| An immunomagnetic separation and polymerase chain reaction (IMS-PCR)-based assay was developed for the detection of Acidovorax avenae subsp. citruli (Aac) in watermelon seeds. IMS detected and recovered Aac from cell suspensions containing 10 CFU/ml, and yielded 10-fold more cells than spread plating. The presence of seed debris had a negative effect on Aac recovery. IMS-PCR was 10-fold more sensitive than direct PCR on Aac cells suspended in sterile distilled water. While watermelon seed-wash inhibited direct PCR, IMS-PCR detected Aac in suspensions containing 10 CFU/ml in 42.8% of the attempts. IMS-PCR also detected Aac in watermelon seedlots with 0.1% infestation. One hundred and nineteen Aac strains were compared using pulse field gel electrophoresis and fatty acid analysis. Both techniques indicated that there were at least two distinct groups (A and B), with group A representing the typical watermelon fruit blotch (WFB) disease pathogen. Group B represented Aac strains from cantaloupe, pumpkin and watermelon and included the American Type Culture Collection type strain, which was present in the US before the initial WFB, outbreaks in 1989. Using artificially inoculated watermelon seeds, the relationship between seedborne Aac inoculum and WFB transmission was studied in the greenhouse. Aac inoculum level had a positive effect on seedling disease but did not affect seedling emergence. Aac inoculum also increased the rate of WFB disease progress. Using IMS-PCR, seedling grow-out and semi-selective media, Aac was detected in seeds from three of 25 symptomless watermelons which developed after blossom inoculation. These preliminary observations suggest that blossom colonization may lead to watermelon seed infestation by Aac. |
