First Report of Pseudomonas syringae pv. aptata Causing Bacterial Leaf Spot on Common Beet (Beta vulgaris) in Ohio

Abstract

In July 2017, common beet leaves (cv. Red Ace) from an open field in Erie County, Ohio, showing foliar blight symptoms were received by the OSU Vegetable Pathology Laboratory. Another beet sample (cv. Boro) from Wayne County, Ohio, displaying the same symptoms was received in June 2018. Symptoms consisted of pale brown circular water-soaked necrotic lesions, 2 to 3 mm in diameter, surrounded by a red halo. Bacterial streaming was observed from the lesions when viewed microscopically. Pieces of symptomatic tissue ∼0.25 cm² were surface sterilized in 70% ethanol for 30 s, rinsed in sterile water, placed in 100 µl of sterile water, and minced with a sterile blade. After 30 s, 10 µl of the resulting suspension was streaked onto Pseudomonas F agar medium (PF) and incubated in the dark for 48 h at 28°C. White creamy colonies fluorescent on PF when exposed to UV light were selected. Isolates from both samples were gram negative, oxidase negative, arginine dihydrolase negative, levan positive, and caused a hypersensitive reaction 24 h after infiltration into tobacco (Nicotiana tabacum) leaves. None of the isolates caused soft rot when inoculated at 10⁷ CFU/ml onto sterile potato slices. Box PCR (Louws et al. 1994) profiles were similar for strain SM200-2017 (Erie County) and SM61-2018 (Wayne County), with an additional band in SM200-2017. Both strains were subjected to whole-cell DNA extraction and multilocus sequence analysis of the housekeeping genes sigma factor 70, rpoD; glyceraldehyde-3-phosphate dehydrogenase, gapA; DNA gyrase B, gyrB; and citrate synthase, gltA (Hwang et al. 2005). Sequences were compared with those of the reference pathotype strain Pseudomonas syringae pv. aptata (Psa) CFBP 1617 deposited in the PAMDB database (Almeida et al. 2010). Both SM200-2017 (MK085510) and SM61-2018 (MK085511) showed 100% identity to the rpoD gene. SM200-2017 gapA sequence (MK085516) shared 99% homology with the gapA sequence of Psa CFBP 1617, and SM61-2018 gapA sequence (MK085517) was 100% identical. Similarly, the gyrB sequences from SM200-2017 (MK085515) and SM61-2018 (MK085514) were 99 and 100% similar to Psa CFBP 1617, respectively. No match was found for SM200-2017 and SM61-2018 gltA sequences (MK085512 and MK085513, respectively) when compared with those of Psa CFBP 1617. However, the sequences were 100% identical to Psa sequence KX226440 (Arabiat et al. 2016) deposited in the NCBI database. Koch’s postulates were confirmed in a greenhouse by spray inoculating 4-week-old beet seedlings (cv. Red Ace) with 10⁶ CFU/ml water suspensions of SM200-2017 or SM61-2018, or with sterile water. Plants were maintained at 27°C with a 14-h photoperiod, in plastic bags for the first 48 h. Symptom development was monitored daily. Water-soaked necrotic lesions similar to those observed on the original samples appeared 10 days after inoculation, whereas mock-inoculated plants did not show any symptoms. Symptomatic leaves were collected and processed as described above. Colonies with the same morphology on PF and with the same biochemical characteristics previously described were isolated. Box PCR profiles matched those of the original strains. To our knowledge, this is the first report of bacterial leaf spot of beet caused by Psa in Ohio. Common beets are cultivated throughout Ohio and can be severely affected under disease-conducive environmental conditions.