Location: Sunflower Improvement Research
Project Number: 3060-21220-034-037-S
Project Type: Non-Assistance Cooperative Agreement
Start Date: Aug 1, 2025
End Date: Dec 31, 2026
Objective:
The aim of this study is to 1) Sequence optimize the virus-derived proteins for their anti-fungal activity., 2) optimize the foliar spray application of REP as protective and/or curative measures against S. sclerotiorum, and 3) test the combination of REP treatment and boscalid fungicide on plants to control S. sclerotiorum isolates that show different aggressiveness or resistance to fungicide.
Approach:
The aim of this project is to delay development of fungicide resistance in soybeans. Specifically, we will 1) Synthesize the full-length SsHADV1 REP with the conserved arginine (K) amino acids in the predicted peptide mutated into a serine (S) to abolish the ATPase activity of the REP and test its relevance in the anti-fungal activity. Using bioinformatic approaches, REP sequences will be selected and synthesized based on the variation of their ATPase domain sequences or any other relevant domain. Each sequence will be expressed in the bacterial secretion system and tested first in plate assays. From additional sequence alignments of the functional sequences, we expect to sequence optimize the protein sequences for high efficiency and stability. 2) test whether the foliar spray of REP cell filtrate can provide protection against Sclerotinia when applied above ground. We will first test the spray application of the REP filtrate on detached leaves of Nicotiana benthamiana followed by inoculation with a plug of actively growing Sclerotinia sclerotiorum. We will measure the size of the necrotic lesions formed on the leaves 48-72 hours post inoculation. The detached leaf assay will allow a quick optimization of the delivery conditions of the anti-fungal on the leaves. We will assess efficacy as a spray as well as stability of the fungal activity. Next, we will perform the REP foliar spray on stem-inoculated soybean plants. Two sets of treatment will be performed to test the efficacy of the spray application of REP to provide preventive or curative protections against S. sclerotiorum. To test the preventive protection of REP, we will spray the cell-free bacterial filtrate containing REP directly on the above ground part of the soybean plants. The spray will be repeated 2 hours later for ensuring full coverage. Next, we will stem-inoculate the plants with fungal hyphae. We will assess disease progression for 10 days. To test the curative protection of REP, soybean plants will be stem-inoculated and next sprayed 2-, 12- and 24-hours later with the bacterial cell filtrates. We will next assess disease progress for 10 days. Then, 3) test whether the REP treatment in combination with low dose of boscalid fungicide can limit aggressiveness of various isolates and disease progression. As an approach, we previously established that the over-expression of the REP protein in Nicotiana benthamiana leaves through agroinfiltration was sufficient to slow down disease progression of Sclerotinia sclerotiorum within 72 hours (Figure 4). Following the transient REP expression assay on detached leaves, we will spray low dose of fungicide and inoculate the leaves with different S. sclerotiorum isolates. We will measure the size of the necrotic lesions formed on the leaves 48-72 hours post inoculation when compared to leaves treated with the 0.1ppm and 0.01ppm fungicide alone. As a second approach (based on optimization in objective 2), we will spray inoculate the leaves with REP in combination with fungicide.