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Coffee leaf rust (CLR), a disease caused by the biotrophic fungus Hemileia vastatrix (Hv), is the main threat to the worldwide production of Arabica coffee. The gradual breakdown of resistance in coffee varieties in the last years has highlighted the need for novel sources of resistance to CLR. This work aimed to unveil the cellular and molecular resistance profile of the Kawisari hybrid (C. arabica x C. liberica), a genotype used as a resistance donor in Arabica breeding programs in India. This coffee genotype was inoculated with two Hv races that triggered either resistance or susceptibility. Progress of infection was monitored using light microscopy. Simultaneously, we conducted a time-course RNA-seq characterization of the transcriptional responses. The microscopic studies showed that the post-haustorial resistance of Kawisari was associated with the hypersensitive response, accumulation of phenolic-like compounds and haustorium encasement with callose. The transcriptomic analysis suggest the downregulation of host primary metabolism genes at the early onset of infection, followed later by activation of genes functionally associated with multiple plant defense responses, including salicylic acid and jasmonate hormonal signaling. Resistance was also accompanied by the differential regulation of genes associated with phenylpropanoid metabolism and lignin biosynthesis. Our results, further validated by qPCR, provide important new insight into the molecular mechanisms underpinning resistance against CLR in this coffee genotype. Foundation for Science and Technology (FCT) and FEDER funds through PORNorte under the project CoffeeRES PTDC/ASPPLA/ 29779/2017 and by FCT UNIT LEAF (UID/AGR/04129/2020). info:eu-repo/semantics/publishedVersion

Plants have evolved sophisticated mechanisms to coordinate carbon metabolism during growth and development both under optimal and stress conditions. In coffee-rust biotrophic interactions, plants try to limit pathogen access to nutrients (e.g., sugars and sugar derivatives) and trigger immune responses, while Hemileia vastatrix (Hv) attempts to circumvent plant defences and control the host's primary metabolism for its own benefit. Previous proteomics data highlighted the up-regulation of proteins from photosynthesis, primary metabolism, and redox-related enzymes along the coffee resistance response. Coffee Kawisari hybrid - Hv interactions (resistant and susceptible reactions) were evaluated using a single sample fractionation method for metabolite and protein extraction. The microscopic evaluation of the Hv infection process revealed that coffee resistance was associated with early hypersensitive response and accumulation of phenolic-like compounds in host cell walls. GC-TOF-MS untargeted metabolomics allowed the identification of metabolic components, such as sugars, sugar derivatives, amino acids, phenylpropanoids, chlorogenic acids, alkaloids, and fatty acids (using the Golm Metabolome database). The overrepresentation of several caffeoylquinic acids in the resistance reaction may be linked to the accumulation of the phenolic-like compounds that were cytologically observed at the infection sites. Furthermore, sugar-related features also played a role in distinguishing between resistant and susceptible reactions, such as glucose and galactose. The cellular availability of mono and disaccharides is the result of the activity of several enzymes, e.g., sucrose synthase and invertases, that can be targeted by Hv in its strategy to manipulate plant carbon metabolism. The activity profile of these enzymes along the infection will be discussed. Proteomic analysis of the same samples (using the single sample fractionation method previously mentioned) is foreseen. The ultimate goal is to establish a connection between the metabolite and protein signatures. Financial support by the Access to Research Infrastructures, Horizon2020 Programme of the EU (EPPN2020 Grant Agreement 731013) and Foundation for Science and Technology (FCT) and FEDER funds through PORNorte under the project CoffeeRES (PTDC/ASP- PLA/29779/2017), UNIT LEAF (UID/AGR/04129/2020), UCIBIO (UIDP/04378/2020; UIDB/04378/ 2020) and i4HB (LA/P/0140/2020). N/A

Coffee leaf rust (Hemileia vastatrix) is the most important disease of Coffea arabica. This project aims to identify coffee’s primary metabolic components essential for growth and development that are simultaneously involved in plant defense responses. We studied the interaction of two contrasting coffee genotypes [C. arabica S4 Agaro and Kawisari hybrid (C. arabica × C. liberica)] inoculated with different H. vastatrix races to establish compatible (susceptible - S) and incompatible (resistant - R) interactions. Mock-inoculated leaves were used as control (C). Leaves were collected during the infection process, and the fungal colonization was cytologically evaluated. Inoculated and mock-inoculated coffee leaves were prepared and, metabolites, proteins, and starch were extracted from a single sample using a fractionated extraction method. At IPK, metabolite analysis by GC-TOF was undertaken while using the Golm Metabolome Database (GMD). The microscopic evaluation of H. vastatrix pre-penetration stages revealed a high rate of urediniospore germination and appressoria differentiation over the stomata which ensured the success of the infection. For both genotypes, starch content increased along the infection process. A preliminary Principal Component Analysis (PCA) was performed on GC-TOF data showing a clear separation of the two coffee genotypes. In the Kawisari genotype, the PCA analysis of sugar-related features revealed an evident split between samples C, R and S, at each time point. The role of these metabolic features in the coffee defense responses will be discussed. info:eu-repo/semantics/publishedVersion

The harvesting, processing, and sale of wild edible mushrooms (WEM) is a relevant economic activity in Angola and a good example of the use of non-wood forest products for food. Although there is deep traditional knowledge about the general properties of WEMs, a huge gap remains in detailed scientific knowledge. Thus, this study aimed to investigate the socio-economic importance of the species sold at local markets in Huila, Angola, from their molecular identification to the assessment of their nutritional, chemical, and bioactive profiles. From the eight WEM morphotypes studied, five were identified based on phenotypical and molecular approaches (four Russula spp., and Amanita loosei). The studied mushrooms proved to be a rich source of carbohydrates, proteins, and ashes, also presenting low amounts of fat. Chemical analyses further revealed mannitol as the main free sugar in all samples, and organic acids, namely, oxalic, quinic, malic, citric, and fumaric acids in low amounts. Additionally, the -tocopherol isoform and monounsaturated fatty acids were predominant. Regarding phenolic acids, protocatechuic, p-hydroxybenzoic, p-coumaric, and cinnamic acids were detected in all mushroom hydroethanolic extracts, being responsible for their antioxidant, antibacterial, and antifungal activities. Our investigation contributes to the identification and knowledge of WEMs as important complementary food sources in Angola, some of which were reported for the first time, promoting their utilization as a basis of nutritional and functional ingredients, as being able to be part of a balanced diet and to be used in new bio-based formulations info:eu-repo/semantics/publishedVersion

XIII. International Symposium on Grapevine Breeding and Genetics, 10th July - 15th July 2022 The biotrophic fungus Erysiphe necator causes Powdery Mildew (PM) in grapevine. Phytohormones are major modulators of defensive responses in plants but the analysis of the hormonome associated with grapevine tolerance and susceptibility against this pathogen has not been elucidated. In this study, changes in hormonal profiling were compared between a tolerant (Vitis rupestris × riparia cv. 101-14 Millardet et de Grasset) and a susceptible (Vitis vinifera cv. Aragonêz) species upon E. necator infection. Control and PM-infected leaves were collected at 0, 6, 24, 96 hours post-infection (hpi), and analysed through LC-MS/MS. The results showed a distinct constitutive hormonome between tolerant and susceptible species. Constitutive high levels of salicylic acid (SA) and indole-3- acetic acid together with additional fast induction of SA within the first 6 hpi as well as constitutive low levels of jasmonates and abscisic acid may enable a faster and more efficient response towards PM. The balance among the different phytohormones seems to be species-specific and fundamental in providing tolerance or susceptibility. These insights may be used to develop strategies for conventional breeding and/or editing of genes involved in hormonal metabolism aiming at providing a durable resistance in grapevine against E. necator. info:eu-repo/semantics/publishedVersion

Grapevine trunk diseases constitute one of the major problems for viticulture worldwide, with Botryosphaeria dieback considered as one of the most important of these diseases. In this work, we aimed to (i) evaluate the effect of the combination of two products, Esquive® (a Trichoderma-based product) and LC2017 (a low-copper-based product), in the control of Lasiodiplodia theobromae, by evaluating the internal lesion length caused by inoculation of this pathogen on greenhouse kept grapevines of cvs Cabernet Sauvignon and Touriga Nacional and, (ii) investigating their elicitor effect on plant defense responses, through the analysis of the expression of a set of genes. The pathogen was always re-isolated from the infected tissues and was able to cause wood discoloration. Touriga Nacional exhibited longer lesions than Cabernet Sauvignon, and the application of both products did not appear to reduce lesion length when compared to LC2017 applied alone. The elicitor effect of LC2017 on plant defense was confirmed by gene expression analysis, and no significant differences were found between plants treated with LC2017 and with both products. Moreover, a specific response related to the cultivar was verified, but this apparently unique interaction between product, cultivar and pathogen remains to be further investigated info:eu-repo/semantics/publishedVersion

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O projeto TCP/GBS/3081 “Apoio à luta contra doenças e pragas do cajueiro na Guiné-Bissau” é uma iniciativa da delegação da FAO na Guiné-Bissau, que conta com a colaboração e apoio técnico-científico dos parceiros portugueses da Universidade de Lisboa, em particular do cE3c (Centre for Ecology, Evolution and Environmental Changes / Faculdade de Ciências) e o LEAF (Linking Landscape, Environment, Agriculture and Food / Instituto Superior de Agronomia). Este projeto tem como objetivo geral identificar os inimigos do cajueiro na Guiné-Bissau, determinar a sua incidência, severidade, e impacto na produtividade dos pomares, com o objetivo final de propor medidas de mitigação e disseminação contra as principais doenças, pragas e parasitas do cajueiro identificados no país info:eu-repo/semantics/publishedVersion

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Doutoramento em Engenharia Agronómica - Instituto Superior de Agronomia - UL Coffee berry disease, caused by the hemibiotrophic fungus Colletotrichum kahawae, is a major constraint to Arabica coffee production in Africa. Coffee variety Catimor 88, which exhibit field resistance in Kenya, was selected to characterize the resistance to C. kahawae, comparatively to the susceptible variety Caturra. Hypocotyls of both varieties were challenged with C. kahawae (isolate Que2 from Kenya) and samples were collected during infection timecourse, simultaneously for analysis of fungal growth and plant responses (light microscopy), evaluation of enzymatic activities (spectrophotometry, electrophoresis, histochemistry) and gene expression analysis (quantitative real-time PCR). The resistance was characterized by restricted fungal growth associated with the hypersensitive reaction and early accumulation of phenolic-like compounds in the cell walls and cytoplasmic contents. Similar responses were detected in the susceptible variety but in a significantly lower percentage of infection sites. Regarding the genes related to the salicylic acid, jasmonic acid (JA) and ethylene (ET) pathways (phytohormones biosynthesis, reception, and responsiverelated genes), this study suggests the involvement of JA in the resistance while ET seems to be more related with the susceptibility. The expression of genes related to recognition and signaling (RLK, LRR-K, CML, PTL) and cell wall modification genes (PME41, MUR4) was induced in both coffee varieties, at early stages of the infection. However, in the resistant variety, a higher expression of recognition and signaling genes was induced together with the PME41 gene during fungal penetration, and the induction of expression of the Lignin-forming anionic peroxidase-like gene (PER4) was supported by the increase of total peroxidase activity and of an anionic isoform. Peroxidase was localized in the walls and cytoplasmic contents of host cells, at the infection sites. The new data obtained enable to identify potential biomarkers of disease resistance that, once validated, will be useful for marker-assisted selection in coffee breeding programmes N/A

Dissertação submetida como requisito parcial para obtenção de grau de Mestre em Publicidade e Marketing. Perante o fenómeno mundial do consumo de tabaco, os organismos governamentais vêm-se no dever de adotar medidas de dissuasão do consumo, tais como a aplicação de advertências de saúde nas embalagens de tabaco comercializadas. Na tentativa de persuadir os fumadores a modificarem o seu comportamento, estas advertências recorrem a estratégias como a utilização do fear appeal. No entanto, são parcos e pouco consensuais os estudos sobre a eficácia destas advertências nos consumidores portugueses. Neste contexto, esta dissertação tem como objetivos centrais percecionar se a utilização do fear appeal exerce influência nos consumidores na alteração do seu comportamento, bem como compreender quais as variáveis que influenciam o sentimento de medo e a adoção dos comportamentos recomendados. Adotou-se uma abordagem quantitativa, de estudo confirmatório, utilizando como instrumento metodológico a aplicação de um inquérito por questionário a 470 indivíduos que consumiram tabaco nos últimos 12 meses. Os participantes foram expostos a um conjunto de 6 advertências de saúde, agrupadas em 2 blocos, segundo a natureza do medo provocado: físico ou social. Após a análise, concluiu-se que a generalidade dos consumidores apresenta índices muito reduzidos no que respeita ao medo gerado pelas advertências e à perceção da eficácia das mesmas na alteração dos seus comportamentos. Foi possível verificar que as mulheres e os consumidores recorrentes são mais suscetíveis às advertências que os homens e os consumidores correntes. Esta investigação permitiu, ainda, delinear uma proposta de modelo explicativo do funcionamento do fear appeal, definindo as variáveis que influenciam o medo gerado pelas advertências, bem como os comportamentos decorrentes do mesmo. ABSTRACT: Facing the global phenomenon of tobacco consumption, government agencies have been obliged to take measures to decrease smoking habits, such as health warnings on tobacco packages. In an attempt to persuade smokers to change their behaviour, these warnings call upon strategies such as the use of fear appeal. However, studies on the efficiency of health warnings are scarce and not consensual in Portugueses’ with smoking habits. This study aims to understand if the use of fear appeal has an effect in consumers changing behaviour and which variables can influence the way fear is felt and in adopting the recommended behaviour. Choosing a quantitive approach, of confirmatory study, we used as methodological instrument a questionnaire survey to 470 individuals, who consumed tobacco in the last 12 months. Participants were exposed to a set of 6 health warnings, arranged in 2 blocks, according to the type of fear they are supposed to cause: physical or social. Analysis of data revealed that most of the consumers show very low levels of fear when facing the health warnings and consider that their effectiveness in changing behaviours is very poor. It was possible to verify that women and repeat users are more susceptible to health warnings than man and ocassional consumers. This investigation led the author to propose an explanatory model of fear appeal action, identifying variables that interfere with fear and behaviour changes, caused by the health warnings. N/A

Pathogen profile Taxonomy and History: Hemileia vastatrix Berk. and Broome (Basidiomycota, Pucciniales) was described in 1869 in eastern Africa and Ceylon as the agent of coffee leaf rust and has spread to all coffee cultivation areas worldwide. Major disease outbreaks in Asia, Africa and America caused and continue to cause severe yield losses, making this the most important disease of Arabica coffee, a cash crop for many tropical and subtropical countries. Life cycle and Disease symptoms: Hemileia vastatrix is a hemicyclic fungus with the urediniosporic life cycle as its most important (if not only) source of inoculum. Chlorotic spots are the first macroscopic symptoms, preceding the differentiation of suprastomatal, bouquet-shaped, orange-coloured uredinia. The disease can cause yield losses of up to 35% and have a polyetic epidemiological impact on subsequent years. Disease control: Although the use of fungicides is one of the preferred immediate control measures, the use of resistant cultivars is considered to be the most effective and durable disease control strategy. The discovery of ‘H ıbrido de Timor’ provided sources of resistance that have been used in several breeding programmes and that have been proven to be effective and durable, as some have been in use for more than 30 years. Genetic diversity and Molecular pathogenicity: Although exhibiting limited genetic polymorphism, the very large genome of H. vastatrix (c. 797 Mbp) conceals great pathological diversity, with more than 50 physiological races. Gene expression studies have revealed a very precocious activation of signalling pathways and production of putative effectors, suggesting that the plant–fungus dialogue starts as early as at the germ tube stage, and have provided clues for the identification of avr genes info:eu-repo/semantics/publishedVersion

Mestrado em Engenharia Agronómica - Instituto Superior de Agronomia Cellular and molecular characterization of resistance to H. vastatrix in coffee lines HDT 832/2 and Sarchimor, was conducted using the non-host resistance model coffee-Uromyces vignae (cow pea rust) for comparison. In both lines, H. vastatrix ceased growth more frequently at the penetration hypha stage, forming few haustoria, inducing hypersensitive reaction (HR), phenol accumulation and haustorium encasement with calose. U. vignae growth was more restricted in HDT 832/2, but failed to form haustoria in both lines, while inducing HR and phenol accumulation. The expression of genes involved in mechanisms of recognition, signaling and defence (rlk, wrky1, pal, chs, 13-lox, gt, pr1b and pr10) was monitored by real time PCR. Both in the host and non-host interactions the expression profile of rlk and wrky1 suggests that the recognition of the fungus and signaling occur during the formation of appressoria over stomata and the differentiation of the first post-penetration infection stages. The first activation peak of 13-lox coincided with the onset of cell death detection. pr1b exhibited high expression levels in the host interaction, being poorly activated in the non-host interaction. These results suggest that both JA and SA pathways may coexist, although SA pathway seams to prevail in the host interaction.