Taxonomnic status and pathogenicity of Rhizoctonia spp. isolates, causing bean stem and root canker in Northern-Khorassan province

Red bean (Phaseolus vulgaris) is an important legume cultivated in various areas with different climates worldwide. Bean damping-off, stem and root canker are caused by several fungal pathogens. Soil-borne pathogenic fungi are the most important pathogens in bean plants, which cause destructive diseases. Rhizoctonia solani is one of the most serious causal agents of stem and root canker that is reported in all kinds of beans. The Rhizoctonia species belong to Basidiomycota which are the causal agents of various diseases different monocots and dicots with high yield losses. The isolates of R. solani are classified into 13 anastomosis groups (AGs). The most common AGs of R. solani which infect bean plants are AG-1 IE, AG-1 IF, AG-2-2, AG-2-3, AG-BI, AG-4 and AG-5. In overall, isolates of AG-4 HG-II were more virulent in peas, soybeans and beans compared to other AGs. Knowledge of taxonomic status of Rhizoctonia spp. causing bean diseases in different geographic regions of Iran is still scarce. However, accurate identification of various taxonomic groups in Rhizoctonia spp. populations is necessary for designing effective disease management strategies. So, we identified the accurate taxonomic status of R. solani isolates causing bean diseases. Also, pathogenicity of these fungal isolates was investigated on red bean. Red bean plants with the symptoms of stem and root canker were sampled during growing season of 2014 in the fields of this crop for characterizing various taxonomic groups of pathogenic Rhizoctonia spp. Symptomatic bean tissues were surface-disinfected with 0.5% sodium hypochlorite solution for 2 min and rinsed three times with sterile distilled water. Then, pieces of the stem or root tissues were dried on sterilized filter paper, placed on a potato dextrose agar (PDA) petri dish containing chloramphenicol, and incubated at 28°C in dark conditions. After 2 to 3 days, cultures were examined microscopically for hyphal characteristics typical of Rhizoctonia spp. All plated samples readily yielded Rhizoctonia spp., and a hyphal tip of each isolate was subcultured onto AWA for further purification. Isolates were transferred to PDA test tube slants and maintained at 28°C. Fungal isolates were initially characterized as either binucleate or multinucleate by the trypan blue staining technique. Colonies of Rhizoctonia on PDA, 2 to 3 days old, were stained to determine nuclear number and were examined for the presence of a septal pore (dolipore) apparatus. The width of the main runner hyphae was investigated from the same preparations. Isolates were subcultured onto PDA petri dishes in triplicate, incubated at 28°C for 3 weeks, and compared for colony morphology and the size and shape of moniloid cells and sclerotia. Rhizoctonia spp. isolates were distinguished using specific primers for PCR amplification based on the ITS region of R. solani. PCR-RFLP of rDNA-ITS region was used to determine the taxonomic group of each R. solani isolate. Genomic DNA of the isolates was used for PCR amplification of the ITS region using a pair of primers, RS1 (5′-CCTGTGCACCTGTGAGACAG-3′) and RS4 (5′-TGTCCAAGTCAATGGACTAT-3′). Restriction polymorphisms were detected using two discriminating restriction enzymes, AvaII and HincII. 32 Rhizoctonia isolates were obtained from culturing the infected plant tissues. Identification of these isolates was carried out using morphological and molecular methods. Among 32 isolates, 29 isolates belonged to multinucleate R. solani and 3 were binucleate Rhizoctonia spp. Investigating restrictesd PCR products on agarose gel was performed and anastomosis gropus and subgroups were determined. Among 29 R. solani isolates, 17 isolates were AG4 HG-I and 12 isolates belonged to AG4 HG-II. Pathogenicity testes on red bean cv. Naz revealed that all isolates of R. solani were pathogenic on bean. Lower level of pathogenicity was observed for the binucleate Rhizoctonia isolates on the root and stem of host plant. Similar levels of pathogenicity were observed for the isolates of AG4 HG-I and AG4 HG-II. This is the first report on exact taxonomic status of Rhizoctonia isolates, on the anastomosis groups and subgroups levels, obtained from bean field of Iran. It can be concluded that R. solani AG4 HG-I is the main taxonomic group of Rhizoctonia, causing stem and root canker on bean plants in the regions investigated in this research. Binucleate Rhizoctonia isolates, which were hypovirulent or avirulent on bean plants bight be used in biocontrol assays to investigate their potential for inducing host defense responses against highly pathogenic R. solani isolates.