Project description:Prior to the identification of Xanthomonas perforans associated with bacterial spot of tomato in 1991, X. euvesicatoria was the only known species in Florida. Currently, X. perforans is the Xanthomonas sp. associated with tomato in Florida. Changes in pathogenic race and sequence alleles over time signify shifts in the dominant X. perforans genotype in Florida. We previously reported recombination of X. perforans strains with closely related Xanthomonas species as a potential driving factor for X. perforans evolution. However, the extent of recombination across the X. perforans genomes was unknown. We used a core genome multilocus sequence analysis approach to identify conserved genes and evaluated recombination-associated evolution of these genes in X. perforans. A total of 1,356 genes were determined to be "core" genes conserved among the 58 X. perforans genomes used in the study. Our approach identified three genetic groups of X. perforans in Florida based on the principal component analysis (PCA) using core genes. Nucleotide variation in 241 genes defined these groups, that are referred as Phylogenetic-group Defining (PgD) genes. Furthermore, alleles of many of these PgD genes showed 100% sequence identity with X. euvesicatoria, suggesting that variation likely has been introduced by recombination at multiple locations throughout the bacterial chromosome. Site-specific recombinase genes along with plasmid mobilization and phage associated genes were observed at different frequencies in the three phylogenetic groups and were associated with clusters of recombinant genes. Our analysis of core genes revealed the extent, source, and mechanisms of recombination events that shaped the current population and genomic structure of X. perforans in Florida.
Project description: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.
Project description:Salmonella enterica rarely grows on healthy, undamaged plants, but its persistence is influenced by bacterial plant pathogens. The interactions between S. enterica, Xanthomonas perforans (a tomato bacterial spot pathogen), and tomato were characterized. We observed that virulent X. perforans, which establishes disease by suppressing pathogen-associated molecular pattern (PAMP)-triggered immunity that leads to effector-triggered susceptibility, created a conducive environment for persistence of S. enterica in the tomato phyllosphere, while activation of effector-triggered immunity by avirulent X. perforans resulted in a dramatic reduction in S. enterica populations. S. enterica populations persisted at ~10 times higher levels in leaves coinoculated with virulent X. perforans than in those where S. enterica was applied alone. In contrast, S. enterica populations were ~5 times smaller in leaves coinoculated with avirulent X. perforans than in leaves inoculated with S. enterica alone. Coinoculation with virulent X. perforans increased S. enterica aggregate formation; however, S. enterica was not found in mixed aggregates with X. perforans. Increased aggregate formation by S. enterica may serve as the mechanism of persistence on leaves cocolonized by virulent X. perforans. S. enterica association with stomata was altered by X. perforans; however, it did not result in appreciable populations of S. enterica in the apoplast even in the presence of large virulent X. perforans populations. Gene-for-gene resistance against X. perforans successively restricted S. enterica populations. Given the effect of this interaction, breeding for disease-resistant cultivars may be an effective strategy to limit both plant disease and S. enterica populations and, consequently, human illness.
Project description:Ralstonia solanacearum is a causative agent of bacterial wilt in many economically important crops, and Xanthomonas perforans is the causal organism of bacterial spot, one of the most important diseases of vegetables. A multiplex PCR protocol has been developed for the simultaneous, specific and rapid identification of R. solanacearum and X. perforans in plant materials. Species-specific primers RS-F-759 and RS-R-760 for R. solanacearum, RST2 and RST3 for X. perforans were used for identification of both pathogens at primer concentrations of 1:4 by optimization of multiplex PCR at annealing temperature of about 61 ± 1 °C. With these primer sets, specific amplification of 281- and 840-bp PCR products was obtained for R. solanacearum and X. perforans, respectively. The multiplex PCR assay was validated with susceptible plants mechanically inoculated with both the pathogens; specific PCR products confirmed the presence of R. solanacearum and X. perforans. The multiplex PCR is valuable in identification as well as primary screening of cultivars of both pathogens. The present study is a rapid and easy method for early identification of pathogens from asymptomatic and symptomatic plant materials.
Project description:Purpose: Investigate genes associated to resistance of Xanthomonas perforans race T4 in tomato line with different resistance level Methods: Resistant and susceptible tomato breeding lines were subjected to the inoculation with Xanthomonas perforans race T4 followed by sample collection at 48 hpi and RNA-seq analysis for screening differential expressed genes associated with inoculation of pathogen. Results: Revealed gene expression profiles associated disease resistance and susceptiblilty. Overall design: Resistant and susceptible tomato breeding lines were subjected to the inoculation with Xanthomonas perforans race T4 followed by sample collection at 48 hpi and RNA-seq analysis for screening differential expressed genes associated with inoculation of pathogen.
Project description:Bacterial spot caused by Xanthomonas perforans is a major disease of tomatoes, leading to reduction in production by 10-50%. While copper (Cu)-based bactericides have been used for disease management, most of the X. perforans strains isolated from tomatoes in Florida and other locations worldwide are Cu-resistant. We have developed DNA-directed silver (Ag) nanoparticles (NPs) grown on graphene oxide (GO). These Ag@dsDNA@GO composites effectively decrease X. perforans cell viability in culture and on plants. At the very low concentration of 16 ppm of Ag@dsDNA@GO, composites show excellent antibacterial capability in culture with significant advantages in improved stability, enhanced antibacterial activity, and stronger adsorption properties. Application of Ag@dsDNA@GO at 100 ppm on tomato transplants in a greenhouse experiment significantly reduced the severity of bacterial spot disease compared to untreated plants, giving results similar to those of the current grower standard treatment, with no phytotoxicity.
Project description:Bacterial spot (BS) is one of the most devastating foliar bacterial diseases of tomato and is caused by multiple species of Xanthomonas. We performed the RNA sequencing (RNA-Seq) analysis of three tomato lines with different levels of resistance to Xanthomonas perforans race T4 to study the differentially expressed genes (DEGs) and transcript-based sequence variations. Analysis between inoculated and control samples revealed that resistant genotype Solanum pimpinellifolium accession PI 270443 had more DEGs (834), followed by susceptible genotype tomato (S. lycopersicum L) breeding line NC 714 (373), and intermediate genotype tomato breeding line NC 1CELBR (154). Gene ontology (GO) terms revealed that more GO terms (51) were enriched for upregulated DEGs in the resistant genotype PI 270443, and more downregulated DEGs (67) were enriched in the susceptible genotype NC 714. DEGs in the biotic stress pathway showed more upregulated biotic stress pathway DEGs (67) for PI 270443 compared to more downregulated DEGs (125) for the susceptible NC 714 genotype. Resistant genotype PI 270443 has three upregulated DEGs for pathogenesis-related (PR) proteins, and susceptible genotype NC 714 has one downregulated R gene. Sequence variations called from RNA-Seq reads against the reference genome of susceptible Heinz 1706 showed that chr11, which has multiple reported resistance quantitative trait loci (QTLs) to BS race T4, is identical between two resistant lines, PI 270443 and NC 1CELBR, suggesting that these two lines share the same resistance QTLs on this chromosome. Several loci for PR resistance proteins with sequence variation between the resistant and susceptible tomato lines were near the known Rx4 resistance gene on chr11, and additional biotic stress associated DEGs near to the known Rx4 resistance gene were also identified from the susceptible NC 714 line.
Project description:Plants depend on innate immune responses to retard the initial spread of pathogens entering through stomata, hydathodes or injuries. These responses are triggered by conserved patterns in pathogen-encoded molecules known as pathogen-associated molecular patterns (PAMPs). Production of reactive oxygen species (ROS) is one of the first responses, and the resulting 'oxidative burst' is considered to be a first line of defense. In this study, we conducted association analyses between ROS production and bacterial spot (BS; Xanthomonas spp.) resistance in 63 genotypes of tomato (Solanum lycopersicum L.). A luminol-based assay was performed on leaf tissues that had been treated with a flagellin 22 (flg22), flagellin 28 and a Xanthomonas-specific flg22 (flg22-Xac) peptide, to measure PAMP-induced ROS production in each genotype. These genotypes were also assessed for BS disease response by inoculation with Xanthomonas perforans, race T4. Although there was no consistent relationship between peptides used and host response to the BS, there was a significant negative correlation (r=-0.25, P<0.05) between foliar disease severity and ROS production, when flg22-Xac was used. This response could potentially be used to identify the Xanthomonas-specific PRR allele in tomato, and eventually PAMP-triggered immunity loci could be mapped in a segregating population. This has potential significance in tomato improvement.
Project description: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.