A sensitive PCR-based assay to detect Neotyphodium fungi in seed and plant tissue of tall fescue and ryegrass species

A polymerase chain reaction (PCR)-based method for detection of Neotyphodium endophytes in seed and plant tissue from tall fescue (Festuca arundinacea Schreb.), Italian (Lolium multifloram Lam.), and perennial (Lolium perenne L.) ryegrasses was developed. The primers were designed to amplify products from a conserved region of the Neotyphodium spp. tubulin 2 gene which is present in N. coenophialum (Morgan-Jones and W. Gams) Glenn, C.W. Bacon, and Hanlin (endophyte of tall fescue), N. lolii (Latch, MJ. Chr., and Samuels) Glenn, C.W. Bacon, and Hanlin (endophyte of perennial ryegrass), and N. occultans C.D. Moon, B. Scott, and M.J. Chr. (endophyte of Italian ryegrass). PCR yielded the expected amplification products from infected seed lots for tall fescue (358 base pairs [bp]), Italian ryegrass (364 bp), and perennial ryegrass (370 bp). Based on DNA mixture tests and bulk seed analysis, the PCR assay was sensitive enough to detect as little as one infected seed per 50 seeds tested. In addition, the printer set detected the Neotyphodium spp. endophyte in all plant tissues except roots. Comparison of the PCR-based assay to microscopic examination and immunoblot detection of endophytes in seed lots showed that all three methods compared favorably to one another. However, none of the three methods could distinguish between viable and nonviable endophyte in seed. This PCR method provides an accurate, sensitive approach for detecting the presence of the endophyte in these grasses, while maintaining enough specificity to discriminate Neotyphodium spp. from related fungal contaminants such as ergot [Claviceps purpurea (Fr.:Fr.) Tul.].