Characterisation of naturally occurring Erwinia amylovora strains lacking the common plasmid pEA29 and their detection with real-time PCR

Abstract

Erwinia amylovora, the causal agent of fire blight, carries the common plasmid pEA29 of 29 kb. To screen for occurrence of natural strains without plasmid pEA29, we applied PCR analysis with primers from the plasmid and the chromosomal ams region. In addition, a described TaqMan probe from pEA29 and newly designed primers from the amsregion were used for identification by real-time PCR. One strain isolated in Iran, one strain from Spain and two strains from Egypt lacked plasmid pEA29. From a recent screening series in southern Germany, in 123 E. amylovora strains from necrotic fire blight host plants, one strain was found without the common plasmid. The strains without pEA29 were virulent in assays with immature pears and on apple seedlings, but showed a reduced growth level in minimal medium without amino acids and thiamine. Transposon-labelled pEA29 was transformed into the plasmid-free strains resulting in restoration of this growth deficiency. The plasmid was stably maintained in these E. amylovora cells. The newly designed chromosomal primers for conventional and for realtime PCR identified E. amylovora strains in field samples lacking pEA29. These variants are apparently rare, but were detected in isolates from different regions in the world with fire blight.

Identification of the Corn Pathogen Pantoea stewartii by Mass Spectrometry of Whole-Cell Extracts and Its Detection with Novel PCR Primers

“…The exact sizes of the products can be calculated from the primer names in the second column of Table 2. The assays were done as described before (12). For qPCR with SYBR green, hot start Taq polymerase (Amplicon) was used.…”

Phenotypic and genetic diversity of Erwinia amylovora: the causal agent of fire blight

“…Such a reaction may indicate an earlier acquisition of pEA29 from E. amylovora. By contrast, a few isolates of E. amylovora without this plasmid were found in Egypt, Iran, Spain and the USA (Carey et al 2011;Llop et al 2006;Mohammadi et al 2009). Also, it was found that another plasmid of approximately 65 kb (pEI70) present in one of these isolates, showed no homology with pEA29.…”

Differentiation of Erwinia amylovora and Erwinia pyrifoliae Strains with Single Nucleotide Polymorphisms and by Synthesis of Dihydrophenylalanine

“…From 1 ml overnight cultures in LB medium was taken, of which 10 ll of culture was lysed in 1 ml 0.1 % Tween 80 by heating for 20 min at 65°C, and 4 ll of these lysates were used as template in PCR. The master mix for the PCR assays has been described recently [28].…”