Two distinct Epichloë species symbiotic with Achnatherum inebrians, drunken horse grass

Abstract

Achnatherum inebrians, colloquially known as drunken horse grass, is associated with livestock toxicity in northern China. Epichloë gansuensis (Eg) was described from endophyte isolates from A. inebrians in Sunan County, Gansu Province, whereas a morphologically distinct variety, E. gansuensis var. inebrians (Ei), was described based on two isolates from A. inebrians seeds collected in Urumqi County, Xinjiang Province. Genome sequencing and alkaloid analyses also distinguish these taxa; the Ei isolates produce neurotropic lysergic acid amides (ergot alkaloids), and an Eg isolate produces paxilline (an indole-diterpene alkaloid). To better elucidate the taxonomic diversity of Epichloë spp. symbiotic with A. inebrians, we surveyed eight populations in Xinjiang, Gansu and Inner Mongolia provinces of China and analyzed their genotypes by multiplex PCR for alkaloid biosynthesis genes and mating-type genes. Genotypes consistent with Ei were present in all eight populations, of which they dominated seven. The Ei isolates were all mating type A and tested positive for the ergot alkaloid gene, dmaW. In contrast Eg isolates were all mating type B and had the indole-diterpene gene, idtG. The genome was sequenced from an Ei isolate from seeds collected in Xiahe County, Gansu, and compared to that of the varietal ex type isolate from Urumqi. Alkaloid genes and four different housekeeping genes were nearly identical between the two sequenced Ei isolates and were distinct from a sequenced Eg isolate. Phylogenetic analysis placed Ei, Eg and Epichloë sibirica into respective subclades of a clade that emanated from the base of the Epichloë phylogeny. Given its chemotypic, genotypic, morphological and phylogenetic distinctiveness, its widespread occurrence in rangelands of northern China, and its importance in livestock toxicity, we propose raising Ei to species rank as Epichloë inebrians.

Keywords: Clavicipitaceae; E. inebrians; Epichloë gansuensis; Hypocreales; endophytes; ergot alkaloids; genome sequence; grasses; phylogenetics; range-lands; symbiosis; toxicosis.