Distribution of the endophytic fungus, Neotyphodium occultans in naturalized Italian ryegrass in western Japan and its production of bioactive alkaloids known to repel insect pests

Abstract Endophytic fungi belonging to the genus Neotyphodium that form mutualistic associations with grasses of the subfamily Pooideae are ecologically and agriculturally important, producing mycotoxins that affect grazing animals and some insect pests. After our first report of naturalized Italian ryegrasses ( Lolium multiflorum , annual ryegrass) infected with a Neotyphodium endophyte in Japan, the identity of this endophyte, the nature of its association with host grasses and its distribution, as well as the possible presence of toxic alkaloids have been of interest. To answer these questions we conducted surveys covering the western part of Japan to determine the distribution of infected Italian ryegrasses. Further, to identify the endophyte we used microscopy to determine its distribution in infected grasses and we applied DNA analysis using the ribosomal RNA gene (rDNA) to compare strains of this endophyte obtained from different geographic locations. Finally, we examined seed obtained from endophyte‐infected Italian ryegrasses using high‐performance liquid chromatograph (HPLC) and gas chromatograph. These studies indicated that naturalized Italian ryegrass in all three islands surveyed; Honshu, Kyushu and Shikoku, was frequently infected with N. occultans. N‐formyl loline, an insect toxin, and peramine, a feeding deterrent were detected in the infected seed samples, but neither ergovaline nor lolitrem B (causal agents of grazing animal toxicity) were detected. These chemical analyses indicated that N. occultans infection of Italian ryegrasses may provide some influence on the insect fauna of the area through the chemicals active against insects, but was unlikely to be toxic to grazing animals.