The genus Xanthomonas contains a large number of strains, which have been characterized by a variety of phenotypic and genotypic classification methods. The Xanthomonas collection constitutes one of the largest groups of bacteria that have been characterized phylogenetically by DNA-DNA homology studies and genomic fingerprinting. Presently, a total genomic DNA-DNA homology value of 70% represents an internationally accepted criterion to define bacterial species levels. However, the complexity of DNA-DNA reassociation kinetics methods precludes the rapid analysis of large numbers of bacterial isolates, which is imperative for molecular microbial diversity studies. Therefore, the aim of this study was to compare more facile PCR-based genomic fingerprinting techniques, such as repetitive-sequence-based (rep)-PCR and AFLP genomic fingerprinting, to DNA-DNA hybridization studies. Using three different primer sets, rep-PCR genomic fingerprint patterns were generated for 178 Xanthomonas strains, belonging to all 20 previously defined DNA-DNA homology groups, and one Stenotrophomonas maltophilia strain. In addition, AFLP genomic fingerprints were produced for a subset of 80 Xanthomonas strains belonging to the 20 DNA-DNA homology groups and for the S. maltophilia strain. Similarity values derived from rep-PCR- and AFLP-generated fingerprinting analyses were calculated and used to determine the correlation between rep-PCR- or AFLP-derived relationships and DNA-DNA homology values. A high correlation was observed, suggesting that genomic fingerprinting techniques truly reveal genotypic and phylogenetic relationships of organisms. On the basis of these studies, we propose that genomic fingerprinting techniques such as rep-PCR and AFLP can be used as rapid, highly discriminatory screening techniques to determine the taxonomic diversity and phylogenetic structure of bacterial populations.

Acibenzolar-S-methyl (CGA 245704 or Actigard 50WG) is a plant activator that induces systemic acquired resistance (SAR) in many different crops to a number of pathogens. Acibenzolar-S-methyl was evaluated for management of bacterial spot (Xanthomonas axonopodis pv. vesicatoria) and bacterial speck (Pseudomonas syringae pv. tomato) of tomato in 15 and 7 field experiments, respectively. Experiments were conducted over a 4-year period in Florida, Alabama, North Carolina, Ohio, and Ontario using local production systems. Applied at 35 g a.i. ha^-1, acibenzolar-S-methyl reduced foliar disease severity in 14 of the 15 bacterial spot and all 7 bacterial speck experiments. Disease control was similar or superior to that obtained using a standard copper bactericide program. Acibenzolar-S-methyl also reduced bacterial fruit spot and speck incidence. Tomato yield was not affected by using the plant activator in the field when complemented with fungicides to manage foliar fungal diseases, but tomato transplant dry weight was negatively impacted. X. axonopodis pv. vesicatoria population densities on greenhouse-grown tomato transplants were reduced by acibenzolar-S-methyl treatment. Bacterial speck and spot population densities on leaves of field-grown plants were not dramatically affected. Acibenzolar-S-methyl can be integrated as a viable alternative to copper-based bactericides for field management of bacterial spot and speck, particularly where copper-resistant populations predominate.

A comprehensive classification framework was developed that refines the current Xanthomonas classification scheme and provides a detailed assessment of Xanthomonas diversity at the species, subspecies, pathovar, and subpathovar levels. Polymerase chain reaction (PCR) using primers targeting the conserved repetitive sequences BOX, enterobacterial repetitive intergenic consensus (ERIC), and repetitive extragenic palindromic (REP) (rep-PCR) was used to generate genomic fingerprints of 339 Xanthomonas strains comprising 80 pathovars, 20 DNA homology groups, and a Stenotrophomonas maltophilia reference strain. Computer-assisted pattern analysis of the rep-PCR profiles permitted the clustering of strains into distinct groups, which correspond directly to the 20 DNA-DNA homology groups(genospecies) previously identified. Group 9 strains (X. axonopodis) were an exception and did not cluster together into a coherent group but comprised six subgroups. Over 160 strains not previously characterized by DNA-DNA hybridization analysis, or not previously classified, were assigned to specific genospecies based on the classification framework developed. The rep-PCR delineated subspecific groups within X. hortorum, X. arboricola, X. axonopodis, X. oryzae, X. campestris, and X. translucens. Numerous taxonomic issues with regard to the diversity, similarity, redundancy, or misnaming were resolved. This classification framework will enable the rapid identification and classification of new, novel, or unknown Xanthomonas strains that are pathogenic or are otherwise associated with plants.

Organic heirloom tomato production is limited in the southeastern United States by foliar and soilborne diseases, thermal stress, and weathered soil structure. Heirloom cultivars command a premium market, but tolerance to disease and abiotic stress is often poor. Organic growers need research that supports the advantages of market niches afforded by heirloom tomatoes through the development of integrated systems to manage pests and reduce risks of associated crop losses or low yields. Two major soilborne diseases common in the southeast, bacterial wilt (caused by Ralstonia solanacearum ) and fusarium wilt (caused by Fusarium oxysporum f.sp. lycopersici ), were effectively managed using susceptible heirloom scions grafted onto resistant rootstock. In naturally infested soil, bacterial wilt incidence for nongrafted ‘German Johnson’ was 79% and 75% in 2005 and 2006, respectively. ‘German Johnson’ showed no symptoms of bacterial wilt in either year when grafted onto the resistant genotypes CRA 66 or Hawaii 7996. Fusarium wilt incidence was 46% and 50%, respectively, in nongrafted and self-grafted ‘German Johnson’ controls. When ‘Maxifort’ rootstock was grafted with ‘German Johnson’, no symptoms of fusarium wilt were seen, and plants with ‘Robusta’ rootstock had an intermediate level of disease (29%). An evaluation of commercially available rootstock was carried out in three separate experiments in diverse organic production systems to determine yield impacts with low disease pressure. ‘Maxifort’ rootstock significantly increased yield in one location ( P = 0.05), but ‘Maxifort’ and ‘Robusta’ rootstock did not consistently impact yield at the other two locations. Grafting is an effective management tool for organic growers in the southeast United States to reduce risk of crop loss resulting from soilborne diseases and will be a valuable component in an integrated pest management program.

Ralstonia solanacearum is a bacterial vascular pathogen causing devastating bacterial wilt. In the field, resistance against this pathogen is quantitative and is available for breeders only in tomato and eggplant. To understand the basis of resistance to R. solanacearum in tomato, we investigated the spatio-temporal dynamics of bacterial colonization using non-invasive live monitoring techniques coupled to grafting of susceptible and resistant varieties. We found four ‘bottlenecks’ that limit the bacterium in resistant tomato: root colonization, vertical movement from roots to shoots, circular vascular bundle invasion, and radial apoplastic spread in the cortex. Radial invasion of cortical extracellular spaces occurred mostly at late disease stages but was observed throughout plant infection. This study shows that resistance is expressed in both root and shoot tissues, and highlights the importance of structural constraints to bacterial spread as a resistance mechanism. It also shows that R. solanacearum is not only a vascular pathogen but spreads out of the xylem, occupying the plant apoplast niche. Our work will help elucidate the complex genetic determinants of resistance, setting the foundations to decipher the molecular mechanisms that limit pathogen colonization, which may provide new precision tools to fight bacterial wilt in the field.