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 endophytes in tissues of the grass Bromus erectus is described. PCR with specific 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 Acremonium strains tested, representing many of the known taxonomic groups, yielded an amplification product, indicating that the MS primers may be useful for in planta detection of a variety of related species, including agronomically important Acremonium coenophialum and Acremonium lolii. No fragments were generated from DNA isolates from uninfected plant material or from unrelated fungi isolated from B. erectus. For diagnostic 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 caused by inhibitory plant compounds present in DNA extracts, For quantitative applications, a heterologous control template with primer binding sites complementary to the MS primers was constructed for use in competitive PCR, allowing direct quantification of Epichloe endophytes in 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 amounts of fungal DNA present in the sheath and blade of a given leaf were correlated, indicating that the degree of infection varied between plant individuals but that individual leaves were colonized in a uniform way.