Host specialization and phylogenetic diversity of Corynespora cassiicola.

The fungus Corynespora cassiicola is primarily found in the tropics and subtropics, and is widely diverse in substrate utilization and host association. Isolate characterization within C. cassiicola was undertaken to investigate how genetic diversity correlates with host specificity, growth rate, and geographic distribution. C. cassiicola isolates were collected from 68 different plant species in American Samoa, Brazil, Malaysia, and Micronesia, and Florida, Mississippi, and Tennessee within the United States. Phylogenetic analyses using four loci were performed with 143 Corynespora spp. isolates, including outgroup taxa obtained from culture collections: C. citricola, C. melongenae, C. olivacea, C. proliferata, C. sesamum, and C. smithii. Phylogenetic trees were congruent from the ribosomal DNA internal transcribed spacer region, two random hypervariable loci (caa5 and ga4), and the actin-encoding locus act1, indicating a lack of recombination within the species and asexual propagation. Fifty isolates were tested for pathogenicity on eight known C. cassiicola crop hosts: basil, bean, cowpea, cucumber, papaya, soybean, sweet potato, and tomato. Pathogenicity profiles ranged from one to four hosts, with cucumber appearing in 14 of the 16 profiles. Bootstrap analyses and Bayesian posterior probability values identified six statistically significant phylogenetic lineages. The six phylogenetic lineages correlated with host of origin, pathogenicity, and growth rate but not with geographic location. Common fungal genotypes were widely distributed geographically, indicating long-distance and global dispersal of clonal lineages. This research reveals an abundance of previously unrecognized genetic diversity within the species and provides evidence for host specialization on papaya.

First Report of a Leaf Spot Disease of Wild Rocket (Diplotaxis tenuifolia) in Florida Caused by Xanthomonas campestris pv. raphani.

Wild rocket, also known as wall rocket or sand rocket (Diplotaxis tenuifolia (L.) DC, family Brassicaceae), is grown in Florida as a salad green and herb, especially for addition to "spring mix" and other bagged salad blends. It is similar in texture and flavor to the more widely known garden arugula (Eruca vesicatoria (L.) Cav. subsp. sativa). During the winter vegetable season of 2006-2007, a leaf spot disease causing severe economic loss was seen in commercial fields of wild rocket near the town of Sebastian in Indian River County, FL. Discrete water-soaked to greasy appearing spots were observed on leaf blades that rarely exceeded 1 mm in diameter with some surrounded by a narrow, yellow halo. There was no evidence of marginal V-shaped lesions suggestive of vascular black rot disease caused by Xanthomonas campestris pv. campestris. A bacterium that formed yellow colonies on nutrient agar was consistently isolated from these lesions. Four strains were isolated, purified, and characterized. All strains were strictly aerobic, gram-negative rods. Strains were positive for esculin hydrolysis and gelatin liquefaction and negative for oxidase, nitrate reduction, urease production, fluorescence on King's B medium, and utilization of asparagine as a sole source of carbon and nitrogen. Proteolysis and an alkaline reaction were observed in inoculated tubes of litmus milk. Colonies were bright yellow and mucoid on plates of yeast extract-glucose-calcium carbonate agar. Carbon source utilization, as revealed by the Biolog system, indicated a match to X. campestris. Fatty acid methyl ester analysis indicated a match with Florida library strains of X. campestris pv. raphani (similarity indices of 0.737 to 0.779). Suspensions (2 × 107 CFU/ml in sterile phosphate-buffered saline) of the four wild rocket strains and a strain isolated in 2003 as a cause of a discrete leaf spot disease of cabbage in southern Florida (1) were sprayed onto plants until runoff with a hand-held plastic mist bottle. Pathogenicity of strains was tested in the greenhouse on seven replicate plants of green cabbage cv. Copenhagen Market, salad arugula cv. Rocket, and wild rocket (an unnamed selection propagated from seed provided by a Florida grower). Symptoms appeared within 6 to 7 days. The wild rocket strains and the cabbage strain were pathogenic on all wild rocket and cabbage test plants, producing small, nonvascular leaf spots. No symptoms were seen on salad arugula or on control plants of wild rocket, cabbage, or arugula sprayed with phosphate-buffered saline. The bacterium was reisolated from infected plants and identified as X. campestris pv. raphani, thus completing Koch's postulates. We have classified the bacterial leaf spot pathogen of wild rocket as X. campestris pv. raphani using the nomenclature of Vincente et al. (2) for X. campestris strains producing nonvascular leaf spots on brassicas. This disease problem seems to be related to widespread use of overhead irrigation in the fields where the disease is prevalent. We have not observed the disease in seepage-irrigated fields of wild rocket. References: (1) K. Pernezny et al. Plant Dis. 87:873, 2003. (2) J. G. Vincente et al. Phytopathology 96:735, 2006.

First Report of Powdery Mildew of Parsley Caused by Erysiphe heraclei in Florida.

Parsley (Petroselinum crispum (Mill.) Nym. ex A.W. Hill) is an important leaf crop in the Everglades Agricultural Area of southern Florida. During the spring of 2005 and 2006, disease signs and symptoms resembling those incited by powdery mildew were observed on parsley at a commercial vegetable farm located 15 km east of Belle Glade. Symptoms consisted of leaf chlorosis, particularly in the dense lower canopy, and desiccation of affected tissue. A dense, white-to-light gray fungal growth was visible macroscopically on the surface of affected leaf tissue. Microscopic examinations revealed ectophytic hyphae with lobed appressoria and hyaline, straight conidiophores bearing single conidia. Conidia were short-cylindrical to cylindrical, measured 33 to 44 µm long and 13 to 16 µm wide, and lacked fibrosin bodies. Conidiophore foot cells were also cylindrical, straight, and measured 27 to 37 × 9 to 10 µm. Ascocarps of the teleomorph were not observed. The fungus closely matched the description of Erysiphe heraclei DC, a pathogen previously reported as attacking parsley on the U.S. West Coast (1,2). Pathogenicity was verified by inoculating adaxial leaf surfaces of 12 plants (cv. Dark Green Italian) with conidia collected from infected tissue by using a small brush. Inoculated plants and 12 noninoculated plants were lightly misted, held in a moist chamber for 48 h (22°C), and then incubated in a growth chamber for 4 weeks at 22°C with a photoperiod of 16 h. Symptoms that developed on inoculated plants were similar to those observed in the field, with no symptoms evident on the controls treated in a similar manner. To our knowledge, this is the first report of powdery mildew on parsley in Florida, even though parsley has been grown in the area for at least six decades. Noted as being somewhat unique among fungal pathogens because it favors dry rather than moist climatic conditions, it is probably no coincidence that powdery mildew was observed both years during the month of April, the height of Florida's dry season. The fact that monthly rainfall totals of 22 and 35 mm were recorded during April of 2004 and 2005, respectfully, well below the historical average of 72 mm, may have been a contributing influence. Glawe et al. (1), in issuing a first report of E. heraclei on carrots and parsley in the state of Washington and observing ascocarps on carrot tissue, mentioned the prospect of contaminated seed serving as a potential source of dissemination. Although they did not observe the teleomorph on parsley, prospects for its occurrence seem likely. With the bulk of parsley seed planted in Florida being produced in Washington, Oregon, or California, the observations reported herein may provide credence to such a hypothesis. References: (1) D. A. Glawe et al. Online publication. doi:10.1094/PHP-2005-0114-01-HN. Plant Health Progress, 2005. (2) S. T. Koike and G. S. Saenz. Plant Dis. 78:1219, 1994.

Outbreak of Choanephora Blight Caused by Choanephora cucurbitarum on Green Bean and Pepper in Florida.

Severe outbreaks of Choanephora blight on green bean (Phaseolus vulgaris cvs. Bronco, Shade, and Gold Mine) and bell pepper (Capsicum annuum cvs. Aristotle, Crusader, and Sentry) were widespread in southwestern (Hendry and Collier counties) and northern Florida (Alachua County) in October and November 2002. Disease incidence, estimated by inspecting 100 randomly selected bean plants in each of four fields, was 40 to 100% and infected fruit ranged from less than 10 to 100%. Incidence estimated similarly on pepper plants in three fields was 35 to 40% with substantial fruit infection observed predominantly around the calyx. Zucchini fruit and a pigweed plant (Amaranthus sp.) were observed with sporulating lesions of Choanephora, indicating that other hosts were affected during the outbreak. Symptoms were blighted leaves, dieback of shoot tips, blighted blossoms, and black, soft-rot lesions on fruit. Choanephora sp. was sporulating in abundance on diseased tissue. Isolates of Choanephora sp. grew readily as pure cultures on acidified potato dextrose agar and malt yeast extract (MYE) agar. C. cucurbitarum (Berk. & Rav.) Thaxter was identified on the basis of shape and ornamentation of the sporangiola (1). The sporangiola of C. cucurbitarum are ellipsoid to broadly ellipsoid, and the wall is usually longitudinally striate. Pathogencity tests consisted of spray inoculation (5,000 spores per ml) of five 6-week-old plants each with and without wounding made by lightly scratching the leaf surface with a needle. Plants were placed in the greenhouse with temperatures ranging from 21 to 26°C, and symptom development was observed as early as 3 days after inoculation. The percentage of infected plants after wounding was 40% for bell pepper ('Enterprise'), 100% for green bean ('Opus'), 0% for watermelon (Citrullus lanatus 'Star Gazer'), 60% for cantaloupe (Cucumis melo 'Vienna'), and 20% for cucumber (Cucumis sativus 'Thunder CY'). Lesions on inoculated leaves were similar to those seen in the field on bean and pepper, and sporulation of C. cucurbitarum was present in the necrotic areas on all symptomatic plants. Pure cultures of C. cucurbitarum were reisolated. C. curcurbitarum was observed and isolated from a few noninoculated bean flowers and two noninoculated bean pods indicating spread to noninoculated plants; otherwise control plants were asymptomatic. Unwounded plants did not develop lesions, indicating that wounding was necessary for infection by this inoculation technique. The mating type was determined by juxtaposing several isolates on MYE agar, and zygospore formation was observed indicating both + and - strains occur in Florida. These outbreaks show that under the proper environmental conditions, such as long periods of high rainfall, high humidity, and high temperatures, crops like bean and pepper that are not usually affected by the disease may experience significant damage. Reference: (1) P. M. Kirk. Mycol. Pap. 152:1-61, 1984.

An Outbreak of a Leaf Spot Disease of Cabbage in Southern Florida Caused by Xanthomonas campestris pv. armoraciae.

From October to December 2001, a leaf spot disease was observed in numerous commercial fields of red and green cabbage (Brassica oleracea var. capitata L.) in the Everglades Agricultural Area, south and east of Lake Okeechobee and in the environs of Immokalee in southwestern Florida. Discrete water-soaked to greasy appearing spots were observed in the leaf blades with no evidence of marginal V-shaped lesions characteristic of black rot caused by Xanthomonas campestris pv. campestris. Profuse bacterial streaming was observed when cut leaf sections were examined microscopically. A bacterium that formed yellow colonies on nutrient agar was consistently isolated from these lesions. Ten bacteria were isolated, purified, and characterized. All strains were aerobic, gram-negative rods. Strains were positive for esculin hydrolysis, proteolysis in litmus milk, and gelatin liquefaction. Strains were negative for urease production, nitrate reduction, oxidase, and utilization of asparagine as a sole source of carbon and nitrogen. Fatty acid methyl ester analysis indicated a match with Florida library strains of X. campestris pv. raphani (similarity indices 0.605-0.738). Suspensions (2 × 107 CFU/ml in phosphate-buffered saline) of two Oklahoma strains identified as X. campestris pv. armoraciae provided by J. P. Damicone (3) and four representative Florida strains were applied to plants using a hand-held sprayer. Pathogenicity of the strains was tested on three replicate greenhouse-grown plants of the following: green cabbage cv. Market Early; red cabbage cv. Salad Delight; radish cv. Red Silk; tomato cv. Sunny; sweet bell pepper cv. Jupiter; and fresh horseradish roots purchased from a retail grocery chain. A strain of X. campestris pv. campestris originally isolated from Homestead, FL was also included in pathogenicity tests. All Florida and Oklahoma strains produced leaf spots, but no V-shaped lesions, on leaves of green cabbage, red cabbage, radish, tomato, and horseradish. Typical black rot symptoms were observed only in radish and green and red cabbage inoculated with the X. campestris pv. campestris strain. On the basis of these results, we identify the Florida strains as X. campestris pv. armoraciae (1,2,3), recognizing the precedent of X. campestris pv. armoraciae over X. campestris pv. raphani based on extensive genetic and serological data (1). Our strains appear to be more similar to those causing outbreaks on crucifers in Oklahoma (3) than those in Ohio (2), because Florida strains were pathogenic on tomato. References: (1) A. M. Alvarez et al. Phytopathology 84:1449, 1994. (2) F. Sahin and S. A. Miller. Plant Dis. 81:1334, 1997. (3) Y. Zhao et al. Plant Dis. 84:1008, 2000.

Occurrence of Bacterial Leaf Spot of Escarole Caused by Pseudomonas cichorii in the Everglades Agricultural Area of Southern Florida.

In the fall of 1997, 1998, and 2000, a leaf spot disease of escarole (Cichorium endivia L.) was widespread among commercial plantings in the Everglades Agricultural Area (EAA), south and east of Lake Okeechobee, FL. Symptoms consisted of dry, dark gray-to-black lesions that expanded to ≈4 cm in diameter. Concentric rings were often observed in mature lesions. Growers and scouts in the area consistently identified this disease as Alternaria leaf spot, because the symptoms closely resembled Alternaria leaf spots seen on a number of other vegetables. Prolific bacterial streaming occurred when cut portions of lesions were observed microscopically. A fluorescent bacterium was consistently isolated when a sterile inoculation needle was pushed through lesions. Eight bacterial strains were isolated, restreaked to obtain pure cultures, and characterized. All strains were aerobic, gram-negative rods that were oxidase positive and arginine dihydrolase negative. Negative reactions were recorded for levan formation and rotting of potato slices. All strains utilized glucose, mannitol, and m-tartrate and were negative for sucrose, sorbitol, benzoate, d-arabinose, l-rhamnose, and cellobiose. Results for utilization of D-aspartate were variable. Based on these results, the causal agent of bacterial leaf spot of escarole was identified as Pseudomonas cichorii. Greenhouse-grown plants of escarole, cv. Full Heart, and Cos lettuce, cv. Tall Guzmaine, were mistinoculated with a suspension (107 CFU/ml) of each test strain from escarole and P. cichorii strain Pc28, originally isolated from celery (1). Plants were bagged for 3 days after inoculation. Symptoms characteristic of this disease were evident on escarole inoculated with all test strains and Pc28 6 days after inoculation. Pure cultures of P. cichorii were recovered from lesions on King's B medium. Three test strains produced mild leaf spot symptoms in Cos lettuce, but the symptoms were distinctly different from those associated with the common bacterial leaf spot of lettuce in Florida caused by Xanthomonas campestris pv. vitians (2). To our knowledge, this is the first report of P. cichorii causing this unusual target spot symptom on escarole in the EAA. References: (1) K. Pernezny et al. Plant Dis. 78:917, 1994. (2) K. Pernezny et al. Plant Dis. 79:359, 1995.

A Serious Outbreak of Race 6 of Xanthomonas campestris pv. vesicatoria on Pepper in Southern Florida.

In the 1997-1998 winter vegetable season, a widespread and serious outbreak of bacterial spot occurred on pepper in commercial fields throughout southern Florida. This was the first serious epidemic of bacterial spot on pepper in southern Florida since the 1993-1994 season. Cultivars affected included those with resistance to races 1-3 of X. campestris pv. vesicatoria, commonly found in previous Florida surveys (1). Field sampling designs, isolation methods, and pathogen identification were carried out as previously described (1). Twenty-seven fields were sampled in Palm Beach, St. Lucie, Martin, Broward, and Collier counties. The race of each strain was determined by infiltration of 3.0 × 108 CFU per ml of sterile tap water suspension into Early Calwonder and three near-isogenic lines of Early Calwonder with genes for vertical resistance to specific races of the pathogen. Test plants were maintained in the greenhouse and observed for hypersensitive and disease reactions over several days. Of a total of 244 X. campestris pv. vesicatoria strains collected, 73.4% were identified as race 6 and 10.2% as race 4. Irrespective of the presence of resistance genes in cultivars sampled, race 6 (eastern Florida) and race 4 (southwestern Florida) predominated in this study. Copper tolerance was widespread among X. campestris pv. vesicatoria strains of all races, with 91% of the strains showing tolerance to 250 g per ml of CuSO4 · 5H2O incorporated into 0.5% (wt/vol) glucose-nutrient agar. Selection pressure resulting from widespread planting of race 1, 2, 3-resistant cultivars may have contributed to this outbreak. High disease pressure also may be related to the unusually wet winter. Rainfall accumulation from December 1997 through March 1998 was 25.6 cm above normal. Reference: (1) K. Pohronezny et al. Plant Dis. 76:118, 1992.

Powdery Mildew of Field-Grown Tomato in Florida.

Powdery mildew was observed affecting mature, field-grown, fresh-market tomatoes in southeastern Florida (St. Lucie, Martin, Palm Beach, and Broward counties). Outbreaks were noted in May of 1996 and throughout the spring of 1997. Affected leaves were generally yellow and, in some cases, developed a purplish cast. Whitish areas of fungal growth were often seen in the middle of lesions, but were less prominent than that reported for powdery mildew of field tomato in Hungary (2). Microscopic examination revealed that conidia were produced in chains, contained no fibrosin bodies, and were not dimorphic. Conidial measurements were 33.2 × 15.6 µm, based on 21 samples of five conidia taken from five different fields. These measurements were similar to those for an Erysiphe sp. causing powdery mildew of greenhouse tomato in New York (1). Greenhouse-grown tomato plants, cv. Sunny, in the fourth-true-leaf stage were inoculated with conidia scraped from lesions gathered in commercial fields. A single-edged razor blade was used to transfer the fungus to the adaxial surface of the test plants. Powdery mildew symptoms were observed within 10 days of inoculation. Morphology and size of conidia taken from greenhouse plants were similar to those for original field samples. This is the first report of powdery mildew of field-grown tomato in Florida. References: (1) D. M. Karasevicz and T. A. Zitter. Plant Dis. 80:709, 1996. (2) L. Kiss. Plant Dis. 80:224, 1996.

Evidence for the Preemptive Nature of Tomato Race 3 of Xanthomonas campestris pv. vesicatoria in Florida.

ABSTRACT Until recently, tomato race 1 (T1) of Xanthomonas campestris pv. vesicatoria was the only race causing bacterial spot of tomato in Florida. In 1991, tomato race 3 (T3) was first identified in 3 of 13 tomato production fields surveyed. By 1994, T3 was observed in 21 of 28 fields and was the only race identified in 14 fields. In field studies, tomato genotypes with resistance to either T1 or T3 or susceptibility to both were co-inoculated with strains of both races. Lesions on 10 plants in each of three replications for each genotype were sampled three times during the experiment; bacterial isolations were made from each lesion, and tomato race identifications were made for each strain. At the third sampling date, T3 was isolated from 97% of the lesions on the susceptible genotype Walter and the T1-resistant genotype Hawaii 7998, while T3 was isolated from 23% of the lesions and T1 from the remaining 77% on the T3-resistant genotypes PI 128216 and PI 126932. In surface population studies done in growth rooms, suspensions of T1 and T3 were applied alone and in combination to the leaf surfaces of susceptible and resistant genotypes. T1 populations were reduced more than 10-fold when applied in combination with T3, compared with populations that developed when T1 was applied alone. T3 populations were not affected when applied in combination with a T1 strain. In greenhouse studies with the T3-resistant genotype Hawaii 7981, disease was significantly reduced in plants inoculated with T3 in combination with T1, compared with plants inoculated with T1 alone. These results clearly demonstrate the competitive nature of T3 in the presence of T1 and help explain the emergence of T3 as a prevalent race in Florida.

Bacterial Leaf Spot of Cilantro in Florida.

In the 1995 to 1996 winter vegetable season, a leaf spot disease of cilantro (fresh coriander) (Coriandrum sativum L.) was widespread throughout commercial plantings in the Everglades Agricultural Area south and east of Lake Okeechobee. Symptoms first appeared as water-soaked spots ≤1 mm in diameter. These spots became dark brown to black and enlarged up to 2 mm in diameter. No chlorotic haloes developed around the lesions. The disorder was observed in numerous plantings of the cultivar Longstanding at four separate locations. Symptoms were apparent throughout each planting, with disease severity ranging from a few individual spots to numerous lesions covering nearly entire leaflets. Severe outbreaks were correlated with heavy precipitation events. In several instances, disease levels were great enough to render entire plantings totally unmarketable. A non-fluorescent bacterium was consistently isolated on King's medium B when a cooled inoculation needle was pushed through lesions. Six representative strains were chosen for further characterization. All strains were aerobic, gram-negative rods, and were oxidase and arginine dihydrolase negative. Levan was produced, but potato slices were not rotted. Tests for utilization of l-tartrate, l-lactate, and erythritol were negative. Biolog analysis identified all strains of the bacterium as Pseudomonas syringae. Highest similarity indices (0.52 to 0.81) were with P. syringae pv. pisi for four of the six strains. Suspensions of each strain were swab inoculated onto leaves of 4-week-old Longstanding cilantro seedlings in the greenhouse. Control plants were swabbed with sterile water only. Plants were covered with clear polyethylene bags for 72 h. Watersoaked spots were evident on test plants when bags were removed. Typical brown, greasy-looking leaf spots were seen by 6 days after inoculation. Control plants were symp-tomless. In a host-range study, cilantro and the following plants were mist-inoculated with a 107 CFU/ml suspension of each of the six test strains: carrot (Daucus carota L. 'Fancy Pack'), celery (Apium graveolens L. var. dulce (Mill.) Pers., 'June Belle'), garden pea (Pisum sativum L. 'Melting Sugar'), snap bean (Phaseolus vulgaris L. 'Pod Squad'), and onion (Allium cepa L. 'Evergreen Bunching'). Six days after inoculation, characteristic symptoms were evident on the cilantro. Four of the six strains produced a few (less than 10 per plant) bacterial leaf spot symptoms in carrot from which P. syringae was readily recovered. Some necrosis was observed on young, emerging leaves of snap bean. No symptoms were recorded for the other host species or the controls. This disease is similar to one reported on cilantro in California (1) and Germany (2). References: (1) D. A. Cooksey et al. Plant Dis. 75:101, 1991. (2) H. M. Toben and K. Rudolph. J. Phytopathol. 144:169, 1996.