The sustainable fungicide choline pelargonate inhibits oomycete and fungal phytopathogens under controlled and field conditions with negligible effects on phyllosphere microbial communities

Chemical fungicides are the most important approaches for plant protection. However, the overuse of chemical fungicides raised concerns about possible negative effects on human health and the environment. Regulations have been implemented in several countries to reduce the use of chemical fungicides, and numerous active substances are under scrutiny for substitution in Europe. Thus, the development of novel sustainable fungicides is of great importance to reduce possible health risks and environmental impacts. Choline pelargonate (CP; also known as trimethyl-ethanolamine nonanoate or choline nonanoate) is a new formulation of pelargonic acid as choline salt for possible use as a sustainable fungicide, but no information is available on the mode of action and effects on plant-associated microorganisms. The aims of this work were i) to summarize current knowledge on the effects of fungicides on the grapevine phyllosphere microorganisms; ii) to investigate the mode of action of CP against oomycete and fungal phytopathogens, focusing on its effects on membrane integrity and lipid metabolism; iii) to evaluate the efficacy of CP against grapevine downy mildew and powdery mildew under greenhouse and field conditions, and to assess its impact on phyllosphere microbial communities; iv) to investigate the efficacy and persistence of CP against tomato grey mold and grapevine downy mildew under controlled and semi-field conditions. The first chapter includes an introduction to the current challenges in plant disease control and the development of biopesticides, and defines the aims of the thesis. The second chapter includes a literature review on the impact of conventional and alternative fungicides on microbial communities of the grapevine phyllosphere. Amplicon sequencing analyses of the grapevine phyllosphere (leaves, bunches, and bark) demonstrated that the taxonomic structure of bacterial and fungal communities is mainly affected by plant compartment, grapevine cultivar, sampling time, and vineyard location. Fungicide treatments can slightly influence the relative abundance of some bacterial and fungal taxa of the grapevine phyllosphere, indicating possible effects of fungicide treatments on grapevine-associated communities, according to the sprayed product. Thus, understanding the possible effects of new fungicides on phyllosphere microorganisms is essential for the further development of sustainable plant protection products. In the third chapter, the mode of action of CP was characterized against Botrytis cinerea and Phytophthora infestans in vitro. CP inhibited mycelial growth and spore germination, causing the leakage of electrolytes and nucleic acids with dose-dependent effects. Ultrastructural alterations of the plasma membrane and cytoplasm were found in CP-treated phytopathogens, causing negative impacts on membrane functionality. Moreover, CP altered the content of free fatty acid, phosphatidic acid, phosphatidylcholine, phosphatidylethanolamine, and triglyceride lipids, as a possible perturbation of lipid metabolism. Thus, CP revealed promising inhibitory activities against oomycete and ascomycete phytopathogens. In the fourth chapter, the efficacy of CP against grapevine phytopathogens was evaluated under controlled and field conditions, and possible side effects on phyllosphere microorganisms were analyzed. CP decreased the severity of grapevine downy mildew and powdery mildew under greenhouse conditions with direct inhibitory effects against P. viticola and E. necator, slight induction of defense-related genes, and no translaminar effects. Field experiments confirmed that CP decreased powdery mildew and downy mildew severity on leaves and bunches. Microbial community analysis revealed that plant compartment, vineyard location, and sampling time were key drivers of microbial community structure, while treatments showed negligible effects on microbial diversity of grapevine leaves and bunches. CP treatment partially modified the relative abundances of some bacterial and fungal taxa, whereas reference fungicides exhibited broad effects. Thus, CP showed promising antifungal activities with minimal effects on microbial communities, highlighting its potential as a sustainable plant protection product that preserves indigenous microbial communities. The fifth chapter includes the evaluation of CP persistence. CP efficacy against tomato grey mold and grapevine downy mildew was decreased by simulated rain under controlled conditions. Semi-field experiments confirmed that CP decreased powdery mildew and downy mildew severity, but the formulation with a drift retardant did not improve the CP efficacy and persistence on leaves, indicating that appropriate CP formulations should be further developed. These results demonstrated that CP can inhibit oomycete and ascomycete phytopathogens by affecting membrane functionality and lipid metabolism. CP can reduce the severity of grapevine downy mildew and powdery mildew under field conditions with negligible effects on microbial communities, highlighting its potential as a sustainable alternative to conventional fungicides. CP sensitivity to rainfall can limit its efficacy under field conditions, and formulations should be optimized for its further development as a plant protection product.