Fungicide Resistance Evolution and Detection in Plant Pathogens: Plasmopara viticola as a Case Study

Abstract

The use of single-site fungicides to control plant pathogens in the agroecosystem can be associated with an increased selection of resistance. The evolution of resistance represents one of the biggest challenges in disease control. In vineyards, frequent applications of fungicides are carried out every season for multiple years. The agronomic risk of developing fungicide resistance is, therefore, high. Plasmopara viticola, the causal agent of grapevine downy mildew, is a high risk pathogen associated with the development of fungicide resistance. P. viticola has developed resistance to most of the fungicide classes used and constitutes one of the most important threats for grapevine production. The goals of this review are to describe fungicide resistance evolution in P. viticola populations and how to conduct proper monitoring activities. Different methods have been developed for phenotyping and genotyping P. viticola for fungicide resistance and the different phases of resistance evolution and life cycles of the pathogen are discussed, to provide a full monitoring toolkit to limit the spread of resistance. A detailed revision of the available tools will help in shaping and harmonizing the monitoring activities between countries and organizations.

Keywords: downy mildew; fungicide resistance; grapevine; oomycete.

Figure 1

Disease cycle of P. viticola: the pathogen survives the winter period as oospores, i.e., the overwintering structures differentiated by sexual reproduction in autumn (A), embedded in dead leaves on the vineyard floor (B). With favorable weather conditions, oospores typically produce sporangia (C) that, in turn, produce zoospores (D). Zoospores are splashed by rain onto leaves and other receptive tissues of the grapevines, originating the primary infections through stomata penetration (D). Disease symptoms, visible as yellow discoloration (oil spots, Ol) on the upper side of the leaves (E), appear at the end of the incubation period and are followed, in high humidity conditions, by the emission of sporangiophores (F) with sporangia (G) that will cause secondary infections through the emission of new zoospores. O = oospore; S = sporangium; st = stoma; Z = zoospore; OI = oil spot symptom on the upper side of the leaf; WS = white sporulation, consisting of sporangiophores and sporangia, on the underside of the leaf.

Figure 2

Global vine-growing areas allocated for the production of wine grapes, table grapes, or dried grapes in 2018 (sources Organisation of Vine and Wine and ood and Agriculture Organization of the United Nations) (A), compared to countries where P. viticola fungicide resistance was reported in 2020 (B) [34,35,36,37,38,39,40,41,42,43,44].

Figure 2

Global vine-growing areas allocated for the production of wine grapes, table grapes, or dried grapes in 2018 (sources Organisation of Vine and Wine and ood and Agriculture Organization of the United Nations) (A), compared to countries where P. viticola fungicide resistance was reported in 2020 (B) [34,35,36,37,38,39,40,41,42,43,44].

Figure 3

Advantages and disadvantages of biological and molecular assays that should be considered when choosing the testing method.

Figure 4

In vivo tests carried out on grapevine plants (A,B) aiming at assessing fungicide resistance through the evaluable 50 value of the P. viticola population. OI = oil spot symptom on the upper side of the leaf; WS = white sporulation, consisting of sporangiophores and sporangia, on the underside of the leaves.

Figure 5

In vitro testing for P. viticola based on leaf disc bioassay (A), zoospore microtiter plates (B), and oospore testing (C). (A) microtiter plate containing leaf discs showing white sporulation (WS). Columns were treated with increasing concentrations of fungicide. (B) Sporangium (S) and free zoospore (Z) in liquid medium. (C) Agar plates containing increasing concentrations of fungicides and inoculated with oospore suspensions. The number of germinated oospores (GO) is counted and used to calculate the germination percentages at each concentration and to estimate the EC₅₀ values of the population or the percentage of resistant oospores at a discriminatory concentration of fungicide.