Effect Of Calcium Carbonate On Growth Of Ralstonia Solanacearum And Detection Of Ralstonia Solanacearum In Soil With Real-time PCR Assay

Tobacco bacterial wilt, which is a kind of bacteria disease and destructive soil-borne disease caused by Ralstonia solanacearum, and greatly affect tobacco product and quality, is hard to control.Therefore, to explore the optimal efficacy of drugs, the relationship between calcium carbonate,calcium chloride and R.solanacearum, RTQ-PCR assay in different layers soil, the research contents in the article include the drug sensitive test, effect of inorganic salt of calcium carbonate and calcium chloride on R.solanacearum, detecting the number of R.solanacearum in soil quantitatively and accurately. That will make us a better understanding about the occurrence and prevention method of the disease.The detailed experimental results are as follows,semiselective medium(PCCG)and apolymerase chain reaction(PCR)technique were combined to detect viable cells of Ralstonia solanacearum in haulm of White Burley from Dazhou city in Sichuan province, and identified the bacterium of biochemical type. As result,23strains Ralstonia solanacearum were isolated, which were all biochemical type Ⅲ. And then with bismerthiazol, ethylicin, streptomycin, lime sulfur,47%Poly-L-lysine hydrochloride,99%kojic acid dealing with R.solanacearum respectively, drag sensitivity were statistics to make sure optimal microbicides on tobacco bacterial wilt bacteria. The results indicated that the bacteria were all susceptible to ethylicin, streptomycin and bismerthiazol, and ethylicin was the optimal drag to control the growth of the Ralstonia solanacearum in the PCCG medium.21conical flasks of soil samples containing different particle sizes and different ratios of calcium carbonate were inoculated with Ralstonis solanacearum and studies of cultures of Ralstonis solanacearum to varying pH and Ca2+concentrations in vitro showed that optimum growth of Ralstonis solanacearum is pH6.0.When the growth environment of pH exceeds8.0, Ralstonis solanacearum can hardly grow.And calcium have certain inhibition on Ralstonis solanacearum population growth,which could rise to78.17%.The in vitro experimengs measuring the population of R.solanacearum showed a stongest(positive) correlation between particle sizes of Calcium calcium and population of R.solanacearum.Suitable particle size of calcium carbonate could make the soil pH increases to8.0and increase soil exchangeable calcium ion concentration,and increased of pH and calcium concentration were the main reason right about the inhibition of R.solanacearum growth, the most suitable calcium carbonate particle size should be less than1mm.The activities of pectinase produced by Ralstonis solanacearum,they were placed in the environment,which contained different concentration of sodium chloride were detected. Variance analysis were conducted showed a significant negative correlation between sodium chloride concentration and activities of pectinase produced by Ralstonis solanacearum(F>Fcrit).There was significant difference compared with the control, and the activities of pectinase produced by Ralstonis solanacearum reduced with increasing sodium chloride concentration.extrapolysaccharide (EPS) produced by Ralstonis solanacearum, they were placed in the environment, which contained different concentration of sodium chloride were detected.The results showed that extrapolysaccharide (EPS) produced by Ralstonis solanacearum had a little relaship with sodium chloride. All treatments had no significant difference compared with control,In order to explore early detection the occurrence of Ralstonis solanacearum of tobacco in naturally infested soil, a SYBR Green I based Real time RTQ-PCR assay was developed.The recombinant plasmid inclued a331bp sequence product was used as the reference and the specification curve was created on that basis. Quantity of Ralstonis solanacearum in different soil layers was monitored by the real time PCR. The result showed that a linear relationship was observed between the amount of input plasmid DNA and cycle threshold (Ct) values over a range of4.16×103copies/μl to4.16×108copies/μl, and correlation coefficient was0.991. PCR amplification efficiency was110%.And in field of planting tobacco, quantity of Ralstonis solanacearum in rhizosphere soil is greater than that of the other soil layers.In soil of tobacco-corn rotation, quantity of Ralstonis solanacearum in non rhizosphere soil is greater than that of rhizonspere.