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United States Department of Agriculture

Agricultural Research Service

Research Project: IDENTIFICATION, CHARACTERIZATION, AND DETECTION OF FOREIGN AND NEWLY EMERGING DOMESTIC BACTERIA

Location: Foreign Disease-Weed Science

Title: An improved assay for detection of Acidovorax avenae subsp. citrulli in watermelon and melon seed

Authors
item Zhao, Tingchang - INST PL PROT BEJING CHINA
item Feng, Jian Jun - CHINA AG UNIV, BEJING
item Sechler, Aaron
item Randhawa, Parmjit - CALIF SEED & PLANT LAB
item Schaad, Norman

Research conducted cooperatively with:
item California Seed & Plant LAB., Inc.

Submitted to: Seed Science and Technology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: February 20, 2008
Publication Date: June 1, 2009
Citation: Zhao, T., Feng, J., Sechler, A.J., Randhawa, P., Schaad, N.W. 2009. An improved assay for detection of Acidovorax avenae subsp. citrulli in watermelon and melon seed. Seed Science and Technology. 37:337-347.

Interpretive Summary: Acidovorax avenae subsp. citrulli (Aac), the causal agent of a watermelon seedling blight and fruit blotch (WFB), has emerged as a serious seedborne pathogen of watermelon, melons, pumpkin, and citron 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 by the seed industry. Seeds contaminated with Aac remain a problem. We describe a combined agar plating, molecular-based assay for detection of Aac in watermelon and melon seeds. The assay detected Aac in extracts containing as few as one cell per ml. When healthy seeds were spiked with a single naturally infested seed containing from 1,040 to 50,000 Aac/seed, all the laboratory assays were positive. This combined agar plating, molecular detection assay should prove useful as a routine assay for detection of Aac in watermelon and melon seeds, even in those samples containing large numbers of saprophytes.

Technical Abstract: Acidovorax avenae subsp. citrulli (Aac), the causal agent of a watermelon seedling blight and fruit blotch (WFB), has emerged as a serious seedborne pathogen of watermelon, melons, pumpkin, and citron. 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 Aac remain a problem for the seed industry. We describe a combined agar plating, real-time PCR assay for detection of Aac in watermelon and melon seeds. The bacteria were extracted by soaking seeds in buffer containing vancomycin and assayed by dilution plating onto ethanol bromothymol blue/brilliant blue R (EBB) agar and EBB agar containing ampicillin (EBBA), direct (without DNA extraction) real-time PCR, and real-time BIO-PCR (enrichment PCR). Using extracts of 1,000 healthy seeds spiked with a pure culture suspension of cells of Aac, the BIO-PCR assay using EBBA agar detected Aac in extracts containing as few as one cell per ml. When replicates of 1,000 healthy seeds were spiked with a single naturally infested seed containing from 1,040 to 50,000 colony forming units of Aac/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 Aac in watermelon and melon seeds.

Last Modified: 4/21/2014
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