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Polymerase Chain Reaction Detection and Phylogenetic Characterization of an Agent Associated with Yellow Vine Disease of Cucurbits

May 1998 , Volume 88 , Number  5
Pages  428 - 436

Francisco J. Avila , Benny D. Bruton , Jacqueline Fletcher , J. L. Sherwood , Sam D. Pair , and Ulrich Melcher

First author: Department of Plant Pathology, University of Nebraska, Lincoln 68583; second and fifth authors: United States Department of Agriculture, Agricultural Research Service, Lane, OK 74555; third and fourth authors: Department of Plant Pathology; and sixth author: Department of Biochemistry and Molecular Biology, Oklahoma State University, Stillwater 74078

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Accepted for publication 23 January 1998.

Diagnosis of yellow vine disease (YVD) in cucurbits, an important disease in the south-central United States, relies on external symptom appearance, phloem discoloration, and the presence of bacterium-like organisms (BLOs) in phloem. Polymerase chain reaction (PCR) amplification of BLO nucleotide sequences was explored as a means to improve diagnostic techniques. PCR, using a primer pair based on sequences of the citrus-greening BLO, amplified a 0.15-kilobase (kb) fragment from the DNA of symptomatic plants, but not from that of asymptomatic plants. Its nucleotide sequence suggested that the DNA amplified was of pro-karyotic origin. A primer pair, designed to amplify nonspecific prokaryotic 16S rDNA, amplified a 1.5-kb DNA fragment in both the symptomatic and asymptomatic plants. The 1.5-kb fragment from the asymptomatic plants corresponded to chloroplast 16S rDNA, and the band from the symptomatic plants was composed of 16S rDNAs from both chloroplasts and a prokaryote. The nucleotide sequence of the prokaryotic DNA was determined and used to design three primers (YV1, YV2, and YV3). Fragments of 0.64 and 1.43 kb were amplified with primers YV1-YV2 and primers YV1-YV3, respectively, from symptomatic plants. Neither primer set yielded fragments from asymptomatic plants, unrelated bacteria, or selected soilborne fungal pathogens of cucurbits. Phylogenetic analysis indicated that the prokaryote is a gamma-3 proteobacterium. The consistent association of the 0.64- and 1.43-kb fragments with symptomatic plants suggests that the gamma-3 proteobacterium may be the causal agent of YVD of cantaloupe, squash, and watermelon.

Additional keywords: Citrullus lanatus , Cucumis melo , Cucurbita pepo , vine decline .

The American Phytopathological Society, 1998