Studies On Bacteria Diversity In The Gut Of Monochamus Alternatzus Hope And Lignin-degradation Characteristics Of Serratia Marcescen

Monochamus alternatus Hope is a severe pest for pine tree,which not only attacks the trees directly,but also is the main vector of pine wilt disease,the most dangerous disease of pine in China.Lignocellulose is the main source of nutrients for wood-feeding insects,and the gut microbiota including bacteria and fungi usually play an important role in helping insects to degrade and absorb the lignocellulose.The diversity of bacteria in the gut of larve,pupa and adult of M.alternatus Hope were measured by the methods of morphology,physiology and molecular biology in this paper.The bacteria with the function of degrading lignin were screened,and the lignin-degradation function of S.marcescens,a dominant gut bacteria was bioassayed.The main results were summarized as follows:1.Through amplifying and sequencing variable region of 16S rDNA-V4 of the bacteria lived in the gut of M.alternatus Hope by the technique of Illumina MiSeq,24 genera bacterium were found totally,and there were 13,10 and 17 genera bacterium distributed in the gut of larva,pupa and adult,respectively.They belong to the phyla of Bacteroidetes,Proteobacteria,Firmicutes,Actinobacteria,Deferribacteres,Verrucomicrobia,Cyanobacteria and Planctomycetes.There were differences observed in species and relative abundances of dominant bacterium among different developmental stage of M.alternatus Hope.The dominant genera(abundance)were Elizabethkingia(61.12%)in larva,Chryseobacterium(61.38%)in pupa and Serratia(43.92%)in adult.The genera of Oscillibacter,Ruminiclostridium and Helicobacter were specific to larva,and Pedobacter,Rhizobium,Variovorax and Sphingopyxis were specific genera to pupa,and Sediminibacterium,Enterococcus,Nubsella,Blautia and Brevundimonas were specific genera to adult.Serratia was found in all three developmental stages which abandunce were 12.6%in larva,0.85%in pupa and 43.92%in adult.The gut bacteria diversity was the highest in adult while followed by larva and pupa.2.Liquid culture experimented with the sole carbon source of filter paper were conducted to screen out bacteria by methods of isolation,purification and molecular identification.Twenty bacterium strains with lignin-degradation function were found from the gut of M.alternatus Hope.The strains belong to fifteen genera that were Proteobacteria,Firmicutes,Actinobacteria and Bacteroidetes,including Serratia,Pseudomonas,Bacillus,Comamonas,Klebsiella,Arthrobacter,Chryseobacterium,Rhizobium,Enterobacter,Stenotrophomonas,Ochrobactrum,Agrococcus,Streptomyces,Achromobacter and Pantoea.Serratia marcescens was proved to be a dominant strain with high efficiency on degrading lignin by the futher experiments of CMC Congo red and re-screen.3.The lignin-degradation characteristic of S.marcescens was measured by the method of liquid medium of kraft lignin.The result illustrated that the degradation rate peaked on 4th day(15.16%),and this bacteria could produced three kinds of extracellular lignin-degrading enzyme including LiP,MnP and Lac simultaneously.Further studied on LiP,the dominant lingin-degrading enzyme were done to explore the influences of different culture conditions on enzymatic activity produced by S.marcescens used the method of single factor that control variable.The results showed that the concentrations of lignin,pH,kinds of nitrogen source,concentration of nitrogen source,mental ions and their concentrations all had significant effects on LiP enzymatic activity.The highest enzymatic activity was found in the conditions of 3 g · L-1 lignin,pH 5,organic nitrogen sources,the nitrogen source of yeast extract with 5 g ·L-1 concentration,and Mg2e,Ca2+,Fee+,Mn2+,K+ with the concentrations of 0.20 g · L-1,0.40 g · L-1,0.15 g · L-1,0.04 g · L-1,0 g · L-1,respectively.The above results could help us to better understand the nutrition utilization mechanism of M.alternatus Hope and its synergetic relationship with the host pine.