A quantitative real-time PCR method for in planta monitoring of Phytophthora infestans growth

• Autores: Llorente, B.; Bravo-Almonacid, F.; Cvitanich, C.; Orlowska, E.; Torres, H.N.; Flawiá, M.M.; Alonso, G.D.
• Año de publicación: 2010
• Idioma: inglés
• Tipo de recurso: artículo
• Fuente: Lett. Appl. Microbiol. 2010;51(6):603-610
• Materia: 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
• Institución: Universidad Nacional de Buenos Aires. Facultad de Ciencias Exactas y Naturales

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.
Fil:Llorente, B. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales; Argentina.
Fil:Torres, H.N. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales; Argentina.
Fil:Flawiá, M.M. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales; Argentina.
Fil:Alonso, G.D. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales; Argentina.