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.

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

Awareness of crop biosecurity and phytosanitation has been heightened since 9/11 and the unresolved anthrax releases in October 2001. Crops are highly vulnerable to accidental or deliberate introductions of crop pathogens from outside U.S. borders. Strategic thinking about protection against deliberate or accidental release of a plant pathogen is an urgent priority. Rapid detection will be the key to success. This review summarizes recent progress in the development of rapid real-time PCR protocols and evaluates their effectiveness in a proposed nationwide network of diagnostic laboratories that will facilitate rapid diagnostics and improved communication.

Advances in molecular-based diagnostics in meeting crop biosecurity and phytosanitary issues.

Direct quantification of fungal DNA from soil substrate using real-time PCR.

SUMMARY New species of Phytophthora such as Phytophthora ramorum, P. kernoviae and P. quercina together with P. citricola are plant pathogens which impact on forest health, natural ecosystem stability and international trade. A real-time multiplex PCR approach based on TaqMan PCR was developed to simultaneously identify and detect these four Phytophthora species. Specific primers and probes labelled with FAM (P. ramorum), Yakima Yellow (P. kernoviae), Rox (P. citricola) and Cy5 (P. quercina) were designed in different regions of the ras-related protein (Ypt1) gene. A new set of Black Hole Quenchers (BHQ), which dissipate energy as heat rather than fluorescence, were utilized. The method proved to be highly specific in tests with target DNA from 72 Phytophthora isolates (35 species). For all pathogens, the detection limit was 100 fg of target DNA and was not improved utilizing a nested approach to provide a first round of amplification with Phytophthora spp.-specific primers. Cycle threshold (Ct) values were linearly correlated with the concentration of the target DNA (correlation coefficients ranged from 0.947 to 0.996) and were not affected by the presence of plant extracts, indicating the appropriateness of the method for qualitative and quantitative analyses. Two universal primers and a TaqMan probe were also developed to evaluate the quality and quantity of extracted DNA and to avoid false negatives. The reliability of the entire procedure was assessed using both artificially and naturally infected leaves of a range of plant species. The method, combined with a rapid procedure for DNA extraction, proved to be rapid, reliable, sensitive and cost effective as multiple pathogens were detected within the same plant extract by using different primer/probe combinations.

http://phytophthoradb.org/diagnostics/Schena_2006.pdf

Detection and quantification of Phytophthora ramorum, P. kernoviae, P. citricola and P. quercina in symptomatic leaves by multiplex real-time PCR

chapter eleven – PLANT DISEASES CAUSED BY FUNGI

A specific and sensitive PCR assay was developed for the detection and identification of Rhizoctonia solani AG-3, the main causal pathogen of stem canker and black scurf of potato. A conventional primer set (Rs1F2 and Rs2R1) was designed from the nuclear ribosomal internal transcribed spacer (ITS1 and ITS2) regions of R. solani. Following PCR amplification, a 0·5-kb product was amplified from DNA of all isolates of AG-3 using primers Rs1F2 and Rs2R1. No product was amplified when DNA from isolates belonging to a range of other R. solani anastomosis groups or from a selection of other potato pathogens was tested, confirming the specificity of the primers for AG-3 only. Rhizoctonia solani AG-3 was also detected in potato tissue with varying black scurf severity, and in soil inoculated with sclerotia of R. solani to a minimum detection level of 5 × 10−4 g sclerotia/g soil. In addition, specific primers RsTqF1 (based on the Rs1F2 sequence) and RsTqR1, and a TaqMan™ fluorogenic probe RQP1, were designed to perform real-time quantitative (TaqMan) PCR. The conventional PCR and real-time PCR assays were compared and combined with direct DNA extraction from soil and a seed-baiting method to determine the most reliable method for the detection and quantification of AG-3 in both artificially inoculated field soil and naturally infested soils. It was shown that direct DNA extractions from soil could be problematic, although AG-3 was detectable using this method combined with the real-time PCR assay. The amplification of Rhizoctonia solani by seed baiting increased the sensitivity of the assay compared with direct extraction of DNA from the soil, and AG-3 was detectable in artificially inoculated and naturally infested soils when seed baiting was combined with either the conventional PCR or the real-time PCR assay. The potential for using these rapid and quantitative AG-3-specific assays to address epidemiological questions and as tools for decision-making in disease management is discussed.

M. J. Nicolson

Papers

Development of conventional and quantitative real‐time PCR assays for the detection and identification of Rhizoctonia solani AG‐3 in potato and soil