Isolation And Identification Of Nitrogen-fixing Bacteria And Actinomycetes From The Gut Of Macrotermes Barneyi

Termite(Blattaria,Insecta),as a social insect,takes wood,litter or humus as food,which are rich in lignocellulose and poor in nitrogen.So,the gut microorganisms play important roles in the lignocellulose digestion and nitrogen fixation.Fungus-growing termite can culture Termitomyces as cultivar,and the termite and comb are easy to be contaminated by other fungi.However,Termitomyces are necessary in a healthy nest of fungus-growing termite.It is hypothesized that termites or the endosymbionts can produce some compounds to inhibit pathogen fungi.Actinomyces as the major active compounds-producer may play a key role.To understand the nitrogen fixation and defense of termite gut microbial,the nitrogen-fixing bacteria and Actinomyces from the gut of a fungus-growing termite Macrotermes barneyi were studied.Firstly,we isolated and identified potential nitrogen-fixing microbial from the whole gut of M.barneyi.Eight strains were isolated on the nitrogen-free plate for further studied by morphological and phylogenetic properities,which belonged to Serratia,Bacillus,Enterobacter,Klebsiella,Salmonella and Kosakonia.It was reported that some of the similar strains shown nitrogen-fixing activity or nitrogenase genes.Except for the Bacillus strain,which belongs to the phylum Firmicutes,other strains belong to Proteobacteria.This research provided strains for the understanding of nitrogen fixation in fungus-growing termites.After that,thirteen Actinomyces were isolated on the Gao’No1 medium from the foregut and midgut of M.barneyi.By morphological and phylogenetic properities,they were belonging to Streptomyces and Kitasatospora.Pathogen strains Escherichia coli,Pseudomonas aeruginosa,Bordetella sp.,Staphylococcus aureus,Bacillus subtilis,Canidia albicans,Metarhizium anisopliae,Beauveria bassiana,Alternaria solani,Fusariu oxysporum,Fusariu sp.and Fusariu oxysporum F0C-R4 were used for the antimicrobial test of Actinomyces strains,and eight strains performed antimicrobial activity.It was the first report about isolated bioactivity Actinomycetes from M.barneyi gut.This research enriched the culturable Actinomyces from termite gut,which may show great potential application value.Then,we analyzed the draft genome of Streptomyces WM-3 by antiSMASH software.We found thirty-seven clusters encoding the synthesis of secondary metabolites,including PKS,Nrps,bacteriocin,siderophore and other nine types.Besides,the medium and extractant was optimized,and the crude extract performed high activity against tested bacteria and fungi.Finally,Streptomyces sp.WF-5 was taken as a putative novel strain based on the 16S rRNA sequence.We sequenced and analyzed the draft genome of WF-5 by antiSMASH software.Twenty-four clusters encoding the synthesis of secondary metabolites were found in the genomic DNA of WF-5,including Nrps,terpene,PKS,Lantipeptide and other six types.Streptomyces sp.WF-5 performed activity against Bordetella sp.in naturel condition and weak activity against 3 plant-pathogen fungi when cultured together on TSB and Bran plates.In brief,eight potential nitrogen-fixing bacteria and thirteen actinomyces were isolated from the gut of M.barneyi.The draft genome of Streptomyces sp.WM-3 and WF-5 was sequenced and the synthesis clusters of secondary metabolites were analyzed.Besides,the bioactivity of these two strains were detected.This research can help to study the functions of the symbionts of fungus-growing termites.And termite gut was taken as a special environment,from which we can obtain novel strains with nitrogen-fixing or anti-microbial activity.