Quantification of Gaeumannomyces graminis var. tritici in infected roots and soil using slot-blot hybridization

Abstract

A slot-blot hybridization assay for quantifying Gaeumannomyces graminis var. tritici, in diseased roots and in soil, using a specific and sensitive DNA probe (pG158), has been developed. The pG158 probe hybridized strongly to pathogenic isolates of G. graminis var. tritici, moderately to G. graminis var. avenae and did not hybridize to non-pathogenic G. graminis var. graminis and to a wide range of other soil fungi. The pG158 contains a dispersed highly repeated sequence which provides the high level of sensitivity needed for a quantitative diagnostic test. The amount of Gaeumannomyces graminis var. tritici in soil can be estimated directly from the test soil without growing the susceptible plants or culturing the fungus. pG158 detects approximately 1·0 ng and 0·3 ng of G. graminis var. tritici DNA in 1 μg total DNA extracted from diseased roots and from highly infested soil organic matter, respectively. Both the high copy clone, pG158 and a single copy clone, pG217, clearly distinguished the three varieties of G. graminis and can be used for accurate intra-specific classifications of G. graminis isolates. The pG158 probe provides a tool for field researchers to determine levels of pathogenic isolates of G. graminis in soils and to study the ecology of this fungus.