Produce Degradation Pathways and Prevention

(Romina) #1

Genomic Exploration of Produce Degradation 11


means. Because of its technical simplicity, RAPD analysis can be used to quickly
compare large numbers of bacterial strains (Chen et al., 1995; Albibi et al., 1998).
DNA bands can be cloned and sequenced. Therefore, the genetic information embed-
ded in the RAPD fragments is determined using information from the genome
sequence database. This is similar to random sample sequencing, but comparison of
DNA bands serves as a prescreening step to efficiently locate the strain-specific
genomic loci.
Toth et al. (1999a) tested 60 strains of Erwinia carotovora subsp. atroseptica
from eight western European countries and found that RAPD analysis was more
sensitive than ERIC and RFLP analyses. RAPD and ERIC analyses are similar in
that the former uses nonspecific primers that bind randomly to regions over the entire
genome and the latter uses primers specific to a repetitive sequence. Because of its
high sensitivity, RAPD analysis can be used to study pathogen host specialization.
Hadas et al. (2001) performed a RAPD analysis using nine arbitrary primers. Cluster
analysis from the 150 distinct DNA characters formed four clusters for the 26
E. carotovora subsp. carotovora isolates from pepper, tomato, potato, and cabbage
across six countries. The clustering pattern is in agreement with the host rather than
geographical origins.
Maki-Valkama and Karjalainen (1994) successfully differentiated E. carotovora
subsp. atroseptica and E. carotovora subsp. carotovora and identified two separate
RAPD clusters along the line of subspecies. Similarity between the 10 E. carotovora
subsp. atroseptica and 10 E. carotovora subsp. carotovora strains was generally
only 10 to 25%. While strain similarity within E. carotovora subsp. atroseptica was
high (> 85%), isolates within the E. carotovora subsp. carotovora group showed
extensive genetic diversity (< 50%).
On the other hand, Parent et al. (1996) explored the use of the RAPD technique
to identify Erwinia carotovora from soft rot-diseased plants. E. caratovora and
pectolytic pseudomonads such as Pseudomonas fluorescens, P. marginalis, and
P. viridiflava are common bacteria associated with soft rot. They found that the
combination of two selected primers was sufficient for adequate distinction of
E. carotovora from pectolytic, fluorescent Pseudomonas species. Furthermore,
E. carotovora subsps. atroseptica and carotovora could also be distinguished from
each other.
While the reproducibility of RAPD has been controversial, its simplicity and
DNA recovering capacity make the technique a good choice to isolate genome-
specific DNA sequences. Maki-Valkama and Karjalainen (1994) selected three
RAPD fragments from the E. carotovora subsps. atroseptica group and used them
as probes for Southern hybridization. All three DNA fragments hybridized only with
E. carotovora subsps. atroseptica isolates, an indication of the sequence specificity
of the three RAPDs. Cloning and sequencing of the unique RAPDs together with
genome sequence comparison can be an effective tool for genetic discovery. This is
particularly suitable for the identification of phages, plasmids, and indels. Interest-
ingly, Toth et al. (1999a) reported that phage typing and RAPD analysis showed a
high level of diversity within Erwinia carotovora subsp. atroseptica compared to
other techniques.

Free download pdf