PLANT DISEASE DIAGNOSIS - The Epidemiology of Plant Diseases - page 16

used as vaccines (Robard, 1987). A monoclonal antibody raised to one synthetic
peptide by Martin et al. (1992a) is able to determine whether tubulin in crude
preparations from fungi is from strains resistant or susceptible to the fungicide
carbendazim, based on only one different amino acid.
It may no longer be essential to inject laboratory animals with antigens each time
a new antibody is required. Splenocytes from non-immunized mice can be stimu-
lated to produce antibodies in vitro. It is also possible to substitute complete anti-
bodies by fragments of antibodies (Fab or F(ab') 2 fragments) comprising only the
heavy and light chain from one arm of a Y-shaped antibody molecule. The genes for
the light and the heavy chains may be extracted from antibody-producing cells and
multiplied by the polymerase chain reaction (PCR) to copy them many times over.
These are stored in bacterial viruses to be expressed in bacteria. As a result, many
more antibodies can be grown much more cheaply, quickly and easily in bacterial
cultures and screened for specificity than if mouse hybridomas were used. If the
antibodies were grown in crop plants in a similar way, antibodies could be produced
at 0.0001 of the cost, which should allow antibodies to be mass-produced cheaply
enough to be used very widely in plant disease epidemiology, diagnosis and control
as well as other far-reaching applications.
Serological tests are restricted by the relative accessibility of the epitopes on the
proteins and polysaccharides. However, although many are inaccessible, there are
sufficient epitopes for the development of a wide range of immunological diagnostic
tests. Others can be created by hapten technology. Since many of these tests are
considered satisfactory, they are currently more widely used for routine practical
diagnosis than methods based on recognizing the similarities between nucleic acids.
According to Robinson (1988), these established immunological tests are not
going to be readily supplanted.
1.5 METHODS BASED ON THE NUCLEIC ACIDS OF PATHOGENS
Many diagnostic methods based on the detection of similarities between nucleic
acids have been successfully adapted for identification of pathogens. Most of these
techniques depend on the use of restriction enzymes to cleave DNA into fragments
at or near a defined recognition sequence. This allows the enzymes to be used in the
differentiation of non-clonal organisms, as each inherits a unique distribution of
restriction sites.
In DNA restriction fragment polymorphisms (RFLPs ), a combination of
hybridization, Southern blot and restriction mapping forms the basis for the genetic
analysis and characterization of pathogens (Botstein et al., 1980). After the DNA
has been digested into fragments, these are separated by agarose gel electrophoresis
and then stained with ethidium bromide, which can be seen under UV light. The
separated bands that result represent a genomic 'fingerprint' which can be analysed
in a way that is somewhat analagous to 'reading' a 'bar chart code'. Although their
chief advantage over antibodies is that they can detect any part of the genome of
the pathogen, however large, nucleic acid techniques are more common for viral