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Erwinia piriflorinigrans is a new pathogenic species of the bacterial genusErwinia that has been described recently in Spain. Accurate detection and identification ofE. piriflorinigrans are challenging because its symptoms on pear blossoms are similar to those caused byErwinia 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 detectE. piriflorinigrans and to differentiate it fromE. amylovora and other species of this genus. These protocols were based on sequences from plasmid pEPIR37, which is present in all strains ofE. piriflorinigrans analyzed. After the stability of the plasmid was demonstrated, the specificities of the protocols were confirmed by the amplification of allE. piriflorinigrans strains tested, whereas 304 closely related pathogenic and nonpathogenicErwinia strains and microbiota from pear trees were not amplified. In sensitivity assays, 103 cells/ml extract were detected in spiked plant material by conventional or real-time PCR, and 102 cells/ml were detected in DNA extracted from spiked plant material by real-time PCR. The protocols developed here succeeded in detectingE. piriflorinigrans in 102 out of 564 symptomatic and asymptomatic naturally infected pear samples (flowers, cortex stem tissue, leaves, shoots, and fruitlets), in necroticPyracantha sp. blossoms, and in necrotic pear and apple tissues infected with bothE. amylovora andE. piriflorinigrans . Therefore, these new tools can be used in epidemiological studies that will enhance our understanding of the life cycle ofE. piriflorinigrans in different hosts and plant tissues and its interaction withE. amylovora .

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