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.

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.

Current management ofXanthomonas perforanson 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 ofX. perforansinoculum 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.

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 10 μg/ml), and DLA (>10% and 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.

Bacterial spot is one of the most detrimental diseases of tomato and is especially severe in the southeast United States when weather conditions (high temperature, high humidity, and rain) become conducive for disease development. This new 8-page publication of the UF/IFAS Plant Pathology Department presents updated information about the causal pathogen and management of bacterial spot on tomato in Florida. Written by Amanda Strayer-Scherer, Ying-Yu Liao, Peter Abrahamian, Sujan Timilsina, Mathews Paret, Tim Momol, Jeff Jones, and Gary Vallad.https://edis.ifas.ufl.edu/pp353

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.