Sustainable agriculture by chitosan nanoparticles: characterisation and functionalisation with doublestranded RNA molecules able to limit Botrytis cinerea mycelium growth

The application of nanomaterials as shuttles for biological agents is one of the most interesting innovations in agricultural research today, aiming to improve plants growth and nutrition or to protect them from stresses. To achieve the latter goal, defence approaches based on molecular techniques, like the RNA-interference (RNAi) mechanism, are also an attracting alternative. One of the main obstacles to develop these technologies, however, is the high cost of nucleotides and their easy degradability: aiming to exploit their maximal potential, a new technological procedure would be needed to extend their lifespan and to reduce the utilized quantity. In our work, we fulfil this issue by employing chitosan nanoparticles (NPs) as vectors for dsRNA sequences with interfering function against Botrytis cinerea. In detail, we have chosen chitosan because it is natural, eco-sustainable and easy to polymerise in nanometric form without hazardous reagents. The NPs obtained were characterised in terms of size, charge and behaviour over time when distributed on leaves with the aid of a surfactant. Their ability to be functionalised and to protect the dopant from photodegradation was then carried out using the generic Green Fluorescent Protein dsRNA sequence as a marker. Finally, an inhibition test of fungal mycelium development was performed on Nicotiana benthamiana leaves, showing that the used sequence (B. cinerea essential genes: BcCYP51, Bcchs1, and BcEF2) was less effective when applied alone than when bound to NPs. The results obtained are encouraging and promising for further development of this technology, which can also be extended to the use of other doping biomolecules or to a different chitosan polymer with intrinsic fungistatic properties, towards an environmentally-friendly crop management.