C. T. Bull

A New Pathovar, Pseudomonas syringae pv. alisalensis pv. nov., Proposed for the Causal Agent of Bacterial Blight of Broccoli and Broccoli Raab

The etiology of three foliar bacterial diseases of crucifers and the relationships between their causal agents were evaluated. Data from LOPAT, carbon utilization tests, and fatty acid analysis indicated that bacterial blights of broccoli and broccoli raab, and leaf spot of broccolini, were caused by strains of Pseudomonas syringae. Data from phage sensitivity, ice nucleation, single carbon source utilization, Polymerase chain reaction using BOXA1R primer (BOX-PCR), and host range analyses were identical for the pathogen causing leaf spot of broccolini and P. syringae pv. maculicola. The broccoli raab and broccoli pathogens infected broccoli raab, all crucifers tested, tomato, and three monocots (California brome, oat, and common timothy). None of the other pathogens tested (P. syringae pv. maculicola, P. syringae pv. tomato, or P. syringae pv. coronafaciens) caused disease on broccoli raab or on both crucifers and monocots. Data from phage sensitivity, ice nucleation, single carbon source utilization, BOX-PCR, and host range analyses were identical for the pathogens from broccoli raab and broccoli, but were different from other pathovars tested, and supported the hypothesis that a new pathovar of P. syringae pv. alisalensis pv. nov. caused a leaf blight on broccoli and broccoli raab.

First Report of Bacterial Blight of Rutabaga (Brassica napus var. napobrassica) Caused by Pseudomonas syringae pv. alisalensis in California

In 2005, commercial, organically grown rutabaga (Brassica napus var. napobrassica) in San Benito County, CA showed symptoms of a previously undescribed disease on approximately 30% of the plants. Initial symptoms consisted of small (1 to 2 mm in diameter), angular, water-soaked flecks that often were surrounded by chlorotic haloes. These flecks enlarged and coalesced into large, irregularly shaped, gray brown lesions that could be as long as 10 mm. Lesions were visible from both adaxial and abaxial leaf surfaces and generally retained the chlorotic borders. A blue-green fluorescing pseudomonad was consistently isolated from lesions on Kings medium B. Eight isolates were characterized and were levan positive, oxidase negative, and arginine dihydrolase negative. Isolates did not rot potato slices but induced a hypersensitive reaction in tobacco (Nicotiana tabacum cv. Samsun). These data indicated that the bacteria belonged to Lelliots LOPAT group 1 (2). This was confirmed with data from fatty acid methyl ester analysis (MIS-TSBA version 4.10; MIDI, Inc., Newark, DE) that showed that the isolates were highly similar (similarity = 0.922 or greater) to Pseudomonas syringae. Amplification of repetitive bacterial sequences (rep-PCR) using the BOXA1R primer and the polymerase chain reaction resulted in identical banding patterns for the rutabaga isolates and the P. syringae pv. alisalensis pathotype strain. Pathogenicity was demonstrated by growing inocula of six isolates in nutrient broth shake cultures for 48 h (24°C), adjusting the bacterial suspension to 106 CFU/ml, and misting the resulting suspensions onto rutabaga (cv. American Purple Top). Plants were enclosed in plastic bags for 24 h and then incubated in a greenhouse (24 to 26°C). Control plants were misted with sterile water and treated the same way. After 5 to 7 days, foliar symptoms similar to symptoms seen in the field developed on all inoculated plants, and reisolated bacteria were characterized and found to be P. syringae pv. alisalensis. Control plants remained symptomless. The results of two sets of pathogenicity tests were the same. To our knowledge, this is the first report of commercially grown rutabaga as a host of P. syringae pv. alisalensis and the first report of a B. napus host of this pathogen. This bacterial pathogen has previously been reported on commercial plantings of arugula (Eruca sativa), broccoli (Brassica oleracea var. botrytis), and broccoli raab (Brassica rapa var. rapa) in California and under experimental conditions it causes disease on additional hosts, including members of the Poaceae (1). References: (1) N. A. Cintas et al. Plant Dis. 86:992, 2002. (2) R. A. Lelliott. J. Appl. Bacteriol. 29:470, 1966.

First report of bacterial blight of Romanesco cauliflower (Brassica oleracea var. botrytis) caused by Pseudomonas syringae pv. alisalensis in California.

In 2005, a new disease was detected on commercial, organically grown romanesco (green) cauliflower (Brassica oleracea var. botrytis) grown in San Benito County, California. Initial symptoms consisted of small (1 to 2 mm in diameter), angular, water-soaked flecks. These flecks developed into tan-to-gray, angular lesions measuring as much as 5 mm in diameter. Lesions were usually surrounded by chlorotic borders. Coalescing lesions caused the leaf to turn papery in texture and have a blighted appearance. A blue-green fluorescing pseudomonad was consistently isolated from lesions on Kings medium B. Strains were levan positive, oxidase negative, and arginine dihydrolase negative. Strains did not rot potato slices but induced a hypersensitive reaction in tobacco (Nicotiana tabacum cv. Samsun). These data indicated that the bacteria belonged to Lelliots LOPAT group 1 (2). This was confirmed with data from fatty acid methyl ester analysis (MIS-TSBA version 4.10, MIDI, Inc., Newark, DE), which showed that the strains were highly similar (similarity = 0.921 or greater) to Pseudomonas syringae. Amplification of repetitive bacterial sequences (rep-PCR) using the BOXA1R primer and the polymerase chain reaction resulted in identical banding patterns for the romanesco strains and the P. syringae pv. alisalensis pathotype strain. Pathogenicity was demonstrated by growing inoculum of six strains in nutrient broth shake cultures for 48 h (24°C), adjusting the bacterial suspension to 106 CFU/ml, and spraying the resulting suspension onto green cauliflower (cv. Romanesco Precoce). Plants were enclosed in plastic bags for 24 h and then incubated in a greenhouse (24 to 26°C). Control plants were misted with sterile water and treated the same way. After 5 days, foliar symptoms identical to symptoms seen in the field developed on all inoculated plants, and reisolated strains were characterized and found to be identical to P. syringae pv. alisalensis by the tests described above. Control plants remained symptomless. The results of two sets of pathogenicity tests were the same. To our knowledge, this is the first report of commercially grown romanesco green cauliflower as a host of P. syringae pv. alisalensis. The infested field had approximately 30% of the plants affected, with perhaps 10% sustaining some crop loss. This bacterial pathogen has previously been reported on commercial plantings of arugula (Eruca sativa), broccoli (Brassica oleracea var. botrytis), and broccoli raab (Brassica rapa var. rapa) and under experimental (greenhouse) conditions causes disease on additional hosts, including members of the Poaceae (1). References: (1) N. A. Cintas et al. Plant Dis. 86:992, 2002. (2) R. A. Lelliott. J. Appl. Bacteriol. 29:470, 1966.

Bacterial Blight on Arugula, a New Disease Caused by Pseudomonas syringae pv. Alisalensis in California

Beginning in 1995, a leaf spot disease has occasionally developed on the leafy crucifer arugula (Eruca vesicaria subsp. sativa) that is grown in coastal California as a fresh market commodity used mostly in bagged salad mixes. Initially, symptoms consist of small (<2 mm in diameter), angular, water-soaked spots that are visible from both sides of the leaf. The spots later enlarge, remain angular in shape, and turn brown to tan. A purple margin sometimes occurs around the spots. An important diagnostic feature is that this disease closely resembles downy mildew infections that have not produced sporangia (3). A blue-green fluorescent pseudomonad was consistently isolated from both types of lesions on Kings medium B. Strains were levan positive, oxidase negative, and arginine dihydrolase negative. Strains did not rot potato slices but induced a hypersensitive reaction on tobacco (Nicotiana tabacum L. cv. Turk). These data indicated that the bacteria belonged to Lelliots LOPAT group 1 (4). This was confirmed with data from fatty acid methyl ester analysis (MIS-TSBA version 4.10; MIDI, Inc., Newark, DE), which indicated that the strains were highly similar (similarity > = 0.758) to Pseudomonas syringae. Amplification of repetitive bacterial sequence-based polymerase chain reaction (rep-PCR) was used to determine the relationship between the P. syringae strains isolated from arugula and two common crucifer pathogens, P. syringae pv. maculicola and P. syringae pv. alisalensis (1). Using the BOXA1R primer, banding patterns for the arugula strains and the P. syringae pv. alisalensis pathotype were similar, differing by only one band. In contrast, the banding patterns of the arugula strains differed significantly from those of P. syringae pv. maculicola. Additionally, the arugula isolates were sensitive to a bacteriophage originally isolated for its ability to lyse P. syringae pv. alisalensis (1). Previously, the pathogen from arugula was reported to be P. syringae pv. maculicola (2). It is the intent of this disease note to clarify this identification. We completed Kochs postulates by confirming pathogenicity on arugula (cv. Rocket Salad). The strains were grown as nutrient broth shake cultures for 48 h at 24°C, adjusted to 108 CFU/ml, and misted onto 2- to 3-week old plants. Control plants were misted with sterile nutrient broth. After 4 to 5 days in a greenhouse (24 to 26°C), large, angular leaf lesions developed on all inoculated arugula plants. Strains were reisolated from symptomatic tissue and identified as P. syringae pv. alisalensis. Control plants remained symptomless. Similar methods confirmed that the host range of the arugula isolates were identical to that of P. syringae pv. alisalensis. The arugula and P. syringae pv. alisalensis isolates caused disease on broccoli (Brassica oleracea var. botrytis cvs. Patriot and Titleist), broccoli raab (B. rapa subsp. rapa cv. Sorento), and oats (Avena sativa cv. Montezuma), while P. syringae pv. maculicola caused disease on broccoli only. Pathogenicity tests were conducted two times with identical results. This confirms that the bacterial blight that has been occurring on commercial plantings of arugula is caused by P. syringae pv. alisalensis. References: (1) N. A. Cintas et al.Plant Dis. 86:992, 2002. (2) S. T. Koike et al. Plant Dis. 80:464, 1996. (3) S. T. Koike. Plant Dis. 82:1063, 1998. (4) R. A. Lelliott, J. Appl. Bacteriol. 29:470, 1966.

First Report of Bacterial Blight of Brussels sprouts (Brassica oleracea var. gemmifera) Caused by Pseudomonas cannabina pv. alisalensis in California

Greenhouse-grown Brussels sprouts (Brassica oleracea L. var. gemmifera) transplants from Monterey County, California showed symptoms in 2006 of a previously undescribed disease. Initial symptoms consisted of small (1 to 2 mm in diameter), angular, water-soaked flecks, some of which were surrounded by chlorotic haloes. These flecks coalesced into large, irregularly shaped, gray brown lesions as large as 10 mm. Lesions were visible from both adaxial and abaxial leaf surfaces and generally retained chlorotic borders. This disease resulted in decreased quality and reduced marketability of the transplants. Gram-negative, blue-green fluorescing bacteria were consistently isolated from lesions on Kings medium B agar. Ten isolates were selected and used in further studies. Isolates were levan positive, oxidase negative, and arginine dihydrolase negative. Isolates did not rot potato slices but induced a hypersensitive reaction in tobacco (Nicotiana tabacum L. cv. Samsun). These data indicated that the bacteria belonged to Lelliots LOPAT group 1 (2). Repetitive extragenic palindromic sequence (REP)-PCR using the BOXA1R primer resulted in identical DNA fragment banding patterns for the Brussels sprouts isolates and the pathotype of Pseudomonas cannabina pv. alisalensis (formerly P. syringae pv. alisalensis). Like P. cannabina pv. alisalensis, the isolates from Brussels sprouts were sensitive to bacteriophage PBS1 (1). All 10 isolates were used in two independent pathogenicity experiments. Inoculum for pathogenicity studies was prepared by growing the bacteria on nutrient agar for 48 h (27°C), suspending the bacteria in 0.01 M phosphate buffer (pH 7.0), and adjusting each suspension to 0.6 OD at 600 nm (approximately 108 CFU/ml). In each experiment, six Brussels sprouts plants were inoculated for each isolate by spraying until runoff or by swabbing a suspension of the appropriate bacterial isolate to which Carborundum had been added. Additionally, four Brussels sprouts isolates were used to spray inoculate six broccoli raab (Brassica rapa subsp. rapa) plants. Positive control plants were inoculated with the pathotype of P. cannabina pv. alisalensis, and the negative control plants were inoculated with sterile buffer or sterile buffer with Carborundum. Inoculated plants were placed in a mist chamber for 48 h and then in a greenhouse (20 to 25°C). After 5 to 7 days, foliar symptoms similar to symptoms observed on the original diseased Brussels sprouts plants developed on all inoculated plants, including the positive control plants inoculated with P. cannabina pv. alisalensis. Negative control plants remained symptomless. In each experiment, bacteria reisolated from symptomatic tissue were identical to the bacteria used to inoculate the plants and to P. cannabina pv. alisalensis for LOPAT reactions, REP-PCR DNA fragment banding pattern, and sensitivity to phage PBS1. To our knowledge, this is the first report of P. cannabina pv. alisalensis causing bacterial blight of Brussels sprouts. References: (1) C. T. Bull et al. Syst. Appl. Microbiol. 33:105, 2010. (2) R. A. Lelliott. J. Appl. Bacteriol. 29:470, 1966.

Holdover inoculum of Pseudomonas syringae pv. alisalensis from broccoli raab causes disease in subsequent plantings

Uniform plots of broccoli raab (Brassica rapa subsp. rapa) seedlings were inoculated with a rifampicin-resistant strain of Pseudomonas syringae pv. alisalensis, the causal agent of bacterial blight on crucifers, resulting in 100% disease incidence in mature plants. Diseased plants were incorporated into the soil at maturity and smaller replicated plots were replanted at various times after incorporation. Rifampicin-resistant fluorescent pseudomonads with rep-PCR profiles identical to P. syringae pv. alisalensis were isolated from lesions on plants grown in soil into which the first diseased crop was incorporated. Disease incidence declined in mature plants as the length of time between incorporation of the first planting and seeding of the replanted plots increased. Bacterial population levels in soil decreased over time and bacteria were no longer detectable 3 weeks after incorporation of the diseased crop. In laboratory tests, population levels of P. syringae pv. alisalensis decreased in untreated soil but not in autoclaved soil. Greenhouse studies demonstrated a direct correlation between population levels of P. syringae pv. alisalensis applied to soil and disease incidence in seedlings. However, the decline in bacterial populations in field soils did not wholly account for the decline in disease incidence with subsequent plantings.

First Report of Bacterial Blight of Cabbage (Brassica oleracea var. capitata) Caused by Pseudomonas cannabina pv. alisalensis in California

In 2008, field-grown cabbage (Brassica oleracea var. capitata L., cv. Grenader) from Monterey County, California showed symptoms on the wrapper leaves of immature plants that had formed heads. Initial symptoms consisted of small, brown, water-soaked flecks surrounded by chlorotic haloes. These flecks later coalesced into large, irregularly shaped, brown-black, necrotic lesions with chlorotic haloes visible on both adaxial and abaxial surfaces of the leaf. This disease resulted in lower quality and reduced marketability of the cabbage. Five gram-negative, blue-green fluorescing bacteria were isolated from separate lesions on different plants on Kings medium B agar. The isolates were positive for levan formation and negative for oxidase and arginine dihydrolase. The isolates did not cause soft rot on potato slices but did induce a hypersensitive reaction in tobacco (Nicotiana tabacum L. cv. Samsun). These data indicated that the bacteria belonged to Lelliots LOPAT group 1 (2). Repetitive extragenic palindromic sequence (rep)-PCR using the BOXA1R primer resulted in identical DNA fragment banding patterns for the cabbage isolates and the pathotype of Pseudomonas cannabina pv. alisalensis (formerly P. syringae pv. alisalensis). Additionally, both P. cannabina pv. alisalensis and the five cabbage isolates were sensitive to bacteriophage PBS1 while the pathotype strain of P. syringae pv. maculicola was not (1). Pathogenicity of the five cabbage isolates was evaluated in two independent experiments. Inoculum was prepared by growing the bacteria on nutrient agar for 48 h (27°C), suspending the bacteria in 0.01 M phosphate buffer (pH 7.0), adjusting each suspension to 0.6 OD at 600 nm (approximately 108 CFU/ml), and adding three to five drops of Tween 20. In each experiment, two cabbage, broccoli raab (Brassica rapa subsp. rapa cv. Sorrento), and oat (Avena sativa cv. Montezuma) plants were inoculated for each isolate by spraying until runoff. Positive control plants were inoculated with the pathotype strain of P. cannabina pv. alisalensis and negative control plants were treated with sterile 0.01 M phosphate buffer. The plants were placed in a mist chamber for 48 to 72 h and then in a greenhouse (20 to 25°C). After 7 to 10 days, foliar symptoms similar to symptoms observed on the original diseased cabbage plants developed on all inoculated plants including the positive control plants inoculated with P. cannabina pv. alisalensis. Additionally severe symptoms on broccoli raab and minor symptoms on oats developed on plants inoculated with cabbage strains or P. cannabina pv. alisalensis. For each experiment, bacteria reisolated from symptomatic tissue were identical to the bacteria used to inoculate the plants and to P. cannabina pv. alsialensis for rep-PCR DNA fragment banding pattern and sensitivity to phage PBS1. There were no symptoms on any of the cabbage and oat negative controls. Additionally, there were no symptoms on any broccoli raab negative controls in the first experiment; however, in the second experiment, a small (<1 mm) lesion was detected on one leaf of one plant. To our knowledge, this is the first report of P. cannabina pv. alisalensis causing bacterial blight of cabbage in California. This disease may have significant impact because of the large acreage of cabbage grown in California (approximately 5,666 ha annually). References: (1) C. T. Bull et al. Syst. Appl. Microbiol. 33:105, 2010. (2) R. A. Lelliott. J. Appl. Bacteriol. 29:470, 1966.

Angular Leaf Spot of Cucurbits is Associated With Genetically Diverse Pseudomonas syringae Strains

Angular leaf spot of cucurbits is generally considered to be caused by Pseudomonas syringae pv. lachrymans. It has a worldwide distribution and has been observed to emerge sporadically under humid and wet conditions. Reports of multiple P. syringae pathovars associated with the disease and lack of molecular analysis has left the true diversity of populations in the United States unclear. In this study, we collected 27 P. syringae strains causing foliar lesions and blighting on watermelon, cantaloupe, and squash in Florida, Georgia, and California over several years. Strains were fluorescent on Kings medium B agar and displayed the typical phenotypic and biochemical characteristics of P. syringae. P. syringae pv. lachrymans is a member of genomospecies 2. However, the genetic profiles obtained through both MLSA (gyrB, rpoD, gapA, and gltA) and BOX-PCR (BOXA1R) identified 26 of the P. syringae strains to be distributed among three clades within genomospecies 1, and phylogenetically distinct from genomospecies 2 member P. syringae pv. lachrymans. A novel MLSA haplotype of the pathogen common to all states and cucurbit hosts was identified. Considerable genetic diversity among P. syringae strains infecting cucurbits is associated with the same disease, and reflects the larger ecological diversity of P. syringae populations from genomospecies 1.

First Report of Bacterial Leaf Spot of Swiss Chard Caused by Pseudomonas syringae pv. aptata in California

From 1999 through 2003, a previously unreported disease was found on commercial Swiss chard (Beta vulgaris subsp. cicla) in the Salinas Valley, (Monterey County) California. Each year the disease occurred sporadically throughout the long growing season from April through September. Initial symptoms were water-soaked leaf spots that measured 2 to 3 mm in diameter. As disease developed, spots became circular to ellipsoid, 3 to 8 mm in diameter, and tan with distinct brown-to-black borders. Spots were visible from the adaxial and abaxial sides. Cream-colored bacterial colonies were consistently isolated from spots. Strains were fluorescent on Kings medium B, levan positive, oxidase negative, and arginine dihydrolase negative. Strains did not rot potato slices but induced a hypersensitive reaction on tobacco (Nicotiana tabacum cv. Turk). The isolates, therefore, belong in LOPAT group 1 (1). Fatty acid methyl esters (FAME) analysis (MIS-TSBA version 4.10, MIDI Inc., Newark, DE) gave variable results that included Pseudomonas syringae, P. cichorii, and P. viridiflava with similarity indices ranging from 0.91 to 0.95. BOX-polymerase chain reaction (PCR) analysis gave identical banding patterns for the chard isolates and for known P. syringae pv. aptata strains, including the pathotype strain CFBP1617 (2). The bacteria were identified as P. syringae. Pathogenicity of 11 strains was tested by growing inoculum in nutrient broth shake cultures for 48 h, diluting to 10 × 6 CFU/ml, and spraying onto 5-week-old plants of Swiss chard cvs. Red, White, Silverado, and CXS2547. Untreated control plants were sprayed with sterile nutrient broth. After 7 to 10 days in a greenhouse (24 to 26°C), leaf spots similar to those observed in the field developed on all inoculated plants. Strains were reisolated from the spots and identified as P. syringae. Control plants remained symptomless. To investigate the host range of this pathogen, the same procedures were used to inoculate three strains onto other Chenopodiaceae plants: five cultivars of sugar beet (B. vulgaris), and one cultivar each of spinach (Spinacia oleracea) and Swiss chard. In addition, five chard strains and strain CFBP1617 were inoculated onto two cultivars of sunflower (Helianthus annuus), and one cultivar each of cantaloupe (Cucumis melo), sugar beet, spinach, and Swiss chard. All Swiss chard, cantaloupe, sunflower, and sugar beet plants developed leaf spots after 7 days. The pathogen was reisolated from spots and confirmed to be the same bacterium using BOX-PCR analysis. Spinach and untreated controls failed to show symptoms. All inoculation experiments were done at least twice and the results were the same. The phenotypic data, fatty acid and genetic analyses, and pathogenicity tests indicated that these strains are P. syringae pv. aptata. To our knowledge this is the first report of bacterial leaf spot of commercially grown Swiss chard in California caused by P. syringae pv. aptata. The disease was particularly damaging when it developed in Swiss chard fields planted for baby leaf fresh market products. Such crops are placed on 2-m wide beds, planted with high seed densities, and are sprinkler irrigated. This disease has been reported from Asia, Australia, Europe, and other U.S. states. References: (1) R. A. Lelliott et al. J. Appl. Bacteriol. 29:470, 1966. (2) J. L. W. Rademaker et al. Mol. Microbiol. Ecol. Man. 3.4.3:1-27, 1998.

First Report of Bacterial Leaf Spot of Italian Dandelion (Cichorium intybus) Caused by a Pseudomonas syringae Pathovar in California

Italian dandelion (Cichorium intybus) is a leafy, nonhead forming chicory plant that is eaten as a fresh vegetable in salads. During the late summer (August through October) of 2002, in the Salinas Valley (Monterey County) in California, a previously unreported disease was found in commercial Italian dandelion fields. Early symptoms were angular, vein delimited, dark, water-soaked leaf spots that measured 2 to 7 mm in diameter. As disease developed, spots retained angular edges but exhibited various irregular shapes. Spots commonly formed along the edges of the leaves; in some cases these spots developed into lesions that measured between 10 and 30 mm long. Spots were visible from adaxial and abaxial sides and were dull black in color. A cream-colored pseudomonad was consistently isolated from leaf spots that were macerated and streaked onto sucrose peptone agar. Fungi were not recovered from any of the spots. Recovered strains were blue-green fluorescent when streaked onto Kings medium B agar. Bacterial strains were levan positive, oxidase negative, and arginine dihydrolase negative. Strains did not rot potato slices but induced a hypersensitive reaction on tobacco (Nicotiana tabacum cv. Turk). These data indicated that the bacteria belonged to LOPAT group 1 of Pseudomonas syringae (1). Pathogenicity of six strains was tested by growing inoculum in nutrient broth shake cultures for 48 h, diluting to 106 CFU/ml, and spraying onto 12 6-week-old plants of Italian dandelion cv. Catalogna Special. Untreated control plants were sprayed with sterile nutrient broth. After 10 to 12 days in a greenhouse (24 to 26°C), leaf spots similar to those observed in the field developed on all inoculated plants. Strains were reisolated from the spots and identified as P. syringae. Control plants remained symptomless. These inoculation experiments were done twice and the results were the same. Amplification of repetitive bacterial sequences (repetitive sequence-based polymerase chain reaction [rep-PCR]) demonstrated that all Italian dandelion strains had the same rep-PCR fingerprint, which differed from fingerprints of P. syringae pv. tagetis and P. syringae pv. tabaci. Additionally, toxin specific primers did not amplify tagetitoxin or tabtoxin biosynthesis genes from Italian dandelion strains. To our knowledge, this is the first report of bacterial leaf spot of commercially grown Italian dandelion in California caused by a P. syringae pathovar. Because fields were irrigated with overhead sprinklers, the disease was severe in several fields and as much as 30% of those plantings were not harvested. Reference: (1) R. A. Lelliott et al. J. Appl. Bacteriol. 29:470, 1966.