Avaliação do efeito do silício na resistência de plantas de castanheiro (castanea sativa mill.) A stresses abióticos e bióticos
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2019-12-10
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As mudanças climáticas que se têm vindo a observar ao longo das últimas décadas, associadas às novas exigências culturais desta cultura, têm levantado novos problemas a nível abiótico e biótico causando graves ameaças à sustentabilidade da cultura em muitas regiões. O presente trabalho teve como objetivos principais avaliar o efeito do tratamento com SiK® através de pulverização em castanheiro. Foram testadas as concentrações 0 mM, 5 mM, 7,5 mM e 10 mM SiK®, em plantas jovens de castanheiro (Castanea sativa) submetidas a stresses bióticos e abióticos, e em plantas adultas visando estudar o seu impacto na produção e qualidade da castanha. A tese encontra-se estruturada em oito capítulos. No Capítulo 1 é apresentado o Estado de arte onde são abordados assuntos relativos à origem, distribuição, ecologia e importância económica da espécie Castanea sativa Mill. Faz-se igualmente referência às alterações climáticas e ao seu impacto nas plantas, assim como à ação mitigadora do silício face aos stresses bióticos e abióticos. Nos capítulos seguintes (Capítulos 2 ao 7) são apresentadas de forma detalhada todas as atividades de investigação desenvolvidas ao longo deste estudo, culminando no Capítulo 8 onde é feito um conjunto de considerações finais. No presente estudo e para cada um dos ensaios (Capítulo 2 ao 6) foram usadas plantas com cerca de 2 meses de idade após germinação provenientes de castanhas da mesma árvore da variedade Sousã (Castanea sativa), existente no banco de germoplasma da Universidade de Trás-os-Montes e Alto Douro. Em todos os ensaios foram formados quatro grupos de plantas correspondestes aos tratamentos a efetuar. Os tratamentos consistiram na fertilização com concentrações crescentes de silício na forma de silicato de potássio (0 mM, 5 mM, 7,5 mM e 10 mM SiK®). Os ensaios iniciaram-se sempre 2 meses após a aplicação do tratamento. No Capítulo 2 foi feito o estudo da influência da fertilização com Si, na resiliência de plantas jovens de castanheiro a temperaturas elevadas. Estas foram submetidas a um ciclo sequencial de temperatura com duração de 1 mês cada, de 25ºC, 32ºC e 25ºC, procurando compreender não só a sua capacidade de tolerância, mas igualmente caracterizar a sua recuperação após um período de calor elevado. Este estudo permitiu concluir que a fertilização com Si nas concentrações 7.5 mM e 10 mM SiK® incrementa a resiliência a temperatura elevada (32ºC), através de uma maior eficiência fotoquímica do fotosistema II, preservando a atividade dos cloroplastos, aumentando a atividade fotossintética e reduzindo a transpiração. Os resultados evidenciam ainda a ação do Si na síntese de compostos fenólicos e tióis, e no incremento da atividade enzimática das enzimas ascorbato peroxidase, catalase e peroxidase, conduzindo à redução do dano oxidativo e consequente maior tolerância ao stresse térmico. Neste estudo, foi ainda observado que as plantas tratadas com Si apresentaram uma recuperação mais rápida após exposição ao período de stresse comparativamente às plantas controlo. De forma a investigar o papel do silício na tolerância do castanheiro à seca, (Capítulo 3), foi realizado um estudo em 2015 e repetido em 2016 complementado com novos parâmetros, de forma a tornar os resultados mais robustos. De forma sequencial, 2 meses após a aplicação do SiK, as plantas foram mantidas num ciclo de adaptação com rega adequada, seguida de ausência de rega até ser atingido o potencial hídrico de -1,2 MPa em algum dos tratamentos, seguido de reposição da rega adequada. As plantas tratadas com 7,5 mM e 10 mM SiK®, apresentaram maior frequência de vasos xilémicos com menor área, incrementando a adesão das moléculas de água às paredes dos vasos condutores, assim como uma menor densidade estomática. Estes resultados sugerem ter sido esta uma estratégia das plantas para reduzir as perdas de água, permitindo desta forma a utilização da água de forma mais eficiente, uma vez que foram verificados valores superiores de potencial hídrico, conteúdo relativo em água e uso eficiente de água. A análise dos parâmetros relativos às trocas gasosas demonstrou que plantas tratadas com Si conseguiram reduzir os valores da taxa de transpiração e condutância estomática, apresentando desta forma maior potencial hídrico e conteúdo relativo em água. Os estudos bioquímicos complementaram o trabalho, sugerindo que a aplicação de Si nas plantas auxilia na melhoria da atividade de defesa das plantas pelo incremento das enzimas ascorbato peroxidase, catalase e peroxidase, assim como na síntese de compostos fenólicos em condições de seca, reduzindo consequentemente o dano oxidativo nas plantas sob stresse. Por fim, nas plantas tratadas com Si observou-se uma retoma mais rápida dos valores de antes da imposição do período de seca, para os parâmetros avaliados, sugerindo assim uma recuperação mais rápida destas em relação às plantas controlo. No Capítulo 4 foi avaliado o efeito da fertilização com silício no incremento da tolerância das plantas ao agente patogénico Phytophthora cinnamomi. Foram efetuados estudos bioquímicos antes da inoculação e aos 15 e 30 dias após a inoculação. Foram igualmente realizados estudos histólogicos, assim como uma avaliação antifúngica do efeito do silício no desenvolvimento da Phytophthora cinnamomi através da inoculação de placas contendo silício no meio PDA. Os tratamentos 7,5 mM e 10 mM SiK® foram os que apresentaram maior capacidade de redução do crescimento da Phythophthora cinnamomi, ocorrendo apenas crescimento reduzido em placa às 144 h. Através da análise histológica das raízes, foi possível observar a presença de fitólitos os quais atuaram como uma barreira mecânica dificultando a entrada das hifas no interior da planta hospedeira. As plantas fertilizadas com 7,5 mM e 10 mM SiK® apresentaram também uma taxa de sobrevivência superior (80%) à das plantas controlo (20%). O Capítulo 5 corresponde ao estudo da influência do silício na tolerância das plantas à Cryphonectria parasitica. A metodologia foi semelhante à descrita no Capítulo 4. Observou-se que os castanheiros tratados com 7,5 mM e 10 mM SiK® apresentaram uma taxa mortalidade de 10% e 0%, respetivamente, tendo as plantas controlo (0 mM SiK®) registado uma taxa de 100%. As plantas tratadas com a maior concentração de Si (10 mM SiK®) apresentaram ainda menor desenvolvimento de necroses no caule. Em relação à análise histológica dos cortes transversais dos caules, os resultados mostraram a presença de fitólitos nos vasos xilémicos em quantidade proporcional à concentração de silício aplicada. Em simultâneo ao estudo dos Capítulo 4 e 5 foram preparados extratos fenólicos a partir de folhas de plantas deixadas sem inoculação provenientes dos 4 tratamentos, sendo os resultados apresentados no Capítulo 6. No Capítulo 6 verificou-se que o ácido gálico e o ácido elágico foram dois dos compostos fenólicos mais predominantes identificados nas plantas fertilizadas com silício, os quais desempenham uma importante ação antifúngica no sistema de defesa das plantas hospedeiras. Foi avaliada a ação in vitro dos compostos fenólicos provenientes de plantas fertilizadas com as diferentes concentrações de Si. Extractos fenólicos foram utilizados para impregenar discos colocados em placas contendo P. cinnamomi e C. parasitica, de forma a avaliar a sua capacidade de inibição. Os resultados demonstraram que a área de inibição antifúngica foi proporcional à concentração de silício aplicado nas plantas. O controlo registou uma área de inibição de 0,63 cm2 , tendo o tratamento 10 mM SiK® mostrado uma área inibição consideravelmente superior (10,56 cm2 ) nas placas com Phytophthora cinnamomi. Comportamento similar foi observado nas placas com Cryphonectria pasitica, 0,81 cm2 e 11,2 cm2 foram as áreas de inibição registadas para o controlo e para o tratamento 10 mM SiK®, respetivamente. Estes resultados sugerem que a combinação dos fenóis com o silício amplifica a capacidade antifúngica do tratamento 10 mM SiK® permitindo um maior controlo/restrição do desenvolvimento dos agentes patogénicos estudados. Neste capítulo estudou-se também a atividade da fenilalanina amónia liase, da polifenol oxidase e da peroxidase nas plantas inoculadas com os dois patogénicos, tendo-se verificado um incremento da sua atividade nos extractos provenientes das plantas inoculadas com qualquer um dos patogénios, sugerindo que o Si apresenta um papel benéfico na amplificação da resposta de defesa bioquímica das plantas perante o ataque destes agentes patogénicos. Por fim, no Capítulo 7 foi feito um estudo em plantas adultas da variedade Martaínha fertilizadas com 10 mM SiK®, no solo ou na folha, de forma a avaliar o impacto deste tratamento em castanha: na morfologia, na eficiência polinização, na composição química e nas caraterísticas organoléticas. Os dados demonstraram que a aplicação de Si promove um maior teor em Si nas folhas quando aplicado por via foliar. A aplicação no solo promoveu um maior teor de Si na castanha. A eficiência da polinização analisada através produção de castanhas bem formadas, aumentou nas plantas tratadas, tendo igualmente aumentado o tamanho destas. Os resultados revelaram ainda que castanhas provenientes de castanheiros tratados apresentaram menor perda de água no período pós-colheita. As castanhas das árvores não tratadas apresentaram maior dificuldade de descasque, não tendo o painel de provadores detetado diferenças organolépticas entre os tratamentos.
The climatic changes that have been observed over the last decades, associated to the new cultural requirements of this culture, have raised new problems in the abiotic and biotic level causing serious threats to the sustainability of the culture in many regions. The present work had as main objectives to evaluate the effect of the treatment in chestnut with SiK ® by spraying. The concentrations of 0 mM, 5 mM, 7.5 mM and 10 mM SiK ® were tested in young chestnut (Castanea sativa) plants submitted to biotic and abiotic stresses and in adult plants to study their impact on the production and quality of chestnut. The thesis is structured in eight chapters. In Chapter 1 the State of art is presented where subjects related to the origin, distribution, ecology and economic importance of the species Castanea sativa Mill are discussed. Reference is also made to climate change and its impact on plants, as well as the mitigating action of silicon in the face of biotic and abiotic stresses. In the following chapters (Chapters 2 to 7) all the research activities developed throughout this study are presented in detail, culminating in Chapter 8 where a set of final considerations is made. In the present study and for each of the trials (Chapters 2 to 6) were used plants with about 2 months of age after germination from the same tree of the Sousã variety (Castanea sativa), from the germplasm bank of the University of Trás -os-Montes and Alto Douro. In all the tests four groups of plants were formed corresponding to the treatments to be performed. Treatments consisted of fertilization with increasing concentrations of silicon in the form of potassium silicate (0 mM, 5 mM, 7.5 mM and 10 mM SiK®). Assays were always started 2 months after treatment. In Chapter 2 the influence of fertilization with Si on the resilience of young chestnut plants at high temperatures was studied. These were subjected to a 1-month sequential cycle of 25 ° C, 32 ° C and 25 ° C, trying to understand not only their tolerability, but also to characterize their recovery after a high heat period. This study allowed us to conclude that Si fertilization at 7.5 mM and 10 mM SiK® increases the resilience at high temperature (32ºC), through a higher photochemical efficiency of photosystem II, preserving chloroplast activity, increasing photosynthetic activity and reducing the sweating. The results also highlight the action of Si on the synthesis of phenolic compounds and thiols and on the enzymatic activity of the enzymes ascorbate peroxidase, catalase and peroxidase, leading to reduction of oxidative damage and consequent greater tolerance to thermal stress. In this study, it was also observed that the plants treated with Si showed a faster recovery after exposure to the stress period compared to the control plants. In order to investigate the role of silicon in chestnut tolerance to drought (Chapter 3), a study was carried out in 2015 and repeated in 2016 supplemented with new parameters, in order to make the results more robust. Sequentially, 2 month after application of SiK®, the plants were maintained in an adaptation cycle with adequate watering, followed by no irrigation until the water potential of -1.2 MPa was reached in any of the treatments, followed by watering. Plants treated with 7.5 mM and 10 mM SiK ® presented a higher frequency of xylem vessels with less area, increasing the adhesion of water molecules to the walls of the conducting vessels, as well as a lower stomatal density. These results suggest that this was a strategy of the plants to reduce water losses, thus allowing the use of water more efficiently, since higher values -1,2 MPa of water potential, relative water content and water efficiency were verified. The analysis of the parameters related to the gas exchange showed that Si treated plants were able to reduce the values of the transpiration rate and stomatal conductance, thus presenting higher water potential and relative water content. The biochemical studies complemented the work, suggesting that the application of Si in plants helps to improve the defense activity of plants by increasing the enzymes ascorbate peroxidase, catalase and peroxidase, as well as in the synthesis of phenolic compounds in drought conditions, consequently reducing the oxidative damage in plants under stress. Finally, in the plants treated with Si, a faster recovery of the values before the imposition of the drought period was observed, for the evaluated parameters, thus suggesting a faster recovery than the control plants. In Chapter 4 the effect of silicon fertilization on the increase of plant tolerance to the pathogen Phytophthora cinnamomi was evaluated. Biochemical studies were performed prior to inoculation and at 15 and 30 days after inoculation. Histological studies, as well as an antifungal evaluation of the effect of silicon on the development of Phytophthora cinnamomi were also carried out by the inoculation of silicon-containing plates in the PDA medium. The 7.5 mM and 10 mM SiK® treatments were the ones that presented the greatest capacity of reduction of the growth of Phythophthora cinnamomi only occurring reduced growth in plate at 144 h. Through the histological analysis of the roots, it was possible to observe the presence of phytoliths which acted as a mechanical barrier making it difficult to enter the hyphae inside the host plant. Plants fertilized with 7.5 mM and 10 mM SiK® also had a higher survival rate (80%) than control plants (20%). Chapter 5 corresponds to the study of the influence of silicon on plant tolerance to Cryphonectria parasitica, the methodology was similar to that described in Chapter 4. Chestnuts treated with 7.5 mM and 10 mM SiK® were found to have a 10% and 0% mortality rate, respectively, while the control plants (0 mM SiK®) registered a rate of 100%. The plants treated with the highest concentration of Si (10 mM SiK®) presented lower development of necroses in the stem. In relation to the histological analysis of the cross sections of the stems, the results showed the presence of phytoliths in xylem vessels proportional to the applied silicon concentration. Simultaneously to the study of Chapter 4 and 5, phenolic extracts were prepared from leaves of plants left without inoculation from the 4 treatments, and the results are presented in Chapter 6. In Chapter 6 it was verified that gallic acid and ellagic acid were two of the most predominant phenolic compounds identified in plants fertilized with silicon, which play an important antifungal action in the host plant defense system. The in vitro action of phenolic compounds from plants fertilized with different concentrations of Si was evaluated. Phenolic extracts were used to impregnate discs placed on plates containing P. cinnamomi and C. parasitica, in order to evaluate its capacity of inhibition. The results demonstrated that the area of antifungal inhibition was proportional to the concentration of silicon applied to the plants. The control had an inhibition area of 0.63 cm2 , with the 10 mM SiK® treatment having a considerably higher inhibitory area (10.56 cm 2 ) on the plates with Phytophthora cinnamomi. Similar behavior was observed on the plates with Cryphonectria pasitica, 0.81 cm2 and 11.2 cm2 were the areas of inhibition recorded for the control and for the treatment 10 mM SiK®, respectively. These results suggest that the combination of the phenols with the silicon amplifies the antifungal capacity of the 10 mM SiK® treatment allowing a greater control/restriction of the development of the pathogens studied. In this chapter we also studied the activity of phenylalanine ammonia lyase, polyphenol oxidase and peroxidase in plants inoculated with the two pathogens, with an increase in their activity in extracts from plants inoculated with any of the pathogens, suggesting that Si has a beneficial role in amplifying the biochemical defense response of plants to the attack of these pathogens. Finally, in Chapter 7 a study was carried out on adult plants of the Martaínha variety fertilized with 10 mM SiK®, in the soil or in the leaf, in order to evaluate the impact of this treatment on chestnut: morphology, pollination efficiency, chemical composition and organoleptic characteristics. The data showed that the application of Si promoted a higher Si content in the leaves when applied by foliar route. The soil application promoted a higher Si content in the nut. The efficiency of the pollination analyzed through the production of well-formed chestnuts increased in the treated plants and also increased their size. The results also revealed that chestnuts from treated chestnut trees had lower water losses in the post-harvest period. The nuts of the untreated trees had the greatest bark difficulty, the panel of tasters did not detect organoleptic differences between treatments
The climatic changes that have been observed over the last decades, associated to the new cultural requirements of this culture, have raised new problems in the abiotic and biotic level causing serious threats to the sustainability of the culture in many regions. The present work had as main objectives to evaluate the effect of the treatment in chestnut with SiK ® by spraying. The concentrations of 0 mM, 5 mM, 7.5 mM and 10 mM SiK ® were tested in young chestnut (Castanea sativa) plants submitted to biotic and abiotic stresses and in adult plants to study their impact on the production and quality of chestnut. The thesis is structured in eight chapters. In Chapter 1 the State of art is presented where subjects related to the origin, distribution, ecology and economic importance of the species Castanea sativa Mill are discussed. Reference is also made to climate change and its impact on plants, as well as the mitigating action of silicon in the face of biotic and abiotic stresses. In the following chapters (Chapters 2 to 7) all the research activities developed throughout this study are presented in detail, culminating in Chapter 8 where a set of final considerations is made. In the present study and for each of the trials (Chapters 2 to 6) were used plants with about 2 months of age after germination from the same tree of the Sousã variety (Castanea sativa), from the germplasm bank of the University of Trás -os-Montes and Alto Douro. In all the tests four groups of plants were formed corresponding to the treatments to be performed. Treatments consisted of fertilization with increasing concentrations of silicon in the form of potassium silicate (0 mM, 5 mM, 7.5 mM and 10 mM SiK®). Assays were always started 2 months after treatment. In Chapter 2 the influence of fertilization with Si on the resilience of young chestnut plants at high temperatures was studied. These were subjected to a 1-month sequential cycle of 25 ° C, 32 ° C and 25 ° C, trying to understand not only their tolerability, but also to characterize their recovery after a high heat period. This study allowed us to conclude that Si fertilization at 7.5 mM and 10 mM SiK® increases the resilience at high temperature (32ºC), through a higher photochemical efficiency of photosystem II, preserving chloroplast activity, increasing photosynthetic activity and reducing the sweating. The results also highlight the action of Si on the synthesis of phenolic compounds and thiols and on the enzymatic activity of the enzymes ascorbate peroxidase, catalase and peroxidase, leading to reduction of oxidative damage and consequent greater tolerance to thermal stress. In this study, it was also observed that the plants treated with Si showed a faster recovery after exposure to the stress period compared to the control plants. In order to investigate the role of silicon in chestnut tolerance to drought (Chapter 3), a study was carried out in 2015 and repeated in 2016 supplemented with new parameters, in order to make the results more robust. Sequentially, 2 month after application of SiK®, the plants were maintained in an adaptation cycle with adequate watering, followed by no irrigation until the water potential of -1.2 MPa was reached in any of the treatments, followed by watering. Plants treated with 7.5 mM and 10 mM SiK ® presented a higher frequency of xylem vessels with less area, increasing the adhesion of water molecules to the walls of the conducting vessels, as well as a lower stomatal density. These results suggest that this was a strategy of the plants to reduce water losses, thus allowing the use of water more efficiently, since higher values -1,2 MPa of water potential, relative water content and water efficiency were verified. The analysis of the parameters related to the gas exchange showed that Si treated plants were able to reduce the values of the transpiration rate and stomatal conductance, thus presenting higher water potential and relative water content. The biochemical studies complemented the work, suggesting that the application of Si in plants helps to improve the defense activity of plants by increasing the enzymes ascorbate peroxidase, catalase and peroxidase, as well as in the synthesis of phenolic compounds in drought conditions, consequently reducing the oxidative damage in plants under stress. Finally, in the plants treated with Si, a faster recovery of the values before the imposition of the drought period was observed, for the evaluated parameters, thus suggesting a faster recovery than the control plants. In Chapter 4 the effect of silicon fertilization on the increase of plant tolerance to the pathogen Phytophthora cinnamomi was evaluated. Biochemical studies were performed prior to inoculation and at 15 and 30 days after inoculation. Histological studies, as well as an antifungal evaluation of the effect of silicon on the development of Phytophthora cinnamomi were also carried out by the inoculation of silicon-containing plates in the PDA medium. The 7.5 mM and 10 mM SiK® treatments were the ones that presented the greatest capacity of reduction of the growth of Phythophthora cinnamomi only occurring reduced growth in plate at 144 h. Through the histological analysis of the roots, it was possible to observe the presence of phytoliths which acted as a mechanical barrier making it difficult to enter the hyphae inside the host plant. Plants fertilized with 7.5 mM and 10 mM SiK® also had a higher survival rate (80%) than control plants (20%). Chapter 5 corresponds to the study of the influence of silicon on plant tolerance to Cryphonectria parasitica, the methodology was similar to that described in Chapter 4. Chestnuts treated with 7.5 mM and 10 mM SiK® were found to have a 10% and 0% mortality rate, respectively, while the control plants (0 mM SiK®) registered a rate of 100%. The plants treated with the highest concentration of Si (10 mM SiK®) presented lower development of necroses in the stem. In relation to the histological analysis of the cross sections of the stems, the results showed the presence of phytoliths in xylem vessels proportional to the applied silicon concentration. Simultaneously to the study of Chapter 4 and 5, phenolic extracts were prepared from leaves of plants left without inoculation from the 4 treatments, and the results are presented in Chapter 6. In Chapter 6 it was verified that gallic acid and ellagic acid were two of the most predominant phenolic compounds identified in plants fertilized with silicon, which play an important antifungal action in the host plant defense system. The in vitro action of phenolic compounds from plants fertilized with different concentrations of Si was evaluated. Phenolic extracts were used to impregnate discs placed on plates containing P. cinnamomi and C. parasitica, in order to evaluate its capacity of inhibition. The results demonstrated that the area of antifungal inhibition was proportional to the concentration of silicon applied to the plants. The control had an inhibition area of 0.63 cm2 , with the 10 mM SiK® treatment having a considerably higher inhibitory area (10.56 cm 2 ) on the plates with Phytophthora cinnamomi. Similar behavior was observed on the plates with Cryphonectria pasitica, 0.81 cm2 and 11.2 cm2 were the areas of inhibition recorded for the control and for the treatment 10 mM SiK®, respectively. These results suggest that the combination of the phenols with the silicon amplifies the antifungal capacity of the 10 mM SiK® treatment allowing a greater control/restriction of the development of the pathogens studied. In this chapter we also studied the activity of phenylalanine ammonia lyase, polyphenol oxidase and peroxidase in plants inoculated with the two pathogens, with an increase in their activity in extracts from plants inoculated with any of the pathogens, suggesting that Si has a beneficial role in amplifying the biochemical defense response of plants to the attack of these pathogens. Finally, in Chapter 7 a study was carried out on adult plants of the Martaínha variety fertilized with 10 mM SiK®, in the soil or in the leaf, in order to evaluate the impact of this treatment on chestnut: morphology, pollination efficiency, chemical composition and organoleptic characteristics. The data showed that the application of Si promoted a higher Si content in the leaves when applied by foliar route. The soil application promoted a higher Si content in the nut. The efficiency of the pollination analyzed through the production of well-formed chestnuts increased in the treated plants and also increased their size. The results also revealed that chestnuts from treated chestnut trees had lower water losses in the post-harvest period. The nuts of the untreated trees had the greatest bark difficulty, the panel of tasters did not detect organoleptic differences between treatments
Descrição
Tese de Doutoramento em Ciências da Terra e da Vida
Palavras-chave
Ação antifúngica , Castanea sativa