Progress 09/15/23 to 09/14/24
Outputs
Target Audience:Our target audience is the fresh market and processing tomato industry that represent a $1.9 billion (NASS, 2020) specialty crop planted to 330 thousand acres across 18 states, and the allied academic and professional plant health extension specialists that cater to this and other specialty crop industries. Changes/Problems:Major project delays have occurred due to personnel changes caused by departures and retirements. Many of these occurred in the first year of the project (2022-23) but additional impacts have lingered into the second year (2023-24) of the project. Changes in project personnel include: UF tomato breeder Co-PDs Samuel Hutton left UF in Nov'23 and was replaced by Dr. Jessica Chitwood-Brown in Jan'24. Although our tomato geneticist CoPD Tong-Geon Lee left UF at the initiation of the project in Sep'22, his replacement Dr. Anuj Sharma joined UF in Dec'23 and will now join the project working on Objective 2. Co-PD Trent Blare left UF in Oct'23 and was originally replaced by Dr. Gilly Evans in Dec'23, but will now be replaced by the new Agricultural Economist, Dr. Malek Hammami, who joined UF in Jun'24 and will take on project duties pertaining to Objectives 4 and 5. Co-PI Dan Egle retired in Jan'24 and has since been replaced by his successor, Dr. Cesar Escalante-Guardad at Purdue University. An important member of our research team, Co-PI Sally Miller, also retired Dec.'23, with her project responsibilities picked up by Co-PI Jonathan Jacobs; however, we anticipate that Dr. Miller's successor will eventually join the team in 2025. Co-PI Gireesh Rajashekara relocated his program from the Ohio State University to the University of Illinois in May 2024, but has continued his efforts pertaining to Objective 3. Additional delays have occurred due to tropical weather events in Florida. In 2023, Hurricane Idalia impact field trials conducted at the Gulf Coast Research and Education Center, spreading marked-Xep strains and causing severe-wind damage to BST field trials that delayed progress in Objective 1 to measure spread. Additional tropical impacts occurred in September and October of 2024 with Hurricanes Helene and Milton that destroyed ALL tomato field trials in progress at the Gulf Coast Research and Education Center which was a major setback to on-going trials for Objectives 1 and 2. In all cases, we are still using initial data collected from trials before and following tropical events, but such weather events still represent significant setbacks to the project; especially breeding efforts in Objective 2. At this point, the personnel changes and weather events have not changed project objectives, but have significantly delayed our original project timeline. We anticipate requesting a No Cost Extension to give us the additional time to complete proposed activities. What opportunities for training and professional development has the project provided?All graduate students and post-doctoral researchers participate in monthly and annual project meetings, and have presented information at several regional and national meetings. We have trained 6 graduate and two undergraduate students in Plant Sciences; the recognition of BST caused by xanthomonads; the design and use of impact and cyclonic samplers; genome sequence analysis; and the design of field and greenhouse experiments. Trained 7 graduate students in genome sequence analysis and 1 in the cloning of bacterial effector genes. Trained one graduate student in developing plasmid constructs for gene editing. Three post-doctoral researchers are currently working on this project to evaluate seed treatments. One post-doctoral researcher (1 FTE) is currently working on this project to generate derivatives of lead compounds and evaluate them for seed and seedling treatment. Two graduate students and a post-doctoral researcher are conducting grower/industry surveys. Six extension agents were trained how to identify and manage bacterial diseases in Puerto Rico. How have the results been disseminated to communities of interest?Project results have been presented at various regional and national venues including:Tomato Disease Workshop, West Lafayette, IN, Nov. 2023;Plant Health Conference, Memphis, TN, Jul. 2023;Annual Meeting of the Southern Division of APS, Columbia, SC, Mar. 2023;Two workshops were offered in Puerto Rico regarding BST management and for surveying growers as part of Obj. 4. In addition, PD Vallad's team has worked directly with a grower cooperator to track a natural BST oubreak at a commercial transplant operation, which has provided an immediate benefits to the stakeholder and the project. Members of ourstakeholder advisory committee have also participated in surveys and in the last Annual Project Meeting in Jul. 2023. What do you plan to do during the next reporting period to accomplish the goals?Per Objective 1: Continue regional BST surveys to monitor outbreaks. Design, build and deploy tower-configured cyclonic air-sampler arrays to measure and model spread of Xep in open-field and commercial transplant facilities during cultural operations; and environmental conditions that favor bioaerosol spread. Complete assessment of Xep and Xhg survival between cropping seasons in FL and OH. Determine survival of Xep and Xhg on inanimate surfaces and weeds common to open-field and transplant facilities. Utilize whole-genome sequencing and metagenome sequencing approaches to detect and monitor spread of BST xanthomonad populations throughout production systems. Per Objective 2: Continue screening the transformations for mutations in candidate genes for bs6 and bs8 resistance and screening the mutants for resistance phenotype. Continue screening wild tomato germplasm with polymorphisms in bs6/bs8 gene. Advance of PTR1 and Zar1/Jim2 resistance introgression lines and develop a trangenic PTR1 resistant lines. Continue advancing bs5, with Rx3 and Xv3/Rx4 major genes and the QTL11 sources of resistance in regionally adapted tomato germplasm. Develop constructs with candidate Xep Type III bacterial effectors to initiate screening of tomato germplasm for novel sources of resistance. Per Objective 3: Conduct testing of both X2-R4 and X12-R7, nano-particles, and novel cold-plasma and ionized hydrogen peroxide against BST on tomato seed and seedlings in Ohio, Florida, and Georgia. Per Objectives 4 & 5: Conduct in-person visits to administer surveys to local growers, including information for economics. Start development of an interactive online eLearning simulation of case study scenarios from field and transplant operations in US and Puerto Rico.
Impacts
What was accomplished under these goals?
Per Objective 1: A series of field (5) and greenhouse trials (5) conducted in Florida and Ohio tracked the movement of marked X. euvesicatoria pv. perforans (Xep) strains in bioaerosols with active 3D-printed cyclonic air samplers and with traditional passive sampling methods. Florida tomato field trials demonstrated the presence of viable Xep in bioaerosols at levels up to 2.5, 5.5, and 3.5 Log10 CFU/m3 of air in respect to active air sampling prior, during, and following a simulated pesticide application to staked tomato plants performed with a high-volume tractor sprayer (100 gal/A @210 psi). Additional passive air sampling performed with open 100 mm petri-dishes with semi-selective media placed perpendicular to field tomato rows recovered Xep up to 7 meters from infected plants. Florida greenhouse trials also demonstrated the presence of Xep in bioaerosols at levels up to 2.5 and 3.5 Log10 CFU/m3 of air collected prior and during overhead irrigation of infected tomato seedlings using active 3D-printed cyclonic air samplers. In addition, high levels of epiphytic Xep were recovered from aymptomatic tomato leaves. Furthermore, we developed a new medium for more precise and sensitive isolation of xanthomonads from field samples down to 1000 CFU. This allowed us to not only track our marked strains but also natural inoculum sources. Results demonstrate the potential importance of bioaerosols and production practices that generate bioaerosols in spreading Xep. Two Florida field trials performed in the winter/spring and summer/fall fallow periods demonstrated the ability of Xep to survive up to 6 and 3 months on infected tomato debris placed at the surface or 15 or 30 cm below the soil surface. Initial studies of Xep survival on inanimate surfaces tested aluminum, finding only a 2-log reduction in recovery over a 10-day period. In Ohio, we were able to track incidence in the field for inoculated Xathomonas hortorum pv. gardneri (Xhg) and other BST agents. Detection of BST strains (e.g. Xep and Xhg) beyond marked strains suggests there are natural field sources in Franklin County, Ohio. We collected approximately 30 field isolates that represent the current natural diversity and are currently being characterized genomically and phenotypically from the Ohio production system. Some of these isolates were from weedy hosts displaying bacterial spot, and we will determine if they are similar to isolates associated with BST epidemics. Similar BST strain collections were conducted during field and transplant production in Georgia, Florida, Indiana, Louisiana, and Puerto Rico; whole-genome sequencing of representative strains are in progress. Our next steps include determining the spatial spread of the bacterial outbreaks in greenhouses that lead to field-level epidemics. In Florida, a BST outbreak that occurred from April to September at a commercial transplant facility was investigated; strain collections from tomato seedlings, culled seedling trays, and bioaerosols are being analyzed. We are also determining the origins of field inoculum that were not inoculated. In Ohio, we are also currently measuring the overwintering of both Xep and Xhg at Waterman farm. The results from Objective 1 underscore the multiple potential sources and pathways for Xanthomonas contamination in tomato production systems. We are developing a genomics-based microbiome pipeline to differentiate epidemiologically relevant lineages of BST pathogens. This information is crucial for developing targeted best management practices (BMPs) to reduce the risk of BST outbreaks and will be used to inform the development of a decision support system (DSS) for BST management and guide extension efforts to promote the adoption of BMPs by tomato growers. Per Objective 2: We continued evaluation of bs5 events against BST caused by Xep and Xhg in field studies, and are employing a Marker-Assisted Selection (MAS) pipeline to combine bs5 events from fresh-market background with Rx3 and Xv3/Rx4 major genes and the QTL11 in processing tomato germplasm. Mutagenesis of candidate pepper genes for bs6 (3 candidates) and bs8 (one candidate) resistance was carried out and transformants are undergoing screening, as well as the screening of tomato germplasm with polymorphisms in bs6/bs8 candidates. We evaluated several Type III bacterial effectors conserved among global Xep populations for effect on in planta bacterial growth. Per Objective 3: A total of 73 potentially Xep-infested seed lots were generated (n:20 tomato seed lots and n: 53 pepper seed lots). Individual seeds (n:20 seeds per seed lot) are being screened for presence of Xanthomonas perforans. Positive seed lots are going to be subjected to novel physical (Cold Plasma) and chemical (Ionized Hydrogen Peroxide) seed treatments. Inoculation of tomato blossoms with nanoparticles and Xep were conducted in field and greenhouse and fruit harvested. Fluorescence microscopic analysis is in progress to localize nanoparticles within different parts of seed. In separate studies, tomato seed, artificially vacuum-infiltrated with Xep and with the nanomaterials (Kocide - 3000, Mg-Cu, FQ-Cu and MgO). A 500 ppm of Mg-Cu, FQ-Cu and MgO significantly reduced Xep populations compared to Kocide 3000 and Xep inoculated controls across two sets of experiments. Subsequent seed germination rates were higher in treated compared to non-treated Xep inoculated seed. For small molecule development, 15 derivatives of X2, X5, and X12 were synthesized in the lab by using imidazole, carbazole, oxazole, pyrimidine, or piperidine scaffolds in basic condition through multiple steps. The synthesized small molecules were confirmed through thin-layer chromatography (TLC), nuclear magnetic resonance (NMR), and mass spectrometry (MS). X2-R4 and X12-R7 demonstrated better inhibition activity against Xhg compared to parent X2 and X12. Large-scale preparations of both X2-R4 and X12-R7 are now available for subsequent seed and seedling testing with PI groups in Ohio, Florida, and Georgia. Per Objectives 4 & 5: Conducted initial grower & industry surveys for perceptions to BST and for assessing economic impact of BST outbreaks in commercial production. These ongoing surveys are needed to explore perceptions towards Best Management Practices (BMPs)for tomato growers in the Midwest and Southeast United States and Puerto Rico; and to subsequently explore the barriers and opportunities for subsequent BMP adoption. Conducted an R2M expert knowledge elicitation with a small group of TomSPOT BST experts and with a small group of additional industry experts. Collected information necessary to lay the groundwork for developing a within-operation decision support system to address priorities for BST management in seedling and open-field production. Consolidated necessary information for project investigators and other baseline information to begin working on TomSPOT website.
Publications
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Progress 09/15/22 to 09/14/23
Outputs
Target Audience:Our target audience is the fresh market and processing tomato industry that represent a $1.9 billion (NASS, 2020) specialty crop planted to 330 thousand acres across 18 states, and the allied academic and professional plant health extension specialists that cater to this and other specialty crop industries. Changes/Problems: Changes in project personnel: Co-PDs Hutton, Lee, and Blare have left UF; Replaced by Sharma (Oct'23); Evans/Ballen (Nov'23); Chitwood-Brown (Jan'24). Anticipate retirement of Co-PIMiller at end of December, project responsibilities to be managed by Co-PI Jacobs. Per Objective 1: Were unable to recover viable bacteria from impact samplers, so pivoted to cyclonic samplers. May revisit the use of impact samplers as a tool for PCR-based detection, but not suitable for tracking strains. What opportunities for training and professional development has the project provided? Trained 2 graduate students in the design and use of impact and cyclonic samplers; genome sequence analysis; and design of field experiments (Ramirez & Giles - UF). Trained 1 graduate student in genome sequence analysis and cloning of bacterial effector genes (Apekshya - UF). Trained one student in developing plasmid constructs for gene editing (Mousami - UF). One post-doctoral researcher (2 FTE) is currently working on this project to evaluate nanoparticle seed treatments. One post-doctoral researcher (1 FTE) is currently working on this project to generate derivatives of lead compounds and evaluate them for seed and seedling treatment. One post-doctoral research associate and three under-graduate students are currently involved in various parts of project. How have the results been disseminated to communities of interest? Demonstration of cyclonic sampler to growers at commercial transplant operation and to stakeholders at the 2023 Florida AgExpo (Nov. 9, Wimauma, FL). Presentation to stakeholders at Florida Tomato Institute (Sep. 13, Labelle, FL) and Gadsen Tomato Forum (Dec. 7, Quincy, FL). Developed protocol for seed disinfection using vacuum infiltration approach and implications of this approach will be discussed with growers and interested stakeholders. We plan to present a poster at the CFAES, OSU annual meeting in spring 2024 on the Synthesis of the active and lead novel small molecule inhibitors and screening them according to preliminary structure-activity relationships (SAR) based on the inhibitory effects of the compounds. Shared project outcomes at 19 stakeholder meetings; 10 county agent trainings; 6 regional meetings, and 1 national meeting. What do you plan to do during the next reporting period to accomplish the goals?Per Objective 1: Large-scale strain collection at regional grower locations using developed cyclonic samplers to identify possible outbreaks. Conduct larger scale evaluation of cyclonic samplers at select grower sites to ascertain Xanthomonas strain movement. Xanthomonas survival on common surfaces, plant debris, and common weeds. Per Objective 2: Screening the transformations for mutations in candidate genes for bs6 and bs8 resistance and screening the mutants for resistance phenotype. Screening wild tomato germplasm with polymorphisms in bs6/bs8 gene. Advancement of PTR1 and Zar1/Jim2 resistance introgression lines. Development of trangenic PTR1 resistant lines. Per Objective 3: Process infested tomato seeds for evaluation of novel physical (Cold plasma) and chemical (Ionized Hydrogen Peroxide) seed treatments; Implement field trials for tomato blossom protection using nanoparticles to prevent Xanthomonas infestation. Standardize the concentrations of materials of interest (nano particles, biocontrol agents, small molecules) for seed coating experiments. Evaluate coating and pelleting of tomato seeds using nano particles, biocontrol agents, small molecules to protect seeds against Xanthomonas tomato leaf spot agents and to promote growth and yield of tomato crop. Synthesize other derivatives of X2, X5, and X12 derivatives and screen them for inhibitory effects [minimal inhibitory concentration (MIC), minimal bactericide concentration (MBC), inhibitory concentration (IC) 50%] on the four species of Xanthomonas (X. gardeneri, X. performance, X. vesicatoria, and X. euvesicatoria) causing BST, using a 96 well plate-based assay and Tecan Sunrise plate reader. Prioritize and sort the synthesized small molecules for establishing preliminary structure-activity relationships (SAR) based on the inhibitory effects of the compounds. Modify the lead derivatives by making them acidic (HCl, H2SO4) and basic (e.g., phenolic OH salts, NaOH, Ca(OH)2) salts to improve ionization and subsequent water solubility. Modify or conjugate the derivatives with hydrophilic groups to enhance water solubility. Treat the selected active synthesized small-molecule inhibitors on naturally and artificially infested seeds through the liquid seed priming process. Encapsulate the selected active compounds in nanoparticles to enhance the efficacy of the inhibitors for seed treatment against the Xanthomonas spp. Per Objectives 4 & 5: Complete all grower surveys and Expert Knowledge Elicitation survey Conduct tomato production economics survey Initiate TomSPOT website to the general public
Impacts
What was accomplished under these goals?
Per Objective 1: Sampledfarm and transplant operations in Georgia, Ohio, Indiana, Puerto Rico, Louisiana, Florida;Strains recovered 34 from GA, LA, FL, OH, and PR;Weeds evaluated for bacterial spot in OH Evaluated impact and cyclonic samplers for collecting viable bacteria from aerosols; found impact samplers were unfit for collecting viable bacteria, while cyclonic samplers were effective at collecting bacteria in aerosols. Evaluated the use of cyclonic samplers for collection of bacteria in aerosols in a series of greenhouse (2 trials), transplant facility (1 trial), and experimental field (1 trial) Dutta lab characterized Pseudomonas capsici isolates collected from tomato leaves in Georgia and compared them with historical isolates. The whole-genome comparison revealed that 13 P. capsici strains from diverse isolation sources that were curated in NCBI were indeed P. capsici indicating a potential wide-host range for this bacterial species. Our greenhouse-based host-range assay also indicated that P. capsici strains were pathogenic on pepper, tomato, eggplant, cabbage, lettuce, and watermelon corroborating a wide-host-range. A phylogenetic tree inferred from the whole genome sequence data showed that the P. capsici strains from Georgia (tomato) were genetically diverse, and were closely related to tomato P. capsici strains from Florida. Per Objective 2: Evaluatedof bs5 events for resistance/susceptibility to X. perforans and X. hortorum pv. gardneri. Expanded the amplicon-based genotyping by sequencing platform from 384 SNPs to >1,000 SNPs. Developeda Marker-Assisted Selection (MAS) pipeline to combine bs5 events from fresh-market background with Rx3 and Xv3/Rx4 major genes and QTL11 in processing tomato germplasm. Conducted two rounds of selection with background genome selection. Mutagenesis of candidate genes for bs6 (3 candidates) and bs8 (one candidate) resistance was carried out. Transformants (NN plants) are undergoing screening. Identified tomato germplasm with polymorphisms in bs6/bs8 candidates. Per Objective 3: Xanthomonas perforans infested tomato seeds are being generated in greenhouse for evaluation of novel physical (cold plasma) and chemical (Ionized Hydrogen Peroxide) seed treatments. Dutta lab is currently optimizing the delivery of antimicrobial nanoparticles to seed via-tomato blossoms. This process may potentially prime these seeds before challenge inoculation with X. perforans or P. capsici. Standardized vacuum infiltration method to introduce materials of interest inside the seeds. In this procedure, tomato seeds were submerged inside a beaker containing the solution of interest and the beaker was attached to vacuum pump to create vacuum inside the beaker. Nano materials (MgO, fixed quat 'Cu', and Mg-Cu) of various concentrations were evaluated for their effect on germination rate of seeds after 7 and 10 days of infiltration with the untreated seeds as control. Different concentrations of MgO (1% and 2%), Mg - Cu (50, 100, 200 and 500 ppm) and fixed quat 'Cu (50, 100, 500, 1,000, 5,000 and 10,000 ppm) were used for vacuum infiltration in this study. All the experiments were conducted twice with three replications per each treatment. Results showed that, 1 % MgO significantly increased the tomato seeds germination as compared to 2 % and control seeds. In addition, vacuum infiltration of 500 ppm of fixed quat 'Cu' showed significant enhancement of germination of tomato seeds as compared with other concentrations and control.Moreover, 100 ppm and 50 ppm in first and second set of Mg - Cu showed higher tomato seed germination. The new synthetic medicinal lab was set up for the synthesis of active analogs of X2, X5, and X12 derivatives which can inhibit Xanthomonas leaf spot diseases. The necessary range of chemicals, including various reagents and solvents, was purchased.The small-molecule inhibitors of the X12 series, including X12A, X12C, X12D, and others, were synthesized in the lab by the reaction of different phenol derivatives and 1-chloro-3-(1H-imidazol-1-yl)propan-2-ol derivatives in basic media. The synthesized small molecules were confirmed through thin-layer chromatography (TLC), nuclear magnetic resonance (NMR), and mass spectrometry (MS). For the purpose of encapsulating the lead molecules with nanoparticles, various blank nanoparticles such as chitosan (size: ~391 nm), chitosan alginate (size: ~557nm), and bovine serum albumin-chitosan nanoparticles (size: ~601 nm) were prepared by optimizing in different conditions. The sizes were analyzed through a zetasizer. The naturally infested tomatoes with Xanthomonas gardeneri and Xanthomonas perforans were harvested in the field with the help of Prof. David Francis. The seeds were extracted from the infested tomatoes. The extracted seeds were dried at room temperature for 72 hr and stored at room temperature in vaccum desiccator. Per Objective 4: Developed grower survey instrument and received ICBR approval. Developed Expert Knowledge Elicitation instrument, awaiting ICBR approval. Per Objective 5: Collected initial materials for development of TomSPOT website; developed website design. Shared project outcomes at 19 stakeholder meetings; 10 county agent trainings; 6 regional meetings, and 1 national meeting.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2023
Citation:
Zhao, M., Gitaitis, R.D., and Dutta, B. 2023. Characterization of Pseudomonas capsici strains from pepper and tomato. Frontiers in Microbiology 14:1267395 doi: 10.3389/fmicb.2023.1267395.
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