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

Abstract

Non-pathogenic binucleate Rhizoctonia species (BNR) belonging to the anastomosis group AG-G are commonly associated with members of the Rhizoctonia solani complex. They provide effective protection to young bean seedlings against root rot caused by R. solani AG-4. Both fungi are morphologically similar and it is difficult to differentiate between them without using laborious 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, cloned and used to probe Southern blots of DNA from: AG-G isolates; isolates from other AGs of binucleate and multinucleate Rhizoctonia species; various heterogeneous 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 DNA from AG-G isolates. Both fragments were nucleotide sequenced and two pairs 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. The SCAR primers were also used to detect AG-G isolates in DNA extracted from bean and soil samples co-inoculated with binucleate and multinucleate Rhizoctonia 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 samples.