Advances towards integrated management of fungal grapevine trunk diseases

Abstract

Grapevine trunk diseases (GTDs) are among the main biotic threats to the economic sustainability of the grapevine industry worldwide, reducing yields, productivity and longevity of vineyards. Several factors such as changes in vineyard cultural practices, low sanitary quality of the grapevine propagation material, the ban of most effective chemical active ingredients, and poor protection of pruning wounds have increased their incidence over last years. Moreover, these diseases are very complex and involve a broad spectrum of taxonomically unrelated fungi making their control very challenging. In this thesis, the development and implementation of novel molecular techniques that lead to the improvement of practical effective strategies to the GTD management were investigated. The assessment of sustainable alternatives to the use of fungicides to increase vineyards lifespan was also performed. Initially, we comparatively assessed the accuracy, efficiency, and specificity of Droplet Digital PCR (ddPCR) and real-time PCR (qPCR) techniques for the detection and quantification of Ilyonectria liriodendri in bulk and rhizosphere soil, as well as, grapevine endorhizosphere obtained from healthy young vines in La Rioja region. The results revealed a strong correlation between both techniques with ddPCR being more sensitive to very low pathogen concentrations. Roots of asymptomatic vines were found to be a microbial niche that is inhabited by black-foot disease pathogens. Then, the spatial dynamics of the fungal communities of these three soil-plant compartments were also characterized by high-throughput amplicon sequencing (HTAS). Soil fungal assemblages had extensive local homogeneity across small distances, and soils with similar climatic and edaphic variables, thus suggesting that vineyard fungal communities are partially conserved in La Rioja region. The diversity and composition of the fungal communities were largely affected by the soil-plant compartment, with diversity decreasing in the endorhizosphere and suggesting that the root tissues entail a barrier for fungal colonization. The results of functional prediction suggested an increase in the relative abundances of potential plant pathogens, endophytes and arbuscular mycorrhiza, and a decrease in the relative abundance of potential wood and/or dung saprotrophs were predicted from bulk soil towards the endorhizosphere. Once more, it was found that the internal root tissues of asymptomatic vines are a microbial niche that is inhabited by GTD fungi. Abstract xiv The HTAS technique was also used to determine the effect of pruning time on the fungal microbiome diversity and composition that colonized pruning wounds naturally over two infection periods (November-February and February-May), in vineyards belonging to three Denominations of Origin (D.O.) in Galicia (Spain). The fungal communities colonizing grapevine pruning wounds were affected in their composition and diversity by the D.O., whereas the spatial variation within each region was low. The results showed the existence of a core community of fungal species conserved in grapevine pruned canes regardless of the infection period. Higher abundances for most GTD genera were detected after pruning in February (winter) than in November (mid-autumn), therefore suggesting a seasonal effect on pruning wounds infections. Additionally, a positive correlation was observed between the accumulated rainfall and the abundance of the total fungal microbiome and Diaporthe genus over eighth and eleventh weeks after pruning. Planting tolerant grapevine cultivars is one of the alternatives to the use of fungicides in controlling GTDs. The tolerance to Phaeomoniella chlamydospora infection of commercial and minority grapevine cultivars from Spanish germplasm collections was evaluated in a rapid and effective detached cutting assay under greenhouse conditions. All cultivars developed internal wood lesions after artificial inoculation with the causal agent of Petri disease and esca, indicating that there is no evidence of qualitative resistance to this fungus. Nevertheless, the severity of internal wood symptoms varied considerably amongst cultivars allowing to classify them according to varying signs of partial tolerance to Pa. chlamydospora. The cultivars ‘Estaldiña’, ‘Albillo Mayor’ or ‘Castañal’ were classified as the most tolerant based on mean lengths of wood discolouration. No foliar symptoms were observed on artificially inoculated plants during the experiments. Regarding disease management strategies, we assessed the effect of several biocontrol agents (BCAs) applied as root treatments at pre- and post-planting to control natural infections caused by black-foot and Petri diseases fungi, and their influence in plant growth parameters. Results showed that BCA effectiveness was dependent on the plant age and the plant part analyzed in reducing the incidence and severity of both diseases. Under specific scenarios, Streptomyces sp. E1 + R4 significantly reduced Dactylonectria torresensis and D. macrodidyma (black-foot) infections, while Pythium oligandrum Po37 and Trichoderma spp. significantly reduced Pa. chlamydospora and Phaeoacremonium minimum (Petri disease) infections. BCAs treatments significantly reduced root weight with respect to the control while no effect was observed on the shoot weight. Abstract xv Finally, we comparatively evaluated the efficacy of various fungicide and BCA formulations registered in Spain to act as pruning wound protectants against Diplodia seriata and Pa. chlamydospora, in the two vineyards of the D.O. Valdeorras (Galicia, Spain). Results showed that BCA-based treatments were less effective than fungicides. The commercial formulation containing pyraclostrobin + boscalid provided a high control of both GTD fungi while Trichoderma-based treatments showed a low efficacy. The poor performance and implantation of treatments based on Trichoderma spp. it is further discussed considering among other factors, the high spore load of GTD fungi used in the artificial inoculations, the short period elapsed between the BCAs treatments application and artificial inoculation with GTD fungi, the effect of unfavourable environmental conditions for the implantation of Trichoderma or the possibility of a low affinity of these fungi for the cultivar 'Godello'.