PLANT DISEASE DIAGNOSIS - The Epidemiology of Plant Diseases - page 20

leaving behind the specific sequences to be recovered (Avery et al., 1980; Smith
et al., 1987; Cook and Sequeira, 1991; Seal et al., 1992). Genomic subtraction has
been used to detect the gene for the distinctive toxin, phaseolotoxin, and hence
specifically diagnose the bacterium Pseudomonas syringae pv. phaseolicola itself
(Schaad et al., 1989).
Often the genetic information responsible for the characteristic pathogenicity of
a bacterial pathovar resides on its indigenous extrachromosomal plasmids, which are
stable enough to be used as natural genetic markers (Lazo and Gabriel, 1987).
Probes may also be developed from chemically synthesized oligonucleotides.
Usually, these are less than 40 nucleotide sequences and can be sufficiently specific
to detect point mutations and single base-pair changes (Wallace et al., 1979) (Fig.
1.1). Such oligonucleotide probes can easily be synthesized cheaply and labelled
non-radioactively - but only when the relevant genetic code has been characterized.
They have been constructed for use as primers for amplification by the polymerase
chain reaction (e.g. Martin et al., 1992b).
The polymerase chain reaction (PCR) is an in vitro method in which a DNA
fragment with known end sequences can be amplified exponentially into billions of
copies, making detection very much easier (Saiki et al., 1985; Mullis and Faloona,
1987). Since an initially very low number of DNA molecules can be multiplied
enormously, PCR has now been widely used in plant pathology as well as numerous
other areas, including criminal forensic medicine. Only a few nanograms of the
initial template DNA is necessary, either in the form of a discrete molecule or as part
of a larger one. Both dsDNA and ssDNA can be amplified by PCR. It is possible to
amplify RNA by reverse transcription into a cDNA copy by RT-PCR (Sambrook
et al., 1989). Synthetic oligonucleotides (primers) can be produced that are comple-
mentary to the end sequences and can hybridize with them. The three stages in
amplification are not complicated. First, the target DNA is melted, then the two
oligonucleotide primers are annealed to the denatured DNA strands and finally the
primer is extended, using the thermostable enzyme Taq polymerase. Since the PCR
process does not depend on the use of purified DNA, the pathogen does not have to
be cultured before its DNA is amplified but any substances that could inhibit the
PCR are easily removed by purification (Steffan and Atlas, 1991). As the choice of
primers determines whether the selectivity of the PCR is either specific or broad,
this flexibility has proved especially useful in disease detection (Henson and French,
1993). The major practical problem experienced with PCR is that contamination
from extraneous sources can cause false positive results, so both positive and nega-
tive controls are essential. A 'hot start' is also often adopted in which the reagents
are heated to avoid premature misreactions (Chou et al., 1992). By introducing a
second round of amplification, nested primers can be used to allow a set of outside
primers to amplify a DNA fragment (Henson et al., 1993), but this is an expensive
option. In addition to PCR, the ligase chain reaction is another technique that is
sensitive enough to detect single base-pair differences (Wiedmann et al., 1992).
Inverse PCR is used to analyse unknown sequences that flank a region that is
known (Triglia et al., 1988). In competitive PCR, the target DNA is co-amplified in
the presence of known quantities of a competitive DNA (Gilliland et al . , 1990a,b),