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.

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.

Genetic and Functional Diversity Help Explain Pathogenic, Weakly Pathogenic, and Commensal Lifestyles in the Genus Xanthomonas.

The genus Xanthomonas has been primarily studied for pathogenic interactions with plants. However, besides host and tissue-specific pathogenic strains, this genus also comprises nonpathogenic strains isolated from a broad range of hosts, sometimes in association with pathogenic strains, and other environments, including rainwater. Based on their incapacity or limited capacity to cause symptoms on the host of isolation, nonpathogenic xanthomonads can be further characterized as commensal and weakly pathogenic. This study aimed to understand the diversity and evolution of nonpathogenic xanthomonads compared to their pathogenic counterparts based on their cooccurrence and phylogenetic relationship and to identify genomic traits that form the basis of a life history framework that groups xanthomonads by ecological strategies. We sequenced genomes of 83 strains spanning the genus phylogeny and identified eight novel species, indicating unexplored diversity. While some nonpathogenic species have experienced a recent loss of a type III secretion system, specifically the hrp2 cluster, we observed an apparent lack of association of the hrp2 cluster with lifestyles of diverse species. We performed association analysis on a large data set of 337 Xanthomonas strains to explain how xanthomonads may have established association with the plants across the continuum of lifestyles from commensals to weak pathogens to pathogens. Presence of distinct transcriptional regulators, distinct nutrient utilization and assimilation genes, transcriptional regulators, and chemotaxis genes may explain lifestyle-specific adaptations of xanthomonads.

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.

Transcriptome profiling of type VI secretion system core gene tssM mutant of Xanthomonas perforans highlights regulators controlling diverse functions ranging from virulence to metabolism.

IMPORTANCE: T6SS has received attention due to its significance in mediating interorganismal competition through contact-dependent release of effector molecules into prokaryotic and eukaryotic cells. Reverse-genetic studies have indicated the role of T6SS in virulence in a variety of plant pathogenic bacteria, including the one studied here, Xanthomonas. However, it is not clear whether such effect on virulence is merely due to a shift in the microbiome-mediated protection or if T6SS is involved in a complex virulence regulatory network. In this study, we conducted in vitro transcriptome profiling in minimal medium to decipher the signaling pathways regulated by tssM-i3* in X. perforans AL65. We show that TssM-i3* regulates the expression of a suite of genes associated with virulence and metabolism either directly or indirectly by altering the transcription of several regulators. These findings further expand our knowledge on the intricate molecular circuits regulated by T6SS in phytopathogenic bacteria.

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.

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.

Xanthomonas infection and ozone stress distinctly influence the microbial community structure and interactions in the pepper phyllosphere.

While the physiological and transcriptional response of the host to biotic and abiotic stresses have been intensely studied, little is known about the resilience of associated microbiomes and their contribution towards tolerance or response to these stresses. We evaluated the impact of elevated tropospheric ozone (O3), individually and in combination with Xanthomonas perforans infection, under open-top chamber field conditions on overall disease outcome on resistant and susceptible pepper cultivars, and their associated microbiome structure, function, and interaction network across the growing season. Pathogen infection resulted in a distinct microbial community structure and functions on the susceptible cultivar, while concurrent O3 stress did not further alter the community structure, and function. However, O3 stress exacerbated the disease severity on resistant cultivar. This altered diseased severity was accompanied by enhanced heterogeneity in associated Xanthomonas population counts, although no significant shift in overall microbiota density, microbial community structure, and function was evident. Microbial co-occurrence networks under simultaneous O3 stress and pathogen challenge indicated a shift in the most influential taxa and a less connected network, which may reflect the altered stability of interactions among community members. Increased disease severity on resistant cultivar may be explained by such altered microbial co-occurrence network, indicating the altered microbiome-associated prophylactic shield against pathogens under elevated O3. Our findings demonstrate that microbial communities respond distinctly to individual and simultaneous stressors, in this case, O3 stress and pathogen infection, and can play a significant role in predicting how plant-pathogen interactions would change in the face of 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.

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.

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.

Corrigendum: Comparative genomic analysis of the lettuce bacterial leaf spot pathogen, Xanthomonas hortorum pv. vitians, to investigate race specificity.

[This corrects the article DOI: 10.3389/fmicb.2022.840311.].

Natural language processing approach to model the secretion signal of type III effectors.

Type III effectors are proteins injected by Gram-negative bacteria into eukaryotic hosts. In many plant and animal pathogens, these effectors manipulate host cellular processes to the benefit of the bacteria. Type III effectors are secreted by a type III secretion system that must "classify" each bacterial protein into one of two categories, either the protein should be translocated or not. It was previously shown that type III effectors have a secretion signal within their N-terminus, however, despite numerous efforts, the exact biochemical identity of this secretion signal is generally unknown. Computational characterization of the secretion signal is important for the identification of novel effectors and for better understanding the molecular translocation mechanism. In this work we developed novel machine-learning algorithms for characterizing the secretion signal in both plant and animal pathogens. Specifically, we represented each protein as a vector in high-dimensional space using Facebook's protein language model. Classification algorithms were next used to separate effectors from non-effector proteins. We subsequently curated a benchmark dataset of hundreds of effectors and thousands of non-effector proteins. We showed that on this curated dataset, our novel approach yielded substantially better classification accuracy compared to previously developed methodologies. We have also tested the hypothesis that plant and animal pathogen effectors are characterized by different secretion signals. Finally, we integrated the novel approach in Effectidor, a web-server for predicting type III effector proteins, leading to a more accurate classification of effectors from non-effectors.

Comparative Genomic Analysis of the Lettuce Bacterial Leaf Spot Pathogen, Xanthomonas hortorum pv. vitians, to Investigate Race Specificity.

Bacterial leaf spot (BLS) of lettuce caused by Xanthomonas hortorum pv. vitians (Xhv) was first described over 100 years ago and remains a significant threat to lettuce cultivation today. This study investigated the genetic relatedness of the Xhv strains and the possible genetic sources of this race-specific pathogenicity. Whole genome sequences of eighteen Xhv strains representing the three races, along with eight related Xanthomonas strains, were included in the analysis. A maximum likelihood phylogeny based on concatenated whole genome SNPs confirmed previous results describing two major lineages of Xhv strains. Gene clusters encoding secretion systems, secondary metabolites, and bacteriocins were assessed to identify putative virulence factors that distinguish the Xhv races. Genome sequences were mined for effector genes, which have been shown to be involved in race specificity in other systems. Two effectors identified in this study, xopAQ and the novel variant xopAF2, were revealed as possible mediators of a gene-for-gene interaction between Xhv race 1 and 3 strains and wild lettuce Lactuca serriola ARM-09-161-10-1. Transposase sequence identified downstream of xopAF2 and prophage sequence found nearby within Xhv race 1 and 3 insertion sequences suggest that this gene may have been acquired through phage-mediated gene transfer. No other factors were identified from these analyses that distinguish the Xhv races.

Phylogenetic Distribution and Evolution of Type VI Secretion System in the Genus Xanthomonas.

The type VI secretion system (T6SS) present in many Gram-negative bacteria is a contact-dependent apparatus that can directly deliver secreted effectors or toxins into diverse neighboring cellular targets including both prokaryotic and eukaryotic organisms. Recent reverse genetics studies with T6 core gene loci have indicated the importance of functional T6SS toward overall competitive fitness in various pathogenic Xanthomonas spp. To understand the contribution of T6SS toward ecology and evolution of Xanthomonas spp., we explored the distribution of the three distinguishable T6SS clusters, i3*, i3***, and i4, in approximately 1,740 Xanthomonas genomes, along with their conservation, genetic organization, and their evolutionary patterns in this genus. Screening genomes for core genes of each T6 cluster indicated that 40% of the sequenced strains possess two T6 clusters, with combinations of i3*** and i3* or i3*** and i4. A few strains of Xanthomonas citri, Xanthomonas phaseoli, and Xanthomonas cissicola were the exception, possessing a unique combination of i3* and i4. The findings also indicated clade-specific distribution of T6SS clusters. Phylogenetic analysis demonstrated that T6SS clusters i3* and i3*** were probably acquired by the ancestor of the genus Xanthomonas, followed by gain or loss of individual clusters upon diversification into subsequent clades. T6 i4 cluster has been acquired in recent independent events by group 2 xanthomonads followed by its spread via horizontal dissemination across distinct clades across groups 1 and 2 xanthomonads. We also noted reshuffling of the entire core T6 loci, as well as T6SS spike complex components, hcp and vgrG, among different species. Our findings indicate that gain or loss events of specific T6SS clusters across Xanthomonas phylogeny have not been random.

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.