Erwinia amylovora: Modern Methods for Detection and Differentiation

Abstract

Erwinia amylovora is the causative agent of fire blight, a very destructive disease of numerous members of the rosaceae. The primary route of infection for host species, including commercially grown apple and pear, is the newly opened blossom. Susceptibility of flowers to infection for only a few days creates narrow window for infection. Not surprisingly, the risk of disease is related to E. amylovora population size. As a result, methods that supply quick, accurate and sensitive quantification of the pathogen population are important tools for determining the need for and the efficacy of disease control intervention. Plating samples and assessing colony-forming units constitutes an accurate and sensitive but slow method. Endpoint PCR is quick and sensitive but is not particularly amenable to quantification. We describe a real-time PCR procedure that provides all requirements. This method is based on chromosomal genes rather than on the pEa29 plasmid and so can be used to measure isolates that have been cured of the plasmid. The method has been used very successfully in directly quantify whole E. amylovora cells, in a variety of tissues from the orchard environment.

Real-time PCR, a method fit for detection and quantification of Erwinia amylovora

“…Analyzing pure bacterial cultures or ooze, colony PCR is a quick and valuable option as most DNA polymerases in use today require an incubation step at 95°C for 5-15 min for enzymatic activation during which bacterial cells added directly to the reaction are disrupted and release DNA. Such approach has been used for analysis of pure cultures (Svircev et al 2009) and is indeed a practical approach for identification of E. amylovora isolates. In a study conducted on pure bacterial cultures, Mohammadi et al (2009), as well as previously Salm and Geider (2004), have used Tween 20 detergent (0.1-1%, with or without heating) to disrupt cells prior to real-time PCR reaction.…”

“…8.6 9 10 3 cells from leaf sample and 1.8 9 10 4 cells from bark sample (Salm and Geider 2004), 3.2 9 10 4 CFU/mL (De Bellis et al 2007), 20 CFU/reaction (Lehman et al 2008), \500 CFU/reaction (Mohammadi et al 2009) and 100 CFUs/reaction (Svircev et al 2009). Taking into account the way the samples are prepared and analyzed these values generally correspond to 10 3 E. amylovora cells/mL of starting plant extract.…”

“…Lehman et al (2008) reported using a simple Direct Plant Extraction Buffer (DiPEB) for DNA extraction from artificially inoculated pear blossoms from which petals and peduncles were removed since it is known that they oxidize quickly and can be a source of potent PCR inhibitors. The same buffer was used by Svircev et al (2009) to isolate E. amylovora from bark, stems, leaves, blossoms, anthers and pollen. However, little data is available on its performance in these different samples.…”

“…Despite successfully increasing sensitivity, the transfer of reaction products from nested to the real-time PCR step increases the possibility of contaminations, and thus this method has not been widely accepted. All other assays were developed with TaqMan probes employing technological advances such as availability of more efficient quencher molecules (Mohammadi et al 2009;Svircev et al 2009) that increase the signal to noise ratio (Marras et al 2002).…”

“…Since its publication, new methods became available or were further developed. In addition to many PCR methods, several real-time PCR assays are now available (Salm and Geider 2004;De Bellis et al 2007;Lehman et al 2008;Mohammadi et al 2009;Pirc et al 2009;Svircev et al 2009), previously reviewed by Dreo (2009). In this review, we complete the data on published real-time PCR assay (including Gottsberger 2010), summarize the use and potential of real-time PCR methods in the detection of fire blight and describe transfer and optimization of a previously developed Ams assay (Pirc et al 2009) onto a faster SmartCycler Ò real-time PCR detection system.…”