ࡱ> Y @bjbjWW -==8]   44448l4d4hCnnnn C C C C C C C$EF^/C9 pt"/C3 nn333" n n C44  C3H36> 0  Cn|&Z8s44$B INFLUENCE OF THE HYBRID AND SEED TREATMENTS BY FUNGICIDES AND SELECTED STRAINS OF MICROORGANISMS TO SUGAR BEET YIELD AND QUALITY M. Ledinski, S. Kristek, A. Kristek, M. Antunovi and B. Sandra Abstract: During two-year investigation on two soil types with presence of pathogenic fungi R. solani sugar beet root decay agent detected from 2001 2004, we studied influence of the hybrid and the seed treatment (a - control seed treated with fungicide Thiram 42 S (600 ml/100 kg seed); b - seed inoculated with P. fluorescens bacterium; c - seed treated with fungicide Thiram 42 S (200 ml/100 kg seed) + inoculated with P. fluorescens bacterium) to the stand and parameters of sugar beet yield and quality. The results obtained depended on the trial year, soil type, degree of the soil infection with the pathogenic fungi R. solani, and mode of the seed treatment applied. Key words: hybrid, fungicide, Rhizoctonia solani, Pseudomonas fluorescens, yield, quality INTRODUCTION During the process of sugar beet cultivation, depending on weather conditions, degree of soil infection with pathogenic microorganisms, and application of soil management, the presence of sugar beet root decay is frequently observed. Pathogenic agents are numerous, inflicting serious damage. Most frequent decay agents on sugar beet seedlings are fungi A. cochlioides, P. ultimum and P. debarianum, as well as R. solani. In sugar beet vegetation phase plant decay is most frequently caused by fungi R. solani and Fusarium ssp. From the root decay agents most damage is inflicted by fungi R. solani. Besides weather conditions that influence this type of root decay, the inadequate soil management irregular soil cultivation resulting in damaged soil structure and rise in soil acidity, is a contributory factor. Fungicide treatment of seeds, application of tolerant hybrids, or proper soil management does not ensure full plant protection against this pathogen. Moreover, heavily infected soils and weather conditions favourable for development of the pathogen, contribute to the infection of higher or lower degree. The development of the fungi is mainly prevented by treating seeds with fungicides; however, they seriously affect human health and environment whilst targeted pathogenic fungi rapidly develop resistance against them. When growing tolerant hybrids, particularly on slightly infected soils, lower yield values and digestion are observed. Apparently, in newer selection of sugar beet hybrids, the difference is slightly lower if comparing to the older ones. Therefore, bearing in mind environmental aspect of the problem (application of high fungicide doses), seed inoculation with bacteria showing antagonism against pathogenic fungi is an acceptable alternative to chemical pesticide application ( HYPERLINK "javascript:popRef('b1')" Andersen etal., 2003). The best results reported in the studies dealing with this issue, were obtained by inoculating seeds with P. fluorescens. ____________________________ Obtain results in this paper are the results of the degree report Influence of the hybrid and seed treatments by fungicides and selected strains of microorganisms to sugar beet yield and quality by Marko Ledinski. P. fluorescens belongs to rod-shaped, asporogenous, gram-negative bacteria that are as saprophytes prevalent in soil and water belong to a group of soil microorganisms crucial to the process of soil denitrification. Due to the production of antimicrobial agents, these bacteria also show a distinguished antibiosis against pathogens causing diseases on arable crops, by inactivating their growth and reproduction ( HYPERLINK "javascript:popRef('b20')" Whipps, 2001) For the capacity to synthesize toxic cyclic lipopeptides ( HYPERLINK "javascript:popRef('b19')" Thrane etal., 2001;  HYPERLINK "javascript:popRef('b1')" Andersen etal., 2003) they are used as effective biological control agents ( HYPERLINK "javascript:popRef('b14')" Nielsen etal., 2002;  HYPERLINK "javascript:popRef('b18')" Sorensen etal., 2001). Many authors have reported that these purified lipopeptides show an antagonistic activity against certain fungi pathogenic on sugar beet roots such as Rhizoctonia solani (Nielsen et al., 2002; Andersen et al., 2003), Aphanomyces cochlioides, (Sorensen et al., 2001), Pythium ultimum and Pythium debarianum ( HYPERLINK "javascript:popRef('b19')" Lee etal., 2000; Nielsen etal., 2002). This suggests that bacteria producing lipopeptides could have a potential role in the biocontrol of fungal diseases, which was approved in both laboratory and field trials (Thrane et al., 2001). Therefore, seed inoculation with bacteria showing antagonism against pathogenic fungi is an acceptable alternative to chemical pesticide application ( HYPERLINK "javascript:popRef('b1')" Andersen etal., 2003). Moreover, considering the heavily infected soils with the pathogen R. solani and the fact that beneficial bacterium Pseudomonas sp. is not sensitive to a low dose fungicide ( HYPERLINK "javascript:popRef('b16')" Pedersen etal. 2002), it is possible to treat the seeds combining low doses of the chemical agents aiming to control growth and reproduction of the pathogenic fungi, which in consequence, produce positive effect on all the parameters of sugar beet yield and quality. MATERIAL AND METHODS The experiment was set up on two soil types - Mollic Gleysols and Eutric Cambisols (Table 1), with the presence of pathogenic fungi Rhizoctonia solani the sugar beet root decay agent detected from 2001 2004. The experiment was set up in 2004 and 2005 in completely randomized block design, with 4 repetitions and 12 different variants, including 2 tolerant and 2 sensitive hybrids to the pathogenic fungi, and 3 various seed treating variants: 1 hybrids A hybrids tolerant to the fungi R. solani: A1 Laetitia (KWS) A2 Solea (Strube) B hybrids sensitive to the fungi R. solani: B1 Belinda (KWS) B2 Sofarizo (Hilleshg) 2 seed treatment C1 control seed treated with fungicide Thiram 42-S (600 ml/100 kg) C2 seed inoculated with Pseudomonas fluorescens bacterium C3 - seed treated with fungicide Thiram 42-S (200 ml/100 kg) + inoculated with P. fluorescens bacterium P. fluorescens was isolated from the sugar beet seedling rhizosphere ( HYPERLINK "javascript:popRef('b19')" Thrane etal., 2000) and cultivated on King's B medium (Pseudomonas F; Difco catalog No. 0448-17-1). Fluorescent Pseudomonas spp. could be detected by illuminating the agar plates with UV light (254 nm) and randomly picking the fluorescent colinies. The inoculum was applied directly to the seed before sowing at a concentration of about 8נ104 bacteria/seed, i.e. 0.81.4נ1010 bacteria/ha. Row seeding was completed at the end of March, with spacing of 50 cm between rows and 20cm within rows. Table 1. Soil characteristics Investigated properties in a fieldType of soilLayer (0 0.3)Mollic GleysolsEutric CambisolspH (H2O)7.426.46pH (KCl)6.445.98Humus (%)3.221.87P (mg per 100 g soil)24.4422.50K (mg per 100 g soil)29.5723.11 Percent of the plants infected with R. solani as well as percent of decayed plants was stipulated in 4-6 true leaves phase. The sugar beet digging in the mid October, was followed by determination of root yield (t/ha) and sugar content (%). RESULTS AND DISCUSSION During 2-year investigations conducted significant differences were shown in the percentage of infected and decayed plants, soil types investigated, tolerant and sensitive hybrids, and hybrids within the two groups (Table 2, Table 3). Table 2. Percentage of the infected plants as a consequence of parasite fungus Rhizoctonia solani infestation in 2 - 4 true leaves phase Hybrid Seed treatmentInfected plants20042005Mollic GleysolsEutric CambisolsMollic GleysolsEutric CambisolsAverage Laetitia121.6022.7623.3325.9023.4028.0310.048.9712.369.85310.508.6611.179.7110.01 Solea123.7524.7424.4426.9224.96210.8112.0313.0114.7012.64312.0610.9914.1513.6212.71 Belinda129.9033.4532.8536.6033.2029.4411.7010.3012.1510.9039.9210.9510.2111.9910.77 Sofarizo125.2827.0527.0730.4527.46210.7012.1211.4012.7311.74311.1011.6512.3512.4011.88 1 - control seed treated with fungicideThiram 42 S (600 ml per100 kg seed) 2 seed inoculated with Pseudomonas fluorescens bacterium 3 - seed treated with fungicideThiram 42 S (200 ml per100 kg seed) + inoculated with P. fluorescens bacterium On Mollic Gleysols soil type the smallest number of the plants infected as well as decayed with pathogenic fungi R. solani was obtained in the variant 2 (seed inoculated with P. fluorescens bacterium), while the variant 3 (seed treated with fungicide Thiram 42 S (200 ml per 100 kg seed) + inoculated with P. fluorescens bacterium) obtained the same on Eutric Cambisols soil type. In variant 1 (seed treated with fungicide Thiram 42 S (600 ml per 100 kg seed) the most discernible difference was observed between tolerant and sensitive hybrids, while in the remaining two variants the differences have disappeared. Table 3. Percentage of the decayed plants as a consequence of parasite fungus Rhizoctonia solani infestation in 2 - 4 true leaves phase Hybrid Seed treatmentInfected plants20042005Mollic GleysolsEutric CambisolsMollic GleysolsEutric CambisolsAverage Laetitia115.3015.7916.4517.9416.3724.485.324.595.815.0536.044.796.675.165.67 Solea116.7017.5117.0319.2617.6325.336.686.248.136.5936.736.047.987.317.02 Belinda126.8129.8829.4633.9530.0324.065.654.815.945.1234.894.815.355.425.12 Sofarizo122.6424.8824.5627.0124.7724.366.024.837.205.6034.905.415.176.115.40 Table 4. Root yield (t ha-1) and sugar content (%) in both investigation years HybridSeed treatmentRoot yield (t ha-1)Sugar content (%)Mollic GleysolsEutric CambisolsMollic GleysolsEutric Cambisols20042005200420052004200520042005 Laetitia177.3166.1270.2164.9013.8412.3013.2111.46282.1971.2776.3568.3414.9012.7613.9612.12381.8072.4580.1071.1214.1112.5414.0112.30 Solea178.0364.2064.1056.4016.0413.9215.9113.08279.1971.2071.6360.2217.0114.9616.4413.97378.2468.3871.2065.1716.8614.8016.8914.25 Belinda178.1871.3274.0067.3815.0713.5814.2612.90282.0577.1675.3173.3817.1614.6015.9313.83380.9274.1178.7075.1116.8414.5416.8114.21 Sofarizo188.0585.4186.1883.1015.4713.1014.9912.56297.6890.2092.1286.0116.9814.5215.6014.01391.9088.1092.3587.3016.0614.4015.9214.56 LSD0.05 0.853 1.260 1.143 1.010 0.213 0.192 0.091 0.110 LSD0.01 1.450 2.077 2.190 2.040 0.380 0.423 0.173 0.2421 - control seed treated with fungicideThiram 42 S (600 ml per100 kg seed) 2 seed inoculated with Pseudomonas fluorescens bacterium 3 - seed treated with fungicideThiram 42 S (200 ml per100 kg seed) + inoculated with P. fluorescens bacterium Root yield (Table 4) showed highly significant positive correlation with sugar content values (r=0.943**). The highest root yield was obtained with Sofarizo hybrid sensitive to R. solani during 2-year trial on both soil types; in the variant 2 on Mollic Gleysols, in relation to the variant 3 on Eutric Cambisols. The same variants on the same soil types obtained the highest sugar content during both years of investigation. CONCLUSIONS Inoculation of sugar beet seed with bacterium P. fluorescens affected root decay agent treated with pathogenic fungi R. solani showed highly positive significant influence to all the elements of sugar beet yield and quality. REFERENCES Andersen, J.B., Koch, B., Nielsen, T.H., Srensen, D., Hansen, M., Nybroe, O., Christophersen, C., Srensen, J., Molin, S. and M. Giskov. 2003. Surface motility in Pseudomonas sp. DSS73 is required for efficient biological containment of the root pathogenic microfungi Rhizoctonia solani and Pythium ultimum. Microbiology 149, 37-46. Lee, C.H., Kempf, H. J., Lim, Y. and H. Cho. 2000. Biocontrol activity of Pseudomonas cepacia AF2001 and anthelmintic activity of its novel metabolite, cepacidine A. J. Microbiol. Biotechnol. 10, 568-571. Nielsen, T. H., Srensen, D., Tobiasen, C., Andersen, J. B., Christophersen, C., Giskov, M. and J. Srensen. 2002. Antibiotic and biosurfactant properties of cyclic lipopeptides produced by fluorescent Pseudomonas spp. from the sugar beet rhizosphere. Appl. Environ. Microbiol. 68, 3416-3423. Pedersen, H.C., Weiergang, I., Pontoppidan, M.M., Jrgensen, L. and A. Svingel. 2002. Danisco Seed, Seed Technology Dept., Hoejbygaardvej 31, DK 4960 Holeby, Denmark. Sorensen, D., Nielsen, T.H., Christophersen, C., Srensen, J. and M. Gajhede. 2001. Cyclic lipoundecapeptide amphisin from Pseudomonas sp. Strain Dss73. Acta Crystallogr. Sect. C Cryst. Struct. Commun. 57, 1123 1124. Thrane, C., Nielsen, M.N., Srensen, J. and S. Olsson. 2001. Pseudomonas fluorescens DR54 reduces sclerotia formation, biomass development, and disease incidence of Rhizoctonia solani causing damping off in sugar beet. Microb. Ecol. 42, 438445. Whipps, J.M. 2001. Microbial interactions and biocontrol in the rhizosphere. Journal of Experimental Botany, 52, 487 511. ABOUT THE AUTHORS: Engineer Marko Ledinski, Faculty of Agriculture, Trg Sv Trojstva 3, 31 000 Osijek, Croatia. Doc.dr.sc. Suzana Kristek, Faculty of Agriculture, Trg Sv Trojstva 3, Croatia, E-mail:  HYPERLINK mailto:skristek@pfos.hr skristek@pfos.hr Prof. dr. sc. Andrija Kristek, Faculty of Agriculture, Trg Sv Trojstva 3, 31 000 Osijek, Croatia, E-mail:  HYPERLINK mailto:akristek@pfos.hr akristek@pfos.hr Prof. dr. sc. Manda Antunovi, Faculty of Agriculture, Trg Sv Trojstva 3, 31 000 Osijek, Croatia, E-mail:  HYPERLINK mailto:mantun@pfos.hr mantun@pfos.hr Sandra Brki, High school of natural science, Vukovarska 209, 31 000 Osijek. 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