Conventional and real-time PCRs for detection of Erwinia piriflorinigrans allow its distinction from the fire blight pathogen, Erwinia amylovora.

Erwinia 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.

Association of 'Candidatus Liberibacter solanacearum' with a vegetative disorder of celery in Spain and development of a real-time PCR method for its detection.

A new symptomatology was observed in celery (Apium graveolens) in Villena, Spain in 2008. Symptomatology included an abnormal amount of shoots per plant and curled stems. These vegetative disorders were associated with 'Candidatus Liberibacter solanacearum' and not with phytoplasmas. Samples from plant sap were immobilized on membranes based on the spot procedure and tested using a newly developed real-time polymerase chain reaction assay to detect 'Ca. L. solanacearum'. Then, a test kit was developed and validated by intralaboratory assays with an accuracy of 100%. Bacterial-like cells with typical morphology of 'Ca. Liberibacter' were observed using electron microscopy in celery plant tissues. A fifth haplotype of 'Ca. L. solanacearum', named E, was identified in celery and in carrot after analyzing partial sequences of 16S and 50S ribosomal RNA genes. From our results, celery (family Apiaceae) can be listed as a new natural host of this emerging bacterium.

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 5888(T). Depending on the method used, strain CFBP 5888(T) 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 5888(T) 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 5888(T) (=CECT 7348(T)).