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Study of the arbuscular mycorrhizal fungi (AMF) associated to the Azorean endemic woody plant Picconia azorica (Tutin) Knobl. and their potential application on sustainable restoration programs
| Name: | Description: | Size: | Format: | |
|---|---|---|---|---|
| Resumo, Índice, Introdução | 280.34 KB | Adobe PDF | ||
| Documento Principal | 5.85 MB | Adobe PDF | ||
| ANEXO IV | 189.18 KB | Adobe PDF | ||
| ANEXO VI | 65.96 KB | Adobe PDF | ||
| Figura 12 | 40.35 KB | Adobe PDF |
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Abstract(s)
Os fungos micorrízicos arbusculares (FMA), simbiontes obrigatórios pertencentes ao filo Glomeromicota, formam relações mutualistas com cerca de 90% das espécies de plantas terrestres. Estes fungos fornecem água e minerais à planta e em troca a planta fornece produtos da fotossíntese. As comunidades de FMA presentes na rizosfera da planta endémica Picconia azorica, de duas ilhas do arquipélago dos Açores, foram estudadas com recurso a métodos morfológicos e moleculares. Os esporos de FMA isolados do solo foram morfologicamente classificados em 46 morfotipos. A caraterização molecular dos esporos foi efetuada por sequenciação de um fragmento de rADN com cerca de 1,5 kb (SSU-ITS-LSU) e resultou na obtenção de 125 sequências. Após análise filogenética estas sequências foram alocadas a 18 filotipos, as quais mostraram pertencer às famílias Acaulosporaceae, Archaeosporaceae, Claroideoglomeraceae, Gigasporaceae e Glomeraceae. Oito destas sequências não agruparam com nenhuma sequência previamente conhecida, o que sugere que correspondem a espécies que ainda não foram molecularmente identificadas ou que constituem novas espécies para a ciência. A análise da ecologia revelou que existem diferenças na comunidade de FMA entre as ilhas Terceira e São Miguel. A comunidade de FMA no solo da ilha Terceira é dominada por elementos das famílias Acaulosporaceae e Glomeraceae, enquanto, que em São Miguel é dominada pelas famílias Glomeraceae e Gigasporaceae. A análise microscópica das raízes de P. azorica revelou que as amostras se encontravam altamente colonizadas (89 – 99%) pelas diferentes estruturas do fungo (hifas, arbúsculos e vesículas), permitindo assim considerar a planta como sendo micotrófica e altamente dependente de FMA. A identificação dos fungos presentes nas raízes de P. azorica foi efetuada com base no fragmento de 1.5 kb do rADN e com recuso ao método de restrição terminal de fragmentos polimórficos (T-RFLP) conhecidos, no programa TRAMPR. A análise identificou 22 filotipos pertencentes às famílias Acaulosporaceae, Claroideoglomeraceae, Diversisporaceae, Gigasporaceae e Glomeraceae. Verificou-se que a espécie Acaulospora brasiliensis estava presente nas raízes de todas as plantas analisadas e que a família Glomeraceae foi a família mais representada com a identificação de 13 filotipos. Esta análise revelou ainda que em ambas as ilhas na colonização das raízes dominavam espécies da família Glomeraceae, sendo que nas raízes da ilha Terceira a presença de Acaulosporaceae foi mais frequente, enquanto, que nenhum membro da família Claroideoglomeraceae foi identificado em amostras de raízes de São Miguel. Os resultados destes estudos sugerem que as comunidades de FMA nas florestas nativas dos Açores são determinadas por fatores históricos e geográficos, bem como pelas propriedades químicas do solo e pelas condições ambientais. Para o estudo da capacidade de colonização de plantas micropropagadas pelas espécies Acaulospora laevis e Acaulospora koskei foram isolados e sequenciados segmentos de quatro genes de P. azorica homólogos a outros previamente relacionados à simbiose e foi analisada a expressão destes genes por qRT-PCR durante os estágios iniciais da simbiose com FMA. Nestes ensaios a indução da expressão por Ac. laevis revelou ser superior à obtida com Ac. koskei. A influência do FMA Funneliformis mosseae sobre o desenvolvimento de plantas de P. azorica micropropagadas foi estudada. Ao fim de 8 meses de tratamento as plantas inoculadas com o FMA revelaram-se maiores e mais robustas que as plantas não inoculadas. Esta é a primeira vez em que se estuda a condição de FMA presentes na rizosfera de P. azorica nas florestas nativas Açorianas. A P. azorica demonstrou elevada dependência pelos FMA o que revela sintonia com o facto de estes FMA terem revelado potencial para serem aplicados como promotores do sucesso na utilização de plantas micropropagadas em programas de conservação e restauração de florestas nativas no arquipélago dos Açores.
ABSTRACT: Arbuscular mycorrhizal fungi (AMF) are obligate symbionts, belonging to the phylum Glomeromycota that form mutualistic symbioses with about 90% of land plant species. They provide the host plant with mineral nutrients and water, in exchange for photosynthetic products. The communities of AMF present in the rhizosphere of endemic tree Picconia azorica from two Azorean islands, Terceira and São Miguel, were assessed by morphological and molecular methods. AMF spores isolated from the soil were morphologically classified in 46 distinguishable morphotypes. Molecular characterisation of a c. 1.5 kb rDNA fragment, comprising SSU-ITS-LSU, resulted in 125 AMF sequences. Phylogenetic analyses assigned the sequences to 18 phylotypes, belonging to the families Acaulosporaceae, Archaeosporaceae, Claroideoglomeraceae, Gigasporaceae and Glomeraceae. Also, 8 phylotypes did not cluster with sequences of any described species implying that they are either species that have not yet been identified or are new to science. Ecological analyses revealed differences in AMF community composition between islands. In Terceira, the rhizospheric soil was dominated by species belonging to Acaulosporaceae and Glomeraceae, while São Miguel was dominated by Glomeraceae and Gigasporaceae. Microscopic survey of P. azorica root samples showed that all studied samples were highly colonised (89 – 99%) by AMF structures (hyphae, arbuscules and vesicles) which allowed us to classify the endemic plant as mycotrophic and highly dependent on AMF. The identity and diversity of AMF colonising the roots of P. azorica were assessed by terminal restriction fragment length polymorphism (T-RFLP) of the c. 1.5 Kb rDNA fragment. Database terminal restriction fragment length polymorphism (T-RFLP) approach in conjunction with TRAMPR package was used to identify the AMF community present in the roots. By that were identified 22 phylotypes belonging to Acaulosporaceae, Claroideoglomeraceae, Diversisporaceae, Gigasporaceae and Glomeraceae. Acaulospora brasiliensis was present in all studied roots of all sampled plants and Glomeraceae showed to be the more diverse family with 13 phylotypes identified. It was also observed differences in AMF community composition between islands. Although in both islands root colonization was dominated by species belonging to Glomeraceae, in Terceira the presence of Acaulosporaceae was more frequent, while no Claroideoglomeraceae were identified in São Miguel root samples. All results suggest that AMF communities in native forest of Azores are determined by geographic and historical factors as well as soil chemical properties and environmental conditions. The ability of Acaulospora laevis and Acaulospora koskei to colonised micropropagated plants of P. azorica was tested by isolating and sequencing of partial sequences of four P. azorica symbiosis-related genes and analyses of gene expression by qRT-PCR during early stages of AMF symbiosis. This revealed that both AMF species varied in their capacity to induce the early stages of the plant symbiotic response, in which Ac. laevis showed superior to Ac. koskei. The influence of Funneliformis mosseae on the growth of micropropagated P. azorica was investigated. After eight months of growth, inoculated plants showed to be bigger and more robust than non-inoculated plants. This is the first time that AMF status in the rhizosphere of P. azorica in native forest of the Azores was investigated. P. azorica showed to be highly dependent on AMF which is in line with the showed potential of AMF application as promoters of plant establishment in conservation and restoration of native forests of the Azores archipelago.
ABSTRACT: Arbuscular mycorrhizal fungi (AMF) are obligate symbionts, belonging to the phylum Glomeromycota that form mutualistic symbioses with about 90% of land plant species. They provide the host plant with mineral nutrients and water, in exchange for photosynthetic products. The communities of AMF present in the rhizosphere of endemic tree Picconia azorica from two Azorean islands, Terceira and São Miguel, were assessed by morphological and molecular methods. AMF spores isolated from the soil were morphologically classified in 46 distinguishable morphotypes. Molecular characterisation of a c. 1.5 kb rDNA fragment, comprising SSU-ITS-LSU, resulted in 125 AMF sequences. Phylogenetic analyses assigned the sequences to 18 phylotypes, belonging to the families Acaulosporaceae, Archaeosporaceae, Claroideoglomeraceae, Gigasporaceae and Glomeraceae. Also, 8 phylotypes did not cluster with sequences of any described species implying that they are either species that have not yet been identified or are new to science. Ecological analyses revealed differences in AMF community composition between islands. In Terceira, the rhizospheric soil was dominated by species belonging to Acaulosporaceae and Glomeraceae, while São Miguel was dominated by Glomeraceae and Gigasporaceae. Microscopic survey of P. azorica root samples showed that all studied samples were highly colonised (89 – 99%) by AMF structures (hyphae, arbuscules and vesicles) which allowed us to classify the endemic plant as mycotrophic and highly dependent on AMF. The identity and diversity of AMF colonising the roots of P. azorica were assessed by terminal restriction fragment length polymorphism (T-RFLP) of the c. 1.5 Kb rDNA fragment. Database terminal restriction fragment length polymorphism (T-RFLP) approach in conjunction with TRAMPR package was used to identify the AMF community present in the roots. By that were identified 22 phylotypes belonging to Acaulosporaceae, Claroideoglomeraceae, Diversisporaceae, Gigasporaceae and Glomeraceae. Acaulospora brasiliensis was present in all studied roots of all sampled plants and Glomeraceae showed to be the more diverse family with 13 phylotypes identified. It was also observed differences in AMF community composition between islands. Although in both islands root colonization was dominated by species belonging to Glomeraceae, in Terceira the presence of Acaulosporaceae was more frequent, while no Claroideoglomeraceae were identified in São Miguel root samples. All results suggest that AMF communities in native forest of Azores are determined by geographic and historical factors as well as soil chemical properties and environmental conditions. The ability of Acaulospora laevis and Acaulospora koskei to colonised micropropagated plants of P. azorica was tested by isolating and sequencing of partial sequences of four P. azorica symbiosis-related genes and analyses of gene expression by qRT-PCR during early stages of AMF symbiosis. This revealed that both AMF species varied in their capacity to induce the early stages of the plant symbiotic response, in which Ac. laevis showed superior to Ac. koskei. The influence of Funneliformis mosseae on the growth of micropropagated P. azorica was investigated. After eight months of growth, inoculated plants showed to be bigger and more robust than non-inoculated plants. This is the first time that AMF status in the rhizosphere of P. azorica in native forest of the Azores was investigated. P. azorica showed to be highly dependent on AMF which is in line with the showed potential of AMF application as promoters of plant establishment in conservation and restoration of native forests of the Azores archipelago.
Description
Tese de Doutoramento, Ciências Agrárias, especialidade em Biotecnologia, 07 de dezembro de 2018, Universidade dos Açores.
Keywords
Picconia azorica Ecologia Vegetal Floresta Nativa Fungos Micorrízicos Arbusculares Identificação Molecular Genética Vegetal Plantas Endémicas Açores AMF Molecular Identification Arbuscular Mycorrhizal Fungi Native Forests Root Colonization
Citation
Luna, Sara Patrícia Bettencourt. "Study of the arbuscular mycorrhizal fungi (AMF) associated to the Azorean endemic woody plant Picconia azorica (Tutin) Knobl. and their potential application on sustainable restoration programs". 2018. 146 p.. (Tese de Doutoramento em Ciências Agrárias, especialidade em Biotecnologia). Angra do Heroísmo: Universidade dos Açores, 2017. [Consult. Dia Mês Ano]. Disponível em www:<http://hdl.handle.net/10400.3/5156>.