Detection And Analysis Of Putative NRPS Genes In China-Native Epichloe Endophytes

Neotyphodium spp. and Epichloe spp. are close related asexual and sexual endophytic fungi endowed in complex evolutionay relationships. They are mutualistic with temperate grass of Pooideae without symptoms (expecting occasional stromata formation) to host while they can bring benefit for host in resistances to drought and insects. These benefits were considered to be mediated by the production of secondary metabolites, especially some heterocyclic alkaloids, part of them are synthesized by non ribosomal peptide synthetase (NRPS). Up to now, there are12NRPS genes and a suspectable NRPS gene reported in Europe and America epichloe fungi. In this study, two types of new Neotyphodium endophytes were identified and distribution of NRPS genes in China-native endophytic fungi of gramineous plant were practiced by PCR. Meanwhile, the whole genome sequences of E. festucae were analyzed for prediction of NRPS genes.Two types of grasses from eastern China were detected to harbour fungal endophyte. Samples of them were identified as Bromus japonicas and Festuca myuros respectively,55.2%and91.2%of infection rates were estimated by using163samples and148samples respectively, and2different Neotyphodium endophytes were identified according to their different morphological features. Isolates from B. japonicas grown in Anhui province grew fast (58-79mm/10days), produced abundant and small (4.3±0.1×2.7±0.1μm) conidia; while isolates from F. myuros in Jiangsu province slow15-20mm/6weeks, with bigger and thinner kidney-shaped conidia (6.9±0.8×3.3±0.4μm). Colonies of these2types of fungal endophyte were round and raised. Phylogenetic analyses based on tefA and tubB fragments. indicated of them had hybrid origins, and their common sexual ancestors might be EBY and ETC. Here, a new species, Neotyphodium huibromum Z. Wang et K. Han sp. nov., was proposed for isolates from B. japonicus, and those from F. myuros were identified as Neotyphodium sinofestucase.According to the specific primers for NRPS genes in epichloe endophytes, NRPS genes were detected from20fungal strains isolated from genera of Agrostis, Brachypodium, Bromus, Calamagrostis, Elymus, Festuca, Poa, and Roegneria. All13reported NRPS genes were detectable in China-native endophytic fungi of gramineous plant while the distribution lacked continuum in all strains. Except Psh6102, all other strains had the NRPS2, NRPS5, NRPS8, NRPS9, CPS1and AC202gene partials while NRPS6distributed in all detected strains. lpsA gene was absent in all China-native endophytic strains. However, lpsB gene had discrete distribution in detected fungal strain. The distribution of other detected NRPS genes indicated differences in strains and geography.The method to detect existence of endophyte in plant mainly relied on dyeing the plant tissue and conducted microscopic detection. This experience-orientation way is both time-consuming and mechanical. In this study, we used specific primer RJ6-R and RJ6-F for NRPS8to detect genus Roegneria with epichloe endophyte infected and free. We proved that the NRPS8gene partials obtained from infected Roegneria plants matched completely with sequence from fungal isolates. A high-specificity and high-efficiency PCR method was proposed to detect existence of endophytic fungi in Roegneria plants.Through investigating the whole genome of E. festucae E2368and identified at least19NRPS genes in the genome, named nefl-nef19, far beyond former reported. Meanwhile,5reported genes were absent from this strain’s genome, so this study proved another11new NRPS gene clusters. Function for only9predicted NRPS had been identified and some NRPS had same function while they were different in sequence structure. Phylogenetic tree was constructed based on the nucleotide sequences of adenylation domain using Neighbor-Joining method. Some adenylation domains from different NRPS gathered in one clade with high bootstrap support while adenylation domains from one NRPS gene divided into several clades. This phylogram supported the limitation of degenerate primer for searching NRPS genes and proved the diversity of adenylation domain within one NRPS gene.In this study, the distribution of NRPS genes in China-native endophyte was surveyed, and identified11potential new NRPS gene clusters from genome of E. festucae E2368. This provided foundamental knowledge for further research in gene structure and products of NRPS involved in China-native epichloe endophytes.