Location: Northwest Irrigation and Soils Research
Project Number: 2054-21220-006-002-T
Project Type: Trust Fund Cooperative Agreement
Start Date: Jul 1, 2023
End Date: Feb 28, 2025
Objective:
Determine the impact of Vibrio gazogenes (Vg)-derived prodigiosin-rich extract (Vg-P) to inhibit sugar beet root rot or restrict L. mesenteroides growth in factory processed sugar beet extracts.
Approach:
A total of 40 randomly picked previously harvested sugar beet roots (cultivar B-7; Betaseed Inc.; Kimberly, ID) will be divided into 5 experimental groups (with n=8 per group) out of which 4 groups will be artificially co-infected with R. solani and L. mesenteroides using protocols developed by ARS. Briefly, a cork borer will be used to create a 10 mm diameter x 24 mm deep hole. A 0.1 mL of a 108 cfu mL-1 suspension of L. mesenteroides inoculum prepared using yeast dextrose calcium carbonate agar 28 will be introduced in that hole along with a 2 x 2 mm piece of mycelial mass of R. solani grown in potato dextrose broth for 10 days at 22°C inside a bench top shaker. After inoculation, the plug will be re-placed and sealed with petroleum jelly (Unilever, Greenwich, CT). Out of the 4 infected groups, one will remain untreated (positive control for infection) and the other 3 treatment groups will receive three different doses of treatments. The positive control group will just receive an equal volume of the DMSO (vector). The fifth group will be a non-infected and untreated control group (the negative control group) which will not be infected but will receive a mock inoculation with 0.1 mL of sterile water and a mock treatment with dimethyl sulfoxide (DMSO) (vector).
Root rot will be evaluated at 2 months post-inoculation by comparison of the extent of fungal growth, root surface discoloration, root weight loss and root sucrose loss in the treated and untreated sugar beets using Northwest Irrigation and Soils Research Laboratory (NWISRL) standardized protocols. Briefly roots will be weighed, and a surface rot evaluation (percent of discolored root surface area) will be conducted before storing the roots post inoculation. Then after the 2-month storage period the roots will be reweighed to determine reduction in root weight during the storage period and surface root rot evaluation will be performed by sectioning the roots vertically, and then measuring the maximum root rot diameter. Also, an evaluation of fungal growth on the surface of each root in each group will be evaluated to determine and compare the percentage of root area covered by fungal growth. To determine sucrose loss during the storage period, a comparison of the percentage sugar loss per root per group will be performed using established NWISRL protocols.
An additional dose-response analysis will be performed to determine the effectiveness of Vg-P for preventing L. mesenteroides growth in factory processed sugar beet extracts. For this experiment, 3 randomly picked different batches of sugar beet extract [representing 3 biological replicates] will be used. Each batch of extract will be divided into 5 equal volume aliquots out of which 4 aliquots will be artificially inoculated with 0.1 mL of a 108 cfu mL-1 suspension of L. mesenteroides.
