Agriculture Reference
In-Depth Information
1992b). Hybridization is invaluable as a diagnostic tool since a selective probe may
detect less of the pathogen's nucleic acid than is present in the inoculum necessary
to infect the host. Universally conserved nucleic acid molecules such as ribosomal
RNA (rRNA) may be adopted because of their wide recognition spectra.
The potential of nucleic acid hybridization to determine the genetic and hence
the taxonomic relatedness of organisms was recognized by Britten and Kohne (1968).
The conditions that favour the reassociation of single-stranded DNA (ssDNA) include
an adequate concentration of cations, a sufficiently high temperature to weaken the
intrastrand secondary structure, sufficiently long incubation time and high con-
centration of DNA to permit an adequate number of collisions and hence the
opportunities to reassociate. The size of the fragments is also important as DNA
sheared into small fragments favours reassociation. If suitable fragments are used,
hybridization is now regarded as the conventional method for determining
subgeneric taxonomic relationships to the species level in bacteria (Schleifer and
Stackerbrandt, 1983).
Hybridization now generally takes place in a solid rather than liquid phase. The
target nucleic acid from the pathogen to be detected is immobilized on a nitrocellu-
lose or nylon membrane. DNA is first cut into fragments by a restriction enzyme,
often following gel electrophoresis and denaturation (Southern, 1975). RNA is
usually transferred by a similar technique known as Northern blotting (Alwine
et
al.,
1977). After it has been blotted onto the support, the target nucleic acid is fixed by
baking or cross-linking under UV light. The labelled probe hybridizes to the target
DNA. After the excess unbound probe has been removed by washing, the hybrid
(target:probe) is detected by a suitable assay.
Most nucleic acid probes for detecting plant pathogens, particularly viruses, are
usually used in routine spot hybridization or 'dot-blot' tests. In many dot-blot diag-
noses, the DNA is neither cut by restriction enzymes nor fractioned but merely
applied as a small drop of sap extracted from the infected plant directly onto a
nitrocellulose sheet, dried, then hybridized with the probe in a sealed plastic bag
using a buffer extract (Maule
et al.,
1983). The nucleic acid is loaded into a
multisample vacuum manifold which is used to spot it onto the membrane. This
boosts sensitivity by increasing the volume of sample that is able to pass through the
area of membrane under the 'dot' until the pores of the membrane become clogged
with material from the plant sap (Robinson, 1988). However, this dot-blot method
may be modified further by lysing cells of the target microorganism
in situ
on the
membrane with alkalis or enzymes, using the colony hybridization method
(Grunstein and Hogness, 1975). It is also possible to hybridize probes directly to
infected plant material, soil or water in order to avoid the need to isolate and thus
include obligate pathogens, and to preserve a clearer idea of their initial abundance
and genetic integrity, as well as saving time. These detection techniques have had
to be modified in order to allow the extraction of sufficient pure DNA to be freed
from inhibition by organic matter, clay and other materials (Steffan
et al.,
1988;
Rasmussen and Reeves, 1992).
At present, routine hybridizations are not as easy as an immunological method
even though prior extraction of nucleic acid from the test sample may be avoided
in some cases. In the laboratory, molecular hybridization can be used after the
