| Previous studies showed that the disease resistance of medicinal plants and the quality of traditional Chinese medicine are closely related to the external and internal microbial environment.Endophytic fungi,as one of the important microbial members in medicinal plants,has formed unique biological characteristics and genetic material in the process of long-term co-evolution with the host plants,and established a good symbiotic relationship with the host like intestinal flora,which has important effects on the growth,metabolism,disease resistance and quality formation of medicinal plants.In this topic,the endophytic fungi of important medicinal plants in our country,Anoectochilus and species were set as the research object;and the tissue culture seedlings of A.roxburghii were considered as a research platform.On the one hand,screening for a target endophytic fungi which can establish a symbiotic relationship with the host and promote the growth of and the target strain which have strong antimicrobial abilities against Fusarium oxysporum were screened out on the other hand.Studying on the effect of target endophytes on secondary metabolites by HPLC and spectrophotometry;studying the infection process of the endophytic fungi on the host plants by immunofluorescence staining techniques;the target endophytes were identified by the morphological and ITS analysis;qRT-PCR technology was used to analyze the expression levels of growth related genes;a large scale functional genomic database of A.roxburghii infected with target strain was established through RNA-Seq technology and bioinformatics analysis.It was expected to reveal the beneficial effects of endophytes on host plants and its mechanisms as well as provide experimental evidences for elucidating how endophytic fungi affect the quality of Chinese medicinal plants.It was hopeful to improve the quality of medicinal plants using biofertilizer made from target endophytes in the future.The main results were as follows:1?277 strains of endophytic fungi were isolated from 5 Anoectochilus and 1 Ludisia species using traditional separation method,among which 53 strains significantly promoted the growth of Arabidopsis thaliana,and only 2 strains(J162 and J211)were symbiotically cocultured with the tissue culture seedlings of A.roxburghii to significantly enhance the biomass.J162 and J211 were further identified as Chaetomium globosum and Colletotrichum gloeosporioides based on the internal transcribed spacer(ITS)region.In addition,49 strains had antibacterial effect on F.oxysporum to a certain degree by using vertical agar plate assay,among which 2 strains showed good biocontrol activity of stem rot disease and identified as Chaetomium globosum(J9)and Colletotrichum crassipes(J246)by ITS sequence analysis.2?The fungal plug punched from fungus-containing PDA of 53 strains of growth-promoting endophytic fungi which significantly promoted the growth of A.thaliana were inoculated into the central position of the tissue seedlings of A.roxburghii and the effects of the target fungi on growth and the biosynthesis of secondary metabolites in A.roxburghii were investigated.The results showed that both of two strains J162 and J211 not only significantly enhance the biomass,induced the biosynthesis and accumulation of flavonoids,kinsenoside and polysaccharides in A.roxburghii but also enhanced the activities of three pathogenesis-related enzymes,including ? 1,3-glucanase,chitinase and superoxide dismutase in the co-cultured 30 days.3?49 isolates obtained from preliminary screening using agar diffusion method were cocultured with the tissue culture seedlings of A.roxburghii.Results showed that J9 and J246 could enhance the disease resistance ability of A.roxburghii and alleviate the effects of pathogens on host growth;the changes of the A.roxburghii seedlings secondary metabolic contents induced by endophytic fungi were investigated by means of UPLC and HPLC,and the results showed the accumulation of flavonoids including rutin,isoquercitrin,narcissin,isorhamnetin-3-O-glucoside and quercetin were induced notably,in addition,the content of kinsenoside,a specific component of A.roxburghii,also had been promoted at different degrees.The phenol-sulfuric acid test results showed the content of polysaccharide was increased by J246.The activity of three pathogenesis-related enzymes including ?-1,3-glucanase,superoxide dismutase and peroxidase were determined by spectrophotocolorimetric method,and the seedlings inoculated with both of endophytic fungi and pathogen showed the highest levels of three enzyme activities,J9 significantly increased the activity of antioxidant defence enzymes and enhanced the host disease resistance in particular.It was clear that J9 and J246 had important influence on growth,disease resistance and the accumulation of active ingredients of A.roxburghii.4?The successful infection of endophytic fungi to A.roxburghii were verified by the means of immunofluorescence staining.The test indicated that J162 and J211 mainly colonized the intercellular gap of xylem parenchyma cells of A.roxburghii roots without obvious harm.It demonstrated that these two strains infested and established a symbiotic relationship with the host successfully,and provide some experimental basis for the future research.5?In addition,qRT-PCR showed that the expression of three growth-related genes,namely,uracil phosphoribosyl transferase(UPRT),amino acid transmembrane transporter(AATMT)and maturase K(matK),were significantly increased in A.roxburghii plants treated with J162,while UPRT and AATMT were enhanced,and matK was decreased in J211-treated samples compared with the control.A large scale functional genomic database of A.roxburghii seedlings which treated by J9 and J246 under stress of fungal plant pathogen was established through RNA-Seq technology and bioinformatics analysis.82515 unigenes with an average length of 463 bp were obtained after de novo assembly,63090 unigenes can be annotated by one of the public databases including Swiss-prot,NR,Pfam,KOG and KEGG.Differentially expressed genes analysis in A.roxburghii transcriptomes with/without endophytes treatment were performed,J9 and J246 were found to regulate or reverse the expression of some genes under the stress of pathogen. |
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