Submitted to: Seed Science and Technology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 1/31/2005
Publication Date: 1/1/2006
Citation: Walcott, R.R., Castro, A.C., Fessehaie, A., Ling, K. 2006. Progress towards a commerical PCS-based seed assay for Acidovorax avenae subsp. citrulli. Seed Science and Technology. 34:101-116. Interpretive Summary: Bacterial fruit blotch caused by Acidovorax avenae subsp. citrulli posts a great threat to the watermelon production in the southeastern United States. The disease management strategy for this devastating disease on watermelon is through the use of pathogen-free seed. Currently seed assay for AAC is through a laborious process of greenhouse seedling grow out. This report describes the development of a molecular technique, called Immunomagnetic Separation Polymerase Chain Reaction (IMS-PCR), and its application to test commercial seed samples for the pathogen. It was concluded that IMS-PCR provided a reliable and sensitive testing on seeds as that of the standard seedling grow-out test.
Technical Abstract: To develop a commercial-scale PCR-based assay for Acidovorax. avenae subsp. citrulli in watermelon seed, parameters for immunomagnetic separation (IMS) were optimized. Optimal conditions for target cell recovery included 40 'g of polyclonal anti-AAC antibody per 108 immunomagnetic beads (IMBs) for coating, ca. 2.5 x 107 IMBs per sample and immunocapture for 1 h. Using these parameters a detection threshold of 10 CFU/ml was consistently observed for IMS-PCR. Enrichment of seed extract in antibiotic-amended Luria-Bertani broth increased IMS sensitivity 100-fold. Using seeds artificially infested with A. avenae subsp. citrulli (104 CFU/seed) IMS-PCR displayed detection frequencies of 25% and 87.5% for seedlots (n=10,000 seeds) with 0.01 and 0.1%, respectively. In contrast the detection frequencies for the seedling grow-out assay (SGO) were 12.5% and 37.5% for similar seedlots. The difference in detection frequencies between SGO and IMS-PCR were not significant. Enrichment did not improve the detection frequency of IMS-PCR for artificially infested seeds; however, IMS-PCR and enrichment IMS-PCR detected A. avenae subsp. citrulli in or 100% of seed lots with 0.01% natural infestation.