Activities Study On Secondary Metabolite From Two Endophytic Fungi

With the use of chemical pesticides increasing year by year,the resistance of target organisms continues to increase,causing certain harm to non-target organisms and affecting the balance and self-regulation of the ecosystem.Therefore,pesticides are undergoing profound changes in the world.Some countries are eliminating chemical pesticides that pollute the environment,and developing a series of biological pesticides those are safe for humans and animals,non-toxic,low-cost,non-pollution,and do not kill natural enemies of pests.Natural products are an important source of drug development due to their diverse structures,rich biological activities,unique effects,easy degradation and good compatibility with the environment.Endophytic fungi will not cause obvious symptoms of the host,but can also protect it from infection and enhance stress resistance.It is mainly achieved through the production of various biologically active substances.These secondary metabolites are of great value for the development of new pesticides.In this paper,the secondary metabolites of Trichoderma sp.EFT2 and Nodulisporium sp.A21 adsorbed by macroporous resin were purified and identified,and a variety of chromatographic analysis methods(column chromatography,column chromatography,HPLC,etc.),and identify the structure of the compounds through their physical and chemical properties and spectral data(nuclear magnetic,mass spectrometry,ECD,etc.),and finally determine the biological activities of the compounds,with the goal of finding a compound with unique structure and good activities.The specific experiment is as follows:Through the isolation and purification of endophytic fungi from the plants collected in Nanjing Zhongshan Botanical Garden,a total of 25 endophytic fungi were obtained.Four common pathogenic fungi(Fusarium graminearum,Sclerotinia sclerotiorum,Rhizoctonia solani and Phytophthora capsici)were tested strains.25 endophytic fungi were subjected to a PDA panel confrontation experiment,and endophytic fungi with inhibitory activity against the pathogen were screened out.The screened endophytic fungi are fermented through 3 different liquid media(Zha’s medium,malt medium,PD medium),and the crude extract obtained by ethyl acetate extraction of the fermentation broth is used for the treatment of four common pathogenic fungi.The antibacterial activity tests,combined with the activity determination results,yield,and nuclear magnetic analysis of the crude extract,finally screened out the strain EFT2 as the target strain,and the malt medium as the target medium.The EFT2 strain was isolated from the stem organs of the Euonymus fortunei,and compared with the endophytic fungus morphology and sequencing results,the strain was identified as Trichoderma sp.Through large-scale fermentation,organic solvent extraction,and vacuum distillation,a total of 138 g of crude extract was obtained.The crude extract of EFT2 fungus was separated and purified by column chromatography and HPLC.The structure of the compounds were identified by spectral data analysis,ECD calculation,and X-ray single crystal diffraction.A total of 14 compounds were identified,of which two new compounds are(-)-Trichodermatrione A(Compound 10)and(+)-Trichodermatrione A(Compound 11).A total of 6 compounds(15-20)were separated and identified from Nodulisporium sp.A21 fermentation product processed by the macroporous resin in situ adsorption technology,of which two new compounds are nodulialkaloid D(compound 19)and nodulialkaloid E(Compound 20).Through the mycelial growth rate method,the phytopathogenic fungi inhibition experiment was carried out,and the results showed that compound 1 has a good inhibitory activity against Botrytis cinerea at a concentration of 50 μg/m L,with an inhibition rate of52.9 %.In the bacterial inhibitory activity test,the racemate compound(±)-Trichodermatrione A has a certain inhibitory effect on the pathogen of Xanthomonas oryzae pv.oryzae and Bacterial Xanthomonas oryzae pv.oryzae.The MIC value measured by MTT method is 64 μg/m L.compound 1 has a significant inhibitory effect on Bacillus subtilis,and its MIC value is 64 μg/m L,and compound 13 has an MIC value of 64 μg/m L for the pathogen of rice bacterial blight.In addition,Eclipta prostrata and Echinochloa crusgalli were selected as the test subjects,and the racemate compound(±)-Trichodermatrione A was tested for activity of plant growth regulation.The results showed that the compound had poor inhibitory activities.