| The endophytic Epichlo? species are symbiotic with cool season grasses in subfamily of Pooideae,forming grass-endophyte associations.The Epichlo? spp.symbiotic with Achnatherum inebrians is one of the three researches focusing on grass-endophytic fungi associations in the world,which is widely distributed in the natural grassland of northern western China.It is reported that the Epichlo? endophyte infected are E.gansuensis and E.inebrins,these two species are the asexual taxa of endophytic fungi.Their transmission mode is mainly vertical transmission through the seed of host drunken horse grass.At the same time,it is also the main mode of vertical transmission on some species seed-borne fungi,these species with fungi often include some important plant pathogenic fungi,some of them often causing the seeding disease.Previous researchesre ported that the symbionts of A.inebrians-Epichlo? sp.have important ecological significance to maintain and restore the degraded grassland species diversity.For the study of grass endophytic fungi on taxonomy,physiological,biochemical and ecological found it plays an important role in the ecosystems,so it is necessary to further clear the evolutionary relationships based on its genome using other methods.The E+ and E-seed of drunken horse grass and their seed-borne fungi as the material for this research.To explore the correlation between endophytic fungi and seed-borne fungi,the effects of the drunken horse grass infected by endophytic fungi on the host rhizosphere soil microorganism community structure and its mechanism were studied.Comparative analysis of their evolution between the model species of E.festucae and E.gansuensis was carried out to provide important scientific basis for the development,utilization and further study of the ecological significance of the drunken horse grass endophytic fungi.The main results of this study are as follows:1.The E+ and E-seeds of drunken horse grass are the material of this research,comparative analysis of the treatment of seed surface disinfection showed it can significantly(P < 0.05)reduce the seed mildew rate and improve the germination rate,germination index and vigor index.Compared with the E-seed,Epichlo? endophyte infection can significantly(P < 0.05)reduce the seed mildew rate and improve the germination rate,germination index and vigor index under the seed without surface disinfection treatment.Under the drought and disinfection treatments,Epichlo? endophyte infection can effectively alleviate drought and unsterilised of seed germination inhibition.Epichlo? endophyte infection can significantly(P < 0.05)reduce the seed mildew rate and improve the germination rate,germination index and vigor index under the drought stress of low concentration(-0.3 MPa)treatment.Under the drought stress of low concentration(-0.3 MPa)treatment,the seed mildew rate of germination period is significantly(P < 0.05)lower than control treatments and the germination increased effectively.With the increasing of the seed moisture content,the germination rate,germination index and vigor index showed varying degrees of decline under storage period.The endophyte could significantly(P<0.05)improve the seed germination percentage,germination index,and vigor index at the seed moisture contents of 5.5% and 9.5%,which was significantly(P<0.05)higher than the other seed moisture treatments.The presence of the endophytic fungi can prolong the storage of seed vigor,which may be associated with antioxidant enzyme system rapid response of drunken horse grass seed induced by endophytic fungi infection.2.Inhibitory interactions between 12 isolates of Epichlo? endophytes,isolated from Achnatherum inebrians,Festuca sinensis and Hordeum brevisubulatum,and 5 pathogenic fungi of grasses: Alternaria alternata,Bipolaris sorokiniana,Curvularia lunata,Fusarium avenaceum and Drechslera erythrospila were studied.The study found that with dual culture testing,the Epichlo? spp.can inhibit the growth of colonies and also spore germination of the five pathogenic fungi.However,the effects differed with different combinations of endophyte isolates and pathogenic fungi.Culture supernatants of the Epichlo? isolates also inhibited the growth of colonies and spore germination of the five pathogenic fungi.Similar effects with the 60 endophyte isolates/ pathogenic fungal species combinations were obtained with both growing procedures.The result showed that the mechanism is associated with pathogenic fungi spore germination inhibited by endophytic fungi.3.The interactive effects of asexual Eg(E.gansuensis),Eb(E.bromicola)and Et(Epichlo? sp.)isolated from Achnatherum inebrians,Hordeum brevisubulatum and Elymus tangutorum,and seed-born fungi on Elymus sibiricus seeds,were determined by an in vitro study using supernatants from liquid cultures of the endophyte strains.The in vitro study was carried out with the seed-borne fungi Alternaria alternaria,Bipolaris sorokinianum,Fusarium avenaceum and a Fusarium sp.isolated from Elymus sibiricus.Different dilution ratios and combinations of the liquid cultures of endophytic fungi enhanced the interim germination,germination rate,length of coleoptile and radicle,and seedling dry weight of E.sibiricus seeds under stress from seed-borne fungi.In the greenhouse study,different concentrations of the supernatant of the endophytes from H.brevisubulatum and E.tangutorum but not A.inebrians,signficantly(P<0.05)enhanced E.sibiricus seed germination.In an eight weeks greenhouse study,the effect on seed imbibing the supernatants prior to sowing on the growth of seedlings was measured.The seeds were treated by the endophytic fungi of Eb03,Et03 and Eg02,there were significant(P<0.05)increases in tiller number(4-8weeks),seedling height(2-8weeks)and dry weight(2-8weeks).The application of Epichlo? endophyte culture supernatants was an effective strategy to improve seed germination and growth under greenhouse conditions.Its mechanism may be related to the alkaloids secondary metabolites alkaloids concentration produced by different endophytic fungi isolates.4.The E +,E-drunken horse grass plants and their rhizosphere soil as the research materials,host grass growth and its rhizosphere microbial diversity influenced by endophytic fungi infection were analyzed.There were no significance(P>0.05)of soil physical and nutritional index between E + and E-drunken horse grass rhizosphere soil,except the soil bulk density,organic carbon and carbon nitrogen ratio(C: N).Based on high-throughput sequencing results,the rhizosphere fungi and bacteria diversity of E+ drunken horse grass were significantly(P<0.05)higher than E-,the protection effect of rhizosphere soil microbial diversity was improved by drunken horse grass infected endophytic fungi.Combination of multiple factors analysis of rhizosphere soil,according to the results that C: N change may be one of the important factors causing the change of rhizosphere microbial diversity of drunken horse grass.In addition,although the soil ph value was no significantly(P>0.05)difference between E+ and E-rhizosphere soil of drunken horse grass,it is also one of the important factors.5.The present study aimed to assess codon usage patterns and biases of the nuclear genome from E.gansuensis and E.festucae,which may be helpful in revealing the constraint factors such as mutation or selection pressure and improving the bioreactor on the cloning,expression,and characterization of some special genes.The results of neutrality and effective number of codons plot analyses showed that both mutational bias and natural selection play roles in shaping codon usage in these two species.We found that gene length is strongly correlated with codon usage and may contribute to the codon usage patterns observed in genes.Nucleotide composition and gene expression levels also shape codon usage bias in E.gansuensis and E.festucae.In addition,we identified 27 and 25 optimal codons that end in a G or C from E.gansuensis and E.festucae.These optimal codons are significantly related to the level of transcription,and it helps us to design of the degenerate primers use this feature,further traced the evolutionary origin of endophytic fungi. |
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