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

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

Erwinia piriflorinigrans sp. nov., a novel pathogen that causes necrosis of pear blossoms

Eight Erwinia strains, isolated from necrotic pear blossoms in València, Spain, were compared with reference strains of Erwinia amylovora and Erwinia pyrifoliae, both of which are pathogenic to species of pear tree, and to other species of the family Enterobacteriaceae using a polyphasic approach. Phenotypic analyses clustered the novel isolates into one phenon, distinct from other species of the genus Erwinia, showing that the novel isolates constituted a homogeneous phenotypic group. Rep-PCR profiles, PCR products obtained with different pairs of primers and plasmid contents determined by restriction analysis showed differences between the novel strains and reference strains of E. amylovora and E. pyrifoliae. Phylogenetic analysis of 16S rRNA, gpd and recA gene sequences showed that the eight novel strains could not be assigned to any recognized species. On the basis of DNA–DNA hybridization studies, the novel isolates constituted a single group with relatedness values of 87–100 % to the designated type strain of the group, CFBP 5888T. Depending on the method used, strain CFBP 5888T showed DNA–DNA relatedness values of between 22.7 and 50 % to strains of the closely related species E. amylovora and E. tasmaniensis. The DNA G+C contents of two of the novel strains, CFBP 5888T and CFBP 5883, were 51.1 and 50.5 mol%, respectively. On the basis of these and previous results, the novel isolates represent a novel species of the genus Erwinia, for which the name Erwinia piriflorinigrans sp. nov. is proposed. The type strain is CFBP 5888T (=CECT 7348T).