Cell wall-degrading enzymes of Didymella bryoniae in relation to fungal growth and virulence in cantaloupe fruit.

Didymella bryoniae is an important pathogen of cucurbits worldwide. Virulence factors of D. bryoniae were investigated in regard to fungal growth and the production of cell wall-degrading enzymes, polygalacturonase (PG), pectate lyase (PL), pectin lyase (PNL), ß-galactosidase (ß-Gal) and cellulase (Cx). Virulence levels of five D. bryoniae isolates were determined by the severity of inoculated cantaloupe fruit decay. The highly virulent isolates had more mycelial growth than the moderately virulent isolates in different media. PG activities produced by the highly virulent isolates in shake cultures and in decayed fruit were greater than those of the moderately virulent isolates. PNL, but not PL, in decayed fruit was higher with the highly virulent isolates compared to the moderately virulent ones. The highly virulent isolates showed higher Cx activity than the moderately virulent ones in decayed fruit and in fruit tissue shake culture. ß-Gal activities of the highly virulent isolates in pectin shake culture and in decayed fruit were greater than those of the two moderately virulent isolates although fruit also produced ß-Gal. Protein analysis showed two fungal ß-Gal isozymes in decayed fruit compared to those of healthy fruit. Correlation analysis indicated that the activities of PG, PNL, ß-Gal and Cx in cultures and in decayed fruit positively correlated with fungal growth and fruit decay severity. The results of this study suggest that PG, PNL, ß-Gal, and Cx appear to be virulence factors of D. bryoniae in cantaloupe decay with PG and ß-Gal as the most predominant fruit decay enzymes.

First Confirmed Report of Tobacco ringspot virus in Cucurbits Crops in Oklahoma.

Cucurbits are major cash crops of vegetable growers in Oklahoma, particularly watermelon, which is the official state vegetable. In 2010, during a survey for cucurbit viruses (1), symptomatic leaf samples of cucumber (Cucumis sativus), cantaloupe (Cucumis melo), pumpkin, (Cucurbita pepo), squash (Cucumis maxima), and watermelon (Citrullus lanatus) showing mild to severe mosaic, mottling, and chlorotic spots were collected in Atoka, Blaine, Jefferson, and Tulsa counties. A total of 161 samples were tested by dot-immunobinding assay (DIBA) (2) using Tobacco ringspot virus (TRSV; genus Nepovirus, family Comoviridae) specific antiserum. Fourteen samples of cantaloupe, pumpkin, and watermelon from Blaine, Jefferson, and Tulsa counties were positive serologically to TRSV. At least one to two samples from each representative cucurbit collected in the field above were used as a source for mechanical inoculation. Sap was extracted from symptomatic leaves using 0.1 M K2HPO4 buffer (pH 7.2) and rub-inoculated to two squash (cv Elite) seedlings at cotyledonary stage pre-dusted with Carborundum. Seven to 10 days post-inoculation, all inoculated plants produced typical TRSV symptoms including chlorotic spots, systemic ringspot, severe leaf deformation, mottling, and stunting. Sap and total RNA was extracted from 10 mechanically inoculated squash seedlings and tested by DIBA and reverse transcription (RT)-PCR using specific TRSV primers (F: 5'-TACAGTGAGGATGCATG-3' and R: 5'-AGTAGCTGCGACAAGCCA-3'). All of the tested samples were positive by DIBA except the negative control. Similarly, all samples from mechanically inoculated plants were also positive by PCR showing the expected 1,039-bp PCR product when analyzed by agarose gel electrophoresis. Total RNA obtained from mock-inoculated squash seedlings used as a control was negative by PCR. Amplified PCR product (1,039 bp) was directly sequenced from three infected squash seedlings. Sequence analysis confirmed that the virus shared 90 to 92% nucleotide and 94% amino acid identities with RNA2 of TRSV isolate from the U.S. (Accession No AY363727) available in the GenBank database. Total RNA extracted from original tissues of 14 DIBA positive samples collected from field were also positive by RT-PCR. The presence of TRSV could pose a serious threat to many vegetable crops, particularly cucurbits and other agricultural crops, due to its wide host range (3). This report confirms the suspected occurrence of TRSV in 1956 from watermelon in Oklahoma (4). References: (1) Ali et al. Plant Dis. 96:243, 2012 (2) A. Ali and J. W. Randles. Plant Dis. 81:343, 1997 (3) M. J. Adams and J. F. Antoniw. Outlooks Pest Manage. 16:268, 2005 (4) R. J. Shephered and F. B. Struble. Phytopathology 46:358, 1956.

First Report of the Cucurbit Yellow Vine Disease Caused by Serratia marcescens in Watermelon and Yellow Squash in Alabama.

More than 3,000 acres of watermelon were planted in Alabama in 2010 with a production value more than $4 million (J. Kemble, personal communication). Symptoms typical of cucurbit yellow vine disease (CYVD) were observed in a 2-ha watermelon field in Crawford, AL on 8 June 2010. Watermelon, cv. Jubilee, exhibited a yellow appearance and some plants were completely wilted. Incidence of affected plants was estimated at 25%. On 24 June, plant samples were collected from a 1-ha watermelon (cv. Jubilee) field near Dadeville, AL. Approximately 30% of the plants exhibited yellowing and wilting, which is symptomatic of CYVD. Samples were also collected from a small planting of yellow crooked-neck squash at the same location. Approximately 20% of the squash plants had symptoms typical of CYVD. Cross-sections of belowground stem and primary root revealed a honey-brown phloem discoloration and a healthy appearing xylem, symptoms consistent with CYVD caused by the phloem-colonizing bacterium, Serratia marcescens Bizio (1). Isolations were made from the crown on four symptomatic watermelon and two squash plants. Approximately 2.5-mm3 tissue pieces from the phloem were excised, surface sterilized in 10% sodium hypochlorite, and ground in 1-ml PBS (phosphate buffer with saline). A 10-µl aliquot of slurry was plated onto nutrient agar (NA) (Difco, Detroit, MI) and the plates were stored at room temperature for 4 days. Individual colonies were selected and purified by serial dilution plating. Isolates from watermelon and squash were consistent with S. marcescens in colony morphology, color, and texture. Three isolates obtained from watermelon were grown on NA and suspended in sterile water at 108 cells per ml for mechanical transmission experiments on 'Lemondrop' squash. Sterile water served as a negative control. After 28 days, plants were cross-sectioned at the juncture of the root and stem and observed for phloem discoloration. Of the 56, 58, and 62 plants inoculated in three replicate studies, 78.6, 56.9, and 62.9% developed CYVD symptoms, respectively, while none of the controls were positive. Cultured bacteria from six of the symptomatic, greenhouse-inoculated plants representing the three watermelon isolates were subjected to multiplex end-point PCR using primer sets YV1/YV4, specific for the species S. marcescens, and a79F/R, which amplifies only the CYVD strains of S. marcescens (3). All six bacteria cultures along with the positive control (reference isolate W01 obtained from watermelon in Texas) were positive, while the negative PBS control was negative. Although rhizosphere-inhabiting and plant growth promoting endophytic strains of S. marcescens have been reported from Alabama (2), to our knowledge, this is the first known report of CYVD and phytopathogenic S. marcescens in Alabama cucurbits. References: (1) B. D. Bruton et al. Plant Dis. 87:937, 2004. (2) J. A. McInroy and J. W. Kloepper. Plant Soil 173:333, 1995. (3) Q. Zhang et al. Appl. Environ. Microbiol. 71:7716, 2005.

Race 3, a New and Highly Virulent Race of Fusarium oxysporum f. sp. niveum Causing Fusarium Wilt in Watermelon.

Three races (0, 1, and 2) of Fusarium oxysporum f. sp. niveum have been previously described in watermelon (Citrullus lanatus) based on their ability to cause disease on differential watermelon genotypes. Four isolates of F. oxysporum f. sp. niveum collected from wilted watermelon plants or infested soil in Maryland, along with reference isolates of races 0, 1, and 2, were compared for virulence, host range, and vegetative compatibility. Race identification was made on the watermelon differentials Sugar Baby, Charleston Gray, Dixielee, Calhoun Gray, and PI-296341-FR using a root-dip, tray-dip, or pipette inoculation method. All four Maryland isolates were highly virulent, causing 78 to 100% wilt on all differentials, one of which was PI-296341-FR, considered highly resistant to race 2. The isolates also produced significantly greater colonization in the lower stems of PI-296341-FR than a standard race 2 reference isolate. In field microplots, two of the isolates caused over 90% wilt on PI-296341-FR, whereas no disease was caused by a race 2 isolate. All four isolates were nonpathogenic on muskmelon, cucumber, pumpkin, and squash, confirming their host specific pathogenicity to watermelon. The Maryland isolates were vegetatively compatible to each other but not compatible with the race 2 isolates evaluated, indicating their genetic difference from race 2. This study proposes that the Maryland isolates belong to a new race, race 3, the most virulent race of F. oxysporum f. sp. niveum described to date.

First Report of Fusarium Wilt Caused by Fusarium oxysporum f. sp. niveum Race 2 in Georgia Watermelons.

Watermelon (Citrullus lanatus (Thunb.) Matsum. & Nakai) is the number one specialty crop grown in Georgia, a state that ranks fourth nationally in watermelon production. In the last 5 years, Fusarium wilt caused by Fusarium oxysporum f. sp. niveum (Fon) has been the greatest yield-limiting disease of watermelon in Georgia. In 2004, a seedless-watermelon field of 'Regency' and 'Tri-X 313' in Berrien County, GA exhibited approximately 40% of wilted plants. Affected plants also exhibited strong discoloration in the crown xylem. Plant samples (cultivars unknown) from a similarly affected field were also tested from Crisp County, GA. Xylem tissue was excised from the main stem of eight diseased plants in the area between the second and third internode, surface sterilized for 1 min in 1% NaOCl, rinsed with 80% ethanol, and plated onto water agar amended with 100 µg/liter of streptomycin sulfate. Fungi with the morphological characteristics of Fusarium oxysporum (4) were consistently recovered from the diseased tissue of all eight plants. The isolates were hyphal tipped and maintained in vials of sterile artificial potting mix until ready for use (1). Isolates were grown on Esposito and Fletcher medium (2) for 10 days, filtered through cheesecloth, and adjusted to 1 × 106 spores/ml. Reference isolates of race 1 and 2 were used as comparisons for race determination of the unknowns. In each of four studies, plants at the two-leaf stage were removed from potting mix, washed gently, and their roots were uniformly trimmed to 2.5 cm. Before repotting, the seedlings were subjected to a 2-min root-dip in the respective spore-containing media. In each study, approximately 40 plants of each watermelon differential were inoculated with the respective isolates. In disease scoring, each plant was considered a rep. 'Black Diamond' is susceptible to races 0, 1, and 2; 'Charleston Gray' is resistant to race 0; 'Calhoun Gray' is resistant to races 0 and 1, and PI-296341-FR (3) is resistant to races 0, 1, and 2 of Fon. Four plants were planted per 15-cm plastic pot, maintained in an air-conditioned headhouse for 24 h, and then placed in the greenhouse in a randomized complete block design. After 30 days, all plants were rated as to healthy, wilted, or dead plants. From eight isolates tested, one isolate from each county was determined to be Fon race 2 on the basis of its ability to wilt/kill a high percentage of the race 1 resistant differential, i.e., 'Calhoun Gray'. Mean disease percentages for the isolates from each of the two counties on the watermelon differentials were 95 and 100% on 'Black Diamond', 68 and 80% on 'Charleston Gray', and 70 and 86% on 'Calhoun Gray.' Because of apparent genetic drift within our PI-296341-FR population, we determined that these data were not useful for identifying race 2. In fact, we observed a range of 17 to 80% wilt/death in the PI-296341-FR over a total of four studies that included a known race 2 isolate (Calg 13(15); E. Vivoda). To our knowledge, this is the first report of race 2 in Georgia and it increases the number of states to seven in which race 2 has been identified. Five of the top 10 watermelon-producing states have now reported race 2 of Fon for which there is no genetic resistance within commercial cultivars. References: (1) B. D. Bruton et al. Plant Dis. 84:907, 2000. (2) R. Esposito and A. Fletcher. Arch. Biochem. Biophys. 93:369, 1961. (3) R. D. Martyn and D. Netzer. HortScience 26:429, 1991. (4) P. E. Nelson et al. Fusarium Species: An Illustrated Manual for Identification. Pennsylvania State University Press, University Park, 1983.

First Report of Verticillium Wilt of Watermelon in the Texas High Plains.

Verticillium dahliae (Kleb.) is known worldwide as a destructive soilborne pathogen with a wide host range (2). Reports of V. dahliae attacking cucurbits are generally limited to 'Casaba' and 'Persian' type melons. During August and September of 2004 to 2006, fields of seedless watermelon (Citrullus lanatus [Thunb.] Matsum. & Nak.) and pollinators in Yoakum County, Texas, exhibited severe symptoms of vine decline. There was no apparent difference between diploid and triploid watermelon cultivars. Night-time temperatures during July, August, and September averaged 20°C or less. Losses were estimated in excess of one-half million dollars. Symptoms consisted of leaf yellowing, wilting, and gradual death of the leaves, but stems generally remained green. The xylem exhibited a uniform tan-to-light brown discoloration that often extended throughout the vine. Dead plants had numerous microsclerotia embedded throughout the root and crown. Crown and root sections (1 cm long) from triploid plants were surface disinfected in 0.5% NaOCl for 30 s, transferred to water agar with 100 ppm of streptomycin sulfate, and incubated at 25°C. Slow-growing colonies were transferred to potato dextrose agar after approximately 72 h. V. dahliae was identified on the basis of morphology (3). Pathogenicity of four selected isolates was determined on the watermelon cultivars used to identify races of Fusarium oxysporum f. sp. niveum (Fon). Flasks containing 100 ml of medium (1) were inoculated with a 1-ml spore suspension at 1 × 105 spores/ml for each isolate and placed on an orbital shaker for 6 days at 100 rpm with continuous near-UV/fluorescent lighting at 25°C. Roots of approximately 40 plants of each of five watermelon cultivars (1 to 2 true-leaf stage) were trimmed to 2 cm long and root dipped for 2 min in the spore suspension (1 × 106/ml) of each isolate. Each cultivar/isolate combination and controls were transplanted into 10 pots (1.5 liter) with four plants per pot. The pots were transferred to the greenhouse where soil temperatures ranged between 15 and 25°C and were fertilized (Jack's fertilizer solution) every 7 days. Plants were rated at the end of 28 days as 1 = healthy, 2 = stunting (≤50% of controls), 3 = wilting, and 4 = dead. Initial wilting was observed within 7 to 10 days postinoculation. All four isolates caused varying degrees of vascular discoloration, stunting, wilting, and plant death. The pathogen was reisolated from symptomatic plants but not the controls. Mean disease ratings for the most virulent Texas isolate (28-040215) on 'Black Diamond', 'Charleston Gray', 'Dixie Lee', 'Calhoun Gray', and 'PI 296341 FR' were 2.7, 3.0, 3.0, 2.9, and 2.9, respectively. All watermelon Fon differentials were equally susceptible to V. dahliae in these studies. Historically, Verticillium wilt has been a problem in this area, which has been in cotton production for approximately 100 years. In the past decade, watermelon production has increased substantially to approximately 3,600 ha in the Texas High Plains. To our knowledge, this is the first known report of Verticillium wilt on watermelon in Texas. References: (1) R. G. Esposito and A. M. Fletcher. Arch. Biochem. Biophys. 93:369, 1961. (2) G. F. Pegg and B. L. Brady. Verticillium Wilts. CABI Publishing, New York, 2002. (3) H. C. Smith. N. Z. J. Agr. Res. 8:450, 1965.

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.

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.

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.

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.

Quantitative Response of Cucumis melo Inoculated with Root Rot Pathogens.

This experiment quantified the effects of three root rot pathogens on muskmelon (Cucumis melo L., var. cantalupensis) growth traits using computerized image analysis. Plants were grown from seed in sand infested with the soilborne pathogen Monosporascus cannonballus, Acremonium cucurbitacearum, or Rhizopycnis vagum. After 28 days in the growth chamber, images of plants were analyzed to quantify their response. Compared to noninoculated muskmelons, inoculated plants had significantly increased mean root diameter (45%), decreased root length (26%, primarily in roots of <0.5 mm diameter), decreased number of root tips (27%), decreased rhizosphere volume (40%), and decreased cumulative and mean surface area of leaves (24%). Effects of M. cannonballus on muskmelon growth were significantly different compared to A. cucurbitacearum and R. vagum. Isolate effects manifested a greater magnitude of difference on muskmelon traits than those observed at the species level. Multivariate analyses of plant responses were more powerful than univariate analyses to differentiate among effects of pathogen species and pathogen isolates. Discriminant analysis were useful to identify groups of plant traits modified by each fungal species or isolate at low disease levels. Digital image analyses proved to be a useful technique in quantitative assessment of plant damage caused by soilborne root rot pathogens.

Assessment of Virulence of Acremonium cucurbitacearum and Monosporascus cannonballus on Cucumis melo.

Assessment of plant response to inoculation with soilborne pathogens is essential for evaluation of fungal virulence. Combined plant responses (damage to hypocotyl, root-stem junction, primary root, secondary roots, and reduction in leaf area) used to derive a disease severity index (DSI) provided a functional assessment of both plant damage and pathogen virulence. Spanish isolates of Acremonium cucurbitacearum ranged from virulent to highly virulent on muskmelon cv. Magnum 45, whereas isolates from the Lower Rio Grande Valley (LRGV) of Texas ranged from weakly virulent to moderately virulent. Spanish isolates of Monosporascus cannonballus ranged from nonpathogenic to weakly virulent, whereas LRGV isolates ranged from weakly virulent to moderately virulent. Regression analysis of multiple inoculum densities established that CFU per gram of soil of 5, 10, 20, and 40 for M. cannonballus and 0.1 × 104, 1 × 104, 2 ×104, and 3 × 104 for A. cucurbitacearum, respectively, were adequate for virulence assessment of isolates of either fungus in greenhouse tests on seedlings. Seed planting depth had a significant effect on DSI. The 4-cm depth generally had smaller standard errors of the mean disease rating, indicating greater precision in determining isolate virulence.

Relationship of Developmental Stage of Cantaloupe Fruit to Black Rot Susceptibility and Enzyme Production by Didymella bryoniae.

Black rot of cantaloupe fruit, caused by Didymella bryoniae, can be severe when environmental conditions and fruit developmental stages are favorable for infection. Symptoms of black rot on cantaloupe fruit varied greatly depending on fruit age. The black rot phase was observed only on mature fruit. Inoculation of cantaloupe fruit at different developmental stages with five D. bryoniae isolates resulted in the greatest amount of decay on 10-day-old fruit compared with 20-, 30-, 40-, or 50-day-old fruit. There was no difference in lesion size among 20-, 30-, 40-, or 50-day-old fruit, although there was variation in lesion size among fungal isolates. Five fungal isolates all produced the greatest polygalacturonase (PG) activity in inoculated 10-day-old fruit compared with 20-, 30-, 40- or 50-day-old fruit. There was a positive correlation between lesion size and total fungal PG activity in decayed tissue. Using a representative D. bryoniae isolate (OK 963096), multiple PG isozymes were detected in both fungal shake culture and decayed fruit. Eleven PG isozymes (pI 4.7 to 7.9) were detected from fungal shake culture using pectin or polygalacturonic acid as the sole carbon source. Twelve PG isozymes (pI 4.7 to 8.7) were detected from decayed tissue of 10-day-old fruit, and 13 PG isozymes (pI 4.2 to 8.7) were observed from decayed tissue of 50-day-old fruit. The activity of D. bryoniae PG produced in vitro and in vivo was optimum at pH 5.0 and 5.5, respectively. The activity of the fungal PG produced in vitro exhibited primarily an endo-mode of action. In contrast, PG extracted from decayed tissue was predominately exo-PG. Thus, PG may play an important role in pathogenesis of D. bryoniae during cantaloupe fruit decay.

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.

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.

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.

Occurrence of Vine Decline Diseases of Muskmelon in Guatemala.

Central American countries have become major production areas of melons (Cucumis melo L.) destined for export to Europe and the United States during the winter months. Double cropping in the same fields year after year has created serious vine decline diseases in melons grown in the Capco area of Guatemala. Vines typically appear healthy until the fruit are approaching maturity, when the crown leaves begin to turn yellow and collapse and the decline gradually radiates outward. There was evidence of slightly water-soaked lesions in the crown of some plants; however, the symptoms were not similar to charcoal rot or gummy stem blight. The roots exhibited some decay, lesions, and discoloration, but were not macerated and rotten. A minimum of 10 affected plants were collected from each of nine fields in the Capco area of Guatemala. Isolations were made from the crown area, primary root, and secondary roots of selected plants by excising 3- to 5-mm pieces and surface sterilizing them for 30 to 60 s with 0.5% sodium hypochlorite. The isolations were made on 2% water agar plus 0.1 g of streptomycin sulfate per liter. Plates were examined for fungal growth daily for 5 days with hyphal tips from all colonies transferred to potato dextrose agar and Synthetischer nährstoffärmer agar (2) and allowed to sporulate. Isolations revealed the presence of several fungi capable of causing vine decline or suspected of being involved in the vine decline complex. Rhizoctonia solani Kühn and Fusarium semitectum Berk. & Ravenel were isolated from more than 50% of plants in some fields. F. semitectum, R. solani, and a Stagonospora-like fungus were isolated from 38, 18, and 17% of the total plants, respectively. R. solani was isolated from the crown more often than from the roots, but its role is not understood. With one exception, F. semitectum was always isolated from the crown area, which may suggest a more significant role in the vine decline complex than suspected previously. The Stagonospora-like fungus, isolated from 30% of the plants from one field, has been reported as a pathogen of cantaloupe that may contribute to the vine decline complex (1). Monosporascus cannonballus Pollack & Uecker and Fusarium oxysporum Schlechtend.:Fr. emend. W. C. Snyder & H. N. Hans. were isolated from 60% of the plants from one field. It was the only field in which M. cannonballus was isolated. Plectosporium tabacinum (van Beyma) M. E. Palm, W. Gams et Nirenberg, a cucurbit pathogen, was isolated infrequently. This is the first report of M. cannonballus, P. tabacinum, and the Stagonospora-like fungus in Guatemala. References: (1) M. E. Miller et al. Phytopathology 86:S3, 1996. (2) H. I. Nirenberg. Mitt. Biol. Bundesanst. Land Forstwirtsch. Berlin-Dahlem. 169:1, 1976.

Occurrence of Vine Decline Diseases of Melons in Honduras.

A survey of vine decline diseases of melons (Cucumis melo L.) was conducted on three geographically separate farms (12 fields) in the area of San Lorenzo, Honduras, during the spring of 1996. Symptoms were typical of the vine declines in that the leaves began yellowing and collapsing in the crown just prior to harvest and the decline radiated outward. Crown lesions, typical of many of the vine declines, were observed infrequently. Most plants exhibited no crown lesion. Roots exhibited brown lesions and root corking, and were generally lacking root hairs, although the roots were not macerated. Fruit were small sized, low in sugars, and sunburned. Isolations were made from the crown area, primary root, and secondary roots of affected plants by excising 3- to 5-mm pieces and surface sterilizing for 30 to 60 s with 0.5% sodium hypochlorite. Four tissue pieces from each plant part were placed on 2.0% water agar plus 0.1 g of streptomycin sulfate per liter and their identity maintained. Plates were examined for fungal growth daily for 5 days and hyphal tips from all colonies were transferred to potato dextrose agar and Synthetischer nährstoffärmer agar (2) to allow for sporulation. Rhizoctonia solani Kühn was the predominant fungus isolated from affected plants in several fields, with an incidence as high as 75%. A Pythium sp. was isolated from primary and secondary roots of 50% of the plants on the Santa Rosa farm. Vines exhibiting tan to light brown crown lesions either on one side of the vine or encompassing the crown, but without gumming, always produced colonies of Fusarium semitectum Berk. & Ravenel. Fusarium solani (Mart.) Appel & Wollenweb. emend. W. C. Snyder & H. N. Hans. was isolated from roots of more than 30% of the plants, although this is not unusually high. Crown lesions typical of charcoal rot were observed in some fields, with Macrophomina phaseolina (Tassi) Goidanich being isolated from 65% of the plants in one field. Charcoal rot was a minor problem in all fields but one. Didymella bryoniae (Auersw.) Rehm was not isolated from any of the plants. A Stagonospora-like fungus, which has been demonstrated recently as a potential contributor to vine decline (1), was isolated on 17% of the plants from five fields on the Embarcadero Farm. Monosporascus cannonballus Pollack & Uecker was isolated infrequently in one field on the Santa Rosa Farm. This is the first report of M. cannonballus and a Stagonospora-like fungus in Honduras. References: (1) M. E. Miller et al. Phytopathology 86:S3, 1996. (2) H. I. Nirenberg. Mitt. Biol. Bundesanst. Land Forstwirtsch. Berlin-Dahlem. 169:1, 1976.