Instability of short-sequence DNA repeats of pear pathogenic Erwinia strains from Japan and Erwinia amylovora fruit tree and raspberry strains

Abstract

An array of short-sequence DNA repeats (SSRs) occurs in the plasmid pEA29 of the fire blight pathogen Erwinia amylovora. A large number of ''fruit tree'' strains, mainly from Central and Western Europe, were screened for their SSR numbers, and the analyses were extended to five raspberry strains from North America and six pear pathogenic Erwinia strains from Japan. The repeat ATTACAGA present in all E. amylovora strains was found to be reiterated 3 to 15 times. The Japanese strains contained the major repeat sequence GGATTCTG, which was reiterated 16 to 24 times. ATTACAGG, which resembles the SSR of E. amylovora, was reiterated two or three times. In a novel approach, sequencing gels were used to visualize the rare occurrence of shorter arrays (down to three repeats) in E. amylovora and the Japanese Erwinia strains. Changes in the repeat numbers in E. amylovora were observed repeatedly when the bacteria had been exposed to stress conditions. The repeat structures of homo-and heteroduplices of PCR-amplified repeats were also analyzed by cleavage of annealed molecules with the single-strand-specific endonuclease from bacteriophage T4. Not only heteroduplexes, but also homoduplexes showed non-matching regions in the SSRs, which could arise from transient formation of loops due to strand slippage during the assays.

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

“…However, our results support the assumption that most Italian E. amylovora strains isolated in the Po valley originated from infected plant material from Belgium (Zhang et al 1998;Jock et al 2002). In fact, nearly all Italian strains used in this study had SSR numbers of four to six SSR units similar to strains from Belgium (Jock et al 2003). Therefore, strain UniBaBA6 with eight SSR units and isolated from Apulia region in southern Italy, might have another origin.…”

“…UK (Jock et al 2003). No other isolates were found in Europe with this specific number of SSR units.…”

“…The fragment length variability is due to the presence of different numbers of shortsequence DNA repeats (SSR) (Schnabel and Jones 1998). The number of SSR units has been recently used to try to type E. amylovora strains (Kim and Geider 1999;Jock et al 2003;Ruppitsch et al 2004), and, in case of a very low or very high number of SSR repeats the traceability of certain strains was possible (Ruppitsch et al 2004).…”

Stability of short sequence repeats and their application for the characterization of Erwinia amylovora strains

“…A disadvantage of this technique is that SSRs were described as unstable entities that undergo frequent variation in length because of slipped strand mispairing during DNA synthesis [7,8,11]. However, our results showed that SSR numbers of several strains remained constant under laboratory conditions.…”

“…possible. However, without this additional knowledge traceability is very difficult and probably impossible for strains with common SSR numbers of 5 or 6 [11]. For strains in culture collections the differentiation into PFGE-type (Pt1 to Pt6) and SSR-type (SSR-3 to SSR-15) represents a valuable tool for strain classification.…”