A leaf spot and blight of greenhouse tomato seedlings incited by a Herbaspirillum sp.

A leaf spot and blighting were observed on leaves of tomato transplants from a producer in Florida in 2001 and 2002. A nonfluorescent bacterium was isolated consistently from affected tissue. The typical bacterium was a gram negative, strictly aerobic, slightly curved rod with one or two flagella. Sequence analysis of the 16S rRNA indicated that two representative strains, F1 and SE1, had greater than 99% nucleotide sequence identity with Herbaspirillum huttiense and H. rubrisubalbicans. The cellular fatty acid composition of the total of 16 tomato strains was very similar to H. huttiense and H. rubrisubalbicans. Based on carbon utilization, six of nine strains tested with the Biolog system were identified as Herbaspirillum spp. The tomato strains were oxidase positive and grew at 40 degrees C, but were negative for levan production, pectate hydrolysis, and arginine dihydrolase activity. Based upon this polyphasic analysis, we concluded that the strains were most closely related to H. huttiense, although placement in this species would require further analyses. However, the tomato strains and H. rubrisubalbicans, but not H. huttiense, caused confluent necrosis when infiltrated at high concentrations into tomato leaves and were able to produce leaf spot symptoms on inoculated tomato seedlings in the greenhouse. Using pulsed-field gel electrophoresis, we determined that there was considerable variability between the strains collected in 2001 and 2002

Multiple Recombination Events Drive the Current Genetic Structure of Xanthomonas perforans in Florida

Prior to the identification of Xanthomonas perforans associated with bacterial spot of tomato in 1991, X. euvesicatoria was the only known species in Florida. Currently, X. perforans is the Xanthomonas sp. associated with tomato in Florida. Changes in pathogenic race and sequence alleles over time signify shifts in the dominant X. perforans genotype in Florida. We previously reported recombination of X. perforans strains with closely related Xanthomonas species as a potential driving factor for X. perforans evolution. However, the extent of recombination across the X. perforans genomes was unknown. We used a core genome multilocus sequence analysis approach to identify conserved genes and evaluated recombination-associated evolution of these genes in X. perforans. A total of 1,356 genes were determined to be “core” genes conserved among the 58 X. perforans genomes used in the study. Our approach identified three genetic groups of X. perforans in Florida based on the principal component analysis (PCA) using core genes. Nucleotide variation in 241 genes defined these groups, that are referred as Phylogenetic-group Defining (PgD) genes. Furthermore, alleles of many of these PgD genes showed 100% sequence identity with X. euvesicatoria, suggesting that variation likely has been introduced by recombination at multiple locations throughout the bacterial chromosome. Site-specific recombinase genes along with plasmid mobilization and phage associated genes were observed at different frequencies in the three phylogenetic groups and were associated with clusters of recombinant genes. Our analysis of core genes revealed the extent, source, and mechanisms of recombination events that shaped the current population and genomic structure of X. perforans in Florida

An Unusual Pseudomonad Isolated From Diseased Parsley Roots in Serbia

In autumn of 1998 and 1999, parsley root rot was observed either in the fields of northern Serbia or during storage. Diseased plants showed total destruction of the leaf rosette and root base resembling soft rot. However, when the remaining part of the root was removed from the soil, reddish brown areas were noticed on the root surface. The tissue was sunken and firm with no cracks visible on the surface. On the root, longitudinal brown discoloration spread from the upper part toward the root tip affecting the vascular cylinder and the surrounding tissue. Thirty-two bacterial isolates were obtained from inner tissue of many roots. All isolates caused a hypersensitive reaction in tobacco leaves within 24 h, were Gram negative, rod-shaped, motile, and produced shiny, greyish-white colonies on King's medium B (KB) without the typical green fluorescent pigment on KB medium in 24 h. They were levan positive but oxidase and arginine dihydrolase negative, non pectolytic, aerobic, utilised sucrose and aesculin, did not reduce nitrates, and did not grow at 37degreesC. Syringomycin and coronatine production was negative and ice nucleation was positive. Based on fatty acid profiles the strains had greatest similarity to Pseudomonas cichorii and P. syringae. Prick-inoculated roots of two-month-old parsley plants caused tissue depression around the point of inoculation and yellowing of older leaves followed by wilting of the foliage one week after inoculation. A week later the roots were pulled out and root rot resembling the symptoms of natural infection was observed. Considering their pathogenic and bacteriological characteristics, investigated bacterial isolates associated with parsley root rot in Serbia, belong to an unusual, non fluorescent Pseudomonas sp

Pseudomonas huttiensis Associated with Leaf Necrosis and Blighting of Tomato Seedlings in the Greenhouse

In October 2001, tomato foliage with blighting and a leaf spot was received from a transplant producer in Florida. The seedlings manifested apical or marginal leaf necrosis or discrete lesions along the leaf veins. Non-fluorescent bacterial strains forming viscous, creamy white colonies on King's medium B and causing a hypersensitive reaction in tomato and pepper leaves was consistently isolated from lesions. Pathogenicity was checked on three-week old tomato plants. The plants were incubated in high humidity for 24 h before and after inoculation. Similar symptoms were observed on the inoculated seedlings. We characterised 12 strains using bacteriological tests. According to the fatty acid profile, the strains displayed highest similarity with the bacterium Pseudomonas huttiensis Leifson 1962. Sequence analysis of the 16S rRNA indicated that this bacterium shows 98.7divided by98.8% homology with two Herbaspirillum species. Thus, our strains were compared with one P. huttiensis and five H. rubrisubalbicans strains. The strains from tomato were Gram-negative, non-fluorescent and oxidase positive, but levan, pectate hydrolysis and arginine dihydrolase negative, and grew at 40degreesC. Based on these results, they had a high degree of similarity with both P. huttiensis and H. rubrisubalbicans

Exploring diversity of Erwinia amylovora population in Serbia by conventional and automated techniquesand detection of new PFGE patterns

Forty Erwinia amylovora strains originating from different host plants and locations in Serbia and one strain from Montenegro were characterized by conventional, automated and molecular techniques. All strains were Gram-negative, nonfluorescent, facultative anaerobes, oxidase negative, levan positive, produced necrotic lesions followed by bacterial exudate on artificially inoculated immature pear fruits and caused HR on tobacco. Based on carbon source utilization, all strains tested with the Biolog system were identified as E. amylovora. Based on fatty acid profiles all tested strains clustered into three groups in which strains from north Serbia differed from strains isolated in central and south parts of the country. Restriction analysis of genomic DNA using XbaI and PFGE resulted in six different patterns differentiating the strains into six groups. Most of the investigated strains clustered in one group having the pattern type similar to Pt2 group described earlier as dominant in East Europe and the Mediterranean region. Two strains showed PFGE pattern similar to the previously described Pt3 pattern and one strain had pattern similar to Pt6. Based on size and number of the bands, new restriction patterns, assigned as Pt7, Pt8 and Pt9 were observed. PFGE results showed that the E. amylovora population in Serbia is not homogenous and was possibly introduced from different directions. This is the first characterization of E. amylovora collection of strains from Serbia using fatty acid analysis and PFGE

Characterization of a Unique Chromosomal Copper Resistance Gene Cluster from Xanthomonas campestris pv. vesicatoria

We characterized the copper resistance genes in strain XvP26 of Xanthomonas campestris pv. vesicatoria, which was originally isolated from a pepper plant in Taiwan. The copper resistance genes were localized to a 7,652-bp region which, based on pulsed-field gel electrophoresis and Southern hybridization, was determined to be located on the chromosome. These genes hybridized only weakly, as determined by Southern analysis, to other copper resistance genes in Xanthomonas and Pseudomonas strains. We identified five open reading frames (ORFs) whose products exhibited high levels of amino acid sequence identity to the products of previously reported copper genes. Mutations in ORF1, ORF3, and ORF4 removed copper resistance, whereas mutations in ORF5 resulted in an intermediate copper resistance phenotype and insertions in ORF2 had no effect on resistance conferred to a copper-sensitive recipient in transconjugant tests. Based on sequence analysis, ORF1 was determined to have high levels of identity with the CopR (66%) and PcoR (63%) genes in Pseudomonas syringae pv. tomato and Escherichia coli, respectively. ORF2 and ORF5 had high levels of identity with the PcoS gene in E. coli and the gene encoding a putative copper-containing oxidoreductase signal peptide protein in Sinorhizobium meliloti, respectively. ORF3 and ORF4 exhibited 23% identity to the gene encoding a cation efflux system membrane protein, CzcC, and 62% identity to the gene encoding a putative copper-containing oxidoreductase protein, respectively. The latter two ORFs were determined to be induced following exposure to low concentrations of copper, while addition of Co, Cd, or Zn resulted in no significant induction. PCR analysis of 51 pepper and 34 tomato copper-resistant X. campestris pv. vesicatoria strains collected from several regions in Taiwan between 1987 and 2000 and nine copper-resistant strains from the United States and South America showed that successful amplification of DNA was obtained only for strain XvP26. The organization of this set of copper resistance genes appears to be uncommon, and the set appears to occur rarely in X. campestris pv. vesicatoria

Exploring diversity of Erwinia amylovora population in Serbia by conventional and automated techniques and detection of new PFGE patterns

Forty Erwinia amylovora strains originating from different host plants and locations in Serbia and one strain from Montenegro were characterized by conventional, automated and molecular techniques. All strains were Gram-negative, nonfluorescent, facultative anaerobes, oxidase negative, levan positive, produced necrotic lesions followed by bacterial exudate on artificially inoculated immature pear fruits and caused HR on tobacco. Based on carbon source utilization, all strains tested with the Biolog system were identified as E. amylovora. Based on fatty acid profiles all tested strains clustered into three groups in which strains from north Serbia differed from strains isolated in central and south parts of the country. Restriction analysis of genomic DNA using XbaI and PFGE resulted in six different patterns differentiating the strains into six groups. Most of the investigated strains clustered in one group having the pattern type similar to Pt2 group described earlier as dominant in East Europe and the Mediterranean region. Two strains showed PFGE pattern similar to the previously described Pt3 pattern and one strain had pattern similar to Pt6. Based on size and number of the bands, new restriction patterns, assigned as Pt7, Pt8 and Pt9 were observed. PFGE results showed that E. amylovora population in Serbia is not homogenous and was possibly introduced from different directions. This is the first characterization of E. amylovora collection of strains from Serbia using fatty acid analysis and PFGE