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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, 65 were identified as Xanthomonas euvesicatoria , and 10 were X. perforans . Thirty-five X. euvesicatoria and 10 X. perforans strains were copper tolerant, whereas only four 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 single-nucleotide polymorphisms 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., Competing Interests: The author(s) declare no conflict of interest.

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., Competing Interests: The author(s) declare no conflict of interest.

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., Competing Interests: The author(s) declare no conflict of interest.

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.

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.

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.

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.

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.