ࡱ> 5@ Tbjbj22 ;XX.+RRRRRRRR>y>y>y8vy"zTR'2{{({{{}}}RTTTTTT$YR^xiR%}}%%xRR{{ %R{R{R %R  RR{v{ t >yA: ,<0' {6 XRRRRRR R }~ $t}}}xxRR$%x$XRRxUSE OF THE BACTERIA BACILLUS MEGATERIUM IN THE CONTROL OF SUGAR BEET ROOT DECAY AGENTS  PYTHIUM ULTIMUM AND PYTHIUM DEBARIANUM Martin EVA I1  Suzana KRISTEK2  Aleksandar STANISAVLJEVI2 1 CBA International, Koturaaka cesta 69, Zagreb, Croatia, e-mail: skristek@pfos.hr 2 Faculty of Agriculture, Trg Sv. Trojstva 3, Osijek, Croatia Abstract: During two-year studies on two different soil types (Mollic Gleysols, Calcaric Fluvisols) in 4 repetitions and 4 different variants, as follows: 1. untreated seed; 2. Dithane S- fungicide treated seed (60% Mancozeb, 800 g 100 kg-1 seed); 3. seed treated with fungicide Dithane S (60% Mancozeb, 400 g 100 kg-1 seed) + inoculated with Bacillus megaterium (0.6 x 1010 bacteria ha-1); 4. seed inoculated with Bacillus megaterium (1.2 x 1010 bacteria ha-1) we investigated influence of different seed treatments to infection with pathogenic fungi Pythium ultimum and Pythium debarianum, as well as, elements of sugar beet yield (root yield, sugar content). The best results were obtained by inoculation of sugar beet seed with the bacterium B. megaterium. Keywords: sugar beet, B. megaterium, P. ultimum, P. debarianum, yield Introduction Agronomists are faced with a growing problem of evolving resistance of soil pathogens to chemical fungicides. Also, there is an increasing sensitivity to the importance of health food production and rising awareness for environmental protection. Namely, all pesticides which are not biodegraded in the soil are being washed out in deeper layers causing eutrophication of underground waters. Therefore, natural resources and processes in the soil are widely used as an alternative to chemical fungicides. Sugar beet is a crop which is from the phases of emergence and germination to the first 2 - 4 true leaves phase (most sensitive phase) attacked by numerous pathogenic fungi that cause root decay of young sugar beet plants. Seed inoculation by the bacteria showing antagonistic activity against pathogenic fungi such as Rhizoctonia solani, P. ultimum, P. debarianum, Phoma betae and Aphanomyces cochlioides account for an alternative to chemical fungicides and an option of solving problem of disease control, not only in sugar beet, but in other, mainly vegetable crops. Many authors (Asaka and Shoda, 1996; Walker et al., 1998; Hsek and Vach, 2006; Kristek et al., 2007; Bali evi et al., 2007) studied control of these pathogens by benefit bacteria Bacillus megaterium and Pseudomonas fluorescens, as well as, benefit fungi Trichoderma harzianum which showed pronounced antagonistic activity against the aforesaid fungi. Since the benefit bacteria do not express sensitivity to the low fungicide doses (Pedersen et al., 2002), on the soils infected with the fungi the root decay agents on sugar beet, positive effect was accomplished by combining seed inoculation with the benefit bacteria and the seed treatment with low doses of fungicides. Pathogenic fungi P. ultimum and P. debarianum are found to be the most serious issue, so the influence of the bacterium B. megaterium to the pathogenic fungi has been investigated. Materials and methods The experiment was set up on two soil types Mollic Gleysols and Calcaric Fluvisols, with the presence of pathogenic fungi P. ultimum and P. debarianum detected from 2001 2005. In 2005 and 2006 the experiment was set up by completely randomised block design in 4 repetitions and 4 various seed treating variants: 1. untreated seed; 2. Dithane S - fungicide treated seed (60% Mancozeb, 800 g 100 kg-1 seed); 3. seed treated with fungicide Dithane S (60% Mancozeb, 400 g 100 kg-1 seed) + inoculated with Bacillus megaterium (0.6 x 1010 bacteria ha-1); 4. seed inoculated with Bacillus megaterium (1.2 x 1010 bacteria ha-1). Soil analyses (Table 1.) were carried out by standard methods: organic matter content was determined by bichromate method, pH in H2O and KCl, phosphorus and potassium content by ammonium-lactate method according to Egner-Riehm-Domingo (Page, 1982). Table 1: Soil properties Investigated field propertiesType of soilMollic GleysolsCalcaric FluvisolspH (H2O)7.197.04pH (KCl)6.496.27Humus (%)3.382.31P (mg per 100 g soil)23.0020.72K (mg per 100 g soil)25.8819.95 Bacteriological culture (B. megaterium) applied in the experiment are from German Collection of Microorganisms and Cell Cultures (DSMZ). B. megaterium No 2894 was transferred from lyophilised into vegetative form, and multiplied on nutrient agar of the following composition: peptone 5.0 g, meat extract 3.0 g, agar 15.0 g, MnSO4 x H2O 10.0 mg, distilled water 1000.0 ml. Hybrid of Georgina (KWS) sugar beet was used in the sowing. In both investigation years sowing was conducted in the second decade of March. The row spacing was 50 cm, with 20 cm within row. Percent of the plants infected with pathogenic fungi P. ultimum and P. debarianum were stipulated in 2-4 true leaves phase. Sugar beet digging conducted in the mid October was followed by determination of root yield (t ha-1) and sugar content (%). Results and discussion The least number of infected and decayed plants in the 2 4 true leaves phase, on both soil types and in two years of investigation (Table 2.) was obtained with the variant 4. The remaining variants obtained statistically very significantly (p<0.01) higher number of infected and decayed plants by pathogenic fungi P. ultimum and P. debarianum. The results obtained are in agreement with those of Whipps (2001) who reported that the plants inoculated with the benefit bacteria were characterized with rapid initial growth that enabled fast undergoing through the most vulnerable phase when the damage of the pathogen affecting was the most pronounced. Due to the pronounced antagonism of the bacterium to the root decay agent of sugar beet fungi P. ultimum and P. debarianum high survival percentage of inoculated plants was obtained in comparison to the non-inoculated ones and decrease in the damage of infected plants, which consequently, influenced sugar beet root yield. Root yield (Table 3.) showed highly significant positive correlation with sugar content values (r = 0.947). On both soil types and in both investigation years the highest average root yield values were obtained with the variant 4, while all remaining variants (except variant 3 in the second year of investigation on Calcaric Fluvisols soil type) recorded statistically very significantly (p<0.01) lower yield values. Table 2. Percentage of the infected and decayed plants as a consequence of P. ultimum and P. debarianum infection in the 2 - 4 true leaves stage Investigated parameterVariantMollic GleysolsCalcaric FluvisolsYearAverageYearAverage2005200620052006Infected plants (%)156.2848.8952.5958.6351.0354.83225.4422.0523.7527.1222.7824.95311.4910.2710.8812.0910.6011.35411.029.5510.2911.359.9810.67Decayed plants (%)149.2141.5045.3650.9043.1047.00218.3116.3817.3519.3116.8518.0838.606.957.788.907.338.1247.955.386.678.126.057.09 LSD0.05 0.26 0.34 0.31 0.38 0.32 0.35 LSD0.01 0.44 0.67 0.55 0.71 0.59 0.64 Table 3. Investigated parameters by soil types and years of investigation Investigated parameterVariantSoil typeAverageMollic GleysolsCalcaric FluvisolsYear2005200620052006Root yield (t ha-1)155.3859.2654.1557.5056.57274.6577.5374.0976.1975.62385.1088.2783.4987.1085.99487.8092.9084.8890.5589.03 LSD0.05 1.08 1.56 1.22 1.61 1.38 LSD0.01 1.96 2.87 2.46 3.01 2.64Sugar content (%)112.4613.0812.2013.0112.69214.3114.5714.1514.4914.38314.6614.9014.6114.8314.75414.9815.2614.8015.1715.05 LSD0.05 0.17 0.19 0.23 0.21 0.21 LSD0.01 0.33 0.37 0.41 0.36 0.35 Variants: 1 untreated seed; 2 seed treated with fungicide Dithane S (60% Mancozeb, 800 g 100 kg-1 seed); 3 seed treated with fungicide Dithane S (60% Mancozeb, 400 g 100 kg-1 seed) + inoculated with Bacillus megaterium (0.6 x 1010 bacteria ha-1); 4 seed inoculated with Bacillus megaterium (1.2 x 1010 bacteria ha-1) The highest sugar content value in both investigation years and on both locations in this parameter was, also, obtained in the variant 4. However, no significant difference (p<0.01) was obtained with the variant 3. Variants 1 and 2 obtained statistically very significantly (p<0.01) lower values in this parameter. Similar results were reported in the studies of Pedersen et al. (2002) and Kristek et al. (2006, 2007). Conclusions Inoculation of sugar beet seed with the bacterium B. megaterium affected root decay agent treated with pathogenic fungi P. ultimum and P. debarianum and showed highly significant influence to all the elements of sugar beet yield and quality. The best results in all parameters tested were obtained by inoculation of sugar beet seed with the bacterium B. megaterium. However, no significant differences (p<0.01) were found between this variant and the variant of combined seed treatment with bacteria B. megaterium and chemical fungicide Dithane S in root yield (first year of investigation, Calcaric Fluvisols) and sugar content values (on both soil types in both years of investigation). Acknowledgements We are grateful to the Ministry of Science, Education and Sports fortheir financial support for theproject Sugar beet nutrition by nitrogen depending on the soil traits genotype" that have enabled scientific research, and the obtained results to be reported in this paper. References Asaka, O.  Shoda, M.: 1996. Biocontrol of Rhizoctonia solani damping-off of tomato with Bacillus subtilis RB14. Applied and Environmental Microbiology, 62: 11. 4081-4085. Bali evi, R.  Paraikovi, M.  `amota, D.: 2007. Control of soil parasites (Pythium debaryanum,Rhizoctonia solani) on tomato by a biological product. Cereal Research Communications, 35: 2. 1001-1004. Hsek, J. Vach, M.: 2006. Application of some biopreparations against fungi of genus Fusarium producing mycotoxins in the system of spring barley growing. Cereal Research Communications, 34: 1. 425-428. Kristek, S. Kristek, A.  Guberac, V.  Glavaa Toki, R.  Pavlovi, H.: 2006. Influence of sugar beet treatment with Pseudomonas fluorescens and low fungicide doses on infection with Pythium and plant yield and quality. Journal of Phytopathology, 154: 10. 622-625. 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