First Report of Bacterial Leaf Spot of Parsley Caused by Pseudomonas syringae pv. coriandricola in Ohio

A severe leaf spot of parsley (Petroselinum crispum L. cvs. Dark Green Italian and Gigante) was observed on ∼1.5 ha in 2007 and 8 ha in 2012 on three vegetable farms in northern Ohio. Tiny, water-soaked spots that enlarged to necrotic lesions (∼5 mm wide) were first observed in June of each year. Lesions often coalesced and leaf marginal necrosis was common. Disease incidence initially ranged from 20 to 50%, and a 1.5-ha field was completely lost in 2012 as a result of the disease. Bacterial streaming was observed microscopically from leaf lesions. Diseased leaf tissue was dipped briefly in 70% ethanol, rinsed in sterile water, and blotted dry. Bacteria were isolated by plating 10-fold serial dilutions of diseased tissue extracts onto yeast dextrose carbonate and Pseudomonas F (PF) agar media. Whitish, opaque, circular colonies were isolated consistently from all samples. One isolate was purified from each of four fields. They were all gram-negative, non-fluorescent on PF medium, levan positive, oxidase negative, arginine dihydrolase negative, potato rot negative, and tobacco hypersensitive reaction positive. Repetitive extragenic palindromic sequence (Rep)-PCR fingerprint profiles using the BOXA1R primer (4) were identical for the four isolates. A pathogenicity test was conducted with strain SM69-07 isolated in 2007. A bacterial culture was suspended in sterile potassium phosphate buffer (0.01M, pH 7.4) and adjusted to 108 CFU/ml. Four 4-week-old plants each of parsley and cilantro (Ferry-Morse Seed Co.) were inoculated by spraying the bacterial suspension on the leaves until runoff. Potassium phosphate buffer was applied as a negative control treatment for each plant species. Plants were kept in a mist room with 100% humidity for 4 h, then transferred to a greenhouse with average maximum and minimum temperatures of 30 and 25°C. Leaf symptoms similar to those on the original plants were observed on the inoculated parsley and cilantro plants within 14 days of inoculation, whereas no symptoms developed on the negative control plants. One bacterial isolate obtained from each inoculated host using the isolation method described above was confirmed to be identical to the original isolates using the LOPAT tests and Rep-PCR DNA fingerprint profiles; no target bacteria were isolated from the negative control plants. Multilocus sequence typing (MLST) of the housekeeping genes gap1, gltA, gyrB, and rpoD was conducted for strain Sm69-07 (2,3). Sequence data were subjected to BLASTn searches in the Plant-Associated Microbes Database (PAMDB, http://genome.ppws.vt.edu/cgi-bin/MLST/home.pl ) (1). The sequences aligned with those of Pseudomonas syringae pv. coriandricola with 100% identity to alleles 101 (gyrB), 123 (rpoD), 7 (gap1), and 64 (gltA). Strain information and sequence alignment results for SM69-07 were submitted to PAMDB and assigned as isolate ID 1138. Based on bacterial culture morphology, LOPAT profile, pathogenicity test results, and MLST, the pathogen was confirmed as P. syringae pv. coriandricola. To our knowledge, this is the first report of bacterial spot of parsley caused by P. syringae pv. coriandricola in Ohio. Due to stringent quality requirements for fresh market parsley, this disease may pose a threat to the economic sustainability of parsley production in Ohio. References: (1) N. F. Almeida et al. Phytopathology 100:208, 2010. (2) C. T. Bull et al. Phytopathology 101:847, 2011. (3) M. S. Hwang et al. Appl. Environ. Microbiol. 71:5182, 2002. (4) J. Versalovic et al. Methods Mol. Cell Biol. 5:25, 1994.