ACTA s AGRICULTURAE SLOVENICA Biotehniška fakulteta Univerze v Ljubljani Biotechnical Faculty University of Ljubljana Acta agriculturae Slovenica • ISSN 1581-9175 • 95 - 1 • Ljubljana, marec 2010 Acta agriculturae Slovenica Volume / Letnik 95 • Number / Številka 1 • 2010 VSEBINA / CONTENTS Valentina SCHMITZER, Franci ŠTAMPAR 5 Substrate pH level effects on anthocyanins and selected phenolics in Rosa x hybrida L. 'KORcrisett' Vpliv pH substrata na antociane in fenole pri Rosa x hybrida L. 'KORcrisett' Helena ROJHT, Christos G. ATHANASSIOU, Bill J. VAYIAS, Nickolas KAVALLIERATOS, Željko TOMANOVIC, Matej VIDRIH, Katarina KOS, Stanislav TRDAN 13 The effect of diatomaceous earth of different origin, temperature and relative humidity against adults of rice weevil (Sitophilus oryzae [L.], Coleoptera, Curculionidae) in stored wheat Vpliv diatomejske zemlje različnega izvora, temperature in relativne vlage pri zatiranju odraslih osebkov riževega žužka (Sitophilus oryzae [L.], Coleoptera, Curculionidae) v skladiščeni pšenici Silvia MELICHÂCOVÂ, Maria TIMORACKÂ, Judita BYSTRICKÂ, Alena VOLLMANNOVÂ, Juraj CÉRY 21 Relation of total antiradical activity and total polyphenol content of sweet cherries (Prunus avium L.) and tart cherries (Prunus cerasus L.) Povezava med antioksidantno aktivnostjo in vsebnostjo vseh polifenolov pri češnjah (Prunus avium L.) in višnjah (Prunus cerasus L.) Špela MECHORA, Mateja GERM 29 Selenium induced lower respiratory potential in Glycine max (L.) Merr. Vpliv selena na nižanje dihalnega potenciala pri soji (Glycine max (L.) Merr.) Sali ALIU, Shukri FETAHU, Ludvik ROZMAN 35 Variation of physiological traits and yield components of some maize hybrid (Zea mays L.) in agroecological conditions of Kosovo Variabilnost fizioloških lastnosti in komponent pridelka nekaterih hibridov koruze (Zea mays L.) v agroekoloških razmerah Kosova Žiga LAZNIK, Jenna L. ROSS, Stanislav TRDAN 43 Massive occurrence and identification of the nematode Alloionema appendiculatum Schneider (Rhabditida: Alloionematidae) found in Arionidae slugs in Slovenia Številčni pojav in identifikacija ogorčice Alloionema appendiculatum Schneider (Rhabditida: Alloionematidae) v lazarjih (Arionidae) v Sloveniji Žiga LAZNIK, Stanislav TRDAN 51 Intraspecific variability of Steinernema feltiae (Filipjev) (Rhabditida: Steinernematidae) as biological control agent of rice weevil (Sitophilus oryzae [L.], Coleoptera, Curculionidae) adults Znotrajvrstna variabilnost entomopatogene ogorčice Steinernema feltiae (Filipjev) (Rhabditida: Steinernematidae) kot biotičnega agensa za zatiranje odraslih osebkov riževega žužka (Sitophilus oryzae [L.], Coleoptera, Curculionidae) Darja KOCJAN AČKO 61 Hop fields in crop rotation Hmeljišča v premeni Barbara AMBROŽIČ TURK, Matej STOPAR 69 Effect of 6-benzyladenine application time on apple thinning of cv. 'Golden Delicious' and cv. 'Idared' Vpliv časa aplikacije 6-benziladenina na redčenje plodičev jablane pri sortah 'Zlati delišes' in 'Idared' Drena GADŽO, Mirha DJIKIC, Teofil GAVRIC, Petra ŠTREKELJ 75 Comparison of tannin concentration in young plants of common and tartary buckwheat Primerjava koncentracije taninov v mladih rastlinah navadne in tatarske ajde Monika CVETKOV, Igor ŠANTAVEC, Darja KOCJAN AČKO, Anton TAJNŠEK 79 Soil organic matter content according to different management system within long-term experiment Vsebnost organske snovi v tleh v odvisnosti od načina gospodarjenja znotraj trajnega poskusa Katarina KOS, Stanislav TRDAN 89 Biotično zatiranje kostanjeve šiškarice (Dryocosmus kuriphilus Yasumatsu, Hymenoptera, Cynipidae) Biological control of chestnut gall wasp (Dryocosmus kuriphilus Yasumatsu, Hymenoptera: Cynipidae) Lučka KAJFEŽ-BOGATAJ, Tjaša POGAČAR, Andrej CEGLAR, Zalika ČREPINŠEK 97 Spremembe agro-klimatskih spremeljivk v Sloveniji v zadnjih desetletjih Trends in agro-climate variables in Slovenia Tomaž BARTOL, Karmen STOPAR 111 Content analysis of the papers in the Acta agriculturae Slovenica Vsebinska obdelava prispevkov v Acta agriculturae Slovenica let. 95 št. 1 115 Navodila avtorjem Notes for authors DOI: 10.2478/v10014-010-0001-5 COBISS Code 1.01 Agrovoc descriptors: rosa, anthocyanins, phenolic compounds, aromatic compounds, soil ph, soil chemicophysical properties, flowers, chemical composition, proximate composition, soil fertility Agris category code: F60, P35 Substrate pH level effects on anthocyanins and selected phenolics in Rosa x hybrida L. 'KORcrisett' Valentina SCHMITZERa, Franci ŠTAMPARb Received October 6, 2009; accepted February 24, 2010. Delo je prispelo 6. oktobra 2009; sprejeto 24. februarja, 2010. ABSTRACT The effect of substrate pH level (4.7, 3.3 and 7.3) on the anthocyanin, quercetin compounds, catechin and phenolic acids concentrations in petals of Rosa x hybrida L. 'KORcrisett' and on the number of flowers per plant was investigated. The phenolic profiles of this plant were established for the first time by the use of HPLC/MS. Plants potted in a substrate with pH 4.7 developed significantly more flowers compared to those planted in an acidic (3.3) and alkaline (7.3) pH levels. However, the concentration of anthocyanins, quercetin compounds, catechin and phenolic acids was always lowest in the petals of 'KORcrisett' rose plants potted in pH level 4.7. Compared to the first sampling, a significant increase in the concentration of major and total anthocyanins and quercetin compounds was measured in the petals of plants potted in pH level 3.3 and 7.3, but not in the plants potted in pH level 4.7, respectfully. Key words: rose, pH, substrate, anthocyanins, phenolic compounds IZVLEČEK VPLIV pH SUBSTRATA NA ANTOCIANE IN FENOLE PRI Rosa x hybrida L. 'KORcrisett' Preučevali smo vpliv pH substrata (4,7, 3,3 in 7,3) na koncentracije antocianov, kvercetinov, katehina in fenolnih kislin v petalih Rosa x hybrida L. 'KORcrisett' ter spremljali število cvetov na posamezno rastlino. Sestava in koncentracija fenolnih spojin je bila pri tej rastlini prvič določena s pomočjo HPLC/MS tehnike. Rastline, ki so bile posajene v substrat s pH 4,7, so razvile statistično značilno več cvetov, kot rastline, posajene v kisel (3,3) oziroma bazičen (7,3) pH, vendar pa so bile koncentracije antocianov, kvercetinov, katehina in fenolnih kislin v pH 4,7 najnižje. V primerjavi s prvim vzorčenjem, je koncentracija prevladujočih in skupnih antocianov v petalih močno narasla pri rastlinah, ki so bile posajene v substrat s pH 3,3 in 7,3. Podobnega trenda nismo opazili pri rastlinah, posajenih v pH 4,7. Ključne besede: vrtnica, pH, substrat, antociani, fenolne spojine 1 INTRODUCTION Roses are one of the most important, diverse and widely planted ornamentals with over 150 species and more than 20.000 cultivars (Cai et al., 2005) with color specter ranging from subtle whites, yellows and pinks to intense purple, orange and red tones. The color of various plant tissues, such as flower petals (Mikanagi et al., 1995) and leaves (Schmitzer et al., 2009a), can be attributed to anthocyanins and other phenolics, for example quercetins, acting as copigments (Eugster and Markifischer, 1991). a) University of Ljubljana, Biotechnical Faculty., Department of Agronomy, Jamnikarjeva 101, SI-1000 Ljubljana, Slovenia, univ. dipl. inž. kraj. arhit., dipl. inž. agr., e-mail: valentina.schmitzer@bf.uni-lj.si b) ibid, prof., Ph.D. Flower pigments accumulate in the epidermal cell vacuoles (Hughes et al., 2007) and their concentration, intensity and hue depends on both internal, such as microenvironment conditions in the vacuoles and developmental stage (Schmitzer et al., 2009b), and external factors. Among the latter light (Cominelli et al., 2007), temperature (Dela et al., 2003), nutrient deficiency (Juszczuk et al., 2004) and substrate pH (Smith et al., 2004a) has been reported to alter anthocyanin and carotenoid concentration in various plant tissues. Root substrate pH also affects nutrient solubility (Smith et al., 2004b; Papafotiou et al., 2007) and influences root formation (Harbage et al., 1998) with a direct impact on overall status of the plant. In roses, root growth was inhibited when plants were exposed to either pH 8 or pH 4 in comparison with plants grown in pH 6. Additionally, plant growth, leaf size and chlorophyll levels were not affected in plants at pH 4, while all these variables were reduced at pH 8 in comparison with plants grown at pH 6 (Zieslin and Snir, 1989). We hypothetisized that the concentration of phenolic compounds (anthocyanins, quercetin compounds, catechin and phenolic acids) in petals of the miniature rose 'KORcrisett' differs according to substrate pH levels. The objective of our study was thus to determine the effects of the substrate pH level on the concentration of secondary metabolites in rose petals, important from the commercial aspect of miniature rose production as well as from the viewpoint of plants response to external stress. As plants grown outside their acceptable pH range show signs of chlorosis and general decline (Smith et al., 2004 a) a reduction in the number of flowers can also be expected in miniature roses potted in acidic or alkaline pH levels. 2 MATERIALS AND METHODS 2.1 Plant material and growth conditions Rosa x hybrida L. 'KORcrisett' plants were planted in 1.3 l plastic pots (14 cm in diameter), containing a growth medium, prepared by mixing 80% of black peat and 20% of mineral component (sand). The substrate pH levels were modified by liming with calcium carbonate; the amount of lime was calculated on the base of a pH curve and three different pH levels were set up; treatment A with pH level 3.3, treatment B with pH level 4.7 and treatment C with pH level 7.3. For each treatment 15 plants were planted; the experiment was a randomized block design on a single bench. Plants were grown from the beginning of August to September 2008 in a controlled environment glass greenhouse at 27/22 °C (day/night) equipped with a cooling system under natural photoperiod. The greenhouse environmental control system was set to start cooling at 27 °C. Relative humidity ranged from 75-85 %. Plants were irrigated daily, using a flood irrigation system with 4 minutes water (18 °C) supply. The number of flowers per plant (buds to senescent flowers) was counted at the beginning of the experiment on 12. Aug. 2008 (day 0) and on 8. Sept. (day 28). At the same time petals (flower developmental stage 3; Muller et al., 1998) for the extraction of phenolics were collected and immediately frozen in liquid nitrogen and stored at -18 °C prior to further analysis. 2.2 Extraction and determination of phenolic compounds For the analysis of phenolic compounds (anthocyanins, quercetin compounds and selected phenolics), frozen petals were ground to a fine powder with liquid nitrogen. A sample of 2 g was extracted with 3 mL methanol containing 3% (v/v) HCOOH and 1% (w/v) 2,6-di-ferf-butyl-4-methylphenol (BHT) in an ultrasonic bath for one hour. After extraction, the treated samples were centrifuged for 7 min at 12,000 gn.. The supernatant was filtered through Chromafil AO-45/25 polyamide filter (Macherey-Nagel, Düren, Germany) and transferred to a vial prior to injection into the highperformance liquid chromatography (HPLC) system. The samples were analyzed using a Thermo Finnigan Surveyor HPLC system (Thermo Scientific, San Jose, CA) with a diode array detector at 280 nm (gallic acid, protocatechuic acid, catechin, p-coumaric acid), 350 nm (quercetins) and 530 nm (anthocyanins). A Phenomenex (Torrance, CA) HPLC column C18 (150 mm x 4.6 mm, Gemini 3^) protected with a Phenomenex Security guard column, operated at 25 °C, was used. The injection volume was 20 ^L and the flow rate was 1mL min-1. The elution solvents were aqueous 1% formic acid (A) and acetonitrile (B). The samples were eluted according to the linear gradient described by Marks et al. (2007): 0-5min, 3-9% B; 5-15 min, 9-16% B; 15-45min, 1650% B; 45-50min, 50% isocratic; and finally washing and reconditioning of the column. The concentrations of selected phenolic compounds were assessed from peak areas and quantified with the use of corresponding external standards and anthocyanins by the use of calibration curve of cyanidin-3,5-di-O-glucoside. Anthocyanins were further identified using a mass spectrometer (Thermo Scientific, LCQ Deca XP MAX, San Jose, USA) with an electroscopy interface (ESI) operating in positive ion mode from m/z 115 to 800. The injection volume was 10 ^L and the flow rate maintained at 1mL min-1. Capillary temperature was 250 °C, the sheath gas and auxiliary gas were 20 and 8 units respectively, the capillary voltage was 26 V and spray voltage 4 V. Multipole Rf amplitude was 550 Vp-p. All compounds were expressed as Mg g-1 FW. 2.3 Chemicals The standards used to determine the phenolic compounds in samples were gallic acid, (+)-catechin, quercetin-3-O-rutinoside, cyanidin-3,5-di-O-glucoside and cyanidin-3-O-glucoside from Sigma-Aldrich (Steinheim, Germany), catechin from Roth (Karlsruhe, Germany), protocatechulic acid from Merck (Darmdstadt, Germany), caffeic acid, p-coumaric acid, quercetin-3-O-glucoside, quercetin-3-O-rhamnoside and peonidin-3-O-glucoside from Fluka (Buchs, Switzerland). The chemicals for the sample preparation and mobile phases were methanol, BHT and acetonitrile from Sigma-Aldrich and formic acid from Fluka. The water used in mobile phase was bidistilled and purified with a Milli-Q water purification system by Millipore (Bedford, MA). 2.4 Statistical analysis Statistical analysis was conducted with the program Statgraphics Plus 4.0 (Statgraphics, Herndon, VA). One-way analysis of variance ANOVA was used for analysis of the effect of substrate pH level on the number of flowers per plant and concentration of anthocyanins, quercetins and selected phenolics in rose petals. Differences in phenolic concentrations among pH treatments were estimated with Duncan's multiple range test (P < 0.05). 3 RESULTS 3.1 The number of flowers On the first sampling, miniature rose plants on average produced 9.25 flowers per plant with no significant differences observed among pH treatments. However, after 28 days, the number of flowers per plant was significantly affected by substrate pH level (Fig. 1). Compared to the first sampling, a decline in the number of flowers per plant was detected, when 'KORcrisett' rose was potted in both alkaline (8.10% less flowers per plant) and acidic pH levels (5.88% less flowers per plant) in contrast to pH 4.7, where plants developed 40% more flowers, respectfully. Figure 1: The effect of substrate pH on the number of flowers per plant at the beginning of the experiment (12 Aug.) and on day 28 (8 Sept.). Values carrying the same letters (a, b) for each set of dates do not differ significantly by Duncan's multiple range test at P < 0.05. 3.2 Anthocyanins The HPLC chromatogram revealed five peaks at 530 nm, corresponding to two pelargonidin-based, two cyanidin-based and one peonidin-based glucoside. On the first sampling, flower petals averagely contained 283.79 ^g g-1 FW pelargonidin-3,5-di-O-glucoside, 67.55 ^g g-1 FW cyanidin-3,5-di-O-glucoside, 20.94 ^g g-1 FW pelargonidin-3-O-glucoside, 10.36 ^g g-1 FW cyanidin-3-O-glucoside, 9.87 ^g g-1 FW peonidin-3-O-glucoside and 380.80 ^g g-1 FW total anthocyanins, with no statistical differences observed among pH treatments (Table 1). After 28 days, significant differences among pH treatments were detected in the concentrations of all anthocyanins with the lowest values obtained from plants potted in 4.7 pH level. The concentration of two major anthocyanins (pelargonidin-3,5-di-O-glucoside and cyanidin-3,5-di-O-glucoside) was 64.20% and 70.01% higher in the acidic pH level and 67.31% to 60.12% higher in alkaline pH level when compared to 4.7 pH level. However, the greatest difference was observed in the concentration of pelargonidin-3-O-glucoside; in both acidic and alkaline pH levels a more than two fold increase was measured when compared to pH level 4.7. Total anthocyanins were 60.88% and 66.73% higher in alkaline and acidic pH levels compared to pH level 4.7 on the second sampling. Generally, no statistical differences in single and total anthocyanins were detected between acidic and alkaline pH levels, except in the concentrations of cyanindin-3-O-glucoside and peonidin-3-O-glucoside, where higher concentrations were detected in alkaline pH levels. Interestingly, the concentration of cyanidin-3,5-di-O-glucoside in petals of plants potted in acidic and alkaline pH level on the second sampling was more than threefold higher than on the first and consequently, the concentration of total anthocyanins in these treatments also increased 66.73% to 60.88%. The concentration of the major anthocyanin (pelargonidin-3,5-di-O-glucoside) and three minor ones, however, were similar to those measured on the first sampling. Table 1: Effect of substrate pH level on the concentration of anthocyanins (^g g-1 FW) in petals of Rosa x hybrida L. 'KORcrisett' on two sampling dates. Anthocyanin1 [mean ± SE (^g g"1)] Sampling date PH Level Pel-di-glu Cy-di-glu Pel-glu Cy-glu Peo-glu Total anthocyanins 3.3 267.12 ± 16.40 a2 69.91 ± 6.00 a 20.33 ± 1.47 a 10.85 ± 0.78 a 10.10 ± 0.84 a 375.57 ± 24.30 a 12 Aug. 4.7 265.17 ± 18.93 a 62.06 ± 6.00 a 19.35 ± 1.48 a 9.65 ± 0.70 a 8.75 ± 0.71 a 357.55 ± 25.53 a 7.3 319.08 ± 21.51 a 70.68 ± 7.34 a 23.15 ± 1.57 a 10.57 ± 0.68 a 10.75 ± 0.86 a 409.28 ± 35.56 a 3.3 324.40 ± 29.65 b 268.33 ± 22.39 b 27.32 ± 3.64 b 10.24 ± 1.67 ab 9.54 ± 1.62 ab 636.04 ± 52.42 b 8 Sept. 4.7 197.56 ± 13.22 a 157.83 ± 13.72 a 13.57 ± 0.66 a 6.19 ± 1.40 a 6.28 ± 0.24 a 381.48 ± 27.75 a 7.3 330.53 ± 34.82 b 253.19 ± 31.60 b 27.74 ± 4.02 b 13.65 ± 2.34 b 12.23 ± 1.60 b 613.74 ± 69.50 b 1 Anthocyanin: Pel-di-glu, Pelargonidin-3,5-di-O-glucoside; Cy-di-glu, Cyanidin-3,5-di-O-glucoside; Pel-glu, Pelargonidin-3-O-glucoside; Cy-glu, Cyanidin-3-O-glucoside; Peo-glu, Peonidin-3-O glucoside. 2 Values carrying the same letters (a-b) for each set of dates do not differ significantly by Duncan's multiple range test at P < 0.05. 3.3 Quercetin compounds and catechin Three quercetin compounds were determined in the petals of Rosa x hybrida L. 'KORcrisett', the predominant quercetin-3-O-rhamnoside and two minor ones (quercetin-3-O-glucoside and quercetin-3-O-rutinoside). On the first sampling petals on average contained 70.87 ^g g-1 FW quercetin-3-O-rhamnoside, 21.81 ^g g-1 FW quercetin-3-O-glucoside and 7.33 ^g g-1 FW quercetin-3-O-rutinoside, with no statistical differences observed among pH treatments (Table 2). On the second sampling, however, the lowest values of major and minor quercetin compounds were obtained from petals of plants, potted in a 4.7 ph level. Statistically significant differences in the concentration of the two most abundant quercetin compounds were also detected between acidic and alkaline pH levels; the concentration of quercetin-3-O-rhamnoside was from 98.31% to 55.77% higher and quercetin-3-O-glucoside from 123.37% to 70.41% higher than in pH level 4.7. Compared to the first sampling, the concentration of all quercetin compounds in petals was higher on the second sampling in both acidic and alkaline pH levels but lower in 4.7 pH level. The concentration of the predominant quercetin compound increased 61.72% and 59.28% in acidic and alkaline pH treatments and decreased by 25.95% in pH level 4.7 and a similar trend was detected for quercetin-3-O-rutinoside. The average concentration of catechin in petals of rose 'KORcrisett' on the first sampling was 2006.66 ^g g-1 FW and after 28 days, the concentration only increased in the petals of plants potted in an acidic pH level. Compared to the first sampling the concentration was 20.50% higher in pH level 3.3 and from 8.05% to 14.51% lower in pH levels 4.7 and 7.3. Table 2: Effect of substrate pH level on the concentration of quercetin compounds and catechin (^g g-1 FW) in petals of Rosa x hybrida L. 'KORcrisett' on two sampling dates. Quercetin compounds1 and catechin [mean ± SE (^g g-1)] Sampling date pH Level Q-rhamn Q-glu Q-rut Catechin 3.3 75.28 ± 6.65 a2 19.64 ± 1.23 a 6.32 ± 0.57 a 1936.52 ± 103.86 a 12 Aug. 4.7 77.30 ± 7.14 a 23.42 ± 1.54 a 7.64 ± 0.60 a 1973.64 ± 72.32 a 7.3 60.04 ± 6.54 a 22.38 ± 1.62 a 8.02 ± 0.54 a 2109.81 ± 82.28 a 3.3 121.74 ± 9.45 c 30.20 ± 1.54 c 13.89 ± 1.52 b 2333.64 ± 179.42 b 8 Sept. 4.7 61.39 ± 3.94 a 13.52 ± 0.86 a 6.08 ± 0.82 a 1826.65 ± 105.03 a 7.3 95.63 ± 7.65 b 23.04 ± 2.32 b 12.76 ± 1.27 b 1842.47 ± 148.71 a 1 Quercetin compounds: Q-rhamn, Quercetin-3-O-rhamnoside; Q-glu, Quercetin-3-O-glucoside; Q-rut, Quercetin-3-O-rutinoside. 2 Values carrying the same letters (a, b,c) for each set of dates do not differ significantly by Duncan's multiple range test at P < 0.05. 3.4 Phenolic acids Four phenolic acids were extracted from the petals of miniature rose 'KORcrisett': gallic acid, protocatechulic acid, caffeic acid and /-coumaric acid (Table 3). On the first sampling rose petals averagely contained 31.37 ^g g-1 FW gallic acid, 121.65 ^g g-1 FW protocatechulic acid, 133.56 ^g g-1 FW caffeic acid and 52.57 ^g g-1 FW /-coumaric acid. After 28 days, the lowest concentrations of all phenolic acids were detected in the petals of plants potted in pH 4.7 and the highest in pH 3.3. The concentration of gallic acid was considerably lower on the second sampling; the decrease was more than seven fold in pH level 4.7, four fold in pH level 7.3 and more than two fold in pH level 3.3. Similarly, the concentrations of protocatechulic acid, caffeic acid and /-coumaric acid were significantly lower on the second sampling in both pH levels 4.7 and 7.3 and remained constant or even increased in the acidic pH level. Table 3: Effect of substrate pH level on the concentration of phenolic acids (^g g-1 FW) in petals of Rosa x hybrida L. 'KORcrisett' on two sampling dates. Phenolic acid [mean ± SE (^g g-1)] Sampling date pH Level Gallic acid Protocatehulic acid Caffeic acid /-Coumaric acid 3.3 27.24 ± 2.02 a1 104.42 ± 9.11 a 123.34 ± 6.51 a 48.04 ± 3.60 a 12 Aug. 4.7 33.04 ± 2.30 a 131.18 ± 11.72 a 137.63 ± 7.52 a 52.57 ± 3.81 a 7.3 33.82 ± 2.17 a 129.34 ± 10.08 a 139.70 ± 4.28 a 57.09 ± 3.42 a 3.3 11.28 ± 2.03 b 104.62 ± 15.07 b 118.79 ± 16.0 b 66.43 ± 7.90 b 8 Sept. 4.7 4.39 ± 0.64 a 59.83 ± 3.78 a 81.29 ± 5.62 a 35.82 ± 3.91 a 7.3 7.82 ± 1.17 ab 69.01 ± 9.90 a 82.27 ± 10.78 a 46.36 ± 4.41 a 1 Values carrying the same letters (a-b) for each set of dates do not differ significantly by Duncan's multiple range test at P < 0.05. 4 DISCUSSION Overall, pH of the substrate had a significant effect on the number of flowers per plant as well as on the phenolic concentration in rose petals, as it affects nutrient uptake into plants (Smith et al., 2004a, Papafotiou et al., 2007) and consequently, reproductive efficiency. Similarly, the production of flowers in Senecio vulgaris L. was fewer in plants, grown in a nutrient deficient substrate (Brown and Molyneux, 1996) and acidic and alkaline pH levels caused significant changes in flowering of tobacco plants (Pasqua et al., 1991). Miniature rose plants developed significantly less flowers when grown in a substrate with pH levels 3.3 and 7.3 compared to pH level 4.7. As early as 1930, a lower substrate pH level (an average of 5.7 is mentioned as optimal) was reported to have a positive effect on the growth of different rose cultivars (Zieslin and Snir, 1989). A clear increase in flower production was also noted when rose plants were grown in a peat and Lelite substrate amended with ammonium. Yields per ft2 of roses increased as the proportion of NH4+ to NO3- increased causing a decrease in pH of the rhizosphere (White ad Richter, 1973; Findenegg et al., 1986). In contrast, the concentration of major and minor anthocyanins in rose petals increased in more acidic and alkaline pH levels. External stressors, such as substrate pH level, promote anthocyanin synthesis as was demonstrated by Hawrylak-Nowak (2008) who reported an increase in anthocyanin concentration dependant on substrate alkalinity in maize (Zea mays L.). Pelargonidin-3,5-di-O-glucoside and cyanidin-3,5-di-O-glucoside were the prevailing anthocyanic pigments in 'KORcrisett' petals, which is in accordance with the results of Biolley et al. (1994) and Mikanagi et al. (1995) who obtained similar results in other rose cultivars. Similarly to the research of Mikanagi et al. (1995) Rosa x hybrida L. 'KORcrisett' petals contained three minor anthocyanic pigments: pelargonidin-3-O-glucoside, cyanidin-3-O-glucoside and peonidin-3-O-glucoside, all significantly affected by substrate pH level. Quercetin-3-O-glucoside, quercetin-3-O-rhamnoside and quercetin-3-O-rutinoside are the major quercetin compounds in rose flowers (Mikanagi et al., 1995; Cai et al., 2005) and, like anthocyanins, their concentration was lowest in flowers of the plants, potted in 4.7 pH level. Among the phenolic acids gallic, protocatechuic acid, caffeic acid and p-coumaric acid were previously reported by Cai et al. (2005) and Kumar et al. (2008) in other rose cultivars and were significantly affected by pH level. According to our research, the optimal pH level of the substrate for increased flowering of miniature rose 'KORcrisett' was 4.7, however when an increase in phenolic concentration is preferential a modified pH level could be used to produce plants with flowers, which contain more anthocyanins and other phenolic compounds. 5 ACKNOWLEDGEMENT This work is part of program Horticulture No. P4-0013-0481, funded by the Slovenian Research Agency (ARRS). 6 REFERENCES Biolley, J.P., Jay, M., Viricel M.-R. (1994): Flavonoid diversity and metabolism in 100 Rosa x hybrida cultivars. Phytochemistry, 35: 413-419. Brown, M.S., Molyneux, R.J. (1996): Effects of water and mineral nutrient deficiencies on pyrrolizidine alkaloid content of Senecio vulgaris flowers. Journal of the Science of Food and Agriculture, 70: 209-211. Cai, Y.-Z., Xing, J., Sun, M., Zhan, Z.-Q., Corke, H. (2005): Phenolic antioxidants (hydrolyzable tannins, flavonols, and anthocyanins) identified by LC-ESI-MS and MALDI-QIT-TOF MS from Rosa chinensis flowers. Journal of Agricultural and Food Chemistry, 53: 9940-9948. Cominelli, E., Gusmaroli, G., Allegra, D., Galbiati, M., Wade, H.K., Jenkins, G.I., Tonelli, C. (2007): Expression analysis of anthocyanin regulatory genes in response to different light qualities in Arabidopsis thaliana. Journal of Plant Physiology, 165: 886-894. Dela, G., Or, E., Ovadia, R., Nissim-Levi, A., Weiss, D., Oren-Shamir, M. (2003): Changes in anthocyanin concentration and composition in 'Jaguar' rose flowers due to transient high-temperature conditions. Plant Science, 164: 333-340. Eugster, C.H., Markifischer, E. (1991): The chemistry of rose pigments. Angewandte Chemie: International Edition in English, 30: 654-672. Findenegg, G.R., van Beusichem, M.L., Keltjens, W.G. (1986): Proton balance of plants: physiological, agronomical and economical implications. Netherlands Journal of Agricultural Science, 34: 371-379. Harbage, J.F., Stimart, D.P., Auer, C. (1998): pH affects 1H-indole-3-butryric acid uptake but not metabolism during the initiation phase of adventitious root induction in apple microcuttings. Journal of the American Society for Horticultural Science, 123: 6-10. Hawrylak-Nowak, B. (2008): Changes in anthocyanin content as indicator of maize sensitivity to selenium. Journal of Plant Nutrition, 31: 1232-1242. Hughes, N.M., Morley, C.B., Smith, W.K. (2007): Coordination of anthocyanin decline and photosynthetic maturation in juvenile leaves of three deciduous tree species. New Phytologist, 175: 675-685. Juszczuk, I.M., Wiktorowska, A., Malusa, E., Rychter, A.M. (2004): Changes in the concentration of phenolic compounds and exudation induced by phosphate deficiency in bean plants (Phaseolus vulgaris L.). Plant and Soil, 67: 41-49. Kumar, N., Bhandari, P., Singh, B., Gupta, A.P., Kaul, V.K. (2008): Reversed phase-HPLC for rapid determination of polyphenols in flowers of rose species. Journal of Separation Science, 31: 262-267. Marks, S.C., Mullen, W., Crozier, A. (2007): Flavonoid and chlorogenic acid profiles of English cider apples. Journal of Science of Food and Agriculture, 87: 719-728. Mikanagi, Y., Yokoi, M., Ueda, Y., Saito, N. (1995): Flower flavonol and anthocyanin distribution in subgenus Rosa. Biochemical Systematics and Ecology, 23: 183-200. Muller, R., Andersen, A.S., Serek, M. (1998): Differences in display life of miniature potted roses (Rosa hybrida L.). Scientia Horticulturae, 76: 59-71. Papafotiou, M., Avajiannelli, B., Michos, C., Chatzipavlidis, I. (2007): Coloration, anthocyanin concentration, and growth of croton (Codiaeum variegatum L.) as affected by cotton gin trash compost use in the potting medium. Hortscience, 42: 83-87. Pasqua, G., Monacelli, B., Altamura, M.M. (1991): Influence of pH on flower and vegetative bud initiation and development invitro. Cytobiosistematics, 68: 111-121. Schmitzer, V., Osterc, G., Veberic, R., Stampar, F. (2009a): Correlation between chromaticity values and major anthocyanins in seven Acer palmatum Thunb. cultivars. Scientia Horticulturae, 119: 442-446. Schmitzer, V., Veberic, G., Osterc, G., Stampar, F. (2009b): Changes in the phenolic concentration during flower development of rose 'KORcrisett'. Journal of the American Society for Horticultural Science, 134: 491-496. Smith, B.R., Fischer, P.R., Argo, W.R. (2004a): Growth and pigment content of container-grown Impatiens and Petunia in relation to root substrate pH and applied micronutrient concentration. Hortscience, 39: 1421-1425. Smith, B.R., Fischer, P.R., Argo W.R. (2004b): Nutrient uptake in container-grown Impatiens and Petunia in response to root substrate pH and applied micronutrient concentration. Hortscience, 39: 1426-1431. White, J.W., Richter, D. (1973): Supplementary fluorescent lighting and low moisture stress improve growth of greenhouse roses. Journal of the American Society for Horticultural Science, 98: 605-607. Zieslin, N., Snir, P. (1989): Responses of rose plants cultivar 'Sonia' and Rosa indica major to changes in pH and aeration of the root environment in hydroponic culture. Scientia Horticulturae, 37: 339-349. DOI: 10.2478/v10014-010-0002-4 COBISS Code 1.01 Agrovoc descriptors: sitophilus oryzae, triticum, cereal crops, wheats, atomite, soil, minerals, pesticidal properties, chemical control, pests of plants, relative humidity, temperature, storage, storage losses, crop losses, postharvest losses Agris category code: H10 The effect of diatomaceous earth of different origin, temperature and relative humidity against adults of rice weevil (Sitophilus oryzae [L.], Coleoptera, Curculionidae) in stored wheat Helena ROJHT1, Christos G. ATHANASSIOU2, Bill J. VAYIAS3, Nickolas KAVALLIERATOS4, Željko TOMANOVIC5, Matej VIDRIH6, Katarina KOS7, Stanislav TRDAN8 Received October 23, 2009; accepted February 24, 2010. Delo je prispelo 23. oktobra 2009; sprejeto 24. februarja 2010. ABSTRACT IZVLEČEK VPLIV DIATOMEJSKE ZEMLJE RAZLIČNEGA IZVORA, TEMPERATURE IN RELATIVNE VLAGE PRI ZATIRANJU ODRASLIH OSEBKOV RIŽEVEGA ŽUŽKA (Sitophilus oryzae [L.], Coleoptera, Curculionidae) V SKLADIŠČENI PŠENICI V laboratorijskih poskusih smo preučevali vpliv diatomejske zemlje (DZ) različnega izvora pri zatiranju enega od najpomembnejših primarnih škodljivcev skladiščenega žita. Ugotavljali smo učinkovitost treh lokalnih tipov DZ (srbskega, grškega in slovenskega) in komercialnega pripravka SilicoSec pri zatiranju odraslih osebkov riževega žužka (Sitophilus oryzae) v skladiščeni pšenici. Insekticidno delovanje DZ smo preučevali pri treh temperaturah (20, 25 in 30 °C), dveh vrednostih relativne vlage (55 and 75 %) in štirih (100, 300, 500 and 900 ppm) koncentracijah vsakega vzorca DZ. Smrtnost hroščev smo ugotavljali 7., 14. in 21. dan po tretiranju (DPT) in je bila največja pri najvišji koncentraciji in 21 DPT. V vseh vzorcih žita je bila smrtnost hroščev večja od 90 % pri koncentraciji 900 ppm ter 21 DPT. Izjemi sta bili slovenski in grški vzorec, ki sta vplivali na 85,3 % smrtnost pri 25 °C in 55 % vlagi ter na 67,6 % smrtnot pri 25 °C in 75 % vlagi. Najbolj učinkovit (100 % smrtnost hroščev) je bil pripravek SilicoSec 14 DPT pri koncentraciji 900 ppm. Slovenski vzorec DE je bil bolj učinkovit pri 55 kot pri 75 % vlagi (7 DPT pri vseh temperaturah). 1 Young researcher, M. Sc., Jamnikarjeva 101, SI-1111 Ljubljana, e-mail:helena.rojht@bf.uni-lj.si 2 Prof., Ph. D., Agricultural University of Athens, Dept. of Plant Science, 75 lera Odos, 11855 Athens, Attica, Greece 3 Ph. D., ibid. 4 Ph. D., Laboratory of Agricultural Entomology, Dept. of Entomology and Agricultural Zoology, Benaki Phytopathological Institute, 8 Stefanou Delta Street, 14561 Kifissia, Attica, Greece 5 Assoc. Prof., Ph. D., University of Belgrade, Faculty of Biology, Dept. of Zoology, Studentski trg 16, SER-11000 Belgrade, Serbia 6 Teach. Assist., Ph. D, Jamnikarjeva 101, SI-1111 Ljubljana 7 Teach. Assist., B. Sc., ibid. 8 Assoc. Prof., Ph. D, ibid. Laboratory experiments were carried out to evaluate the impact of diatomaceous earth (DE) samples of different origin with their insecticidal properties to control one of the most important primary pest in stored grain. We tested the efficacy of three local DE, from Serbia, Greece and Slovenia, and commercial formulation SilicoSec against the rice weevil, Sitophilus oryzae, adults in stored wheat. The experiments were carried out at three temperatures (20, 25 and 30 °C) and two relative humidity (RH) levels (55 and 75 %). Mortality of pest was counted 7, 14 and 21 days after exposure (DAT) at the following DE dose rates: 100, 300, 500 and 900 ppm. The mortality of adults normally increased with increasing dose rates and DAT. In all samples the mortality of rice weevil adults (dose rate 900 ppm, 21 DAT) was above 90 %, except at Slovenian DE (at 20 °C and 55 % RH) and Greek DE (at 25 °C and 75 % RH), when the mortality was 85.3 and 67.6 %, respectively. With 100 % mortality (14 DAT and at 900 ppm) the most effective was SilicoSec. Slovenian DE was more effective at 55 % RH than at 75 % RH (7 DAT at all temperatures). Key words: Sitophilus oryzae, diatomaceous earth, origin, stored wheat, temperature, relative humidity Ključne besede: Sitophilus oryzae, diatomejska zemlja, izvor, pšenica, temperatura, relativna vlaga 1 INTRODUCTION Diatomaceous earth (DE) is a light weight, porous sedimentary rock made up of the diatoms (Bacillariophyta), unicellular aquatic plants. Their shells walls are made of amorphous hydrated silica (SiO2xnH2O), therefore main constituent of DE is amorphous silica (SiO2) with small amounts of other elements such as aluminum, iron oxide, calcium hidroxide, magnesium and sodium (Round et al., 1990). Geological deposits of DE can be hundreds of meters thick (Ross, 1981). Residual insecticides are the most common agents for protection of stored products against stored products pests. Because of their several negative properties, such are mammal toxicity, residues on grain and increased resistance of pests on some residual insecticides (Arthur, 1996), some researchers decided to evaluate several new alternative protection methods for stored products (Athanassiou et al., 2005). Among them was also the use of DE, which is classified as environmental acceptable method for controlling stored products pests (Arthur, 1996). For decades, DE was intensively investigated for protection of stored products. Insecticidal properties of DE in majority depend on geological origin, SiO2 content, tapped density, oil absorbency, particle size and pH (Golob, 1997; Korunic, 1998) and also of other factors such as days after treatment (DAT), moisture and temperature (Arthur, 2001). Different theories about control activity of DE have been proposed: 1.) blockage of stigmata and tracheae (Webb, 1945); 2.) impairment of the digestive tract (Smith, 1969); 3) water loss from insect's body through desiccation resulting in death (DE particles absorb or/and abrasive waterproof epicuticular lipids from insect's cuticle) (Ebeling, 1971). Therefore the control activity is a physical and possibility of occurring the physiological resistance is very low (Golob, 1997; Korunic, 1998; Subramanyam and Roesli, 2000). DE has a several advantages above traditional grain protectants, because it has low mammalian toxicity, does not break down rapidly and does not affect end-use quality (Korunic et al., 1996), it can be applied to the commodity with approximately the same technology as traditional residual insecticides, it can provide long-term protection against insects pest (Stathers et al., 2004, Athanassiou et al., 2005) and it can be easily removed from grain during processing (Golob, 1997; Korunic, 1998; Subramanyam and Roesli, 2000). The rice weevil, Sitophilus oryzae (L.), Coleoptera, Curculionidae, is a cosmopolitan pest, which is considered to be one of the most destructive and widespread species in stored grain, also in Europe (Maceljski, 1999). This pest has developed a considerable level of resistance to some insecticides and it is considered to be the most resistant to piretroids (Arthur, 1996). That's why it is impossible to suppress him by application in dose rates which is effective against most other stored-grain beetle (Arthur, 1999). Rice weevil is classified as a primary pest. That means that beside damage, caused by feeding and multiplication, rice weevil can easily infest sound seeds and enable spreading secondary pests (Maceljski, 1999). In this work we evaluated insecticidal activity of DE of different origin (three local DEs and commercial product SilicoSec) against rice weevils adults at three temperatures and two relative humidity levels. Also the comparison of insecticidal efficiency of Slovenian local DE sample with other DEs was made. Efficacy of Slovenian and Serbian DE was evaluated for the first time. 2 MATERIALS AND METHODE 2.1 Wheat and Insect Origin Wheat was produced in 2007 in Horjul surroundings (latitude 46° 01N, longitude 14°31'E, 339.8 m above sea level, Slovenia). Rice weevil adults (<21 days old) which were used for the test, were taken from a population that was kept in the Laboratory for Entomology on Chair of Phytomedicine, Agricultural Engineering, Crop Production, Grassland, and Pasture Management (Biotechnical Faculty, Ljubljana, Slovenia) for several years at 25±1°C on wheat. 2.2 Diatomaceous Earth Formulations Three local DEs and commercial DE formulation were used in the test: 1. Slovenian sample was taken in a place near the village Bela cerkev (45 52 N, 15°16E, 190.4 m). 2. Greek sample was from surroundings of town Elassona at the foothills of the Olimp mountain (39°52' N 22°10' E, 308.0 m). 3. Serbian sample was from town Kolubara (44°22' N, 20°15' E, 161.0 m). 4. Commercial DE formulation SilicaSec (Biofa GmbH, Münsingen, Germany) is of freshwater origin containing approximately 92 % SiO2 (Athanassiou et al., 2003). Previous studies have shown that SilicoSec is effective against several stored pests (Athanassiou et al., 2003; Athanassiou et al, 2005) and therefore it was used for positive control. Particle sizes of all four DE were approximately 12 ^m. 2.3 Laboratory Bioassay Exposure studies were carried out at 20, 25 and 30 °C and at 55 and 75 % relative humidity (RH). Four concentrations of each DE were used (100, 300, 500 and 900 ppm). Single glass jar contained 1 kg of wheat grain and individual dose of DE. An additional jar of untreated wheat was used as control. All jars were shaken manually for approximately 5 minutes to achieve distribution of the DE in the entire grain mass (Kavallieratos et al., 2007). For each treating, 3 samples of 50 g each were taken from each jar and each sample was placed in a vial. Then, 30 rice weevil adults were introduced into each vial, after that vials were closed with a slender net to prevent the insects from escaping and were placed into a rearing chamber with the dark: light ratio of 24:0. Mortality of the exposed adults was measured after 7, 14 and 21 days of exposure. The entire procedure was repeated 3 times. 2.4 Data Analysis The data obtained from the experiment were subjected to analysis of variance to determine differences in mortality rates (%) of rice weevil adults, reared under conditions of different treatments at three different temperatures (20, 25 and 30 °C) and two levels of relative humidity (55 and 75 %) (GenStat 7.1; VSN International Ltd., Hemel Hempstead, UK) and Duncan's post hoc test was used to determine significant differences between the treatments (Hoshmand, 2006). Before the analysis, each variable was tested for homogeneity of treatment variances. Mortality rate data were corrected for control mortality using Abbott's formula (Abbott, 1925), the arcsine square-root being transformed before analysis. The data are presented as untransformed means. 3 RESULTS All main effects as well as their associated interactions for mortality levels were significant at the P < 0.05 level. Generally the most efficient was SilicoSec with 88.1 % mortality, following Serbian and Greek DE with 87.0 % and 71.9 % mortality respectively. The lowest mortality (64.0 %) was observed with Slovenian sample. At 20 °C and after 7 DAT the highest mortality (97.9 % and above) was at SilicoSec at the highest dose rates (Figure 1). Mortality at Serbian Sample was 89.1 % and above at the highest dose rates. Greek and Slovenian samples were less efficacious with the lowest mortality (55.6 and 41.3 %). At 500 ppm only SilicoSec reached 100 % mortality and at 20 °C, 55 % RH and 30 °C, 75 % RH. At this temperature level DE samples caused in general better mortality at higher RH level. (7 d) (14 d) (21 d) A 100 ^ 60 ro "C 40 O a I b b ,bb b b SRB GR SI SilicoSec SRB GR SI SlllcoSec SRB GR SI SilicoSec bb c b b c b b b b b 100 a b d b 80- 80- 80- a c 60 60 b b b a 40- 40- a a a 20 20 20 a 0 0 0 B 100- Co 0s >-. 60-TO "C 40 ab ,1 a/ I SRB GR SI SilicoSec SRB GR SI SilicoSec DE formulation bb SRB GR SI SilicoSec b b bb b b ab a b b 100- 100- a a a a a a a a a 80- 80- a a a bc 60- 60- b b 40 40 a 20- 20- 20- 0 0 0 Figure 1: Mean ± SE mortality (%) of S. oryzae adults exposed for 7, 14 and 21 d on wheat treated with four DE formulations and at four concentrations ff - 100, W - 300, □ - 500, ■ - 900 ppm) in 55 % RH (A) and 75 % RH (B) at 20 °C (means at the same DE formulation and concentration followed by the same latter are not significantly different; Duncan test, at P = 0.05). At 25 °C mortality activity of all DE samples decreased. Total mortality was reached at SilicoSec at highest dose rate and mortality of Serbian samples was above 96.9 % at the same dose rate (Figure 2). Greek sample also give a good results at the 900 ppm (mortality above 99.0 %). Slovenian sample was better at 75 % RH than at 55 % RH. At higher RH and at the 900 ppm the mortality was at least 97.6 % and at 55 % RH was under 81.7 %. (7 d) (14 d) (21 d) A 10080- vP . ^ 60--i—' 1Č "C 40- B 80- vP . ^ 60--i—' "C 40- SI SilicoSec b b b i SRB GR SI SilicoSec 100- b 80- b h i / / / / 60- / / / / / / / 40- / / / / / / / / / 20- / a / / / / i / - 0- i / / b bJl t/ I E/ / iS SI SilicoSec SRB GR SI SilicoSec DE formulation SRB GR SI SilicoSec Figure 2: Mean ± SE mortality (%) of S. oryzae adults exposed for 7, 14 and 21 d on wheat treated with four DE formulations and at four concentrations ff - 100, W - 300, □ - 500, ■ - 900 ppm) in 55 % RH (A) and 75 % RH (B) at 25 °C (means at the same DE formulation and concentration followed by the same latter are not significantly different; Duncan test, at P = 0.05). c b b bbb a a a 80- a 60- b 40- 20 20 0 0 b b cb b c c a a 20 a a 0 At 30 °C the mortality of rice weevils was higher than at 20 or 25 °C. At highest dose rates the mortality was at all trials above 90 % except at Slovenian sample (20 °C and 55 % RH) and Greek sample (at 25 °C and 75 % RH) where it was 85.3 and 67.6 % respectively (Figure 3). The mortality above 90 % at 500 ppm was at SilicoSec, Greek and Serbian samples, except at one treating (Greek DE, 25 °C, 75 % RH) where the mortality was only 67.6 %. Slovenian sample was the less efficient and mortality was between 36.8 and 90.6 % at the same dose rate. DE formulation Figure 3: Mean ± SE mortality (%) of S. oryzae adults exposed for 7, 14 and 21 d on wheat treated with four DE formulations and at four concentrations ff - 100, W - 300, □ - 500, □ - 900 ppm) in 55 % RH (A) and 75 % RH (B) at 30 °C (means at the same DE formulation and concentration followed by the same latter are not significantly different; Duncan test, at P = 0.05). 4 DISCUSSION Insecticidal efficacy of DE is highly influenced by several factors such as temperature, type of DE formulation and dose rate (Kavallieratos et al. 2007). Important factor which affect the rice weevil mortality is also exposure interval. DE has physical action, since the DE particles grasp insect cuticle meanwhile weevils are moving on treated wheat (Athanassiou et al, 2005). Further exposure means active contact with DE particles, which damages weevil wax layer and insect dies through desiccation (Korunic, 1998). The lowest mortality between rice weevil adults were 7 days after treatment and the highest mortality was obtained after 21 days of exposure. Some investigations showed that mortality of tested insects increased with increasing temperature (Fields and Korunic, 2000; Athanassiou et al, 2005). At higher temperatures the insects are more mobile, which increases contact with the DE particles and therefore greater damage of the insect cuticle. Increased water loss via spiracles due to increased respiration at higher temperatures is also observed (Arthur, 2000; Fields and Korunic, 2000). In our investigation the lowest mortality was obtained at 25 °C and not at the lowest temperature (20 °C). We believe that the rearing temperature (25 ±1 °C) of rice weevils before treatments is responsible for this result. The results of our research showed that the relative humidity had some effects on mortality of rice weevil adults, because at 55 and 75 % relative humidity the mortality was 80.2 % and 75.4 % respectively. This data confirm the fact that efficacy of DE decreased with increase in relative humidity (Mewis, 1998; Athanassiou et al., 2007). On the contrary, at Slovenian DE the mortality was with a few exceptions at 75% RH much higher as was at 55% RH. Effective dose rate of SilicoSec for successfully rice weevil treating in wheat is between 500 and 900 ppm (Athanassiou et al, 2005). In our study the mortality of rice weevil was at dose rate 900 ppm more than 97.9 % and at dose rate 500 ppm the mortality was above 90 %, except after 7 DAT at 25 °C, 75 % RH and at 30 °C, 55 % RH, where the mortality was 84.1 and 76.4 % respectively. In our investigation SilicoSec and Serbian DE have satisfactory effect on rice weevil adults at dose rates which are acceptable to environmental, people health and it has no influence on quality of stored products (Korunic et al, 1996; Korunic, 1998). Therefore it can be used for control of this pest on stored wheat. The mortality of beetle increased with increasing dose rates. Although Slovenian DE shows some insecticidal properties against rice weevils, it has to be applied at concentrations higher than 900 ppm. Concentrations higher than 1000 ppm are inappropriate for pest control, because they influence several physical and mechanical properties of grain (Korunic et al, 1996; Subramanyam and Roesli, 2000). On the base of our investigation on rice weevil, species which belongs to DE sensitive pests (Fields and Korunic, 2000), we concluded that Slovenian and Greek diatomaceous sediments have no appropriated insecticidal potential for control of stored products against stored insects pests. 5 ACKNOWLEDGEMENT Funding for this study was provided by the multilateral project SEE-ERA.NET 9902 «Development of a nontoxic, ecologically compatible, natural-resource based insecticide from diatomaceous earth deposits of South Eastern Europe to control stored-product insect pests» and by national project L4-1013 »Development, optimization and implementation of sustainable control methods against plant pests«. We thank Aleksander Horvat from the Faculty of Natural Sciences and Engeneering in Ljubljana for showing us the only location of local diatomaceous earth in Slovenia and Aleksander Bobnar from Biotechnical Faculty for technical assistance. 6 REFERENCES Abbott, W. S. 1925. A method of computing the effectiveness of an insecticide. J. Econ. Entomol., 18: 265-267. Arthur, F. H. 1996. Grain protectants: current status and prospects for the future. J. Stored Prod. Res.: 32, 293302. Arthur, F. H. 1999. Knockdown, mortality and progeny production of lesser grain borers (Coleoptera: Bostrychidae) and rice weevils (Coleoptera: Curculionidae) exposed for short intervals on wheat treated with cyfluthrin. J. Econ. Entomol., 92: 1198-1205. Arthur, F. H. 2000. Toxicity of diatomaceous earth to red flour beetles and confused flour beetles (Coleoptera: Tenebrionidae): effects of temperature and relative humidity. J. Econ. Entomol., 93: 526-532. Arthur F. H. 2001. Immediate and delayed mortality of Oryzaephilus surinamensis (L.) exposed on wheat treated with diatomaceous earth: effects of temperature, relative humidity, and exposure interval. J. Stored Prod. Res., 37: 13-21. Athanassiou, C. G., Kavallieratos N. G., Tsaganou F. C., Vayias B. J., Dimizas C. B., Buchelos C. Th. 2003. Effect of grain type on the insecticidal efficacy of SilicoSec against Sitophilus oryzae (L.) (Coleoptera: Curculionidae). Crop Prot., 22: 1141-1147. Athanassiou, C. G., Kavallieratos N. G., Economou L. P., Dimizas C. B., Vayias B. J., Tomanovic S., Milutinovic M. 2005. Persistence and efficacy of three diatomaceous earth formulations against Sitophilus oryzae (Coleoptera: Curculionidae) on wheat and barley. J. Econ. Entomol., 98: 1404-1412. Athanassiou C. G., Kavallieratos N. G., Peteinatos G. G., Petrou S. E., Boukouvala M. C., Tomanovic Z. 2007. Influence of temperature and humidity on insecticidal effect of three diatomaceous earth formulations against larger grain borer (Coleoptera: Bostrychidae). J. Econ. Entomol., 100: 599-603. Bardossy G., Hajos M. 1963. Caracteristiques lithologiques et geochemiques des complewes a Diatomees de Szurdokpuspoki. Fold. Int. Evi.Jel. (1960-rol): 121-146, pl. 1-3, Budapest. Bostrom, K., Joensuu Valdes, S., Riera, M. 1972. Geochemical history of south Atlantic Ocean sediments since Late Cretaceous. - Marine Geology, 12: 85-121. Ebeling W. 1971. Sorptive dust for pest control. Annu. Rev. Entomol., 16: 123-158. Fields P., Korunic Z. 2000. The effect of grain moisture content and temperature on the efficacy of diatomaceous earths from different geographical locations against stored-product beetles. J. Stored Prod. Res., 36: 1-13. Golob P., 1997. Current status and future perspectives for inert dusts for control of stored product insects. J. Stored Prod. Res., 33: 69-79. Horvat, A., Misic M. 2004. Mineralogy and sedimentology of diatomaceous sediments of Slovenia. RMZ - Materials and Geoenvironment, 51: 2145-2161. Hoshmand, A.R. 2006. Design of experiments for agriculture and the natural sciences. 2nd Edition. Chapman and Hall, CRC Press: 437 p. Kavallieratos N. G., Athanassiou, C. G., Vayias B. J., Maistrou S. N. 2007. Influence of temperature on susceptibility of Tribolium confusum (Coleoptera: Tenebrionidae) populations to three modified diatomaceous earth formulations. Fla. Entomol., 90: 616625. Korunic Z., Fields P. G., Kovacs M. I. P., Noll J. S., Lukow O. M., Demianyk C. J., Shibley K. J. 1996. The effect of diatomaceous earth on grain quality. Postharvest Biol. Technol., 9: 373-387. Korunic Z. 1997. Rapid assessment of the insecticidal value of diatomaceous earths without conducting bioassays. J. Stored Prod. Res., 33: 219-229. Korunic Z. 1998. Diatomaceous earths, a group of natural insecticides. J. Stored Prod. Res., 34: 87-97. Maceljski M. 1999. Poljoprivredna entomologija. Zrinski, Cakovec: 464 p. Mewis I. 1998. Morphologische und physiologische Wirkungsweise amorpher Diatomeenerden auf ausgewählte vorratsschädliche Insekten. Diplomarbeit, Biologische Bundesanstalt für Land-und Forstwirtschaft/freie Universität Berlin. Round F. E., Crawford R. M., Mann D. G. 1990. The diatoms. Cambrige University Press, Cambridge, 747 p. Ross T. E. 1981. Diatomaceous earth as a possible alternative to chemical insecticides. Agric. Environ., 6: 43-51. Subramanyam, Bh., Roesli, R., 2000. Inert dusts. In: Subramanyam, Bh., Hagstrum, D.W. (Eds.), Alternatives to Pesticides in Stored-Product IPM. Kluwer Academic Publishers, Dordrecht, pp. 321-380. Smith B. C. 1969. Effects of silica on the survival of Coleomegilla maculate lengi (Coleoptera: Coccinellidae) and Leptinotarsa decemlineata (Coleoptera: Chrysomelidae). Can. Entomol., 101: 460-462. Stathers, T. E., M. Denniff, and P. Golob. 2004. The efficacy and persistence of diatomaceous earth admixed with commodity against four tropical stored product beetle pests. J. Stored Prod. Res., 40: 113-123. Webb J. E. 1945. The penetration of Derris Through the Spiracles and cuticle of Melophagus ovinus, L. Bull. Entomol. Res., 36: 15-22. DOI: 10.2478/v10014-010-0003-3 COBISS Code 1.01 Agrovoc descriptors: prunus avium, prunus cerasus, cherries, extracts, antioxidants, phenolic compounds, aromatic compounds, chemical composition, proximate composition Agris category code: F60 Relation of total antiradical activity and total polyphenol content of sweet cherries (Prunus avium L.) and tart cherries (Prunus cerasus L.) Silvia MELICHÁČOVÁ1, Mária TIMORACKÁ1, Judita BYSTRICKÁ1, Alena VOLLMANNOVÁ1, Juraj ČÉRY1 Received January 27, 2010; accepted March 2, 2010. Delo je prispelo 27. januarja 2010, sprejeto 2. marca 2010. ABSTRACT In present study the quantification of total phenolics content (TPC), total antioxidant activity (TAA) and their relation in sweet and tart cherries were studied. Aqueous and pure polar solvents were used to compare the yield of present phenolic compounds in prepared extracts. The solubility of phenolics was the most effective in sweet cherry extracts with using of 50 % methanol and in tart cherry extracts with using of 50 % acetone. The yield of TPC of both tested cherry fruit extracts was higher with pure methanol in comparison to pure acetone. Substantial TPC assessed with Folin-Ciocealteu assay in tart cherry extracts were in range from 70.6±8.46 mg to 241.4±7.26 mg GAE/100 g FW. Extracts from lyophilised tart cherries (methanolic and water-acetone mixtures) contain in average 2-times higher amount of polyphenols than ethanol extracts. The DPPH antiradical efficiency values of the both tested fruit extracts were higher in extracts of tart cherries (from 5.4 to 9.9 % of inhibition of DPPH radical) when compared to those of sweet cherries (from 2.4 to 3.5 % of inhibition of DPPH radical). Total antioxidant activity (TAA) of sweet cherry extracts (using 70 % ethanol and with 70 % methanol) and of tart cherry extracts (with 50 % methanol) depended on phenolics content. Key words: total phenolics, sweet cherries, tart cherries, total antioxidant activity IZVLEČEK POVEZAVA MED ANTIOKSIDANTNO AKTIVNOSTJO IN VSEBNOSTJO VSEH POLIFENOLOV PRI ČEŠNJAH (Prunus avium L.) IN VIŠNJAH (Prunus cerasus L.) Pri tej raziskavi je bila proučevana celotna vsebnost polifenolov (TPC), celotna antioksidantna aktivnost (TAA) in povezava med njima pri češnjah in višnjah. Uporabljeni so bili vodni ekstrakti in ekstrakti s polarnimi topili ter primerjana vsebnost ekstrahiranih fenolnih snovi. Ta je bila največja pri češnjah, ekstrahiranih s 50 % metanolom in pri višnjah z uporabo 50 % acetona. TPC je bilo več pri češnjah ekstrahiranih s 100 % metanolom, v primerjavi z acetonom. TPC ugotovljeni s Folin-Ciocealteu-jevim reagentom so pri višnjah dali vrednosti od 70,6±8,46 mg do 241,4±7,26 mg GAE/100 g sveže teže. Ekstrakti liofiliziranih višenj so pri ekstrahiranju z metanolom ali mešanico vode in acetona v povprečju vsebovali dvakrat višjo količino polifenolov kot etanolni ekstrakti. Antioksidativna učinkovitost DPPH je imela višje vrednosti pri višnjah (od 5,4 do 9,9 % inhibicija DPPH radikalov) v primerjavi z ekstrakti pri češnjah (od 2,4 do 3,5 % inhibicije DPPH radikalov). Celotna antioksidativna aktivnost (TAA) ekstraktov češenj (z uporabo 70 % etanola in 70 % metanola) in pri ekstraktih višenj (s 50 % metanolom) sta odvisni od vsebnosti fenolnih snovi. Ključne besede: skupni fenoli, češnje, višnje, celotna antioksidativna aktivnost 1 INTRODUCTION Commonly consumed raw materials of plant origin are the most important sources of phenolic compounds from a health standpoint and from the aspect of consumption by the whole population. Due to reported epidemiological studies providing data about correlations between fruit consumption and reduced risk of chronic diseases (He et al., 2007), fruits have gained an important place in human nutrition. The combination of vitamins, fibre, phenolics and other 1 Department of Chemistry, Slovak University of Agriculture in Nitra, Department of Chemistry, Slovak University of Agriculture in Nitra, e-mail address: melichac@afnet.uniag.sk antioxidants are probably reliable for these effects (Ruxton et al., 2006). Parallel with this recognition, the consumption of fruits has increased all over the world. Sweet and tart cherries are natural sources of antioxidants influencing the incidence of degenerative diseases. Mentioned stone fruits from family Rosaceae and genus Prunus have been part of the human diet for several thousands year. Both sweet and tart cherries are widely grown in Slovakia. Local people from some regions have been using sweet and tart cherry fruits as fresh fruit, also for jams, marmalade and compote preparation. In spite of their short consumption period, they are very preferable. Sweet cherry (Prunus avium L.) contains besides water, saccharides, vitamins A, B and C, pectin, organic acids, also minerals mainly potassium, magnesium, phosphor and calcium (Kalyoncu et al., 2009). Sweet cherries are from ancient times associated with better function of organism and for instance, Wang et al. (1999) reported that the consumption of sweet cherries alleviates arthritis and gout-related pain. Reduction of the proliferation of human colon cancer cells (Kang et al., 2003) has been specifically associated with the consumption of cherries (Serrano et al., 2005). Recently, also tart cherries with their health benefits have been preferred with unique composition of anthocyanins and related polyphenolics (Gao and Mazza, 1995; Kim et al., 2005; Piccolella et al., 2008). Tart cherry fruits (Prunus cerasus L.) contain also phenolic acids and quercetin (Seeram et al., 2001; Seymour et al., 2008). Polyphenolic compounds found in fruit contribute to total antioxidant activity. The large group of plant polyphenols attracts major interest because of their potential antioxidant properties presumably based on their function of natural free radicals scavengers (Sellappan et al., 2002). Although many studies on the phenolics contents of plant sources have been reported from different countries, data about the relations between their amount and antioxidants activity are still insufficient. In this paper polyphenolic compounds contents and antiradical activity have been quantified in sweet cherry and tart cherry fruits samples grown in Slovakia. Correlations between polyphenolic compounds and total antioxidant activity were evaluated. The objective was to survey also the effect of pure and aqueous organic solvents and extraction times on the yield of polyphenols from sweet and tart cherry extracts. Efficiency of extractions was tested using the following extraction solvents: water, methanol (100 %, 70 %, 50 % v/v), ethanol (70 %, 50 % v/v), acetone (100 %, 70 %, 50 % v/v) and two different time duration of extractions (1h, 8h). 2 MATERIALS AND METHODS Chemicals as Folin-Ciocalteau assay, gallic acid were purchased from Merck (Germany). Sodium carbonate, methanol, ethanol were obtained from Sigma (USA) and 2,2-diphenyl-1-picrylhydrazyl radical from Organics (USA). Preparation of the samples Sweet and tart cherry fruits (unknown cultivars) were obtained from local market in mature stage in June 2009. The fruits were selected, pitted and processed for laboratory analyses. Organic solvents: methanol (100 %, 70 % and 50 % v/v), ethanol (70 %, 50 % v/v), acetone (100 %, 70 %, 50 % v/v) and water were used for extraction of polyphenols. At first, fresh samples (0.5 g) with 50 mL of solvents were shaken at room temperature for 1 hour. Then it was filtered through filter paper (Filtrak No. 392). Afterwards obtained extracts were analyzed. Another part of mixed samples were lyophilised and gained material (0.5 g) was extracted in the Twisselmann apparatus with organic solvents (100 mL) for 8 hours. Determination of the antioxidant activity (TAA) The antioxidant activity of sweet and tart cherry extracts was measured in terms of radicalscavenging ability, using the DPPH method (Brand-Williams et al., 1995). The stock solution was prepared by dissolving DPPH with methanol, and then the working solution was obtained by mixing 10 mL stock solution with 90 mL methanol. Fruit extracts (100 ^L) were allowed to react with DPPH solution. The decrease in absorbance of DPPH at 515 nm was measured in intervals until the absorbance stabilised (10 min.). Results were expressed as the percentage of inhibition of free radical by present antioxidants. Determination of total polyphenol content (TPC) The total polyphenol content was assessed by the method of Lachman et al. (2003) employing the reduction of a phosphowolframate-phosphomolybdate complex to blue products by phenolic compounds. Briefly, an aliquot of the extract, blank or standard was placed in a 50 mL flask, where the Folin-Ciocalteu assay (2.5 ml) was added and the mixture was allowed to react for 3 min under continuous stirring before a solution of sodium carbonate (7.5 mL) was added and mixed thoroughly. The volume was then made up to 50 ml with distilled water and left standing at room temperature for 2 h. The absorbance was measured at 765 nm using Shimadzu UV-1800 spectrophotometer (Japan). Results were expressed as mg gallic acid equivalents (GAE) per 100 g fresh matter (or dry weight when calculating results of TPC of lyophilised tart cherries). Statistics Data was analyzed with the programme Statistica (Statistica 6.0 CZ). All analyses were done in triplicate, and expressed as average values ± standard deviation. Results were processed by using a one-way analysis of variance (ANOVA). Significance was determined at p < 0.01 using t-test for correlating samples. 3 RESULTS Analyses for surveying the effect of different solvents on the yield of extracted polyphenols from cherry fruits were carried out and evaluated. The solubility of polyphenols was highest in sweet cherry extracts with using of 50 % methanol and 70 % ethanol, followed by 100 % methanol ~ 70 % methanol, 50 % acetone, 50 % ethanol, water, 70 % acetone and 100 % acetone. It seems that 50 % and 70 % solvents (except for ethanol) gave higher phenol yields than pure solvents or water (Figure 1). On the contrary, the highest content values of phenolics were in tart cherries extracts when using of 50 % acetone, followed by 70 % acetone, 50 % methanol, 100 % methanol, 70 % methanol, water, 50 % ethanol, 70 % ethanol and 100 % acetone. Our results showed that 50 % methanol as extracting solution is the most efficient solvent by sweet and tart cherry extracts. This solvent represents better experimental conditions for polyphenol analysis of tested fruits. Interesting results were obtained by the extraction of tart cherry fruits samples with aqueous acetone which provided the highest yield of total phenolics. We suppose this is due to possible extraction of tannins that are responsible for a bitter taste of some fruits (mainly of tart cherries in this case) and which are soluble in acetone. The total polyphenol contents (TPC) of tart cherries extracts were higher than those of sweet cherry ones and ranged from 70.7±8.46 mg gallic acid equivalents (GAE)/100 g FW to 241.4±7.26 mg GAE/100 g FW. Marinova et al. (2005) reported higher total polyphenols for tart cherries (429.5 mg GAE/100 g FW) as compared to the samples analysed in the present study. TPC of sweet cherry extracts were in range from 27.8±9.74 mg GAE/100 g FW to 105.8±10.5 mg GAE/100 g FW. Marinova et al. (2005) assessed similar TPC in sweet cherries extracts (78.8 mg GAE/100 g FW). Literature differs in TCP of sweet cherry extracts, from 23 mg GAE/100 g FW to 168 mg/100 g FW (Vangdal and Slimestad, 2006), those reported by Usenik et al. (2008) ranged from 44.3 to 87.9 mg GAE/100 g FW, while Kim et al. (2005) reported the average of four sweet cherry cultivars 110 mg GAE/100 g FW. There could be some discrepancies when comparing foreign study works with ours due to various extraction solvents as well as various extraction conditions. There could be also many other factors influencing results, because the presence of polyphenols in various plant foods is greatly determined by genetic factors, environmental conditions, degree of ripeness, variety, etc. (Bravo, 1998). o o £ 300 250 200 150 100 50 0 fi I i 100% 70% 50% Water Methanol 100% 70% 50% 70% 50% 100% 70% Ethanol tit 100% 70% 50% Acetone □ Sweet cherries □ Tart cherries Figure 1: Comparison of total phenolics contents (TPC) in sweet and tart cherry extracts with different solvents. Tart cherry extracts (methanolic and water-acetone mixtures) contained in average 2-times higher amount of polyphenols than ethanol extracts (Figure 1). Water appeared to be better solvent for phenolics yield of tart cherry extracts in comparison to sweet cherry extracts. The best solubility of phenolic compounds was achieved in tart cherry extracts with 50 % acetone and in sweet cherry extracts with 50 % methanol. Literature has also provided data about antioxidant profile in both fresh and lyophilised material. For this reason we decided to lyophilise tart cherry fruits and then to extract them by polar solvents (100 %, 70 %, 50 % methanol and 70 %, 50 % ethanol for 8 hours), because of the lack of sweet cherry fruits. Fresh material was also extracted for 1 hour Results were evaluated and compared with those from lyophilised tart cherries samples (Table 1). Table 1: Polyphenol contents (mg gallic acid equivalent/100 g FW) in extracts of tart cherries with used polar solvents. Extraction medium Extraction time/temperature TPC in extracts of tart cherries, SD Methanol 100 % (v/v) 1 hour/room temp. 196.7±4.21 8 hours/room temp. 165.3 ±2.03 Methanol 70 % (v/v) 1 hour/room temp. 190.6±3.65 8 hours/room temp. 134.9±0.25 Methanol 50 % (v/v) 1 hour/room temp. 200.5±2.09 8 hours/room temp. 166.4±5.21 Ethanol 70 % (v/v) 1 hour/room temp. 93.7±1.76 8 hours/room temp. 46.3±2.45 Ethanol 50 % (v/v) 1 hour/room temp. 104.3±5.67 8 hours/room temp. 83.3±4.23 Values presented are means ± standard deviation (SD) of triplicate analysis of each duplicate extraction. When considering polar solvents and the yield of total phenolics in tart and sweet cherry extracts, methanol was obviously more efficient than ethanol in extracts from fresh and from lyophilised material. Because of higher content of polyphenols in extracts of fresh material compared to lyophilised material, we can sum up that the assessment of the polyphenols in fresh material using 50 % methanol is sufficient. The same results were mentioned by Timoracka et al. (2009) who determined similar results in redcurrants and raspberries. Total phenolics from tart cherry extracts were higher after 1 hour extraction using 50 % methanol and 50 % ethanol, compared to extracts of lyophilised tart cherries. Antioxidative capacity is an important fruit quality parameter. The DPPH radical scavenging assay is commonly employed to evaluate the ability of antioxidant to scavenge free radicals. Figure 2: Values of total antioxidant activity (TAA) in extracts of sweet and tart cherry fruits with different solvents 12.0000 10.0000 8.0000 % 6.0000 4.0000 2.0000 0.0000 iir . 70 ifl Jft % 50% \m Water 100% 70% 50% Methanol 4ft % 100% 70% ft 50% I Sweet cherries □ Tart cherries 70% 50% 100% 70% 50% Ethanol Acetone The DPPH antiradical efficiency values of tart cherry extracts were higher when compared to sweet cherries (Fig. 2). Tannins released by acetone in tart cherry extracts also exert antiradical activity and it is obviously reflected in the inhibition of DPPH radical. Values of total antioxidant activity (TAA) of tart cherry extracts ranged from 5.4 to 9.9 % of inhibition of DPPH. Values of TAA were relatively high in tart cherries extracts with aqueous acetone and in sweet cherries extracts with pure methanol and pure acetone. The lowest inhibition of DPPH radical was determined in sweet cherry extracts using water and in tart cherry extracts using 100 % acetone. The relation between TPC and TAA was studied. The dependence of variables (TPC and TAA in sweet cherry extracts using 50 % acetone was highly significant at P<0.05. There was an inverse correlation between TPC and TAA in sweet cherry extracts (r=-0.7233) using 100 % of acetone. Antioxidant activity in the case of sweet cherry extracts using 70 % ethanol depends on phenolics. Analyzed TPC and TAA showed strong dependence in sweet cherry extracts using 70 % methanol and in tart cherry extracts using 50 % methanol. Contrary results were gained for tart cherry extracts, where positive correlation was found for 100 % acetone. Weak correlations were found between TPC and TAA in sweet and tart cherry fruit extracts using 100 % methanol. Figure 3: Relation of total phenolics (TPC) and total antioxidant activity (TAA) of sweet cherries extracts using 70 % of ethanol Correlation: r = 0,81250 7.4 7.2 7.0 6.8 6.6 6.4 6.2 6.0 5.8 5.6 2100 TPC in mg/100 g 2200 2300 2400 95% confidence interval Figure 4: Relation of total phenolics contents (TPC) and total antioxidant activity (TAA) of tart cherry extracts using 50 % methanol Positive correlation of variables (TCP and TAA) in sweet cherry extract using 70 % ethanol was evaluated at P < 0.05 (Fig. 3). From the correlation analysis, it can be summarized that total phenolic content had positive correlation with antioxidant capacity in extracts of tart cherries using 50 % methanol (Fig. 4). 4 DISCUSSION AND CONCLUSION The highest extraction yields of phenolics were determined using 50 % methanol in sweet cherry extracts. The most efficient extraction solvent in tart cherry extracts was 50 % acetone. In our research, 100 % acetone has proved to be the least effective solvent for the gained yield of phenolics of both cherry extracts. Aqueous 70 % acetone provided higher yield of polyphenols in tart cherry extracts in comparison to pure acetone, in spite that Rodtjer et al. (2006) reported that quantification of the total amount of phenolics in the extracts showed that 70 % solvent-water mixtures extracted phenolics more efficiently than pure solvent extracts did. But, in our paper it is obvious that 50 % solvent-water mixtures gave higher extraction yield than pure solvents. The solubility of TPC of both tested cherry fruit extracts was higher with 100 % methanol in comparison to 100 % acetone. There were significant differences of TPC in both stone fruit extracts when evaluating the content values of one organic solvent, so we can sum up that the phenol contents of the extracts were dependent on the solvent used. Also Turkmen et al. (2006) have reported that the phenolics contents of extracts are strongly dependent on the type of the solvent as well as on different concentrations of solvent. Our results showed that tart cherry extracts have substantial amounts of total phenolics which were higher than those of sweet cherry ones, what had reflected on the strong antiradical activity. This fruit can be considered in fresh state as good source of dietary phenols. Despite the TPC in extracts of lyophilised tart cherries samples were lower than those of extracts form fresh material, using of 50 % methanol as extraction solvent for lyophilised tart cherry samples showed good extraction ability. Total antioxidant activity (TAA) of sweet cherry extracts (with used 70 % ethanol and with 70 % methanol) and of tart cherry extracts (with 50 % methanol) depended on phenolics content. This shows that both tested stone fruits are source of natural antioxidants. However, further studies about relation of antioxidant activity and phenolics content are necessary also for other fruits. 5 ACKNOWLEDGEMENT This research work was supported by KEGA 3/5081/07, VEGA 1/0030/09 and APVV SK-SI-0008-08. 6 REFERENCES Brand-Williams, W., Cuvelier, M.E. and Berset, C. 1995. Use of a free radical method to evaluate antioxidant activity, Lebensmittel-Wissenschaft und Technologie 28: 25-30. Bravo, L. 1998. Polyphenols: chemistry, dietary sources, metabolism and nutritional significance. Nutrition Reviews 56: 317-333. Gao, L. and Mazza, G., 1995. Characterization, quantitation and distribution of anthocyanins and colorless phenolics in sweet cherries. Journal of Agricultural and Food Chemistry 43: 343-346. He, F., Nowson, C., Lucas, M. and Macgregor, G. 2007. Increased consumption of fruit and vegetables is related to a reduced risk of coronary heart disease: Metaanalysis of cohort studies. Journal of Human Hypertension 09509240/07: 1-12. Kalyoncu, I.M., Ersoy, N. and Yilmaz, M. 2009. Some physico-chemical properties and mineral contents of sweet cherry (Prunus avium L.) type grown in Konya. African Journal of Biotechnology 8: 2744-2749. Kang, S.Y., Seeram, N.P., Nair, M.G. and Bourquim, L.D., 2003. Tart cherry anthocyanins inhibit tumor development in APC min mice and reduce proliferation of human colon cancer cells. Cancer Letters 194: 13-19. Kim, D.O., Heo, H.J., Kim, Y.J., Yang, H.S. and Lee, C.Y. 2005. Sweet and sour cherry phenolics and their protective effects on neuronal cells. Journal of Agricultural and Food Chemistry, 53, 26: 9921-9927. Lachman, J., Hejtmankova, A. and Dudjak, E. 2003. Content polyphenolic antioxidants and phenolcarboxylic acids in selected parts of yacon. In: Proceedings of Vitamins 2003 - Prirodni antioxidanty a volne radikaly. Univerzita Pardubice, 89-97 p. Marinova, D., Ribarova, F. and Atanassova M. 2005. Total phenolics and total flavonoids in Bulgarian fruits and vegetables. Journal of the University of Chemical Technology and Metallurgy 40, 3: 255-260. Piccolella, S., Fiorentino, A., Pacifico, S., D'Abrosca, B., Uzzo, P. and Monaco, P. 2008. Antioxidant properties of sour cherries (Prunus cerasus L): Role of colorless phytochemicals from the methanolic extract of ripe fruits. Journal of Agricultural and Food Chemistry 56: 19281935. Redtjer, A., Skibsted, L.H. and Andersen, M.L. 2006. Antioxidative and prooxidative effect of extracts made from cherry liqueur pomace. Food Chemistry 99:6-14. Ruxton, C., Gardner, E. and Walker, D. 2006. Can pure fruit and vegetable juices protect against cancer and cardiovascular disease too? A review of the evidence. International Journal of Food Science and Nutrition 57: 249-272. Seeram N.P, Momin R.A, Nair M.G. and Bourquin L.D. 2001. Cyclooxygenase inhibitory and antioxidant cyanidin glycosides in cherries and berries. Phytomedicine 8(5): 362-369. Sellapan, S., Akoh, C. and Krewer, G. 2002. Phenolic Compounds and Antioxidant Capacity of Terbia-Grown Blueberries and Blackberries. Journal of Agricultural and Food Chemistry 50: 2432-2438. Serrano, M., Guillen, F., Martinez-Romero, D., Castillo, S. and Valero, D. 2005. Chemical constituents and antioxidant activity of sweet cherry at different ripening stages. Journal of Agricultural and Food Chemistry 53: 2741-2745. Seymour E., Lewis S., Urcuyo-Llanes, D. and Bolling S. 2008. The effect of tart cherry enriched diets on abdominal fat gene expression in rats. Journal of American Diet Association 108(9): A14 - A14. Timoracka, M., Melichacova, S. and Cery, J. 2009. Determination of antioxidants in small fruit - comparison of methods. Acta fytotechnica et zootechnica (Special issue of Slovaca Universitas Agriculturae Nitriae) 1: 652660. Turkmen, N., Sari, F. and Velioglu, Y.S. 2006. Effect of extraction solvents on concentration and antioxidant activity of black and black mate polyphenols determined by ferrous tartrate and Folin-Ciocalteu methods. Food Chemistry 99: 838-841. Usenik, V., Fabcic, J. and Stampar, F., 2008. Sugars, organics acids, phenolic composition and antioxidant activity of sweet cherry (Prunus avium L.). Food Chemistry 107: 185-192. Vangdal, E. and Slimestad, R. 2006. Methods to determine antioxidative capacity in fruit. Journal of Fruit and Ornamental Plant Research 14: 123-131. Wang H., Nair, M.G., Straburg, G.M., Booren, A.M. and Gray, J.I. 1999. Antioxidant polyphenols from tart cherries (Prunus cerasus). Journal of Agricultural and Food Chemistry 47: 840-844. DOI: 10.2478/v10014-010-0004-2 COBISS Code 1.01 Agrovoc descriptors: glycine max, soybeans, selenium, foliar application, fluorescence, chlorophylls, nutrient transport, respiration Agris category code: F62 Selenium induced lower respiratory potential in Glycine max (L.) Merr. Špela MECHORA1, Mateja GERM2 Received Nov. 24, 2009; accepted Jan. 18, 2010. Delo je prispelo 24. novembra 2009, sprejeto 18. januarja 2010. ABSTRACT Effect of selenium (Se) was studied in soybean (Glycine max (L.) Merr.) cv. Olna in Ljubljana, Slovenia. Se was added to plants as foliary spraying. Quantum yield of photosystem II (PSII) and respiratory potential measured as ETS activity of mitochondria were measured two times in the growing season. Respiratory potential was higher in young plants compared to mature plants. Se induced the lowering of respiratory potential. Addition of Se had no effect on quantum yield of photosystem II. Key words: chlorophyll a fluorescence, electron transport system activity, Glycine max, selenium IZVLEČEK VPLIV SELENA NA NIŽANJE DIHALNEGA POTENCIALA PRI SOJI (Glycine max (L.) Merr.) Preučevali smo vpliv selena na fiziološke lastnosti soje (Glycine max (L.) Merr.) cv. Olna. Rastline so bile foliarno gnojene s selenom. Meritve fotosintezne učinkovitosti in respiratornega potenciala so bile opravljene dvakrat v rastni sezoni. Respiratorni potencial, merjen s pomočjo aktivnosti terminalnega elektronskega sistema je bil višji pri mladih rastlinah. Respiratorni potencial je bil nižji pri rastlinah, foliarno gnojenih s selenom v primerjavi s kontrolo. Fotosintezna učinkovitost ni bila odvisna od obravnavanja s selenom. Ključne besede: aktivnost elektronskega transportnega sistema, Glycine max, fluorescenca klorofila a, selen Abbreviations - Okrajšave: ETS - electron transport system; F0 - minimal chlorophyll a fluorescence yield in dark adapted samples; Fm - maximal chlorophyll a fluorescence yield in dark adapted samples; Fm' - the maximal fluorescence of an illuminated sample; Fv - variable fluorescence; INT - iodo-nitro-tetrazolium-chloride; PPFD - photo synthetic photon flux density; PSII - photosystem II. 1 INTRODUCTION Selenium (Se) is a naturally occurring trace element found in the Earth's crust, soils and minerals (Simmons and Wallschlager, 2005). It is an essential trace nutrient important to humans and animals, whose deficiency and toxic concentrations are very close to each other (Navarro-Alacron and Cabrera-Vique, 2008). Se has been recognized as an integral component of different enzymes, such as glutathione peroxidase and thioredoxin reductase (Birringer et al., 2002). Chemically it is similar to sulphur (S), leading to nonspecific replacement of S by Se in proteins and other sulphur components (Nowak et al., 2004). This results in non-functional proteins and enzymes. In plants, Se functions as an antioxidant (Hartikainen, 2000). Plants take up Se from the soil primarily as selenate (SeO42-) or selenite (SeO32-) (Ellis and Salt, 2003). Se can increase the tolerance of plants to UV-induced oxidative stress, delay senescence and promote the growth of ageing seedlings (Xue et al., 2001). 1 Department of Biology, Biotechnical Faculty, Večna pot 111, SI-1000 Ljubljana, Slovenia, e-mail: spela.mechora@bf.uni-lj.si 2 Department of Biology, Biotechnical Faculty, Večna pot 111, SI-1000 Ljubljana, Slovenia In Se rich areas plants that accumulate large amounts of Se are found. They are called accumulators. Actively growing tissues usually contain the largest amounts of Se (Kahakachchi et al., 2004). Astragalus bisulcatus is reported to be the best Se accumulator. However, when absorbed in higher concentrations, Se can be harmful and catalyse the oxidation of thiols and simultaneously generate superoxide (O2-), which means it acts as a prooxidant (Stewart et al., 1999). The availability of Se for plants depends on soil properties including pH, salinity and the content of CaCO3 (Kabata Pendias, 2001). Temperature is also important factor affecting Se availability. In soils low with Se, plants absorb more Se at temperature higher than 20 °C. Plants from arid regions have more Se in their tissues as those from wet regions. Se content of soils ranges from deficit quantities of 0.01 mg kg-1 at the Russian Plane to heavily toxic values of 1200 mg kg-1 in organic soils at Meath, Ireland (Nowak et al., 2004). Slovenian soils are poor with Se (Kreft et al., 2002). In acid soils Se is mainly present in form of selenite, which has very low solubility and plant aviability. In alkaline soils, Se is oxidized to selenate, which is more soluble and more available for uptake (Navarro-Alacron and Cabrera-Vique, 2008). Se levels in soil generally reflect its presence in food and Se levels consumed by human populations. Deficiency of Se can cause a heart disease, hypothyroidism and a weakened immune system (Ellis and Salt, 2003). Kahakachchi et al. (2004) reported that Se has a cancer chemopreventive properties for humans. The daily intake of Se depends on its concentration level in food and on the amount of food consumed. The recommended intake is 80 ^g/day for men and 55 ^g/day for women (National Research Council, 2000). Plants, which accumulate Se, may be useful as a "Se-delivery system" to supplement the mammalian diet in many areas that are deficient in Se (Terry et al., 2000). Seed soaking or spraying of plants with Se solution may enrich the utilisable plant parts with Se compounds in concentrations of nutritional importance (Germ et al., 2007). Glycine max (L.) Merr. (soybean) is one of the most important crops. It is one of the most important vegetables for dietary purposes. It is a member of the pea family (Fabaceae). Soybean plants height is from 20 cm to 2 m. Steam, leaves and fruit are covered with hairs, which protect plants against drought. The leaves are trifoliolate. The fruit grows in clusters. Seeds contain very high levels of protein, including all essential amino acids (Nenadic, 1985). The general metabolic activity of individual organisms can be assessed from terminal electron transport system (ETS) activity in mitochondria. Under stress condition organisms increase their need for energy. The ability to cope with stress in vital plants is therefore related to respiratory potential (ETS activity) of certain tissue. When stress is too big, the antioxidant metabolism is defeated. Strong stress causes reduced vitality of tissue, that reflects in lower respiratory potential (Germ and Gaberscik, 2003). Lower photosynthetic activity could be a consequence of low photochemical efficiency of PSII, as shown by its lower quantum yield (Pieters and El Souki, 2005). Photochemical quantum yield of PSII is a measure of stress in plants. Any deviation from the maximum quantum yield may be considered as an integrative measure of stress to which a sample is exposed in a given situation (Schreiber et al., 1995). The aim of present work was to study the influence of Se on the respiratory potential and quantum yield of photosystem II (PSII) in soybean. 2 MATERIALS AND METHODS Plant Material and Growth Conditions Soybean (Glycine max (L.) Merr.) cv. Olna plants were grown outdoors in Ljubljana, Slovenia (320 m above sea level, 46°35' N, 14°55' E), in a Se-poor soil. Plants emerged on May 21, 2005 and were foliary sprayed with an aqueous solution containing 10 mg Se/L in the form of Na selenate on June 5. Fluorescence and ETS measurements were performed on 13th of June and 20th of July, 1st and 26th of July 2005, respectively. ETS activity The respiratory potential of mitochondria was measured in plants via terminal electron transport system (ETS) activity as described by Packard (1971) and Kenner and Ahmed (1975). Plant material was homogenised in an ice-cold homogenisation buffer and sonicated with an ultrasound homogeniser (40W, 4710, Cole-Parmer, Vernon Hills, IL, USA). The homogenate was than centrifuged (8500*g, 4 min, 0 °C) in a top refrigerated ultracentrifuge. 1.5 mL of substrate solution and 0.5 mL of iodo-nitro-tetrazolium-chloride (INT) were added to triplicates of the supernatant (0.5 mL) and incubated at 20 °C for 40 min. During the incubation, INT instead of oxygen was reduced to formazan. After stopping the reaction with a stopping solution (formaldehyde and phosphoric acid, 1:1), the formazan absorption at 490 nm was determined. ETS activity was measured as the rate of tetrazolium dye reduction, and converted to equivalent oxygen as described by Kenner and Ahmed (1975). Photochemical efficiency Chlorophyll fluorescence was measured in situ using a fluorometer (OS-500, Opti-Sciences, Tyngsboro, MA, USA). The potential quantum yield was evaluated in terms of the ratio Fv/Fm. Measurement of minimal (F0) and maximal (Fm) chlorophyll fluorescence were made after 15 min of darkness, provided by dark-adaptation clips. Fluorescence was excited with a saturating beam of "white light" (PPFD = 8 000 ^mol m-2 s-1, 0.8 s). With a saturating pulse of "white light" (PPFD = 9 000 ^mol m-2 s-1, 0.8 s) the effective quantum yield was determined. The yield coefficient was defined as Y = (Fm' -F0')/ Fm', where Fm' is the maximum and F0' the minimum fluorescence of an irradiated sample (Schreiber et al., 1995). Statistical Analysis The data were evaluated by ANOVA (Statgraphics Version 4) and significance accepted at p < 0.05. 3 RESULTS The effects of Se were studied in soybean plants. Se exerted no effect on potential or effective photochemical quantum yield of PSII (Table 1). The values of potential as well as effective photochemical efficiency were very similar comparing control and Se treated plants. ANOVA test showed no statistical significant difference between control and Se treated plants at the 95 % confidence level. Values of Fv/Fm were close to 0.8, which indicates the plant vitality. ETS activity of untreated and treated plants was higher in the first measurement, comparing to the second measurement. Foliar treatment with Se had an effect on the ETS activity in soybean plants (Figure 1). Compared to the untreated plants, treated plants had lower ETS activity. ANOVA test revealed statistical significant difference between control and Se treated plants at the 95 % confidence level. Table 1. Potential - Fv/Fm and effective - AF/Fm' photochemical quantum yield of PSII on 13th of June and on 20th of July 2005. Se0 - control, without added Se, Se1 - with added Se. Means±SD (n=5-14). 13.6. 20.7. Fv/Fm Se0 0.73±0.03 0.69±0.07 Se1 0.75±0.03 0.66±0.05 AF/Fm' Se0 0.28±0.06 0.42±0.08 Se1 0.22±0.07 0.42±0.07 Figure 1. Terminal electron transport system (ETS) activity on 1st (left panel) and on 26th (right panel) of July 2005. White - control, without added Se, gray - with added Se. Means±SD (n=4). 4 DISCUSSION Selenium exerted no influence on potential (Fv/Fm) or effective (AF/Fm') photochemical quantum yield of PSII (Table 1). For the purpose of comparison we made a table of Se impact taken from other studies (Table 2). It comprises the impact of selenium and its species on physiological parameters in leaves of different plants. Our results were in agreement with results obtained for common buckwheat (Tadina et al., 2007), chicory (Germ et al., 2007) and Cucurbita pepo (Germ et al., 2005), as shown in this table (Table 2). Values of Fv/Fm were close to 0.8, which indicate an undamaged antenna complex (Bischof et al., 1998). The effective quantum yield was lower than the potential quantum yield of PSII. Germ and co-workers (2007) report, that the values of potential photochemical efficiency close to the theoretical maximum indicate reversible disturbance rather than damage to the reaction centre. Terminal electron transport system activity was higher at the beginning of the growth period, because plants needed more energy during intensive growth in order to develop essential structural components (Figure 1). Smillie (1962) reports the decreasing of respiratory rates of pea leaves during leaf development. Author suggests that this may be a result of decreasing ability of the cells to use fully their potential enzymic capacity, or it may be the direct consequence of a decrease in the respiratory enzymic capacity of the leaf. Higher ETS activity in young plants comparing to mature plants were also reported for Fagopyrum esculentum and F. tataricum (Breznik et al., 2005; Tadina et al., 2007), for Pisum sativum (Smrkolj et al., 2006), and for Potamogeton crispus (Mazej and Gaberscik, 1999). ETS activity is a measure of the metabolic potential of organisms and vary among plant species. ETS activity was lower in plants, treated with Se in both measurements (Figure 1). The opposite results are given by Germ and Osvald (2005) for Eruca sativa. ETS activity was higher in Se-treated plants as well in some other plants (Table 2). Increased ETS activity indicates that plants are under stress. When the stress is too strong and plants cannot cope with it any more, the respiratory potential drops (Ozbolt et al., 2008), as shown in Figure 1. However, potential photochemical efficiency of PSII in soybean was close to 0.8 in Se treated and untreated plants. This indicated that Se negatively affected respiratory chain but not photosynthetic processes. Table 2. Literature data on impact of selenium and its species on physiological parameters in leaves of different plants. Plant Se concentration Way of application of Se Parameter Effects Se Literature Fagopyrum 1 g Se Foliar spray Fv/Fm - Breznik et al., esculentum F. tataricum AF/Fm' î 2005 Cichorium intybus 1 mg Se/L Foliar spray 2x Fv/Fm - Germ et al., AF/Fm' - 2007 ETS î Fagopyrum esculentum 10 mg Se/L Soaked seeds ETS 4 Germ, 2006 Cucurbita pepo 1.5 mg Se/L Foliar spray ETS 4 Germ, 2005 Fagopyrum 1g Se Foliar spray Fv/Fm - Tadina et al., esculentum AF/Fm' - 2007 ETS 4 Pisum sativum 10 mg Se Foliar spray 1x, 2x ETS î(2x) Smrkolj et al., 2006 Cucurbita pepo 1.5 mg Se/L Foliar spray Fv/Fm - Germ et al., AF/Fm' - 2005 ETS î Eruca sativa 10 mg Se/L Soaked seeds Fv/Fm î Germ and AF/Fm' - Osvald, 2005 ETS î Solanum 10 mg Se/L Foliar spray Fv/Fm î Germ et al., tuberosum AF/Fm' î 2007 ETS î Fragaria x ananassa Hordeum vulgare 0.1 mg Se/L 1 mg Se/L Foliar spray Fv/Fm î Valkama, 2003 Legend: t - increased, [ - decreased, - no effect, 1x - once foliary sprayed, 2x - twice foliary sprayed. 5 ACKNOWLEDGEMENT This research was financed by the Ministry of Higher Education, Science and Technology of the Republic of Slovenia through the programs "Biology of plants" (P1- 0212), "Young researchers" (32059) and the project (J4-2041). 6 REFERENCES Birringer M., Pilawa S., Flohc L. 2002. Trends in selenium biochemistry. Nat. Prod. Rep. 19: 693-718. Bischof K., Hanelt D., Wiencke C. 1998. UV-radiation can affect depth-zonation of Antarctic macroalgae. Mar. Biol. Berlin 131: 597-605. Breznik B., Germ M., Gaberscik A., Kreft I. 2005. Combined effects of elevated UV-B radiation and the addition of selenium on common (Fagopyrum esculentum Moench) and tartary (Fagopyrum tataricum (L.) Gaertn.) buckwheat. Photosyntetica 43-4: 583-589. Ellis D.R., Salt D.E. 2003. Plants, selenium and human health. Curr. Opin. Plant Biol. 6: 273-279. Germ M., Gaberscik A. 2003. Dihalni potencial - kazalnik stresa pri rastlinah. Zb. Bioteh. Fak. Univ. Ljublj. Kmet. 81-2: 335-339. Germ M., Osvald J. 2005. Selenium treatment affected respiratory potential in Eruca sativa. Acta agriculturae Slovenica 85-2: 329 - 335. Germ M. 2005. Combined impact of solar UV-B radiation and selenium treatment on respiratory potential in pumpkins (Cucurbita pepo L.). Acta agriculturae Slovenica 85-2: 337-342. Germ M., Kreft I., Osvald J. 2005. Influence of UV-B exclusion and selenium treatment on photochemical efficiency of photosystem II, yield and respiratory potential in pumpkins (Cucurbita pepo L.). Plant physiol. Biochem. 43: 445-448. Germ M. 2006. The effect of UV-B radiation and selenium on respiratory potential in common buckwheat (Fagopyrum esculentum). Fagopyrum 23: 91-93. Germ M., Stibilj V., Osvald J., Kreft I. 2007. Effects of selenium foliar application on chicory (Cichorium intybus L.). J. of Agric. Food Chem. 55: 795-798. Germ M., Kreft I., Stibilj S., Urbanc-Bercic O. 2007. Combined effects of selenium and drought on photosynthesis and mitochondrial respiration in potato. Plant Physiol. Biochem. 45: 162-167. Germ M. 2008. The response of two potato cultivars on combined effects of selenium and drought. Acta agriculturae Slovenica 91-1: 121-137. Germ M., Stibilj V., Kreft S., Gaberscik A., Pajk F., Kreft I. 2009. Selenium concentration in St. John's wort (Hypericum perforatum L.) herb after foliar spraying of young plants under different UV-B radiation levels. Food Chem. 117: 204-206. Hartikainen H., Xue T., Piironen V. 2000. Selenium as an antioxidant and prooxidant in ryegrass. Plant. Soil. 225: 193-200. Kabata Pendias A. 2001. Trace elements in soils and plants, 3rd edn. Boca Raton, FL, CRC Press: 241-252. Kahakachchi C., Boakye H.T., Uden P.C., Tyson J.F. 2004. Chromatographic speciation of anionic and neutral selenium compounds in Se-accumulating Brasica juncea (Indian mustard) and in selenized yeast. Chromatogr. A 1054: 303-312. Kenner R.A., Ahmed S.I. 1975. Measurements of electron transport activities in marine phytoplankton. Mar. Biol. 33: 119-127. Kreft I., Stibilj V., Trkov Z. 2002. Iodine and selenium content in punpkin (Cucurbita pepo L.) oil and oil-cake. Eur. Food Res. Technol., 215: 279-281. Mazej Z., Gaberščik A. 1999. ETS-activity as a measure of vitality of different macrophyte species. 2nd Slovenian Symposium on Plant Physiology with international participation / issue editors Vilhar B., Grill D., Guttenberger H. Apud Ferdinandum Berger & Fil. 39-3: 181-185. National Research Council, Recommended Dietary Allowances, National Academy Press, Washington, DC, 2000. Navarro-Alarcon M., Cabrera-Vique C. 2008. Selenium in food and the human body: A review. Sci. Tot. Environ. 400: 115-141. Nenadic N. 1985. Soja. Ljubljana, ČZP Kmečki glas. Nowak J., Kaklewski K., Ligocki M. 2004. Influence of selenium on oxidoreductive enzymes activity in soil and in plants. Soil Biol. Biochem., 36: 1553-1558. Ožbolt L., Kreft S., Kreft I., Germ M., Stibilj V. 2008. Distribution of selenium and phenolics in buckwheat plants grown from seeds soaked in Se solution and under different levels of UV-B radiation. Food chem. 3-110: 691-696. Packard T.T. 1971. The measurement of respiratory electron-transport activity in marine phytoplankton. J. Mar. Research 29: 235-243. Pieters A.J., El Souki S. 2005. Effects of drought during grain filling on PS II activity in rice. J. Plant Physiol. 62: 903911. Schreiber U., Bilger W., Neubauer C. 1995. Chlorophyll fluorescence as a nonintrusive indicator for rapid assessment of in vivo photosynthesis. - In: Schulze E.D., Caldwell, M.M. (ed.): Ecophysiology of Photosynthesis. pp. 49-70. Springer-Verlag, Berlin - Heidelberg - New York. Simmons D.B.D., Wallschlager D. 2005. A critical review of the biogeochemistry and ecotoxicology of selenium in lotic and lentic environments. Environ. Toxicol Chem. 246: 1331-1343. Smillie R.M. 1962. Photosynthetic and respiratory activities of growing pea leaves. Plant Physiol. 37: 716-721. Smrkolj P., Germ M., Kreft I., Stibilj V. 2006. Respiratory potential and Se compounds in pea (Pisum sativum L.) plants grown from Se-enriched seeds. J. Exp. Bot. 57-1: 3595-3600. Stewart M.S., Spallholz J.E., Neldner K.H., Pence B.C. 1999. Selenium compounds have disparate abilities to impose oxidative stress and induce apoptosis. Free Radic. Biol. Med. 26: 42-48. Tadina N., Germ M., Kreft I., Breznik B., Gaberscik A. 2007. Effects of water deficit and selenium on common buckwheat (Fagopyrum esculentum Moench.) plants. Photosyntetica 45-3: 472-476. Terry N., Zayed A., De Souza M.P., Tarun A.S. 2000. Selenium in higher plants. Annu. Rev. Plant Physiol. Plant Mol. Biol. 51: 401-432. Valkama E., Kivimäenpää M., Hartikainen H., Wulff A. 2003. The combined effects of enhanced UV-B radiation and selenium on growth, chlorophyll fluorescence and ultrastructure in strawberry (Fragaria x ananassa) and barley (Hordeum vulgare) treated in the field. Agric. For. Meteor. 120: 267-278. Xue T.L., Hartikainen H., Piironen V. 2001. Antioxidative and growth-promoting effects of selenium on senescing lettuce. Plant Soil 237: 55-61. COBISS Code 1.01 Agrovoc descriptors: zea mays, maize, hybrids, leaves, leaf area, foliage, harvest index, yield components, crop yield, growth rate, biological development, plant breeding Agris category code: F60, F62, F30 Variation of physiological traits and yield components of some maize hybrid (Zea mays L.) in agroecological conditions of Kosovo Sali ALIU1, Shukri FETAHU1, Ludvik ROZMAN2 Received October 30, 2009; accepted February 24, 2010 Prispelo 30. oktobra 2009; sprejeto 24. februarja 2010. ABSTRACT The aim of this study was to investigate some physiological traits and yield of different maize hybrids in growth conditions of Kosovo. The field experiment was conducted in 2006 and 2007 in Kosovo, near Prishtina. The experiment was based on a randomized complete block design with 3 replications. For calculating and statistical analysis 10 plants per each plot were randomly chosen. In the study, seven commercial maize hybrids belonging to different FAO groups (FAO 300, 400 and 600), originating from two breeding institutions: Maize Dept. of Bc Institute Rugvica - Croatia (Jumbo 48 [H-1], BC418 [H-2], BC408 [H-3], BC288 [H-4], BC394 [H-5]) and from Pioneer Hi-Bred Int. (Austria) (Pregia [H-6] and Colombo [H-7]), were included. For traits ear leaf area (LA), absolute growth rate (AGR), crop growth rate (CGR) and biological dry matter (BDM) higher values were obtained in the 2nd year in comparison to the 1st year, however the harvest index (HI) of the 1st year was of a higher value than the 2nd year. Also, for all investigated traits, except for HI, statistically significant differences were obtained among the studied maize hybrids. The highest values for all traits, except for HI, expressed the H-6 and these values were statistically significant higher than those of all other hybrids, but not for BDM. Our results indicated that the H-6 was the most appropriate for cultivation in agroecological conditions in Kosovo, while the H-4 and H-5 were less appropriate in the given conditions. Keywords: maize, Zea mays, leaf area, absolute growth rate, crop growth rate, harvest index, yield. Abbreviations: LA, ear leaf area; AGR, absolute growth rate; CGR, crop growth rate; BDM, biological dry matter; HI, harvesting index. IZVLEČEK VARIABILNOST FIZIOLOŠKIH LASTNOSTI IN KOMPONENT PRIDELKA NEKATERIH HIBRIDOV KORUZE (Zea mays L.) V AGROEKOLOŠKIH RAZMERAH KOSOVA Namen raziskave je proučiti nekatere fiziološke lastnosti in pridelek različnih koruznih hibridov za pridelovanje v agroekoloških razmerah Kosova. Poljski poskus je bil izveden v letih 2006 in 2007 v Prištini na Kosovu po metodi naključnih blokov v 3 ponovitvah. Za obračunavanje in stastistične analize je bilo iz vsake parcelice naključno odbranih 10 rastlin. V proučevanje je bilo vključenih 7 hibridov različnih zrelostnih razredov (FAO 300, 400 in 600), iz dveh žlahtniteljskih institucij in sicer iz Zavoda za kukuruz Bc Inštituta iz Rugvice - Hrvaška (Jumbo 48 [H-1], BC418 [H-2], BC408 [H-3], BC288 [H-4], BC394 [H-5]) in iz Pioneer Hi-Bred Int. (Austria) (Pregia [H-6] in Colombo [H-7]). Za lastnosti listna površina lista ob storžu (LA), rastna stopnja na rastlino (AGR), rastna stopnja na enoto površine (CGR) in skupno suho snov (BDM) smo v drugem letu dobili višje vrednosti kot v prvem letu; medtem ko so za žetveni indeks (HI) bile višje vrednosti v prvem letu. Za vse proučevane lastnosti, razen za HI, smo ugotovili statistično značilne razlike med hibridi. Za vse lastnosti, razen za HI, je imel najvišje vrednosti H-6, ki so bile statistično značilno višje od vseh drugih hibridov; samo za BDM je H-6 bil statistično značilno enak H-2 in H-3. Rezultati nakazujejo, da je za pridelovanje v rastnih razmerah Kosova najbolj primeren hibrid H-6, medtem ko sta hibrida H-4 in H-5 manj primerna. Ključne besede: koruza, Zea mays, listna površina, rastna stopnja na rastlino, rastna stopnja na enoto površine, žetveni indeks, pridelek. Okrajšave: LA, listna površina; AGR, rastna stopnja na rastlino; CGR, rastna stopnja na enoto površine; BDM, skupna suha snov; HI žetveni indeks. 1 The University of Prishtina, Faculty of Agriculture, Kosovo, e-mail: s_aliu@hotmail.com 2 University of Ljubljana, Biotechnical Faculty, Slovenia, e-mail: ludvik.rozman@bf.uni-lj.si 1 INTRODUCTION Maize (Zea mays L.) is an important field crop and a major component of numerous animal feeds. Based on the area and the production it ranks on the second place among field crops in Kosovo. During 2005-2006, maize was grown on a total area of 60,000-70,000 ha with an average yield of 4.5 t ha-1 (MAFRD, 2008). In Kosovo, there are two major agriculture regions and both have good soil and climatic conditions for maize production. However, yield per hectare is lower when compared to other maize growing countries of the region. Among the main reasons are inappropriate seed material and inappropriate hybrids, limited use of fertilizers and lack of adoption of modern technologies. Maize growth and productivity depends on many factors such as genetic structure, environmental characteristics and production technology. If environment is not optimal (i.e., in areas with marginal rainfall such as <600 mm per year) the production of maize is considered less reliable (Robertson et al., 2003). The success of genetic improvement of maize yield depends on several factors such as initial sources of genes (initial parental material), improvement method, types of the gene actions involved in yield control, inheritance and genetic control of related traits such as capacity of production (Rezaei et al., 2004). Biomass accumulation in kernels begins shortly after fertilisation and can be represented by a sigmoidal pattern in which a lag and a linear growth phase can be distinguished (Duncan et al., 1965). Growth analysis is one approach to the analysis of yield-influencing factors and plant development (Berzsenyi and Lap, 2004). In Kosovo, about 95 % of arable land (cca 66,000 ha) is planted by different types of hybrids. The activities aimed at the development of hybrids in territory of Kosovo started during 1970s, and more intensive work took place during 1980-1987. This work was not very successful and for this reason the research was reestablished after 1999 (Aliu, 2006). The new program considered physiological parameters as crucial in the development of productive lines and hybrids. Physiological parameters, especially the leaf area and related components such as LA, AGR and CGR are crucial factors in photosynthesis. The environmental factors and their interactions with plant characteristics and with cultural practice are also very important (Aliu et al., 2008). The main objective of this study was to determine the most appropriate genotypes regarding to physiological parameters associated with leaf area and yield suitable for agro-ecological conditions of Kosovo. 2 MATERIALS AND METHODS Experimental site The experiment was conducted during 2006-2007, 5 km southwest of the capital Prishtina (570 m a.s.l., 42°38'97'' N latitude, 21°08'45'' E longitude), with an average rainfall of 613.3 mm per year and the mean annual temperature is 10.27 °C (HMIK, 2008). Summer temperatures in this region are sometimes exceeding 35°C resulting in high evapotranspiration. Plant material and experimental design The experimental design was based on a randomized complete block design (RCBD) with three replications. The size of individual plots was 28 m2. The distance between rows was 70 cm, whereas the distance between plants within rows was 30 cm (47,000 plants ha-1). Seven commercial maize hybrids originating from two different institutions and belonging to FAO groups 300, 400 and 600 were included. Five hybrids originated from the Maize Dept. of Bc Institute Rugvica -Croatia (Jumbo 48 [H-1], BC418 [H-2], BC408 [H-3], BC288 [H-4], BC394 [H-5]) and two from the Pioneer Hi-Bred Int. (Austria) (Pregia [H-6] and Colombo [H-7]). Measurements The measurements took place in field conditions and laboratory. They included leaf area (LA), absolute growth rate (AGR), crop growth rate (CGR), biological dry matter (BDM), yield and harvest index (HI). The basic data were obtained from samples of 10 plants, randomly chosen in the middle rows of each plot. Regarding to CGR and AGR up to silking time the maize plants were randomly chosen from middle rows of experimental plots, harvested manually, and cut in small partitions. Plants' weight was measured in grams per plant and determined in laboratory for Plant production in Agriculture Faculty in Prishtina at temperature 60oC for 24 hours. LA was determined according to the formula by Montgomery (1911), A = LxWx0.75, where L is the leaf length, W is the leaf width, 0.75 is the factor of recalculation for maize. The same formula was used by several researchers, e.g., Whigham et al., 1974; Pearce et al., 1975; Aliu et al., 2008. Other parameters were calculated using the formulas by Radford (1967): Wp , , AGR = (g plant-1 day-1), Ds AGR - absolute growth rate, Wp - weight per plant (biomass), Ds - number of days to silking, and Wp CGR = —— x No. ofplants m-2 (g m-2 day"1) Ds CGR - Crop Growth Rate. Yield and yield components For the analysis of yield, data were collected at biological maturity from 10 plants and their corresponding ears from each replication. Data associated with biological yield or biomass were obtained by adding the weight of ears and stalk. Harvest Index (HI), was calculated as a percentage for each plot using the following formula: AGR and CGR were also measured at physiological maturity using formulas: Wp , , AGR = (g plant-1 day4), Dm Dm - number of days to maturity and CGR = Wp Dm x No of plants m"2 (g m"2 day"1). Gy HI = (%), By Gy - grain yield, By - biological yield. Statistical analysis The collected data were analysed by ANOVA considering LSD at p=0.05 and p=0.01 using MINITAB-14 and Microsoft Excel programs. 3 RESULTS Ear leaf area The green ear leaf area (LA) and its duration are important factors associated with photosynthesis, especially for maize. Highly significant differences (p=0.01) were found among the seven maize genotypes (Table 1). The analysis of LA in our study showed that maize genotypes produced larger ear leafs (larger area of ear leaf) in the second year (Y2), than in the first year (Y1). An average minimal value for the two experimental years was obtained in H-4 (540.5 cm2 plant-1), while the highest LA was recorded in H-6 (752.4 cm2 plant-1). Individual comparison of H-4 and H-6 showed that differences were + 211.90 cm2 plant-1. Many researchers have reported different results for LA. According to observation data by Aliu et al. (2008), the total average value for LA was 677.8 cm2 plants-1. Comparing to our study the results differ for -55.2 cm2 plant-1. The coefficient of variation for LA in maize genotypes was 12.9 % respectively. Table 1: Values of ear leaf area (LA) of different maize genotypes included in the study. Hybrid FAO group Y1 Y2 X " Homog. groups* H-1 400 475.5 636.0 555.8 ab x H-2 400 578.0 677.3 627.6 b xy H-3 400 567.9 685.1 626.5 b xy H-4 300 477.5 603.5 540.5 a x H-5 400 562.3 581.0 571.7 abc xy H-6 600 797.0 707.9 752.4 d z H-7 400 642.5 727.9 685.2 cd yz Mean LSD p=0.05 LSD p=0.01 585.8 659.8 622.8 77.8 117.9 * - the same letters indicate the same homogenous groups: - a-d - homogenous groups at p=0.05. - x-z - homogenous groups at p=0.01. 2 Absolute growth rate (AGR) and crop growth rate (CGR) The AGR and CGR are the instruments to measure dry matter accumulation for plant per unit time. Our results indicated the significant differences among the studied maize genotypes also for AGR and CGR (Table 2). The experimental average value of AGR up to silking was 6.62 g plant-1 day-1, while for CGR was 31.04 g m-2 day-1. This is a relatively high value for AGR and CGR. The maize genotypes H-6 produced highest value of AGR (8.58 g plant"1 day"1), and CGR (40.33 g m"2 day"1); both of them were statistically significant higher (at p=0, 05 and at p=0, 01) than other genotypes. Other genotypes had statistically same value for AGR (all genotypes, except H-6, form one homogeneous group), while for CGR all genotypes, except H-6 again, form two homogeneous groups. Tollenaar (1991) and Otegui et al. (1995) emphasized the importance of the total dry matter accumulation and plant growth rate within 10 to 20 days after silking. The total mean value of AGR and of CGR up to silking was higher in the Y2 than in the Y1. The coefficients of variation for AGR and CGR were 14.69 and 14.82 %, respectively. Pearson's coefficient of correlation between AGR and CGR up to silking was highly significant (r=0.99**). The lowest mean values for AGR and CGR expressed the H-4 (5.03 g plant"1 day"1 and 23.62 g m2 day"1, respectively). While the highest mean values for AGR and CGR expressed the H"6 (8.24 g plant"1 day"1 and 38.71 g m"2 day"1, respectively) (Table 3). For both investigated traits (AGR and CGR), the H"6 showed significantly higher values than all other investigated hybrids; while the H"4 with lowest values was significantly equal to one or to six other hybrids, depending on traits and on probability level. As up to silking, the AGR and CGR were also highly correlated up to maturity. About different results of AGR and CGR reported also other researchers. Berzsenyi and Lap (2004), reported different results of AGR (6.97 g plant"1 day"1) as the results of increasing plant density. Late sowings increased CGR during the vegetative period because of high radiation use efficiency and higher percent radiation interception as suggested by Cirilo and Andrade (1994). CGR depends on the amount of intercepted photosynthetically active radiation, where the leaf area index play an important role (Subedi and Ma, 2005). According to data of Buttery (1970) the mean relative growth rate (RGR) and mean net assimilation rate (NAR) increased with higher density, while CGR and leaf area ratio (LAR) decreased. Also Molnarova and Szucs (2009) reported different results (18.38 till 22.05 g m"2 day"1) on CGR at some maize hybrids. Table 2. Values of Absolute Growth Rate (AGR) and Crop Growth Rate (CGR) up to silking of studied maize genotypes. FAO ■ group AGR (g plant"1 day"1) CGR (g m"2 day"1) Hybrid Y1 Y2 X Homog. groups* Y1 Y2 X Homog. groups* H"1 400 5.16 7.73 6.45 a x 24.3 36.34 30.30 ab x H"2 400 6.12 7.51 6.82 a xy 28.8 35.30 32.03 b x H"3 400 5.73 7.59 6.66 a x 26.9 35.68 31.30 ab x H"4 300 4.79 6.68 5.74 a x 22.5 31.40 26.95 ab x H"5 400 4.70 6.57 5.64 a x 22.1 30.80 26.44 a x H"6 600 6.95 10.21 8.58 b y 32.7 47.99 40.33 c y H"7 400 5.85 7.13 6.49 a x 26.4 33.52 29.95 ab x Mean 5.61 7.63 6.62 26.2 35.86 31.04 LSD p=0.05 1.19 5.29 LSD p=0.01 1.80 8.02 * " the same letters indicate the same homogenous groups: ■ a-c - homogenous groups at p=0.05; ■ x-y - homogenous groups at p=0.01. Table 3: Values of Absolute Growth Rate (AGR) and Crop Growth Rate (CGR) up to maturity of studied maize genotypes. FAO group AGR (g plant-1 day-1) CGR (g m-2 day-1) Hybrid Y1 Y2 X Homog. groups* Y1 Y2 X Homog. groups* H-1 400 5.01 7.50 6.26 bc xy 23.55 35.25 29.40 bc x H-2 400 5.99 7.35 6.67 c y 28.16 34.55 31.36 c xy H-3 400 5.75 6.68 6.22 bc xy 27.03 31.40 29.22 abc x H-4 300 4.57 5.48 5.03 a x 21.48 25.76 23.62 a x H-5 400 4.48 6.27 5.38 ab xy 21.06 29.47 25.27 ab x H-6 600 6.67 9.80 8.24 d z 31.35 46.06 38.71 d y H-7 400 5.56 7.10 6.33 bc xy 26.14 33.37 29.76 bc x Mean 5.44 7.17 6.31 25.54 33.70 29.62 LSD p= 0.05 1.00 5.66 LSD p= =0.01 1.51 8.09 * - the same letters indicate the same homogenous groups: - a-d - homogenous groups at p=0.05; - x-z - homogenous groups at p=0.01. Biological Dry matter (BDM), yield and harvest index (HI) The mean values of BDM and yield were significantly different among maize genotypes, but not for HI (Table 4). The maize hybrids H-6 produced significantly higher BDM (1029.16 g plant-1), while the hybrids H-4 realized the lowest value for BDM (635 g plant-1). H-4 was found to be equal to two (p=0, 05) or to all other hybrids, except to H-6, (p=0, 01). The differences between hybrids regarding extreme values were +394.16 g plant-1 (51.01 %). The studied maize genotypes produced significantly more BDM in Y2 (893.1 g plant-1) than in Y1 (652.5 g plant-1), the difference was + 240.6 g plant-1. The coefficient of variation for BDM in maize genotypes was 16.80 %. Morgado and Willey (2008) observed some trend for physiological parameters, and in their study they found that the total biomass yield of maize under different cropping systems and nitrogen levels was from 7887 to 10513 kg BDM per ha-1. Also, Khan et al., (2003) presented different results for biological yield in some maize genotypes from 9.62 to 20.04 t ha-1, while Birch (2004) reported different results of biomass from 11.4 to 28.18 t ha-1. The final grain yield (t ha-1) in maize is considered to be a combined effect of various yield components, like number of ear bearing plants, number of ears per plant, number of grains per ear, weight of grains per ear and 1000 grain weight. The total mean value of yield for all investigated maize hybrids was 8.69 t ha-1 and that was relatively high and could guarantee a high yield. Results of the grain yield exhibited significant differences among the investigated hybrids (Table 4). The H-6 had significantly the highest mean grain yield (9.9 t ha-1), while H-4 the lowest (7.8 t ha-1). The difference between H-6 and H-4 was +2.10 (24.2 %) while distinction between years was +0.96 t ha-1. Table 4. Values of Biological Dry matter (BDM), yield and harvest index (HI) of maize genotypes. FAO group BDM (g plant-1) Yield (t ha-1) HI (%) Hybrid Y1 Y2 X Homog. groups* Y1 Y2 X Homog. groups* Y1 Y2 X Homog. groups* H-1 400 595.3 891.7 743.5 ab x 7.8 9.1 8.5 bc xy 42.5 31.9 37.2 a x H-2 400 706.0 866.3 786.2 cd x 8.2 9.3 8.8 c xy 30.8 36.4 33.6 a x H-3 400 678.3 898.3 788.3 cd x 8.8 9.5 9.2 c yz 38.3 33.3 35.8 a x H-4 300 530.0 740.0 635.0 a x 7.1 8.5 7.8 a x 44.5 35.6 40.1 a x H-5 400 541.6 758.0 649.8 a x 7.4 8.7 8.0 ab x 42.6 35.8 39.2 a x H-6 600 833.3 1225.0 1029.2 d y 9.7 10.1 9.9 d z 36.5 23.2 29.9 a x H-7 400 683.3 873.0 778.1 bc x 8.4 9.1 8.8 c xy 37.0 35.8 36.4 a x Mean 652.5 893.1 772.8 8.2 9.2 8.7 38.9 33.1 36.0 LSD p= 0.05 135.7 0.66 23.0 LSD p= =0.01 205.6 1.01 32.8 * - the same letters indicate the same homogenous groups: - a-d - homogenous groups at p=0.05; - y-z - homogenous groups at p=0.01. Pearson's correlation coefficient between BDM and obtained grain yield from 6.1 to 11.5 t ha-1. The grain yield was strong (r=0.96**). Birch (2004) physiological efficiency of maize plants to convert the total dry matter to grain yield can be estimated by calculating the harvest index (HI). The value of HI varied from 29.87 % (H-6) to 40.07 % (H-4), but no significant differences were found among these hybrids, while rather high difference was found between years (Y1 - 38.90 %, Y2 - 33.14 %). Many researchers have reported different results about HI. HI obtained by Worku and Zelleke (2007) varied from 31.1 % to 45.0 % whereas results by Subedi and Ma (2005) varied from 0.49 to 0.55 %. 4 CONCLUSION The investigated seven maize hybrids differed significantly in all studied parameters, except for HI. The investigated hybrids come from two institutions and involve distinct genetic and morphological backgrounds. However, they were cultivated in same agroecological conditions. H-6 exhibited higher values of all measured traits. The hybrids realized more productivity for parameters AGR, CGR, BDM and yield in the 2nd than in the 1st year, because of more precipitations. However, physiological parameters' analysis include LA, AGR, CGR, HI and provide an excellent opportunity to monitor the independent and interactive effects of different factors affecting maize yields and open the way to managing these factors in plant. 5 REFERENCES Aliu, S. 2006. The study of GCA and SCA for some maize inbred lines in agro-ecological conditions of Kosovo. PhD Thesis, Agriculture University of Tirana, 52. Aliu, S., Fetahu, Sh., Rozman, L., Salillari, A. 2008. General and specific combining ability studies for leaf area in some maize inbreds in agroecological conditions of Kosovo. Acta agriculturae Slovenica. 91: No. 1: 67-73. Berzsenyi, Z., Lap, Q. 2004. Studies on the effect of plant density on maize growth using Richards function. 4th International Crop Science Congress, 513-516. Birch, C.J. 2004. Performance and reliability of two maize simulation models in a range of environments. AUSIMM. The university of Queensland, Australia. 344-349. Buttery, B.R. 1970. Effects of variation in Leaf area index on growth of maize and soybeans. Crop Sci. 10: 9-13. Madison, WI 53711 USA. Cirilo, A.G., Andrade, F.H. 1994. Sowing date and maize productivity. Crop Growth and dry matter partitioning. Crop Sci. 34: 1039-1043. Duncan, W.G., Hatfield, A.L., Ragland, J.L. 1965. The growth and yield of corn. II. Daily growth of corn kernels. Agron. J. 57: 221-223. Francis, C.A., Rutger, J.N., Palmer, A.F.E. 1969. A rapid method for plant leaf area estimation in maize. Crop Sci. 9: 537-539. HMIK. 2008. Hydro-meteorological Institute of Kosovo; Data base for sum of Temperatures and Rainfall. Khan, B.M., Asif, M., Aman, M. 2003. Response of some maize (Zea mays L.) genotypes to different irrigation levels. Int. J. Agri. Biol., 5, No 1, 17-18. MAFRD, 2008. Ministry of Agriculture, Forestry and Rural Department of Kosovo. Molnarova, J., Szucs G. 2009. The maize production potential and yield in dependence from year. 44 Croatian & 4 International symposium on Agriculture, Opatija. 589592. Montgomery, J.Z., Doak, P.B. 1970. Diallel analysis of leaf area and relationships to yield in maize. Crop Sci. 2: 178180. Morgado, L.B., Willey R.W. 2008. Optimum plant population for maize-bean intercropping system in the brazilian semi-arid region. Sci. Agric. 65: 474-480. Otegui, M.E., Andrade, F.H., Suero, E.E. 1995. Growth, water use and kernel abortion of maize subjected to drought at silking. Field Crops Res. 40: 87-94. Pearce, R.B., Mock, J.J., Bailey, T.B. 1975. Rapid method for estimating leaf area per plant in maize. Crop Sci. 15: 691694. Radford, P.J. 1967. Growth analysis formulae - Their use and abuse. Crop Sci. 7: 171-175. Rezaei, A., Yazdisamadi, B., Zali, A., Tallei, A., Zeinali H., Rezaei, A. 2004. Estimate of heterosis and combining ability in maize (Zea mays L.) using diallel crossing method. In Proceedings of the 17th XVII EUCARPIA General Congress: Genetic variation for plant breeding, 8-11 September, Vienna 2004. 395-398. Robertson, M.J., Cawthray S., Birch C.J., Bidstrup R., Crawford M., Dalgleish, N.P., Hammer G.L. 2003. Managing the risk of growing dry land maize in the northern region. In 5th Australian Maize Conference: Versatile Maize, Golden Opportune. (Eds Birch C.J., Wilson S.). Maize Association of Australia; Darlington Point. 112-119. Tollenaar, M. 1991. Physiological basis of genetic improvement of maize hybrids in Ontario from 1959 to 1988. Crop Sci. 31: 119-124. Sadek, S.E., Ahmed M.A., Abd El-Ghany, H.M. 2006. Genetic differences of product energy of four parents inbred lines and five yellow hybrid maize (Zea mays L.) genotypes grown in Egypt. J. Appl. Sci. Res. 2 (3): 153158. Subedi, K.D., Ma, B.L., 2005. Ear position, leaf area, and contribution of individual leaves to grain yield in conventional and leafy maize hybrids. Crop Sci. 45: 2246-2257. Whigham, D.K., Wooley, D.G. 1974. Effect of leaf orientation, leaf area and plant densities on corn production. Agron. J. 66: 482-486. Worku, M., Zelleke, H. 2007. Advances in improving harvest index and grain yield of maize in Ethiopia. East Afr. J. Sci. 1: 233 p. COBISS Code 1.01 Agrovoc descriptors: nematoda, life cycle, slugs, mortality, parasites, identification, diagnosis, monitoring Agris category code: H10 Massive occurrence and identification of the nematode Alloionema appendiculatum Schneider (Rhabditida: Alloionematidae) found in Arionidae slugs in Slovenia Žiga LAZNIK1, Jenna L. ROSS2, Stanislav TRDAN3 Received October 26, 2009; accepted January 18, 2010. Delo je prispelo 26. oktobra 2009; sprejeto 18. januarja 2010. ABSTRACT In the period from June to October 2008 we collected 500 slugs from the genus Arion in the area of Ljubljana and Prekmurje (Slovenia). By means of dissection we determined the presence of parasitic nematodes in slug cadavers. Identification of the nematodes was made by a molecular technique (PCR). In these slugs we did not find the parasitic nematode Phasmarhabditis hermaphrodita, however the presence of Alloionema appendiculatum in larger quantities was confirmed. The most infected was a Spanish slug, Arion lusitanicus. In Petri dishes younger slugs showed a satisfactory mortality rate already on the fourth day after the application of the nematode suspension. Unfortunately, we can not confirm with certainly that the nematode A. appendiculatum undergoes a complete life cycle in A. lusitanicus, which is otherwise typical for Phasmarhabditis hermaphrodita. Key words: Alloionema appendiculatum, Arion lusitanicus, parasitic nematodes IZVLEČEK ŠTEVILČNI POJAV IN IDENTIFIKACIJA OGORČICE Alloionema appendiculatum Schneider (Rhabditida: Alloionematidae) V LAZARJIH (Arionidae) V SLOVENIJI V obdobju od junija do oktobra 2008 smo na območju Ljubljane in Prekmurja nabrali 500 polžev iz rodu Arion. Polže smo secirali in ugotavljali zastopanost ogorčic v njihovem telesu. Identifikacija ogorčic je bila opravljena z molekulsko tehniko (PCR). V nobenem polžu nismo našli parazitske ogorčice Phasmarhabditis hermaphrodita, medtem ko smo zastopanost ogorčice Alloionema appendiculatum, predvsem v predstavnikih vrste Arion lusitanicus, potrdili v večjem številu. V petrijevkah so mladi polži pokazali zadovoljivo stopnjo smrtnosti že četrti dan po nanosu suspenzije ogorčic. Žal pa z našo raziskavo ne moremo potrditi, da ogorčica A. appendiculatum v polžu A. lusitanicus razvije popolni parazitski razvojni krog, kot je to značilno za ogorčico P. hermaphrodita. Ključne besede: Alloionema appendiculatum, Arion lusitanicus, parazitske ogorčice polžev 1 INTRODUCTION More than 25,000 species of nematodes have been described up to the present, of which around 4,000 are marine free-living nematodes, 6,000 terrestrial free-living nematodes, 12,000 parasitic nematodes of vertebrates, and 3,500 parasitic nematodes of invertebrates (Poulin and Morand, 2000; Hugot et al., 2001). Hitherto, in terrestrial slugs nematodes from eight families have been determined. Among them are parasitic species (of the order Rhabditida) which spend their complete life cycle inside slugs, as well as nematodes (of the order Strongylida) which use slugs as intermediate hosts (Grewal et al., 2003). When compared with entomopathogenic nematodes -the natural enemies of insects (Kaya and Gaugler, 1993) - these nematodes, which are related to slugs, are poorly studied (Wilson and Grewal, 2005). However, among those which have been studied up to now, specificity to 1 Young researcher, B. Sc., Jamnikarjeva 101, SI-1111 Ljubljana, email: ziga.laznik@bf.uni-lj.si 2 Ph. D, University of Aberdeen, Institute of Biological and Environmental Sciences, AB24 3UU, UK 3 Assoc. Prof., Ph. D, Jamnikarjeva 101, SI-1111 Ljubljana host species as well as diversity among individual species of nematodes are known. Slug-parasitic nematodes were found in all organs of the body cavity and also on foot muscles. Infection mechanisms are different, from entering through the body wall (infective juveniles of nematodes which are found in the soil) to the transference of infective juveniles between slugs during mating. It is well known that slug-parasitic nematodes can be an important biotic factor which balances the slug population in the natural environment (Grewal et al, 2003). The most intensively studied species of slug-parasitic nematode is Phasmarhabditis hermaphrodita (Schneider) (Rhabditidae), and in some countries this species is also used in the biological control of slugs (Wilson et al., 1993ab). The natural hosts of P. hermaphrodita are numerous slug species which can also be found in Slovenia: Deroceras leave (Müller), D. reticulatum (Müller), Arion distinctus Mabille, A. lusitanicus Mabille, A. silvaticus Lohmander, Tandonia budapestensis (Hazay) and T. sowerbyi (de Ferussac) (Vaupotic and Velkovrh, 2002; Morand et al, 2004). Alloionema appendiculatum Schneider is classified into the family Alloionematidae and it consists of two genera, Rhabitophanes Fuchs, the species of which are most frequently associated with insects, and the genus Alloionema Schneider, whose only representative, A. appendiculatum, is linked with gastropod molluscs (Morand et al., 2004). The former species was discovered for the first time in 1859 (Schneider). Its presence has hitherto been confirmed in Europe and Australia (Cabaret et al., 1988). In Slovenia this species was confirmed as early as in the 1970s, when Hržič studied the nematological fauna of Slovenia. The nematode was found in the soils of a vineyard situated in the Primorska region of Slovenia (Hržič, 1969; Urek et al., 2003). With our research we also wanted to determine if in Slovenia this nematode species is also present in slugs, as they are becoming increasingly more important pest organisms of some cultivated plants (Barker, 2001). 2 MATERIAL AND METHODS In September and October 2008 we collected 500 slugs in the area of Prekmurje and Ljubljana from the genus Arion (Arionidae). We dissected the slugs and with electronic magnification searched for the possible presence of nematodes inside them. We confirmed the presence of nematodes in the slugs collected in Ljubljana. With a pipette we separated the nematodes from the slug tissue and cleaned them according to the prescribed protocol (Riddle 1988). We infected new slugs with the obtained nematode suspension. We put 1 ml of a nematode suspension with a concentration of 300 IJ/ml into a Petri dish (diameter = 9 cm) and added 5 smaller slugs of the species Arion lusitanicus Mabille. After 4 days the slugs died and we dried the dead individuals at air temperature for 10 days. On the 10th day we placed the dried cadavers into a so-called White trap (Bedding and Akhurst 1975) in order to separate the nematodes from the dead slugs. The nematodes started to leave the cadavers on the 4th day after being exposed to the White trap. We washed the nematodes according to a procedure using a centrifuge and a 5 % concentration of sodium hypochlorate. The aim of this process was to obtain infective juveniles from the suspension. The nematode suspension was sent to the Institute of Biological and Environmental Sciences, University of Aberdeen, for further analysis. DNA was extracted using a QIAmp DNA Mini Kit. The 18S gene was amplified using a combination of universal primer pairs: G18S4, 26R, 24F, and 18P (Blaxter et al. 1998). An additional primer pair, 22F, and a newly designed primer, 1080R (TCC-TGG-TGG-TGC-CCT-TCC-GTC-AAT-TTC), were also used to ensure complete coverage of the 18S region (J. Ross, unpubl.). PCR cycling parameters involved primary denaturation at 94°C for 5 minutes, followed by 35 cycles of 94°C for 60 s, 55°C for 90 s, and 72°C for 2 min. Postamplification extension occurred at 72°C for 10 min (J. Ross, unpubl.). The PCR products were visualized on a 1% agarose gel and cleaned using a Qiagen QIAquick® PCR Purification Kit (fig. 1). Sequencing was carried out using an ABI3730 sequencer. The sequences were assembled using Sequencer 4.1 (Genes Codes Corp. Ann Arbor, Michigan, USA). The results were then compared with the GenBank Database, (http://www.ncbi.nlm.nih.gov/), using the BLASTn search tool (Altschul et al. 1990). The Alloionema appendiculatum sequence was deposited in the NCBI GenBank under FJ665982. Figure 1: 1% TAE buffered agarose gel, in the 1st lane; GeneRuler 100 bp DNA Hyperladder (Fermentas), in the 3rd, 6th, and 9th lane; the PCR product of Phasmarhabditis hermaphrodita (Becker Underwood commercial strain), which was used as a positive control, using the primer pair specified in the text; the 4th, 7th and 10th lane: the PCR product of the sample slug nematode - Alloionema appendiculatum. 3 RESULTS AND DISCUSSION The genetic studies proved that this nematode species is Alloionema appendiculatum Schneider (1859). The closest matches to this Alloionema appendiculatum sequence were Rhabditoid sp. (DQ531722.1) (QVarnstrom et al, 2007) with 92% identity over 89% coverage, followed by Strongyloides ratti (U81581.1) (Frisse et al., unpubl.) with 87% identity over 100% coverage (Fig. 2). The ITS1-5.8S-ITS2 region, including the partial 18S and 28S rRNA genes (flanked by the above primers) of the Slovenian isolate of A. appendiculatum is 1425 bp long (fig. 2). FJ665982 1 AACTGAGGTAATTCTTGAGCTAATACACGC--T--T--TA-ATGCCACATT-CGTGGTGC 52 U81581 86 ..............................T-. — . — .T-..A.......-A....... 138 FJ665982 53 U81581 139 GTTTATTTGACTAGA-C-A---T-C-A-TA-T-T-GGTTGACTCAAAATATCCTTGCT-A 100 ..........T..A.C.-.---.---T-.T-.-.C...................C...-G 187 FJ665982 101 DQ531722 1 U81581 188 atttttttttta-aaaat-atgccgtatgagtatctgctttatcaactttcgatggtagg 15 8 ...... 6 .....G..AC. — ....C-..A.......T.......G...................... 244 FJ665982 159 DQ531722 7 U81581 245 GTATTGGCCTACCATGG-TGTTGACGGATAACGGAGAATTAGGGTTCGACTCCGGAGAGG 217 -.A.. T..-. 65 303 FJ665982 218 DQ531722 66 U81581 304 GAGCCTGAGAAACGGCTACCACATCCAAGGAAGGCAGCAGGCGCGAAAATTACCCAATCT 277 ..........................................................T. 125 ..........................................................T. 363 FJ665982 DQ531722 U81581 FJ665982 DQ531722 U81581 FJ665982 DQ531722 U81581 FJ665982 DQ531722 U81581 FJ665982 DQ531722 U81581 278 TAG-TT-AAAGGAGGTAGTGACGAAAAATGACAAAG-C-AA-CACT-TT-TTAAG-T-GT 328 126 ...-..-...A.........................C.-.T-G...-..-.. — .T.-A. 176 364 ...-..-...A.....................-..C-.A..-T.T.A..-..T..-.AT. 416 32 9 TGTTATTGGAAATCTTGC-AA-CCTAAAT-A-GTTGCTTGGTAAAGGAGAGGGCCAGTCT 384 177 G................T-.G-TG.....-.-AC.A................................................232 417 ..-G....A.......-.-..GTT.....A.-C...-...........A.....A..........471 385 GGTGCCAGCAGCCGCGGTAATACCAGCTCTCCAAGTGCATAAAATGATTGTTGTGGTTAA 4 44 233 ........................................................................................................................292 472 ............................T...............................531 445 AAAGCTCGTA-GTTGGATT-T----ATTG---AATA-A-G—T-TCA-GA-G---T-C— 482 293 ..........-........-.----....---.G..-.G. — .-.-.-.T-.T—.-G— 331 532 ..........-......-.-.----.-.A---..G.TT-. — .G.A.T..-.CA-.-.— 572 483 —A-T-GT-A-T-ATG-GCT — T—C — TGA-A-AT-G-TT---CA-TA-C—T-TT — T 514 332 —.C.-..-G-. — .A-A.A—. — . — .-.-.C..-.C..AAAT.T..-G—.A.. — . 369 573 —T-.-.G-.-.GT.A-TT. — . — .A-.T.CC-..-C-..----.-.T-T—.-.. — . 6ü6 FJ665982 DQ531722 U81581 515 -G-C---A-A---TT-AG---C-A-AT-A-A-T—T-GC-CT-GTTACTTTGAATAAATC 551 37ü -T-T---T-T---..A.A---.G.-.A-.-.-A—.-A.-..-................. 4ü8 6ü7 AT-T---.T.---..-..---A-.-..A.T.-.AA.-AA-..-................. 649 FJ665982 DQ531722 U81581 552 4ü9 65ü AGAGTGTTCAAA—A—CA-G---TC-A---T-ATGAC-TG-A—AT-A-T-CCTAGCAT ....G.......—C—..G.CA-.T-.---.-...-.T..-T—..GG-. —....... 591 448 692 FJ665982 DQ531722 U81581 592 GGAATAACACTATA-GAAGTATATG-TAGTGCGGTGTTTCA-C—ACA-T-TACTTCATG 644 44 9 ..............-.......-.T-.....T.........TT—.-.-A-......... 5üü 693 ..............-... — .-.A.T.....-T.-......-.---T.-.C.TT...... 741 FJ665982 DQ531722 U81581 FJ665982 DQ531722 U81581 645 5ü1 742 7ü4 56ü 8ü1 GTTAATA-GAAACAAACGGGGGCATTCGTATCGATACGTTAGAGGTGAAATTCTTGGACC A......-.G.......................C.......................... GTATCGAGACGTCCTACTGCGAAAGCATTTGCCAAGAATGTTTTCATTAATCAAGAACGA ...G........................................................ 7ü3 559 8üü 763 619 8 6ü FJ665982 DQ531722 U81581 764 62ü 861 AAGTTAGAGGTTCGAAGGCGATCAGATACCGCCCTAGTTCTAACCGTAAACTATGCCTAC ...................................................G........ 823 679 92ü FJ665982 DQ531722 U81581 82 4 TAGATGTTTG-A-TGGTA-AT-GTA-AAT-TA-TTA-TTGA-GCATCTTCTCGGAAACGA 874 68ü .......A..-.-.A...-..-...-...-..-...-...T-.................. 73ü 921 ...G...A..A.-. — ..-T.A..—T..-A.-.A.-..T.T...C.............. 971 FJ665982 DQ531722 U81581 875 731 972 AAGTCTTTCGGTTCCGGGGGAAGTATGGTTGCAAAGCTGAAACTTAAAGGAATTGACGGA 934 79ü 1ü31 FJ665982 DQ531722 U81581 935 791 1ü32 AGGGCACCACCAGGAGTGGAGCCTGCGGCTTAATTTGACTCAACACGGGAAAACTCACCC .....................................N...................... 994 85ü 1ü91 FJ665982 DQ531722 U81581 995 GGGCCGGACA-TAAG-AAGGATTGACAGATTTAGAGCTCTTTCAAGATTTTA-TGGTTGG 1ü51 851 ..........-CG.T-......AA.........T........TT......CG-....... 9ü7 1ü92 ..........C..T--...............G.T..........T.....-.G.......1148 FJ665982 DQ531722 U81581 1ü52 9ü8 1149 TGGTGCATGGCCGTTCTTAGTTCGTGGATATGATTTGTCTGGTTGATTCCGATAACGAAC ..........................................................G. 1111 967 12ü8 FJ665982 DQ531722 U81581 FJ665982 DQ531722 U81581 FJ665982 DQ531722 U81581 FJ665982 DQ531722 U81581 FJ665982 DQ531722 U81581 FJ665982 DQ531722 U81581 1112 GAGACTGTTAA-T-T-TAT-T---T---T-AC—AC-G-TGGGGG-ATTC-T-T—A-A- 1149 968 .......C...-.-A-...-.---.---.-.G—.T-A-.ACATT-...TA.-.—G-.- 1006 1209 ......T... — .G.-...-A---.---.-.A—.T---.ATATT-...T-.-.T-.-.- 1246 1150 -TCC---T—T-TA-ACGGTT—AATAA-ATGATTAGT-GA-TGTTTAA-TCACTTGAGA 1195 1007 -.AG---.—GA..T.. — .. — ....T-.........-.GC.-....G-.......... 1052 1247 -.AA---.A-.-..-.TT-.. —.....C.-.....A.A. —.......C.-.T...... 1292 1196 AAGAGCAATAACAGGTCTGTGATGCCCTTAGATGTCTGGGGCTGCACGCGCGCTACAATG 1255 1053 .G.......................................................... 1112 1293 G.....G.............A...............C.......................1352 125 6 TAGTGAGCA-CTATGTTCCTGATTC-GAGAGGATTGGGTAAACCTTGAAAGCATTACGTA 1313 1113 .........T.-.............-..................................1170 1353 ......T..-T..........T..A-....TA.A.........A......A......... 1410 1314 ACTGGGAGTGTAACTTGCAATTATGTTACATGAACGAGGAATTCCAAGTAAACGTGAA-T 1372 1171 ......................................................C...-. 1229 1411 ..........A................T...........................-..G. 1469 1373 CATTAG-TTCACATTGATTACGTCCCTGCCCTTTGTACACACCGCCCGTCGCTG 1425 1230 ......-...G...........................................1282 1470 ......C..-............................................1522 Figure 2: Multiple sequence alignment of the ITS rDNA region (including partial fragments of the 18S and 28S rRNA genes) of 3 nematode species. Code FJ665982 corresponds to the Slovenian isolate of Alloionema appendiculatum. Codes DQ531722 and U81581 refer to the Rhabditoid sp. and Strongyloides ratti, respectively. 4 CONCLUSIONS For the nematode Alloionema appendiculatum it is acknowledge that it has a free-living and parasitic life cycle (Cabaret and Morand, 1990). In the parasitic part, the larvae of the third larval stage (L3) enter into the slug's body through its foot, where they moult and undergo a fourth larval stage (L4) and which stays encapsulated in foot muscles. The encysted larvae engorge (mechanism unknown) and then exit the slug and molt into immature adults (L5), which are free-living (Morand and Daguzan, 1986). Adult nematodes which undergo a series of molts in the parasitic life cycle are two to three times longer than those (from 1 to 2 mm) which grow in a free-living life cycle. The nematode A. appendiculatum has hitherto been found in different species of slugs; Deroceras sp. (Schneider, 1859), Arion ater (Schuuramns -Stekhoven, 1950), A. circumscriptus (Mengert, 1953), A. intermedius (Cabaret et al., 1988), A. silvaticus (Morand and Bonnet, 1989), A. subfuscus (Cabaret and Morand, 1990), Cantareus aspersus (Charwat and Davies 1999), and Prietocella barbara (Morand, 2004). Research studies have not yet found a natural host in which the nematode A. appendiculatum undergoes a complete life cycle and which causes it to have a higher mortality rate (Morand et al., 2004). Morand and Daguzan (1986) report that the nematode A. appendiculatum does not appear very frequently in natural slug populations, meanwhile its presence in snails which have been bred is more common (Cabaret et al, 1988). A research study which was conducted by Cabaret and Morand (1990) indicates that a positive interaction exists between the nematodes Muellerius capillaris (Strongylida: Strongyloididae) and A. appendiculatum, as with the species M. capillaris previously infected slugs attain a higher mortality rate when infected with A. appendiculatum afterwards. The application of A. appendiculatum in practice might be carried out in soils where a larger number of nematodes from the family Strongyloididae is present and for which it is known that slugs are only intermediate hosts (Grewal et al., 2003). But for now, no such co-infection has been discovered (Morand et al., 2004). This is the first large-scale record of the mentioned nematode in slugs from the genus Arion, among which Arion lusitanicus Mabille predominated in terms of the number of individuals. Younger slugs of the mentioned species showed a satisfactory mortality rate already on the fourth day of the nematode suspension application in Petri dishes. But because these results are merely preliminary, we can not yet confirm for certain that the nematode A. appendiculatum is capable of developing a complete life cycle inside the slug A. lusitanicus, although this is common for Phasmarhabditis hermaphrodita (Wilson et al, 1993a). The presence of P. hermaphrodita has not yet been confirmed in Slovenia, thus only chemical plant protection products with limacide activity can be used to control slugs and snails (Ozimic et al., 2007). Due to the fact that the Regulations on Biological Plant Protection prohibit the introduction of exotic organisms into the natural environment, we will continue our research on the presence of slug-parasitic nematodes. Possible future identification of the nematode P. hermaphrodita could entail an alternative to chemical limacides, as due to the milder winters and humid summers, slugs and snails are increasingly important pests on cultivated plants in Slovenia as well as in many other countries (Kozowski et al., 2006; Grubisic et al., 2007). 5 ACKNOWLEDGEMENT This work was carried out within Horticulture No P4-0013-0481, a program funded by the Slovenian Research Agency, and within the L4-1013 project funded by the Slovenian Research Agency and Ministry of Agriculture, Food, and Forestry of the Republic of Slovenia and the company Unichem d.o.o. Part of the research was funded within Professional Tasks from the Field of Plant Protection, a program funded by the Ministry of Agriculture, Forestry, and Food of Phytosanitary Administration of the Republic of Slovenia. This study was also supported by Becker Underwood and BBSRC. 5 REFERENCES Altschul, S.F., Gish, W., Miller, W., Myers, E.X., Lipman, D.J. 1990. Basic local alignment search tool. J. Mol. Biol. 215: 403-410. Barker, G.M. 2001. The biology of terrestrial gastropods. CAB Int., Wallingford: 468 pp. Bedding, R. A., Akhurst, R. J. 1975. Simple technique for the detection of insect parasitic rhabditid nematodes in soil. Nematologica 21: 109-110. Blaxter, M.L., De Ley, P., Garey, J. 1998. A molecular evolutionary framework for the phylum Nematoda. Nature 392: 71-75 Cabaret, J., Morand, S., Aubert, C., Yvoré, P. 1988. Snail farming: a survey of breeding management, hygiene and parasitism of the garden snail, Helix aspersa Müller. J. Mollus. Stud. 54: 209-214. Cabaret, J., Morand, S. 1990. Single and dual infection of the land snail Helix aspersa with Muellerius capillaris and Alloionema appendiculatum (Nematoda). J. Parasitol. 76: 579-580. Charwat, S.M., Davies, K.A. 1998. A nematode isolate for biocontrol of pest snails in south Australia. Nematologica 44: 469-470. Grewal, S.K., Grewal, P.S., Hammond, R.B. 2003. Susceptibility of North American native and non-native slugs (Mollusca: Gastropoda) to Phasmarhabditis hermaphrodita (Nematoda: Rhabditidae). Biocontrol Sci. Technol. 13: 119-125. Grubisic, D., Gotlin-Culjak, T., Vitas, I. 2007. Puzevi golaci u ratarskim kulturama - procjena rizika i mjere suzbijanja. Glasnik biljne zastite 7: 394-398. Hrzic, A. 1969. Investigation of nematodes as direct plant pests and vectors of plant diseases. Agricultural Institute of Slovenia, Ljubljana, Technical Report, 26 pp. Hugot, J.P., Baujard, P., Morand, S. 2001. Biodiversity in helminths and nematodes as a field of study: an overview. Nematology 3: 1-10. Mengert, H. 1953. Nematoden und Schnecken. Z. Morphol. Okol. Tiere 4: 311-349. Kaya H.K., Gaugler R. 1993. Entomopathogenic nematodes. Ann. Rev. Entomol. 38: 181-206. Kozowski, J., Zielinska, M., Pawowska, A., Kozowska, M. 2006. Susceptibility of some vegetable species to feeding of Cepaea hortensis (Muller) and Arion rufus (Linnaeus). J. Plant Prot. Res. 46: 231-239. Morand, S., Bonnet, J.C. 1989. Importance des nématodes en héliciculture et méthode de prophylaxie. Haliotis 19: 6975. Morand, S., Daguzan, J. 1986. Contribution à l'éscargot petit-gris (Helix aspersa Müller): Premiers résultats concernants l' acarien Riccardoella limacum (Schrank) et le nématode Alloionema appendiculatum (Schneider). Haliotis 15: 31-39. Morand, S., Wilson, M.J., Glen, D.M. 2004. Nematodes (Nematoda) Parasitic in Terrestrial Gastropods. In Barker, G.M. (ed.) Natural Enemies of Terrestrial Molluscs. CABI Publishing, Wallingford, UK, 644 pp. Poulin, R., Morand, S. 2000. The diversity of parasites. Q. Rev. Biol. 75: 277-293. Riddle, D.L. 1988. The dauer larva, p. 393-412. In W.B. Wood (ed.), The nematode Caenorhabditis elegans, Cold Spring Harbor Laboratory Press, Plainview, New York, NY. Qvarnstrom, Y., Sullivan, J.J., Bishop, H.S., Hollingsworth, R., Da Silva, A.J. 2007. PCR-Based Detection of Angiostrongylus cantonensis in Tissue and Mucus Secretions from Molluscan Hosts. Appl. Environ. Microbiol. 73: 1415-1419. Schneider, A. 1859. Monographie der Nematoden. Druck und Verlag Von Georg Reimer, Berlin. Schuuramns-Stekhoven, L.H. 1950 Alloionema appendiculatum. Acta Zool. Lilloana 9: 481-485. Urek, G., Sirca, S., Karssen, G. 2003. A review of plant-parasitic and soil nematodes in Slovenia. Nematology 5: 391-403. Vaupotic, M., Velkovrh, F. 2002. Slugs (Gastropoda: Pulmonata: Milacidae, Limacidae, Boettgerillidae, Agriolimacidae, Arionidae) of Slovenia. Acta Biol. Slov. 45: 35-52. Wilson, M.J., Grewal, P.S. 2005. Biology, Production and Formulation of Slug-parasitic Nematodes. In Grewal, P.S., Ehlers, R.-U. And Shapiro-Ilan, D.I. (eds.) Nematodes as biocontrol agents. CABI Publishing, Wallingford, UK, 505 pp. Wilson, M.J., Glen, D.M., George, S.K. 1993a. The rhabditid nematode Phasmarhabditis hermaphrodita as a potential biological-control agent for slugs. Biocontrol Sci. Technol. 3: 503-511. Wilson, M.J., Glen, D.M., George, S.K., Butler, R.C. 1993b. Mass cultivation and storage of the rhabditid nematode Phasmarhabditis hermaphrodita, a biocontrol agent for slugs. Biocontrol Sci. Technol. 3: 513-521. COBISS Code 1.01 Agrovoc descriptors: steinernema feltiae, nematoda, insect nematodes, biological control, sitophilus oryzae, torage, storage losses, crop losses Agris category code: H10 Intraspecific variability of Steinernema feltiae (Filipjev) (Rhabditida: Steinernematidae) as biological control agent of rice weevil (Sitophilus oryzae [L.], Coleoptera, Curculionidae) adults Žiga LAZNIK1, Stanislav TRDAN2 Received December 28, 2009, accepted February 26, 2010. Delo je prispelo 28. decembra 2009; sprejeto 26. februarja 2010. ABSTRACT The efficacy of three strains (B30, B49 in 3162) of Steinernema feltiae to control adults of rice weevil (Sitophilus oryzae) was tested in a laboratory experiment in 2009. The activity of entomopathogenic nematodes was assessed at five different concentrations (125, 250, 500, 1000 and 2000 infective juveniles/individual) and four different temperatures (15, 20, 25 and 30 °C). Results demonstrated that all strains acted most effective at 25 °C and at highest concentration of nematode suspension, meanwhile the lowest mortality of rice weevil adults was attained at 30 °C. The results of our research showed that at high concentrations entomopathogenic nematodes are an effective biological agent for controling the studied primary stored products pest. Key words biological control, entomopathogenic nematodes, Steinernema feltiae, Sitophilus oryzae, strains, laboratory experiment, stored products pest IZVLEČEK ZNOTRAJVRSTNA VARIABILNOST ENTOMOPATOGENE OGORČICE Steinernema feltiae (Filipjev) (Rhabditida: Steinernematidae) KOT BIOTIČNEGA AGENSA ZA ZATIRANJE ODRASLIH OSEBKOV RIŽEVEGA ŽUŽKA (Sitophilus oryzae [L.], Coleoptera, Curculionidae) V letu 2009 smo pri laboratorijskem poskusu preizkušali učinkovitost treh ras (B30, B49 in 3162) entomopatogene ogorčice Steinernema feltiae na odrasle osebke riževega žužka (Sitophilus oryzae). Delovanje entomopatogenih ogorčic smo preizkušali pri petih različnih koncentracijah (125, 250, 500, 1000 in 2000 infektivnih ličink/osebek) in štirih različnih temperaturah (15, 20, 25 in 30 °C). Rezultati so pokazali, da so vse rase najbolj učinkovito delovale pri 25 °C in najvišji koncentraciji suspenzije ogorčic, medtem ko smo najmanjšo smrtnost odraslih osebkov riževega žužka ugotovili pri 30 °C. Rezultati naše raziskave so pokazali, da so v visokem številu entomopatogene ogorčice učinkovit biotični agens za zatiranje preučevanega primarnega skladiščnega škodljivca. Key words: entomopatogene ogorčice, Steinernema feltiae, Sitophilus oryzae, biotično varstvo, laboratorijski poskus, skladiščni škodljivec 1 INTRODUCTION On stored agricultural products, especially cereal grains and legume seeds, several pests appear which can cause a large amount of damage. While they feed on stored grains, the yield quantity is reduced and the pests especially lower product quality (Neethirajan et al, 2007). Annual damage due to storage pests on stored products amounts to from 1.25 to 2.5 billion USD (Scholler et al, 2006). Storage pest insects cause stored 1 Young researcher, Teach. Assist., B. Sc., Jamnikarjeva 101, SI-1111 Ljubljana, e-mail: ziga.laznik@bf.uni-lj.si 2 Assoc. Prof., Ph. D., ibid. products to heat up or that their moisture level increases with the consequence of grain infection due to microorganisms (Rees, 1998). We also consider the rice weevil (Sitophilus oryzae [L.], Curculionidae) to be among the most important pests regarding stored grain in Europe in recent years (Stejskal et al, 2003). It is a known fact that the insect developed a resistance to the insecticide malation already in the 1960s (Haliscak & Beeman, 1983; van Graver & Winks, 1994). Only four years passed from the appearance of resistance until the total ineffectiveness of malation in controlling the rice weevil (van Graver & Winks, 1994). Despite data which shows that the usage of the insecticide in controlling storage pest is not notably hazardous to consumers, researchers are still of the opinion that insecticide residues in food are harmful to human health, as are insecticide residues in feed to animal health and indirectly as well to human beings (Acuff, 1993; South, 1993; Bryne et al, 1994). Consumers seem willing to pay more for food produced without the use of chemical products (Ott, 1990; Brewer et al, 1994). The application of entomopathogenic nematodes (EPNs) as biological control agents in controlling pest insects is well documented (Kaya & Gaugler, 1993; Helyer et al, 1995; Trdan et al, 2007). EPNs live in symbiosis with bacteria which after infection are released by nematodes into the hemolymph system of the host (Gaugler, 2002). The ability to infect the host holds only for infective juveniles (IJs) which carry mutualistic bacteria in special intestine vesicles (Kaya, 2000). A host in which the nematodes manage to penetrate usually die due to septicemia or failure of some insect organs in 24 to 72 hours after infection (Smart, 1995; Forst & Clarke, 2002). The results of many research studies have demonstrated that EPNs at high concentrations together with favourable abiotic factors (high humidity, optimal temperature) can be effective biological agents in controlling adults from the order Coleoptera (Journey & Ostlie, 2000; Trdan et al., 2006). Recent research has confirmed their efficacy in controlling adults of the Japanese beetle Popillia japonica Newman (Lacey et al, 1993; Grewal et al, 2002; Koppenhofer & Fuzy, 2007), the western corn rootworm (Diabrotica virgifera virgifera LeConte) (van der Burgt et al, 1998; Toepfer, 2005), the hairy fungus beetle (Typhaea stercorea [L.]) (Svendsen & Steenberg, 2000), the granary weevil (Sitophilus granarius [L.]) (Trdan et al., 2006), the sawtoothed grain beetle (Oryzaephilus surinamensis [L.]) (Trdan et al, 2006), the flea beetle (Phyllotreta spp.) (Trdan et al., 2008), and some others which belong to the order Coleoptera. In previous years many research studies were carried out in connection with EPNs ability to control storage pests (Trdan et al., 2006; Ramos-Rodriguez et al, 2007; Athanassiou et al., 2007), yet data on their effectiveness in controlling the rice weevil are limited (Ramos-Rodriguez et al., 2006). Because in Europe EPNs have not yet been tested for controlling the rice weevil -which appears to be an increasingly harmful species in grain warehouses (Hamel, 2007) - we decided to study the activity of one of the generally most effective species for controlling pest insects, the nematode Steinernema feltiae (Filipjev) (Scheepmaker et al., 1998; Cuthbertson et al., 2003) with regard to the above mentioned storage pest. The aim of our research was to determine which of three tested strains (B30, B49, and 3162) is the most effective and how its effectiveness is related to temperature and suspension concentration. Namely, the most efficient strain could possibly be applied in the near future for practical purposes, i.e. for controlling this pest in grain storehouses. 2 MATERIALS AND METHODS 2.1 Sitophilus oryzae and entomopathogenic nematodes In this experiment we studied nematode pathogenicity against adults of the rice weevil (Sitophilus oryzae). Adults of the mentioned insect had been in culture at the Chair of Phytomedicine, Agricultural Engineering, Crop Production, Grassland and Pasture Management (University of Ljubljana, Biotechnical Faculty, Dept. of Agronomy) in Ljubljana, Slovenia. If required, we added undamaged wheat grain in a two week intervals in a rearing vessels to provide optimal conditions for the studied insect species to feed and multiply. The rearing vessels were stored in darkness and at room temperature. We included three strains of Steinernema feltiae EPNs in the experiment. We isolated strains B30 and B49 in Slovenia (Laznik et al., 2008), meanwhile strain 3162 was isolated in Hungary (Toth, 2006). The two Slovenian strains were tested for the first time in this experiment, while strain 3162 has been proven to be very effective in many laboratory experiments (Lakatos, unpubl.). All EPN strains were reared using late instar larvae of Galleria mellonella L. (Bedding and Akhurst, 1975). We used only infective juveniles which were less than 2 weeks old. During the experiment, which was repeated three times, we stored the infective juveniles at 4 °C. 2.2 Laboratory bioassay We tested the efficacy of the EPNs in controlling adults of the rice weevil Sitophilus oryzae by exposing individuals to either 0, 250, 500, 1000, or 2000 IJ/adult. We determined the number of infective juveniles in a previously prepared unknown concentration of nematode suspension by counting the number of such in droplets (5 ^l x 5) and by diluting (adding M9 solution) or by concentrating (reduction to an adequate volume with the assistance of centrifugation). In this manner we obtained the selected concentrations of nematode suspensions (0, 2500, 5000, 10000, and 20000 IJ/ml). We carried out the experiment according to the procedure described in the paper of Trdan et.al., 2006. The following procedure was performed with a time interval in three replications. We placed adults in glassy Petri dishes (diameter = 9 cm) with each containing 10 adults. Prior to this, we put filter paper into each Petri dish (the same diameter as the former Petri dish) and 30 wheat grains. Each treatment in the experiment was repeated 10 times. The assigned nematode concentration was added to the filter paper with a pipette (1 ml). If needed, we additionally moistened the filter paper every second day of the experiment. The Petri dishes were put in a rearing chamber (type: RK-900 CH, producer: Kambic Laboratory equipment, Semic, Slovenia) with a volume of 0.868 m3 (width x height x depth = 1000 x 1400 x 620 mm). We tested the efficacy at four different temperatures (15, 20, 25, and 30 °C) and at a relative humidity of 80 %. The number of dead adults of S. oryzae was determined 4, 6, and 8 days after treatment. The dead individuals were dissected to determine if the nematodes were present. In such a manner we wanted to prove that the insects died due to the EPNs' activity. 2.3 Statistical analysis A multifactor analysis of variance (ANOVA) was conducted to determine the differences in mortality rates (%) between the adults of S. oryzae reared in 48 different treatments (three strains of S. feltiae - each with four different concentrations at four different temperatures). Before the analysis, the mean mortality was tested for the homogeneity of treatment variances. Mortality rate data were corrected for control mortality, using Abbott's formula (Abbott, 1925). The arcsine square-root was transformed before this analysis. Duncan's multiple range test (P < 0.05) was used to separate mean differences among the parameters in all the treatments. All statistical analyses were performed with Statgraphics Plus for Windows 4.0 (Manugistics, Rockville, MD, USA) and the figures were created with MS Office Excel 2003. The data are presented as untransformed means ± SE. 3 RESULTS 3.1 Group analysis Group analysis indicated that the mortality percentage of granary weevil (Sitophilus oryzae) adults was statistically significantly influenced by the concentration of the nematode suspension (F= 151.09; df= 4, 720; P<0.0001), temperature (F= 61.43; df= 3, 720; P<0.0001), DAT (F= 54.75; df= 2, 720; P<0.0001), nematode strain (F=4.15; df=2, 720; P=0.0162), interaction between DAT and the concentration of the nematode suspension (F=7.23; df=8, 720; P<0.0001), interaction between the concentration of the nematode suspension and the strain (F=5.33; df=8, 720; P<0.0001), interaction between the concentration of the nematode suspension and temperature (F=14.57; df=12, 720; P<0.0001), interaction between the strain and temperature (F=9.77; df=6, 720; P<0.0001), interaction between DAT, strain, and temperature (F=2.12; df=12, 720; P=0.0142) and interaction between the concentration of the nematode suspension, strain, and temperature (F=2.95; df=24, 720; P<0.0001). Interaction between DAT and strain (F=1.03; df=4, 720; P=0.3893), interaction between DAT and temperature (F=1.45; df=6, 720; P=0.1920), interaction between DAT, the concentration of the nematode suspension, and the strain (F=0.32; df=16, 720; P=0.9951), interaction between DAT, the concentration of the nematode suspension, and temperature (F=1.08; df=24, 720; P=0.3587), and interaction between DAT, the concentration of the nematode suspension, strain, and temperature (F=0.45; df=48, 720; P=0.9995) did not statistically significantly influence beetle mortality. In all treatments total mortality was significantly different from the control treatment. Corrected mortality was therefore calculated. Among the individual strains of EPNs (3162, B49, and B30) we did not observe statistically significant differences in pathogenicity, while the average values of beetle mortality treated with them were 17.71 ± 1.08, 16.60 ± 0.79, and 15.33 ± 0.77 %, respectively. But we confirmed statistically significant differences between different temperatures. The studied strains were the most active at 25 °C (22.34 ± 1.19 %), while no difference was observed at 15 and 20 °C (15.75 ± 0.98 in 18.33 ± 1.11 %). The lowest efficacy was noted at 30 °C (9.75 ± 0.52 %) (Figure 1). Statistically significant differences were confirmed also between the concentration of the nematode suspension. At the highest concentration of the suspension beetle mortality was the highest (31.79 ± 1.53 %), less effective were nematodes at both lower concentrations (8.8 ± 0.66 and 11.19 ± 0.63 %, respectively). 100 90 80 i? 70 60 UJ w 50 +i i 40 c ro Jj 30 20 10 0 S. feltiae B30 S. feltiae B49 S. feltiae 3162 Entomopathogenic nematode strains Fig. 1: Mean adult mortality of Sitophilus oryzae treated with three different strains of the entomopathogenic nematode Steinernema feltiae depending on rearing temperature. The data shown are corrected for control mortality and analyzed by multifactor ANOVA. Capital and lower-case letters correspond to the grouping of means by Duncan's multiple range test (P<0.05) for EPN strains and temperature, respectively. The same letters do not differ significantly. Table 1: Mean adult mortality (± SE) of Sitophilus oryzae treated with five different concentrations of three entomopathogenic nematode Steinernema feltiae strains at 15, 20, 25, and 30 °C at 8 DAT. The data shown are corrected for control mortality. Tempe- S. feltiae Nematode concentration (IJs/adult) rature strain 125 250 500 1000 2000 (°C) 15 B30 6.80 ± 3.39 14.89 ± 4.75 10.63 ± 4.25 36.16 ± 7.52 38.29 ± 6.20 B49 10.86 ± 4.06 15.21 ± 5.32 13.04 ± 3.43 23.91 ± 9.09 41.30 ± 8.13 3162 5.95 ± 4.96 5.52 ± 2.48 14.04 ± 5.28 16.16 ± 8.20 31.91 ± 7.21 20 B30 16.66 ± 4.65 14.58 ± 3.89 11.24 ± 4.77 16.66 ± 3.29 49.99 ± 6.91 B49 8.69 ± 4.34 8.69 ± 2.66 19.56 ± 2.66 15.21 ± 2.17 43.47 ± 7.98 3162 3.26 ± 1.99 14.69 ± 4.88 18.36 ± 3.22 22.44 ± 2.50 65.30 ± 6.12 25 B30 11.81 ± 3.89 11.36 ± 2.27 11.81 ± 3.89 20.45 ± 3.59 45.45 ± 4.25 B49 24.44 ± 2.22 22.22 ± 3.51 17.77 ± 2.72 48.88 ± 4.44 42.21 ± 5.44 3162 11.81 ± 5.29 20.45 ± 5.08 20.45 ± 3.59 40.90 ± 9.77 72.72 ± 5.79 30 B30 9.78 ± 3.19 6.37 ± 2.12 10.63 ± 2.60 10.63 ± 4.25 17.01 ± 2.12 B49 6.37 ± 2.12 8.50 ± 2.60 10.63 ± 4.25 14.89 ± 4.75 17.01 ± 3.98 3162 10.20 ± 2.04 16.32 ± 2.04 18.36 ± 3.22 20.40 ± 2.04 16.32 ± 3.81 B We determined statistically significant differences also between days after treatment (DAT). At 8 DAT we confirmed the significantly highest beetle mortality (20.49 ± 1.00 %), and 6 and 4 DAT gave the significantly lowest mortality rate of rice weevil adults (17.21 ± 0.91 and 11.93 ± 0.66 %, respectively). Between all three time treatments there were statistically significant differences. 3.2 Individual analysis At 15 °C, 8 DAT, and the highest concentration of the nematode suspension, strains B49 and B30 demonstrated the highest mortality (41.30 ± 8.13 % and 38.29 ± 6.20 %, respectively). Similar effectiveness was attained for strain B30 at a lower concentration of the nematode suspension (1000 IJ/adult), namely (36.16 ± 7.52 %). At the lowest concentrations (125 IJ/adult and 250 IJ/adult) of the nematode suspension the best pathogenicity was noted in strain B49 (10.86 ± 4.06 %; 15.21 ± 5.32%, respectively). The results showed that at the lowest temperatures the weakest efficacy was for strain 3162 at all concentrations of the nematode suspension (from 5.52 ± 2.48 to 31.91 ± 7.21) (8 DAT) (Table 1). At 20 °C, 8 DAT, and at the highest concentration of the nematode suspension, the highest mortality rate was noted in strain 3162 (65.30 ± 6.12 %). Also strains B30 and B49 were satisfactorily effective at the highest concentration of the nematode suspension (49.99 ± 6.91 %; 43.47 ± 7.98 %, respectively) (8 DAT). At the lower concentration of the nematode suspension their pathogenicity was less satisfying (from 3.26 ± 1.99 to 22.44 ± 2.50 %). At 25 °C and 8 DAT the effectiveness of all studied concentrations of the nematode suspension was the highest. Strain 3162 pathogenicity at the highest concentration led to 72.72 ± 5.79 % mortality of the studied insect. Less effective were strains B30 and B49 (45.45 ± 4.25 %; 42.21 ± 5.44 %, respectively). Also the lowest concentration (1000 IJ/adult) of strain B49 demonstrated satisfactory pathogenicity (48.88 ± 4.44 %). At all studied concentrations strain 3162 was the most effective (from 11.81 ± 5.29 to 72.72 ± 5.79 %), except for the better performance at the lowest concentration of strain B49 (24.44 ± 2.22 %) (Table 1). At 30 °C and 8 DAT the effectiveness of all studied strains was the lowest (from 6 to 20 %). The highest mortality was reached by strain 3162 at a suspension concentration of 1000 IJ/adult (20.40 ± 2.04 %), while the lowest mortality was attained by strain B30 at a suspension concentration of 250 IJ/adult (6.37 ± 2.12 %). Strain 3162 demonstrated the best infectivity at this temperature (from 10.20 ± 2.04 to 20.40 ± 2.04 %), while strain B30 was the least infective (from 6.37 ± 2.12 to 17.01 ± 2.12 %) (Table 1). Strain 3162 (5.95 ± 4.96) and strain B30 (6.80 ± 3.39) at the lowest concentration of the nematode suspension (125 IJ/adult ) and at 15 °C and 8 DAT caused statistically significantly lower mortality of rice weevil adults, while strain B49 (6.37 ± 2.12) acted as weakly at 30 °C as at the lowest concentration of the nematode suspension (Table 1). The highest mortality of Sitophilus oryzae adults was noted in strain 3162 (72.72 ± 5.79) at 25 °C and the highest concentration of the nematode suspension (2000 IJ/adult) (8 DAT), which holds true also for strain B49 (48.88 ± 4.44). Strain B30 reached the highest mortality of the studied insect (49.99 ± 6.91) at 20 °C and at the highest concentration of the nematode suspension (2000 IJ/adult) (8 DAT). 4 DISCUSSION The results of our research showed that the mortality of rice weevil (Sitophilus oryzae) adults is mostly affected by temperature in connection with the concentration of the nematode suspension, the nematode strain, and DAT. All three studied strains (B30, B49, and 3162) caused the highest mortality of the storage pests 8 days after treatment at 25 °C (45 %; 48 %, and 72 %, respectively) and at the two highest concentrations of the nematode suspension (1000 and 2000 IJ/adult). The lowest mortality rate was recorded at 30 °C, at which none of the strains with none of the concentrations of the nematode suspension exceeded 20 % efficacy. Sufficient efficacy at the lowest temperature (15 °C) was reached only by strain B49 (41 %), while the pathogenicity of strain 3162 at this temperature was less satisfactory (from 6 to 31 %). One of the reasons for such ineffectiveness of this strain may lie in the origin of this strain, which was not isolated in Slovenia. Namely, this strain originates from Hungary, where the climate is typically continental and the average temperatures higher than in Slovenia (Peel et al, 2007). Confirmation of the above mentioned thesis is also demonstrated in that strain 3162 was the best (72 %) of all the studied strains. Also, the calculated LC50 value achieved was the lowest when compared at the highest temperature. A similar conclusions was also arrived at by Hazir et al. (2001), when they established that EPNs which were isolated from areas of warmer climate had better pathogenicity at higher temperatures. The weaker efficacy of all three strains at 30 °C can be attributed to the fact that the interval of optimal control for the majority of EPNs is between 20 and 26 °C (Kaya et al, 1993; Belair et al, 2003; Trdan et al, 2008), yet this characteristic is specific to either species or strain (Grewal et al, 1994; Hazir et al, 2001). It is well known that temperatures which fall below 0 °C or rise above 40 °C are fatal for the majority of EPNs (Brown & Gaugler, 1996). The reason for studying the activity of EPNs also at 30 °C is a certain characteristic of the species Sitophilus oryzae, which is a distinctive thermophilus insect species with the ability to multiply more successfully at higher temperatures (from 25 to 35 °C) (Fields, 1992). It is know for the majority of storage pests that temperature has a great influence on their survival ability (Ileleji et al., 2004), while most of them feed and multiply less intensively at temperatures lower than 18 °C (Howe, 1965). In a similar research study (Ramos-Rodriguez et al., 2006) it was determined that the EPN Steinernema riobrave controls storage pests at 32 °C better than some other species of EPNs. And this is because this species tolerates very high temperatures (up to 35 °C) and in such conditions demonstrates a very high rate of pathogenicity (Cabanillas et al., 1994). Such an EPN species has not been found in Europe yet. Until now it has only been found in the USA (Cabanillas et al, 1994). Previous research studies showed the efficacy of the EPN Steinernema feltiae in controlling some other storage pests from the order Coleoptera: Sitophilus granarius (Trdan et al., 2006), Tribolium confusum Jacquelin du Val (Athanassiou et al., 2007), Tenebrio molitor L., Tribolium castaneum (Herbst), Trogoderma variabile Ballion, Sitophilus oryzae (Ramos-Rodriguez et al., 2006), and Oryzaephilus surinamensis (Ramos-Rodriguez et al, 2006; Trdan et al., 2006). Some authors (Chen et al, 2003; Arthurs, 2004; Singh-Somvanshi, 2006) established that the efficacy of EPNs depends to a large extent on temperature and the concentration of the nematode suspension, but others established that only temperature influences the efficacy of EPNs (Trdan et al, 2008). Research studies have also shown that EPNs can more or less effectively control some other beetles (Jaworska & Ropek, 1996; Trdan et al, 2006, 2008), while their control of larvae is much more substantial (Pezowicz, 1992; Nadasy et al, 1999; Arthurs et al, 2004). All hitherto research has demonstrated that the sensitivity of insects to EPN activity is a complex process which does not depend only on the momentary vitality of the insect, but also on the developmental stage, size, and aggressiveness of the nematode species (Jian et al, 2002). Several insects developed mechanisms during their evolution which serve as protection against natural enemies, including EPNs. In this way it is known that some larvae from the family Scarabaeidae lower the possibility of infection from EPNs by more frequent excreta secretion, which lessens the ability of infective juveniles to enter through the anus. It is characteristic of some representatives from the order Diptera and Lepidoptera that the anus is closed by a muscle and the spiracles are covered by setae or are too narrow for the EPNs to enter. Some insects do not activate their defence mechanisms until the nematode has already entered into their body. They start to form hoops of melanin around the EPN in order to isolate it before it releases mutualistic bacteria into the host's hemolymph. In this way the isolated nematode decays and the insect eliminates it from its body (Koppenhoffer, 2000). Hitherto no indication of a similar defence mechanism in Sitophilus oryzae has been reported by any researcher, however we can attribute the lower efficacy of the studied strains of EPNs in our experiment also perhaps to special defence mechanisms of the studied insect. In Slovenia only the EPNs Steinernema feltiae (Laznik et al., 2008a) and Steinernema carpocapsae (Laznik et al., 2008b) have the status of indigenous species, while other discovered species of EPNs in Slovenia are for the present in the process of being removed from the national list of exotic species. That means that other species of EPNs in Slovenia for the time being can only be tested under laboratory conditions. But it is important to have in mind that results obtained from laboratory research can not be uncritically transferred to natural conditions, especially because of the many limiting factors which affect the survival of EPNs (Berry, 1993). In the future we will use the Steinernema feltiae 3162 strain in combination with biotechnical methods (Trdan et al., 2006) also in a trial in a grain warehouse, as the above mentioned strain demonstrated the greatest efficacy among the studied strains in our laboratory bioassay. 5 ACKNOWLEDGEMENT This work was done within Professional Tasks in the Field of Plant Protection, a program funded by the Ministry of Agriculture, Forestry, and Food of the Republic of Slovenia Phytosanitary Administration. This research was conducted in the framework of bilateral collaboration between Slovenia and Hungary (BI-HU/08-09-009). 6 REFERENCES Abbott, W.S. 1925. A method of computing the effectiveness of an insecticide. J. Econ. Entomol. 18: 265-267. Acuff, G. 1993. The scare that wasn't (National Academy of Science (NAS) report on pesticides in the diets of infants and children; editorial). Am. Fruit Grower 113: 34. Arthur, F.H. 1996. Grain Protectants: current status and prospects for the future. J. Stored Prod. Res. 32: 293-302. Arthurs, S., Heinz K.M., Prasifka J.R. 2004. An analysis of using entomopathogenic nematodes against above-ground pests. Bull. Entomol. Res. 94: 297-306 Athanassiou, C.G., Palyvos, N.E., Kakouli-Duarte, T. 2007. Insecticidal effect of Steinernema feltiae (Filipjev) (Nematoda: Steinernematidae) against Tribolium confusum du Val (Coleoptera: Tenebrionidae) and Ephestia kuehniella (Zeller) (Lepidoptera: Pyralidae) in stored wheat. J. Stored Prod. Res. 44: 52-57. Bedding R.A., Akhurst R.J. 1975. Simple technique for the detection of insect parasitic rhabditid nematodes in soil. Nematologica 21: 109-110. Belair, G., Fournier, Y., Dauphinais, N. 2003. Efficacy of steinernematid nematodes against three insect pests of crucifers in Quebec. J. Nematology 35: 259-265. Berry, E. C. & Lewis, L. C. 1993. "Interactions between nematodes and earthworms: Enhanced dispersal of Steinernema carpocapsae". J. Nematol. 25: 189-192. Brewer, M.S., Sprouls, G.K., Russon, C. 1994. Consumer attitudes toward food safety issues. J. Food Safety 14, 6376. Brown, I.M., Gaugler, R. 1996. Cold tolerance of steinernematid and heterorhabditid nematodes. J. Thermal Biol., 21: 115-121. Bryne, P.J., Bacon, J.R., Toensmeyer, U.C. 1994. Pesticide residue concerns and shopping location. Agribusiness 10: 41-50. Cabanillas, H.E., Poinar, G.O., Raulston, J.R. 1994. Steinernema riobravis n. sp. (Steinernematidae and Heterorhabditidae) infective-stage jumping behaviour. Nematology 4: 471-482. CGC. 1998. Canadian grains industry statistical handbook 98. Winnipeg, MB: Canada Grains Council: pp. 259. Chen, S., Li, J., Han, X., Moens, M. 2003. Effect of temperature on the pathogenicity of entomopathogenic nematodes (Steinernema and Heterorhabditis spp.) to Delia radicum. Biological Control 48: 713-724. Cuthbertson, A.G.S, Head, J., Walters, K.F.A., Gregory, S.A. 2003. The efficacy of the entomopathogenic nematode, Steinernema feltiae, against the immature stages of Bemisia tabaci. J. Invertebr. Pathol. 83: 267-269. Fields, P.G. 1992. The control of stored-product insects and mites with extreme temperatures. J. Stored Prod. Res. 28: 89-118. Forst, S., Clarke, D. 2002. Bacteria-nematode symbiosis. In: Entomopathogenic Nematology (Gaugler, R. ed.). Wallingford, CABI: 57-77. Gaugler R. 2002. Entomopathogenic Nematology. New Jersey, CABI Publishing: 372 p. Grewal, P.S., Selvan, S., Gaugler, R. 1994. Thermal adaptation of entomopathogenic nematodes: Niche breadth for infection, establishment, and reproduction. J. Thermal Biol.19: 245-253. Grewal, P.S., Grewal, S.K., Malik, V.S., Klein, M.G. 2002. Differences in susceptibility of introduced and native white grub species to entomopathogenic nematodes from various geographic localities. Biol. Control 24: 230-237. Haliscak, J.P., Beeman, R.W. 1983. Status of malathion resistance in five genera of beetles infesting farm-stored corn, wheat, and oats in the United States. J. Econ. Entomol. 76, 717-722. Hamel, D. 2007. Storing maize in stores and protection from pests. Glasnik biljne zaštite 7: 344-349. Hazir, S., Stock, S.P., Kaya, H.K., Koppenhofer A.M., Keskin, N. 2001. Developmental temperature effects on five geographic isolates of the entomopathogenic nematode Steinernema feltiae (Nematoda: Steinernematidae). J. Invertebr. Pathol. 77: 243-250. Helyer, N.L., Brobyn, P.J., Richardson, P.N., Edmonson, R.N. 1995. Control of western flower thrips (Frankliniella occidentalis Pergande) pupae in compost. Ann. App. Biol. 127: 405-412. Hosmand, A.R. 2006. Design of experiments for agriculture and the natural science (2nd ed.). Chapman & Hall, CRC Press. Howe, R.W. 1965. A summary of estimates of optimal and minimal conditions for population increase of some stored products insects. J. Stored Prod. Res. 1: 177-184. Ileleji, K.E., Maier, D.E., Woloshuk, C.P. 2004. Maize Weevil, Sitophilus zeamais (Motschulsky) adult Survival, reproduction and control in stored corn under three temperature management strategies (Summer 2003 PilotBin Trials). 2004 International Quality Grains Conference Proceedings: 1-11. Jaworska, M., Ropek, D. 1996. Effect of biotic factors on control of Sitona lineatus by nematodes. Bull. OILB/SROP 19: 124-127. Jian, F., Jayas, D.S., White, N.D.G., Muir, W.E. 2002. Temperature and geotaxis preference by Cryptolestes ferrugineus (Coleoptera: Laemophloeidae) adults in response to 5 °C/m temperature gradients at optimum and hot temperatures in stored wheat and their mortality at high temperature. Environ. Entomol. 31: 816-826. Journey, A.M., Ostlie, K.R. 2000. Biological control of western corn rootworm (Coleoptera: Crysomelidae) using the entomopathogenic nematode, Steinernema carpocapsae. Biol. Control 29: 822-831. Kaya, H.K., Gaugler, R. 1993. Entomopathogenic nematodes. Ann. Entomol. 38: 181-206. Kaya, H.K., Burlando, T.M., Thurston, G.S. 1993. Two entomopathogenic nematode species with different search strategies for insect suppression. Environ. Entomol. 22: 859-864. Kaya H.K. 2000. Entomopathogenic nematodes and their prospects for biological control in California. In: California conference on biological control. Hoddle M.S. (ed). Riverside, California: 38-46 p. Koppenhöfer A.M. 2000. Nematodes. In: Field manual of techniques in invertebrate pathology. Kaya H.K. (ed.). The Netherlands, Kluwer Academic Publishers: 283-301. Koppenhöfer, A.M., Fuzy, E.M. 2007. Soil moisture effects on infectivity and persistence of the entomopathogenic nematodes Steinernema scarabaei, S. glaseri, Heterorhabditis zealandica, and H. bacteriophora. Appl. Soil Ecol. 35: 128-139. Lacey, L., Bettencourt, A., Garrett, F., Simoes, N., Gaugler, R. 1993. Factors influencing parasitism of adult Japanese beetles by entomopathogenic nematodes. Entomophaga 38: 501-509. Laznik, Z, Toth, T., Lakatos, T., Trdan, S. 2008a. Entomopathogenic nematode Steinernema feltiae (Filipjev) (Rhabditida: Steinernematidae) recorded for the first time in Slovenia. Acta Agric. Slovenica 91: 37-45. Laznik, Z, Toth, T., Lakatos, T., Trdan, S. 2008b. Entomopathogenic nematode Steinernema carpocapsae (Weiser) (Rhabditida: Steinernematidae), a new member of Slovenian fauna. Acta Agric. Slovenica 91: 351-359. Nadasy, M., Saringer, G.Y., Lucskai, A., Fodor, A., Samu, Z., Ignacz, J., Budai, C.S., Klein, M. 1999. Effect of entomopathogenic nematodes from the genera Steinernema and Heterorhabditis on caterpillars of two pest insect species (Pieris brassicae L. and Mamestra brassicae L.) that damage cruciferous vegetable crops. IOBC Bull. 22: 127-135 Neethirajan, S., Karunakaran, C., Jayas, D.S., White, N.D.G. 2007. Detection techniques for stored-product insects in grain. Food Control 18: 157-162. Ott, S.L. 1990. Supermarket shoppers' pesticide concerns and willingness to purchase certified pesticide residue-free fresh produce. Agribusiness 6: 593-602. Peel, M.C., Finlayson, B.L., McMahon, T.A. 2007. Updated world map of the Köppen-Geiger climate classification. Hydrol. Earth Syst. Sci. 11: 1633-1644. Pezowicz, E. 1992. Migrations of invasive larvae of Steinernema carpocapsae (Weiser) towards their host insects. Entomonematologia 2: 7-15. Ramos-Rodriguez, O., Campbell, J.F., Ramaswamy, S.B. 2006. Pathogenicity of three species of entomopathogenic nematodes to some major stored-product insect pests. J. Stored Prod. Res. 42: 241-252. Ramos-Rodriguez, O., Campbell, J.F., Ramaswamy, S.B. 2007. Efficacy of the entomopathogenic nematode Steinernema riobrave against the stored-product insect pests Tribolium castaneum and Plodia interpunctella. Biol. Control 40: 15-21. Rees, D. 1998. Pest trends in the Australian grain bulk handling system. Australian Postharvest Technical Conference: 39-42. Schöller, M., Flinn, P.W., Grieshop, M.J., Zd'arkova, E. 2006. Biological control of stored product pests. In: Heaps, J. (Ed.), Insect management for food storage and processing, second ed. American Association of Cereal Chemists, pp. 67-87. Scheepmaker, J.W.A., Geels, F.P., Griensven, L.J.L.D., Smits, P.H. 1998. Susceptibility of larvae of the mushroom fly Megaselia halterata to the entomopathogenic nematode Steinernema feltiae in bioassays. BioControl 43: 201214. Singh-Somvanshi, V., Ganguly, S., Paul, A.V.N. 2006. Field efficacy of the entomopathogenic nematode Steinernema thermophilum Ganguly and Singh (Rhabditida: Steinernematidae) against diamondback moth (Plutella xylostella L.) infesting cabbage. Biol. Control 37: 9-15. Smart G.C. Jr. 1995. Entomopathogenic nematodes for the biological control of insects. J. Nematol. 27: 529-534. South, L. 1993. Believe it: kids are safe (no link found between pesticides on produce and disease in children). Am. Vegetable Grower 41: 45-46. Stejskal, V., Hubert, J., Kucerova, Z., Munzbergova, Z., Lukas, J., Zd'arkova, E. 2003. The influence of the type of storage on pest infestion of stored grain in the Czech Republic. Plant Soil and Environ. 49: 55-62. Stejskal, V., Kosina, P., Kanyomeka, L. 2006. Arthropod pests and their natural enemies in stored crops in northern Namibia. J. Pest Sci. 79: 51-55. Svendsen, T.S., Steenberg, T. 2000. The potential use of entomopathogenic nematodes against Typhaea stercorea. Biocontrol 45: 97-111. Toepfer, S., Gueldenzoph, C., Ehlers, R.-U., Kuhlmann, U. 2005. Screening of entomopathogenic nematodes for virulence against the invasive western corn rootworm, Diabrotica virgifera virgifera (Coleoptera: Chrysomelidae) in Europe. Bull. Entomol. Res. 95: 473482. Toth, T. 2006. Collection of entomopathogenic nematodes for the biological control of insect pests. J. Fruit Ornam. Plant Res. 14: 225-230. Trdan, S., Vidrih M., Valic N. 2006. Activity of four entomopathogenic nematode species against young adults of Sitophilus granarius (Coleoptera: Curculionidae) and Oryzaephilus surinamensis (Coleoptera: Silvanidae) under laboratory conditions. J. Plant Dis. Prot. 113: 168173. Trdan, S., Znidarcic, D., Vidrih, M. 2007. Control of Frankliniella occidentalis on greenhouse-grown cucumbers: an efficacy comparison of foliar application of Steinernema feltiae and spraying with abamectin. Russ. J. Nematol.15: 25-34. Trdan, S., Vidrih, M., Valic, N., Laznik, Z. 2008. Impact of entomopathogenic nematodes on adults of Phyllotreta spp. (Coleoptera: Chrysomelidae) under laboratory conditions. Acta Agri. Scand. Section B - Soil and Plant Sci. 58: 169-175. van der Burgt, W.A.C.M., Budai, C., Fodor, A., Lucskai, A. 1998. Parasitism of western corn rootworm adults by Steinernema spp. and Heterorhabditis spp. Proceedings of the Section Experimental and Applied Entomology of the Netherlands Entomological Society 9: 165-170. van Graver, S.J., Winks, R.G. 1994. A brief history of the entomological problems of wheat storage in Australia. In: Stored-product protection. Proceedings of the 6th International Conference on Stored-product Protection. Highley E., Wright E.J., Banks H.J. & Champ B.R. (eds.), Canberra, Australia. 2, 1250-1258, CAB International, Wallingford, UK. COBISS Code 1.01 Agrovoc descriptors: rotational cropping, hops, humulus lupulus, plant protection, land management, integrated plant production, farms, animal husbandry, farm size, Agris category code: F08, E11 Hop fields in crop rotation Darja KOCJAN ACKO1 Received August 10, 2009; accepted January 20, 2010. Delo je prispelo10. avgusta 2009; sprejeto 20. januarja 2010. ABSTRACT A number of factors influenced the increased number of crops on Slovenian farms legal limitation of maize production to two years on the same field, introduction of integrated crop production and certain measures of the Slovene Agri-Environmental Programme. Crop rotation on hop fields that has already been analyzed on 22 hop growing and livestock farms in Spodnja Savinjska dolina in 2008 show that the maize monoculture has been supplemented by some other crops like wheat, barley, lucerne, clover-grass mixtures and high beans; compared to the conventional production in the past, the crop rotation is now longer. Phytosanitary adequacy of two- and three-year rotation could be improved with supplementary crops, that is with fodder crucifers (fodder rapeseed and rape, forage kale), green manure (white mustard, oil radish), legume crops (soya, peas, vetch, field bean, white lupin, crimson clover, Persian clover, Egyptian clover) and compound fodders like Landsberger mixture and mixtures of vetch with oat or barley. Prohibition to sow broad-leafed plants in the quarantine rotation due to hop wilting limits the selection of crops to grasses (Italian rye-grass) and fodder grains (maize, barley), but the farmers could increase the selection of grains with oats, rye, triticale, mixture of wheat and rye, millet, sorghum and Sudan grass. With the use of recommended crop rotations in two-, three- and four-year rotation, we can expect the increased influence of crop rotation on improved health condition of the new hop plantations in the future with simultaneous reduction of the use of pesticides as well. Key words: hop field, crop rotation, length of crop rotation, species in the crop rotation, phytosanitary importance of crop rotation, fodder supplementary crops, integrated crop production, hop growing and livestock farms IZVLEČEK HMELJIŠČA V PREMENI Zakonsko omejena pridelava koruze na dve leti, uvedba integrirane pridelave in nekateri ukrepi slovenskega kmetijskega okoljskega programa so vzrok, da se povečuje število poljščin na kmetijah. Premena na hmeljiščih, ki smo jo leta 2008 analizirali na 22 hmeljarsko-živinorejskih kmetijah v Spodnji Savinjski dolini, kaže, da so monokulturo koruze razbremenili posevki pšenice, ječmena, lucerne, deteljno-travne mešanice in visoki fižol, premena pa je daljša kot pri konvencionalni pridelavi v preteklosti. Fitosanitarno ustreznost dve- in triletne premene bi lahko izboljšali z dosevki, to je križnicami za krmo (krmna ogrščica in repica, krmni ohrovt) in za podor (bela gorjušica, oljna redkev), metuljnicami (soja, grah, grašica, bob, bela lupina, inkarnatka, perzijska in aleksandrijska detelja) in krmnimi mešanicami, kot so grašljinka (= landsberška mešanica), ovsiga (oves + grašica) in ječmiga (ječmen + grašica). Prepoved setve širokolistnih rastlin v karantenski premeni zaradi hmeljeve uvelosti omejuje posevke na trave (mnogocvetna ljuljka) in krmna žita (koruza, ječmen), kljub temu pa bi kmetje lahko povečali izbor žit z ovsom, ržjo, tritikalo, soržico, prosom, sirkom in sudansko travo. Pri uporabi predlaganih kolobarjev v dve-, tri- in štiriletni premeni lahko v prihodnje pričakujemo večji vpliv premene na boljše zdravstveno stanje novega nasada hmelja ob hkratnem zmanjšanju porabe fitofarmacevtskih sredstev. Ključne besede: hmeljišče, premena, dolžina premene, vrste poljščin v premeni, fitosanitarni pomen premene, krmni dosevki, integrirana pridelava, hmeljarsko-živinorejske kmetije 1 INTRODUCTION Hop (Humulus lupulus L.) is a perennial crop and a years of exploitation period on modern plantations. In creeper with 40 years lifespan and twelve to fifteen Slovenia it is recently produced on approximately 1600 1 Assis. Prof. Ph.D., Jamnikarjeva 101, SI-1111 Ljubljana, e-mail: darja.kocjan@bf.uni-lj.si ha, mainly in Savinjska dolina, the average yield of cones is 1400 kg/ha (Statistical yearbook, 2008; Statistic information, 2009). Production is based on experience of generations of hop growers and supported by technological advice from the professional experts of the Slovenian Institute for hops research and brewing. Slovene hops, valued by brewers for its high quality biter resins (a-acids), is almost all (90%) sold abroad. The technology of hop production is increasingly oriented in sustainable production and practice shows that a number of hop producing farms has already reached and fulfilled the demands of integrated production and gained the status of integrated producer (Rožič Plazovnik et al., 2008). Breeding of domestic animals on hop growing farms is traditionally connected with fertilization of hop fields. As a root crop, hop needs a lot of organic matter or humus for optimum growth and manure also improves the quality of the soil. Similar to other monoculture crop production hop monoculture also is a cause of higher portion of specific pathogens and pests and increased weediness (Friškovec in Škerbot, 2007). Previous experiences have shown that some time off is needed between the time of grubbing-up the old degenerated plantation and planting the new one. The term »rotation« (alternation, replacement; Slovar..., 1993) was recognised and established for the field that is temporary intended for the production of other culture(-s). In the past hop growers regularly used crop rotation and the duration of the rotation was up to seven years (Sadar, 1961). Phytosanitary benefits of the rotation are increasing with its duration, but only if the broad-leafed plants are being replaced by spring grains and crops from different botanic families (Spanring, 1959; Sadar, 1961; Kocjan Ačko, 1992, 2002, 2004; Plazovnik Rožič, 2008). In addition to the usual crop rotation, the quarantine rotation is also well known in practice and is used after the destruction of the infected hop production. Only crops that do not serve as a host to the hop pathogens (Zakon o..., 2001) can be used in this rotation. The duration of the quarantine rotation is determined according to the survival capacity of the fungus in the soil without a presence of the host plants (Radišek et al., 2006). The quarantine rotation introduced as a consequence of hops wilting, caused by the funghy Verticillium albo-atrum Reinke at Berthold and Verticillium dahliae Klebahn, shall last at least four years and only narrow-leaf non-host plants, such as spring grains, maize and grasses can be used in this crop rotation (Čerenak et al., 1999). In the time of intensive specialization and intensive use of chemicals, experts (Oset, 1991; Veronek, 1995; Friškovec et al., 2002) have been strongly recommending at least two year rotation before planting the virus free seedlings, but on the other side the economic interest forced hop growers to use the shortest rotation possible (Veronek, 1997). Plants used for the rotation give a lot of organic mass for green manure and need cultivation to improve soil structure and air and water retention capacity, they also increase the share of humus and on the other side reduce soil hardness, weediness and the number of pathogens and pests. It is also highly beneficial that the main crop is followed by a supplementary one that remains on the field till the next main crop. 2 MATERIAL AND METHODS We have studied the crop rotation on hop fields in 2008 by visiting twenty-two selected farms in Spodnja Savinjska dolina. Farmers, skilled hop growers and livestock breeders have answered our questions and provided information on the size of their fields, crops, crop rotation, fertilization and livestock production. Based on these notes we have determined the duration of crop rotation, which crops are used in the rotation and calculated the total production of individual crops. We believed that important differences would be detected between the farms in the duration of rotation and in the number and species of crops used for the rotation. Due to the livestock production we expected that the fodder crops would be predominantly used in the rotation instead of the crops for human consumption. We have processed the answers with methods of analysis, synthesis, generalization and specialization and used referrals to domestic and foreign realizations, principles of crop sequence and results of similar research. Using the correct order of rotation we have composed two-, three and four-year biologically balanced crop rotations that can bring a lot of benefits to hop growers in the future. 2.1 Description of hop growing and livestock farms in Savinjska dolina Hops is produced on all farms that we have studied, being the main crop on half of them; twenty-one farms also have the livestock production which reflects the close interconnectedness of both types of production. On these hop and livestock producing farms there is 276.7 ha of hop fields, 192.7 ha of other crops (80 ha less than hops) and the least -107.1 ha of meadows. Our calculations show that the average arable land per individual farm is 23.6 ha, out of that - 12.5 ha is under hops. More than half of farms have over thirty ha of land including forests with the average of 6.5 ha per farm. On the majority of farms (82%) there is over one hundred years tradition in hop growing, so the family members have different generation connections to various technologies of hop growing and are excellent connoisseurs of hop growing history in Savinjska dolina. Due to financial benefits more than half of farms have fields and hop plantations included in the measures of integrated crop production. On the livestock farms they breed young male bovine animals for fattening (total of 269 animals), the total number of milking cows is 197, total number of calves - 100 and 90 breeding heifers. Seventeen farms have pigs and five have horses. There is an intensive poultry production on two farms - turkeys in two and chickens in five terms. To fertilize hop fields all farms mostly use manure, six of them using liquid manure and slurry. Twenty farms use mineral fertilizers during the growing period, mainly nitrogen, till now - green manure was sown only on six farms. 3 RESULTS AND DISCUSSION 3.1 Length of crop rotation and number of crops in the rotation More than half of farmers grubb-up hops at fifteen years of age, the rest of them at twenty years. The majority of plantations have been grubbed due to reduced yields and low fertility of older plantations, also the introduction of a new variety is a common reason for grubbing old plantations. Introduction of quarantine rotation is becoming more common cause for grubbing the whole or part of the plantation - we have recorded it on thirteen farms. Despite knowing and realising the phitosanitary importance of a longer crop rotation on new hop plantations, fourteen growers used two-year rotation and seven growers used three-year crop rotation. One-year rotation was recorded at one grower who only works in plant production. The main cause for prolonging the time of the rotation is the introduction of measures of integrated crop production that demand two-year rotation (Tehnološka navodila za IPP, 2008). Under the conditions of market economy the short term economic interest contradicts the recommendations of experts for the multiannual rotation that is why it will not be easy for the hop growers to fully implement and use the crop rotation. On the studied farms they produce two to four crops within the rotation, mostly as the main crops. For closed and biologically harmonised crop order the supplementary crops, mostly fodder, manure and supplementary crops are missing from the rotation. 3.2 Species of crops in crop rotation Studied farms are different also considering the species of crops used within the rotation influenced mainly by the type and age of animals they breed, the purpose of their production and the duration of the rotation. Although maize (Zea mays L.) and other root crops as sugar beet (Beta vulgaris L. var. altissima) use a lot of nutrients from the soil and due to the late harvest with heavy machinery destroy the soil structure, maize is energetically and in terms of income an irreplaceable fodder crop. It is sown on one third of hop fields in rotation (31%), out of which 23% of land is used for the production of silage maize. We assume that the western corn rootworm (Diabrotica virgifera virgifera LeConte), which is the cause for legal limitation for the maize production of two years on the same grounds, is less harmful to the maize being sown within the crop rotation compared to the production outside the hop fields. It is true that corn borer (Ostrinia nubilalis L.) also affects maize and hops. Unploughed maize and hop remains are the cause of extensive multiplication of corn borer larvae that can also cause damage on beans, peppers, tomatoes and some ornamental plants (Majer and Zolnir, 2002; Rak Cizej et al., 2009). On some fields in crop rotation juicy remains of corn stalks in the ground have been the cause of increased numbers of May beetle larvae (Melolontha melolontha L.) during some previous years. From grains (23.5% of fields) wheat (Triticum aestivum L.) and barley (Hordeum vulgare L.) are the dominant ones within the rotation, mostly used as pig or poultry fodder. With the exception of few fields of spring barley and oats there was no rye (Secale cereale L.), triticale (Triticosecale Wittm.) or mixture of wheat and rye in the crop rotation. Millet (Panicum miliaceum L.), maize, cut green as grass, sorghum (Sorghum bicolor L.) and Sudan grass (Sorghum sudanense (Piper) Stapf.) could be sown as supplementary crops. The use of high beans (Phaseolus vulgare L.) in the rotation (20.5%) is important not only for its favourable influence on soil fertility, but also for the use of hop wires. Jabelski strocnik variety of beans (known to consumers and farmers also as »the grey bean«) is becoming very popular for its culinary quality and for the income that it brings. High beans are economically important crop in the rotation on fourteen out of twenty-two farms. Hop growers do not have much work growing these beans, late maturity is also a good feature since it ripens after hop is already picked and maize has been silaged. Other legumes could also be tested for sowing in the rotation, such as field peas (Pisum sativum L.), especially varieties with tendrils so it needs no support, vetch (Vicia sp. L.), soya (Glycine max L.), field bean (Vicia faba L.) and white lupin (Lupinus albus L.). Varieties with shorter growing season or those sown in combination with other crops that can be used as hay or silaged in vax maturity of grains are more suitable for the crop rotation. Fodder legumes, on their own or in mixtures with grasses, are now used on 10.3% of fields in rotation. For lucerne (Medicago sativa L.) with four-to five-year use, the optimum use exceeds the duration of rotation that is the reason that it is rarely used on the fields in crop rotation. In addition to favourable characteristics for the soil fertility, also detrimental affects of the lucerne crop on a new hop plantation can be detected. There is an increased danger of underground pests, such as vole (Arvicola terrestis L.), that remains in the ground after planting of a new hop plantation and in numerous cases destroys a plantation by gnawing the hop rootstock. More suitable selection would be red clover (Trifolium pratense L.) and white clover (Trifolium repens L.) with slightly shorter exploitation period. The selection of fodder legumes for sowing in the rotation could be variegated by supplementary crops with short growing period such as crimson clover (Trilolium incarnatim L.), Persian clover (Trifolium resupinatum L.), Egyptian clover (Trifolium alexandrinum L.) and Landsberger mixture, that is the mixture of crimson clover, vetch and Italian rye-grass. With the goal to produce more protein feeding stuffs on their own fields, the growers in Savinjska dolina could again start sowing previously known mixtures of common or winter vetch with grains like oats, barley, rye, wheat or triticale. The oilseeds - pumpkin (Cucurbita pepo L.) and rapeseed (Brassica napus L. var. napus) and root crops -sugar beet and cabbage (Brassica oleracea L.) were only seen on few fields - 14.7% of total fields in rotation. After closure of the Ormož sugar factory in 2005, farmers stopped growing sugar beet. At first it seemed that the rapeseed will replace the sugar beet on Slovene fields (Friškovec in Škerbot, 2007), but the production of rapeseed for biodiesel did not take on on hop farms. Compared to rapeseed production for biodiesel - fodder cruciferous like winter fodder rapeseed (Brassica napus L.), turnip rape (Brassica rapa L.) and forage kale (Brassica oleracea L. convar. acephala) , and also root crops like oil radish (Raphanus sativus L. var. oleiformis Pers.) and white mustard (Sinapis alba L.) have proven to be more interesting crops for livestock farms. For green manure phacelia (Phacelia tanacetifolia Benth.) could also be used. Fooder rapeseed was used for the green manure in the crop rotation only on one farm, but as a green manure -it is valued on hop plantations when sown between the rows. Pumpkins for production of oil are grown on two farms; they used the pumpkin oil at home or sold it, and feed the pericarp and fleshing to the pigs. On one farm they started shelling pumpkin seeds and selling them fresh or toasted. All farmers are informed about the quarantine rotation. Due to the hop wilting it was introduced on thirteen farms, that is more than half (59%) of all studied farms. Winter wheat and winter barley are the prevailing crops in the quarantine rotation, followed by maize and sown grass, mainly Italian rye-grass (Lolium multiflorum Lam.). Farmers could also include oats, rye, triticale, mixture of wheat and rye, millet, sorghum and Sudan grass. 3.3 Proposals for the crop rotations on hop fields Although the multiannual grasses and clovers have most favourable influence on physical, chemical and biological structure of the soil, their growing period hinders thorough processing and sanitation of fields due to certain pests and disease agents on hops. Since the duration of the rotation will not be importantly prolonged in the future, we have set up six recommended rotations for two-, three- and four year rotation (Tables 1, 2 and 3). Four year rotation (in the 1st and 4th case) follows the example of biologically harmonised Norfolk crop rotation with 50% share of cereals and 25% share of root crops and legumes (Butorac, 1999; Diepenbrock et al., 2005). Table 1: Recommended crop rotations for two-year crop rotations on hop fields Order of rotation 1. rotation 2. rotation 3. rotation 1. year winter wheat or triticale or rye + fodder rape or white mustard for the green manure grain maize or silage maize oil pumpkins or cabbage 2. year winter barley + Landsberger mixture spring barley or spring oats + winter fodder rapeseed or oil radish for green manure spring oats + fodder rapeseed for animal feed or green manure, in case of cabbage -phacelia for green manure (also honey plant) Order of 4. rotation 5. rotation 6. rotation rotation 1. year spring barley + red clover (sub-crop) spring or winter barley + maize for green forage or sorghum or Sudan grass triticale or rye + phacelia for green manure (also honey plant) 2. year red clover high beans or soya or field beans or white lupin for grains mixed crop of field peas and barley for grains or field peas with tendrils as pure crop Table 2: Recommended crop rotations for three-year crop rotations on hop fields Order of 1. rotation 2. rotation 3. rotation rotation 1. year winter wheat + forage kale or fooder rapeseed grain maize or silage maize oil pumpkins or cabbage 2. year high beans spring barley + millet spring oats + white mustard for the green manure 3. year spring barley or oats + phacelia for green manure mixture of oat and vetch + fodder rapeseed Persian or Egyptian clover Order of 4. rotation 5. rotation 6. rotation rotation 1. year winter wheat + forage kale grain or silage maize oil pumpkins 2. year spring barley + red clover (sub-crop) high beans or soya or field bean for grains mixed crop of fodder peas and grains barley or fodder peas with tendrils as pure crop 3. year red clover spring or winter barley + Landsberger mixture Winter rye or triticale + phacelia for the green manure (also honey plant) Table 3: Recommended crop rotations for four-year crop rotations on hop fields Order of rotation 1. rotation 2. rotation 3. rotation 1. year grain or silage maize + crimson clover grain or silage maize Cabbage 2. year spring barley + sorghum or Sudan grass for forage spring barley + fodder carrots spring oats + turnip 3. year high beans or grain soya Persian clover spring oat and common vetch 4. year winter wheat or triticale + forage kale or fodder rapeseed or oil radish for the green manure winter wheat or triticale + phacelia or white mustard for the green manure winter triticale or rye + forage kale or fooder rapeseed Order of rotation 4. rotation 5. rotation 6. rotation 1. year grain or silage maize grain or silage maize oil pumpkins 2. year spring barley + red clover (sub-crop) high beans or soya or field bean for grains winter wheat + Italian ryegrass or grass-clover mixture 3. year red clover grain or silage maize Italian rye-grass or grass-clover mixture 4. year winter wheat + phacelia spring or winter barley + Landsberger mixture spring barley + oil radish for the green manure When looking for new crops that could be included in the crop rotation some people are contemplating the introduction of hemp (Cannabis sativa L.) that belongs to the same botanic family (Cannabaceae) as hops. We have to mention the increased danger of hemp flea beetle (Psylliodes attenuatus Koch) attack that can completely destroy the first year plantation (Friskovec et al., 2002). 4 CONCLUSIONS Following are the conclusions we have reached based on the analysis of the crop rotation and crops used in the rotation on hop fields: • In recent years the hop growers gave increased attention to improvement of the soil fertility after grubbing up the old plantations and to all necessary phytosanitary measures before planting a new plantation. They are prolonging the crop rotation; on more than half of studied hop growing and livestock farms they used two-year rotation, on seven farms they used three-year rotation and one-year rotation on only one farm. • Since 2003, when majority of farms joined the measures of integrated crop production, the selection of crops in the rotation is improving. Before that year mostly maize was sown in the rotation, which further depleted and wearied the soil, now - due to the environmental measures - farmers also decide to sow the other cereals, beans, grasses, clover-grass mixtures, lucerne, pumpkins and cabbage. • Despite the fact that maize as a root crop is not best suited for the crop rotation, due to the livestock production it still remains the most sown crop, on second place we can find barley and wheat, mostly used for animal feed. The production of high beans is becoming more important; hop growers can use hop wires for its production. Also other grain legumes, like field peas with tendrils, field beans and soya as pure crops or mixed with cereals could serve as source of proteins in animal feed. Hop growers should only be informed of the technology to reduce the fear of technical difficulties of their production and harvest. Multiannual hay crops (lucerne, mixtures of grasses and clovers) could be substituted or supplemented with fodder crops, that is with fodder crucifers (fodder rapeseed and rape, forage kale) and green manure (white mustard, oil radish), legumes (soya, field beans, vetch, phacelia, crimson clover, Persian or Egyptian clover) and fodder mixtures like Landsberger mixture and mixture of vetch with some cereals. Introduction of winter oil rapeseed as a favourable crop in the rotation shows that we could not successfully establish rapeseed production for biodiesel. Hop growers realised the positive influence of sowing fodder rapeseed between the rows against weeds, erosion and for green manure. Most likely they will use it for these purposes also in the future (Ur. 1. RS st. 18/07 in 124/07). Wheat and barley are the main crops in the quarantine rotation, maize is on the second place and grasses and grass mixtures on the third. Selection of narrow-leafed crops could be increased by oats, rye, triticale, mixture of wheat and rye, millet, sorghum and Sudan grass, some of them sown as additional crops. 5 REFERENCES Butorac, A. 1999. Sustavi biljne proizvodnje. V: Opca agronomija, Školska knjiga, Zagreb: 537-574. Diepenbrock, W., Ellmer, F., Leon, J. 2005. Ackerbau, Pflanzenbau und Pflanzenzüchtung. Verlag Eugen Ulmer Stuttart, 366 str. Bodennutzungssysteme: 31-87. Čerenak, A., Dolinar, M., Rak, M. 1999. Izolacija in identifikacija povzročiteljic hmeljeve uvelosti (Verticillium albo-atrum Reinke & Berthold in Verticillium dahlie Klebahn). Hmeljar, 3-4: 37-38. Friškovec I., Škerbot I. 2007. Oljna ogrščica kot posevek v premeni. V: 44. seminar o hmeljarstvu, Žalec, 16. feb. 2007. Žalec, IHPS: 11. Friškovec, I., Zmrzlak, M., Knapič, M. 2002. Zasnova novega nasada. V: Priročnik za hmeljarje. Majer D. (ur.). Žalec, IHPS: 137-145. Kocjan Ačko, D. 1992. Kolobar. V: Kmetijski priročnik, ČZD Kmečki glas: 116-127. Kocjan Ačko, D. 2002. Mikrobiologija tal in kolobar. Sklop 6: Sonaravno kmetijstvo in gozdarstvo. V: Svetovanje v kmetijstvu in gozdarstvu: 8 p. Kocjan Ačko, D. 2004. Izročilo prednikov predaleč, lastnih izkušenj premalo. Sodobno kmetijstvo, 4: 37-39. Majer, D. Žolnir, M. 2002. Priročnik za hmeljarje. Hmeljevi škodljivci. Žalec, Inštitut za hmeljarstvo in pivovarstvo: 70-71. Oset, F. 1991. Hmeljni kolobar. Hmeljar, 61 avgust: 34. Pravilnik fitosanitarnih ukrepih za preprečevanje širjenja koruznega hrošča. Ur. l. RS, št. 21/2004 in št. 106/2006. Program razvoja podeželja Republike Slovenije za obdobje 2007-2013. 2006. MKGP, 320 s. Uredba o plačilih za kmetijsko okoljske ukrepe iz Programa razvoja podeželja Republike Slovenije 2004-2006 v letih 2007-2010. Ur. l. RS, št. 19/07 in 124/07. Radišek, S., Leskošek, G., Žveplan, S., Zmrzlak, M., Knapič, V. 2006. Hmeljeva uvelost v slovenskih hmeljiščih. Žalec, IHPS, Oddelek za varstvo rastlin: 19-20. Rak Cizej, M., Leskošek G., Radišek S. 2009. Koruzna vešča v slovenskih hmeljiščih. 46. seminar o hmeljarstvu z mednarodno udeležbo, zbornik seminarja, Portorož, 2009. Inštitut za hmeljarstvo in pivovarstvo Slovenije: 107-113. Rožič Plazovnik, M. 2008. Krmne poljščine v slovenskih hmeljiščih v premeni. Dipl. delo. Mentorica: Darja Kocjan Ačko, Biotehniška fakulteta, Oddelek za zootehniko, 52 s. Rožič Plazovnik, M., Kocjan Ačko D., Šantavec I. 2008. Premena v hmeljiščih. Novi izzivi v poljedelstvu 2008, Zbornik simpozija, Slovensko agronomsko društvo, Rogaška Slatina: 68-73. Slovar slovenskega knjižnega jezika. 1993. Ljubljana, Državna založba Slovenije: 1000 Statistični letopis Republike Slovenije 2007. Statistični urad republike Slovenije, Ljubljana: 294 Statistične informacije. Kmetijstvo in ribištvo. 2008. Statistični urad republike Slovenije, Ljubljana: št. 14-15. Sadar, V. 1961. Poljski kolobar in kolobarjenje. Univerza v Ljubljani, Fakulteta za agronomijo, gozdarstvo in veterinarstvo, 104 s. Spanring, J. 1959. Pregled poljščin in predlog nekaterih kolobarjev za Slovenijo. Kmetijski inštitut Slovenije, 24 s. Tehnološka navodila za integrirano pridelavo poljščin. 2008. MKGP: 43-49. Ur. l. RS št. 18/07 in 124/07 Veronek, M. 1997. Premena hmelj za hmeljem kot včasih. Hmeljar, 1-2: 15-17. Zakon o zdravstvenem varstvu rastlin. Ur. l. RS št. 454990/01. COBISS Code 1.01 Agrovoc descriptors: apples, malus pumila, fruit, fruits, thinning, defruiting, cultivation, flowering, flowers, diameter, dimensions, plant developmental stages, phenology Agris category code: F50, F62 Effect of 6-benzyladenine application time on apple thinning of cv. 'Golden Delicious' and cv. 'Idared' Barbara AMBROZIC TURK1, Matej STOPAR2 Received: July 21, 2009; accepted: January 18, 2010. Delo je prispelo: 21. julija 2009; sprejeto: 18. januarja 2010. ABSTRACT Apple thinning of cv. 'Golden Delicious' and cv. 'Idared' was studied after the application of 6-benzyladenine (BA) 100 mgl-1 and 50 mgl-1 respectively, applied at different time of blooming period and over a wider range of fruitlet diameter. When BA 100 mgl-1 was applied on cv. 'Golden Delicious', significant reduction in final fruit number per tree was observed if applied at the end of blooming period or when fruitlet diameters were 6 mm, 15 mm or 20 mm, but thinning was not significant if applied at full bloom or at 10 mm or 25 mm fruitlet diameter. The share of bigger fruits of cv. 'Golden Delicious' (> 65mm) was increased at all BA application time treatments, but significantly only in the treatment of hand thinned trees. Regarding the BA applications carried out on cv. 'Idared', significant thinning response was noticed in the case of BA sprayed at the end of bloom and at 10 mm of fruit diameter. The mean fruit weight of cv. 'Idared' apples was increased significantly only if BA was applied at 10 mm fruitlet diameter. Although the thinning results were inconsistent for two studied cultivars it was demonstrated from the trial that BA could be active as a thinner if applied over a wider phenological interval, at the end of blooming period up to 20 mm fruitlet diameter. Key words: apple (Malus x domestica Borkh.), 6-benzyladenine, flower thinning, fruit thinning, fruitlet diameter IZVLEČEK VPLIV ČASA APLIKACIJE 6-BENZILADENINA NA REDČENJE PLODIČEV JABLANE PRI SORTAH ZLATI DELIŠES' IN 'IDARED' Redčenje plodičev jablane smo proučevali po aplikaciji pripravka 6-benziladenina (BA) v koncentraciji 100 mgl-1 pri sorti 'Zlati delišes' ter 50 mgl-1 pri sorti 'Idared', škropljenega ob različnem času cvetenja ter pri različni velikosti plodičev. Pri sorti 'Zlati delišes' je tretiranje z BA vplivalo na statistično značilno zmanjšanje števila plodov ob obiranju, če je bilo izvedeno ob koncu cvetenja ter pri velikosti plodičev 6 mm, 15 mm in 20 mm, medtem ko v primeru tretiranja v času polnega cvetenja ter pri velikosti plodičev 10 mm in 25 mm redčenje ni bilo statistično značilno. Pri sorti 'Zlati delišes' se je delež plodov večjih od > 65 mm sicer povečal pri vseh časovnih BA aplikacijah, vendar je bilo povečanje deleža le-teh statistično značilno le v primeru obravnavanja z ročnim redčenjem. Pri sorti 'Idared' je tretiranje z BA v različnem času povzročilo statistično značilno redčenje pri aplikaciji BA ob koncu cvetenja ter pri 10 mm premera plodičev. Povprečna masa plodov ob obiranju pri sorti 'Idared' pa se je statistično značilno povečala le pri tretiranju z BA pri 10 mm premera plodičev. Kljub temu, da se rezultati redčenja razlikujejo pri posameznih BA obravnavanjih med proučevanima sortama, je iz poskusa razvidno, da je BA učinkovito sredstvo za redčenje plodičev pri uporabi v širšem fenološkem intervalu, od konca cvetenja pa do 20 mm premera plodičev. Ključne besede: jablana (Malus x domestica Borkh.), 6-benziladenin, redčenje cvetov, redčenje plodičev, premer plodičev 1 M.Sc., Agricultural Institute of Slovenia, Hacquetova 17, SI-1000 Ljubljana, e-mail: barbara.a.turk@kis.si 2 Ph.D., Agricultural Institute of Slovenia, Hacquetova 17, SI-1000 Ljubljana, e-mail: matej.stopar@kis.si 1 INTRODUCTION Thinning of flowers or fruitlets is the most important technique in apple growing practice to improve fruit quality, increase return bloom and reduce biennial cropping (Link, 2000). Chemicals and their concentrations, the timing of their application, the environmental factors encountered before, during and after application as well as tree factors, they all influence the ultimate thinning response (Greene, 2002). The cytokinin 6-benzyladenine (BA) has been suggested to be used as an effective thinning compound for apples (McLaughlin and Greene, 1984). BA increased the fruit weight as a result of reducing crop load (ElfVing and Cline, 1993; Ferree, 1996), but it also increased the fruit weight additionally, beyond the effect attributable to chemical thinning (Greene, 1993; Greene, 2005). BA influenced larger fruit size or weight by increasing the number of cells per fruit through the stimulation of cell division (Wismer et al., 1995). It is also well documented in the literature that thinning with BA had a positive effect on increasing return bloom the following year (Greene et al., 1990; Bound et al., 1991; ElfVing and Cline, 1993; Wismer et al., 1995). The studies have shown that both, concentrations and timing of application, can influence the thinning effectiveness of BA (Greene et al., 1990). Although it was reported that BA had a thinning response over a wider range of fruitlet diameter (ElfVing and Cline, 1993), it was mostly demonstrated that BA was the most effective when applied at about 10 mm of fruitlet diameter (Greene, 1993; Basak, 1996; Stopar and Lokar, 2003). However, the variable thinning response of BA may be attributed, partly, to the differences in environmental conditions at the time of application. Bound et al. (1997) indicated that BA thinning efficacy is dependent on temperature at the time of application, with the lower levels of thinning achieved when the temperature was low. Furthermore, when the average temperature during 10 days after treatments was higher than before the treatment, good thinning results were recorded compared to the results obtained when temperature decreased during the 10 days after treatments (Buban and Lakatos, 2000). The aim of this study was to evaluate the thinning efficacy of BA sprayed on two apple cultivars at various phenological stages, i.e. applied at different time of blooming period and over a wider range of fruitlet diameter. 2 MATERIAL AND METHODS The experiments were conducted in the experimental orchard of Agricultural Institute of Slovenia on mature, 2.5 m high and 1.0 m wide apple trees of cv. 'Golden Delicious'/ M.9 and cv. 'Idared'/ M.9. The trees were planted at a density of 3300 trees per hectare and trained to a slender spindle form. Standard commercial practices for fertilization and pest control were performed during the experiments. The trees selected for the experiment were of similar growth vigor and bloom density, approximately 190-220 flower clusters per tree for cv. 'Golden Delicious' and 95-120 flower clusters per tree for cv. 'Idared'. The experiments were designed as a complete randomized block with six and eight replications for cv. 'Golden Delicious' and cv. 'Idared', respectively and a single tree as the experimental unit per treatment. Non thinned and hand thinned trees were compared with trees in which BA was applied at full bloom and at the end of the blooming period as well as at different fruitlet diameters (6 mm, 10 mm, 15 mm, 20 mm, 25 mm). The treatments were sprayed at a BA concentration rate of 100 mgl-1 for cv. 'Golden Delicious' and 50 mgl-1 for cv. 'Idared' with the product Exilis (2% 6-benzyladenine, Fine Agrochemicals, Whittington, Great Britain). Spraying was performed to the drip point with a hand sprayer to whole tree. About half a liter of water per tree was used to fully wet the crown. Hand thinned treatment was done at the end of June drop. Fruits were collected at harvest, counted, weighed and graded by diameter into two size classes (<65 mm, >65 mm). The return bloom was estimated the following year at full bloom time with the scale 1 - 10 (1 = no flowers, 10 = snow ball). Data were statistically evaluated using analysis of variance (ANOVA) followed by means separation using Duncan's multiple range test at P= 0.05. All calculations were performed using the statistical program Statgraphics 5.0 (STSC, Rockwille, USA).. 3 RESULTS AND DISCUSSION 3.1 Cv. 'Golden Delicious' Hand thinning performed at the end of June drop period reduced the final fruit number from 254 fruits (control) to 142 fruits per tree, i.e. for about 44 % (Table 1). The consequence was a significant increase in the mean fruit weight for about 38 % and also the number of bigger sized fruits (fruits > 65mm) increased significantly compared to the control, non thinned trees. Spraying of BA 100 mgl-1 caused significant thinning of cv. 'Golden Delicious' (Table 1 - fruit no ./tree) if BA was applied at the end of blooming period or if applied when fruitlet diameters were 6 mm, 15 mm or 20 mm. If final fruit number per 100 flower clusters was taken into consideration there was a significant reduction as well as when BA was applied at the end of bloom or at 15 mm fruit diameter. The thinning response was not significant if BA was applied at full bloom or at 10 mm or 25 mm fruitlet diameter. The results obtained in this experiment with BA sprayed at 10 mm did not support the findings of many reports saying that the best application time coincides with about 10 mm stage of fruit development (Greene, 1993; Basak, 1996; Buban and Lakatos, 1997). It is known that different factors like weather conditions, especially temperature, may influence the thinning efficiency (Williams, 1994; Green, 2002) and that BA needs high temperature for a good effect (Bound et al., 1997). This cannot be confirmed by our experiment, since high morning temperature (20°C) recorded at the time of 10 mm BA application and prolonged sunny weather on the day when the treatment was done did not induce appropriate thinning of cv. 'Golden Delicious'. However, it can be seen from our results that BA can be effective as a thinner, if applied at a very wide phenological interval, since significant thinning was obtained if BA was used at the end of bloom time and later at 6 mm fruit diameter as well as at greater fruitlet diameters (15 mm and 20 mm), which could be very interesting for the practice. BA applications did not have any significant influence on the total yield per tree or on the mean fruit weight at all application time treatments performed when compared to the non thinned trees (Table 1). Only hand thinning resulted in significantly greater mean fruit weight and enhanced the share of bigger fruits. The mean fruit weight of BA treated apple trees of cv. 'Golden Delicious' did not increase significantly irrespective of the time of BA application, even at the treatments in which BA significantly reduced the final fruit retention. According to the literature this is quite unusual since BA has been shown to increase the fruit size even in the absence of fruit thinning (Greene, 1993; Wismer et al., 1995; Basak, 1996). A possible reason for such small mean fruit weight could be found in dry weather conditions present in the studied year. However, the share of bigger size fruits (> 65mm) was enhanced on BA applied trees, but not significantly. Return bloom estimated the following spring showed very low bloom density for cv. 'Golden Delicious' even for the treatments, in which significant thinning occurred the previous year, with the exception of the treatment applied at 6 mm fruitlet diameter, the return bloom of which was slightly but significantly better compared to the control (Table 1). However, very high crop load, likely too high in some thinned trees, could be the reason for unsuccessful flower bud formation on experimental trees. 3.2 Cv. 'Idared' BA thinning experiment on cv. 'Idared' showed an even weaker thinning response than that on cv. 'Golden Delicious'. Hand thinning of cv. 'Idared' was done according to the needs, the final fruit number was reduced almost to the half and the mean fruit weight was increased (Table 1). There was a significant reduction of the total yield as well. BA treatments applied on cv. 'Idared' at different time of blooming period and at different size of fruitlet development at the concentration of 50 mgl-1 caused significant thinning response only if treated at the end of the bloom or at 10 mm fruitlet diameter (Table 1 - fruit no./tree) while there was no significant thinning for the remaining BA time application treatments. Although there was a weak increase of cv. 'Idared' mean fruit weight at different time of BA application treatments compared to the control, it was not significant, except for the application performed at 10 mm fruitlet diameter. Anyhow, the mean fruit weight was still insufficient and does not satisfy the commercial demand. The total yield as well as the yield of bigger fruits (> 65mm) of all BA treatments did not differ significantly compared to the control trees (Table 1). In comparison with cv. 'Golden Delicious' better return bloom was estimated for cv. 'Idared', although only the treatment, in which BA was applied at the end of the bloom showed significantly better return bloom compared to the control (Table 1). Considering that the size of the trees of both cultivars was approximately the same, the trees of cv. 'Idared' had only a half of flower clusters per tree when starting the experiment and also the final fruit retention was about half of that compared to the trees of cv. 'Golden Delicious'. This could be the reason for much better return bloom observed on the trees of cv. 'Idared'. 4 CONCLUSIONS It was observed in the experiments performed that it may result in insufficient mean fruit weight as well. inadequate thinning occurred for both cultivars and that This could be influenced by the dry weather conditions present at the studied year. Also, BA applied at fruit size of 10 mm showed inconsistent thinning results for the two investigated cultivars, since thinning was poor at 10 mm application for cv. 'Golden Delicious', but it was significant for cv. 'Idared'. However, it was demonstrated that BA can be active as a thinner in a wider period of phenological stages, from the end of bloom up to 20 mm of fruit diameter and not mainly around the 10 mm stage of fruit development as indicated by many reports. Table 1: The number of flower clusters per tree when starting the experiment, the final fruit retention, yield and fruit quality parameters at harvest and return bloom in cv. 'Golden Delicious'/ M.9 and cv. 'Idared'/ M.9 thinning experiments Cultivar / No. Fruit (final) Yield Mean Fruit Return Treatment flower no./tree no./100 (kg/ fruit number bloom clusters/ clusters tree) weight > 65mm (1-10) tree (g) 'Golden Delic.' No thinning 218 a 254 d 121 c 18.7 ab 74 ab 10 a 1.0 a Hand thin 190 a 142 a 75 a 14.5 ab 102 c 59 b 1.2 ab BA full bloom 218 a 255 d 116 bc 19.4 b 74 ab 38 ab 1.0 a BA end of bloom 217 a 190 abc 89 ab 16.0 ab 88 bc 38 ab 1.3 ab BA 0 = 6 mm 197 a 185 abc 94 abc 15.2 ab 83 ab 38 ab 2.3 b BA 0 = 10 mm 203 a 213 bcd 108 bc 15.7 ab 73 ab 23 ab 1.7 ab BA 0 = 15 mm 207 a 174 ab 89 ab 13.9 ab 80 ab 22 a 2.0 ab BA 0 = 20 mm 203 a 195 abc 98 abc 13.5 a 70 a 12 a 1.3 ab BA 0 = 25 mm 202 a 243 cd 120 c 19.2 b 78 ab 44 ab 1.5 ab 'Idared' No thinning 103 ab 119 c 120 b 11.3 b 96 ab 54 ab 3.5 abc Hand thin 110 ab 68 a 64 a 8.3 a 124 d 47 a 2.9 ab BA full bloom 120 b 126 c 107 b 11.7 b 93 a 53 ab 2.7 ab BA end of bloom 100 ab 89 ab 95 b 9.5 ab 110 bcd 52 ab 5.6 d BA 0 = 6 mm 106 ab 109 bc 107 b 11.9 b 110 bcd 63 ab 4.9 cd BA 0 = 10 mm 97 a 89 ab 92 b 10.0 ab 115 cd 61 ab 4.9 cd BA 0 = 15 mm 111 ab 109 bc 102 b 11.5 b 106 abc 59 ab 2.4 a BA 0 = 20 mm 103 ab 120 c 120 b 12.3 b 102 abc 69 b 4.1 bcd BA 0 = 25 mm 110 ab 116 c 106 b 12.1 b 104 abc 69 b 2.8 ab * Mean separation within column by Duncan's multiple range test, P = 0.05 5 REFERENCES Basak, A. 1996. Benzyladenine (BA) as an apple fruitlets thinning agent - preliminary results. Horticultural Science, 28: 54-57. Bound, S.A., Jones, K.M., Koen, T.B., Oakford, M.J. 1991. The thinning effect of benzyladenine on red 'Fuji' apple trees. Journal of Horticultural Science, 66: 789-794. Bound, S.A., Jones, K.M., Oakford, M.J. 1997. Post-bloom thinning with 6- benzyladenine. Acta Horticulturae, 463: 493-499. Buban, T., Lakatos, T. 1997. Benzyladenine for treating trees of hard to thin apple cultivars. Acta Horticulturae, 463: 509-515. Buban, T., Lakatos, T. 2000. Contributions to the efficacy of benzyladenine as a fruit thinning agent for apple cultivars. Acta Horticulturae, 514: 59-67. Elfving, D.C., Cline, R.A. 1993. Benzyladenine and other chemicals for thinning 'Empire' apple trees. Journal of the American Society for Horticultural Science, 118: 593598. Ferree, D.C. 1996. Performance of benzyladenine as a chemical thinner on eight apple cultivars. Journal of Tree Fruit Production, 1: 33-50. Greene, D.W., Autio, W.R., Miller, P. 1990. Thinning activity of benzyladenine on several apple cultivars. Journal of the American Society for Horticultural Science, 115: 394400. Greene, D.W. 1993. A review of the use of benzyladenine (BA) as a chemical thinner for apples. Acta Horticulturae, 329: 231-236. Greene, D.W. 2002. Chemicals, timing and environmental factors involved in thinner efficacy on apple. HortScience, 37: 477-481. Greene, D.W. 2005. Effects of repeated yearly application of chemical thinners on 'Mcintosh' apples. HortScience, 40: 401-403. Link, H. 2000. Significance of flower and fruit thinning on fruit quality. Plant Growth Regulation, 31: 17-26. McLaughlin, J.M., Greene, D.W. 1984. Effects of BA, GA4+7 and daminozide on fruit set, fruit quality, vegetative growth, flower initiation and flower quality of 'Golden Delicious' apples. Journal of the American Society for Horticultural Science, 109: 34-39. Stopar, M., Lokar, V. 2003. The effect of ethephon, NAA, BA and their combinations on thinning intensity of 'Summered' apples. Journal of Central European Agriculture, 4: 399-403. Williams, M.W. 1994. Factors influencing chemical thinning and update on new chemical thinning agents. Compact Fruit Tree, 27: 115-122. Wismer, P.T., Proctor, J.T.A., Elfving, D.C. 1995. Benzyladenine affects cell division and cell size during apple fruit thinning. Journal of the American Society for Horticultural Science, 120: 802-807. COBISS Code 1.01 Agrovoc descriptors: fagopyrum esculentum, buckwheat, fagopyrum tataricum. pseudocereals, tannins, polyphenols, metabolites, antioxidants, leaves, proximate composition, chemical composition Agris category code: F60 Comparison of tannin concentration in young plants of common and tartary buckwheat Drena GADŽO1, Mirha DJIKIC1, Teofil GAVRIC1, Petra ŠTREKELJ2 * Received: July 15, 2009; accepted: September 25, 2010. Delo je prispelo: 15. julija, 2009; sprejeto: 25. septembra, 2010. ABSTRACT The aim of this study was to compare the tannin concentration of young common and tartary buckwheat plants. Tannins are a group of polyphenols, formed as secondary metabolites in plants. Tannins are known as antioxidants and have chemoprotective potential. They occur in many fruits and drinks, such as tea, beer, wine and juices, making them significant in human nutrition. Concentration of tannins was high in upper leaves of tartary buckwheat and in young plants of common buckwheat cv. Bosanka. There are interesting differences between tannin concentration in Bosanka young plants and tartary and Darja buckwheat young plants. Key words: common buckwheat, tartary buckwheat, tannins, fertilization IZVLEČEK PRIMERJAVA KONCENTRACIJE TANINOV V MLADIH RASTLINAH NAVADNE IN TATARSKE AJDE Namen raziskave je primerjava koncentracije tanina v rastlinah navadne in tatarske ajde. Tanini spadajo v skupino polifenolov in so sekundarni produkti metabolizma rastlin. Tanini so močni antioksidanti in delujejo zavirajoče proti nekaterim vrstam raka. Najdemo jih v sadju, čaju, vinu in sokovih, raziskava taninov je pomembna za prehrano ljudi. Koncentracija taninov je bila visoka v zgornjih listih tatarske ajde in mladih rastlinah Bosanke. Pomembne so razlike med koncentracijami taninov pri mladih rastlinah Bosanke in mladih rastlinah tatarske ajde ter Darje. Ključne besede: navadna ajda, tatarska ajda, tanini, gnojenje 1 INTRODUCTION Tartary buckwheat growing Common buckwheat is grown in many countries around the world, in Asia, Europe and South Africa, in Canada, USA, Brazil and in some other places. A large variety of buckwheat foods are being traditionally produced for centuries. Dishes made from buckwheat seed are generally classified in two groups, flour dishes and groats dishes. Other products made from buckwheat are buckwheat floral honey, green buckwheat tea, buckwheat sprouts, and fresh green plant parts used as a vegetable. Buckwheat herb is especially known as a rich source of tannis and other polyphenols (Kreft et al. 2002; Kreft et al., 2006, Kalinova et al., 2006; Kalinova & Vrchotova, 2009). In Europe buckwheat has been grown for centuries and is now, one of the important alternative crops, suitable for ecological growing, without the use of fertilizers or pesticides. It is used for flour and groats products in central and eastern Europe. For many years, cultivation of buckwheat declined, but recent interest in old, traditional foods and a re-evaluation of typical regional products has led to a resurgence in its cultivation. 1 Faculty of Agriculture and Food Science, University of Sarajevo, Zmaja od Bosne 8, BiH 71000 Sarajevo, Bosnia and Herzegovina 2 Biotechnical Faculty, University of Ljubljana, Jamnikarjeva 101, SI-1000 Ljubljana, Slovenia * Corresponding author, e-mail: petra.strekelj@gmail.com Common buckwheat is grown in all parts of Slovenia and in several regions of Bosnia-Herzegovina. Common and tartary buckwheat was grown in Bosnia-Herzegovina at least before the year 1940 but, since about 1980, this mainly ceased. In Europe, it is traditional growing of tartary buckwheat mostly spread in the cross border region Islek - which covers northern Luxemburg, the Westeifel (Germany) and the border area of the German-speaking part of Belgium. In Bosnia-Herzegovina tartary buckwheat was grown in past ten or twenty years at least in a small extent in the mountain plains south and north of Sarajevo, up to the altitude of about 1000 m (Se photo on the title page of this journal in issue 93-3, 2009). This crop is in Bosnia-Herzegovina now coming back because of the interest for healthy food. Health importance of tannins in the diet The cellular injury caused by oxidative stress and excess of free radicals has been associated with aging and linked to clinical disorders, including cancer, heart disease, and liver damage. Plant derived polyphenols are known for their strong antioxidant potency (Bialonska et al., 2009 b). Tannins are a group of polyphenols that are formed as secondary plants metabolites. They occur in human diet, such as tea, beer, wine and juices, making them significant in human nutrition (Frazier et al., 2010). The antimicrobial activity of tannins is associated with capability to form stable complexes with proteins, starch, and physiological metals, thereby disturbing the metabolic activity of bacterial enzymes, nutrient availability, and functionality of biological membranes (Bialonska et al., 2009 a). The antioxidant properties of tannin are well documented, but the interactions between tannins and proteins is fundamental for their biological activities. Therefore, a better understanding of this interaction will enable clearer explanations for the biological and pharmacological activities of tannin. Frazier et al. (2010) documented that interactions with proteins were exotermic and involved multiple binding sites on the protein. Bialonska et al. (2009 a) reported a potent antioxidant activity of tannin components from pomegranate extracts. The consumption of pomegranate products leads to a significant accumulation of ellagitannins in the large intestines, where they interact with complex gut bacteria. Results showed that pomegranate byproducts and punicalagins inhibited the growth of pathogenic clostridia and Staphylococcus aureus. Awika et al. (2009) reported that tannin containing sorghum extracts have strong chemoprotective potential (cancers in the gastrointential tract, especially esophageal cancer). The antioxidant activity of buckwheat tannins are not studied yet, therefore the investigations of buckwheat tannins are important. The aim of this study was to compare the tannin concentration in green parts of common and tartary buckwheat, and to evaluate their value as possible functional foods. 2 MATERIALS AND METHODS Material: Plants were grown at the experimental field of Faculty of Agriculture and Food Science, University of Sarajevo, in Sarajevo in 2008. Domestic cv. Bosanka, from Bosnia, Slovenian buckwheat cv. Darja and tartary buckwheat from Luxemburg were used in a study, performed in Sarajevo in 2008. Plants were grown without fertilization, or with a standard NPK fertilization; namely granulated mineral fertilizer, N:P:K in relation 4 : 12 : 20, amount 50g/m2. Samples for analyses were collected from young plants (two proper leaves) and at the beginning of flowering separately for lower and upper leaves of the plant. Determination of tannin: Total tannin contents were determined spectrophotometrically using vanillin-HCl reagent, as previously reported (Kreft et al., 2002). Each sample was analyzed in triplicate. The data were evaluated by multifactor ANOVA (Statgraphics Version 4) and significance accepted at P < 0.05. 3 RESULTS AND DISCUSSION Results presented in Fig. 1 show that concentration of tannins was high in upper leaves of tartary buckwheat and low in young plants of Darja. This probably depends on higher impact of sunshine radiation on young plants and on higher positioned leaves of flowering plants. Results in Bosanka are different. Here the tannins buckwheat. The concentration of tannins in Darja was concentration are high in young plants and not in upper lower in comparison to Bosanka and tartary buckwheat. leaves as we observed in tartary buckwheat and Darja Tannins, Bosanka 5 4 3 2 1 0 rh r*rh jn Young plants Lower Upper □ Non-fertilized □ Fertilized Tannins, Tartary rh rh j -- □ Non-fertilized □ Fertilized Young plants Lower Upper Tannins, Darja □ Non-fertilized □ Fertilized Young plants Lower Upper Fig. 1. Tannins in young plants (two leaves), lower and upper leaves in flowering Bosanka - Bosnia domestic common buckwheat variety, tartary buckwheat and common buckwheat Darja; without and with NPK fertilization, respectively (g in 100 g of dry matter). 5 0 Fertilisation had no effect on the concentration of studied substances. We may conclude that fertilization with standard NPK fertilization had no clear impact on the concentration of tannins in buckwheat samples. Studied tartary buckwheat in the upper, light exposed leaves had higher concentration of tannins in comparison to common buckwheat. Except in common buckwheat cv. Bosanka, young plants had lower concentration of tannins in comparison to the upper leaves of flowering plants. In any way, different varieties of buckwheat react differently on environmental factors, influencing the concentration of tannins in green parts of buckwheat plants. 4 ACKNOWLEDGEMENTS This research was financed by Federal Ministry of Education and Science of Bosnia and Herzegovina - No 03-39-5980-95-2/08, and the Slovenian Research Agency through projects J4-9673 and J7-9805. Analyses were performed in the laboratories of the Faculty of Pharmacy, Chair of Pharmaceutical Biology, University of Ljubljana. 5 REFERENCES Awika J.M., Yang L.Y., Browning J.D., Faraj A. 2009. Comparative antioxidant, antiproliferative and phase II enzyme inducing potential of sorghum (Sorghum bicolor) varieties. LWT-Food Science and Technology. 42 : 10411046. Bialonska D., Kasimsetty S.G., Schrader K.K., Ferreira D. 2009 a. The effect of pomegranate (Punica granatum L.) byproducts and ellagitannins on the growth of human gut bacteria. Journal of Agricultural and Food Chemistry. 57: 8344-8349. Bialonska D., Kasimsetty S.G., Khan SI., Ferreira D. 2009 b. Urolithins, intestinal microbial metabolites of pomegranate ellagitannins, exhibit potent antioxidant activity in a cell-based assay. Journal of Agricultural and Food Chemistry. 57: 10181-10186. Frazier R.A., Deaville E.R., Green R.J., Stringano E., Willoughby I., Plant J., Mueller-Harvey I. 2010. Interactions of tea tannins and condensed tannins with proteins. Journal of Pharmaceutical and Biomedical Analysis. 51: 490-495. Germ M., Vollmannova A., Timoracka M., Melichacova S., Stibilj V., Vogrincic M., Kreft I. 2009. Antioxidative substances of tartary buckwheat sprouts and impact of Se and Zn on the sprout development. V: Park, C. H. , Kreft, I. (Eds.). Proceedings of the International symposium on buckwheat sprouts. Development and utilization of buckwheat sprouts as medicinal natural products: ISBS 2009, Sep. 7-9, Bongpyoung, Korea. 46-53. Kalinova J., Triska, J., Vrchotova N. 2006. Distribution of vitamin E, squalene, epicatechin and rutin in common buckwheat plants (Fagopyrum esculentum Moench). Journal of Agricultural and Food Chemistry . 54, 5330533. Kalinova J., Vrchotova N. 2009. Level of catechin, myricetin, quercetin and isoquercitrin in buckwheat (Fagopyrum esculentum Moench.), changes of their levels during vegetation and their effect on the growth of selected weeds. Journal of Agricultural and Food Chemistry. 57 (7), 2719-2725. Kreft S., Štrukelj B., Gaberščik A., & Kreft I. 2002. Rutin in buckwheat herbs grown at different UV-B radiation levels: comparison of two UV spectrophotometric and an HPLC method. Journal of Experimental Botany. 53 (357), 1801-1804. COBISS Code i.Gi Agrovoc descriptors: soil fertility, nutrient availability, chemical composition, fertilizer application, mineral content, inorganic fertilizers, farmyard manure, straw, humus, soil organic matter, nitrogen Agris category code: P35, P33 Soil organic matter content according to different management system within long-term experiment Monika CVETKOV1, Igor ŠANTAVEC2, Darja KOCJAN AČKO3, Anton TAJNŠEK4 Received October 12, 2009; Accepted January 19, 2010. Prispelo 12. oktobra, 2009; sprejeto 19. januarja, 2010. ABSTRACT IZVLEČEK Within the long-term field experiments at IOSDV Rakican, Slovenia, the impact of organic matter management system and mineral nitrogen fertilization on the soil organic matter content was studied in the period 1994-2008. The annual balance of Corg was calculated on the basis of the quantity of added organic fertilizers ("Bavarian method", "VDLUFA method"), while the "Swiss method" also consider the quantity of Corg in the topsoil in the calculation. The following management systems were selected: system A - no organic matter, system B - farmyard manure ploughing in, system C -straw/catch crop ploughing in. Four different mineral N rates (N0, N1, N2, N3) were evaluated. In 2008 the Corg content in topsoil (0-25 cm) was measured according to ISO 10694. Farmyard manure (FYM) fertilization significantly influenced the content of Corg, while the straw application did not result in the significant increase of Corg content. Mineral nitrogen fertilization did not impact Corg content within system A. In system B and system C positive effect of nitrogen fertilization VSEBNOST ORGANSKE SNOVI V TLEH V ODVISNOSTI OD NAČINA GOSPODARJENJA ZNOTRAJ TRAJNEGA POSKUSA V statičnem poskusu IOSDV Rakičan, Slovenija smo preučevali vpliv gospodarjenja z organskimi gnojili in vpliv gnojenja z mineralnimi dušikom na vsebnost organske snovi v tleh v letih 1994 do 2008. Letna bilanca Corg je bila izračunana na podlagi količin dodanih organskih gnojil pri „Bavarski metodi" in „ VDLUFA metodi", medtem ko se je pri „ Švicarski metodi" v izračunih upoštevalo tudi stanje Corg v tleh. Vključeni sistemi gospodarjenja so bili: sistem A -gospodarjenje brez organskega gnojenja, sistem B - gnojenje s hlevskim gnojem, sistem C - zaoravanje slame/podorin. Preučevane so bile štiri stopnje gnojenja z mineralnim dušikom: N0, N1, N2 in N3. V letu 2008 je bila izmerjena vsebnost Corg v globini od 0 do 25 cm po standardu ISO 10694. Gnojenje s hlevskim gnojem je značilno povečalo on the Corg content was detected. However, statistically vsebnost Corg, medtem ko gnojenje s slamo ni imelo takšnega significant impact of mineral N on a higher Corg content was not determined. All three methods underestimated the actual analysed results, although, we can determine the "Swiss method" as the most precise and appropriate for this site-specific location. Key words: organic fertilizers, farmyard manure, straw, N fertilizers, Corg content, humus balance, humus balance calculation methods vpliva. V sistemu brez organskih gnojil gnojenje z mineralnim dušikom ni doprineslo k povečanju vsebnosti Corg. Kljub pozitivnemu vplivu gnojenja z mineralnim dušikom na vsebnost Corg v sistemu B in C, pa vpliv ni bil statistično značilen. Izračuni vseh treh metod podcenijo dejanske rezultate laboratorijskih analiz posameznega vzorca. Še najbolj se omenjenim rezultatom približajo vsebnosti Corg, izračunane s „Švicarsko metodo", zato lahko povzamemo, da je slednja najbolj primerna za določitev Corg za preučevano lokacijo. Ključne besede: organska gnojila, hlevski gnoj, slama, mineralni dušik, vsebnost Corg, bilanca humusa, metode humusa izračunavanja bilance 1 University of Ljubljana, Biotechnical Faculty, Jamnikarjeva 101, SI-1111 Ljubljana, e-mail: monika.kunaver@bf.uni-lj.si 2 ibid., igor.santavec@bf.uni-lj.si 3 ibid., darja.kocjan@bf.uni-lj.si ibid., tone.tajnsek@bf.uni-lj.si 4 1 INTRODUCTION Land use and agricultural management practices such as crop rotation, soil tillage and organic amendment, depending on the site-specific conditions can affect soil organic matter (SOM). Changes in the organic carbon (Corg) content in soil occur almost exclusively in its decomposable part. The tendency of the decomposable carbon (Cdec) changes also depends on its initial level. Application of the same fertilization and cropping system can cause a decrease of the SOM content if its initial level was high, as well as an increase of the SOM if its initial level was low (Korschens, 2002). As the organic substances interact with clay in soil to form complexes and micro-aggregates, which make the organic matter less accessible to decomposers, Corg tend to increase and mineralization rate decrease with the clay content (Bosatta and Ágren, 1997; Korschens, 2004; Diekow et al., 2005). The content of organic carbon (Corg) is controlled by changes in management via the annual input of organic matter and the rate at which decays (Jenkinson et al., 1999). The amount of SOM is generally higher in the fertilized soils than in the unfertilized soils (Ellmer et al., 2000; Hoffmann et al., 2002; Berecz et al., 2004; Goyal et al., 2006). Data from several long-term comparative experiments on the effect of FYM and mineral fertilization prove that FYM increases the organic matter content of the soil to a greater extent than mineral fertilization (Korschens, 1997; Kuzyakov and Domanski, 2000; Berecz et al., 2004; Brodowski et al., 2007; Simon, 2007). On the other hand organic C stocks had barley changed in response to very considerable changes in management in the experiment by Jenkinson et al. (1999). In case of omitted mineral N fertilization, the humus content in the soil decreased rapidly (Stumpe et al., 2000; Beschow and Merbach, 2004; Winkelmann et al., 2006). Incorporation of harvest residues also increased Corg content in soil (Buyanovsky and Wagner, 1998; Triberti et al., 2008). Not only the quantity, also the structure of SOM varies depending on the rate of fertilizer (Ellerbrock et al., 1999; Dorado et al., 2003). By using methods of calculating the balance of humus we are given an opportunity to control the SOM content in arable soils in order to achieve higher yields and simultaneously avoid environmental pollution. Since the most Corg is bound in soil humic matter, the mineralization and humification of plant carbon in soil should be monitored (Filip and Kubat, 2004). There are several humus balance calculation methods and models, however, in many models, humification is not considered as a part of the process of transformation of organic debris into humus, and the role of humus in the kinetics of this process is evidently underestimated (Chertov et al., 2007). Three humus balance calculation methods, which take into account a humification and mineralization rates and are believed to be appropriate for European Central site-conditions are investigated: "Swiss method" determined by Diez and Krauss (1992), the "Bavarian method" determined by Bavarian working group (Anonymous, 1998) and the "VDLUFA method" determined by Korschens et al. (2004). The aim of our study was to examine, with the application of mentioned methods, the impact of organic and mineral fertilization on the humus content in the soil according to particular crop rotation at ISODV Rakican location. As the soil analyses were conducted every year since the establishment of the trial, the results calculated by each method could be compared with the analysed results, therefore a most appropriate method for this site-specific condition could be selected. 2 MATERIAL AND METHODS 2.1 Experimental layout As part of the "International Long-term Experiments for Investigating the Effect of Organic and Inorganic Fertilizers" (IOSDV), field experiment was set up at Rakican, Slovenia (46°38'N, 14°11'E, Pannonian climate, sandy silt) in 1993. The trial was set up as a permanent experiment related to crop rotation with ten different fertilization combinations as a block trial with three repetitions. First, the trial area was divided into three plots, on which each year crops were sown in the following order: corn, winter wheat, barley. Each plot was further divided into two subplots, on which different systems of fertilization with organic management were studied. Each subplot thus represented five variants differing according to the rate of fertilization with mineral nitrogen in the three repetitions. The basic plot size was 30 m2 (5 x 6 m). Ten different treatments were included in the investigation: - management system with no organic fertilizers (system A) and two different mineral rates (N0, N3), - management system with farmyard manure (FYM) ploughing in (system B) and four different mineral N rates (N0, N1, N2, N3), - management system with straw ploughing in (system C) and four different mineral N rates (N0, N1, N2, N3). Fertilizing plan for the nutrition of arable crops is shown in Table 1. Fertilization with phosphor and potassium was uniform for all mineral nitrogen rates (N0=0 N kg/ha, N1=73 N kg/ha, N2=147 N kg/ha, N3= 220 N kg/ha): 75 kg/ha P2O5 and 160 kg/ha K2O. At the harvest time, yield and straw quantities were measured for each plot. After harvesting every year soil samples from each plot were taken at a depth of 0-25 cm. Table 1: Management systems, mineral N fertilization with regard to the crop, the average amount of mineral N in the three-year crop rotation (N-minaver.) at IOSDV Rakican location N-min Maize Wheat Barley/ N-minaver. rates/ (kg/ha N) (kg (kg (kg Treatment /ha N) /ha N) /ha N) A No organic / / / m fertilizers O t s NO (ANO) 0 0 0 0 y in NB (AN3) 300 195 165 22Q FYM ploughing in 30 t/ha FYM* / / B (t/ha) m e NO (BN0) 0 0 0 0 t s N1 (BN1) 100 65 55 7B y N2 (BN2) 200 BQ 11Q 147 N (BN3) 300 195 165 22Q Straw/catch crop Barley straw + Maize Wheat 6Q** C ploughing in (t/ha) fodder radish straw straw m e NO (CN0) 0 0 0 0 t s N1 (CN1) 100 65 55 7B y m N2 (CN2) 200 BQ 11Q 147 N (CN3) 300 195 165 22Q * FYM is applied every third year. ** Mineral N is added after barley and before fodder radish is sown, every third. 2.2 Weather and soil conditions The soil type, soil properties and some climatic characteristics of the experimental site are listed in Table 2 (Tajnsek, 2003). Annual precipitation and the average annual temperature for the period 1994-2008 in comparison with the long-term average precipitation and the long-term average temperature for the period 1960-1990 are shown in Figure 1. Table 2: Soil properties of the experimental site (Tajnšek, 2QQB) Long. East1 Lat. North2 Prec3. (mm) Temp4. (°C) Soil type (FAO classification) Clay (<2.0^m) (%) pH (KCl) C ^org (%) N org (%) C/N Ratio 14°11' 46°B8' 814 9.2 Eutric Fluviosol (ELe) 14.67 7.Q4 Q.926 Q.Q98 9.5 'Longitude; 2 Latitude; 3 Precipitation, the long-term average precipitation in the period 1961-1990; 4 Temperature, the long-term average temperature in the period 1961-1990 Weather conditions for IOSDV Rakičan 1200 1000 800 iS c %E 600 400 200 • • 14 12 10 8 3 ¡0 a ~ 6 EE 0 I I I I I I 1 I 1 11 I I I I I I 11 I 1 0 1994 1995 1996 1997 1998 1999 2000 2001 2002 2003 2004 2005 2006 2007 2008 Years ]P (61-90) P (94-08) -T (61-90) T (94-08) Figure 1: Annual precipitation and the average annual temperature for the period 1994-2008, the long-term average precipitation (1961-1990) and the long-term average temperature (1961-1990) for the IOSDV Rakičan (weather station Murska Sobota) location 2.3 Humus balance calculation methods In our investigation, there are three methods of calculating the balance of humus involved: - The method determined by Diez and Krauss (1992) named as the "Swiss method" or "S". The annual balance is calculated on the basis of the ploughed-in quantity of organic matter (manure, straw, catch crop, harvest residues) with the corresponding humification coefficient, taking into account the quantity of humus in the soil (H) with the appropriate mineralization coefficient. Results are given in matter inputs: manure, straw and catch crop. The organic matter decay is represented in the coefficients of depleting and increasing crop species. While for the major intensive forms of agricultural managements the lower values of annual humus balance under specific crop are recommended by DirektZahlVerpflV (Korschens et al., 2004), we have chosen the lower values for further calculation. Results are given in the Corg value (t/ha), which is calculated on the basis of content Humus-C (kg/ha) multiplied by factor 0.001 (kg/ha^t/ha). the Corg value (t/ha), which is calculated on the basis of 2. 4 Chemical analyses humus content (t/ha) multiplied by factor 0.58. The method determined by Bayerische Landesanstalt für Bodenkultur und Pflanzenbau, Freising-München (Anonymous, 1998) named as the »Bavarian method« or »B«. The humus balance is calculated on the basis of humification rate of individual crop in crop rotation, taking into account the quantity of added organic matter: manure, straw and catch crop. In this method humification factor varies due to soil texture, therefore at IOSDV Rakican a humification factors for soil type sandy silt (Eutric Fluviosol [ELe]) are chosen. Results are given in the Corg value (t/ha), which is calculated on the basis of humus content (dt/ha) multiplied by factor 0.58 (humus In the year of the establishment of the experiment (1993), as well as in all subsequent years, the soil analyses were conducted at the laboratories UFZ Leipzig-Halle, Germany (Tajnsek, 2003). The Corg content was determined according to ISO 10694, 1996-08. In the calculation of humus balance we considered initial value of Corg content in 1993. content^Corg) and by factor 0.1 (dt/ha^t/ha). The method determinated by working group Korschens et al. (2004) named as the »VDLUFA method« or »V«. With the "VDLUFA method" an organic matter surplus is calculated by addition of specific humification coefficients using for organic matter depleting crop species and for the organic 2.5 Processing of statistical data Statistical analysis was conducted with the Statgraphics Plus 4.0 program. Before analysis each treatment was tested for homogeneity of treatment variances. If variances were not homogeneous, data was transformed to log (Y) before ANOVA. Multifactor ANOVA was used in order to analyze the effect of different management systems on the humus content in the soil. Differences among treatments were detected by Duncan's Multiple Range Test (p < 0.05). Data is presented as untransformed means±SE. 4 2 3 RESULTS 3.1 The humus balance in the period 1993-2008 Results showing the Corg content of the analysed soil samples at IOSDV Rakican in the beginning of the trial and the Corg content calculated by different methods for the year 2008 are given in Table 3. The initial value of Corg in 1993 was 37.27 t/ha Corg. FYM fertilization significantly influenced the content of Corg, while the straw application did not result in a significant increase of the Corg content. Results show that mineral N fertilization had no significant impact within all three management systems. content in the management without organic fertilization (system A). Moreover, with the application of the average amount of N in a three-year crop rotation (220 kg/ha N), the Co!g content resulted in a decrease of 2.3 %, i.e. 0.94 t/ha Corg. This is comparable to the results of the experiment by Beschow and Merbach (2004), where a comparison of the highest mineral nitrogen (N) with no-N resulted in a merely 1.2-fold higher value of the Corg content in the topsoil. Likewise, in the case of mineral N fertilization, the content of Corg was slightly higher (1.4 to 1.5 %) than without any N fertilization (1.3 %) (Stumpe et al., 2000; Triberti et al., 2008). As demonstrated in Table 3, mineral nitrogen fertilization did not contribute to an increase of the Corg Table 3: Corg content (t/ha) of analysed soil samples in 1993 and 2008, the Corg content (t/ha) in 2008, calculated by »Swiss« (S), »Bavarian« (B) and »VDLUFA« (V) methods and the difference between analysed and calculated Corg content (t/ha) in 2008 for ten different treatments (IOSDV Rakican) Tre. Corg1993 Corg2008(anal.) Corg2008(cal.) Corg2008(anal.) - C org2008(cal.) (anal.) (t/ha) (t/ha) (t/ha) (t/ha) »S« »B« »V« »S« »B« »V« AN0 41.73± 1.28 37.06±0.02 33.28±0.00 31.67±0.00 4.67 8.45 10.06 a* a a a AN3 40.79±1.10 37.26±0.08 33.28±0.00 31.67±0.00 3.53 7.51 9.12 a a a a BN0 46.96±1.36 38.19±0.02 36.76±0.00 37.67±0.00 8.77 10.20 9.29 bcde c b b BN1 48.97±2.16 38.25±0.02 36.76±0.00 37.67±0.00 10.72 12.21 11.30 de cd b b BN2 49.37±0.58 38.37±0.04 36.76±0.00 37.67±0.00 11.00 12.61 11.70 37.27 e e b b BN3 47.63±0.75 38.35±0.00 36.76±0.00 37.67±0.00 9.28 10.87 9.96 cde de b b CN0 40.79±1.68 37.90±0.11 37.63±0.06 37.61±0.08 2.89 3.16 3.18 a b c b CN1 42.53±1.36 37.92±0.07 38.67±0.16 39.08±0.23 4.61 3.86 3.45 ab b d c CN2 44.41±1.10 37.94±0.10 39.27±0.18 39.91±0.25 6.47 5.14 4.50 abcd b e d CN3 43.74±2.11 38.18±0.03 39.90±0.03 40.80±0.04 5.56 3.84 2.94 abc c f e 1 Xpovp.| 6.75 7.79 7.55 a-f The same letter in the column indicates thet there is no significant difference among treatments (Duncan Multiple Range Test, P<0.05). In the system with FYM (system B), Corg was were not significant. The Corg content increased by significantly higher in all the treatments when compared 5.23 t/ha (BN0), 7.24 t/ha (BN1), 7.64 t/ha (BN2) and to AN0 and AN3. The application of mineral N 5.90 t/ha (BN3). The highest amount of mineral N increased the Co!g content; however, the differences (BN3) combined with 10 t/ha.yr FYM resulted in a slightly lower Corg content. Ellmer reported that the total carbon content between 600 and 800 mg/100g can be achieved by organic fertilization (15 t/ha.yr FYM) and with appropriate crop rotation (Ellmer et al., 2000). An average demand for FYM was determined to be 10 t/ha in a year (Korschens et al., 2004). In the management with ploughing in of straw (system C), there was a decrease of the Corg content by 0.94 t/ha in the treatment without mineral N (CN0). Although the Corg content increased with higher fertilizer rates of mineral N, a significant impact of N fertilizing could not be confirmed among the treatments. The Corg content increased by 0.80 t/ha (CN1), 2.68 t/ha (CN2) and 2.01 t/ha (CN3). In the experiment by Triberti et al. (2008), the SOC stock did not change in unfertilized plot and N fertilized plot, while it increased at a mean rate of 0.16, 0.18 and 0.36 t/ha in a year with the incorporation of residues, slurry and manure. was confirmed. Compared to the unfertilized plot, the FYM application resulted in a 8.2 % higher total organic carbon content than the equivalent NPK fertilization according to Hoffmann et al. (2006). In the experiment by Berecz et al. (2004), both types of organic manuring (FYM, straw or green manure) resulted in significantly higher Corg contents compared to the mineral N fertilization without manuring or incorporation of crop residues. Application of FYM combined with mineral N resulted in the highest Corg content among all the treatments. This is in accordance with the results given by other authors (Filip and Kubat, 2004; Goyal et al., 2006). The increase of the C content in the soil during a 50-year period required a four times (e.g. FYM on loamy soil) to 12 times (e.g. straw fertilization on sandy soil) higher input C, depending on the local conditions and the type of primary organic matter (Korschens et al., 2004). In both systems (system B, system C), the application of the highest amount of mineral N (BN3, CN3) resulted in a lower Corg content. The contribution of FYM to the maintenance of the Corg content is greater than the input of straw. According to AN0, where the Corg content amounted to 41.73 t/ha, the Corg content in system B increased in the range of 5.23 to 7.64 t/ha Corg, i.e. 12.5 % to 18.3 %. In system C, where no mineral N was added (CN0), the Corg content decreased by 0.9 t/ha, while in increased by 0.80 to 2.68 t/ha Corg, %. other treatments it i.e. by 1.9 % to 6.4 According to different management system, the same fertilizing rate of mineral N significantly influenced the Corg content only in system B. In comparison with AN0, the change of the Corg content was higher for 12.5 % in BN0, while in system C the Corg content decreased for 2.3 % (CN0). When comparing the treatments with the highest mineral N rate, an increase of the Corg content by 16.7 % in BN3 and by 7.2 % in CN3 according to AN3 3.2 Calculated and analysed Corg content in 2008 In calculation by "Swiss method", the Corg content increased by application of mineral nitrogen (AN3). The results calculated by "Bavarian method" and "VDLUFA method" within the systems are equal; this was expected as the calculation takes into account the amount of added organic matter which does not differentiate between treatment AN0 and AN3. As it is seen from figure 2, method which most closely approximated the analysed Corg content in the system with no organic matter was "Swiss" method. The difference between analysed Corg2008 content and calculated Corg2008 content is called deviation from analysed Corg content. Deviation from analysed Corg content was higher by using "Bavarian" and " VDLUFA" methods, deviation ranged "Bavarian" method and VDLUFA" method. from 7.51 to 8.45 t/ha Corg at from 9.12 to 10.06 t/ha Corg at Figure 2: Corg content of analysed soil samples (CorgM), Corg content, calculated by »Swiss« (CorgS), »Bavarian« (CorgB) and »VDLUFA« (CorgV) methods in 2008 for two treatments in management system with no organic fertilizers (IOSDV Rakican) Rakican, 2008 46 -, 44 42 _ 40 (0 £ 38 E? o u 36 34 -32 -30 AN0 AN3 □ Corg S o Corg B A Corg V x Corg M The application of mineral nitrogen in system with FYM statistically increased Corg content in results calculated by "Swiss" method at BN2 and BN3, while within treatments in "Bavarian" and "VDLUFA" methods there were no differences. Deviation from analysed Corg content ranged from 8.77 to 11.00 t/ha Corg at "Swiss", from 10.20 to 12.61 t/ha Corg at "Bavarian", from 9.29 to 11.70 t/ha Corg at "VDLUFA" method. For this system method which most closely approximated the analysed Corg content was again "Swiss" method (Figure 3). Rakican, 2008 54 52 50 48 46 44 42 40 38 36 34 32 30 g ♦ BN0 g ♦ BN1 à ♦ BN2 & ♦ BN3 □ Corg S 0 Corg B A Corg V x Corg M Figure 3: Corg content of analysed soil samples (CorgM), Corg content, calculated by »Swiss« (CorgS), »Bavarian« (CorgB) and »VDLUFA« (CorgV) methods in 2008 for four treatments in management system with FYM ploughing in (IOSDV Rakican ) 50 48 46 44 £ 42 S" 40 t» o 38 36 34 32 30 Rakičan, 2008 5 o CN0 CN1 CN2 CN3 □ Corg S ♦ Corg B A Corg V X Corg M Figure 4: Corg content of analysed soil samples (CorgM), Corg content, calculated by »Swiss« (CorgS), »Bavarian« (CorgB) and »VDLUFA« (CorgV) methods in 2008 for four treatments in management system with straw ploughing in (IOSDV Rakican) In the system with straw ploughing in application of mineral nitrogen significantly increased the Corg content in »Bavarian« and »VDLUFA« method, while in »Swiss« method the impact of mineral nitrogen was notices only in treatment with the highest mineral rate (CN3). In this system the most precise method seemed to be »VDLUFA« method, where the deviation from analysed Corg ranged from 2.94 to 4.50 t/ha Corg (Figure 4). Using "Swiss" method the mentioned deviation ranged from 2.89 to 6.47 t/ha Corg, using "Bavarian" method it was in interval from 3.16 to 5.14 t/ha Corg. 4 CONCLUSIONS After the fifteen-year experiment at IOSDV Rakican, the application of organic fertilizers (farmyard manure, straw) influenced the Corg content. However, the impact was significant only in the system with farmyard manure. Mineral nitrogen fertilization contributed to an increase of the Corg content in all three management systems (system A, system B, system C). However, the increase was not significant. With the application of mineral nitrogen in the system with no organic fertilizers the Corg content resulted in a decrease of 2.3%, i.e. 0.94 t/ha Corg. In the system with farmyard manure ploughing in the Corg content increased by 5.23 t/ha (BN0), 7.24 t/ha (BN1), 7.64 t/ha (BN2) and 5.90 t/ha (BN3). In the system with straw ploughing in the Corg content increased by 0.80 t/ha (CN1), 2.68 t/ha (CN2) and 2.01 t/ha (CN3). The comparison of Corg contents calculated by different humus balance methods in 2008 shows that all three methods underestimated the actual analysed results. "Swiss" method's results most closely approximated the analysed Corg in the systems A and system B, while in the system C the most appropriate method was "VDLUFA". As the average of absolute values of deviations from analysed Corg contents was the lowest by using "Swiss" method (6.75 t/ha Corg), we can conclude that this method is most appropriate method for this site-specific location. 5 ACKNOWLEDGEMENT This work is part of the "Genetics and modern technologies of agricultural plants" programme (P4- 0077) funded by the Slovenian Research Agency (ARRS). 6 REFERENCES Anonymous, (1998): Leitfaden für die Düngung von Acker-und Gründland, 6. uberarbeitete Auflage 1997, Bayerische Landesanstalt für Bodenkultur und Pflanzenbau, Freising - München: 9-12 p. Berecz, K., Kismanyoky, T., Debreczeni, K. (2004): Studying the effect of organic matter recycling combined with mineral N fertilization in long-term field and model pot experiments. Archives of Agronomy and Soil Science, 50: 65 - 72. Beschow, H., Merbach, W. (2004): Entwicklung der Organischen Bodensubstanz (OBS) auf Löss in Abhängigkeit von unterschiedlicher Düngung am Beispiel des Bodenbildungsversuches in Halle/Saale. Archives of Agronomy and Soil Science, 50: 59 - 64. Bosatta, E., Ägren, G.I. (1997): Theoretical analyses of soil texture effects on organic matter dynamics. Soil Biology and Biochemistry, 29: 1633 - 1638. Brodowski, S., Amelung, W., Haumaier, L., Zech, W. (2007): Black carbon contribution to stable humus in German arable soils. Geoderma, 139: 220 - 228. Buyanovsky, G.A., Wagner, G.H. (1998): Changing role of cultivated land in the global carbon cycle. Biology and Fertility of Soils, 27: 242 - 245. Chertov, O.G., Komarov, A.S., Nadporozhskaya, M.A. (2007): Analysis of the Dynamics of Plant Residue Mineralization and Humification in Soil. Eurasian Soil Science, 40: 140 - 148. Diekow, J., Mielniczuk, J., Knicker, H., Bayer, C., Dick, D.P., Kögel-Knabner, I. (2005): Carbon and nitrogen stocks in physical fractions of a subtropical Acrisol as influenced by long-term no-till cropping systems and N fertilisation. Plant and Soil, 268: 319 - 328. Diez, T., Krauss, M. (1992): Berechnung von Humisbilanzen, SuB 06: III-9-III-11. Dorado, J., Zancada, M.-C., Almendros, G., Löpez-Fando, C. (2003): Changes in soil properties and humic substances after long-term amendments with manure and crop residues in dryland farming systems. Journal of Plant Nutrition and Soil Science, 166: 31 - 38. Ellmer, F., Peschke, H., Köhn, W., Chmielewski, F.-M., Baumecker, M. (2000): Tillage and fertilizing effects on sandy soils. Review and selected results of long-term experiments at Humboldt-University Berlin. Journal of Plant Nutrition and Soil Science, 163: 267 - 272. Ellerbrock, R.H., Höhn, A., Rogasik, J. (1999): Functional analysis of organic matter as affected by long-term manurial treatment. European Journal of Soil Science, 50: 65 - 71. Filip, Z., Kubat, J. (2004): Mineralisation and humification of plant matter in soil samples as a tool in the testing of soil quality. Archives of Agronomy and Soil Science, 50: 91 - 97. Goyal, S., Sakamoto, K., Inubushi, K., Kamewada, K. (2006): Long-term effects of inorganic fertilization and organic amendments on soil organic matter and soil microbial properties in Andisols. Archives of Agronomy and Soil Science, 52: 617 - 625. Hoffmann, S., Csitari, G., Hegedüs, L. (2002): The humus content and soil biological properties as a function of organic and mineral fertilization. Archives of Agronomy and Soil Science, 48: 141 - 146. Hoffmann, S., Schulz, E., Csitari, G., Bankö, L. (2006): Influence of mineral and organic fertilizers on soil organic carbon pools. Archives of Agronomy and Soil Science, 52: 627 - 635. ISO 10694, 1996-08 Soil quality - Determination of organic and total carbon after dry combustion (elementary analysis). Jenkinson, D.S., Harris, H.C., Ryan, J., McNeill, A.M., Pilbeam, C.J, Coleman, K. (1999): Organic matter turnover in a calcareous clay soil from Syria under a two-course cereal rotation. Soil Biology and Biochemistry, 31: 687 - 693. Körschens, M. (1997): Abhängigkeit der Organischen Bodensustanz (OBS) von Standort und Bewirtschaftung sowie Ihr Einfluss auf Ertrag und Bodeneigenschaften. Archives of Agronomy and Soil Science, 41: 435-463. Körschens, M. (2002): Importance of soil organic matter (SOM) for biomass production and environment (a review). Archives of Agronomy and Soil Science, 48: 89 - 94. Körschens, M. (2004): Soil organic matter and environmental protection. Archives of Agronomy and Soil Science, 50: 3 - 9. Körschens, M., Rogasik, J., Schulz, E., Böning, H., Eich, D., Ellerbrock, R., Franko, U., Hülsbergen, K.-J., Köppen, D., Kolbe, H., Leithold, G., Merbach, I., Peschke, H., Prystav, W., Reinhold, J., Zimmer, J. (2004): Humusbilanziehrung - Methode zur Beurteilung und Bemessung Humusversorgung von Ackerland. Verband Deutscher Landwirtschaftlichcher Untersuchungs- und Forschungsnastalten (VDLUFA) (ed.): 12 p. Kuzyakov, Y., Domanski, G. (2000): Carbon input by plants into the soil. Review. Journal of Plant Nutrition and Soil Science, 163: 421 - 431. Stumpe, H., Wittenmayer, L., Merbach, W. (2000): Effects and residual effects of straw, farmyard manuring, and mineral fertilization at Field F of the long-term trial in Halle (Saale), Germany. Journal of Plant Nutrition and Soil Science, 163: 649 - 656. Simon, T. (2007): Quantitative and qualitative characterization of soil organic matter in the long-term fallow experiment with different fertilization and tillage. Archives of Agronomy and Soil Science, 53: 241 - 251. Tajnšek, A. (2003): Vsebnost humusa in nekatere fizikalne lastnosti tal v odvisnosti od gnojenja z organskimi gnojili in dušikom v IOSDV Jable in IOSDV Rakičan. In: Deset let poskusov IOSDV v Sloveniji, Jable in Rakičan 1993-2003. Zbornik posveta. Tajnšek, A., Čeh Brežnik, B., Kocjan Ačko, D. (ur.) Ljubljana: 25-34. Triberti, L., Nastri, A., Giordani, G., Comellini, F., Baldoni, G., Toderi, G. (2008): Can mineral and organic fertilization help sequestrate carbon dioxide in cropland? European Journal of Agronomy, 29: 13 - 20. Winkelmann, W., Ellmer, F., Zeitz, J. (2006): Einfluss differenzierter organisch-mineralischer Düngung auf die partikuläre organische Substanz des Bodens. Archives of Agronomy and Soil Science, 52: 365 - 376. COBISS koda 1.02 Agrovoc descriptors: dryocosmus kuriphilus, control biologico see biological control, biological control organisms, natural enemies, control methods, damage, crop losses, identification, monitorin Agris category code: H10 Biotično zatiranje kostanjeve šiškarice (Dryocosmus kuriphilus Yasumatsu, Hymenoptera, Cynipidae) Katarina KOS1, Stanislav TRDAN2 Received August 10, 2009; accepted January 20, 2010. Delo je prispelo10. avgusta 2009; sprejeto 20. januarja 2010. IZVLEČEK Kostanjevo šiškarico, Dryocosmus kuriphilus (Hymenoptera: Cynipidae), štejemo med najpomembnejše škodljivce kostanja (Castanea sp. in Castanopsis sp.) na različnih območjih sveta. Ličinke te osice povzročjo nastanek šišk na vrhu poganjkov in na listnih žilah, kar močno poslabša zdravstveno stanje kostanja in zmanjša pridelek plodov. Kostanjeva šiškarica izvira s Kitajske, v Evropo in po svetu pa se je razširila z napadenim rastlinskim materialom. V Sloveniji se je prvič pojavila leta 2004 na Goriškem, v sosednji Italiji pa se je pojavila že tri leta prej in se je že razširila po skoraj celi državi. V nasadih kostanja v Italiji že povzroča veliko škodo. Za najbolj učinkovit ukrep zatiranja kostanjeve šiškarice velja vnos parazitoidnih osic, ki parazitirajo kostanjevo šiškarico in tako pomembno omejujejo škodo, nastalo zaradi tega škodljivca. Najbolj učinkovita je osica Torymus sinensis (Hymenoptera: Torymidae), ki izvira s Kitajske in so jo že uspešno vnesli v nasade kostanja na Japonskem. Znani so tudi drugi parazitoidi, ki pa so manj učinkoviti biotični agensi za zatiranje kostanjeve šiškarice. 15 vrst parazitoidov, ki so jih doslej našli v šiškah kostanjeve šiškarice, je prisotnih tudi v sosednji Italiji. V Sloveniji zaenkrat še nismo odkrili domorodnih vrst parazitoidov kostanjeve šiškarice, vendar raziskave potekajo, saj je velika nevarnost, da se bo škodljivec v prihodnjih letih razširi po vsej Sloveniji. Ključne besede: kostanjeva šiškarica, Dryocosmus kuriphilus, biotično varstvo, naravni sovražniki, parazitoidi, Torymus sinensis ABSTRACT BIOLOGICAL CONTROL OF CHESTNUT GALL WASP (Dryocosmus kuriphilus Yasumatsu, Hymenoptera: Cynipidae) The chestnut gall wasp, Dryocosmus kuriphilus (Hymenoptera: Cynipidae), is considered to be one of the most important pest of chestnut (Castanea sp. and Castanopsis sp.) worldwide. The larvae of chestnut gall wasp feed on plant tissue, causing formation of galls on the shoots and leaf veins, resulting in severe reduction of fruit yield and vitality of plant. The wasp is of Chinese origin and it invaded Europe and the rest of the world with infected plant material. In Slovenia, chestnut gall wasp appears in Goriška region in 2004 for the first time, but in Italy it appeared three years earlier and is yet widespread in chestnut orchards and forests causing great damage. Introducing parasitoid wasps of chestnut gall wasp seems to be the most promising biological control method to reduce this pest and its damage. The most effective parasitoid species is Torymus sinensis (Hymenoptera: Torymidae), which originate from China and it has been yet successfully introduced and widespread in Japan. The chestnut gall wasp has many parasitoids, but they are not that effective in reducing the population of the pest. 15 species of chestnut gall wasp are presented in Italy. In Slovenia, we have not found any native chestnut gall wasp parasitoids yet, but the research is in process, because of the danger of spreading this important pest throughout Slovenia chestnut orchards and forests. Key words: chestnut gall wasp, Dryocosmus kuriphilus, biological control, natural enemies, parasitoids, Torymus sinensis 1 asist., univ. dipl. inž. agr., Katedra za fitomedicino, kmetijsko tehniko, poljedelstvo, pašništvo in travništvo, Jamnikarjeva 101, SI-1111 Ljubljana, e-pošta: katarina.kos@bf.uni-lj.si 1 Izr. prof. dr., prav tam 1 UVOD Ose šiškarice, kamor spada tudi kostanjeva šiškarica (Dryocosmus kuriphilus Yasumatsu (Hymenoptera: Cynipidae) (slika 1), so obligatni paraziti rastlin, ki bodisi same povzročijo tvorbo šišk (slika 2, levo) na rastlinah ali pa se naselijo v šiške drugih os šiškaric. Tvorba rastlinske šiške je posledica izločkov jajčec ali ličink v rastlinskem meristemskem tkivu. Vsaka ličinka se razvija v lastni kamrici v šiški, v rastlinski šiški pa je lahko več kamric. V pleme Cynipini oz. hrastovih os šiškaric spada od 900 do 1000 vrst, med njimi je tudi kostanjeva šiškarica (Stone in sod., 2002). Rod Dryocosmus ima 25 vrst in prvotno naseljuje cer (Quercus cerris L.), lahko pa so gostitelji tudi vrste iz dveh rodov kostanja, Castanea in Castanopsis (Stone in sod., 2002). Hrastove ose šiškarice iz rodov Andricus, Disholcaspis in Dryocosmus tvorijo šiške, ki izločajo nektar in tako privlačijo mravlje; te lahko pomembno zmanjšajo napad parazitoidov in drugih vsiljivcev na šiške os (Stone in sod., 2002). Kostanjeva šiškarica izvira s Kitajske, kjer pa ima mnogo domorodnih naravnih sovražnikov. Zato tam ni gospodarsko pomemben škodljivec. Napad škodljive osice lahko zmanjša pridelek plodov za 50-75 %, ob močnem napadu pa je zmanjšana vitalnost drevesa, prirast lesa, takšno drevo lahko celo propade. Doslej so preizkušali že številne metode za omejevanje populacij kostanjeve šiškarice. Uporaba kemičnih insekticidov se ni izkazala za učinkovit način, saj so jajčeca in ličinke zavarovane v rastlinskih šiškah in jih insekticid ne doseže. Mehanične metode, kot so odstranjevanje napadenih poganjkov in zaščita mladih poganjkov z mrežami, so sicer učinkovite, vendar to niso praktične rešitve. Žlahtnjenje kostanja za odpornost proti kostanjevi šiškarici so 20 let uspešno izvajali na Japonskem, vendar so se kmalu pojavile nove virulentne rase osice, ki so premagale odpornost. Zaenkrat je edina učinkovita metoda omejevanja populacij in škode zaradi napada kostanjeve šiškarice uporaba parazitoidov iz reda kožekrilcev (Hymenoptera) kot biotičnih agensov (Aebi et al, 2007). Izmenjava kultivarjev kostanja med gojitelji in partenogenetsko razmnoževanje sta omogočila hitro širjenje kostanjeve šiškarice v Aziji, Severni Ameriki in Evropi (Aebi et al. 2006). Kostanjeva šiškarica je v svetu eden od najpomembnejših škodljivih organizmov na vrstah z rodu Castanea in spada med karantenske organizme Evropske in sredozemske organizacije za varstvo rastlin (EPPO) (EPPO, 2005). V Evropi so tega karantenskega škodljivca zasledili najprej v Italiji leta 2002 (Brussino et al, 2002), nato pa v Sloveniji (Seljak, 2006) in Franciji (EPPO, 2007). 'M > Slika 1: Samica kostanjeve šiškarice (foto: K. Kos). Slika 2: Prerez skozi šiško pravega kostanja (Castanea sativa Mill.) (levo) in buba kostanjeve šiškarice (Dryocosmus kuriphilus) (desno) (foto: K. Kos). 2 PREGLED MOŽNOSTI BIOTIČNEGA ZATIRANJA KOSTANJEVE ŠIŠKARICE S PARAZITOIDI 2.1 AZIJA Na Kitajskem kostanjeva šiškarica ni pomemben škodljivec kostanja, majhne populacije te žuželčje vrste pa so rezultat parazitiranja in plenjenja s strani naravnih sovražnikov. Znanih je 11 vrst parazitoidnih osic iz petih družin (Torymidae, Ormyridae, Eurytomidae, Eulophidae in Eupelmidae), ki so na Kitajskem domorodne (preglednica 1) in tam omejujejo populacije šiškarice (Murakami in sod., 1980). Prve napade kostanjeve šiškarice na Japonskem so opazili že leta 1941 v Okay ami, od leta 1946 naprej pa se je napad hitro širil. Tako je postala kostanjeva šiškarica eden od najpomembnejših škodljivcev kostanja na Japonskem. Kdaj je bil škodljivec vnesen na Japonsko, ni znano, predvidevajo pa, da je bil nehote vnesen iz Kitajske (Murakami et al, 1977). Japonci so leta 1975 nabrali 69 šišk na kostanju in jih prenesli na Japonsko. Iz šišk je izletelo 7 osebkov parazitoidov iz rodu Torymus (Syntomaspis), v šiškah pa so našli še pet vrst parazitoidov: Megastigmus sp., Ormyrus sp., Eurytoma brunniventris, Eudecatoma sp. in en osebek neidentificiranega parazitoida. Parazitizem v kostanjevem gozdu, kjer je bil material nabran, je bil ocenjen na 60 do 80 %. Na raziskovalni postaji Hiratsuka so opravili preliminarni test izpusta 3 samic in dveh samcev parazitoidov iz rodu Torymus. Rezultati so pokazali, da je bilo povprečje potomcev ene samice parazitoida 10.3 in so tako namigovali na možnost uspešne introdukcije tega parazitoida na Japonsko. Med kitajskimi parazitoidi je bila vrsta Torymus sp. edina, ki je bila gostiteljsko specifična in je bila razvojno sinhrona s kostanjevo šiškarico (Murakami et al.., 1977). Preglednica 1: Domorodne vrste parazitoidov kostanjeve šiškarice (Dryocosmus kuriphilus) na Kitajskem (Aebi et al., 2006: cit. po Murakami et al., 1980). Vrsta parazitoida Družina Eupelmus urozonus Dalman Eupelmidae Eurytoma brunniventris Ratzeburg Eurytomidae Eurytoma setigera Mayr Eurytomidae Megastigmus maculipennis Yasumatsu & Kamijo Torymidae Megastigmus nipponicus Yasumatsu & Kamijo Torymidae Ormyrus pomaceus (Geoffroy) (=O. punctiger) Ormyridae Sycophila concinna (Boheman) Eurytomidae Sycophila variegata (Curtis) Eurytomidae Tetrastichus sp. Eulophidae Torymus geranii (Walker Torymidae Torymus sinensis Kamijo Torymidae Leta 1979 so na Kitajskem v raziskovalnem inštitutu Hoipei Fruit Tree ugotavljali vrstno pestrost parazitoidov kostanjeve šiškarice. Vrsta Torymus sp. je bila identična vrsti, ki je izletela iz kostanjevih šišk nabranih leta 1975. Omenjena vrsta je sorodna z vrsto Torymus (Syntomaspis) beneficus Yasumatsu et Kamijo, ki je parazitoid kostanjeve šiškarice in ose šiškarice na hrastu (Andricus sp.) na Japonskem. Tudi tam je bil parazitoid Torymus sp. najbolj dominanten, njegov razvojni krog pa je sovpadal z razvojnim krogom kostanjeve šiškarice; ličinke so prezimovale v posušenih šiškah in iz njih so se v aprilu in v začetku maja razvile odrasle osice. Ostali parazitoidi so izletavali šele od junija do avgusta iz na novo nastalih šišk (Murakami et al., 1980) in njihov razvoj ni bil tako dobro skladen z razvojem kostanjeve šiškarice. Zanje je zato znano, da potrebujejo alternativne gostitelje, kot so ose šiškarice na hrastu in druge (Otake et al., 1982). Že leta 1959 je Torii objavil študijo o biotičnem zatiranju kostanjeve šiškarice, kjer je ocenil, da prvi parazitoidi na območju Kamiina na Japonskem izletijo okoli 20. junija, prvi osebki kostanjeve šiškarice pa letajo vsako leto od 1. julija do 5. avgusta, torej izletijo približno od 10 do 15 dni pozneje kot njeni parazitoidi. Omenjeni je ocenil tudi najbolj optimalen čas za izvajanje biotičnega zatiranja kostanjeve šiškarice s parazitoidi, in sicer je ta od 4. do 12. julija. V tem času namreč izleti 75 % parazitoidov in le 10 % vseh os šiškaric (Torii, 1959). V Fukuoki na Japonskem so se odrasli osebki vrste T. beneficus iz suhih šišk vrst D. kuriphilus in Andricus sp., ki so nastale prejšnjo pomlad, razvili od marca do začetka aprila. Disekcija mladih šišk kostanjeve šiškarice, nabranih od konca marca do sredine aprila, je pokazala, da so bila jajčeca parazitoida odložena na notranjo steno kamrice ličinke v šiški ter da so bile ličinke kostanjeve šiškarice v teh kamricah že parazitirane. To pomeni, da samica parazitoida paralizira gostiteljsko ličinko med odlaganjem jajčec (Murakami in Tokuhisa, 1985). Na Japonskem kostanjevo šiškarico parazitira kar 24 vrst parazitoidov. V tej državi in na Kitajskem imajo 10 skupnih vrst parazitoidov, med katerimi je 8 znanih tudi kot parazitoidov hrastove šiškarice Andricus sp. na Japonskem (preglednica 2). Na Japonskem je 18 vrst parazitoidov, ki parazitirajo kostanjevo šiškarico, povezanih s hrastovo šiškarico, kar kaže na menjavo gostiteljev z domorodne hrastove šiškarice na kostanjevo šiškarico (Aebi et al, 2006). Vendar pa domorodni parazitoidi niso uspeli omejiti širjenja populacij kostanjeve šiškarice, zato je bil s Kitajske vnesen parazitoid Torymus sinensis (Otake in sod., 1984), ki je v nekaj letih močno zmanjšal populacijo škodljivca. To je poseben primer, kjer je parazitoidna vrsta, ki je navadno generalist, nastopila kot učinkovita monofagna vrsta na izredno veliki populaciji ene vrste gostitelja. Pojavlja se vprašanje ali bi se ta parazitoid podobno odzval tudi v skupnosti več gostiteljev (Stone et al, 2002). Na Japonskem so preučevali tudi vpliv domorodnih parazitoidov na učinkovitost vnesenih parazitoidov. Preučevali so komplekse domorodnih parazitoidov kostanjeve šiškarice na dveh lokacijah (Miyagi in Okayama), kjer se parazitoid Torymus sinensis še ni pojavil. Rezultate te raziskave so primerjali z rezultati iz območja nasada kostanjev Kumamoto, kamor je bil parazitoid T. sinensis že vnesen in se je že ustalil. Struktura kompleksa parazitoidov in velikost sta se razlikovali glede na lokacije. Dominantni parazitoid na obeh območjih je bila vrsta T. beneficus, vendar se je stopnja dominiranosti razlikovala glede na lokacijo. Tudi hiperparazitiranost s strani fakultativnih hiperparazitoidov je bila različna glede na lokacijo; na območju Miyagi so imeli fakultativni hiperparazitoidi le majhen vpliv na smrtnost vrste T. beneficus, na območju Okayama pa je bil ta vpliv zelo velik. Glede na te rezultate so predvidevali, da bo populacija vrste T. sinensis, ki je zelo podobna vrsti T. beneficus, na območju Okayama potrebovala veliko časa, da se poveča in to zaradi učinka obratne odvisnosti gostote fakultativnih hiperparazitoidov, medtem ko bi se populacija na območju Miyagi lahko hitro povečala (Murakami et al, 1994). Preglednica 2: Parazitoidi, ki na Japonskem parazitirajo kostanjevo šiškarico (Dryocosmus kuriphilus). Vrste, ki so znane tudi na Kitajskem, so pisane krepko, oznaka * pa pomeni vrste, ki so povezane s hrastovo šiškarico (Andricus sp.) na Japonskem (Aebi et al.., 2006). Vrsta parazitoida Družina Amblymerus sp. Pteromalidae Arthrolytus usubai Kamijo * Pteromalidae Caenacis peroni Kamijo* Pteromalidae Cecidostiba fushica Kamijo* Pteromalidae Cecidostiba semifascia (Walker)* Pteromalidae Cynipencyrtus flavus Ishii* Encyrtidae Cynipencyrtus sp. Encyrtidae Eupelmus urozonus Dalman* Eupelmidae Eupelmus sp. Eupelmidae Eurytoma brunniventris Ratzeburg* Eurytomidae Eurytoma schaeferi Yasumatsu et Kamijo* Eurytomidae Eurytoma setigera Mayr* Eurytomidae Megastigmus maculipennis Yasumatsu & Kamijo* Torymidae Megastigmus nipponicus Yasumatsu & Kamijo * Torymidae Mesopolobus yasumatsui Kamijo* Pteromalidae Ormyrus flavitibialis Yasumatsu & Kamijo* Ormyridae Ormyrus pomaceus (Geoffroy) (=O. punctiger)* Ormyridae Pteromalus apantelophagus (Crawford)* Pteromalidae Sycophila variegata (Curtis)* Eurytomidae Tetrastichus sp. Eulophidae Torymus beneficus Yasumatsu & Kamijo* Torymidae Torymus geranii (Walker)* Torymidae Torymus sinensis Kamijo Torymidae Torymus sp. Torymidae Aspilota yasumatsui WATANABE Braconidae Synergus sp. Cynipidae 2.2 EVROPA V Evropi so kostanjevo šiškarico prvič našli leta 2002 v Piemontu v Italiji (Brussino et al, 2002). Do leta 2005 se je osica razširila v 5 dotlej nenaseljenih italijanskih provincah (Lacio, Campania, Toscana, Abruzzo in Marche), ki predstavljajo precejšen del polotoka (Aebi et al, 2006). Leta 2008 so iz Italije poročali o novih najdiščih napadenih kostanjev, in sicer skupaj že iz 11 provinc. Kostanjeva šiškarica je tako napadla tudi najbolj zgodovinsko pomembna območja gojenja kostanja v Italiji (Graziosi in Santi, 2008). Leta 2004 je bilo iz Piemonta v Slovenijo uvoženih 1250 sadik pravega kostanja. Pregledanih je bilo 47 % rastlin, zaradi napadenosti pa je bilo uničenih 10 rastlin (Aebi et al., 2006). V Italiji so zaradi hitrega širjenja škodljive osice ukrepali z vnosom parazitoidne osice T. sinensis, ki je bila kot domorodna vrsta najdena tako na Kitajskem kot tudi na Japonskem. Po dveh letih (2003 in 2004) neuspešnih poskusov vnosa zaradi prezgodnjega izleta parazitoidnih osic glede na razvoj gostitelja (le 0,5-1,6 % parazitiranost), so leta 2005 na tri lokacije v Italiji prvič izpustili 90 oplojenih samic vrste T. sinensis iz Japonske. Omenjene osebke so hladili in na ta način umetno sinhronizirali izlet osice in razvoj šišk gostitelja (Aebi et al, 2006). Leta 2006 so izpustili 1058 parov osic na 11 lokacij, izvorni material pa je predstavljalo 25.500 šišk iz Japonske. Da bi olajšali nadaljnje izpuste osice T. sinensis, so začeli s poskusi njenega masovnega gojenja na mladih napadenih drevesih kostanja v Italiji (Aebi et al., 2007). V italijanskih laboratorijih so raziskovali bionomijo in gostitelj sko specifičnost parazitoida T. sinensis. Za te raziskave so uporabili parazitoidne osice, ki so iz šišk izletele mesec pred nastankom šišk na kostanjih v Italiji. Leta 2004 so gojili osice v šiškah pri sobni temperaturi in ugotovili, da je izlet osic veliko prezgoden, saj se šiške še ne formirajo in tako osice niso uspešne pri odlaganju jajčec. V letih 2005 in 2006 so bile osice hranjene v šiškah pri nizki temperaturi do začetka nastajanja novih šišk na kostanju, nato pa pri zunanjih razmerah. Šiške so se začele oblikovati 14. aprila, kar so ocenili iz povprečja nastajanja šišk iz treh let opazovanja (Quacchia et al., 2008). V Italiji so našli 15 parazitoidov kostanjeve šiškarice in le 4 vrste so skupne vrstam na Kitajskem, Japonskem in v Koreji (preglednica 3). Vse vrste parazitoidov, ki napadajo vrsto D. kuriphilus v Italiji, so znane kot parazitoidi hrastove šiškarice v Evropi in veliko jih je razširjenih na območju zahodnega Palearktika. Najbolj učinkovit parazitoid v Italiji je bila vrsta Eupelmus urozonus, ki je tudi najbolj konstantno napadala šiške kostanjeve šiškarice (preglednica 3) (Aebi et al., 2006) Preglednica 3 Vrste parazitoidov, ki napadajo kostanjevo šiškarico (Dryocosmus kuriphilus) v Italiji, leto prvega odkritja vrste v šiškah kostanjeve šiškarice in geografska razširjenost v zahodnem Palearktiku (WP: zahodni Palearktik; M: južna Evropa in Sredozemlje). Vrste parazitoidov, ki so skupne Italiji in trem azijskim državam, so pisane krepko (Aebi et al, 2006). Vrsta parazitoida Družina Geografska razširjenost Sycophila iracemae Nieves Aldrey Eurytomidae M Sycophila variegata (Curtis) Eurytomidae WP Sycophila biguttata (Swederus) Eurytomidae WP Eurytoma pistacina Rondani Eurytomidae M Eurytoma brunniventris Ratzeburg Eurytomidae WP Mesopolobus mediterraneus Mayr Pteromalidae M Mesopolobus sericeus (Forster) Pteromalidae WP Mesopolobus tarsatus (Nees) Pteromalidae M Torymus auratus (Müller) Torymidae WP Torymus flavipes (Walker) Torymidae WP Torymus scutellaris (Walker) Torymidae WP Megastigmus dorsalis (Fabricius) Torymidae WP Eupelmus urozonus Dalman Eupelmidae WP Baryscapus pallidae Graham Eulophidae M Ormyrus pomaceus (Geoffroy) (=O. punctiger Westwood) Ormyridae WP Vrste, ki so parazitirale kostanjevo šiškarico v Italiji, vključujejo skoraj vse parazitoidne vrste, ki naseljujejo različne gostitelje šiškaric. Te vrste (Baryscapus pallidae Graham, Eurytoma pistacina Rondani, Mesopolobus sericeus [Forster], Sycophila biguttata [Swederus], Sycophila iracemae Nieves Aldrey in Torymus flavipes [Walker]) napadajo šiškarice na najmanj dveh vrstah hrasta, na platanah in na vrtnicah. Takšen širok spekter vrst parazitoidov, ki napadajo različne gostitelje in izkoriščajo različne rastlinske sestoje, omogoča hitro detekcijo in izkoriščanje kostanjeve šiškarice kot gostitelja teh mnogih parazitoidnih vrst (Aebi et al., 2006). 3 PARAZITOIDA Torymus (Syntomaspis) beneficus IN Torymus sinensis Obe vrsti sta univoltilni, prav tako kot kostanjeva šiškarica. Morfološko se vrsti zelo težko ločita, saj sta si zelo podobni. Parazitoida Torymus beneficus lahko razdelimo na dve rasi glede na čas izleta (zgodnja in pozna rasa). Odrasle osice izletijo zgodaj spomladi iz posušenih šišk, ki so nastale v pomladi prejšnjega leta. Odrasle samice odlagajo jajčeca v novonastale šiške. Ko se ličinke osic izležejo iz jajčec v šiškah, se ličinke prve stopnje hranijo z mehkih rastlinskim tkivom stene kamrice ličinke šiškarice v šiški. Ko se ličinka razvije do druge razvojne stopnje, se hrani kot ektoparazit na odrasli ličinki kostanjeve šiškarice. Po mesecu dni se ličinke parazitoida razvijejo do stopnje odrasle ličinke in vstopijo v dolgo obdobje diapavze - ta traja do jeseni. Pozimi se zabubijo. Parazitoid se razvija tudi v šiškah na hrastu, kjer je gostitelj osa šiškarica (Andricus sp.), ki naj bi bil prvotni gostitelj zgodnje rase vrste T. beneficus (Murakami, 1988). Biotično zatiranje kostanjeve šiškarice (Dryocosmus kuriphilus Yasumatsu, Hymenoptera, Cynipidae) 4 ZAKLJUČKI Močan pojav kostanjeve šiškarice v Sloveniji lahko pomeni odlično izhodišče za domorodne vrste parazitoidov os šiškaric, ko bodo kostanjevo šiškarico sprejeli kot gostitelja. Pri nas so ose šiškarice zelo razširjene in kostanjevi šiškarici predstavljajo konkurenco v pridobivanju gostiteljev, prav tako pa zagotovo obstajajo domorodne vrste parazitoidov os šiškaric, ki bi lahko pomembno vplivale na manj intenzivno širjenje tega škodljivca, če bi ga sprejele za njihovega gostitelja. Ob močnem pojavu kostanjeve šiškarice in nevarnosti njenega širjenja v nasadih pravega kostanja ter v gozdovih, kjer raste pravi kostanj, bi bilo smiselno uporabiti parazitoida Torymus sinensis, ki je bil s podobnim namenom že vnesen na Japonsko, kjer je pokazal precejšnjo učinkovitost. Če bo omenjena parazitoidna osica uspela v Evropi sinhronizirati njen razvoj z razvojem kostanjeve šiškarice kot gostitelja, bo lahko predstavljala pomemben biotični agens pri zatiranju kostanjeve šiškarice in bo kot takšen uporaben tudi v Sloveniji. 5 ZAHVALA Prispevek je nastal s finančno pomočjo Ministrstva za kmetijstvo, gozdarstvo in prehrano RS - Fitosanitarne uprave RS v okviru strokovnih nalog s področja zdravstvenega varstva rastlin in s finančno pomočjo Javne agencije za raziskovalno dejavnost RS v okviru programa Hortikultura P4-0013. 5 VIRI Aebi A., Schönrogge K., Melika G., Alma A., Bosio G., Quacchia A., Picciau L., Abe Y., Moriya S., Yara K., Seljak G., Stone G.N. (2006): Parasitoid recruitment to the globally invasive chestnut gall wasp Dryocosmus kuriphilus. V: Ozaki K., Yukwa J., Ohgushi T., Price P.W. (ur.) Ecology and evolution of galling arthropods and their associates. Springer-Verlag, Tokyo:103-121. Aebi A., Schönrogge K., Melika G., Alma A., Stone G.N. (2007): Native and introduced parasitoids attacking the invasive chestnut gall wasp Dryocosmus kuriphilus. EPPO Bull., 37: 166-171. Brussino G., Bosio, G., Baudino, M., Giordano, R., Ramello, F., Melika, G. (2002): Pericoloso insetto esotico per il castagno europeo. Infor. Agrar., 58: 59-61. Quacchia, A., Moriya, S., Bosio, G., Scapin, I., Alma, A. (2008): Rearing, release and settlement prospect in Italy of Torymus sinensis, the biological control agent of teh chestnut gall wasp Dryocosmus kuriphilus. BioControl, 53: 829-839. EPPO (2005): Dryocosmus kuriphilus. EPPO Bull., 35: 422424. EPPO (2007): Dryocosmus kuriphilus found in the south of France (Alpes Maritimes). EPPO Reporting service -Pests & Diseases, 5 (086): 2. online URL: http//archives.eppo.org/ Graziosi, I., Santi, F. (2008): Chestnut gall wasp (Dryocosmus kuriphilus): spreading in Italy and new records in Bologna province. Bull. Insect., 61: 343-348. Murakami, Y., Umeya, K., Oho N. (1977): A preliminary Introduction and release of a parasitoid (Chalcidoidea, Torymidae) of the Chestnut Gall Wasp, Dryocosmus kuriphilus Yasumatsu (Cynipidae) from China. Jpn. J. Appl. Entomol. Zool., 21: 197-203. Murakami, Y., Ao, H.B., Chang, C.H. (1980): Natural enemies of the chestnut gall wasp in Hoipei Province, China (Hymenoptera: Chalcidoidea). Jpn. J. Appl. Entomol. Zool., 15: 184-186. Murakami, Y., Tokuhisa, E. (1985): Behavioural sequences of oviposition and host-feeding of Torymus (Syntomaspis) beneficus Yasumatsu et Kamijo (Hymenoptera: Torymidae), a native parasitoid of Dryocosmus kuriphilus Yasumatsu (Hymenoptera: Cynipidae). Jpn. J. Appl. Entomol. Zool., 20: 43-49. Murakami, Y. (1988): Ecotypes of Torymus (Syntomaspis) beneficus Yasumatsu et Kamijo (Hymenoptera: Torymidae) with different seasonal prevalences of adult emergence. Jpn. J. Appl. Entomol. Zool., 23: 81-87. Murakami, Y., Hiramatsu, T., Maeda, M. (1994): Parasitoid complexes of the Chestnut gall wasp (Hymenoptera: Cynipidae) in two localities before introduction of Torymus (Syntomaspis) sinensis (Hymenoptera: Torymidae), with special reference to prediction of results after release of the parasitoid. Jpn. J. Appl. Entomol. Zool., 38: 29-41. Otake, A., Shiga, M., Moriya, S. (1982): A study on parasitism of the chestnut gall wasp, Dryocosmus kuriphilus Yasumatsu (Hymenoptera: Cynipidae) by parasitoids indigenous to Japan. Bull. Fruit Tree Res. Stn. A 9: 177-192. Otake, A., Shiga, M., Moriya, S. (1984): Colonization of Torymus sinensis Kamijo (Hymenoptera: Torymidae), a parasitoid of the chestnut gall wasp, Dryocosmus kuriphilus Yasumatsu (Hymenoptera: Cynipidae) introduced from China. Appl. Entomol. Zool., 19: 111114. Seljak, G. (2006): Chestnut gall wasp - Dryocosmus kuriphilus Yasumatsu. Report - Phytosanitary Administration of the Republic of Slovenia. [online] URL: http://www.furs.si/ Stone, G.N., Schonrogge, K., Atkinson, R.J., Bellido, D., Pujade-Villar, J. (2002): The population biology of oak gall wasps (Hymenoptera: Cynipidae). Annu. Rev. Entomol., 47: 633-668. Torii, T. (1959): Studies on the biological control of the chestnut gall wasp, Dryocosmus kuriphilus Yasumatsu (Hym.: Cynipidae), with particular reference to the utilization of its indigenous natural enemies. J. Fac. Agric., Shinshu Univ. 2: 71-149. COBISS koda 1.02 Agrovoc descriptors: climatic change, climatic factors, meteorological elements, meteorological observations, data collection, precipitation, evapotranspiration, temperature, seasons, adaptation, trends, environmental factors Agris category code: P40 Spremembe agro-klimatskih spremeljivk v Sloveniji v zadnjih desetletjih Lučka KAJFEŽ-BOGATAJ1 , Tjaša POGAČAR2, Andrej CEGLAR3, Zalika ČREPINŠEK4 Prispelo: 21.septembra 2009; sprejeto 30. decembra 2009 Received: September 21, 2009; accepted December 30, 2009 IZVLEČEK Kmetijstvo je v veliki meri odvisno od vremena oz. od podnebnih razmer. Kmetijstvo ima ob vse izrazitejših podnebnih spremembah pomembno vlogo pri njihovem blaženju, hkrati pa se mora nanje tudi prilagajati. Za razvoj strategij prilagajanja je potrebno poznavanje vzorcev spreminjanja različnih agro-meteoroloških kazalcev, zato je namen tega prispevka ovrednotiti spremembe ključnih agro-meteoroloških spremenljivk nad območjem Slovenije. Obravnavali smo spremembe temperatur, števila hladnih, toplih ter vročih dni, evapotranspiracije, količine padavin in drugih izvedenih spremenljivk. Za izračun sprememb smo niz meteoroloških podatkov razdelili v izhodiščno obdobje (1961 - 1990) ter primerjalno obdobje (1991 - 2007), za izračun trendov pa smo uporabili kar celoten niz meteoroloških podatkov med leti 1961 ter 2007. Povprečna letna temperatura zraka se je v primerjalnem obdobju povečala med 0,7 °C ter 1,4 °C, izrazit pa je tudi dvig povprečne maksimalne temperature zraka. Izrazito je tudi povečanje števila vročih dni ter zmanjšanje števila hladnih dni. Pri padavinah so opazne spremembe izrazito sezonsko ter regionalno pogojene; tako se je količina padavin zmanjšala pozimi, jeseni pa povečala. Izrazito pozitiven trend je razviden pri poletni evapotranspiraciji, pri čemer so vrednosti trenda najvišje julija (na Goriški ter Obalno-kraški regiji celo 30 mm/10 let). Ključne besede: podnebne spremembe, prilagajanje, agro-meteorološki kazalci, trend, temperatura, padavine, evapotrans-piracija, Winklerjev indeks, Huglinov indeks ABSTRACT TRENDS IN AGRO-CLIMATE VARIABLES IN SLOVENIA Agriculture crucially depends on climate conditions. It has a significant role in mitigating climate change but it also has to adopt certain adaptation measures in order to preserve sustainability. Development of adaptation measures is based on knowledge about agro-meteorological variables. The purpose of this work is therefore to assess changes of most important agro-meteorological variables over Slovenia. These variables include temperature, precipitation, number of hot, warm and cold days, evapotranspiration, precipitation amount and other indices. For the calculation purposes we divided time series of meteorological variables into comparison period (1991 - 2007) which was compared to the base period (1961 -1990). Average annual temperature has risen for about 0.7 °C to 1.4 °C, the rise in maximum temperature is even more pronounced. The number of hot days has increased whereas the number of cold days has decreased. Precipitation has decreased in winter and increased in autumn, changes in spring and summer exhibit spatial dependency and are in general lower. Summer evapotranspiration exhibits significant positive trend, which is most pronounced in south-eastern part of Slovenia (30 mm/10 years). Key words: climate change, adaptation, agro-meteorological variables, trends, air temperature, precipitation, evapotranspi-ration, Winkler's index, Huglin's index 1 prof. dr., Univerza v Ljubljani, Biotehniška fakulteta, 1000 Ljubljana, Jamnikarjeva 101, lucka.kajfez.bogataj@bf.uni-lj.si 2 univ.dipl.ing.meteor., Agencija republike Slovenije za okolje, 1000 Ljubljana, Vojkova 1b 3 univ.dipl.ing.meteor., Univerza v Ljubljani, Biotehniška fakulteta, 1000 Ljubljana, Jamnikarjeva 101 4 doc. dr., Univerza v Ljubljani, Biotehniška fakulteta, 1000 Ljubljana, Jamnikarjeva 101 1 UVOD Kmetijstvo je usodno odvisno od vremena oziroma podnebnih razmer, saj imajo temperatura zraka in tal, sončno obsevanje, zračna vlaga, količina in razporeditev padavin, pogostnost in intenzivnost vremenskih ujm odločilen vpliv na kmetijsko pridelavo. Podobno je z gozdom v Sloveniji, ki se s 60 odstotnim deležem gozdov uvršča v tretjo najbolj gozdnato državo v Evropi. A podnebje na zemlji se zaradi človeških posegov v podnebni sistem vse hitreje spreminja (Luterbacher in sod., 2004). Z veliko zanesljivostjo lahko trdimo, da je izrazitemu dvigu temperature v zadnjih nekaj desetletjih botroval predvsem človek s spreminjanjem rabe tal in zlasti z emisijami toplogrednih plinov (TGP) ter aerosolov, ki spreminjajo sestavo ozračja in s tem vplivajo na energijsko bilanco Zemlje kot celote. Mnogo gospodarskih sektorjev je močno odvisnih od podnebnih razmer in posledice podnebnih sprememb bodo neposredno občutile predvsem kmetijske dejavnosti (Črepinšek in sod., 2009). Zmanjšana razpoložljivost vode, škoda zaradi vetra, višje temperature, suše, večje število gozdnih požarov in večje tveganje za bolezni bodo povzročile škodo pri gozdovih (Simončič in sod., 2001). Čeprav imajo podnebne spremembe nekaj pozitivnih vidikov (npr. kmetijska proizvodnja v nekaterih omejenih predelih Evrope), utegnejo negativne posledice prevladati. Napredek znanosti je omogočil, da podatki o podnebju danes segajo že daleč v preteklost. Čeprav se je podnebje našega planeta v preteklosti že mnogokrat spreminjalo, pa je danes pojem podnebne spremembe vezan na človekov vpliv na podnebni sistem. Človek vse hitreje prek izpustov toplogrednih plinov (TGP) spreminja sestavo ozračja, s spremenjeno rabo tal in sekanjem gozdov pa tudi značilnosti zemeljske površine. Na primer vsebnost CO2 je danes v atmosferi najvišja v zadnjih 650.000 letih. TGP povzročajo učinek tople grede in ker povečujemo njihovo vsebnost v ozračju, s tem zmanjšujemo prepustnost ozračja za sevanje površja. Kar povzroča dvig temperature površja, je torej povečani učinek tople grede. Posledice podnebnih sprememb so v Evropi že precejšnje in izmerljive. Zaradi nelinearnosti podnebnih vplivov in občutljivosti ekosistemov imajo lahko že majhne temperaturne spremembe zelo velike posledice (Črepinšek in sod., 2006). Evropa se je v prejšnjem stoletju segrela za skoraj 1 °C, kar je hitreje od svetovnega povprečja. Toplejše ozračje vsrka več vodne pare, vendar se novi vzorci padavin močno razlikujejo glede na regijo. Na kopnem v zmernih geografskih širinah se je izrazito zmanjšalo število hladnih dni, še zlasti so se zvišale minimalne nočne temperature zraka. V poletnem času se je povečalo število toplih noči. Navkljub globalnemu povečanju padavin, se je pogostnost suš povečevala, predvsem kot posledica spremenjene splošne cirkulacije zraka. Te spremembe se odražajo v okrepljenih zahodnih vetrovih in premiku lege polarne fronte. Okrepljeni zahodni vetrovi vplivajo na prenos oceanskih zračnih mas nad kontinente. Tako lahko zahodni deli kontinentov postajajo toplejši kot vzhodnješi, kar je še posebej očitno v zimskem času. Slovenija je podnebno raznolika dežela, na kar še zlasti vplivata njen razgibani relief in lega med Alpami, Sredozemljem in Panonsko nižino. Glede podnebnih sprememb Slovenija seveda ni izjema. Analiza dolgoletnih meteoroloških meritev kaže številne spremembe v časovnem poteku posameznih podnebnih značilnosti, še posebej pa naraščanje temperatur zraka. Najbolj opazne spremembe beležimo po letu 1990 (Črepinšek in Kajfež Bogataj, 2003). Tudi na Kredarici temperatura zraka narašča, kar kaže, da gre za globalna dogajanja in ne za krajevne spremembe temperature, ki jih lahko povzročijo tudi drugi vzroki - na primer učinek toplotnega otoka v mestnih okoljih. V prispevku se ukvarjamo zlasti z vplivom podnebnih sprememb na agrometeorološke kazalce, ki so nujna podlaga za strategije blaženja podnebnih sprememb in prilagajanja, in sicer spremembe povprečne, minimalne in maksimalne temperature zraka, števila hladnih, toplih in vročih dni, akumulirane temperaturne vsote (efektivne in aktivne), spremenjeno tveganje zaradi pomladanske pozebe, ocena povečane evapotranspiracije ter nove vrednosti klimatskih indeksov za vinogradništvo. Za bazno obdobje smo uporabili tridesetletje 1961-1990 (Schonwiese in Rapp, 1997) in s tedanjimi razmerami primerjali stanje v obdobju 1991-2007 ter tako ponazorili spremembe, ki se že dogajajo. 2 METODE IN MATERIAL Padavinski in temperaturni podatki, izmerjeni na padavinskih ter klimatoloških postajah so bili dobljeni iz arhivov Urada za meteorologijo na Agenciji Republike Slovenije za Okolje (ARSO). Prostorsko interpolacijo podatkov smo naredili z računalniškim programom GSTAT, ki je namenjen geostatističnemu modeliranju, napovedi ter simuliranju, slike prostorske interpolacije pa so bile narisane s pomočjo programa ArcGIS. Velik problem pri izračunih predstavljajo manjkajoči podatki v nizu meritev (bodisi zaradi izpada merilne postaje, ukinitve postaje ...). Število padavinskih ter klimatoloških postaj v Sloveniji je v upadanju, kar na tako razgibanem terenu pomeni vse slabše možnosti za kvalitetno prostorsko obdelavo podatkov. V letu 1961 je delovalo 123 klimatoloških postaj, leta 1971 112, leta 1981 89, leta 1991 68, v letu 2007 pa jih je bilo le še 39. Nekaj takih postaj smo uporabili tudi v našem izračunu. Manjši delež manjkajočih podatkov smo nadomestili s preverjenimi interpolacijskimi shemami, postaje z daljšim nizom manjkajočih podatkov (več let), pa smo v izračunu izpustili. Karte smo pripravili s pomočjo statističnih metod prostorske interpolacije, ki temeljijo na statistični analizi prostorskih podatkov in statističnem modeliranju porazdelitve spremenljivk v prostoru. Uporabili smo metodo splošnega kriginga z upoštevanjem nadmorske višine (Cressie, 1993), ki je implementirana v geostatističnem programskem paketu GSTAT. Ob manjšem številu postaj se pojavi vprašanje reprezentativnosti tovrstne prostorske interpolacije podatkov na območju Slovenije. POTENCIALNA EVAPOTRANSPIRACIJA Referenčna evapotranspiracija je po FAO definirana kot količina vode, ki izhlapi iz površine tal, ki jih pokriva ekstenzivna travna ruša visoka 0,12 m, z albedom 0,23 in konstantno površinsko upornostjo 70 s/m. Površina je zelena, dobro preskrbljena z vodo in enakomerno pokriva celotna tla. Potencialna evapotranspiracija je zmožnost atmosfere, da odstrani določeno količino vode iz tal, poraščenih s poljubno rastlino, pri neomejeni količini vode v tleh. Dejanska evapotranspiracija se iz potencialne izračuna z upoštevanjem lastnosti rastline in tal. Meritev v Sloveniji ni, zato ima izbira dobre računske metode še poseben pomen. Največkrat uporabljamo kombinirano Penman-Moteithovo metodo, ki pa je zelo kompleksna in zahteva velik nabor vhodnih podatkov, ki jih za daljše obdobje in večje število lokacij težko dobimo in uskladimo. Njeno najboljše nadomestilo za Slovenijo je precej enostavnejša Antalova metoda (Kurnik, 2002). Temelji na povezavi s povprečno dnevno temperaturo in povprečno dnevno relativno vlago: / T \ 4'8 1 + — V k 2 J 0,7 ET0 = k1(ea - ed)0, pri čemer je T > 0: c1 = 6,108hPa, c2 = 17,08, c3 = 234,175°C in pri T < 0: cj = 6,107hPa, c2 = 22,44, c3 = 272,44°C (Kurnik, 2002). Ko računamo za zgoraj opisano ekstenzivno travno rušo, sta izraza referenčna in potencialna evapotranspiracija enakovredna, uporabljali bomo slednjega. VODNA BILANCA Najbolj preprost, a vseeno učinkovit način določanja sušnih obdobij je izračun vodne bilance (VB), to je vodnega presežka oz. primanjkljaja. Določimo jo tako, da od dnevne količine padavin (RR) odštejemo dnevno količino potencialne evapotranspiracije (ETp) in za rezultat vzamemo vodno bilanco zadnjega dne v mesecu: VBt = VBt-1 + RRt - ETpt , kjer prvi dan v mesecu upoštevamo vodno bilanco prejšnjega meseca, vendar brez presežkov in primanjkljajev, torej 0 < VBi — < PK , pri čemer je PK poljska kapaciteta tal. Ta označuje količino vode, ki ostane v tleh po tem, ko iz z vodo nasičenih tal del odteče odteče zaradi gravitacije. Tla s srednjo zadrževalno sposobnostjo vode so predstavljena s PK = 100 mm. V severovzhodni Sloveniji prevladujejo tla s srednjo zadrževalno sposobnostjo (poljska kapaciteta med 80 in 150 mm), v zahodni Sloveniji imamo večinoma tla z majhno zadrževalno sposobnostjo (poljska kapaciteta med 30 in 80 mm), v severozahodni celo z zelo majhno (poljska kapaciteta do 30 mm). Veliko zadrževalno sposobnost (poljska kapaciteta med 150 in 230 mm) imajo le tla v jugovzhodnem delu in izjemoma še v posameznih katastrskih občinah po Sloveniji. BIOKLIMATSKI INDEKSI Izbiramo lahko med mnogo različicami bioklimatskih indeksov, ki nam glede na temperaturo zraka ter lahko tudi glede na količino padavin določajo primernost razmer za različne kulture. Izbrali smo dva, ki se največkrat pojavljajata v strokovni literaturi. Iz povprečnih temperatur zraka (T [°C]) računamo Winkleijev indeks po enačbi: 31.10. ET0 [mm/dan] dnevna referenčna evapotranspiracija, T = ^ (T 10 C), [°C] povprečna dnevna temperatura zraka na višini 2 m, mm L = 0,736- k2 = 273°C. e - e, a d dan ■ hPa0'7 [hPa] predstavlja razliko med nasičenim in dejanskim parnim tlakom, ki jo izračunamo iz relativne vlage Rh [%] po enačbi: e a - ed = Cl| 1 - Rh 100 • exp V c3 + T J kjer seštevamo povprečne dnevne temperature, ki so višje od 10 °C, v obdobju od začetka aprila do konca oktobra. Klasifikacija vinorodnih klimatskih regij glede na Winklerjev indeks je primerna za izračun kjerkoli v svetu. Tabela 1 podaja razrede vinogradniške klime glede na Winklerjev indeks ter primer pripadajočih vinorodnih okolišev. Slovenija ustreza področju I ter II, ki sta naprej razdeljena na pet con (Tabela 2). konstante pa so glede na arhiv ARSO (2008) pri 1.4 Tabela 1: Razredi vinogradniške klime glede na Winklerjev indeks (Riou, 1994). Table 1: The classification of the viticultural climatic regions according to Winkler climatic index (Riou, 1994) . predstavljena klasifikacija glede na Huglinov indeks, ki se večinoma uporablja v vinogradništvu. Tabela 3: Razredi vinogradniške klime glede na heliotermalni Huglinov indeks (Huglin, 1986). Table 3: Winegrowing regions according to Huglin climatic index (Huglin, 1986). področje vrednosti IW [°C] nekaj vinorodnih okolišev iz tega področja I < 1390 Ženeva, Dunaj, Bordeaux, Geisenheim, Dijon II 1391 - 1670 Budimpešta, Santiago (Cile), Napa (ZDA) III 1671 - 1950 Milano, Montepellier IV 1951 - 2220 Benetke, Mendoza (Argentina), The cape (J Afrika) V > 2220 Split, Palermo, Alžir razred vinogradniške klime oznaka vrednosti HI [°C] zelo topla HI+3 > 3000 topla HI+2 2400 - 3000 zmerno topla HI+1 2100 - 2400 zmerna HI-1 1800 - 2100 hladna HI-2 1500 - 1800 zelo hladna HI-3 <= 1500 Tabela 2: Razredi vinogradniške klime za Slovenijo glede na prilagojen Winklerjev indeks (Sušnik in sod., 2005). Table 2: Winegrowing regions according to modified Winkler climatic index as used in Slovenia (Sušnik et al., 2005). cona vrednosti IW [°C] 1 < 1150 2 1150 - 1250 3 1251 - 1350 4 1351 - 1450 5 > 1450 Huglinov heliotermalni indeks računamo glede na letne vrednosti spremenljivk. Uporabili smo enačbo H = g(T - 10) + Orna, - 10) . d , 1.4. 2 kjer je T povprečna temperatura zraka v °C, Tmax maksimalna temperatura zraka v °C in d koeficient dolžine dneva, odvisen od geografske širine (d = 1,04). Seštevamo po vegetacijskem obdobju (od 1. aprila do 30. septembra). V Tabela 3 je POZEBA Pozeba, ki se pojavi spomladi, ko je rastlina v fazi cvetenja, lahko povzroči veliko znajšanje pridelka. Pri pozebi se osredotočimo na minimalno temperaturo, saj so od nje odvisne poškodbe na cvetovih ali plodičih. Temperature, nižje od -2 °C, povzročijo zmerno, nižje od -3 °C močno ter nižje od -4 °C hudo pozebo. V naši študiji smo kot temperaturo praga definirali 0 °C ter pozebo definirali kot zadnji dan spomladi, ko minimalna temperatura pade pod 0 °C in je rastlina že v fazi cvetenja. Za obravnavo smo določili v Sloveniji precej razširjeno sadno vrsto, to je domačo češpljo. Začetek fenološke faze cvetenja je določen z akumulirano toplotno energijo, ki jo določimo na osnovi vsote aktivnih temperatur (to so temperature nad 0 °C). Za domačo češpljo se vrednosti temperaturne vsote nekoliko razlikujejo po regijah, odvisno od klimatskih značilnosti posamezne regije. Tako znašajo vrednosti povprečne vsote aktivnih temperatur za domačo češpljo v začetni fazi cvetenja v Ratečah 341 °C, v Celju 420 °C, v Biljah 400 °C itd. Toplejši kraji imajo praviloma tudi višjo vsoto aktivnih temperatur, ki so potrebne za začetek fenološkega razvoja rastline. Pri samem izračunu smo vzeli povprečno vrednost vsote aktivnih temperatur za Slovenijo, ki pri domači češplji znaša 420 °C. 3 REZULTATI IN DISKUSIJA TEMPERATURA ZRAKA Povprečna dnevna temperatura zraka se zelo očitno povečuje. Poleti je razlika med obdobjema 1961-1990 in 1991-2007 od 1 do 1,8 °C, najmanj temperatura narašča jeseni, letna razlika se giblje od 0,7 do 1,4 °C. Segrevanje na Kredarici je primerljivo z nižinskimi postajami. Dvig ni značilno odvisen od prvotnega povprečja v obdobju 1961-1990, saj so rezultati med seboj primerljivi. Variabilnosti pri maksimalnih dnevnih temperaturah so večje kot pri povprečnih in se z zviševanjem povprečij še povečajo, a v veliki odvisnosti od lokacije - na grafu (Slika 1) vidimo izredno veliki variabilnosti za Novo mesto in Mursko Soboto. Sicer pa je dvig povprečja nekoliko večji od tistega pri povprečnih dnevnih temperaturah zraka. Največje spremembe so v vzhodnem in severovzhodnem delu Slovenije. 20.00- q icn "o o nj 2 18.00- N CÜ V a E o nj 16.00-cz nj E 'in k nj E 14.00- a > o a obdobje Bilje -1- Ljubljana Novo mesto Murska Sobota postaja Slika 1: Okvirji z ročaji za Bilje, Ljubljano, Novo mesto in Mursko Soboto, ki predstavljajo porazdelitev letnih povprečij maksimalnih dnevnih temperatur zraka za obdobji 1961-1990 (svetli) in 1991-2007 (temni). Figure 1: Boxplots of yearly averages of maximal daily tamperatures for Bilje, Ljubljana, Novo mesto and Murska Sobota for the periods 1961-1990 (bright) and 1991-2007 (dark). Pri minimalnih temperaturah opazimo podobno naraščanje kot pri povprečnih dnevnih temperaturah zraka, torej nekoliko manjše kot pri maksimalnih. Največje razlike se sicer kažejo poleti in najmanjše pozimi, a so kljub temu zanimivejše zimske spremembe, ko rastline potrebujejo mraz za mirovanje. ŠTEVILO VROČIH, TOPLIH IN HLADNIH DNI Z naraščanjem temperature zraka je povezano naraščanje števila vročih (maksimalna temperatura nad 30 °C) in toplih dni (maksimalna temperatura nad 25 °C) ter upadanje števila hladnih dni (minimalna temperatura pod 0 °C). Za fenološki razvoj rastlin je ključnega pomena, da je dovolj hladnih dni, preveliko število toplih ali celo vročih dni pa deluje stresno. Vroči dnevi spadajo med bolj ekstremne dogodke, kar se odraža predvsem pri večji variabilnosti. Izredno velik razpon v Murski Soboti (Slika 2) lahko pripišemo dejstvu, da je zaradi večjega števila let z več vročimi dnevi leto 2003 ostalo maksimum in ne osamelec kot pri ostalih postajah. Na grafih lahko tudi vidimo, kako ekstremno je zares bilo leto 2003 z izjemno velikim številom toplih in vročih dni. Dejstvo, da je bilo leta 2003 v Biljah 79 vročih dni, je prav neverjetno. 2003.00 * obdobje □ 1961-1990 □ 1991-2007 >o o .M 40.00- o C >o OD ži > o o. 2003.00 o I 2003.00 o 1988.0t O 1 Bilje Ljubljana Novo mesto Murska Sobota postaja 80.00- 60.00- 20.00- Slika 2: Okvirji z ročaji za Bilje, Ljubljano, Novo mesto in Mursko Soboto, ki predstavljajo porazdelitev poletnih povprečij števila vročih dni (Tmaks > 30 °C) za obdobji 1961-1990 (svetli) in 1991-2007 (temni). Figure 2: Boxplots of summer averages of number of hot days (Tmaks > 30 °C) for Bilje, Ljubljana, Novo mesto and Murska Sobota for the periods 1961-1990 (bright) and 1991-2007 (dark). PADAVINE Pozimi opazimo občutno zmanjšanje povprečne količine padavin v zahodni polovici Slovenije z največjim odstopanjem v severozahodnem delu. Kljub dosedanjim napovedim, da naj bi se pozimi količina padavin zviševala, opažamo drugačne spremembe. V Gorenjski in Goriški regiji se je tako količina padavin v zadnjem obdobju glede na tridesetletje 1961-1990 zmanjšala celo do 30 odstotkov. Pri spomladanski količini padavin je opaziti upad na celotnem območju Slovenije, kar smo tudi pričakovali glede na napovedi ob trenutnem povečevanju toplogrednih plinov v ozračju. Kot pozimi pa je tudi spomladi odstopanje izrazitejše v zahodni polovici Slovenije, kjer je povprečna količina padavin v obdobju 1991-2006 za okoli 20 odstotkov manjša kot v tridesetletnem obdobju 1961-1990. Količina padavin se je spomladi najmanj zmanjšala v severovzhodnem delu ter predelih južne Slovenije. Poleti se vpliva raznolikosti reliefa in vremenskih situacij še bolj odražata na rezultatih, ki so zato zelo raznoliki glede na lokacije po Sloveniji. Odstopanja so manjša kot spomladi, večinoma gre za okoli 10 odstotno zmanjšanje količine padavin. Vendar pa se izjemoma na nekaterih lokacijah kaže tudi pozitivno odstopanje, kar že izgleda kot začetna znamenja jesenskega dogajanja. Jeseni je opazen dvig količine povprečnih padavin glede na obdobje 1961-1990. Porast količine padavin je najbolj intenziven v osrednjem delu Slovenije, kjer imamo do 20 odstotna odstopanja, ter v Posočju in na povsem vzhodnem robu Prekmurja z okoli 15 odstotnim dvigom. Na splošno skoraj lahko rečemo, da so tam, kjer so pozimi odstopanja povprečja količine padavin v obdobju 1991-2006 najbolj negativna, jeseni najbolj pozitivna, z izjemo pasu od Ljubljane proti Zasavju. Podobne prostorske vzorce kot pri odstopanjih pričakujemo tudi pri trendih padavin. Spomladi je trend v večini Slovenije negativen, količina padavin upada za okoli 5 odstotkov, seže pa tudi do 10 odstotkov na 10 let. Pozimi je največji trend upadanja padavin v severozahodni in deloma v jugozahodni Sloveniji. V omenjenih predelih je trend upadanja količine padavin do 20 odstotkov na 10 let. Proti vzhodu se trend upadanja nekoliko zmanjšuje, še posebej proti jugovzhodu Slovenije, kjer trenda praktično ni. Slika 3: Trend spreminjanja količine poletnih padavin v obdobju 1971-2006 v odstotkih glede na povprečje 19712006 na izbrani lokaciji. Figure 3: The trend of summer precipitation sum in the period 1971-2006 according to the average 1971-2006. Slika 4: Trend spreminjanja količine jesenskih padavin v obdobju 1971-2006 v odstotkih glede na povprečje 19712006 na izbrani lokaciji. Figure 4: The trend of the summer precipitation sum in the period 1971-2006 according to the average 1971-2006. Pri prostorski karti spreminjanja količine padavin poleti (Slika 3) je za razliko od ostalih sezon izrazit vpliv nadmorske višine, kar se jasno odraža tudi pri sami strukturi karte. Poleg tega je zanimivo pasovno naraščanje negativnih trendov od severa proti jugu. Sicer količina poletnih padavin po celi Sloveniji upada, a je na severu trend komaj zaznaven, medtem ko gre v osrednji Sloveniji za upadanje za okoli 5 odstotkov na 10 let, na jugu pa tja do 7 odstotkov na 10 let. Pri trendih jesenskih padavin (Slika 4) se povečevanje količine padavin v osrednji Sloveniji ne kaže v taki meri kot smo lahko opazili pri odstopanju povprečja zadnjih let. Najbolj se količina padavin povečuje na severozahodu Slovenije, kjer so trendi skoraj do 20 odtotkov na 10 let. Proti jugovzhodu in vzhodu se trendi povečevanja zmanjšujejo, v osrednji Sloveniji se količina padavin povečuje za okoli 10 odstotkov na 10 let, najmanjše naraščanje padavin pa je okoli Novega mesta in Kočevja. Proti severovzhodu Slovenije se trendi naraščanja padavin spet povečujejo in dosežejo na skrajnem robu okoli 10 odstotkov na 10 let. Slika 5: Trend spreminjanja količine jesenskih padavin v obdobju 1971-2006 v odstotkih glede na povprečje 19712006 na izbrani lokaciji. Figure 5: The trend of the summer precipitation sum in the period 1971-2006 according to the average 1971-2006. POTENCIALNA EVAPOTRANSPIRACIJA IN VODNA BILANCA Odstopanje povprečnih vrednosti potencilane evapotranspiracije v obdobju 1991-2007 glede na obdobje 1961-1990 je ponekod celo 50 odstotno, kar je povsem izven naših predstav. Poleg tega se poleti potencialna evapotranspiracija povečuje prav povsod in ni odstopanje nikjer manjše od 15 odstotkov. Po vseh lokacijah imamo v obdobju 1971-2006 precej izrazito naraščujoče trende, od 5 odstotkov na 10 let pa do skoraj 30 odstotkov na 10 let. Povečevanje evapotranspiracije povzroča večje izsuševanje tal, kar je posebej problematično na Goriškem pa tudi v Podravju in Pomurju. Pozitivni trendi so opazni tudi v vseh ostalih letnih časih, a so precej nižji. Skupni učinek padavin in potencialne evapotranspiracije nam predstavljata vodno bilanco, ki nam da jasnejšo informacijo o stanju po Sloveniji. Julija (Slika 6) so vrednosti trendov najvišje. Tako imamo v Goriški in Obalno-kraški regiji trend do 30 mm na 10 let, prav tako tudi na skrajnem severovzhodu. Tako v večini osrednje Slovenije kot tudi na Gorenjskem in Koroškem ostaja vodna bilanca skozi obdobje 1971-2006 bolj ali manj nespremenjena. Karte za različna tla so bile skoraj identične, torej je vpliv večje poljske kapacitete v najbolj sušnem mesecu praktično zanemarljiv. Slika 6: Absolutni trend spreminjanja julijskega primanjkljaja vode v tleh v obdobju 1971-2006 za tla s poljsko kapaciteto 100 mm. Figure 6: Absolute trend of water deficit in July in the period 1971-2006 for the soil with 100 mm field capacity. Slika 7: Klasifikacija Slovenije po povprečnih vrednostih Winklerjevega indeksa v obdobju 1961-1990. Figure 7: The classification of Slovenia according to average Winkler index values in the period 1961-1990. VINOGRADNIŠKI INDEKSI Pri izračunih Winklerejevega indeksa že povprečno povišanje temperatur zraka za 0,5 °C skoraj vedno prinese prerazporeditev v višjo cono. Slika 7 predstavlja klasifikacijo Slovenije glede na povprečen Winklerjev indeks za obdobje 1961-1990. Skoraj celotna Slovenija se uvršča v 1. cono, v 5. cono se uvrščajo najbolj zahodne lokacije, v vmesne cone pa pretežno jugozahodna in severovzhodna Slovenija (delno tudi jugovzhodna in osrednja), kjer je vinogradništvo tudi najbolj razširjeno. Slika 8 pa obdobju 1991-2007. S povečevanjem temperature zraka prikazuje, kakšna je bila nova razporeditev v cone v se višje cone širijo v notranjost države. Slika 8: Klasifikacijo Slovenije po povprečnih vrednostih Winklerjevega indeksa v obdobju 1991-2007. Figure 8: The classification of Slovenia according to average Winkler index values in the period 1991-2007. Slika 9: Spremembe povprečnega zaporednega dne v letu, ko v povprečju nastopi fenološka faza cvetenja pri domači češplji, za obdobje 1991-2007 glede na 1961-1990. Figure 9: Changes in average Julian day of flowering of plum tree for the period 1991-2007 according to 1961-1990. Huglinov indeks je v obdobju 1991-2007 glede na višji razred vinogradniške klime. Najmanjše so opažene referenčno obdobje 1961-1990 že višji za približno 200 spremembe na Vojskem in Brniku, največje pa v do 250 °C, kar na več lokacijah že prinese uvrstitev v Bizeljskem in Murski Soboti. POZEBA Višanje temperatur zraka prinaša tudi časovne premike doseganja določenih temperaturnih pragov. Največji trend premikanja zadnjega dne spomladi s pojavom minimalne temperature pod 0 °C v koledarskem letu nazaj imajo Veliki Dolenci in Brnik (-6 dni / 10 let), najmanjši pa Kredarica, Slap in Črnomelj (pod -1 dan / 10 let). V 40-ih letih prinesejo taki trendi že zelo očitne spremembe, tako da se v nekaterih krajih lahko v povprečju zadnjič pojavijo negativne temperature že zelo zgodaj v koledarskem letu. Izračunali smo tudi zaporedne (julijanske) dni v letu, ko nastopi fenološka faza cvetenja pri domači češplji (vsota aktivnih temperatur doseže 420 °C). V obdobju 19611990 je cvetenje v večini Slovenije (od osrednje do severvzhodne) nastopilo v mesecu aprilu (med dnevoma 91 in 112), zelo pozno je vsota dosežena v hribovitih predelih, zgodaj pa v jugozahodni in zahodni Sloveniji. Spremembe v obdobju 1991-2007 glede na 1961-1990 so predstavljene na karti (Slika 9). Razlike so že sedaj tedenske ali še večje, z nadaljnjim segrevanjem pa lahko pričakujemo tudi 20 ali več dni hitrejši nastop cvetenja domače češplje. Zanimajo nas pozebe po nastopu fenološke faze cvetenja. Pomemben določitelj pozebe je zadnji dan spomladi, ko minimalne temperature padejo pod 0 °C. Slika 10 nam prikazuje graf za zadnji dan spomladi, ko je minimalna temperatura padla pod 0 °C, ter za zaporedni dan v letu, ko je nastopila fenofaza cvetenja domače češplje. Fenološki razvoj rastline se zaradi višjih povprečnih temperatur začenja vse bolj zgodaj. To seveda pomeni, da cvetenje pomika v obdobje, ko je nevarnost prodorov hladnega zraka in s tem pozebe višja. Slika 10: Zaporedni dan v letu, ko minimalna temperatura (spomladi) zadnjič pade pod 0 °C (svetlo) ter zaporedni dan v letu, ko nastopi fenofaza cvetenja (temno) v Murski Soboti. Figure 10: Julian day on which the minimum temperature falls the last time in the spring under 0 °C (bright) and julian day on which flowering begins (dark) for Murska Sobota. 4 SKLEPI Agro-klimatske spremenljivke so se v obdobju 19912007 glede na obdobje 1961-1990 v večini že zaznavno spremenile, kar postaja bistvenega pomena predvsem za kmetijstvo. Že opažene spremembe nam dajejo potrditev, da so tudi napovedi po različnih scenarijih podnebnih sprememb upravičene. Povprečne dnevne temperature zraka so se že izrazito povečale, nekoliko bolj in lokacijsko bolj raznoliko so se povečale povprečne minimalne temperature zraka ter še malenkost več povprečne maksimalne temperature zraka. S segrevanjem se spreminjajo tudi ostale spremenljivke, vezane na temperaturo. Število vročih in toplih dni se povečuje, število hladnih dni pa zmanjšuje, kar ima pomemben vpliv na fenološki razvoj rastlin. Večinoma se je bolj povečalo število toplih dni - za do 24 dni, število vročih dni pa za do 18. Pri padavinah so v obdobju 1971-2006 opaženi trendi pozimi drugačni od napovedi, po katerih naj bi bili pozitivni, a so v večini Slovenije močno negativni, kar velja tudi za spomladanske in poletne trende. Le pri jesenskih padavinah se kažejo pozitivni trendi. Pri potencialni evapotranspiraciji zasledimo med letoma 1971 in 2006 izrazito naraščajoče trende - od 5 pa do skoraj 30 odstotkov na 10 let. Najbolj kritičen del vegetacijskega obdobja je julij, ko se nam primanjkljaj vode v tleh že sedaj precej izrazito povečuje. To ima največje posledice na Goriškem, Primorskem in v Prekmurju. Po Winklerjevem indeksu za obdobje 1961-1990 se skoraj celotna Slovenija uvršča v 1. cono, v 5. cono se uvrščajo le najbolj zahodne lokacije. S povečevanjem temperature zraka se višje cone širijo v notranjost države. V obdobju 1991-2007 je glede na referenčno obdobje 1961-1990 Huglinov indeks že višji za približno 200 do 250 °C. Ob dodatnem segrevanju pričakujemo celo uvrstitev Slovenije za dva razreda višje. Pri obravnavanju pozebe so spremembe vezane na minimalno temperaturo, saj so od nje odvisne poškodbe na cvetnih brstih v različnih razvojnih fazah. Verjetnost za pojavljanje minimalne temperature, nižje od 0 °C, se večinoma niža. Pas večjega trenda premikanja zaporednega dne, ko se še pojavi negativna minimalna temperatura, po koledarskem letu nazaj, poteka nekako od jugozahoda do severovzhoda Slovenije. Največji trend je -6 dni / 10 let. Pri primerjavi izračunov za obdobji 1961-1990 in 1991-2007 zaporednega (julijanskega) dne v letu, ko nastopi fenološka faza cvetenja pri domači češplji, so razlike tedenske ali še večje. Regionalni scenariji podnebnih sprememb za Slovenijo kažejo na dvig temperature zraka (Bergant, 2003, Kajfež-Bogataj in Bergant 2005). Velikost temperaturnih sprememb je zelo odvisna od izbranega scenarija izpustov. V obdobju od leta 2001 do 2030 se bodo v Sloveniji temperature zraka predvidoma povečale za 0,5 do 2,5 °C. Količina padavin poleti se bo lahko zmanjšala za do 20 odstotkov. Opisane spremembe nas zato spodbujajo k razmišljanju o možnih prilagoditvah v kmetijstvu, ki bodo vsekakor potrebne. Poleg tega pa so nujne tudi nadaljnje raziskave v tej smeri, saj dosedanji izračuni predstavljajo le osnovo. Kot smo že omenili, sta obstoječi klimatološka in fenološka mreža postaj za natančnejšo analizo prostorskih značilnosti agro-klimatskih spremenljivk premalo gosti. Glede na izračunane kazalce sprememb, ki zahtevajo kompleksno obravnavo, še enkrat opozarjamo, da bi se moralo za kakovostne klimatološke analize število klimatoloških postaj vsaj podvojiti. 5 LITERATURA Arhiv meteoroloških podatkov Agencije republike Slovenije za okolje. 2008. Podatki za obdobje 1961-2007. Ljubljana, Agencija republike Slovenije za okolje (izpis iz baze podatkov). ARSO, 2006. Agencija Republike Slovenije za okolje http://www.arso.gov.si/podro~cja/vreme_in_podnebje/po dnebje/. Bergant, K., 2003. Projekcije simulacij globalne klime na lokalni nivo in njihova uporaba v agrometeorologiji. Doktorska disertacija. Ljubljana: Univerza v Ljubljani, Biotehniška fakulteta, Oddelek za agronomijo. 170 str. Cressie, N. A. C. 1993. Statistics for spatial data. Wiley series in probability and mathematical statistics. Applied probability and statistics section. Str. 211 - 275. Črepinšek, Z. in Kajfež-Bogataj, L. 2003. Spring phenological trends in Slovenia. Ann, Ser. hist. nat., 13 (1): 57-64. Črepinšek, Z., Kajfež-Bogataj, L. in Bergant, K. 2006. Modelling of weather variability effect on fitophenology. Ecol. model., 194 (1-3): 256-265. Črepinšek, Z., Solar, M., Štampar, F. in Solar, A. Shifts in walnut (Juglans regia L.) phenology due to increasing temperatures in Slovenia. The journal of horticultural science & biotechnology, 84 (1): 59-64. Huglin, P. 1986. Biologie et écologie de la vigne. Paris, Editions Payot Lausanne: 372 str. Kajfež-Bogataj, L., Bergant, K., 2005. Podnebne spremembe v Sloveniji in suša. Ujma (19): 37-41. Kajfež-Bogataj, L., Bergant, K., 2005. Kakšno bo podnebje v Sloveniji v tem stoletju? Ujma (19): 218-223. Kajfež-Bogataj, L., Bergant, K., 2005. Podnebne spremembe v Sloveniji in suša = Climate change and drought in Slovenia. Ujma (Ljublj.), 2005, št. 19, str. 37-41. Kurnik, B. 2002. Primerjava različnih metod za izračun referenčne evapotranspiracije v Sloveniji. Diplomsko delo. FMF, Univerza v Ljubljani. 66 str. Luterbacher, J., Dietrich, D., Xoplaki, E., Grosjean, M. in Wanner, H., 2004. European seasonal and annual temperature variability. Trends and extremes since 1500. Science 303, pp. 1499-1503. Riou, C. 1994. Le déterminisme climatique de la maturation du raisin: application au zonage de la teneur en sucres dans la communauté européeenne. 319 pp. Office des Publications Officielles des Communautés Européennes, Luxembourg. Schonwiese, C.D. in Rapp, J., 1997. Climate trend atlas of Europe. Kluwer Academic publishers, 228 str.. Simončič, P., Kobler, A., Kranjc, N., Medved, M., Torelli, N. in Robek, R., 2001. Podnebne spremembe in slovenski gozdovi, Gozdarski vestnik 59, Ljubljana, s. 184-202 Sušnik, A., Kajfež-Bogataj, L., Bergant, K., Cegnar, T., Črepinšek, Z., Gregorič, G., Rogelj, D. in Žust, A. 2006. Climate change: it is not too late if farmers act now. Ljubljana, Environmental Agency of the Republic of Slovenia, 40 str. Susnik, A., Kajfez-Bogataj, L. in Kurnik, B. 2005. GIS assessment of climate warming impact on wine growing regions distribution in Slovenia. Ljubljana. Predstavitev za 'Workshop on climatic analysis and mapping for agriculture', Bologna, Italija. CONTENT ANALYSIS OF THE PAPERS IN THE ACTA AGRICULTURAE SLOVENICA VSEBINSKA OBDELAVA PRISPEVKOV V ACTA AGRICULTURAE SLOVENICA let. 95 št. 1 Tomaž BARTOLa, Karmen STOPARb, SUBJECT INDEX BY AGROVOC DESCRIPTORS PREDMETNO KAZALO PO DESKRIPTORJIH AGROVOC adaptation 97-109 animal husbandry 61-67 anthocyanins 5-11 antioxidants 21-28 aromatic compounds 5-11, 21-28 atomite 13-20 biological control 51-59, 89-96 biological control organisms 89-96 biological development 35-41 cereal crops 13-20 chemical composition 5-11, 21-28, 79-88 chemical control 13-20 cherries 21-28 chlorophylls 29-34 climatic change 97-109 climatic factors 97-109 control methods 89-96 crop losses 13-20, 51-59, 89-96 crop yield 35-41 damage 89-96 data collection 97-109 diagnosis 43-49 dryocosmus kuriphilus 89-96 environmental factors 97-109 evapotranspiration 97-109 extracts 21-28 farm size 61-67 farms 61-67 farmyard manure 79-88 fertilizer application 79-88 flowers 5-11 fluorescence 29-34 foliage 35-41 foliar application 29-34 glycine max 29-34 growth rate 35-41 harvest index 35-41 hops 61-67 humulus lupulus 61-67 humus 79-88 hybrids 35-41 identification 43-49, 89-96 a Ph. D., M. Sc.., B. Sc., Jamnikarjeva 101, SI-1000 Ljubljana, P. O. Box 95 b B.Sc., M.Sc., ibid Acta agriculturae Slovenica, 93 - 3, september 2009 str. 111 - 113 112 Tomaž BARTOL, Karmen STOPAR inorganic fertilizers 79-88 insect nematodes 51-59 integrated plant production 61-67 land management 61-67 leaf area 35-41 leaves 35-41 life cycle 43-49 maize 35-41 meteorological elements 97-109 meteorological observations 97-109 mineral content 79-88 minerals 13-20 monitoring 43-49, 89-96 mortality 43-49 natural enemies 89-96 nematoda 43-49, 51-59 nitrogen 79-88 nutrient availability 79-88 nutrient transport 29-34 parasites 43-49 pesticidal properties 13-20 pests of plants 13-20 phenolic compounds 5-11, 21-28 plant breeding 35-41 plant protection 61-67 postharvest losses 13-20 precipitation 97-109 proximate composition 5-11, 21-28 prunus avium 21-28 prunus cerasus 21-28 relative humidity 13-20 respiration 29-34 rosa 5-11 rotational cropping 61-67 seasons 97-109 selenium 29-34 sitophilus oryzae 13-20, 51-59 slugs 43-49 soil 13-20 soil chemicophysical properties 5-11 soil fertility 5-11, 79-88 soil organic matter 79-88 soil ph 5-11 soybeans 29-34 steinernema feltiae 51-59 storage 13-20, 51-59 storage losses 13-20, 51-59 straw 79-88 temperature 13-20, 97-109 trends 97-109 triticum 13-20 wheats 13-20 yield components 35-41 zea mays 35-41 SUBJECT INDEX BY AGRIS CATEGORY CODES VSEBINSKO KAZALO PO SKUPINAH ZNANJA (PREDMETNIH KATEGORIJAH) E11 Land economics and policies 61-67 F08 Cropping patterns and systems 61-67 F30 Plant genetics and breeding 35-41 F60 Plant physiology and biochemistry 5-11, 21-28, 35-41 F62 Plant physiology-growth and development 29 -34, 35-41 H10 Pests of plants 13-20, 43-49, 51-59, 89-96 P33 Soil chemistry and physics 79-88 P35 Soil fertility 5-11, 79-88 P40 Meteorology and climatology 97-109 NAVODILA AVTORJEM Prispevki Sprejemamo izvirne znanstvene članke, predhodne objave in raziskovalne notice s področja agronomije, hortikulture, rastlinske biotehnologije, raziskave živil rastlinskega izvora, agrarne ekonomike in informatike ter s sorodnih področij v slovenskem, angleškem in nemškem jeziku, znanstveno pregledne članke samo po poprejšnjem dogovoru. Objavljamo prispevke, podane na simpozijih, ki niso bili v celoti objavljeni v zborniku simpozija. Če je prispevek del diplomske naloge, magistrskega ali doktorskega dela, navedemo to in tudi mentorja na dnu prve strani. Navedbe morajo biti v slovenskem in angleškem jeziku. Pri prispevkih v slovenskem jeziku morajo biti preglednice, grafikoni, slike in priloge dvojezični, povsod je slovenščina na prvem mestu. Naslovi grafikonov in slik so pod njimi. Slike in grafikoni so v besedilu. Priloženi morajo biti tudi jasno označeni izvirniki slik. Na avtorjevo željo jih vračamo, s tem da je želja pisno sporočena ob oddaji gradiva in ponovno v teku 30 dni po izidu. Latinske izraze pišemo ležeče. V slovenščini uporabljamo decimalno vejico, v angleščini decimalno piko. Prispevki v angleščini morajo imeti povzetek v slovenščini in obratno. Prispevki v nemščini morajo imeti tudi povzetka v slovenščini in angleščini. Prispevki naj bodo strnjeni, kratki, praviloma največ 12 strani. Uporabljamo Microsoft Word 97 (Windows); pisava Times New Roman, velikost strani 16,2 x 23,5 cm, velikost črk besedila 10, v obsežnih preglednicah je lahko 8; izvlečki in metode dela Arial velikost 8, levi in desni rob 2,1 cm, zgornji rob 1,3 cm, spodnji rob 1,6 cm, Prva stran Na prvi strani prispevka na desni strani označimo vrsto prispevka v slovenščini in angleščini, sledi naslov prispevka, pod njim avtorji. Ime avtorjev navedemo v polni obliki (ime in priimek). Vsak avtor naj bo označen z indeksom, ki ga navedemo takoj pod avtorji, in vsebuje polni naslov ustanove ter znanstveni in akademski naslov; vse v jeziku prispevka. Navedemo sedež ustanove, kjer avtor dela. Če je raziskava opravljena drugje, avtor navede tudi sedež te inštitucije. Na željo avtorjev bomo navedli naslov elektronske pošte. Pod naslovi avtorjev je datum prispetja in datum sprejetja prispevka, ki ostaneta odprta. Sledi razumljiv in poveden izvleček z do 250 besedami. Vsebuje namen in metode dela, rezultate, razpravo in sklepe. Sledijo ključne besede. Izvlečku v jeziku objave sledi naslov in izvleček s ključnimi besedami v drugem jeziku. Viri V besedilu navajamo v oklepaju avtorja in leto objave: (priimek, leto). Če sta avtorja dva, pišemo: (priimek in priimek, leto), če je avtorjev več, pišemo: (priimek in sod., leto). Sekundarni vir označimo z "navedeno v" ali "cv.". Seznam virov je na koncu prispevka, neoštevilčen in v abecednem redu. Vire istega avtorja, objavljene v istem letu, razvrstimo kronološko z a, b, c. Primer: 1997a. Navajanje literature naj bo popolno: pri revijah letnik, leto, številka, strani; pri knjigah kraj, založba, leto, strani. Za naslove revij je dovoljena uradna okrajšava, za okrajšanimi besedami naj bodo vedno pike. Navedbo zaključimo s piko. Za primere upoštevajte objave v Zborniku BFUL. Oddaja Avtorji prispevke oddajo v dveh izvodih, enega z dvojnim razmakom med vrsticami in največ 35 vrst na strani, in na disketi. Priložijo tudi izjavo s podpisi vseh avtorjev, da avtorske pravice v celoti odstopajo reviji. Prispevke recenziramo in lektoriramo. Praviloma pošljemo mnenje prvemu avtorju, po želji lahko tudi drugače. Če uredniki ali recenzenti predlagajo spremembe oz. izboljšave, vrne avtor popravljeno besedilo v 10 dneh v dveh izvodih, enega z dvojnim razmakom. Ko prvi avtor vnese še uredniške pripombe, odda popravljeno besedilo v enem izvodu in na disketi ter vrne izvod z uredniškimi popravki. Prispevke sprejemamo vse leto. NOTES FOR AUTHORS Papers We publish original scientific papers, preliminary communications and research statements on the subject of agronomy, horticulture, plant biotechnology, food technology of foods of plant origin, agricultural economics and informatics; in Slovenian, English and German languages while scientific reviews are published only upon agreement. Reports presented on conferences that were not published entirely in the conference reports can be published. If the paper is a part of diploma thesis, master of science thesis or dissertation, it should be indicated at the bottom of the front page as well as the name of the supervisor. All notes should be written in Slovenian and English language. Papers in Slovenian language should have tables, graphs, figures and appendices in both languages, Slovenian language being the first. Titles of graphs and figures are below them. Figures and graphs are part of the text. Clearly marked origins of figures should be added; they can be returned if author desires. Latin expressions are written in italics. Decimal coma is used in Slovenian and decimal point in English. Papers in English should contain abstract in Slovenian and vice versa. Papers in German should contain abstracts in German, Slovenian and English. The papers should be condensed, short and usually should not exceed 12 pages. 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