| At present,the research on Cinnamomum camphora is still focused on reproductive technology and bioecology,but there is less research on microbiology on the Cinnamomum camphora,especially on Cinnamomum camphora(L.)Presl.The essential oil components analysis,cutting technology and dwarf afforestation of C.camphora have been studied.However,how identification and separation of endophytes and the relationship between endophytes producing and C.camphora is largely unclear.Therefore,this study used 16 srDNA high-throughput sequencing technology to sequence endophytes from three seasons of C.camphora,to study the physiological and biochemical characteristics,endophytes diversity and secondary metabolism of C.camphora in different seasons.And secondary metabolism,and Screening out the dominant endophytes that promote the production of essential oils,and provide a theoretical basis for studying the function and application of endophytes.We analyzed the physiological properties,biochemical indexes and essential oil content and component of the leaves of five selected clones to explore the relationship between endophytes and physiological properties,biochemical indexes and essential oil content and component.Tissue block separation method was adopted to isolate and purify C.camphora bacteria and fungi,after being determined primarily by microscopy.The representative bacteria and fungi were further analyzed using molecular identification strategy.After purification of endophytes by solid-phase microextraction,the metabolites were extracted and separated;the components of metabolites were determined,and compared with the components of C.camphora oil,to screen for endophytes that promote the essential oil content of the leaves.Results obtained are summarized below.At different sampling times,10,844,124 effective sequences from the leaves were obtained.Shannon index indicated that the diversity of the bacterial community was low over the sampling periods.The richness indices indicate that for Nanping area sampling,the samples taken in October was higher than that taken in July.For Fuqing area sampling,however,the July sample was higher than April sample in terms of richness.Using RDP classifier to identify the classification status of the sequences,it was observed that these sequences belonged to 9 major phyla: Proteobacteria,Firmicutes,Bacteroidetes,Actinobacteria,Gemmatimonadetes,Acidobacteria,Planctomycetes,Chloroflexi and Fusobacteria.In each sample,Proteobacteria had the highest proportion,ranging from 35.15% to 89.72%,indicating that these bacteria were the dominant species.The various sequences mainly belonged to 10 orders which included Rhizobiales,Clostridiales,Peseudomonadales,Burkholderiales,Bacteroidales,Enterobacteriales,Rhodocyclales,Sphingomonadales,Lactobacillales and Bacillales.However,there was no firm sequence at the order level,which suggested the presence of other new taxa with high taxonomic status.There were 40 bacteria isolates from NP 187,NP 195,NP 209,NP MD-1 and NP PC-1,belonging to 10 genera and 25 species.By comparing the physiological and biochemical indexes and essential oil contents of the five clones,it was found that there were significant differences between NP 187,NP 195,NP 209,NP MD-1 and NP PC-1,and that chlorophyll,soluble sugars,soluble proteins and MDA contents,as well as SOD,POD and CAT activities of NP 209 sample were significantly higher than those of the control group.The number of Bacillus anthracis in NP 209 sample was significantly higher than that of the other two clones and there were different bacteria in NP 209 sample.Among the clones screened,NP 209 sample had more bacterial density and oil content.The results showed that the subsamples NP 209-1and 209-5 had higher bacterial density and oil content at p<0.05 compared to the control group.This showed that the NP 209-1 and NP 209-5 samples had remarkable promoting effect and the two associated endophytic bacteria were Pseudomonas trivialis and Bacillus anthracis,respectively.The main fungi identified were Aspergillus niger and Penicillium sp.The metabolites of the endophytic fungi were mainly olefin,alcohols and lipid.Also found were Limonene,O-cymene,3-Carene,1,3,8-p-Menthatriene,α-Terpineol,β-Curcumene which was consistent with the composition of Linalool camphor oil compound.The screened NP 209-L1,and NP PC-1-L5,had a great promoting effect in the content of the essential oil of C.camphora.We therefore constructed phylogenetic trees which showed that NP 209-L1 had the highest similarity with Aspergillus nige r whereas NP PC-1-L5 was most closely related to Penicillium sp.Furthermore,the C.camphora endophytic bacteria Pseudomonas trivialis and Bacillus anthracis as well as its endophytic fungi,Aspergillus niger and Penicillium sp.,had positive influence on camphor oil content of the plant.The camphor endophytes promoting essential oil production fully reflect co-evolution between the microbes and the host which provided suitable living environment and metabolic substances for the development of its endophytes.Endogenous bacteria,on the other hand,promoted not only the growth of the camphor but also its essential oil content either by participating in the process of camphor oil production or by providing certain precursor substances necessary for oil production. |
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