PCR ASSAY BASED ON A MICROSATELLITE-CONTAINING LOCUS FOR DETECTION AND QUANTIFICATION OF EPICHLOE ENDOPHYTES IN GRASS TISSUE

A PCR assay which allows detection and quantification of Epichloe endo phytes in tissues of the grass Bromus erectus is described. PCR with s pecific primers flanking a microsatellite-containing locus (MS primers ) amplified fragments 300 to 400 bp in length from as little as 1.0 pg of fungal genomic DNA in 100 ng of DNA from infected plant material, When annealing temperatures were optimized, all Epichloe and Acremoniu m strains tested, representing many of the known taxonomic groups, yie lded an amplification product, indicating that the MS primers may be u seful for in planta detection of a variety of related species, includi ng agronomically important Acremonium coenophialum and Acremonium loli i. No fragments were generated from DNA isolates from uninfected plant material or from unrelated fungi isolated from B. erectus. For diagno stic applications, a B. erectus-specific primer pair was designed for use in multiplex PCR to allow simultaneous amplification of plant and fungal DNA sequences, providing an internal control for PCR failure ca used by inhibitory plant compounds present in DNA extracts, For quanti tative applications, a heterologous control template with primer bindi ng sites complementary to the MS primers was constructed for use in co mpetitive PCR, allowing direct quantification of Epichloe endophytes i n plant DNA extracts, The fungal DNA present in infected leaves of B. erectus varied between 1 and 20 pg per 100 ng of leaf DNA, but the amo unts of fungal DNA present in the sheath and blade of a given leaf wer e correlated, indicating that the degree of infection varied between p lant individuals but that individual leaves were colonized in a unifor m way.