Development of RNA silencing as a novel disease control strategy to protect pines from Dothistroma needle blight : a thesis presented in the partial fulfilment of the requirements for the degree of Master of Science (MSc) in Genetics at Massey University, Manawatu, New Zealand

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Pinus radiata, the main commercial forest species grown in New Zealand, is one of many pine species worldwide that are susceptible to Dothistroma needle blight (DNB), caused by the fungus Dothistroma septosporum. New methods are needed to help manage and control this disease over current control measures such as fungicide spraying, pruning, and thinning. RNA silencing is a radical new approach to directly combat pathogens. Due to the success of many studies in controlling agricultural and horticultural crops, by exogenously applying dsRNA molecules targeting virulence genes, this has raised the question of whether forest pathogens can also be controlled with this method. RNA silencing has the potential to silence genes specific to a fungal pathogen, rendering it less virulent, and reducing disease symptoms on affected host plants. The aims of this work were to create an RNA spray targeting individual genes specific to D. septosporum, and to determine if spray applications of the RNA can reduce pathogen virulence and protect pines from fungal infection. As proof of concept, a spray application of a 737 nt eGFP-dsRNA was used to target an enhanced green fluorescent protein gene in D. septosporum. Also, 509 nt and 408 nt DsAflR-dsRNAs were synthesised targeting two different regions of the dothistromin pathway regulatory protein gene, named DsAflR 1-dsRNA and DsAflR 2-dsRNA. The DsAflR gene is involved in the production of the virulence factor dothistromin. All three dsRNAs were labelled with fluorescein to detect its uptake into cells, which was successful. RNA silencing was detected by reduced gene expression levels in vitro for samples treated with DsAflR 1-, DsAflR 2-dsRNA, as well as the RNAi control eGFP. There was a statistically significant reduction in DsAflR gene expression by applying DsAflR 1-dsRNA to D. septosporum grown on agar; however not all the reductions seen for treatment with each respective dsRNA were statistically significant and a lot of variability was observed between replicates. In planta silencing trials, in which pine shoots were treated with dsRNA and inoculated with D. septosporum spores, revealed reductions in fungal biomass in dsRNA treated samples in some cases, although more replicates are needed to confirm these results. Nevertheless this study has contributed new knowledge for the development of spray applications of dsRNA to reduce DNB disease. It provides a starting point for more research in controlling forest pathogens and could ultimately help to replace existing chemical-based forest management practices. Furthermore, the knowledge gained is applicable to a diverse range of pathogens and plant hosts, for which this ground-breaking technology holds great promise.
Permission was obtained for the re-use of Figures 1.1, 1.3 -1.8, 1.10, 1.11 & 3.1, and Tables 1.2 & 1.3. Figures 1.12 & 6.1 are re-used under a CC Attribution-NonCommercial-NoDerivatives 4.0 International (CC-BY-NC-ND 4.0) license. Figures 1.9, 2.9, 5.11 & 6.2 are re-used under a CC Attribution 4.0 International (CC BY 4.0) license.