During the summer of 2010, onions (Allium cepa L.) of several cultivars growing in muck-land soils in Orange, Genesee, Orleans, and Oswego counties of New York exhibited leaf dieback and bulb decay consistent with disease symptoms caused by Enterobacter cloacae as described previously (1,3,4). Isolations of bacteria from symptomatic tissues and muck soil were made using onion extract medium (OEM), which contains extracts of autoclaved onions, salts, and inhibitors of fungi and gram-positive bacteria. Some presumptive strains of E. cloacae were isolated; 5 from symptomatic onions growing in Genesee County, 2 from muck-land soil, and 27 from bulbs stored for ~2.5 months in a farm storage facility in Oswego County. Tentative identification was based on colony morphology (convex, cream-color colonies, 2 to 3 mm in diameter following incubation at 28°C for 1 day on OEM), which was similar to the morphology of reference strains of E. cloacae ATCC 23355, ATCC 13047, and strain 310 (gift of H. F. Schwartz, which was derived from reference 4; personal communication). Strains were gram-negative rods, negative for oxidase and indole, positive for nitrate reductase and catalase; produced acid from glucose aerobically and anaerobically. Also, all strains produced PCR products from the 16S-23S internal transcribed spacer (ITS) DNA region of the predicted sizes using primers T5A and T3B designed for identification of E. cloacae (2). The growth of eight of the isolated strains and strains ATTC 23355 and 310 were evaluated on several carbon sources with RapiD 20E test strips (bio Mérieux, Inc, Durham, NC). All strains were positive for β-d-galactosidase, ornithine decarboxylase, utilization of citrate and malonate, and production of acetoin. Hydrolysis of esculin by β-glucosidase differed among the eight. All strains were negative for lysine decarboxylase, urease, para-phenylalanine deaminase, indole, and oxidase. All produced acid from arabinose, xylose, rhamnose, cellobiose, melibiose, saccharose, trehalose, raffinose, and glucose; no strains produced acid from adonitol. These characteristics are consistent with published data for E. cloacae. Surface-disinfested onion bulbs and sets were inoculated with 50 to 100 μl of bacterial suspensions containing ~108 CFU/ml, injected with hypodermic needles and syringes, and incubated at 37°C for 2 weeks. Bisected onions revealed dry brown discoloration in each of the four bulbs and sets that had been inoculated with each presumptive strain. Symptoms were indistinguishable from those apparent in onions inoculated with the authentic strains mentioned. Strains recovered on OEM were identified as E. cloacae based on the stated biochemical properties and analysis of the 16S rRNA gene amplified by PCR as above. The sequence of the amplicon from the isolated strains was identical to that of reference strains ATCC 23355 and 310. Amplicon sequences of the 16S rRNA gene of New York strains Ecl3, Ecl6, and Ecl7 were deposited in GenBank as JF832951, JF832952, and JF832953, respectively. The strains were accessioned as ATCC BAA-2271, ATCC BAA-2272, and ATCC BAA-2273, respectively. To our knowledge, this is the first published report of E. cloacae causing Enterobacter bulb decay of onion in New York. References: (1) A. L. Bishop and R. M. Davis. Plant Dis. 74:692, 1990. (2) M. M. Clementino et al. J. Clin. Microbiol. 39:3865, 2004. (3) B. K. Schroeder and L. J. du Toit. Plant Dis. 93:323, 2009. (4) H. F. Schwartz and K. Otto. Plant Dis. 84:808, 2000.
Pseudomonas knackmussii sp. nov.