DRUG DELIVERY NANOSYSTEMS AS PLANT “VACCINES”: FABRICATION AND ASSESSMENT OF THEIR USE FOR PLANT PROTECTION AGAINST BROAD HOST-RANGE NECROTROPHIC PATHOGENS

Drug-delivery nano-systems enhances the potency of bioactive molecules due to its increase membrane permeability, as a result of their sub-cellular size. The concept of engineered nano-carriers may be a promising route to address confounding challenges in agriculture that could lead to an increase in crop production while reducing the environmental impact associated with crop protection and food production. A key motivation of this work is to evaluate the potential use of drug delivery nanosystems in agriculture, especially in the area of disease control. To this end, identifying the most suitable materials to serve as carrier and cargo is imperative. Understanding their bioactive properties and their physical-chemical characteristics is critical because these influences not only their biological effects on plants and environmental impact, but also, the fabrication process and potential scaling-up, enabling practical and relevant field applications in the future.

In this work, chitosan was selected as nano-carrier material because of its biological and chemical properties. The chemical structure of chitosan allows spontaneous assemble of core-shell like nanostructures via ionic gelation, has enabled it to be used as nano-carrier biomaterial intended for delivery of bioactive cargo. In agriculture, the use of chitosan is of special interest due to its immune-modulatory activity elicited in plants. However, due to its inherent molecular heterogenicity, the formulation and fabrication of stable and low inter-batch variability chitosan nanocarriers via ionic gelation is difficult and time consuming.

A myriad of different bioactive molecules has been tested as payload, encapsulated into chitosan-based delivery nano-systems for a range of purposes ranging from biomedicine, pharmaceutical, food and agriculture. In this work plant derived essential oils were selected as bioactive payload. Essential oils are at the core of the plant communication process with their phytobiome, including plant pathogens. Molecules from essential oils can carry an air-borne message serving as a plant-to-plant communication system (a phenomenon known as allelopathy) that activate the plant defense mechanisms. Encapsulation of essential oils into chitosan nanocarriers is only possible by forming nano-emulsions.

Despite the potential benefits from the use of chitosan and essential oils in agriculture, its use at a large scale has been hindered by the overwhelming inconsistencies in the current literature, regarding their formulation and fabrication. This work addresses these problems and presents evidence that support the feasibility of producing highly chitosan nanocarriers loaded with essential oils, in a facile and rapid way, using FDA-grade materials only, without the need of expensive or specialized instrumentation.

The plant-pathogen compatible interaction between A. thaliana and B. cinerea was used as biological model to test the hypothesis that chitosan nano-carriers and essential oil nano-emulsions can enhance the quantitative disease resistance of plants against broad host-range necrotrophic pathogens. We found that these treatments display a dose-dependent response in plants triggering a systemic immune response. Image-based phenotyping analysis showed that chitosan nanoparticles alone, as well as loaded with d-limonene, significantly enhanced the disease resistance of A. thaliana against B. cinerea. Nano-emulsions using essential oils from cinnamon, clove, coriander and red thyme also produced similar effects on the defense response in the pathosystem under study. Functional analysis of the differentially expressed genes from treated plants revealed that these treatments up-regulated the biological process involved in “stress management”, while down-regulated the biological process required for normal growth and development during ideal, non-stressful conditions.