Mutagenesis And Breeding Of High-yield Forsythin Endophytic Fungus Colletotrichum Gloeosporioides

Endophytic fungi are microorganisms that live inside the tissues and organs of healthy plants without causing discernible manifestation of disease.It has been recognized that endophytic fungi have the capability to produce biologically active constituents which are identical or similar to that produced by their host plants.Recent studies have indicated that the fungal endophytes also have a substantial effect on the growth and yield of ingredients of the host plants.Forsythia suspensa(Thunb.)Vahl is a climbing plant and distributed in shanxishaanxi and henan province in our country.As one of the main active components of F.Suspensa,phillyrin is recorded in "China Pharmacopoeia" as the quality control indexes of the medicinal materials of " Lianqiao".Phillyrin was reported to have various biological activities,such as antioxidant,anti-inflammatory,anti-hyperlipidemia and antipyretic activities.In the previous study,an endophytic fungus numbered G10,which is isolated from the fruit of F.suspensa,was demonstrated to produce low levels of phillyrin.This paper aimed to acquire a mutant with high production of phillyrin by the mutagenic techniques and medium optimization.The main results are as follows:(1)The regular mutagenesis and mutant screening.The production of phillyrin of the wild type G10 by TU-1810 UV/Vis spectrophotometer is 5.09 mg per g.The phillyrin production were efficiently improved by three techniques,including G10 mutation by ultraviolet radiation(UV),G10 mutation by sodium nitrite(NaNO2)and G10 mutation by a combination of both UV and NaNO2.Three isolates(UV 2.5-2?Na 5-1 and S1.5-2)increased production of phillyrin by the regular mutagenesis.The results showed that the mutating methods of the combination of UV and NaNO2 exhibited higher efficiency in improving of phillyrin production in G10.The more applicable mutanting condition is that the strain was set in UV 2min and NaNO2 30 g per L.An improved mutant,labeled as S1.5-2,was obtained from the combination of UV and NaNO2.We found one isolate(S1.5-2)exhibited higher production of phillyrin(12.62 mg per g),which is 2.48 times than the production of G10 by TU-1810 UV/Vis spectrophotometer.The HPLC result showed that the content of phillyrin produced by S1.5-2 held a relative high value of 0.79±0.22 mg per g,and the production of phillyrin is 2.19 times than that of G10.The high yield of the strain can stable inheritance after serial passages.(2)The identification of UV2.5-2?Na5-1 and S1.5-2.The agreement of the classical examination and molecular analysis identified that UV2.5-2?Na5-1 and S1.5-2 belonged to Colletotrichum gloeosporioides.(3)Random amplified polymorphic DNA assay revealed genotypic differences between the mutagenic strains and the wild type strain.RAPD band pattern was analyzed using the Nei similarity index.A dendrogram was constructed based on the similarity matrix data by applying the cluster analysis method.The mutagenic strains were segregated,and high producers(Na5-1 and S1.5-2)were clustered into a subgroup.Based on the dendrogram,close homology existed in those strains that the similarity coefficient of the four strains varied from 0.37 to 0.67.The mutagenic strains UV2.5-2 was closest to G10(their genetic similarity coefficient was 0.67).The specific bands and dendrogram of cluster analysis obtained in our study showed that genetic information was transferred from the wild type strain to the mutagenic strains.(4)In order to produce more phillyrin by the mutant strain S1.5-2,the optimization tests were applied to gain the best culture medium.Through the test of different basic culture mediums,we found that bean sprouts liquid medium was propitious to S1.5-2 both in mycelium growth and phillyrin accumulation.Mycelium growth of bean sprouts liquid medium attain to maximum(0.72 g),which is 1.2 times than mycelium growth of PDA medium when the 8 days of culture.Phillyrin accumulation of bean sprouts liquid medium attain to maximum(13.5 mg per g),which is 1.08 times than phillyrin accumulation of PDA medium when the 8 days of culture.By the analysis with SPSS 17.0,the best carbon resource was approved to be glucose,and the best nitrogen resource was approved to be yeast extract.The results of optimization showed that:the concentration of glucose was 6%,the concentration of yeast extract was 0.25%,pH was 7.0.