Development of protocols for detection of Colletotrichum acutatum and monitoring of strawberry anthracnose using real‐time PCR

Abstract

Real-time PCR (TaqMan®) assays were developed for the specific detection and discrimination of Colletotrichum spp., C. acutatum and C. gloeosporioides causing anthracnose in strawberry using the most divergent area of the internal transcribed spacers (ITS1 and ITS2) and 5·8S ribosomal RNA (rRNA) gene region. The specificity of the new assays was tested using DNA from six species of Colletotrichum and nine fungal species commonly found associated with strawberry material, and additionally by comparing the sequences with those from databases using a blast search. The sequences only showed identity with homologous sequences from the desired target organisms. The new assays were 10-100 times more sensitive than conventional PCR methods previously published for the diagnosis of strawberry anthracnose. When real-time PCR was compared with ELISA methods, PCR improved the sensitivity of the identification by obtaining positive results for samples of strawberry plant material that tested negative with ELISA. The development of C. acutatum was monitored using artificially infected strawberry crowns from two strawberry cultivars (Camarosa and Ventana) and a real-time PCR assay specific for this species between January and June 2006. The amount of C. acutatum detected using real-time PCR varied significantly by month ( P < 0·001), but not by cultivar ( P = 0·394). The new assays were shown to be useful tools for rapid detection and identification of these pathogens and to allow rapid and accurate assessment of the casual agents of anthracnose in strawberry.

Phylogenetic relationships and genome organisation of Colletotrichum acutatum causing anthracnose in strawberry

“…have been focused on just one or two aspects of the disease: traditionally, with cultural or morphological studies (Afanador-Kafuri et al 2003;Denoyes-Rothan and Baudry 1995;Smith and Black 1990), and nowadays, using molecular techniques including isoenzyme comparisons, Restriction Fragment Length Polymorphisms (RFLP) analyses of mitochondrial DNA, Amplified Fragment Length Polymorphism (AFLP), AT rich analyses, Random Amplified Polymorphic DNA (RAPD), and ITS sequence analyses for specific PCR identification (Buddie et al 1999;Garrido et al 2007Garrido et al , 2009Freeman et al 1993;Sreenivasaprasad et al 1996;Talhinhas et al 2005). These techniques have improved the accuracy and reduced the time necessary for the identification and classification of Colletotrichum spp.…”

Real-time PCR applied to study on plant pathogens: potential applications in diagnosis - a review

“…electrophoresis, colorimetric reaction or hybridisation). Secondly, real-time PCR commonly amplifies the short DNA fragments (70-100 bp), which favours a higher level of efficiency and sensitivity (Garrido et al 2009). Generally, the main advantage of real-time PCR over cPCR is the increased sensitivity and the ability to perform quantitative measurements, making it suitable for studying pathogen biology, epidemiology, and ecology.…”

“…The inhibitory substances can be removed using different columns and resins such as gel filtration (also known as size exclusion) resins, agarose gel electrophoresis, template dilution (Miller 2001), and commercial kits. Several practical DNA extraction methods such as isopropanol, silica-columns, magnetic beads, lyophilisation, freeze-grind and heat treatment have been used for extraction of high-quality DNA from microorganisms such as fungi and bacteria in order to minimise the influence of the extraction in the quantification of a low copy number of target (Cullen & Hirsch 1998;Reeleder et al 2003;Ippolito et al 2004;Weller et al 2007;Garrido et al 2009;Williams et al 2009;Bilodeau et al 2012). Also, to extract DNA from soil samples, a variety of extraction kits are available.…”

“…As the first available real-time chemistry, SYBR Green I can be used in a wide range of plant pathogens, whereas the TaqMan chemistry has an inherently higher degree of specificity, reliability, and performance, as discussed in the previous sections. The specific hybridization between probe and target DNA that is required to generate a fluorescent signal is the main advantage of fluorogenic probes over DNA binding dyes (Okubara et al 2005;Garrido et al 2009). A search of relevant published articles indicates that the relative frequency of chemistries applied in realtime PCR technique to date is, in increasing order: Molecular beacons < Scorpion < SYBR Green I < TaqMan (Schena et al 2013).…”

“…Quantitative real-time PCR (qPCR) technology allows accurate detection and/or quantification of pathogens that cannot be extracted or cultured easily from host tissue, or are presented at low inoculum load in samples. In fact, quantification based on culturing techniques is considered relatively inaccurate, while quantification using real-time PCR provides a reliable estimation of the pathogen load (Garrido et al 2009). Also, real-time PCR technology provides conclusive results as it can discriminate between closely related organisms and is therefore a versatile method for the accurate, reliable, and high throughput quantification of target DNA in various biological fields such as botany and genetics (Cooke et al 2007;Schena et al 2013).…”