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.
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