Esca is a grapevine trunk disease with worldwide distribution. The disease is destructive, without effective treatment, leading to high costs in the viticultural sector. Esca disease is complex, being associated with fungal infection of the xylem, as well as with environmental factors. External symptom appearance is inconstant, and esca-related fungi may live within the plant without causing apparent disease. Water stress is thought to be a factor involved in esca symptom appearance. The objective of this project was to better understand the factors involved in the pathogenicity of esca-related fungi, specifically how water stress could facilitate esca disease. A metabolomic analysis of xylem sap from plants inoculated with esca-related fungi and concomitantly subjected to drought revealed an overall concentration increase of several amino acids, sugars and other small molecules in xylem sap. Most of these changes were associated with the water stress, but the presence of fungal infection also contributed to the concentration increase of specific metabolites. The results suggest the increase of nutrients in xylem sap associated with drought may be involved in esca disease progression. Techniques used in this project included greenhouse experiments, microbiological techniques, controlled inoculations with live pathogen, proton Nuclear Magnetic Resonance (1H-NMR) spectroscopy.
Plants and animals require iron as an essential nutrient. Iron deficiency adverse effects on agriculture include reduced yield and quality of crops, and lower nutritional value of plant foods. Besides, iron deficiency is a public health concern causing iron deficiency anemia, which affects over 30% of the world population (those relying on plant-based diets being more affected). In this project, plant responses to iron deficiency were investigated by analyzing changes in mineral composition of root and shoot tissues, gene expression, enzyme activity, proteome and metabolome. Several leaf metabolic pathways were shown to be affected by iron deficiency, and some metabolites were implicated for the first time in the response to iron deficiency. Root proteome analysis revealed the existence of a fine tuning of root metabolism associated with ferric iron reduction. Additionally, a human intestinal mucosa cell line model, Caco-2 cells, was used to analyze the bioavailability of iron in common beans. Caco-2 cells showed higher iron concentration and increased iron uptake from cooked beans, suggesting the presence of components that enhance iron intestinal absorption after cooking. Techniques used in this project included hydroponics, molecular biology, animal cell culture, gel-based proteomics, and 1H-NMR spectroscopy.
Pine Wilt Disease is caused by Bursaphelenchus xylophilus, a nematode recently introduced to Europe. The disease affects conifer forests with a high mortality rate. The objective of this project was to assess the impact of the nematode in autochthonous plant species of Portuguese forests and to investigate the mechanism(s) of resistance. Although species of the genus Pinus are the primary host, some autochthonous non-Pinus species appeared to be compatible with the nematode suggesting other species could act as a repository of the nematode in an ecosystem. Additionally, a large-scale susceptibility screening test was developed and used to screen pine trees of different geographic origins. Trees from different locations presented different degrees of resistance to the nematode. Furthermore, nematode infection induced differential volatile production in pine trees within a week after inoculation, suggesting these compounds could be used as disease biomarkers. Techniques used in this project included controlled inoculations with live pathogen, Scanning Electron Microscopy, and Solid-Phase MicroExtraction-Gas Chromatography/Mass spectrometry (SPME-GC/MS).
Esca is serious disease of grapevine for which no effective treatment exists. The disease is complex; it involves infection by several fungi and is influenced by environmental factors. In this project, a grapevine cell suspension culture challenged with Phaeomoniella chlamydospora was used to study the plant’s molecular and biochemical defense responses. The cells increased the production of phenolic compounds, some with recognized antifungal activity, and up-regulated several defense-related genes in response to fungal elicitation. This in vitro model also suggested a role for methyl jasmonate, and the involvement of reactive oxygen species and calcium signaling in the defense mechanism. Leaves from plants naturally infected with esca were also analyzed. Diseased leaves increased phenolic production, confirming the results obtained in vitro, and appeared to reroute carbon and energy from primary to secondary metabolism. Results suggested that the leaf phenolic compounds could be used to develop an early detection (non-destructive) esca disease diagnostic method. Techniques used in this project included plant tissue culture, microbiologic and molecular biology techniques, High Performance Liquid Chromatography (HPLC), and proton Nuclear Magnetic Resonance (1H-NMR) spectroscopy.