Location: Plant Genetic Resources Conservation Unit
Project Number: 6046-21000-013-040-S
Project Type: Non-Assistance Cooperative Agreement
Start Date: Aug 1, 2024
End Date: Aug 1, 2025
Objective:
The primary objectives of this project are aimed at comprehensively evaluating the health of okra germplasm:
1) To evaluate the presence of seed-borne pathogens on okra germplasm through blotter tests and assess their damage to seed germination and seedling growth.
2) To assess the effectiveness of published PCR and qPCR systems and identify reliable techniques for rapidly detecting major seed-borne pathogens in okra seeds.
3) To screen a portion of the USDA/ARS collection of okra germplasm (~300 accessions) using PCR and/or qPCR techniques identified in Objective 2. Emphasis will be placed on accessions introduced from countries outside of the United States that have not been previously grown for seed regeneration.
Approach:
Two-hundred seeds of selected okra accessions (total approximately 300 accessions) from the USDA Genebank will be packaged and sent to the Cooperator's plant pathology lab. A total of 25x4 seeds (i.e., 25 seeds per replication with 4 replications) for each accession will be used for blotter tests to assess seed borne pathogen presence. Pathogens cultured on the seeds will be isolated, purified, and identified based on microscopy observation and ITS/16S rDNA sequencing. Seed germination will be recorded, and seedling disease severity will be evaluated based on the published scale.
The genomic DNA of targeted pathogens, along with that of other pathogens and non-pathogens isolated from the seeds, will be amplified to evaluate the specificity of previously published PCR/qPCR assays. Identified assays will then be used to evaluate the presence of major seedborne pathogens on the rest of 25x4 seeds by amplifying seed-extracted DNA. Samples testing positive for pathogens, even if occurring in one replication, will be flagged in the GRIN database as 'infected'. The inventory will no longer be distributed, and the seed will be subject to appropriate treatment. Inventories testing negative will be designated as such in the database. It is important to note that in some cases, compounds within the seeds can inhibit DNA amplification, resulting in false negatives. Therefore, this work utilizes commercial DNA extraction kits, which have been reported to efficiently remove most PCR inhibitors.
