FUNGICIDE RESISTANCE IN GRAPEVINE DOWNY MILDEW MANAGEMENT: PHENOTYPIC AND GENOTYPIC CHARACTERIZATION OF PLASMOPARA VITICOLA POPULATIONS FOR RESISTANCE TO FUNGICIDES

Viticulture plays a role of prime importance within the world agricultural panorama. In 2020 the total world area planted with vineyards for all intended uses (wine and juices, table grapes and raisins), including young vines and not yet in production, was estimated at 7.3 Million ha by the International Organization of Vine and Wine in 2020. Within this vast production panorama, the Eurasian grapevine (Vitis vinifera L.) is the most cultivated species of grapevine due to the high quality of its grapes. One of the main problems affecting this species is that it is highly susceptible to infections of the phytopathogenic oomycete Plasmopara viticola (Berk. et Curt.) Berl. & De Toni. This pathogen is native to North America and with favorable climatic conditions can cause considerable damages to the grapevine production both from a qualitative and a quantitative point of view. P. viticola is a polycyclic pathogen, able to carry out numerous cycles of infection during a single vegetative grapevine season. Consequently, given the high susceptibility of V. vinifera cultivars to this pathogen in areas with frequent rainfall and moderate temperatures during the growing season, the cultivation of traditional varieties is not conceivable without frequent applications of fungicides. Fungi and fungal-like organisms, such as the oomycetes, are highly adaptable to different environmental conditions, to host defense mechanisms and to fungicide selection. Repeated treatments with selectively active, site-specific fungicides, is frequently followed by the development of the phenomenon of fungicide resistance, which represents one of the major threats for downy mildew control and for modern agriculture in general, because it potentially leads to a reduction of disease control in the field (practical resistance). In order to preserve the effectiveness of such compounds, fungicide resistance must be carefully managed, and to this purpose proper disease control strategies have to be implemented by reducing the selection pressure associated to the fungicide use. The implementation of sound anti-resistance strategies is based on many factors: the risk of a particular fungicide class to evolve resistance, the risk related to the pathogen features, the agronomic risk associated to specific climatic conditions and the results obtained in sensitivity monitoring activities, that allow to characterize the fungicide sensitivity of pathogen strains or populations through bio tests and molecular diagnostic tools. In particular, monitoring of P. viticola populations for their sensitivities to the different active principles plays a key role in fungicide resistance management. However, for some fungicides these data are currently not yet present or incomplete, and actual testing methodologies to investigate some life stages of the pathogen are quite limited because they can provide only a qualitative description of resistance status. Considering these current challenges, the aims of my PhD project were: a) to evaluate the sensitivity profile of P. viticola populations to different fungicide classes subjected to different disease pressure levels and spray programs in open field; b) to develop a new method based on flow cytometry and single-cell sorting for isolation of single sporangia in order to obtain a precise estimation of the percentage of sporangia able to positively infect grapevine plants in the population; c) to characterize possible resistant strains for the mechanism of resistance and pathogenicity. In general, among the populations tested we found a good sensitivity for the fungicides under investigation. Sporadic cases of resistance were detected, and for some of them it was possible to link the resistant phenotype to single point mutations in the gene encoding the target protein. Depending on the life stages of the pathogen under investigation, more quantitative or qualitative data were obtained according to the testing methods available. This made it necessary to develop a further tool to make the investigations on the asexual reproduction stages of the pathogen more homogeneous to those of sexual reproduction. Flow cytometry and single-cell sorting have proven to be excellent technologies to bridge this gap, and the quantitative method here developed and proposed could be positively implemented in future for large-scale monitoring investigations of fungicide resistance.