| In this paper,we studied on the endophytes diversity of Cinnamomum camphora chvar.Borneol in three seasons using 16S rDNA high-throughput sequencing technology.Adopting the method of spread plate method,combined with 16S rDNA sequence analysis,we seperated the endophytes from Borneol Cinnamomum camphora.Then we screened and returned the dominant endophytes after analysing the physiological,biochemical indexes and essential oil content of three cloned,hadving compared the difference of essential oil content with the contrast.Adopt tissue block separation method to separate and purify camphora fungus,after determined primarily by the microscopy fungi type,then further determined the species of the representative form of fungus with molecular identification.After purification of the endophytic fungi,using the method of solid phase microextraction separated and extracted metabolites,we screened of endophytic fungi that promote the content of essential oils,after compared volatile components of metabolites with borneol camphor oil composition.The results showed:(1)We got 10,917,170 effective sequences from leaves of different growing period of camphor.Using RDP classifier to identify the classification status of sequence,the results showed that these sequences belonged to 20 orders:Rhizobiales,Clostridiales,Pseudomonadales,Burkholderiales,Enterobacteriales,Bacteroidales,Sphingomonadales,Rhodocyclales,Xanthomonadales,Lactobacillales,Rhodobacterale,Bacillales,Caulobacterales,Desulfovibrionales,Flavobacteriales,Gemmatimonadals,Sphingomonadales,Rhodospirillales,Erysipelotrichales,Myxococcales.HB sample seasonal endophytes community structure in two regions just had little difference,while the other two clones were significantly different across the growth period.The endophytes community structure have changed and the diversity have been more and more significant with the continuous growth and development of Bomeol Cinnamomum camphora.(2)There were 43 bacterium isolates from HB,HN and JX,which belonged to 10 genus,25 species.By comparing physiological and biochemical indexes and essential oil contents of three clones,it was found that there were significant differences between JX and HB,HN,and that contents of chlorophyll,SOD,POD,CAT,soluble sugar and soluble protein of JX sample showed significant advantages.Compared with the control group,the number of Bacillus in JX sample was significantly higher than that the other two clones,and there were unique bacteria in JX sample.Screened and tieback JX sample dominant 5 bacteria J1,J2,J3,J4,J5,we have compared with controls.The results found that screening of bacteria group showed advantages,especially the J1,J2,J5 three bacteria which P<0.05 in analysis of variance with the control group.It meant J1,J2,J5 had remarkable promoting effect,The three endogenic bacteria were Staphylococcus pasteuria,Pseudomonas trivialis,and Bacillus.(3)The main fungi were Pestalotiopsis,Xylariales and Nigrospora.The metabolites of endophytic fungi mainly were olefin,alcohols and lipid,also found Limonene,alpha Pinene,alpha terpineol and 1,8eucalyptus oil,alpha myrcene,alpha caryophyllene which was consistent with the composition of borneol camphor oil compound.Have screened JX-Y1,JX-Y5,JX-Z3 had a great promoting effect in the content of the essential oil of camphor camphor,we determined and constructed phylogenetic trees.It showed JX-Y1 had the highest similarity with Pestalotiopsis anacardiacearum,JX-Y5 had the highest similarity with Guignardia mangiferae,and JX-Z3 had the highest similarity with Uncultured Xylariales.Results show that:the borneol camphor endogenous bacteria Staphylococcus pasteuri,Pseudomonas trivialis and a strain of Bacillus,and the endophytic fungi a strain of Pestalotiopsis anacardiacearum,Guignardia mangiferae,and a strain of Uncultured Xylariales all had positive effect on camphor oil content.The borneol camphor endophytes promoting essential oil content fully reflected the co-evolution between microbes and hosts,which borneol camphor provided suitable living environment and metabolic substances for its endophytes,endogenous bacteria on the one hand,promoted the growth of borneol camphor to increased content of essential oil,on the other hand,by participating in process of borneol camphor oil or provided certain substances to impacted on oil content. |
