Tese de Doutoramento em Biociencias, no ramo de Biologia Celular e Molecular, apresentada ao Departamento de Ciencias da Vida da Faculdade de Ciencias e Tecnologia da Universidade de Coimbra

Little is known of the response of Portuguese Dao wine appellation's most common grapevine cultivars to the causal agents of esca and Petri diseases, despite the high incidence of both diseases in the region and the consequent economic losses. Phaeomoniella chlamydospora has been considered one of the major causal agents of these diseases in that region. The present study evaluated the responses of four of the most propagated Dao's grapevine cultivars - Alfrocheiro, Aragonez, Jaen and Touriga Nacional - to infection by three different Portuguese isolates of P. chlamydospora. Field trials were conducted in 2012, 2013 and 2015. The cultivar Alfrocheiro was the most susceptible to P. chlamydospora while cv. Jaen was the least. Variation in parameters such as lesion length and pathogen recovery from infected spurs (within trial years) suggest relation of pathogenicity with weather data, particularly temperature. Differences in aggressiveness among isolates were also detected, with one, a non-native, being the most aggressive. These results provide valuable information for local winegrowers, identifying, for the first time, susceptibility differences among local cultivars to P. chlamydospora, and suggesting adjustments to recommended pruning strategies, specifically to leave long spurs and avoid late winter pruning, thus reducing grapevine trunk colonization by P. chlamydospora.

Black rot on grapevine is a fungal disease caused by Phyllosticta ampelicida (syn. Guignardia bidwellii ) affecting grape leaves as well as clusters. A novel black rot decision support system termed VitiMeteo Black rot was assembled based on existing sub-models and incorporated into the established VitiMeteo forecast and decision support platform. Based on local weather data and a 5-day weather forecast, VitiMeteo Black rot simulates the relative susceptibility of grape clusters, the occurrence and severity of infection events as well as the duration of incubation periods. Data sets obtained in extended international (14 case studies; eight monitoring locations; 11 cultivars; seven countries in Europe and North America) field monitoring campaigns in 2012 and 2013 were used to evaluate the model predictions of newly expressed symptoms on leaves. In the case of the Vitis vinifera cultivars, on average 26.3 disease assessments took place per season. On average, 9.9 predictions were classified as true positive, 8.0 as true negative, 5.2 as false positive and 3.2 as false negative. Model precision, sensitivity and accuracy were on average 64, 77 and 67 %. Potential reasons for false positive and false negative predictions are discussed. VitiMeteo Black rot is freely available for several locations in Germany, Luxembourg and Austria on the internet via the VitiMeteo platform and might be expanded to other regions in the future.

Sixty-eight isolates of Phaeomoniella chlamydospora obtained from symptomatic esca and Petri-diseased grapevines, and mostly from within the Portuguese Dao appellation, were investigated for phenotypic and molecular diversity, in order to determine intraspecific variability and population structure. In vitro growth features, including colony texture, colour, growing margin zonation, hyphal morphology and colony growth rate, were evaluated. Molecular characterization was performed through the sequencing of the total ITS region, and molecular analyses were used to infer phylogenetic relationships, using the Maximum Likelihood approach. Isolates were separated in two groups, supported by phenotypic and molecular analyses, but no clear correspondence was found between the two approaches. Nevertheless, both analyses revealed strong homogeneity among all isolates, despite their diverse geographical origin, year of isolation and scion/rootstock combination, supporting the clonal reproduction strategy described for this species.

Esca and Petri disease, two of the most important fungal trunk diseases of grapevine, are responsible for significant losses by causing premature decline and dieback in vineyards worldwide. The Portuguese Dao wine region is no exception. Local winegrowers' knowledge on Grapevine Trunk Diseases (GTD) in general and of esca and Petri disease in particular is incomplete. The real scope of those problems has been based largely on individual perceptions rather than on a methodical evaluation of the situation. In order to get a full picture of the diseases impact, a leaflet with color pictures was produced and issued to winegrowers, accompanied by a simple questionnaire. The results of this survey represent a first indication of the extent of grapevine trunk diseases in the Dao wine region, specifically its economic impact and relevance to the local wine industry. In conjunction with the survey, several samples of wood collected from esca and Petri disease symptomatic vines, identified during the survey throughout the entire region, were processed and a collection of isolates of Phaeomoniella chlamydospora obtained. To determine the intra-specific variability among these isolates, morphological, cultural and molecular characteristics were evaluated. A protocol to study the pathogenicity with P. chlamydospora was conducted, consisting on the inoculation of cv. Touriga Nacional's spurs with a previously studied P. chlamydospora isolate.

During three years, from 2002 to 2004, a vineyard was surveyed for esca incidence and spatial distribution of deceased/symptomatic vines. Individual plants were recorded as diseased when characteristic esca symptoms were visible in summer surveys or in winter surveys. Some of the symptomatic vines were uprooted every year and fungi most commonly isolated from those plants were listed. Two dimensional maps referring the presence/absence of diseased plants were drawn every year and a cumulative map was obtained on the last year of the survey. Based on the former map, spatial distribution of esca symptomatic plants was analysed in order to confirm the null hypothesis of randomness on the disease spread. Our results showed that: i) internal lesions of uprooted vines were classifiable in two main categories, according to the observed decays; ii) with the exception of Eutypa lata, fungi commonly associated with esca were present in the uprooted vines; iii) the number of esca symptomatic plants increased every year of the survey; iv) esca foliar symptoms were discontinuous from year to year and v) the distribution of infected vines on the plot was random.