Bioresources And Detection Of Alkaloid Biosynthesis Genes In Epichloae Endophytes Native To China

The symbionts of epichloae endophytes-grasses can produce a series of alkaloids. The related biosynthetic genes had also been detected in endophytes. In this study, we surveyed epichloae endophytes within grasses (Poaceae) in north of China. Then, the biosynthetic genes of four classes alkaloids in isolated strains were detected by PCR method. At last, the potential synthesis of four classes alkaloids were predicted for epichloae endophytes within the grasses to produce, which provide basic information for evaluating the toxicity of these grasses.From 2012 to 2013,5028 plants of at least 9 tribe 19 genera, were collected from Jiangsu, Henan, Shandong, Liaoning, Inner Mongolia and Gansu. Thirty-five point six percent of these plants were detected to harbor fungal endophytes. The infection level had significant difference among genera and species. Moreover, the infection level of the same host was also varied in different places. The endophytes were detected in Melica scabrosa collected from different places in north of China. It was firstly reported as a new endophytic symbiont with Melica scabrosa in China. Seventy strains were obtained from the infected grasses.The taxonomy of fifty-one endophytes from Melica scabrosa were identified in different places. Based on the morphological characteristics, the endophytes from M. scabrosa were divided into five types:type I (17 strains), type II (10 strains), typeIII(18 strains), typeⅣ(2 strains), type V (4 strains). They were different in morphology of colonies, conidia and phialides. The colonies of types I,Ⅱ and III were raised in the centre and loosely connected with the culture medium; the colony margins of type IV were irregular; the colonies of type V were large and flat, closely connected with the culture medium. The shape of conidia and phialides of types I, II and III were similar, but different in size; Those of type IV were relatively smaller and more productive. Those of type V were significantly different from other types. Single alleles of tubB, tefA and actG were confirmed within the strains from M. scabrosa. Phylogenetic trees based on tubB, tefA and actG fragments revealed that the strains of types I, Ⅱ, III, IVclustered together in a distinct subclude within N. gansuense, the strains of type V clustered with E. yangzii. Together with the geographical distribution of hosts, morphological and phylogenetic characteristics of fungal endophytes, we identified the endophytes of types I,Ⅱ,Ⅲ and IV as N. gansuense. The relationship between the endophytes of type V and E. yangzii remained further study.Based on the morphological features, the endophytes from Poa angustifolia in some places in Dalian were divided into types Pls and Pdl. The significant differences exhibited in their colonies, conidia and phialides. Moreover, Pls and Pdl strains were confirmed single alleles of tubB and tefA fragments. Phylogenetic tree based on tubB and tefA fragments revealed that Pls and Pdl strains clustered with the second alleles of E. liyangensis. Morphological, phylogentic properties, the geographical distribution and the host species of Pls and Pdl strains were different from E. liyangensis. All these results indicated the divergence between the isolated strains and E. liyangensis. They could be a new endophyte group.The biosynthesis genes of four different alkaloids for forty-three strains were detected by PCR method based on previously reported specific primers. Some of them were sequenced to verify the existence of the biosynthesis genes. The results indicated that the endophytic strains contain one or more alkaloids synthesis-related genes cluster. Distribution of alkaloids synthesis-related genes clusters were different in various species. LTM genes cluster were detected in N. gansuense, N. sibiricum and some endophytes from P. angustifolia and Roegneria spp. plants. LOL genes cluster were detected in the endophytes from P. angustifolia and some N. stromatolongum strains. The key genes of EAS cluster, dmaW, lpsA, lpsB were detected in the endophytes from P. angustifolia, N. stromatolongum and some strains from Roegneria spp.. We detected per A in the strains except N. gansuense and N. sibiricum. The universality and diversity of the synthesis-related genes of alkaloids in domestic epichloae endophytes were revealed. Together with the biosynthesis paths of four types alkaloids, it was predicted that our native epichloae endophytes may produce terpendole C, N-formylloline(NFL), peramine and ergopeptine. It is worthwhile further study of epichloae endophytes producing alkaloids.