Protection of canola from pathogenic fungi using RNA interference technologies

Project Summary

Background

Canola is Canada’s most important oilseed crop, contributing $26.7 billion to the country’s economy [1]. Canola crops are threatened by a variety of fungal pathogens, but one of the most damaging is Sclerotinia sclerotiorum. This fungus occurs in all canola growing regions of Canada and throughout the world [2-4], causing stem rot of the plants. Annual losses due to this fungus are highly variable, ranging from 5 to 100%; in 2010, 90% of Canadian canola crops showed some level of Sclerotinia infection and the loss to growers was estimated at $600 million [5]. With no available Sclerotinia-resistant cultivars available, damage from this fungus is mitigated primarily by crop rotations and foliar fungicides. Unfortunately, under damp climatic conditions, such methods are insufficient to control the disease. In addition, there is increasing public concern over the risk that chemicals pose to the environment and human health. Together, these present compelling reasons to find safer (fungal or species-specific) alternatives to control this costly fungal pathogen.

RNA interference (RNAi) is a method of reducing a targeted gene’s expression through the application of double-stranded RNA (dsRNA). Recently, the research team had identified dsRNA molecules that can inhibit Sclerotinia growth, and have observed that topical application of these dsRNAs, under laboratory conditions, can reduce Sclerotinia infections in canola. Due to RNAi’s high degree of specificity, the research team can design dsRNAs to target just the pathogenic fungus or related pathogenic fungi, and not affect beneficial species.  

Purpose

This research is aimed at developing and field-test a new generation of dsRNA-based, species-specific foliar fungicides that can be designed to target Sclerotinia sclerotiorum, to reduce our reliance on broad-spectrum fungicides and provide canola growers with safer alternatives to existing conventional chemistries. The development of topically-applied RNAi technologies will reduce excessive chemical inputs and promote agro-ecological health. Implementation of RNAi technologies that prevent Sclerotinia outbreaks will be made accessible to researchers and producers, thus enhancing the resiliency of the agriculture sector.

Objectives

1. Identification and nomination of Sclerotinia bioactive dsRNA molecules.

• Conduct RNAseq to identify fungal genes associated with pathogenicity, infection, or proliferative growth in different plant tissues and at different stages of the infection cycle.
• Establish a prioritized list of the most effective dsRNAs, based on gene expression profiles and species-specificity.

2. Synthesis of dsRNAs and screening for fungicidal activity against Sclerotinia and non-target effects.

• Using high throughput RNAi bioassay screening methods, over 200 dsRNAs, synthesized in house, will be tested for their efficacy to suppress Sclerotinia.
• Testing the 20 most potent dsRNAs that control Sclerotinia on non-target benign or non-pathogenic fungi, to assess species-specificity.

3. Development and testing of topical formulations for dsRNA adhesion to leaves and durability under different environmental conditions.

• Test adjuvants and microcarriers for adhesion to plant leaves under various UV exposures, watering regimens, wind and abrasive compounds within the laboratory.
• Test adjuvants and microcarriers for enhanced bioactivity, including assessing whether the dsRNAs can penetrate plant tissues under different application methods.
• Test the best performing application formulations under field conditions.

4. Assess persistence of dsRNAs in the soil

• Test persistence of dsRNA in soil using different formulations, dsRNA structures, and soil types.

Results

For the field trials, researchers seeded and fertilized a commercial canola cultivar on June 9, 2022 at a facility near Minto, Manitoba. They applied two test applications of dsRNA – July 15 at the early flowering and July 22 at late flowering. They also oversprayed the trial with disease inoculum on July 16, 21 and 25 and misted the trial with water regularly to promote disease development. Disease levels were high throughout the trial, with sclerotinia being the main disease present.

Of the 100 dsRNA molecules tested on leaves at a dose of 200 nanograms, 63 showed a reduction in lesion size when applied with the adjuvant Silwet-77. The best target, Sclero-1703, showed an 85 per cent reduction in lesion size.

Formulation testing showed that spreader molecules that include Stilwet are compatible with top performing dsRNAs. Penetrant oil based molecules are not compatible, and nanoparticles are compatible but do not result in improved RNAi-mediated transcript knockdown or improved protection against fungal lesion size.

Field trial results did not show strong performance of dsRNA applications. The night following the first dsRNA application, the field sustained heavy rains, lasting for several days, which most likely led to loss of the dsRNA from the plants, as no sticking agent or penetrant was provided in the formulation. The disease severity and incidence on canola plants treated with either of the negative control treatments (Silwet or GS1+Silwet) showed no significant difference to the treatment with the sclerotinia-specific dsRNA (1703+Silwet), regardless of whether the plants were treated once or twice.

Overall, the treatment with Sclero-1703 applied at the first spraying showed the lowest incidence and severity of disease. However, the differences between treatments were slight. There were no significant differences in yield between treatments.

Consequently, researchers observed no significant impacts of the dsRNA on disease severity, incidence or seed yield. The only noteworthy and possibly favourable result was that the highest seed yield, albeit only marginally higher than the controls, was seen in plants that had two Sclero-dsRNA sprayings. For future field trials, adjuvants that improve adhesion onto or penetration into the leaves will be needed.

The persistence test, done in the lab on five Manitoba soils, showed that dsRNAs were not detectable after six hours mixed in the soil. Further tests are planned to evaluate if the dsRNA is detectable at earlier times, to determine the half-life of the dsRNA with the different soil types.

References

1.www.canolacouncil.org; 2. Baharlouei et al., 2011, African J Biotech 10: 5785-5794; 3. Litholdo et al., 2010, Genetics Molec Res 10: 868-877; 4. Hu et al., 2011, Can J Microbiology 57: 539-546; 5.www.gov.mb.ca;