10 Produce Degradation: Reaction Pathways and their Prevention
analysis of hybridization profiles resulted in phylogenetic dendograms providing
species-level to strain-level resolution. The method does not require laborious cross-
hybridizations and can provide an open database of hybridization profiles.
1.3.2 RANDOM GENOME SAMPLING
Genome sequencing of a large number of bacterial strains is currently cost-prohib-
itive. Alternatively, whole-genome comparisons could be achieved using random, or
presumptively random, sampling approaches. Some of the approaches that have been
applied to study soft rot erwinia include AFLP (amplified fragment length polymor-
phism), RAPD (random amplified polymorphic DNA), RFLP (restriction fragment
length polymorphism), and ERIC (enterobacterial repetitive intergenic consensus).
Mostly, these techniques are PCR-based and they are specifically designed to dif-
ferentiate closely related strains. Because of their simplicity, multiple random sam-
pling approaches are commonly used or are used in combination with other genotypic
and phenotypic techniques (Darrasse et al., 1994b; Toth et al., 1999a; Dellagi et al.,
2000; Hadas et al., 2001; Seo et al., 2002; Yahiaoui-Zaidi et al., 2003).
Traditionally, RFLP analysis involves blotting restriction enzyme-digested DNA
on nylon membrane. DNA polymorphisms are reviewed by hybridization with
labeled probes (Chen et al., 1992). In the report of Toth et al. (1999a), RFLPs of
genomic DNA of 60 strains of E. carotovora subsp. atroseptica from eight western
European countries were evaluated with a DNA probe. Digestion with EcoRI offered
no differentiation between the E. carotovora subsp. atroseptica strains. However,
when EcoRI was replaced by HindIII, hybridization patterns divided the strains into
two groups. Another type of RFLP study is PCR-based: PCR amplicons are digested
with restriction enzymes. DNA polymorphisms are commonly resolved through
agarose gel electrophoresis. When combining with specific PCR such as those using
primers from the rrn operon, RFLP analysis is an effective tool to differentiate
closely related bacterial strains (Darrasse et al., 1994b; Helias et al., 1998; Toth et al.,
1999a; Hadas et al., 2001; Fessehaie et al., 2002; Seo et al., 2002; Yahiaoui-Zaidi et
al., 2003).
Similar to classical RFLP anslysis, AFLP analysis is based on random genome
sampling of restriction sites. The digested DNA fragments are ligated with specially
designed oligomers that can be used as priming sites for PCR amplification. Based
on AFLP profiles, Avrova et al. (2002) identified four clusters of soft rot erwinia.
Cluster 1 contained Erwinia carotovora subsp. carotovora and Erwinia carotovora
subsp. odorifera; cluster 2 contained Erwinia carotovora subsp. atroseptica and
Erwinia carotovora subsp. betavasculorum; and Clusters 3 and 4 contained Erwinia
carotovora subsp. wasabiae and E. chrysanthemi strains. E. carotovora subsp. caro-
tovora and E. chrysanthemi showed a high level of molecular diversity (23 to 38%
mean similarity). Others showed considerably less (56 to 76% mean similarity).
RAPD analysis compared similarities and differences among PCR-generated
genomic DNA fragments flanked by defined 10-base sequences. Fragments amplified
are generally ca. 0.1 to 2 kb. The electrophoretic profiles of DNA fragments based
on size are scored in a binary format and variations are summarized by statistical