Isolation And Identification Of Potato Black Leg And Establishment Of A Fluorescent Quantitative PCR Detection System In Ningxia

As one of the main food crops in Ningxia,potato diseases were also getting worse and worse with the expansion of continuous planting areas.Black leg was such a bacterial disease that caused by one or several pathogenic bacteria.It could occur throughout the whole development stage of potatoes,seriously affecting the production and restricting the development of potatoes in Ningxia.In this study,we investigated black leg in major potato production areas,the mountainous regions of south-central Ningxia,then isolated the pathogen causing this disease for identification,and established a fluorescent quantitative monitoring system for the main pathogenic bacteria in the soil,and finally clarified the impact of bacterial content in the soil and disease occurrence,providing a theoretical basis for risk assessment,and prediction and control of potato black leg in Ningxia.The test results were as follows:1.A total of 16 villages and towns were investigated,including Guyuan,Wuzhong,Zhongwei and Sizuishan,of which the highest incidence of 35%was found in Guanzhuang Town Longde County Guyuan,and the lower incidence of 5%-3%in Hongyao of Xigi,Haoshui of Longde and Xiamaguan of Tongxin,no potato black shank plants were found in Zhangyi Town,Honghe Town of Yuanzhou District,Malian village of Xiji County or Fengjigou Village of Yanchi.2.The collected specimens were isolated for identification,with a total of 5 strains of Pectobacterium carotovorum subsp.brasiliensis,8 strains of Pectobacterium atrosepticum,14 strains of pseudomonas grimontii and 1 strain of stenotrophomonas rhizophila.The pathogenicity test showed that the pathogenic bacteria causing potato black shank in Ningxia were P.carotovorum subsp.brasiliensis and P.atrosepticum.3.According to the reported specific primers for P.atrosepticum amplification strain TY-1-1,the specific primer paf4/par4 suitable for quantitative test was designed through specific fragments.The genomic DNA of pathogenic bacteria was amplified,using that primer,to obtain the specific fragment of 152bp.After conjugating the fragment with the carrier pClone007 and taking transformed recombinant plasmid as the standard,the standard curve of real-time fluorescent quantitative PCR plasmid of P.atrosepticum was developed.The regression equation was y=0.996x+36.813 with the correlation coefficient R2 of 0.996 and the amplification efficiency of 107.3%.The lower limit of detection was 1.39×101 copies·μL-1.The linear regression equation between the Lg value of bacterial concentration in soil and the Lg value of recombinant plasmid copy number was established as y=0.8258x-0.8798 with the correlation coefficient R2=0.9925,and bacterial concentration in soil could be detected was 7.7×102cfu·g-1.