Bacterial Mutation During Seasonal Epidemics.

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

Phylodynamic Insights into Global Emergence and Diversification of the Tomato Pathogen Xanthomonas hortorum pv. gardneri.

The emergence of plant pathogens is often associated with waves of unique evolutionary and epidemiological events. Xanthomonas hortorum pv. gardneri is one of the major pathogens causing bacterial spot disease of tomatoes. After its first report in the 1950s, there were no formal reports on this pathogen until the 1990s, despite active global research on the pathogens that cause tomato and pepper bacterial spot disease. Given the recently documented global distribution of X. hortorum pv. gardneri, our objective was to examine genomic diversification associated with its emergence. We sequenced the genomes of X. hortorum pv. gardneri strains collected in eight countries to examine global population structure and pathways of emergence using phylodynamic analysis. We found that strains isolated post-1990 group by region of collection and show minimal impact of recombination on genetic variation. A period of rapid geographic expansion in X. hortorum pv. gardneri is associated with acquisition of a large plasmid conferring copper tolerance by horizontal transfer and coincides with the burgeoning hybrid tomato seed industry through the 1980s. The ancestry of X. hortorum pv. gardneri is consistent with introduction to hybrid tomato seed production and dissemination during the rapid increase in trade of hybrid seeds.

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

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

Xanthomonas as a Model System for Studying Pathogen Emergence and Evolution.

In this review, we highlight studies in which whole-genome sequencing, comparative genomics, and population genomics have provided unprecedented insights into past and ongoing pathogen evolution. These include new understandings of the adaptive evolution of secretion systems and their effectors. We focus on Xanthomonas pathosystems that have seen intensive study and improved our understanding of pathogen emergence and evolution, particularly in the context of host specialization: citrus canker, bacterial blight of rice, and bacterial spot of tomato and pepper. Across pathosystems, pathogens appear to follow a pattern of bursts of evolution and diversification that impact host adaptation. There remains a need for studies on the mechanisms of host range evolution and genetic exchange among closely related but differentially host-specialized species and to start moving beyond the study of specific strain and host cultivar pairwise interactions to thinking about these pathosystems in a community context.

Effect of Cucurbit Host, Production Region, and Season on the Population Structure of Pseudoperonospora cubensis in Florida.

Pseudoperonospora cubensis, the causal agent of Cucurbit downy mildew (CDM), is one of the most important diseases affecting cucurbit production in the United States. This disease is especially damaging to Florida production areas, as the state is a top producer of many cucurbit species. In addition, winter production in central and south Florida likely serves as a likely source of P. cubensis inoculum for spring and summer cucurbit production throughout the eastern United States, where CDM is unable to overwinter in the absence of a living host. Over 2 years (2017 and 2018) and four seasons (spring 2017, spring 2018, fall 2017, and fall 2018), 274 P. cubensis isolates were collected from cucurbit hosts at production sites in south, central, and north Florida. The isolates were analyzed with 10 simple sequence repeat (SSR) markers to establish population structure and genetic diversity and further assigned to a clade based on a qPCR assay. Results of population structure and genetic diversity analyses differentiated isolates based on cucurbit host and clade (1 or 2). Of the isolates assigned to clade by qPCR, butternut squash, watermelon, and zucchini were dominated by clade 1 isolates, whereas cucumber isolates were split 34 and 59% between clades 1 and 2, respectively. Clade assignments agreed with isolate clustering observed within discriminant analysis of principal components (DAPC) based on SSR markers, although watermelon isolates formed a group distinct from the other clade 1 isolates. For seasonal collections from cucumber at each location, isolates were typically skewed to one clade or the other and varied across locations and seasons within each year of the study. This variable population structure of cucumber isolates could have consequences for regional disease management. This is the first study to characterize P. cubensis populations in Florida and evaluate the effect of cucurbit host and clade-type on isolate diversity and population structure, with implications for CDM management in Florida and other United States cucurbit production areas.

Strength in Numbers: Density-Dependent Volatile-Induced Antimicrobial Activity by Xanthomonas perforans.

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.

Effective Management of Powdery Mildew in Cantaloupe Plants Using Nighttime Applications of UV Light.

Ultraviolet light at wavelengths from 254 to 283 nm/has been reported to effectively suppress powdery mildews in several crops, including some cucurbits. Its use to suppress powdery mildew (Podosphaera xanthii) specifically in cantaloupe has not been previously reported. We evaluated the foregoing technology in cantaloupe fields for suppression of powdery mildew and possible effects on plant growth and yield. In a controlled laboratory study, greenhouse-grown cantaloupe plants were exposed to a gradient of UV-C (254 nm) doses during darkness, and the effects upon powdery mildew development and the plant were evaluated. We also evaluated the efficacy of nighttime applications of UV-C at 100 and 200 J/m2 against powdery mildew on adaxial leaf surfaces in greenhouse, high-tunnel, and open-field plantings. UV-C at the foregoing doses reduced sporulation and germination of P. xanthii conidia without damaging plants. On cantaloupe seedlings in the greenhouse, disease severity was equivalently suppressed at all doses and frequencies of applications of the light. In high-tunnel and open-field experiments, the most effective control of powdery mildew was provided by UV-C applied at 200 J/m2 twice every week, where suppression provided by UV-C was generally equal to and sometimes better than the fungicide treatment. The foregoing UV-C dose and frequency of application also provided the highest yield under field conditions, indicating that UV-C treatment is a promising technology for commercially relevant suppression of powdery mildew on cantaloupe in a variety of growing systems.

Duration of Downy Mildew Control Achieved with Fungicides on Cucumber Under Florida Field Conditions.

Cucurbit production in Florida is impacted by downy mildew on a yearly basis. Cucurbit downy mildew (CDM), caused by Pseudoperonospora cubensis, is one of the most devastating cucurbit diseases and can lead to complete yield loss. Nearly continuous production of cucurbits occurs temporally throughout Florida, which puts extensive pressure on the pathogen population to select for individuals that are resistant to fungicides in use labeled for CDM. Loss of efficacy as a result of fungicide resistance developing is becoming a major concern for Florida cucurbit growers who rely on these products to manage CDM. This study was established to evaluate the field activity of 11 utilized fungicides by determining their duration of activity when applied at various intervals for the management of CDM in cucumber under Florida field conditions. By comparing levels of percent CDM control and area under the disease progress curve values, the fungicide's duration of field activity was established. Field activities were <1 week for dimethomorph and fluopicolide; 1 week for cymoxanil; 1 to 2 weeks for chlorothalonil and mancozeb; 2 weeks for ethaboxam; 1 to 3 weeks for propamocarb, cyazofamid, and ametoctradin + dimethomorph; and 2 to 4 weeks for oxathiapiprolin and fluazinam. Knowledge of duration of field activity can potentially improve the development of CDM management programs and slow the resistance selection.

Supplemental Fumigant Placement Improves Root Knot and Fusarium Wilt Management for Tomatoes Produced on a Raised-Bed Plasticulture System in Florida's Myakka Fine Sand.

Fresh-market tomatoes are produced on a raised-bed plasticulture system that relies heavily on soil-applied preplant fumigants for the management of soilborne pathogens, nematodes, and weeds. Since the transition from methyl bromide to alternative fumigants, growers have experienced a resurgence of several soilborne pests and pathogens, including root-knot nematode caused by Meloidogyne spp. and Fusarium wilt caused by Fusarium oxysporum f. sp. lycopersici race 3. This resurgence is attributed to the inability of the alternative fumigants to effectively disperse through the soil in the same manner as methyl bromide. Two supplemental fumigation strategies, the application of chloropicrin (PIC) below bed edges (herein "supplemental PIC") and broadcast deep-shank applications of 1,3-dichloropropene (1,3-D), were evaluated in conjunction with standard raised-bed applications of Pic-Clor 60, Pic-Clor 80, and Pic 100 covered with a virtually impermeable film or a totally impermeable film. Large-plot replicated studies were conducted in two separate commercial tomato fields with a history of production losses caused by root-knot nematode and Fusarium wilt. Deep-shank 1,3-D applications significantly reduced the recovery of root-knot and total parasitic nematodes across field sites before the preparation of raised beds. Both supplemental PIC and deep-shank 1,3-D reduced root-knot galling and Fusarium wilt incidence, but the latter supplemental treatment statistically had the greatest impact. Fumigant applied within raised beds or plastic film had no significant effect on root-knot galling or Fusarium wilt. Although both supplemental fumigation strategies had a significant effect on pest and disease pressure, neither statistically improved tomato yields based on small subplot harvests. Controlled laboratory experiments confirmed the fungicidal activity of 1,3-D against F. oxysporum f. sp. lycopersici, with 75, 90, 95, and 99% lethal doses corresponding to estimated field application rates of 56.1, 93.5, 121.6, and 184.7 liters/ha, respectively. The results demonstrate how fumigant placement can improve pest and disease control activity with current fumigant alternatives to methyl bromide and further support the broader pesticidal activity of some chemical fumigants.

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

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

Interaction Studies Between Meloidogyne Javanica and Fusarium Oxysporum f. Sp. lycopersici (Fol) Race 3 on Different Isolines of Tomato Cv. Tasti Lee.

The Mi gene in tomato confers resistance to Meloidogyne javanica, M. incognita, and M. arenaria, the most common tropical root-knot nematode (RKN) species found in Florida. Fusarium wilt (Fol) is another major problem in Florida tomatoes which may interact with RKN and cause more plant damage. To study the interactions between RKN, Fusarium, and Mi in tomato, two greenhouse experiments were conducted. Both experiments used different isolines (with and without I-3 and Mi genes) of the tomato cultivar Tasti Lee®. In the first experiment, all four isolines were subjected to two levels of RKN (~10,000 eggs/pot and no eggs) and two levels of Fol (1000 cc soil with 1,000 cfu/g at planting and no Fol), both applied at planting. In the second experiment, the two isolines without I-3 were exposed to the same two levels of RKN as described above and three levels of Fol (50 ml Fol with 1×106 cfu/m at planting, at 10 DAT, and no Fol). Fol reduced root-knot infection and reproduction when both Fol and RKN were inoculated at planting but not when Fol was inoculated 10 days later. Plant damage from Fol was exacerbated in the presence of RKN, especially when both pathogens were present at planting. Isolines with I-3 grew better in Fol-inoculated soil but had no effect when Fol and RKN were both present. Isolines with Mi gene reduced RKN infection and reproduction but did not affect plant damage caused by Fol. In summary, while RKN reproduction was reduced in the presence of Fol, the overall plant damage was more severe when both pathogens were present.

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

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

Characterization and elimination of linkage-drag associated with Fusarium wilt race 3 resistance genes.

KEY MESSAGE: Reducing the size of the I-3 introgression resulted in eliminating linkage-drag contributing to increased sensitivity to bacterial spot and reduced fruit size. The I-7 gene was determined to have no effect on bacterial spot or fruit size, and germplasm is now available with both the reduced I-3 introgression and I-7. Tomato (Solanum lycopersicum) production is increasingly threatened by Fusarium wilt race 3 (Fol3) caused by the soilborne fungus, Fusarium oxysporum f. sp. lycopersici. Although host resistance based on the I-3 gene is the most effective management strategy, I-3 is associated with detrimental traits including reduced fruit size and increased bacterial spot sensitivity. Previous research demonstrated the association with bacterial spot is not due to the I-3 gene, itself, and we hypothesize that reducing the size of the I-3 introgression will remedy this association. Cultivars with I-7, an additional Fol3 resistance gene, are available but are not widely used commercially, and it is unclear whether I-7 also has negative horticultural associations. To characterize the effect of I-3 on fruit size, segregating populations were developed and evaluated, revealing that the large I-3 introgression decreased fruit size by approximately 21%. We reduced the I-3 introgression from 5 to 140 kb through successive recombinant screening and crossing efforts. The reduced I-3 introgression and I-7 were then separately backcrossed into elite Florida breeding lines and evaluated for effects on bacterial spot sensitivity and fruit size across multiple seasons. The reduced I-3 introgression resulted in significantly less bacterial spot and larger fruit size than the large introgression, and it had no effect on these horticultural characteristics compared with Fol3 susceptibility. I-7 was also found to have no effect on these traits compared to Fol3 susceptibility. Together, these efforts support the development of superior Fol3-resistant cultivars and more durable resistance against this pathogen.

Magnesium Oxide Nanomaterial, an Alternative for Commercial Copper Bactericides: Field-Scale Tomato Bacterial Spot Disease Management and Total and Bioavailable Metal Accumulation in Soil.

Copper (Cu) is the most extensively used bactericide worldwide in many agricultural production systems. However, intensive application of Cu bactericide have increased the selection pressure toward Cu-tolerant pathogens, including Xanthomonas perforans, the causal agent of tomato bacterial spot. However, alternatives for Cu bactericides are limited and have many drawbacks including plant damage and inconsistent effectiveness under field conditions. Also, potential ecological risk on nontarget organisms exposed to field runoff containing Cu is high. However, due to lack of alternatives for Cu, it is still widely used in tomato and other crops around the world in both conventional and organic production systems. In this study, a Cu-tolerant X. perforans strain GEV485, which can tolerate eight tested commercial Cu bactericides, was used in all the field trials to evaluate the efficacy of MgO nanomaterial. Four field experiments were conducted to evaluate the impact of intensive application of MgO nanomaterial on tomato bacterial spot disease severity, and one field experiment was conducted to study the impact of soil accumulation of total and bioavailable Cu, Mg, Mn, and Zn. In the first two field experiments, twice-weekly applications of 200 µg/mL MgO significantly reduced disease severity by 29-38% less in comparison to a conventional Cu bactericide Kocide 3000 and 19-30% less in comparison to the water control applied at the same frequency (p = 0.05). The disease severity on MgO twice-weekly was 12-32% less than Kocide 3000 + Mancozeb treatment. Single weekly applications of MgO had 13-19% higher disease severity than twice weekly application of MgO. In the second set of two field trials, twice-weekly applications of MgO at 1000 µg/mL significantly reduced disease severity by 32-40% in comparison to water control applied at the same frequency (p = 0.05). There was no negative yield impact in any of the trials. The third field experiment demonstrated that application of MgO did not result in significant accumulation of total and bioavailable Mg, Mn, Cu, or Zn in the root-associated soil and in soil farther away from the production bed compared to the water control. However, Cu bactericide contributed to significantly higher Mn, Cu, and Zn accumulation in the soil compared to water control (p = 0.05). This study demonstrates that MgO nanomaterial could be an alternative for Cu bactericide and have potential in reducing risks associated with development of tolerant strains and for reducing Cu load in the environment.

Epidemiology, diversity, and management of bacterial spot of tomato caused by Xanthomonas perforans.

Tomato is an important crop grown worldwide. Various plant diseases cause massive losses in tomato plants due to diverse biotic agents. Bacterial spot of tomato (BST) is a worldwide disease that results in high losses in processed and fresh tomato. Xanthomonas perforans, an aerobic, single-flagellated, rod-shaped, Gram-negative plant pathogenic bacterium, is one of the leading causes of BST. Over the past three decades, X. perforans has increasingly been reported from tomato-growing regions and became a major bacterial disease. X. perforans thrives under high humidity and high temperature, which is commonplace in tropical and subtropical climates. Distinguishing symptoms of BST are necrotic lesions that can coalesce and cause a shot-hole appearance. X. perforans can occasionally cause fruit symptoms depending on disease pressure during fruit development. Short-distance movement in the field is mainly dependent on wind-driven rain, whereas long distance movement occurs through contaminated seed or plant material. X. perforans harbors a suite of effectors that increase pathogen virulence, fitness, and dissemination. BST management mainly relies on copper-based compounds; however, resistance is widespread. Alternative compounds, such as nanomaterials, are currently being evaluated and show high potential for BST management. Resistance breeding remains difficult to attain due to limited resistant germplasm. While the increased genetic diversity and gain and loss of effectors in X. perforans limits the success of single-gene resistance, the adoption of effector-specific transgenes and quantitative resistance may lead to durable host resistance. However, further research that aims to more effectively implement novel management tools is required to curb disease spread. KEY POINTS: • Xanthomonas perforans causes bacterial spot on tomato epidemics through infected seedlings and movement of plant material. • Genetic diversity plays a major role in shaping populations which is evident in loss and gain of effectors. • Management relies on copper sprays, but nanoparticles are a promising alternative to reduce copper toxicity.

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

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

Dynamics and Spread of Bacterial Spot Epidemics in Tomato Transplants Grown for Field Production.

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.

First Report of Neopestalotiopsis rosae causing foliar and fruit spots on pomegranate in Florida.

Foliar and fruit spots were observed on pomegranate trees (Punica granatum L.) planted in orchards located at Balm and Zolfo Springs, FL, in 2019. Symptoms on leaves and fruits included dark brown to black irregular spots, ranging from 0.2 to 1.5 cm, with gray centers. Visible pycnidia were present in the center of the lesions. Leaves became chlorotic and prematurely dropped from the trees. In a disease survey performed on 24 pomegranate cultivars, all of the trees were infected and the disease severity ranged from 2 to 80%. The cultivars Bhagwa and Mridula were the most susceptible. Symptoms on the fruit were similar to those on the leaves; however incidence on the fruits was less than one percent. To isolate the pathogen, small pieces (5 mm2) of symptomatic leaves and fruits were excised from the area between diseased and healthy tissue. Excised tissue pieces were surface disinfested in 70% ethanol solution for 30 seconds, followed by 10% sodium hypochlorite (NaOCl) solution for 2 minutes, rinsed in sterilized distilled water three times, dried on a paper towel, placed onto potato dextrose agar and incubated at 25°C under 12-hour photoperiod for seven days. Fungal cultures formed white cottony aerial mycelium with undulated edges and abundant black pycnidia. Four-septate conidia were fusiform or clavate, straight or slightly curved measuring on average 29.1 (23.3- 34.4) µm long × 6.3 (5.1- 6.8) µm wide (n = 100). Conidia had three median cells which were dark brown, with a single basal hyaline appendage, 5.9 (4.1- 7.7) µm long, and two or four (usually three) apical hyaline appendages 20.3 (14.8- 24.5) µm long. Morphological features were consistent with those of the genus Neopestalotiopsis (Maharachchikumbura et al., 2014). Single-spore cultures were obtained and genomic DNA was extracted from seven isolates (one from fruit, GEV3523, and six from leaf, GEV3426 - GEV3431). Internal transcribed spacer (ITS) region, partial sequences of elongation factor (TEF), and ß-tubulin (TUB2) were amplified with the respective primer pairs ITS4 and ITS5, EF1-1567 and EF1-536, and Bt-2a and Bt-2b (Maharachchikumbura et al., 2014). BLAST analysis of the fruit isolate, GEV3523, showed homology of 100% for ITS, 100% for TEF and 99.8% for TUB2 to fungal pathogen Neopestalotiopsis rosae isolate CBS 101057 obtained from a rose plant in New Zealand (Accession Nos. MT587806, MT605118 and MT597152, respectively), whereas the six leaf isolates were 100% identical, GEV3426- GEV3431, showing homology of 100% for ITS, 99.8% for TEF and 98.4% for TUB2 to the same fungal species. Accession numbers: GEV3423 (MT587806, MT605118, MT597152) and GEV3427 (MT587804, MT605120, MT597150); for ITS, TEF and TUB2, respectively. Maximum likelihood phylogenetic analysis, based on the combined alignment of ITS, TEF, and TUB2, differentiated the leaf isolates from other species, suggesting a new Neopestalotiopsis cryptic species. Three-month-old rooted pomegranate cuttings, cultivars Bhagwa and Mridula, were inoculated with two isolates GEV3523 and GEV3527. Each cutting was treated as two distinct halves; one half was wounded by gently rubbing sterilized sand on the leaves to cause abrasions and the other half remained intact. Four cuttings from each pomegranate cultivar were spray inoculated with a spore suspension (105 conidia mL-1) and four control plants were sprayed with sterilized water. The experiment was performed once on each cultivar. Inoculated cuttings were covered with a transparent plastic bag to maintain 100% humidity and incubated for 36 hours at 26°C under 12-hour photoperiod in a growth chamber. Leaves began to show symptoms of small, irregular, brown spots with a gray center three days after inoculation. The pathogen started to produce pycnidia in the center of the lesions one week after inoculation. Symptoms developed on both wounded and unwounded inoculated leaves with 100% disease incidence on both pomegranate cultivars inoculated separately with GEV3527 and GEV3523. Disease severity on wounded leaves ranged from 10 - 25% for 'Bhagwa' and 30 - 60% for 'Mridula' and on intact leaves from 2 - 5% and 5 - 12%, respectively. Furthermore, the fruit isolate, GEV3523, caused an average disease severity of 8 and 3.5 %, on wounded leaves of 'Bhagwa' and 'Mridula', respectively, whereas the leaf isolate GEV3427 caused 19 and 45% disease severity, respectively. No symptoms were observed on control plants and no fungal growth was observed on the re-isolations performed on the control plants. Neopestalatiopsis spp. were re-isolated from leaves fulfilling Koch's postulates. To our knowledge, this is the first report of Neopestalotiopsis rosae infecting pomegranate in Florida as well as in the United States. This pathogen could represent a threat to pomegranate production in Florida due to its ability to cause premature defoliation.

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

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

Molecular Epidemiology of Xanthomonas perforans Outbreaks in Tomato Plants from Transplant to Field as Determined by Single-Nucleotide Polymorphism Analysis.

Outbreaks of bacterial spot on tomato (BST) caused by Xanthomonas perforans are a major concern for sustainable crop production. BST is a common occurrence in tomato transplants grown for field production. We hypothesized that BST outbreaks in commercial fields originate from X. perforans strains inadvertently introduced from commercial transplant facilities. To test this hypothesis, we used a genome-wide single-nucleotide polymorphism (SNP) analysis to characterize X. perforans strains recovered from tomato transplant facilities and fields in commercial production areas. X. perforans strains were isolated from symptomatic transplants prior to roguing at two commercial transplant growers. Then, the same groups of transplants were tracked to commercial fields to recover X. perforans strains from diseased plants prior to harvest. Whole-genome sequencing was carried out on 84 strains isolated from transplant and field plants from Florida and South Carolina. SNPs were called using three reference strains that represented the genetic variation of the sampled strains. Field strains showing genetic similarity to transplant strains had a difference of 2 to 210 SNPs. Transplant and field strains clustered together by grower within each phylogenomic group, consistent with expectations. The range of genetic divergence among strains isolated from field plants was similar to the range obtained from strains on transplants. Using the range of genetic variation observed in transplants, we estimate that 60% to 100% of field strains were an extension of the transplant strain population. Our results stress the importance of BST management to reduce X. perforans movement from transplant to field and to minimize subsequent disease outbreaks.IMPORTANCE Current management of Xanthomonas perforans on tomato plants mainly relies on the frequent application of pesticides. However, the lack of effective pesticides and the development of strain tolerance to certain bactericides limit the ability to control outbreaks in production fields. Better knowledge of probable sources of X. perforans inoculum during tomato production is required to refine management strategies. Tomato plants are typically established in the field using transplants. This study aimed to determine if strains from field epidemics were coming from transplant facilities or resulted from local field outbreaks. The overall goal was to identify potential sources of inoculum and subsequently develop strategies to reduce carryover from transplant production to the field. Our results indicate that tomato producers should shift disease management efforts to transplant facilities to reduce disease in the field. Improved transplant health should reduce the likelihood of bacterial spot outbreaks and subsequently reduce pesticide usage in the field.