Source: SYS_ORG - UNIVERSITY OF GEORGIA submitted to NRP
ONION-IQ (IMPROVE QUALITY): IMPROVING QUALITY, RESILIENCE, SOIL HEALTH AND FOOD SAFETY IN ORGANIC ONION IN THE SOUTHEASTERN AND SOUTHWESTERN U.S.
Sponsoring Institution
National Institute of Food and Agriculture
Project Status
ACTIVE
Funding Source
OTHER GRANTS
Reporting Frequency
Annual
Accession No.
1031157
Grant No.
2023-51300-40913
Cumulative Award Amt.
$3,214,975.00
Proposal No.
2023-04359
Multistate No.
(N/A)
Project Start Date
Sep 1, 2023
Project End Date
Aug 31, 2027
Grant Year
2023
Program Code
[113.A]- Organic Agriculture Research & Extension Initiative
Recipient Organization
SYS_ORG - UNIVERSITY OF GEORGIA
(N/A)
null,GA null
Performing Department
(N/A)
Non Technical Summary
Soil-borne bacterial pathogens are the significant critical limiting factors for sustainable organic onion production in the southern US. Most conventional and organic management practices for pathogen control are either ineffective or have not been rigorously evaluated for these issues. Hence, the University of Georgia and the Texas A&M University will coordinate an integrated multi-regional effort to improve the quality of organically-grown onions by managing soil-borne bacterial diseases (sour skin and slippery skin) through the optimization of nitrogen fertilization and the use of cultural practices (bio-fumigation and soil solarization). We will develop research-based tools to improve soil fertility, soil health, and food safety in organic onions grown in the southeastern and southwestern U.S. Using an integrated approach, we will optimize organic nitrogen-fertilization scheduling in organic onions that will increase productivity, improve soil health (decrease in pathogenic bacterial populations and increase in beneficial microflora), and improve food safety. We will integrate bio-fumigation and soil-solarization with nitrogen fertilization strategies, soil-biome [both micro and macro (insect natural enemies and predators)] assessments, and evaluations of plant-parasitic and beneficial nematodes, weeds and soil-borne bacterial diseases that will improve quality of organic onion production. We will integrate economic assessments, extension and education efforts into each objective that will further strengthen our project. This project meets the legislatively mandated OREI focus areas of "developing and evaluating systems-based integrated management programs to address diseases, nematodes, and weeds related problems for organically grown crops in southern US" and "Conduct on-farm crop, research and development that emphasize innovation for organic farms".
Animal Health Component
80%
Research Effort Categories
Basic
20%
Applied
80%
Developmental
(N/A)
Classification

Knowledge Area (KA)Subject of Investigation (SOI)Field of Science (FOS)Percent
21201101100100%
Knowledge Area
212 - Pathogens and Nematodes Affecting Plants;

Subject Of Investigation
0110 - Soil;

Field Of Science
1100 - Bacteriology;
Goals / Objectives
Objectives: Five-broad and integrated objectives will aim at addressing critical needs for organic onion growers in the southeastern and southwestern U.S.Objective 1:Evaluate cultural practices that reduce soil-borne bacterial pathogens and weeds and increase/sustain growth of beneficial microflora, and predators and natural enemies (20% effort).Objective 2:Evaluate and optimize N-fertilization programs in organic onions that will increase productivity and reduce N loss and improve food safety (20% effort).Objective 3: Integrate activities from objectives 1 and 2 to improve soil health (decrease in sour skin and slippery skin; evaluation of soil-microbiome and predators and natural enemies) and improve food safety (30% effort).Objective 4:Conduct economic analysis (10% effort). Objective 5: Conduct, outreach activities and enhance organic education opportunities (20% effort).
Project Methods
Objective 1. Evaluate cultural practices that reduce soil-borne bacterial pathogens and weeds (digital scouting for weeds and diseases) and increase/sustain growth of beneficial microflora, and predators and natural enemies (20% effort).(i) Assessing robustness of a qPCR assay that differentiate onion-disease-associated vs. saprophytic Burkholderia spp.in soil:(ii) Evaluate cultural practices. We propose to monitor the effects of cultural practices in separate field trials in GA and TX (solarization duration treatments and biofumigant-crop treatments) on the populations of onion-disease-associated Burkholderia spp. in soil. (ii a) Soil-solarization: We will work with organic onion producers from GA and TX to assess if soil solarization can effectively reduce populations of onion-disease-associated Burkholderia spp. in soil. A total of four field trials in organic certified fields (two in each state) over a period of two years (Y1 and Y2) in GA and TX will be conducted.Experimental design: We will evaluate the effectiveness of a 60-day duration of solarization in reducing both bacterial pathogens in soil, thereby reducing sour skin and slippery skin incidence/severity in onion bulbs (pre- and post-harvest).(ii b) Biofumigation: We will assess if biofumigation with two known biofumigant Brassica species, namely Sinapis alba (white mustard) and Brassica juncea (brown mustard) can effectively reduce B. cepacia and B. gladioli pv. alliicola in soil.Experimental design: We will evaluate three cultivars that will include a cultivar each from B. juncea (cv. Kodiak) and S. alba (cv. Ida Gold) and a blend of S.albaandB. juncea (cv. Caliente 199) for their biofumigation abilities to reduce both bacterial pathogens in soil compared with a no treatment control plot and thereby reducing sour skin and slippery skin incidence/severity in onion bulbs (pre- and post-harvest). Hence, we will utilize a randomized complete block design with four replicates/plots. Each field trial will consist of plots with dimension (50 ft × 6 ft) with 6 ft to 10 ft borders on all sides. Each brassica cover crop for biofumigation purposes will be grown and maintained according to the UGA and TAMU Cooperative Extension Recommendations. At flower initiation, these crops will be flail mowed and immediately rototilled in the soil. Prior to mowing, brief over-head irrigation will be employed. Immediately after incorporation, a transparent 30µM-thick, low density polyethylene mulch will be laid in strips using a tractor.Objective 2: We will focus on evaluating and optimizing N-fertilization programs in organic onion that increase productivity, improve soil health, and improve food safety (20% effort). Evaluation of two organic fertilizer sources (PL, and COF) and assessment of cool season N-release patterns.Field sites: There will be four organic certified field sites two will be in GA: Tifton, Athens and two will be in TX (TAMU): Overton, College Station. The project will conduct studies over two consecutive years (Y1 and Y2), providing a total of 4 sites for each year under varied conditions of climate and soil in the southeastern and southwestern US. The field study will evaluate N release from two organic fertilizer sources (PL, and COF) as a function of temporal application patterns. The PL will be locally available broiler litter and the COF will be a Nature Safe 10-2-8.factorial design and 4 replications of each combination plus a non-amended control (36 plots total) (Right). Plots will be 20-ft long (on-raised beds), containing 4 rows each of onions, separated by 10-ft alleys for adjacent plots within the same row.(i) Nutrient assessments: Preplant and at-harvest samples will be collected for routine and micronutrient soil tests for all 4 sites each year (2 TX and 2 GA). Soil samples will be collected at the 0-15 cm and 15-30 cm depths by hand sample probe. Routine soil analyses will be conducted on all samples as well as, SOM (loss on ignition) total C and N (combustion), active C as PMO×C (49), NO3-N and NH4-N (50). Organic fertilizers will be analyzed for total nutrient content (acid digestion and ICP-AES), total C and N, NO3-N and NH4-N soluble carbohydrates, cellulose/hemicellulose, and lignin to provide supporting data for prediction of N mineralization.Influence of organic N-fertilizer sources on food safety; Salmonella Newport survival in soils. BSL2 level-growth -chamber assessment: Food safety aspect in this proposal will be conducted only in GA under controlled BSL2 facility at the Center for Food Safety, UGA, Griffin. This aspect will be led by Co-PIs Dunn and Kumar. The survival of Salmonella enterica serovar Newport (onion outbreak isolate) will be evaluated in soil obtained from organic onion cultivation fields. The soil (100g portions) will be seeded with Salmonella Newport (approx. 2 and 5 log CFU/g). Soil will be amended with raw and treated amendments of poultry origin (used by organic growers). Traceability of the Salmonella strains will be facilitated by developing resistance to ampicillin and streptomycin (100 µg/ml) as described earlier (53). Survival will be evaluated over a duration of 120 days (10-day intervals) using culture-based enumeration on selective media and enrichment-based presence/or absence tests to assess if population declined below the limit of detection. Non-amended soils will be used as a comparative control. Objective 3: Integration of organic production practices (organic N sources, biofumigation, and solarization) on onion-disease-associated Burkholderia spp., crop quality, food safety, nutrient cycling, and the soil microbiome (30% effort).Experimental design: In Y3 and Y4, integrated field trials will be conducted, one in each of the two locations (GA and TX). Three main treatments will be used: biofumigation, solarization, and non-treated control. Within each main treatment, four fertility sub-treatments will be applied: F1). poultry litter (PL) applied at the beginning of the disease-reducing techniques (at planting of mustard for biofumigation, at tarping for the solarization, and to the soil in the non-treated control); F2). PL applied at onion planting, F3) commercial organic fertilizer (COF; Nature Safe 10-2-8 organic mix) applied at onion planting; and F4). non-fertilized control. PL and COF will be applied at a rate equivalent to 200 lbs N/acre.Objective 4: Analyze the economic feasibility of the proposed integrated production approaches for organic onion in Southern U.S. (10% effort) (Greg Colson; Economist).In Y1, a survey will evaluate economic losses due to sour skin and slippery skin in organic onion growers and packers across the southeastern and southwestern U.S. The survey will also gather information on management, variety selection, fertility and irrigation regimes, and organic pest and disease programs in their region. Project team members and stakeholder advisory panel (SAP) will analyze the results. Economic analysis will be calculated annually (Y1 and Y2 assessments of individual components, and Y3 and Y4 assessments of integrated components).Objective 5: Extension, outreach and education (20%): We will extend our information at local and regional meetings focusing on organic onion production, and disease management after the first year of trials in GA and TX. Based on our experience with Extension and outreach (seven Extension specialists on the team, including PD and several Co-PIs), we are confident of identifying numerous opportunities to present information in a face-to-face format and also reach a large contingent of vegetable growers. The event that will capture a large cohort of vegetable growers is the SE Regional Fruit and Vegetable Conference (annually in Savannah, GA) and the GA Organics Conference (annually in Georgia).

Progress 09/01/24 to 08/31/25

Outputs
Target Audience:Primary audience of the project is the organic onion producers in the southern United States. This includes organic onion producers, packers, shippers, agronomists, crop consultants, farm managers, field workers, seed companies, and dealers; and onion storage and shipping/transport personnel and companies. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?Training opportunities were provided for postdocs, graduate students, and undergraduates in plant pathology, genomics, bioinformatics, and extension. These opportunities include conducting thesis or dissertation research, participating in computational, laboratory, greenhouse, or field projects, contributing to scientific or extension publications, and presenting findings to industry and scientific audiences. Undergraduate and graduate students learned to identify sour skin and slippery skin symptoms onleaves and onion bulbs, how to quantify TTG-containing Burkholderia spp., and how to isolate pathogenic bacteria from diseased plant samples and preserve them as purified cultures. How have the results been disseminated to communities of interest?Yes, a total of 15 county extension meetings in Georgia and Texas, along with dissemination of outputs at the national, regional and state research or growers' conferences were done. What do you plan to do during the next reporting period to accomplish the goals?For the next reporting period, we will integrate treatments from objectives 1 and 2 and conduct trials in TX and GA.

Impacts
What was accomplished under these goals? Objective 1: Assessing robustness of a qPCR assay that differentiates onion-disease-associated vs. saprophytic Burkholderia spp.in soil: Soils from GA and TX trials were assessed by TaqMan qPCR for quantities of TTG+ Burkholderia. In GA, trials were conducted for solarization in Tifton, and biofumigation in Athens. In TX, trials were conducted for solarization and fertility. TX samples were DNA extracted in TX and shipped to Athens for qPCR testing. When possible, a positive control (genomic DNA of a TTG+ Burkholderia strain without soil, standardized to 40ng per well) was included to confirm no miscalculation of TTG quantities, and generally adhered to a result of 4.2 log10 cfu/ml. In total, 300 samples have been tested. The use of competitive internal positive control (CIPC) allowed for accurate monitoring of and adjustment for any PCR-inhibition associated with co-extraction of PCR inhibitors from soil. GA soil-solarization: the trial bed was established to test five treatments over four blocks, with three sampling collections on August 12, 2024, and January 7 and May 13, 2025. In January, the soil was sampled at two depths (15 and 30cm), yielding 80 samples tested total. Quantities of TTG remained high (6X log10 cfu/ml) and did not change in these soils regardless of treatment or depth. TX soil-solarization: one trial bed was shared by two groups and thus was tested in duplicate over similar collection dates (August 12 and 22, 2024, November 19 and 14, 2024). A third, single collection was done January 24, 2025. Five treatments were tested over four blocks, for a total of 100 samples tested in this trial. TX showed much lower TTG quantities in their soils compared to GA, ranging from 2-4 log10 cfu/ml. However, qPCR results were extremely variable such that any differences from treatments were lost in analysis between blocks and even replicate PCR wells. The low quantities of detectable TTG also tend to contribute to variability in qPCR results. These issues were not observed in GA soil qPCR analyses. In Georgia, soil solarization influenced the composition and abundance of soil nematode communities across several trophic groups. Among the herbivores, prior to solarization, root-knot, spiral, stubby-root, and ring nematodes were detected, with spiral and ring nematodes showing the greatest abundance. Although significant differences were not observed post-solarization, root-knot nematode juvenile populations were substantially lower in clear plastic treatments compared to bare-ground and black plastic. However, similar trends were not observed for the ectoparasitic herbivores. No significant differences in free-living nematodes, primarily bacterivores and fungivores, were observed between post-solarization treatments and the bare-ground treatment. However numerically, solarization treatments had lower free-living nematode populations than bare-ground, with black plastic treatments exhibiting lower populations than clear plastic. GA Biofumigation: The trial bed was established to test three treatments over four blocks at two depths (15 and 30cm), with four collections on July 1 and October 15, 2024, and January 9 and May 16, 2025, yielding 96 samples tested. The quantities of TTG also did not change in these soils regardless of treatment, however the quantities of TTG increased by nearly a full log10 cfu/ml at the final collection. A full biofumigation trial was conducted in Watkinsville, GA during the 2024-25 growing season. The variety Pacific Gold was grown and terminated at 30-50% flowering in October 2024. Treatments consisted of no mustard control, standard biofumigation and biofumigation with plastic mulch applied immediately after incorporation. Then organic onions were planted and harvested. Weed growth, weed seedbank, disease, yield, survival of food safety pathogens, nematodes, and the Burkholderia pathogen were evaluated. and flavor quality were evaluated. No yield differences were present among treatments but the biofumigant plots had significantly lower cull rates than the control. Furthermore, the bio-fumigant treatments had a significant impact on preplant weeds. At planting, bacterivores and fungivores increased substantially across all treatments, with significant differences observed among treatments (p < 0.05). While mustard-treated plots without plastic exhibited the highest average levels of these decomposer groups, with bacterivores increasing more than sixfold and fungivores nearly twentyfold relative to pre-treatment levels, these increases were not statistically different from the control. However, mustard + plastic plots had significantly higher bacterivore and fungivore populations than the control, suggesting that the combination of biofumigation and plastic mulch may enhance decomposer communities more effectively than biofumigation alone. Objective 2: TX fertility trial: A trial was established to test four treatments over three blocks, with two collections on Feb 24 and June 16, 2025, for a total of 24 samples. Similar to the solarization trial, these samples yielded Burkholderia TTG quantities equivalent to the 2-4 log10 cfu/ml range, but variability between samples and replicate PCR wells yielded no measurable differences between treatments. GA fertility trial: Organic onions were planted in Feb 2025, with one side dress of fertilizers and poultry litter applied in March 2025.A later planting was targeted based on feedback that many organic growers in northern Georgia use intermediate day onion varieties and plant in January or February for a May/June harvest. A 10-2-8 fertilizer along with poultry litter were applied. Ammonia volatilization was measured every 3 days for 14 days after applications, soils sampled for inorganic nitrogen, and onions measured total nutrient uptake. In collaboration with Dr. Laurel Dunn, we have designed a mesocosm experiment to determine the survivability of food borne pathogens that may be introduced with the addition of poultry litter and commercial organic fertilizers that will simulate field conditions in our trial.Onions were harvest in June 2025, graded, and disease incidence determined. Total nutrient uptake and pungency were determined at harvest. Data are being analyzed currently. Economic analysis: To prepare for the completion and availability of second-year field trials conducted by the research team, the necessary data for the timely completion of economic analyses, including market price data for multiple states, input prices, and other production costs, have been collected. As the data from the project team is finalized and ready for use by the economics team, analysis of the separate field trials, in addition to cross-state comparisons, will be conducted. Co-PI Dev Kumar assessed cross contamination of onions by Salmonella entericaNewport from soilin a BSL2 setting. The study indicated a potential for contamination in onion bulbs, stems, and leaves. A comparitive study with onion outbreak isolate is being conducted.

Publications

  • Type: Conference Papers and Presentations Status: Other Year Published: 2024 Citation: Ramachandran, R., and Mowrer, J. 2024. Biofumigation with mustard (BRASSICACEAE) can alter nitrogen mineralization and nitrification of organic fertilizers, University of Agriculture and Veterinary Medicine, Life Sciences for Sustainability Conference, Cluj Napoca, RO
  • Type: Conference Papers and Presentations Status: Other Year Published: 2025 Citation: Ramachandran, R., Mowrer, J., and Isakeit, T. 2024. Does Mustard Biofumigation Inhibit Nitrogen Dynamics ? ASA CSSA SSSA CANVAS International Meetings San Antonio, Texas.
  • Type: Peer Reviewed Journal Articles Status: Published Year Published: 2024 Citation: Paudel, S, Dutta, B., and Kvitko, B. 2024. Onion?pathogenic Burkholderia species: Role and regulation of characterized virulence determinants. Plant Pathology 73: 2281-2297.
  • Type: Peer Reviewed Journal Articles Status: Published Year Published: 2025 Citation: Paudel, S., Franco, Y., Zhao, M., Dutta, B. , Kvitko, B.H. 2025. Distinct Virulence Mechanisms of Burkholderia gladioli in Onion Foliar and Bulb Scale Tissues. Molecular Plant-Microbe Interactions
  • Type: Peer Reviewed Journal Articles Status: Published Year Published: 2025 Citation: Myers, B., Lamichhane, A., Kvitko, B., and Dutta, B. 2025. NLP-like Deep Learning Aided in Identification and Validation of Thiosulfinate Tolerance Clusters in Diverse Bacteria. mSphere 10:e00023-25
  • Type: Conference Papers and Presentations Status: Published Year Published: 2025 Citation: Mitchell, B., Okeh, M. Widmer, J, Cassity-Duffey, K, Coolong, T, and Dunn L. 2025. Control of Escherichia coli TVS 353 in certified organic soils using a biofumigant mustard cover crop. Intl. Assoc. Food Protection Annual Meeting.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2025 Citation: Pulliam, J., Coolong, T., and Cassity-Duffey, K. 2025. Biofumigation blast: Battling soil-borne bacteria organically. Amer. Soc. Hort. Sci. Annual Meeting.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2025 Citation: Mphande, K., Jackson, D., Malik, A.A., and Dutta, B. 2025. Diverse Species of soil-borne Burkholderia are associated with bulb rot in Vidalia onions in Georgia, USA. Annual Plant Health Meeting, Honolulu, HI
  • Type: Conference Papers and Presentations Status: Published Year Published: 2025 Citation: Mphande, K., Jackson, D., Malik, A.A., and Dutta, B. 2025. 2. Soil-borne Burkholderia species are associated with onion bulb rot in Vidalia onions in Georgia. Southeast Regional Fruit and Vegetable Conference, Savannah, GA
  • Type: Conference Papers and Presentations Status: Published Year Published: 2024 Citation: Neupane P., Somenahally A., Gentry T., Aguilar C. 2024. Evaluating disease suppression and soil health properties under organic practices for vegetable production. ASA, CSSA and SSSA International Annual Meetings, San Antonio, TX
  • Type: Conference Papers and Presentations Status: Awaiting Publication Year Published: 2025 Citation: Isakeit, T. 2025. Using Solarization to Control Bacteria-Vectoring Nematode Populations. Annual Plant Health Meeting, Honolulu, HI
  • Type: Theses/Dissertations Status: Published Year Published: 2024 Citation: Myers, B. 2024. Informatics approaches exploring both virulence factors of Pantoea ananatis and the host-resistance to center rot in Allium spp. Ph.D. Dissertation.
  • Type: Other Status: Other Year Published: 2024 Citation: Dutta, B. Perilous Pantoea: Lessons learned in bacterial diseases of onion in Georgia, Kansas State University, Invited presentation.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2025 Citation: Dutta, B. 2025. Diverse virulence factors in a range of bacterial pathogens affecting onion. Plenary Session Speaker, Annual Conference of Indian Phytopathological Society, Nagpur, India.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2025 Citation: Dutta, B. 2025. Update on the management of bacterial diseases on onion. Annual Meeting of the Southeast Fruit and Vegetable Conference, Savannah, GA.
  • Type: Conference Papers and Presentations Status: Other Year Published: 2024 Citation: Dutta, B. Management of bacterial diseases in organic onions. Vidalia Onion Production Meeting, Lyons, GA


Progress 09/01/23 to 08/31/24

Outputs
Target Audience:Primary audience of the project is the organic onion producers in the southern United States. This includes organic onion producers, packers, shippers, agronomists, crop consultants, farm managers, field workers, seed companies, and dealers; and onion storage and shipping/transport personnel and companies.Primary audience of the project is the organic onion producers in the southern United States. This includes organic onion producers, packers, shippers, agronomists, crop consultants, farm managers, field workers, seed companies, and dealers; and onion storage and shipping/transport personnel and companies.Primary audience of the project is the organic onion producers in the southern United States. This includes organic onion producers, packers, shippers, agronomists, crop consultants, farm managers, field workers, seed companies, and dealers; and onion storage and shipping/transport personnel and companies. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?CO-PIs Kvitko, Isakeit, Mowrer trained their graduate students in experimental design and helped them in establishing field trials. Co-PI Kumar's graduate student was trained in basic culturing and detection methods for Salmonella. The student was trained on the analysis of growth curve data to calculate lag phase duration, growth rate, maximum cell growth. Student was trained on electroporation and methodology to evaluate plasmid stability in both growth medium and environmental matrices The graduate student presented this data at three conferences- AOAC, ASM and IAFP. How have the results been disseminated to communities of interest?PD-Dutta and Co-PD Coolong extended some of the initial outcomes of the project at various conferences including Southeast Fruit and Vegetable Conference, Annual Meeting of American Phytopathological Society and American Society of Horticultural Sciences. Co-PD Coolong and Co-PI Cassity-Duffey presented information on mustard performance and biofumigation practices in organic systems at two winter cover crop programs (Feb. 16, Gulf Shores Alabama and March 15, Griffin GA) with approximately 100 attendees. What do you plan to do during the next reporting period to accomplish the goals?We plan to finish our on-going field trials related to objectives in Georgia and Texas. Data obtained will be analyzed and presented at various meetings and conferences. In the meantime our stakeholder advisory panel will be meeting on Jan 9, 2025 to provide their assessments on our Year 1 trials.

Impacts
What was accomplished under these goals? To develop a qPCR assay to quantify Burkholderia TTG in onion field soils, we first designed a primer and probe set that would amplify within the TTG region for all common genotypes under TaqMan conditions. We assembled a collection of Burkholderia spp. type and field strains to verify their TTG status. From this group we selected three strains, LMG 1222 (B. cepacia, a TTG+ type strain), 20GA0385 (B. gladioli pv alliicola, a TTG+ well-studied GA field strain) and LMG 10929 (B. vietnamensis, a TTG- type strain) as controls for assay development. Genomic DNA was isolated from HortHill Farm, UGA, Tifton field soils with no history of onion cultivation spiked with standardized concentrations of control inocula using Qiagen's DNEasy PowerSoil Pro kit. The two TTG+ strains showed titratable quantities via qPCR, and the TTG- strain showed amplification on par with un-spiked soil. Since DNA isolation from soils tend to carry over inhibitors (soilborne chemicals which impede amplification), we incorporated the use of a competitive internal positive control (CIPC) to measure shifts in amplification due to soil quality. The CIPC will allow us to mathematically correct under-estimations of TTG quantities due to PCR inhibition. The CIPC is a synthetic, short double-stranded DNA fragment of Green Fluorescent Protein DNA flanked with recognition sites for the TTG forward and reverse primers. The TaqMan probe for the CIPC is specific to GFP and labelled with a different fluorophore than the TTG probe. Thus, we can multiplex detection of both TTG and CIPC in the same PCR reaction. We are currently optimizing these two probes and the reaction to ensure they do not interfere with each other's detection. We also are currently testing a variety of onion and non-onion field soils against a standardized dilution series of bacterial gDNA to quantify TTG+ Burkholderia within each field. This qPCR assay will next be used to track changes in TTG+ Burkholderia populations in fields undergoing elimination treatments (fumigation and solarization) in support of this project. Co-PI Mowrer in Texas has established solarization trial at College Station, Texas. Soil is mapped as a Chazos loamy fine sand. Activities under this objective include tillage to remove weeds, loosen soil, and smooth field for plastic installation. Field has been squared and plots laid out and labelled according to plant. Nematode soil sampling were performed. Soil sampling for nutrients, weed seed bank, and DNA were performed. Overhead irrigation has been installed. Inoculation with onion bulbs is pending as is installation of the plastic. Starting in September, 2023 Co-PD Coolong evaluated eight mustard varieties for suitability for biofumigation in replicated plots. Plots were 150-ft long each so that termination and incorporation could better simulate biofumigation processes. Because of the tight production window and high temperatures during the cover crop growing period (Aug-Oct) we needed to identify a mustard variety that could germinate, grow, mature and produce significant biomass in the early fall in Georgia. We identified Pacific Gold as the variety with the best combination of maturity index, biomass, sulfur accumulation (as a surrogate for glucosinolate concentrations). The trial repeated in Spring 2024 with similar results. Organic biofumigation trials are currently underway in Fall 2024 using the selected mustard. Co-PI Mowrer in Texas conducted a soil incubation study on three soils to assess the effect of reagent grade allyl isothiocyanate and two mustard meals at multiple rates on the mineralization and nitrification of organic N from organic fertilizer. Data shows clear inhibition of both mineralization and nitrification and that this inhibition is dependent upon soil texture. Trials are underway in Fall 2024 to evaluate PL and COF in organic production systems in Georgia. Greenhouse trials are in progress to evaluate poultry manure mineralization at Texas A&M AgriLife Research, Overton TX. The goal is to establish N availability timeframe for both 3 and 5 ton/ha PM. We evaluated and standardized methodologies to extract, identify, and quantify plant-parasitic nematodes and free-living nematodes. Preliminary soil samples were collected from fields naturally infested with nematodes. Soil sampling involved collecting 8-10-inch-deep soil cores from 10-15 sampling points to generate each composite sample. The samples were then transported and stored at 4°C until processing. Nematodes were then extracted from each soil samples using the centrifugal sucrose floatation method. Plant-parasitic nematodes were then identified and enumerated to genus level, based on morphological features, under the microscope. We were also able to identify the free-living nematode to their trophic level. Additionally, we have evaluated several free-living nematode identification keys and found that the web-based "Interactive Diagnostic Key to Plant Parasitic, Free-living and Predaceous Nematodes" developed by the University of Nebraska Nematology Team can be utilized to identify free-living nematodes to their family or genus level. Thus far, we collected pre- fumigation and pre-solarization soil samples from each of the trial plots. Nematodes were extracted from these samples and currently being processed following the procedures described above.Utilizing taxonomic keys is time consuming and tedious for processing large number of samples. Hence, we are working on a functional group identification scheme and also preparing a workflow to metabarcoding soil DNA samples and DNA extracted from nematodes isolated soil to compare and use for free living nematode identification.To accomplish this, we first reviewed the literature on metabarcoding primers currently in practice for nematode eDNA work, and optimized them in the lab during May-July 2024. The primers are amplifying common entomopathogenic nematode (EPNs) species. The next step is creating mock communities in soil extractions, and isolating DNA from these standardized soil extractions. We will use a combination of EPN species, free-living, and plant pathogenic species in these nanocosm tests. Following results, we will conduct a controlled set of positive controls for many different species of common nematodes. Co-PI Schmidt has also worked with a variety of arthropod primers, and in the last sequencing runs has observed a few primer sets that are capable of amplifying both microbial (bacterial and fungal) DNA. These will be applied to soil samples and a set of arthropod specific primers will be used to detect changes in soil arthropod communities in relation to biofumigation, using eDNA as an indication of activity of soil arthropods in relation to treatments. The work to characterize Burkholderia species in plots under solarization and biofumigation has already started. We are in the process of extracting microbial genomic DNA from pre-planting/pre-treatment samples. The characterization of the Bulkholderia species will be carried out with amplicon sequencing targeting the recA gene. The California onion outbreak strain was obtained and characterized for its growth rate through comparative studies with cattle isolate of S. Newport. Plasmid (pGFPuv) stability was evaluated in this strain both with and without the presence of selective pressure (ampicillin) in growth medium. Further, the plasmid stability and survival of the strain in soil was evaluated through comparative survival studies with a cattle isolate of S. Newport. The soil used for this experimental study was from an onion farm.

Publications

  • Type: Journal Articles Status: Published Year Published: 2024 Citation: Paudel, S., Zhao, M., Dutta, B., and Kvitko, B. 2024. Thiosulfinate tolerance gene clusters are common features of Burkholderia onion pathogens. Molecular Plant Microbe Interactions 37: 507-519.
  • Type: Journal Articles Status: Awaiting Publication Year Published: 2024 Citation: Paudel, S., B. Dutta, and B. Kvitko. 2024. Onion?Pathogenic Burkholderia Species: Role and Regulation of Characterized Virulence Determinants. Plant Pathology
  • Type: Conference Papers and Presentations Status: Published Year Published: 2024 Citation: Neupane P., Somenahally A., Gentry T., Aguilar C. 2024. Evaluating disease suppression and soil health properties under organic practices for vegetable production. ASA, CSSA and SSSA International Annual Meetings, Nov 10-12, 2024. San Antonio, TX.