Concluding remarks on the future of RNAi in agriculture

Insect pest control by specific, insecticidal dsRNAs is generally still on the verge of commercialization.

So far, only one product, the GM-maize SmartStax PRO expressing dssnf7 amongst other traits (Head et al., 2017), gained approval and awaits its launch in the United States of America (ISAAA website).

However, RNAi-based products in agriculture in general have been on the market for more than twenty years. Initially in 1995, the squash cultivar Freedom II was the first crop with RNAi traits to be commercialized (Fuchs et al., 1998; Schultheis and Walters, 1998). Shortly afterwards in 1998, transgenic papaya targeting a coat protein of the papaya ringspot virus were commercialized in Hawaii to confer resistance against this devastating plant disease and are estimated to make up 77% of Hawaiian papaya plants in 2017 (Ferreira et al., 2002; ISAAA, 2017). More crops were genetically modified to utilize the RNAi mechanism to combat viral diseases, adjust nutritional value, improve crop quality or modulate secondary metabolite contents with subsequent variety approval and/or

92

commercialization such as bean (Bonfim et al., 2007), rice (Iida et al., 1993; Kusaba et al., 2003), potato (Waltz, 2015), tomato (Gupta et al., 2013), alfalfa, soybean, plum and apple (Baranski et al., 2019;

ISAAA website; ISAAA, 2017). RNAi-based insect pest control can therefore arguably be considered an extension of this plant protection technology, with new benefits and risks.

The above-mentioned crop examples all used a transgenic approach. Sprayable RNAi for insect control as it was investigated in more detail in this thesis circumvents several problems of GM crops such as lacking public support of this technology especially in Europe, long registration processes or difficulties in the efficient generation of transgenic plants (Altpeter et al., 2016). With foliarly applied dsRNA, adjustments to target new or multiple pest species can be realized more quickly. Considering the fast degradation of dsRNA in the environment leaving no residues (only nucleotides), the possibility to create selective measures targeting individual pest species beneficial to non-target arthropods, inferred low health risks (Aliabadi et al., 2012; Petrick et al., 2015; Witwer and Hirschi, 2014) and low application rates (chapters 2 and 3), this control measure would be attractive not only for conventional agriculture, but particularly for organic farming – as dsRNA molecules are natural compounds and do not belong to synthetic chemical insecticides.

One drawback of RNAi as a control strategy is its rather slow action taking several days to elicit insecticidal effects (chapters 2 and 3), during which the insects can still directly damage the crop or transmit diseases. For this reason, dsRNA is not suitable for insect control in ornamental plants or cut flowers where pristine appearance is desired. Another drawback is the limited number of insect species that are efficiently targeted by RNAi. Stabilization of dsRNA with the focal aim to improve oral delivery to the respective insect was attempted by complexation with numerous nanoparticle types (Avila et al., 2018; Castellanos et al., 2019; Christiaens et al., 2018; He et al., 2013; Parsons et al., 2018;

Zhang et al., 2010). While these formulations show promise, they may not be available to organic farming and need to be tested whether improved delivery also extends to mammals.

Additionally, prices for dsRNA products need to be comparable to current insecticides. For example, the “Decis forte” formulation with the pyrethroid deltamethrin as active ingredient costs approximately 50-60€/l depending on the vendor (e. g. Avagrar; myAGRAR). This corresponds to roughly 3-4€ per hectare at a field application rate of 5-7.5g/ha in potato or cereal crops (BVL, 2020a, 2020b, 2020c). Advances in the production of long dsRNA considerably dropped production costs below 0.5$/g (Maxwell et al. from GreenLight Biosciences). The results from this thesis indicates that field rates of 10g/ha may be enough to manage insect pests (depending on the species and target gene), as this rate was sufficient for P. cochleariae control with e. g. dsrpn7 or dsrpt3 (comparable to lowest rate used in chapter 2) and exceeded the rate necessary to control CPB larvae from different

93

locations (ten times higher than the rate used in chapter 3). Together, a production cost of at least 5$/ha anticipates much higher costs for the finished, marketable product. Prices of approximately 20-40€/ha for the Bt toxin-based product “Xentari” requiring application rates of at least 324g/ha in vegetable crops like cabbage, root vegetable or tomato raise hope that sprayable dsRNA products could be competitive (Avagrar; BVL, 2020d, 2020e, 2020f, 2020g)

Fast degradation of dsRNA is not just an advantage, but also represents a problem if a species has numerous generations per season making additional treatments necessary which are likely to be much more expensive than conventional insecticide sprays. Greenhouse-grown crops avoid one predominant source of degradation - UV light – deeming it the most favorable environment for foliarly applied dsRNA. Nevertheless, companies are pursuing field application of sprayable dsRNA as well with apparently encouraging results in field trials (GreenLight Biosciences; Syngenta).

Taken together, the future of sprayable RNAi in agriculture is difficult to predict and the balance can still tip either way. It continues to face many limitations and therefore might end up only as a niche product for specific pest control problems or as a putative resistance-breaking agent, despite its positive qualities. Instead, the focus of insecticidal RNAi could shift from agriculture to other insect nuisances such as ants and termites in domestics (Choi et al., 2012; Raje et al., 2018; Zhou et al., 2008) and mosquitoes as vectors of human diseases (Hapairai et al., 2017; Kumar et al., 2013; Mysore et al., 2019). Depending on the development of political and regulatory frameworks in Europe and other regions, RNAi may find its niche in some agricultural and horticultural production systems as a future alternative to chemical insecticides.

94