sproščanjem komercialno dostopnih vrst parazitoidnih os iz rodu Trichogramma Westwood, 1833 (Hymentoptera: Tri- chogrammatidae) kot potencialno učinkovitih in okolju pri- jaznih metod. Poleg vrst iz rodu Trichogramma se v biotičnem varstvu stalno preiskušajo mnogi drugi koristni organizmi, kot so tenčicarice (Chrysopa spp., Neuroptera: Chrysopidae) in plenilske stenice iz rodu Orius (Hemiptera: Anthocoridae). Zmanjševanje uporabe kemikalij in njihovo nadomeščanje z biotičnim varstvom rastlin je popolnoma v skladu z razvojno strategijo kmetijstva in je potrjeno lahko doseženo v praksi. Še posebej je pomembno uporabiti te metode v velikopovr- šinski pridelavi kmetijskih rastlin, ki hkrati predstavlja tudi enega izmed največjih onesnaževalcev okolja. Ključne besede: koristne žuželke; biotični nadzor; Tri- chogramma spp.; veliko površinsko gojene kmetijske rastline; IPM Acta agriculturae Slovenica, 118/2 – 20222 A. IVEZIĆ et al. 1 INTRODUCTION A combination of high commodity crop prices, rising global food demand and technological advances, has transformed the scale of global crop production (Hochman et al., 2014). Certain crops are becoming more prevalent taking into consideration monocul- ture mainstreaming in agricultural production globally. These large-scale crops which occupy most of the glob- al agricultural area, have already transformed the land use dynamics and changed the ecosystems worldwide, and are still causing major impact on global agricul- tural diversity. Wheat (Triticum spp.), rice (Oryza spp.), corn (Zea spp.) and soybean (Glycine spp.) are prime examples. These four crops alone occupy approximately 50 % of the world’s entire agricultural lands, while the remaining crops cover the rest (Ben Ari & Makowski, 2016). Large scale farming (or intensive farming) is gen- erally defined as highly mechanized and commercial- ized cropping activities with much greater use of exter- nal inputs (Tittonell et al., 2020). It also includes wide range of synthetic substances (fertilizers, fungicides, in- secticides, and herbicides). These agrochemicals, used to prevent crop of diseases, manage the weeds and pests and boost plant growth, are very often applied irration- ally and excessively. In such conditions side effects of- ten occur. Inadequate use of pesticides can lead to pests’ resistance, excessive pesticide residues in food, pollu- tion of agroecosystems and suppression of beneficial organisms. Among all, these chemical agents are toxic for both wildlife and humans. Additionally, agrochemi- cals are often associated with reduction of populations of birds, amphibians and insects (bees, butterflies) by destroying their food source, contaminating soil and ground waters (Feshchenko, 2019). As the volume and production of agriculture continue to grow to new global records, so does the environmental awareness of societies. There is an in- creasingly pronounced demand for production of high- quality food without pesticide residues and other toxic substances. There is also an ecological issue underlining the use of renewable energy sources and preservation of natural resources and environment. Modern trend of sustainable agricultural production imposes the need to change technological process of production with the application of techniques that pollute the environment less and contribute to the health security in general. Numerous studies and social initiatives are calling for conversion to more sustainable agricultural practices due to their favorable effect on ecosystems, biodiversity and human health (Siebrecht, 2020). Given the prevalence of large-scale farming in world agriculture, it is necessary to begin the transition from conventional crop protection to integrated pest management (hereinafter: IPM). IPM is based on ex- tremely controlled and justified use of chemical agents with the emphasis on the use of alternative ways for pest control, like biological control measures. 1.1 BIOLOGICAL CONTROL MEASURES One of the most important components of IPM are biological control measures. Biological control or biocontrol is defined as a set of methods significant for pest control (insects, mites, weeds and plant diseases etc.) using their natural enemies. It relies on predation, parasitism, herbivory, or other natural mechanism, but also involves an active human influence (Flint & Dre- istadt, 1998). Since the existing form of natural balance between pests and beneficial organisms is usually in- sufficient to achieve expected results in intensive farm- ing, biological control requires the manipulation of beneficial insects by people to reduce the population of agricultural pests (Raspudić et al., 1999). In a strict ecological sense, applied biological control can be con- sidered as a strategy to restore functional biodiversity in agroecosystems by adding missing entomophagous insects through classical and/or augmentative biocon- trol techniques, but it can also be considered as a way of enhancing naturally occurring predators and para- sitoids through conservation and habitat management (Altieri, 1994). Biological control is a self-sustaining strategy through which farmers rely on pest control through ecological services provided by restored func- tional biodiversity, thus avoiding dependence on costly pesticides (Polanczyk & Pratissoli, 2009). The aim of this paper is to emphasize wide pos- sibility of implementation of beneficial insects in large crop farming, to point out their great potential in ag- ricultural practice and to discuss their high diversity worldwide. 1.2 TRADITIONAL KNOWLEDGE AND HISTORY OF BIOLOGICAL CONTROL The use of natural enemies to reduce the impact of pests has a long history. There are antecedent historical events that trace the evolution of some of the funda- mental concepts in the development of biological con- trol, and several of these events show the remarkable and perceptive insight of man into the workings of na- ture (Den Bosch et al., 1982). The first description of use of biological control dates from around 300 AD, when Acta agriculturae Slovenica, 118/2 – 2022 3 The usage of beneficial insects as a biological control measure in large-scale farming - a case study review on Trichogramma spp. predatory ants were used for control of pests in citrus orchards in China, a method which is still used today in Asia. In the 1750s, the British and French transported mynah birds from India to Mauritius to control locusts. Early applied biological control programs began under the USDA’s Department and later Bureau of Entomol- ogy established in 1881 (Polanczyk & Pratissoli, 2009). The first introduction of an exotic braconid wasp parasite, Cotesia glomerata (Linnaeus, 1758) (Hyme- noptera: Braconidae), against the imported cabbage- worm, Pieris rapae (Linnaeus, 1758) (Lepidoptera: Pieridae), into the United States occurred in 1883 and the introduction of the famous predaceous vedalia bee- tle Rodolia cardinalis (Mulsant, 1850) (Coleoptera: Coc- cinellidae) to control the cottony cushion scale Icerya purchasi Maskell, 1878 (Hemiptera: Margarodidae), fol- lowed in 1888 (Clausen 1978). The Department’s first large-scale biological control program did not begin until 1905 and involved explorations in Europe and Japan for natural enemies of the gypsy moth Lyman- tria díspar Linnaeus, 1758 (Lepidoptera: Lymantriidae), and browntail moth, Euproctis chrysorrhoea Linnaeus, 1758 (Lepidoptera: Lymantriidae), introduced into New England (Vail et al., 2001). It must be pointed out that from 1930 to 1940 there was a peak in biological con- trol activity in the world with 57 different natural en- emies established at various places, but World War II caused a sharp drop in biological control activity, and it did not regain popularity after war due to production of relatively inexpensive synthetic organic insecticides (Polanczyk & Pratissoli, 2009). Today, there are hundreds of biological control products commercially available for pest control, but not all are sufficiently effective for large-scale farming. One of the potentially most successful and environmen- tally friendly methods for biological control of pests is the augmentative release of commercially available spe- cies of the genus Trichogramma Westwood, 1833 (Hy- mentoptera: Trichogrammatidae) (Stouthamer, 1993). These organisms have been identified and successfully used for inundative biological control of lepidopteran pests for more than 120 years (Smith, 1996; Van Len- teren, 2000). Trichogramma are egg parasitoids that at- tack more than 200 lepidopteran host species, including pest groups of borers, webworms, loopers, leafworms, fruitworms, cutworms, bollworms and armyworms (Knutson, 1998). More than a thousand scientific papers have been published on Trichogramma and its usage as a biologi- cal control agent, making it one of the most researched natural enemies in the world. As a result, they have been widely used in inundative and inoculative biological control programs in more than 30 countries in agricul- tural crops (e.g. corn, cotton, sugarcane, rice, soybean, fruit trees, vegetables) and natural forests (Knutson, 1998). 1.3 Trichogramma spp. LIFE CYCLE Trichogramma wasps primarily parasitize eggs of moths and butterflies (Lepidoptera). However, certain species of Trichogramma also parasitize eggs of beetles (Coleoptera), flies (Diptera), true bugs (Heteroptera), other wasps (Hymenoptera), lacewings and their rela- tives (Neuroptera) (Knutson, 1998). The adult female wasp uses chemical and visual clues to locate a host egg (Nordlund et al., 1981). Once a female finds a host egg, she drills a hole through the chorion (eggshell) and in- serts two to three eggs into the host egg. Eggs of para- sitoid hatch in about 24 hours and the parasite larvae develop very quickly. Larvae develop through three instars. During the third instar, dark melanin granules are deposited on the surface of the egg chorion, causing the host egg to turn black. Larvae than transform to the inactive pupal stage. After about 4-5 days, the adult wasps emerge from the pupae and escape the host egg by chewing a circular hole in the eggshell. The black layer inside the chorion and the exit hole are evidence of parasitism by Trichogramma (Ruberson et al., 1993). 2 Trichogramma IN MAIZE Maize (Zea spp.) is one of the most abundantly produced cereal in the world. It is grown in every conti- nent except Antarctica (Eckhoff et al., 2003). This large- scale crop has many pests and the European corn borer Ostrinia nubilalis Hubner, 1796 (Lepidoptera: Crambi- dae) is considered the major one worldwide (Mutuura & Monroe, 1970). The egg stages of many corn pests including O. nubilalis, are attacked by various species of Trichogramma (Ivezić & Trudić, 2021). More than 15 species and strains of native and exotic Trichogramma were evaluated in field and laboratory tests to deter- mine those with a high preference for O. nubilalis egg clusters or other lepidopteran corn pests (Wang et al., 1999). Several species of Trichogramma have been iden- tified as promising biological control agents of O. nubi- lalis, including T. brassicae Bezdenko, 1968 and T. eva- nescens Westwood, 1833 (Bigler, 1986; Hassan, 1993). Both species appear to be widespread across Europe and reared in commercial facilities for release as bioag- ents. In South Europe, these two Trichogramma species are considered to be the most abundant Trichogramma species in maize (Bohinc et al., 2015; Ivezić et al., 2021). Acta agriculturae Slovenica, 118/2 – 20224 A. IVEZIĆ et al. In order to ensure production of effective para- sites, mass rearing facilities developed rearing tech- niques with stringent quality control procedures. This requires controlled environments, artificial diets and ovipositional substrates, mechanized equipment and operations performed by work units (Knutson, 1998). This process selects the strain of Trichogramma with the most efficient ability to fly, locate and parasitize the eggs of a targeted host. After selecting the best strain/colony, Trichogramma pupae is used to colonize the crop. Pu- pae can be programmed to enter a condition of arrested development called diapause. Once in diapause, wasp pupae can be stored for up to 9 months so the large demand for Trichogramma during the summer can be met (Bigler, 1994). Cardboard capsules containing host eggs with developing Trichogramma are applied to the corn field and can be distributed from the ground or air. Capsules either fall to the ground or are caught in the corn plant. The capsules protect the Trichogramma from predators and weather extremes until the adults emerge from the host egg and escape through tiny holes in the capsules. Released Trichogramma are at different developmental stages so that adults emerge from the capsules over several days. This increases the time in- terval between applications (Knutson, 1998). Releases of Trichogramma are performed manu- ally or mechanically (ground and air) (Li, 1994). Since manual applications have shown as time-consuming process, aerial applications are more acceptable option. One such experiment was performed in Poland, where the use of ultralight aircraft proved to be an effective option (Bzowska-Bakalarz et al., 2020). The results in- dicate that the low-height aerial application allows pre- cise dosing and satisfactory distribution of bioagents. The efficacy of 60–85 % (depending on the year) of the gyroplane-based spraying operations was comparable with those monitored for ground application. These re- sults show a promising alternative for the application of Trichogramma, especially in large-scale crops, because the ground application in intensive agriculture requires too much time and energy (Bzowska-Bakalarz et al., 2020). Other caterpillar pests of corn, such as the south- western corn borer Diatraea grandiosella Diar, 1911 and American cotton bollworm Helicoverpa zea Bod- die, 1850, are attacked by native or introduced species of Trichogramma (T. pretiosum Riley, 1879, T. deion Pinto and Oatman, 1986, T. thalense Pinto and Oatman, 1985) throughout the world (Manandhar & Wright, 2015). Some companies sell Trichogramma for control of these pests, but research to support this usage is lacking. At this point, European corn borer is still the most controlled pest of maize with wasps of the genus Trichogramma. 3 Trichogramma IN RICE Rice (Oryza spp.) is one of the most important crops in the world, being produced in many locations and under a variety of climatic conditions. Since sizable portions of certain crops are used for purposes other than human consumption, rice is the most important food crop, directly feeding more people than any other crop (Rao et al., 2017). Traditionally, countries in Asia have the largest share in world rice production, but this crop is becoming increasingly important in Africa and Latin America. Meanwhile, the expansion of rice crops poses a challenge for agricultural workers as they con- stantly face many obstacles, such as pests and diseases (Afifah et al., 2019). Key pests of rice are striped rice stemborer Chilo suppressalis Walker 1863 (Lepidoptera: Crambidae), yellow stem borer Scirpophaga incertulas Walker 1863 (Lepidoptera: Crambidae), pink stem borer Sesamia inferens Walker 1856 (Lepidoptera: Noctuidae), rice leafroller Cnaphalocrocis medinalis Guenee, 1854 (Lepidoptera: Crambidae), rice planthopper Nilapar- vata lugens Stal, 1854 (Hemiptera: Delphacidae) and rice green semilooper Naranga aenescens Moore, 1881 (Lepidoptera: Noctuidae) (Tang et al., 2017). Among these, yellow stem borer is considered to be the most important pest of rain-fed lowland and flood-prone rice ecosystems (Barthakur, 2010; Ko et al., 2014). Popu- lations of these pests substantially increased within one decade, therefore, the appropriate, effective, and inex- pensive control measures are needed for the continuity of high rice production (Gao et al., 2012). Optimized methods with less environmental impact and high sus- tainability are in demand, such as releasing biological control agents. As one of the most important natural enemies worldwide, the use of Trichogramma wasps in rice fields is the subject of constant research (Afifah et al., 2019). Although Trichogramma has been studied for management of key lepidopteran pests in rice, these wasps aren’t still commercially used in intensive rice production. Recent findings from China indicate that Trichogramma releases may be considered practical for control of striped rice stemborer and rice leafrol- ler. However, it is less clear whether yellow stem borer can also be controlled by Trichogramma wasps as less studies have been done on this species so far (Tang et al., 2017). From field surveys conducted in Indian rice fields, there are indicators that yellow stem borer eggs may not be effectively parasitized under natural condi- tions (Hikjm, 1988; Chakraborty, 2012). On the other hand, more positive results have been reported from a Acta agriculturae Slovenica, 118/2 – 2022 5 The usage of beneficial insects as a biological control measure in large-scale farming - a case study review on Trichogramma spp. field survey in China showing rather high parasitism rates of yellow stem borer eggs in the range of 46.7 % to 79.1 % (Guo et al., 2002; Samara et al., 2008). In general, there have been a very few attempts to control yellow stem borers by inundative releases of Trichogramma (Tang et al., 2017). Positive results were obtained from Indonesia where T. japonicum Ashmead, 1904 showed the potential to become candidate for control of white rice stem borer (Yunus, 2018), while promising results were also obtained in Egypt in the control of rice stem borer Chilo agamemnon Bleszynski, 1962 (Lepidoptera: Crambidae) due to inundative release of T. evanescens (Sherif et al., 2008). There are rich communities of beneficial insects, spiders, and diseases that attack insect pests of rice, but just a few of them are commercially applied. Certain results indicate that the application of Trichogramma wasps shows promising results in the control of rice pests, which has consequently aroused great interest among consumers and rice producers. However, for wider commercial application of these organisms in rice production positive results are lacking, and the use of these organisms is mainly done for the purpose of research. 4 Trichogramma IN SOYBEAN The largest producers of soybean (Glycine spp.) in the World are Brazil, the USA, Canada, China and Argentina (https://www.fas.usda.gov/commodities/ soybeans). There is a significant variety of insects that may be found in soybean fields at any given time of the season, and among them is a large number of different pests, many of which can cause significant yield losses. As in the above-mentioned crops, different species of the genus Trichogramma are present in soybean fields, both as native populations and as introduced biocon- trol agents (Bueno et al., 2008). Some of these species are important natural enemies of key pests of soybean production such as sunflower looper Rachiplusia nu Guenee, 1852 (Lepidoptera: Noctuidae), velvetbean cat- erpillar Anticarsia gemmatalis Hubner, 1818 (Lepidop- tera: Erebidae), soybean budborer Crocidosema apore- ma Walsingham, 1914 (Lepidoptera: Tortricidae) and cotton earworm Helicoverpa armigera Hubner, 1808 (Lepidoptera: Noctuidae) (Bortolotto et al., 2015). In Southern Brazil three species of Trichogramma spp. are well known and used for controlling the num- ber of velvetbean caterpillar: T. pretiosum, T. acacioi Brun, Moraes and Soares, 1984 and T. rojasi Nagaraja and Nagarkatti 1973 (Foerster et al., 2015). In Uruguay, six species of Trichogramma have been identified from collections of different crops (Basso et al., 2020). Among the reported species, T. pretiosum is the most widely distributed in Uruguay and parasitizing a great number of lepidopteran pests (Basso et al., 1999a; Basso et al., 1999b; Basso & Pintureau, 2004). Given the prevalence, in last decades this species was introduced as biological agent in soybean crops (Basso et al., 2020). The selec- tion of T. pretiosum was based on the fact that, in the laboratory, it presented the highest fertility parasitiz- ing eggs of A. gemmatalis deposited on soybean plants, when compared to T. exiguum Pinto and Platner, 1978 and T. galloi Zucchi, 1988, species also present in Uru- guay (Basso et al., 2020). In this country a multi-year study was conducted to compare conventional practice with different doses of the egg parasitoid. Although the best results were obtained with the application of chemical insecticides, two releases of T. pretiosum by terrestrial methods, 20 days apart, or 4 weekly appli- cations by means of a drone, reached the best results below the thresholds of sanitary intervention, both op- tions with 200,000 parasitoids per hectare (Basso et al., 2020). The application of T. pretiosum under the inun- dative biological control method appears as a real al- ternative to chemical insecticides for the control of the main lepidopteran pests in soybean crops in Uruguay (Basso et al., 2020). These results showed that biological tool such as egg parasitoids of the genus Trichogramma can differentiate and value production of soybean. Be- side Latin America, T. pretiosum is present in almost all biogeographic regions in the world. With 240 host re- cords in the Americas, it is one of the most commonly collected species, especially in agricultural and other disturbed habitats (Pinto, 1998). The efforts of large soybean producers to imple- ment alternative ways and eco-friendly methods for pest control management indicate that environmental awareness is constantly growing, but also that future production of large- scale crops should be much more based on the use of beneficial insects and biological control measures in general. 5 Trichogramma AS A BIOAGENT IN OTHER CROP SPECIES Besides in large scale crops, augmentation of Trichogramma has been promoted for pest control in cotton, apple, spruce, avocado, tomato and potato pro- duction (Olkowski & Zhang, 1990). In Europe, T. evane- scens is widely used for control of codling moth Cydia pomonella Linnaeus, 1758 (Lepidoptera: Tortricidae) in apples (Knutson 1998), but also as an effective tool to decrease population of potato tuber moth Phthori- Acta agriculturae Slovenica, 118/2 – 20226 A. IVEZIĆ et al. maea opercullea Zeller, 1873 (Lepidoptera: Gelechiidae) (Saour, 2004). Three Trichogramma species, T. cacoeciae Marchal, 1927, T. evanescens, and T. principium Sug- onjaev and Sorokina, 1976, are proved as effective can- didates in parasitizing potato tumber moth eggs (Saour, 2004). In the USA, parasitism of tomato fruit pests (H. armigera or tomato leafminer Tuta absoluta Meyrick, 1917 (Lepidoptera: Gelechiidae) by native T. pretiosum in tomatoes is considered in the treatment thresholds for these pests with insecticides (Hoffman et al., 1990). Augmentation of T. pretiosum is an effective control tactic in Mexico and is a part of the integrated pest pro- gram for fresh market tomatoes (Trumble & Alvarado- Rodriguez, 1991). In some countries like China, the Philippines, India, and Taiwan, T. chilonis Ishii, 1941 is already being used as a biological control agent in sug- arcane plantations. In Indonesia (Grieshop et al., 2014), T. chilonis was first developed to address the problem of stem borer in several sugarcane plantations in Java which was later introduced to Lampung after similar problems arose (Afifah et al., 2019). The results showed that the release of 150,000 eggs Trichogramma spp. per hectare could reduce the population of sugarcane shoot borer Chilo infuscatellus Snellen, 1890 (Lepidoptera: Crambidae) while 250,000 eggs are required per hec- tare to control sugarcane stem borer C. terrenellus Pa- genstecher, 1900 (Lepidoptera: Crambidae) (Cascone et al., 2015). Besides being able to parasitize Chilo spp. T. chilonis is also capable of parasitizing Agrotis spp. (Lepi- doptera: Noctuidae), sugarcane gray borer Tetramorea schistaceana Snellen, 1891 (Lepidoptera: Tortricidae), rice leafroller Cnaphalocrosis medinalis Guenee, 1854 (Lepidoptera: Crambidae), H. armigera, soyabean pod borer Leguminivora glycinivorella Obraztsov, 1960 (Lep- idoptera: Tortricidae) and beet armyworm Spodoptera exigua Hubner, 1808 (Lepidoptera: Noctuidae) (Li- Ying, 1994). In California, two avocado pests, the omnivorous looper Sabulodes aegrotata Guenee, 1857 (Lepidoptera: Geometridae) and the avocado leafroller Amorbia cu- neana Walsingham, 1879 (Lepidoptera: Tortricidae), can be managed by releasing T. platneri Nagarkatti, 1975 in every fourth avocado tree (Olkowski & Zhang, 1990). Large field studies in Canada have shown that two releases, each with 30 million T. minutum individu- als per acre, resulted in 60 to 80 % egg parasitism of spruce budworm Choristoneura fumiferana Clemens, 1865 (Lepidoptera: Tortricidae) in white spruce stands (Olkowski & Zhang, 1990). The actual rates of release vary considerably, even for the same pest, crop, and country. This range is prob- ably related to the range in dimensional volume of the crop. For example, the total rates of release for T. bras- sicae alone, which is reared from small host eggs against European corn borer in Europe, range from 150,000 to 2.8 million wasps/ha (El-Wakeil et al., 2020). Rates Species* Strain Host and taxonomy* Country of Origin Trichogrammatoidea bactrae Nagaraja, 1979 Bac-1 Plutella xylostella Linnaeus, 1758 (Lepidoptera: Plutellidae) Thailand Trichogramma bourarachae Pintureau and Babaul, 1988 Bou-1 Vanessa cardui Linnaeus, 1785 (Lepidoptera: Nymphalidae) Morocco Trichogramma bourarachae Bou-2 Helicoverpa armigera (Lepidoptera: Noctuidae) Portugal Trichogramma buesi Voegele, 1982 Bue-1 Ephestia kuehniella Zeller, 1879 (Lepidoptera: Pyralidae) Canada Trichogramma chilonis Chi-1 Plutella xylostella Japan Trichogramma chilonis Chi-3 Ephestia kuehniella Taiwan Trichogramma dendrolimi Matsumara, 1926 Den-1 Lobesia botrana Denis and Schiffermu ller, 1775 (Lepidoptera: Tortricidae) Italy Trichogramma evanescens Eva-1 Pectinophora gossypiella Saunders, 1884 (Lepidoptera: Gelechiidae) Egypt Trichogramma oleae Voegele and Pointe, 1979 Ole-1 Prays oleae Bernard, 1794 (Lepidoptera: Plutellidae) France Trichogramma ostriniae Ost-2 Ephestia kuehniella Moldova Trichogramma principium Sugonjaev and Sorokina, 1976 Pri-1 Earias insulana Boisduval, 1833 (Lepidoptera: Nolidae) Syria Table 1: Host and country of origin of the Trichogramma species and strains (adapted from Tabone et al., 2010) *Detailed explanation of the species (author and taxonomy) is provided only for those species which are not mentioned in previous text Acta agriculturae Slovenica, 118/2 – 2022 7 The usage of beneficial insects as a biological control measure in large-scale farming - a case study review on Trichogramma spp. in several millions of wasps/ha are generally cited in arboreal situations such as forestry, and in fruit or nut orchards, whereas those in agricultural crops such as corn, cotton, and tomato, range from 500 to more than 1 million wasps/ha, with averages of 200,000-600,000 wasps/ha (El-Wakeil et al., 2020). China often reports lower rates than other countries, possibly because of the frequent use of large host eggs (Wang, 2013). When determining the effectiveness of certain Trichogramma species in biological control, it was found that the rate of parasitism does not only depend on the selected Trichogramma species, but also on the choice of the appropriate strain. Certain strains within the same species may have different laboratory and field performances, or different preferences towards the same or different hosts. Tabone et al. (2010) analyzed the rate of parasitism of different Trichogramma spe- cies and strains and pointed out significant differences within the diversity of Trichogramma species in certain regions and on different hosts (Table 1.). These results very effectively represent the preference of Trichogram- ma parasitoids for Lepidoptera, both among different species and among different strains within the same Trichogramma species. 6 GENERALIST PREDATORS The scientific community uses all available re- sources in order to explore the natural potential of entomofauna and introduce less-toxic solutions in pest management programs. Many other beneficial organ- isms are constantly being tested as potential biocon- trol agents such as species of the genus Chrysopa spp. (Neuroptera: Chrysopidae) and Orius spp. (Hemiptera: Anthocoridae). Chrysopids commonly known as lacewings, occur in numerous agricultural and horticultural zones of the northern hemisphere. Adults are free-living and usually non-predatory in nature, surviving on nectar and pol- len, while three larval stages are highly predatory (Bel- lows & Fisher, 1999). They are active predators of a wide variety of pests including aphids, chinch bugs, mealy- bugs, scales, whiteflies, leafhoppers, lepidopterous eggs and larvae, and mites (Principi & Canard, 1984). The efficacy in biological control of aphids as well as other arthropod pests has been recognized for more than 250 years (Dhandapani et al., 2016). Inundative releases of the common green lacewing Chrysoperla carnea Stephens, 1836 (Neuroptera: Chrys- opidae) on cotton provided effective results in control of American cotton bollworm Helicoverpa zea Boddie, 1850 (Lepidoptera: Noctuidae) and tobacco budworm Heliotihis virescens Fabricius, 1777 (Lepidoptera: Noc- tuidae) (Ridgway & Jones, 1969). Releases of C. carnea eggs in field cages at rates of 50,000 and 100,000 per acre can significantly reduce the population of tobacco budworm and increase the yield (Ridgway & Jones, 1969). Green lacewing has also been effective on potato aphid Macrosiphum euphorbie Thomas, 1878 (Hemip- tera: Aphididae) and buckthorn aphid Aphis nastrurtii Kaltenbach, 1843 (Hemiptera: Aphididae) (Capinera, 2001). A species that is very common in corn fields is Chrysoperla oculata Ruzicka, 1997. The most suitable prey for C. oculata in corn fields is corn leaf aphid Rho- palosiphum maidis Fitch, 1856 (Hemiptera: Aphididae) an important pest of corn (Bellows & Fisher, 1999). Many species of the genus Chrysopa, such as red-lipped green lacewing C. rufilabris Burmeister, 1839, C. externa Hagen, 1861 and C. perla Linnaeus, 1758 are important predators and are used as biological control agents worldwide. Among the above-mentioned species, most attempts have evaluated the efficacy of C. carnea in aug- mentation releases in the field or in the greenhouses (Ridgway & Mcmuphy, 1984; Nordlund et al., 2001). In the context of biological control, the genus Ori- us includes several species that have found their place in commercial pest control in agriculture. This genus is represented by very tiny true bugs commonly known as minute pirate bugs and flower bugs (Riudavets & Cas- tane, 1994). They play a key role in the management of various agricultural pests in greenhouse and field envi- ronments. They can be found in numerous crops, pas- tureland and surrounding areas (cotton, soybean, bean, potato, wheat, alfalfa, maize, orchards, other vegetables and ornamental crops), as well as in trees, shrubs, weeds and many wild plants. They prey on thrips, aphids, mites, whiteflies, moths and other tiny arthropods and insect eggs. Orius are very effective predators and can thus provide biological pest control in a variety of crop- ping systems (Brust & Yurchak, 2021). The species insidious flower bug Orius insidiosus Say, 1832 (Hemiptera: Anthocoridea) is one of the most important predators in corn field. The ability of O. insid- iosus to search, find and destroy European corn borer and corn earworm eggs was investigated in numerous studies. O. insidiosus is an important natural enemy of corn earworm in corn, cotton and sorghum. A study conducted in the USA, revealed European corn borer larvae sustain high mortality in field corn and that O. insidiosus was the most important predator of these lar- vae in western Maryland (Brust & Yurchak, 2021). Their population peak coincides with corn pollen-shedding and sulking, during which they feed on second-gener- ation of European corn borer larvae and corn pollen. Therefore, successful biological control of European Acta agriculturae Slovenica, 118/2 – 20228 A. IVEZIĆ et al. corn borer larvae by O. insidiosus is linked to arthropod prey and corn pollen (Brust & Yurchak, 2021). Orius insidiosus adults and nymphs are common in soybean fields. Its population dynamics in soybean fields have been linked to thrips population levels and soybean flowering. Nymphs and adults eat soybean aphids in the field. Experimental findings suggest that under certain conditions, O. insidiosus can effectively suppress aphid population growth and that they may be key factors influencing aphid population dynamics in soybeans in some areas within the USA. In addition to soybean aphids, soybean thrips are believed to be one of the most important thrips prey of O. insidiosus in soybean. It is believed that soybean thrips serve as an important prey resource for O. insidiosus in soybeans and may be important in sustaining O. insidiosus popu- lations before the arrival of soybean aphids. O. insidio- sus is known to feed on eggs and first instar of green cloverworm Hypena scabra Fabricius, 1789 (Lepidop- tera: Erebidae) as well (Brust &Yurchak, 2021). Rice predators include spiders, ants, some insect families such as Carabidae, plant bugs, amphibians, dragonflies and other beetles and water bugs. However, the most abundant ones are the spiders (Wopereis et al., 2008). Their ability to hunt in a variety of habitats in combination with high abundance, positions spiders as potentially effective biocontrol agents (Symondson et al., 2002). Dispersal by running and ballooning allows spiders to colonize agricultural fields soon after distur- bance due to agricultural practices such as ploughing and seed sowing (Radermacher et al., 2020). This applies in particular to agricultural systems with multiple crop- ping cycles per year and asynchronous planting prac- tice (Marc et al., 1999). In fact, spiders are among the most abundant arthropod predators in rice ecosystems and assumed to contribute to the control of pest species such as plant and leafhoppers (Sigsgaard, 2007). With the ability to capture prey of different feeding guilds, including herbivores and detritivores, spiders may play an important role soon after planting rice fields when herbivore populations still are low (Radermacher et al., 2020). Generalist predators in agricultural systems such as spiders may link aboveground herbivore and below- ground detrital systems using prey of both systems (Scheu, 2001; Snyder & Wise, 2001; Wise et al., 2006). 7 CONCLUSIONS Although the use of beneficial insects may seem simple at first, effective pest biocontrol is determined by many factors. Those factors are adequate selection of parasites and predators, the quality and fitness of used bioproduct, the numbers released and the tim- ing of the release, the release method, complex interac- tions between the parasite/predator, the target pest, the crop and environmental conditions (Knutson, 1998). However, a fundamental step in the development of any biological control program utilizing beneficial in- sects is the identification and choice of species and/or strains to use; not all species (or populations) perform equally well, in terms of mass rearing or field disper- sal and performance (Ivezić et al., 2018). Therefore, the first step for the implementation of beneficial insects in the program of biological control of pests is the ac- curate identification and genetic characterization of na- tive species, since autochthonous species are likely to be best adapted to environmental conditions in a specific ecosystem (Whitman & Nordlund, 1994). In addition to native populations of beneficial in- sects, biological control also involves the introduction of natural enemies that are not typical of certain ar- eas. The introduction of natural enemies is used when a pest of exotic origin is the target of the biocontrol program (White, 2019). Pests are constantly being im- ported into countries where they are not native, either accidentally, or in some cases, intentionally. Many of these introductions do not result in establishment or if they do, the organism may not become pest. However, it is not uncommon for some of these introduced organ- isms to become pests due to a lack of natural enemies to suppress their population number. In these cases, in- troduction of natural enemies can be highly effective. Once the country of origin of the pest is determined, exploration in the native region can be conducted to search for promising natural enemies (White, 2019). If such enemies are identified, they may be evaluated for potential impact on the pest organism in the na- tive country or alternatively, introduced into the new country for further study. They first need to be placed in a quarantine for one or more generations to be sure that no undesirable species are accidentally imported (diseases, hyperparasitoids etc.). Additional permits are required for interstate shipment and field release (White, 2019). Besides inundative and inonculative activities for commercial usage of beneficial insects, it is also neces- sary to preserve the autochthonous residential species populations. Conservation of habitats and preservation of biodiversity as one of the main prerequisites for suc- cessful implementation of biological control strategies can be defined as an identification and modification of human influence that allows natural enemies to express their potential to suppress pests (Rechcigl & Rechcigl, 2020). Numerous studies and experience have shown that conserving natural enemies is of tremendous im- Acta agriculturae Slovenica, 118/2 – 2022 9 The usage of beneficial insects as a biological control measure in large-scale farming - a case study review on Trichogramma spp. portance in the safe and economical management of in- sect pests and doing so has to be a major component of a producer’s management activities (Rechcigl & Rech- cigl, 2020). Likewise, the most important component of biological control is establishing the list of indigenous species of organisms for biological control, which in- cludes only those beneficial species that are indigenous or ubiquitous in specific region. Such an approach has been done in Slovenia and represents an additional safeguard that practically prevents the use of beneficent species that could in any way endanger the common domestic flora and fauna (Trdan et al., 2020). Over the last decades large scale farming led to increasing production, but also caused substantial en- vironmental degradation as such increases were mostly based on its expansion onto natural areas and greater use of external inputs and other forms of intensified use (IPBES, 2019). Achieving sustainability of agricultural production is one of the key challenges for humanity. Minimizing the use of chemicals and replacing them with biological plant protection is firstly fully in line with the agriculture’s development strategy and sec- ondly, confirmed to be achievable in practice. It is es- pecially important to apply such tactical decisions in the production of large-scale crops, which, at the same time, represent the biggest polluters of the environment in general. The use of beneficial insects in biological control is common worldwide but its potential has not been explored for many pests and in many geographic regions. Even in Europe certain countries with very in- tensive agriculture do not currently use the full poten- tial of these organisms in their agroecological systems. In addition, a recent survey found that very few gov- ernmental Extension Services currently provide recom- mendations for controlling pests with Trichogramma spp. or any other beneficial insects (Ivezić & Trudić, 2021). The successful use of augmentative releases of biocontrol agents in pest management programs will depend on a sound and thorough research program, favorable economics, commercial investment, and the development of an extension program to transfer this technology to crop consultants and large scale grow- ers. Mainstreaming agroecology and its biological con- trol alternatives among large scale farmers is urgently needed, but it requires addressing specific questions in research, technology and policy development to sup- port sustainable transitions. 8 ACKNOWLEDGEMENT This review paper is done under the scope of RIS fellowship programme (September 2021-December 2021, Warsaw, Poland) of The European Institute of In- novation and Technology (EIT), University of Warsaw, Warsaw, Poland. 9 REFERENCES Afifah, L., Muhammad Bayfurqon, F. and, Latifatus Siriyah S. (2019). Control of Rice Stem Borer Scirpophaga sp. using Trichogramma sp. Indonesian Journal of Community Engage- ment. 5(1), 99-107. http://doi.org/10.22146/jpkm.34180 Afifah, L., Fawzy, B., and Siti, S. (2019). Control of Rice Stem Borer Scirpophaga sp. Using Trichogramma sp. Journal Pengabdian Kepada Masyarakat (Indonesian Journal of Community Engagement), 5(99), https://doi.org/10.22146/ jpkm.34180 Altieri, M.A. (1994). Biodiversity and pest management in agro- ecosystems. New York, Haworth Press. 213p. Basso, C., Grille, G., and Pintureau, B. (1999a). Eficacia de Trichogramma exiguum Pinto & Platner y de T. pretiosum Riley en el control de Argyrotaenia sphaleropa (Meyrick) y de Bonagota cranaodes (Meyrick) en la vid en el Uruguay. Agrociencia Uruguay, 3, 20-6. Basso, C., Pintureau, B., and Grille, G. (1999b). Taxonomic study of two Trichogramma species from Uruguay (Hym: Trichogrammatidae). Boletin de Sanidad Vegetal Plagas, 25, 373-82. Basso, C. and Pintureau, B. (2004). Las especies de Trichogram- ma del Uruguay (Hymenoptera: Trichogrammatidae). Revista de la Sociedad Entomológica Argentina, 63(1-2), 71-80. Basso, C., Chiaravalle, W., and Maignet, P. (2020). Effectiveness of Trichogramma pretiosum in controlling lepidopterous pests of soybean crops. Agrociencia Uruguay. https://doi. org/10.31285/AGRO.24.419 Bellows, T.S. and Fisher, T.W. (1999). Handbook of Biological Control. Principles and Application of Biological Control, Academic Press San Diego. California, 1047p. Ben Ari, T. and Makowski, D. (2016). Analysis of the trade-off between high crop yield and low yield instability at the global scale. Environmental Research Letters, https://doi. org/10.1088/1748-9326/11/10/104005 Bigler, F. (1986). Mass production of Trichogramma maidis Pint. et Voeg. and its field application against Ostrinia nu- bilalis Hbn. in Switzerland. Journal of Applied Entomology, 101, 23–29. Bigler, F. (1994). Quality control in Trichogramma produc- tion. In Biological Control with Egg Parasitoids, eds. E. Wajnberg and S. A. Hassan, pp. 93-111. Oxon, U.K.: CAB International. https://doi.org/10.1111/j.1439-0418.1986. tb00829.x Bohinc, T., Schmidt, S., Monje, J.C., Trdan, S. (2015). Prva na- jdba parazitoidne ose Trichogramma brassicae Bezdenko, 1968 (Hymenoptera, Trichogrammatidae) v Sloveniji. Acta agriculturae Slovenica, 105(2), 323-327. https://doi. org/10.14720/aas.2015.105.2.15 Bortolotto, O.C., Pomari-Fernandes, A., Bueno, RCOF., Bueno, AF., Cruz, YKS., Sanzovo, A., Ferreira, R.B. (2015). The Acta agriculturae Slovenica, 118/2 – 202210 A. IVEZIĆ et al. use of soybean integrated pest management in Brazil: A review. Agronomy Science and Biotechnology, 1(1), 25-32. https://doi.org/10.33158/ASB.2015v1i1p25 Brust, G. and Yurchak, V. (2021). Minute Pirate Bug: A Ben- eficial Generalist Insect Predator. University of Meriland Extension. https://extension.umd.edu/resource/minute- pirate-bug-beneficial-generalist-insect-predator Bueno, A.F., Bueno, RCOF., Parra, JRP., and Vieira S.S. (2008). Effects of pesticides used in soybean crops to the egg par- asitoid Trichogramma pretiosum. Ciência Rural, 38, 1495– 1503. https://doi.org/10.1590/S0103-84782008000600001 Bzowska-Bakalarz, M., Bulak, P., Beres, P.K., Czarnigowska, A., Czarnigowski, J., Karamano, B., Pniak, M., and Bieganow- ski, A. (2020). Using gyroplane for application of Tricho- gramma spp. against the European corn borer in maize. Pest Management Science. https://doi.org/10.1002/ps.5762 Capinera, J. (2001). Handbook of Vegetable Pests. Gulf Profes- sional Publishing. 729 p. Cascone, P. et al. (2015). Improving the efficiency of Tricho- gramma achaeae to Control Tuta absoluta. BioControl, 60(6), 761-771. https://doi.org/10.1007/s10526-015-9684- 1 Chakraborty, K. (2012). Relative composition of egg parasi- toid species of yellow stem borer Scirpophaga incertulas Wlk. in paddy field at Uttar Dinajpur, West Bengal, India. Current Biology, 6, 42–52. Clausen, C.P. (1978). Introduced parasites and predators of arthropod pests and weeds. USDA, Agriculture Handbook, 454p. Deka, S. and Barthakur, S. (2010). Overview on current status of biotechnological interventions on yellow stem borer Scirpophaga incertulas (Lepidoptera: Crambidae) resis- tance in rice. Biotechnology Advances, 28, 70–81. https:// doi.org/10.1016/j.biotechadv.2009.09.003 Dhandapani, N., Sarkar, P., and Mishra, G. (2016). Chrysop- ids. In: Ecofriendly Pest Management for Food Security. Academic Press, Elsevier, Inc. pp. 311-327. https://doi. org/10.1016/B978-0-12-803265-7.00010-5 Eckhoff, S. R., M.R. Paulsen, and S. C. Yang. (2003). ‘MAIZE’. In Encyclopedia of Food Sciences and Nutrition (Second Edition), edited by Benjamin Caballero, 3647–53. Ox- ford: Academic Press. https://doi.org/10.1016/B0-12- 227055-X/00725-2 El-Wakeil, N., Mahmoud, S., and Mohamed, A.H. (2020). Cottage Industry of Biocontrol Agents and Their Appli- cations Practical Aspects to Deal Biologically with Pests and Stresses Facing Strategic Crops: Practical Aspects to Deal Biologically with Pests and Stresses Facing Strategic Crops. https://doi.org/10.1007/978-3-030-33161-0 Feshchenko, A. (2019). Large Scale Agriculture. Impact of In- tensive Farming on Biodiversity. https://www.largescalea- griculture.com/home/news-details/impact-of-intensive- farming-on-biodiversity/ Flint, M.L. and Dreistadt, S.H. (1998). The Illustrated Guide to Biological Pest Control. In Natural Enemies Handbook. Clark, JK (ed). University of California Press. ISBN 978- 0-520-21801-7. Foelix, R.F. (2011). Biology of spiders, 3rd ed. Oxford Univer- sity Press, New York. Foerster, M.R., Marchioro, C.A., and Foerster, L.A. (2015). How Trichogramma survives during soybean offseason in Southern Brazil and the implications for its success as a biocontrol agent. BioControl, 60, 1–11. https://doi. org/10.1007/s10526-014-9616-5 Gao, X.H., Tian, J.H., and Chen, P. (2012). Population dynam- ics of Tryporyza incertulas (Walker) in Menghai, Yunnan from 2001–2011. Journal of Yunnan University, 34, 367– 372. Giraud, M. (2015). Trichogramma achaeae as an IPM tool in tomato greenhouses. Pure final congress, Poznan, Poland. Grieshop, M., Flinn, P., Nechols, J. and Schöller, M. (2014). Host-foraging success of three species of Trichogramma (Hymenoptera: Trichogrammatidae) in a simulated retail environment’. Journal of Economic Entomology, 100, 591– 98. https://doi.org/10.1603/0022-0493(2007)100[591:HS OTSO]2.0.CO;2 Guo, H.F., Fang, J.C., Xie, Y.F., Du, Z.W. (2002). Egg parasit- ism of rice stem borers in regions with different rice stem borer occurring patterns. Chinese Journal of Biological Control, 18, 13–16. Hassan, SA. (1981). Mass-production and utilization of Trich- ogramma: 2. Four years successful biological con- trol of the European corn borer. Mededelingen van de Faculteit Landbouwwetenschappen. Rijksuniversiteit Gent, 46(2), 417–427. Hassan, S.A. (1993). The mass rearing and utilization of Trich- ogramma to control lepidopterous pests: achievements and outlook. Pesticide Science, 37(4), 387–391. https://doi. org/10.1002/ps.2780370412 Hikjm, I.S. (1988). Seasonal parasitism by egg parasites of the yellow rice borer, Scirpophaga incertulas (Lepidop- tera Pyralidae). Entomophaga, 33, 115–124. https://doi. org/10.1007/BF02372319 Hochman, G., Deepak, R., Govinda, T., and Zilberman, D. (2014). Quantifying the causes of the global food com- modity price crisis. Biomass and Bioenergy, 68, 106–14. https://doi.org/10.1016/j.biombioe.2014.06.012 Hoffman, M.P., Wilson, L.T., Zalom, F.G., and Hilton, R.J. (1990). Parasitism of Heliothis zea (Lepidoptera: Noctui- dae) eggs: effect on pest management decision rules for processing toma- toes in the Sacramento Valley of Cali- fornia. Environmental Entomology, 19, 753-763. https:// doi.org/10.1093/ee/19.3.753 IPBES 2019. Global Assessment Report on Biodiversity and Ecosystem Services of the Intergovernmental Science- Policy Platform on Biodiversity and Ecosystem Services. Ivezić, A., Rugman-Jones, P., Stouthamer, R., and Ignjatović- Ćupina, A. (2018). Molecular identification of Trichogram- ma egg parasitoids of Ostrinia nubilalis in the north east- ern Serbia. Archives of Biological Sciences, 70(3), 425-432. https://doi.org/10.2298/ABS171103002I Ivezić, A. and Trudić, B. (2021). Parasitoids of the genus Trich- ogramma (Hymenoptera: Trichogrammatidae), natural enemies of European corn borer Ostrinia nubilalis (Hüb- ner, 1796) (Lepidoptera: Crambidae). Journal of Central European Agriculture (in press). https://doi.org/10.5513/ JCEA01/22.4.3247 Knutson, A. (1998). The Trichogramma Manual. Texas (TX): Acta agriculturae Slovenica, 118/2 – 2022 11 The usage of beneficial insects as a biological control measure in large-scale farming - a case study review on Trichogramma spp. The Texas A&M University Systems. Ko, K. et al. (2014). Evaluation for potential Trichogramma (Hymenoptera: Trichogrammatidae) strains for control of the striped stem borer (Lepidoptera: Crambidae) in the greater Mekong subregion. Journal of economic entomol- ogy, 107(3), 955-963. https://doi.org/10.1603/EC13481 Kot, J. (1964). Experiments in the biology and ecology of spe- cies of the genus Trichogramma Westw. and their use in plant protection. Ekologia Polska – Seria A, 12(15), 1-61. Li-Ying, Li. (1994). Worldwide Use of Trichogramma for Bio- logical Control on Different Crops: A Survey. In Hassan. S. A. & Wajnberg. E. (Ed.), Biological Control with Egg Parasi- toids (pp. 37-51). Wallingford: CAB International. Manandhar, R. and Mark, G. Wright. (2015). Enhancing bio- logical control of corn earworm, Helicoverpa and Thrips through habitat management and inundative release of Trichogramma pretiosum in corn cropping systems. Bio- logical Control, 89, 84–90. https://doi.org/10.1016/j.bio- control.2015.05.020 Marc, P., Canard, A., and Ysnel, F. (1999). Spiders (Araneae) useful for pest limitation and bioindication. Agriculture, Ecosystems & Environment, 74, 229–273. https://doi. org/10.1016/S0167-8809(99)00038-9 Murali-Baskaran, R.K., Sharma, K.C., Sridhar, J., Jain, L., Ku- mar, J. (2021). Multiple releases of Trichogramma japoni- cum Ashmead for biocontrol of rice yellow stem borer, Scirpophaga incertulas Walker). Crop Protection. https:// doi.org/10.1016/j.cropro.2020.105471 Mutuura, A. and Monroe, E. (1970). Taxonomy and distribu- tion of the European corn borer and allied species: genus Ostrinia (Lepidoptera: Pyralidae), pp. 1-112 in Memoirs of the Entomological Society of Canada, edited by D. P. Pie- lou. Entomological Society of Canada, Ottawa. https://doi. org/10.4039/entm10271fv Nordlund, D.A., Cohen, A.C., and Smith, R.A. (2001). Mass-rearing. Release Techniques and Augmentation. In Lacewings in the Crop Environment. Mcewen PK, New TR, Whittington AE (eds). Cambridge Univer- sity Press, Cambridge, 303-319. https://doi.org/10.1017/ CBO9780511666117.015 Nordlund, D.A., W. J. Lewis, H.R. Gross, Jr., and M, Beevers. (1981). Kairomones and their use for management of en- tomophagus insects. XII. The stimulatory effects of host eggs and the importance of host-egg density to the ef- fective use of kairomones for Trichogramma pretiosum. Riley. Journal of Chemical Ecology, 7, 909-17. https://doi. org/10.1007/BF00987616 Nurindah, D.A. and Sunarto, S. (2020). Overview of the use of biocontrol agents in the control of Indonesian sugar- cane borers. IOP Conference Series: Earth and Environ- mental Science, 418, 012061. https://doi.org/10.1088/1755- 1315/418/1/012061 Obrycki, J.J., Hamid, M.N., Sajap, A.S., and Lewis, L.C. (1989). Suitability of corn insect pests for development on surviv- al of Chrysoperla carnea and Chrysoperla oculata (Nuoro- ptera: Chrysapidae). Environmental Entomology, 18, 1126- 1130. https://doi.org/10.1093/ee/18.6.1126 Olkowski, W. and A. Zhang. (1990). Trichogramma- a modern day frontier in biological control. The IPM Practitioner, 12, 1-15. Pappas, M. and Broufas Koveos, D.S. (2011). Chrysopid preda- tors and their role in biological control. Journal of Ento- mology. https://doi.org/10.3923/je.2011.301.326 Pinto, J.D. (1998). Systematics of the North American species of Trichogramma Westwood (Hymenoptera: Trichogrammati- dae). Washington (DC): Entomological Society of Wash- ington, 289p. (Memoirs of the Entomological Society of Washington; 22). Polanczyk, R.A. and Pratissoli, D. (2009). Biological control of agricultural pests: Principles and field applications. Re- vista Ceres, 56(4), 410-419. Principi, M.M. and Canard, M. (1984). Feeding Habits. In Biol- ogy of Chrysopidae. Canard M, Semeria Y, New TR (eds). Dr W. Junk Publishers, The Hague, The Nederlands 76-92. Radermacher, N., Hartke, T.R., Villareal, S., et al. (2020). Spi- ders in rice-paddy ecosystems shift from aquatic to ter- restrial prey and use carbon pools of different origin. Oecologia, 192, 801–812. https://doi.org/10.1007/s00442- 020-04601-3. Radermacher, N., Hartke, T.R., Villareal, S., and Scheu, S. (2020). Spiders in ricepaddy ecosystems shift from aquatic to terrestrial prey and use carbon pools of diferent origin. Oecologia, 192, 801–812. https://doi.org/10.1007/s00442- 020-04601-3. Rao, A. N., S. P. Wani, M. S. Ramesha, and J. K. Ladha. (2017). Rice Production Systems. In Rice Production Worldwide, edited by Bhagirath S. Chauhan, Khawar Jabran, and Gulshan Mahajan, 185–205. Cham: Springer International Publishing. https://doi.org/10.1007/978-3-319-47516-5_8. Raspudić, E., Ivezić, M., and Mlinarević, M. (1999). Utjecaj transgenih Bt hibrida kukuruza na kukuruznog molj- ca (Ostrinia nubilalis Hübner). Sažeci priopćenja 43. seminara iz Zaštite bilja Opatija, Glasnik zaštite bilja, 1, 6-7. Rechcigl, J.E. and Rechcigl, N.A. (2000). Biological and biotech- nological control of insects pest. Boca Raton: CRC Press; 2000. 375 p. https://doi.org/10.1201/9781439822692 Ridgway, R. and Jones, S.L. (1969). Inundative releases of Chrysopa carnea for control of Heliothis on cotton. Jour- nal of Economic Entomology. https://doi.org/10.1093/ JEE/62.1.177 Ridgway, R.L. and Mcmuphy, W.L. (1984). Biological Control in the Field. In: Biology of Chrysopidae. Canard M, Semeria Y, New TR (eds). Dr W. Junk Publishers, The Hague, The Nederlands 220-228. Riechert, S.E. and Lockley, T. (1984). Spiders as biological control agents. Annual Review of Entomology, 29, 299–320. https://doi.org/10.1146/annur ev.en.29.010184.001503 Riudavetes, J. and Castane, C. (1994). Abundance and host plant preferences for oviposition of Orius spp. (Heter- optera: Anthocoridae) along the Mediterranean coast of Spain. IOBC/wprs Bulletin, 17(5), 230-236. Ru, N., Whitcomb, W.H., and Murphey, M. (1976). Cultur- ing of Chrysopa rufilabris (Neuroptera: Chrysopidae). The Florida Entomologist, 59(1), 21-26. https://doi. org/10.2307/3493163 Ruberson, J.R. and Kring, T.J. (1993). Parasitism of develop- ing eggs by Trichogramma pretiosum (Hymenoptera: Acta agriculturae Slovenica, 118/2 – 202212 A. IVEZIĆ et al. Trichogrammatidae): Host age preference and suitabil- ity. Biological Control, 3, 39-46. https://doi.org/10.1006/ bcon.1993.1007 Samara, R.Y., Monje, J.C., and Zebitz, C.P.W. (2008). Compari- son of different European strains of Trichogramma auro- sum (Hymenoptera: Trichogrammatidae) using fertility life tables. Biocontrol Science and Technology, 18, 75–86. https://doi.org/10.1080/09583150701749789 Saour, G. (2004). Efficacy assessment of some Trichogramma species (Hymenoptera: Trichogrammatidae) in control- ling the potato tuber moth Phthorimaea operculella Zell. (Lepidoptera: Gelechiidae). Journal of Pest Science, 77, 229–234. https://doi.org/10.1007/s10340-004-0058-1 Scheu, S. (2001). Plants and generalist predators as links between the below-ground and above-ground sys- tem. Basic and Applied Ecology, 13, 3–13. https://doi. org/10.1078/1439-1791-00031 Scheu, S. (2002). The soil food web: structure and perspectives. The European Journal of Soil Biology, 38, 11–20. https://doi. org/10.1016/S1164-5563(01)01117-7 Sherif, M.R., Hendawy, A., and El-Habashy, M.M. (2008). Utilization of Trichogramma evanescens (Ashmead) for controlling rice stem borer, Chilo agamemnon Bles. in rice fields in Egypt. Egyptian Journal of Biological Pest Control, 18, 11–16. Siebrecht, N. (2020). Sustainable agriculture and its imple- mentation gap - overcoming obstacles to implementation. Sustainability. http://dx.doi.org/10.3390/su12093853 Sigsgaard, L. (2007). Early season natural control of the brown planthopper, Nilaparvata lugens: the contribution and in- teraction of two spider species and a predatory bug. Bul- letin of Entomological Research, 97, 533– 544. https://doi. org/10.1017/S0007485307005196. Smith, S.M. (1996). Biological control with Trichogramma: advances, successes, and potential of their use. Annual Re- view of Entomology, 41, 375–406. https://doi.org/10.1146/ annurev.en.41.010196.002111 Snyder, W.E. and Wise, D.H. (2001). Contrasting trophic cas- cades generated by a community of generalist predators. Ecology, 82, 1571–1583. https://doi.org/10.1890/0012- 9658(2001)082[1571:CTCGBA]2.0.CO;2 Stouthamer, R., Breeuwert, J.A., Luck, R., and Werren, J. (1993). Molecular identification of microorganisms associated with parthenogenesis. Nature, 361(6407), 66–68. https:// doi.org/10.1038/361066a0 Symondson, WOC., Sunderland, K.D., and Greenstone, M.H. (2002). Can generalist predators be effective biocontrol agents? Annual Review of Entomology, 47, 561–594. htt- ps://doi.org/10.1146/annurev.ento.47.091201.145240 Tabone, E., Bardon, C., Desneux, N., and Wajnberg, E. (2010). Parasitism of different Trichogramma species and strains on Plutella xylostella L. on greenhouse cauliflower. Jour- nal of Pest Science, 83, 251–256. https://doi.org/10.1007/ s10340-010-0292-7 Tang, R., Babendreier, D., Zhang, F., Kang, M., Song, K., and Hou, M.L. (2017). Assessment of Trichogramma japoni- cum and T. chilonis as potential biological control agents of yellow stem borer in rice. Insects, 8(1), 19. https://doi. org/10.3390/insects8010019 Tittonell, P., Piñeiro, G., Garibaldi, L.A., Dogliotti, S., Olff, H., and Jobbagy, E.G. (2020). Agroecology in large scale farm- ing - A research agenda. Frontiers in Sustainable Food Sys- tems, 214. https://doi.org/10.3389/fsufs.2020.584605 Trdan., S, Laznik, Ž., Bohinc, T. (2020). Thirty years of research and professional work in the field of biological control (predators, parasitoids, entomopathogenic and parasitic nematodes) in Slovenia: A review. Applied Sciences, 10(21), 1-12. https://doi.org/10.3390/app10217468 Trumble, J.T. and Alvarado-Rodriguez, B. (1991). Develop- ment and economic evaluation of an IPM program for fresh market tomato production in Mexico. Agriculture, Ecosystems and Environment, 43, 267-284. https://doi. org/10.1016/0167-8809(93)90091-3 Vail, P.V., Coulson, J.R., Kaufmann, W.C. and Dix, M.E. (2001). History of biological control programs in the United States Department of Agriculture. American Entomologist, 47, 24-49. https://doi.org/10.1093/ae/47.1.24 Van den Bosch, R., Messenger, P.S., and Gutierrez, A.P. (1982). The History and Development of Biological Control. In: An Introduction to Biological Control. Springer, Boston, MA. https://doi.org/10.1007/978-1-4757-9162-4_3 Van Driesche, R.G. and Bellows, T.S. (1996). Biological con- trol. New York (NY): Chapman and Hall. https://doi. org/10.1007/978-1-4613-1157-7 Van Lenteren, J.C., Babendreier, D., Bigler, F., Burgio, G., Hok- kanen, H.M.T., Kuske, S., Loomans, A.J.M., Menzler-Hok- kanen, I., van Rijn, PCJ., and Thomas M.B., et al. (2003). Environmental risk assessment of exotic natural enemies used in inundative biological control. BioControl, 48(1), 3–38. https://doi.org/10.1023/A:1021262931608 Van Schelt, J. and Ravensberg, W.J. (1991). Some aspects on the storage and application of Trichogramma maidis in corn. In: Wajnberg E, Vinson SB, editors. The Third Inter- national Symposium on Trichogramma and other Egg Para- sitoids; San Antonio, USA; p. 239–242. Van Lenteren, JC. (2000). Success in biological control of ar- thropods by augmentation of natural enemies. In: Gurr GM, Wratten SD, editors. Biological control: measures of success. Dordrecht, Netherlands: Kluwer Academic Pub- lishers; p. 77–103. https://doi.org/10.1007/978-94-011- 4014-0_3 Wang, B., Ferro, D.N., and Hosmer, D.W. (1999). Effectiveness of Trichogramma ostriniae and T. nubilale for controlling the European corn borer Ostrinia nubilalis in sweet corn. Entomologia Experimentalis et Applicata, 91, 297–303. https://doi.org/10.1046/j.1570-7458.1999.00496.x Zhenying, W., He, K., Zhang, F., Lu, X., and Babendreier, D. (2014). Mass rearing and release of Trichogramma for biological control of insect pests of corn in China. Bio- logical Control, 68, 136–44. https://doi.org/10.1016/j.bio- control.2013.06.015 White, B. (2019). Biological Control of Insects Pests. ED-Tech Press, United Kingdom 298. Whitman, D. and Nordlund, D.A. (1994). Plant chemicals and the location of herbivorous arthropods by their natural enemies. In: Ananthakrishnan TN (editor). Functional Dynamics of Phytophagous Insects. New Delhi, India: Ox- ford and IBH Publishing, pp. 207–248. Acta agriculturae Slovenica, 118/2 – 2022 13 The usage of beneficial insects as a biological control measure in large-scale farming - a case study review on Trichogramma spp. Wise, D.H., Moldenhauer, D.M., and Halaj, J. (2006). Using stable isotopes to reveal shifts in prey consumption by generalist predators. Ecological Applications, 16, 865–876. https://doi.org/10.1890/1051-0761(2006)016[0865:USITR S]2.0.CO;2 Wopereis, M.C.S., Defoer, T., Idinoba, P., Diack, S., and Dugué, M.J. (2008). Participatory Learning and Action Research (PLAR) for Integrated Rice Management (IRM) in Inland Valleys of Sub-Saharan Africa: Technical Manual. AfricaRi- ce Training Series. Cotonou, Benin: Africa Rice Center. 128 pp. Yuan, X., Song, L., Zhang, J., Zang, L., Zhu, L., Ruan, C., and Sun, G. (2012). Performance of four Chinese Trichogram- ma species as biocontrol agents of the rice striped stem borer, Chilo suppressalis, under various temperature and humidity regimes. Journal of Pest Science, 85, 497–504. https://doi.org/10.1007/s10340-012-0456-8 Yunus, M. (2018). Effectiveness of Trichogramma japoni- cum Utilization for Biological Control Agents on Scirpo- phaga Incertulas in Indonesia’. https://doi.org/10.3923/ ajcs.2018.31.39