Characterization and selection of microbial symbionts of faba bean (Vicia faba L.) and cowpea (Vigna unguiculata (L.) Walp.) for development of inoculants
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2020-06-09
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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.
Global agriculture has to double food production by 2050 in order to feed the world’s growing population. In this sense, food with a high nutritional value, such as the leguminous plants (protein, minerals, vitamins and bioactive compounds) appear as an answer to this need. However, it is crucial to increase its productivity. At the same time, it is also necessary to reduce the application of inorganic fertilizers, due to the high negative impact they have on the environment. To achieve these goals, it is essential to take advantages from the multiple beneficial interactions that occur between plants and microorganisms. Beneficial microorganisms present in the soil, namely rhizobia and arbuscular mycorrhizal fungi, in symbiosis with leguminous plants, results in a tripartite symbiosis and can be a biological tool to enhance crop production, through biological nitrogen fixation and phosphorus uptake from soil. This symbiosis also increases the resistance of crops to drought stress and high temperatures, improves crop productivity and quality and soil fertility and decreases the incidence of weeds, diseases and pests, without the negative impacts in the environment provoked by chemical fertilizer inputs. In this sense, the objectives of this work were to select improved strains of Rhizobium leguminosarum and Bradyrhizobium spp. for enhanced biological nitrogen fixation and field performance on cultivars of faba beans and cowpeas, and to perform the phenotypic and genotypic characterization of microbial symbionts using a polyphasic approach based on phenotypic properties and molecular analysis. In the present work, the molecular identification of rhizobial bacteria present in cowpea and faba bean plants collected from several regions of Portugal with different climatic conditions and different types of soil was performed using a Multilocus Sequence typing” (MLST) approach with 9 genes (housekeeping and symbiotic genes), to obtain information at species and symbiovars level, since the amplification of 16SrDNA region alone did not provide enough resolution power. After the molecular identification, in vitro studies were performed to check the ability of the isolates to nodulate other plants (Koch's postulates) and to select the best inoculants for each crop, which were after tested under greenhouse conditions, with the purpose of evaluating the effects of single and co-inoculation with the selected microorganisms on the growth, yield and protein content of the respective leguminous plants. High diversity of rhizobial bacteria was identified in different fields and regions. For cowpea plants, were selected two rhizobial strains, Bradyrhizobium sp. and Bradyrhizobium elkanii. For faba bean, were selected Rhizobium laguerreae and Burkholderia sp.. Regarding the mycorrhizal inoculants, a mix of Rhizophagus irregularis BEG140, Funneliformis geosporum BEG199 and Claroideoglomus claroideum BEG210 (1 : 1 : 1) was developed and prepared by Symbiom (Sázava, Czech Republic) for the faba bean crop. For cowpea, the mycorrhizal fungi (Claroideoglomus claroideum BEG210) was provided by Dr. Rui Oliveira, from de University of Coimbra, Portugal. In the greenhouse work developed with inoculation and co-inoculation with Rhizobium laguerreae and AMF, faba bean plants single inoculated with the bacteria showed a significant increase in the number of leaves, leaf area, leaf mass per area and leaf area ratio, as well as in all evaluated yield parameters. Single inoculation of these plants with AMF also significantly increased the yield parameters. Co-inoculation showed significant improvement in leaf area ratio and in all productivity parameters when compared with the control, but it was not significantly different from the individual inoculations. In the studies with cowpea, under greenhouse conditions, using non-sterilized soil, the co-inoculation of plants with Rhizobium sp. and AMF, Bradyrhizobium elkanii and AMF and Bradyrhizobium sp. and AMF increased the crude protein content of the seeds in plants under drought stress (25% of field capacity) in 13, 17 and 30%, respectively. Considering all analyses performed in this work in both crops, it is possible to conclude that single and combined inoculation of leguminous plants with selected microorganisms showed great potential as a biological tool to improve the growth and yield of leguminous plant under abiotic stress, mitigating the effects of climate change and reducing the need for chemical fertilizer inputs.
Global agriculture has to double food production by 2050 in order to feed the world’s growing population. In this sense, food with a high nutritional value, such as the leguminous plants (protein, minerals, vitamins and bioactive compounds) appear as an answer to this need. However, it is crucial to increase its productivity. At the same time, it is also necessary to reduce the application of inorganic fertilizers, due to the high negative impact they have on the environment. To achieve these goals, it is essential to take advantages from the multiple beneficial interactions that occur between plants and microorganisms. Beneficial microorganisms present in the soil, namely rhizobia and arbuscular mycorrhizal fungi, in symbiosis with leguminous plants, results in a tripartite symbiosis and can be a biological tool to enhance crop production, through biological nitrogen fixation and phosphorus uptake from soil. This symbiosis also increases the resistance of crops to drought stress and high temperatures, improves crop productivity and quality and soil fertility and decreases the incidence of weeds, diseases and pests, without the negative impacts in the environment provoked by chemical fertilizer inputs. In this sense, the objectives of this work were to select improved strains of Rhizobium leguminosarum and Bradyrhizobium spp. for enhanced biological nitrogen fixation and field performance on cultivars of faba beans and cowpeas, and to perform the phenotypic and genotypic characterization of microbial symbionts using a polyphasic approach based on phenotypic properties and molecular analysis. In the present work, the molecular identification of rhizobial bacteria present in cowpea and faba bean plants collected from several regions of Portugal with different climatic conditions and different types of soil was performed using a Multilocus Sequence typing” (MLST) approach with 9 genes (housekeeping and symbiotic genes), to obtain information at species and symbiovars level, since the amplification of 16SrDNA region alone did not provide enough resolution power. After the molecular identification, in vitro studies were performed to check the ability of the isolates to nodulate other plants (Koch's postulates) and to select the best inoculants for each crop, which were after tested under greenhouse conditions, with the purpose of evaluating the effects of single and co-inoculation with the selected microorganisms on the growth, yield and protein content of the respective leguminous plants. High diversity of rhizobial bacteria was identified in different fields and regions. For cowpea plants, were selected two rhizobial strains, Bradyrhizobium sp. and Bradyrhizobium elkanii. For faba bean, were selected Rhizobium laguerreae and Burkholderia sp.. Regarding the mycorrhizal inoculants, a mix of Rhizophagus irregularis BEG140, Funneliformis geosporum BEG199 and Claroideoglomus claroideum BEG210 (1 : 1 : 1) was developed and prepared by Symbiom (Sázava, Czech Republic) for the faba bean crop. For cowpea, the mycorrhizal fungi (Claroideoglomus claroideum BEG210) was provided by Dr. Rui Oliveira, from de University of Coimbra, Portugal. In the greenhouse work developed with inoculation and co-inoculation with Rhizobium laguerreae and AMF, faba bean plants single inoculated with the bacteria showed a significant increase in the number of leaves, leaf area, leaf mass per area and leaf area ratio, as well as in all evaluated yield parameters. Single inoculation of these plants with AMF also significantly increased the yield parameters. Co-inoculation showed significant improvement in leaf area ratio and in all productivity parameters when compared with the control, but it was not significantly different from the individual inoculations. In the studies with cowpea, under greenhouse conditions, using non-sterilized soil, the co-inoculation of plants with Rhizobium sp. and AMF, Bradyrhizobium elkanii and AMF and Bradyrhizobium sp. and AMF increased the crude protein content of the seeds in plants under drought stress (25% of field capacity) in 13, 17 and 30%, respectively. Considering all analyses performed in this work in both crops, it is possible to conclude that single and combined inoculation of leguminous plants with selected microorganisms showed great potential as a biological tool to improve the growth and yield of leguminous plant under abiotic stress, mitigating the effects of climate change and reducing the need for chemical fertilizer inputs.
Descrição
This work was produced as original thesis for the degree of Doctor in AgriChains-From Fork to Farm at the University of Trás-osMontes and Alto Douro