Strctures And Bioactivities Of Secondary Metabolites From Strain A21,an Endophytic Fuguns Of Nodulisporium Sp.

Pesticides play a crucial role in agricultural production and ensure a good harvest of crops.However,with the long-term and unreasonable use of chemical pesticides,the resistance of target organisms is gradually enhanced and the non-target organisms are also killed.At the same time,the natural ecological balance and self-regulation ability are seriously broken.Excavation an efficient and low risk pesticides which can slow down the development of resistance and have new mechanisms of action and targets becomes an important task for the development of new pesticides.Natural products have a wide variety of chemical structures,diverse biological activities,unique action mode,easy degradation and friendly compatible with environment,providing a variety of precursor configurations for the research of new pesticides.The leading compounds with natural products have provided or are providing a variety of lead chemical structures for active pesticides.Endophytic fungi are the microorganisms that grow inside healthy plants tissue.Due to the special growing environment,the secondary metabolites are diverse in structures and activities,which considered as important sources of active natural lead compounds.Nodulisporium sp.are common plant endophytic fungi and often exist as an asexual form of some other genera.Many secondary metabolites from Nodulisporium sp.have novel structures and present antifungal and inseticides activities.Nearly,more than one hundred species of secondary metabolites have been reported,among which there are many compounds with novel structures and new activities.In paper,an endophytic fungus A21 of Nodulisporium sp.which had significant antifungal activity was isolated from the leaves of Ginkgo biloba.The secondary metabolites of A21 were isolated by means of chromatography and identified by MS,NMR and X-ray crystal diffraction.The agricultural activities of some compounds about antifungal acivities and herbicidal activities were measured.This study can provide active lead compounds for the synthesis of new pesticides.1.Twenty-five endophytic fungi were isolated from the healthy leaves of G.biloba.Their fermentation broth and ethyl acetate crude extract were tested about antifungal activities against Rhizoctonia solani,Sclerotinia sclerotiorum and Fusarium graminearum.The strain A21 was selected because of it significant antifungal activities and had a high yield of ethyl acetate crude extract.The strain A21 was identified as Nodulisporium sp.depending on its morphology characteristic and ITS sequencing.The target fungus A21 was subjected to liquid fermentation and the culture broth was extracted by organic solvent petroleum ether and ethyl acetate.The crude extract was isolated used the methods of column chromatography,HPLC and fourteen compounds were obtained.The chemical structures of compounds(1?3?4?5?7?9?13?16)were identified by NMR MS and X-ray.Among them,there are eleven new compounds(4,5,6,8,9,10,11,13,14,15,16),included two new skeleton compounds(8,9).2.To study the biosynthetic pathway of the isolated compounds,the isotope-labeled experiments were carried out.Five labeled compounds were obtained from the fermentation adding CH313COONa and 13CH3COONa and verify the fatty acid biosynthetic pathway.3.The results of biological activities indicated that the known compound 1(sporothriolide)had better antifungal activities against R.solani and S.sclerotiorum with EC50 of 3.04 and 2.78?g/mL,respectively.Compound 1 also had significant activities in inhibiting the germination of pathgentic spores and could inhibite the germination of Magnaporthe oryzae and F.graminearum at 1.5?g/mL and Botrytis cinerea at 5.0 ?g/mL.In vivo protection experiments,the inhibition rate of compound 1 was 71.7%at the concentration of 200 ?g/mL,which was equivalent to inhibition rate of 72.2%about positive control validamycin at the concentration of 100 ?g/mL on rice sheath blight.The inhibition rate of compound 1 was 41.5%at 250 ?g/mL,which was equivalent to inhibition rate of 42.5%about positive control carbendazim at 125 ?g/mL on rape sclerotinia rot using detached rape leaves.The inhibition rate was 85.6%against rice blast at the concentration of 50 ?g/mL.The inhibition rate was 74.0%against tomato fruit gray mold at 200 ?g/mL and is equivalent to the positive control which the inhibition rate was 79.4%at the same concentration.Compound 3,4,5,11,12 were measured their herbicidal activities against four weeds species,such as Echinochloa crusgalli,Eclipta prostrate,Alopecurus japonicas and Veronica persica.The results showed they can promote the root growth in a low range concentration of 6.25-100 ?g/mL and inhibite the growth in the range of 200-400?g/mL.Compound 3 and 4 had a selection about monocotyledonous and diocotyledonous weeds,and had a high toxicity to diocotyledonous weeds at high concentration.Compound 5 had a general promotion effect on the root growth of all the weeds and the most promotion effect reached 44.5%about E.prostrate.4.Because of the significant antifungal activities of compound 1,the single factor and orthogonal experiments were designed to optimize the liquid fermentation condition of fungus A21 based on the concentration of compound 1.The optimized object were different liquid medium,carbon source,fermentation days,temperature,pH and the volume of liquid medium.The results showed that the optimal conditions for liquid fermentation were PD medium,25?,125mL/250 mL,pH=8 and the fermentation days are 12 d and the concentration of compound 1 reached 12.5 mg/L.5.The volatile organic compounds produced by fungus A21 after growth on PDA 5 days had antifungal activities against R.solani,S.sclerotiorum and F.sulphureum with inhibition rate 50.0%,53.3%and 31.6%,respectively.Forty-two constituents were determined by SPME-GC-MS,and the main compounds were 1,3-trans,5-cis-octatriene,2-methyl-bicyclo[2.2.1]hept-2-ene,caryophyllene and so on.This result indicated that fungus A21 had antagonistic activities against a variety of plant pathogenic fungi and the volatile organic compounds were aslo worked.The above study showed that endophytic fungus Nodulisporium sp.A21 can produce a variety of secondary metabolites and part of them had significant bioactivies.Compound 1 had excellent antifungal activities and Compound 3 and 4 had obviously herbicidal activities.These natural products could provide new clue for discovery new lead of pesticides.