Investigation On Bio Active Secondary Metabolites Of Symbionts From Odontotermes Formosanus And Mecopoda Elongata

It is a common phenomenon in nature for the symbiosis between microorganisms and insects.There are many different kinds of insects on Earth,and the diversity of insects means the diversity of insect symbionts.According to the reported research papers,insect symbionts can produce new natural products,which have complicated structures and notable active natural products.Insect symbionts are an important resource of active secondary metabolites.However,the research on this aspect is still not deep enough.Therefore,the dissertation takes this kind of microbial flora as the research object,which aims to provide important information for fully exerting the insect symbionts' secondary metabolites.In this paper,Odontotermes formosanus and Mecopoda elongata were the mainly study objects,and the symbiotic fungi isolated from them were preliminarily studied.The variety of symbiotic fungi of O.formosanus was analyzed by dilution plate method,morphological observation and molecular biological identification.The diversity of the symbiotic bacteria of the termite commensal fungi was preliminarily determined by 16S rDNA sequencing.Three strains of symbiotic fungi BYCDW4,BYCDW8 and BYSTW3 from O.formosanus and one fungal strain FZNCD-1 of M.elongata were selected as the research objects.Silica gel column chromatography,Sephadex LH-20 column chromatography,semi-preparative HPLC,Reverse phase column(C18 column)and other chromatographic separation techniques were used to separate and purify the secondary metabolites of target strains.The structures of the metabolites were analyzed and identified by spectroscopic techniques such as 1H-NMR,13C-NMR,HMQC,HMBC,1H-1HCOSY and HR-ESI-MS.Nine compounds were obtained,one of which was a new natural product.Briefly described as follows:1?The diversity of fungi connected with the O.formosanus were studied by dilution-plate method,combined with morphological characteristics and 5.8s rDNA gene sequencing.The results showed that 39 strains of symbiotic fungi were isolated and purified from O.formosanus,which were belonging to 2 phylums,4 classes and 15 different genus,also including 14 potential new fungi.Nine bacterial 16S rDNA sequences were obtained from 39 strains of fungi,which derived from genomic DNA extractions of fungal mycelia.Using the genomic DNA of symbiotic fungi as a template and 27F and 1492R as primers for 16s rDNA sequence amplification,nine endophytic bacteria(9/39)were obtained.All the bacteria members were geared to 2 main Phylums:Firmicutes and Proteobacteria,those all strains were contained in Bacillus.Antibacterial activity of all endophytic fungi culture extracts were tested using the filter paper method against Escherichia coli(ATCC 8739),Bacillus subtilis(ATCC 6633),Staphylococcus aureus(ATCC 6538)and Canidia albicans(ATCC 10231).The results exhibited that about 46%of the strains had good antibacterial activity.About 62%of the strains had good antibacterial effect on Staphylococcus aureus,of which BYCDW8,BYCDW4,BYCDW13 and BYCDW19 had remarkable inhibitory activity.About 31%,39%and 15.4%of the strains showed good antibacterial effects respectively against on E.coli,B.subtilis,C.albicans.2?The bioactive metabolites of Pleosporales sp.BYCDW4?BYSTW3 and Didymosphaeria variabile BYCDW8,were isolated and identified.The activity test showed that the crude extracts of BYCDW4 and BYCDW8 had significant inhibitory effects on the growth of S.aureus(21.88 mm and 22.33 mm in diameter of inhibition zone,respectively)at the test concentration of 30 ?g/filter paper,which were stronger than that of positive gentamicin sulfate(21.33 mm),and the two extracts also had good inhibitory effects on B.subtilis(18.45 mm and 14.00 mm,respectively).The extract of BYCDW4 has good inhibitory activity on E.coli(21.08 mm).Antifungal activity results showed that the crude extract of BYSTW3 had good inhibitory activities against radical growth of Valsa mali and Fusarium oxysporum f.sp.mornordicae with the inhibition rates of 54.5%and 53.4%relatively under the tested concentration of 100 ?g/mL.A major metabolite was isolated from the ethyl acetate extract of BYCDW4 and the weight was about 5.4 g.This natural product was analyzed as a mixture of 5-hydroxyramulosin(1):Biatriosporin M(2)= 2:1.The compounds1?2 exhibited moderate growth inhibitory activities against E.coli,B.subtilis,S.aureus and C.albicans.More specifically,the inhibitory effect on E.coli was the most significant with the diameter inhibition zone of 13.7 mm.Three compounds were obtained by separation and purification from the strain BYSTW3:Physcion(3)?FK-17-p2a(4)and Citrinin(5).The antibacterial test showed that the compound 3 exhibited great activity against S.aureus,B.subtilis and E.coli with the inhibition zone of 23.0 mm,24.2 mm and 20.7 mm,The compounds 3-5 showed weak phytotoxic activities against Echinochoia crusgalli L with inhibition rates of between 14%to 25%.A new compound 4-hydroxy-2-(2,6-dihydroxyphenyl)-butan-l-one(6)was isolated from the fermentation broth of BYCDW8.The new compound 6 showed moderate growth inhibitory activity against B.subtilis and S.aureus with the diameter of 8.32 mm and 9.13 mm respectively.3?Chemical investigations of the intestinal symbiotic fungi Curvularia verruculosa FZNCD-1 from M.elongata were studied.The antifungal assays indicated that the crude extract of FZNCD-1 had notable growth inhibitory activity against various plant pathogenic fungi.In particular,the extract had the most significant inhibitory activity against V.mali with IC50<0.1 ?g/mL.Three active natural products,Lunatin(7),Rheoemodin(8)and Cytoskyrin A(9),were isolated and purified from the crude extract of FZNCD-1.Under the tested concentration of 100 ?g/mL,the compounds 7-9 exhibited extremely significant growth inhibitory activities against V.mali with the inhibition rates above 80%.Specifically,the compound 9 had the potent antifungal effect against the V.mali with the inhibitory rate of 91.7%.The compounds 8-9 showed significant growth inhibitory activities against Dothiorella gregaria with the inhibitory rates of 94.9%and 98.8%respectively,which were similar to that of the positive cycloheximide.Compound 7 showed only moderate growth inhibitory activity against D.gregaria,with an inhibitory rate of 66.9%.The compounds 8-9 presented moderate inhibitory effects against Alternaria solan and F.oxysporum f.sp.cucumerium with the inhibition rates of 50.9%and 69.5%,respectively.