DAGRO - Teses de Doutoramento

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  • ItemAcesso Embargado
    Exploring the potential of Prunus lusitanica L.: From chemical characterization to bioactivities aiming the development of new products
    2023-05-05 - Abraão, Ana Cristina dos Santos; Silva, Amelia Maria Lopes Dias da; Barros, Ana Isabel Ramos Novo Amorim de; Domínguez-Perles, Raúl
    Prunus is a genus classified within the family Rosaceae, comprising over 400 species of deciduous and evergreen trees or shrubs. Prunus lusitanica L., commonly known as "Azereiro" or "Loureiro de Portugal," is considered a survivor of the relict Lauroid fog forests that once populated the Mediterranean basin during the Tertiary period. This rare species possesses significant ecological and ornamental value, and is currently classified as "near threatened" in the "Red List of Vascular Flora of Mainland Portugal." Prior to the works described in this thesis, only two studies had been conducted on the phytochemical composition and bioactive properties of this species, both of which focused on the leaves. However, given the diversity of phytochemical compounds identified in other Prunus species, particularly those phylogenetically related to P. lusitanica, such as Prunus padus and Prunus laurocerasus, and the bioactivities attributed to them leads us to believe that it is scientifically relevant to study the fruits of P. lusitanica. This approach may lead to the development of new strategies to enhance the value of these fruits and expand this species geographic range. The main objective of this thesis was to explore different strategies for the valorisation of Prunus lusitanica L. fruits. In this sense, specific objectives were drawn, namely to assess the nutritional composition (Chapter 3), phenolic composition (both qualitative and quantitative), and antioxidant capacity (Chapter 4) of Prunus lusitanica fruits harvested over a four-year period, and also, to evaluate the in vitro bioactive potential of the fruits extracts from different regions of Portugal concerning their anti-proliferative/cytotoxic, anti-inflammatory, anti-diabetic, anti-aging, and neuroprotective activities (Chapter 5). Based on the findings presented in Chapter 3, it has been observed that P. lusitanica fruits are abundant in a variety of nutrients, including proteins, fats, carbohydrates, dietary fibers, amino acids, and minerals. Furthermore, the nutritional composition of these fruits was found to be primarily influenced by the year factor and was found to be strongly correlated with environmental variables such as precipitation and temperature. It is noteworthy that the nutritional contents of P. lusitanica fruits are significant in comparison to other fruits of the same genus, particularly with respect to the protein (ranging from 2.73 to 6.45 g/100 g fw) and total dietary fiber (ranging from 6.68 to 12.08 g/100 g fw) contents, among others, regardless of the year of study. Concerning the results described in Chapter 4, it was observed that P. lusitanica fruits are abundant sources of phenolic compounds. In total, twenty-eight compounds from different classes were identified and quantified, comprising twenty-one hydroxycinnamic acids (60.3%), two flavan-3-ols (27.9%), two anthocyanins (10.5%), two flavonols (1.0%), and one secoiridoid (0.3%). Furthermore, in addition to their phenolic composition, the fruits exhibited a high antioxidant capacity, as determined by ABTS, DPPH, and FRAP assays, with values ranging from 7.88 - 10.69 mmol TE (millimoles of trolox equivalents (mmol TE))/100 g fw, 5.18-8.17 mmol TE/100 g fw, and 8.76-11.76 mmol TE/100 g fw, respectively. Furthermore, a strong positive correlation was observed between the antioxidant capacity and the phenolic composition of the fruits. The findings presented in Chapter 5 have shown that hydroethanolic extracts derived from P. lusitanica fruits obtained from three distinct locations possessed comparable phytochemical profiles and bioactive properties, albeit with minor disparities in the levels of specific compounds. Extracts obtained from P. lusitanica fruits harvested in Vila Real (PL1) exhibited higher levels of total phenolic compounds and hydroxycinnamic acids, whereas total flavan-3-ols were present in lower concentrations. In contrast, samples from Mata da Margaraça (PL3) had higher concentrations of flavan-3-ols (over two-fold higher than Vila Real samples) and anthocyanins (more than two-fold higher than Pardieiros (PL2) samples). We have provided evidence for the first time that P. lusitanica fruit extracts possess antiproliferative activity, which was more pronounced in HepG2 cells, as well as significant anti-inflammatory and neuroprotective potential, with moderate anti-aging and anti-diabetic effects. Overall, the combined results of the studies conducted in this thesis demonstrate that P. lusitanica L. fruits are rich sources of nutrients and phenolic compounds that possess promising bioactive properties related to several pathophysiological processes. Based on these findings, it was demonstrated that P. lusitanica fruits hold great potential for developing new products or ingredients to be used in multiple industries, such as food, cosmetics, and/or (phyto) pharmaceuticals. However, since the nutritional, phytochemical, and functional characterizations of P. lusitanica have been conducted for the first time, further research on this plant species is imperative to determine its potential as a source of bioactive compounds that promote health. Additional characterizations are required to explore its full potential in a near future.
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    Contribution to the knowledge of Port Wine oxidative ageing process: study of chromatic characteristics and aroma profile of Tawny and White Port Wines
    2023-03-02 - Ferreira, Juliana Milheiro; Nunes, Fernando Hermínio Ferreira Milheiro; Martins, Maria Fernanda Gil Cosme
    White (WPW) and Tawny Port wines (TPW) are two of the most important Port wines styles produced in the Douro Demarcated Region, made from authorised white and red grape varieties where after a short fermentation period is fortified with non-rectified wine spirit up to 20% of ethanol and aged in small used oak barrels (~ 600 L) for at least three years. These conditions allow the contact with oxygen, and therefore, these wines are aged under oxidative conditions. Port wines with indication of age (IAC) are one of the most important categories of WPW and TPW. These are blend wines made up by older and newer aged Port wines to obtain the sensory characteristics of a WPW or TPW that would been aged for the time indicated. The certification of these categories is made by the sensory panel of the “Instituto dos Vinhos do Douro e Porto”. Colour of the different styles of Port wines and IAC are a distinctive quality parameter dependent on the grapes used, ageing process, and time. The impact of the phenolic composition of the different Port wine styles and IAC is largely unknown. We characterised the chromatic characteristics (CIEL*a*b*) and relation with the phenolic composition including monomeric, small (SPP) and large polymeric pigments (LPP) and tannins of WPW, TPW and Ruby Port wines (RPW) and evaluated the feasibility of its use for their discrimination. Port wine styles can be discriminated using the chromatic characteristics. WPW presented the highest L* values, RPW presented the highest a* values and TPW presented the highest b* values. Their chromatic characteristics were related to their pigment/phenolic composition. RPW contained the highest levels of phenolic compounds. For all Port wines the majority of the total pigments at 520 nm and 420 nm are presented as polymeric pigments (PP, 71%). On the other hand, RPW and TPW presented the PP as LPP, higher. The same trend was observed for SPP 420 nm and LPP 420 nm. The more oxidative ageing of TPW when compared to RPW, results in wines with lower levels of pigment/phenolic compounds, this decrease increasing with the IAC of TPW resulting in an increase in L* and b* and decrease in a*. On the contrary for WPW the pigments are developed and increased with the IAC, resulting in a decrease in the L* value and increase in a* and b* values. The higher b* values, corresponding to the brownish/yellowish colour of TPW and WPW belonging to higher IAC seems to be related to the sugar browning than oxidative change in phenolic compounds, although this phenomenon is important for decreasing the red colour (a* value) of TPW for the higher IAC. The aroma profile of TPW and WPW with increasing IAC was determined to study the similarities and differences in the oxidative ageing of these two related but different matrixes, being quantified thirty-one and twenty-eight aroma compounds, respectively. Sotolon increased for both wine styles being important for the aroma of Port wines with higher IAC, where it presented an olfactory activity value (OAV) higher than unity, nevertheless, ethyl hexanoate, ethyl octanoate, ethyl decanoate had also OAVs >1, especially in TPW where their concentration increased with increasing IAC, and decrease in importance for WPW, as they decrease for higher IAC. TDN and -damascenone appear to have also an important contribution to the aroma due to their very low odour threshold. The differences in aroma evolution pattern observed for these two wine styles can be due to the differences in the wine matrix composition preventing the loss of aroma compounds by evaporation in TPW as that observed for the WPW with increasing IAC. Sotolon, HMF, and -ketobutyric acid increased with the increase IAC with small differences for WPW and TPW, and that there is probably a common origin of these three compounds. Results obtained from model systems allowed to support the hypothesis that fructose is the main precursor of sotolon, HMF and -ketobutyric acid, that increase for TPW and WPW with increasing IAC. The presence of amino acids reduced the amount of sotolon and increased the amount of -ketobutyric acid and HMF formed from fructose. On the other hand, metal catalysts (iron and copper) reduced Sotolon yield but increased the b* value of Port wine like models. These results show that in Port wines obtained by oxidative ageing, either made from white or red grape varieties, fructose caramelisation is the main pathway for sotolon formation and also -ketobutyric acid and HMF, although for these last two the Maillard reaction is the most efficient pathway.
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    Valorisation of Cistus ladanifer L.: from production to evaluation of raw materials
    2022-09-23 - Frazão, David Passos Morgado Franco; Silva, Amélia Maria Lopes Dias da; Sousa, Fernanda Maria Grácio Delgado Ferreira de; Gonçalves, José Carlos Dias Duarte
    A esteva, Cistus ladanifer L. (Cistaceae), é um recurso subvalorizado largamente distribuído na Península Ibérica. Devido à sua abundância, a esteva nunca foi cultivada na sua área de distribuição endémica, mas poderá ser relevante em termos de produção, fito-remediação, ocupação e recuperação de solos, uso ornamental, e investigação. A valorização dos produtos de esteva e a criação de uma cadeia de valor sustentável são necessárias para compensar a gestão dos seus matos naturais ou do seu cultivo. Atualmente, só o óleo essencial e a resina lábdano são valorizados nos setores das fragrâncias e perfumaria, mas, outros extratos e a biomassa de esteva têm sido estudados para serem valorizados. Embora a resina e as sementes de esteva, tradicionalmente, tenham sido usadas medicinalmente e como alimento, respetivamente, não existem estudos que abordem essa valorização. Este trabalho teve como objetivos: testar uma prática de gestão do esteval natural de modo a reduzir a biomassa combustível; testar o cultivo da esteva aplicando a mesma prática de gestão; usar técnicas de cultura de tecidos vegetais in vitro para a propagação clonal de esteva e produção de metabolitos; avaliar o valor nutricional das sementes e avaliar o potencial do lábdano como ingrediente cosmético e medicinal, de modo a contribuir para uma valorização integrada da esteva e contribuir para uma economia circular. O corte a 0,5 m efetuado no verão, no esteval silvestre e no cultivado, mostrou ser adequado para a gestão destes dois tipos de campos de esteva, inibindo a acumulação de biomassa lenhosa combustível e rendendo uma produção anual de biomassa verde fotossintética de 1500 kg/ha (esteval silvestre) e de 5500 kg/ha (esteval cultivado) e uma produção anual de lábdano, para uma colheita no final do verão, de 150 kg/ha (esteval silvestre) e 550 kg/ha (esteval cultivado). Para uma produção significativa de cápsulas e sementes foi necessário um crescimento por dois anos consecutivos tendo, os dois estevais, atingido uma produção de 270 kg/ha de cápsulas e 80 kg/ha de sementes. Por técnicas de cultura in vitro, a partir de segmentos caulinares internodais e segmentos foliares foi possível induzir a regeneração de rebentos (38%: explantes caulinares e 6-benzilaminopurina, BAP), a formação de callus rizogénicos (100%: dois explantes e ácido diclorofenoxiacético, 2,4-D, e BAP) ou a formação de callus com potencialidade para a regeneração de rebentos (100%: explantes caulinares e BAP e ácido 1-naftalenoacético) que poderão ser usados para a produção de metabolitos in vitro. As sementes de esteva mostraram ser uma fonte de proteína (16,2 %), cálcio (238 mg/100g) e zinco (3,22 mg/100g) e uma alta fonte de fibra (20,9%), xiv ácidos gordos insaturados (74%, gordura: 13%), fósforo (452 mg/100g), ferro (5,18 mg/100g) e magnésio (232 mg/100g). A extração de lábdano pelo processo Andaluz (2-11% peso resina seca/material fresco) foi mais eficiente que pelo processo Zamoreano (0-2% peso resina seca/material fresco), ambos produzindo um efluente aquoso com grande quantidade de fenóis (1-12 gGAE/L). As duas resinas renderam cerca de 70% do seu peso em extrato absoluto. O absoluto de lábdano obtido pelo processo Andaluz mostrou ser, principalmente, constituído por diterpenoides e flavonoides enquanto o absoluto de lábdano obtido pelo método Zamoreano mostrou ser constituído, principalmente, por fenilpropanoides, ácidos gordos e diterpenoides. O absoluto Andaluz mostrou atividades de proteção UV (Fator de proteção solar 5) e anti-inflamatória (95% de inibição de produção de NO em células RAW 264.7, a 15 μg/mL), devido principalmente aos flavonoides, o que é relevante para o seu uso como ingrediente em produtos cosméticos, dermocosméticos e medicamentos tópicos, mas mostrou fraca inibição da elastase e atividades antioxidante (DPPH, FRAP, and ABTS), e atividade antimicrobiana unicamente para Staphylococcus aureus ATCC 25923 (MIC 1,2 mg/mL). Este absoluto revelou 70% de inibição da acetilcolinesterase a 0,5 mg/mL, a fração de diterpenos revelou 30% de inibição da α-amilase a 0,5 mg/mL, e o absoluto revelou atividade citotóxica (IC50 (24h de exposição): 77 μg/mL e 53 μg/mL em células HepG2 e CACO2) o que é relevante para sua valorização como ingrediente medicinal e fonte de fármacos. Resumidamente, os estevais podem ser geridos de modo a obter produtos de valor acrescentado como lábdano (p.e., ingrediente cosmético e medicinal), extratos ricos em compostos fenólicos (p.e., aditivos alimentares), sementes (como alimento humano), biomassa verde (p.e., forragem) e restos de cápsulas e biomassa lenhosa (p.e., fonte de energia), ao mesmo tempo que se reduz a produção de biomassa combustível e se mantém a cobertura e recuperação de solos.
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    Estratégias de modelação do ecossistema vitícola
    2022-05-27 - Reis, Samuel Castro Maciel; Santos, João Carlos Andrade dos; Carlos, Cristina da Conceição Ribeiro; Torres, Laura Monteiro
    A fenologia, quer da traça-da-uva, Lobesia botrana (praga-chave da vinha na Região Demarcada do Douro), quer da videira (seu hospedeiro) tem-se alterado ao longo das últimas décadas em resposta ao aumento da temperatura. Por isso, simulações baseadas em modelos da fenologia são cruciais para prever os possíveis impactos deste aumento de temperatura na fenologia de ambos, conhecimento que é útil para os viticultores planificarem corretamente as várias operações, isto é, operações culturais na vinha, métodos de proteção contra a praga, entre outras. Nesse contexto, o objetivo da presente Tese, foi o de avaliar o potencial impacto das alterações climáticas na fenologia da videira (hospedeiro) e no desenvolvimento de uma das principais pragas da videira, a traça-da-uva, na Região Demarcada do Douro. A partir do objetivo geral, os seguintes pontos foram estudados: i) desenvolvimento de um modelo generalizado (GSM) para cada estado fenológico (abrolhamento, floração e pintor) e sua validação, para o conjunto das castas selecionadas em quatro regiões vitícolas (Douro, Dão, Vinhos Verdes e Lisboa). Consequentemente, pretendeu-se aplicar os modelos numa plataforma de apoio à decisão ao viticultor; ii) avaliação do impacto das alterações climáticas na fenologia do voo de L. botrana (daqui em diante esta terminologia utilizada) na Região Demarcada do Douro, disponibilizando informações úteis para o viticultor otimizar as medidas de proteção contra a praga. Esta abordagem traduziu-se no uso dos modelos para prever os voos de L. botrana em cenários de alterações climáticas (concentração representativa de gases de efeito estufa, RCP4.5 e RCP8.5) para as parcelas selecionadas e avaliar a existência de alterações nas datas de início e pico dos três voos do inseto. Por último, realização de uma análise preliminar, em uma parcela, relativamente à infestação e voo; iii) elaboração de mapas espácio-temporais sobre a fenologia da videira (cv. Touriga Nacional e Touriga Franca) e L. botrana cobrindo toda a Região Demarcada do Douro em malha de ~1 km para o histórico e futuro. Consequentemente, avaliou-se a partir dos modelos da fenologia disponíveis (com dados da região), a interação entre os níveis tróficos (videira e L. botrana). Foram desenvolvidos modelos tendo em consideração dados da fenologia e meteorologia de quatro regiões vitícolas. Para cada estado fenológico, usou-se o centroide dos parâmetros estimados (d e e), e consequentemente os três modelos sigmoide generalizados (GSM) foram construídos (abrolhamento: d = −0.6, e = 8.6; floração: d = −0.6, e = 13.7; pintor: d = −0.5, e = 13.2). Além disso, de acordo com outros estudos, a capacidade preditiva para o abrolhamento é menor relativamente à floração e pintor. As datas simuladas (DOY), correspondentes aos principais eventos do voo (início e pico das capturas de machos em cada voo; três voos) de L. botrana obtiveram-se em parcelas selecionadas para os períodos histórico e futuro, usando os modelos de voo com dados exclusivos da região. A partir dos resultados obtidos, os voos ocorrerão mais cedo em todas as parcelas durante o período de 2021−2080, sendo de 7 a 12 dias de antecipação no cenário RCP4.5, e 15 a 24 dias no RCP8.5, comparativamente com os valores históricos (2000−2019) e independentemente do voo. Os resultados sugerem que, um quarto voo completo será provável no futuro, e que L. botrana tornar-se-á uma espécie tetravoltina na região. Além disso, a validação numa parcela de estudo permitiu verificar que o número total de capturas de machos durante o segundo e terceiro voos diminui, assim como a percentagem de cachos atacados na segunda e terceira gerações, com o aumento do número de dias em que a temperatura máxima esteve acima de 33°C. Em última análise, verificar-se-á uma antecipação nos estados fenológicos da videira (cv. Touriga Nacional e Touriga Franca) em toda a região (numa malha de resolução ~1 km) para 2051–2080 em RCP8.5. Deste modo, o abrolhamento antecipará até 14 dias, a floração e o pintor até 10 dias (principalmente em cotas baixas, junto ao rio Douro). Quanto a L. botrana, uma antecipação generalizada será expectável ocorrer ao longo da região para os três voos, embora o terceiro tenha uma antecipação mais significativa (poderá antecipar até 22 dias no pico). Por último, as simulações sugerem que existirá sincronia entre o terceiro voo e o pintor em ambas as castas (cv. Touriga Nacional e Touriga Franca) em 2051–2080 para RCP8.5.
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    Spectroscopy data-driven models for grape quality assessment in precision viticulture
    2022-04-05 - Gomes, Véronique Medeiros; Melo-Pinto, Pedro; Rovira Más, Francisco; Reis, Marco Paulo Seabra dos
    Currently, the wine industry is facing a serious competitive challenge that involves raising the production paradigm to new levels of excellence. Grape ripeness assessment, based on the evolution of enological parameters over time, is one of the most important factors for ensuring and improving wine quality, playing a decisive role in the definition of the optimal time for harvesting and in the selection of grapes according to the desired characteristics of the final product. In this context, precision viticulture technologies have been explored to accomplish a faster, non-destructive, non-invasive and ultimately more sustainable assessment of grape maturity. In particular, hyperspectral imaging has emerged as a promising cost-effective alternative to the conventional physical and chemical methods to predict important enological parameters and assist in harvesting critical decisions. However, the application of such technology involves large volumes of data that need to be explored and transformed into useful information so that it can be used as a basis for decision-making. Such large amount of data, together with the large variability associated, raise computational challenges for data-drivenmodelling, making the selection of proper models a complex and cumbersome task. Therefore, the overall objective of this thesis was outlined in order to provide consolidated insights for the development of precision viticulture, boosting the application of a non-invasive and nondestructive solutionsfor monitoring the grapes ripeness. The base technology was hyperspectral imaging of proximity in reflectance mode, with the development of data-driven models being the main focus of this thesis. Sugar content, pH and anthocyanin concentration were the parameters considered for grape ripeness assessment. More specifically, and in order to take advantage of the highly complex and informative datasets, various machine learning methods were developed, tested and compared, whose performance was assessed through crossvalidation and independent test sets for generalization ability evaluation, using samples from different vintages, varieties and growth conditions not used during the training process. Another topic discussed in this thesis was the development of more complex models, namely deep learning algorithms, in order to assess their generalization ability with new samples compared to classical machine learning methods, and also to investigate the need for more training data to properly train such class of complex models. To do so, two one-dimensional convolutional neural networks architectures were proposed, one simpler and another more complex. Additionally, a transfer learning mechanism based on the more complex architecture was xviii implemented to evaluate improvements in the model’s generalization. The effect of different spectral preprocessing techniques was also investigated for both approaches, machine learning and deep learning. Overall, the results obtained demonstrated that the large differences in terroir, between varieties and between vintages have a great impact on grape ripeness and, consequently, on the generalization robustness of the predictive models developed. Moreover, the results for models’ generalization ability showed good performances under lab conditions (laboratory-acquired data), with deep learning outperforming the machine learning methods and achieving very good predictions, demonstrating the effectiveness and robustness of the methodology. Finally, the evaluation of important spectral regions corresponding to the enological parameters under analysis was another topic covered in this thesis. For machine learning approaches, this was done through the analysis of regression coefficients, while for deep learning approach, it was proposed a model explainability technique, based on regression activation maps, to discriminate between regions in the spectrum that the deep learning model has more intensively used to perform the predictions. Here, the results obtained provided good indicators, highlighting important spectral regions and opening new ways to reduce the cost of the equipment and the dimensionality of the data without losing predictive power. As a final remark, the results of this thesis contributed to the progress in the knowledge regarding the application of imaging spectroscopy combined with appropriate predictive methods, and how the models are influenced by the natural variability of the samples and the specific characteristics that may be present inside a vineyard, representing an important step towards a more sustainable grape quality assessment.
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    Innovative strategies to mitigate effects of climate change for sustainable hazelnut production
    2020-09-24 - Cabo, Sandra Cristina Santos do; Macedo, Berta Maria de Carvalho Gonçalves; Silva, Ana Paula Calvão Moreira da; Pascual Seva, Núria
    Climate change affects directly several economic sectors, namely agriculture, forestry, energy consumptions, tourism among others. In agriculture, the climate change has a huge impact on crop growth and yield. This takes great importance in perennial fruit crops due to their higher sensitivity to environmental conditions, particularly on floral differentiation, blossom, pollination, fruit setting and growth stages. Temperate fruit treesrequire some chilling during the development, however climate change is making climates warmer, which significantly influence the crops growth and yield. Furthermore, the average global temperature will move plant species to new areas with more favourable climate conditions. These difficulties will be transversal to all perennial fruit species being hazelnut (Corylus avellana L.) one of them, especially those growing in Mediterranean countries. The main objective of this Doctoral Thesis is to contribute with strategies to mitigate effects of climate change and on the other hand, to enhance the quality and production of hazelnut by different approaches. To achieve these goals, an evaluation of the suitability of preharvest foliar spray treatments was performed, gaining a further insight in the agricultural management strategies by understanding the plant physiological and biochemical response, enhance hazelnut quality, environmental sustainability and economic viability for hazelnut production. In addition, regarding the massive amounts of bio-wastes, namely the hazelnut husks, the determination of the suitability of husks (hazelnut by-product) as a source of bioactive compounds was also studied. In this sense, two experiments were carried out, the first experiment consisted in spraying treatments combined with irrigation: kaolin (K, 4%); natural bioestimulant Ascophyllum nodosum (An, 0.15%); salicylic acid (SA, 0.01 %); K with irrigation (Ki); An with irrigation (Ani); SA with irrigation (SAi); control with irrigation (Ci) and control (C), during 2016 and 2017, in a hazelnut orchard with adult trees in full production using an economically important hazelnut cultivar ‘Grada de Viseu’, located in Moimenta da Beira (Northern Portugal). A positive effect of the spray treatment on ‘Grada de Viseu’ yield, physiological and biochemical responses was shown. Results showed that K and An were effective for reducing hazelnut heat and drought stresses by increasing the relative water content, net CO2 assimilation rate, water use efficiency and by reducing the leaf mass per area and electrolyte leakage. Moreover, concentration of abscisic acid, malondialdehyde, proline, lipid peroxidation and the average content of antioxidant enzymes was lower, suggesting that under K and An treatments, plants have a better physiological performance than control trees. Under K, An and SA with and without irrigation treatments, the content of total sugars decreased and the content of starch, salicylic acid, thiols and the ratio between total chlorophylls and total carotenoids increased, demonstrating that these treatments can be effective against drought. These treatments affected positively the biometric parameters, namely nut and kernel sizes, vitamin E levels, antioxidant activity and some phenolic compound contents. The sensorial attributes were not affected by the treatments, this way the application of K, AN and SA improve the hazelnut tree physiological response to the climate change, without compromising the hazelnut chemical and sensorial quality. Other experiment was carried in a hazelnut orchard with adult trees in full production in Vila Real, Portugal. Hazelnuts husks were obtained from four cultivars: ‘Butler’, ‘Grada de Viseu’, ‘Morell’ and ‘Lansing’, and the bioactive potential of hazelnut husks was determined as a function of their cultivar source and extraction solvent. A total of 11 phenolics were identified. The results showed that cultivar and extraction solvent influenced significantly the extraction yield. Methanolic husk extracts exhibited the greatest antioxidant potentials followed by water and acetone. ‘Grada de Viseu’ husks presented the highest content of individual phenolics and antioxidant activity, while the husks from the cultivar ‘Lansing’ showed the lowest levels. The valorization of hazelnuts husks gives an important contribution for the isolation and purification of bioactive molecules. In conclusion, this thesis contributed to increase knowledge of the use of promising preharvest compounds for the improvement of the hazelnut physiological and biochemical performance as well the nut quality. Nonetheless lower and non-significant differences between treatments with and without irrigation were observed revealing that K, An and SA can be used without irrigation to mitigate summer stress effects being these compounds useful strategies to mitigate climate change effects for sustainable hazelnut production
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    Estudo da produtividade do souto (Castanea sativa Miller) e avaliação do efeito da inoculação com fungos ectomicorrízicos e bactérias no crescimento das plantas e na resistência ao oomiceta Phytophthora cinnamomi Rands
    2020-07-23 - Henriques, Cândido Alexandre dos Santos Pereira; Marques, Guilhermina Miguel da Silva
    Em Portugal, o castanheiro (Castanea sativa Miller) encontra-se amplamente distribuído no Norte e Centro do País, sendo Trás-os-Montes a principal região produtora de castanha. Para além da produção de castanha, nos soutos pode fazer-se o aproveitamento de cogumelos comestíveis, tendo-se observado nas últimas décadas um incremento da sua coleta e comercialização. A disponibilidade de água no solo, a associação de fungos micorrízicos e bactérias promotoras do crescimento influenciam o desenvolvimento do castanheiro e a frutificação dos fungos. Com períodos cada vez mais alargados sem precipitação, para a salvaguarda de uma boa produção de castanha e de cogumelos, a rega do souto torna-se indispensável nos períodos mais críticos, nomeadamente no verão, durante o período de formação da castanha. Este trabalho, teve como objetivo contribuir para a caracterização do setor de cogumelos silvestres e sua comercialização na Região Norte de Portugal, bem como, contribuir para uma das estratégias do Setor da Castanha, nomeadamente o incremento da rentabilidade do souto através da defesa do castanheiro face às ameaças abióticas decorrentes das alterações climáticas e às ameaças bióticas tais como a doença da tinta. A caracterização do setor de cogumelos silvestres na região Norte de Portugal foi feita através da elaboração e aplicação de inquéritos a coletores e recetores/concentradores. Os resultados evidenciam que os concelhos mais próximos da fronteira com Espanha possuem uma maior tradição de apanha sobretudo para autoconsumo, embora cerca de um terço dos coletores afirme proceder à sua venda, manifestando assim obterem uma receita significativa relativamente ao seu rendimento anual. A coleta incide em 26 espécies de cogumelos comestíveis, todas com valor gastronómico e a maior parte com interesse comercial, sendo mais referenciadas as seguintes: Macrolepiota procera, Lactarius deliciosus, Amanita caesarea, Boletus edulis e Cantharellus cibarius. Apesar da variação quase diária do preço, estabelecido maioritariamente por intermediários que adquirem os cogumelos, verifica-se que não existem problemas de escoamento do produto, antes pelo contrário, a procura é elevada e o pagamento é feito praticamente sempre no ato da entrega. Ao longo dos últimos anos, verificou-se um aumento do preço e um acréscimo de coletores, havendo a perceção de diminuição do recurso em quantidade e em número de espécies. No que diz respeito aos recetores/concentradores, verificou-se o estabelecimento destes em 12 concelhos, a maior parte há mais de seis anos, atuando geralmente a nível individual, usando instalações simples e equipamento rudimentar, sem cuidados tanto na conservação, como no transporte e no armazenamento. Estes procedem basicamente à pesagem, embalamento por vezes precedido de uma seleção prévia e armazenamento dos cogumelos até o comprador chegar e os carregar. A maioria dos cogumelos rececionados é comercializada em fresco, sendo o preço normalmente, negociado com o concentrador. Verificou-se que a venda direta ao consumidor final e a transformação são ainda práticas muito incipientes e, em resultado disso, grande parte das mais valias não ficam na região. Em 2017 e 2018, num souto da região de Bragança, implantado há cerca de 25 anos com a variedade Judia, foi quantificada a produtividade ao nível de cogumelos comestíveis (número de espécies, número de exemplares e produção) e castanhas (curva de crescimento do fruto, peso, calibre, relação C/L, densidade, volume, coloração da casca, coloração do miolo, firmeza do miolo, análise bioquímica, análise sensorial e produção) nos sistemas de maneio delineados: sem rega ao longo de todo o ciclo e com rega gota-a-gota duplo, entre os meses de julho e setembro, ativada sempre que o potencial mátrico do solo a 30-60 cm era superior a 130 cbar. No ensaio com rega, verificou-se nos dois anos, um aumento da produção de cogumelos, tanto na época de primavera como na de outono e, em simultâneo, uma produção superior de castanha de melhor calibre, parâmetro que mais contribui para a sua valorização comercial. Tendo em conta apenas o ano mais húmido (2018), o tratamento com rega registou, considerada apenas a época de outono, um incremento de produção de cogumelos de 119% e um aumento de rendimento bruto potencial de 321%. Quanto à produção de castanha, o tratamento com rega registou um incremento mínimo de produção e de rendimento bruto de 33%. Através de dois ensaios em vaso estabelecidos em bancada, com cinco tratamentos e quatro repetições com cinco castanheiros da variedade Judia (n = 20), estudou-se o efeito da micorrização dos castanheiros com fungos das espécies Boletus edulis e Pisolithus tinctorius em associação com duas bactérias promotoras do crescimento previamente selecionadas, no desenvolvimento vegetativo do castanheiro e na proteção sanitária da planta, nomeadamente, na tolerância ao oomiceta Phytophthora cinnamomi Rands. Para o efeito, num dos ensaios foi inoculado P. cinnamomi. As duas bactérias isoladas pertenciam ao subgrupo da espécie Bacillus velezensis, espécie formadora de endósporos que promovem o crescimento das plantas e que podem suprimir o crescimento de patógeneos. No ensaio sem o patógeno P. cinnamomi durante o período da sua realização não ocorreu a morte de nenhum castanheiro, sendo que a condição vegetativa global dos castanheiros foi melhor (4,55) nos tratamentos com B. edulis + Bactérias e apenas com P. tintorius. No índice de performance, o tratamento B. edulis + Bactérias foi também o que registou o valor mais elevado (10,59). Ao nível do crescimento em altura, o tratamento B. edulis + Bactérias registou o valor mais elevado com 45,65 cm, seguindo-se o tratamento P. tintorius + Bactérias com 45,10 cm, tratamento este que registou maior eficiência no uso da água (4,869 µmol CO2 mmol H2O) e o valor mais elevado de fotossíntese líquida (2,010 µmol CO2 m-2 s-1). No ensaio em que se inoculou P. cinnamomi, no parâmetro da condição vegetativa global, o tratamento que registou o valor mais elevado (3,75) foi o tratamento B. edulis + Bactérias + P. cinnamomi. Já no índice de performance, foi o tratamento P. tintorius + Bactérias + P. cinnamomi (6,12). Ao nível do crescimento em altura, o controlo2 (P. cinnamomi) registou o valor mais baixo (39,25 cm) e o tratamento B. edulis + Bactérias + P. cinnamomi não só atingiu a maior altura (46,65 cm), como obteve o valor mais elevado de fotossíntese líquida (3,395 µmol CO2 m-2 s-1). O tratamento B. edulis + P. cinnamomi foi o que registou uma maior eficiência do uso da água (2,520 µmol CO2 mmol H2O). Ao nível da taxa de sobrevivência dos castanheiros, o controlo2 (P. cinnamomi) foi o primeiro a registar mortalidades superiores a 50%, revelando-se a combinação B. edulis + Bactérias, a que teve um efeito mais desfavorável no desenvolvimento de P. cinnamomi. Pelos efeitos verificados, as bactérias obtidas neste estudo são potenciais candidatas a agentes de controlo biológico do fitopatogéneo oomiceta P. cinnamomi.
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    Characterization and selection of microbial symbionts of faba bean (Vicia faba L.) and cowpea (Vigna unguiculata (L.) Walp.) for development of inoculants
    2020-06-09 - Pereira, Sandra Isabel Ribeiro; Marques, Guilhermina Miguel da Silva; Rosa, Eduardo Augusto dos Santos
    A agricultura global deve duplicar a produção de alimentos até 2050 por forma a alimentar a crescente população mundial. Neste sentido, alimentos com um elevado valor nutritivo, como por exemplo as leguminosas (proteína, minerais, vitaminas e compostos bioativos) aparecem como uma resposta a esta necessidade. É, no entanto, essencial aumentar a sua produtividade. Ao mesmo tempo, é também necessário reduzir a aplicação de fertilizantes inorgânicos, devido ao elevado impacto negativo que estes têm para o ambiente. Para atingir estes objetivos, é essencial tirar proveito das múltiplas interações benéficas que ocorrem entre as plantas e os microrganismos. Os microrganismos benéficos presentes no solo, nomeadamente os rizóbios e os fungos micorrízicos arbusculares, em simbiose com plantas leguminosas, resultam numa simbiose tripartida e podem ser uma ferramenta biológica para melhorar a produção das culturas, através da fixação biológica de azoto e da absorção de fósforo do solo. Esta simbiose também aumenta a resistência das culturas à seca e às altas temperaturas, melhora a produtividade e a qualidade das culturas e a fertilidade do solo e diminui a incidência de ervas daninhas, doenças e pragas, sem os impactos negativos para o ambiente provocados pela aplicação de fertilizantes químicos. Neste sentido, os objetivos deste trabalho foram selecionar estirpes melhoradas de Rhizobium leguminosarum e Bradyrhizobium spp. para melhorar a fixação biológica de azoto e o desempenho das cultivares de fava e feijão-frade, e efetuar a caracterização fenotípica e genotípica dos simbiontes microbianos usando uma abordagem polifásica baseada em propriedades fenotípicas e na análise molecular. No presente trabalho, foi assim efetuada a identificação molecular dos rizóbios presentes em plantas de feijão-frade e fava recolhidas em diversas regiões de Portugal com diferentes condições climáticas e diferentes tipos de solo, utilizando uma abordagem de “Multilocus Sequence typing” (MLST) com 9 genes (“housekeeping” e simbióticos), a fim de obter informações ao nível da espécie e da simbiovar, uma vez que a amplificação da região 16SrDNA isoladamente não providenciou poder de resolução suficiente. Após a identificação molecular, foram realizados estudos in vitro para verificar a capacidade infectiva dos isolados (postulados de Koch) e para selecionar os melhores inóculos para cada cultura, os quais foram depois testados em condições de estufa, com o objetivo de avaliar os efeitos das inoculações simples e das co-inoculações com os microrganismos selecionados no crescimento, produtividade e conteúdo em proteína da respetiva leguminosa. Foi identificada uma elevada diversidade de rizóbios nos diferentes campos e regiões. Para o feijão-frade, foram selecionadas duas estirpes de rizóbios, Bradyrhizobium sp. e Bradyrhizobium elkanii. Para a faveira, foram selecionadas as bactérias Rhizobium laguerreae e Burkholderia sp.. Relativamente aos inóculos micorrízicos, uma mistura de Rhizophagus irregularis BEG140, Funneliformis geosporum BEG199 e Claroideoglomus claroideum BEG210 (1: 1: 1) foi desenvolvida e preparada pela Symbiom (Sázava, República Checa) para a cultura da faveira. Para o feijão-frade, o fungo micorrrízico (Claroideoglomus claroideum BEG210) foi cedido pelo Dr. Rui Oliveira, da Universidade de Coimbra, Portugal. No trabalho realizado em estufa com inoculação e co-inoculação com Rhizobium laguerreae e AMF, as plantas de faveira inoculadas com a bactéria mostraram um aumento significativo no número de folhas, área foliar, massa foliar por área e razão de área foliar, bem como em todos os parâmetros de produtividade avaliados. A inoculação simples dessas plantas com AMF também aumentou significativamente os parâmetros de produtividade. A co-inoculação mostrou melhorias significativas na proporção da área foliar e em todos os parâmetros de produtividade quando comparado com o controlo, mas não foi significativamente diferente das inoculações individuais. Nos estudos com feijão-frade, em condições de estufa, usando solo não esterilizado, a co-inoculação das plantas com Rhizobium sp. e AMF, Bradyrhizobium elkanii e AMF e Bradyrhizobium sp. e AMF aumentaram o teor de proteína das sementes em plantas sujeitas a déficite hídrico (25% da capacidade de campo) em 13, 17 e 30%, respetivamente. Considerando todas as análises realizadas neste trabalho em ambas as culturas, é possível concluir que a inoculação simples e combinada de plantas leguminosas com os microrganismos selecionados mostrou ter um grande potencial como ferramenta biológica para melhorar o crescimento e a produtividade das plantas leguminosas sujeitas a stress abiótico, mitigando os efeitos das alterações climáticas e reduzindo a necessidade de aplicação de fertilizantes de síntese.
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    Enhancement of wheat establishment and drought stress resistance through seed priming and foliar fertilisation
    2020-01-21 - Pavia, Ivo Miguel Meneses; Correia, Carlos Manuel; Brito, José Eduardo Lima
    O trigo (Triticum spp.) é uma das culturas mais importantes do mundo. A seca é um fator crítico na segurança alimentar mundial, e um dos mais importantes fatores limitantes na produtividade dos cereais. No contexto das alterações climáticas à escala global, esperam-se aumentos da temperatura média e da severidade da seca nas principais regiões produtoras de trigo, pelo que é urgente aumentar a resistência da cultura ao stresse hídrico. A aplicação exógena de microminerais e antioxidantes às plantas, através do pré-tratamento de sementes ou via aplicação foliar, é considerada uma estratégia eficaz para aumentar a resistência ao stresse e o conteúdo nutricional das plantas. Esta tese foca-se principalmente nas consequências do pré-tratamento de sementes e aplicação foliar de zinco (Zn) e ácido ascórbico (AsA) e, secundariamente, nos efeitos da aplicação de ferro (Fe) e piridoxina (Pyr). Neste estudo demonstramos que as cultivares antigas de trigo possuem características que lhes permitam germinar e resistir ao stresse hídrico. Em todo o caso, para atingir os objetivos do trabalho, selecionamos uma cultivar moderna - ‘Jordão’ - com rendimento elevado e estável, e boas características de germinação em vários potenciais hídricos. O pré-tratamento de sementes com Zn induziu inibição do crescimento, instabilidades citogenéticas e alterações na atividade nucleolar, tendo estes efeitos sido exacerbados em condições de stresse hídrico. A combinação de Zn e Fe na solução de pré-tratamento de sementes contribuiu para a redução das anomalias citogenéticas causadas pelo Zn. Esta combinação leva a uma diminuição nas anomalias citogenéticas e a efeitos benéficos a longo prazo, como o melhoramento do afilhamento e do rendimento. O pré-tratamento de sementes com AsA e Pyr também promoveu melhorias a longo prazo, melhorando o crescimento do trigo. Considerando que as alterações climáticas devem alterar a data de sementeira e reduzir o período de crescimento do trigo, esta melhoria pode ser valiosa. Também se demonstrou que o stresse hídrico severo induz a dissipação não regulada de energia, o que produz danos duradouros nos fotossistemas. O pré-tratamento de sementes isolado e / ou acoplados à aplicação foliar de Zn ou AsA foram capazes de melhorar a fotoproteção durante a seca, aumentando a dissipação regulada do excesso de energia e promovendo uma melhor recuperação das plantas de trigo após o alívio do stresse.
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    Efficient tools to simulate main crops in Portugal for decision support systems
    2019-11-27 - Chenyao Yang; Santos, João Carlos Andrade dos; Van Ieperen, Wim; Fraga, Helder
    Agricultural systems are inherently vulnerable to climate variability and climate change is expected to increase this vulnerability. Various studies warn the anthropogenic-driven global warming with elevated CO2 concentration and altered regional precipitation pattern, are expected to negatively affect local crop productivity and thus exacerbate food insecurities in many regions worldwide, particularly for Mediterranean basin. Mediterranean basin is one of the most prominent climate change “hotspot” due to ongoing and projected changes in both climate means and variabilities, comprising a robust climate change signal of an overall warming and drying trend, accompanied by more frequent occurrence of severe drought and extreme high temperatures. Specifically, these projected changes are expected to be more pronounced in southern Europe, such as in Portugal, where annual mean temperature has increased at a rate more than double the global warming rate in the past decades, along with the observed decreases in precipitation and its enhanced inter-annual variability. Therefore, it is urgently needed to carry out the assessment of climate change impacts on agricultural production and explore suitable adaptation strategies, whereas the related studies so far remain scarce in Portugal. We had chosen three important cropping systems for Portuguese agriculture, i.e. irrigated maize, rainfed wheat and perennial forage grassland, while representative study sites in their current principal growing regions were identified accordingly. The overall methodology follows combined use of climate and crop models, where the spatially-downscaled bias-corrected climate change projections from climate models were utilized to drive crop model simulations at study sites, which were prior calibrated using local observed weather, soil and management data. For employed process-based crop models, both STICS and AquaCrop were applied for the irrigated maize production, whereas the other two cropping systems were only analyzed using STICS model. It was noteworthy one major strength from current studies consisted in, on top of projected mean climate changes, we had consistently incorporated the effects of potential changes in climate variability and its associated extreme weather events into the simulated impacts (e.g. yield changes) for a more reliable assessment. The results indicate threats and risks of future climate change are substantially high for agriculture production in Portugal. Because an overall negative climate change impact from the mid until the end of 21st century is obtained for all three important cropping systems, corresponding to moderate-to-severe yield losses with increased inter-annual variabilities. Yield losses are greater in magnitude with higher year-to-year variability, in the second half of the century than in the first half, and in a high emission pathway than in a low emission scenario. The CO2 fertilization effect is unlikely to compensate these yield reductions, where it brings more yield increment for C3 species (wheat and defined grass mixture) than for C4 (maize). Specifically, majority of negative impacts are derived from the shortened growth duration for irrigated maize under a warmer climate, and from intensified drought and heat stresses during a sensitive period (grain-filling) for rainfed wheat or during an unfavorable summer period for perennial grassland. These aspects correspond to the vulnerabilities of cropping systems facing climate change. It is interesting to note though higher temperature is clearly detrimental to irrigated maize production, it facilitates advanced phenology of perennial grass shifting towards the favorable cool and wet winter period for enhanced production or it may also help rainfed wheat crop to mature earlier to avoid excessive terminal stresses. Yet the magnitude of climate change impacts on agricultural productivity remains uncertain, varying with analyzed cropping systems, locations and management practices, applied climate models (including downscaling approaches) and crop models (including partial or full calibration), selected time periods and emission pathways. Adaptation strategies provide potential to mitigate these negative impacts, and development of appropriate and risk-focused adaptation policy should address previously identified vulnerabilities and prioritize available options for an integrated and comprehensive strategy. For annual cereal crops, increased irrigation amount at various levels has been firstly tested for irrigated maize cropping system under climate change, taking into account crop water demand and projected seasonal rainfall distribution. Though increased irrigation is able to mitigate yield reductions and maintain current yield levels, crop WUE considerably declines as a result of diminished yield responsiveness to seasonal water input with shorter growth duration. In view of increasing risks of water scarcity and decreasing portion of fresh water available for agriculture in the Mediterranean basin, solely increased irrigation supply might not be a feasible strategy, whereas the adaptive response for maize should be prioritized to promote water-saving techniques and maximize WUE for stabilizing yields (marginal reductions allowed). Combining optimized irrigation strategy (e.g. deficit irrigation) and installed efficient facilities (e.g. drip irrigation system) with other adaptation options, including introducing longer cycle cultivars and advanced sowing dates to counterbalance the shortened growing duration, is recommend, but should be further rigorously examined. For the rainfed wheat cropping system, adaptation priority should address the exacerbated risks of drought and heat stresses during the sensitive anthesis and grain-filling periods. The terminal stress escaping strategy is proposed by firstly testing early flowering cultivars (also known as short-cycle genotypes), where the trade-off between lower risk of exposure to terminal stress and higher risk of reduced yield potential tends to be positive, leading to net yield gains. Still, this option needs to be combined with other adaptation opportunities including early sowing date, wheat cultivars with less or no vernalization requirement (e.g. using spring wheat) and supplementary irrigation during the sensitive stage. Early sowing is expected to achieve the same stress escaping goals by anticipation of growth cycle. But winter warming during early sowing window could potentially slow vernalization fulfillment, with limited benefits to advance the susceptible stages. Using earlyflowering spring wheat cultivars (the earliness threshold must be carefully defined) thus can help advocating early sowing practice that potentially make use of more autumn-winter rainfall. Nevertheless, the proposed stress escaping strategy is found to be comparatively more useful to avoid enhanced terminal heat stress (>38º last over a short period) than prolonged terminal drought stress, where the latter can be alleviated with optimized supplemental irrigation. Adaptation strategy for perennial forage grassland should take advantage of opportunity and tackle the challenge, both arising from climate change. Benefiting from advanced phenology towards winter and early spring with alleviated cold stress and enriched ambient CO2 concentration, adaptation measures should focus on maximizing growth potential during this favorable period. These include optimized resource use (balanced early fertilization strategy with limited N leaching) and using grass-legume mixture for flexible forage utilization and better exploiting the stimulated CO2 responsiveness. In contrast, to cope with the challenge of exacerbated risks of summer heat and drought stresses, future breeding programs should ensure a diversification (intra- and interspecific variations) of available germplasms in phenology (fit new seasonal climate pattern), heat tolerance and dehydration tolerance for principal forage species. Specifically, continuous improvement of drought persistence and summer dormancy traits should gain more importance for rainfed Mediterranean grassland. Moreover, these drought survival traits should be integrated into plant materials with deeper root system to enhance water uptake (e.g. more of tall fescue), but it may raise forage quality issues that remain unassessed. Besides, we also hypothesize it is possible to adapt to summer drought from a management perspective without the needs to improve and diversify the species and variety mixture. The findings suggest that provided minimum soil moisture is guaranteed by supplemental irrigation to ensure adequate drought survival rate and standing density, breeding efforts should be more motivated towards heat tolerance, particularly in southern Portugal. Meanwhile, this measure is likely to result in a considerable increase in irrigation need, rendering a similar water-restriction issue facing irrigated maize. Crop yield projections and explored adaptation strategies are essential to assess the regional food security prospects and provide crucial information to support planning and implementing suitable adaptation strategies for farmers and policymakers in Portugal and in Mediterranean basin that is known to be susceptible to climate change. Despite the uncertainties in the magnitude of yield impacts and quantitative effectiveness of adaptations, the proposed and recommended adaptation strategies can represent promising opportunities to maintain or increase production in future climate while minimize environment impacts. Future research efforts should be directed towards using multi-model ensembles (both crop and climate models) to quantify the uncertainties and make the estimations more robust and reliable, but sustained and extensive international cooperation is required. Moreover, stronger link of field experimentation with crop modelling is essential for a more mechanistic understanding of crop response to climate change, as well as the integration of crop model into economic modelling for complex farm-level assessment. These shall all contribute to appropriate manage the climate risks and comprehensively improve the resilience of cropping system
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    Pre and postharvest innovative strategies to enhance cherry quality
    2019-11-08 - Correia, Sofia Mendes Moreira; Gonçalves, Berta; Silva, Ana Paula Calvão Moreira Da; Schouten, Rob
    In the context of modern fruit growing, the cherry tree (Prunus avium L.) has a high economic importance, so the concern is to produce high quality fruits. Cherries are attractive fruits, due to their colour, flavour, nutritional value and potential health benefits. However, cherry tree is susceptible to damage caused by low winter temperatures, spring frosts and cracking of fruit during ripening. Moreover, the increasing frequency of intense preharvest rainfall resulting from climate change will aggravate the effects of cherry cracking. Cherries are also highly perishable, with a relatively short shelf-life, which makes it difficult to export these small fruits to distant markets. In this sense, the main objective of this Doctoral Thesis is to contribute to enhancing the quality and production of sweet cherries by different approaches, focusing on an evaluation of the suitability of preharvest foliar spray treatments and also of the application of compounds postharvest, to extend the shelf-life. Moreover, we aim to achieve further insight into preharvest spray treatments as part of orchard management practices, by understanding the physiological and biochemical response of the plant, and also to mitigate the impact environmentally stressful conditions, to reduce the severity of cherry cracking. In that regard, the experiment consisted of repeated sprayings of calcium (Ca, 0.5 %), gibberellic acid (GA3, 10 ppm), abscisic acid (ABA, 10 μM), salicylic acid (SA, 1 mM), glycine betaine (GB, 0.1 %), biostimulant Ascophyllum nodosum (AN, 0.05 %) and water as control, without addition of Ca in 2015, whereas in 2016 Ca was added. The studies in this work focused on six-years old sweet cherry trees of two economically important cherry cultivars, Skeena and Sweetheart, during the 2015 and 2016 seasons in an orchard located in Northern Portugal. The impact of foliar spray treatments on sweet cherry quality, cracking incidence and plant behaviour, was assessed. The effect of postharvest application of chitosan (CH, 1 %), Aloe vera (AV, 0.33 %) and calcium (CaCl2, 1 %) on quality attributes, bioactive compounds and antioxidant systems of cherries, was also studied. Fruits at the commercial maturity stage were dipped into these solutions, stored at 4 °C for 14 days, and then kept at 20 °C for three days to assess shelf-life. To achieve this goal, several activities were undertaken involving field trials and phytochemical, physiological, biochemical and molecular biology approaches. A positive effect of spray treatments on ‘Skeena’ cherry yield and physiological and biochemical response was shown, displaying a boost of relative water content and reduced cell membrane damage. Furthermore, AN and ABA increased soluble sugars content and yield, while GA3 increased net CO2 assimilation rate, and when associated to Ca also improved soluble sugars content and yield. GB in combination with Ca increased net CO2 assimilation rate, chlorophyll content and yield. When studying the impact of spray treatments on cherry quality attributes as health promoting compounds (phenolics, anthocyanins, carotenoids, ascorbic acid), the highest variability was noticed in the seasonal differences. Higher solar radiation combined with higher temperatures and less precipitation in 2015 compared to 2016, likely contributed to this shift in health promoting contents. Additionally, GA3 and SA increased fruit weight and firmness, while GB with Ca increased fruit weight, appearance and colour development. Soluble sugars content was not affected by spray treatments, while AN and SA with Ca increased total organic acid levels. GA3, ABA, SA and GB sprays increased anthocyanin content, and GA3 and GB also induced lower carotenoid and lower ascorbic acid content. AN appears to induce an effect opposite to GB, increasing carotenoid and ascorbic acid but lowering phenolic content. The impact of spray treatments conjugated with Ca on the incidence of ‘Sweetheart’ cherry cracking, was also assessed. All spray treatments reduced cracking incidence compared to the control, with the least cracking observed for ABA and GB treated fruits including less cheek cracks. ABA and GB sprays showed higher wax content and higher cuticle and epidermal thickness compared to control, including increased expression of wax synthase (ABA) and expansin 1 (GB). At the fruit ripening stage, larger cell size of the epidermis, hypodermis and parenchyma cells lowers cracking incidence, pointing at the importance of flexibility and elasticity of the epidermis. Finally, the application of postharvest compounds as a tool for the extension of the shelf-life of cherries, was shown to be an effective way of delaying the cherry fruits’ postharvest ripening and senescence processes by increasing of the antioxidant systems activity. In conclusion, the work conducted contributed to increased knowledge of the use of promising pre- and postharvest compounds to improve the quality and increase bioactive compounds of the cherries. GA3 and GB sprays seem to be more beneficial for the physiological performance of the trees and to increase cherry quality attributes, thus could be a new strategy in the fruit production system. Foliar application of ABA and GB in association with Ca are therefore likely promising cherry cracking mitigation strategies. CH, AV and CaCl2 can also be an effective tool for maintaining cherry quality during storage with higher concentration of bioactive compounds and antioxidant activity.
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    Composting of agro-food residues: chemical composition, greenhouse gases emissions and compost agronomic value
    2019-10-18 - Santos, Cátia Inês Rodrigues dos; Trindade, Henrique Manuel Da Fonseca; Cortez, Maria Isabel Mendes Guerra Marques; Jensen, Lars Stoutmann
    Composting has been widely described and accepted as one of the most feasible and viable tecnhologies for processing and valorising agro-industry wastes. Portugal, as member of the European Union needs acomplish the Community goals reducing the amount of waste generate and/or valorising their wastes. Beyond the recycling advantage, the application of the end product improve the soil quality acting as biologic fertilizer, enables carbon sequestration and may allow their use as a sustainable toll on management of soil-borne diseases. There exists, however an environmental risk associated to the process, such a hight warming potential (GWP) owing to the emission of greenhouse gases (GHGs), such as carbon dioxide (CO2), nitrous oxide (N2O) and methane (CH4) and other gases as ammonia (NH3) and nitric oxide (NO). One of the objectives of the present thesis was to assess the effect of the chemical composition (phenolic and lignocellulosic contents) of the eight diferentt raw materials used on the composting process and on the gases emmited. This evaluation was fullfilled through the physical-chemical analysis of the raw materials, samples mixtures collected along the composting process and final composts. Composting was studied in two sets on 135 L insulated reactors with forced air circulation and with a system that allow the measurement of gases emissions and temperature during the process. In the first composting set, that use different proportions of spent coffee grounds (0 to 40%), all tratments revealed a substantial decrease in total phenolics and total tannins and an important increase in gallic acid. The GHGs emissions were very low and no significant difference between the treatments was registered. Is possible to conclude that that all spent coffee grounds treatments are suitable for composting, but the proportion of 40 % led to better conditions for composting and lower GHGs emissions. In the second composting set, that use different agro-industry wastes (broccoli, chestnut, red grape marcs, white grape marcs, olive leaves) significant variations were observed over time and between the different wastes. The compost obtained with Chestnut wastes and olive leaves led to higher carbon dioxide, methane and nitrous oxide emissions. A positive correlation was found between phenolics and nitric oxide (r = 0.63; p < 0.01) and between lignocellulose and nitrous oxide (r = 0.51; p < 0.05). Its possible to obsserve that a higher content of hemicelluloses in the wastes seemed to entail higher N2O emissions. These results suggest the potential use of wastes rich in lignocelluloses in composting for the purpose of reducing N losses by NH3 and NO emissions After obtaining the final composts we also studied the effect of eight different composts and their chemical composition on C and N mineralization and GHGs emissions by determination of ammonium (NH4 + ) and nitrate (NO3 - ) and measurement of carbon dioxide (CO2), nitrous oxide (N2O) and methane (CH4) fluxes from amended soils under laboratory conditions. Throuthg the results its possible conclude that mineralization of the C added to the soils was directly correlated to C/N, total phenolic/N, gallic acid/N and NH4 + and negatively correlated with total organic nitrogen (TON) and lignin. N mineralization was only correlated with C/N. All composts enhanced N2O emissions and no significant difference in cumulative emissions were observed between treatments. Finally a biological assay was performed with the objective of test the suppressive potential of four composts (Chestnut, Grape marc, Coffee grounds and Olive) against the soilborne Botrytis cinerea and Fusarium oxysporum with significant impact on lettuce cultivation. The suppressive effect was noticed in Chestnut and Olive with no significant difference between the two doses applied. These treatments also showed significantly higher values of dry matter yields of leaves and roots which denotes their bio-fungicide and bio-fertilizer potential. The results of this thesis could be a useful instrument for planning field scale experiments giving an idea of quantification and quantification of the GHG and soil C and N pools. Overall, the results pinpointed out that the wastes with high lignocellulose contents have additional benefits on the environment in relation to mitigating nitrogen losses. All the composts applied in pot experiments, were found to be beneficial to plant growth. Besides, composts obtained from chestnut wastes and olive leaves are promisors wastes as bio-pesticides supressing Botrytis cinerea and Fusarium oxysporum wilt diseases.
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    Diversity and ecology of entomopathogenic fungi in the Douro wine region of Portugal
    2019-05-07 - Sharma, Lav; Marques, Guilhermina Miguel Da Silva; Torres, Laura Monteiro
    Investigations studying diversity and factors affecting the abundance of entomopathogenic fungi (EPF) populations, the natural enemies of insects, are limited. In this work, culture based techniques and molecular biology tools were applied to investigate the natural diversity of EPF in the vineyards of the Douro wine region (DWR) of Portugal. Two different approaches were undertaken in this direction. First approach was the sampling of naturally mycosed insect-pest, i.e., vine mealybug Planococcus ficus, which is a major grapevine pest worldwide, and whose numbers are increasing in the DWR. Vine mealybugs were observed in two of the four farms searched for their presences. Out of 183 vine mealybugs collected, 58 were dead of which 25 presented symptoms of mycosis and 13 were parasitized. Subculturing and subsequent pathogenicity confirmation led to the isolation of 22 fungi including yeasts. Yeast Meyerozyma (=Pichia) guilliermondii, and EPF Sarocladium kiliense and Purpureocillium lilacinum were the most abundant, i.e., 18.18%, 13.64% and 13.64%, respectively. To our knowledge, this work reported (a) the first isolations of EPF from vine mealybug worldwide, and, (b) first isolation of Pseudocosmospora rogersonii in Europe, and as EPF worldwide. It was noticed that P. ficus mortalities originated by mycoses were significantly higher than by its parasitoids (P=0.026). Second approach was the sampling of 183 soils from the vineyards and adjacent hedgerows of the six wine farms, Arnozelo, Aciprestes, Carvalhas, Cidrô, Granja and S. Luiz, to isolate EPF using common bait-insect Tenebrio molitor (TM). Moreover, to study the effect of insect baiting on EPF recovery, 81 of these soils were also baited with Galleria mellonella (GM). Twelve species were found in 44.26%±3.67% soils. Clonostachys rosea f. rosea was found in maximum soils (30.05%±3.38%), followed by Beauveria bassiana (12.57%±2.37%), P. lilacinum (9.29%±2.14%) and Metarhizium robertsii (6.01%±1.75%). Beauveria pseudobassiana (P=0.038) and M. robertsii (P=0.003) were isolated significantly more using GM and TM, respectively. Principal component analysis revealed that M. robertsii was associated both with TM baiting and cultivated habitats, however, B. bassiana was slightly linked with GM baiting only. To us, this was thefirst report of: C. cicadae isolation from Mediterranean soils; Purpureocillium lavendulum as an EPF worldwide; and significant recoveries of M. robertsii using TM over GM. Based on these results, a ‗Galleria-Tenebrio-bait method‘ was also advocated for EPF isolations. Moreover, this research focussed on how soil physicochemical properties affect EPF occurrences, as previous studies in this direction are very few. The work was divided based on the EPF of interest. The soil properties studied were: organic matter content, total organic carbon, total nitrogen, available potassium, available phosphorus, exchangeable cations such as K+ , Na+ , Ca2+ and Mg2+, pH, total acidity, degree of base saturation, and effective cation exchange capacity. Soils which yielded any of the four common EPF, i.e., B. bassiana, P. lilacinum, M. robertsii and C. rosea f. rosea, were studied and, in total, 42 soils were assessed. Beauveria bassiana was negatively affected by higher available P (P=0.02), exchangeable K-ions (P=0.016), and positively affected by higher soil pH_H2O (P=0.021). High exchangeable K-ions inhibited P. lilacinum (P=0.011) and promoted C. rosea f. rosea (P=0.03). Moreover, high available K also suppressed P. lilacinum (P=0.027). Metarhizium robertsii was inhibited by organic matter content (P=0.009), C:N (P=0.017), total N (P=0.007), and exchangeable Mg-ions (P=0.026), and promoted by exchangeable Na-ions (P=0.003). As soil chemistry affected B. bassiana and M. robertsii the most, a principal component analysis was performed to cluster three groups of fungi, i.e., (a) B. bassiana, (b) M. robertsii and (c) others. First three factors, F1, F2 and F3, could explain 73.81% of the variance, i.e., 38.10%, 20.62% and 15.09%, respectively. Nonetheless, it was noticed that soil properties such as higher soil pH and exchangeable Ca-ions have contrasting effects on EPF occurrences, as they promoted B. bassiana and inhibited M. robertsii. Fusarium oxysporum is generalised as an opportunistic insect-pathogen colonising weak or dead insects. Here, in other works, authors convincingly reviewed and argued the entomopathogenicity of F. oxysporum, and, using a generalised linear mixed model approach and 80 soils, reported that soil physicochemical properties hardly affect its natural abundance. Besides, in another parallel work, authors recommended amplifying region of translational elongation factor 1-alpha subunit for identifying any random ascomycetous fungus. Nonetheless, this research provides important insights into the ecology and diversity of EPF in the DWR, and would facilitate soil quality predictions and subsequent soil amendment practices, especially for a conservation biological control viewpoint.
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    Influência da temperatura de armazenamento na idade fisiológica da batata-semente
    2018-12-07 - Rodrigues, Paula Maria Fidalgo; Martins, Fernando Manuel Coelho Franco
    Em Portugal, a cultura da batata é ainda uma cultura representativa do ponto de vista económico dos sistemas de agricultura da região de Trás-os-Montes. As preocupações centram-se sobretudo na manipulação da batata-semente de modo a obter a maior produtividade, dado o seu peso no custo da produção. No entanto, a compreensão de que produções de qualidade podem ser obtidas também pela manipulação dos ciclos de produção, idade fisiológica e condições de armazenamento tem levado à procura de quais as melhores condições de armazenamento da batata-semente, por forma a permitir a sua adequada utilização com redução de custos por perdas de qualidade. É neste contexto que surge esta dissertação, onde apresentamos um conjunto de trabalhos que visa a avaliação do comportamento fisiológico de três cultivares de batata-semente (Désirée, Kennebec e Jerla), produzidas em duas zonas diferentes de Trás-os-Montes (Portugal) e na Holanda; quando submetidas a dois diferentes regimes de temperatura (4 ºC e temperatura ambiente) e 165 dias de armazenamento. Neste âmbito, observou-se os efeitos nos hábitos fisiológicos da batata-semente, na capacidade de abrolhamento, no período de incubação e o seu comportamento no campo, através de um ensaio preliminar. Os resultados obtidos permitiram observar que combinando regimes de temperatura e duração de conservação é possível influenciar de forma significativa a idade fisiológica, para além de que cultivares diferentes requerem condições de armazenamento diferentes. Observamos que aumentos de temperatura tendem a provocar um maior grau de abrolhamento combinado com maiores perdas de peso do tubérculo eventualmente por aumento da respiração e transpiração, sendo favoráveis à antecipação da idade fisiológica, situação favorável quando temos calendários de produção de ciclos curtos em que se deseja precocidade. Em situações de ciclo de produção mais longo ou em plantações mais tardias serão preferíveis situações de conservação com temperaturas mais baixas, pois estas permitem uma evolução mais lenta da idade fisiológica da batata-semente, logo uma maior preservação da qualidade do tubérculo disponível para a plantação. Das cultivares estudadas, a Jaerla foi aquela que esgotou mais cedo a sua capacidade de abrolhamento sendo por isso mais adequada para calendários de produção de ciclo curto, enquanto as cultivares Désirée e Kennebec, fisiologicamente mais jovens e ativas durante mais tempo, para ciclos mais longos, possuindo assim a vantagem de serem armazenadas durante mais tempo e com mais baixos custos de armazenamento.
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    Genetic diversity and molecular responses to drought stress in Vigna unguiculata L. Walp.
    2018-12-19 - Carvalho, Márcia Raquel Gomes de; Carnide, Valdemar Pedrosa; Neto, Maria Teresa Lino
    Climate change is considered as one of the major threats to agriculture sustainability and biodiversity. Drought is a severe environmental stress with major impacts on plant development and productivity. The use and improvement of crops with the ability to mitigate the effects of drought will be a key step for future crop sustainability. Cowpea (Vigna unguiculata L. Walp) is a warm-season grain legume, considered as an interesting crop, due to its high adaptability to heat and drought, as well as to its association with nitrogen fixing rhizobia. As other legumes, cowpea plays a major role in the global food security by providing an affordable dietary source of nutrients mainly proteins. The thesis main objective is to contribute for a higher cowpea production in Europe, anticipating the upcoming climate changes. To achieve this goal, multidisciplinary approaches were undertaken involving field trials and molecular genetics, physiology and biochemistry approaches. Regarding genetic diversity, the morphological and agronomical characterization of 24 Iberian Peninsula cowpea genotypes was performed, thus emphasizing the high genetic diversity among genotypes. From this characterization, ten cowpea genotypes were selected and further used for determining the stability of morphological and agronomical traits in three different environments (two in Portugal and one in Spain), during two consecutive years. A high interaction between genotype and environment was found and Elvas (Portugal) revealed to have the most appropriated environment for the production of this set of cowpea genotypes. The recently developed Cowpea iSelect Consortium Array (Illumina, Inc.) provided an excellent opportunity for further determination of cowpea genetic diversity. This array contains 51,128 SNPs and was used in a set of 96 cowpea genotypes, 43 of which from Iberian Peninsula and 23 from 22 other worldwide countries. Cowpea genotypes were clustered in four subpopulations, mainly differentiated by their geographical origin, allowing the suggestion of a new hypothesis about cultivated cowpea dispersion routes. Most of Iberian Peninsula genotypes and those from other Southern European and Northern African countries were grouped in the same subpopulation, indicating a high genetic similarity among them. However, three Iberian Peninsula cowpea genotypes did not belong to this subpopulation, being two of them classified as ‘admixed’ and another from a different subpopulation. These genotypes could be considered as interesting sources of diversity for future cowpea breeding programs. To get new insights on cowpea drought stress responses, the selection of the best approachesfor screening cowpea genotypes with enhanced drought tolerance is fundamental. Four cowpea genotypes (two Portuguese and two international tolerant references) were submitted to three different watering regimens, during 15 days. Several physiological, biochemical and molecular approaches were tested, revealing that stomatal function parameters, free proline and anthocyanins contents were the most effective in discriminating cowpea tolerance levels. Furthermore, two drought-related genes (VuCPRD14 and VuHsp17.7) were identified as the most effective for drought tolerance selection. For screening cowpea genotypes with enhanced drought tolerance, a worldwide collection of cowpea genotypes (58 genotypes) was tested for seed germination, seedling emergence and proline content under different osmotic potentials. A total of seven drought tolerant genotypes were suggested, which could represent starting material for future cowpea breeding programs. This thesis gave a good contribution for increasing cowpea production in Europe, being the selection of more productive and drought tolerant genotypes the first step. These genotypes could be integrated into breeding programs for enhancing cowpea resilience to climate change. Furthermore, the methodologies tested and proposed in this study allow an effective and fast screening of cowpea genotypes drought tolerance.