Understanding the metabolic changes activated by volatile organic compounds in grapevine leaves against downy mildew

Plants can produce a wide variety of volatile organic compounds (VOCs), which are mediators of plant responses against stresses. Abiotic and biotic factors can modulate VOC emission dynamics from plants, suggesting a key role of these compounds in plant defense processes. Although grapevine VOCs responsible for wine aroma and plant-insect communications are well characterized, the functional properties of VOCs produced in response to phytopathogens were less studied. The aims of this work were i) to summarize the information available in the literature on emission patterns and possible biological functions of VOCs produced by grapevine plants in response to abiotic and biotic factors, ii) to investigate the defense mechanisms activated by volatile linalool in grapevine leaves against downy mildew caused by Plasmopara viticola, and iii) to identify metabolic changes associated with VOC-induced resistance activated by 2-phenylethanol and β-cyclocitral against downy mildew. The first chapter includes an introduction to VOCs in plant communication and analytical methods for metabolic detections, while the second chapter defines the aims of the project. In the third chapter, the literature on grapevine VOCs was reviewed and volatile molecules emitted in response to the exogenous stimuli were discussed. The key findings of this chapter are that specific VOC emission patterns can be stimulated by phytopathogens, resistance inducers, beneficial microorganisms, and abiotic factors, suggesting the precise involvement of airborne signals in grapevine responses to abiotic and biotic stresses. In particular, VOCs with inhibitory activities against grapevine pathogens are known to be emitted mainly by resistant grapevine genotypes upon pathogen inoculation, and some of these VOCs were proposed as biomarkers of grapevine resistance traits. In the fourth chapter, defense responses activated by volatile linalool in grapevine leaves were analyzed. Linalool treatment reduced downy mildew severity on leaf disks of susceptible grapevines (cultivar Pinot noir) and stimulated callose deposition at the sites of P. viticola infection. Moreover, the upregulation of defense-related genes was found in linalool-treated leaf disks, indicating the activation of grapevine defense mechanisms related to salicylic acid and jasmonic acid pathways. By ultra-high pressure liquid chromatography-electrospray ionization-high-resolution quadrupole time of flight-mass spectrometry it was demonstrated that linalool treatment caused changes in the leaf metabolome of mock-inoculated and P. viticola-inoculated samples at one and six days post inoculation (dpi). In the fifth chapter, a deep metabolomic analysis of VOC-induced grapevine resistance was carried out by ultra-high pressure liquid chromatography-heated electrospray ionization-high-resolution Orbitrap-mass spectrometry, which allowed accurate mass detection, fragment and isotope pattern matching, and metabolite annotation. In this study, 2-phenylethanol and β-cyclocitral treatment reduced downy mildew severity on leaf disks of susceptible grapevines (cultivar Pinot noir) causing complex changes in the leaf metabolome of mock-inoculated and P. viticola-inoculated samples at 1 dpi and 6 dpi. Metabolic features with significant increases in abundance in 2-phenylethanol- and β-cyclocitral-treated samples mainly belonged to putative carbohydrates and conjugates, carboxylic acids and derivates, carbonyl compounds, phenylpropanoids, and terpenoids, including molecules possibly associated with plant defense against pathogens. Thus, the chemical classification of annotated compounds revealed that VOCs treatments can activate specific metabolic responses in grapevine leaves that include the accumulation of defense-related compounds. These results demonstrated that linalool, 2-phenylethanol, and β-cyclocitral induce grapevine resistance against downy mildew, acting as volatile signaling molecules for plant resistance induction. Although further studies on formulation and efficacy under controlled and field conditions are required, these VOCs have great potential for the further development of sustainable products for downy mildew control.