Genomic Exploration of Produce Degradation 7
Alternatively, PCR methods are developed. Toth et al. (1999a) developed a one-step
PCR-based method for the detection of five subspecies of E. carotovora, including
subsp. carotovora and subsp. atroseptica, and all pathovars/biovars of E. chrysan-
themi on plant tissue culture material.
The 16S rDNA sequence approaches are generally considered to have limited
sensitivity below the species level (Vandamme et al., 1996). However, there are excep-
tions. Single nucleotide polymorphisms (SNPs) in this conserved region provide an
opportunity to explore strain variations because the primers used are species- or
subspecies-specific. Seo et al. (2002) differentiated 87 strains of E. carotovora subsp.
carotovora into two groups by analysis of 16S rDNA restriction fragment length
polymorphism (RFLP) generated by HinfI. They found that most strains from Korea
and Japan belonged to the same group. In the RFLP format, SNPs are located by
restriction enzyme sites. SNPs can be included in PCR primers for strain differen-
tiation. With real-time PCR technology, SNPs can be placed in the fluorescence-
labeled probe.
In contrast, sequence polymorphisms in the 16S–23S rDNA spacer regions are
more commonly recommended to differentiate closely related bacterial strains. Fes-
sehaie et al. (2002) reported that the 16S–23S rDNA spacer regions of Erwinia spp.
varied considerably in size and nucleotide sequence. Phylogenetic analysis showed
a consistent relationship among the Erwinia strains tested and was roughly in
agreement with the 16S rDNA data. Toth et al. (2001) studied the variation of
16S–23S rDNA spacer of soft-rot erwinia. Isolates of E. carotovora subspecies,
E. chrysanthemi, and the closely related E. cacticida yielded unique banding patterns
that clearly distinguished them from other Erwinia and non-Erwinia species tested.
Within the soft rot erwinias, three PCR groups were distinguished based on differ-
ences in their banding patterns. Group I comprised E. carotovora subsp. atroseptica
and subsp. betavasculorum. Group II comprised E. carotovora subsp. carotovora,
subsp. odorifera, and subsp. wasabiae and E. cacticida. Groups I and II were clearly
related based on the banding patterns. Group III comprised all E. chrysanthemi
isolates.
1.2.4 OTHER GENES
Exoenzymes such as pectate lyase, pectin lyase, and polygalaturonase are the primary
virulence factors of soft rot erwinia (Collmer and Keen, 1986; Hugouvieux-Cotte-
Pattat et al., 1996; Py et al., 1998; Chen, 2002). Because of their economic impor-
tance, exoenzyme genes are among the earliest to be sequenced from the soft rot
erwinia genome. Based on these sequences, several PCR primer sets were developed
for pathogen detection (Table 1.1). Darrasse et al. (1994b) designed a primer set,
Y1-Y2, based on a pectate lyase gene (pel) common to some strains of E. carotovora
and Yersinia pseudotuberculosis (Y family). An amplified fragment of 434 bp was
obtained from strains of E. carotovora subspecies except for E. carotovora subsp.
betavasculorum, E. chrysanthemi, and other Erwinia species. Differentiation of
subspecies was achieved through RFLPs of the amplicons. Another primer set, Y45-
Y46 (Frechon et al., 1995), which is also from the same Pe l gene, specifically