Diversity Of Endophytic And Epiphytic Bacteria And Allelopathic Potential Of Their Metabolites In Differently Aged Casuarina Equisetifolia Litter

Casuarina equisetifolia L.is cultivated as a coastal species in Hainan,Guangdong,Guangxi and other provinces and islands in the South China Sea.At present,most Casuarina equisetifolia forests have difficulties in secondary regeneration.According to previous studies,the accumulation of allelochemicals is the main reason for the difficulties in forest regeneration.Among them,Casuarina equisetifolia extract,soil microorganisms and endophytic flora are all the sources of allelochemicals.The litter of Casuarina equisetifolia forest is generally present in the forest land,and the microbes on the litter are also the accumulation phase of allelochemicals.Therefore,the diversity of bacteria inside and outside the litter of Casuarina equisetifolia was analyzed by high-throughput sequencing of Illumina Miseq,and the culturable bacteria were isolated by dilution coating method,and some of the bacterial fermentation broth was identified by GC-MS to explore its secondary metabolites.The results of this study are as follows:1.Based on high-throughput sequencing,589096 effective sequences were obtained from the litter of Casuarina equisetifolia.239092 and 350004 effective sequences were obtained from the DNA of endophytic bacteria and epiphytic bacteria.The diversity,community composition and function prediction of microorganisms inside and outside the litter were analyzed.The results showed that the diversity of epiphytic bacteria was higher than that of endophytic bacteria.Moreover,the diversity and richness of bacteria inside and outside the litter of Casuarina equisetifolia in the intermediate-aged forest were the highest,followed by the young-aged forest and the mature-aged forest.The analysis of microbial community composition showed that the dominant community composition of endophytic bacteria in young and intermediate age forests was more similar.The endophytic bacteria of litter are greatly affected by forest age,while epiphytic bacteria in litter are greatly affected by environmental factors,but the bacteria inside and outside litter are significantly related to N content,the results showed that the bacteria inside and outside the litter may participate in the decomposition of N element in the litter to a certain extent and play a certain role in the formation of soil nutrients in Casuarina equisetifolia forest land.There were 21 subfunctions of the same dominant flora in the secondary functional layer.The diversity of bacteria inside and outside litter is not only affected by forest age,but also by environmental factors,and the same dominant bacteria have different gene expression.2.The bacteria epiphytic and endophytic the litter of Casuarina equisetifolia of three forest ages by dilution coating method.The morphology and Gram staining of the bacteria were observed.The culturable bacteria were identified by molecular biology combined with 16 S rDNA gene sequence analysis technology.According to identification,116 strains of epiphytic and endophytic bacteria were isolated from Casuarina equisetifolia litter,belonging to 31 genera and 51 species in total;60 strains of endophytic bacteria were isolated,belonging to 22 genera and 35 species in total;56 strains of epiphytic bacteria were isolated,belonging to 22 genera and 31 species in total.According to different forest ages,39 strains of bacteria were isolated from young-aged forest,belonging to 16 genera and 26 species in total;52 strains of bacteria were isolated from intermediate-age forest,belonging to 19 genera and 29 species in total;25 strains of bacteria were isolated from mature-aged forest,belonging to 18 genera and 19 species in total.The isolates were mainly distributed in Actinobacteria,Bacteroides,Firmicutes and Proteobacteria.3.The allelopathy index of Casuarina equisetifolia seeds was determined by the bacterial fermentation liquid epiphytic and endophytic bacteria the litter of different forest ages.The results showed that the allelopathy index of seeds treated by Bacillus amyloliquefaciens was the strongest,which was-0.708;The allelopathy index of the seeds treated with Luteibacter yeojuensis fermentation liquid was 0.087,and its fermentation liquid could promote seed germination.4.By GC-MS analysis of four kinds of fermentation liquid of Bacillus amyloliquefaciens,Burkholderia plantarii,Pantoea ananatis and Luteibacter yeojuensis,2,2'-methylenebis(6-tert-butyl-4-methyl-phenol),2,4-Di-tert-butylphenol and other secondary metabolites were also identified from Casuarina equisetifolia litter extract in the early stage,indicating that the litter bacteria participated in the synthesis of allelochemicals.In conclusion,the diversity of bacterial community structure epiphytic and endophytic of Casuarina equisetifolia litter is related to the age of forest and environmental factors.In addition,the secondary metabolites of epiphytic and endophytic bacteria can exert allelopathic effecs on seed of Casuarina equisetifolia,indicating that the epiphytic and endophytic bacteria of Casuarina equisetifolia can directy produce secondary metabolites to participate in the synthesis of Casuarina equisetifolia allelopathic substances.Exploring the microbe-by-allelochemical and plant interactions of Casuarina equisetifolia land has positive meaning from the perspective of allelopathy.