Isolation,Screening And Applied Research On Rhizospheric Microorganism And Endophyte Of Ginger

Ginger bacterial wilt,a bacterial soil-borne vascular bundle disease caused by Ralstonia solanacearum,is difficult to be prevented for its complicated infection mechanism and fast pathophoresis.Existing associated researches still focus on biological control,therefore,it is of important significance to employ probiotics to carry on study of biocontrol.In this paper,microorganism were separated from fresh tuber and rhizosphere soil?Laiwu,Shandong?and potential microbial resources were explored through evaluation of biocontrol.Based on the previous work,immobilization and colonization ability of the rhizospheric microorganism BJW-1 were discussed,and,the research also studied preliminarily marking effect of enhanced green fluorescent protein?EGFP?on endophytic strain BJ-1.Research results demonstrated that:In this paper,a total of 113 microorganism were separated from fresh tuber and rhizosphere soil of Laiwu ginger,including 66 rhizobacterial strains and 47 endophytic strains.Endophyte were separated from ginger tuber by optimized surface disinfection method.It found that the average quantity of endophyte in ginger tuber is 2.86×10³cfu/g.fw and most of them are bacteria,followed by actinomycetes and fungus successively.R₂A medium is the best culture medium for separation of endophyte in ginger.R₂A medium,KB medium and TSA medium can separate satisfactorily endophyte from ginger.In this study,biocontrol potential of some strains against pathogenic fungus were discussed preliminarily,finding that 11 actinomycetes and 8endogenous spore bacteria have good antagonistic activity.SJW-1,BJW-5 and BJ-7 all can inhibit growth of Fusarium spp.36194 and Fusarium oxysporum,which might be good biocontrol agents against Fusarium-induced plant diseases.Bacillus is an important biocontrol resource to control ginger bacterial wilt.Two excellent biocontrol strains?BJW-1 and BJ-1?were screened separately from rhizosphere soil and tuber through inhibition zone and pot experiment.The disease control effects of BJW-1 and BJ-1 are 66.45%and 61.02%,respectively.Based on morphological and physiological-biochemical analysis as well as molecular biological identification,BJW-1 was determined Bacillus amyloliquefaciens and BJ-1 was Bacillus subtilis.BJW-1 was embedded by immobilized microbe technology,aiming to increase its colonization in soil and provide basis for the development of microbial fertilizer.Firstly,the proportion and concentration of basic ingredients was:sodium alginate solution:bacterial solution=1:1 and 3.0%calcium chloride by the experiment of embedding survival rate and proliferation capacity on liquid medium.In liquid medium,viable count of BJW-1 presents an inverted“V-shaped”variation trend and viable bacterial of BJW-1 at the 8d is 53.26%higher than that of the control group.Secondly,colonization capacity of BJW-1 was analyzed in soil under laboratory which was immobilized by compound biological polysaccharide,thus determining primarily the optimal complex formula:sodium alginate solution:bacterial solution=1:1,3.0%calcium chloride,1.5%porous starch and 2%molasses.At 30d of tracking test,the survival rate changed stablely and the average survival rate was 160%.The survival rate of BJW-1 was 122%after 30d,which was 8.13 times higher than that of the control group.Finally,colonization capacity of the optimal complex formula was affirmed with field experiments,the max of which was 12.4 times higher than that of the control group.At last,we used EGFP as a fluorescent biomarker to label endophytic bacterial BJ-1 to clarify the colonization of endophytic bacterial and mechanism of Biocontrol.A new plasmid PHY-P43-egfp was constructed and transformed into BJ-1 by electroporation.In the study,we obtained an engineering bacterial in which the egfp gene was included.Therefore,this study provide a foundation for further study of egfp-tagged endophytic strain and its colonization in plant.