CHARACTERIZATION OF ANASTOMOSIS GROUPS OF BINUCLEATE RHIZOCTONIA SPECIES USING RESTRICTION ANALYSIS OF AN AMPLIFIED RIBOSOMAL-RNA GENE

Seven U.S. and 16 Japanese binucleate Rhizoctonia anastomosis tester isolates, representing 21 different anastomosis groups, were characterized by restriction analysis of a ribosomal RNA (rRNA) gene. Genomic DNA was extracted from each isolate and a region of DNA coding for a portion of the 25S rRNA (rDNA) was amplified using the polymerase chain reaction. Five tester isolates (CAG1, AGF, AGI, AGJ, and AGK) produced either two or three bands ranging from 1.4 to 1.8 kilobases (kb), whereas five other tester isolates (CAG5, AGBa, AGC, AGD, and AGH) produced a single, 1.8-kb fragment. The remaining 13 tester isolates produced a single, 1.4-kb fragment. Amplified rDNA from 64 additional binucleate isolates produced either a single 1.4- or 1.8-kb fragment or a combination of both fragments. Amplified rDNA was isolated and digested with eight restriction enzymes to determine restriction fragment length polymorphisms (RFLPs). RFLPs of CAG2, AGA, and AGBo were identical with all enzymes tested. Also, RFLPs of CAG6, CAG7, and AGE were identical or nearly identical with all enzymes tested. The RFLP patterns of CAG1 and AGD were also similar with all enzymes tested, and were distinct from all other anastomosis groups. Restriction analysis of nuclear encoded rDNA with four enzymes (HhaI, HpaII, Sau3AI and TaqI), allowed the separation of 13 of 21 anastomosis groups of binucleate Rhizoctonia spp. into distinct groups and were found to be consistent with prior groupings based on hyphal anastomosis.