Origin of Enriched Regulatory T Cells in Patients Receiving Combined Kidney-Bone Marrow Transplantation to Induce Transplantation Tolerance.

We examined tolerance mechanisms in patients receiving HLA-mismatched combined kidney-bone marrow transplantation (CKBMT) that led to transient chimerism under a previously published nonmyeloablative conditioning regimen (Immune Tolerance Network study 036). Polychromatic flow cytometry and high-throughput sequencing of T cell receptor-ß hypervariable regions of DNA from peripheral blood regulatory T cells (Tregs) and CD4 non-Tregs revealed marked early enrichment of Tregs (CD3+ CD4+ CD25high CD127low Foxp3+ ) in blood that resulted from peripheral proliferation (Ki67+ ), possibly new thymic emigration (CD31+ ), and, in one tolerant subject, conversion from non-Tregs. Among recovering conventional T cells, central memory CD4+ and CD8+ cells predominated. A large proportion of the T cell clones detected in posttransplantation biopsy specimens by T cell receptor sequencing were detected in the peripheral blood and were not donor-reactive. Our results suggest that enrichment of Tregs by new thymic emigration and lymphopenia-driven peripheral proliferation in the early posttransplantation period may contribute to tolerance after CKBMT. Further, most conventional T cell clones detected in immunologically quiescent posttransplantation biopsy specimens appear to be circulating cells in the microvasculature rather than infiltrating T cells.

Genetic relatedness and recombination analysis of Allorhizobium vitis strains associated with grapevine crown gall outbreaks in Europe.

AIMS: To analyse genetic diversity and epidemiological relationships among 54 strains of Allorhizobium vitis isolated in Europe during an 8-year period and to assess the relative contribution of mutation and recombination in shaping their diversity. METHODS AND RESULTS: By using random amplified polymorphic DNA (RAPD) PCR, strains studied were distributed into 12 genetic groups. Sequence analysis of dnaK, gyrB and recA housekeeping genes was employed to characterize a representative subcollection of 28 strains. A total of 15 different haplotypes were found. Nucleotide sequence analysis suggested the presence of recombination events in A. vitis, particularly affecting dnaK locus. Although prevalence of mutation over recombination was found, impact of recombination was about two times greater than mutation in the evolution of the housekeeping genes analysed. CONCLUSIONS: The RAPD analysis indicated high degree of genetic diversity among the strains. However, the most abundant RAPD group was composed of 35 strains, which could lead to the conclusion that they share a common origin and were distributed by the movement of infected grapevine planting material as a most common way of crossing long distances. Furthermore, it seems that recombination is acting as an important driving force in the evolution of A. vitis. As no substantial evidence of recombination was detected within recA gene fragment, this phylogenetic marker could be reliable to characterize phylogenetic relationships among A. vitis strains. SIGNIFICANCE AND IMPACT OF THE STUDY: We demonstrated clear epidemiological relationship between majority of strains studied, suggesting a need for more stringent phytosanitary measures in international trade. Moreover, this is the first study to report recombination in A. vitis.

First Report of Broccoli Soft Rot Caused by Pectobacterium carotovorum subsp. carotovorum in Serbia.

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.

First Report of Brown Rot Caused by Monilinia fructicola on Stored Apple in Serbia.

Monilinia fructicola (G. Winter) Honey is a causal agent of brown rot of stone fruits, occasionally affecting pome fruits as well. The pathogen is commonly present in North and South America, Oceania, and Asia, but listed as a quarantine organism in Europe (4). After its first discovery in France in 2001, its occurrence has been reported in Germany, Hungary, Italy, Poland, Romania, Slovenia, Spain, Switzerland, Austria, and the Slovak Republic (1). In February 2011, during a survey for fungal postharvest pathogens in cold storage conditions, apple fruits (Malus domestica Borkh.) grown and stored in the Grocka Region, Serbia, were collected. All pathogens from symptomatic fruits were isolated on potato dextrose agar (PDA). One isolate from apple fruit cv. Golden Delicious with brown rot symptoms was identified as M. fructicola based on morphological and molecular characters. Colonies cultivated on PDA at 22°C in darkness were colorless, but later became grayish, developing mass of spores in concentric rings. Colony margins were even. Conidia were one-celled, limoniform, hyaline, measured 12.19 to 17.37 (mean 13.8) × 8.62 to 11.43 µm (mean 9.9), and were produced in branched monilioid chains (3). Morphological identification was confirmed by PCR (2) using genomic DNA extracted from the mycelium of pure culture, and an amplified product of 535 bp, specific for the species M. fructicola, was obtained. Sequence of the ribosomal (internal transcribed spacer) ITS1-5.8S-ITS2 region was obtained using primers ITS1 and ITS4 and deposited in GenBank (Accession No. JN176564). Control fruits were inoculated with sterile PDA plugs. After 3 days of incubation in plastic containers with high humidity at room temperature, typical symptoms of brown rot developed on inoculated fruits, while control fruits remained symptomless. The isolate recovered from symptomatic fruits showed the same morphological and molecular features of the original isolate. To our knowledge, this is the first report of M. fructicola in Serbia. Further studies are necessary for estimation of economic importance and geographic distribution of this quarantine organism in Serbia. References: (1) R. Baker et al. European Food Safety Authority. Online publication. www.efsa.europa.eu/efsajournal . EFSA J. 9(4):2119, 2011. (2) M.-J. Côté et al. Plant Dis. 88:1219, 2004. (3) J. E. M. Mordue. CMI Descriptions of Pathogenic Fungi and Bacteria. No. 616, 1979. (4) OEPP/EPPO. EPPO A2 List of Pests Recommended for Regulation as Quarantine Pests. Online publication. Version 2010-09. Retrieved from http://www.eppo.org/QUARANTINE/listA2.htm , June 27, 2011.

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.

Ten-year data on 138 patients with endometrial carcinoma and postoperative vaginal brachytherapy alone: no need for external-beam radiotherapy in low and intermediate risk patients.

OBJECTIVE: To evaluate long-term outcome, risk factors, and causes of death in stage I-IIIA endometrial carcinoma (EC) patients treated only with adjuvant vaginal brachytherapy (VB) and to clarify for which subgroups of patients it is safe to omit external-beam radiotherapy (EBRT). METHODS: Out of 224 EC patients receiving postoperative radiotherapy between 1990 and 2002, 138 had VB alone in curative intent (FIGO [2002]: 85%I, 12%II, 3%IIIA; 18 low risk [IA G1-2, IB G1], 103 intermediate risk [IB G2-3, IC G1-2, IIA-B G1-2], 17 high risk [IC G3, IIIA]). After surgery+/-lymphadenectomy, HDR-brachytherapy prescription (in 95.7% of patients) was 3x10 Gy to the surface or 3x5 Gy at 5 mm tissue depths. RESULTS: Median follow-up was 107 months (range 3-185). Three intermediate and 7 high risk-patients relapsed. The 10-year vaginal control was 99.2%, locoregional control was 95.2% (low/intermediate/high risk: 100%/98.9%/68.8%), and disease-free survival (DFS) was 91.7% (100%/96.8%/55.2%). Risk factors for poor DFS were lymphovascular space invasion, > or = 50% myometrial invasion (univariate, p<0.05), pathological FIGO-stage, and grade 3 (uni-/multivariate, p<0.05). Leading causes of deaths (n=41) were cardiovascular disease (29%) and other malignancies (24%) ahead of EC (19.5%). The 10-year overall survival was 68.5% and the disease-specific survival was 92.4%. Thirty-five secondary tumors in 31 patients led to a higher actuarial death rate (10-year 9.9%, 15-year 17.7%) than EC (7.6%). CONCLUSIONS: Restricting adjuvant therapy to VB alone seems to be safe in low and intermediate risk EC and can be recommended. As death rarely relates to early-stage EC, value of adjuvant therapy is probably better reflected by DFS rather than by overall survival.

Bacteriophages for plant disease control.

The use of phages for disease control is a fast expanding area of plant protection with great potential to replace the chemical control measures now prevalent. Phages can be used effectively as part of integrated disease management strategies. The relative ease of preparing phage treatments and low cost of production of these agents make them good candidates for widespread use in developing countries as well. However, the efficacy of phages, as is true of many biological control agents, depends greatly on prevailing environmental factors as well as on susceptibility of the target organism. Great care is necessary during development, production and application of phage treatments. In addition, constant monitoring for the emergence of resistant bacterial strains is essential. Phage-based disease control management is a dynamic process with a need for continuous adjustment of the phage preparation in order to effectively fight potentially adapting pathogenic bacteria.

Integration of Biological Control Agents and Systemic Acquired Resistance Inducers Against Bacterial Spot on Tomato.

Two strains of plant growth-promoting rhizobacteria, two systemic acquired resistance inducers (harpin and acibenzolar-S-methyl), host-specific unformulated bacteriophages, and two antagonistic bacteria were evaluated for control of tomato bacterial spot incited by Xanthomonas campestris pv. vesicatoria in greenhouse experiments. Untreated plants and plants treated with copper hydroxide were used as controls. The plant growth-promoting rhizobacteria or a tap water control were applied as a drench to the potting mix containing the seedlings, while the other treatments were applied to the foliage using a handheld sprayer. The plant growth-promoting rhizobacteria strains, when applied alone or in combination with other treatments, had no significant effect on bacterial spot intensity. Messenger and the antagonistic bacterial strains, when applied alone, had negligible effects on disease intensity. Unformulated phage or copper bactericide applications were inconsistent in performance under greenhouse conditions against bacterial spot. Although acibenzolar-S-methyl completely prevented occurrence of typical symptoms of the disease, necrotic spots typical of a hypersensitive reaction (HR) were observed on plants treated with acibenzolar-S-methyl alone. Electrolyte leakage and population dynamics experiments confirmed that acibenzolar-S-methyl-treated plants responded to inoculation by eliciting an HR. Application of bacteriophages in combination with acibenzolar-S-methyl suppressed a visible HR and provided excellent disease control. Although we were unable to quantify populations of the bacterium on the leaf surface, indirectly we determined that bacteriophages specific to the target bacterium reduced populations of a tomato race 3 strain of the pathogen on the leaf surface of acibenzolar-S-methyl-treated plants to levels that did not induce a visible HR. Integrated use of acibenzolar-S-methyl and phages may complement each other as an alternative management strategy against bacterial spot on tomato.

First Report of Pear Decline Phytoplasmas on Pear in Serbia.

During August of 2004, pear (Pyrus communis L.) plants with typical symptoms of pear decline (PD) were observed in orchards in central Serbia. The affected plants showed premature reddening and upward rolling of leaves that often showed down-turned petioles. In some cases, premature defoliation was observed. Although a similar decline of pear was observed earlier, until now, the causal agent had not been identified. DNA was extracted with a chloroform/phenol procedure from fresh leaf midribs and branch phloem scrapes of four symptomatic and one asymptomatic pear plants separately. A nested polymerase chain reaction assay (PCR) was used for phytoplasma detection (first PCR round with P1/P7 (4) phytoplasma universal primer pair, followed by nested PCR with group 16SrX specific primers f01/r01) (3). With these primers, the expected products from phloem scrapes and midrib extracts of symptomatic plant samples were obtained. Restriction fragment length polymorphism (RFLP) analyses of the f01/r01 amplicon, with RsaI and SspI restriction enzymes, discriminating among 16SrX subgroup phytoplasmas, showed profiles corresponding to those of the apple proliferation phytoplasma group, 16SrX-C subgroup, "Candidatus Phytoplasma pyri" (2). A 1,155-bp sequence of 16S rDNA gene for one of the PA2f/r (1) amplicons obtained in nested PCR on P1/P7 products from one of the leaf midrib samples was deposited in GenBank (Accession No. AY949984); both strands of the fragment were sequenced with the Big Dye Terminator reaction kit (Applied Biosystems, Foster City, CA). The sequences were analyzed with the Chromas 1.55 DNA sequencing software (Technelysium, Queensland, Australia) and aligned with BLAST software ( http://www.ncbi.nlm.nih.gov ). The blast search showed 100% homology of this sequence with that of PD strain Y16392, confirming the identity with PD of the phytoplasma detected. To our knowledge, this is the first report of pear decline phytoplasmas in Serbia. References: (1) M. Heinrich et al. Plant Mol. Biol. Rep. 19:169, 2001. (2) IRPCM Phytoplasma/Spiroplasma Working Team-Phytoplasma Taxonomy Group. Int. J. Syst. Evol. Microbiol. 54:1243, 2004. (3) K.-H. Lorenz et al. Phytopathology 85:771, 1995. (4) Schneider et al. Pages 369-380 in: Molecular and Diagnostic Procedures in Mycoplasmology. Vol I. S. Razin and J. G. Tully, eds. The American Phytopathological Society, 1995.

Management of Tomato Bacterial Spot in the Field by Foliar Applications of Bacteriophages and SAR Inducers.

Various combinations of the harpin protein, acibenzolar-S-methyl, and bacteriophages were compared for controlling tomato bacterial spot in field experiments. Harpin protein and aciben-zolar-S-methyl were applied every 14 days beginning twice before transplanting and then an additional four applications throughout the season. Formulated bacteriophages were applied prior to inoculation followed by twice a week at dusk. A standard bactericide treatment, consisting of copper hydroxide plus mancozeb, was applied once prior to inoculation and then every 7 days, while untreated plants served as an untreated control. Experiments were conducted in north and central Florida fields during fall 2001, spring 2002, and fall 2002. In three consecutive seasons, acibenzolar-S-methyl applied in combination with bacteriophage or bacteriophage and harpin significantly reduced bacterial spot compared with the other treatments. However, it did not significantly affect the total yield compared with the standard or untreated control. Application of host-specific bacteriophages was effective against the bacterial spot pathogen in all three experiments, providing better disease control than copper-mancozeb or untreated control. When results of the disease severity assessments or harvested yield from the bacteriophage-treated plots were grouped and compared with the results of the corresponding nonbacteriophage group, the former provided significantly better disease control and yield of total marketable fruit.

Improved Efficacy of Newly Formulated Bacteriophages for Management of Bacterial Spot on Tomato.

Bacteriophages are currently used as an alternative method for controlling bacterial spot disease on tomato incited by Xanthomonas campestris pv. vesicatoria. However, the efficacy of phage is greatly reduced due to its short residual activity on plant foliage. Three formulations that significantly increased phage longevity on the plant surface were tested in field and greenhouse trials: (i) PCF, 0.5% pregelatinized corn flour (PCF) + 0.5% sucrose; (ii) Casecrete, 0.5% Casecrete NH-400 + 0.5% sucrose + 0.25% PCF; and (iii) skim milk, 0.75% powdered skim milk + 0.5% sucrose. In greenhouse experiments, the nonformulated, PCF-, Casecrete-, and skim milk-formulated phage mixtures reduced disease severity on plants compared with the control by 1, 30, 51, and 62%, respectively. In three consecutive field trials, nonformulated phage caused 15, 20, and 9% reduction in disease on treated plants compared with untreated control plants, whereas plants treated with PCF- and Casecrete-formulated phage had 27, 32, and 12% and 30, 43, and 24% disease reduction, respectively. Plants receiving copper-mancozeb treatments were included in two field trials and had a 20% decrease in disease in the first trial and a 13% increase in the second one. Skim milk-formulated phage was tested only once and caused an 18% disease reduction. PCF-formulated phage was more effective when applied in the evening than in the morning, reducing disease on plants by 27 and 13%, respectively. The Casecrete-formulated phage populations were over 1,000-fold higher than the nonformulated phage populations 36 h after phage application.

First Report of a Wilt and Stem Rot of Muskmelon and Watermelon Transplants Incited by Pseudomonas cichorii in Serbia.

In 1989, a wilt and stem rot of muskmelon (Cucumis melo L.) was reported on seedlings grown under plastic in northern Serbia (1). In 1998, a similar disease of watermelon (Citrullus vulgaris L.) transplants occurred in central Serbia, resulting in losses estimated at 20% of plants grown in the nursery. Initial symptoms appeared as water-soaked lesions on the stem and lower surfaces of cotyledons and first true leaves. Necrosis started from leaf water-soaked areas and spread over the entire plant. Many stems softened and toppled. Wilting was also associated with the latter stages of the disease. Six representative strains isolated from water-soaked stem lesions on muskmelon in 1989 (three strains) and on watermelon in 1998 (three strains) were characterized. All strains were gram negative, strictly aerobic, motile, rod-shaped, and fluorescent on King's B medium, but negative for levan production, soft rot of potato, and arginine dihydrolase activity. Furthermore, strains were oxidase positive and induced a strong hypersensitive reaction in tobacco leaves. All of these reactions are characteristic of Pseudomonas cichorii (2). All six strains were confirmed to be P. cichorii based on analysis of cell-wall fatty acid profiles using the Microbial Identification System (MIDI, Newark, DE). Similarity indices ranged from 0.87 to 0.97. Pathogenicity of the P. cichorii strains was confirmed by prick inoculation of the hypocotyl of 2-week-old plants of domestic muskmelon cv. Sezam and watermelon cv. Rosa. Six plants of each cultivar were pricked with a dissecting needle dipped into a bacterial suspension (108 CFU/ml) prepared from each of the six strains and placed in a humidity chamber at 26°C for 48 h. Water-soaking occurred on all plants around the inoculation site after 24 h. The upper part of the plants lost turgor and wilted. Eventually, the hypocotyl and leaf veins became water-soaked, and the stem tissue softened, resulting in toppling of plants. The symptoms were identical to those described on muskmelon in 1989 and observed on watermelon in 1998. No symptoms developed on control plants. The bacterium was reisolated from the inoculated plants and shown to be identical to the original strains, confirming that P. cichorii was responsible for the disease of muskmelon and watermelon seedlings in 1989 and 1998, respectively. References: (1) M. Arsenijevic and V. Stojsin. Contemp. Agric. 9:487, 1989. (2) R. A. Lelliott et al. J. Appl. Bacteriol. 29:470, 1966.

First Report of Black Rot of Cauliflower and Kale Caused by Xanthomonas campestris pv. campestris in Yugoslavia.

In Yugoslavia, Xanthomonas campestris pv. campestris was isolated from forage kale in 1964 and cabbage in 1997 (1). Recently, the incidence and severity of black rot symptoms on cabbage, cauliflower, and kale have increased. Gram-negative, rod-shaped, motile bacteria were isolated from the diseased leaf and vascular tissues of cauliflower and kale plants collected from 1995 to 1998. The isolates formed yellow, convex, mucoid colonies on yeast dextrose chalk medium, metabolized glucose oxidatively, grew at 37°C, hydrolyzed gelatin and esculin, produced acids from d-arabinose, glucose, sucrose, and trehalose, and did not reduce nitrates. They were nonfluorescent, amylolytic and pectolytic, oxidase negative and catalase positive, and tolerant to 5% NaCl but not to 0.1% triphenyl tetrazolium chloride. Koch's postulates were completed by injecting bacterial suspensions (108 CFU/ml) into leaf petioles of cabbage, cauliflower, and kale seedlings (2- to 3-leaf stage). Dark green watersoaking of petioles and leaf veins followed by yellowing and collapse of inoculated plants was observed after 3 to 5 days. When compared with published information (2), the isolates were identified as X. campestris pv. campestris. This is the first occurrence of this bacterium in cauliflower and kale in Yugoslavia. References: (1) O. Jovanovic et al. Plant Prot. Belgrade 221:175, 1997. (2) N. W. Schaad. 1988. Laboratory Guide for Identification of Plant Pathogenic Bacteria. 2nd ed. The American Phytopathological Society, St. Paul, MN.