Overwintering squash bugs harbor and transmit the causal agent of cucurbit yellow vine disease.

Since 1988, cucurbit crops, particularly watermelon, cantaloupe, and squash, grown in Oklahoma and Texas have experienced devastating losses from cucurbit yellow vine disease (CYVD), caused by the phloem-limited bacterium Serratia marcescens Bizio. Squash bug, Anasa tristis (De Geer), is a putative vector of the pathogen. In 2000-2001, overwintering populations of squash bug collected from DeLeon, TX, were tested for their ability to harbor and transmit the bacterium. Individual squash bugs (n = 73) were caged serially for periods of up to 7 d on at least four squash seedlings. Two studies were conducted, one with insects collected in November 2000 placed on first true leaf-stage seedlings and the second with insects from an April 2001 collection, placed on 3-5 true leaf-stage squash. Controls consisted of squash seedlings caged without insects. Squash bug transmission rates of the pathogen in studies I and II were 20 and 7.5%, respectively. Overall, 11.0% of the squash bugs harbored and successfully transmitted the bacterium to squash seedlings. All control plants tested negative for S. marcescens and did not exhibit CYVD. Female squash bugs killed a significantly greater proportion of young first leaf-stage seedlings than males. Feeding on 3-5 leaf-stage squash resulted in no plant mortality regardless of squash bug gender. This study demonstrated that the squash bug harbors S. marcescens in its overwintering state. The squash bug-S. marcescens overwintering relationship reported herein greatly elevates the pest status of squash bug and places more importance on development of integrated strategies for reducing potential overwintering and emerging squash bug populations.

Identification, Phylogenetic Analysis, and Biological Characterization of Serratia marcescens Strains Causing Cucurbit Yellow Vine Disease.

ABSTRACT A serious vine decline of cucurbits known as cucurbit yellow vine disease (CYVD) is caused by rod-shaped bacteria that colonize the phloem elements. Sequence analysis of a CYVD-specific polymerase chain reaction (PCR)-amplified 16S rDNA product showed the microbe to be a gamma-proteobacterium related to the genus Serratia. To identify and characterize the bacteria, one strain each from watermelon and zucchini and several noncucurbit-derived reference strains were subjected to sequence analysis and biological function assays. Taxonomic and phylogenetic placement was investigated by analysis of the groE and 16S rDNA regions, which were amplified by PCR and directly sequenced. For comparison, eight other bacterial strains identified by others as Serratia spp. also were sequenced. These sequences clearly identified the CYVD strains as Serratia marcescens. However, evaluation of metabolic and biochemical features revealed that cucurbit-derived strains of S. marcescens differ substantially from strains of the same species isolated from other environmental niches. Cucurbit strains formed a distinct cluster, separate from other strains, when their fatty acid methyl ester profiles were analyzed. In substrate utilization assays (BIOLOG, Vitek, and API 20E), the CYVD strains lacked a number of metabolic functions characteristic for S. marcescens, failing to catabolize 25 to 30 compounds that were utilized by S. marcescens reference strains. These biological differences may reflect gene loss or repression that occurred as the bacterium adapted to life as an intracellular parasite and plant pathogen.

Genotyping of Serratia marcescens Strains Associated with Cucurbit Yellow Vine Disease by Repetitive Elements-Based Polymerase Chain Reaction and DNA-DNA Hybridization.

ABSTRACT The bacterium that causes cucurbit yellow vine disease (CYVD) has been placed in the species Serratia marcescens based on 16S rDNA and groE sequence analysis. However, phenotypic comparison of the organism with S. marcescens strains isolated from a variety of ecological niches showed significant heterogeneity. In this study, we compared the genomic DNA of S. marcescens strains from different niches as well as type strains of other Serratia spp. through repetitive elements-based polymerase chain reaction (rep-PCR) and DNA-DNA hybridization. With the former, CYVD strains showed identical banding patterns despite the fact that they were from different cucurbit hosts, geographic locations, and years of isolation. In the phylogenetic trees generated from rep-PCR banding patterns, CYVD strains clearly were differentiated from other strains but formed a loosely related group with S. marcescens strains from other niches. The homogeneity of CYVD strains was supported further by the DNA relatedness study, in that labeled DNA from the cantaloupe isolate, C01-A, showed an average relative binding ratio (RBR) of 99%, and 0.33% divergence to other CYVD strains. Used as a representative strain of CYVD, the labeled C01-A had a RBR of 76%, and a 4.5% divergence to the S. marcescens type strain. These data confirm the previous placement of CYVD strains in S. marcescens. Our investigations, including rep-PCR, DNA-DNA hybridization, and previous phenotyping experiments, have demonstrated that CYVD-associated strains of S. marcescens cluster together in a group significantly different from other strains of the species.

Serratia marcescens, a Phloem-Colonizing, Squash Bug -Transmitted Bacterium: Causal Agent of Cucurbit Yellow Vine Disease.

Cucurbit yellow vine disease (CYVD), which can inflict heavy losses to watermelon, pumpkin, cantaloupe, and squash in U.S. production areas from the midwest to northeastern states, causes phloem discoloration, foliar yellowing, wilting, and plant decline. Bacteria were cultured from the phloem of crown sections of symptomatic plants of Citrullus lanatas and Cucurbita pepo. Those bacteria testing positive in CYVD-specific polymerase chain reaction (PCR) were all gram negative and appeared morphologically identical, producing creamy white, smooth, entire, convex colonies on Luria-Bertani or nutrient agar. Characterized cucurbit-derived strains of Serratia marcescens were introduced into greenhouse-grown squash plants by puncture inoculation and into field-grown squash plants by enclosure with S. marcescens-fed squash bugs, Anasa tristis. Up to 60% of the bacteria-inoculated plants in the greenhouse and up to 17% of field plants caged with inoculative squash bugs developed phloem discoloration and tested positive for S. marcescens by CYVD-specific PCR. None of the controls developed phloem discoloration or tested positive by PCR. Of the diseased field plants, 12% (2 of 35) also yellowed, wilted, and collapsed, exhibiting full symptom development of CYVD. However, neither plant collapse nor decline was observed in the greenhouse-grown, puncture-inoculated plants. The morphology, growth habit, and PCR reaction of bacteria cultured from crown tissue of a subset of plants in each experimental group were indistinguishable from those of the inoculum bacteria. Evidence presented from our studies confirms that the squash bug can transmit S. marcescens, the CYVD causal bacterium. The S. marcescens-A. tristis relationship described here is the first instance in which the squash bug has been identified as a vector of a plant pathogen. Our experiments represent a completion of the steps of Koch's postulates, demonstrating that S. marcescens is the causal agent of CYVD and that the squash bug, A. tristis, is a vector of the pathogen.

Effect of Insect Exclusion on the Incidence of Yellow Vine Disease and of the Associated Bacterium in Squash.

Yellow vine (YV) of cucurbits, associated with a phloem-limited bacterium, causes rapid wilting and death in affected plants. In a previous study, experimental insecticide-treated plots had a lower incidence of YV than untreated plots, suggesting that insects were involved in the transmission of the bacterium. In the study reported here, we compared the incidence of YV and polymerase chain reaction (PCR) detection of the YV bacterium in noncovered squash plants (Cucurbita pepo var. melopepo) with plants covered with fine-mesh fabric secured in such a way that insects were excluded. Rows of squash were covered with row mesh cover that was stretched over hoops and anchored in the soil. The row cover was removed after 40 or 50 days, at which time all plants were sampled destructively by harvesting the crown and root. In the first experiment, 3% of the noncovered plants had foliar symptoms, 7% were positive with the use of Dienes' stain, and 25% were positive when analyzed by PCR with specific primers. No covered plants were positive by any detection method, and no plants in the second experiment had foliar symptoms or tested positive with Dienes' stain. However, 20% of noncovered and 0% of covered plants were PCR positive. These data support the hypothesis that insects were involved in the transmission of the bacterium.

Detection of Cucurbit Yellow Vine Disease in Squash and Pumpkin in Massachusetts.

Commercial plantings of summer squash in Charlemont, Franklin County, MA, were decimated in 1999 by 100% incidence of a yellowing disease resembling cucurbit yellow vine disease (CYVD) (1). Both plantings were established in the same field during the third week of May, one with transplants and the second by direct-seeding. Each planting consisted of four 30-m rows each of yellow zucchini (Cucurbita pepo cv. Gold Rush), summer squash (C. pepo cv. Seneca Prolific), and zucchini (C. pepo cv. Condor). Crops were produced organically and pyrethrum was used to control a high infestation of squash bugs, Anasa tristis (De Geer) (Heteroptera:Coreidae), a putative vector of CYVD (3). Just prior to fruit set, during the first two weeks of June, plants began showing symptoms of foliar chlorosis, plant stunting, or both. All of the plants in the field eventually wilted and collapsed. Cross-sections of the below-ground stem and primary root revealed a honey-brown phloem discoloration and healthy appearing xylem, symptoms characteristic of CYVD. Plants yielded marketable fruit for only about 1 week. When plant samples were tested by polymerase chain reaction (PCR) with CYVD bacterium specific primers (2), a band of the expected size for the CYVD bacterium, identified as Serratia marcescens based on 16s rDNA and groE sequence analyses (4), was amplified in every case. Since all plant samples collected were symptomatic and PCR positive for S. marcescens, asymptomatic greenhouse plants were run simultaneously as a control. All control plants tested negative. A third planting, similar to the two disease-affected plantings and containing the same three squash cultivars from the same seed lot, was established at about the same time approximately 3 km away. No symptoms of CYVD occurred at this site, further evidence that the pathogen is not seed-borne (1). Furthermore, squash bugs were not observed in this field. In 2000, the disease was observed in a planting of 'Atlantic Giant' pumpkin in Erving, Franklin County, MA, and confirmed by PCR. Until now, CYVD has been reported only in the states of Oklahoma, Texas, and Tennessee. Confirmation of the disease in Massachusetts significantly increases the known geographical range of CYVD to include the New England area. References: (1) B. D. Bruton et al. Plant Dis. 82:512-520, 1998. (2) U. Melcher et al. Phytopathology 89:S95, 1999. (3) S. D. Pair et al. Pages 145-148 in: Proc. 19th Ann. Hort. Conf., Okla. State Univ. (4) J. Rascoe et al. Phytopathology 90:S63, 2000.

Yellow Vine of Watermelon and Pumpkin in Tennesse.

Yellow vine (YV) is a recently recognized decline of cucurbits expressed as plant yellowing, phloem discoloration, and death of vines as fruit approach maturity. In severely affected fields, YV incidence can range from 50 to 100% with similar yield loss. The disease has been associated with a phloem-limited, walled bacterium belonging to the gamma-3-proteobacteria (1), for which specific polymerase chain reaction (PCR) primers have been developed and used in diagnosis (2). First observed in 1988 in Oklahoma and Texas squash and pumpkin, YV was not detected in watermelon and cantaloupe until 1991. The disease has never been detected in cucumber. Efforts to date have been unsuccessful in transmitting the disease with dodder, grafting, or selected insects. Initially, the geographic range of the disease appeared to be generally confined to central and northeastern Oklahoma and north central Texas, an area known as the Cross Timbers Region. In 1997 to 1998, YV was diagnosed in commercial fields of watermelon and muskmelon from east Texas (Post Oak Savannah) and all cucurbit-growing areas of Oklahoma. In late summer 1998, symptoms similar to those of YV were observed in one watermelon (Hardeman County) and three pumpkin (Rhea and Morgan counties) fields in Tennessee where the leaves turned yellow and chlorotic and affected plants exhibited phloem discoloration. Estimated incidence of YV ranged from less than 1 to 20% of the plants in affected fields. PCR, with the YV-specific primers (2), amplified a band of the expected size (409 bp) from all watermelon and pumpkin plants exhibiting phloem discoloration. In contrast, no bands were amplified from asymptomatic (no phloem discoloration) watermelon or pumpkin. The nucleotide sequence of the DNA fragment amplified from a Tennessee watermelon and pumpkin plant was identical to that of the YV bacterium. The occurrence of YV outside of the Cross Timbers Region, and in a location as distant as Tennessee, suggests that the disease may be much more widespread than previously recognized. Diagnosis and monitoring of YV in all cucurbit-growing areas is critical for determining the geographic distribution and losses caused by this emerging disease. References: (1) F. J. Avila et al. Phytopathology 88:428, 1998. (2) U. Melcher et al. (Abstr.) Phytopathology. 89(suppl.):S95, 1999.

Association of a Phloem-Limited Bacterium with Yellow Vine Disease in Cucurbits.

Since 1991, a new disease of cucurbits in central Texas and Oklahoma, designated yellow vine, has resulted in the decline and plant death of watermelon, cantaloupe, squash, and pumpkin. Affected plants are characterized by leaf yellowing, phloem discoloration, and plant collapse. Year-to-year variation in disease incidence has ranged from spotty outbreaks to complete crop loss in early-planted watermelon fields. A systematic investigation to determine the causal agent of the disease included pathogen isolation attempts, transmission tests, serological assays with various antisera (enzyme-linked immunosorbent assay and Western blotting), and DNA hybridizations with selected probes (dot and Southern blots). None of these tests revealed a consistent relationship between the expression of yellow vine symptoms and the presence of a particular microorganism or virus in the plant. However, transmission electron microscopic examination showed the consistent presence of a bacterium in the phloem sieve elements of symptomatic plants. The rod-shaped bacteria, observed only in symptomatic cucurbits, measured 0.25 to 0.5 µm in width and 1.0 to 3.0 µm in length and were surrounded by a triple-layered cell envelope.

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.