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.
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