First Report of Bacterial Leaf Blight on Mustard Greens (Brassica juncea) Caused by Pseudomonas cannabina pv. alisalensis in Mississippi.

In 2010, a brassica leafy greens grower in Sunflower County, MS, observed scattered outbreaks of a leaf blight on mustard greens (Brassica juncea) in a 180-ha field. A severe outbreak of leaf blight occurred on mustard greens and turnip greens (B. rapa) in the same field in 2011 with more than 80 ha affected. The affected field, established in 2010, had no prior history of being cropped to brassica leafy greens. Symptoms appeared on the 6-week-old transplants as brown to tan necrotic spots with faint chlorotic borders and associated water-soaking. Lesions varied from 4 mm to 3 cm in diameter and often coalesced to cover >90% of older leaves. Whole plants of the mustard greens cv. Florida Broadleaf were collected in 2011 from the symptomatic field. Leaves were surface-disinfested with 0.5% NaOCl for 5 min, rinsed twice in sterilized distilled water [(sd)H2O], macerated in sdH2O, then streaked onto nutrient agar (NA), pseudomonas agar F (PAF), and potato dextrose agar (PDA). Little or no bacterial growth was observed on PDA, while on NA and PAF the majority of bacterial growth appeared to be a single colony type. All strains collected (25 total, one per plant) were gram-negative and fluoresced blue-green under UV light after 48 h at 28°C on PAF. All 25 strains were identified as belonging to Pseudomonas group 1a using Lelliot's determinative assay (2). Ten of the 25 strains were tested for pathogenicity on Florida Broadleaf, and turnip greens cv. Alamo. Bacteria were grown on PAF for 48 h, and a bacterial suspension was prepared and adjusted to an optical density of 0.1 at 600 nm. Three-week-old plants (three plants per cultivar) were sprayed with the appropriate bacterial suspension to runoff, placed at 100% relative humidity for 48 h, and then put in a growth chamber at 28°C with a 16-h diurnal light cycle for 14 days. Additionally, three plants each of Florida Broadleaf and Alamo were either sprayed with H2O or inoculated with Pseudomonas cannabina pv. alisalensis (Pca) pathotype strain BS91 (1). All 10 strains, as well as the Pca pathotype strain, were pathogenic on both cultivars and caused symptoms similar to those observed in the field. Symptoms were not observed on H2O-sprayed plants. Comparative rep-PCR analysis using the BOXA1R primer showed the 10 strains had identical DNA-banding profiles and were identical to that of Pca BS91 (5). Five strains tested using a Pca-specific, 'light-tagged' reporter bacteriophage gave a strong positive reaction, while a negative control strain, P. syringae pv. maculicola, gave no signal (3). From these tests, the isolated bacteria were determined to be Pca. Bacteria re-isolated on PAF from the inoculated Florida Broadleaf plants had identical rep-PCR profiles with those of the strains used for inoculations. Over the past 10 years, Pca has been found in numerous states in the United States, as well as in Europe, Australia, and Japan (4). As brassica leafy greens production expands to new fields and new states, leaf blight caused by Pca appears to become a problem rapidly. Since resistant cultivars and highly effective bactericides are lacking, growers are extremely concerned about the rapid spread of this disease into existing and new brassica leafy greens regions. References: (1) N. A. Cintas et al. Plant Dis. 86:992, 2002. (2) R. Lelliott. J. Appl. Bacteriol. 29:470, 1066. (3) D. Schofield et al. Appl. Environ. Microbiol. 78:3592, 2012. (4) F. Takahashi et al. J. Gen. Plant Pathol. 79:260, 2013. (5) J. Versalovic et al. Methods Mol. Cell Biol 5:25, 1994.

Expression of Bacterial Blight Resistance in Brassica Leafy Greens Under Field Conditions and Inheritance of Resistance in a Brassica juncea Source.

Brassica leafy greens are one of the most economically important vegetable commodity groups grown in the southeastern United States, and more than 28,000 metric tons of these crops are harvested in the United States annually. Collard and kale (Brassica oleracea Acephala group), mustard green (B. juncea), and turnip green (B. rapa) are the most commonly planted members of the brassica leafy greens group. In the last 10 years, numerous occurrences of bacterial blight on these leafy vegetables have been reported in several states. One of the pathogens responsible for this blight is designated Pseudomonas cannabina pv. alisalensis. Two B. rapa (G30710 and G30499) and two B. juncea (PI418956 and G30988) plant introductions (PIs) that exhibited moderate to high levels of resistance to this pathogen in greenhouse studies were tested for field resistance in comparison with eight commercial cultivar representatives of turnip green, mustard green, collard, and kale. The two B. juncea PIs and one of the B. rapa PIs (G30499) were found to have significantly less disease than all tested cultivars except 'Southern Curled Giant' mustard green (B. juncea) and 'Blue Knight' kale (B. oleracea). Inheritance of resistance studies performed with populations derived from the resistant G30988 and two susceptible PIs provided some evidence that resistance may be controlled by a single recessive gene.

Soil Solarization and Biological Control for Managing Mesocriconema xenoplax and Short Life in a Newly Established Peach Orchard.

The effects of soil solarization, with and without a Pseudomonas spp. cocktail or wheat rotation as alternatives to chemical control of Mesocriconema xenoplax, were investigated from 2004 to 2011. Preplant solarization and soil fumigation (67% methyl bromide + 33% chloropicrin mixture; henceforth, referred to as MBr) was initiated in 2004 in an orchard infested with M. xenoplax and a history of peach tree short life (PTSL). Plots consisted of nine treatments: (i) nonsolarized soil-alone, (ii) nonsolarized soil with bacteria cocktail (nonsolar-bacteria), (iii) nonsolarized soil with wheat (nonsolar-wheat), (iv) nonsolarized soil with bacteria cocktail and wheat (nonsolar-bacteria-wheat), (v) solarized soil-alone, (vi) solarized soil with bacteria cocktail solar-bacteria), (vii) solarized soil with wheat (solar-wheat), (viii) solarized soil with bacteria cocktail and wheat (solar-bacteria-wheat), and (ix) preplant MBr fumigation. Peach trees were planted into all plots in 2005. Nematode populations were suppressed 20 months longer after orchard establishment in solar-alone and solar-wheat plots than solar-bacteria and solar-bacteria-wheat plots. Pseudomonas spp. cocktails did not have a pronounced effect in suppressing M. xenoplax in this study. Fumigation effect on M. xenoplax population density dissipated 24 months after application. Solar-wheat-treated soil was as effective as preplant MBr fumigation in increasing tree survival from PTSL for at least 6 years after orchard establishment.

First Report of Bacterial Leaf Blight on Broccoli and Cabbage Caused by Pseudomonas syringae pv. alisalensis in South Carolina.

In May of 2009, leaf spot and leaf blight symptoms were observed on broccoli (Brassica oleracea var. italica) and cabbage (B. oleracea var. capitata) on several farms in Lexington County, the major brassica-growing region of South Carolina. Affected areas ranged from scattered disease foci within fields to entire fields. Initial infection symptoms on leaves of both crops included circular and irregular-shaped necrotic lesions that were 3 to 10 mm in diameter, often with yellow halos and water soaking. As the disease progressed, the lesions tended to coalesce into a general blight of the entire leaf. Diseased leaves from both broccoli and cabbage were collected from each of four fields at different locations in the county. Leaves were surface disinfested, macerated in sterile distilled water, then aliquots of the suspension were spread on King's medium B (KB) agar. All samples produced large numbers of bacterial colonies that fluoresced blue under UV light after 24 h of growth. In total, 23 isolates (13 from broccoli and 10 from cabbage) were collected. These isolates were gram negative, levan production positive, oxidase negative, pectolytic activity negative, arginine dihydrolase negative, and produced a hypersensitive response on tobacco, thus placing them in the Pseudomonas syringae LOPAT group (2). Two broccoli and two cabbage isolates were selected at random and tested for pathogenicity to cabbage cv. Early Jersey Wakefield, broccoli cv. Decicco, turnip cv. Topper, broccoli raab cv. Spring, collard cv. Hi-Crop, and oat cv. Montezuma in greenhouse tests. Bacteria were grown on KB agar for 24 h and a bacterial suspension was prepared and adjusted to an optical density of 0.1 at 600 nm. Three-week-old plants were spray inoculated to runoff and held at 100% relative humidity for 12 h after inoculation, prior to return to the greenhouse bench (4). P. syringae pv. maculicola strain F18 (4) and the pathotype strain of P. syringae pv. alisalensis BS91 were included as controls, along with a water-inoculated negative control. Plants were evaluated at 14 days postinoculation. The four unknown bacterial isolates and BS91 were pathogenic on all brassica plants tested, as well as on oat. In contrast, the P. syringae pv. maculicola strain F18 was not pathogenic on broccoli raab or oat. Symptoms produced by all isolates and strains tested were similar to those observed in the field. No symptoms were observed on water-inoculated plants. Comparative repetitive sequence-based (rep)-PCR DNA analysis using the BOXA1R primer (3) resulted in a DNA banding pattern of each of the isolates from the South Carolina fields (23 isolates), as well as those reisolated from inoculated plants, that was identical to P. syringae pv. alisalensis BS91 and differed from the P. syringae pv. maculicola F18 strain. On the basis of the rep-PCR assays and the differential host range (1), the current disease outbreak on broccoli and cabbage in South Carolina is caused by the bacterium P. syringae pv. alisalensis. Broccoli is a relatively new, albeit rapidly expanding, production vegetable in South Carolina; this disease may represent a limiting factor to future production. References: (1) N. A. Cintas et al. Plant Dis. 86:992, 2002. (2) R. A. Lelliott et al. J. Appl. Bacteriol. 29:470, 1966. (3) J. Versalovic et al. Methods Mol. Cell. Biol. 5:25, 1994. (4) Y. F. Zhao et al. Plant Dis. 84:1015, 2000.

Simultaneous detection of Acidovorax avenae subsp. citrulli and Didymella bryoniae in cucurbit seedlots using magnetic capture hybridization and real-time polymerase chain reaction.

To improve the simultaneous detection of two pathogens in cucurbit seed, a combination of magnetic capture hybridization (MCH) and multiplex real-time polymerase chain reaction (PCR) was developed. Single-stranded DNA hybridization capture probes targeting DNA of Acidovorax avenae subsp. citrulli, causal agent of bacterial fruit blotch, and Didymella bryoniae, causal agent of gummy stem blight, were covalently attached to magnetic particles and used to selectively concentrate template DNA from cucurbit seed samples. Sequestered template DNAs were subsequently amplified by multiplex real-time PCR using pathogen-specific TaqMan PCR assays. The MCH multiplex real-time PCR assay displayed a detection threshold of A. avenae subsp. citrulli at 10 CFU/ml and D. bryoniae at 10(5) conidia/ml in mixtures of pure cultures of the two pathogens, which was 10-fold more sensitive than the direct real-time PCR assays for the two pathogens separately. Although the direct real-time PCR assay displayed a detection threshold for A. avenae subsp. citrulli DNA of 100 fg/microl in 25% (1/4 samples) of the samples assayed, MCH real-time PCR demonstrated 100% detection frequency (4/4 samples) at the same DNA concentration. MCH did not improve detection sensitivity for D. bryoniae relative to direct real-time PCR using conidial suspensions or seed washes from D. bryoniae-infested cucurbit seed. However, MCH real-time PCR facilitated detection of both target pathogens in watermelon and melon seed samples (n = 5,000 seeds/sample) in which 0.02% of the seed were infested with A. avenae subsp. citrulli and 0.02% were infested with D. bryoniae.

First Report of Severe Outbreaks of Bacterial Leaf Spot of Leafy Brassica Greens Caused by Xanthomonas campestris pv. campestris in South Carolina.

Severe outbreaks of leaf spot disease of leafy vegetable brassica crops have occurred from early spring to late fall for at least the past 7 years in Lexington County, South Carolina, the major growing region for leafy greens in the state. Significant economic losses to this disease totaling $1.7 million have been incurred by large and small growers. In 2005, Pseudomonas syringae pv. maculicola was reported as one of the causal organisms of leaf spot disease in South Carolina (2). Investigations during 2006 and 2007 have led to the isolation of another bacterium causing leaf spotting of brassica crops. Symptoms in the field were nearly identical to symptoms caused by P syringae pv. maculicola, i.e., small, brown necrotic spots, often with chlorotic halos that expand and coalesce to cover the leaves. Colonies recovered from diseased tissues were xanthomonad like, nonfluorescent on Pseudomonas Agar F, mucoid on yeast extract dextrose chalk medium, grew at 35°C, hydrolyzed starch, positive for protein digestion, alkaline in litmus milk, and produce acid from arabinose. Sequence data from the 16S rDNA and fatty acid methyl ester analysis gave the best homology to Xanthomonas campestris pv. campestris with a similarity score index of >0.98 and >0.70, respectively, confirming genus and species. Excised-cotyledon assays, used to differentiate between pathovars campestris and armoraciae, confirmed the pathovar as campestris (1). Pathogenicity assays with spray inoculations (1 × 107 CFU/ml) (3) on eight plants each of 'Topper' and 'Alamo' turnip, 'Early Jersey Wakefield' cabbage, and 'Money maker' tomato produced leaf-spot symptoms within 10 days in the greenhouse and growth chamber on the turnip and cabbage plants, but not the tomato. X. campestris pv. campestris, which is common throughout the world, also is the causal agent of black rot in brassica. Typical black rot symptoms are seen often in Lexington County fields in summer and are quite different from the leaf spot symptoms observed. Leaf-spotting X. campestris pv. campestris (LS) strains and black rot (BR) strains, recovered from black rot-symptomatic plants lacking leaf spots, from the same fields were compared in greenhouse pathogenicity assays on six plants each of 'Topper' turnip and 'Early Jersey Wakefield' cabbage. Spray inoculations with 20 individual LS strains and 10 individual BR strains, collected from 2005 to 2007, produced symptoms unique to each group. These symptoms included chlorotic 'V'-shaped lesions initiating from the leaf margins with black veining when plants were inoculated with BR strains, versus rapid and severe leaf spotting followed by chlorotic 'V'-shaped lesions typically lacking black-veining 10 to 16 days postinoculation associated with LS strains. Additional inoculation tests gave similar results. To our knowledge, this is the first report of a severe leaf spotting disease of field-grown brassica leafy greens caused by X. campestris pv. campestris in South Carolina. These findings may have importance in differentiation of bacterial leaf spot pathogens in brassica crops. References: (1) A. M. Alvarez et al. Phytopathology 84:1449, 1994. (2) A. P. Keinath et al. Plant Dis. 90:683, 2006. (3) W. P. Wechter et al. Hortic Sci. 42:1140, 2007.

First Report of Bacterial Leaf Spot on Leafy Brassica Greens Caused by Pseudomonas syringae pv. maculicola in South Carolina.

As of 2001, South Carolina ranked second in the United States in acreage of turnip greens (Brassica rapa) and collard (B. oleracea) and third in acreage of mustard (B. juncea). In June 2001, a leaf disease was found on turnip greens (cv. Alamo), mustard (cvs. Southern Giant Curled and Florida Broadleaf), and rape salad greens (B. napus var. napus cv. Essex) on a commercial farm in Lexington County, South Carolina. Symptoms appeared after a heavy rainstorm that included blowing sand. The disease was found in May and June 2002 on three additional farms in the same county on turnip greens cv. Topper and Royal Crown and collard cv. Top Bunch. Symptoms included small tan spots, water soaking, yellowing, and brown necrosis of leaves after spots coalesced on the lower halves of plants. Yellowing was more prevalent on older than on younger leaves. Leaf samples were collected in 2001 and 2002 from the affected hosts on the four farms. Bacterial streaming was evident from these samples and 27 strains were isolated on nutrient agar or King's medium B (KMB). All strains were gram negative and fluoresced bluegreen or yellow under UV light after 48-h growth at 28°C on Pseudomonas agar F (PAF). On the basis of LOPAT tests, the strains were identified as P. syringae (2). All 27 strains were tested for pathogenicity to rape salad greens cv. Essex and then to turnip greens cv. Topper. Plants were grown in peat-vermiculite potting mix in 10-cm-diameter pots in a greenhouse. P. syringae pv. maculicola F41, isolated from turnip in Oklahoma, and P. syringae pv. tomato F33, isolated from tomato in Oklahoma, were included as positive and negative controls along with a noninoculated control. Bacteria were grown on KMB for 48 h at 24°C, and bacterial suspensions were prepared and adjusted to 0.1 optical density at 600 nm. Three-week-old plants were held at 95 to 100% relative humidity (RH) for 48 h before they were sprayed just to runoff with inoculum and then held at 95 to 100% RH for 48 h after inoculation (4). After an additional 5 to 8 days in a greenhouse, nine strains and F41 caused symptoms on both Topper and Essex similar to symptoms observed in the field. No symptoms were observed on noninoculated plants or plants inoculated with F33. On the basis of repetitive sequence-based polymerase chain reactions with the BOXA1R primer, the DNA fingerprint of each of the nine pathogenic strains from South Carolina was nearly identical to that of F41. Bacteria isolated from inoculated, symptomatic turnip leaves had identical LOPAT and BOXA1R profiles to the corresponding original strains. Pathogenic strains had bluegreen fluorescence on PAF, whereas nonpathogenic strains fluoresced yellow. Five pathogenic strains, as well as F41, were further identified to species and pathovar with fatty acid methyl ester profiles as P. syringae pv. maculicola. To our knowledge, this is the first report of P. syringae pv. maculicola from South Carolina. Over the past 10 years, P. syringae pv. maculicola has been found in Oklahoma (4), California (1), and Ohio (3). Bacterial leaf spot has occurred yearly in South Carolina since the initial outbreaks. Currently, it is the disease that causes the greatest yield losses of leafy brassica greens in the state. References: (1) N. A. Cintas et al. Plant Dis. 85:1207, 2001. (2) R. A. Lelliott et al. J. Appl. Bacteriol. 29:470, 1966. (3) M. L. Lewis Ivey et al. Plant Dis. 86:186, 2002. (4) Y. F. Zhao et al. Plant Dis. 84:1015, 2000.

Physical mapping, BAC-end sequence analysis, and marker tagging of the soilborne nematicidal bacterium, Pseudomonas synxantha BG33R.

A bacterial artificial chromosome (BAC) library was constructed for the genome of the rhizosphere-inhabiting fluorescent pseudomonad Pseudomonas synxantha BG33R. Three thousand BAC clones with an average insert size of 140 kbp and representing a 70-fold genomic coverage were generated and arrayed onto nylon membranes. EcoRI fingerprint analysis of 986 BAC clones generated 23 contigs and 75 singletons. Hybridization analysis allowed us to order the 23 contigs and condense them into a single contig, yielding an estimated genome size of 5.1 Mb for P. synxantha BG33R. A minimum-tile path of 47 BACs was generated and end-sequenced. The genetic loci involved in ring nematode egg-kill factor production in BG33R Tn5 mutants, 246 (vgrG homolog), 1122 (sensor kinase homolog), 1233 (UDP-galactose epimerase homolog), 1397 (ferrisiderophore receptor homolog), and 1917 (ribosomal subunit protein homolog), have been mapped onto the minimum-tile BAC library. Two of the genetic regions that flank Tn5 insertions in BG33R egg-kill-negative mutants 1233 and 1397 are separated by a single BAC clone. Fragments isolated by ligation-mediated PCR of the Tn5 mutagenized regions of 29 randomly selected, non-egg-kill-related, insertion mutants have been anchored onto the ordered physical map of P. synxantha.

Biological control of the phytoparasitic nematode Mesocriconema xenoplax on peach trees.

Seven fluorescent Pseudomonas spp. capable of inhibiting reproduction of Mesocriconema xenoplax have been isolated from soil sites that suppress both nematode multiplication and Peach Tree Short Life (PTSL). One of these seven strains, Pseudomonas sp. BG33R, inhibits M. xenoplax multiplication in vivo and egg hatch in vitro. Mesocriconema xenoplax populations on peach seedlings inoculated with BG33R and planted into soil-solarized field plots remained at or below the economic threshold for nematicide treatment in South Carolina for nearly 18 months. Soil solarization alone induced a shift toward a microbial community that was suppressive to nematode multiplication. Additionally, five Tn5 mutants of BG33R, lacking the ability to kill eggs, have been generated. The Tn5 insertion site in each mutant has been cloned and sequenced. DNA sequence analysis has revealed a high degree of homology to several genes of interest because of their potential involvement in the production of the egg-kill factor. These Tn5 egg-kill negative mutants also no longer produce protease or salicylic acid while producing nearly twice the amount of fluorescent siderophore as the wild type parent.