First Report of Bacterial Blight of Sugar Beet Caused by Pseudomonas syringae pv. aptata in Georgia, USA

Sugar beet (Beta vulgaris L.) is not currently a commercial crop in Georgia, but experimental plantings as a winter rotational crop are promising in terms of yield and industrial sugar production (T. Brenneman, personal communication). A disease outbreak of suspected bacterial origin occurred in sugar beet plots (experimental lines Beta Seed energy beet 'BTS ENC115,' 'BTS EGC184,' 'BTS EGC195,' and 'BTS 1EN6702') in Tift Co., GA, in December 2012, at ~35% incidence. Foliar symptoms included circular to irregular spots, each with a tan center and dark margin. Ten leaves/experimental line with leaf spot symptoms were collected, and bacterial isolations made on King's B agar medium. After 48 h of incubation, cream-colored, fluorescent yellow, round colonies with smooth margins were isolated. The isolates were each gram negative, oxidase negative, non-pectolytic on potato, arginine dihydrolase negative, produced levan, and gave a hypersensitivity response (HR) on tobacco. These characteristics indicated that the isolates belonged to Pseudomonas syringae van Hall LOPAT group Ia (3). The 16S-23S rRNA (internal transcribed regions) (1) from four foliar isolates (SB-1, SB-2, SB-3, and SB-4), one/experimental line, was amplified, and the resultant PCR products were sequenced and BLAST searched in GenBank. The 16S-23S rRNA sequences matched those of P. syringae pv. syingae (Pss) (KF023189) and P. syringae pv. aptata (Psa) (AY342167.1) with 96 to 98% and 97 to 99% sequence identity, respectively. Also, the percent similarity of the 16S-23S rRNA sequences among the four isolates was >99% (KJ922021 to 24 for SB-1 to SB-4, respectively). The four test isolates also had ≤89 and ≤99% similarity with Pss and Psa, respectively, when tested with BIOLOG (Hayward, CA). In addition, four sugarbeet isolates along with a type strain of Psa (NCPPB 3539) were amplified using a PCR primer pair that detected the presence of the avrPphE gene, an avirulence gene present in Psa but absent in Pss (2). The type strain of Pss (NCPPB 1770) was not amplified using this primer pair. BOX-PCR analysis gave identical banding patterns for the four isolates as that of a type strain of Psa. In two independent experiments, 3-week-old seedlings of the sugar beet cv. Beta EGR099 (n = 10 seedlings/isolate/experiment) were spray-inoculated with a sterilized water suspension of 1 × 108 CFU/ml of each of the isolates. All of the inoculated seedlings developed symptoms (water-soaked lesions that developed into necrotic spots) 10 days after inoculation (DAI) in greenhouse conditions (~30°C and ~80% RH). All of the seedlings inoculated with the type strain of Psa also produced typical bacterial blight symptoms at 10 DAI. In contrast, five control seedlings inoculated with sterilized water remained asymptomatic, and target bacterial colonies were not re-isolated from the leaves of these plants. Bacterial colonies were re-isolated from symptomatic seedlings, and showed similar characteristics based on physiological tests, BIOLOG profile, BOX-PCR analysis, and positive amplification with the avrPphE PCR assay, which indicated that these strains were Psa. To our knowledge, this is the first report of Psa in sugarbeet in Georgia. The fact that a Psa strain was also isolated from a sugar beet seed lot (data not shown) suggested that the pathogen may have been introduced on contaminated seeds. Knowledge of the presence of Psa in the agro-ecosystem of Georgia may encourage scientists to implement integrated management practices for this pathogen. References: (1) C. Guasp et al. Int. J. Syst. Evol. Microbiol. 50:1629, 2000. (2) Y. Inoue and Y. Takikawa. Page 687 in: Presentations 6th Int. Conf. Pseudomonas syringae Pathovars and Related Pathogens, 2003. (3) R. A. Lelliot et al. J. Appl. Bacteriol. 29:470, 1966.