Characterization of double-stranded RNA (dsRNA) from Rhizoctonia solani

The existence, nature and possible functions of double-stranded RNA (dsRNA) were studied in strains of Rhizoctonia solani anastomosis group (AG) 3, which infects potatoes, and in some other AGs of R. solani. The aim was to determine whether dsRNA might be exploited as a basis for reducing the virulence of R. solani strains, as occurs in Cryphonectria parasitica, a pathogen of chestnut trees. Isolates of R. solani AG 3 were obtained from potato tubers from a single field site, and from geographically distant sites. DsRNA was found to be ubiquitous, with multiple elements present in each strain, as determined by CF 1 cellulose chromatography. Similar gel banding patterns were observed between strains isolated from separate tubers within a single field site; however, banding patterns differed between isolates from diverse sources. All the AG 3 isolates were assessed as being weakly virulent in seedling assays on six host crops (carrot, cress, lettuce, onion, radish, tomato). Attempts to "cure" strains of dsRNA by repeated hyphal tip subculturing or by growing strains in the presence of cycloheximide were generally unsuccessful; although some individual dsRNA bands were lost, they sometimes reappeared, potentially indicating the presence of a chromosomally, integrated copy of the dsRNA. Partially cured strains were unaltered in virulence compared with their respective parental strains. To determine whether dsRNA elements might be transmitted throughout field populations by hyphal anastomosis, strains were paired in various combinations on agar and examined microscopically. Strains from single tubers were compatible with one another; but isolates from different tubers showed a high degree of incompatibility with one another, and isolates from separate fields were incompatible with each other. Pathogenicity-related enzymatic activities were compared between different strains. Activity of phenol oxidases and pectic enzymes was similar between dsRNAcontaining isolates. Cellulolytic activity varied up to 3 fold between isolates. Further examination of cellulolytic activity between isolates from all but two (AG 10 and BI) AGs of R. solani indicated different levels both within and between AGs. Plasmid pXH9 containing complementary DNA of hypovirus CHV1-713 of C. parasitica was used as a probe in hybridization studies with dsRNA of R. solani, to determine potential sequence similarity. At low stringency (42°C) no hybridization was observed, indicating no homology between C14VI-713 and dsRNA elements of R. solani. A protoplast production protocol, using Novozyme 234 with 1.0 M MgSO 4 as an osmotic stabilizer, was successfully developed for R. solani. The protoplasts were transformed with plasmids pES200 and pA)(HY2, both of which encode hygromycin B resistance. Following PEG-mediated transformation, the regenerated strains were hygromycin resistant. However, transformation was non-integrative, with the hygromycin resistance phenotype being lost upon subculture. Overall, the study indicated the ubiquity of dsRNA in R. solani strains, but no firm correlation could be found between the presence of dsRNA and the degree of virulence of strains. Moreover, vegetative incompatibility seen in this study would indicate the difficulty of achieving transmission of any hypovirulent dsRNA in field populations of R. solani AG 3. This would form a major barrier to the potential use of dsRNA for biological control of this important plant pathogen.