Control biológico de "Phaeoacremonium minimum" mediante el uso de "Trichoderma" spp. en plantas de vid

Résumé

This doctoral thesis evaluates the possibility of using autochthonous biological control agents to face grapevine trunk diseases. In particular, the use of fungi from the genus "Trichoderma" against "Phaeoacremonium minimum", which is considered a pioneer fungus of esca and Petri diseases. Nowadays, the main problem is the lack of effective products against these diseases. This is due to the withdrawal of chemical products to reduce the pernicious effects of pests and diseases and it is motivated by a greater concern about environment, and health of consumers and farmers. European policies look for ecological and sustainable solutions, adapted to climate change and respectful to animal, human and environmental health. However, there is a lack of consistency of commercial biological products and a limited number of chemical ones that, in addition, can induce resistance to pathogens. Therefore, the main objective of this thesis is based on looking for solutions based on the use autochthonous biocontrol "Trichoderma" spp. agents adapted to the ecological niche of the vineyards from Castilla y León region. Here we show that the isolate "Trichoderma" sp. T154, selected and characterized as a biological control agent adapted to the ecological conditions of this region, which acts efficiently against "Phaeoacremonium minimum", being also able to colonize pruning wounds under climate change conditions. First, a survey of vineyards was carried out in Castilla y León. A new method was used to isolate "Trichoderma" strains from bark of grapevine plants. It was confirmed that the older grapevine plants are, the higher is the diversity of "Trichoderma" spp. isolates and also, a negative correlation was identified between the number of fungicides sprayed per campaign and the diversity of "Trichoderma" spp. isolates. Moreover, 25 isolates of "Trichoderma" were identified from the 10 sampled plots. The antagonistic capacity of these isolates was evaluated using "in vitro" confrontation dual assays against "Phaeoacremonium minimum" Y038-05-3a. The isolates T75, T79, T84 and T154 were selected by their high biocontrol activity. Subsequently, the isolate T154 was used for evaluating biocontrol mechanisms against the isolate "Phaeoacremonium minimum" CBS 100398. This autochthonous "Trichoderma" isolate was transformed using the protoplast procedure in order to express the red protein tdTom. Thus, mycoparasitism under "in vitro" conditions and niche exclusion in wounds of Vitis vinifera cv. Tempranillo were identified as main mechanisms of biocontrol performed by this biological control agent. Finally, "Trichoderma" strain T154 among others indigenous "Trichoderma" strains were evaluated at different temperature conditions and also were inoculated in pruning wounds to evaluate their capacity of colonization. This strain demonstrated a great production of spores at 25ºC under "in vitro" conditions. Moreover, "Trichoderma" strain T154 was able to significantly colonize pruning wounds of Vitis vinifera cv. Tempranillo under semi-field conditions during winter. Besides, this strain was able to remain at least 12 weeks pos inoculation in plant, adapted to simulated climate change conditions and inoculated based on One health approach. Future implications derived from this thesis should be the spraying of this autochthonous strain, "Trichoderma" sp. T154, under field conditions to study its interaction with the rest of microbiota and its effects on wine production.