Secondary Metabolites Of Colletotrichum Gloeosporioides BWH-1:Isolation,Identification And Biological Activities Study

Plant pathogenic fungi can produce many secondary metabolites with biological activity of pesticides,which has huge development potential.We previously found that the crude ethyl acetate extracts of the fermentation broth of plant pathogenic fungus Colletotrichum gloeosporioides BWH-1 show herbicidal activity.Therefore,in order to further clarify the main active component in the secondary metabolites of strain BWH-1 and the biological activity of the active component,based on the activity tracking strategy,multiple methods,including thin layer chromatography,silica gel column chromatography,reverse chromatography column chromatography and high performance liquid chromatography,were used in this study to further isolate and purify the target active component in the crude extracts.The structure of the target active compound after purification was identified by NMR,MS and IR methods,and the biological activity of the target compound was further studied.In this study,a main biologically active component was isolated from the secondary metabolite produced by C.gloeosporioides BWH-1 strain,and the structural analysis results indicated that the active component was dirhamnolipid（Rha-Rha-C10-C10）.The herbicidal activity test results of dirhamnolipid show that the IC₅₀values of dirhamnolipid for primary root length inhibition rate on Bidens pilosa,Mikania micrantha,Capsella bursa-pastoris,Echinochloa crusgalli and Alopecurus aealisqua are 47.34 mg·L^-1,57.13mg·L^-1,65.68 mg·L^-1,88.77 mg·L^-1and 145.23 mg·L^-1,respectively;the IC₅₀values of dirhamnolipid for fresh plant weight inhibition rate on B.pilosa,M.micrantha,C.bursa-pastoris,E.crusgalli and A.aealisqua are 79.56 mg·L^-1,123.72 mg·L^-1,98.25mg·L^-1,136.03 mg·L^-1and 217.71 mg·L^-1,respectively;the IC₅₀values of dirhamnolipid for primary root length inhibition rate on Oryza sativa is 779.44 mg·L^-1;the IC₅₀values of dirhamnolipid for fresh plant weight inhibition rate on O.sativa is above 1000 mg·L^-1.The above herbicidal activity results indicate that dirhamnolipid exhibits better herbicidal activity against dicot weeds than monocot weeds and dirhamnolipid has no inhibitory effect on rice.The synergistic herbicidal activity of dirhamnolipid was further evaluated by mixing dirhamnolipid（Di RL）with commercial herbicides cyhalofop-butyl（CB）and penoxsulam（PEN）.The test results of mixed herbicidal activity show that the inhibition rate of Di RL+CB mixed treatment on the root length and fresh weight of B.pilosa reached 94.79%and 71.74%,respectively;the inhibition rate of Di RL+PEN mixed treatment on M.micrantha root length and fresh weight reached 100%and 79.78%,respectively.The herbicidal activity of the mixed treatment is significantly better than that of Di RL,CB or PEN alone.The results indicate that the mixed treatment of dirhamnolipid with the commercial herbicides cyhalofop-butyl and penoxsulam shows synergistic herbicidal activity.The test results of mycelium growth inhibition rate of dirhamnolipid on Rhizoctonia solani showed that:at 10 mg·L^-1and 20 mg·L^-1treatment concentrations,the inhibition rates of positive control azoxystrobin on R.solani were 19.26%and 35.74%,respectively,which were significantly higher than those of the positive control hymexazol 0%and 0%,and dirhamnolipid 0%and 5.52%.At the treatment concentration of 50 mg·L^-1,the inhibitory rates of azoxystrobin and dirhamnolipid on the mycelial growth of R.solani reached 39.37%and 39.15%,respectively,but there was no significant difference,but they were both significantly better than those of hymexazol 30.19%.At 100 mg·L^-1and 200mg·L^-1treatment concentrations,the mycelial growth inhibition rates of dirhamnolipid on R.solani reached 55.74%and 80.96%,respectively,which were significantly higher than the inhibitory rate of hymexazol on the mycelial growth of R.solani 51.19%and 58.18%,and the mycelial growth inhibition rate of hymexazol on R.solani is significantly higher than that of azoxystrobin 42.93%and 46.70%.The results of mycelial growth inhibition rate of R.solani showed that dirhamnolipid had a relatively high inhibitory effect on the mycelial growth of R.solani under the high concentration treatment.The test results of sclerotia formation inhibition rate of dirhamnolipid on R.solani show that at 50 mg·L^-1and 200mg·L^-1treatment concentrations,the azoxystrobin inhibited the sclerotia formation of R.solani 71.27%and 100%,respectively,which was significantly better than dirhamnolipid’s68.05%and 88.75%.At the same time,the inhibition rate of dirhamnolipid on the sclerotia formation of R.solani was significantly better than the inhibition rate of hymexazol 57.91%and 80.42%.The test results of sclerotia germination inhibition rate of dirhamnolipid on R.solani show that at the treatment concentration of 50 mg·L^-1,the inhibition rates of azoxystrobin,hymexazol and dirhamnolipid on the germination of R.solani sclerotia were all 0%.At the treatment concentration of 200 mg·L^-1,the inhibitory rate of azoxystrobin and hymexazol on the germination of R.solani sclerotia were still 0%,but the inhibition rate of dirhamnolipid on R.solani sclerotia reached 68.89%,which shows that the high concentration of dirhamnolipid treatment on R.solani sclerotia germination has a higher inhibitory effect,and this inhibitory effect is significantly better than the positive control azoxystrobin and hymexazol.The protective and therapeutic effects of dirhamnolipid against R.solani showed that the protective and curative effects of dirhamnolipid on R.solani were better than those of positive control azoxystrobin and hymexazol,and the protective effect was better the therapeutic effect.The possible mechanism of dirhamnolipid’s biological activity is to observe the changes in the morphology of the root tips of B.pilosa and the mycelium of R.solani after treatment with dirhamnolipid using scanning electron microscope.The observation results show that both the root tips of B.pilosa and the mycelium of R.solani treated with dirhamnolipid showed the same dehydration and atrophy.Therefore,combined with the references,the possible action mechanism of dirhamnolipid’s biological activity was inferred that the possible mechanism of bioactivity of dirhamnolipid is due to the strong affinity between dirhamnolipid’s hydrophilic and lipophilic structure and the plasma membrane of the tissue,leading to the destruction of the phospholipid bilayer of the tissue cell membrane,leading to the leakage of the substance in the membrane,and finally triggering the procedural death of biological organism.To sum up,dirhamnolipid（Rha-Rha-C10-C10）is the main active ingredient in the secondary metabolites of C.gloeosporioides BWH-1,which causes rotting and death of bawanghua,and dirhamnolipid has great potential to be further exploited as a biological pesticide or as a pesticide additive.In this study,the future space for the exploitation of functional active components in secondary metabolites of plant pathogenic fungi as biopesticides was expanded and the research and development of biopesticides was further promoted.