DNA based detection of Epichloe/Neotyphodium endophytes from host grasses with combined use of FTA card and genera/species specific PCR primers

Festuca-Lolium grasses infected with Epichloe/Neotyphodium endophytes have been introduced as forage and/or turf to Japan mainly from the USA and European countries for about a century, but they also have been invading the islands as weeds from various sources and spreading rapidly. For better application and management of those grasses both in agro-ecosystems and nature, detection and identification of the endophytes are increasingly important in the islands. Using Whatman FTA-PlantSaver Cards and PCR primers designed for rDNA of the fungi, we tried their detection and identification from growing infected plants. Basal parts of fresh, infected plant tillers were crushed on the card with a pestle, air dried, and 2-mm disks from the card was used as PCR templates following the supplier's instruction. With combined use of two different forward primers designed for the rDNA ITS-1 region of the fungi, one designed for N. occultans and N. uncinatum, and the other for other related species including N. lolii, N. coenophialum and several Epichloe species, together with an universal reverse primer (ITS4), we could amplify the target region from the card and distinguish those two groups. The former group is not toxic to grazing animals, but the latter group contains some toxic species. However, maybe due to low biomass of the endophytic fungi throughout the host tissue, the target was amplified from only about half of the infected samples tested. The simplicity of the DNA preparation and storage method, which requires no major instrumentation or refrigeration, would be a great advantage for related studies, but further improvement to increase the efficiency will be needed.