The Establishment Of Rapid Detection Method For The Pathogen Of Crucifer Bacterial Black Spot

Crucifer bacterial black spot, caused by Pseudomonas syringae pv. maculicola, is one of bacterial diseases on crucifer all over the world. The disease severity is growing up in recently years, which threaten the productions of crucifer vegetable seriously. Crucifer bacterial black spot is a typically seed-borne disease, and the important prevention and cure method is the quarantine of the seeds with bacteria. However, most kinds of detection methods are time-consuming, or in defect of low precision or low specificity. So in this study, a rapid and precise detection method was constructed for the detection of the bacteria in crucifer vegetable seeds.In this study, a selective culture medium was confected in order to enrich and culture the target bacteria exclusively while inhibiting growths of non-target bacteria. Antiserum to Pseudomonas syringae pv. maculicola was made and used for indirect ELISA, which stimulated the detection of bacteria in seeds. A set of specific primers for Pseudomonas syringae pv. maculicola was chosen and used in PCR. The precision of bacteria detection by PCR was app. 3×105 CFU/ml. A practicable TaqMan probe was designed and composed and the precision of bacteria detection by real-time PCR was app. 3×103 CFU/ml. Then the immuno-detection was combined with PCR by using the superparamagnetic beads to form IMS-PCR, which increase the precision of bacteria detection to app. 3×102 CFU/ml.In this study, the integrated method of bacterial detection,which was called BIO-IMS-(real-time)PCR, was formed by combining the bacterial isolation with PBS, the selective culture medium, IMS and (real-time) PCR. One seed with bacteria in 1000 commercial seeds could be detected using this integrated method.