Detection of Acidovorax avenae subsp. citrulli in Watermelon Seed Using Immunomagnetic Separation and the Polymerase Chain Reaction

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Abstract
An immunomagnetic separation and polymerase chain reaction (IMS-PCR)-based assay was developed for detecting Acidovorax avenae subsp. citrulli in watermelon seed. IMS yielded a 10-fold increase in recovery of A. avenae subsp. citrulli over direct spread-plating on King's Medium B; however, the presence of seed debris reduced IMS efficiency. Synthetic oligonucleotide primers were designed based on the 16S rRNA gene of a known A. avenae subsp. citrulli strain and tested for specific DNA amplification by PCR. The primers amplified DNA from all A. avenae subsp. citrulli strains tested but also yielded amplicons with several closely related bacteria. IMS-PCR resulted in a 100-fold increase in A. avenae subsp. citrulli detection sensitivity over direct PCR and was unaffected by PCR inhibitors in watermelon seed. The threshold of A. avenae subsp. citrulli detection for IMS-PCR was 10 CFU/ml in watermelon seed wash, and seedlots with 0.1% infestation were consistently detected. IMS-PCR represents an efficient and sensitive approach to detecting A. avenae subsp. citrulli in watermelon seedlots.