Study On The Effect Of Promote Growth On Host And Fermentation Products Of Endophytic Fungi From Huperzia Serrata

Huperzia serrata is a kind of important Chinese medicinal herb,and at present the large-scale cultivation has not been reported.The symbiosis of H.serrata with endophtic fungi is its important biological characteristic.In order to understand plant biology and physiology,provide the theory reference for cultivation,in this paper,the isolation technology of endophytic fungi,its morphology and the effects on cuttings rooting,growth and development of H.serrata were studied.The main contents and conclusions are as follow:(1) Isolation method of endophytic fungal.The optimal method for isolation of endophytic fungal:Stem surface-sterilization with 75%alcohol(2min),1%NaClO(4min) and leaf surface-sterilization with 75%alcohol(0.5min),l%NaClO(3min).The sample treatment with dissection method and used 1/2PDA as isolation medium.Aseptic water method was used to isolate endophytic fungi from the root.(2) Results of the isolation of endophytic fungi:There were 59 strains endophytic fungi isolated from H.serrata,17 strains isolated from H.serrata of Jiangxi Yihuang;20 strains isolated from H.serrata of Anhui Jinxian and 22 strains isolated from H.serrata of Anhui Xuancheng.The endophytic fungal species from root were more than that from stem and leaf.(3) The mycorrhiza structure of H.serrata:A large number of root hairs was found around the roots,fungi hypha was observed in root hairs.The results suggested that mycorrhiza of H.serrata belonged to endotrophic mycorrhiza.(4) Morphological studying on endophytic fungal.The identification result showed the biodiversity of endophytic fungi in H.serrata.There were 56%endophytic fungi can produce spore on the PDA medium,they were identified as Trichoderma, Aspergillus,Penicillium,Rhizopus etc.There were 9 strains that belong to Penicillium, account for 15%,8 strains that belong to Glomerularia,account for 14%,5 strains that belong to Trichoderma,account for 8%in totals isolaled endophytic fungi。(5) The method of cutting was established in the laboratory.The cutting test simulated June climate of the habitat in artificial climate box.Humidity was setted at 90%,illumination was setted at 33%(800-1200Lx),temperature was setted at the day 22℃in day(14h),and 14℃(10h)in night.Cutting was found rooting after 44 days.The rooting rate of spring cutting is 77%.(6) The effect and mechanism of endopythe on cutting rootings.The cuttings of H.serrata were planted on soilⅠof primary habitat,on soilⅡwhich had been inoculated with endophytic fungi,on soilⅢwhich had been autoclaved.The rooting rate was examined,the change of endogenous substances contents during rooting were determined too.Results showed that the rooting rate of H.serrata planted on SoilⅢincreased by 10%and 16%compared with SoilⅠand SoilⅡ.The effect of endophy fungi on the content of Phenols,IAA and the activity of POD and PPO was observed. The endopythe could increase the rooting rate and the number of roots.The endophytic fungi promoted callus formation and cell differentiation then could increase rooting rate.The close relation between endopythe and H.serrata can be demonstrated.(7) Effects of endophytic fungi on physiological and biochemical indexes of H.serrata.The cuttings of H.serrata were planted on soilⅠof primary habitat,on the soilⅡhad been inoculated endophytic fungi,on the soilⅢhad been autoclaved.Soluble protein and soluble sugar content,activities of PPO and POD,flavonoids and huperzine A contents were determined after cutting 120d.Results:SoilⅠincreased the soluble protein content of stem of H.serrata(P＜0.05),soilⅠincreased the soluble sugar contents of leaves and stem of H.serrata(P＜0.05),soilⅠand soilⅡincreased the flavonoids contents of H.serrata(P＜0.05).Soil microbes from primary habitat, endophytic fungi promote rooting,they also increase plant metabolism level of H.serrata.(8) The study on plant-active substance in fermentation products by endopytic fungi.7 strains of endopythe(xj1,xj2,xj3,xj4,xjS,dg5,dg14)which have specifical symbiosis with H.serrata were researched.Results showed that two of the 7 strains were able to produce IAA,three of them strains have activity of PPO,three of them have POD activity.(9) The AchE inhibitory effect of the fermentation products by endopythe was studied.Results showed that 9 strains endopythe could produce alkaloid,and 4 strains of them have the inhibitory effect on AchE.The strongest inhibitory effect was the alkaloid produced by xj14,its IC50 is 134.8280μg.mL^-1.(10) Molecular identification of endophytic fungi.The strain xj14 was suggested belonging to Trichoderma 7 strains endopythe that have specifical symbiosis with H.serrata belonged to Zygomycetes and ascomycetes.3 strains belong to Trichoderma,2 strains belong to Mortierella,1strain belongs to Rhizomucor.