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AI/n. Rev. Phytopathol. 1988. 26:409-32

MOLECULAR DIAGNOSIS OF PLANT

DISEASE*
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Sally A. Miller

Agri-Diagnostics Associates, 2611 Bram:h Pike, Cinnaminson, New Jersey 08077

Robert R. Martin

Agriculture Canada, 6660 Northwest Marine Drive, Vancouver, British Columbia,

Canada V6T lX2

INTRODUCTION

The early and accurate diagnosis of plant disease is a crucial component of

any crop-management system . Plant diseases can be managed most effective­
ly if control measures are introduced at an early stage of disease development.
Reliance on symptoms is often not adequate in this regard, since the disease
may be well underway when symptoms first appear, and symptom expression
can be highly variable. Biological techniques for disease diagnosis and
pathogen detection are usually highly accurate but too slow and not amenable
to largc-scale application.
Recent advances in molecular biology and biotechnology are being applied
to the development of rapid, specific, and sensitive tools for the detection of
plant pathogens. This review focuses on the development and use of im­
munological and nucleic-acid hybridization-based methods for pathogen de­
tection in crop systems.

IMMUNOASSAYS IN PLANT PATHOGEN DETECTION

Immunoassay Technology
Serological methods for detection of pathogens, particularly plant viruses,
have been available to plant pathologists for many years (126, 139). Howev-

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copyright covering this paper.

409
 4lO MILLER & MARTIN

er, the introduction into plant pathology of more advanced immunodiagnostic

methods (18, 74, 149) has expanded the scope of application to the diagnosis
of diseases caused by many different viruses, bacteria, spiroplasmas, myco­
plasma-like organisms (MLOs), and fungi. The basic features of the most
commonly used and most widely applicable immunoassays are described
below. More detailed information can be obtained from a number of review
articles (18, 74, 75, 89, 139, 140).
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ELISA During the last ten years, ELISA (Enzyme Linked Immunoabsorbent
Annu. Rev. Phytopathol. 1988.26:409-432. Downloaded from www.annualreviews.org

Assay) has become widely accepted as an immunodetection method for use

with plant pathogens that provides high sensitivity, ease of use, speed, and the
ability to quantify pathogen biomass in plant tissue and other matrices. The
binding of antigen (pathogen) and specific antibody is made visible through
the use of a "tag" enzyme, which acts upon its substrate and generates a
colored product. A common mode of execution is the direct, double-antibody
sandwich ELISA (19), in which untagged antibody is bound to a solid phase,
such as the wells of a polystyrene microtiter plate. The test sample, enzyme­
labelled antibody (conjugate), and enzyme substrate are added sequentially,
while unbound material is removed after each step by washing . In a positive
test, the substrate solution will become colored, and the intensity of the color,
determined spectrophotometrically, is proportional to the amount of conjugate
bound, and consequently, to the amount of antigen present. In a negative test,
the solution remains colorless. The sandwich ELISA is especially useful for
detecting antigens in complex mixtures, such as soil or plant extracts, because
the bound antibody specifically captures the antigen(s) of interest, while
irrelevant material is removed in the wash step.
In a variation of the method, indirect ELISA, the specific primary anti­
serum is used untagged, and bound antibody is detected using an enzyme­
conjugated second antibody reactive with the immunoglobulin (Ig) species of
the primary antibody. Primary antibody (IgG) can also be detected by means
of staphylococcal protein A conjugated to an enzyme (42, 77). Immunoassay
sensitivity can be enhanced by the use of avidin-biotin amplification systems
(43, 46, 63). The indirect approach is sometimes considered less strain­
specific than the sandwich ELISA for virus detection, possibly as a result of
more complete antigen-binding capacity of the unconjugated virus-specific
antibody (8, 71, 145).
Other assay formats have been developed that combine the benefits of
indirect assays and antibody sandwich assays. An indirect sandwich ELISA,
utilizing specific immunoglobulins from two different animal species (8),
confers the advantagcs of a capture antibody without requiring the conjuga­
tion of enzyme to each antigen-specific Ig. Antigens can also be captured by
F(ab'h fragments of specific IgG immobilized on a solid phase; these frag-
 MOLECULAR DISEASE DIAGNOSIS 41 1

ments are the specific antigen binding portions of immunoglobulins which

have been cleaved enzymatically from the Fe or constant region of the
antibody. Bound antigens are detected using whole specific IgG, which in
tum is detected by an enzyme conjugate specific for the Fe portion of the IgG
molecule (11). The antibody layers of the sandwich can thus be derived from
the same antiserum, if that is desired.
A number of solid phase supports, in addition to polystyrene microtiter
plates or cuvettes, are suitable for ELISA . Assays in which antibodies or
antigens are bound to nitrocellulose membrane filters (7, 67. 77. 115. 131,
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134) or cellulose filters (77, 130) have been used to detect plant-pathogenic
bacteria and viruses in infected plants. Immunoblot (also called dot-ELISA,
dot-immunobinding) assays tend to be rapid, easy to execute, conservative of
reagents, and often more sensitive than ELISAs carried out in microtiter
plates using the same immunoreagents (7, 58, 59, 115, 134). Indirect assays
of infected plant material, in which the plant extract is bound directly to the
membrane, may be unsuitable as a result of interference by chlorophyll and
other plant components (77, 85). However, removal of such components
(115) or release of the pathogen into water or buffer without extracting the
tissue (77) can reduce or eliminate plant interference. A double-antibody
sandwich ELISA will serve the same purpose, since plant components are not
bound to the primary, pathogen-specific antibody and are eliminated during
the first wash step (7, 134). Immunoblot assays utilize the same types of
reagents used in microtiter plate ELISAs, except that the substrate required
forms an insoluble product that precipitates onto the membrane. Positive
reactions can be determined visually or by using a reflectance densitometer
(134).
As a result of the tremendous growth in the last decade of the medical
diagnostics industry, a number of procedures and devices have been de­
veloped that increase speed, sensitivity and ease of use of immunoassays in
nonlaboratory situations. Immunoassays utilizing beads as the solid phase
greatly increase the surface area available for binding of antibodies or anti­
gens (88). Magnetic beads or particles have the additional advantage that they
can be conveniently separated from the reaction mixtures without centrifuga­
tion or filtration steps. Devices have also been developed that utilize absor­
bent materials to cause the reagents to flow through the reaction areas; this
greatly improves reaction kinetics (142). Finally, dry-chemistry technology
has resulted in the production of assays in which the reagents are immobilized
and dried in a layered porous matrix prior to use. This eliminates the need for
many of the liquid reagents (74). Adaptation of these new technologies to
immunoassays used for agricultural applications should make it possible to
detect very small amounts of a target pathogen rapidly and in field situations.
One adaptation of an immunoassay suitable for field use is the "dipstick"
 412 MILLER & MARTIN

variation. Initially developed for the physician's office and home use, this
method is being used to detect fungal plant pathogens in turfgrass (74, 99)
under field conditions. Primary antibodies are bound to a membrane attached
to the end of a dipstick, which is incubated with the extracted plant material
and the enzyme-conjugated antibody. The dipstick is then rinsed and trans­
ferred to substrate solution. After a short incubation period, the dipsticks are
dried, and the intensity of the color development is quantified using a
handheld reflectometer.
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COLLOIDAL GOLD Colloidal gold has been used as an immunohistochemi­

cal marker in electron microscopic studies of pathogenic and mycorrhizal
fungi (12, 106) but is also a useful antibody tag for dot-immunobinding and
related assays (64, 74). Owing to the optical characteristics of the colloidal
gold label, binding of antigen and gold-conjugated antibody can be made
visible directly on the membrane as a red signal, which eliminates the need
for additional steps to make the antigen-antibody complex visible. How­
ever, increased sensitivity can be obtained through silver enhancement ( 103,
104).

IMMUNOFLUORESCENCE ASSAY In direct immunofluorescence (IFA),

pathogen-specific antibodies are conjugated with fluorescent dye molecules,
commonly fluorescein isothiocyanate (FITC) or rhodamine isothiocyanate
(RITC) . Antigens present in samples bound to microscope slides are made
visible by means of a fluorescence microscope (44). Instruments are also
available that measure emissions from samples bound to other solid surfaces,
e.g. the wells of a microtiter plate . Indirect IFA, like indirect ELISA, uses a
second, tagged antibody to detect specific antibody-antigen binding. A type
of double-antibody sandwich IFA has also been described ( 148). Im­
munofluorescent assays have been particularly useful in detecting and localiz­
ing fungi in plant material and soil ( 17, 29, 34, 72, 81, 83, 84, 117, 153) and
to diagnose diseases caused by plant-pathogenic bacteria (26, 96, 97, 125,
127, 148). Quantitative results can be obtained when the number of fluoresc­
ing units (i.e. bacterial cells or fungal spores) can be counted, but this tends to
be very tedious and restricts the number of samples that can be evaluated.
Automated, computer-driven microscope systems are being developed to
provide quantitative estimates of cell popUlations and may make IFA more
amenable to large-scale use (S. De Boer & J. Hall, personal communication).
Autofluorescence of plant material and fungicides in soil samples (148),
adsorption of tagged antibodies to soil particles (84), and potential cross­
reactivity with other microorganisms can affect the interpretation of results
and must be analyzed individually for each system.
 MOLECULAR DISEASE DIAGNOSIS 413

RADIOIMMUNOASSAY Radioimmunoassay (RIA) is used routinely in clini­

cal microbiology laboratories (140) but infrequently for detection of plant
pathogens (40, 124). Many executions of RIAs are similar to the variations of
ELISA, except that radioisotopes are used to tag the detection antibody .
Antibody binding is quantified using a scintillation counter. Radioimmunoas­
say has found limited application in plant pathology, primarily because the
appropriate equipment, which tends to be quite expensive, has not been
available, and owing to the short half-life of some isotopic reagents and to
hazards associated with handling and disposing of radioactive material.
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Immunoreagents
Antibodies are the most critical components of any immunoassay, and stan­
dardized antibody reagents with the appropriate specificity and sensitivity are
required for consistent, interpretable results. Polyclonal antisera have been
used routinely in immunoassays for plant pathogens; they are fairly easy and
inexpensive to prepare, depending on the antigen selected, and can be pro­
duced in a short time (3-6 months) . Because polyclonal antisera result from a
generalized immune response, they contain a popUlation of antibodies that
may react with many different determinants of the immunogen. This is
advantageous in instances where a population of variable or potentially
variable target pathogens must be detected. The population of antibodies in a
polyclonal antiserum is also heterogeneous with respect to antigen-binding
affinities and suitability for covalent attachment of enzymes, fluorochromes,
etc. Thus, while some individual antibodies in the population may have low
affinity or lose binding ability upon covalent modification, the reactivity of
the antiserum, on the whole, may not be affected (43).
However, undesired cross-reactivity has hindered the practical application
of immunoassays for some pathogens, particularly fungi . Cross-reactivity in
polyclonal antisera can be reduced by (a) using highly purified, well­
characterized specific antigens for immunization; (b) diluting high titer anti­
serum to eliminate low levels of cross-rcactivity, while retaining activity for
the specific reaction; and (c) absorbing antiserum with heterologeous antigens
to remove cross-reacting antibodies. In instances where the above measures
are insufficient to provide the appropriate specificity, monoclonal antibodies
(MABs) may be useful .
The theory and practice of MAB production are well described in a number
of books and review articles (37, 43, 53, 90, 156). Briefly, monoclonal
antibodies are homogeneous populations of antibodies produced by fusing
antibody-secreting cells (usually from the spleen) from an immunized animal,
with myeloma cells from a specially developed cell line. The cells are
transferred to a selective medium that will allow only the hybrid cells, or
 414 MILLER & MARTIN

hybridomas, to grow. Individual hybridomas are cloned by limiting dilution,

and antibodies secreted into the culture medium are tested for antigen-binding
characteristics. Individual hybridomas that secrete antibodies demonstrating
desirable sensitivity and specificity attributes are then scaled up for further
study. Hybridomas can be frozen and stored in liquid nitrogen or at -70°C,
then revived later to ensure continuous production of the MABs. In this way,
a constant source of uniform antibodies is available, and batch-to-batch
variation, which may be observed with polyclonal antisera, is not a factor.
Although the potential advantages of MABs make them very attractive as
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immunoreagents, the development of MAB-based diagnostic tests is not

necessarily a straightforward procedure. Monoclonal antibodies are notorious
for "assay specificity," and a MAB selected using one approach may not
function in another (50, 53, 90, 14 1). A fairly high proportion of MABs,
secreted by hybridomas produced on a typical experiment, can be expected to
have low affinities, which will affect the sensitivity of any assay in which they
are used (43, 48, 61). Covalent binding or conjugation of MABs with
enzymes or other labels may also reduce sensitivity (92, 131). The im­
munoglobulin class and subclass of tbe MAB will also be a factor, as different
isotypes respond differently to purification protocols, assay formats, etc.
(43). Because MABs react with a single determinant of an antigen, an
extremely high degree of specificity is possible. A large number of isolates
must be screened to ensure, with a reasonable degree of confidence, that the
breadth of specificity is sufficient to detect all isolates within a particular
taxonomic unit. The specificity and sensitivity needed for practical applica­
tions may be achieved by mixing MABs of different specificities to create a
synthetic polyclonal reagent (27, 49, 53, 102, 116).
Virologists were the first among plant pathologists to take advantage of
hybridoma technology, and the production and use of MABs specific for plant
viruses have been reviewed recently (53, 62, 75, 89, 90). A major advantage
of MABs over polyclonal antisera with respect to detection of viruses is the
elimination of background, the undesired generation of positive signal,
caused by the presence of antibodies to host-plant tissue. In addition, the
range of specificities possible with MABs can overcome some of the limita­
tions of polyclonal antisera. Monoclonal antibodies have proved to be superi­
or to polyclonal antisera as broad-spectrum probes for the detection of barley
yellow dwarf virus (BYDV) in infected tissue (30). Polyclonal antisera
consistently differentiated isolates of BYDV, requiring the use of several
different antiserum preparations to detect the virus. The MABs detected
MAV, PAY, and RPV isolates of BYDV, apparently binding with antigenic
determinants common to all three isolates. However, other, highly specific
antibodies have been developed for diagnostic purposes. In a five-year study
of potato samples for the presence of the tobacco veinal necrosis strain of
 MOLECULAR DISEASE DIAGNOSIS 415

potato virus Y (pVyN), a MAB against PVyN was more effective than
polyclonal antibodies in differentiating PVyN from the ordinary strain
(PVyo) ( 12 1). A commercially produced MAB specific for PVyN was also
successful in this regard.
Plant-pathogenic bacteria present more challenges in the production of
MABs than do viruses, primarily because bacterial antigens are complex and
have not been well characterized. However, MABs have been produced for
many important genera of phytobacteria (2, 27, 28, 82). Using whole cells as
immunogens, Alvarez et al (2) produced MABs against Xanthomonas cam­
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pestris pv. campestris (Xcc) that were specific at the genus, pathovar, and
strain levels. Later, a MAB was developed that distinguished Xcc isolates
from nonpathogenic xanthomonads recovered from crucifer seeds (3). Other
approaches, utilizing cellular and extracellular fractions as immunogens, have
also been successful in developing specific MABs. Monoclonal antibodies
that reacted with the O-side chain of purified lipopolysaccharide of Erwinia
carotovora subsp. atroseptica (Eca) were produced when a crude outer-cell­
membrane fraction or a soluble antigen extract were used as immunogens
(27). The MABs were specific for Eca strains in serogroups I and XXII, and
did not react with other serogroups of Eca, E. carotovora subsp. carotovora
(Ecc), or E. chrysanthemi. Purified endopcctate lyase (PL) from Ecc, used as
an immunogen, resulted in the production of MABs that reacted with PL from
isolates of all Ecc serogroups tested (M. Klopmeyer & A. Kelman, personal
communication) . Whole cells and cell wall antigens were used in the develop­
ment of MABs to detect the ring-rot pathogen of potato, Corynebacterium
sepedonicum (28) . Most of the MABs cross-reacted slightly with other
Corynebacterium spp., but one MAB was highly specific for C. sep­
edonicum. The usefulness of this MAB in detecting latent ring-rot infections
in potato by IFA has been demonstrated (26). Hybridoma technology has also
been successfully applied to the detection of spiroplasmas (25, 79) and MLOs
(80).
The issue of antigen or immunogen complexity and lack of characterization
is greatly magnified for plant-pathogenic fungi. There is no general consensus
in the literature regarding the type of immunogen (whole cells, cell walls,
soluble antigen, extracellular components, etc) likely to result in the most
specific polyclonal antiserum (34, 39, 60, 107-1 10, 124) . Production of
highly specific MABs has also been difficult to achieve (5, 6, 66), but recent
studies have demonstrated that species-specific and sub-specific distinctions
in fungi can be made using MABs. Mitchell (101) described MABs that
detected isolates of Sirococcus strobilinus from spruce and pine but not from
western hemlock. Some hybridoma cell lines secreted MABs that detected all
S. strobilinus isolates, regardless of host. Species-specific MABs have also
been developed for Pythium aphanidermatum and Sclerotinia homoeocarpa
 416 MILLER & MARTIN

(F. Petersen & S. Miller, unpublished data) . Hardham et al (56, 57) used
zoospores and cysts of Phytophthora cinnamomi as immunogens to produce
MABs with the following classes of specificity: isolate-specific, species
specific, genus-specific, and general reactivity (among Pythiaceous fungi
tested). Some of the antibodies also demonstrated specificity for antigens
associated only with zoospores or cysts. Of the MABs that reacted with
zoospores, several were restricted in binding to specific structures such as
flagellae and a portion of the zoospore groove.

Immunoassay Applications
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Immunoassays are being used routinely for the detection of plant pathogens in
vegetatively propagated material and seeds in conjunction with quarantine
seed-testing, seed-certification, and pathogen-indexing programs (53, 55, 75,
100, 1 18, 129, 138, 146, 147) . While the majority of these programs are
directed toward the detection of viruses and bacteria, immunoassays have
been developed recently that detect fungi in seeds (67a, 102) and vegetative
tissue (32, 39, 98, 99, 108, 124) . Tests such as ELISA and IFA have
demonstrated the sensitivity and specificity required to replace such time­
consuming and expensive bioassays as indicator-plant inoculation, growing­
on tests, and dilution plating. ELISA kits for detection of plant-pathogenic
viruses, bacteria, and fungi are now commercially available (80a).
One critical application of immunoassay technology is the monitoring of
pathogen spread in a crop system. ELISA was used to follow the spread of
PAV- and RPV-like isolates of BYDV in cereals in Indiana, and provided a
much more reliable indication of infection than symptom expression (20).
Dipstick ELISA kits developed to detect Pythium spp., Rhizoctonia solani,
and Sclerotinia homoeocarpa in golfcourse turfgrass were used to monitor
changes in pathogen populations resulting from management practices and
environmental conditions (99; S. Miller, unpublished data; B. Clarke, per­
sonal communication). Pathogen-biomass changes were clearly reflected in
the test results, which could be correlated with symptom expression, environ­
mental parameters, and fungicide use.
Immunoassays have the potential to detect and quantify pathogen pro­
pagules in soil and other complex matrices (32, S. Miller & M. Klopmeyer,
unpublished data) . In the short term, immunoassays may be used in conjunc­
tion with baiting assays, in which pathogens are "baited" from soil samples
using susceptible plant material, to confirm quickly and accurately the col­
onization of bait material by a particular pathogen (32). Direct detection and
quantification of pathogens in soil may be more difficult to achieve as a result
of low propagule counts, soil complexity, and other factors. However, highly
sensitive tests combined with appropriate soil preparation measures will likely
overcome some of these obstacles to provide information that can be used in
making crop-management decisions.
 MOLECULAR DISEASE DIAGNOSIS 417

NUCLEIC-ACID HYBRIDIZATION-BASED PATHOGEN

DETECTION

Nucleic-acid hybridization, which depends on the high degree of specificity

inherent in the pairing of nucleotide base sequences, is a well-established tool
in molecular biology. As a result of this specificity, the technique is being
applied for diagnostic purposes, including the detection of plant pathogens.
Labeling one of the nucleotide sequences provides the necessary signal
required to follow the hybridization. The most commonly used label is 32p,
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incorporated into the "detecting" strand of nucleic acid. However, the de­
velopment of nonradioactive probes will greatly enhance the applicability of
nucleic-acid probes as diagnostic tools.
A cloned DNA probe is defined as a strand of DNA produced when DNA is
the starting material for cloning, while probes cloned from an RNA template
are known as complimentary DNA (cDNA) probes. Probes for plant viruses
are nearly all cDNA, since the genomes of most plant viruses are RNA.
Development of transcription vectors to produce RNA probes in vitro pro­
vides a system for the production of RNA: DNA or RNA: RNA hybrids.
These hybrids are more stable than DNA: DNA hybrids and may give cleaner
assays with less background. However, RNA probes have the disadvantage of
susceptibility to RNAse digestion (54).
Nucleic-acid probes offer an advantage over serological methods in that a
test can be made for a whole set of genes within a given pathogen (entire
genomic information can be probed). For plant-pathogenic viruses, only
about 2-5% of the nucleic acid of viral genomes are represented in the
antigenic determinants of the coat protein (51, 65). Strain differences or large
group affinities may be found by using probes that represent other parts of the
genome. In the case of bacteria, fungi, and nematodes, the antigens present at
one stage of development may differ from antigens present at other de­
velopmental stages; this situation requires that a composite antiserum be
available to detect all stages of these organisms, since the entire genetic
information is present at each stage of development.
This discussion will focus on the use of nucleic-acid probes and restriction
fragment length polymorphisms (RFLPs) for detection and diagnosis of plant
pathogens. Developments in the use of nonradioactive labels will also be
reviewed, as these are essential to the large-scale application of nucleic-acid
technology in agriculture.

Nucleic Acid-Based Detection Techniques

Large scale nucleic acid-based plant pathogen detection systems likely will
make use of cloned eDNA or DNA probes in a dot-blot or related assay as
demonstrated for potato spindle tuber viroid (PSTV) ( 1 12). These probes have
the potential to detect single nucleotide differences, as in the case of the
 418 MILLER & MARTIN

prenatal detection of sickle-cell anemia in humans (16). This degree of

specificity may be essential to differentiate between mild and severe strains of
a virus or different races of pathogenic fungi, nematodes, MLOs, or bacteria.

DOT-BLOT ASSAY A recent development in nucleic-acid hybridization tech­

nology that offers considerable potential for plant-pathogen detection is the
dot-blot assay (95) . The target nucleic acid from a plant- or insect-vector
sample is spotted onto a solid matrix, commonly nitrocellulose, and bound by
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baking. Free binding sites on the nitrocellulose are then blocked with
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nonhomologous DNA (usually salmon sperm or calf-thymus DNA) and a

protein source (usually bovine serum albumin (BSA) or nonfat dried milk).
Thereafter, hybridization with a labeled probe is carried out. The label is then
detected by autoradiography, for 32p labels, or a colorimetric reaction if an
enzyme label is used. The steps are quite similar to those in ELISA, although
the basis of binding is quite different. While ELISA is based on the binding of
antibody and antigen, hybridization is based on the annealing of com­
plementary strands of nucleic acids The sensitivity of dot-blot hybridization
.

is about the same as that of ELISA (9, 94, 128). A modification of the
dot-blot assay, squash blotting, has been used to detect maize streak virus
-

(MSV), the bee spiroplasma, and com stunt spiroplasma in single leafhoppers
(13).
The basic parameters of the dot-blot assay are given here to help the reader
to understand the applications and limitations of this technique (for more
detail, see Ref. 95). The basis of the technique is straightforward: two
complementary strands of nucleic acid will anneal at specific temperature and
salt concentrations. At higher temperatures, the bonds holding the duplex
together will break, and the strands will separate. The temperature at which
half the strands are disassociated is called the melting temperature (Tm),
which is affected by salt concentration, and by length and composition of
nucleic acid . The maximum rate of hybridization is found at about 25°C
below the Tm, at 1.0 M NaCl. The temperature for optimal hybridization is
lower for probes shorter than 150 bases. Hybridization rate is not affected
significantly as salt concentrations vary from 0.4-1.0 M, or as the pH of the
hybridization solution varies from 5 to 9 at these salt concentrations (95).
Hybridizations are often carried out at 65°C in 0.3-0.7 M NaCI for plant
pathogens (23, 36, 65). Even MLO nucleic acids, which are AT-rich and thus
should have a lower temperature for maximum hybridization, give excellent
results at 65°C (13, 69). Addition of formamide to the hybridization mix
reduces the temperature of optimum hybridization by 0 . 7°C for each one
percent of formamide in the solution. The Tm of DNA hybrids decreases by
1°C with every 1 percent of mismatched base pairs. By adjusting the
hybridization conditions (temperature, salt concentration, or formamide con-
 MOLECULAR DISEASE DIAGNOSIS 419

centration), the stringency or amount of mismatched base pairs allowed can

be controlled (95). This is a critical consideration, since the amount of
hybridization observed with related organisms can be controlled by the strin­
gency of the assay conditions.

NONRADIOACTIVE LABELS Radioactive probes for nucleic-acid hybridiza­

tion are limited by the same inherent disadvantages that affect the use of
radioactive labels in immunoassay. These problems have slowed the develop­
ment of nucleic-acid probes for disease diagnosis and have also resulted in the
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search for nonradioactive labels. To be acceptable, nonradioactive labels must

approach the sensitivity of 32p, have a long shelflife and be suitable in
rapid-assay systems. Several approaches have been taken in the development
of nonradioactive probes.
The label or tag can be introduced into the nucleic acid chemically (35,
120, 136) or enzymatically (47, 73, 78). The most promising nonradioactive
tag at this time is biotin (vitamin H), which binds very tightly to avidin (a
68,000 dalton glycoprotein; Kdis = 10-15 M) and to streptavidin . Avidin is
coupled to either phosphatase or peroxidase enzymes (35, 143) and hybridiza­
tion is followed by the addition of a colored precipitating substrate.
The biotinylated probes are hybridized to target nucleic acid in the same
manner as are 32P-Iabeled probes. After hybridization, the filter is washed to
remove excess probe and incubated with avidin-enzyme conjugate, which
binds to the biotin . The filters are washed and precipitating substrate is added.
The colored precipitate indicates hybridization between probe and target
DNA. The first nonradioactive probe used in clinical laboratories was based
on biotin-streptavidin (143). The biotin can be incorporated into DNA by nick
translation as a biotin analog of TTP, in which the biotin is separated from the
TIP by an eleven-carbon spacer arm, making the biotin more accessible to the
avidin (73). Biotinylated-l l-UTP can be incorporated into RNA in in vitro­
transcription reactions. RNA or DNA probes prepared with biotinylated
nucleotides detect as little as 1 pg of membrane-bound DNA. Kits for
preparing nick-translation probes with biotinylated nucleotides are available
commercially (i.e. Bethesda Research Laboratories, Bethesda, MD).
Nucleic acids have also been biotinylated chemically with photobiotin,
which is biotin coupled with a photoactivatable group that will react with any
organic material under intense light (35). The advantage of this method is that
no expensive enzymes are required. It labels double-stranded (ds) or single­
stranded (ss) DNA or RNA and may be a better means of labeling dsRNA
than the currently used end-labelling with 32p. Photobiotin-Iabeled plasmid
DNA with a PAV-BYDV cDNA insert was used to detect the virus in infected
plant sap with the same level of sensitivity as 32P-labeled probes (52).
Another approach in developing nonradioactive probes is to couple alkaline
 420 MILLER & MARTIN

phosphatase or horseradish peroxidase to polyethyleneimine, giving a posi­

tively charged tail to the enzyme (120) . This tail binds electrostatically to
nucleic acids and is covalently linked by the addition of glutaraldehyde . DNA
labeled in this way can be added directly to a hybridization reaction. After
hybridization and washing, the precipitating substrate is added to make the
bound probe visible. The detection limit of this method is in the 1-5 pg range .
A third approach is to prepare antibodies to chemically modified DNA (47,
136). N-acetoxy-N-2-acetylaminofluorene (AAF) , or N-2-(guanosin-8-yl)
acetylaminofluorene (Guo-AAF) bound to DNA, or 5-bromo-deoxyuridine
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incorporated into DNA have bcen used as immunogens to produce antibodies

specific for the modified DNA. Any DNA probe thus modified can be
detected with these antibodies. This method detected target DNA at the pg
level; it thus has a degree of sensitivity similar to the two methods described
above. Ds- and ssRNA and DNA could be labeled by this method. Monoclo­
nal antibodies specific for dsRNA offer another potential source of broad­
spectrum nonradioactive probes for those plant RNA viruses with dsRNA
genomes, RNA viruses which produce dsRNA replicative intermediates, and
perhaps for some fungi with associated dsRNAs (111).
A novel nonradioactive probe that could eliminate the need to bind target
nucleic acids to a solid matrix and make detection with nucleic-acids probes a
one-step method is being developed (68). This procedure involves cutting a
DNA probe at an internal-restriction site. A photon-emitting group (e . g .
luciferase) i s added at one side o f the restriction site and a photon-absorbing
group (e. g . porphorin), which also emits light, is bound at the other side of
the restriction site . When a hybrid between the probe and target nucleic acid is
formed, these two compounds are placed in close proximity; the photon
emitted by luciferase (400 nm) is captured by the porphorin; and a 500--550
nm range photon is emitted. If no hybrid is formed, the luciferase and
porphorin will not be close enough for the photon transfer to occur. Emission
of light in the 500-550 nm range would indicate the formation of a hybrid.
This could be done in liquid phase with no need to remove excess probe; and
would make it a one-step procedure that could be performed in a few hours.

RESTRICTION FRAGMENT LENGTH POL YMORPHISMS This technique

makes use of restriction enzymes to fragment DNA, which is then separated
by agarose-gel electrophoresis. Each restriction enzyme recognizes a specific
nucleotide sequence (usually � bp long), and cuts the DNA spccifically
every time the sequence occurs. The fragmented DNA is separated by gel
electrophoresis, and the banding pattern is made visible by staining (ethidium
bromide) or by autoradiography. When mitochondrial DNA (mtDNA), which
ranges from 10--200 kilobase pairs (kbps) in length, from different species are
examined, differences in the size and number of restriction fragments can be
 MOLECULAR DISEASE DIAGNOSIS 421

detected. These differences can result from inserts or deletions between

existing restriction-enzyme sites or mutations that destroy or create restriction
sites. The observed differences in restriction fragments are known as restric­
tion fragment length polymorphisms (RFLPs) . RFLP analysis, though used
for diagnosis of human diseases and genetic disorders, is too tedious, time­
consuming and expensive (tests for sickle-cell anemia, based on RFLP analy­
sis combined with a specific probe, cost about $300 each) for large-scale plant
pathogen detection. However, it may help to identify regions of genomic or
mtDNA that could be used in the development of species- or race-specific
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probes.

Nucleic Acid Probes

UNCLONED PROBES Isolation of large molecular weight, double-stranded

RNAs (dsRNA) from virus-infected plants and fungi (31) is a means of
detecting viruses. Caution must be used with this method, since there are
several well-documented cases of non-viral-associated large-molecular­
weight dsRNAs in seemingly healthy plants (150). Many fungi have been
shown to contain dsRNA, but in most cases no association with virus infec­
tion has been demonstrated. These fungal dsRNAs offer a potential means of
detecting fungi when the dsRNA is diagnostic for a particular fungus (111,
119, 135, 155). DsRNA also offers potentially useful templates for the
generation of cDNA probes specific for recalcitrant viruses using the cloning
techniques developed for animal reoviruses (15) and Ml dsRNA of yeast
(133). The major problem associated with cloning these nucleic acids is their
great stability, which makes strand separation difficult. It may be beneficial to
denature the dsRNA by boiling and snap cooling in liquid nitrogen, which has
been successful for in vitro-translation studies of dsRNA genome of beet
cryptic virus 1 (1). dsRNA can be denatured readily in 20 mM methylmercu­
ric hydroxide for 10 min, then cloned by standard methods (R. Martin,
unpublished). The methodology and application of dsRNA analysis has been
reviewed recently (31) and will not be discussed further here.
The preparation of probes varies, depending on the type of nucleic acid
used as starting material. For plant viruses (most have RNA genomes) the
starting material is usually single-stranded RNA (ssRNA), which may or may
not be poly (A) tailed (93). The RNA can be labeled directly with T4
polynucleotide kinase and gamma-labeled ATP (86, 123). With this method
only the 5' end of the RNA is labeled, yielding probes of low specific
activity. The activity can be increased by partial hydrolysis of the RNA prior
to labeling, which provides additional 5' ends. Care must be taken that the
RNA is not cleaved to the point that specificity is jeopardized by making
probes too short (54) . This method has been used to make probes from
 422 MILLER & MARTIN

dsRNA (31), but is not widely applied because the specific activity of the
probes is low and the RNA template must be purified frequently.
Probes of high specific activity (l08 cpm per ug of RNA) can be made
directly from RNA with RNA-dependent DNA polymerase (reverse transcrip­
tase) (45, 86, 113, 114). With this labeling method, the RNA serves as a
template for making a complementary DNA. If one of the nucleotides is
labeled, on the average every fourth nucleotide in the probe will be labeled.
For probes prepared in this manner, random primers should be used rather
than a specific primer to ensure that the entire genome is represented in the
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synthesized cDNA (45). Probes prepared in this manner are ideal for examin­
ing relationships between viruses since most of the genome is represented (45,
94, 113, 144). DNA probes, either cloned or uncloned, are not as vulnerable
to nuclease digestion as ssRNA probes (54). This is an important considera­
tion, especially in view of the development of nonradioactive . probes that have
long shelflife.

SYNTHETIC PROBES The development of synthetic oligonucleotides offers

the potential for both strain-specific and broad-spectrum diagnostic probes.
Synthetic oligomers representing two regions of the citrus exocortis viroid
(CEV) genome known to have sequences in common with PSTV and chrysan­
themum stunt viroid hybridize with CEV-infected Citrus medica grown in
growth chambers (9, 10). On the other hand, diagnosis of sickle-cell anemia
in humans using oligonucleotide probes is based on the detection of a single­
point mutation (16). As more strains of plant viruses and other pathogens are
sequenced, synthetic strain-specific probes will become a reality.

CLONED PROBES AND RFLPS For purposes of virus detection, cloned

probes have many advantages over probes prepared from random primed
RNA or nick-translated uncloned DNA. The advantages are similar to those
outlined above for monoclonal antibodies as compared to polyclonal antibod­
ies, including the unlimited supply of uniform probe, and potentially better
specificity. The basic prinCiples for cloning from viral RNA templates or the
cloning of DNA will be referred to here, but details of these procedures are
given in several excellent molecular-biOlogy laboratory manuals (54, 86,
154). Most of the procedures require a considerable familiarity with molecu­
lar biology and the handling of nucleic acids; therefore, the beginner can save
time and effort by working with someone who has training in molecular
biology, or by starting with a commercially available cloning kit.

Viruses and viroids Viroids are the only plant pathogens that have been
detected on a large scale by using nucleic-acid probes (lOS, 112, 132). This
has come about by necessity, since viroids lack any protein component and
 MOLECULAR DISEASE DIAGNOSIS 423

cannot be detected by serological methods. In the course of basic research,

cloned cDNA probes have been prepared for a wide range of plant viruses.
Hybridization of plant viral nucleic acids was originally carried out with target
and detecting nucleic acids in a liquid phase. The technique has been very
useful for studying plant-virus relationships (45), but is not adaptable to
routine detection of plant viruses.
Strain-specific probes can be developed by careful screening of a cDNA
library prepared from one strain of a pathogen, using different strains of the
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pathogen. Testing of a cDNA library of citrus tristeza virus (CTV) prepared

Annu. Rev. Phytopathol. 1988.26:409-432. Downloaded from www.annualreviews.org

by random priming has shown that strain-specific cDNA clones can be

obtained in this manner (122). These cDNA probes differentiated between
T-36, a severe Florida isolate, and other biologically distinct isolates of CTV
that could not be differentiated serologically by means of polyclonal antisera
(122). From a library of cDNA clones of BYDV, some clones specific for the
RPV or PA V strains were identified, while other probes hybridized to both
serotypes of BYDV, beet western yellows, potato leaf roll, and soybean
dwarf viruses, all members of the luteovirus groups ( 151). These results
suggest the possibility of developing group-specific as well as strain-specific
probes.

Mycoplasma-like organisms and bacteria Recent work with western-X

MLO demonstrates that specific DNA probes can be produced even for these
difficult-to-study organisms (69). Kirkpatrick and co-workers used to their
advantage the high titer that the westem-X MLO reached in its insect vectors.
Total DNA was purified from MLO-enriched fractions obtained from infected
leafhoppers. Diseased leafhoppers yielded two bands of linearized DNA in
equilibrium CsC l gradients, whereas healthy leafhoppers yielded a single
DNA band. The extra band was digested with EcoRI and Hind III restriction
enzymes, size-fractionated by electrophoresis on agarose gels, and cloned
into an EcoRI- and Hind III-cut pUC 8 plasmid. After colony hybridization,
24 recombinant plasmids that hybridized with DNA from westem-X diseased
plants and insects, but not with DNA from healthy plants and insects, were
obtained. These cloned DNA probes were also used successfully in dot-blot
assays to detect Western-X MLO in infected plants and insects (69).
Another potentially useful probe for MLOs is the general probe developed
from cloned fragments of the 23S ribosomal gene of Mycoplasma hyorhinis,
an animal mycoplasma (41). This DNA probe hybridized to five species of
Mycoplasma, as well as to Acholeplasma laidlawii, but not to E. coli or Hela
DNA. The probe has not been evaluated for hybridization to plant MLOs but
may be a useful broad-spectrum probe for this group of plant pathogens. It
could be much more objective than fluorescent staining by DNA-binding dyes
such as diaminophenyl indol (DAPI), and much faster then electron micros-
 424 MILLER & MARTIN

copy of ultra thin sections, the two broad-spectrum methods currently used to
detect plant-pathogenic MLOs.
Bacterial plasmids are excellent candidates for probe development. Most
strains of Xanthomonas campestris that contained plasmids could be differen­
tiated at the pathovar level on the basis of plasmid profiles (76). Plasmid­
specific probes should be very useful in epidemiological studies of plant­
pathogenic bacteria and also for studying the ecology of various bacteria,
including non-ice-nucleating bacteria used for frost protection. Since the
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bacterial genome is relatively small, preparation and screening of bacterial

Annu. Rev. Phytopathol. 1988.26:409-432. Downloaded from www.annualreviews.org

DNA libraries is quite feasible, as demonstrated in studies using Salmonella.

Fifty-four clones were selected that did not cross-hydribize to E. coli from a
DNA library of Salmonella typhimurium. Of these 54 clones, 10 did not react
to any of 32 isolates of other genera of bacteria. Two of the 10 clones
hybridized to each of hundreds of Salmonella isolates tested in dot-blot
hybridization assays (33) and are being used in field studies. A similar
approach should be successful in the development of DNA probes specific for
bacterial plant pathogens.

Fungi The use of RFLPs has been increasing in fungal taxonomy in recent
years (4, 21, 36, 38, 70, 87, 119). RFLPs of genomic DNA combined with
random probes allowed for the differentiation of species, formae speciales,
races, and even isolates of Fusarium (21, 87). RFLPs detected in mtDNA of
F. oxysporum corresponded with forma specialis (70). Restriction enzyme­
digested fungal genomic DNA generally yields a smear on agarose gels after
electrophoresis, with discernible bands from repetitive DNA. RFLP analysis
of ribosomal DNA nuclear genes (rDNA) have given variable results. In the
case of F. oxysporum, the rDNA appeared highly conserved (70), while in
Cochliobolis heterostrophus, rDNA RFLPs were specific for species or even
strains (38). Most RFLP work with fungi has concentrated on the mtDNA,
which is much smaller than genomic DNA (4,36,38,70). MtDNA cut with a
single restriction enzyme (with a 6-base recognition sequence) yields only
10-20 fragments that give distinct bands after electrophoresis on agarose gels.
It has been demonstrated that RFLPs obtained with mtDNA (4, 36) and with
total DNA (21, 87) are useful for taxonomic purposes.

Nematodes RFLPs of repetitive DNA have been used to differentiate

Meloidogyne incognita, M. javanica, M. arenaria, and M. hapla (22, 23,
24), Globodera rostochiensis and G. pallida (14), and Bursaphelenchus
xylophilus and B. mucronates (D. Bailie & J. Webster, personal communica­
tion). The differentiation of one population of nematode or fungus of a given
species from a second population of another species by RFLPs does not
necessarily demonstrate that the RFLP maps obtained are distinctive for each
 MOLECULAR DISEASE DIAGNOSIS 425

species. Many populations of each species, jorma specialis, or any taxonomic

group must be characterized by RFLP maps before a particular map has any
taxonomic or diagnostic value (22). Moreover, while RFLP mapping com­
bined with Southern transfers to DNA binding paper and subsequent probing
with DNA probes may be useful for taxonomic purposes, the technique is too
tedious for detection and diagnostic purposes.
Perhaps the most important contribution of RFLP analysis to plant­
pathogen detection is the identification of regions of DNA that may be good
candidates for development into diagnostic probes. If DNA from two closely
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related pathogens is cut with one restriction enzyme and an RFL difference is
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cloned, this cloned probe can be used to determine if other restriction en­
zymes give RFL differences in the same region. Regions of DNA that show
RFLPs with several restriction enzymes are good candidates to produce
specific probes, since multiple nucleotide differences occur in this small
identifiable region of DNA. After the region of DNA is cloned by ligating into
a suitable plasmid, the cloned DNA can be used in hybridization assays with
many populations of the pathogen and related organisms to evaluate the
specificity of the probe. In this way, it should be possible to produce highly
specific probes. Such an approach was taken in the development of a probe to
differentiate B. xylophilus and B. mucronates. These two species of nema­
todes live in conifer wood and are difficult to differentiate morphologically;
however, only B. xylophilus is pathogenic. RFLP mapping was used to
identify a region of DNA that was quite diverse in these two nematode
species. Cloning the diverse region of DNA yielded a DNA probe that could
be used to differentiate these species in a dot-blot assay. The probe has been
100% successful in separating these two species in double-blind tests (D.
BailIe & J. Webster, personal communication).

CONCLUDING REMARKS

- Recent developments in molecular detection technology resulting in more

convenient, effective, and specific assays have opened the door to greater use
of these tests for detecting plant pathogens. These assays will help to free
growers, crop consultants, and plant-health-care professionals from having to
rely excessively on symptomatology and/or time-consuming diagnostic pro­
cedures, and permit early detection of pathogens. However, they should be
viewed as management tools, to be used in conjunction with other diagnostic
procedures, knowledge of the crop. and understanding of the biology of the
pathogen and the ecology of the disease. For example, viruses and MLOs can
be detected in individual leafhoppers (13, 69) and aphids (91, 137) by
immunoassay. Acyrthosiphon solani, Myzus persicae, and Macrosiphum eu­
phorbiae collected from potato leaf roll-infected potatoes all tested positive
 426 MILLER & MARTIN

for potato leaf roll virus (PLRV) by ELISA, using monoclonal antibodies (R.
Martin, unpublished data) . However, of these aphids, only Myzus persicae
efficiently transmitted PLRV, which demonstrates that detection of pathogens
in insects is meaningless unless the insect can vector the pathogen. Similarly,
sensitive serological or cDNA probes used to detect other plant pathogens
must be used with the biology of the system in mind. Bursaphelenchus
xylophilus is a serious pathogen of conifers in Japan and recently also in the
South Central United States, and its presence in the northern United States,
Canada, and northern Europe in economically important conifer species has
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been confirmed recently ( l 23a). However, it does not cause disease in these
Annu. Rev. Phytopathol. 1988.26:409-432. Downloaded from www.annualreviews.org

areas, probably because the environment is too cold for replication at rates
required for disease development. As the sensitivity of detection increases,
many pathogens may be found at levels below economic thresholds. Where
inoculum-threshold data have been developed, assays that quantify pathogen
biomass can be used to determine the point at which control measures should
be implemented .

ACKNOWLEDGMENTS
The authors thank G . D. Grothaus, M . J . Klopmeyer, F. P. Petersen, J. H .
Rittenburg, R . K. Lankow, R . Stace-Smith, R . I . Hamilton, and P. Ellis for
helpful discussions and critical review of this manuscript.

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