Investigation of action mechanisms of natural bioactive compounds in grapevine: antifungal activity and induction of plant defense responses

Traditionally, plant disease management strategies have relied heavily on chemical treatments, particularly synthetic fungicides, to control pathogen outbreaks and maintain crop productivity. However, the extensive and prolonged use of these chemicals has raised serious concerns due to their detrimental effects on the environment, the emergence of resistant pathogen strains, and potential risks to human and animal health. In response to increasing regulatory restrictions and growing consumer demand for sustainable and environmentally friendly agricultural practices, research efforts have progressively shifted toward the development of alternative plant protection strategies. Within this framework, both public and private research institutions have focused on the discovery and characterization of eco-friendly bioactive compounds capable of enhancing plant defense mechanisms, by offering a promising approach to reducing the reliance on conventional chemical fungicides. The objective of this research was to investigate the mode of action of newly identified bioactive compounds for potential use in grapevine defense. By elucidating their direct and/or indirect effects, this work aims to provide innovative and sustainable tools to protect grapevines against major fungal pathogens while minimizing environmental impact. Chapter 2 presents a study designed to elucidate biochemical changes occurring in the bark and foliage of cypress clones inoculated with Seiridium cardinale. This work was essential for optimizing the methods used to determine phytohormones and signaling molecules, which were then applied in the following chapters. Chapters 3, 4 and 5 report the studies conducted to evaluate the mechanism of action of UPSIDE® (ABE-IT 56, S. cerevisiae strain DDSF623), a novel formulation developed by Kwizda Agro (Vienna, Austria; co-funder of the PhD grant). The work generally explored the investigation of resistance mechanisms activated by the treatment in grapevine leaves. Specifically, chapter 3 reports the results due to the application of UPSIDE® sprayed at full canopy on healthy potted grapevines (one week for three consecutive weeks, with leaf sampling performed every 24 hours after each treatment until the 5th day) kept under environmental controlled conditions. Samples were analysed for the major biochemical and molecular markers of induction of the signaling defense. In particular, phytohormones (i.e., Et, JA, SA and ABA) and oxidative stress markers (i.e., H2O2 and superoxide anion) were representative for the biochemical compounds, while for molecular analysis, genes encoding for the putative hypersensitive response marker hsr1, and for PR proteins, particularly pr1, pr2 or pr5, were considered. Other genes, such as pal1 or chi1b, encoding for phenylalanine-ammonia lyase and chitinase 1b, respectively, have been reported due to their early activation after biostimulant application. Chapter 4 investigates whether the application of UPSIDE® can induce biochemical changes in potted grapevines inoculated with Botrytis cinerea during early stages of infection (i.e., 1-, 3-, 24- and 48 hours post inoculation) and aims at evaluating also the potential antifungal activity by conducting in vitro test (i.e., inoculation of B. cinerea on growing substrates amended with UPSIDE®, at the concentration suggested by the company). In addition, microscopic pictures were taken, by staining in vivo samples with lactophenol cotton blue solution and observing them under optical microscope. Chapter 5 illustrates outcomes from an experimental trial aimed at elucidating the induction of plant defense responses in potted grapevines treated with UPSIDE® and then inoculated with Plasmopara viticola under environmental controlled conditions. Both biochemical and molecular analysis were carried out at early stages of infection (2-, 5-, 24- and 72 hours after inoculation) and, to evaluate the potential inhibitory effect of UPSIDE® on P. viticola disease severity, a leaf disc assay was also performed. Chapter 6 and 7 report the studies aimed at evaluating the effects of exposing sweet basil plants to monochromatic LED light supplementation (i.e., polychromatic, narrowband green, blue and red) or ozonated water (OW). Specifically, chapter 6 describes the characterization of chemical composition and yield of the essential oils extracted from plants subjected to these controlled stresses, as well as the results of in vitro tests assessing the potential antifungal activity of the oils against B. cinerea. Chapter 7 focuses on basil essential oil ability to inhibit the in planta development of the B. cinerea mycelium, while also examining the direct effects of the essential oil application on grape quality. Finally, chapter 8 reports a general conclusion and highlights the main results obtained overall the PhD work.