Anastomosis Groups, Pathogenicity and Sensitivity to Fungicides of Rhizoctonia solani Isolates Collected on Potato Crops in France

A collection of 241 isolates of Rhizoctonia solani obtained from potato plants grown in different areas in France was characterized for anastomosis grouping, symptomatology on tubers of different cultivars and sensitivity to three fungicides. Most isolates collected belonged to (anastomosis groups (AGs)) AG 3, but 2% and 4% of the isolates were AG 5 and AG 2-1. AG 3 and AG 2-1 isolates were mostly obtained from sclerotia on tubers, but all AG 5, some AG 3 and some AG 2-1 isolates were recovered from superficial tuber alterations, like deformations, corky or scabby lesions. Sclerotia were formed on tubers produced by healthy stem cuttings grown in soil artificially infested with AG 3, but not on tubers grown in soil infested with either AG 5 or AG 2-1. No variation in susceptibility to sclerotial formation was observed among five potato cultivars. In all cases, a large proportion of tubers showed superficial corky lesions, often associated with deformations. The proportion of tubers with lesions and deformations was highest in soil infested with AG 2-1 and significantly lower on cv. Samba in all treatments. All isolates were highly sensitive to flutolanil, iprodione and pencycuron, except the AG 5 isolates, moderately sensitive to pencycuron. These results show that, although AG 3 is the most common R. solani group on potato in France, AG 5 and AG 2-1 may be present. Isolates differed for pathogenicity. In vitro sensitivity to fungicides varied among AGs.     

Genetic variability and pathogenicity of Rhizoctonia solani associated with black scurf of potato in New Zealand

Isolates (a total of 129) of Rhizoctonia solani were collected from black scurf on potato tubers from different potato‐growing regions in New Zealand. Sequence analysis of the nuclear ribosomal DNA internal transcribed spacer (rDNA–ITS) regions from these isolates identified three anastomosis groups (AGs), AG‐3PT, AG‐2‐1 and AG‐5. Isolates classified as AG‐3PT were widely distributed, whereas AG‐2‐1 and AG‐5 were confined to distinct locations. Sequence heterogeneity was identified in the ITS regions of 100 AG‐3PT and AG‐2‐1 isolates. Variation in the sequence and length of the rDNA–IGS1 region was also observed for selected isolates of AG‐3PT and AG‐2‐1. Phylogenetic studies found all AG‐2‐1 isolates belong to AG‐2Nt, a subset of AG‐2‐1 previously associated with solanaceous crops in other countries. AG‐2‐1 isolates were consistently more aggressive than those of AG‐3PT. Delayed emergence, severe infection on stolons, formation of aerial tubers and considerable yield losses were associated with AG‐2‐1, but they caused negligible black scurf. In contrast, AG‐3PT caused black scurf on progeny tubers but variable effects on stem emergence and stolons. Furthermore, AG‐2‐1 isolates caused severe tuber malformation, but isolates of other AGs did not. This is the first report on the AG composition, genetic variability and pathogenicity of R. solani isolates associated with black scurf of New Zealand potatoes.     

Infection of potato by Rhizoctonia solani: effect of anastomosis group

Glasshouse and field experiments showed that the pathogenicity and disease type on potato varied between different anastomosis groups (AGs) of Rhizoctonia solani. For example, severe stem and stolon disease developed in plants inoculated with a single isolate of AG3PT and AG5. Severe root disease was observed with single isolates of AG8 and to a lesser extent AG3PT, but rarely with single isolates of the other AGs tested. In both field and glasshouse experiments the AG2‐1 isolate (X81) produced only small lesions (<5 mm). However, this was not representative of two other AG2‐1 isolates. When AG2‐1 isolates of the three different rDNA IGS1 types were tested in a glasshouse trial, one caused more severe stem and stolon infection than AG3PT. In the field experiment, the yield of tubers, by weight, was significantly less (P < 0·05) in all inoculated plants than for uninoculated (control) plants. Yield losses were greatest and tuber numbers smallest in plots inoculated with an AG8 isolate, suggesting that root infection is important in determining quantitative yield loss. The incidence of black scurf was greatest in the progeny tubers in plots inoculated with AG3PT (83·9%), whereas only very small amounts of black scurf developed on tubers from plants infected with AG2‐1 (510 bp) or AG5 isolates. This is supported by laboratory tests, where isolates of AG3PT produced significantly more sclerotia on potato dextrose agar than isolates of AGs 2‐1, 4, 5 and 8.     

Detection and recovery of Rhizoctonia solani in naturally infested glasshouse soils using a combined baiting, double monoclonal antibody ELISA

A combined baiting, double monoclonal antibody immunoassay was developed that allows specific and sensitive detection of the economically important soil-borne plant pathogen Rhizoctonia solani in naturally infested soils. The assay is quick, taking only three days to complete from receipt of soil samples and the immunoassay format allows recovery of Rhizoctonia isolates from colonized baits for determination of anastomosis group (AG) affiliation and pathogenicity. The assay was tested on naturally infested soils from commercial glasshouses used to grow lettuce. Using the immunoassay, conventional anastomosis tests against known AG isolates, and pathogenicity tests, it was shown that R. solani isolates recovered from soil samples were pathogenic towards lettuce and belonged to AG4. Furthermore, those isolates that exhibited strong pathogenicity towards lettuce were recovered from sites that had experienced severe Rhizoctonia damage in previous lettuce crops. The possibility of developing a preplanting test to predict damage to specific crop plants due to the presence of particular AGs in the soil is discussed.     

Anastomosis group and pathogenicity of isolates of Rhizoctonia solani from potato crops in South Australia

Isolates of Rhizoctonia collected from the stems, roots, tuber sclerotia and soil of potato crops in Virginia and Lenswood, South Australia, were identified to anastomosis groups (AG). Of the 301 multinucleate isolates of Rhizoctonia solani tested, 90% were AG-3, 7% were AG-4 and 2% were AG-5; 12 isolates were binucleate Rhizoctonia spp. This is the first report of isolates of AG-4 and AG-5 causing disease in potato crops in South Australia. All AG-3, AG-4 and AG-5 isolates tested caused rhizoctonia disease symptoms on the potato cultivar Coliban in pathogenicity trials conducted under glasshotise conditions. Both AG-3 and AG-5 isolates caused black scurf and stem cankers, although symptoms of black scurf were less severe with AG-5. AG-4 isolates produced the most severe stem and stolon cankers of all isolates tested. The pathogenicity of tuber-borne inoculum was confirmed by growing plants from sclerotia-infested tubers. AG-8 isolates from diseased barley and wheat produced severe root cankers and caused loss of feeder roots on inoculated potato plants. Results suggest that rhizoctonia disease in potato fields in South Australia is caused by a combination of different anastomosis groups and this has important implications for crop rotations.     

Characterization ofRhizoctonia solani isolates from potatoes in turkey and screening potato cultivars for resistance to AG-3 isolates

A total of 304Rhizoctonia solani isolates and 60 binucleateRhizoctonia-like fungi were recovered from stems and tubers of infected potato plants over a 2-yr period in northeast Turkey.R. solani isolates were identified to 11 anastomosis groups (AGs): AG-1 (0.66%), AG-2-1 (5.6%), AG-2-2 (0.99%), AG-3 (83.9%), AG-5 (4.6%), AG-6 (0.66%), AG-8 (1.32%), AG-9 (0.33%), AG-10 (1.32%), AG-12 (0.33%), and AG-13 (0.33%). In the greenhouse tests, most of the AG-3 isolates were significantly more virulent than isolates belonging to other AGs on potato cv. Batum. Isolates of other anastomosis groups differed in their virulence. Results indicated that AG-3 is an important pathogen on potatoes grown in the study area. Five of 22 commercial and local potato cultivars evaluated for their reaction toR. solani AG-3 isolates (TP-2) under greenhouse conditions were highly resistant; the remaining cultivars exhibited different levels of susceptibility to the pathogen isolate. http://www.phytoparasitica.org posting July 14, 2005.     

A One-Step, Immunochromatographic Lateral Flow Device Specific to Rhizoctonia solani and Certain Related Species, and Its Use to Detect and Quantify R. solani in Soil

ABSTRACT A murine hybridoma cell line GD2 secreting an immunoglobulin (Ig)M monoclonal antibody (MAb) was produced against surface antigens from an anastomosis group (AG) 4 isolate of Rhizoctonia solani (teleomorph: Thanatephorus cucumeris). Ascites were produced in mice using GD2 hybridoma cells and used to develop a rapid immunochromatographic lateral flow device (LFD) for the detection of antigens from R. solani and certain related Rhizoctonia spp. The LFD was tested for specificity against surface antigens from related and unrelated soil fungi. Antigens from representative isolates of R. solani AGs 1, 2-1, 2-3, 2-t, 3, 4, 5, 6, 7, 8, 9, 10, 11, and BI gave a positive response in LFD tests, as did antigens from Thanatephorus orchidicola, T. praticola, R. fragariae (teleomorph: Ceratorhiza fragariae), Ceratorhiza goodyerae-repentis, Ceratobasidium cornigerum, and binucleate AGE. Antigens from R. solani AGs 2-2, 2-2IIIB, and 2-2IV and from the related fungi R. carotae, R. cerealis (teleomorph: Ceratobasium cereale), R. crocorum (teleomorph: Helicobasidium brebissonii), R. oryzae (teleomorph Waitea circinata), and R. zeae gave negative responses, as did antigens from a range of unrelated fungi and oomycetes including Fusarium, Gliocladium, Trichoderma, Pythium, and Phytophthora spp. The usefulness of the LFD to detect R. solani was demonstrated in soils naturally infested with R. solani AG3. There was close agreement between results of LFD tests and conventional plate enrichment tests employing selective medium. The specificity of the technique was confirmed by polymerase chain reaction PCR using R. solani AG3-specific primers and by analyses based on sequences of the internal transcribed spacer (ITS)1-5.8S-ITS2 rRNA-encoding regions of unrelated fungi recovered from soil samples. The LFD was used to quantify R. solani AG4 in artificially infested soil samples (chopped potato soil inoculum). Estimates of CFU per gram of soil were derived using a most-probable number technique, which was based on the presence or absence of a detectable signal in the LFD. Estimates of CFU obtained in LFD tests and those obtained in a plate-trapped antigen enzyme-linked immunosorbent assay incorporating MAb GD2 were identical (449 CFU g(-1) of soil).     

A TaqMan real-time PCR assay for <Emphasis Type="Italic">Rhizoctonia cerealis</Emphasis> and its use in wheat and soil

Rhizoctonia cerealis causes sharp eyespot in cereals and the pathogen survives as mycelia or sclerotia in soil. Real-time Polymerase Chain Reaction (qPCR) assays based on TaqMan chemistry are highly suitable for use on DNA extracted from soil. We report here the first qPCR assay for R. cerealis using TaqMan primers and a probe based on a unique Sequence Characterised Amplified Region (SCAR). The assay is highly specific and did not amplify DNA from a range of other binucleate Rhizoctonia species or isolates of anastomosis groups of Rhizoctonia solani. The high sensitivity of the assay was demonstrated in soils using a bulk DNA extraction method where 200 μg sclerotia in 50 g of soil were detected. DNA of the pathogen could also be amplified from asymptomatic wheat plants. Using the assay on soil samples from fields under different crop rotations, R. cerealis was most frequently detected in soils where wheat was grown or soil under pasture. It was detected least frequently in fields where potatoes were grown. This study demonstrates that assays derived from SCAR sequences can produce specific and sensitive qPCR assays.     

Anastomosis groups and pathogenicity of binucleate Rhizoctonia isolates associated with stem canker of potato in China

Potato stem canker caused by Rhizoctonia solani commonly occurs in potato-growing regions around the world, but little is known about whether binucleate Rhizoctonia (BNR) can incite this disease on potato plants in China. In the present study, a total of 69 BNR isolates were recovered from cankered subterraneous stems of potato collected from 17 provinces, autonomous regions, and municipalities of China. Based on the morphological characteristics and sequence analysis of the internal transcribed spacers of the ribosomal DNA, 48 isolates were classified as anastomosis group (AG)-A with a ratio of 69.56 %, 15 isolates (21.74 %) were AG-K, four isolates (5.80 %) were AG-F, one isolate (1.45 %) was AG-I and one isolate (1.45 %) was AG-U. Pathogenicity tests under glasshouse conditions revealed that all BNR isolates, except for the AG-I isolate, could induce symptoms of stem canker on potato plants with disease incidence ranging from 5.26 to 55.56 % and disease index ranging from 1.32 to 13.89, respectively. To our knowledge, this is the first detailed report about BNR AGs causing stem canker on potato plants in China.     

Detecting Migrants in Populations of Rhizoctonia solani Anastomosis Group 3 from Potato in North Carolina Using Multilocus Genotype Probabilities

ABSTRACT The relative contribution of migration of Rhizoctonia solani anastomosis group 3 (AG-3) on infested potato seed tubers originating from production areas in Canada, Maine, and Wisconsin (source population) to the genetic diversity and structure of populations of R. solani AG-3 in North Carolina (NC) soil (recipient population) was examined. The frequency of alleles detected by multilocus polymerase chain reaction-restriction fragment length polymorphisms, heterozygosity at individual loci, and gametic phase disequilibrium between all pairs of loci were determined for subpopulations of R. solani AG-3 from eight sources of potato seed tubers and from five soils in NC. Analysis of molecular variation revealed little variation between seed source and NC recipient soil populations or between subpopulations within each region. Analysis of population data with a Bayesian-based statistical method previously developed for detecting migration in human populations suggested that six multilocus genotypes from the NC soil population had a statistically significant probability of being migrants from the northern source population. The one-way (unidirectional) migration of genotypes of R. solani AG-3 into NC on infested potato seed tubers from Canada, Maine, and Wisconsin provides a plausible explanation for the lack of genetic subdivision (differentiation) between populations of the pathogen in NC soils or between the northern source and the NC recipient soil populations.     

Characterization of Rhizoctonia solani from potato in Great Britain

One hundred and thirty five isolates of Rhizoctonia solani were obtained from British potato crops between 2001 and 2003. Isolates were assigned to anastomosis group (AG) using conventional PCR assays for AG2-1 or AG3 or through the observation of hyphal interactions, where appropriate. A previously published primer set was modified in this study to enhance specificity for AG3PT. Most of the isolates (92·6%) belonged to AG3PT whilst some (6·7%) belonged to AG2-1. Only one isolate recovered (0·7%) belonged to AG5. Isolates of AG2-1 were diverse, with variation in both the length of the rDNA intergenic spacer 1 (IGS1) region and the categories of hyphal interaction observed between pairings of AG2-1 isolates. No variation in the length of the rDNA IGS1 region was observed amongst the AG3 isolates collected. Tests carried out on potato stems with a sub-set of the isolates revealed a wide range of aggressiveness amongst AG2-1 isolates. Sequencing of the rDNA internal transcribed spacer (ITS) region of the AG2-1 isolates and construction of a neighbour joining tree with other AG2-1 sequences available indicated that AG2-1 isolates with the short IGS1 region were closely related. This is the first investigation which provides evidence of the relative AG composition of R. solani populations causing disease in potato crops in Great Britain.     

In vitro analysis of host plant specificity in Rhizoctonia solani

Rhizoctonia solani is a plant pathogenic fungus with a wide host range. Host plant specificity within R. solani was analysed on seedlings grown aseptically on agar, which allowed continuous observation of both the fungus and the whole plant without disturbing the interaction. Symptom development on cauliflower, Arabidopsis , eggplant, tomato and potato by 32 R. solani isolates, belonging to six different anastomosis groups (AGs), was studied. Host plant specificity of isolates, as analysed by similarity clustering, was similar to AG-related host plant specificity as observed in the field, with AG3 isolates (except two avirulent strains) separating from the other isolates. Two R. solani isolates with a reciprocal pathogenicity on cauliflower and tomato were selected for further studies. These showed that in the pathogenic combination, R. solani isolates grew over the plant, adhered and formed infection structures, while in the nonpathogenic combination isolates grew over the plant, but neither adhesion nor the formation of infection structures occurred. From these data, it was concluded that host plant specificity is mediated in the early steps of the infection process.     

Characterization of AG-13, a Newly Reported Anastomosis Group of Rhizoctonia solani

ABSTRACT Rhizoctonia solani anastomosis group (AG)-13 was collected from diseased roots of field grown cotton plants in Georgia in the United States. Isolates of AG-13 did not anastomose with tester isolates of AG-1 through AG-12. Mycelium of all isolates of AG-13 were light brown but darkened as cultures aged. All isolates produced aerial mycelium. Concentric rings were visible after 3 to 4 days of growth but disappeared as cultures aged and darkened. Individual sclerotia were up to 1.5 mm in diameter, similar in color to the mycelium, and generally embedded in the agar. Clumps of sclerotia up to 5 mm in diameter were produced on the agar surface. All attempts to induce basidiospore production were unsuccessful. The 5.8S region of the rDNA from isolates of AG-13 was identical in length and sequence to isolates of all other AGs of R. solani. Length and sequence of the internal transcribed spacer (ITS) regions of rDNA from isolates of AG-13 were unique among AGs of R. solani. Similarity between AG-13 and other AGs of R. solani ranged from 68 to 85% for ITS region 1 and 85 to 95% for ITS region 2. Selected isolates of AG-13 caused minor or no damage to barley, cauliflower, cotton, lettuce, potato, and radish in laboratory or greenhouse studies.     

Postharvest dark skin spots in potato tubers are an oversuberization response to Rhizoctonia solani infection

Israeli farmers export 250,000 tons of potato tubers annually, ≈40,000 tons of which are harvested early, before skin set. In recent years, there has been an increase in the occurrence of dark skin spots on early-harvested potato tubers ('Nicola') packed in large bags containing peat to retain moisture. The irregular necrotic spots form during storage and overseas transport. Characterization of the conditions required for symptom development indicated that bag temperature after packing is 11 to 13°C and it reaches the target temperature (8°C) only 25 days postharvest. This slow decrease in temperature may promote the establishment of pathogen infection. Isolates from typical lesions were identified as Rhizoctonia spp., and Koch's postulates were completed with 25 isolates by artificial inoculation performed at 13 to 14°C. Phylogenetic analysis, using the internal transcribed spacer sequences (ITS1 and ITS2) of rDNA genes, assigned three isolates to anastomosis group 3 of Rhizoctonia solani. Inoculation of wounded tubers with mycelium of these R. solani isolates resulted in an oversuberization response in the infected area. With isolate Rh17 of R. solani, expression of the suberin biosynthesis-related genes StKCS6 and CYP86A33 increased 6.8- and 3.4-fold, respectively, 24 h postinoculation, followed by a 2.9-fold increase in POP_A, a gene associated with wound-induced suberization, expression 48 h postinoculation, compared with the noninoculated tubers. We suggest that postharvest dark spot disease is an oversuberization response to R. solani of AG-3 infection that occurs prior to tuber skin set.     

Suppression of Rhizoctonia solani in potato fields. 1. Occurrence

A search was made forRhizoctonia solani-suppressive soils by establishing many small experimental plots, half of which were planted withRhizoctonia-infected seed potatoes and the other half with disinfected seed stock. The sclerotium index of the harvested tubers was compared witht that of the seed potatoes. In suppressive soils, the sclerotium index of the harvest is much lower than that of the seed potatoes. None of the plots on holocene marine soils (loamy sand, sandy loam, clay loam and clay) proved to be suppressive in 1978 and 1979. Only on pleistocene, slightly acid sandy soil suppressiveness was observed. In 1978, four out of twelve plots showed suppressiveness when the plots were planted with seed potatoes produced on a sandy soil. In 1979, only two out of thirtyone plots were slightly suppressive when planted with seed potatoes produced on a young clay loam from a new polder. A higher percentage of sclerotia on tubers from sandy soils proved to be infected with antagonistic fungi (73%) than of those on tubers from marine clay or loam soils (25%). Factors that influence suppressiveness are suggested.     

Molecular tools to investigate Rhizoctonia solani distribution in soil

Real-time PCR protocols were developed to detect and discriminate 11 anastomosis groups (AGs) of Rhizoctonia solani using ribosomal internal transcribed spacer (ITS) regions (AG-1-IA, AG-1-IC, AG-2-1, AG-2-2, AG-4HGI+II, AG-4HGIII, AG-8) or β-tubulin (AG-3, AG-4HGII, AG-5 and AG-9) sequences. All real-time assays were target group specific, except AG-2-2, which showed a weak cross-reaction with AG-2^tabac. In addition, methods were developed for the high throughput extraction of DNA from soil and compost samples. The DNA extraction method was used with the AG-2-1 assay and shown to be quantitative with a detection threshold of 10⁻⁷ g of R. solani per g of soil. A similar DNA extraction efficiency was observed for samples from three contrasting soil types. The developed methods were then used to investigate the spatial distribution of R. solani AG-2-1 in field soils. Soil from shallow depths of a field planted with Brassica oleracea tested positive for R. solani AG-2-1 more frequently than soil collected from greater depths. Quantification of R. solani inoculum in field samples proved challenging due to low levels of inoculum in naturally occurring soils. The potential uses of real-time PCR and DNA extraction protocols to investigate the epidemiology of R. solani are discussed.     

云南省马铃薯黑痣病病原菌融合群鉴定及8种药剂对其的毒力

由立枯丝核菌引起的马铃薯黑痣病是一种常见的土壤和种薯传播病害,在世界各地均有发生。本试验对分离纯化的67个丝核菌菌株进行融合群测定,结果表明:67个菌株都属于AG3融合群;对其中来自6个不同地域的11个菌株进行ITS测定,11个菌株的相似度为99%。8种药剂对马铃薯黑痣病菌的毒力测定结果表明,20%氟胺·嘧菌酯水分散粒剂50mg/L和25g/L咯菌腈悬浮种衣剂50mg/L的抑菌效果较好,室内抑菌效果可达100%。     

Characterisation of Rhizoctonia solani Anastomosis Groups Causing Bottom Rot in Field-Grown Lettuce in Germany

Bottom rot caused by Rhizoctonia solani is an increasing problem in field-grown lettuce in Germany. During the growing seasons of 1999 and 2000, 95 isolates of R. solani from lettuce plants with bottom rot symptoms were collected from eight locations. The isolates were characterised using hyphal anastomosis, pectic zymograms and morphological characteristics. Ninety-three isolates were identified as anastomosis group (AG) 1-IB, one as AG 1-IC and one as AG 2-1. Optimum hyphal growth was measured over a temperature range of 20–30 °C with an optimum at 25 °C. Aggressiveness of the AG 1-IB isolates varied from weak to strong when tested on detached lettuce leaves. The pathogenic potential of six AG 1-IB isolates was determined on 14 plant species in comparison with lettuce under conditions favourable for the fungus. Radish, broccoli, kohlrabi, spinach and millet seedlings were as severely infected as lettuce seedlings. The same isolates caused little symptoms on maize, tomato and onion. Knowledge about the host range of AGs of R. solani are important for planning an effective crop rotation as part of a control management system.     

Antagonism of Azotobacter chroococcum isolates to Rhizoctonia solani

Antagonism of isolates ofAzotobacter chroococcum toRhizoctonia solani on agar plates was studied, and isolates were tested for their ability to controlR. solani infection of potato sprouts in sterilized and unsterilized soil. The degree of antagonism exhibited varied strongly among the isolates and was also found to be temperature-dependent. At 25, 20 and 15°C, all but one strongly antagonisticAzobacter isolate effectively prevented infection of sprouts of potatoes planted in a soil heavily infected with a pathogenic isolate ofR. solani. At 10°C none was effective.     

Rhizoctonia spp. Recovered from Strawberry Roots in Central Coastal California

ABSTRACT Rhizoctonia spp. were commonly recovered from the roots of strawberry plants growing in nonfumigated soil in the central coastal region of California. With the exception of one multinucleate isolate of R. solani (frequency of recovery of 0.8%), all other isolates were binucleate and were in anastomosis groups (AG) A, G, or I. AGs-A and -I were recovered from all five collection sites, whereas AG-G was recovered from only two sites. AG-A was the most commonly isolated AG, followed by AGs-I and -G. Similar levels of virulence were observed among the different AGs, but differences in virulence were observed among isolates in the same AG. Evaluating anastomosis grouping by pairing isolates recovered from strawberry with known tester isolates did not always yield a positive anastomosis reaction, even though both isolates anastomosed with other members of the same AG. Subsequent investigations with multiple isolates in the same AG from the same collection location confirmed that there was a lack of anastomosis or weak anastomosis reactions for some combinations of pairings, highlighting the need for to use multiple tester isolates or molecular techniques for AG determination. Restriction fragment length polymorphism (RFLP) analysis of a polymerase chain reaction-amplified region of the rDNA was effective for differentiating AGs. Sixteen RFLP groups were observed after cluster analysis with data for the size of the amplified products and fragment sizes after digestion with four restriction enzymes. Although each AG had isolates in multiple RFLP groups, any one individual RFLP group contained isolates of only a single AG. There was no consistent correlation between RFLP group and location of isolate collection.