Update on the biopesticide regulation and development of protein-based products for the biocontrol of crop diseases

In the last decades, concerns on the negative drawbacks of chemical pesticides on human health and environment have raised interest in safer alternatives; biopesticides, such as biogenic elicitors, represent an encouraging solution. The regulatory approach in the European Union does not distinguish biopesticides as a specific category of plant protection, and for this reason they are subjected to the same regulations as synthetic chemicals, requiring several authorization steps for the final approval and marketing. Among others, protein-based products and peptide fragments can stimulate plant growth, and represent a wide category of elicitors able to reduce the symptoms of common crop diseases, by acting as stimulators of plant defence and influencing systemic resistance processes, which can be regarded as effective alternative to synthetic chemical pesticides. The final objective of the current doctoral project was to provide new insights on the use of bioactive protein-based products against crop diseases, to further develop new sustainable strategies for organic agricultural practices. More in details, as well as providing an updated overview of the regulatory procedures for the authorization of biopesticides, the specific goals of the present research were i) to characterize the mode of action of protein-based products against crop diseases and ii) to optimize a method for the low-cost production of bioactive protein-based products. For this purpose, we analysed the mechanisms of action of a protein derivative called nutrient broth (NB) against grapevine downy mildew (caused by Plasmopara viticola), focusing on its roles as resistance inducer and nutritional source for phyllosphere microbial populations (Chapter 2). We showed that NB reduced downy mildew symptoms and induced the expression of defence-related genes in greenhouse- and in vitro-grown plants, indicating the activation of grapevine defence processes. Furthermore, NB increased the number of culturable phyllosphere bacteria, and altered the composition of bacterial and fungal populations on grapevine leaves. Thus, modifications in the structure of leaf populations caused by NB application could partially contribute to downy mildew control by competition for space/nutrients with the pathogen or other biocontrol strategies. Particularly, changes in the abundance of phyllosphere microorganisms may provide a cont ribution to the resistance induction, partially affecting the hormone-mediated signalling pathways involved. Later, we optimized an experimental procedure to develop low-cost protein hydrolysates starting from plant agro-industrial by-products, since animal-derived protein derivatives may create concerns about food safety (Chapter 3). Particularly, we compared the effect of enzymatic and acid hydrolysis on different plant protein sources (soybean, rapeseed and guar protein meals), in terms of efficacy against the powdery mildew of Cucurbitaceae (caused on courgette plants by Podosphaera xanthii), investigating the potential contribution of amino acids and peptide fragment s generated during the hydrolysis to the activation of plant resistance. Our results showed that the original protein source affected the biocontrol properties of protein hydrolysates, and two hydrolysis processes improved the functional properties of guar protein meal against powdery mildew. A positive correlation was found between the efficacy and degree of hydrolysis of guar acid hydrolysates, suggesting that the hydrolysis method may enhance the functional properties of the original protein source. In addition, significant correlations were revealed between the efficacy of guar hydrolysates and concentrations of specific peptide fragments and amino acids, which may be involved in the regulation of the plant defence response. Specifically, guar enzymatic hydrolysates did not present a direct toxic effect against the germination of pathogenic conidia, suggesting a mode of action mainly based on the stimulation of plant resistance mechanisms, as observed for NB. The possibility of controlling crop diseases with the preventive foliar application of protein-based products represents an innovative approach, especially in a view of reducing harmful chemical pesticides in integrated pest management programs. However, further studies are required to fully clarify their modes of action and the impact on phyllosphere microorganisms under field conditions.