The Diversity Of Endophytic Fungi In Morus Alba L.and Their Allelopathic,Antiphytopathegenic And Antioxidative Activities

Endophytes are microbes which reside in the inner tissues or organs of health plants,not only promote the growth of host plants but also enhance the plant resistances under biotic or abiotic stresses.The rich bioactivities of endophtes are due to their unique habitat and mutual relationship with host plants.Recently the greet potential of secondary metabolites from endophytes has been developed for medical application and biocontrol on agricultural pests.Endophytes,one of microbes in special habitat are being new resource for natural bioactive products.Mulberry trees(Morus alba L.)are important economical plants cultivated in China,whose leaves are used to feed silkworms and roots are used in traditional Chinese medicine.The mulberry endophtic microbes have been discovered recently.However,their distribution,diversity and biological function are less studied.In present studies,we isolated fungal endophytes from mulberry tree.Then the stains with important biological activities including the allelopathic effects(herbicidal activity and algal inhibition),antiphytopathegenic and antioxidative activities have been screened from those endophytes.The molecular identification for isolated endophytes and the GC-MS analysis on fungal metabolites have been taken.The physiologicalmechanism of thosebiological functions has been explored.The main results are showed below.It is a first time to reveal the diversity of fungal endophytes in mulberry treeswith the tissue isolating and metagenomic r DNA sequence analysis.The strains of endophytes(132)have been isolated from mulberry tissues,including endophytic fungi(106),bacteria(23)and actinobacteria(3).The number sequence of endophytesisolated is roots>stems>leaves and summer>autumn>spring>winter.The fungal endophytes(86stains)are subjected to 18 genera of 5 orders.The dominant strains are from Moniliales(28.3%),Tuberculariaceae(22.64%)and Dematiaceae(16.04%).In genus level,thedominant endophytes in mulberry trees are from Fusarium genus(24 stains,22.64)in roots and Alternaria(16 stains,15.1%)in stems and leaves.On the other hand,we extracted and purified themetagenomic DNA from mulberry and used 16S r DNA and 18S r DNA to analyze the endophyte diversity.The index of Shannon Wiener and Chao was greater than 4,and 12800,respectively according to microbial r DNA information,indicating the rich and diversity of endophytes in mulberry trees.The dominate species of endophytic bacteria are from Hymenobacter(5.65%),Pseudomonas(4.61%)?Methylobacterium(3.55%),Bacteroides(2.42%)?and Sphingomonas(2.05%).The mainly uncultural fungi were revealed to subject to Knufia(20.75%),Cantharellula(4.99%)and Myriangium(2.30%).The results enriched the information on fungal endophytes in mulberry trees and provided the resource for develop functional metabolites of those endophytes.The ferments of mulberry endophytes have been screened on allelopathic potentials on some vegetables and weeds includingcucumber,lettuce,fescue and barnyard grass.The significant effects on seed germination growth were observed in the ferments from the endophytic strains(SZ-21,SZ-42,SZ-55,SZ-60,SZ-83 and SZ-102).The ferments from stain SZ-21 could inhibit significantly the seed germination of lettuce,fescue and barnyard grass,especially retarded the growth of stem and root growth by 53.85%and 71.87%,respectively.The growth of most vegetables and turf plants were not inhibited by such ferments,suggesting a good potential for being herbicides.Eth OAc extracts of cultural medium for SZ-21(diluted by 5 times)could decrease germination rate,index and vigor index of barnyard grass by 19.82%?23.69%and 90.37%while the metabolite diisobutyl phthalatedecreased those indexes by 46.38%?21.43%and 78.84%.The growth of the seedlings were also inhibited by the extract(81.79%inhibition on rootsand 34.85%inhibition on stems)while diisobutyl phthalatedecreased those indexes by 87.64%and 75.37%.Another metabolite from the strain diisooctyl phthalate had a weaker action to the weed.The phthalate derivate are allelopathiccompounds on the weeds.Combined with morpholocial observation and r DNAITS sequence the stain was identified as Phyllostictasp.SZ-21.We further investigated on the growth inhibition of those endophytes on freshwater cyanobacteria including Microcwis denginowa FACHB-912,M.flos-aquae FACHB-1028 and Pseudanabaena sp.The obvious inhibition was observed in the cultures of freshwater cyanobacteria with endophytes(SZ-5,SZ-11,SZ-18,SZ-21,SZ-24?SZ-67 and SZ-69).Phyllostictasp.SZ-21 inhibited all cyanobacterial growth significantly.Our results demonstrated that the strain SZ-21 could inhibit growth of M.flos-aquae FACHB-1028 by mycelium contactingand M.denginowa FACHB-912 by macro molecules such as proteins.Diisobutyl phthalate inhibited cyanobacterial growth by 62.26%,while the inhibition rate by diisooctyl phthalate reached 52.83%on day 8 of the treatment.Our results provided the potential of Phyllostictasp.SZ-21 to be a green herbicide and anti-algae agent.To obtain biocontrol endophytic fungus for mulberryand human diseases,the antimicrobial effects of endophytic fungi were screened and evaluated byusing dual-culture,inhibition zone assay and colony diameter assay methods with mulberrypathogenic fungi and humanpathogenic bacteria.We found the antifungal capacity of SZ-30?SZ-48 and SZ-74(inhibition rate 20-80%)with a wide antimicrobial spectrum.SZ-48 stain could inhibit the growth of pathogenic fungi over50%.The MIC value on Eth OAc extract of Botrytis cinerea,Fusarium oxysporum and Myrothecium rorideumis ranged from 0.5-1.0 mg/m L,suggesting a powerful antifungalactivity of SZ-48 stain.The GC-MS analysis demonstrated there were 23compounds from the ferments of the stain.Among them were cyclo(leucylprolyl),cyclo(penprolyl)(51.31%),3-benzyl-pyrrolopiperazine-1,4-dione(10.53%),tetratriacontane(6.6%),tetracontane(4.74%),tetratetracontane(3.68%)and diisobutyl phthalate(1.33%).Cyclo(leucylprolyl),cyclo(penprolyl)and diisobutyl phthalate could be the antifungal metabolites of the stain.We explored the antifungal mechanism of Eth OAc extracts of SZ-48 stain on mulberrypathogenic fungus M.rorideum.The inhibition on spore germination by the extract was found.Also the reactive oxygen species(ROS)generation in mycelium of M.rorideumwas induced by the extract,resulting in the cell membrane lipid peroxidation,membrane injury and cell death.Combined with morphological observation and r DNAITS sequence the stain was identified as Alternaria sp.SZ-48.Alternaria sp.SZ-2,an endophytic fungus of mulberry trees,has been found to synthesized exopolysaccharide(EPS).With the treatment of trypsin and Sevag method to eliminate proteins,the yield rate and purity of EPS reached 6.96%and 92.4%.The homogeneous EPS 2 and 3 were further purified from crude EPS via column chromatography of anion-exchange(DEAE-52)and gel filtration(Sephacryl S-400).The molecular weight of EPS 2 and 3 was determined to be 5.8×10⁴ Da and 1.5×10⁵Da,respectively.The rate of galactose,glucose,mannose,rhamnose and glucuronic acid in the digested EPS 2 and 3 was 8.5:9.2:1.1:1:1.3 and 12:16:1:2.4:0.5,respectively.EPS2 has a stronger capacity of radical eliminating.The eliminating rate on superoxide,hydroxyl radical and DPPH could reache 38.2-60.3%,68.2-85.2%and10.7-49.6%at 1-4 mg/m L.The non site-specific DNA strand-breaking induced by Fenton reaction could be blocked by EPS2,suggesting the attenuatingcapacity on DNA injury by·OH.The protective of EPS2 on H₂O₂-injuried PC12 cell was proved.The cell survival rate was enhanced to more than 61.2%after the treatment of EPS2 at 2-10mg/m L while 150 mol/L H₂O₂ treatment of 1 h could cause the survival rate of PC12cell to 30.5%.EPS2 could catalyze H₂O₂via recovering the activities of glutathione peroxidase(GSH-Px)and catalase(CAT).On the other hand,EPS showed stronger inhibition on?-glucosidasein vitro.The results showed that inhibitionability is in a mode of content dependent and the half inhibitory concentration IC50value was 6.98mg/m L in a typical competitive pattern.The EPS of Alternaria sp.SZ-2 could be used to develop newtype?-glucosidase inhibitorfor reducing blood sugar.To summarize,in the current study,we isolated and identified fungal endophytes from mulberry tree and analyzed the diversity and distribution of endophyte in mulberry.Mulberry endophytes have been proved to have rich biological activities.The study on allelopathic effects(herbicidal activity and algal inhibition),antiphytopathegenic and antioxidative activities provided methods to screen functional strains from mulberry trees.The work on molecular identification and GC-MS analysis gave a clue to recognize endophyte species and secondary metabolites of the endophytes.Our results will benefit for understanding the mechanism of allelopathic effects,antiphytopathegenic and antioxidative activities of the endophytes,substance basis for developing green herbicides,anti-algal andantifungal agents,and anti-oxidative drug.Our study elucidated a new way for exploring mulberry resources.