Submitted to: Crop Science
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 12/5/2005
Publication Date: 6/30/2006
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. Crop Science.46:1064-1070. Interpretive Summary: Cool season grasses such as tall fescue, annual and perennial ryegrasses can be infected with the endophytic fungi, Neotyphodium spp. The presence of the fungus confers to the host plant, increased vigor as well as enhanced tolerance to a variety stresses. However, many of these fungi also produce toxic alkaloids that diminish forage quality and cause livestock toxicosis. While the beneficial effects are desirable for turf applications, the harmful side effects of the plant/fungus symbiosis are undesirable for use in forage and pasture applications. Therefore reliable methods are needed for testing plant tissue and seed for the presence of endophytes. A method for the detection of endophyte, using polymerase chain reaction (PCR) in infected plants and seed was developed. Endophyte detection is performed by amplifying a specific segment of DNA present in the fungal genome isolated from infected seed or plant tissue. This general PCR method provides an accurate and sensitive approach of determining the presence of endophyte in different grass species.
Technical Abstract: A polymerase chain reaction (PCR)-based method for detection of Neotyphodium endophytes in seed and plant tissue from tall fescue, perennial and annual ryegrass was developed. The primers were designed to amplify products from a region of the Neotyphodium spp. tubulin 2 gene isolated from 3 plant species. PCR yielded the expected amplification products from infected seed lots for tall fescue (358 base pairs), annual ryegrass (364 base pairs) and perennial ryegrass (370 base pairs). 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 was able to detect the Neotyphodium spp. endophyte in all plant tissues except roots. Comparison of the PCR based assay to microscopic examination and the immunoblot detection of endophytes in selected seed lots showed that all 3 methods compared favorably to one another. However, the PCR method may be more sensitive in detecting endophytes in cases of low hyphal density. In addition all 3 methods were unable to distinguish between viable and inviable endophyte in seed. This PCR method provides an accurate and sensitive approach for detecting the presence of the endophyte in different grass species, while maintaining enough specificity to readily discriminate against related fungal contaminants such as ergot.