Real-time quantitative PCR: a new technology to detect and study phytopathogenic and antagonistic fungi

Abstract

Real-time PCR technologies open increasing opportunities to detect and study phytopathogenic and antagonistic fungi. They combine the sensitivity of conventional PCR with the generation of a specific fluorescent signal providing real-time analysis of the reaction kinetics and allowing quantification of specific DNA targets. Four main chemistries are currently used for the application of this technique in plant pathology. These chemistries can be grouped into amplicon sequence non-specific (SYBR Green I) and sequence specific (TaqMan, Molecular beacons, and Scorpion-PCR) methods. Amplicon sequence nonspecific methods are based on the use of a dye that emits fluorescent light when intercalated into doublestranded DNA. Amplicon sequence specific methods are based on the use of oligonucleotide probes labelled with a donor fluorophore and an acceptor dye (quencher). The fluorescent signal eliminates the requirement for post-amplification processing steps, such as gel electrophoresis and ethidium bromide staining. This significantly reduces time and labour required for the analysis and greatly increases the throughput of PCR testing as an automated diagnostic system, making it suitable for large-scale analyses. Furthermore, the use of different fluorescent dyes facilitates the detection of several target microrganisms in a single reaction (multiplex-PCR). Real-time PCR makes possible an accurate, reliable and high throughput quantification of target fungal DNA in various environmental samples, including hosts tissues, soil, water and air, thus opening new research opportunities for the study of diagnosis, inoculum threshold levels, epidemiology and host-pathogen interactions. Moreover, the quantification of specific mRNA transcription by real-time PCR is being increasingly applied to the study of changes in gene expression in response to phytopathogenic and antagonistic fungi.

Verticillium wilt of olive: a case study to implement an integrated strategy to control a soil-borne pathogen

“…It is more accurate and less time consuming than conventional, end-point quantitative PCR because it monitors PCR products before reaction components become limiting. Currently, RT-qPCR technology is commonly used in plant pathology for the precise detection and quantification of pathogens in infected plants and infested soils (reviewed by Schena et al 2004b;Ma and Michailides 2007), and it has been used to quantify V. dahliae DNA as well (Schena et al 2004a;Atallah et al 2007;Gayoso et al 2007). RTqPCR has also been successfully implemented to quantify the biomass of V. dahliae D and ND pathotypes in infected olive plants (Mercado-Blanco et al 2003a;Markakis et al 2009).…”

Molecular Tools for Detection of Plant Pathogenic Fungi and Fungicide Resistance

“…It is frequently used in PCR-based methods when there are not enough differences available across the ITS. Primers in this region have been designed to detect and identify Verticillium dahliae and V. alboatrum (Schena et al, 2004) and to distinguish pathogenic and non-pathogenic Fusarium oxysporum in tomato (Validov et al, 2011). As another example, Inami et al (2010) differentiated Fusarium oxysporum f. sp lycopersici, and its races using primers and TaqMan-MGB probes based on IGS and avirulent SIX genes.…”

Phylogeny analysis of Indian strains of Rhizoctonia solani isolated from chickpea and development of sequence characterized amplified region (SCAR) marker for detection of the pathogen

“…To avoid the above limitations, a new era of DNA based markers system (De Curtis et al, 2004;Schena et al, 2004) begun with the RAPD markers (Williams et al, 1990). Random amplified polymorphic DNA (RAPD) offers a promising, versatile and informative molecular tool to detect genetic variation within population of plant pathogens (Sharma et al, 2005).…”