| Casuarina equisetifolia is one of the primary species of tree responsible for protection forests along the coast of Hainan province.However,C.equisetifolia forests present self-sustaining problems with increasing age,and the main reason is the accumulation of allelopathic substances.However,most of the research on allelopathic substance sources focuses on C.equisetifolia plant.C.equisetifolia is a symbiotic nutrient plant rich in endophytes.Endophytes can produce the same or similar secondary metabolites as host plants during the long coevolution process.Therefore,the metabolites produced by endophytes may be an important source of allelopathic substances in C.equisetifolia forest land.Therefore,high-throughput sequencing platform was used in this paper to analyze the diversity of endophytic bacterial community structure of C.equisetifolia at different forest ages;traditional methods were used to isolate and purify root endophytic bacteria of different forest ages,and 16 s DNA was used for strain identification;the seeds of C.equisetifolia were treated with different fermentation broth,so as to explore the allelopathic potential of different fermentation broth to seed germination of C.equisetifolia;three endophytic bacteria with the strongest allelopathic potential for C.equisetifolia seeds were selected,and the methanol and n-hexane extracts from the fermentation broth were identified by GC-MS technology,and the results were compared with those obtained from the roots and soil extracts of C.equisetifolia.The results can not only clarify the theoretical basis of endophytic bacteria participating in allelopathy in C.equisetifolia,but also provide practical guidance for the subsequent renewal and transformation of C.equisetifolia forests using engineering strains or microbial preparations.The results of this study are as follows:1.A total of 330447 sequences with an average length of about 430-448 bp were obtained by 16 s rDNA directional sequencing of the metagenomic DNA of C.equisetifolia root in young-,intermediate-and mature-aged forest trees by high-throughput sequencing.The species composition of endophytic bacteria in C.equisetifolia root was abundant in different stands.The diversity of endophytic bacteria in roots of intermediate-aged forest was the highest,followed by mature-aged forest and the lowest in young-aged forest.In addition,there were some differences in the community structure of endophytic bacteria in three forest ages,among which the community composition of endophytic bacteria in roots of young-aged and mature-aged forests was similar,which was different from that of intermediate-aged forests.Young-aged and mature-aged forest was dominanted by Proteobacteria and Firmicutes at the phylum level,but intermediate-aged forest was dominanted by Proteobacteria and Actinobacteria.The physicochemical properties of soil,such as organic matter,nitrate nitrogen and ammonium nitrogen,had significant effects on the composition of endophytic bacteria in the roots of C.equisetifolia at different forest ages.The results showed that the community structure diversity of endophytic bacteria in C.equisetifolia root was affected by forest age and environmental factors.2.Endophytic bacteria were isolated from the roots of C.equisetifolia of three forest ages by dilution coating method.The morphology of the isolated endophytic bacteria was observed,and the sequence of 16 S rRNA was determined and the homology was analyzed.The results showed that 36 strains of endophytic bacteria belonged to 21 species of 9 genera.Among them,there were 6 species of Bacillus,accounting for 28.57% of the total bacterial species,as the dominant species.Acinetobacter and Staphylococcus were the three species,each accounting for 14.29% of the total bacterial species,as the subdominant species.In addition,there were 8 species of 5 genera in the young-aged forest,10 species of 6 genera in the intermediate-age forest and 11 species of 6 genera in the mature-aged forest,accounting for 38.10%,47.62% and 52.38% of the total bacterial species respectively.3.The endophytic bacterial fermentation broth of C.equisetifolia root at different ages inhibited the germination of the C.equisetifolia seeds to different degrees and reduced the germination rate of the seeds.Among them,the fermentation broth of Bacillus amyloliquefaciens and Bacillus aryabhattai had the strongest inhibitory effect on seed germination,and the allelopathic effect indexes were-1 and-0.99,respectively.However,the inhibitory effect of bacillus cereus fermentation broth was the weakest,and the allelopathy index was-0.008.4.Using GC-MS technology to identifiy and analyze the secondary metabolites of endophytic bacteria of C.equisetifolia roots,and the organic compounds such as phenols,ketones,esters,organic acids,ethers,aldehydes,alcohols and pyridines were detected.Among them,2,2'-methylenebis[6-(1,1-dimethylethyl)-4-methyl-Phenol and 1,2,3,4-Butanetetrol were the metabolites of endophytic bacteria shared with root and soil extracts.However,the 5 compounds of 4-Methoxy-3,5-dihydroxybenzoic acid,3,4,5-trihydroxy-Benzoic acid,,1,4-Benzenediamine,3-amino-Phenol and 3,5-dimethoxy-Phenol were unique to endophytic bacterial metabolites.The above results showed that the diversity of endophytic bacteria in C.equisetifolia roots varied with the age of the forest and was closely related to environmental factors such as organic matter,nitrate nitrogen and ammonium nitrogen.In addition,the secondary metabolites of culturable endophytic bacteria can exert allelopathic effects on seed of C.equisetifolia,indicating that the endophytic bacteria of C.equisetifolia can directly produce secondary metabolites to participate in the synthesis of C.equisetifolia allelopathic substances.And it is also possible that endophytic bacteria are involved in the synthesis of allelopathic substances through the interaction with C.equisetifolia plants such as the transmission of genetic information or gene exchange. |
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