Symptoms typical of center rot of onion (Allium cepa L.) were observed in farmers' fields in spring of 2009 and 2010 in Hamyang, South Korea, at an incidence of 30 to 50% (five affected fields representing approximately 6 ha). The symptoms were identical to those reported from infected onions in Georgia in 1997 (1). Harvested bulbs of symptomatic plants had reddish, collapsed scales near the neck. Symptomatic bulb tissues were surface-sterilized by immersing sections of the tissue for 30 s in 1% NaOCl, then rinsing the sections with sterilized, distilled water. Tissues were then macerated in 1 ml of sterilized, distilled water in a 1.5 ml Eppendorf tube using a sterile scalpel. The macerated tissue was left to soak for 10 min, after which a 5 μl suspension from each section was streaked onto plates of nutrient agar (NA). Gram-negative, rod-shaped, yellow bacteria were consistently recovered on NA. Three bacterial isolates recovered were each facultative anaerobes and induced a hypersensitive reaction on tobacco leaves. The biochemical test, API 20E (Biomérieux, Marcy l'Etoile, France), was also used for identification. All three strains tested positive for β-D-galactosidase, utilization of citrate, and production of acetoin, catalase, and indole. All three strains tested negative for ornithine decarboxylase, lysine decarboxylase, urease, and oxidase. All produced acid from arabinose, glucose, mannitol, and sorbitol; while none produced acid from melibiose, inositol, and rhamnose. These characteristics are consistent with those of P. ananatis (1,2). Five bulbs were each surface-disinfested with 70% ethanol, dried, and injected with 50 μl of the appropriate bacterial suspension containing ~108 CFU/ml, using a syringe. The bulbs were placed in plastic boxes with four sheets of wet paper towel to maintain the relative humidity at 100%, and incubated at 25°C for 2 weeks. Three onion bulbs treated similarly but injected with sterilized, distilled water served as replicates of the control treatment. After 1 week of incubation, inoculated onion bulbs developed a brown discoloration and decay of the internal, fleshy scales matching those observed in the original farmers' fields. The control onion bulbs remained asymptomatic. Bacteria reisolated from lesions in the fleshy bulb scales of the inoculated bulbs had the same characteristics as the original isolates inoculated, proving Koch's postulates. Bacteria were not reisolated from any of the control bulbs. To confirm identity of the isolated bacteria, 16S rRNA and recA genes were amplified with primers 27mF: 5′-AGAGTTTGATCMTGGCTCAG-3′ and 1492mR: 5′-GGYTACCTTGTTACGACTT-3′, and PAGRECA21: 5′-GGTGAAGACCGCTCAATGGA-3′ and PAGRECA621: 5′-CACCGATACGGCGGATATCA-3′, respectively (3). Amplification of the 16S rRNA gene generated a 1,506-bp consensus sequence (GenBank Accession No. JQ762264), and amplification of the recA gene generated a consensus sequence of 601 bp (JQ762265). The 16S rRNA and recA gene sequences shared 99% nucleotide identity with those of a P. ananatis strain in GenBank (DQ195523 and AY219004, respectively). Based on symptoms, biochemical tests, and molecular analyses, the bacterium responsible for the onion symptoms in Korea was identified as P. ananatis. To our knowledge, this is the first report of center rot of onion caused by P. ananatis in Korea.
References: (1) R. D. Gitaitis and J. D. Gay. Plant Dis. 81:1096, 1997. (2) H. G. Truper and L. de Clari. Int. J. Syst. Bacteriol. 47:908, 1997. (3) A. Wensing et al. Appl. Environ. Microbiol. 76:6248, 2010.