Study On Diversity And Enzyme-producing Activities Of Endogenous Bacteria In Three Stored Grain Pests

Sitophilus oryzae,Rhizopertha dominica and Tribolium castaneum are three common storage grain pests in China,which seriously damage the quality of wheat,rice and other cereals.In this study,Illumina MiSeq sequencing technology was used to analyze the diversity of endogenous bacteria of the three stored grain pests.At the same time,the gut bacteria of the last instar larvae and adult of S.oryzae,R.dominica and T.castaneum were isolated and identified by traditional microbial isolation and 16S rDNA sequence analysis.Furthermore,we explored the biological characteristics of cultivable gut bacteria of the three storage grain pests,including enzyme-producing activities of these bacteria.These results would provide theoretical basis for further exploration on endogenous bacteria,functional bacteria selection and control of storage grain pests.The main results are as follows:1.The diversity of endogenous bacteria were significant differences among three storage grain pests when all fed on wheat.Endogenous bacteria of R.dominica was mainly consisted by 410 genera of 72 classes,which was significantly more than that of S.oryzae(70 genera of 20 classes)and T.castaneum(156 genera of 32 classes).The proportion of endogenous bacteria was also different on class level from the three stored grain pests.Actinobacteria and Gammaproteobacteria were the dominant bacteria in R.dominica and T.castaneum,whose proportion were 69.95%and 13.72%,and 24.51%and 85.90%,respectively.The proportion of Gammaproteobacteria in S.oryzae was absolute dominance,up to 99.78%,and the other bacteria were very low.At the order level,Enterobacterales was the dominant bacteria in both S.oryzae and T.castaneum,accounting for 99.71%and 82.61%,respectively.Different from those,Corynebacteriales was the dominant bacteria order in R.dominica,accounting for 69.38%.The dominant families of bacteria in the three stored grain pests were significantly different.The dominant family of S.oryzae was Pectobacteriaceae,which accounted for 99.71%.The dominant families of R.dominica were Corynebacteriaceae and Burkholderiaceae,which accounted for 68.69%and 22.95%,respectively.Morganellaceae was the main family in T.castaneum,which accounted for 82.60%.At the genus level,there was only one dominant genus in S.oryzae,Sodalis,accounting for 99.71%.The dominant genera of R.dominica were the unclassified genus of Corynebacteriaceae and Ralstonia,accounting for 68.68%and 22.21%,respectively.The dominant bacteria genera of T.castaneum were Proteus and the unclassified genus of Corynebacteriaceae,accounting for 82.60%and 13.38%,respectively.Furthermore,there are 28 shared Orders and 37 shared genera in three stored grain pests.Enterobacterales was the dominant shared order among the three stored grain pests.The dominant shared genus of them was Ralstonia.2.Combining traditional microbial isolation and purification,and 16S rDNA sequence analysis,we isolated and identified the gut bacteria of the late instar larvae and adults of S.oryzae,R.dominica and T.castaneum.It was found that the species of cultivable gut bacteria isolated from R.dominica were more abundant than S.oryzae and T.castaneum,and more abundant in three adults than in late instar larvae.Eleven and 16 bacteria species were isolated from the intestines of the last instar larvae and adults of S.oryzae,respectively,which were assigned to 4 genera,Bacillus,Enterobacter,Staphylococcus and Desulfovibrio.Fifteen and 26 bacteria were isolated from the last instar larvae and adults of R.dominica,which belonged to 7 bacterial genera,Enterobacter,Staphylococcus,Bacillus,Streptomyces,Sphingomonas,Kocuria and Rhizobium.Ten and 17 bacteria were isolated from the last instar larvae and adults of T.castaneum,which belonged to 8 bacterial genera,Bacillus,Staphylococcus,Desulfovibrio,Enterobacter,Kocuria,Sphingomonas,Rhizobium and Streptomyces.The following 5 species were shared by S.oryzae,R.dominica and T.castaneum in both last instar larvae and adults stage:S.warneri,B.velezensis,B.amyloliquefaciens,B.cereus and B.subtilis.3.The enzyme-producing activities of 36 isolated strains were detected by extracellular enzyme screening medium.It was found that 34 strains of them produce protease,27 strains produce amylase,and 15 strains produce cellulase,accounting for 94.4%,75%and 41.7%of the total strains,respectively.Among of them,27 strains produce both amylase and protease,15 strains produce both amylase and cellulase,and 15 strains produce both protease and cellulase.In addition,fourteen strains of them simultaneously produce the above three enzymes.It should be noted that none strains produce lipase.