An improved assay for detection of Acidovorax citrulli in watermelon and melon seed

Acidovorax citrulli (Ac), the causal agent of a bacterial seedling blight and fruit blotch, has emerged as a serious seedborne pathogen of watermelon and melons world-wide. Although attempts have been made to develop a simple routine laboratory seed assay to detect the organism in seeds, none is routinely used. Seeds contaminated with Ac remain a problem for the seed industry. We describe a combined agar plating, real-time PCR assay for detection of Ac in watermelon and melon seeds. The bacteria were extracted by soaking seeds in buffer containing vancomycin and assayed by dilution plating onto semiselective ethanol bromcresol purple /brilliant blue R (EBB) agar and onto EBB agar containing ampicillin (EBBA), by direct real-time PCR. and by real-time BIO-PCR (enrichment PCR). Using extracts of 1,000 healthy seeds spiked with a pure culture suspension of cells of Ac, the BIO-PCR assay using EBBA agar detected Ac in extracts containing as few as one cell per ml. When replicates of 1,000 healthy seeds were spiked with a single naturally infected seed, all the laboratory assays, including plating onto EBB and EBBA agar, direct PCR, and BIO-PCR were positive. This combined agar plating, real-time PCR protocol should prove useful as a routine assay for detection of Ac in watermelon and melon seeds, even in those samples containing relatively high levels of saprophytic bacteria.