Spray-Induced Gene Silencing of SsAgo2 Confers Field Protection Against White Mold in Sunflower.

Abstract

Sclerotinia sclerotiorum, the causal agent of white mold, is a globally distributed fungal pathogen responsible for major yield losses in many crops. Spray-induced gene silencing (SIGS) has emerged as a promising alternative for disease control. Building on prior research, this study advanced SIGS by targeting S. sclerotiorum argonaute 2 (SsAgo2), using FF4 (PAZ domain to 5'-PIWI domain) and FF5 (PIWI central domain) fragments which are important regions involved in RNA binding and cleavage. We optimized spray tank mix/dilution in the greenhouse and scaled up dsRNA production using E. coli. The greenhouse results demonstrated that dsRNA with nanosheets significantly inhibited white mold disease on tomato and N. benthamiana compared to empty vector control (p = 0.03 and 0.004, respectively). To evaluate off-target effects, SIGS was tested on Botrytis cinerea, a related necrotrophic pathogen. No significant differences in lesion size were observed between SsAgo2 dsRNA-treated and water-treated Nicotiana benthamiana leaves. Gene ontology analysis of differential expressions in sunflower leaves revealed no significant differences between SsAgo2-FF4 or SsAgo2-FF5 compared to control. Field trials in 2024 demonstrated effectiveness. Parameters assessed included disease severity, wet weight, head width, and seed weight. Area under the disease progress curve (AUDPC) analysis revealed that dsRNA sprays, applied before and after manual inoculation or during natural infection, significantly reduced infection levels with p < 0.1 as the cut off. Sunflower fresh weight increased by 104.4 g (p = 0.04) and 96.7 g (p = 0.07), while head width increased by 2.5 cm (p = 0.03) and 1.5 cm (p = 0.10), respectively. This report demonstrates that the dsRNA mixture has potential to incorporate SIGS into field applications for the management of white mold disease.