RESUMEN
In September 2012, soft rot symptoms on broccoli (Brassica oleracea L. var. italica Plenck) were observed in several commercial fields in the western part of Serbia. Following the first harvest, water-soaked areas developed on broccoli stem tissue and progressed into soft rot decay of entire plants. The incidence of disease was approximately 30%. In Serbia, broccoli is grown on smaller fields compared to other vegetables, but its production and consumption increased significantly in recent years. From the diseased tissue, shiny, grayish white, round colonies were isolated on nutrient agar. Six non-fluorescent, gram-negative, facultative anaerobic, oxidase-negative, and catalase-positive bacterial strains were chosen for further identification. All strains caused soft rot on potato and carrot slices and did not induce hypersensitive reaction on tobacco leaves. They grew at 37°C and in yeast salts broth medium containing 5% NaCl (2), did not produce acid from α-methyl glucoside, but utilized lactose and trehalose, and did not produce indole or lecitinase. Investigated strains formed light red, 1.5-mm-diameter colonies on Logan's medium (2), and did not produce blue pigmented indigoidine on glucose yeast calcium carbonate agar (2) nor "fried egg" colonies on potato dextrose agar. Based on biochemical and physiological characteristics (1) and ITS-PCR and ITS-RFLP analysis (4), the strains were identified as Pectobacterium carotovorum subsp. carotovorum. The 16S rRNA gene sequence from two strains (GenBank KC527051 and KC527052) showed 100% identity with sequences of P. carotovorum subsp. carotovorum previously deposited in GenBank (3). Pathogenicity of the strains was confirmed by inoculation of broccoli head tissue fragments. Three florets per strain were inoculated by pricking the petals with a syringe and hypodermic needle and depositing a droplet of bacterial suspension (approx. 1 × 108 CFU/ml) at the point of inoculation. Sterile distilled water was used as a negative control. Inoculated florets were placed in a sealed plastic container and incubated in high humidity conditions at 28°C. Tissue discoloration and soft rot developed around the inoculation point within 48 to 72 h. No symptoms developed on control florets. Identity of bacterial strains reisolated from inoculated plant tissues was confirmed by ITS-PCR using G1/L1 primers followed by digestion of PCR products with Rsa I restriction enzyme (4). In Serbia, P. carotovorum subsp. carotovorum has been isolated from potato, some vegetable crops, and ornamentals, but not from broccoli until now. References: (1) S. H. De Boer and A. Kelman. Page 56 in: Laboratory Guide for Identification of Plant Pathogenic Bacteria, 3rd ed. N. W. Schaad et al., eds. The American Phytopathological Society, St. Paul, MN, 2001. (2) P. C. Fahy and A. C. Hayward. Page 337 in: Plant Bacterial Diseases: A Diagnostic Guide. P. C. Fahy and G. J. Persley eds. Academic Press, New York, 1983. (3) S. Nabhan et al. J. Appl. Microbiol. 113: 904, 2012. (4) I. K. Toth et. al. Appl. Environ. Microbiol. 67:4070, 2001.
RESUMEN
In November 2010, a serious outbreak of crown gall disease was observed on 3-year-old grapevine (Vitis vinifera L.) cv. Cabernet Sauvignon grafted onto Kober 5BB rootstock in two commercial vineyards located in the South Banat District in Serbia. Large, aerial tumors were visible above the grafting point on grapevine trunks, and in most cases, the tumors completely girdled the trunk. From the gall tissues, white, circular, and glistening bacterial colonies were isolated on yeast mannitol agar medium. Eight, nonfluorescent, gram-negative, and oxidase-positive strains were isolated from seven tumor samples and selected for further identification. PCR assays with A/C' (1) and VCF3/VCR3 (4) primers corresponding to the virD2 and virC genes yielded 224- and 414-bp fragments, respectively, confirming that the strains harbored the plasmid responsible for pathogenicity. The strains were differentiated to the species/biovar level with a multiplex PCR assay targeting 23S rRNA gene sequences (3) and were identified as Agrobacterium vitis. The 16S rDNA gene sequence from one isolate (GenBank Accession No. JN185718) showed 99% identity to the sequences of A. vitis previously deposited in NCBI GenBank database. The physiological and biochemical test results corresponded to the results of genetic analysis (2). The strains grew at 35°C and in nutrient broth supplemented with 2% NaCl. They were negative in 3-ketolactose, acid clearing on PDA supplemented with CaCO3, and ferric ammonium citrate tests; nonmotile at pH 7.0; pectolytic at pH 4.5; utilized citrate; produced acid from sucrose and alkali from tartarate. Pathogenicity was confirmed by inoculation of three plants per bacterial strain on grapevine cv. Cabernet Franc and on a local cultivar of tomato (Lycopersicon esculentum L.). The plants were inoculated on the stem by pricking one to three times through a drop of inoculum (108 CFU/ml) at three inoculation sites. Sterile distilled water was used as a negative control. Inoculated plants were maintained in a greenhouse at 24 ± 3°C. Typical tumors developed at the inoculation sites on tomatoes 3 weeks after inoculation and on grapevine 6 weeks after inoculation. No symptoms were observed on the control plants. Bacteria were reisolated from tumorigenic tissues and identified as pathogenic A. vitis by PCR. Crown gall disease was sporadically observed in vineyards in Serbia in previous years, but did not cause significant damage. Therefore, the causal agent was not studied in detail. To our knowledge, this is the first report of A. vitis determined as the causal agent of grapevine crown gall in Serbia. References: (1) J. H. Haas et al. Appl. Environ. Microbiol. 61:2879, 1995. (2) L. W. Moore et al. Page 17 in: Laboratory Guide for Identification of Plant Pathogenic Bacteria. 3rd ed. N. W. Schaad et al., eds. The American Phytopathological Society, St. Paul, MN, 2001. (3) J. Pulawska et al. Syst. Appl. Microbiol. 29:470, 2006. (4) K. Suzaki et al. J. Gen. Plant Pathol. 70:342, 2004.