Physiological responses of pepper (Capsicum annum) to combined ozone and pathogen stress.

Tropospheric ozone [O3] is a secondary air pollutant formed from the photochemical oxidation of volatile organic compounds in the presence of nitrogen oxides, and it is one of the most damaging air pollutants to crops. O3 entry into the plant generates reactive oxygen species leading to cellular damage and oxidative stress, leading to decreased primary production and yield. Increased O3 exposure has also been shown to have secondary impacts on plants by altering the incidence and response to plant pathogens. We used the Capsicum annum (pepper)-Xanthomonas perforans pathosystem to investigate the impact of elevated O3 (eO3) on plants with and without exposure to Xanthomonas, using a disease-susceptible and disease-resistant pepper cultivar. Gas exchange measurements revealed decreases in diurnal photosynthetic rate (A') and stomatal conductance ( g s ' $$ {g}_{\mathrm{s}}^{\prime } $$ ), and maximum rate of electron transport (Jmax) in the disease-resistant cultivar, but no decrease in the disease-susceptible cultivar in eO3, regardless of Xanthomonas presence. Maximum rates of carboxylation (Vc,max), midday A and gs rates at the middle canopy, and decreases in aboveground biomass were negatively affected by eO3 in both cultivars. We also observed a decrease in stomatal sluggishness as measured through the Ball-Berry-Woodrow model in all treatments in the disease-resistant cultivar. We hypothesize that the mechanism conferring disease resistance to Xanthomonas in pepper also renders the plant less tolerant to eO3 stress through changes in stomatal responsiveness. Findings from this study help expand our understanding of the trade-off of disease resistance with abiotic stresses imposed by future climate change.

Complete functional analysis of type IV pilus components of a reemergent plant pathogen reveals neofunctionalization of paralog genes.

Type IV pilus (TFP) is a multifunctional bacterial structure involved in twitching motility, adhesion, biofilm formation, as well as natural competence. Here, by site-directed mutagenesis and functional analysis, we determined the phenotype conferred by each of the 38 genes known to be required for TFP biosynthesis and regulation in the reemergent plant pathogenic fastidious prokaryote Xylella fastidiosa. This pathogen infects > 650 plant species and causes devastating diseases worldwide in olives, grapes, blueberries, and almonds, among others. This xylem-limited, insect-transmitted pathogen lives constantly under flow conditions and therefore is highly dependent on TFP for host colonization. In addition, TFP-mediated natural transformation is a process that impacts genomic diversity and environmental fitness. Phenotypic characterization of the mutants showed that ten genes were essential for both movement and natural competence. Interestingly, seven sets of paralogs exist, and mutations showed opposing phenotypes, indicating evolutionary neofunctionalization of subunits within TFP. The minor pilin FimT3 was the only protein exclusively required for natural competence. By combining approaches of molecular microbiology, structural biology, and biochemistry, we determined that the minor pilin FimT3 (but not the other two FimT paralogs) is the DNA receptor in TFP of X. fastidiosa and constitutes an example of neofunctionalization. FimT3 is conserved among X. fastidiosa strains and binds DNA non-specifically via an electropositive surface identified by homolog modeling. This protein surface includes two arginine residues that were exchanged with alanine and shown to be involved in DNA binding. Among plant pathogens, fimT3 was found in ~ 10% of the available genomes of the plant associated Xanthomonadaceae family, which are yet to be assessed for natural competence (besides X. fastidiosa). Overall, we highlight here the complex regulation of TFP in X. fastidiosa, providing a blueprint to understand TFP in other bacteria living under flow conditions.

Phylodynamic Insights into Global Emergence and Diversification of the Tomato Pathogen Xanthomonas hortorum pv. gardneri.

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.

Xanthomonas as a Model System for Studying Pathogen Emergence and Evolution.

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.

Open Access and Reproducibility in Plant Pathology Research: Guidelines and Best Practices.

The landscape of scientific publishing is experiencing a transformative shift toward open access, a paradigm that mandates the availability of research outputs such as data, code, materials, and publications. Open access provides increased reproducibility and allows for reuse of these resources. This article provides guidance for best publishing practices of scientific research, data, and associated resources, including code, in The American Phytopathological Society journals. Key areas such as diagnostic assays, experimental design, data sharing, and code deposition are explored in detail. This guidance aligns with that observed by other leading journals. We hope the information assembled in this paper will raise awareness of best practices and enable greater appraisal of the true effects of biological phenomena in plant pathology.

Influence of Co-occurring Weakly Pathogenic Bacterial Species on Bacterial Spot Disease Dynamics on Tomato.

Mixed infections caused by multiple pathogenic and weakly pathogenic strains inhabiting the same host plants are common in nature and may modify pathogen dynamics. However, traditional plant pathogen studies have mostly focused on the binary interaction between a single host and a single pathogen. In this study, we have looked beyond this binary interaction and evaluated the impact of coinfection on disease dynamics on tomato using the bacterial spot pathogen Xanthomonas perforans (Xp), the co-occurring weakly pathogenic strain of X. arboricola (Xa), and the co-occurring potential weak pathogenic strain of Pseudomonas capsici (Pc). Time-series coinfection experiments monitoring disease severity and within-host population dynamics revealed higher disease severity in coinfection by three species compared with infection by Xp alone. However, coinfection by dual species, Xp and Pc, or Xa resulted in lower disease severity compared with Xp alone. Thus, coinfection outcomes depend on interacting species. Weak pathogens could exploit Xp to colonize the host plant as indicated by their higher populations in coinfection. However, Xp population dynamics were dependent on the coinfecting partner. While resource competition might be a possible explanation for lower Xp population in dual coinfection, interaction of Pc with the host was found to influence Xp population. Interestingly, Xp population was higher in the presence of three-species interaction compared with Xp and Xa coinfection, suggesting potential modulation of cooperative interactions among Xp and Xa in three-species coinfection rather than competitive interactions. Humidity played a significant role in population dynamics of the three species. Overall, this study highlighted the importance of coinfection dynamics in studying plant disease outbreaks.

Unexpected Diversity of Pseudomonads Associated with Bacterial Leaf Spot of Cucurbits in the Southeastern United States.

Bacterial leaf spot of cucurbits (BLS) is an emerging disease in the southeastern United States that is capable of causing widespread outbreaks under conducive conditions. Historically attributed solely to the bacterium Pseudomonas syringae pv. lachrymans, recent studies have identified additional P. syringae pathovars as causal agents of the disease. To further investigate the identity and diversity of P. syringae strains associated with BLS in the southeastern United States, 47 bacterial isolates were recovered from symptomatic cucurbits from Florida, Alabama, and Georgia. Strains were characterized using the LOPAT testing scheme, fluorescence, and pathogenicity to watermelon and squash seedlings. Thirty-eight fluorescent isolates underwent whole-genome sequencing and were further characterized with 16S rRNA, four gene multilocus sequence analysis (MLSA) phylogeny, and average nucleotide identity analysis. Thirty-four isolates were identified as members of the P. syringae species complex, including P. syringae sensu stricto (12), P. alliivorans (12), P. capsici (nine), and P. viridiflava (one). An additional four isolates were found to belong to the Pseudomonas genus outside of the syringae species complex, though they did not share 95% or greater average nucleotide identity to any validly published species and are believed to belong to three novel Pseudomonas species. These results reveal an unpredicted level of diversity of Pseudomonas strains associated with BLS in the region and show the benefits of whole-genome sequencing for strain identification. Identification of P. capsici, which is capable of causing disease at higher temperatures than P. syringae, as a causal agent of BLS may also affect management strategies in the future.

Crosstalk and trade-offs: Plant responses to climate change-associated abiotic and biotic stresses.

As sessile organisms, plants are constantly challenged by a dynamic growing environment. This includes fluctuations in temperature, water availability, light levels, and changes in atmospheric constituents such as carbon dioxide (CO2 ) and ozone (O3 ). In concert with changes in abiotic conditions, plants experience changes in biotic stress pressures, including plant pathogens and herbivores. Human-induced increases in atmospheric CO2 levels have led to alterations in plant growth environments that impact their productivity and nutritional quality. Additionally, it is predicted that climate change will alter the prevalence and virulence of plant pathogens, further challenging plant growth. A knowledge gap exists in the complex interplay between plant responses to biotic and abiotic stress conditions. Closing this gap is crucial for developing climate resilient crops in the future. Here, we briefly review the physiological responses of plants to elevated CO2 , temperature, tropospheric O3 , and drought conditions, as well as the interaction of these abiotic stress factors with plant pathogen pressure. Additionally, we describe the crosstalk and trade-offs involved in plant responses to both abiotic and biotic stress, and outline targets for future work to develop a more sustainable future food supply considering future climate change.

Genome-Wide Association to Study the Host-Specificity Determinants of Xanthomonas perforans.

Xanthomonas perforans and X. euvesicatoria are the causal agents of bacterial spot disease of tomato and pepper, endemic to the Southeastern United States. Although very closely related, the two bacterial species differ in host specificity, where X. perforans is the dominant pathogen of tomato and X. euvesicatoria that of pepper. This is in part due to the activity of avirulence proteins that are secreted by X. perforans strains and elicit effector-triggered immunity in pepper leaves, thereby restricting pathogen growth. In recent years, the emergence of several pepper-pathogenic X. perforans lineages has revealed variability within the bacterial species to multiply and cause disease in pepper, even in the absence of avirulence gene activity. Here, we investigated the basal evolutionary processes underlying the host range of this species using multiple genome-wide association analyses. Surprisingly, we identified two novel gene candidates that were significantly associated with pepper-pathogenic X. perforans and X. euvesicatoria. Both candidates were predicted to be involved in the transport/acquisition of nutrients common to the plant cell wall or apoplast and included a TonB-dependent receptor, which was disrupted through independent mutations within the X. perforans lineage. The other included a symporter of protons/glutamate, gltP, enriched with pepper-associated mutations near the promoter and start codon of the gene. Functional analysis of these candidates revealed that only the TonB-dependent receptor had a minor effect on the symptom development and growth of X. perforans in pepper leaves, indicating that pathogenicity to this host might have evolved independently within the bacterial species and is likely a complex, multigenic trait.

A Brief Evaluation of a Copper Resistance Mobile Genetic Island in the Bacterial Leaf Spot Pathogen Xanthomonas euvesicatoria pv. perforans.

Due to the continuous use of copper containing bactericides without effective alternative bactericides, copper resistance has become more prevalent in plant pathogens, including Xanthomonas euvesicatoria pv. perforans (formerly Xanthomonas perforans), a predominant cause of bacterial leaf spot disease of tomato and pepper in the Southeastern United States. Previously, reports of copper resistance have been associated with a large conjugative plasmid. However, we have characterized a copper resistance genomic island located within the chromosome of multiple X. euvesicatoria pv. perforans strains. The island is distinct from a previously described chromosomally encoded copper resistance island in X. vesicatoria strain XVP26. Computational analysis revealed the genomic island to contain multiple genes associated with genetic mobility, including both phage-related genes and transposase. Among copper-tolerant strains of X. euvesicatoria pv. perforans isolated from Florida, the majority of strains were found to have the copper resistance chromosomally encoded rather than plasmid borne. Our results suggest that this copper resistance island may have two modes of horizontal gene transfer and that chromosomally encoded copper resistance genes may provide a fitness advantage over plasmid-borne resistance.

Evaluation of Meloidogyne Incognita and Rotylenchulus Reniformis Nematode-resistant Cotton Cultivars with Supplemental Corteva Agriscience Nematicides.

Meloidogyne incognita- and Rotylenchulus reniformis-resistant new cotton cultivars have recently become available, giving growers a new option in nematode management. The objectives of this study were: (i) to determine the yield potential of the new cultivars PHY 360 W3FE (M. incognita-resistant) and PHY 332 W3FE (R. reniformis-resistant) in nematode-infested fields and (ii) to evaluate the effects of combining the nematicides Reklemel (fluazaindolizine), Vydate C-LV (oxamyl), and the seed treatment BIOST Nematicide 100 (heat killed Burkholderia rinojenses and its non-living spent fermentation media) with resistant cotton cultivars on nematode population levels and lint yield. Field experiments in 2020 and 2021 indicated M. incognita population levels were 73% lower on PHY 360 W3FE (R) and 80% lower for R. reniformis on the PHY 332 W3FE (R) at 40 days after planting. Nematode eggs per gram of root were further reduced an average of 86% after the addition of Reklemel and Vydate C-LV when averaging both cultivars over the two years. Tests with BIOST Nematicide 100 + Reklemel + Vydate C-LV (0.56 + 2.5 L/ha) in both M. incognita and R. reniformis fields produced higher lint yields. Overall, planting PHY 360 W3FE (R) and PHY 332 W3FE (R) improved yields an average of 364 kg/ha while limiting nematode population increases. The addition of the nematicides further increased yields 152 kg/ha of the nematode-resistant cultivars.

Mutation of a Single Core Gene, tssM, of Type VI Secretion System of Xanthomonas perforans Influences Virulence, Epiphytic Survival, and Transmission During Pathogenesis on Tomato.

Xanthomonas perforans is a seedborne hemibiotrophic pathogen that successfully establishes infection in the phyllosphere of tomato. While most studies investigating mechanistic basis of pathogenesis have focused on successful apoplastic growth, factors important during asymptomatic colonization in the early stages of disease development are not well understood. In this study, we show that tssM gene of the type VI secretion system cluster i3* (T6SS-i3*) plays a significant role during initial asymptomatic epiphytic colonization at different stages during the life cycle of the pathogen. Mutation in a core gene, tssM of T6SS-i3*, imparted higher aggressiveness to the pathogen, as indicated by higher overall disease severity, higher in planta growth, and shorter latent infection period compared with the wild-type upon dip inoculation of 4- to 5-week-old tomato plants. Contribution of tssM toward aggressiveness was evident during vertical transmission from seed to seedling, with wild-type showing reduced disease severity as well as lower in planta populations on seedlings compared with the mutant. Presence of functional TssM offered higher epiphytic fitness as well as higher dissemination potential to the pathogen when tested in an experimental setup mimicking transplant house high-humidity conditions. We showed higher osmotolerance being one mechanism by which TssM offers higher epiphytic fitness. Taken together, these data reveal that functional TssM plays a larger role in offering ecological advantage to the pathogen. TssM prolongs the association of hemibiotrophic pathogen with the host, minimizing overall disease severity yet facilitating successful dissemination.

Taxonomic Refinement of Xanthomonas arboricola.

Xanthomonas arboricola comprises a number of economically important fruit tree pathogens classified within different pathovars. Dozens of nonpathogenic and taxonomically unvalidated strains are also designated as X. arboricola, leading to a complicated taxonomic status in the species. In this study, we have evaluated the whole-genome resources of all available Xanthomonas spp. strains designated as X. arboricola in the public databases to refine the members of the species based on DNA similarity indexes and core genome-based phylogeny. Our results show that, of the nine validly described pathovars within X. arboricola, pathotype strains of seven pathovars are taxonomically genuine, belonging to the core clade of the species regardless of their pathogenicity on the host of isolation (thus the validity of pathovar status). However, strains of X. arboricola pv. guizotiae and X. arboricola pv. populi do not belong to X. arboricola because of the low DNA similarities between the type strain of the species and the pathotype strains of these two pathovars. Thus, we propose to elevate the two pathovars to the rank of a species as X. guizotiae sp. nov. with the type strain CFBP 7408T and X. populina sp. nov. with the type strain CFBP 3123T. In addition, other mislabeled strains of X. arboricola were scattered within Xanthomonas spp. that belong to previously described species or represent novel species that await formal description.

Population Genomic- and Phylogenomic-Enabled Advances to Increase Insight Into Pathogen Biology and Epidemiology.

Population genetics has been a key discipline in phytopathology for many years. The recent rise in cost-effective, high-throughput DNA sequencing technologies, allows sequencing of dozens, if not hundreds of specimens, turning population genetics into population genomics and opening up new, exciting opportunities as described in this Focus Issue. Without the limitations of genetic markers and the availability of whole or near whole-genome data, population genomics can give new insights into the biology, evolution and adaptation, and dissemination patterns of plant-associated microbes.

Genome-resolved metagenomics to study co-occurrence patterns and intraspecific heterogeneity among plant pathogen metapopulations.

Assessment of pathogen diversity in agricultural fields is essential for informing management decisions and the development of resistant plant varieties. However, many population genomic studies have relied on culture-based approaches that do not provide quantitative assessment of pathogen populations at the field-level or the associated host microbiome. Here, we applied whole-genome shotgun sequencing of microbial DNA extracted directly from the washings of pooled leaf samples, collected from individual tomato and pepper fields in Alabama that displayed the classical symptoms of bacterial spot disease caused by Xanthomonas spp. Our results revealed that while the occurrence of both X. perforans and X. euvesicatoria within fields was limited, evidence of co-occurrence of up to three distinct X. perforans genotypes was obtained in 7 of 10 tomato fields sampled. These population dynamics were accompanied by the corresponding type 3 secreted effector repertoires associated with the co-occurring X. perforans genotypes, indicating that metapopulation structure within fields should be considered when assessing the adaptive potential of X. perforans. Finally, analysis of microbial community composition revealed that co-occurrence of the bacterial spot pathogens Pseudomonas cichorii and Xanthomonas spp. is common in Alabama fields and provided evidence for the non-random association of several other human and plant opportunists.

Molecular Epidemiology of Xanthomonas perforans Outbreaks in Tomato Plants from Transplant to Field as Determined by Single-Nucleotide Polymorphism Analysis.

Outbreaks of bacterial spot on tomato (BST) caused by Xanthomonas perforans are a major concern for sustainable crop production. BST is a common occurrence in tomato transplants grown for field production. We hypothesized that BST outbreaks in commercial fields originate from X. perforans strains inadvertently introduced from commercial transplant facilities. To test this hypothesis, we used a genome-wide single-nucleotide polymorphism (SNP) analysis to characterize X. perforans strains recovered from tomato transplant facilities and fields in commercial production areas. X. perforans strains were isolated from symptomatic transplants prior to roguing at two commercial transplant growers. Then, the same groups of transplants were tracked to commercial fields to recover X. perforans strains from diseased plants prior to harvest. Whole-genome sequencing was carried out on 84 strains isolated from transplant and field plants from Florida and South Carolina. SNPs were called using three reference strains that represented the genetic variation of the sampled strains. Field strains showing genetic similarity to transplant strains had a difference of 2 to 210 SNPs. Transplant and field strains clustered together by grower within each phylogenomic group, consistent with expectations. The range of genetic divergence among strains isolated from field plants was similar to the range obtained from strains on transplants. Using the range of genetic variation observed in transplants, we estimate that 60% to 100% of field strains were an extension of the transplant strain population. Our results stress the importance of BST management to reduce X. perforans movement from transplant to field and to minimize subsequent disease outbreaks.IMPORTANCE Current management of Xanthomonas perforans on tomato plants mainly relies on the frequent application of pesticides. However, the lack of effective pesticides and the development of strain tolerance to certain bactericides limit the ability to control outbreaks in production fields. Better knowledge of probable sources of X. perforans inoculum during tomato production is required to refine management strategies. Tomato plants are typically established in the field using transplants. This study aimed to determine if strains from field epidemics were coming from transplant facilities or resulted from local field outbreaks. The overall goal was to identify potential sources of inoculum and subsequently develop strategies to reduce carryover from transplant production to the field. Our results indicate that tomato producers should shift disease management efforts to transplant facilities to reduce disease in the field. Improved transplant health should reduce the likelihood of bacterial spot outbreaks and subsequently reduce pesticide usage in the field.

Genome-Wide Association Study of Resistance to Xanthomonas gardneri in the USDA Pepper (Capsicum) Collection.

Bacterial spot Xanthomonas species cause significant disease outbreaks on tomato and pepper in tropical and subtropical regions throughout the world. Host resistance has been one of the key components of integrated disease management approaches to mitigate plant pathogens. Although a number of resistance genes have been identified in pepper against bacterial spot xanthomonads, emergence of bacterial spot pathogen variants capable of overcoming these sources and changing pathogen distributions reinforce the importance of identifying novel candidates to incorporate into breeding programs. We conducted a genome-wide association study (GWAS) on a diverse U.S. Department of Agriculture collection of pepper germplasm including different species of Capsicum to identify novel sources of disease resistance against a highly virulent X. gardneri strain isolated from a recent outbreak. GWAS identified highly significant single nucleotide polymorphisms associated with defoliation in response to infection with X. gardneri. Functionally relevant candidate genes encoded products involved in disease resistance/susceptibility, hormone signaling, and basal resistance against multiple pathogens in various host-pathogen systems. The X. gardneri-resistant genotypes and quantitative trait loci identified in this study provide alleles that could be used for a resistance gene pyramiding effort against different species of bacterial spot xanthomonads in pepper.

Pacbio sequencing of copper-tolerant Xanthomonas citri reveals presence of a chimeric plasmid structure and provides insights into reassortment and shuffling of transcription activator-like effectors among X. citri strains.

BACKGROUND: Xanthomonas citri, a causal agent of citrus canker, has been a well-studied model system due to recent availability of whole genome sequences of multiple strains from different geographical regions. Major limitations in our understanding of the evolution of pathogenicity factors in X. citri strains sequenced by short-read sequencing methods have been tracking plasmid reshuffling among strains due to inability to accurately assign reads to plasmids, and analyzing repeat regions among strains. X. citri harbors major pathogenicity determinants, including variable DNA-binding repeat region containing Transcription Activator-like Effectors (TALEs) on plasmids. The long-read sequencing method, PacBio, has allowed the ability to obtain complete and accurate sequences of TALEs in xanthomonads. We recently sequenced Xanthomonas citri str. Xc-03-1638-1-1, a copper tolerant A group strain isolated from grapefruit in 2003 from Argentina using PacBio RS II chemistry. We analyzed plasmid profiles, copy number and location of TALEs in complete genome sequences of X. citri strains. RESULTS: We utilized the power of long reads obtained by PacBio sequencing to enable assembly of a complete genome sequence of strain Xc-03-1638-1-1, including sequences of two plasmids, 249 kb (plasmid harboring copper resistance genes) and 99 kb (pathogenicity plasmid containing TALEs). The pathogenicity plasmid in this strain is a hybrid plasmid containing four TALEs. Due to the intriguing nature of this pathogenicity plasmid with Tn3-like transposon association, repetitive elements and multiple putative sites for origins of replication, we might expect alternative structures of this plasmid in nature, illustrating the strong adaptive potential of X. citri strains. Analysis of the pathogenicity plasmid among completely sequenced X. citri strains, coupled with Southern hybridization of the pathogenicity plasmids, revealed clues to rearrangements of plasmids and resulting reshuffling of TALEs among strains. CONCLUSIONS: We demonstrate in this study the importance of long-read sequencing for obtaining intact sequences of TALEs and plasmids, as well as for identifying rearrangement events including plasmid reshuffling. Rearrangement events, such as the hybrid plasmid in this case, could be a frequent phenomenon in the evolution of X. citri strains, although so far it is undetected due to the inability to obtain complete plasmid sequences with short-read sequencing methods.

Genomic Inference of Recombination-Mediated Evolution in Xanthomonas euvesicatoria and X. perforans.

Recombination is a major driver of evolution in bacterial populations, because it can spread and combine independently evolved beneficial mutations. Recombinant lineages of bacterial pathogens of plants are typically associated with the colonization of novel hosts and the emergence of new diseases. Here we show that recombination between evolutionarily and phenotypically distinct plant-pathogenic lineages generated recombinant lineages with unique combinations of pathogenicity and virulence factors. Xanthomonas euvesicatoria and Xanthomonas perforans are two closely related lineages causing bacterial spot disease on tomato and pepper worldwide. We sequenced the genomes of atypical strains collected from tomato in Nigeria and observed recombination in the type III secretion system and effector genes, which showed alleles from both X. euvesicatoria and X. perforans Wider horizontal gene transfer was indicated by the fact that the lipopolysaccharide cluster of one strain was most similar to that of a distantly related Xanthomonas pathogen of barley. This strain and others have experienced extensive genomewide homologous recombination, and both species exhibited dynamic open pangenomes. Variation in effector gene repertoires within and between species must be taken into consideration when one is breeding tomatoes for disease resistance. Resistance breeding strategies that target specific effectors must consider possibly dramatic variation in bacterial spot populations across global production regions, as illustrated by the recombinant strains observed here.IMPORTANCE The pathogens that cause bacterial spot of tomato and pepper are extensively studied models of plant-microbe interactions and cause problematic disease worldwide. Atypical bacterial spot strains collected from tomato in Nigeria, and other strains from Italy, India, and Florida, showed evidence of genomewide recombination that generated genetically distinct pathogenic lineages. The strains from Nigeria and Italy were found to have a mix of type III secretion system genes from X. perforans and X. euvesicatoria, as well as effectors from Xanthomonas gardneri These genes and effectors are important in the establishment of disease, and effectors are common targets of resistance breeding. Our findings point to global diversity in the genomes of bacterial spot pathogens, which is likely to affect the host-pathogen interaction and influence management decisions.

Long read and single molecule DNA sequencing simplifies genome assembly and TAL effector gene analysis of Xanthomonas translucens.

BACKGROUND: The species Xanthomonas translucens encompasses a complex of bacterial strains that cause diseases and yield loss on grass species including important cereal crops. Three pathovars, X. translucens pv. undulosa, X. translucens pv. translucens and X. translucens pv.cerealis, have been described as pathogens of wheat, barley, and oats. However, no complete genome sequence for a strain of this complex is currently available. RESULTS: A complete genome sequence of X. translucens pv. undulosa strain XT4699 was obtained by using PacBio long read, single molecule, real time (SMRT) DNA sequences and Illumina sequences. Draft genome sequences of nineteen additional X. translucens strains, which were collected from wheat or barley in different regions and at different times, were generated by Illumina sequencing. Phylogenetic relationships among different Xanthomonas strains indicates that X. translucens are members of a distinct clade from so-called group 2 xanthomonads and three pathovars of this species, undulosa, translucens and cerealis, represent distinct subclades in the group 1 clade. Knockout mutation of type III secretion system of XT4699 eliminated the ability to cause water-soaking symptoms on wheat and barley and resulted in a reduction in populations on wheat in comparison to the wild type strain. Sequence comparison of X. translucens strains revealed the genetic variation on type III effector repertories among different pathovars or within one pathovar. The full genome sequence of XT4699 reveals the presence of eight members of the Transcription-Activator Like (TAL) effector genes, which are phylogenetically distant from previous known TAL effector genes of group 2 xanthomonads. Microarray and qRT-PCR analyses revealed TAL effector-specific wheat gene expression modulation. CONCLUSIONS: PacBio long read sequencing facilitates the assembly of Xanthomonas genomes and the multiple TAL effector genes, which are difficult to assemble from short read platforms. The complete genome sequence of X. translucens pv. undulosa strain XT4699 and draft genome sequences of nineteen additional X. translucens strains provides a resource for further genetic analyses of pathogenic diversity and host range of the X. translucens species complex. TAL effectors of XT4699 strain play roles in modulating wheat host gene expressions.