First report of Pseudomonas syringae pv. Aptata causing bacterial leaf spot on sugar beet in Serbia

Abstract

A severe bacterial leaf spot was observed during June and July 2013 on commercial cultivars of sugar beet (Beta vulgaris var. saccharifera) in the Vojvodina Province of Serbia. Serbia is a major sugar beet production area in southeastern Europe, with 62,895 ha and 3 million tons of sugar beet yield in 2013. A foliar leaf spot observed in 25 commercial sugar beet fields surveyed ranged from 0.1 to 40% severity. Symptoms were characterized as circular or irregular, 5- to 20-mm diameter, white to light brown necrotic spots, each with a dark margin. Diseased leaves were rinsed in sterilized, distilled water (SDW) and dried at room temperature, and leaf sections taken from the margin of necrotic tissue were macerated in SDW. Isolations from 48 symptomatic leaves onto nutrient agar with 5% (w/v) sucrose (NAS) produced bacterial colonies that were whitish, circular, dome-shaped, and Levan-positive. Representative isolates (n = 105) were Gram negative; aerobic; positive for catalase, fluorescence on King's medium B, and tobacco hypersensitivity; and negative for oxidase, potato rot, and arginine dehydrolase. These reactions corresponded to LOPAT group Ia, which includes Pseudomonas syringae pathovars (2). Repetitive extragenic palindromic sequence (rep)-PCR was used for genetic fingerprinting the isolates using the REP, ERIC, and BOX primers. Twenty-five different profiles were obtained among the strains. From each profile group, one representative strain was sequenced for the gyrB gene (1). Four heterogenic groups were observed, and representative gyrB gene sequences of each group were deposited in the NCBI GenBank (Accession Nos. KJ950024 to KJ950027). The sequences were compared with those of pathotype strain P. syringae pv. aptata CFBP 1617 deposited in the PAMDB database; one strain was 100% homologous, and the other three were 99% homologous. To fulfill identification of the Serbian sugar beet isolates, gltA and rpoD partial gene sequences were determined (1), and the sequences were deposited as Accession Nos. KM386838 to KM386841 for gltA and KM386830 to KM38683033 for rpoD. The sequences were 100% homologous with those of pathotype strain CFBP 1617. Pathogenicity of each of four representative bacterial strains was tested on 3-week-old plants of the sugar beet cultivars Marinela, Serenada, and Jasmina (KWS, Belgrade, Serbia) and Lara (NS Seme, Novi Sad, Serbia) by atomizing a bacterial suspension of ~106 CFU/ml of the appropriate isolate onto the abaxial leaf surface of three plants per cultivar until water-soaking of the leaf surface was observed. Three plants of each cultivar atomized similarly with P. syringae pv. aptata CFBP 2473 and SDW served as positive and negative control treatments, respectively. Inoculated plants were kept in a clear plastic box at 80 to 100% RH and 17 ± 1°C and examined for symptom development over 3 weeks. For all test isolates and the control strain, inoculated leaves first developed water-soaked lesions 7 days after inoculation (DAI). By 10 to 14 DAI, lesions were necrotic and infection had spread to the petioles. By 21 DAI, wilting was observed on more than 50% of inoculated plants. Negative control plants were symptomless. Bacteria re-isolated onto NAS from inoculated leaves had the same colony morphology, LOPAT results, and gyrB partial gene sequences as described for the test strains. No bacteria were re-isolated from negative control plants. Based on these tests, the pathogen causing leaf spot on sugar beet in Serbia was identified as P. syringae pv. aptata. References: (1) P. Ferrente and M. Scortichini. Plant Pathol. 59:954, 2010. (2) R. A. Lelliott et al. J. Appl. Bacteriol. 29:470, 1966.