Investigation On Antimicrobial Activity Of Pantala Flavescens And Harpalus Sinicus Symbionts

In this world,insects are well known as one of the most diverse species of organisms,widely distributed in every corner of the globe.In comparison with insect species research,there are currently few researches focus on insect symbionts and their metabolites.Insect symbionts exhibit distinct distinctly physiological and biochemical characteristics due to the diversity of their host living environments,and they are important sources of active secondary metabolities.Consequently,it is necessary to investigate such special microorganisms and their active metabolites.In this study,the symbionts from the larvae of Pantala flavescens and Harpalus sinicus and their metabolites were preliminarily studied.An intestinal fungus QTYC-77 was isolated and identified from Pantala flavescens larvae.Its activity against Fusarium oxysporum f.sp.cucumerium was investigated and the antimicrobial mechanism was initially explored.The secondary metabolites of target strain were separated and purified by silica gel and sephadex LH-20 column chromatography,and four metabolites were obtained.The structures of metabolites were analyzed by ¹H-NMR,¹³C-NMR,HMQC,HMBC,¹H-¹HCOSY and HR-ESI-MS,three chenmical compounds were identified,one of which was new.In addition,35 symbiotic actinomycetes were isolated from Harpalus sinicus using the plate dilution method.The inhibitory activity of the fermentation broth against four pathogenic microbes was determined by the Oxford cup method.The result showed that the BJ37 strain processed significant antimicrobial activity.Then,the BJ37 strain was identified and its antimicrobial activity of the different polar fractions extracted from the fermentation broth was compared.The major research works were mentioned as follows:1.A fungus QTYC-77 was isolated from the intestinal tract of the larvae of P.flavescens,and it was initially identified as Trichoderma aureoviride according to the morphological characteristics and ITS-rDNA gene sequencing.The antifungal test of the plate presented that QTYC-77 could effectively inhibit the growth of F.oxysporum f.sp.cucumerium with the inhibition rate of 71%.The biological control effect of QTYC-77 against F.oxysporum f.sp.cucumerium in pot culture was determined by root irrigation method.The control effect was as high as 67%.Simultaneously,the QTYC-77 could significantly promote the growth of stems and leaves of cucumber seedlings.The antifungal mechanism of QTYC-77 was further tested.Microscopic observation showed that QTYC-77 could parasitize on the hyphae of F.oxysporum f.sp.cucumerium,of which hyphae was ruptured.The QTYC-77was tested for chitinase,β-1,3-glucanase and extracellular protease activity at different fermentation times.The results showed that the activities of chitinase,β-1,3-glucanase and extracellular protease presented a trend of initially increasing and then decreasing.At 5 d,the activities of three enzyme reached the maximum with0.1529 U/mL,1.3481 U/mL and 31.4261 U/ml,respectively.The experiment indicated that QTYC-77 parasitized on F.oxysporum f.sp.cucumerium by secreted enzymes.The metabolital antifgungal test proved that there were antagonistic substances in the fermentation broth of QTYC-77 which inhibited the growth of F.oxysporum f.sp.cucumerium.2.Three compounds were isolated and two compounds,including new compound azaphilone D（1）,2H-1-Benzopyran-2-one,8-Hydroxy-3-（2-hydroxypropyl）-6-methoxy（2）were identified from the ethyl acetate extract of QTYC-77fermentation broth.Furtherly,the compound niacin（3）was isolated from the strain,which was subjected to epigenetic modification of niacinamide.Subsequently,the compounds 1,2,and 3 were tested for antimicrobial activity.The activity test showed that the compound 1 was weakly resistant to Staphylococcus aureus and Escherichia coli at a concentration of 30μg/filter paper,and the diameter of the inhibition zone was 7.3 mm and 7.0 mm respectively.Compounds 2 and 3 were also weakly resistant to S.aureus with diameters of 7.2 mm and 7.5 mm,respectively.3.The 35 symbiotic actinomycetes of the Harpalus sinicus were isolated and purified by the dilution plate method.The inhibitory activity of the fermentation broth and extract against the growth of four pathogenic microbes was tested by the Oxford cup method（100μL per cup）.The results showed that fermentation broth of 34actinomycetes had antimicrobial activity against at least one test organism.BJ37 had a strong antimicrobial effect against B.subtilis and C.albicans,with the diameter of the inhibition zone of 49.2 mm and 30.0 mm,respectively.Antimicrobial experiments of different polar extracts of BJ37 fermentation broth showed that the ethyl acetate extract of fermentation broth had obvious antimicrobial effect against B.subtilis and C.albicans with the diameter of the inhibition zone of 34.1 mm and 29.3mmrespectively under the concentration of 100μg/mL.The antagonistic activity of other parts was poor.The BJ37 strain was identified as Streptomyces venezuelae by the morphological characteristics and 16s rDNA gene sequencing.