Abstract

It is well known that bacterial fruit blotch (BFB) of watermelon caused by Acidovorax avenae subsp. citrulli threatens watermelon production. There is no pathogen enrichment or concentrated steps before detection in conventional PCR-based diagnostic technique, which consequently leads to the lower detection sensitivity. In the present study, two different bacterial suspensions were prepared, and four pathogen enrichment protocols including Biological Preparation (BP), Membrane Filtration (MF), Immune-Magnetic Separation (IMS) and Immune-Capture Preparation (ICP) were employed to prepare the PCR template for the pathogen detection. Double Antibody Sandwich Enzyme Linked Immunosorbent Assay (DAS-ELISA) was also conducted as a parallel comparison. The results showed that IC-PCR was the optimum method through comparing the detection limits, time and expenditure among those methods. The detection limit of IC-PCR for both bacterial suspension and seeds suspension can reach 10² CFU/ml, which was 10 times lower than that of IMS-PCR when the seed suspension was used as template. In addition, the results of IC-PCR showed higher degree of precision than those of IMS-PCR. The time and expenditure of IC-PCR were comparable with other methods. The present study demonstrated that the procedures of immune-capture PCR is a sensitive, specific, rapid, reproducible, and economical method to detect A. avenae subsp. citrulli in watermelon seeds.

Key words: Bacterial Fruit Blotch, watermelon seed, pathogen enrichment, PCR.