Spray-induced gene silencing for improving chestnut tolerance to pathogens

Sweet chestnut (Castanea sativa Mill.) is a tree species of great importance in Europe, where it is severely threatened by ink disease caused by the oomycete Phytophthora cinnamomi Rands. Spray-Induced Gene Silencing (SIGS), which employs double-stranded RNA (dsRNA) to silence specific genes, is an eco-friendly approach used to improve plant tolerance against several fungal pathogens. The effectiveness of dsRNA to control ink disease is currently unknown. Our study tested whether some dsRNA-based products can reduce the mycelial growth of P. cinnamomi by targeting both pathogen effector genes and plant susceptibility genes, which are involved in plant-pathogen interactions during the infection process. Specifically, we silenced the DICER-LIKE (DCL) gene of the pathogen, a key gene involved in its pathogenicity. DCL:dsRNAs were synthesized and applied to P. cinnamomi colonies grown in Petri plates on V8 medium. A solution containing 3 µg of dsRNA was applied every 12 h for 3 days, and the mycelial growth was monitored at 24, 48, and 72 h post-treatment. Results showed a significant reduction (30%) in mycelial growth compared to water-treated controls, suggesting that dsRNA can be effective against P. cinnamomi in vitro, although further studies are needed to test whether dsRNA can be effective in planta as well. Future studies will also evaluate additional pathogen effector genes to achieve higher tolerance and will test dsRNA targeting susceptibility genes, such as pmr4 (powdery mildew resistant 4) and dmr6 (downy mildew resistance 6), as a preventive strategy to enhance the plant’s tolerance to P. cinnamomi. The development of nanoparticle-based delivery systems could also improve dsRNA stability and effectiveness by protecting RNA molecules from degradation and heat sensitivity, extending the window of protection, reducing application frequency, and improving overall efficiency.