Conventional and Real-Time PCRs for Detection of Erwinia piriflorinigrans Allow Its Distinction from the Fire Blight Pathogen, Erwinia amylovora

Abstract

bErwinia piriflorinigrans is a new pathogenic species of the bacterial genus Erwinia that has been described recently in Spain. Accurate detection and identification of E. piriflorinigrans are challenging because its symptoms on pear blossoms are similar to those caused by Erwinia amylovora, the causal agent of fire blight. Moreover, these two species share phenotypic and molecular characteristics. Two specific and sensitive conventional and real-time PCR protocols were developed to identify and detect E. piriflorinigrans and to differentiate it from E. amylovora and other species of this genus. These protocols were based on sequences from plasmid pEPIR37, which is present in all strains of E. piriflorinigrans analyzed. After the stability of the plasmid was demonstrated, the specificities of the protocols were confirmed by the amplification of all E. piriflorinigrans strains tested, whereas 304 closely related pathogenic and nonpathogenic Erwinia strains and microbiota from pear trees were not amplified. In sensitivity assays, 10 3 cells/ml extract were detected in spiked plant material by conventional or real-time PCR, and 10 2 cells/ml were detected in DNA extracted from spiked plant material by real-time PCR. The protocols developed here succeeded in detecting E. piriflorinigrans in 102 out of 564 symptomatic and asymptomatic naturally infected pear samples (flowers, cortex stem tissue, leaves, shoots, and fruitlets), in necrotic Pyracantha sp. blossoms, and in necrotic pear and apple tissues infected with both E. amylovora and E. piriflorinigrans. Therefore, these new tools can be used in epidemiological studies that will enhance our understanding of the life cycle of E. piriflorinigrans in different hosts and plant tissues and its interaction with E. amylovora. Erwinia piriflorinigrans is a Gram-negative plant-pathogenic bacterium causing pear blossom necrosis (1). The disease affects pear production and can reduce fruit yield. It was first observed in Valencia, Spain, in 1999 (2, 3). After the first identification, the disease was observed at the same place in later years (1). Due to its recent discovery, the biology, ecology, and life cycle of this new species are still poorly understood.Unlike Erwinia amylovora, the causal agent of fire blight, E. piriflorinigrans affects blossoms and not other parts of plants (3). Nevertheless, E. piriflorinigrans colonies can be easily mistaken for E. amylovora in culture media, e.g., CCT medium (4), King's medium B (5), and sucrose nutrient agar (SNA) or Levan medium, which are usually utilized and recommended for the isolation of E. amylovora (6, 7). These two pathogens show similar metabolic profiles by use of the commercial API 20E, API 20NE, API ZYM, and API 50CH strips (bioMérieux, France), as well as close fatty acid profiles. E. piriflorinigrans can react with several antisera, and even with one of the monoclonal antibodies, used to detect E. amylovora (3). Moreover, E. piriflorinigrans reacts positively in PCR using the 23S rRNA gene sequence-based primers de...

Development of a real-time PCR method for the specific detection of the novel pear pathogen Erwinia uzenensis

“…piriflorinigrans by Barbé et al . [3], using 10 6 cfu ml -1 bacterial suspensions. Additionally, sensitivity and specificity assays of the developed primers and probe were performed in bacterial mixtures consisting of E .…”

“…Recent records of E . piriflorinigrans in new hosts in Spain [3] or in new areas in Switzerland (Smits, T., personal communication), as well as the discovery of E . pyrifoliae in strawberry plants in The Netherlands [4] suggest that the likelihood of spread of E .…”

“…amylovora and E . piriflorinigrans [3], should be taken into account when examining disease outbreaks.…”

PCR-based methods for detection of Erwinia psidii on guava

“…Bacterial detection using serology with a specific polyclonal antibody has been described as well (Teixeira et al 2008); however, due to its low sensitivity, an enrichment step in plant extracts was necessary to detect bacterial cells in small numbers. PCR, either conventional or real time, has been broadly employed for successful detection and diversity studies of plant pathogenic bacteria, including Pantoea stewartii (Wensing et al 2010), Erwinia amylovora (Powney et al 2011;Dreo et al 2012;Gehring and Geider 2012;Wensing et al 2012;Hannou et al 2013) and E. piriflorinigrans (Barbé et al 2014). Several genomic regions, including PCR fragments obtained from rep-PCR profiles (Rico et al 2008), plasmidial sequences (Bereswill et al 1992;Barbé et al 2014) and draft whole genome sequences (Pritchard et al 2013), have been used for the development of specific identification tools for plant pathogenic enterobacteria.…”

“…PCR, either conventional or real time, has been broadly employed for successful detection and diversity studies of plant pathogenic bacteria, including Pantoea stewartii (Wensing et al 2010), Erwinia amylovora (Powney et al 2011;Dreo et al 2012;Gehring and Geider 2012;Wensing et al 2012;Hannou et al 2013) and E. piriflorinigrans (Barbé et al 2014). Several genomic regions, including PCR fragments obtained from rep-PCR profiles (Rico et al 2008), plasmidial sequences (Bereswill et al 1992;Barbé et al 2014) and draft whole genome sequences (Pritchard et al 2013), have been used for the development of specific identification tools for plant pathogenic enterobacteria. The recombinase A (recA) gene is a valuable marker for the major bacterial groups (Eisen, 1995) and for differentiating species in Erwinia and related genera (Waleron et al 2002;Young and Park 2007;Parkinson et al 2009;Wensing et al 2010).…”