Screening, Identification And Fermentation Conditions Of Antagonistic Germ Q6 Against Ralstonia Solanacearum

Ginger bacterial wilt is a destructive disease caused by Ralstonia solanacearum.The prevention against the disease was always through agricultural and chemicalmeasures since long before, but the effect of prevention was not good. Applicationof chemical pesticides brought entironment pollution. Biological control by naturalantagonistic organisms is a potential measure against plant diseases. So in our test aantagonistic germ was isolated from ginger soil and was studied.108 strains of germs were isolated by tube dilution assay from the soil of gingerin Qingmu town,Bazhong city. Q6 strain was screened from these strains of germsthough the first screened and rescreened double with deck agar method andimproved aperture-strikeing method. Adoptingthe method of improvedaperture-strikeing, compared to chloromy (10mg/ml), phytomycin (2mg/ml),pesticide Jiang-fu (1/500), Jun-sha-de(1/800), The result showed that it haddistinctive lyinhibitive effect on Ralstonia solanacearum.It'smorphological,cultural physiological, biochemical characteristics and 16S rDNAsequences analysis were studied. The result showed that it can utilize cane sugar,glucose,D-galactose,D(+)xylose,D-fructose,tartrate,citrate,Malonate,etc,cannotD-arabinose,L-arginine,etc,hydrogen sufide assay,V-P assay and nitrate reductionis masculine,indole assay M. R assay, amylohydrolisis,cellulose-lysis is negative.A phylogenetic tree was constructed by comparing with the published 16S rDNA sequences of the related bacteria species. In the phylogenetic tree the overallsimilarity value between strain Q6 and 11 type Pantoea are 95.54～99 %.The effects of a number of factors on the production of germ Q6 werestudied ,including pH ,volume of culture media ,ultural temperature and time,carbon source ,nitrogen source. The results showed that the optimal carbon sourceand nitrogen source were amidulin,CaCO₃ and yeast.The optimization pH 7.0～8.0,cultural temperature:30—32℃,and germ Q6 was cultured for 48h inbroad-openning flask with 20% volume medium. On the basis of these, the recipeof fermentation medium was optimized through uniform-design.A suitablefermentation condition was found for antibacterial production of germ Q6. Theoptimal conditions were as follows: amidulin 1.25% ,CaCO₃ 0.05%, yeast0.6%,NaC1 0.7%,KH2PO4 0.3%.The stability of antagonistic substance produced by Q6 was studied. Theactivity of the inhibitive substance was influenced when time and temperaturewaxing. In 100℃for 30 minutes,the inhibitive zone diminished distinctly, but thesubstance still had inhibitive effect on the pathgen. In pH 3.0～above 9.5 forovernight or treated by Proteinase K, the inhibitive zone is not changed. Resultsindicate that the antagonistic substance is stable against heat and acid and alkali andenzyme.