Ph. D. Alexia GRAU

GRAPEVINE TRUNK DISEASES: UNDERSTANDING OF TOLERANCE MECHANISMS IN PLANTS AND STUDY OF BOTRYOSPHAERIACEAE EFFECTORS

Thesis defended on 13th December, 2024 in Colmar.

Thesis supervisor: Pr. Julie CHONG, University of Haute Alsace

Thesis co-supervisor: Ph. D. Romain PIERRON, Associate Professor, University of Haute Alsace and Ph. D. Mary-Lorene GODDARD, Research Engineer, University of Haute Alsace.

Abstract:

Botryosphaeria dieback, one of the three main grapevine trunk diseases, is a worrying threat to the entire wine industry. They are caused by complexes of fungi in the Botryosphaeriaceae family, and can give rise to a variety of symptoms ranging from the appearance of foliar discolorations to the apoplectic form, which leads to the death of the vine. Although they are present in vineyards throughout the world, no truly effective treatment is available following the ban of sodium arsenite.

Although there are no completely tolerant grape cultivars, different levels of susceptibility depending on the grape variety have been observed in the vineyard. The first objective was therefore to study more precisely the interactions between a fungal pathogen associated with Botryosphaeria dieback, Neofusicoccum parvum, and 3 grapevine varieties, Vitis vinifera cv. Chardonnay, Riesling (more tolerant), and Gewurztraminer (more susceptible). Studies carried out on detached internodes showed that the kinetics of necrotic symptom development in the wood were the same in the first 5 days following infection for the 3 cultivars. On the other hand, quantification of fungal DNA by qPCR in wood showed more rapid colonization by N. parvum in Riesling. These results suggest a better tolerance to infection in Riesling based on a lower development of symptoms in relation to the amount of mycelium present.

A metabolomic study measuring the response of Chardonnay and Gewurztraminer grape varieties to infection at 4, 8, and 14 weeks post-inoculation with N. parvum was then carried out on potted plants. Results from wood, the infection area, showed that the main response to infection by the fungal pathogen was stilbene synthesis. This response differed between grape varieties, with Chardonnay appearing to give a more rapid and constant response.

Thus, the establishment of tolerance is a complex mechanism that seems to involve host control of necrotic symptom development and the establishment of a rapid and adapted response, notably via the synthesis of stilbene polymers. Further studies on primary metabolism and the establishment of a link between metabolite synthesis and the level of wood colonization will be investigated in the future.

In parallel, the aggressive mechanisms of N. parvum were studied through the characterization of three small cysteine-rich proteins selected on the basis of their affinity for sodium arsenite: these proteins were found to belong to the Cerato-platanin family, NpCP1, and the Alt-a1-llike protein family, NpAA1.1 and NpAA1.2. Homologs of these proteins have been identified in other plant pathogens, and some are already known to be virulence factors. Our study concluded that they were effectors secreted in detached internode wood rapidly after the onset of infection. This secretion differed depending on the grape variety studied: Chardonnay, Riesling, and Gewurztraminer. A transient expression assay of these effectors in Nicotiana benthamiana and Vitis vinifera leaves revealed the induction of a defense response involving modulation of the oxidative stress pathway for all 3 effectors, as well as synthesis of PR protein, STS, PAL, CHS, and modulation of proteins involved in ethylene synthesis for one of the two NpAA1s.

The greater response in V. vinifera leaves seems to reflect a co-evolution of N. parvum and its grapevine host. Further studies involving the creation of KO mutant strains are planned to further investigate the role of these effectors.

Key words: Grapevine Trunk Disease; Botryosphaeriaceae; Neofusicoccum parvum; Vitis vinifera; grape cultivar; virulence factors; tolerance factors; specialized metabolism; sodium arsenite