First Report of Agrobacterium vitis as the Causal Agent of Grapevine Crown Gall in Serbia.

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.

Development of a semi-nested PCR based method for sensitive detection of tumorigenic Agrobacterium in soil.

AIMS: To develop a specific, sensitive and rapid PCR-based method for detection of tumorigenic Agrobacterium in soil. METHODS AND RESULTS: Three newly designed primers complementary to tms2 gene amplified DNA of only the tumor-inducing agrobacteria of 113 strains tested, resulting in 617 bp and 458 bp products in the first and second rounds of semi-nested PCR respectively. As optimized method of pre-incubation of soil suspensions on selective medium, DNA isolation and two-round semi-nested PCR enabled detection of 1-2 bacterial cells in 1 g of soil. Using this method tumour-inducing Agrobacterium was detected in 67 of 69 samples of naturally infested soil originating from the field, where plants with crown gall symptoms occurred. The pathogen was detected only in two samples of 15 tested, collected from a nursery where crown gall symptoms were not observed. CONCLUSIONS: The semi-nested PCR-based method allowed for sensitive and rapid detection of tumorigenic agrobacteria in soil. SIGNIFICANCE AND IMPACT OF THE STUDY: The method is proposed for testing of soil in fields intended for nursery production of fruit trees, roses or other plants susceptible to crown gall, as well as a tool for ecological studies.

Phylogenetic analysis of 23S rRNA gene sequences of. Agrobacterium, Rhizobium and Sinorhizobium strains.

The phylogenetic relationship among twelve Agrobacterium, four Rhizobium, and two Sinorhizobium strains originating from various host plants and geographical regions was studied by analysis of the 23S rDNA sequences. The study included Agrobacterium strains belonging to biovars 1, 2 (with tumor- or hairy-root inducing and non-pathogenic strains), A. vitis, A. rubi; representative species of the Rhizobium genus: R. galegae, R. leguminosarum and R. tropici and Sinorhizobium meliloti strains. The phylogenetic analysis showed that within Agrobacterium, the biovar designation was reflected in the 23S rDNA similarity and that strains of Agrobacterium and Rhizobium are closely related to each other. The results suggest that the taxonomic definition of Agrobacterium and Rhizobium should be considered for revision and that the Agrobacterium-biovar identity is probably a reliable taxonomic trait.

[Mixed connective tissue disease--etiology, pathogenesis, clinical significance, treatment]. / Mieszana choroba tkanki lacznej--etiologia, patogeneza, znaczenie kliniczne, leczenie.

Some patients have features of more than one rheumatic disease and thus do not fit into traditional classification. Patients with combination of clinical finding similar to those of systemic lupus erythematosus (SLE), progressive systemic sclerosis (PSS), polymyositis, rheumatoid arthritis (RA) and with unusually high titers of circulating antinuclear antibody with specificity for nuclear ribonucleoprotein (RNP) are considered to have mixed connective tissue disease (MCTD). The overlap was described by Sharp and colleagues in 1972. During the post 20 years many studies exposed the clinical correlates of this antibody system (now called anti U1RNP). Controversy arose about whether MCTD was a distinct entity or would be better defined as subset of SLE. Anti RNP antibodies precipitate three proteins uniquely associated with U1RNP. Clinical correlates considered to be distinctive of MCTD are associated with 68 kD antigen specificity. Its to be expected that T cells receptors and HLA molecules are involved in the generation of these antibodies. Several observations have indicated, that 68 kD anti U1RNP antibody response in associated with HLA DR 4 and DR2 phenotype. Several studies have pointed a role of viruses initiating an antibody response against URNPs. Initial observations of MCTD suggested infrequent renal disease, a good response to corticosteroids and favourable prognosis. Future study has shown that some patients may require aggressive and prolonged pharmacologic therapy and that pulmonary involvement is common. Pulmonary hypertension associated proliferative vascular lesions may be serious complication with not always favourable prognosis.