Highly specific and sensitive non-radioactive molecular identification of Phytophthora cinnamomi

Abstract

In response to the need for a faster, more reliable method for identifying Phytophthora cinnamomi in cork oak soils in Portugal, a simple, fast, sensitive molecular identification method is described. It is based on a colorimetric assay which involves an oligonucleotide capture probe covalently immobilised on microtitration wells, a multi-biotinylated oligonucleotide detection probe and the PCR-amplified target DNA. The target DNA is a 349 bp DNA fragment partially covering the 3h-translated and 3h-untranslated regions of the cinnamomin gene. When the specificity of the PCR reaction was evaluated in vitro using isolates of P. cinnamomi and eight other Phytophthora species, including the related P. cambivora, it was specific to P. cinnamomi. When 30 isolates of P. cinnamomi from oak roots in southern Portugal were assayed, 26 gave a strong positive response. The assay has a sensitivity of about 2-5 genome equivalents of P. cinnamomi. The reason for the negative response of four isolates remains unclear.

Involvement of the β-cinnamomin elicitin in infection and colonisation of cork oak roots by Phytophthora cinnamomi

“…Both media were sterilized at 121°C for 20 min. The strains were identified by their morphological characters as well as by a colorimetric molecular assay (Coelho et al 1997).…”

Pathways to false positive diagnoses using molecular genetic detection methods; Phytophthora cinnamomi a case study

“…Currently, Phytophthora can be detected using a variety techniques, including direct isolation on Phytophthora selective media (Tsao and Ocana 1969), baiting and isolation onto selective media ), immuno-detection assays (Cahill and Hardham 1994), conventional PCR by guest on January 16, 2017 http://femsle.oxfordjournals.org/ Downloaded from (Coelho et al 1997;Dobrowolski and O'Brien 1993;Judelson and Messenger-Routh 1996;O'Brien et al 2009), nested PCR ), restriction fragment length polymorphism analysis (RFLP) (Martin and Tooley 2004), qPCR (Martin and Tooley 2004;Tooley et al 2006), TaqMan Real Time PCR (Bilodeau et al 2007), and digital PCR (Blaya et al 2014;Sanders et al 2011). Briefly, conventional methods include direct isolation from diseased material, or indirectly by baiting infected plant tissues, water and soil with known host plants and isolation of the pathogen from infected baits (Cooke et al 2007).…”

Touchdown nested multiplex PCR detection of Phytophthora cinnamomi and P. cambivora from French and English chestnut grove soils

“…targeting ribosomal RNA (rRNA) spacer regions which exist in high copy number such as the Internal Transcribed Spacer (ITS) regions (Bonants et al 1997;Tooley et al 1997;Trout et al 1997;Ristaino et al 1998;Schubert et al 1999;Winton & Hansen 2001) and the intergenic-spacer regions (Liew et al 1998), low-to-middle copy sequences ( Ersek et al 1994;Niepold & Sch€ ober-Butin 1995;Schubert et al 1999;Schena et al 2008), as well as, exceptionally high copy sequences (Judelson & Tooley 2000). Although PCR primers have been previously reported for P. cinnamomi based on the ITS 2 region (Lee et al 1993), the cinnamomin gene (Coelho et al 1997), or the Lpv putative storage protein genes (Kong et al 2003), and P. cambivora, through an anonymous RAPD-PCR amplicon (Schubert et al 1999), none have been used for the detection of these species direct from soils with the exception of the recent report of Williams et al (2009) for P. cinnamomi, although the work presented here was completed prior to publication of the Williams et al study.…”