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Xanthomonas citri pv. citri (Xcc) causes the devastating citrus canker disease. Xcc is known to have been introduced into Florida, USA in at least three different events in 1915, 1986 and 1995 with the first two claimed to be eradicated. It was questioned whether the Xcc introduction in 1986 has been successfully eradicated. Furthermore, it is unknown how Xcc has spread throughout the citrus groves in Florida. In this study, we investigated the population structure of Xcc to address these questions. We sequenced the whole genome of 343 Xcc strains collected from Florida groves between 1997 and 2016. Our analysis revealed two distinct clusters of Xcc. Our data strongly indicate that the claimed eradication of the 1986 Xcc introduction was not successful and Xcc strains from 1986 introduction were present in samples from at least 8 counties collected after 1994. Importantly, our data revealed that the Cluster 2 strains, which are present in all 20 citrus-producing counties sampled in Florida, originated from the Xcc introduction event in the Miami area in 1995. Our data suggest that Polk County is the epicenter of the dispersal of Cluster 2 Xcc strains, which is consistent with the fact that three major hurricanes passed through Polk County in 2004. As copper-based products have been extensively used to control citrus canker, we also investigated whether Xcc strains have developed resistance to copper. Notably, none of the 343 strains contained known copper resistance genes. Twenty randomly selected Xcc strains displayed sensitivity to copper. Overall, this study provides valuable insights into the introduction, eradication, spread, and copper resistance of Xcc in Florida. Author summary: Sequencing and analyzing the genomes of 343 Xcc strains collected from 1997 to 2016 have enabled us to gain a deeper understanding of its introduction, spread, and eradication. This information is critical for optimizing strategies for responding to the invasion of quarantine pathogens. We have conclusively demonstrated that the Xcc introduction in 1986 was not successfully eradicated, three major hurricanes in 2004 play a critical role in spreading Xcc throughout Florida, and Polk County is the epicenter of Xcc dispersal. Additionally, none of the tested Xcc strains displayed resistance to copper, consistent with the absence of copper resistance genes in their genome. [ABSTRACT FROM AUTHOR]

Eastern gamagrass (Tripsacum dactyloides (L.) L. [Poaceae]) is a native bunchgrass that once occurred throughout most of the US and northern Mexico. Considered a high-quality forage crop for livestock by early settlers, its importance declined due to overgrazing and land conversion to crop fields. There has been a renewed interest in the species as a forage and for conservation and restoration purposes. The USDA NRCS Plant Materials Program has focused on evaluation of eastern gamagrass ecotypes, and several cultivars or germplasm have been released from various Plant Material Centers (PMC) in cooperation with other state and federal programs based primarily on material collected from Arkansas, Kansas, Oklahoma, Mississippi, Tennessee, and Texas. The only Florida eastern gamagrass ecotypes, St Lucie Germplasm and Martin Germplasm, were collected from St Lucie and Martin Counties in Florida, respectively, and were released in 2000 as vegetatively propagated ornamental types for the Florida nursery industry. Interest in a Florida ecotype eastern gamagrass for wildlife habitat and other conservation uses was addressed with the further evaluation of more than 70 Florida gamagrass accessions. As a result, Sunrise Germplasm (tested as accession 9059266) was identified for joint release by the USDA NRCS Brooksville PMC and University of Florida, Institute of Food and Agricultural Sciences Extension. [ABSTRACT FROM AUTHOR]

Bacterial spot of tomato continues to pose a significant problem to tomato production worldwide. In Florida, bacterial spot of tomato caused by Xanthomonas perforans is one of the most important diseases responsible for tomato yield loss. This disease is difficult to control, and new strategies are continually being investigated to combat the devastating effect of this disease. Recent efforts focusing on essential oils based on small molecules have spurred interests in the utilization of this class of chemicals for disease management. In this study, we evaluated the efficacy of eugenol for the management of bacterial spot of tomato caused by X. perforans. In the greenhouse experiments, eugenol applied as a foliar spray significantly (p < 0.5) reduced bacterial spot disease compared to the untreated control. In the field experiments, the area under the disease progress curve (AUDPC) was significantly (p < 0.5) lower in the plots treated with eugenol or eugenol combined with the surfactant Cohere than in the untreated control plots, and it was comparable to the copper-based treatments. To provide additional insights into the possible pathways of eugenol activities, we applied a liquid chromatography mass spectrometry (LC-MS)-based metabolomic study using a thermo Q-Exactive orbitrap mass spectrometer with Dionex ultra high-performance liquid chromatography (UHPLC) on X. perforans strain 91–118 treated with eugenol. Our results showed that eugenol affected metabolite production in multiple pathways critical to bacterial survival. For example, treatment of cells with eugenol resulted in the downregulation of the glutathione metabolism pathway and associated metabolites, except for 5-oxoproline, which accumulation is known to be toxic to living cells. While the peaks corresponding to the putatively identified sarmentosin showed the most significant impact and reduced in response to eugenol treatment, branched-chain amino acids, such as L-isoleucine, increased in production, suggesting that eugenol may not negatively affect the protein biosynthesis pathways. The results from our study demonstrated the efficacy of eugenol in the management of bacterial spot of tomato under greenhouse and field conditions and identified multiple pathways that are targeted. [ABSTRACT FROM AUTHOR]

Changes in Xanthomonas race and species composition causing bacterial spot of tomato have occurred throughout the world and are often associated with epidemics. Knowledge of bacterial population structure is key for resistance discovery and deployment. We surveyed Xanthomonas spp. composition from processing tomato fields in the Midwestern United States over a 4-year period between 2017 and 2020, compared these to strains collected previously, and found that X. perforans is currently the most prevalent species. We characterized 564 X. perforans isolates for sequence variation in avrXv3 to distinguish between race T3 and T4 and validated race designation using hypersensitive response (HR) assays for 106 isolates. Race T4 accounted for over 95% of X. perforans isolates collected in the Midwest between 2017 and 2020. Whole genome sequencing, Average Nucleotide Identity (ANI) analysis, core genome alignment and single nucleotide polymorphism (SNP) detection relative to a reference strain, and phylogenomic analysis suggest that the majority of Midwestern X. perforans strains collected between 2017 and 2020 were nearly identical, with greater than 99.99% ANI to X. perforans isolates collected from Collier County, Florida in 2012. These isolates shared a common SNP variant resulting an a premature stop codon in avrXv3. One sequenced isolate was identified with a deletion of avrXv3 and shared 99.99% ANI with a strain collected in Collier Co., Florida in 2006. A population shift to X. perforans T4 occurred in the absence of widely deployed resistance, with only 7% of tomato varieties tested having the resistant allele at the Xv3 / Rx-4 locus. The persistence of nearly identical strains over multiple years suggests that migration led to the establishment of an endemic population. Our findings validate a genomics-based framework to track shifts in X. perforans populations due to migration, mutation, drift, or selection based on comparisons to 146 genomes. [ABSTRACT FROM AUTHOR]

Bacterial spot is a destructive disease of tomato in Florida that prior to the early 1990s was caused by Xanthomonas euvesicatoria. X. perforans was first identified in Florida in 1991 and by 2006 was the only xanthomonad associated with bacterial spot disease in tomato. The ability of an X. perforans strain to outcompete X. euvesicatoria both in vitro and in vivo was at least in part associated with the production of three bacteriocins designated Bcn-A, Bcn-B, and Bcn-C. The objective of this study was to characterize the genetic determinants of these bacteriocins. Bcn-A activity was confined to one locus consisting of five ORFs of which three (ORFA, ORF2 and ORF4) were required for bacteriocin activity. The fifth ORF is predicted to encode an immunity protein to Bcn-A based on in vitro and in vivo assays. The first ORF encodes Bcn-A, a 1,398 amino acid protein, which bioinformatic analysis predicts to be a member of the RHS family of toxins. Based on results of homology modeling, we hypothesize that the amino terminus of Bcn-A interacts with a protein in the outer membrane of X. euvesicatoria. The carboxy terminus of the protein may interact with an as yet unknown protein(s) and puncture the X. euvesicatoria membrane, thereby delivering the accessory proteins into the target and causing cell death. Bcn-A appears to be activated upon secretion based on cell fractionation assays. The other two loci were each shown to be single ORFs encoding Bcn-B and Bcn-C. Both gene products possess homology toward known proteases. Proteinase activity for both Bcn-B and Bcn-C was confirmed using a milk agar assay. Bcn-B is predicted to be an ArgC-like serine protease, which was confirmed by PMSF inhibition of proteolytic activity, whereas Bcn-C has greater than 50% amino acid sequence identity to two zinc metalloproteases. [ABSTRACT FROM AUTHOR]

Bacterial spot, caused by Xanthomonas perforans , is one of the major diseases in fresh market tomato production in Florida. It is reported that X. perforans has developed resistance to copper, an element of major bactericides used for management of this disease in Florida and worldwide. Continuous efforts are necessary for providing the tomato industry with new effective approaches to managing this important disease. A small molecule 3-indolylacetonitrile (IAN) that can inhibit biofilm formation has been reported to be effective against many medical bacterial pathogens. In this study, we evaluated the efficacy of IAN in planta for the control of bacterial leaf spot on tomato, the effectiveness of IAN when applied at different times from pathogen inoculation, how IAN efficacy compared to a copper bactericide (Kocide 3000), and if IAN could be applied as a tank mixture with copper bactericide. In vitro testing in liquid media was also completed to further investigate observations made in planta. Our results showed that IAN was effective in reducing disease severity of bacterial leaf spot on tomato between 0.5 and 1.0 mg/ml. However, phytotoxicity to tomato plants occurred when the concentration was higher than 1.0 mg/ml. IAN significantly (P < 0.05) reduced the disease when it was foliar applied during the period from 3 days prior to inoculation to 1 day after inoculation. When IAN was applied in combination with a copper bactericide Kocide 3000, additive effect was observed in reducing both disease severity and bacterial growth, compared to either IAN or copper alone, for both copper-sensitive and copper-resistant X. perforans strains. Results from this study indicated that IAN significantly reduced bacterial leaf spot on tomato and enhanced the efficacy of copper against X. perforans. Specifically, IAN significantly improved the control of bacterial leaf spot caused by a copper-resistant X. perforans strain. • 3-Indolylacetonitrile (IAN) reduced bacterial leaf spot on tomato in greenhouses. • Bacterial spot of tomato was further reduced by IAN in combination with Kocide 3000. • IAN increased efficacy of Kocide 3000 against copper-resistant X. perforans strains. • IAN inhibited in vitro bacterial growth of X. perforans in liquid media. [ABSTRACT FROM AUTHOR]

Objective:We examined the incidence and predictors of peritraumatic distress and dissociation after one of the most common forms of civilian trauma exposure: motor vehicle collision (MVC).Method:In this study, patients presenting to the emergency department after MVCs who were without serious injury and discharged to home after evaluation (n= 935) completed an emergency department interview evaluating sociodemographic, collision-related, and psychological characteristics.Results:The incidence and predictors of distress (Peritraumatic Distress Inventory score ≥23) and dissociation (Michigan Critical Events Perception Scale score >3) were assessed. Distress was present in 355 of 935 patients (38%), and dissociation was present in 260 of 942 patients (28%). These outcomes showed only moderate correlation (r= .45) and had both shared and distinct predictors. Female gender, anxiety symptoms prior to the MVC, and vehicle damage severity predicted both distress and dissociation. Higher socioeconomic status (higher education, higher income, full-time employment) had a protective effect against distress but not dissociative symptoms. Better physical health and worse overall mental health were associated with increased risk of dissociation but not distress. Distress but not dissociation was associated with lower patient confidence in recovery and a longer expected duration of recovery.Conclusion:There are unique predictors of peritraumatic distress and dissociation. Further work is needed to better understand the neurobiology of peritraumatic distress and dissociation and the influence of these peritraumatic outcomes on persistent psychological sequelae. [ABSTRACT FROM PUBLISHER]

Abstract: Phosphorous acid salts (PASs) were evaluated alone or in combination with other products for managing bacterial spot of tomato in greenhouse and field experiments in Florida during a 3-year period. Field treatments included a weekly schedule of PAS alone, PAS combined with standard copper-bactericide at full rate or half rate, PAS alternated with a standard copper-bactericide, and PAS every week plus biweekly applications of acibenzolar-S-methyl (ASM). Field data showed that disease control with PAS combined with standard copper-bactericide full rate, PAS alternated with a standard copper-bactericide, and PAS every week plus biweekly applications of ASM was similar to that obtained using the standard copper-bactericide program. In greenhouse experiments PAS alone had a similar level of disease compared to the standard program, whereas PAS combined with standard copper-bactericide full rate was significantly better than the standard program. Yields in the field experiments were not affected by any of these treatments. Phytotoxicity was observed when PAS was applied to the foliage of tomato seedlings under greenhouse conditions. These data suggest that PAS combined with standard copper-bactericide full rate and PAS plus ASM could be used for managing bacterial spot of tomato in the field in Florida. The mode of action of PAS is still unclear, as it only slightly affected multiplication of Xanthomonas perforans in vitro, and was also ineffective as a plant activator. [Copyright &y& Elsevier]

We provide the first conclusive evidence that Xanthomonas axonopodis pv. citri Asiatic strain (Xac-A) and, in particular, Xac-Aw, a unique citrus canker A strain isolated from Key lime in Wellington, Florida, induces a hypersensitive reaction (HR) in grapefruit leaves. Using the heterologous tomato pathogen X. perforans, as a recipient of the Xac-Aw genomic library, we identified a 1599-bp open reading frame responsible for HR in grapefruit, but not Key lime, and designated it avrGf1. Xac-AwΔ avrGf1 produced typical, although visibly reduced, citrus canker symptoms (i.e. raised pustules) in grapefruit and typical canker symptoms in Key lime. We also determined that the X. perforans transconjugant carrying an Xac-Aw hrpG elicited HR in grapefruit and Key lime leaves, and that xopA in X. perforans was partly responsible for HR. Xac-A transconjugants carrying the X. perforans xopA were reduced in ability to grow in grapefruit leaves relative to wild-type Xac-A. The X. perforans xopA appears to be a host-limiting factor. An avrBs3 homologue, which contained 18.5 repeats and induced HR in tomato, was designated avrTaw. This gene, when expressed in a pustule-minus Xac-Aw, did not complement pustule formation; however, pthAw, a functional pthA homologue, complemented the mutant strain to produce typical pustules in Key lime, but markedly reduced pustules in grapefruit. Both avrBs3 homologues, when expressed in a typical Xac-A strain, resulted in typical citrus canker pustules in grapefruit, indicating that neither homologue suppressed pustule size in grapefruit. Xac-Aw contains other unidentified factors that suppress development in grapefruit. [ABSTRACT FROM AUTHOR]

A leaf spot and blighting were observed on leaves of tomato transplants from a producer in Florida in 2001 and 2002. A nonfluorescent bacterium was isolated consistently from affected tissue. The typical bacterium was a gram negative, strictly aerobic, slightly curved rod with one or two flagella. Sequence analysis of the 16S rRNA indicated that two representative strains, F1 and SE1, had greater than 99% nucleotide sequence identity with Herbaspirillum huttiense and H. rubrisubalbicans. The cellular fatty acid composition of the total of 16 tomato strains was very similar to H. huttiense and H. rubrisubalbicans. Based on carbon utilization, six of nine strains tested with the Biolog system were identified as Herbaspirillum spp. The tomato strains were oxidase positive and grew at 40°C, but were negative for levan production, pectate hydrolysis, and arginine dihydrolase activity. Based upon this polyphasic analysis, we concluded that the strains were most closely related to H. huttiense, although placement in this species would re- quire further analyses. However, the tomato strains and H. rubrisubalbicans, but not H. huttiense, caused confluent necrosis when infiltrated at high concentrations into tomato leaves and were able to produce leaf spot symptoms on inoculated tomato seedlings in the greenhouse. Using pulsed-field gel electrophoresis, we determined that there was considerable variability between the strains collected in 2001 and 2002. [ABSTRACT FROM AUTHOR]

Weeds were sampled in commercial vegetable fields in Palm Beach County, FL in August 2001, December 2001, and March 2002 for the presence of Phytophthora capsici. Fields sampled had a recent history of this plant pathogen. P. capsici was successfully isolated from the roots of six of 42 Carolina geranium (Geranium carolinianum) plants, four of 28 American black nightshade (Solanum americanum) plants, and two of 130 common purslane (Portulaca oleracea) plants. All but one of the 12 isolates were of the A1 mating type. All 12 isolates were resistant to mefenoxam, although at different levels. All but one isolate were strongly pathogenic on pepper seedlings. When two or three isolates recovered from each weed were inoculated onto the roots of their weed host of origin, P. capsici was recovered from the roots. Isolates of P. capsici were tested on four other solanaceous weeds of importance or potential importance to agricultural fields in Florida: Solanum nigrum, S. ptycanthum, S. carolinense, and S. capsicoides. Recovery of P. capsici from roots varied with weed species and isolate tested. P. capsici caused disease mortality on S. nigrum, and no reisolation of P. capsici was possible with S. capsicoides. This is the first report of S. americanum and G. carolinianum being alternative hosts for P. capsici under field conditions. This study also validated P. oleracea as an alternative host. In Florida, and perhaps elsewhere, weeds may contribute to pathogen survival in the absence of a host crop or when propagules may not readily survive in soil or plant debris. [ABSTRACT FROM AUTHOR]

Southern highbush blueberry (interspecific hybrids of Vaccinium corymbosum L.) is cultivated near wild V. corymbosum as well as closely related species in Florida, USA. The expansion of blueberry cultivation into new areas in Florida and deployment of new cultivars containing viruses can potentially increase the diversity of viruses in wild and cultivated V. corymbosum. In this study, viral diversity in wild and cultivated blueberries (V. corymbosum) is described using a metagenomic approach. RNA viromes from V. corymbosum plants collected from six locations (two cultivated and four wild) in North Central Florida were generated by high throughput sequencing (HTS) and analyzed using a bioinformatic analysis pipeline. De novo assembled contigs obtained from viromes of both commercial and wild sites produced sequences with similarities to plant virus species from a diverse range of families (Amalgaviridae, Caulimoviridae, Endornaviridae, Ophioviridae, Phenuiviridae, andVirgaviridae). In addition, this study has enabled the identification of blueberry latent virus (BlLV) and blueberry mosaic associated ophiovirus (BlMaV) for the first time in Florida, as well as a tentative novel tepovirus (blueberry virus T) (BlVT) in blueberry. To the best of our knowledge, this is the first study that compares viral diversity in wild and cultivated blueberry using a metagenomic approach. [ABSTRACT FROM AUTHOR]

Bacterial spot disease caused by Xanthomonas euvesicatoria is one of the major constraints for pepper production in Florida and worldwide. The common prevalence of copper (Cu)-tolerant X. euvesicatoria strains has reduced the efficacy of Cu based bactericides against bacterial spot of pepper. In this study, we evaluated antimicrobial activity of three Cu-based nanocomposites, namely, core-shell Cu (CS–Cu), fixed-quat Cu (FQ-Cu) and mixed-valence Cu (MV-Cu), along with Kocide® 3000, which is a micron size metallic Cu bactericide and is commercially available, both in vitro and in greenhouse trials, against a Cu-tolerant strain of X. euvesicatoria. In repeated in vitro tests, X. euvesicatoria growth was completely inhibited 24 h after exposure of bacterial cells to 500 and 1000 μg/ml of MV-Cu and all concentrations of FQ-Cu and CS-Cu (100, 200, 500, and 1000 μg/ml) treated for 1 h. Micron size copper, even at 1000 μg/ml, had no statistical difference in growth in comparison to the untreated control (P = 0.05; LSD). In the repeated greenhouse experiments, plants treated with all the copper composites exhibited less bacterial spot severity than untreated control and similar or significantly less than Cu-mancozeb (P < 0.05). However, MV-Cu was the only Cu composite with no phytotoxicity on plants under controlled conditions. This study shows potential to use Cu-based nanoparticles for efficient management of bacterial spot on pepper. [ABSTRACT FROM AUTHOR]

Bacterial spot of tomatoes (BST) caused by Xanthomonas perforans , is a ubiquitous disease of tomato in field and transplant operations in Florida. For disease management, producers rely heavily on copper or copper-mancozeb sprays, which are often ineffective due to the high prevalence of copper-tolerant Xanthomonas strains. In this study, 19 different chemical agents, biological control agents, plant defense activators, and novel products were evaluated for their ability to manage bacterial spot on tomato seedlings and in the field. Efficacies of single rotations or tank mixes of several compounds were compared to a non-treated control and a copper standard on seedlings. Stand-alone applications of acibenzolar-S-methyl (ASM), copper octanoate, quinoxyfen, oxysilver nitrate, and pentasilver hexaoxoiodate significantly reduced disease compared to inoculated controls. Seedling programs that included either ASM or copper octanoate significantly reduced disease compared to the control. Oxytetracycline and streptomycin were ineffective at reducing disease severity under high disease pressure in seedling trials. Although there were significant differences in disease severity among treatments, there were no significant difference in the overall yield among treatments across field trials. Overall, ASM provided significant disease reduction as a stand-alone or in a program with other compounds in both the greenhouse and field. However, copper octanoate alone or as a tank mixture with other compounds was only effective for seedling production. The use of alternative compounds should improve tomato health, reduce the introduction of disease inoculum into fields, and reduce residual copper in the environment. • Copper hydroxide and sulphate resulted in variable disease management. • Acibenzolar S-methyl and copper octanoate were suitable disease suppressants in tomato transplants. • Efficacy of field treatments were variable and did not improve tomato yields. • There was no silver bullet treatment for management of bacterial leaf spot. [ABSTRACT FROM AUTHOR]

Xanthomonas perforons--the dominant causal agent of bacterial leaf spot of tomato--is an emerging pathogen of pepper, indicative of a potential host expansion across the southeastern United States. However, studies of the genetic diversity and evolution of X. perforons from pepper remain limited. In this study, the whole-genome sequences of 35 X. performs strains isolated from pepper from four fields and two transplant facilities across southwest Florida between 2019 and 2021 were used to compare genomic divergence, evolution, and variation in type III secreted effectors. Phylogenetic analysis based on core genes revealed that all 35 X. perforons strains formed one genetic cluster with pepper and tomato strains from Alabama and Turkey and were closely related to strains isolated from tomato in Indiana, Mexico, and Louisiana. The in planta population growth of tomato strains isolated from Indiana, Mexico, Louisiana, and Turkey in pepper leaf mesophyll was on par with pepper X. perforons and A. euvesicatoria strains. Molecular clock analysis of the 35 Florida strains dated their emergence to approximately 2017. While strains varied in copper tolerance, all sequenced strains harbored the avrHahl transcription activation-like effector located on a conjugative plasmid, not previously reported in Florida. Our findings suggest that there is a geographically distributed lineage of X. perforons strains on tomato that has the genetic background to cause disease on pepper. Moreover, this study clarifies potential adaptive variants of X. perforons on pepper that could help forecast the emergence of such strains and enable immediate or preemptive intervention. [ABSTRACT FROM AUTHOR]

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. perforons (formerly Xanthomonas perforons), 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. perforons 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. perforons 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. [ABSTRACT FROM AUTHOR]

For most of the 20th century, Xanthomonas euvesicatoria was the only known bacterium associated with bacterial spot of tomato in Florida. X. perforans quickly replaced X. euvesicatoria, mainly because of production of three bacteriocins (BCNs) against X. euvesicatoria; however, X. perforans outcompeted X. euvesicatoria even when the three known BCNs were deleted. Surprisingly, we observed antimicrobial activity against X. euvesicatoria in the BCN triple mutant when the triple mutant was grown in Petri plates containing multiple spots but not in Petri plates containing only one spot. We determined that changes in the headspace composition (i.e., volatiles) rather than a diffusible signal in the agar were required for induction of the antimicrobial activity. Other Xanthomonas species also produced volatile-induced antimicrobial compounds against X. euvesicatoria and elicited antimicrobial activity by X. perforans. A wide range of plant pathogenic bacteria, including Clavibacter michiganensis subsp. michiganensis, Pantoea stewartii, and Pseudomonas cichorii, also elicited antimicrobial activity by X. perforans when multiple spots of the species were present. To identify potential antimicrobial compounds, we performed liquid chromatography with high-resolution mass spectrometry of the agar surrounding the spot in the high cell density Petri plates where the antimicrobial activity was present compared with agar surrounding the spot in Petri plates with one spot where antimicrobial activity was not observed. Among the compounds identified in the zone of inhibition were N-butanoyl-L-homoserine lactone and N-(3-hydroxy-butanoyl)-homoserine lactone, which are known quorum-sensing metabolites in other bacteria. [ABSTRACT FROM AUTHOR]

In the Wellington and Lake Worth areas of Palm Beach County, FL, citrus canker appeared on Key/Mexican lime (Citrus aurantiifolia) and alemow (C. macrophylla) trees over a period of about 6 to 7 years before detection, but nearby canker-susceptible citrus, such as grapefruit (C. x paradisi) and sweet orange (C. sinensis), were unaffected. Colonies of the causal bacterium, isolated from leaf, stem, and fruit lesions, appeared similar to the Asiatic group of strains of Xanthomonas axonopodis pv. cirri (Xac-A) on the nutrient agar plate, but the growth on lima bean agar slants was less mucoid. The bacterium produced erumpent, pustule-like lesions of typical Asiatic citrus canker syndrome after inoculation into Key/Mexican lime, but brownish, flat, and necrotic lesions on the leaves of Duncan grapefruit, Madame Vinous sweet orange, sour orange (C. aurantium), citron (C. medica), Orlando tangelo (C. reticulata x C. x paradisi), and trifoliate orange (Poncirus trifoliata). The bacterium did not react with the Xac-A specific monoclonal antibody A1 using enzyme-linked immunosorbent assay (ELISA) and could not be detected by polymerase chain reaction (PCR)-based assays using primers selected for Xac-A. DNA reassociation analysis confirmed that the pathogen, designated as Xac-AW, was more closely related to Xac-A and Xac-A∗ strains than X. axonopodis pv. aurantifolii or the citrus bacterial spot pathogen (X. axonopodis pv. citrumelo). The strain can be easily differentiated from Xac-A and Xac-A∗ using ELISA, PCR-based tests, fatty acid analysis, pulsed-field gel electrophoresis of genomic DNA, and host specificity. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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 (HI, 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 [IcicF], 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. [ABSTRACT FROM AUTHOR]

A bacterial spot of rose (Rosa spp.) caused by a xanthomonad was observed in Florida and Texas. Ten representative strains collected from the two states between 2004 and 2010 were used to determine the taxonomic position of this rose pathogen. Fatty acid methyl ester analysis was performed and a nearly 2-kb 16S-23S rRNA intergenic spacer along with flanking portions of the 16S and 23S rRNA genes were sequenced for selected strains, showing that they were members of the genus Xanthomonas. Multilocus sequence typing and analysis (MLST/MLSA) and pathogenicity tests were conducted to further characterize the Xanthomonas strains. The MLSA, based on six gene fragments--fusA, gapA, gltA, gyrB, lacF, and lepA--showed that the rose strains fell into Xanthomonas axonopodis subgroup 9.2 and shared the highest similarity values (98.8 to 99.7%) with X. alfalfae subsp. citrumelonis of the subgroup. However, principal coordinate analysis grouped the rose strains into a unique cluster distinct from other members of the subgroup according to virulence phenotypes on 11 plant species belonging to five plant families (Araceae, Euphorbiaceae, Rosaceae, Rutaceae, and Solanaceae). Moreover, the rose strains were aggressive on rose and Indian Hawthorn (Rhaphiolepsis indica). On the basis of the MLSA and virulence phenotypes, the pathovar epithet rosa is proposed. [ABSTRACT FROM AUTHOR]

Studies were conducted at three locations in Florida to evaluate the effects of kasugamycin alone, in alternation, or as a tank-mix partner with copper bactericides and other fungicides against bacterial spot of tomato. In greenhouse trials, kasugamycin, formulated as Kasumin® 2L, reduced bacterial spot severity by up to 37.5% compared with a non-treated control. Little advantage in disease control was observed by mixing kasugamycin with other fungicides. Kasugamycin was assessed in six field trials. In the four field trials that tested kasugamycin alone, it was as effective as the standard copper + mancozeb treatment for the control of bacterial spot. In four trials, no benefit was observed in applying kasugamycin as a mixture with copper + mancozeb, and only one of three trials did alternating kasugamycin with copper + mancozeb improve bacterial spot control over either the copper + mancozeb standard or kasugamycin alone. Although kasugamycin was effective for the control of bacterial spot in greenhouse and field trials, rapid development of resistance in field populations of X. perforans may shorten the effective use of this antibiotic. [ABSTRACT FROM AUTHOR]

In 2003 and 2004, 15 isolates of Ralstonia solanacearum were obtained from wilting plants of field-grown pepper (Capsicum annuum) in south Florida and from pot-grown hydrangea (Hydrangea paniculata and H. macrophylla) and geranium (Pelargonium x hortorum) in commercial nurseries and retention ponds in north Florida. Diagnostic immunoassays and polymerase chain reaction (PCR) analyses identified all the isolates as R. solanacearum but not race 3 biovar 2. Pathogenicity studies on tomato, pepper, and tobacco revealed that all 15 strains had similar high virulence on tomato and all caused wilting of tobacco, although there were significant differences among the strains in aggressiveness on tobacco. An indigenous Florida tomato strain, race 1 biovar 1 (Rs5), caused no disease on tobacco and little or none on pepper. The three pepper strains were more aggressive than Rs5 or two hydrangea strains on all three pepper cultivars studied. Phylogenetic analysis based on an endoglucanase gene sequence indicated that these strains had three distinct origins. The three pepper strains belonged to phylotype I biovar 3 and clustered with strains from diverse hosts in Asia belonging to sequevar 13. The six geranium strains and four of the hydrangea strains were closely related to strains in sequevar 5, a distinct subcluster of phylotype II biovar 1 strains isolated from the French West Indies and Brazil. Two other biovar 1 strains from hydrangea and strains K60, AW, and Rs5 belonged to sequevar 7 in phylotype II and probably are native to North America. None of the Florida isolates belong to the highly regulated Select Agent race 3 biovar 2 subgroup, according to both the DNA sequence analysis and the biovar phenotypic test results. However, the race 3 biovar 2-specific B2 primers weakly amplified a product from some race 1 biovar 1 strains in real-time PCR, indicating that this assay may give false positives under some conditions. Given the high cost of a misdiagnosis, it seems advisable to use at least two independent diagnostic methods to confirm that a suspect isolate is R. solanacearum R3B2. This is the first report of the presence of R. solanacearum race 1 biovar 3 or pbylotype I strains in North America, and the first report confirming R. solanacearum causing natural infection of hydrangea in Florida. Thus, R. solanacearum strains that are quite distinct from presumably indigenous strains are present and can infect diverse hosts in Florida. [ABSTRACT FROM AUTHOR]

Bacterial leaf spot of pepper (BSP), primarily caused by Xanthomonas euvesicatoria (Xe ), poses a significant challenge to pepper production worldwide. Despite its impact, the genetic diversity of this pathogen remains underexplored, which limits our understanding of its population structure. To bridge this knowledge gap, we conducted a comprehensive analysis using 103 Xe strains isolated from pepper in southwest Florida to characterize genomic and type III effector (T3E) variation in this population. Phylogenetic analysis of core genomes revealed a major distinct genetic lineage associated with amylolytic activity. This amylolytic lineage was represented in Xe strains globally. Molecular clock analysis dated the emergence of amylolytic strains in Xe to around 1972. Notably, non-amylolytic strains possessed a single base pair frameshift deletion in the ⍺-amylase gene yet retained a conserved C-terminus. GUS assay revealed the expression of two open reading frames in non-amylolytic strains, one at the N-terminus and another that starts 136 base pairs upstream of the ⍺-amylase gene. Analysis of T3Es in the Florida Xe population identified variation in 12 effectors, including two classes of mutations in avrBs2 that prevent AvrBs2 from triggering a hypersensitive response in Bs2 -resistant pepper plants. Knowledge of T3E variation could be used for effector-targeted disease management. This study revealed previously undescribed population structure in this economically important pathogen.IMPORTANCEBacterial leaf spot (BSP), a significant threat to pepper production globally, is primarily caused by Xanthomonas euvesicatoria ( Xe ). Limited genomic data has hindered detailed studies on its population diversity. This study analyzed the whole-genome sequences of 103 Xe strains from peppers in southwest Florida, along with additional global strains, to explore the pathogen's diversity. The study revealed two major distinct genetic groups based on their amylolytic activity, the ability to break down starch, with non-amylolytic strains having a mutation in the ⍺-amylase gene. Additionally, two classes of mutations in the avrBs2 gene were found, leading to susceptibility in pepper plants with the Bs2 resistance gene, a commercially available resistance gene for BSP. These findings highlight the need to forecast the emergence of such strains, identify genetic factors for innovative disease management, and understand how this pathogen evolves and spreads., Competing Interests: The authors declare no conflict of interest.

In Florida, angular leaf spot, caused by Xanthomonas fragariae , was the only known bacterial disease in strawberry, which is sporadic and affects the foliage and calyx. However, from the 2019-2020 to 2023-2024 Florida strawberry seasons, unusual bacterial-like symptoms were observed in commercial farms, with reports of up to 30 % disease incidence. Typical lesions were water-soaked and angular in early stages that later became necrotic with a circular-ellipsoidal purple halo, and consistently yielded colonies resembling Pseudomonas on culture media. Strains were pathogenic on strawberry, fluorescent, oxidase- and arginine-dihydrolase-negative, elicited a hypersensitive reaction on tobacco, and lacked pectolytic activity. Although phenotypic assays, such as fatty acid methyl profiles and Biolog protocols, placed the strains into the Pseudomonas group, there was a low similarity at the species level. Further analysis using 16S rRNA genes, housekeeping genes, and whole genome sequencing showed that the strains cluster into the Pseudomonas group but do not share more than 95 % average nucleotide identity compared to representative members. Therefore, the genomic and phenotypic analysis confirm that the strains causing bacterial spot in strawberry represent a new plant pathogenic bacterial species for which we propose the name Pseudomonas fragariae sp. nov. with 20-417 T (17 T =LMG 32456 T =DSM 113340 T ) as the type strain, in relation to Fragaria × ananassa , the plant species from which the pathogen was first isolated. Future work is needed to assess the epidemiology, cultivar susceptibility, chemical sensitivity, and disease management of this possible new emerging strawberry pathogen.

Bacterial spot caused by Xanthomonas spp. is a significant disease that challenges pepper growers worldwide and is particularly severe in a hot and humid environment. Understanding the pathogen's population biology is critical for sustainable disease management. The goal of this study was to characterize the species, race, and bactericide sensitivity of bacterial spot-associated Xanthomonas collected from pepper in Florida. A survey of pepper production fields in southwest Florida between 2019 and 2021-covering two counties, eight farms, and two transplant facilities-resulted in the isolation of 542 Xanthomonas euvesicatoria and 35 Xanthomonas perforans strains. Four races were identified on pepper, of which most strains were race P1 (42%), race P6 (26%), race P3 (24%), and less common was race P4 (8%). All X. perforans strains were characterized as race P1 and showed a compatible reaction on tomato. Sixty-two and 96% of strains were sensitive to copper sulfate and streptomycin, respectively. One farm that did not use copper to manage the disease contained only copper-sensitive strains and was the only farm with race P3 strains. Strains were assayed for starch hydrolysis activity of which a third of X. euvesicatoria strains were strongly amylolytic, a characteristic not typically observed in X. euvesicatoria . All X. perforans strains produced bacteriocins against X. euvesicatoria in vitro. The Xanthomonas population causing bacterial spot on pepper in southwest Florida is diverse and dynamic; thus, regular monitoring provides pertinent information to plant breeders and growers for designing disease management strategies., Competing Interests: The author(s) declare no conflict of interest.

Tomato transplants are the primary means of establishing commercial tomato production fields in the eastern United States. Transplants are often suspected as the source of inoculum for major outbreaks in production fields of bacterial spot of tomato (BST) caused by Xanthomonas perforans ( Xp ). A combination of high plant densities with overhead irrigation, high humidity, and high temperatures are conducive to BST outbreaks during transplant production. In addition to chemical control, transplant growers use roguing to remove diseased transplants, as a primary way to manage BST during transplant production. The value of roguing is often questioned, because information about the rate of pathogen spread and the incubation period between infection and symptom development is limited. In this study, we evaluated the extent of X. perforans spread on tomato transplants relative to symptom development by using a rifampicin-resistant X. perforans strain and conducting experiments in an environmentally controlled greenhouse simulating grower practices and also at a commercial transplant facility in Florida. BST symptom development typically lagged behind X. perforans dispersal by at least 5 to 7 days depending on environmental conditions. Furthermore, X. perforans was capable of aerosolization, which resulted in long-distance dispersal of ≤2 m under highly favorable conditions. Growers should rogue diseased plants and surrounding nonsymptomatic plants by >1 and ≤3 m, depending on outbreak severity, to limit disease spread. As a result, proper disease management should reduce introduction of nonsymptomatic transplants into the field and subsequently reduce pesticide applications.

Field trials were conducted at two locations in Florida to evaluate transgenic tomato expressing the ELONGATION FACTOR TU RECEPTOR (EFR) gene from Arabidopsis thaliana, the Bs2 gene from pepper, or both Bs2 and EFR (Bs2/EFR) for managing bacterial wilt caused by Ralstonia solanacearum and bacterial spot caused by Xanthomonas perforans. Expression of EFR or Bs2/EFR in the susceptible genotype Fla. 8000 significantly reduced bacterial wilt incidence (50 to 100%) and increased total yield (57 to 114%) relative to lines expressing only Bs2 or the nontransformed Fla. 8000 control, although the marketable yield was not significantly affected. Following harvest, surviving symptomatic and nonsymptomatic plants were assessed for colonization by R. solanacearum. There were no significant differences in the population at the lower stem. Interestingly, in the middle stem, no bacteria could be recovered from EFR or Bs2/EFR lines but viable bacterial populations were recovered from Bs2 and nontransformed control lines at 10 2 to 10 5 CFU/g of stem tissue. In growth-chamber experiments, the EFR transgenic tomato lines were found to be effective against seven different R. solanacearum strains isolated from the southeastern United States, indicating utility across the southeastern United States. In all of the bacterial spot trials, EFR and Bs2/EFR lines had significantly reduced disease severity (22 to 98%) compared with the Fla. 8000 control. The marketable and total yield of Bs2/EFR were significantly higher (43 to 170%) than Fla. 8000 control in three of four field trials. These results demonstrate for the first time the potential of using the EFR gene for field management of bacterial wilt and bacterial spot diseases of tomato.

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., (Copyright © 2018 American Society for Microbiology.)

Studies on genetic diversity and recombination in bacterial pathogens are providing a better understanding of the mechanisms shaping bacterial diversity, which can affect disease control. Xanthomonas campestris pv. vitians, causal agent of bacterial leaf spot of lettuce, is a threat to the worldwide lettuce industry. We examined the genetic variation within a sample of 83 strains from California, Florida, and Ohio using multilocus sequence typing of six housekeeping genes, totaling 2.7 kb. Additionally, polymorphism in two virulence-related genes, hrpB2 and a putative glycosyl hydrolase, were examined. Based on housekeeping genes, we found three genetic groups of strains that were all able to induce the disease. These included strains collected from weeds and irrigation water that had haplotypes identical to strains from diseased lettuce. High linkage disequilibrium across the sequenced loci indicates that the pathogen is predominantly clonal but recombination has contributed to the observed sequence variation. Although there was significant genetic variation in X. campestris pv. vitians within and among sampled states, identical haplotypes were observed across all three states. This finding suggests that seedborne inoculum may contribute to the diversity of X. campestris pv. vitians in the United States. Knowledge of the genetic structure of the pathogen may be used for developing resistant lettuce varieties.

Background: Motor vehicle collisions (MVCs) are the second most common injury mechanism resulting in emergency department (ED) visits by older adults. MVCs result in substantial pain and psychological distress among younger individuals, but little is known about the occurrence of these symptoms in older individuals. We describe the frequency of and characteristics associated with pain, distress, and anticipated time for physical and emotional recovery for older adults presenting to the ED after MVC in comparison to younger adults., Methods: In-person interviews were conducted for adults presenting to one of eight EDs after MVC without an obvious fracture or injury requiring admission as part of two prospective studies. Pain severity was assessed using a 0-10 verbal scale. Distress was assessed using the Peritraumatic Distress Inventory (range 0-52). Patients were asked to estimate their expected time for physical and emotional recovery; these responses were dichotomized to <30 or ≥30 days. ED pain and distress and associations between patient and collision characteristics and ED pain and distress were examined for patients age 65 years and older and patients age 18 to 64., Results: Older (n = 96) and younger (n = 943) adults had the same mean pain scores (5.5, SD 2.5 vs. 5.5, SD 2.4). Distress scores were lower in older than in younger adults (15.5, SD 9 vs. 19.2, SD 10). A higher percentage of older adults than younger adults had an anticipated time to physical recovery ≥30 days (41%, 95% confidence interval [CI] 28%-55% vs. 11%, 95% CI 9%-13%). Similarly, older adults were more likely to have an anticipated time for emotional recovery ≥30 days (45%, 95% CI 35%-55% vs. 17%, 95% CI 15%-20%). Older adults were less likely than younger adults to have moderate or severe neck pain (score ≥4) (25%, 95% CI 23% to 41% vs. 54%, 95% CI 48% to 60%) or back pain (31%, 95% CI 23% to 46% vs. 56%, 95% CI 51 to 62%) but more likely to have moderate or severe chest pain (42%, 95% CI 32% to 50% vs. 20%, 95% CI 16 to 23%). Pre-MVC depressive symptoms and pain catastrophizing were positively associated with pain and distress in both older and younger adults., Conclusions: In our cohort, older adults who presented to the ED after MVC experienced similar pain severity as younger patients and less distress but were more likely to estimate their times for physical and emotional recovery to be 30 days or more. Increased emergency provider awareness of acute pain and distress symptoms among older patients experiencing MVC may improve outcomes for these patients.

ABSTRACT We determined that multiple and independent introductions of copper resistance genes have taken place for strains of Xanthomonas citri subsp. citri from Argentina and strains of X. alfalfae subsp. citrumelonis from Florida. This study compared the partial nucleotide sequences of principal copper resistance genes copL, copA, and copB from X. citri subsp. citri and X. alfalfae subsp. citrumelonis to strains of other Xanthomonas spp. resistant to copper that were isolated from 12 different countries or territories. The survey confirmed that the copLAB gene cluster is present in many species of Xanthomonas from different parts of the world. Alignment of partial nucleotide sequences of copper resistance genes among the copper-resistant (Cu(R)) strains of Xanthomonas detected homology of ≥92, ≥96, and ≥91% for copL, copA, and copB, respectively. Grouping of strains based on branching patterns of phylogenetic trees was similar for copL and copA but differed for copB. When the three genes were concatenated and analyzed using various phylogenetic methods, it appeared that the plasmid had been horizontally transferred and various populations were mutating based on selection pressure unique to geographic regions. Although high homology of the genes among the strains indicated that the copper resistance in xanthomonads has a common origin, the slight differences in nucleotide sequences within groups of strains indicated that Cu(R) genes have been independently exchanged among species of Xanthomonas throughout the world.

Copper sprays have been widely used for control of endemic citrus canker caused by Xanthomonas citri subsp. citri in citrus-growing areas for more than 2 decades. Xanthomonas alfalfae subsp. citrumelonis populations were also exposed to frequent sprays of copper for several years as a protective measure against citrus bacterial spot (CBS) in Florida citrus nurseries. Long-term use of these bactericides has led to the development of copper-resistant (Cu(r)) strains in both X. citri subsp. citri and X. alfalfae subsp. citrumelonis, resulting in a reduction of disease control. The objectives of this study were to characterize for the first time the genetics of copper resistance in X. citri subsp. citri and X. alfalfae subsp. citrumelonis and to compare these organisms to other Cu(r) bacteria. Copper resistance determinants from X. citri subsp. citri strain A44(pXccCu2) from Argentina and X. alfalfae subsp. citrumelonis strain 1381(pXacCu2) from Florida were cloned and sequenced. Open reading frames (ORFs) related to the genes copL, copA, copB, copM, copG, copC, copD, and copF were identified in X. citri subsp. citri A44. The same ORFs, except copC and copD, were also present in X. alfalfae subsp. citrumelonis 1381. Transposon mutagenesis of the cloned copper resistance determinants in pXccCu2 revealed that copper resistance in X. citri subsp. citri strain A44 is mostly due to copL, copA, and copB, which are the genes in the cloned cluster with the highest nucleotide homology (≥ 92%) among different Cu(r) bacteria.