Development of reliable molecular markers to detect non-pathogenic binucleate Rhizoctonia isolates (AG-G) using PCR

Non-pathogenic binucleate Rhizoctonia species (BNR) belonging to the anasto mosis group AG-G are commonly associated with members of the Rhizoctonia so lani complex. They provide effective protection to young bean seedlings aga inst root rot caused by R. solani AG-4. Both fungi are morphologically simi lar and it is difficult to differentiate between them without using laborio us conventional techniques. RAPD assays were carried out on a, large range of isolates of binucleate Rhizoctonia species to identify markers common to all AG-G isolates. Two fragments of 1368 bp and 882 bp were isolated, clon ed and used to probe Southern blots of DNA from: AG-G isolates; isolates fr om other AGs of binucleate and multinucleate Rhizoctonia species; various h eterogeneous pathogens known to infect bean plants; and co-inoculated bean plants with BNR AG-G and R. solani AG-4. The fragments hybridized only to D NA from AG-G isolates. Both fragments were nucleotide sequenced and two pai rs of SCAR (sequence characterized amplified region) primers (BR1a F/R and BR1b F/R) were generated for use in PCR. Two fragments of anticipated size were generated following PCR of all isolates of AG-G and not from any range of other fungal species associated with root and leaf diseases of beans. T he SCAR primers were also used to detect AG-G isolates in DNA extracted fro m bean and soil samples co-inoculated with binucleate and multinucleate Rhi zoctonia species. The assays were capable of detecting as little as 2.6 pg of fungal DNA in extracts of soil samples. This system offers the potential to determine the presence of AG-G isolates in infected soil and plant samp les.