Quantitative Detection Of The Cotton Verticillium Dahliae Microsclerotia Of Cotton In Soil By Molecular Analysis

Cotton wilt disease, caused by a soilborne phytopathogenic fungus Verticillium dahliaeKleb. is a widespread, destructive disease in most cotton-growing countries. It is especiallydifficult to control V. dahliae because microsclerotia, the surviving structures of V. dahliaemay persist in soil for many years in the absence of a host plant. The dormant microsclerotiaare the primary infectious propagules, and quantifying the density of microsclerotia in soil isimportant for the development of disease prediction systems and to assess the effects ofcontrol tactics. Traditional methods used for quantifying microsclerotia of V.dahliae in soilwere plating (dry and wet plating), But these methods involving plating a small amount of soilon semi-selective medium were highly soil-type dependent, time consuming, labor intensiveand inconsistent. Advances in PCR offer the prospect of rapid detection and identification ofmicro-organisms without resorting to culturing. The present study, we successfully establishedthe conventional PCR and Real-time PCR systems for quantitative analysis of V. dahliae inthe soil. The aims were to set up a reliable method to predict the likelihood of cotton wiltdisease infection in agricultural fields and provide integrated methods which giving priority tocultivation. The main results were as follows:1. Comparing five methods for soil DNA extract, the standardized procedures, using wetsieving based extraction of microsclerotia followed by soil DNA extracted kit, wasestablished. This method improved10-20times using soil doses than conventional method(0.5g-1g). Yielding DNA of superior quality can used for PCR amplification directly.2. The routine PCR detection systems were developed. The specific primer pair P1/P2were designed based on the ITS regions of rDNA of V. dahlia, and the production is324bp.The detecting sensitivity for the routine PCR detection systems was1.5microsclerotia pergram soil and0.6microsclerotia per reaction for the artificially infected soil.3. The SYBR Green I PCR detection assay for quantification of V. dahliae wassuccessfully developed with the specific premier pair P1/P2. The standard curve generatedfrom recombinant plasmids were Y=-3.563X+37.837(R2=0.999) with90.8%PCRamplification efficiency. The detecting sensitivity for the SYBR Green I PCR detection assaywas10-fold higher than that of the conventional PCR. According this calibration curve, the number of microsclerotia in soil samples can be quantified.4. The relationship between gene copy numbers and microsclerotia number wasstudied.the relationship was: N=e7.3-Ct/3.905. The relationship between gene copy numbers andwilt disease was also studied. Pots were inoculated with a serious amounts of microsclerotia.Viable cotton seeds were planted in these pots with three replicates in glasshouse, and thediseases severity was surveyed at regular intervals. Meanwhile, aliquots of soil samples werecollected from all treatments for molecular detection. The result indicated that there was asignificant positive correlation between the estimates of the number of V. dahliaemicrosclerotias and the disease severity.In a word, the developed SYBR Green I real time detecting system can be applied in riskassessment of cotton wilt disease and to for providing a technical support and theoreticalfoundation in comprehensive control of this disease. Meanwhile, this study can also providetheoretical and technical references for the detection of other plant pathogens in soil.