Construction Of Engineered Bacterium Burkholderia Pyrrocinia JK-SH007 And Its Effects On Poplar Rhizosphere Environment

Along with poplar(Populus spp.)cultivation expanding,and improper management,protection and prevention in the process of poplar afforestation,occurrences of diseases and pests of poplar were becoming more serious,which hinderd the forestry production and development in China.At present,the prevention and control management of poplar canker disease and pest are still dependent on chemical in China.However,it is unsatisfactory that long-term using chemical methods not only make control effects worse,but cause natural environment pollution and harmful material residue.Therefore,using microorganisms instead of chemicals to control plant diseases and pests is becoming more popular for forestry protection researchers.Endophytic bacteria colonize in healthy plant tissues,and form a harmonious relationship with its hosts during a long coevolutionary process.Endophytic bacteria have potential biocontrol abilities,such as disease resistance,growth promoting,nitrogen fixation and bioremediation,and are potential natural sources for forestry diseases and pests biological control.Besides,endophytic bacteria also can be used as the carrier of foreign disease resistant or insectical gene,to improve the ability of plant resistance to diseases and pests without any changes of plant genome.Therefore,the application of beneficial endophytic bacteria and its engineered bacteria to improve plant resistances to diseases and pests has become research focus.In our previous study,we had screened an endophytic bacterium Burkholderia pyrrocinia JK-SH007 from poplar stem.It had showed certain biological control effect on poplar canker disease as well as growth-promoting effect on poplar.Moreover,JK-SH007 has no potential threats on plant,animal and people,and can be as a safe biological control agent for poplar canker disease.Several strains of Burkholderia cepacia complex(Bcc)have antagonistic effects on many plant pathogenic fungi,and have been widely used to control plant diseases.However,there have little reports about the construction of engineered Bcc at home and abroad now.In current study,foreign disease resistant genes and insecticidal genes were separately introduced into JK-SH007 to construct efficient engineered bacteria with multiple biocontrol abilities.Moreover,micro-ecological effects of engineered JK-SH007E1 on soil microorganisms were assessed after its release into poplar rhizosphere,and evaluated its biosecurity.According to the principle of homologous recombination,Bacillus subtilis Chi113 gene,Bacillus thuringiensis cry218 gene,B.thuringiensis vip3 A gene and Bursaphelenchus xylophilus Bxchi gene were separately inserted into the region between promoter and gfp gene on vector pHKT2,and successfully constructed recombinant plasmids pHKT2-Chi113,pHKT2-cry218,pHKT2-vip3 A and pHKT2-Bxchi.Genetic stability of recombinant plasmids showed that all the recombinant plasmids had high genetic stability except of pHKT2-Bxchi,and pHKT2-Chi113 had the highest genetic stability.The four recombinant plasmids were transformed into JK-SH007 competent cells respectively,and engineered bacteria were named as JK-SH007E1,JK-SH007E2,JK-SH007E3 and JK-SH007E4 separately.The four engineered bacteria were all detected bright green fluorescence glow under fluorescence microscope,and the four foreign genes were overexpressed at mRNA level.But only engineered JK-SH007E1 was detected forein gene expression at protein level.Compared with the wild type JK-SH007,chitinase activity,antifungal activity and antagonism to poplar canker pathogens of engineered JK-SH007E1 were improved significantly.Colonization dynamic research showed that JK-SH007E1 can rapidly invade into poplar tissues and stably colonize for a long term.To study the colonization of engineered bacterium JK-SH007E1 in poplar using GFP marker,It showed that JK-SH007E1 was distributed both in intracellular and intercellular space of poplar tissues.Moreover,JK-SH007E1 can significantly enhance photosynthesis capacity and growth of poplar.Growth characteristic of JK-SH007E1,JK-SH007E2,JK-SH007E3 and JK-SH007E4 were studied and compared with that of JK-SH007,the results showed that the introduction of foreign gene to JK-SH007 did not affect its original colony and cell morphologies,but the transformation of recombinant plasmid to a certain extent slowed the growth of the host cell.As an engineered bacterium,the biosafety of JK-SH007E1 must be fully evaluated before its release into natural environment.In this study,after releasing engineered JK-SH007E1 into poplar rhizosphere,its micro-ecological effects on soil microbial communities were investigated by using biochemical method,Biolog EcoPlate method and 16 S rRNA sequencing technology.Determination of four main soil enzymes activities showed that introduction of JK-SH007E1 improved soil enzymes activities expecially for soil dehydrogenase.And soil fertility was promoted by JK-SH007E1,which contributed to growth-promoting effect on poplar.Biolog EcoPlate analysis showed that engineered JK-SH007E1 enriched soil bioactivity,soil microbial population and its functional diversity to a certain degree,but the positive effects were gradually weakened with the extension of inoculation time.16 S rRNA high throughput sequencing results showed that introduction of engineered JK-SH007E1 changed soil microbial community structure,but the effect was gradually diminished in a long term.All the results suggested that there has no threat or little threat of genetically engineered JK-SH007E1 on biological diversity and community structure of soil microorganisms in a long term.Consequently,the release of JK-SH007E1 to natural environment is safe and feasible.This research provides an more efficient biocontrol agent for poplar canker disease,which lays a theoretical foundation for security application of engineered JK-SH007E1 in the future.