Ph. D. Alessandro PUCA

Enzymatic and Non-Enzymatic Wood Degradation by Basidiomycetes in the Esca Complex of Grapevine

Thesis defended on 4th July, 2025 in Florence.

Thesis supervisors: Prof. Christophe BERTSCH, University of Haute Alsace and Prof. Laura MUGNAI, Florence

Abstract

Esca Complex of Diseases (ECD) is widespread in viticultural areas worldwide, posing a serious risk to modern viticulture. The role of basidiomycetes in the onset of foliar symptoms has recently been reconsidered, highlighting a link between the reduction in these symptoms and the presence of Fomitiporia mediterranea in vines. This pathogen is considered the main decay agent on the grapevine in the European basin and is the most studied among white rot fungi affecting grapevines. However, the global spread of ECD requires a broader perspective, considering other wood-decay fungi associated with grapevines that are much less known than F. mediterranea.

Wood degradation is central to many of the hypotheses explaining the development of foliar symptoms, making investigating the biomolecular mechanisms underlying wood decay essential to understanding ECD etiology. With the recent re-evaluation of the distinction between brown and white rot fungi, the first study of this thesis explored the non-enzymatic degradation capabilities of F. mediterranea (Chapter II).

To address the knowledge gap regarding other fungal species and to identify commonalities among white rot agents, an investigation of the degradation capabilities of F. capensis (South Africa ), F. australiensis (Australia), F. langloisii, F. polymorpha, Tropicoporus texanus and Inonotus vitis (North America), and Stereum hirsutum (Europe) was conducted. The analyses focused on lignocellulolytic enzyme activity and the potential implementation of nonenzymatic mechanisms by these fungi (Chapter III). Moreover, the phenotypical characteristics of the fungal collection were investigated regarding the in vitro degradation capabilities of grapevine wood and temperature preferences (Chapter IV).

Under nutrient-restricted conditions, F. mediterranea was found to produce low molecular weight compounds (LMWC) capable of driving all the steps of the chelator-mediated Fenton (CMF) mechanism. These steps include acidifying the surrounding environment, reducing ferric iron, producing hydrogen peroxide in the process, and ultimately generating hydroxyl radicals.

Importantly, this ability was found to be shared among the studied white rot agents, as all the LMW fractions of their secretomes triggered a radical-generating CMF mechanism. Analyses of the lignocellulolytic activity of fungal secretomes revealed a low production of peroxidase activity, suggesting the importance of the CMF mechanism as a secondary degradation method potentially acting synergistically with the enzymatic pool. This study provides critical insights into the degradation strategies of ECD-associated white rot agents, offering potential new perspectives on studying ECD symptomatology and developing targeted control strategies. The findings emphasize the need to consider the potential role of the CMF mechanism in ECD development. This understanding could potentially be applied to control strategies, as disrupting various phases of the CMF mechanism might represent a novel control strategy on the grapevine. Future research should focus on in planta studies to further evaluate the role of wood degradation in ECD symptoms development.