Eop1 from a Rubus strain of Erwinia amylovora functions as a host-range limiting factor.

Strains of Erwinia amylovora, the bacterium causing the disease fire blight of rosaceous plants, are separated into two groups based on host range: Spiraeoideae and Rubus strains. Spiraeoideae strains have wide host ranges, infecting plants in many rosaceous genera, including apple and pear. In the field, Rubus strains infect the genus Rubus exclusively, which includes raspberry and blackberry. Based on comparisons of limited sequence data from a Rubus and a Spiraeoideae strain, the gene eop1 was identified as unusually divergent, and it was selected as a possible host specificity factor. To test this, eop1 genes from a Rubus strain and a Spiraeoideae strain were cloned and mutated. Expression of the Rubus-strain eop1 reduced the virulence of E. amylovora in immature pear fruit and in apple shoots. Sequencing the orfA-eop1 regions of several strains of E. amylovora confirmed that forms of eop1 are conserved among strains with similar host ranges. This work provides evidence that eop1 from a Rubus-specific strain can function as a determinant of host specificity in E. amylovora.

First Report of Enterobacter Bulb Decay of Onions Caused by Enterobacter cloacae in New York.

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 ß-d-galactosidase, ornithine decarboxylase, utilization of citrate and malonate, and production of acetoin. Hydrolysis of esculin by ß-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.

First Report of Bulb Disease of Onion Caused by Pantoea ananatis in New York.

In winter 2007, disease symptoms were observed in stored yellow onion bulbs (Allium cepa) grown in New York (NY) in 2006. Similar symptoms were observed in bulbs produced in 2007, 2008, and 2009. Symptoms were associated with one to three bulb scales near the midsection. Infected scales were light brown to brown, not macerated, and lacking foul odors typical of onion bulbs infected with Burkholderia cepacia. Onion grower-packers located in Orange County, NY were concerned that onion lots were rejected following grading by inspectors who cut bulbs to check market quality. Extent of the problem statewide is not currently clear. Isolation attempts were made from symptomatic tissues onto nutrient agar plates (3), with incubation for 24 h at 26 to 28°C, and PA-20 (2), a semiselective medium for the isolation of Pantoea ananatis, with similar incubation for 4 to 6 days. Most strains that grew on PA-20 were gram negative and yellow pigmented with dark centers. Isolated strains were tentatively identified as P. ananatis on the basis of growth on PA-20, a positive indole and negative oxidase test, positive tests for catalase, fermentation of glucose, Voges-Proskauer, and citrate utilization; negative for phenylalanine deaminase, urease, nitrate reductase, methyl red tests, and hypersensitive response induction in tobacco. The BIOLOG (Hayward, CA) system indicated that all presumptive strains of P. ananatis utilized d-mannose, d-cellobiose, d-melibiose, l-inositol, d-arabinose, cellulose, glycerol, d-arabitol, and sucrose, but not glycogen, N-acetyl-d-galactosamine, malonic acid, l-fucose, or xylitol. Strains of P. ananatis recovered from diseased onions in Georgia (GA) (1) were included in all tests as positive controls. We used PCR primers suggested by R. D. Gitaitis (University of Georgia): PanITS1 (5'-GTC TGA TAG AAA GAT AAA GAC-3') and AS2b (5'-TTC ATA TCA CCT TAC CGG CGC-3'). Together, they amplify the 16S-23S rDNA internal transcribed spacer region of 398 bp; the nucleotide sequences of six NY and three GA strains are identical to each other and 99.3% identical to P. ananatis LMG 20103 (GenBank CP001875) and 93.3% identical to P. stewartii (AJ311838). Pathogenicity tests were done in onion leaves. For inoculation, strains were grown on nutrient agar for 24 h and bacterial suspensions of ~108 CFU/ml were prepared in sterile water. Tips of healthy, greenhouse-grown onion leaves were cut and inoculum was applied to the cut surfaces with cotton swabs. Plants were incubated in a greenhouse for up to 2 weeks. Plants mock inoculated with water were symptomless. Bacteria were recovered from all lesions induced by artificial inoculation with the presumptive strains of P. ananatis. Recovered bacteria had characteristics of P. ananatis. Pathogenic strains from NY and GA produced off-white lesions that extended the length of the leaf, which was consistent with previous studies of the pathogenicity of P. ananatis (1). On the basis of microbiological and molecular analyses and pathogenicity tests, 14 NY strains, each isolated from a different diseased bulb, were identified as P. ananatis. To our knowledge, this is the first published report of P. ananatis causing a disease of onion in New York. References: (1) R. D. Gitaitis et al. USA Crop Prot. 21:983, 2002. (2) T. Goszczynska et al. J. Microbiol. Methods. 64:22, 2006. (3) N. W. Shaad et al, eds. Laboratory Guide for Identification of Plant Pathogenic Bacteria. 3rd ed. The American Phytopathological Society, St. Paul, MN, 2000.