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

Authors

Francisco J. Avila, University of Nebraska-Lincoln
Benny D. Bruton, USDA-ARSFollow
Jacqueline Fletcher, Oklahoma State University
J. L. Sherwood, Oklahoma State University
Sam D. Pair, USDA-ARS
Ulrich Melcher, Oklahoma State University

Document Type

Article

Date of this Version

5-1998

Citation

Avila, F. J., Bruton, B. D., Fletcher, J., Sherwood, J. L., Pair, S. D., and Melcher, U. 1998. Polymerase chain reaction detection and phylogenetic characterization of an agent associated with yellow vine disease of cucurbits. Phytopathology 88:428-436.

Comments

Copyright 1998 The American Phytopathological Society. Used by permission.

Abstract

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 prokaryotic 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.