In August of 2006, onion plants of cv. Redwing exhibiting premature dieback and bulb rot were obtained from a commercial onion crop under center pivot irrigation in the Columbia Basin of Washington State. High temperatures during the summer were similar to those in 2004, which preceded significant outbreaks of Enterobacter rot of onion bulbs in storage. Fungal pathogens of onion were not observed. Bacteria from infected bulb tissue were isolated and purified on nutrient broth yeast extract (NBY) agar, and 537 isolates were evaluated for the ability to ferment glucose anaerobically. Of the facultative anaerobes (~50% of all isolates), 48 isolates were arginine dihydrolase positive, indole negative, and unable to degrade pectin, i.e., characteristics typical of the genus Enterobacter (2), which includes Enterobacter cloacae, a bacterial pathogen reported to cause onion bulb rot in California and Colorado (1,3). Sixteen of the putative Enterobacter isolates, along with four strains of E. cloacae known to be pathogenic on onion (1) (ATCC 23355 and ATCC 13047, 310 (H. F. Schwartz, Colorado State University), and E6 (J. Loper, USDA ARS), were tested for pathogenicity on onion bulbs (8 to 10 cm in diameter; cv. Tamara). The isolates were grown overnight in NBY broth at 28°C, harvested by centrifugation and resuspended to an OD600 = 0.3 (~108 CFU/ml) in sterile distilled water. After the outermost fleshy scale of each bulb was removed, each bulb was surface disinfected in 0.6% NaOCl for 2 min, dipped in sterile distilled water, and then dipped in 95% ethanol. Each bulb was air dried before a 0.5-ml aliquot of bacterial suspension was injected into the shoulder of the bulb with a 20-gauge needle. Three bulbs were inoculated for each isolate, placed in individual plastic bags, sealed, and incubated at 30°C in the dark. Three bulbs injected with water and three noninjected bulbs served as controls. After 14 days, each bulb was sliced through the center and rated for rot. Thirteen isolates induced rot symptoms on the inner fleshy scales of all inoculated bulbs. Of these, seven also caused tan-to-brown discoloration of the inner fleshy scales; similar symptoms were caused by the four pathogenic reference strains of E. cloacae (1). No symptoms were observed in any of the controls. Symptoms were not observed when the bacteria, prepared as described above, were infiltrated into onion leaves. Bacteria were reisolated from the symptomatic inoculated bulb tissue and confirmed to be Enterobacter spp. by the above physiological tests. In addition, an isolate designated ECWSU2 and the corresponding strain recovered from one of the inoculated symptomatic bulbs, along with the four reference strains, were evaluated for anaerobic growth on a variety of carbon sources by using API 50 CHE test strips (bio Mérieux Vitek, Inc., Hazlewood, MO). The physiological test data along with sequence analysis of a portion of the 16S rRNA gene of each isolate confirmed all of these isolates to be E. cloacae (4; Ribosomal Database Project [http://rdp.cme.msu.edu/]). To our knowledge, this is the first report of E. cloacae causing a bulb rot of onion in Washington State.
References: (1) A. L. Bishop and R. M. Davis. Plant Dis. 74:692, 1990. (2) J. G. Holt et al. Bergey's Manual of Determinative Bacteriology. Williams and Wilkins, Baltimore, MD, 1994. (3) H. F. Schwartz and K. Otto. Plant Dis. 84:808, 2000. (4) L. Verdonck et al. Int. J. Syst. Bacteriol. 37:4, 1987.
