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

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.

Host-mediated gene silencing of a single effector gene from the potato pathogen Phytophthora infestans imparts partial resistance to late blight disease

“…The qRT-PCR estimation allowed detection of femtogram (fg) quantity of Pi08 DNA in the infected leaf samples (Llorente et al 2010), as well as in terms of high copy number (14,000 copies/nucleus; Judelson and Tooley 2000).…”

Loop‐mediated isothermal amplification for detection of the tomato and potato late blight pathogen, P hytophthora infestans

“…Highly repetitive DNA sequences have also been targeted to improve detection sensitivity over ITS‐based assays (Judelson and Tooley ; Llorente et al . ). The detection of P. infestans inoculum in soil samples was demonstrated using conventional PCR (Hussain et al .…”

Pathogen recognition in compatible plant-microbe interactions

“…The biotrophic phase (48 hpi) was estimated by macro and microscopical assessments, and comparative gene expression studies with so-called transition (96 hpi) and necrotrophic phases (144 hpi) 8 . Depending on the experimental conditions, first symptoms appeared on the potato and tomato leaves at approximately 48–72 hpi 7, 9 . Oidium in tomato is caused by the obligate biotrophic fungus Oidium neolycopersici , which invades the epidermal cells by means of appressoria formation 10 .…”