PLANT DISEASE DIAGNOSIS - The Epidemiology of Plant Diseases - page 14

et al., 1984; Hardham et al., 1985; Clarke et al., 1986; Fox and Hahne, 1988; Dewey
et al., 1989a; Estrada-Garcia et al., 1989), research on fine structure of pathogens
(Hardham et al., 1985; Day et al., 1986) or the detection of their toxins (Benhamou
et al., 1985a,b).
The monoclonal antibodies that have been raised against fungi (Hardham et al .,
1986; Dewey, 1988; Wong et al ., 1988; Dewey et al ., 1989a,b; Estradia-Garcia
et al ., 1989), including seedborne fungi (Mitchell and Sutherland, 1986), form the
basis of many effective diagnostics. Several double antibody sandwich assays
(DAS-ELISA) based on monoclonal antibodies and polyclonal antisera have been
developed as commercial kits for the detection of turf grass diseases by Miller et al .
(1988, 1990) for Agri-Diagnostics.
1.4.4 Immunohistology
A sensitive indirect 'sandwich' method of immunolabelling a distinctive protein
in situ can improve the light or electron microscopic examination of a pathogen in
its host (Polak and Vardnell, 1984). In this technique, a suspension of MCA binds to
the specific antigen epitope on the pathogen, then a second antibody against the first
is added which is conjugated with a fluorochrome, enzyme substrate or heavy metal.
If the fluorochrome fluorescein isothiocyanate (FITC) is used, a microscope with
an expensive adaptation for fluorescence using ultraviolet light is required and only
sections cut from fresh or frozen material can be used. When peroxidase-
antiperoxidase (PAP) from horseradish is conjugated to the second antibody in the
presence of hydrogen peroxide and diaminobenzene (DAB), it liberates a
permanent brown polymeric pigment which stains the sections so that they can be
counterstained. However, DAB is a suspected carcinogen, and safer techniques have
now been developed by conjugating heavy metals (such as gold) to the specific
monoclonal antibody, which produces a reddish appearance under the light
microscope (e.g. Benhamou et al., 1985a,b). The subsequent addition of silver
enlarges the particles and at the same time makes them appear blacker and hence
more distinct.
As the cell wall and tissues of plants prevent antibody probes from reaching the
antigens of their pathogens, immunocytochemical techniques are less popular in
plant pathology than in medical and animal pathology (Finan, 1984; Bullock and
Petrusz, 1985-1989). Despite this, immunolabelling is now a routine procedure in
the light and electron microscopy of plants (Pertot-Rechenmann and Gadal, 1986) as
a variety of techniques for tissue preparation have been devised to overcome these
constraints. It is far easier and more practical to squeeze out some sap from a plant
to inspect for virus particles directly under the transmission electron microscope
(TEM) than to go through the process of fixing, embedding and cutting sections.
However, when virus particles are present in low numbers, this effective technique
can be greatly enhanced (Barton, 1985) by immunosorbent electron microscopy
(ISEM or IEM). In this procedure, an electron microscope grid coated with carbon is
floated on a drop containing labelled antibodies which become strongly bound to it
before it is placed on a drop of the sap of the host plant to be tested. Virus particles