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Título: Monitoring Clavibacter michiganensis SUBSP. michiganensis GFP – marked strain inoculated in tomato seeds
Título Alternativo: Monitoramento de um isolado de Clavibacter michiganensis subsp. michiganensis marcado com GFP inoculado em sementes de tomate
Autor(es): Souza, Ricardo Magela de
Wolf, Jan Martin Van der
Moura, Andréa Bittencourt
Figueira, Antonia dos Reis
Ferreira, Maria Alves
Santos, Heloísa Oliveira dos
Assunto: Bacterial canker
Confocal laser scanning microscopy
Epifluorescence stereomicroscopy
Green fluorescent protein
TaqMan assay
Cancro bacteriano
Microscopia de escaneamento com laser confocal
Microscopia de epifluorescência
Proteína verde fluorescente
Ensaio TaqMan
PCR em tempo real
Data de publicação: 9-Dez-2015
Publicador: Universidade Federal de Lavras
Referência: RIBEIRO, D. H. Monitoring Clavibacter michiganensis SUBSP. michiganensis GFP – marked strain inoculated in tomato seeds. 2015. 129 p. Tese (Doutorado em Agronomia/Fitopatologia)-Universidade Federal de Lavras, Lavras, 2015.
Resumo: Clavibacter michiganensis subsp. michiganensis (Cmm) is an economicallyimportant seed-borne phytobacteria that causes bacterial canker and bacterial wilt, the most important seed-borne disease of tomato. For studies on colonization of tomato seeds, seed treatments, seed transmission and for evaluation of diagnostic methods with Cmm, there is a need for seed that is internally infected with Cmm, how it is observed in natural infections . In this study, a procedure was developed to generate tomato seed (internally-) infected with Cmm. These seeds can be used to evaluate methods for detection of Cmm in seed extracts, to study the effect of seed treatments and to study transmission from seed to seedlings. Seeds were subsequently wounded by scarification, softened by incubation on wetted blotting paper, and inoculated with a spontaneous rifampicin resistant Cmm strain suspension by vacuum-infiltration. A GFP-tagged Cmm strain was used to visualize Cmm in planta. The population dynamics of Cmm on or in (disinfected) seeds and seedlings were studied by plating, a non-invasive monitoring system for GFP-tagged bacteria in plants (PathoScreen), Confocal laser scanning microscopy (CLSM), Epifluorescence stereomicroscopy (ESM) and a TaqMan assay. Also, dead and viable seeds were used to observe the Cmm behavior on and in different seed tissues. To enhance growth of the Cmm in the seeds, they were incubated on wetted blotting paper or on a Cmm-selective medium/broth. Directly after inoculation, a few single Cmm cells could be spotted into the seeds and incubation of seeds resulted in a strong increase of Cmm populations. The increase of the bacteria into the seed was confirmed with the monitoring of the GFP signal by the PathoScreen analyzes. Transected and undivided seeds analyzed with CSLM and ESM indicated that during incubation, Cmm colonized trichome hairs, the outer testa layers, the endosperm and embryo. The GFP signal could also be observed by epifluorescence stereomicroscopy in germinated seeds on the seed coat, cotyledons, stem and roots. Colonization of xylem vessels in stems and roots of seedlings was observed with confocal laser scanning microscopy, indicating systemic infections of germinated seeds. Previous incubation of infected seeds in semi-selective media improved the detection of Cmm by a TaqMan assay. This enhancement was obtained with the increasing of low population densities of Cmm in tomato seeds.
URI: http://repositorio.ufla.br/jspui/handle/1/10672
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