First Report of a Field Outbreak of a Bacterial Leaf Spot of Cantaloupe and Squash Caused by Pseudomonas syringae pv. syringae in Georgia.

In March 2000, a leaf spot was reported affecting yellow summer squash (Cucurbita pepo) and cantaloupe (Cucumis melo) in commercial fields in Colquitt, Echols, and Grady counties in Georgia. All of the crops affected were reported within a 10-day period, and average temperatures during that time were 8 to 22.5°C, which is very close to the 50-year normal temperatures for these areas located in southwest Georgia. Incidence in affected fields was 100%. Lesions on squash leaves appeared irregularly shaped, dark, water soaked, somewhat vein restricted, and were 5 to 10 mm in diameter. Lesions on cantaloupe were angular, light tan, and necrotic with a lesion diameter of 2 to 5 mm. A general chlorosis was observed around lesions of both crops. Leaf distortion was observed on squash. Four isolates collected were used in biochemical, pathogenicity, and physiological tests. Gram-negative, rod-shaped bacteria were isolated from diseased tissue from squash and cantaloupes. Bacteria were aerobic, catalase-positive, fluorescent on King's medium B (1), oxidase-negative, nonpectolytic on potato, arginine dihydrolase-negative, utilized sucrose as a carbon source, produced levan, and gave a hypersensitivity response on tobacco (HR). Analysis of fatty acid methyl ester (FAME) profiles using the Microbial identification system (Sherlock version 3.1, Microbial identification system, Newark, DE) characterized representative strains as Pseudomonas syringae (similarity indices 0.65 to 0.80). Upon further characterization, the strains were negative for l (+)-tartarate utilization but utilized l-lactate and betaine and also exhibited ice nucleation activity. These characteristics are consistent with those of Pseudomonas syringae pv. syringae. Squash and cantaloupes were grown in a greenhouse for 4 weeks. Bacteria were grown in nutrient broth, resuspended in sterile tap water, and standardized using a spectrophotometer. Plants were inoculated by infiltrating leaves with 1 ml of bacterial suspensions (1 × 107 CFU/ml) using sterile syringes. Sterile water was used as a negative control, and 1 ml was infiltrated into leaves of squash and cantaloupes. Water-soaked lesions developed in 4 to 6 days on squash and cantaloupes inoculated with bacterial suspensions, and Pseudomonas syringae pv. syringae was isolated from diseased tissue. No symptoms developed on squash and cantaloupes used as negative controls. This outbreak of Pseudomonas syringae pv. syringae did not cause significant economic damage to either crop as symptoms subsided once daily high temperatures reached 28 to 32°C. This disease has been isolated from several cucurbit transplants reared in greenhouses, but to our knowledge, this is the first report of this disease occurring in the field. Reference: (1) E. O. King et al. J. Lab. Clin. Med. 44:301, 1954.

First Report of Cabbage leaf curl virus (Family Geminiviridae) in Georgia.

Cabbage and collard greens were inflicted with a previously undescribed virus-like disease during the fall 2000. Symptoms on leaves were yellow spots, vein clearing, mosaic, curling, and puckering. Symptomatic plants were widespread in Brooks, Colquitt, Grady, and Pierce counties in Georgia. Disease incidence ranged from 10 to 20% in the majority of the fields surveyed but some fields had 100% incidence. Fields were heavily infested by Bemisia argentifolii and the symptoms were suggestive of a whitefly-transmitted geminivirus infection. A polymerase chain reaction (PCR)-based diagnostic test for geminivirus was conducted. Total DNA was extracted from symptomatic cabbage and collard green plants collected from commercial fields. The two primers, 5'-GCCCACATYGTCTTYCCNGT-3' and 5'- GGCTTYCTRTACATRGG-3' (2,3), are "universal" for genus Begomovirus of family Geminiviridae. The primer pair could amplify a part of the replicase-associated protein and coat protein and the complete common region of DNA-A. The PCR gave a DNA band of expected size (1.1 kb) from both symptomatic cabbage and collard green samples, whereas no such product was obtained from healthy samples, suggesting that the causal agent could be a geminivirus. To establish the identity of the virus, the 1.1 kb PCR product was cloned into pGEM-T Easy (Promega) and sequenced. GenBank search showed that the geminivirus isolated in Georgia was most closely related (98% sequence identity) to Cabbage leaf curl virus (accession number U65529) reported from Florida (1). The virus was mechanically transmitted to healthy cabbage and collard green plants under experimental conditions. To our knowledge, this is the first report of Cabbage leaf curl virus from Georgia. References: (1) A. M. Abouzid et al. Phytopathology 82:1070, 1992. (2) S. S. Pappu et al. Plant Dis. 84:370, 2000. (3) M. R. Rojas et al. Plant Dis. 77:340-347, 1993.

Outbreak of Tomato yellow leaf curl virus (Family Geminiviridae) in Georgia.

Tomato yellow leaf curl virus (TYLCV) of the family Geminiviridae is a serious production constraint to tomato (3). In the southeastern United States the virus has been largely confined to Florida. The disease appeared in the southern most Georgia county (Decatur) in 1998, at an incidence rate of less than 1% (2). During the fall of 1999, tomato plants showing symptoms indicative of TYLCV were observed in commercial fields in Grady, Colquitt, and Lowndes counties and the experimental plots of the Coastal Plain Experiment Station in two locations in Tift County, GA. The 12-acre commercial field in Grady County had a disease incidence of 15%. In Tift County, in both experimental plots (≈5 miles apart), TYLCV incidence ranged from 15 to 20%. Bemisia argentifolii populations in southern Georgia, based on the observed high incidence of silverleaf symptoms in squash and the intensity of adult migrations during August and September, were the highest in more than 5 years. TYLCV infection was verified by polymerase chain reaction (PCR) amplification with degenerate primers (5'-GCC CAC ATY GTC TTY CCN GT-3' and 5' -GGC TTY CTR TAC ATR GG-3') specific to the DNA A component (4). A simplified and faster DNA extraction procedure was used to obtain PCR-ready templates. Leaf tissue was homogenized in 300 µl of extraction buffer (1), followed by one phenol and one chloroform/ isoamyl alcohol (24:1) extraction. The supernatant was purified using a QiaPrep MiniPrep purification kit (Qiagen, Valencia, CA) and was used in PCR amplification. The procedure yielded highly consistent PCR-quality template. The resulting ≈1.3-kb PCR product was cloned in pGEM-T vector (Promega Corp., Madison, WI) and completely sequenced. Sequence comparisons indicated 98% identity with known TYLCV isolates from Spain (GenBank Accession no. AJ223505), the Dominican Republic (GenBank Accession no. AF024715), and Israel (GenBank Accession no. X15656). Using PCR followed by restriction digestion analysis, three symptomatic plants from one field each in Colquitt and Lowndes counties were TYLCV positive. The higher incidence of TYLCV in the Georgia counties of Tift and Grady and its concurrent occurrence in Colquitt and Lowndes counties indicates its rapid spread in the southeastern United States. References:(1) I. B. Dry et al. J. Gen. Virol. 74:147, 1993. (2) M. T. Momol et al. Plant Dis. 83:487, 1999. (3) J. E. Polston et al. Plant Dis. 83:984, 1999. (4) M. R. Rojas et al. Plant Dis. 77:340, 1993.

Natural Outbreak of a Bacterial Fruit Rot of Cantaloupe in Georgia Caused by Acidovorax avenae subsp. citrulli.

In April and July 1999, cantaloupe plants (Cucumis melo) from commercial greenhouses and fields in Grady, Colquitt, Mitchell, and Tift counties, GA, exhibited severe foliar necrosis and a fruit rot. Foliar symptoms were V-shaped, necrotic lesions occurring at the margin of the leaf and extending inward toward the midrib. Symptoms on the fruit surface were observed after net development and occurred randomly as round, necrotic, sunken spots or cracks a few millimeters in diameter. A soft rot originating from lesions on the surface of the fruit expanded into the flesh. Approximately 5% of the fruits were affected. Bacteria recovered from cantaloupe fruit and leaf tissues produced nonfluorescent, smooth, off-white colonies on King's medium B. Characteristic of Acidovorax avenae subsp. citrulli, the bacteria produced pits in carboxymethyl cellulose media (WFB 44), and reduced Tween 80 to give a visible precipitate on WFB 68 media (1). Based on fatty acid analysis, all strains were identified as A. avenae subsp. citrulli by Microbial Identification System software, version 3.6 (MIDI, Newark, DE), and similarity indices of 0.06, 0.79, 0.21, and 0.43 were recorded for strains recovered form Grady, Tift, Colquitt, and Mitchell counties, respectively. Using specific oligonucleotide primers (WFB 1/2) (2), PCR conducted on DNA from each strain yielded a 390-bp DNA fragment, confirming similarity to A. avenae subsp. citrulli. Indirect enzyme-linked immunosorbent assay with genus-specific antibodies also verified that the bacteria were Acidovorax spp. Pathogenicity of the A. avenae subsp. citrulli strains was confirmed by inoculating and observing symptom development on 2-week-old watermelon seedlings. Although all strains were identified and confirmed as A. avenae subsp. citrulli, restriction fragment length polymorphism data indicated that the Tift County strain was distinguishable from the others, suggesting that inoculum for these outbreaks may have originated from at least two different sources. References: (1) R. D. Gitaitis. 1993. Development of a seedborne assay for watermelon fruit botch. Pages 9-18 in: Proc. 1st Int. Seed Testing Assoc. Plant Dis. Commit., Ottawa, Canada. (2) R. R. Walcott and R. D. Gitaitis. (Abstr.) Phytopathology 88(suppl.):S92, 1998.