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

Plant Disease ◽  
2006 ◽  
Vol 90 (5) ◽  
pp. 683-683 ◽  
Author(s):  
A. P. Keinath ◽  
W. P. Wechter ◽  
J. P. Smith

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.

Plant Disease ◽  
2002 ◽  
Vol 86 (8) ◽  
pp. 921-921 ◽  
Author(s):  
S. T. Koike ◽  
H. R. Azad ◽  
D. C. Cooksey

In 2000 and 2001, a new disease was observed on commercial spinach (Spinacia oleracea) in the Salinas Valley, Monterey County, CA. Initial symptoms were water-soaked, irregularly shaped leaf spots (2 to 3 mm diameter). As the disease developed, spots enlarged to as much as 1 to 2 cm, were vein-delimited, and turned dark brown. Faint chlorotic halos sometimes surrounded the spots. Death of large areas of the leaf occurred if spots coalesced. Spots were visible from the adaxial and abaxial sides of leaves, and no fungal structures were observed. The disease occurred on newly expanded and mature foliage. No fungi were isolated from the spots. However, cream-colored bacterial colonies were consistently isolated on sucrose peptone agar, and these strains were nonfluorescent on King's medium B. Strains were positive for levan and negative for oxidase, arginine dihydrolase, and nitrate reductase. Strains did not grow at 36°C, did not rot potato slices, but induced a hypersensitive reaction in tobacco (Nicotiana tabacum cv. Turk). These results suggested the bacterium was similar to Pseudomonas syringae. Fatty acid methyl ester (FAME) analysis (MIS-TSBA 4.10, MIDI Inc., Newark, DE) indicated the strains were highly similar (80.1 to 89.3%) to P. syringae pv. maculicola. However, in contrast to P. syringae pv. maculicola, the spinach strains did not utilize the carbon sources erythritol, L+tartrate, L lactate, and DL-homoserine. Pathogenicity of 10 strains was tested by growing inoculum in nutrient broth shake cultures for 48 h, diluting to 106 CFU/ml, and spraying 4-week-old plants of spinach cv. Bossanova. Control plants were sprayed with sterile nutrient broth. After 5 to 8 days in a greenhouse (24 to 26°C), leaf spots identical to those observed in the field developed on cotyledons and true leaves of inoculated plants. Strains were reisolated from the spots and identified as P. syringae. Control plants remained symptomless. The 10 strains were also inoculated on beet (Beta vulgaris), Swiss chard (Beta vulgaris subsp. cicla), cilantro (Coriandrum sativum), and spinach. Spinach showed leaf spots after 8 days; however, none of the other plants developed symptoms. Two strains were inoculated onto spinach cvs. Califlay, Lion, Nordic IV, Polka, Resistoflay, Rushmore, RZ 11, Spinnaker, Springfield, Viroflay, and Whitney. Leaf spot developed on all cultivars, and the pathogen was reisolated. Because the FAME data indicated a similarity between the spinach pathogen and P. syringae pv. maculicola, we inoculated sets of spinach cv. Bolero, cabbage (Brassica oleracea subsp. capitata cv. Grenedere), and cauliflower (Brassica oleracea subsp. botrytis cv. White Rock) with three P. syringae pv. maculicola and three spinach strains. Cabbage and cauliflower developed leaf spots only when inoculated with P. syringae pv. maculicola; spinach had leaf spots only when inoculated with the spinach strains. All inoculation experiments were done twice, and the results of the two tests were the same. To our knowledge, this is the first report of bacterial leaf spot of spinach in California caused by a nonfluorescent P. syringae, and the first record of this disease in the United States. Biochemical characteristics and limited host range of the pathogen indicate the California strains are likely the same as the P. syringae pv. spinaciae pathogen that was reported in Italy (1) and Japan (2). References: (1) C. Bazzi et al. Phytopathol. Mediterr. 27:103, 1988. (2) K. Ozaki et al. Ann. Phytopathol. Soc. Jpn. 64:264, 1998.


Plant Disease ◽  
2002 ◽  
Vol 86 (2) ◽  
pp. 186-186 ◽  
Author(s):  
M. L. Lewis Ivey ◽  
S. Wright ◽  
S. A. Miller

In 2000, circular water-soaked lesions typical of bacterial leaf spot were observed on leaves of collards (Brassica oleracea L. var. viridis) throughout commercial fields in northwest Ohio. Light brown, rectangular, water-soaked lesions were observed on turnip leaves (Brassica rapa L.). Bacterial streaming from lesions on both crops was observed microscopically. Cream colored, fluorescent colonies were isolated from diseased tissues on Pseudomonas F medium, and eight representative colonies (four from collards and four from turnip) were selected and purified. Fatty acid methyl ester analysis was performed on all of the isolates. Two from collards and two from turnip were identified as Pseudomonas syringae pv. maculicola (mean similarity index = 0.82 [MIDI Inc., Newark, DE]). DNA extracts from pure cultures of the P. syringae pv. maculicola strains were used as template in a polymerase chain reaction (PCR) assay with primers derived from the region of the coronatine gene cluster controlling synthesis of the coronafacic acid moiety found in P. syringae pv. tomato and P. syringae pv. maculicola (CorR and CorF2) (D. Cuppels, personal communication). DNA from P. syringae pv. tomato strain DC3000 and P. syringae pv. maculicola strain 88–10 (2) served as positive controls, while water and DNA from Xanthomonas campestris pv. vesicatoria strain Xcv 767 were used as negative controls. The expected 0.65-kb PCR product was amplified from three of four strains (two from turnip and one from collards) and the positive control DNA, but not from the negative controls. Pathogenicity tests were performed twice on 6-week-old turnip (‘Forage Star’, ‘Turnip Topper’, ‘Turnip Alamo’, ‘Turnip 7’), collard (‘Champion’) and mustard (Brassica juncea L. ‘Southern Giant Curl’) seedlings using the three PCR-positive strains. Premisted seedlings were spray-inoculated separately with each of the three strains (2 × 108 CFU/ml, 5 ml per plant) and a water control. Greenhouse temperatures were maintained at 20 ± 1°C. For both tests, all strains caused characteristic lesions on all of the crucifer cultivars within 5 days after inoculation; the control plants did not develop symptoms. To satisfy Koch's postulates, one of the turnip strains was reisolated from ‘Turnip Topper’ plants, and the collard strain was reisolated from ‘Champion’ plants. The three original and two reisolated strains induced a hypersensitive response in Mirabilis jalapa L. and Nicotiana tabacum L. var. xanthia plants 24 h after inoculation with a bacterial suspension (1 × 108 CFU/ml). The original and reisolated strains were compared using rep-PCR with the primer BOXA1R (1). The DNA fingerprints of the reisolated strains were identical to those of the original strains. To our knowledge, this is the first report of bacterial leaf spot on commercially grown collards and turnip greens in Ohio. References: (1) B. Martin et al. Nucleic Acids Res. 20:3479, 1992. (2) R. A. Moore et al. Can. J. Microbiol. 35:910, 1989.


Plant Disease ◽  
2021 ◽  
Author(s):  
Lei Li ◽  
Yishuo Huang ◽  
Yanxia Shi ◽  
A LI CHAI ◽  
Xuewen Xie ◽  
...  

Coriander (Coriandrum sativum L.) or Chinese parsley is a culinary herb with multiple medicinal effects that are widely used in cooking and traditional medicine. From September to November 2019, symptoms were observed in 2-month-old coriander plants from coriander fields in Lanzhou and Wenzhou, China. The disease developed rapidly under cold and wet climatic conditions, and the infection rate was almost 80% in open coriander fields. Typical symptoms on leaves included small, water-soaked blotches and irregular brown spots surrounding haloes; as the disease progressed, the spots coalesced into necrotic areas. Symptomatic leaf tissue was surface sterilized, macerated in sterile distilled water, and cultured on nutrient agar plates at 28 °C for 48 h (Koike and Bull, 2006). After incubation, six bacterial colonies, which were individually isolated from collected samples from two different areas, were selected for further study. Colonies on NA plate were small, round, raised, white to cream-colored, and had smooth margins. All bacterial isolates were gram-negative, rod-shaped and nonfluorescent on King's B medium. The bacteria were positive for levan production, Tween 80 hydrolysis, and tobacco hypersensitivity but negative for oxidase, potato slice rot test, arginine dihydrolase, ice nucleation activity, indole production and H2S production. The suspension of representative isolate for inoculating of plants was obtained from single colony on King's B medium for 2-3 days at 28 °C. DNA was extracted from bacterial suspensions of YS2003200102 cultured in 20 ml of King’s B medium broth at 28 °C for 1 day. Extraction was performed with a TIANamp Bacterial DNA Kit (TIANGEN, China) according to the manufacturer’s recommendations. The pathogen was confirmed by amplification and sequencing of the glyceraldehyde-3-phosphate dehydrogenase A (gapA) gene, the citrate synthase (gltA) gene, the DNA gyrase B (gyrB) gene and the RNA polymerase sigma factor 70 (rpoD) gene using gapA-For/gapA-Rev, gltA-For/gltA-Rev, gyrB-For/gryB-Rev, rpoD-For/rpoD-Rev primers, respectively (Popović et al., 2019). The sequences of the PCR products were deposited in GenBank with accession numbers MZ681931 (gapA), MZ681932 (gltA), MZ681933 (gyrB), and MZ681934 (rpoD). Phylogenetic analysis of multiple genes (Xu and Miller, 2013) was conducted with the maximum likelihood method using MEGA7. The sequences of our isolates and ten published sequences of P. syringae pv. coriandricola were clustered into one clade with a 100% confidence level. To confirm the pathogenicity of isolate YS2003200102, 2-month-old healthy coriander plants were inoculated by spraying the leaves with a bacterial suspension (108 CFU ml−1) at 28 °C incubation temperature and 70% relative humidity condition, and sterile distilled water was applied as a negative control treatment (Cazorla et al. 2005). Three replicates were conducted for every isolate, and each replicate included 6 coriander plants. After twelve days, only the inoculated leaves with bacterial suspension showed bacterial leaf spot resembling those observed on naturally infected coriander leaves. Cultures re-isolated from symptomatic leaves showed the same morphological characteristics and molecular traits as those initially isolated from infected leaves in the field. This bacterium was previously reported causing leaf spot of coriander in India and Spain (Gupta et al. 2013; Cazorla et al. 2005). To our knowledge, this is the first report of P. syringae pv. coriandricola causing leaf spot disease on coriander in China. Studies are needed on strategies to manage P. syringae pv. coriandricola in crops, because its prevalence may cause yield loss on coriander in China.


Plant Disease ◽  
2021 ◽  
Author(s):  
Marilen Nampijja ◽  
Mike Derie ◽  
Lindsey J. du Toit

Arizona is an important region of the USA for winter production of baby leaf crops such as spinach (Spinacia oleracea), table beet (Beta vulgaris subsp. vulgaris Condivita Group), and Swiss chard (B. vulgaris subsp. vulgaris Cicla Group). In the winter of 2019, severe leaf spots were observed at 80% incidence and 40% severity per plant in a 1-ha baby leaf Swiss chard crop of an (unknown cultivar) in Arizona. The lesions were circular to irregular, necrotic, water-soaked, and 1 to 5 mm in diameter. Symptomatic leaf sections (1-cm2) were surface-sterilized with 0.6% NaOCl, rinsed, and macerated in sterilized, deionized water. An aliquot of each macerate was streaked onto King’s B (KB) agar medium. Cream-colored, non-fluorescent colonies typical of Pseudomonas were isolated consistently, and all were non-fluorescent. A dozen isolates selected randomly were all negative for potato soft rot, oxidase, and arginine dihydrolase, and positive for levan production and tobacco hypersensitivity, which is typical of fluorescent P. syringae isolates, but can also include non-fluorescent strains (Lelliot et al. 1966). Three isolates were tested for pathogenicity on the table beet cv. Red Ace and Swiss chard cv. Silverado. Strain Pap009 of P. syringae pv. aptata (Psa), demonstrated previously to be pathogenic on Swiss chard and table beet, served as a positive control strain (Derie et al. 2016; Safni et al. 2016). Each isolate was grown inoculated into medium 523 broth and incubated on a shaker at 175 rpm overnight at 25°C. Each bacterial suspension was adjusted to an optical density (OD) of 0.3 at 600 nm (108 CFU/ml), and diluted in 0.0125M phosphate buffer to 107 CFU/ml. Thirty-day-old seedlings grown in Redi-Earth Plug and Seedling Mix in a greenhouse at 22 to 26°C were inoculated by rubbing the abaxial and adaxial leaf surfaces of each plant with a cotton swab dipped in inoculum to which Carborundum had been added (0.06 g/10 ml). The negative control plants were treated similarly with phosphate buffer with Carborundum. The experiment was set up as a randomized complete block design with 4 replications per treatment and 6 seedlings per experimental unit. In both trials, leaf spots resembling those on the original plants developed on all table beet and Swiss chard plants inoculated with the Arizona isolates and Pap009, but not on negative control plants. Disease severity was greater on Swiss chard (average 39% leaf area with spots) than on table beet (14%). Re-isolates obtained from inoculated seedlings using the same method as the original isolations resembled Psa. Multilocus sequence analysis (MLSA) was carried out for the original three Arizona isolates and the re-isolates using DNA amplified from the housekeeping genes gyrB, rpoD, gapA, and gltA (Hwang et al. 2005; Sarkar and Guttman 2004). Sequence identities of these genes of the Arizona isolates (GenBank accession numbers MW291615 to MW291618 for strain Pap089; MW291619 to MW291622 for Pap095; and MW291623 to MW291626 for Pap096 for gltA, gyrB, rpoD, and gapA, respectively) and the re-isolates ranged from 98 to 100% with those of Psa pathotype strain CFBP 1617 in the PAMDB database (Almeida et al. 2010; Altschul et al. 1997). Based on Koch’s postulates, colony characteristics, and MLSA, Psa was the causal agent of leaf spots in the Arizona Swiss chard crop. To our knowledge, this is the first report of bacterial leaf spot on chard in Arizona. The pathogen could have been introduced on infected seed as Psa is readily seedborne and seed transmitted.


Plant Disease ◽  
2001 ◽  
Vol 85 (11) ◽  
pp. 1207-1207 ◽  
Author(s):  
N. A. Cintas ◽  
C. T. Bull ◽  
S. T. Koike ◽  
H. Bouzar

In 1998, a new disease was detected on 3-week-old commercial broccolini (Brassica oleracea L. var. botrytis × B. alboglabra) transplants in a Salinas Valley, Monterey County, CA greenhouse. Initial symptoms were small (2 to 4 mm diameter) circular to angular, water-soaked spots. As the disease progressed, spots remained relatively small, but turned tan to brown. When diseased tissues were macerated and streaked on King's medium B, a blue-green fluorescent pseudomonad was consistently isolated. 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. ‘Turk’). Fatty acid methyl ester analysis (MIS-TSBA, version 4.10, MIDI Inc., Newark, DE) indicated that strains had a high similarity index (0.82 or higher) to Pseudomonas syringae, and GN (version 3.50, Biolog, Inc., Hayward, CA) profiles also identified strains as P. syringae. The bacterium associated with the disease, therefore, was identified as P. syringae van Hall. Pathogenicity was demonstrated by growing inoculum in nutrient broth shake cultures for 48 h, misting the broth cultures (1×106 CFU/ml) onto broccolini (cv. Aspabrock), and subjecting the plants to 48 h of high humidity. Control plants were misted with sterile nutrient broth. After 4 to 5 days in a greenhouse, leaf spot symptoms developed on all inoculated broccolini plants, and reisolated strains were characterized and found to be P. syringae. Control plants remained symptomless. The results of two sets of pathogenicity tests were the same. Repetitive sequence-based polymerase chain reaction using the BOXA1R primer resulted in identical banding patterns for the broccolini pathogen and for known isolates of P. syringae pv. maculicola from crucifers. In host range testing, P. syringae pv. maculicolawas pathogenic to broccolini plants. The broccolini isolates and P. syringae pv. maculicola isolates had the same pathogenicity results when crucifers and tomatoes were tested as hosts; broccoli and cauliflower (B. oleracea var. botrytis) were infected, and tomato results were variable. These tests suggest that the broccolini pathogen is the bacterial leaf spot pathogen, Pseudomonas syringae pv. maculicola, that occurs on broccoli and cauliflower transplants (1). To our knowledge, this is the first report of this pathogen causing a disease on commercially grown broccolini. Reference: (1) S. T. Koike et al. Plant Dis. 82:727, 1998.


2005 ◽  
Vol 153 (3) ◽  
pp. 181-184 ◽  
Author(s):  
F. M. Cazorla ◽  
M. A. Vazquez ◽  
J. Rosales ◽  
E. Arrebola ◽  
J. Navarro ◽  
...  

Plant Disease ◽  
2003 ◽  
Vol 87 (11) ◽  
pp. 1397-1397 ◽  
Author(s):  
S. T. Koike ◽  
D. M. Henderson ◽  
C. T. Bull ◽  
P. H. Goldman ◽  
R. T. Lewellen

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 King's 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.


Plant Disease ◽  
2017 ◽  
Vol 101 (2) ◽  
pp. 380-380 ◽  
Author(s):  
E. V. Porotikova ◽  
U. D. Dmitrenko ◽  
E. E. Atapina ◽  
Y. A. Volkov ◽  
V. I. Risovannaya ◽  
...  

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