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

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 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.

From Environment to Man: Genome Evolution and Adaptation of Human Opportunistic Bacterial Pathogens

“…Intimate associations with plants predispose phytopathogens to frequent encounters with herbivorous insects that could evolve to become specific vectors for pathogens or alternative primary hosts for phytopathogenic bacteria [44]. S. marcescens is a phloem-resident pathogen that causes yellow vine disease of pumpkins and squashes transmitted by the squash bug, Anasa tristis [45]. Not only adapted to plants and insects, S. marcescens is also an emerging human pathogen that is predominantly involved in HAIs, particularly in neonatal intensive care units [46] and also implicated in a range of ocular infections [47].…”

THE THREE DS OF PCR-BASED GENOMIC ANALYSIS OF PHYTOBACTERIA: Diversity, Detection, and Disease Diagnosis

“…Pathogen isolation attempts, serology protocols, transmission tests, and DNA-DNA hybridization experiments failed to ascertain the identity of the causal agent (18). PCR amplification of 16S rDNA, complemented with DNA sequence analysis, however, provided a phylogenetic framework to identify the causal agent as a proteobacterium (6). Specific primers were subsequently designed to amplify diagnostic fragments from the 16S rDNA for efficient diagnosis of YVD-symptomatic plants (6).…”

Overwintering Squash Bugs Harbor and Transmit the Causal Agent of Cucurbit Yellow Vine Disease

“…A plant was considered positive for CYVD if phloem discoloration was observed. In addition, a plant tissue sample was taken from the same area for bacterial isolation and polymerase chain reaction (PCR) by using methods developed by Avila et al (1998) and modiÞed by Melcher et al (1999). Tissue samples (3 mm) for isolation were surface sterilized in 10% Clorox solution for 30 s, placed on a paper towel, and sprayed with 80% ethanol; ground using a sterilized mortar and pestle; and a 10-l pipetted subsample was spread onto nutrient agar.…”