RNAi-based protection of East African highland banana against nematodes and weevils

Banana production worldwide is severely constrained by the banana weevil (Cosmopolites sordidus) and plant-parasitic nematodes, leading to significant yield losses. This thesis investigates RNA interference (RNAi) as a targeted pest management strategy, silencing genes critical for the survival and development of these pests. A multi-pronged approach was employed, encompassing artificial diet assays, carrot disc assays, and greenhouse evaluations of transgenic banana plants. An artificial diet assay, supporting the complete life cycle of banana weevil larvae, was developed using banana corm flour and agar. This enabled controlled testing of RNAi constructs targeting key weevil genes, including Snf7 (Sucrose non-fermenting), Rps13 (Ribosomal protein S13), Laccase-2 (cuticle pigmentation), and Mad-1 (Mitotic Arrest Deficient). Constructs targeting Snf7 and Rps13 were most effective, causing nearly complete larval mortality within 9 and 13 days, respectively. Moderate mortality rates were observed for laccase-2 (30–40%), with albino phenotypes confirming gene silencing. Mad-1 silencing had partial effects, reducing survival and prolonging development. Carrot disc assays evaluated dsRNA constructs targeting nematode-specific genes, including Rps13, Chs-2, Pat-10 (paralyzed arrest at two-fold), Unc-87 (uncoordinated), and Eng1a (beta-1,4-endoglucanase). Constructs targeting Rps13 achieved an 8-fold reduction in nematode populations compared to controls, while silencing Pat-10 and Eng1a reduced nematode viability by 30–40%. In the greenhouse, 540 plants were evaluated for banana weevil resistance, with 180 plants serving as controls (90 non-transformed and 90 expressing non-target dsRNA targeting Unc-87). The remaining 360 plants, comprising 90 plants for each weevil-target dsRNA construct (Snf7, Rps13, and Laccase-2), were assessed for resistance. Lines expressing Snf7 dsRNA reduced corm damage by up to 85% compared to controls, while Rps13-dsRNA lines showed moderate reductions. Laccase-2-silenced lines reduced weevil-induced corm damage by 40–50%. For nematode resistance, 540 plants were also evaluated, with 180 plants serving as controls (90 non-transformed controls and 90 expressing non-target dsRNA Laccase-2, a weevil-specific gene). The remaining 360 plants, comprising 90 plants for each nematode-target dsRNA construct (Rps13, Chs-2, Pat-10, and Unc-87), were assessed for resistance. Lines expressing Rps13 dsRNA reduced nematode populations by 55%, while dsChs-2-expressing lines reduced nematode-associated root necrosis by 30%. Lines targeting Pat-10 and Unc-87 demonstrated moderate reductions in nematode multiplication and viability, supporting their utility in RNAi strategies. This study underscores the potential of RNAi technology for sustainable management of banana weevils and nematodes by targeting essential pest genes. The findings provide a robust foundation for field validation, biosafety evaluations, and broader adoption of RNAi strategies to enhance banana production systems.