Development of a PCR assay to identify and quantify Microdochium nivale var nivale and Microdochium nivale var majus in wheat

Microdochium nivale isolates were subjected to random amplified polymorphic DNA (RAPD) assay. Amplification products common to isolates of var. majus and/or var. nivale were cloned and used as probes to determine the level of cross hybridization to isolates of the opposite variety. Two clones hybridizing to either var. majus or var. nivale were sequenced and primers generated for use in conventional PCR. The primer pair derived from a var. majus specific probe amplified a product only from DNA of var. majus isolates. Similarly, the primer pair generated from a var. nivale specific probe amplified a product only from DNA of var. nivale isolates. This is the first molecular evidence to indicate that members of the diverse nivale variety may form a single group. Both sets of primers were used for the specific detection of the two varieties in infected wheat seedlings. A quantitative assay was developed from these primer pairs, using competitive PCR, for each variety of M. nivale. Competitive PCR was also used to determine the level of colonisation of wheat seedlings by isolates of each variety. (C) 1996 Academic Press Limited