Differentiation of Erwinia amylovora strains from Bulgaria by PCR-RFLP analysis

Abstract

Fifty strains of Erwinia amylovora isolated in Bulgaria from different host plants and locations as well as in different years were analysed by RFLP analysis of the pEA29 PstI amplified fragment with HpaII. All the strains formed three well-resolved fragments (large-from 365 to 440 bp, mediumabout 341 bp and small-about 180 bp).The strains were classified into three RFLP groups based on the polymorphism in the length of the largest fragment. This fragment was of intermediate size for 63% of the strains, and it was the longest (from 410 to 440 bp) for 29% of the strains. The variable region was sequenced for five strains. The DNA sequence analysis confirmed the different size of the largest fragment. Ten or more than ten SSRs were found for the strains in the group with the largest size of the largest fragment. Some correlation between the RFLP profiles and the origin of the strains was revealed. The RFLP profiles displayed stability in certain strains isolated from the same trees and orchards, but in different years. The number of SSRs was different in strains isolated from one and the same host plant, orchard and year, and also in strains isolated from the same host plant and orchard, but in different years. This could indicate that under natural conditions the fire blight symptoms might be caused by a mixture of E. amylovora strains with different SSR numbers, and so coexistence of distinguishable strains or a change in the population could be assumed.

Characterization of Erwinia amylovora Strains from Bulgaria by Pulsed-Field Gel Electrophoresis

“…This profi le is characterized by the shift of one band from 400 kbp to about 414 kbp, the appearance of one new 348-kbp band, and the lack of two bands (146 kbp and 299 kbp). All these strains showed the same RFLP profi le of the pEA29 plasmid PstI-amplifi ed fragment digested with HpaII (Atanasova et al, 2009). Zhang and Geider (1997) described a PFGE profi le of a strawberry E. amylovora strain identical to the Maloideae strains and different from the profi le established by us, but emphasized that the strain was isolated from plantations near apple orchards.…”

“…The strains of this major group originated from different locations, host plants, and orchards and were isolated in different years, which indicates genomic stability, as well as homogeneity of the Bulgarian population of E. amylovora. Additionally, the strains of this group possessed different RFLP profi les of the pEA29 plasmid PstI-amplifi ed fragment digested with HpaII and a different number of SSR (short sequence repeats) -8, 10, 11, 12, and 13 repeats (Atanasova et al, 2009). Halupecki et al (2006) found that the type Pt2 was characteristic for the strains of E. amylovora isolated in Croatia.…”

“…The recent historical distribution of the pathogen may also be a reason for this homogeneity in the genomes of individual strains (Zhang and Geider, 1997). However, several molecular techniques such as polymerase chain reaction (PCR)-ribotyping (McManus and Jones, 1995;Jeng et al, 1999), random amplifi ed polymorphic DNA (RAPD) (Momol et al, 1995), restriction fragment length polymorphism (RFLP) (Lecomte et al, 1997;Kim and Geider, 1999;Jock et al, 2003;Ruppitsch et al, 2004;Barionovi et al, 2006;Atanasova et al, 2009), amplifi ed fragment length polymorphism (AFLP) (Rico et al, 2004;Donat et al, 2007), and pulsed-fi eld gel electrophoresis (PFGE) (Zhang and Geider, 1997;Zhang et al, 1998;Jock et al, 2002a;Jock and Geider, 2004;Halupecki et al, 2006;Donat et al, 2007) have proven useful in the determination of intraspecifi c diversity within strains from different geographical origins and hosts. Some of these approaches like RAPD and PCR-ribotyping allowed differentiation between Maloideae and Rosoideae strains (Momol et al, 1995;McManus and Jones, 1995;McGhee et al, 2002).…”