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ARS Home » Pacific West Area » Corvallis, Oregon » Forage Seed and Cereal Research Unit » Research » Publications at this Location » Publication #206632
Title: A sensitive PCR-based assay to detect Neotyphodium fungi in seed and plant tissue of tall fescue and ryegrass spp.

Author
item Dombrowski, James
item Baldwin, James
item Azevedo, Mark
item Banowetz, Gary

Submitted to: Seed Production Research at Oregon State University
Publication Type: Experiment Station
Publication Acceptance Date: 3/31/2006
Publication Date: 4/30/2006
Citation: Dombrowski, J.E., Baldwin, J.C., Azevedo, M.D., Banowetz, G.M. 2006. A sensitive PCR-based assay to detect Neotyphodium fungi in seed and plant tissue of tall fescue and ryegrass spp. Seed Production Research at Oregon State University. EXT/CRS V 125 4/06 P. 52-55.

Interpretive Summary: 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 multiflorum Lam.) and perennial (Lolium perenne L.) ryegrasses was developed. 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 primer set detected the 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 non-viable 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).

Technical Abstract: 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 multiflorum Lam.) and perennial (Lolium perenne L.) ryegrasses was developed. 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 primer set detected the 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 non-viable 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).