Bacterial Mutation During Seasonal Epidemics.

Rapidly evolving bacterial pathogens pose a unique challenge for long-term plant disease management. In this study, we investigated the types and rate of mutations in bacterial populations during seasonal disease epidemics. Two phylogenetically distinct strains of the bacterial spot pathogen, Xanthomonas perforans, were marked, released in tomato fields, and recaptured at several time points during the growing season. Genomic variations in recaptured isolates were identified by comparative analysis of their whole-genome sequences. In total, 180 unique variations (116 substitutions, 57 insertions/deletions, and 7 structural variations) were identified from 300 genomes, resulting in the overall host-associated mutation rate of ∼0.3 to 0.9/genome/week. This result serves as a benchmark for bacterial mutation during epidemics in similar pathosystems. [Formula: see text] Copyright © 2024 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

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.

Whole-Genome-Sequence-Based Classification of Xanthomonas euvesicatoria pv. eucalypti and Computational Analysis of the Type III Secretion System.

Xanthomonas spp. infect a wide range of annual and perennial plants. Bacterial blight in young seedlings of Eucalyptus spp. in Indonesia was originally identified as X. perforans. However, these strains failed to elicit a hypersensitive response (HR) on either tomatoes or peppers. Two of the strains, EPK43 and BCC 972, when infiltrated into tomato and pepper leaves, failed to grow to significant levels in comparison with well-characterized X. euvesicatoria pv. perforans (Xp) strains. Furthermore, spray inoculation of 'Bonny Best' tomato plants with a bacterial suspension of the Eucalyptus strains resulted in no obvious symptoms. We sequenced the whole genomes of eight strains isolated from two Eucalyptus species between 2007 and 2015. The strains had average nucleotide identities (ANIs) of at least 97.8 with Xp and X. euvesicatoria pv. euvesicatoria (Xeu) strains, both of which are causal agents of bacterial spot of tomatoes and peppers. A comparison of the Eucalyptus strains revealed that the ANI values were >99.99% with each other. Core genome phylogeny clustered all Eucalyptus strains with X. euvesicatoria pv. rosa. They formed separate clades, which included X. euvesicatoria pv. alangii, X. euvesicatoria pv. citrumelonis, and X. euvesicatoria pv. alfalfae. Based on ANI, phylogenetic relationships, and pathogenicity, we designated these Eucalyptus strains as X. euvesicatoria pv. eucalypti (Xee). Comparative analysis of sequenced strains provided unique profiles of type III secretion effectors. Core effector XopD, present in all pathogenic Xp and Xeu strains, was absent in the Xee strains. Comparison of the hrp clusters of Xee, Xp, and Xeu genomes revealed that HrpE in Xee strains was very different from that in Xp and Xeu. To determine if it was functional, we deleted the gene and complemented with the Xee hrpE, confirming it was essential for secretion of type III effectors. HrpE has a hypervariable N-terminus in Xanthomonas spp., in which the N-terminus of Xee strains differs significantly from those of Xeu and Xp strains.

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.

Phenotypic and genetic diversity of xanthomonads isolated from pepper (Capsicum spp.) in Taiwan from 1989 to 2019.

Bacterial spot caused by Xanthomonas spp. is an economically important disease of pepper causing significant yield losses in Taiwan. Monitoring the pathogen population on a continuous basis is necessary for developing disease management strategies. We analyzed a collection of xanthomonad strains isolated from pepper in Taiwan between 1989 and 2019. Among the sequenced genomes, sixty-five were identified as Xanthomonas euvesicatoria and ten were X. perforans. Thirty-five X. euvesicatoria and ten X. perforans strains were copper tolerant, whereas only five X. euvesicatoria and none of the X. perforans strains were tolerant to streptomycin. Nine X. euvesicatoria strains were amylolytic, which is considered an unusual characteristic for X. euvesicatoria. Bayesian analysis of the population structure based on core gene SNPs clustered the strains into five clusters for X. euvesicatoria and three clusters for X. perforans. One X. perforans cluster, designated as TP-2019, appears to be a novel genetic cluster based on core genes, accessory gene content, and effector profile. This knowledge of pathogen diversity with whole genomic information will be useful in future comparative studies and in improving breeding programs to develop disease-resistant cultivars and other disease management options.

Curtobacterium allii sp. nov., the actinobacterial pathogen causing onion bulb rot.

A Gram-stain-positive, aerobic, and non-spore-forming bacterial strain, 20TX0166T, was isolated from a diseased onion bulb in Texas, USA. Upon testing its pathogenicity on onion bulb, it produced pathogenic response which makes it first species of pathogen belonging to the phylum actinobacteria detected in onion. Phylogenetic analysis of the 16S rRNA gene sequence revealed that the strain belonged to the genus Curtobacterium and was most similar to Curtobacterium flaccumfaciens LMG 3645T (100%), C. pusillum DSM 20527T (99.5%), and C. oceanosedimentum ATCC 31317T (99.5%). The estimated genome size of the novel species was 4.0 Mbp with a G + C content of 70.8%. The orthologous ANI (orthoANIu), ANI based on blast (ANIb), and dDDH values between the novel strain and the closest relative, C. flaccumfaciens LMG 3645T, were 95.7%, 95.4%, and 63.3%, respectively. These values were below the recommended species cut-off threshold of 96% (ANI) and 70% (dDDH), suggesting the strain may be a novel species. Physiologic and phenotypic characters of this novel strain were also unique when compared with the closely related species. The major cellular fatty acids of this strain were anteiso-C15:0 and anteiso-C17:0. Using a polyphasic approach based on phenotypic and genotypic analyses, strain 20TX0166T represents a novel species of the genus Curtobacterium, and the name Curtobacterium allii sp. nov. is proposed. The type strain is 20TX0166T (= LMG 32517T = CIP112023T = NCIMB 15427T).

Pseudomonas uvaldensis sp. nov., a bacterial pathogen causing onion bulb rot.

A Gram-stain-negative, aerobic and non-spore-forming bacterial strain, designated 20TX0172T, was isolated from a rotting onion bulb in Texas, USA. The results of phylogenetic analysis based on the 16S rRNA sequence indicated that the novel strain represented a member of the genus Pseudomonas and had the greatest sequence similarities with Pseudomonas kilonensis 520-20T (99.3 %), Pseudomonas corrugata CFBP 2431T (99.2 %), and Pseudomonas viciae 11K1T (99.2 %) but the 16S rRNA phylogenetic tree displayed a monophyletic clade with Pseudomonas mediterranea CFBP 5447T. In the phylogenetic trees based on sequences of four housekeeping genes (gap1, gltA, gyrB and rpoD), the novel strain formed a separate branch, indicating that the strain was distinct phylogenetically from known species of the genus Pseudomonas. The genome-sequence-derived average nucleotide identity (ANI) and digital DNA-DNA hybridization (dDDH) values between the novel isolate and P. mediterranea DSM 16733T were 86.7 and 32.7 %, respectively. These values were below the accepted species cutoff threshold of 96 % ANI and 70 % dDDH, affirming that the strain represented a novel species. The genome size of the novel species was 5.98 Mbp with a DNA G+C content of 60.8 mol%. On the basis of phenotypic and genotypic characteristics, strain 20TX0172T represents a novel species of the genus Pseudomonas. The name Pseudomonas uvaldensis sp. nov. is proposed. The type strain is 20TX0172T (=NCIMB 15426T=CIP 112022T).

Pseudomonas californiensis sp. nov. and Pseudomonas quasicaspiana sp. nov., isolated from ornamental crops in California.

Five bacterial strains were isolated from symptomatic leaves of Achillea millefolium, Delphinium sp. and Hydrangea sp. in California. Colonies isolated on King's medium B (KMB) appeared white, mucoid and round, similar to Pseudomonas species. Phylogenetic analyses based on 16S rRNA, rpoB, rpoD and gyrB genes placed the bacteria into three distinct groups within Pseudomonas that were most closely related to Pseudomonas viridiflava, Pseudomonas cichorii or Pseudomonas caspiana. To further characterize the strains, phenotypic analyses and the following tests were performed: fatty acid methyl ester composition, LOPAT, fluorescence on KMB, Biolog assay, and transmission electron microscopy. Finally, whole genome sequencing of the strains was conducted, and the sequences were compared with reference genomes of Pseudomonas species based on average nucleotide identity (ANI). The first group, which consists of three strains isolated from delphinium, hydrangea and achillea, had 95.6-96.9 % pairwise ANI between each other; the second group consists of two strains isolated from delphinium that had 100 % pairwise ANI. Although comparisons of the two groups with publicly available genomes revealed closest relationships with P. viridiflava (91.6 %), P. caspiana (88.3 %) and P. asturiensis (86.7 %), ANI values were less than 95 % compared to all validly published pseudomonads. Combining genomic and phenotypic data, we conclude that these strains represent two new species and the names proposed are Pseudomonas quasicaspiana sp. nov. (type strain DSMZ 11 30 42T=LMG 32 434T) for the strains isolated from delphinium, achillea and hydrangea and Pseudomonas californiensis sp. nov. (DSMZ 11 30 43T=LMG 32 432T) for the two strains isolated from delphinium. The specific epithets quasicaspiana and californiensis were selected based on the close phylogenetic relationship of strains with P. caspiana and on the geographic location of isolation, respectively.

A Long-Amplicon Viability-qPCR Test for Quantifying Living Pathogens that Cause Bacterial Spot in Tomato Seed.

Bacterial spot is one of the most serious diseases of tomato. It is caused by four species of Xanthomonas: X. euvesicatoria, X. gardneri, X. perforans, and X. vesicatoria. Contaminated or infected seed can be a major source of inoculum for this disease. The use of certified pathogen-free seed is one of the primary management practices to reduce the inoculum load in commercial production. Current seed testing protocols rely mainly on plating the seed extract and conventional PCR; however, the plating method cannot detect viable but nonculturable cells, and the conventional PCR assay has limited capability to differentiate DNA extracted from viable or dead bacterial cells. To improve the sensitivity and specificity of the tomato seed testing method for bacterial spot pathogens, a long-amplicon quantitative PCR (qPCR) assay coupled with propidium monoazide (PMA-qPCR) was developed to quantify selectively the four pathogenic Xanthomonas species in tomato seed. The optimized PMA-qPCR procedure was evaluated on pure bacterial suspensions, bacteria-spiked seed extracts, and seed extracts of inoculated and naturally infected seed. A crude DNA extraction protocol also was developed, and PMA-qPCR with crude bacterial DNA extracts resulted in accurate quantification of 104 to 108 CFU/ml of viable bacteria when mixed with dead cells at concentrations as high as 107 CFU/ml in the seed extracts. With DNA purified from concentrated seed extracts, the PMA-qPCR assay was able to detect DNA of the target pathogens in seed samples spiked with ≥75 CFU/ml (about 0.5 CFU/seed) of the viable pathogens. Latent class analysis of the inoculated and naturally infected seed samples showed that the PMA-qPCR assay had greater sensitivity than plating the seed extracts on the semiselective modified Tween Medium B and CKTM media for all four target species. Being much faster and more sensitive than dilution plating, the PMA-qPCR assay has potential to be used as a standalone tool or in combination with the plating method to improve tomato seed testing and advance the production of clean seed.

Need for speed: bacterial effector XopJ2 is associated with increased dispersal velocity of Xanthomonas perforans.

Bacterial spot caused by Xanthomonas perforans (Xp) is an economically important disease in tomato. Previous studies have shown that the recently isolated Xp strains have acquired and retained the effector gene, xopJ2, which has been reported to increase fitness of the pathogen in the field. To elucidate the fitness benefit of xopJ2, we quantified the effect of xopJ2 on the dispersal and evolution of Xp populations on tomato. We compared movement of two wild-type Xp strains expressing xopJ2 to their respective xopJ2 mutants when co-inoculated in the field. We developed a binary logistic model to predict the presence of Xp over spatial and temporal dimensions with or without xopJ2. Based on the model, wild-type bacteria were dispersed approximately three times faster than the xopJ2 mutants. In a simulation experiment, the selective advantage due to increased dispersal velocity led to an increase in the frequency of xopJ2 gene in the Xp population and its apparent fixation within 10 to 12 cropping seasons of the tomato crop. Our results show that the presence of a single gene can affect the dispersal of a bacterial pathogen and significantly alter its population dynamics.

Assessing Changes and Associations in the Xanthomonas perforans Population Across Florida Commercial Tomato Fields Via a Statewide Survey.

Before 1991, Xanthomonas euvesicatoria was the causal agent of bacterial spot of tomato in Florida but was quickly replaced by X. perforans. The X. perforans population has changed in genotype and phenotype despite lack of a clear selection pressure. To determine the current Xanthomonas population in Florida, we collected 585 Xanthomonas strains from 70 tomato fields, representing 22 farms across eight counties, in the Florida tomato production region. Strains were isolated from 23 cultivars across eight seed producers and were associated with eight transplant facilities during the fall 2017 season. Our collection was phenotypically and genotypically characterized. Only X. perforans was identified, and all strains except one (99.8%) were tolerant to copper sulfate and 25% of strains were resistant to streptomycin sulfate. Most of the strains (99.3%) that were resistant to streptomycin sulfate were sequence type 1. The X. perforans population consisted of tomato races 3 (8%) and 4 (92%) and all three previously reported sequence types, ranging from 22 to 46% frequency. Approximately half of all strains, none of which were sequence type 2, produced bacteriocins against X. euvesicatoria. Effector profiles were highly variable among strains, which could impact the strains' host range. The effector xopJ4, which was previously thought to be conserved in X. perforans tomato pathogens, was absent in 19 strains. Nonmetric multidimensional scaling and network analyses show how strains and strain traits were associated with production system variables, including anonymized farms and transplant facilities. These analyses show that the composition of the Florida X. perforans population is diverse and complex.

Widespread QoI Fungicide Resistance Revealed Among Corynespora cassiicola Tomato Isolates in Florida.

Target spot of tomato caused by Corynespora cassiicola is one of the most economically destructive diseases of tomato in Florida. A collection of 123 isolates from eight counties in Florida were evaluated for sensitivity to azoxystrobin and fenamidone based on mycelial growth inhibition (MGI), spore germination (SG), detached leaflet assays (DLAs), and sequence-based analysis of the cytochrome b gene (cytb). Cleavage of cytb by restriction enzyme (Fnu4HI) revealed the presence of a mutation conferring a glycine (G) to alanine (A) mutation at amino acid position 143 (G143A) in approximately 90% of the population, correlating with quinone outside inhibitor (QoI) resistance based on MGI (<40% at 5 µg/ml), SG (<50% at 1 and 10 µg/ml), and DLA (<10% severity reduction). The mutation conferring a phenylalanine (F) to leucine (L) substitution at position 129 (F129L) was confirmed in moderately resistant isolates (#9, #19, and #74) based on MGI (40 to 50% at 5 µg/ml), SG (<50% at 1 µg/ml and >50% at 10 µg/ml), and DLA (>10% and <43% severity reduction) for both QoI fungicides, whereas sensitive isolates (#1, #4, #7, #28, #29, #46, #61, #74, #75, #76, #91, #95, and #118) based on MGI (>50% at 5 µg/ml), SG (>50% at 1 µg/ml and 10 µg/ml), and DLA (>50% severity reduction) correlated to non-mutation-containing isolates or those with a silent mutation. This study indicates that QoI resistance among C. cassiicola isolates from tomato is widespread in Florida and validates rapid screening methods using MGI or molecular assays to identify resistant isolates in future studies.

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.

Phenotypic and Genetic Diversity of Xanthomonas perforans Populations from Tomato in North Carolina.

Bacterial spot caused by Xanthomonas spp. is one of the most devastating diseases of tomato in North Carolina (NC). In total, 290 strains of Xanthomonas spp. from tomato in NC collected over 2 years (2015 and 2016) were analyzed for phenotypic and genetic diversity. In vitro copper and streptomycin sensitivity assays revealed that >95% (n = 290) of the strains were copper tolerant in both years, whereas 25% (n = 127) and 46% (n = 163) were streptomycin tolerant in 2016 and 2015, respectively. Using BOX repetitive element PCR assay, fingerprint patterns showed four haplotypes (H1, H2, H3, and H4) among the strains analyzed. The multiplex real-time quantitative PCR on a subset of representative strains (n = 45) targeting the highly conserved hrcN gene identified Xanthomonas strains from tomato in NC that belonged to X. perforans. Race profiling of the representative strains (n = 45) on tomato and pepper differentials confirmed that ∼9 and 91% of strains are tomato races T3 and T4, respectively. Additionally, PCR assays and sequence alignments confirmed that the copL, copA, copB (copLAB copper tolerance gene cluster), and avrXv4 genes are present in the strains analyzed. Phylogenetic and comparative sequence analyses of six genomic regions (elongation factor G [fusA], glyceraldehyde-3-phosphate dehydrogenase A [gapA], citrate synthase [gltA], gyrase subunit B [gyrB], ABC transporter sugar permease [lacF], and GTP binding protein [lepA]) suggested that 13 and 74% of X. perforans strains from NC were genetically similar to races T3 and T4 from Florida, respectively. Our results provide insights that bacterial spot management practices in tomato should focus on deploying resistance genes to combat emerging pathogenic races of X. perforans and overcome the challenges currently posed by intense use of copper-based bactericides.

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.

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.

The Type III Effector AvrBsT Enhances Xanthomonas perforans Fitness in Field-Grown Tomato.

Type III secretion system effectors contribute to pathogenicity through various mechanisms. Recent surveys showed an increasing prevalence of the type III secretion effector avrBsT among Xanthomonas perforans strains. We hypothesized that the acquisition of avrBsT has a fitness advantage for the pathogen. The contribution of avrBsT to fitness on tomato was evaluated based on disease severity, in planta growth, competition, and recovery rates of wild-type (WT) and avrBsT mutant strains in greenhouse and field plants. GEV872 and GEV1001, representative strains of two phylogenomic groups of X. perforans, were selected for generating avrBsT mutants. Disease severity was higher for WT strains compared with the avrBsT mutant strains. X. perforans WT and avrBsT mutant strains did not differ following leaf infiltration of greenhouse plants in direct competition and in planta growth assays. The effect of avrBsT on pathogen fitness was noticeable under field conditions. Differences in strain recovery were significant, with WT being recovered two to eight times more than avrBsT mutant strains in the case of both strains GEV872 and GEV1001. WT strains were capable of spreading longer distances across field plots compared with avrBsT mutant strains. Findings suggest that the functional AvrBsT affects the fitness of X. perforans under field conditions, making it an ideal candidate for bacterial spot resistance breeding efforts in tomato.

Bacterial Gall of Loropetalum chinense caused by Pseudomonas amygdali pv. loropetali pv. nov.

In 2012, stem gall samples on Loropetalum chinense were sent to Florida diagnostic labs from Alabama and Florida nurseries. A fluorescent pseudomonad was consistently isolated from the galls. The organism was originally identified in Alabama based on 16S rRNA sequencing as Pseudomonas savastanoi, which causes a production-limiting disease of olive. The loropetalum strains and reference strains were compared using LOPAT, Biolog, fatty acid analysis, multilocus sequence analysis (MLSA), and pathogenicity tests. The LOPAT tests placed the loropetalum strains within Pseudomonas syringae. Biolog and fatty acid analysis placed the strains in various pathovars of P. syringae and P. savastanoi, respectively. MLSA of a set of housekeeping genes separated the loropetalum strains from the olive knot-inducing strains. Our work indicates there is a need to use more tests than 16S rRNA to accurately diagnose new bacterial diseases. In pathogenicity tests, the loropetalum strains produced galls only on loropetalum, but not on olive, mandevilla, or almond, indicating this strain is not a threat to the olive industry. Based on the pathogenicity assays and molecular tests, loropetalum strains represent a distinct and new pathovar, P. amygdali pv. loropetali pv. nov., for which the strain PDC13-208 (= DSMZ 105780PT) has been designated as the pathotype 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.

Analysis of Sequenced Genomes of Xanthomonas perforans Identifies Candidate Targets for Resistance Breeding in Tomato.

Bacterial disease management is a challenge for modern agriculture due to rapid changes in pathogen populations. Genome sequences for hosts and pathogens provide detailed information that facilitates effector-based breeding strategies. Tomato genotypes have gene-for-gene resistance to the bacterial spot pathogen Xanthomonas perforans. The bacterial spot populations in Florida shifted from tomato race 3 to 4, such that the corresponding tomato resistance gene no longer recognizes the effector protein AvrXv3. Genome sequencing showed variation in effector profiles among race 4 strains collected in 2006 and 2012 and compared with a race 3 strain collected in 1991. We examined variation in putative targets of resistance among Florida strains of X. perforans collected from 1991 to 2006. Consistent with race change, avrXv3 was present in race 3 strains but nonfunctional in race 4 strains due to multiple independent mutations. Effectors xopJ4 and avrBs2 were unchanged in all strains. The effector avrBsT was absent in race 3 strains collected in the 1990s but present in race 3 strains collected in 2006 and nearly all race 4 strains. These changes in effector profiles suggest that xopJ4 and avrBsT are currently the best targets for resistance breeding against bacterial spot in tomato.