In this review, we highlight studies in which whole-genome sequencing, comparative genomics, and population genomics have provided unprecedented insights into past and ongoing pathogen evolution. These include new understandings of the adaptive evolution of secretion systems and their effectors. We focus on Xanthomonas pathosystems that have seen intensive study and improved our understanding of pathogen emergence and evolution, particularly in the context of host specialization: citrus canker, bacterial blight of rice, and bacterial spot of tomato and pepper. Across pathosystems, pathogens appear to follow a pattern of bursts of evolution and diversification that impact host adaptation. There remains a need for studies on the mechanisms of host range evolution and genetic exchange among closely related but differentially host-specialized species and to start moving beyond the study of specific strain and host cultivar pairwise interactions to thinking about these pathosystems in a community context.

The widespread use of antimicrobials that target bacterial pathogens has driven evolution of resistance, compromising the efficacy of these bactericides. Understanding the emergence and spread of resistance genes via mobile genetic elements is crucial for combating antimicrobial resistance. Copper resistance (CuR) in Xanthomonas euvesicatoria pv. perforans has severely affected the efficacy of copper-based bactericides for controlling bacterial leaf spot disease of tomato and pepper. Here, we investigated the evolutionary pathways of CuR acquisition and dissemination in X. euvesicatoria pv. perforans using an extensive collection of strains. We determined that chromosomally encoded CuR predominates over plasmid-borne CuR in multiple distinct phylogenetic groups of X. euvesicatoria pv. perforans . Our analysis revealed a single site of chromosomal integration by a CuR genomic island, although the genomic island showed sequence variation among phylogenetic groups. While chromosomal CuR was more prevalent, strains with plasmid-borne resistance conferred greater copper tolerance. Additionally, we identified strains carrying two copies of CuR genes, on plasmid and chromosome, that exhibited increased copper tolerance. Strains of X. euvesicatoria pv. perforans from the USA shared identical CuR gene sequences whether on plasmids or chromosome while different alleles were found in strains from other countries. In contrast to X. euvesicatoria pv. perforans , plasmid-borne CuR predominated in closely related pathovar, X. euvesicatoria pv. euvesicatoria . Overall, these findings contribute to a better understanding of the evolution and persistence of CuR in X. euvesicatoria pv. perforans and its closest relatives. IMPORTANCE: The emergence of antimicrobial resistance is a significant threat to agricultural production as it reduces the efficacy of various antimicrobials including copper-based bactericides that are widely used to control plant diseases. The challenge of increasing antimicrobial resistance entering a production system necessitates a deeper understanding of the dynamics and mechanisms by which pathogens acquire resistance. As a result of this research, we have identified different mechanisms of copper resistance acquisition as well as levels of copper resistance in a devastating plant pathogen, X. euvesicatoria pv. perforans . The evolution and dissemination of copper resistance in strains through plasmid or chromosomally integrated genomic island or both presents barriers to current management approaches, where growers rely heavily on copper-based bactericides to manage disease outbreaks. This knowledge is crucial when considering the continued use of existing antimicrobials or adopting alternative antimicrobials in efforts to implement enhanced antimicrobial stewardship strategies in agriculture.

The emergence of plant pathogens is often associated with waves of unique evolutionary and epidemiological events. Xanthomonas hortorum pv. gardneri is one of the major pathogens causing bacterial spot disease of tomatoes. After its first report in the 1950s, there were no formal reports on this pathogen until the 1990s, despite active global research on the pathogens that cause tomato and pepper bacterial spot disease. Given the recently documented global distribution of X. hortorum pv. gardneri, our objective was to examine genomic diversification associated with its emergence. We sequenced the genomes of X. hortorum pv. gardneri strains collected in eight countries to examine global population structure and pathways of emergence using phylodynamic analysis. We found that strains isolated post-1990 group by region of collection and show minimal impact of recombination on genetic variation. A period of rapid geographic expansion in X. hortorum pv. gardneri is associated with acquisition of a large plasmid conferring copper tolerance by horizontal transfer and coincides with the burgeoning hybrid tomato seed industry through the 1980s. The ancestry of X. hortorum pv. gardneri is consistent with introduction to hybrid tomato seed production and dissemination during the rapid increase in trade of hybrid seeds. [ ] Copyright © 2024 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license .

Plasmids are key drivers of horizontal gene transfer. These genetic elements promote diversification and rapid adaptation of bacterial populations to changing environments by transferring beneficial traits within and between bacterial species. Xanthomonas euvesicatoria pv. perforans is a devastating plant pathogen that causes bacterial spot disease in tomato and pepper. The pathogen population in Florida contains several distinct genetic lineages that differ in relative frequency. The objective of this study was to characterize plasmid diversity and gene content and plasmid distribution in relation to chromosomal phylogeny. Our in silico-based plasmid prediction revealed the presence of diverse plasmids ranging from approximately 16 to 235 kb. A network approach based on shared k-mer content uncovered 10 distinct plasmid groups with high genetic similarity (cliques). Interestingly, these plasmid cliques were confined to specific phylogenetic clusters, suggesting potential incompatibility or restricted plasmid movement between clusters. Some of the predicted plasmids carry virulence genes coding for type III secretion effectors, including transcription activator-like effectors and genes related to biocide resistance, such as copper. We also identified gene reshuffling between the plasmids, likely carried out by transposons present within them. Overall, these results provide foundational insights into plasmid diversity in X. euvesicatoria pv. perforans with implications for the role of these mobile genetic elements in genome dynamics and pathogen adaptation.

Xanthomonas spp. are increasingly recognized as a global threat to agriculture, impacting a broad range of economically important crops. We report the whole-genome sequences of six Xanthomonas strains isolated from tomato and pepper plants in Turkey that were experiencing symptoms of bacterial spot disease. Phylogenomic analysis with representative Xanthomonas genomes from each species revealed that three of these strains belonged to Xanthomonas perforans , two to Xanthomonas euvesicatoria and one to Xanthomonas campestris . We then analysed the phylogenomic relatedness of these strains with other strains from these respective species and characterized their type III secreted effector content. These genomic data represent a valuable resource for understanding the genetic diversity and local epidemiology of bacterial spot disease in Turkey.

The management of bacterial spot of tomato is challenged by the widespread occurrence of copper resistance among its causal pathogens. In Xanthomonas euvesicatoria pv. perforans , a major causal agent, copper resistance is conferred by cop genes organized in an operon located on either the chromosome or plasmid. In the United States, copper resistance is predominantly chromosomally encoded. Plasmid-borne resistance is limited to a few phylogenetic groups but is prevalent in other bacterial spot pathogens. We hypothesized that plasmid-encoded copper resistance genes exhibit higher expression than chromosomally encoded genes but may be unstable in the absence of selective pressure for resistance. To test this hypothesis, we examined how the chromosomal versus plasmid-borne cop operon influences gene copy number and expression, as well as plasmid stability. Relative to strains with only chromosomal resistance, strains carrying plasmid-encoded resistance exhibited an increased copy number of copB , a key gene in the operon, while strains carrying both chromosomal and plasmid-encoded resistance showed an even greater increase in gene copy number. Correspondingly, copB expression was elevated in both groups. During in vitro growth without copper, partial or complete plasmid loss occurred in a subset of strains, and genome sequencing of colonies with reduced plasmid size revealed transposase-mediated plasmid rearrangements. These findings demonstrate that plasmid-borne copper resistance increases gene copy number and expression of copper resistance genes but can be unstable without copper selection in some strain backgrounds, highlighting the role of mobile genetic elements in shaping Xanthomonas euvesicatoria pv. perforans adaptation to changing copper environments. IMPORTANCE: Plasmids are key players in the dissemination of antimicrobial resistance. Although copper resistance is widespread among Xanthomonas euvesicatoria pv. perforans strains, copper-based bactericides remain at the forefront of disease management. We previously demonstrated that distinct genetic elements, including plasmid- and chromosomally encoded genomic islands, contribute to the acquisition of copper resistance and the occurrence of multicopy resistance in X. euvesicatoria pv. perforans . Here, we show that the localization of copper resistance genes on a plasmid, chromosome, or both influences gene copy number and expression of copper resistance genes. Furthermore, rearrangements between co-resident plasmids affect plasmid stability, leading to the loss of copper resistance in X. euvesicatoria pv. perforans . These findings highlight how plasmid dynamics can shape bacterial evolvability and adaptation to selective environments.