Isolation Of Cymbopogon Citratus(DC.) Stapf Endophytic Bacteria And Their Affect On The The Citral Production In Host

Cymbopogon citratus(DC.)Stapf is an important spice plant.The primary composition of its volatile organic compounds(VOCs)is citral,which is widely used to produce flavors and fragrances.However,citral from aromatic plants can not meet the needs of highly developed spices industry.Thus,the approach to efficiently induce citral production in C.citratus is worth exploring.Endophytes composed an important part of the microbial ecosystems,but their effect on the VOCs of C.citratus was never reported.This study concentrated on the impact of endophyic bacteria on citral production of the host C.citratus.The main contents in this article are as follows:(1)Isolated and analyzed endophytic bacteria of C.citratus grown at different seasons:Endophytic bacteria of C.citratus grew in different seasons were isolated by surface sterilization and four isolation media.The result showed that the distribution of culturable endophytic bacteria was:autumn>winter>summer>spring.One of the endophytic bacteria named CcLf-2 showed a dominant distribution in different seasons.It was clustered within the genus Bacillus identified by the morphological characterization and the 16S rRNA sequence analysis.The volatile organic compounds(VOCs)of CcLf-2 were identified to contain gamma-Undecalactone,alpha-Ionone and other valuable spice material.Besides by sensory evaluation the fragrance-producing characters of endophytic bacteria were found to be opposite to their abundance pattern,that is,that is,when the endophytic bacteria appeared high frequencies in the host,their ability to produce fragrance was not necessarily strong.The VOCs of C.citratus grew in different seasons were further detected by HS-GC-MS and the results showed that the citral production appeared evidently higher in spring and summer than that in autumn and winter.This result was consistent with the distribution of the fragrance-producing endophytic bacteria,as well as the distribution rate of dominant endophytic bacteria CcLf-2.So,we inferred that the endophytic bacteria of C.citratus might have effect on the production of host VOCs,especially the main citral component.(2)Treated the C.citratus aseptic seedlings with fragrance-producing endophytic bacteria and analyzed the expression levels of relevant genes:The C.citratus aseptic seedlings were obtained by tissue culture technique.Then dominant endophytic bacterium CcLf-2 and Pantoea ananatis CcSh-1,an endophytic bacterium isolated from C.citratus previously with citral-produceing ability,were then inoculated into C.citratus aseptic seedlings for 10 days.The VOCs of treated,untreated and wild C.citratus seedlings were detected.Meanwhile,the mRNA levels of citral backbone synthesis related genes in seedlings were evaluated by quantitative real-time PCR(qRT-PCR).The results showed that endophy bacteria CcLf-2 and CcSh-1 may enhance the citral production by modulating the expression of genes related to biosynthesis.Although CcSh-1 has the same secondary metabolite with the host plant,the dominant bacterium CcLf-2 had stronger ability to induce citral production in C.citratus.In addition,after the C.citratus aseptic seedlings were treated with CcLf-2 for different times,the citral productions were significantly increased for 5 days and 10 days,and the expression levels of biosynthesisrelated genes were up-regulated too.While the citralcontent in C.citratus aseptic seedlingswas decreased after 15 days treatment with CcLf-2,although the gene expression levels werestill up-regulated(3)Preliminary study on the citral synthesis pathway of CcSh-1:CcSh-1 can produce citral,but its ability to induce citral production in C.citratus is not very strong.The genome of CcSh-1 was sequenced,and the key genes of MEP synthesis pathway were found.Then two key genes in CcSh-1,DXS and DXR,which might related to the MEP synthesis pathway were cloned and constructed into the recombinant vectors under identifiedstrong promoters,pUC19.After DXS and DXR were overexpressed in the host strain CcSh-1 respectively,the citral content of pUC19-DXS-CcSh-1 was 0.172?g/ml,which was higher than that of the control pUC19-CcSh-1 0.157?g/ml,while the content of citral in pUC19-DXR-CcSh-1 was reduced to 0.144?g/ml.The results demonstrated that DXS and DXR genes involved in the synthesis of citral in the MEP pathway in CcSh-1.