Research and Development of new Biological Alternatives to Control Downy Mildew of Grapevine (Plasmopara viticola) and Implement of new Integrated Pest Management Strategies

The plant pathogenic oomycete Plasmopara viticola represents one of the major threats to viticulture, causing heavy and diffused yield losses if not properly controlled. Nowadays, the control of P. viticola relies mainly on repeated fungicide application, with a remarkable impact on the environment and human health. For these reasons, many active ingredients have been excluded from the market, or their use has been drastically limited. On the other hand, the high occurrence of mutations in P. viticola populations leads to an outburst of resistant mechanisms worldwide towards single-site chemical fungicides, making appropriate disease management even more challenging. Hence, there is urgent need for new sustainable candidates, such as plant extracts or biocontrol agents, to widen the portfolio of active ingredients available on the market and provide new tools to farmers to develop efficient plant protection strategies. At the same time, it is essential to deepen the knowledge on the structure of P. viticola populations and constantly monitor the occurrence of fungicide resistances. In this challenging scenario, the aim of the thesis is to present and characterize new sustainable biofungicides for the control of P. viticola and to offer an insight on the populations ́ structure in the Trentino Alto Adige region, with a further investigation on the resistance to fungicides. Glechoma hederacea aqueous plant extract has significantly inhibited activity towards P. viticola. Moreover, the purification of the extract by chromatographic techniques lead to the isolation of six pure metabolites with anti-oomycete activity, identified as carvacrol, caffeic acid and methyl caffeate, the flavonoids cirsimaritin and apigenin, and the polyphenolic acid rosmarinic acid. Furthermore, Gowan proposed three active ingredients as potential biofungicides, namely the plant extracts 12035 and 12110 and the bacterial BioControl Agent (BCA) Lysobacter capsici AZ78. Following their remarkable plant protection efficacy and rain fastness, the mode of action has been partially characterized. All the candidates showed acute toxic activity on the sporangia and the induction of reactive oxygen species production in grapevine leaf discs. Moreover, 12110 and L. capsici AZ78 stimulated the production of callose on the stomata ́s guard cells. Finally, the metabolites responsible for the L. capsici AZ78 anti-oomycete activity were isolated and identified, namely dihydromaltophylin and maltophylin. On the other hand, P. viticola samples have been collected in 18 vineyards with different disease management strategies in Trentino Alto Adige in 2021 and 2022. Genotyping has been performed on the isolates to identify the structure of P. viticola populations in the region using 22 microsatellite loci. However, the analysis did not highlight a genetic differentiation among the different P. viticola populations, according to location or disease management strategy. Finally, 11 single-site chemical fungicides were tested on the isolates to assess the presence of fungicide resistances. The bioassay highlighted frequent resistant isolates towards cymoxanil in the Region and mandipropamid in Alto Adige province. A moderate adaptation was shown in isolates treated with ametoctradin, amisulbrom, and fluopicolide. On the other hand, the isolates were susceptible to azoxystrobin, dimetomorph, zoxamide and oxathiapiprolin. This study presents several valid candidates for the control of P. viticola, with multi-level mode of action, which can represent a promising alternative to conventional fungicides. However, further studies on product ́s formulation need to be performed, since it plays a crucial role in disease management in commercial vineyards. Moreover, the investigation on P. viticola populations displayed a very limited genetic variability in Trentino Alto Adige, but at the same time diffused adaptation towards single-site fungicide, suggesting an extremely high risk of outburst of resistant isolates. This underscores the importance of detailed and constant monitoring, which is a key proactive measure in the fight against P. viticola.