Characterization of Erwinia amylovora strains from different host plants using repetitive-sequences PCR analysis, and restriction fragment length polymorphism and short-sequence DNA repeats of plasmid pEA29

Abstract

Aims: The three main aims of the study were the assessment of the genetic relationship between a deviating Erwinia amylovora strain isolated from Amelanchier sp. (Maloideae) grown in Canada and other strains from Maloideae and Rosoideae, the investigation of the variability of the PstI fragment of the pEA29 plasmid using restriction fragment length polymorphism (RFLP) analysis and the determination of the number of short‐sequence DNA repeats (SSR) by DNA sequence analysis in representative strains. Methods and Results: Ninety‐three strains obtained from 12 plant genera and different geographical locations were examined by repetitive‐sequences PCR using Enterobacterial Repetitive Intergenic Consensus, BOX and Repetitive Extragenic Palindromic primer sets. Upon the unweighted pair group method with arithmetic mean analysis, a deviating strain from Amelanchier sp. was analysed using amplified ribosomal DNA restriction analysis (ARDRA) analysis and the sequencing of the 16S rDNA gene. This strain showed 99% similarity to other E. amylovora strains in the 16S gene and the same banding pattern with ARDRA. The RFLP analysis of pEA29 plasmid using MspI and Sau3A restriction enzymes showed a higher variability than that previously observed and no clear‐cut grouping of the strains was possible. The number of SSR units reiterated two to 12 times. The strains obtained from pear orchards showing for the first time symptoms of fire blight had a low number of SSR units. Conclusions: The strains from Maloideae exhibit a wider genetic variability than previously thought. The RFLP analysis of a fragment of the pEA29 plasmid would not seem a reliable method for typing E. amylovora strains. A low number of SSR units was observed with first epidemics of fire blight. Significance and Impact of the Study: The current detection techniques are mainly based on the genetic similarities observed within the strains from the cultivated tree‐fruit crops. For a more reliable detection of the fire blight pathogen also in wild and ornamentals Rosaceous plants the genetic features of deviating E. amylovora strains have to be studied in detail.

Diversity, Evolution, and Functionality of Clustered Regularly Interspaced Short Palindromic Repeat (CRISPR) Regions in the Fire Blight Pathogen Erwinia amylovora

“…Long-distance dispersal occurs principally via infected plant host material, limiting the number of invasion events into new regions. E. amylovora, even strains from the area of origin in North America (42), has been considered a genetically low-diversity species based on previous molecular approaches, including pulsed-field gel electrophoresis (PFGE) (27,28), PCR fingerprinting based on PCR ribotyping (11,37), rep-PCR (1,46), amplified fragment length polymorphism (AFLP) analysis (47), random amplified polymorphic DNA fragment (RAPD) analysis, and amplified rRNA gene restriction analysis (ARDRA) (42). While strain patterns derived using these methods distinguish strains from Rubus (subfamily Rosoideae) and other Spiraeoideae plants (1,37) and indicate a North American origin based on the slightly greater diversity of strains from this region (47), they provide poor discrimination of strains within local infested regions, which is required for effective implementation of phytosanitary control measures.…”

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

“…Nevertheless, strains from Europe and the Mediterranean region can be distinguished by their PFGE pattern after XbaI restriction [18]. Further distinction was also possible by SSR analysis [3,21]. The usefulness of these and similar approaches, such as CRISPR sequences, may be limited by frequent changes due to random recombination within these DNA elements of the genome.…”

Molecular typing of bacteria for epidemiological surveillance and outbreak investigation / Molekulare Typisierung von Bakterien für die epidemiologische Überwachung und Ausbruchsabklärung

“…The final result is the number of repeat units that is calculated from the fragment size. In our laboratory, SSR analysis was used for typing of E. amylovora the etiologic agent of fire blight Barionovi et al, 2006). SSR typing data revealed that the spreading of fire blight from the western provinces to the east of Austria and onward to Hungary was only an apparently continuous event.…”