PLANT DISEASE DIAGNOSIS - The Epidemiology of Plant Diseases - page 12

pathogen present in sap squeezed from the host plant permeate through the plastic,
where it is detected after incubation with the antibody-enzyme conjugate, followed
by washing, then dipping in substrate, further washing, and finally adding the
stopping solution. Hence ELISA reactions can be carried out in situ quickly and
easily in the field as the dip-stick becomes coloured if the specified pathogen is
present. If the test specimen is healthy the dip-stick remains colourless. Both
positive and negative controls should be included (Mitchell et al., 1988). In other
variations, an ELISA reaction can be carried out in a similar way on membrane
enzyme/substrate systems. A drop of the test sample is added to a 'dot' containing
the specific MCA which has already been absorbed on the membrane and then
blotted. Plant disease epidemiologists can benefit from the numerous advantages of
portability and adaptability shared by dot-blot and dip-stick tests (Hill, 1984; Cooper
and Edwards, 1986), as well as the extra bonus that both the protein and
carbohydrate constituents of fungi can be detected.
The detection of prokaryotes and viruses in diseased plants is not prone to a
number of serious difficulties that have delayed the widespread application of
ELISA to fungi. Most antisera against mycelial fragments, extracts from lyophilized
mycelium, surface washings of solid cultures or culture filtrates, cross-react widely
with host tissues or extracts as well as related and unrelated fungi when tested by
ELISA or similar techniques. The same antisera may appear species-specific when
tested by immunodiffusion. Non-specific antigens may be common to both the
insoluble and soluble fractions of fungal material (Chard et al., 1985a,b). Efforts
have been made to improve specificity by diluting out non-specific antibodies or
cross-absorbing antisera with related fungi (Kitagawa et al., 1989). Non-specific
immunodominant carbohydrates or glycoproteins induce non-T cell stimulated
responses. Better results have been obtained with antisera raised against protein
precipitates from either culture filtrates or mycelial extracts (Gerik et al., 1987;
Gleason et al., 1987; Barker and Pitt, 1988; Mohan, 1989) or specific fungal
fractions such as enzymes, toxins or soluble carbohydrates (Johnson et al., 1982;
Notermans et al., 1987). Where the fungus is present on or near the surface of the
infected tissue, overnight soaking enables detection by polyclonal antisera to be
effected at very low levels (Gleason et al., 1987). This is important as specific
antigens at the species- and subspecies-specific level may often be present in the
walls and cross-walls of the hyphae but not the spores (Dewey et al., 1989b).
While it is conceivable that there is an almost infinite supply of both epitopes on
fungal protein and polysaccharide antigens and hence a virtually inexhaustible
potential for the specific antibodies to them, this also implies that there would be an
increased chance of cross-reactivity with fungi and other eukaryotes than with the
simpler viruses and prokaryotes which consist of far fewer antigenic sites.
Nonetheless, many popular serological techniques are still based on reactions
between as yet unidentified antigens with relatively crude immune sera (antisera).
Although these and other simple methods involving purified antibody mixtures
(polyclonal antibodies) are still widely used, pure single (monoclonal) antibodies
can be used to attain greater levels of specificity. However, with increased selectiv-
ity there is a concomitant loss of potential binding sites and hence often a fainter
reaction. For this reason, polyclonal antibodies are often preferred. These are