SHORT COMMUNICATION GENETIC CHARACTERIZATION BY REP-PCR OF MYANMAR ISOLATES OF RHIZOCTONIA spp., CAUSAL AGENTS OF RICE SHEATH DISEASES

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1 Journal of Plant Pathology (2010), 92 (1), Edizioni ETS Pisa, SHORT COMMUNICATION GENETIC CHARACTERIZATION BY REP-PCR OF MYANMAR ISOLATES OF RHIZOCTONIA spp., CAUSAL AGENTS OF RICE SHEATH DISEASES S.S. Aye and M. Matsumoto Institute of Tropical Agriculture, Kyushu University, Fukuoka , Japan SUMMARY Sheath disease of rice is one of the major constraints of rice production in Myanmar. Forty-four isolates of Rhizoctonia solani, 30 isolates of R. oryzae and 29 isolates of R. oryzae-sativae were recoverd from diseased samples collected from three different regions of Myanmar namely, Mandalay, Pyinmana and Hmawbe. Repetitive-element Polymerase Chain Reaction (Rep-PCR) was conducted using the BOXA1R and ERIC2 primers separately and a combined dendrogram was constructed for the isolates on the basis of the different fingerprint patterns generated by each primer. Two types of R. solani AG1, two types of R. oryzae and three types of R. oryzae-sativae were differentiated by rep-pcr amongst the analyzed isolates. The results indicate the presence of genetically diverse populations of R. solani, R. oryzae and R. oryzae-sativae in Myanmar. Key words: Rhizoctonia solani, R. oryzae, R. oryzaesativae, sheath disease of rice, detection, survey. Rhizoctonia solani, R. oryzae and R. oryzae-sativae are the causal agents of sheath blight and sheath blight-like symptoms on rice (Matsumoto et al., 1997). The presence of these pathogens has become a major factor associated with diseased rice plants and decreasing yields in rice-growing countries. Among them, R. solani AG-1 IA, the causal agent of sheath blight, causes the most serious damage and is a significant pathogen with a major economic impact in many major rice-growing countries (Kobayashi et al., 1997). Rice sheath diseases caused by Rhizoctonia species are relatively difficult to diagnose by visual observation alone due to the similarity of the symptoms with those caused by other disorders. Moreover, various Rhizoctonia species have been isolated from rice sheaths showing similar symptoms. R. oryzae, the causal agent of bordered sheath spot and R. oryzae-sativae, the causal Corresponding author: M. Matsumoto Fax: mmatsu@agr.kyushu-u.ac.jp agent of aggregate sheath spot have been reported on rice from eastern and south-eastern Asia (Inagaki, 1996; Inagaki et al., 2004). These pathogens produce very similar symptoms in the field. In Myanmar, scientific information on rice sheath blight and related diseases is scanty and the population diversity of the causal agents has not yet been surveyed. However, knowledge of the populations of pathogenic Rhizoctonia species is essential for integrated control strategies; along with the understanding of the influence of other characteristics, including pathogenicity, host range and adaptability to environmental conditions. Various methods have been used to study the population structures of different Rhizoctonia species including morphological characteristics (Vijayan and Nair, 1985), intra and extracellular enzymes and proteins (Liu and Sinclair, 1993; Zuber and Manibhushanrao, 1982), total cellular fatty acid analysis (Matsumoto and Matsuyama, 1999) and molecular techniques (Matsumoto et al., 1997; Matsumoto, 2002). However, in the absence of distinctive morphological characteristics, the identification of Rhizoctonia spp. proved to be very difficult and tedious. Recently, molecular techniques have been developed to address the problems associated with the study of genetic diversity in Rhizoctonia spp. Repetitive-element Polymerase Chain Reaction (Rep-PCR) primers (such as the BOX, ERIC and REP primers), which were originally designed for repeated elements in prokaryotic genomes, have been used to characterize several fungal genera including Verticillium, Fusarium, Stagonospora and Septoria (Arora et al., 1996; Czembor and Arseniuk, 1999; Edel et al., 1995). Recently, ERIC2 and BOXA1R primers have been used to characterize clonal populations of Rhizoctonia fungi because of their potential of distinguishing fungal isolates, and because PCR-based fingerprinting has the advantage of being relatively simple and widely applicable in any laboratory with PCR capability. This study was out to investigate the diversity of populations of R. solani, R. oryzae and R. oryzae-sativae within a distinct field population in order to obtain a clearer picture of the relationships of such population types. Diseased rice sheath tissues were collected from three different locations, namely, Pyinmana, Hmawbe, and Mandalay in October 2006 (Fig. 1). These locations

2 256 Genetic characterization of Rhizoctonia Journal of Plant Pathology (2010), 92 (1), Fig. 1. Myanmar map showing the sampling sites for Rhizoctonia spp. Numbers in parentheses represent the total number of isolates collected from each area. differ in their climate and geographically. Pyinmana is located approximately 320 km north of Hmawbe (Yangon), whilst Mandalay is located 716 km north of Yangon and lies at the center of Myanmar s dry zone. Diseased rice sheath tissues were isolated in water agar (WA) media and identified according to morphological characteristics of the Rhizoctonia species as described by Sneh et al. (1991). The cultured fungal mass on potato dextrose agar (PDA) plates was then used for DNA extraction. Lyophilized fungal mycelia (100 mg) was ground in liquid nitrogen, transferred into a microtube containing 600 µl of extraction buffer (10 mm Tris-HCl, ph 7.5, 100 mm EDTA, 0.5% SDS, 100 mm LiCl) and incubated at 65 C for 30 min. After vortexing for 2 min, the samples were centrifuged at 11,000 g for 15 min and the supernatant was transferred into a new tube and extracted with 600 µl of phenol-chloroformisoamyl alcohol (25:24:1) by centrifuging at 11,000 g for 15 min. DNA was precipitated with an equal volume of isopropyl alcohol followed by centrifugation at 11,000 g for 15 min., washed with 70% ethanol, dried in an evaporator for 30 min and dissolved in 100 µl of TE buffer (10 mm Tris-HCl, ph 7.5, 1 mm EDTA). Two µl of RNase (10 mg/ml) were added and the solution was incubated at 37 C for 1 h. DNA was extracted sequentially with Tris-saturated phenol, phenol-chloroformisoamyl alcohol, chloroform-isoamyl alcohol (24:1) and diethyl ether by centrifugation at 11,000 g for 15 min. The supernatant was precipitated with a half volume of isopropyl alcohol at -20ºC for 30 min, and the precipitated DNA was washed with 70% ethanol and dissolved in 200 µl of TE buffer. Primers, BOXA1R (5 -CTACGGCAAGGC- GACGCTGACG-3 ) and ERIC2 (5 -CTACGGCAAG- GCGACGCTGACG-3 ) were used separately for Rep- PCR analysis. Amplifications were done in a 25 µl reaction volume containing 2 µl of template DNA, 2.5 µl of 10 x reaction buffer, 2 µl of dntps (2 mm), 1.5 µl of the primer (100 pm), 0.25 µl Taq DNA polymerase (5 units/sample) and µl of MillQ water. Amplifications were performed in a Thermal cycler with slight modifications to the temperature profiles reported by Toda et al. (1999), i.e., one cycle of initial denaturation (95ºC, 7 min), followed by 30 cycles of denaturation (94ºC, 1 min), annealing (52ºC for 1 min) and extension (72ºC for 8 min) with a final extension at 72ºC for 16 min. PCR products were electrophoresed in 1.0% agarose gels stained with ethidium bromide and the gels were visualized under UV light. Based on the finger- Table 1. Population composition of each type of Rhizoctonia spp. at three different sampling sites in Myanmar. Origin/Type RS Type 1 RS Type2 Total a (RS) RO Type 1 RO Type 2 Total a (RO) ROS Type 1 ROS Type 2 ROS Type 3 Total a (ROS) Hmawbe (56.8) (13.3) (17.2) Pyinmana (34.1) (86.7) (79.3) Mandalay (9.1) (0) (3.4) Total b 30 (68.1) 14 (31.8) (83.4) 5 (16.7) (57.7) 11 (37.9) 3 (10.3) 29 a Total number of each species at respective area; numbers in parentheses in the same column represent the percentage of each species at the respective area b Total number of each type from each species; numbers in parentheses in the same row represent the percentage of each type of the respective species

3 Journal of Plant Pathology (2010), 92 (1), Aye and Matsumoto 257 prints, a total of 10 distinct bands generated by the BOXA1R primer and 12 distinct bands generated by the ERIC2 primer were used for similarity comparison tests. The polymorphic bands were scored as 1 and 0 according to the presence or absence of a band at a certain position for each isolate. The resulting binomial matrix data was analysed by statistixl software and a combined dendrogram was constructed by hierarchical clustering of the variance using the furthest neighbouring method. Fifteen isolates of R. solani, 26 isolates of R. oryzae and 23 isolates of R. oryzae-sativae were obtained from Pyinmana samples; four isolates of R. solani and 1 isolate of R. oryzae-sativae were recovered from Mandalay; and 25 isolates of R. solani, 4 isolates of R. oryzae and 5 isolates of R. oryzae-sativae came from Hmawbe. The total number of isolates of R. solani, R. oryzae and R. oryzae-sativae was 44, 30 and 29, respectively. Based on the dendrogram, the Myanmar isolates of R. solani AG-1 IA were clustered into two groups and denoted RS Type 1 and RS Type 2 (Fig. 2). Examples of the ERIC2 and BOXA1R generated fingerprints for each RS Type are shown in Fig. 3. A total of 30 isolates were classified as RS Type 1 and 14 as RS Type 2 at the variance value of 0.21 (Fig. 2). Isolates of the RS Type 1 formed the major population type of R. solani AG-1 IA in Myanmar (Table 1). Populations of RS Type 1 and RS Type 2 were found in all the sampled locations (Table 1). For example, isolates such as M1 (Hmawbe) and M58 (Mandalay), clustered closely in the RS Type 1. In addition, different types were found at the same location. For example, isolates from the same area (Hmawbe) showed different banding patterns and clustered in different RS Type1 and RS Type 2 (Fig. 3) subgroups. Most isolates of R. solani AG-1 IA in Myanmar were distributed predominantly in the Hmawbe area, which is more humid than the other sampling sites. Thirty isolates of R. oryzae from Myanmar were characterized by the rep-pcr assay and variations were observed in the fingerprints generated. The dendrogram showed that two different population structures existed, which were named RO Type 1 and RO Type 2, based on the banding patterns that were generated by the BOXA1R and ERIC2 primers. Twentyfive isolates of RO Type 1 and 5 isolates of RO Type 2 were classified at a variance value of 0.19 (Fig. 4). The RO Type 1 populations prevailed as they comprised 83.4% of the total isolates (Table 1). This RO Type 1 was mainly present in the Pyinmana area, with only a few isolates (3) found in Fig. 2. Dendogram produced from polymorphic fingerprint patterns observed for 44 Myanmar isolates of R. solani. Numbers in parentheses indicate the total number of isolates classified into each population type. Fig. 3. Fingerprint patterns generated by the ERIC2 primer and the BOXA1R primer for representative samples of each RS Type.

4 258 Genetic characterization of Rhizoctonia Journal of Plant Pathology (2010), 92 (1), Fig. 4. Dendogram produced from polymorphic fingerprint patterns observed for 30 Myanmar isolates of R. oryzae. Numbers in parentheses indicate the total number of isolates classified into each population type. the Hmawbe area. R. oryzae isolates did not occur in the central dry Mandalay area, likely because of the parameters, including weather, cultural practices and epidemiological conditions that affect the dispersal and distribution of R. oryzae in Myanmar. R. oryzae-sativae populations were differentiated into three different subgroups denoted ROS Type 1, ROS Type 2 and ROS Type 3 (Fig. 5). A total of 15 isolates were classified as ROS Type 1, 11 isolates as ROS Type 2, and 3 isolates as ROS Type 3 at a variance of 0.15 based on the fingerprints. The ROS Type 1 population was found to be predominant and was obtained from all sampling sites in Myanmar (Table 1). In this survey, the R. oryzae-sativae isolates prevailed in Pyinmana, which accounted for 23 out of the total of 29 R. oryzae-sativae isolates. The prevalence of Rhizoctonia species can vary as a result of environmental conditions or geographical location. The highest percentage of R. solani was isolated from the samples from Hmawbe, whilst the highest number of R. oryzae and R. oryzae-sativae isolates came from Pyinmana. The lowest percentage of all Rhizoctonia species was isolated from Mandalay where a total of 4 isolates of R. solani, no isolates of R. oryzae and 1 isolate of R. oryzae-sativae were recovered. There is the possibility of natural suppression of these pathogens in this area, and the influence of the environment on disease development is an interesting subject for future epidemiological studies. Datta (1981) reported that the occurrence and distribution of rice sheath disease is influenced by weather factors, physiological responses and agricultural practices. Our results revealed the presence of different population structures in the three different locations of sampling characterized by different environmental conditions. Nevertheless, further studies are required to provide a more extensive analysis of the existing population structures of the species at different rice growing areas in Myanmar. Many researchers have used different methods to differentiate the Rhizoctonia isolates. Matsumoto et al. (1996) described a total cellular fatty acid protocol and successfully differentiated between isolates of R. solani Ag1-1A, AG2-IIIB, R. oryzae, R. oryzae-sativae and R. fumigate. Taheri et al. (2007) studied the genetic relationships among Rhizoctonia species by using fingerprint patterns generated by four primers. Matsumoto et al. (1997) reported the detection of Rhizoctonia from diseased plant tissue by PCR-RFLP analysis. In this present report, we successfully used Rep-PCR to characterize and differentiate among different types within the same species to identify different populations of three Rhizoctonia species. Genetic relatedness among and within different Rhizoctonia solani anastomosis groups has been studied by Toda et al. (1999), who presented the polymorphic fingerprint patterns generated by the ERIC primers. In our research, the polymorphic fingerprint patterns generated by the ERIC2 and BOXA1R were detected in different PCR samples. Simple banding patterns were produced by ERIC2 and complex banding patterns by BOXA1R. According to Versalovic et al. (1994), the optimal number of bands for Rep-PCR is 8 to 15. Fingerprints generated by BOXA1R consisted of more than 15 bands some of which seemed to overlap adjacent bands. The overall results of BOXA1R and ERIC2 amplification indicate that the ERIC2 generates more efficient fingerprints for differentiating Rhizoctonia population types. So, for future studies we suggest that the ERIC2 primer is best to use for classification of R. solani, R. oryzae and R. oryzae-sativae. Taheri et al. (2007) stated that geographic region is significant in determining the genetic structure of the Rhizoctonia population in India. In the present study, genetic structure was diverse even within the same geographical region, which agrees with the findings of Ducan et al. (1993). For example, isolates M10, M5 and

5 Journal of Plant Pathology (2010), 92 (1), Aye and Matsumoto 259 ACKNOWLEDGEMENTS We acknowledge to Prof. Dr. K. Ogata, Director, Institute of Tropical Agriculture, Kyushu University for his valuable comments. REFERENCES Fig. 5. Dendogram produced from polymorphic fingerprint patterns observed for 29 Myanmar isolates of R. oryzae-sativae. Numbers in parentheses indicate the total number of isolates classified into each population type. M79 were collected from the same region of Hmawbe, but these isolates showed diverse fingerprints and were identified as different types. Linde et al. (2005) described the possibility of gene flow by some R. solani genotypes up to a distance of 280 m. Our evidence supports this. In fact, the occurrence of isolate M89 at Mandalay and of several isolates at Hmawbe with the same RS Type 2 group suggests a possibility of gene flow of up to a distance of 716 km, highlighting the importance of extensive sampling of sheath diseases from different locations. In summary, although our investigaton disclosed the presence of genetically diverse populations of R. solani, R. oryzae and R. oryzae-sativae in Myanmar, further comparative studies for the grouping of such species by different methods seem advisable to help with the solution of the the taxonomic problem of the Rhizoctonia genus in Myanmar. Arora D.K., Hirsch P.R., Kerry B.R., PCR-based molecular discrimination of Verticillium chlamydosporum isolates. Mycological Research 100: Czembor P.C., Arseniuk E., Study of genetic variability among monopycnidal and monopycnidiospore isolates derived from single pycnidia of Stagonospora spp. and Septoria tritici with the use of RAPD-PCR, MP-PCR and Rep- PCR techniques. Journal of Phytopathology 147: Datta D.S.K., Principles and Practices of Rice Production. John Wiley & Sons, New York, NY, USA. Ducan S., Barton J.E., O Brien P.A., Analysis of variation in isolates of Rhizoctonia solani by random amplified polymorphic DNA assay. Mycological Research 97: Edel V., Steinberg C., Avelange I., Laguerre G., Alabouvette C., Comparison of three molecular methods for the characterization of Fusarium oxysporum strains. Phytopathology 85: Inagaki K., Distribution of strains of rice bordered sheath spot fungus, Rhizoctonia oryzae, in paddy fields and their pathogenicity to rice plants. Annals of the Phytopathological Society of Japan 62: Inagaki K., Qingyuan G., Masao A., Overwintering of rice sclerotial disease fungi, Rhizoctonia and Sclerotium spp. in paddy fields in Japan. Plant Pathology Journal 3: Kobayashi T., Mew T.W., Hashiba T., Relationship between incidence of rice sheath blight and primary inoculum in the Philippines: mycelia in plant debris and sclerotia. Annals of the Phytopathological Society of Japan 63: Linde C.C., Zala M., Paulraj R.S.D., McDonald B.A., Gnanamanickam S.S., Population structure of rice sheath blight pathogen Rhizoctonia solani AG-1 IA from India. European Journal of Plant Pathology 122: Liu Z.L., Sinclair J.B., Differentiation of intraspecific groups within anastomosis group 1 of Rhizoctonia solani using ribosomal DNA internal transcribed spacer and isozyme comparison. Canadian Journal of Plant Pathology 15: Matsumoto M., Furuya N., Takanami Y., Matsuyama N., A study of fatty acid analysis as a new taxonomic tool for differentiating Rhizoctonia spp. Journal of the Faculty of Agriculture, Kyushu University 40: Matsumoto M., Furuya N., Takanami Y., Matsuyama N., Rapid detection of Rhizoctonia species, causal agent of rice sheath diseases, by PCR-RFLP analysis using an alkaline DNA extraction method. Mycoscience 38: Matsumoto M., Matsuyama N., Grouping of isolates in AG2 of Rhizoctonia solani by total cellular fatty acid analysis. Mycoscience 40:

6 260 Genetic characterization of Rhizoctonia Journal of Plant Pathology (2010), 92 (1), Matsumoto M., Trials of direct detection and identification of Rhizoctonia solani AG1 and AG2 subgroups using specifically primed PCR analysis. Mycoscience 43: Sneh B., Burpee L., Ogoshi A., Identification of Rhizoctonia Species. APS Press, St. Paul, MN, USA. Taheri P., Gnanamanickam S., Hofte M., Characterization, genetic structure, and pathogenicity of Rhizoctonia spp. associated with rice sheath diseases in India. Phytopathology 97: Toda T., Hyakumachi P., Arora D.K., Genetic relatedness among and within different Rhizoctonia solani anastomosis groups as assessed by RAPD, ERIC and REP-PCR. Microbiological Research 154: Versalovic J., Schneider M., de Bruijn F.J., Lupski J.R., Genomic fingerprinting of bacteria using repetitive sequence-based polymerase chain reaction. Methods in Molecular and Cell Biology 5: Vijayan M., Nair C.M., Anastomosis group of isolates of Rhizoctonia solani (Thanatephorus cucumeris) causing sheath blight of rice. Current Science 54: Zuber M., Manibhushanrao K., Studies on comparative gel electrophoretic patterns of proteins and enzymes from isolates of Rhizoctonia solani causing sheath blight disease in rice. Canadian Journal of Microbiology 28: Received June 1 st, 2009 Accepted July 23, 2009