Zbornik Matice srpske za prirodne nauke / Proc. Nat. Sci., Matica Srpska Novi Sad,¥ 113, 161—171, 2007

UDC 633.63:632.25

V e r a S t o j š i n1 , D r a g a n a B u d a k o v2 ,B a r r y J a c o b s e n3 , E v a G r i m m e3 ,F e r e n c B a g i1 , S t e v a n J a s n i ã1

1 Faculty of Agriculture, Department of Environmental and Plant protection,Trg Dositeja Obradoviãa 8, 21000 Novi Sad, Serbia

2 Faculty of Agriculture, Department of Environmental and Plant protection,Trg Dositeja Obradoviãa 8, 21000 Novi Sad, Serbia — grant studentof National Ministry of Science and Environmental Protection

3 Montana State Universty, Department of Plant Sciences and Plant Pathology,Bozeman, MT 59715 MT, USA

IDENTIFICATION OF RHIZOCTONIA SOLANI ISOLATESFROM SUGAR BEET ROOTS BY ANALYSING THE

ITS REGION OF RIBOSOMAL DNA

ABSTRACT: Rhizoctonia solani (Kühn) is one of the most important sugar beet pat-hogens. Rhizoctonia solani anastomosis groups (AGs) 2-2 and 4 are proven to be the mostcommon pathogenic strains on sugar beet. AG 2-2 (intraspecific groups IIIB and IV) cancause root and crown rot while damping-off of seedlings is most frequently attributed toAG 4. Four isolates of R. solani from sugar beet roots showing characteristic crown and ro-ot rot symptoms, collected from different localities in Vojvodina Province, were chosen andcompared to the well-characterized R. solani isolate R9, AG 2-2 IV, from the USA. AllVojvodinian isolates showed medium level of pathogenicity and were able to cause crownand root rot symptoms on inoculated sugar beet roots. Based on anastomosis reaction, isola-tes from Vojvodina did not belong to the AG 2-2 group. Sequencing of the ITS (internaltranscribed spacer) region of ribosomal DNA was performed on the Vojvodinian isolatesfrom R9 in order to determine their relatedness. Sequence analysis showed that these isola-tes were different than R9 and were closely related (99—100% sequence homology) to ana-stomosis group 4, subgroup HG II.

KEY WORDS: Sugar beet, Rhizoctonia solani, anastomosis group, ITS region, PCR

INTRODUCTION

Rhizoctonia solani Kühn, teleomorph Thanatephorus cucumeris (Frank)Donk is a soilborne fungus with a worldwide distribution. In the USA morethan 24% of acres planted with sugar beet have an economic damage from thispathogen, while in Europe only 5—10% of the planted area is considered tohave economic losses from this pathogen (J a c o b s e n, 2005). There are no

161

data about economic damages of Rhizoctonia crown and root rot in Vojvodina(J a s n i ã et al., 2006). However, this fungus was isolated from 0—18.2% ofbeets showing root rot during the period from 2000 to 2005 (S t o j š i n et al.,2006). This specy is highly heterogeneous and produces damping-off, root rotand foliar blight symptoms on many plant species. Rhizoctonia is typically asterile fungal genus and has been characterized by division into binucleate andmultinucleate groups. Rhizoctonia solani is multinucleate and on the basis ofhyphal anastomosis between different isolates, it is divided into 14 anastomo-sis groups: AG 1-13 and AG BI (C a r l i n g et al., 2002). Groups -1, -2, -3,-4, -6, -7, -8 and -9 are additionally divided into subsets (intraspecific groups)based on their morphological characteristics, frequency of hyphal fusion, viru-lence, host range, nutritional requirements, biochemical characteristics, thia-mine requirement, pectic isozymes, fatty acids and molecular characteristics( C a r l i n g et al., 2002; G u i l l e m a u t et al., 2003). On sugar beet, thisfungus causes damping-off, root, and crown root as well as foliar blight of su-gar beet. According to W i n d e l s and N a b b e n (1989) R. solani anasto-mosis groups -1, -2-2, -4 and -5 can cause damping-off of sugar beet. Additio-nally, AG 3 and AG 5 were isolated from sugar beet with symptoms of darkdiscoloration on petiole basis (W i n d e l s et al., 1997). The causal agent ofRhizoctonia crown and root rot of sugar beet is typically characterized as be-longing to AG 2-2 with individual isolates being placed into intraspecificgroups IIIB and IV. Both of these intraspecific groups are found worldwide,although AG 2-2 IIIB is more common on sugar beet in Europe in rotationwith maize (J a c o b s e n, 2005). Recently, besides the system of anastomosisgrouping of R. solani isolates based on hyphal fusion, different molecularmethods have been developed and proven to be very useful for analysis of theevolutionary homology between isolates in the R. solani complex. Genetic he-terogenicity between, and within anastomosis groups was evaluated by F e -n i l l e et al., 2003, using sequence analysis of the internal transcribed spacer(ITS) region of the ribosomal DNA. Comparison of the ITS region is signifi-cant not only for the determination of anastomosis groups, but these sequencesare also useful for verifying subsets. Polymorphism between AGs was revealedin the ITS1 and ITS2 sequences of ribosomal DNA, while 5.8s rDNA sequen-ce is comletely conservative across all AGs (K u n i n a g a et al., 1997).

In this study, the ITS region of four R. solani isolates, which originatedfrom sugar beet root with typical symptoms of crown and root rot, wasanalysed in order to determine sequence variations between the isolates, and toidentify the anastomosis group and subset of examined isolates.

MATERIALS AND METHODS

Four isolates of Rhizoctonia solani: RhKZ, RhBG, RhGL and RhVRwere isolated from the sugar beet plants with typical symptoms of Rhizoctoniacrown and root rot in Vojvodina Province. These isolates were pathogenic onthe susceptible sugar beet variety Delta (B u d a k o v et al., 2006), and causedcharacteristic crown and root rot symptoms.

162

Characterization of the isolates. Isolates were checked for number ofnuclei by staining mycelia with safranin-0 and observed microscopically at400X (B a n d o n i, 1971). Anastomosis tests were performed using the met-hod which was set up by L i u and S i n c l a i r (1991). Before microscopicexamination, samples were dyed with cotton blue in dilute lactophenol (P a r -m e t e r et al., 1969). These isolates were tested for anastomosis with determi-ned culture of Rhizoctonia solani, which was isolated from sugar beet in theUSA and belonged to AG 2-2 IV. Hyphae were checked in, at least, 15 micro-scopic fields at a magnification of 400X, while anastomosis was rated at 600X(V i c o, 1997). Examination of the reaction between hyphae was categorizedaccording to C a r l i n g (1996) — Table 1.

Tab. 1 — Categories of hyphal interaction in Rhizoctonia solani

Category Hyphal interaction

C0 No interactionC1 Hyphal wall contactC2 Hyphal wall and membrane fusion, death in fused and adjacent cells

C3 Hyphal wall and membrane fusion, no death in fused and adjacent cells(perfect fusion)

DNA isolation. Prior to DNA extraction, R. solani isolates were culturedon potato-dextrose agar (PDA, Difco Laboratories, Detroit, MI) at 25°C for4—5 days. The mycelium was harvested by removing excess of the solid me-dia using steril scalpel and then ground up in liquid nitrogen. DNA wasextracted from 100 mg of ground fungal tissue using the E.Z.N.A. FungalDNA Kit (Omega Bio-Tek Inc., Lilburn, GA, USA) and following the proto-col recommended by the manufacturer.

PCR amplification. The ITS region of the rDNA was amplified usingITS 1F and ITS 4 primers (Table 2). The PCR reaction was performed in 50ml total volume consisting of 25 ml of PCR Master Mix (Promega Corporation,Madison, WI, USA), 5 ml of MgCl2, 2 ml of each primer (concentration 10pmol/ml), 2 ml of DNA template and 14 ml of PCR Grade water. The amplifi-cation was performed in PCR thermal cycler (Whatman Biometra, Goettingen,Germany). The cycle parameters were as follows: an initial denaturation at95°C for 2 minutes, followed by 38 cycles consisting of denaturation at 95°Cfor 1 minute, annealing at 55°C for 45 seconds, and extension at 72°C for 1minute. Final extension was at 72°C for 5 minutes. Following the PCR reac-tion, the amplified products were loaded in a 2% agarose gel stained with ethi-dium-bromide, together with 100 bp DNA marker (Promega Corporation, Ma-dison, WI, USA). Before loading, both samples and marker were dyed withBlue/Orange 6X Loading Dye used for tracking migration during electrophore-sis. Electrophoresis was run at 80 V for 2 hours. The DNA bands were visua-lised using a AlphaImager 2200 Imaging System (Alpha Innotech Corporation,San Leandro, CA).

163

Tab. 2 — Primer ITS 1F and ITS 4 sequences, base count, molecular weight, melting temperatureand GC content

Sequence (5' to 3') Basecount

Molecularweight

Meltingtemperature

(°C)

GCcontent

(%)

ITS 1F CTT GGT CAT TTA GAGGAA GTA A 22 6813.32 56.60 36.37

ITS 4 TCC TCC GCT TAT TGATAT GC 20 6033.78 61.50 45.00

DNA sequencing. After the amplification of the ITS region of the rDNA,each product was purified using the QIAquick PCR Purification Kit and proto-col (Qiagen Inc., Valencia, CA, USA). The purified rDNA was shipped byovernight mail to DNA Sequencing Facility at University of California, Ber-keley where isolates were sequenced.

Data analysis. ITS sequence analysis was performed using on-line soft-wares CLUSTAL W and BLAST via http://www.ncbi.nlm.nih.gov, www.bio-servers.org. CLYSTAL W is a multiple sequence alignment program whichcalculates the best match for the selected sequences, and lines them up so thatsimilarities and differences can be seen. The Basic Local Alignment SearchTool (BLAST) finds regions of local similarity between sequences. The pro-gram compares nucleotide sequences to the sequence databases and calculatesthe statistical significance of the matches. BLAST can be used to infer functio-nal and evolutionary relationships between sequences, as well as to identifythe members of the gene families. Sequence database from the National Centerfor Biotechnology Information (NCBI), which was downloaded fromhttp://www.ncbi.nlm.nih.gov, was used for sequence information on selectedR. solani isolates (Table 3).

Tab. 3 — Rhizoctonia solani isolates whose ITS sequences were recovered from the GenBank(National Center for Biotechnology Information — NCBI) and used for determining complemen-tarity with tested Vojvodinian isolates RhKZ, RhBG, RhGL and RhVR

AG and subset Host and geographic origin GenBank accession numberof ITS sequence

AG 1-IA Oryza sativa, Japan AB000017AG 1-IB Beta vulgaris, Japan AB000038AG 1-IC Beta vulgaris, Japan AB000029AG 2-1 Solanum tuberosum, USA AB000026AG 2-2IIIB Beta vulgaris, USA AB054856AG 2-2 IV Beta vulgaris, USA AB054859AG 2-3 Glycine max, Japan U57740AG 2-4 Zea mays, USA AB054878AG 3 Beta vulgaris, USA AB019006AG 3PT Solanum tuberosum, USA AB019013AG 3TB Nicotiana tabacum, USA AB000001AG 4 HGI Beta vulgaris, Japan AB000028AG 4 HGII Beta vulgaris, Japan AB000033AG 4 HGIII Beta vulgaris, USA AF354075AG 5 Beta vulgaris, Japan AF153777

164

AG 6 Pterostylis acuminata, Australia AF153784AG 6GV Soil, Japan AF354101AG 6HGI Soil, Japan AG153779AG 7 Soil, Japan AB000003AG 8 Triticum aestivum, Australia AB000011AG 8ZGI-1 Soil, Australia AF153795AG 8ZGI-2 Soil, Australia AF153797AG 8ZGI-3 Hordeum vulgare, Australia AF354068AG 8ZGI-4 Hordeum vulgare, Scotland AF354066AG 9 Solanum tuberosum, USA AF354109AG 9TX Solanum tuberosum, USA AB000037AG 9TP Solanum tuberosum, USA AB000046AG 10 Hordeum vulgare, Australia AF354071AG 11 Glycine max, USA AF354114AG 12 Pterostylis acuminata, Australia AF153803AG BI Soil, Japan AB000044

RESULTS

Anastomosis reaction. Rhizoctonia solani isolates RhKZ, RhBG, RhGLand RhVR were multinucleate and did not anastomose with a well-characte-rized isolate R9, AG 2-2 IV, from the USA (Figure 1.b). However, isolatesRhKZ, RhBG, RhGL and RhVR anastomosed with each other (Figure 1.a) in-dicating that they belonged to the same anastomosis group (Table 4).

Tab. 4 — Categories of hyphal interacton between all isolates

Isolate Rh KZ Rh GL Rh BG Rh VR Rh R9

Rh KZ C3 C3 C3 C3 C0Rh GL C3 C3 C3 C0Rh BG C3 C3 C0Rh VR C3 C0Rh R9 C3

165

Fig. 1 — a) Hyphal fusion between two Vojvodina isolates RhKZ and RhVR at 400X;b) Contact between hyphae belonging to isolates RhR9 and RhGL at 400 X

Gel elecrophoresis. After PCR reaction, the presence of amplified pro-ducts were tested by running an agarose gel in TAE buffer. DNA bands wereapproximately 740—750 base pairs long (Figure 2).

rDNA sequence analysis. Sequencing of rDNA of isolates RhKZ, RhBG,RhGL and RhVR showed that sizes of the ITS region varied from 713 to 716base, pairs and their sequences were identical (100%) in the ITS region. Simi-liarity of the ITS region of Vojvodinian isolates and the American isolate R9(AG 2-2 IV) was 93%. The ITS sequences of 31 isolates of R. solani, whichbelonged to AG 1-12 and AG BI were recovered from the GenBank (Table 4).The comparison between sequences registered in GenBank and sequences ofVojvodinian tested isolates showed that all isolates had sequence homologybetween 90 and 100% with the referent isolates (Table 5). ITS sequences ofthe tested isolates showed 100% complementarity with representative of ana-stomosis group 4, intraspecific group HGII.

Tab. 5 — Sequence similarity between isolates RhKZ, RhBG, RhGL and RhVR and other AGsisolates of Rhizoctonia solani

AG and subset(GenBank)

ISOLATES

RhKZ RhBG RhGL RhVR

AG 1-IA 94% 94% 94% 94%AG 1-IB 91% 91% 91% 91%AG 1-IC 93% 93% 93% 93%AG 2-1 94% 94% 94% 94%AG 2-2IIIB 92% 92% 92% 92%AG 2-2 IV 93% 93% 93% 93%AG 2-3 93% 93% 93% 93%

166

Fig. 2 — Amplification of DNA from Rhizoctonia solani isolatesRhKZ, RhBG, RhGL and RhVR with primers ITS 1F and

ITS 4 shows bands 740 base pairs long (line 1 — 100 bp marker,line 2 — control, line 3 — RhKZ, line 4 — RhBG,

line 6 — RhGL and line 7 — RhVR)

AG 2-4 93% 93% 93% 93%AG 3 93% 93% 93% 93%AG 3PT 93% 93% 93% 93%AG 3TB 95% 95% 95% 95%AG 4 HG-I 96% 96% 96% 96%

AG 4 HG-II 100% 100% 100% 100%

AG 4 HG-III 91% 91% 91% 91%AG 5 92% 92% 92% 92%AG 6 94% 94% 94% 94%AG 6-GV 95% 95% 95% 95%AG 6HG-I 93% 93% 93% 93%AG 7 95% 95% 95% 95%AG 8 95% 95% 95% 95%AG 8ZGI-1 95% 95% 95% 95%AG 8ZGI-2 95% 95% 95% 95%AG 8ZGI-3 95% 95% 95% 95%AG 8ZGI-4 91% 91% 91% 91%AG 9 94% 94% 94% 94%AG 9TX 94% 94% 94% 94%AG 9TP 94% 94% 94% 94%AG 10 90% 90% 90% 90%AG11 92% 92% 92% 92%AG12 93% 93% 93% 93%AG BI 94% 94% 94% 94%

DISCUSSION

The present study shows that ITS sequencing is a powerful tool in under-standing and determinating the relationship between anastomosis groups andsubgroups of R. solani. Techniques which rely on molecular markers are veryimportant in understanding R. solani complex because they are more accurate,easier and more rapid than conventional techniques for determining anastomo-sis group. The aim of the research was to analyse the less conserved part ofthe rDNA region, since it is proven that 5.8s rDNA sequence is completelyconserved across all AGs, whereas the ITS1 and ITS2 rDNA sequences showsignificant differences between AGs (F e n i l l e et al., 2003). The sequencehomology in the ITS regions is above 96% for isolates of the same subgroup,66—100% for isolates of different subgroups within an AG, and 55—96% forisolates of different AG (K u n i n a g a, 1997). The four isolates from Vojvodi-na Province which were isolated from sugar beet root with symptoms ofcrown and root rot, were shown to belong to AG 4 HGII, with ITS sequencehomology with referent AG 4 HGII isolates of 100%. In general, AG 4 isola-tes are known to be pathogenic on wide variety of hosts (K u n i n a g a, 1997),among which is sugar beet (W i n d e l s and N a b b e n, 1989). AG 4 HG-IIis pathogenic on soybean seedlings, causing damping-off and hypocotyl rot( F e n i l l e et al., 2000), on turfgrass (H s i a n g, 2000), coffee seedlings( K u r a m a e et al., 2000), and sugar beet (S n e h et al., 1991; G o n z a l e zet al., 2006). Besides sequencing of the ITS part of the rDNA, other molecularbiology techniques have been developed. For example, restriction fragment

167

length polymorphism (RFLP) analysis of ITS region of rDNA was develo-ped for a rapid and accurate analysis of large number of isolates of R. solani( G u i l l e m a u t et al., 2003). RFLP analysis can be very fast and can becarried out with 10 to 40 isolates simultaneously (B u r n s et al., 1991). Ano-ther example is the use of random amplified polymorphic DNA-RAPD usedfor detection of differences between Rhizoctonia solani AGs (D u n c u n etal., 1993), then for determinating AG 3 and its subsets (J u s t e n s e n et al.,2003; B o u n o u et al., 1999), and for identification of a uninucleate Rhizo-ctonia sp. (L i l j a et al., 1996). Analysis of the ITS region of ribosomal DNAhas proven in this instance to be an excellent tool for the identification and de-termination of R. solani, on the level of anastomosis group and subset. Sinceconventional methods for assigning AGs, such as observation of hyphal ana-stomosis, can be complicated, time-consuming and subjective, PCR assays pro-vide a more accurate and rapid detection. This research shows the importanceof identifying the correct AG of R. solani associated with crown and root rotof sugar beet, since the host range of AG 2-2 IIIB and IV are much differentthan AG 4 (S n e h et al., 1991; W i n d e l s and N a b b e n, 1989). AG 2-2has a reported host range that includes sugar beet, bean, soybean, corn, rice,mat rush, turf grasses, ginger, gladiolus, burdock, chrysantemum, konjack andchinese yam, whereas AG 4 has a host range that includes sugar beet, tomato,pea, spinach, potato, slash and lobolly pine, and snap bean. These differencescan affect the recommended crop rotations used to control partially the crownand root rot disease of sugarbeet. For example, potato would be a host for AG4, but not AG 2-2.

ACKNOWLEDGEMENTS

We thank the National Ministry of Science and Environmental Protection,Belgrade, Serbia for the partly financial support that allowed Ms. Budakov toconduct the molecular aspects of this research at Montana State University,Department for Plant Sciences and Plant Pathology under the supervision ofDr. Barry Jacobsen. This research was also supported in part by a grant fromthe Western Sugar Joint Research Commission. We also thank Dr. Alice Pil-geram (Montana State University, Department for Plant Sciences and PlantPathology, Bozeman) for providing expertise in molecular biology work.

REFERENCES

B a n d o n i, R. J. (1971): Safranin-0 as a rapid nuclear stain for fungi, Mycologia 63:873—874.

B u d a k o v, D., S t o j š i n, V., J a s n i ã, S., B a g i, F., Ÿ u p u n s k i, V. (2006): Vi-

rulence of Rhizoctonia solani (Kühn) isolates from sugar beet root, XVII. Czechand Slovak Plant Protection Conference, 12th—14th September 2006 at the CzechUniversity of Agriculture in Prague (CUA) Book of abstracts, 214.

168

B o u n o u, S., J a b a j i - H a r e, S. H., H o g u e, R. and C h a r e s t, P. (1999):Polymerase chain reaction-based assay for specific detection of Rhizoctonia so-

lani AG-3 isolates, Mycol. Res. 103 (1): 1—8.B u r n s, T. D., W h i t e, T. J. and T a y l o r, J. W. (1991): Fungal Molecular Syste-

matics, Annual Review of Ecology and Systematics, Vol. 22, pp. 525—564.C a r l i n g, D. E. (1996): Grouping in Rhizoctonia solani by hyphal anastomosis re-

action. In: Rhizoctonia species. Taxonomy, molecular biology, ecology, pathology

and disease control, eds B. Sneh, S. Jabaji-Hare, S. Meate and G. Dijst, pp,37—47. Kluwer Academic: Dordrecht, London.

C a r l i n g, D. E., K u n i n a g a, S., B r a i n a r d, K. A. (2002): Hyphal Anastomosis

Reactions, rDNA-Internal Transcribed Spacer Sequences, and Virulence Levels

Among Subsets of Rhizoctonia solani Anastomosis Group-2 (AG-2) and AG-BI,Phytopathology 92: 43—50.

D u n c u n, S., B a r t o n, J. E. and O ' B r i e n, P. A. (1993): Analysis of variation in

isolates of Rhizoctonia solani by random amplified polymorphic DNA assay,Mycol. Res. 97: 1075—1082.

F e n i l l e, R. C., I z i o k a - K u r a m a e , E. E., S o u z a, N. L. (2000): Cytomorpho-

logical, molecular and pathogenic characterization of Rhizoctonia solani asso-

ciated with soybean from Brazil; Annal International Symposium on Rhizoctonia;Abstracts; III International Symposium on Rhizoctonia; Taichung; India.

F e n i l l e, R. C., C i a m p i, M. B., K u r a m a e, E. E. and S o u z a, N. I. (2003):Identification of Rhizoctonia solani Associated with Soybean in Brazil by rDNA-

-ITS Sequences, Fitopatologia Brasileira 28: 413—419.G o n z a l e z, D., C u b e t a, M. A. and V i l g a l y s, R. (2006): Phylogenetic utility of

indels within ribosomal DNA and beta-tubulin sequences from fungi in the Rhi-

zoctonia solani species complex, Mol. Phylogenet. Evol. 40 (2), 459—470.G u i l l e m a u t, C., E d e l - H e r m a n, V., C a m p o r o t a, P., A l a b o u v e t t e,

C., R i c h a r d - M o l a r d, M. and S t e i n b e r g, C. (2003): Typing of anasto-

mosis groups of Rhizoctonia solani by restriction analysis of ribosomal DNA,Can. J. Microbiol. 49: 556—568.

H s i a n g, T. (2000): Anastomosis grouping of Rhizoctonia solani isolates from turf-

grass based on ITS sequencing, Annal International Symposium on Rhizoctonia;Abstracts; III International Symposium on Rhizoctonia; Taichung; India.

J a c o b s e n, J. B. (2006): Root rot diseases of sugar beet, Proceedings for NaturalSciences, Matica Srpska, Novi Sad, No. 110, 9—19.

J a s n i ã, S., B a g i, F. i S t o j š i n, V. (2006): Rhizoctonia solani prouzrokovaå mrke

truleÿi korena šeãerne repe — rasprostranjenost i znaåaj, Zbornik radova Nauå-nog instituta za ratarstvo i povrtarstvo, sv 42: 275—282.

J u s t e n s e n, A. F., Y o h a l e m, D., B a y, A. and N i c o l a i s e n, M. (2003): Ge-

netic diversity in potato field populations of Thanatephorus cucumeris AG-3, re-

vealed by ITS polymorphism and RAPD markers, Mycol. Res. 107 (11): 1323—1331.

K u n i n a g a, S., N a t s u a k i, T., T a k e u c h i, T. and Y o k o s a v a, R. (1997):Sequence variation of the rDNA ITS regions within and between anasomosis

groups in Rhizoctonia solani, Curr. Genet. 32: 237—243.K u r a m a e, E. E., S u s s e l, A. A. B., F e n i l l e, R. C. and d e S o u z a, N. L.

(2000): Isolates of Rhizoctonia solani HGII AG4 anastomosis group causing dam-

169

ping-off in seedlings of Coffea arabica in Brazil, Annal International Symposiumon Rhizoctonia; Abstracts; III International Symposium on Rhizoctonia; Taichung;India.

L i l j a, A., H i e t a l a, A. M. and K a r j a l a i n e n, R. (1996): Identification of a uni-

nucleate Rhizoctonia sp by Pathogenicity, Hyphal Anastomosis and RAPD Ana-

lysis, Plant pathology 45: 997—1006.L i u, Z. and S i n c l a i r, J. B. (1991): Isolates of Rhizoctonia solani anastomosis

group 2-2 pathogenic to soybean, Plant Dis. 75: 682—687.P a r m e t e r, Jr., J. R., S h e r w o o d, R. T. and P l a t t, W. D. (1969): Anastomosis

Grouping Among Isolates of Thanetephorus cucumeris, Phytopathology 59: 1270—1278.

S n e h, B., B u r p e e, L. and O g o s h i, A. (1991): Identification of Rhizoctonia spe-

cies, APS Press, St. Paul. 133p.S t o j š i n, Vera, B a g i, F., J a s n i ã, S., B a l a ÿ, F. and B u d a k o v, Dragana

(2006): Root rot of sugar beet in the Vojvodina Province, Matica Srpska Pro-ceedings for Natural Sciences. No. 110, 103—108.

V i c o, I. (1997): Prilog taksonomiji fitopatogenih gljiva roda Rhizoctonia DC. ex Fr.,doktorska disertacija, Univerzitet u Beogradu, Poljoprivredni fakultet, Zemun, Sr-bija.

W i n d e l s, C. E. and N a b b e n, D. J. (1989): Characterization and Pathogenicity of

Anastomosis Groups of Rhizoctonia solani isolated from Beta vulgaris, Phyto-pathology 79: 83—88.

W i n d e l s, C. E., K u z n i a, R. A. and C a l l , J. (1997): Characterization and Pa-

thogenicity of Thanatephorus cucumeris from Sugarbeet in Minnesota, Plant Dis.81: 245—249.

IDENTIFIKACIJA IZOLATA RHIZOCTONIA SOLANI SA KORENAŠEÃERNE REPE ANALIZOM ITS REGIONA RIBOZOMALNE DNK

Vera Stojšin1, Dragana Budakov2, Barry Jacobsen3,Eva Grimme3, Ferenc Bagi1, Stevan Jasniã1

1 Poqoprivredni fakultet, Departman za zaštitu biqa i ÿivotne sredine,Trg Dositeja Obradoviãa 8, 21000 Novi Sad, Srbija.

2 Poqoprivredni fakultet, Departman za zaštitu biqa i ÿivotne sredine,Trg Dositeja Obradoviãa 8, 21000 Novi Sad, Srbija — stipendista

Ministarstva nauke i zaštite ÿivotne sredine.3 Montana State Universty, Department of Plant Sciences and Plant Pathology,

Bozeman, 59715 MT, USA

Rezime

Rhizoctonia solani (Kühn) je jedan od najvaÿnijih patogena šeãerne repe.Poznato je da su anastomozne grupe (AG) 2-2 i 4 najpatogenije na šeãernoj repi.AG 2-2 (intraspecifiåne grupe ¡¡¡B i ¡¢) mogu prouzrokovati truleÿ korena iglave šeãerne repe, dok je paleÿ i propadawe klijanaca karakteristiåno za AG4. Odabrana su åetiri izolata R. solani izolovanih sa korena šeãerne repe sakarakteristiånim simptomima mrke truleÿi i koji su poreklom sa razliåitihlokaliteta u Vojvodini. Ovi izolati su uporeðeni sa R. solani izolatom R9, AG

170

2-2 ¡¢, poreklom iz SAD. Svi izolati poreklom iz Vojvodine su pokazalisredwi nivo patogenosti i prouzrokovali simptome mrke truleÿi na inokuli-sanom korenu šeãerne repe. Na osnovu reakcije anastomoze utvrðeno je da izo-lati iz Vojvodine ne pripadaju AG 2-2. U ciqu utvrðivawa srodnosti izmeðuizolata iz Vojvodine i SAD izvršeno je sekvencionirawe ITS (internal transcri-bed spacer) regiona ribozomalne DNK. Utvrðeno je da su naši izolati drugaåijiod R9, a da su bliski (homologija sekvenci 99—100%) izolatima iz anastomoznegrupe 4, podgrupe HG ¡¡.

171