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.

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 centenary for bacterial spot of tomato and pepper.

DISEASE SYMPTOMS: Symptoms include water-soaked areas surrounded by chlorosis turning into necrotic spots on all aerial parts of plants. On tomato fruits, small, water-soaked, or slightly raised pale-green spots with greenish-white halos are formed, ultimately becoming dark brown and slightly sunken with a scabby or wart-like surface. HOST RANGE: Main and economically important hosts include different types of tomatoes and peppers. Alternative solanaceous and nonsolanaceous hosts include Datura spp., Hyoscyamus spp., Lycium spp., Nicotiana rustica, Physalis spp., Solanum spp., Amaranthus lividus, Emilia fosbergii, Euphorbia heterophylla, Nicandra physaloides, Physalis pubescens, Sida glomerata, and Solanum americanum. TAXONOMIC STATUS OF THE PATHOGEN: Domain, Bacteria; phylum, Proteobacteria; class, Gammaproteobacteria; order, Xanthomonadales; family, Xanthomonadaceae; genus, Xanthomonas; species, X. euvesicatoria, X. hortorum, X. vesicatoria. SYNONYMS (NONPREFERRED SCIENTIFIC NAMES): Bacterium exitiosum, Bacterium vesicatorium, Phytomonas exitiosa, Phytomonas vesicatoria, Pseudomonas exitiosa, Pseudomonas gardneri, Pseudomonas vesicatoria, Xanthomonas axonopodis pv. vesicatoria, Xanthomonas campestris pv. vesicatoria, Xanthomonas cynarae pv. gardneri, Xanthomonas gardneri, Xanthomonas perforans. MICROBIOLOGICAL PROPERTIES: Colonies are gram-negative, oxidase-negative, and catalase-positive and have oxidative metabolism. Pale-yellow domed circular colonies of 1-2 mm in diameter grow on general culture media. DISTRIBUTION: The bacteria are widespread in Africa, Brazil, Canada and the USA, Australia, eastern Europe, and south-east Asia. Occurrence in western Europe is restricted. PHYTOSANITARY CATEGORIZATION: A2 no. 157, EU Annex designation II/A2. EPPO CODES: XANTEU, XANTGA, XANTPF, XANTVE.

Xanthomonas diversity, virulence and plant-pathogen interactions.

Xanthomonas spp. encompass a wide range of plant pathogens that use numerous virulence factors for pathogenicity and fitness in plant hosts. In this Review, we examine recent insights into host-pathogen co-evolution, diversity in Xanthomonas populations and host specificity of Xanthomonas spp. that have substantially improved our fundamental understanding of pathogen biology. We emphasize the virulence factors in xanthomonads, such as type III secreted effectors including transcription activator-like effectors, type II secretion systems, diversity resulting in host specificity, evolution of emerging strains, activation of susceptibility genes and strategies of host evasion. We summarize the genomic diversity in several Xanthomonas spp. and implications for disease outbreaks, management strategies and breeding for disease resistance.

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.

Corrigendum: Inference of Convergent Gene Acquisition Among Pseudomonas syringae Strains Isolated From Watermelon, Cantaloupe, and Squash.

[This corrects the article DOI: 10.3389/fmicb.2019.00270.].

Inference of Convergent Gene Acquisition Among Pseudomonas syringae Strains Isolated From Watermelon, Cantaloupe, and Squash.

Pseudomonas syringae sensu stricto (phylogroup 2; referred to as P. syringae) consists of an environmentally ubiquitous bacterial population associated with diseases of numerous plant species. Recent studies using multilocus sequence analysis have indicated the clonal expansion of several P. syringae lineages, located in phylogroups 2a and 2b, in association with outbreaks of bacterial spot disease of watermelon, cantaloupe, and squash in the United States. To investigate the evolutionary processes that led to the emergence of these epidemic lineages, we sequenced the genomes of six P. syringae strains that were isolated from cucurbits grown in the United States, Europe, and China over a period of more than a decade, as well as eight strains that were isolated from watermelon and squash grown in six different Florida counties during the 2013 and 2014 seasons. These data were subjected to comparative analyses along with 42 previously sequenced genomes of P. syringae stains collected from diverse plant species and environments available from GenBank. Maximum likelihood reconstruction of the P. syringae core genome revealed the presence of a hybrid phylogenetic group, comprised of cucurbit strains collected in Florida, Italy, Serbia, and France, which emerged through genome-wide homologous recombination between phylogroups 2a and 2b. Functional analysis of the recombinant core genome showed that pathways involved in the ATP-dependent transport and metabolism of amino acids, bacterial motility, and secretion systems were enriched for recombination. A survey of described virulence factors indicated the convergent acquisition of several accessory type 3 secreted effectors (T3SEs) among phylogenetically distinct lineages through integrative and conjugative element and plasmid loci. Finally, pathogenicity assays on watermelon and squash showed qualitative differences in virulence between strains of the same clonal lineage, which correlated with T3SEs acquired through various mechanisms of horizontal gene transfer (HGT). This study provides novel insights into the interplay of homologous recombination and HGT toward pathogen emergence and highlights the dynamic nature of P. syringae sensu lato genomes.

Pseudomonas floridensis sp. nov., a bacterial pathogen isolated from tomato.

An unusual fluorescent pseudomonad was isolated from tomato exhibiting leaf spot symptoms similar to bacterial speck. Strains were fluorescent, oxidase- and arginine-dihydrolase-negative, elicited a hypersensitive reaction on tobacco and produced a soft rot on potato slices. However, the strains produced an unusual yellow, mucoid growth on media containing 5 % sucrose that is not typical of levan. Based on multilocus sequence analysis using 16S rRNA, gap1, gltA, gyrB and rpoD, these strains formed a distinct phylogenetic group in the genus Pseudomonas and were most closely related to Pseudomonas viridiflava within the Pseudomonassyringae complex. Whole-genome comparisons, using average nucleotide identity based on blast, of representative strain GEV388T and publicly available genomes representing the genus Pseudomonas revealed phylogroup 7 P. viridiflava strain UASW0038 and P. viridiflava type strain ICMP 2848T as the closest relatives with 86.59 and 86.56 % nucleotide identity, respectively. In silico DNA-DNA hybridization using the genome-to-genome distance calculation method estimated 31.1 % DNA relatedness between GEV388T and P. viridiflava ATCC 13223T, strongly suggesting the strains are representatives of different species. These results together with Biolog GEN III tests, fatty acid methyl ester profiles and phylogenetic analysis using 16S rRNA and multiple housekeeping gene sequences demonstrated that this group represents a novel species member of the genus Pseudomonas. The name Pseudomonas floridensis sp. nov. is proposed with GEV388T (=LMG 30013T=ATCC TSD-90T) as the type strain.

A Novel Phylogroup of Pseudomonas cichorii Identified Following an Unusual Disease Outbreak on Tomato.

Recently, in Central Florida tomato production fields, tomato foliage and fruit were observed with symptoms similar to bacterial speck. Fluorescent pseudomonads were consistently isolated and the strains were characterized by standard LOPAT tests, pathogenicity tests, and genetic characterization using 16S ribosomal RNA (rRNA) sequences and multilocus sequence analysis (MLSA) of conserved housekeeping genes. LOPAT test results indicated that the strains were likely Pseudomonas cichorii. These strains were pathogenic on tomato and were also pathogenic on lettuce, the host for the type strain of P. cichorii. Likewise, strains of P. cichorii isolated in Florida since the early 1980s from hosts other than tomato, along with the type strain, were also pathogenic on tomato. Genetic characterization using 16S rRNA and MLSA confirmed that the strains were most closely related to P. cichorii but varied significantly from the type strain. The Florida P. cichorii strains formed a separate phylogenetic group along with P. cichorii strains isolated from tomato in Tanzania. These strains were different from the previously described morphotypes and genomovars of P. cichorii. Our results indicate the presence of a genetically distinct group of multihost pathogenic P. cichorii strains that have been present in Florida since at least the early 1980s.

Genetic Characterization of Didymella bryoniae Isolates Infecting Watermelon and Other Cucurbits in Florida and Georgia.

Gummy stem blight caused by Didymella bryoniae (anamorph Phoma cucurbitacearum) is a major fungal disease of watermelon (Citrullus lanatus) and other cucurbits. Thirty-five isolates of Didymella and Phoma spp. associated with symptoms of gummy stem blight on watermelon, Canary melon (Cucumis melo), muskmelon (C. melo), and winter squash (Cucurbita maxima) from Florida and Georgia were characterized based on morphology on agar media, pathogenicity on 'Melody' watermelon, the internal transcribed spacer (ITS) sequence of ribosomal DNA (rDNA), random amplified polymorphic DNA (RAPD) analysis, and polymerase chain reaction (PCR) restriction fragment length polymorphism (RFLP) analysis. All of the isolates were pathogenic on watermelon but differed in virulence. RAPD and ITS sequence analysis indicated genetic variability among the isolates but PCR-RFLP analysis did not show any variability. ITS sequence phylogenetic analysis identified two isolates, DB-05 and DB-33, which had a greater identity to that of D. bryoniae isolates from China (98 to 100% sequence homology) than other isolates from Florida and Georgia (95 to 98%). These two isolates possessed a single nucleotide substitution of A to G at position 131 of the ITS1 region. The study characterized the genetic profile of a collection of D. bryoniae isolates from Florida and Georgia in relation to isolates from other U.S. states and countries.