Abstract The infection of Neotyphodium gansuense in drunken horse grass (Achnatherum inebrians) has caused great losses for livestock production in China. An effective molecular method for detection of N. gansuense is quite necessary to set up to replace the present identification method based on morphology and isolation culture. In this study, the N. gansuense isolated from the seed of drunken horse grass collected from Xinjiang, China was confirmed by aniline blue staining microscopy and used as the environmental samples. The reference strain collection used in this study included six species of Neotyphodium (N. gansuense, N. coenophialum, N. lolii, N. huerfanum, N. chisosum and N. aotearoae), and all of them had highly similar morphological patterns. A fragment of about 430 bp in size of the β-tubulin gene was first amplified and sequenced from each strain used. The universal Taqman probe and its primers for genus Neotyphodium, and specific Taqman probe and its primers for N. gansuense, were designed according to the sequence alignment results. The results showed the detection method based on the double-colored Real-time fluorescence PCR developed in this study was an effective, rapid and sensitive molecular method for detection of N. gansuense, which allowed us to detect the mycelia of N. gansuense from a single grass seed, and the detection duration only needed about seven hours. This method can be used to direct detect the infection rate of N. gansuense from the seeds of animal husbandry and lawn.
