Phytoparasitica 25:1, 1997 3
Molecular Detection Tools in Integrated Disease
Management: Overcoming Current Limitations
Taxonomy continues to be the cornerstone of any pest management research or
applications. It is imperative that pest organisms be identified properly so that judicious
use of the literature can be made and management strategies can be designed as quickly as
possible. Numerous pathogens are difficult to identify by morphological characteristics
and require extensive, time-consuming work with pure cultures and/or pathogenicity
tests. It is challenging for most plant pathologists to identify isolates routinely from
fungal genera such as Fusarium and Pythium because each includes a range of plant
pathogenic and saprophytic species. In entomology, great improvements in Integrated Pest
Management practices have been achieved by the use of pheromone traps which attract
only individuals from a single pest species. Mechanical trapping devices which capture
fungal spores are available to plant pathologists; however, the usefulness of these tools is
limited by the painstaking microscopic sorting and identification required after trapping.
Selective media can help but in most cases they go only as far as genus selectivity. Baiting
methods used for trapping motile stages of certain water moulds can reduce the range of
isolates to be sorted out but the selectivity of the baits used for the water moulds is poor
and the taxonomy of these fungi is so complex that identifying what colonized a given bait
still remains difficult. Technologies that would enable pathologists to identify pathogens
from plants, traps, baits or in soil samples rapidly and accurately would be very useful in
epidemiological and ecological studies as well as for detecting initial inoculum in disease
forecasting systems.
Although numerous publications describe novel molecular techniques for faster, more
accurate and reliable detection of pathogens, there are a few instances where these new
tools are being used in disease management. Applications and limitations of molecular
techniques for diagnosis of bacterial diseases were discussed recently (6). Are there recent
advances that might accelerate the adoption of molecular technologies into integrated
disease management programs? What are the challenges that these new technologies will
have to address to become more widely used?
The number of molecular detection techniques has increased to such an extent in
the recent past that some journals now refuse to publish incremental additions to these
technologies unless there is also some novelty in the techniques (2). Many of the published
reports described species-specific applications of the polymerase chain reaction (PCR), a
revolutionary technique by which specific regions of DNA are copied or amplified over a
million-fold (9). If the DNA of the pathogen is present, the species-specific primers (short
chains of nucleotides that determine the region of DNA to be copied) will bind to the DNA
and will be amplified during the PCR reaction. One can use multiple pairs of primers
in a single reaction and use the size difference of the PCR products for each different
pair to test for several pathogens in a single PCR reaction (16). An interesting variation
on this technology was developed recently. This technology, called TaqMan , uses
different fluorescent labels attached to nested primers and can detect as well as quantify in
a single reaction up to three pathogens at a time without having to run an agarose gel (1).
The hardware with this technology also allows dynamic monitoring of the PCR reaction,
facilitating quantitation, one of the most difficult issues with molecular detection systems.
A potential limitation of most current, antibody or DNA-based, detection technologies
is that only a single (or a few) species is detected per assay. Although convenient when
a large number of samples must be assessed for the presence of one pathogen, they are
inefficient when samples must be assessed for several different pathogens. Quarantine
testing, the first line of defense in pest management, is only one area that would benefit
greatly from the availability of multi-pathogen assays. As free-trade agreements between
countries become the norm, rapid testing for possible food contamination from a wide
range of quarantined organisms (nematodes, fungi, viruses, bacteria and even human
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pathogens such as amoebae) will be in high demand. For a wide range of disease
management applications, there is a need for comprehensive diagnostic kits that can detect
the presence of numerous pathogens in a single test. One could have a kit that could
identify all the pathogenic Fusarium species and formae speciales present in a given
sample. Kits could also be host-based by having the capability of concurrently testing
for all the key pathogens of a given host. Medical laboratories working on cystic fibrosis
have designed a method that can detect several disease-related mutations in a single assay
4 C.A. L´evesque
(7). This technique, called the reverse dot-blot, involves the use of multiplex PCR to
amplify and label simultaneously the regions of the DNA that contain the mutations. The
PCR products are then used as probes for hybridization with a membrane that contains an
array of oligonucleotides representing several different mutations and their corresponding
normal sequences. The hybridization results are read like a checklist, where the mutations
and normal alleles in a patient are determined from the positive reactions in the test. A
similar approach was designed to monitor bacteria from environmental samples (15). Such
approaches bring us closer to the kind of comprehensive pathogen testing that could lead
to new practical applications in integrated disease management.
Although computers have been used in molecular biology since its inception and are
now an integral component in data analysis and automation of complex tasks such as
automated sequencing, only recently have applications resulted from the direct merging
of microchip fabrication and DNA-based technologies (5). The goal of this merger is the
integration of all the steps of a diagnostic test (from DNA extraction and amplification
to hybridization and/or electrophoresis) into a microchip. Over 10 oligonucleotides/cm
can be attached to a solid support in arrays that can be used like the reverse dot-blot
assay discussed above (8). The microfabricated lab or ‘mini-lab on a microchip’ is only
a few years away. Recent developments are primarily targeting the medical field; however,
several applications could be adapted to plant pathology. The single most important
limitation in applying medical technology to plants occurs with DNA extraction. Plant
pathologists must extract DNA from substrates which vary greatly in composition: from
the soft, easily lysed tissue of ripe fruits to the hard, lignified tissue of trees. It is unlikely
that the automated DNA extraction devices being developed for human samples will be
easily adapted to plant tissues unless this technology expands in handling capacity to all
types of human tissue including bone. However, even if DNA extraction does not become
miniaturized, improvements to existing protocols to make them faster and cheaper are
being made regularly (13).
Many of the technologies discussed above are expensive to develop and to use. This
issue is particularly critical for developing countries that would probably benefit the most
from the new technologies because of the diversity of their pathosystems. The majority
of new applications in DNA-based technologies comes from medical research. Several
of these new technologies, including PCR, have been patented with human applications in
mind. Use of these technologies in the agricultural sector will necessitate the consideration
of market differences between human and agriculture diagnostics by the companies
granting licenses. However, one of the possible benefits from microfabrication technology
might be a reduction in costs. The replacement of expensive pieces of equipment with
disposable microchips, along with the drastically lowered (ca 10 ) requirements of
reagents, will aid in the implementation of these molecular diagnostic technologies to a
wider range of laboratories. The development of new tests will still depend largely on DNA
sequencing and oligonucleotide design: technologies that will likely be available only in
well funded laboratories.
Molecular diagnostic and detection techniques do what the name says: they detect
molecules. What is the significance of finding in a given sample some DNA known to
belong to a pathogen? The condition of these molecules is a function of the condition
of the organism from which they were isolated. Degradation of DNA varies depending
on soil type (10) and moisture. Slower rates of DNA degradation were observed in
desiccated soil than in control soil (4). Therefore, population studies of soil-dwelling
pathogens sampled from drier or cooler soils may be more biased towards detection of
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dead organisms than samples conducted in humid soils, where the DNA is more readily
degraded. Further data on DNA or RNA degradation in natural systems will be required to
improve correlation of molecular detection with populations of living organisms. Proper
controls will have to be incorporated to detect the PCR inhibition often observed with
processing of environmental samples. Protocols such as the BIO-PCR (12) can be used
to minimize this problem. Complex extrapolations will have to be performed with most
molecular technologies to make them quantitative, increasing the risk for error. The factors
used for these extrapolations can also have their own large variation, unlike traditional
techniques such as dilution plating, where the error of the dilution is negligible compared
with the biological variation in the propagule counts. In order to make proper use of
quantitative data from new detection technologies, it will be imperative to incorporate
proper error terms along with the quantitative estimates. Replication will always be
required for any population study no matter how sophisticated the technology.
GenBank, a widely used database of molecular sequences, has more than doubled its
entries in the past year alone (3). The new on-line phylogenetic taxonomy of the fungi,
one of the different ways to access GenBank through the World Wide Web, is a very
exciting recent innovation (14). As the database increases and as insightful interpretations
of the data allow updates of the on-line classification, integration of more organisms
into detection systems and identification of the causal agents of new diseases are likely
to become simpler tasks. The decline of financial support for taxonomic work will
undoubtedly have an impact on the analysis of the increasing body of molecular data and
ultimately on disease management. Some of these issues were recently reviewed (11).
However, one hopes that the negative impact of the worldwide decline in funding for
taxonomy and its related services will be somewhat mitigated by new technologies that
will facilitate routine detection and identification for disease management.
C. Andr´e L´evesque
Pacific Agri-Food Research Centre
Agriculture and Agri-Food Canada
Summerland, B.C., Canada, V0H 1Z0
[Fax: +1-250-494-0755; e-mail: levesqueca@em.agr.ca]
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