DEVELOPMENT AND USE OF A PCR ASSAY TO DETECT RHIZOCTONIA CEREALIS, THE CAUSE OF SHARP EYESPOT IN WHEAT

摘要

Random amplified polymorphic DNA assays were carried out on a range of isolates of Rhizoctonia cerealis to identify markers common to all isolates, Two fragments were isolated, cloned and used to probe Southern blots of DNA from R. cerealis and isolates from a range of anastomosis groups of Rhizoctonia solani. The two fragments hybridized specifically to DNA of R. cerealis and not to DNA of any of the isolates of R. solani. Both fragments were partially sequenced and two pairs of primers were generated for use in the polymerase chain reaction (PCR). Amplification of a fragment of the anticipated size occurred following PCR of all isolates of R. cerealis and not from any of a range of fungal species associated with disease of the stem base of cereals. The primer pairs for R. cerealis were also used, along with those for Microdochium nivale and W and R-type of Pseudocercosporella herpotrichoides, to detect these pathogens in extracts from field-grown wheat plants exhibiting symptoms of sharp eyespot, eyespot, foot rot or a combination of the diseases. No relationship was found between visual disease assessment of sharp eyespot at growth stage 37 and the results of PCR. However, the results of PCR and visual disease assessment al growth stage 75 were similar, indicating that visual disease assessment may not be reliable until later growth stages. This system offers the potential to detect the presence of R. cerealis in cereals and avoid problems commonly associated with conventional diagnosis of this disease and isolation of the pathogen.