Molecular Detection, Genotypes And Chemotypes Of Epichloe Endophytes In Achnatherum Inebrians

Drunken horse grass(Achnatherum inebrians) is intoxicating perennial gramineous grass, the secondary metabolism alkaloids produced by endophyte symbionts with drunken horse grass is the reason of livestock toxicity. In this paper, 8 seed populations collected from Gansu, Inner Mongolia and Xinjiang Province(Autonomous Region) were used as materials, obtained Epichlo? gansuensis(Eg) and Epichlo? gansuensis var. inebrians(Ei) strains, carried out the morphology comparison of Eg and Ei, genome sequencing and comparison, inoculate A. inebrians with Eg type isolate e7080, alkaloids analysis of the symbiot plants, analysis the diversity of symbionts and phylogenetics. The results are as follows:1. Two types Ei and Eg endophytes have been isolated from 8 seed-populations of A. inebrians: 28 isolates of Ei, e7478、e818-1……e818-10、e817-1……e817-10、ep4914、ep4916、NFR-2、eAIM-1、eXH-B1、eXH-B2 and eXH-C;8 isolates of Eg, eA168、eA169、eA170、eA171、eA172、eA173、eA185 and eA186. Ei genotype is in all 8 populations, and 6 of 8 populations seed pamples all showed as Ei genotype, whereas only the seed samples SNB-1、SNB-4 and SNB-5 of population SNB showed Eg genotype.2. Established PCR molecular detection methoths for endophytes in Achnatherum inebrians, including the specific primers for ergot alkaloids biosynthesis gene dma W and idtG, mating type genes mtA and mtB, and housekeeping gene tefA. Obtained the specific primers of dmaW, idtG, mtA and mtB. Different mating types have been found in Ei and Eg, Ei is mt A, whereas Eg is mtB.3. Genome sequencing of Ei isolates e818-5 and e7478, comparative genome analysis with previous sequenced Eg isolate e7080, and the results showed the genome size of e818 and e7478 isolates are similar, 29.8Mb and 25.4Mb respectively, the genome size of e7080 isolate is 39.9Mb 。 Ei genome has ergot alkaloids biosynthesis gene clusters EAS1 and EAS2(Genebank: JX072969 and JX273434), but no IDT cluster; whereas Eg has indole diterpenes biosynthesis gene cluster IDT, but no EAS clusters. Only single copy of housekeeping genes has been found in Ei/Eg.4. Inoculated A. inebrians with strain e7080, obtained the symbionts plant and seeds. MS analysis and comparison of alkaloids profiles in A. inebrians symbiotic with Ei, A. inebrians symbiotic with Eg7080 and A. inebrians without endophyte(E-), the results indicate A. inebrians symbiotic with Ei produce a bunch of ergonovine and Lysergic acid α–hydroxyethylamide, small amount of lysergic acid amide and chanoclavine; A. inebrians symbiotic with Eg produce the most amount of paspaline, small amount of 13-desoxypaxilline, paxilline, and little bit of paspaline B; neither ergot alkaloids nor indole diterpenes detected in A. inebrians without endophyte. The genotype of Ei and Eg are consistent with the chemotype of the symbionts.5. Phylogenetic analysis placed Ei isolates e818, e817 and e7478 always in the same clade; Placed Ei, Eg and Epichlo? sibirica into respective subclades of a clade that emanated from the base of the Epichlo? phylogeny. Ei and Eg close related with Achnatherum pekinense, Achnatherum sibiricum and Epichlo? guerinii.In summary, given its morphological, genotypic, chemotypic and phylogenetic distinctiveness, and its widespread occurrence in Achnatherum inebrians collected from rangelands of northern China, and its importance in livestock toxicity, raise Ei to species rank as Epichlo? inebrians.