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Abstract:

Aims: To establish a reliable and rapid protocol to simultaneously obtain high quality DNA from an infected host plant and the infecting pathogen. To develop an accurate and sensitive low-cost assay for the quantification and in planta monitoring of Phytophthora infestans growth.Methods and Results: In this study, we describe a SYBR Green-based quantitative real-time PCR (qPCR) method for the quantification of P. infestans. The method is based on a simultaneous plant-pathogen DNA purification followed by a qPCR in which the relative quantification of pathogen and plant DNA is performed. Besides assuring an accurate quantification, the use of a plant gene provides a reliable indicator of sample quality, allowing the exclusion of inappropriate samples. By applying this methodology, we were able to detect P. infestans in potato leaf and tuber tissue before the first symptoms of the disease were observed and to monitor the in planta growth of the pathogen for 6 days.Conclusions: This is a reliable low-cost assay that provides rapid, accurate and sensitive quantification of the late blight pathogen, allowing the in planta monitoring of P. infestans growth.Significance and Impact of the Study: The quantitative nature of the assay described in this study may be useful in plant breeding programmes and basic research. The method is appropriate for the comparison of cultivars with different, and even subtle, degrees of pathogen resistance and in the screening of new anti-oomycete compounds. The method can be easily adapted to tomato and the model plant Nicotiana benthamiana. © No claim to Argentinean Government works. Letters in Applied Microbiology 51, 603-610 © 2010 The Society for Applied Microbiology.

Registro:

Documento: Artículo
Título:A quantitative real-time PCR method for in planta monitoring of Phytophthora infestans growth
Autor:Llorente, B.; Bravo-Almonacid, F.; Cvitanich, C.; Orlowska, E.; Torres, H.N.; Flawiá, M.M.; Alonso, G.D.
Filiación:Instituto de Investigaciones en Ingeniería Genética, Biología Molecular (INGEBI-CONICET), Buenos Aires, Argentina
Departamento de Ciencia y Tecnología, Universidad Nacional de Quilmes, Bernal, Buenos Aires, Argentina
Department of Molecular Biology, Aarhus University, Aarhus, Denmark
Departamento de Fisiología, Biología Molecular y Celular, Facultad de Ciencias Exactas y Naturales, Universidad de Buenos Aires, Buenos Aires, Argentina
Palabras clave:Biomass monitoring; Late blight; Phytophthora infestans; Potato crop; Quantitative real-time PCR; Solanum tuberosum; adaptation; bioassay; cultivar; disease resistance; fungal disease; growth rate; host plant; host-pathogen interaction; monitoring system; plant breeding; polymerase chain reaction; real time; symptom; tuber; article; DNA purification; fungus growth; monitoring; nonhuman; nucleotide sequence; pathogenesis; Phytophthora infestans; plant; plant disease; plant gene; plant leaf; potato; quantitative analysis; real time polymerase chain reaction; DNA; DNA Primers; DNA, Plant; Organic Chemicals; Phytophthora infestans; Plant Diseases; Plant Leaves; Plant Tubers; Polymerase Chain Reaction; Solanum tuberosum; Species Specificity; Lycopersicon esculentum; Nicotiana benthamiana; Phytophthora infestans; Solanum tuberosum
Año:2010
Volumen:51
Número:6
Página de inicio:603
Página de fin:610
DOI: http://dx.doi.org/10.1111/j.1472-765X.2010.02942.x
Título revista:Letters in Applied Microbiology
Título revista abreviado:Lett. Appl. Microbiol.
ISSN:02668254
CODEN:LAMIE
CAS:DNA, 9007-49-2; DNA Primers; DNA, Plant; Organic Chemicals; SYBR Green I, 163795-75-3
PDF:https://bibliotecadigital.exactas.uba.ar/download/paper/paper_02668254_v51_n6_p603_Llorente.pdf
Registro:https://bibliotecadigital.exactas.uba.ar/collection/paper/document/paper_02668254_v51_n6_p603_Llorente

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Citas:

---------- APA ----------
Llorente, B., Bravo-Almonacid, F., Cvitanich, C., Orlowska, E., Torres, H.N., Flawiá, M.M. & Alonso, G.D. (2010) . A quantitative real-time PCR method for in planta monitoring of Phytophthora infestans growth. Letters in Applied Microbiology, 51(6), 603-610.
http://dx.doi.org/10.1111/j.1472-765X.2010.02942.x
---------- CHICAGO ----------
Llorente, B., Bravo-Almonacid, F., Cvitanich, C., Orlowska, E., Torres, H.N., Flawiá, M.M., et al. "A quantitative real-time PCR method for in planta monitoring of Phytophthora infestans growth" . Letters in Applied Microbiology 51, no. 6 (2010) : 603-610.
http://dx.doi.org/10.1111/j.1472-765X.2010.02942.x
---------- MLA ----------
Llorente, B., Bravo-Almonacid, F., Cvitanich, C., Orlowska, E., Torres, H.N., Flawiá, M.M., et al. "A quantitative real-time PCR method for in planta monitoring of Phytophthora infestans growth" . Letters in Applied Microbiology, vol. 51, no. 6, 2010, pp. 603-610.
http://dx.doi.org/10.1111/j.1472-765X.2010.02942.x
---------- VANCOUVER ----------
Llorente, B., Bravo-Almonacid, F., Cvitanich, C., Orlowska, E., Torres, H.N., Flawiá, M.M., et al. A quantitative real-time PCR method for in planta monitoring of Phytophthora infestans growth. Lett. Appl. Microbiol. 2010;51(6):603-610.
http://dx.doi.org/10.1111/j.1472-765X.2010.02942.x