ࡱ> 5@ bjbj22 ,XXG-+88888\69b2V:l:(::::::b b b b b b b$dRSfPDbEg?::g?g?Db::bXXXg? ::bXg?bXXX[\:J: ?)8J [\Db0b[,fWf\f\:;XN<<:::DbDb->1duXX>1USE OF THE BACTERIA PSEUDOMONAS FLUORESCENS IN THE CONTROL OF SUGAR BEET ROOT DECAY AGENT  RHIZOCTONIA SOLANI Andrija KRISTEK1  Suzana KRISTEK1 - Milan POSPI`IL2 - Martin EVA I3 - Stjepan GALOVI4 1Faculty of Agriculture, Trg Sv. Trojstva 3, Osijek, Croatia, e-mail:  HYPERLINK "mailto:akristek@pfos.hr" akristek@pfos.hr 2 Faculty of Agriculture, Svetoaimunska cesta 25, 10 000 Zagreb, Croatia 3 CBA International, Koturaaka cesta 69, Zagreb, Croatia 4 akovatina d.d.., Elektri ne centrale 12, akovo, Croatia Introduction In Republic of Croatia sugar beet is grown on approximately 30 000 ha, with an average yield of 37 t ha-1 and sugar content of 14,4% (Kristek et al., 2003). Reasons for such a low production results after the Patriotic War are found in inadequate soil management, devastated soil structure, and climatic conditions. The result of the stated is high degree of disease development. Apart from beet leaf spot (C. beticola) and the disease induced by Rhyzomanie virus, the most serious issue is root rot of beet induced by pathogenic fungi (P. betae, P. ultimum, P. debarianum, R. solani, Fusarium spp., and A. cochlioides). Depending on the soil and climatic conditions, 10-50% of plants have been decaying being attacked by the pathogenic fungi. Growth of the surviving plants is slowed significantly giving them no possibility to reach normal size, which finally reflects on yield and sugar content values. Major measure in the control of sugar beet root decay agent is application of fungicides in the course of seed processing. However, though chemical fungicides prevent development of the fungi to some degree, they are at a disadvantage affecting human health and environment (being exposed to washing out, they give rise to underground water eutrophication). Moreover, pathogenic fungi have ability to rapidly develop resistance to the fungicides applied. Acceptable alternative to the application of chemical pesticides is seed inoculation with the bacteria P. fluorescens and T. harzianum that express antagonism against pathogenic fungi (Hsek and Vach, 2006; Kristek and Kristek, 2005; Thrane et al., 2000, 2001). Since the benefit bacteria do not express sensitivity to the low fungicide doses (Pedersen et al., 2002), on the soils heavily infected with the fungi the root decay agents on sugar beet, positive effect was accomplished by combining seed inoculation with the bacterium P. fluorescens and the seed treatment with low doses of fungicides. Pathogenic fungi R. solani is found to be the most serious issue, so the influence of the bacterium P. fluorescens to the pathogenic fungi the root decay agent of sugar beet, has been investigated. Material and methods The experiment was set up on two soil types - Mollic Gleysols and Eutric Cambisols, 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 (A1 - hybrids tolerant to the R. solani - Laetitia (KWS); Solea (Strube); A2 - hybrids sensitive to the R. solani - Belinda (KWS), Sofarizo (Hilleshg); 2 seed treatment (B1 control seed treated with fungicide Thiram 42-S (600 ml per 100 kg seed); B2 seed inoculated with P. fluorescens bacterium; B3 - seed treated with fungicide Thiram 42-S (200 ml per 100 kg seed) + inoculated with P. fluorescens). Pseudomonas fluorescens was isolated from the sugar beet seedling rhizosphere (Thrane et al., 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 per seed, i.e. 0.81.4נ1010 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 characteristics Investigated properties in a fieldType of soilLayer (0 0.3 m)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 Row seeding was completed at the end of March, with spacing of 50 cm between rows and 20cm within rows. Percent of the plants infected with pathogenic fungi R. solani was stipulated in 2-4 true leaves phase. The sugar beet digging, conducted in the mid October, was followed by determination of root yield (t ha-1) and sugar content (%). Results and discussions During 2-year investigations conducted significant differences were shown in the percentage of infected plants, soil types investigated, tolerant and sensitive hybrids, and hybrids within the two groups (Table 2). On Mollic Gleysols soil type the smallest number of the plants infected 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. Root yield (Table 3) showed highly significant positive correlation with sugar content values (r=0.922**). 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 Table 2. Percentage of the infected plants as a consequence of parasite fungus Rhizoctonia solani infestation in 2 - 4 true leaves phase Investigated parametar Hybrid Seed treatmentSoil type AverageMollic GleysolsEutric CambisolsYear2004200520042005 Infected plants (%) Laetitia1 2 321.60 8.03 10.5023.33 8.97 11.1722.76 10.04 8.6625.90 12.36 9.7123.40 9.85 10.01 Solea1 2 323.75 10.81 12.0624.44 13.01 14.1524.74 12.03 10.9926.92 14.70 13.6224.96 12.64 12.71 Belinda1 2 329.90 9.44 9.9232.85 10.30 10.2133.45 11.70 10.9536.60 12.15 11.9933.20 10.90 10.77 Sofarizo1 2 325.28 10.70 11.1027.07 11.40 12.3527.05 12.12 11.6530.45 12.73 12.4047.31 11.74 11.88 Table 3. 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 Laetitia1 2 377.31 82.19 81.8066.12 71.27 72.4570.21 76.35 80.1064.90 68.34 71.1213.84 14.90 14.1112.30 12.76 12.5413.21 13.96 14.0111.46 12.12 12.30 Solea1 2 378.03 79.19 78.2464.20 71.20 68.3864.10 71.63 71.2056.40 60.22 65.1716.04 17.01 16.8613.92 14.96 14.8015.91 16.44 16.8913.08 13.97 14.25 Belinda1 2 378.18 82.05 80.9271.32 77.16 74.1174.00 75.31 78.7067.38 73.38 75.1115.07 17.16 16.8413.58 14.60 14.5414.26 15.93 16.8112.90 13.83 14.21 Sofarizo1 2 388.05 97.68 91.9085.41 90.20 88.1086.18 92.12 92.3583.10 86.01 87.3015.47 16.98 16.0613.10 14.52 14.4014.99 15.60 15.9212.56 14.01 14.56 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 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. The highest sugar content was obtained with Belinda hybrid sensitive to R. solani, on Mollic Gleysols soil type in variant 2, in relation to the Eutric Cambisols in variant 3. However, no statistically significant differences were found between the aforementioned hybrid and Solea hybrid tolerant to R. solani. Solea hybrid also showed the highest values of the parameter on Mollic Gleysols in the variant 2, and in variant 3 on Eutric Cambisols due to the heavier infection on Eutric Cambisols soil type with the pathogenic fungi. This is in agreement with the trial of Pedersen et al. (2002) who have reported that soils heavily infected with pathogenic fungi R. solani, and regarding the fact that beneficial bacterium Pseudomonas sp. is usually not especially fungicides sensitive, seed is possible to be treated in combination with low doses of the fungicide aiming at effective control of pathogenic fungi growth and reproduction resulting in a positive effect on all sugar beet yield and quality indicators. Conclusion Inoculation of sugar beet seed with the bacterium P. fluorescens affected root decay agent treated with pathogenic fungi R. solani and showed highly significant influence to all the elements of sugar beet yield and quality. The highest values of the elements investigated on Mollic Gleysols soil type were obtained in variant 2 (seed inoculated with P. fluorescens bacterium). On Eutric Cambisols soil type the same was obtained in variant 3 (seed treated with fungicide Thiram 42 S (200 ml per 100 kg seed) + inoculated with P. fluorescens bacterium). In variant 1 (seed treated with fungicideThiram 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. References 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, vol. 34 no. 1 425-428pp. Kristek A. - Kristek S. - Antunovi, M. : 2003. Proizvodne vrijednosti linija aeerne repe i njihovih kri~anaca ovisno o ploidnosti - Agriculture Scientific and Professional Review, vol. 9 no. 2 38-44 pp. Kristek S. - Kristek A. : 2005. Inoculation(*V " $ F H h j ( * t v x  : øøøøøÞre^Z^Z^hq h,hLh,hL0JmH sH #jh,hLUmH sH jh,hLUmH sH h,hLmH sH h,hLH*mH sH hZN_hL6H*mH sH hL6H*mH sH hL6mH sH hWVhL56mH sH h2chL56mH sH hL56mH sH h2chL5mH sH " : $ & @  6C$x$Ifa$gd^ $$Ifa$gd^$a$gd^$a$gd @ F ^     x &'Ont} 9MO\^giuxĹsssssssh2chL6mH sH hP$hLmH sH h |hLmH sH h~E@hLmH sH hsH*mH sH hsmH sH hLmH sH h2chLmH sH  hL5h#hL5 h,hLh,hLH*mHsHhLmHsHh,hLmHsHh,hLH*-?Yvmxz+AHT6023>VWXY|}¶ͶhcP,hL6mH sH hcP,hLmH sH hL6mH sH h2chL6mH sH hP$hLmH sH hLmH sH h2chLmH sH G%7!`alopz  ),-սմɫիիմɫէ՜ՂhPhLH*mH sH h)hL56mH sH h6>#hLmH sH h)hL6mH sH hLH*mH sH h)hL5mH sH hPhL6mH sH hLmH sH hPhLmH sH  hL5h)hL5h W<hLmH sH 3-:Vabjostv{|   2ij~cdٷ˦hLh` hL6 h` hLhhLmH sH h hL6 h>hLh>GhL6mH sH hLmH sH >E!R""""@#A#N#X#Y#`#a#f#p#z#{##$x$Ifa$gdD $$Ifa$gdD$8^8`a$gdL$8^8`a$gdL$a$gdL########J>>>>>> $$Ifa$gdDkdT$$Ifl4rms 2"```c`* t0644 lal#####7+++ $$Ifa$gdDkd$$Ifl4ֈms  2"   cNP * t0644 lal########>kd$$Ifl4rms 2"   c * t0644 lal $$Ifa$gdD##### $$Ifa$gdD##kd$$Ifl4ִms  2"   c''(( * t06    44 lal##############$$ $$$$#$($.$4$9$?$D$$x$Ifa$gdD $$Ifa$gdDD$J$ $$Ifa$gdDJ$K$kd$$Ifl4\ִms  2"`c''((* t06    44 lalK$L$M$S$U$W$Y$_$e$k$q$w$}$$$$$$$$$$ $$Ifa$gdD$$kdh$$Ifl4\ִms  2" c''((* t06    44 lal$$$$$$$$$$$$$$$$$% %%%% $$Ifa$gdD%%kdF $$Ifl4\ִms  2" c''((* t06    44 lal%%%(%*%,%.%4%:%@%F%L%R%X%^%d%j%p%v%|%%% $$Ifa$gdD%%kd$ $$Ifl4\ִms  2" c''((* t06    44 lal%%%%%%%&&$x$Ifa$gdD $$Ifa$gdD$a$gdL&&&&+&<&L&]&]QQQQQQ $$Ifa$gdDkd $$Ifl4\iOt"``d 5 t0t"644 lal]&^&kd $$Ifl4ֈiOt"  dzzy t0t"644 lalp<^&_&`&e&j&o&t&y&~&&&&&&&&&&&&&&&&&&&&Ff $$Ifa$gdD&&&&&&&' ''''#')'*'+'1'3'5'7'='C'I'O'U'['a'g'Ff $$Ifa$gdDg'm's'y'''''''''''''''''''''''''Ff $$Ifa$gdD''(( ((((%(+(1(7(=(C(I(O(U([(a(g(h(i(r(t(v(x(~((Ff $$Ifa$gdD((((((((((((((((((((()) )s))Ffd $$Ifa$gdD))/*l***+y/z//2223yy$h^h`a$gdL$a$gdLnkd$$Iflt"t" t0t"644 lalp *"*I*`*j*l*******++++,,--------...".>.?.T.d.x/z/////////00000ƻỜ|黜h)hL5hPhLB*mH phfsH hL6mH sH hPhL6mH sH h=mH sH hK`hLmH sH hPhLmH sH h:xhLmH sH h>GhL6mH sH h)hLmH sH hLh>hL6 h>hLh'000000000114171@1B1t1y111111111111221282223 334556-=̌όɽh"=hLmH sH  h"=hL hL6Uh)hLmH sH hL5mH sH hP$hLmH sH hLh=h' h>hLh'mH sH hL6mH sH hPhL6mH sH hPhLmH sH hLmH sH 435ďŏǏȏʏˏ͏ΏϏЏяҏݏޏߏ &`#$gdb(gd&n $da$gdL$a$gdL$^`a$gdL of sugar beet seed by the bacterium P. fluorescens - chemical fungicides alternative - Listy Cukrovarnicke a Reparske, vol. 122 no. 2 49-53 pp. Page A.L. : 1982. Methods in Soil Analysis - American Society of America, Madison, Wisconsin. Pedersen H. C. Weiergang I. - Pontoppidan M. M. - Jrgensen L. - Svingel A. :2002. Danisco Seed -Seed Technology Dept. Hoejbygaardvej 31, DK 4960 Holeby, Denmark. Thrane C. - Nielsen T. H. - Nielsen M.N. - Srensen J. Olsson S. :2000. Viscosinamide producing Pseudomonas fluorescens DR54 exerts a biocontrol effect on Pythium ultimum in sugar beet rhizosphere - FEMS Microbiol Ecol., vol. 33 no. 2 139-146 pp. Thrane C. - Nielsen M. N. - Srensen J. - Olsson S. : 2001. Pseudomonas fluorescens DR54 reduces sclerotia formation, biomass development, and disease incidence of Rhizoctonia solani causing damping off in sugar beet - Microb. 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