Molecular characterization of Erwinia amylovora strains from different host plants through RFLP analysis and sequencing of hrpN and dspA/E genes

A total of 73 Erwinia amylovora strains obtained from 13 Maloideae host species and from Rubus spp., and isolated from different geographic areas, were assessed using RFLP and DNA sequencing analysis of the 3'hrpN gene and/or of a fragment of 1341 bp of the dspA/E region. An Erwinia pyrifoliae strain, used as outgroup, was checked in the same way. For the three strains isolated from Rubus spp. and for one strain from Amelanchier sp., RFLP analysis of the hrpN gene using the RsaI enzyme yielded a PCR product 60 bp smaller than that of all the other strains. Sequence analysis of the gene revealed this was due to the absence of a 60 bp fragment in the noncoding region downstream of the gene. The strain PD 2915, isolated from Amelanchier sp. grown in Canada, showed five same-sense substitutions and one missense substitution at position 868 of the hrpN gene, converting aspartic acid into asparagine. Also, restriction analysis of a fragment of 613 bp of the dspA/E region with CfoI revealed an RFLP pattern suitable for differentiating the E. amylovora strains isolated from Rubus spp. and Amelanchier sp. from all the others. In the dspA/E coding region, the four strains showed 13-14 missense point mutations, in some cases yielding drastic amino acid substitutions. In addition, partial sequencing of the dspA/E region of PD 2915 from Amelanchier sp. indicated a higher similarity to E. amylovora strains isolated from Rubus spp. than towards strains from other Maloideae hosts. The E. pyrifoliae strain showed 23 single nucleotide substitutions along the hrpN gene and 88% of nucleotide identity with E. amylovora strains in the portion of dspA/E region. Artificial inoculations on immature pear fruits and young shoots of Maloideae and Ruboideae showed a restricted pathogenicity for the strains from Rubus and Amelanchier, with the latter inciting blight symptoms only on Amelanchier.