An RNA interference (RNAi) target with potential to control Dothistroma needle blight

Abstract

Spray-induced gene silencing (SIGS) involves exogenous spray applications of double-stranded RNA (dsRNA) molecules targeting specific genes, such as those essential to pathogens. This technique has shown great potential for controlling fungal pathogens of horticultural and agricultural plants, but few studies have evaluated its application to forest pathogens. Here we report the first demonstration of exogenous application of dsRNA molecules targeting a gene required for virulence in the foliar pine pathogen Dothistroma septosporum, the causal agent of Dothistroma needle blight (DNB). Constructs expressing sense and antisense dsRNAs were generated targeting a 509 bp fragment of the dothistromin toxin regulatory gene (DsAflR) in D. septosporum. DsAflR is required for the production of dothistromin, a phytotoxin essential for full pathogen virulence. We found that exogenous dsRNA is taken up and the RNA silencing mechanism is functional in D. septosporum through a series of in vitro and in planta trials, such as monitoring the uptake of fluorescently labelled DsAflR-dsRNA into the cell by confocal microscopy and quantifying the extent of gene silencing by quantitative Polymerase Chain Reaction (qPCR). In vitro applications of DsAflR-dsRNA to D. septosporum reduced the accumulation of DsAflR mRNA compared to controls and, in some cases, were found to be statistically significant, despite a high level of variability in the results. Our study also demonstrated a reduction in DNB symptoms on infected pine needles sprayed with DsAflR-dsRNA, which was further supported by a significant reduction in fungal biomass. This preliminary study highlights the potential for SIGS as a future management scheme for controlling the forest pathogen D. septosporum, as well as other forest pathogens.