Separated And Preliminary Function Of Wild Soybeans Endophytic Fungi Under Different Environments In Heilongjiang Province

In this research,wild and cultivated soybeans under different environments in Heilongjiang Province were used as experiment material.Separated and purified endophytic fungi from roots,stems and leaves were identified by morphology and molecular biology methods,and phylogenetic tree were built by ITS sequences from different endophytic fungi in order to clarify the their species specificities and taxonomic status.Meanwhile,by analyzing the contents of polysaccharides,flavonoids,triterpenoids and phenols from fermented fungi strains,as well as by analyzing the tolerance of fungi strains to acid,alkali,salt,cadmium ion and the inhibition of strains to pathogens,the diversity of endophytic fungi and their bio functions from wild soybean were detected.Above all,this research could provide useful information for agricultural,medical and industrial active compounds selection.The main research results are as blow:1?By using 6 different wild soybeans and 6 cultivated soybeans as material,302 endophytic fungi strains were seperated.Wild soybeans(inculding wild pea and semi-wild soybean)have more endophytic fungi,which are 215 strains,while cultivated soybeans have 87 strains;24.17%of 302 fungi strains are from roots,28.8%are from stems and 47.01%are from leaves,which indicated leaves have more fungi strains but differed in type.By using semi-solid culture method,the morphology and microstructure of strains are observed and identified,with obtaining their microstructures and species specificities.2?ITS sequences of 162 soybean endophytic fungi were identified and analyzed by using PCR method.Through sequential analysis,endophytic fungi from wild soybean roots mainly belonged to Fusarium sp.;the ones from stems mainly belonged to Ascomycota clone while partially belonged to Fusarium sp.;the ones from leaves mainly belonged to Alternaria sp.while partially belonged to Fusarium sp..The result from Blast analysis showed that the homogeneities of 5 endophytic fungi sequence were lower than 95%,which suspected them as new strains,and they were WDL2(Dothideomycetes sp.similarity 95%),24(Dothideomycetes sp.similarity 92%),Y2S1(Gibberella moniformis similarity 91%),Y5L13(Pleosporales sp.similarity 95%),Y5R4(Fusarium sp.similarity 76%).3?Phylogenetic tree were built according to the ITS sequences of 162 separated and purified soybean endophytic fungi by using NJ method of software MEGA 6.0.The obtained sequenced strains distributed in 25 species.73 strains belonged to Fusarium sp.,which take 45.06%of all strains,and they were found in roots,stems and leaves in different soybean materials,which leaded to a conclusion that Fusarium sp.could be regarded as dominant species in number and range.The following species with more strains founded were Epicoccum sp.and Gibberella moniformis with 20 strains and 24 strains.4?The 60 wild soybean endophytic fungi strains were separated and purified by fermented culture,while detected the secondary metabolite contents in endophytic fungi mycelium and fermentation liquid.The results showed:1.Strains Y2S9,WDL2,WDL3,YSYS1 had higher polysaccharides content,with average content 49.013mg/g,among which Y2S9 had the highest content as 85.51mg/g;2.Strains Y1S9,Y6S8,WDS7,WDS8 had higher flavonoids content,among which Y6SS had the highest content as 2.989mg/g.The top three strains with the highest flavonoids content from broth were Y6R2,Y1S8,SYS4 with contents,among which SYS4 had the highest content as 4.89 mg/L;3.Strains Y6S8,Y2R1,Y5R15 had higher triter:penoids content,among which Y6S8 had the highest content as 25.548mg/g.The top three strains with the highest triterpenoids content from broth were YSYL2,Y1S8,WDL3 with contents,among which WDL3 had the highest content as 42.975 mg/L;4.Strains WDS7,Y5S6,Y1S5 had higher phenols content,among which WDS7 had the highest content as 22.651mg/g.T he top three strains with the highest phenols content from broth were Y4S1,Y2L5,WDL3 with the highest content as 3.679mg/L.Finally,22 strains with high production of secondary metabolite were obtained.5?The results of 15 wild soybean endophytic fungi strains tolerance analysis to cadmium ion,salt,acid and alkali showed:1.The most tolerant strains to cadmium ion were Y2R14,RWDL4-1,Y1S5,Y5R11,which could still grow under 400mg/L cadmium chloride,and the strain Y2R14 had the most tolerance to cadmium.All the cadmium ion tolerant strains had high production of secondary metabolite,which implicated that the accumulation of secondary metabolite could raise the tolerance to cadmium ion.2.The salt tolerance selection result showed that strain Y5L11 had 21.39%higher mycelium growth than control in PDB liquid medium with 5%NaCl.3.The acid and alkali tolerance selection result showed that strains YSYS1(L),SY2S2-1(E),ZY1S-8(A),S20-2(J)had a significant higher mycelium growth under PH=3 compared to control environment(PH=7)with increases as 19.27%,2.36%,1.64%andl.29%,While PH=9,strain Y2R5(G)had the highest mycelium growth with 38.87%higher than control environment(PH=7).The strains Y5R11(I)and Z3(M)were sensitive to PH value.6?By using 18 wild soybean endophytic fungi strains with richer secondary metabolite as material,antagonistic experiment against 4 pathogens(Sclerotinia sclerotiorum 15-2,Phytophthora race 1,Magnaporthe grisea P-46-2,Exserohilum turcicum p51-2 were went on,and the results showed 7 strains showed antagonistic effects,among which 4 endophytic fungi strains(YSYL2,Y6R1,Y6R15 and YSYL3)had strong antagonistic effects against Magnaporthe grisea P-46-2 and Sclerotinia sclerotiorum 15-2,and these strains could be developed and used as bio control bacteria.7?To the 5 suspected new strains WDL2,Z4,Y5R4,Y5L13,Y2S1 obtained in this research,the result of their tolerance experiments showed that,strain WDL2 was tolerant to cadmium ion and acid,strain Z4 was tolerant to cadmium ion,strain Y5R4 was tolerant to acid,strain Y5L13 was tolerant to alkali,strain Y2S1 was tolerant to both acid and alkgali.The result of their antibacterial experiments showed that,strains Z4 and WDL2 could inhibite the growth of E.coli Trans T1 and P.aeruginosa LBA4404,strain Y5L13 could inhibite the growth of E.coli Trans T1.