Diversity Of Several Insect-associated Bacteria And Exploration Of Antimicrobial And Herbicidal Compounds

Insects are the most diverse animal group in the world.Their rich diversity means the diversity of insect symbionts.However,compared with the diversity of insect symbionts,there are only a small percentage of studies on these special environmental microorganisms,and less researchs on their active secondary metabolites.In this dissertation,the bacteria composition of intestinal of Locusta migratoria manilensis and termites’nests were investigated by amplicons sequencing technology.Further studies were carried out on the intestinal bacterium DYFHCD-18 from the L.migratoria manilensis,two strains of bacteria YC-9 and YT1-1 related to nest of Odontotermes formosanus,and the intestinal bacterium W14 from the Periplaneta americana.The secondary metabolites of the above four bacteria were separated by thin layer chromatography（TLC）,silica gel column chromatography and LH-20 dextran gel chromatography.The structures of the isolated compounds were identified by 1D-NMR(¹H-NMR,¹³C-NMR,DEPT-135),2D-NMR（¹H-¹H COSY,HMQC,HMBC,ROESY）and HR-ESI-MS.A total of 28 compounds were obtained and 26 compounds were identified,of which 2 were new compounds.At the same time,the antibacterial and herbicidal activities of some isolated compounds were further tested.The specific results are as follows:1.16S rDNA amplicon sequencing analysis showed that the bacterial diversity structure in the intestinal tract of L.migratoria manilensis infected with Beauveria bassiana changed significantly.After the infection,the intestinal bacteria Weissella decreased significantly,but Lactococcus,Hafnia-Obesumbacterium,Enterococcus and Serratia increased significantly.A strain DYFHCD-18 with antifungal activity against B.bassiana was isolated from the intestine of L.migratoria manilensis.It was identified as Pseudomonas aeruginosa.15compounds were further isolated from P.aeruginosa DYFHCD-18,and 13 known compounds（1-13）were identified,including pyoluteorin（1）,phenylacetic acid（2）,cyclo（l-phe-trans-4-oh-l-pro）（3）,3-indolylacetic acid（4）,2-heptyl-3-hydroxy-4（1H）quinolone（5）,（E）-2-non-1-en-1-yl-4（1H）quinolone（6）,2-n-Octyl-4-hydroxyquinoline（7）,cyclo-（L-Pro-L-Leu）（8）,cyclo-（4-hydroxyl-S-Pro-S-Leu）（9）,dirhamnolipid（10）,o-aminoacetophenone（11）,1-hydroxyphenazine（12）,carisphthalate（13）.It was found that metabolite 1 showed significant inhibitory effect against Phytophthora parasitica var.nicotianae,with the IC₅₀value of 4.0μg/m L,which was similar to that of the positive cycloheximide(IC₅₀=3.8μg/m L).Compounds 4 and 12 showed significant inhibitory effects on the conidia germination of P.parasitica var.nicotianae at the concentration of 50μg/m L,with the inhibition rates of 98.2%and 85.2%,respectively.Compound 1 showed significant antibacterial activity against Staphylococcus aureus,Micrococcus tetragenus,Bacillus subtilis and Escherichia coli at the concentration of 30μg/6mm disc,with the diameter of inhibition zone of 26.2,39.5,33.8,and 24.7 mm,respectively,which were better than those of positive gentamicin sulfate.Compound 1 was also founded have significant inhibitory effects on the root’s growth of Echinochloa crusgalli with the IC₅₀ value of 11.9μg/m L.Furtherly,the whole genome sequencing analysis of DYFHCD-18 was carried out,and a total of 18 secondary metabolite gene clusters were annotated.Among them,the gene clusters that synthesize pyoverdin,pyocyanine,oxalomycin B and lankacidin C have low similarity,which has a certain mining value.2.The results of 16S rDNA amplicon sequencing showed that termite nest related bacteria belonged to 77 phyla,168 classes,330 orders,472 families and 760 genera;Bacterial diversity analysis showed that Bacillus and Myxococcaceae had high abundance in termite nests.Two bacterial strains were further isolated from the nest of O.formosanus and identified as Bacillus siamensis YC-9 and Myxococcus stipitatus YT1-1 respectively.Five known compounds（14-18）were isolated and identified from B.siamensis YC-9,including hexadecanoic（14）,cyclo（Gly-D-Phe）（15）,cyclo（L-trans-Hyp-L-Leu）（16）,C₁₅ surfactin（17）and macroactin A（18）.It was found that metabolites 14-18 showed inhibitory effects on the conidia germination of Fusarium oxysporum f.sp cucumerinum（FOC）at the concentration of 50μg/m L,with the inhibition rates of 35.4%、7.3%、18.4%、100%and61.1%,respectively.Among them,the IC₅₀ value of the best activity of 17 on FOC spores was 5.1μg/m L,which was similar to that of the positive cycloheximide(IC₅₀=2.6μg/m L).At the concentration of 30μg/6mm disc,compound 17 showed weak antibacterial activity against M.tetragenus with the diameter of inhibition zone of 10.2 mm.which was significantly less than that of the positive gentamicin sulfate with the inhibition zone diameter of 33.8 mm.Furtherly,the whole genome sequencing analysis of YC-9 was carried out,and a total of 17 secondary metabolite gene clusters were annotated.Among them,the gene clusters that synthesize butirosin A/butirosin B,surfactin,rhizocticin,plipastatin,surfactin and fengycin have low similarity,which has a certain mining value.The activity test showed that strain M.stipitatus YT1-1 had strong inhibitory activity against Geratobasidium cornigeru and the roots growth of E.crusgalli.Two compounds（19-20）were isolated and identified from M.stipitatus YT1-1,including one new compound 19 and one known compound cyclo-（Leu-Tyr）（20）.It was found that compound 19 showed inhibitory effects on the conidia germination of FOC at the concentration of 50μg/m L,with the inhibition rates of 45.7%.3.Six compounds（21-26）were isolated and identified from the intestinal bacterium Brevibacterium epidermidis W14 from Periplaneta americana,of which 21 was new compound.Known compounds included cyclo（L-Pro-L-Leu）（22）,bis-（2-ethylhexyl）-benzene-1,2-dicarboxylate（23）,2,7-dimethyl-5,10-butenophenazine-11,14-dione（24）,cyclo[L-（4-hydroxyprolinyl）-L-leucine]（25）and cyclo（L-Pro-Gly）（26）.It was found that compounds 21,22 and 26 showed inhibitory effects on the conidia germination of FOC at the concentration of 50μg/m L,with the inhibition rates of 25.3%,5.1%,and 53.6%,respectively.The above results show that insect related bacteria,as a kind of less studied special environmental microorganisms,are an important source of novel active natural products,which is worthy of further research.