Publicação
Assessment of Erwinia amylovora diversity and virulence: from strain molecular typing to immuno-flow cytometry of infected Pyrus fruits
| Resumo: | Erwinia amylovora is a phytopathogenic bacterium and the causative agent of fire blight, a destructive disease that affects several members of the Rosaceae family. Pear and apple are particularly susceptible hosts of this bacterium, representing a major concern due to their socioeconomic value. The devasting nature of this disease has led to the classification of E. amylovora as a quarantine organism. Although this bacterium is widespread throughout the world, its emergence in Portugal happened relatively recently when compared to other European countries. Since its first report in 2006 in Fundão region, the number of outbreaks has increased, leading Portugal to loss its statute of Integral Protected Area within the European Union, in 2019. Factors such as the absence of a cure, false-negatives results and the need to understand unknown aspects about the life cycle of E. amylovora, lead to the interest in conducting characterization studies and developing alternative laboratory methods as an attempt to make preventive measures more effective. A set of Portuguese and foreign E. amylovora isolates was characterized by molecular, pathogenicity and virulence studies. Molecular characterization used CRISPR-PCR and genomic fingerprintings, whereas pathogenicity and virulence were assessed by biological tests on immature fruitlets of Pyrus communis cv. “Rocha”. In addition, a flow cytometry and an immuno-flow cytometry protocols were developed using pure and mixed bacterial cultures for the detection and cell viability assessment of E. amylovora in planta. A total of two CRISPR genotypes, A and D, was observed revealing a low genetic diversity among the E. amylovora isolates tested. Genomic fingerprinting reinforced the high homogeneity of this species. In contrast, a wide diversity in virulence was displayed. Flow cytometry and immuno-flow cytometry protocols were validated, revealing the capacity to detect and distinguish different viability states of E. amylovora artificially inoculated in pear fruitlets. In the future, the established protocols may help shed some light over previously undiscovered aspects of E. amylovora life cycle. |
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| Autores principais: | Costa, Telma Patrícia Natário Guedes da |
| Assunto: | Fogo bacteriano CRISPR-PCR Perfis genómicos Ensaios de virulência Citometria de fluxo Imuno-citometria de fluxo Teses de mestrado - 2020 |
| Ano: | 2020 |
| País: | Portugal |
| Tipo de documento: | dissertação de mestrado |
| Tipo de acesso: | acesso aberto |
| Instituição associada: | Universidade de Lisboa |
| Idioma: | inglês |
| Origem: | Repositório da Universidade de Lisboa |
| Resumo: | Erwinia amylovora is a phytopathogenic bacterium and the causative agent of fire blight, a destructive disease that affects several members of the Rosaceae family. Pear and apple are particularly susceptible hosts of this bacterium, representing a major concern due to their socioeconomic value. The devasting nature of this disease has led to the classification of E. amylovora as a quarantine organism. Although this bacterium is widespread throughout the world, its emergence in Portugal happened relatively recently when compared to other European countries. Since its first report in 2006 in Fundão region, the number of outbreaks has increased, leading Portugal to loss its statute of Integral Protected Area within the European Union, in 2019. Factors such as the absence of a cure, false-negatives results and the need to understand unknown aspects about the life cycle of E. amylovora, lead to the interest in conducting characterization studies and developing alternative laboratory methods as an attempt to make preventive measures more effective. A set of Portuguese and foreign E. amylovora isolates was characterized by molecular, pathogenicity and virulence studies. Molecular characterization used CRISPR-PCR and genomic fingerprintings, whereas pathogenicity and virulence were assessed by biological tests on immature fruitlets of Pyrus communis cv. “Rocha”. In addition, a flow cytometry and an immuno-flow cytometry protocols were developed using pure and mixed bacterial cultures for the detection and cell viability assessment of E. amylovora in planta. A total of two CRISPR genotypes, A and D, was observed revealing a low genetic diversity among the E. amylovora isolates tested. Genomic fingerprinting reinforced the high homogeneity of this species. In contrast, a wide diversity in virulence was displayed. Flow cytometry and immuno-flow cytometry protocols were validated, revealing the capacity to detect and distinguish different viability states of E. amylovora artificially inoculated in pear fruitlets. In the future, the established protocols may help shed some light over previously undiscovered aspects of E. amylovora life cycle. |
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