Development of a PCR assay to identify and quantify Microdochium nivalevar. nivaleand Microdochium nivalevar. majusin wheat

Abstract

Microdochium nivaleisolates were subjected to random amplified polymorphic DNA (RAPD) assay. Amplification products common to isolates of var. majusand/or var. nivalewere cloned and used as probes to determine the level of cross hybridization to isolates of the opposite variety. Two clones hybridizing to either var. majusor var. nivalewere sequenced and primers generated for use in conventional PCR. The primer pair derived from a var. majusspecific probe amplified a product only from DNA of var. majusisolates. Similarly, the primer pair generated from a var. nivalespecific probe amplified a product only from DNA of var. nivaleisolates. This is the first molecular evidence to indicate that members of the diverse nivalevariety 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.