Identification And Antimicrobial Metabolites Of Gut Fungi Isolation From Dragonflies

Insect symbionts are kinds of microorganisms coevolved with insects,which possess huge diversities of species and metabolites.Insect gut micrbes are one of the insects symbionts as important microbial resources.Up to now,it has been confirmed that insect intestinal microorganisms micrbes are important sources of new microbial species and new active metabiolites.In this paper,the gut fungi of Crocothemis servilia and Pantala flavescens were studied.The diversity and antibacterial activity of intestinal fungi from C.servilia were studied using the methods of dilution culture,morphology,and molecular biology.In addition,the antimicrobial activity of the gut fungi of C.serville and Trichoderma sp.QTYC44 from P.flavescens were measured by in vitro and in vivo bioassay.A total of 9 compounds,in which7 compounds were authenticated,isolated from Phoma sp.QTH17 from C.serville and Trichoderma sp.QTYC44 from P.flavescens were further studied using the methods of column chromatography and extensive spectroscopic analysis.The details are as follows:1.The diversity of gut fungi of Croothemis serville were investigated using dilution culture,morphology,and molecular biological identification based on the internaltranscribed-spacer sequence analysis.A total of 42 included in 5 classes and 13 different genera.The generas are as follows: Thalaromyces(21.43%),Aspergillus(19.05%),Penicillium(7.14%),Trichoderma(19.05%),Curvularia(7.14%),Fusarium(2.38%),Peristalsis(2.38%),Bipolaris(2.38%),Arthrinium(2.38%),Cladosporium(2.38%).Mucor(7.14%),Rhizomucor(2.38%).Alternaria(4.76%,Phoma(2.38%).2.The antagonistic activity of gut fungi of C.serville against Beauveria bassiana was tested.Trichodema virens QTY2 and Trichodema sp.QTH36 showed strong antagonistic effects on B.bassiana,and the inhibition diameter of the strains was 68.9 mm and 74.1 mm,respectively.The further study showed that the products of PKS and NRPS genes might play an important role in the antagonistic activity for QTH36.Bipolaris sp.QTH21,Talaromyces pinophilus QTH28,and Talaromyces sp.QTH38 had moderate antagonistic effects on B.bassiana,and the inhibition diameter of the strains was 33.32 mm,25.47 mm and 23.94 mm,respectively.Besides,the antibacterial activities of ethyl acetate extracts of gut fungi of C.serville against three pathogenic bacteria were further tested.The results showed that the inhibitory effect of the gut fungi extracts was obvious on Gram-positive bacteria.Four extracts(belonging to Aspergillus sp.QTH15,Arthrinium sp.QTY15,Phoma sp.QTH17,Bipolaris sp.QTH21)had moderate inhibitory effects on Micrococcus tetragenus with inhibition zone diameters of 8.3,10.5,10.8,and 9.2 mm,respectively.The inhibition zone of the positive control(gentamicin sulfate)was 16.5 mm.The extracts of Phoma sp.QTH17,Bipolaris sp.QTH21 also showed moderate inhibitory effect on Staphylococcus aureus with inhibition zone diameters of 8.7,and 8.2 mm,respectively,which were slightly weaker than that of the positive control(12.3 mm).3.Five compounds were isolated from the gut fungus Phoma sp.QTH17 from C.servilleand identified as ergosterol(1),3-chlorogenital alcohol(2),epoxydon(3),epoxydon6-methylsalicylate ester(4),and mannitol(5),respectively.Compound 3 showed the best inhibition activity against methicillin-resistant S.aureus with an inhibition zone diameter of19 mm,which was comparable with that of the positive levofloxacin activity(21.5 mm).The compound 3 showed moderate antibaterial activity against Escherichia coli,M.tetragenus and susceptible S.aureus with inhibition zone diameters of 7,14 and 12.5 mm,respectively.Both compounds 2 and 4 also showed moderate antibaterial activity against M.tetragenus and susceptible S.aureus with inhibition zone diameters of 9 and 11 mm,respectively.4.Trichoderma sp.QTYC44 from P.flavescens showed good antimicrobial activities against phytopathogenic fungi.The results of in vitro bioassay showed that Trichoderma sp.QTYC44 had potent antifungal activites with 94.3% inhibitory rate against Fusarium graminearum and more than 80% inhibitory rate against F.oxysporum f.sp.cucumerinum and Alternaria alternate.The pot test results showed that Trichoderma sp.QTYC44 exhibited up to 72.58% control efficacies of against cucumber fusarium wilt.The chitinase activity of Trichoderma sp.QTYC44 with culture times showed that the highest activity was only 0.1429 U / ml on the 5th day,and the chitinase activity maybe not the main mechanism of its antifungal activity.Four compounds were further isolated from the metabolites of Trichodema sp.QTYC44,in which two metabolites,palmitic acid(6)and uracil(7),were identified.The other two monomer compounds were still to be further charactered.