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ANNUAL
REVIEWS
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New Grower-Friendly
Methods for Plant
Pathogen Monitoring∗
Solke H. De Boer1 and Marı́a M. López2
1
Charlottetown Laboratory, Canadian Food Inspection Agency, Charlottetown, PE,
C1A 5T1 Canada; email: [email protected]
2
Plant Protection and Biotechnology Center, Instituto Valenciano de Investigaciones
Agrarias (IVIA), 46113 Moncada, Valencia, Spain; email: [email protected]
Annu. Rev. Phytopathol. 2012. 50:197–218
Keywords
First published online as a Review in Advance on
May 15, 2012
detection, diagnosis, identification, laboratory services, on-the-spot
diagnostics, surveillance
The Annual Review of Phytopathology is online at
phyto.annualreviews.org
This article’s doi:
10.1146/annurev-phyto-081211-172942
For the Canadian Food Inspection Agency
c 2012 Her Majesty The Queen in Right of
Canada (Canadian Food Inspection Agency).
All rights reserved. Use without permission is
prohibited.
0066-4286/12/0908-0197$20.00
∗
This paper was authored by an employee of the
Canadian Government as part of their official
duties and is therefore subject to Crown
Copyright.
Abstract
Accurate plant disease diagnoses and rapid detection and identification of plant pathogens are of utmost importance for controlling plant
diseases and mitigating the economic losses they incur. Technological
advances have increasingly simplified the tools available for the identification of pathogens to the extent that, in some cases, this can be done
directly by growers and producers themselves. Commercially available
immunoprinting kits and lateral flow devices (LFDs) for detection of
selected plant pathogens are among the first tools of what can be considered grower-friendly pathogen monitoring methods. Research efforts,
spurned on by point-of-care needs in the medical field, are paving the
way for the further development of on-the-spot diagnostics and multiplex technologies in plant pathology. Grower-friendly methods need
to be practical, robust, readily available, and cost-effective. Such methods are not restricted to on-the-spot testing but extend to laboratory
services, which are sometimes more practicable for growers, extension
agents, regulators, and other users of diagnostic tests.
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INTRODUCTION
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Accurate detection and identification of
pathogens underlie successful mitigation and
control measures for plant diseases. The history
of plant pathology has been shaped as much by
efforts to accurately identify and detect causal
agents of disease as it has by studies on the
ecology of disease-inciting microorganisms,
epidemiology, host-pathogen interactions,
and control methods. The chief means for
disease diagnosis and pathogen identification
has been description of symptoms of disease
and characterization of pathogenic species.
Disease diagnosis and pathogen detection and
identification have until recently been in the
domain of discipline specialists, taxonomists,
and plant pathologists. Led by developments
in point-of-care diagnostic devices in human
medicine, simplified testing devices using
serological or molecular strategies are now also
making their appearance for rapid plant disease
diagnosis directly in the field by nonexperts.
Technological developments suggest we are
on the cusp of a new paradigm in which
plant diseases can be quickly diagnosed in
cost-effective ways right in the field or simply
in locally accessible laboratory facilities. Also,
advancements in sample management and processing make large-scale testing increasingly
feasible.
WHO NEEDS GROWERFRIENDLY PATHOGEN
MONITORING METHODS?
The importance of timely and accurate plant
disease diagnoses has recently been ably
described (81). Reliable diagnosis is necessary
for disease management and decision-making,
but there is also a diagnostic component in
efforts to safeguard our plant resources and to
maintain biosecurity. The urgency for having
on-site diagnostic methods has dramatically
increased with the globalization of markets
for agricultural products, the exchange of
plant reproductive materials, and the need for
real-time information to meet the threats of old
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and emerging plant diseases. Direct or indirect
losses due to various invasive plant diseases
could be mitigated if grower-friendly methods
were utilized in strategies to prevent new
disease introductions and their progression
(6, 15, 111, 130). Also, the implementation
of Directive 2000/29 and its modifications
in the European Union (EU) have made it
necessary that accurate screening for a large
number of pathogens be made by public or
private laboratories or directly by the growers
themselves to obtain a phytosanitary passport.
Grower-friendly pathogen detection and
monitoring methods are just beginning to be
used, and although data on their actual usage is
still lacking, it is anticipated that it will dramatically increase. Availability of new grower- and
user-friendly tools is on the increase, and they
are being actively marketed to the agricultural
industry. Potential users cover a large spectrum
of activities ranging from those of regulatory
agencies to individual growers and producers.
Regulatory Agencies, Importers,
and Exporters
The lack of rapid methods for disease diagnosis and pathogen detection at international
borders results in unacceptable delays for
perishable material when samples must first be
sent to expert laboratories for testing (15). The
problems could be solved with simple devices
for on-site testing and the use of portable
equipment at the point of inspection. This is
especially important for quarantine pathogens
because the risk of their entry, establishment,
and spread must be minimized to avoid serious
economic consequences (55, 74). If a quarantine pathogen is detected in an imported
commodity, it is usually refused entry into
the country, causing immediate and serious
long-term economic consequences due to loss
of markets, increased competitiveness, and
more restrictive import requirements (15). To
protect their markets, importers usually prefer
not only to inspect the products visually but also
to test commodities for pathogens in the field,
in the packinghouse, or at the point of export.
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Both exporters and importers would benefit
from on-site diagnostic tools that can rapidly
distinguish symptoms caused by quarantine
organisms from those of lesser consequence.
To improve the efficiency of inspections, some
rapid test devices would be very useful, for
example, to differentiate Tomato mosaic virus
from Pepino mosaic virus in imported tomatoes
in the EU, and to distinguish symptoms of
Xanthomonas citri subsp. citri, causal agent of
citrus canker, from other fruit diseases in packinghouses to facilitate international trade (47).
Another example is the value of on-site testing
for rapid detection of Bursaphelenchus xylophilus,
the pine wood nematode, in materials such as
pallets, firewood, and wood chips (62).
Producers of Seed and Seed Potatoes
There are many old and emerging seedtransmitted diseases, especially among vegetable crops, for which simple rapid tests are
urgently required. New on-site methods being
officially considered for seed testing should be
validated by the International Seed Testing Association or another international organization,
but they could initially be used for preliminary
screening by seed companies directly in seed
production fields. Research on several fronts
has led to development of sampling protocols
and enhanced detection methods for several
seed-transmitted pathogens, such as Tilletia indica, causal agent of karnal bunt, that represent
a great risk for grain imports (116). Methods
for sampling airborne inoculum have also
advanced considerably in recent times, resulting in sophisticated but portable and flexible
systems adaptable for on-site testing (54, 133).
Potato is another crop for which various validated and established analytical protocols have
been developed for detecting viruses and other
pathogens (9, 12, 40). In the EU, compulsory
and validated protocols for sampling and analyses have been published for ring rot caused
by Clavibacter michiganensis subsp. sepedonicus
and brown rot caused by Ralstonia solanacearum
(34, 36), but they did not include on-site testing
methodologies. However, several commercial
on-site devices are now available for rapid
analysis of viral and bacterial infections in
symptomatic plants or tubers, although they
are not yet sensitive enough for detecting
latent infections. Field observation of visual
symptoms by growers and inspectors to meet
seed-potato certification requirements is widely
done and could be replaced to some extent by
analytical tests conducted directly in the field
or in a laboratory for increased accuracy (28).
Fruit Tree, Citrus, and Olive
Nurserymen and Orchardists
A long list of viral pathogens is included in
certification schemes for pome and stone fruit,
citrus, and olive trees in many countries because of the losses they cause (50). In addition,
there are also many pathogenic fungi and
bacteria that limit productivity of these crops.
Among them, Monilinia fruticola, Xanthomonas
arboricola pv. pruni, and Erwinia amylovora,
causal agents of peach brown rot, bacterial spot,
and fireblight, respectively, represent serious
threats to fruit tree crops. Guignardia citricarpa,
X. citri subsp. citri, and “Candidatus Liberibacter spp.,” causing black spot, citrus canker, and
huanglongbing (HLB), respectively, must also
be avoided in citrus nurseries and orchards.
Verticillium dahliae, which is responsible for
wilt of olive trees, is currently the biggest problem for olive nurseries and new plantations
(57, 87). Although in many cases only visual
inspections are used to assess disease status,
the availability of kits or protocols for their
rapid analysis would represent an important
benefit because of the role these could play
in preventing further economic losses caused
by the spread and dissemination of infected
material. Orchardists would also benefit from
the availability of field-friendly detection possibilities to identify the etiology of symptoms
more accurately and to facilitate monitoring
of pests, thereby improving the selection of
strategic control treatments. In particular,
simplified monitoring of viruliferous aphids,
i.e., aphid vectors carrying plant viruses, would
aid early detection and subsequent timely
implementation of control programs (18).
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Grapevine Nurserymen
and Viticulturists
Grapevine quality plays an important role
in wine production. Many nematodes, fungi,
bacteria, and viruses infecting grapevines can
affect yield and quality and consequently the
winemaking process. Although millions of
dollars are spent annually in grape-growing regions on fungicides to control fungal diseases,
accurate and rapid diagnosis of new diseases is
also required, particularly in new plantations,
to support control efforts for preventing or
delaying development of diseases that would
eventually kill the vines if left untreated.
Phaeomoniella chlamydospora, Phaeoacremonium
spp., Phomopsis spp., Botryosphaeria spp, Cylindrocarpon spp., and Campylocarpon spp., which
are agents of grapevine decline syndrome
(48, 49), and Agrobacterium vitis, Xylophylus
ampelinus, and Xylella fastidiosa, which are
agents of tumors, bacterial blight, and Pierce’s
disease, respectively, as well as Grapevine fan
leaf virus, Grapevine leaf rot virus 1–3, Grapevine
fleck virus, and Arabis mosaic virus require early
diagnosis to avoid their spread within and
among vineyards. Early and correct diagnosis,
as well as monitoring, of these and other
diseases is of utmost importance for those
who have responsibility for maintaining and
growing grapes for the highly valued wine and
table grape industries (48, 102, 132).
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PCR: polymerase
chain reaction
Extension Agents
Economic production of different crops
requires appropriate disease management systems, and extension services have responsibility
for providing advice about control programs.
To meet individual needs, rapid diagnosis
of disease and pathogen monitoring is most
essential (111). Calendar-based spray schedule
systems are seldom the most appropriate
control measure and are expensive (60). The
reduced benefits of unwarranted application
of pesticides to increase production, legislation limiting use of toxic products, and
chemical residue restrictions in food crops are
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increasingly leading extension services to use
the most precise tools possible for disease avoidance and control. This requires rapid and accurate diagnoses best achieved with on-site assays.
National Agencies
Preventing Bioterrorism
The role of first responders in the event of a biological attack on agricultural crops or forests
is of great importance. The tools required by
such first responders to make accurate identification of bioagents and their detection are
no different from those required by growers
guarding against unintentional invasion of plant
pathogens (41). Field portable real-time polymerase chain reaction (PCR) equipment would
be an appropriate analytic tool for responding
to this kind of attack, although various other
detection and identification technologies could
also be valuable if they were to be made available for potential bioterrorist candidates (104).
CHARACTERISTICS OF
GROWER-FRIENDLY PATHOGEN
MONITORING METHODS
Grower-friendly
methods
to
monitor
pathogens may mean different things to
different people. In this review, any method
for plant disease diagnosis, as well as for identification and detection of plant pathogens,
that is affordable and readily accessible to
producers of agricultural and ornamental
crops is considered to be grower-friendly.
This definition includes rapid methodologies
available through local laboratories and commercially available test devices. In the context
of considering what constitutes a suitable test,
it is important to reflect on the objective of the
test because the answer obtained is a function
of the question asked. The adage that “you
will only find what you are looking for” holds
true for many so-called diagnostic tests. For
example, an immunostrip for detection of
Hosta virus X (26) determines whether or not
the specific virus is present in the sample, and a
PCR test for C. michiganensis subsp. sepedonicus
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(66) determines whether the bacterial ring
rot pathogen is present at detectable levels in
the sample, but nothing else. Most PCR- and
immuno-based tests are targeted, i.e., they are
designed to detect or identify a specific plant
pathogenic species and answer the question:
Is this pathogen present? This is in contrast
to the question: What is this disease, or what
is causing these symptoms? To answer this
question, a diagnostic test is needed that
interrogates a number of different possibilities.
Array technologies are good diagnostic tools
because they can simultaneously interrogate
a large number of potential causal agents to
determine which of any number of pathogens
is present.
Grower-Friendly Field Methods
In the world of human medicine, point-of-care
service is an increasingly important concept
that is driving a move toward production of
test kits and devices that can be used by health
care givers or by patients themselves. Stimulated by the success of devices such as those
used to measure blood glucose levels and pregnancy tests, much research effort and many resources have been devoted to development of
in-the-field or on-the-spot diagnostics in plant
pathology.
There are a number of criteria that characterize tests considered to be grower friendly and
suitable for in-the-field use. A most important
one is cost effectiveness. No matter how good a
test may be, if it does not provide a grower with
information that enhances the sustainability
of his operation, it is not marketable. Closely
aligned with cost is a long shelf life. Detection
devices or kits need to be stable over an entire
growing season, or longer, because pathogen
testing cannot always be timed precisely. Other
essential features of grower-friendly methods
include simplicity and robustness. Manipulations in many of the newer detection methods
require accurate pipetting of very small volumes, which is not particularly easy to do in
a field environment. Consequently, growerfriendly methods must provide predispensed
reagents, have a minimum number of manipulative steps, and be sufficiently simple not to require extensive training. Tests need to be robust
in the sense that they need to remain unaffected
by external conditions such as temperature,
humidity, and dust. Grower-friendly and fieldadapted tests probably also ought to consist of
disposable units to avoid cross-contamination
between samples. Particularly when a test is inherently sensitive, as many of the tests involving
DNA amplification are, cross-contamination is
a considerably important problem. The need
to avoid cross-contamination pertains equally
to the process of acquiring and processing
samples as it does to the test itself.
Tests that take longer than an hour or so are
not practical for field use. Results of growerfriendly tests should be available rapidly, but
sensitivity is not necessarily paramount if
the purpose is disease diagnosis or pathogen
identification. Sensitivity is important if the
objective is detection to determine freedom
of seed or plants from a specific pathogen
and must be adequate to detect its presence
at economic levels. Often expensive and
technically demanding molecular methods are
required for accurate detection of pathogens
in asymptomatic plant samples (76). Specificity
is, of course, essential and needs to be broad
enough to detect all variants of the target taxon
but narrow enough to avoid cross-reactions
with nontarget organisms.
ELISA: enzymelinked immunosorbent
assay
Laboratory-Based Methods
Laboratory-based methods are included among
grower-friendly diagnostic and detection methods because sometimes it is more practical
for growers to have tests done by experts in a
laboratory than directly in the field. To be considered grower friendly, laboratory-based tests
also need to conform to certain requirements,
of which cost-effectiveness and accessibility are
key considerations. Low cost of testing can be
achieved in laboratories by parallel testing of
multiple samples for multiple targets. Enzymelinked immunosorbent assays (ELISAs) have
long been efficiently used to test multiple
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samples. Today, multiplex real-time PCR
configurations and microarray technologies
exemplify the kind of assays in which a large
number of samples can be tested for multiple
targets in a single run (73, 74, 77). A significant
advantage of laboratory-based testing is the
possibility of doing tests under a quality control
program. Although field-based testing units
can be manufactured under quality control, implementation of quality standards in field applications is challenging at best. Accuracy of field
test results is largely a function of the test’s robustness and simplicity. In a laboratory setting,
certification to the standards of the International Organization for Standardization (ISO)
provides a level of confidence not possible in the
field. Of course, results obtained by field testing
can always be confirmed by laboratory testing
if the seriousness of the disease/pathogen
warrants.
LFD: lateral flow
device
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LFI: lateral-flow
immunoassay
WHAT IS AVAILABLE NOW
Although conventional culture methods have
played a very important role in the detection and identification of plant pathogens, immunoassays are presently the basis of many
grower-friendly tests, and nucleic acid–based
methods are increasingly being used (76, 130).
Lateral Flow Devices
Perhaps the most readily available growerfriendly diagnostic tools today are lateral flow
devices (LFDs). The advantages of these devices are that they are simple to use and results
are quick, usually in less than 10 min. LFDs
have been particularly useful for the detection
of plant viruses, and for many of them, LFDs
are commercially available. They are based on
the serological specificity of polyclonal or monoclonal antibodies, and their general advantages and disadvantages have been discussed
elsewhere (98).
Although there are a number of different
immunoassay platforms, the LFD formats are
the most common ones that are commercially
available. LFDs typically consist of a porous
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nitrocellulose membrane bound to a narrow
plastic strip on which pathogen-specific antibodies are immobilized in a band partway up
the strip. Species-specific antibodies bound to
microparticles of latex, colloidal gold, or silica
are placed between the band of immobilized
antibodies and a sample application pad.
Immersion of the sample pad, located at one
end of the strip, in a sample suspension allows
sample liquid to wick up the strip by capillary
forces. Antigens of the target pathogen bind to
the particle-bound antibodies in the liquid flow
and carry them to the band of immobilized
antibodies trapping the specific targeted antigens along with the bound reporter particles.
Accumulation of bound particles makes the
band visible to the naked eye and is indicative
of a positive reaction (27, 98). Anti-antibody
immunoglobulin immobilized in a separate
distal zone of the membrane traps particles that
bypass the first band of immobilized antitarget
antibodies to serve as an internal control. Thus,
in the most common types of devices, two
bands appear on the lateral flow strip for positive samples, whereas only the positive control
band becomes visible for negative samples.
In another platform option, the nucleic acid
lateral-flow immunoassay (LFI), the analyte is
a target-specific, double-stranded nucleic acid
amplified with tagged primers, which is then
detected using tag-specific antibodies (98).
The sensitivity of LFDs varies with target
and type of antibody used. Monoclonal antibodies are well suited for such devices because
their specificity and properties can be tailored
to a considerable extent. An LFD-based test
using an IgM monoclonal antibody to detect
Rhizoctonia solani can detect as little as 3 ng ml−1
of antigen, equal to the sensitivity of standard
ELISA procedures (120). This study was particularly interesting because it targeted a soilborne
plant pathogenic fungus, whereas most commercial LFD-based tests target plant viruses
and bacterial pathogens for which specific antibodies are generally widely available. Development of species-specific antibodies to fungi has
been a greater challenge but, as noted above,
has been successfully achieved for some targets.
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Simplicity of LFD use is further enhanced
by the availability of LFD readers (38, 98).
Various systems have been devised for such
instruments based on handheld reflectometry,
charge-coupled device-based imaging systems,
scanners, or fluorescence visualizers. LFD
readers increase accuracy, provide potential
for quantitation, and allow electronic capture
of data (98). A portable LFD reader with numerical output and Bluetooth functionality to
export data is presently marketed for use with
some brands of plant pathogen–targeting LFDs
(http://www.forsitediagnostics.com/forsite_
lfdr101_reader).
Unfortunately, the relatively high cost per
LFD unit is a current limitation for their use
in routine analysis of large numbers of samples because they are designed for individual
tests rather than high-throughput screening.
The advantages of LFDs lie in the fact that
they are a one-step assay; are fast, simple, and
versatile; and have prolonged shelf life without refrigeration (98). The weakness of LFDs
is their general unsuitability for detecting latent infections because of their relatively low
sensitivity.
Luminex
Luminex X MAP is a registered multiplexing platform technology with capacity for
analyzing many analytes per test. It utilizes
color-coded microspheres covalently linked
to specifically designed reagents that capture
target pathogen–specific antigens or oligonucleotides. Addition of a labeled target-specific
probe to the microbead suspension after the
antigen capture step is completed allows
a dedicated analyzer to identify individual
microspheres with its captured antigen and
reporting probe (100). Luminex assays are
carried out in microplates with each well
handling a multiplicity of beads with different
specificities. Up to 100 parameters can be analyzed simultaneously in a single run. Although
Luminex technology is just beginning to be
used for plant pathology applications (9, 97),
its enormous multiplexing capacity and rapid
analysis make it as attractive a technology for
laboratory-based testing of grower samples
as it has been for medical samples in clinical
settings (http://www.luminexcorp.com).
Portable Polymerase Chain
Reaction Machines
LATE-PCR: linear
after the exponential
polymerase chain
reaction
PCR, first developed more than 30 years ago,
has been an enormously useful tool not only for
basic genomic research but also for detection
and identification (74, 77, 94). The Cepheid
SmartCycler was the first attempt to move PCR
technology (in real-time format) from the laboratory to the field. Its portability and compatibility with standard laptop computers provided
an opportunity to carry out PCR assays outside
of the conventional research laboratory but still
required a great deal of manipulation of very
small volumes of relatively unstable reagents
with a considerable degree of manual dexterity and expertise. The SmartCycler was used
experimentally for field detection of Phytophthora ramorum by PCR (123). The next level of
field-applicable PCR technology was a handheld real-time thermal cycler (51).
A fully field-compatible handheld PCR
device, called Bio-Seeq Plus, designed to
specifically detect and identify biowarfare
agents, such as anthrax, tularaemia, plague, and
orthopox, is commercially available. The device
uses a PCR technology known as LATE-PCR
(linear after the exponential PCR), an asymmetrical method that generates single-stranded
DNA products but is rationalized to improve
efficiency to the level of symmetrical PCR
by optimizing primer melting temperature
and concentrations (103). Designed for the
nontechnical user, Bio-Seeq Plus can be used in
the field, is battery powered, and provides results in approximately one hour (http://www.
smithsdetection.com/Bio-Seeq_PLUS.php).
Data generated using the Bio-Seeq compared
favorably with laboratory-based PCR, suggesting that the handheld unit has promise for
detecting microorganisms in field applications
(30). Because of the complexity and cost of the
device, its application has been restricted to
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highly dangerous microorganisms and would,
no doubt, be far too costly for routine use
in agriculture. Just recently the Palm PCR,
another handheld PCR device, has appeared
on the market, but its utility for plant pathological applications still needs to be tested
(http://www.ahrambio.com).
Laboratory Services
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Although the number of private companies
offering identification services for plant
pathogens is increasing, the market is not as
large as that for human pathogens, and their
number remains limited. Private companies
generally offer on-demand services and follow
either their own protocols or official ones
developed by various international bodies.
As the recommended test in many cases is a
PCR-based method, the nucleic acid extraction
procedure is the bottleneck for rapid and
low-cost analyses (73). However, other simpler
approaches include tests such as the growerfriendly tissue printing or squash tests for
viruses and bacteria in which freshly cut stems
of infected plants or arthropods are imprinted
or squashed onto membranes for development
by a serological or molecular-based protocol
(19). Field-printed samples can be sent to a
laboratory for immediate processing or stored
at room temperature for up to several years,
making this an extremely useful and safe
method for handling samples suspected of
being infected with quarantine organisms.
Laboratories usually perform analyses using
only a single method because of cost, although
an integrated approach that combines several
techniques for maximum accuracy is advised
(5, 130). For example, two positive test results,
based on different methodologies, are required
in Canada for a positive potato bacterial
ring rot diagnosis (28). Most European and
Mediterranean Plant Protection Organization
(EPPO) protocols also require the use of two
different techniques to determine officially the
presence or absence of a particular pathogen.
This approach to pathogen testing ensures
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greater reliability for determining the presence
of a pathogen or the health status of a plant
consignment (76, 128).
There is a need for accreditation of private laboratories and international agreement
on required standards. A recent initiative is
the business chain system Good Seed and
Plant Practices for analyses of tomato seeds
for C. michiganensis subsp. michiganensis in
which laboratories are audited by certifying
organizations in France and the Netherlands
(http://www.gspp.eu). Also in several EU
countries, private laboratories have official accreditation for performing analyses
of C. michiganensis subsp. michiganensis and
R. solanacearum, following the official EU protocols (34, 35). In Canada, several private companies test for potato pathogens to ISO standards under the auspices of the national plant
protection organization (28).
WHAT ABOUT PRACTICAL
EXPERIENCES?
The Case of Tristeza
(Citrus Tristeza Virus)
Citrus tristeza virus (CTV), one of the most
harmful viral diseases of citrus, causes decline
or tristeza syndrome, a bud-union disease,
which occurs when scions from an infected
cultivar are grafted on sour orange rootstock.
Aggressive CTV strains can also cause stem
pitting and cause poor production and fruit
quality. CTV is often introduced into new
geographic areas with imported budwood.
Preventive control of CTV is based on
strict quarantine regulations and certification
programs that limit grafting of virus-free
scions onto CTV-tolerant rootstocks (129).
Availability of sensitive, specific, and reliable
CTV detection methods is critically important for these programs. Indexing assays
on Mexican lime seedlings is sensitive, but
the procedure takes too long (more than
six months) and is expensive. Conventional
ELISA or tissue-printing ELISA with specific
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monoclonal antibodies is recommended in
the EPPO protocol (31). In fact, the direct
tissue blot immunoassay (DTBIA) (71), also
called direct immuno-printing-ELISA or
tissue print–ELISA (21, 44), is one of the main
strategies for avoiding trade in CTV-infected
plants. More than four million DTBIA tests
have been done in Spain since 1994 by citrus
nurserymen themselves after a complete tissue
print-ELISA kit (PlantPrint Diagnostics,
http://www.plantprint.net) became available
(20). This grower-friendly approach popularized accurate CTV detection and greatly
contributed to the well-being of the economically important citrus industry. Recently,
several real-time reverse transcriptase (RT)PCR-based protocols were developed to detect
CTV directly in plant tissues and aphid vectors
without the need for preparation of plant
extracts or purifying nucleic acids (10, 18, 128).
These user-friendly direct methods of sample
preparation for molecular assays that aim to
reduce the time and cost of analyses have been
validated, and their diagnostic parameters have
been calculated for large-scale use (22, 128).
The Case of Sharka (Plum Pox Virus)
Sharka caused by the Plum pox virus (PPV) is a
devastating disease of stone fruit trees, such as
apricot, peach, and plum. The main pathway
for new PPV introductions is long-distance
transport of infected plant material. Once the
virus is introduced into an area with susceptible
Prunus hosts, it spreads locally by aphid transmission, and there are few control possibilities,
so eradication is advised (43). The need for
simple but accurate and validated methods for
PPV diagnosis and detection is crucial, not only
for analyses at the points of entry but also for
eradication programs to prevent the spread of
sharka disease. The first method developed for
screening large numbers of samples was based
on double antibody sandwich (DAS)- and double antibody sandwich indirect (DASI)-ELISA
using specific monoclonal antibodies (19, 33).
Recent PPV eradication programs in Canada
and the United States were based on results
from several million of such tests and exemplify
the need for accurate routine laboratory assays
(119). A highly efficient user-friendly real-time
RT-PCR technique in which crude plant extracts or spots immobilized on membranes can
be tested directly has now also been validated
and is one of the selected techniques in the new
International Plant Protection Convention
(IPPC) protocol for PPV detection (22, 53,
128).
The Case of Fireblight
(Erwinia amylovora)
E. amylovora, the bacterial agent of fireblight
of Rosaceae species, is a quarantine pathogen in
the EU, Australia, and many countries in South
America, Africa, and Asia. Fireblight is a major
economic threat for pear, apple, quince, and loquat production, requiring efficient phytosanitary controls at the orchard, nursery, and border
levels. The eradication programs in several EU
countries and restrictions in international trade
of fruits and plants from areas where the disease
occurs justify the development of new tools for
its rapid detection. Current diagnostic methods
for fireblight follow international standards,
such as those of EPPO, and are based on isolation, immunofluorescence, ELISA, and PCR
(32). The new EPPO and IPPC protocols being
prepared will include two commercial LFD devices (http://www.bioreba.ch, http://www.
pocketdiagnostic.com), which were ring
tested by 14 laboratories (75). Both LFDs
used polyclonal antibodies, although at least
one of them cross-reacted with a closely
related Erwinia species (16). A loop-mediated
isothermal amplification (LAMP) protocol has
also been evaluated for fireblight detection
(118). Because these methods all have a sensitivity of approximately 105 –106 CFU ml−1 ,
they are recommended only for use on symptomatic plant tissues (16). More sensitive
diagnostic tools for detecting latent infections
of E. amylovora would be most welcome for
surveillance and monitoring applications.
www.annualreviews.org • Plant Pathogen Monitoring
DTBIA: direct tissue
blot immunoassay
RT-PCR: reverse
transcriptase
polymerase chain
reaction
DAS-ELISA: double
antibody sandwich
ELISA
DASI-ELISA: double
antibody sandwich
indirect ELISA
LAMP: loopmediated isothermal
amplification
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The Case of Huanglongbing or
Citrus Greening (“Candidatus
Liberibacter spp.”)
M.M. Lopez, S. Lopes, J. Avres, and J. Bove,
unpublished results).
HLB, also called citrus greening, is currently
considered one of the most destructive diseases
of citrus and its relatives, being responsible for
yield reductions reaching as high as 100%. The
disease is associated with three different species
of the phloem-limited bacterium, “Candidatus
Liberibacter asiaticus,” “C. L. africanus,”
and “C. L. americanus,” and occurs in Asia,
Africa, Central and South America, and the
United States (14, 46, 65). The bacterium is
transmitted by the psyllid vectors Diaphorina
citri and/or Trioza erytreae. Although HLB is
difficult to detect because the disease displays
a long latency period and the bacterium is
unevenly distributed at low concentrations in
the plant, the application of accurate diagnostic methods plays a very significant role in
eradicating and delimiting disease outbreaks.
Initially diagnosed by biological indexing, electron microscopy, and serological techniques,
current HLB diagnosis relies on PCR and
real-time PCR analyses. In Brazil and Florida,
several PCR protocols have been developed
and evaluated but not comparatively (24). A
1-h real-time PCR assay including a plant
cytochrome oxidase–based primer probe as
an internal control has been optimized on a
portable PCR machine for quantification of the
bacterium in host and vector samples (65, 79).
A LAMP assay has also been developed (92).
In citrus-producing areas of Florida and Brazil,
all plants from nurseries and other propagation
sources as well as suspect trees in orchards are
tested annually for “C. L. asiaticus” to prevent
the spread of HLB. Taken together, the tests
conducted for regulatory control of HLB probably constitute the greatest use of real-time
PCR assays in plant disease control today. Recently, a real-time PCR procedure for analysis
of membrane-spotted or squashed preparations
of plant samples and individual psyllid vectors,
similar to the test for CTV, has been developed
as a more grower-friendly option to monitor for
the presence of HLB (E. Bertolini, M. Cambra,
The Case of Potato Diseases
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Potato is plagued with more regulated diseases
than probably any other crop because it is an
annual crop propagated by vegetative tubers.
Potato crops designated for use as seed for the
subsequent production of table and processing
crops are usually grown under the auspices of
a seed potato program in which the health status of the crop is closely monitored. Although
initially based solely on visual crop inspection,
increasingly seed potato programs utilize diagnostic laboratory tests. Laboratory-based tests,
and in some cases field tests, are available for
detecting the common potato viruses, such as
potato viruses A, S, X, and Y; potato leaf roll
virus; potato mop top virus; and the potato
spindle tuber viroid. In addition to seed certification, diagnostic tests are also increasingly
used to ensure seed potatoes meet the requirements of export markets. Extensive use of diagnostic tests is also made to prevent the spread
of regulated bacterial diseases, such as brown
rot (R. solanacearum) in Europe and bacterial
ring rot (C. michiganensis subsp. sepedonicus) in
Canada (28, 55). In regions where potato wart
(Synchytrium endobioticum) and potato cyst nematodes (Globodera rostochiensis and Globodera
pallida) occur, a considerable amount of soil
testing is conducted to delimit the areas infected
with these quarantine pests (8). The ongoing
need for diagnostic tests in the potato industry
drives the market for increasingly simpler and
more grower-friendly technologies that can be
used directly by the producer and by certification agencies to meet production, regulatory,
and market requirements.
WHAT THE FUTURE HOLDS
On-the-Spot Diagnostics
It is anticipated that diagnosis of plant diseases and identification of pathogens will be
revolutionized over the next decade or so by
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developments in on-the-spot testing technologies. Although the appearance of LFDs has
signaled the beginning and is likely to remain
an important and valued technology, it is likely
that new tests will make use of molecular DNA
amplification strategies. However, some major
challenges to bringing the lab-on-a-chip and
similar ideas to reality still need to be resolved.
Challenges include sample preparation methods, system integration strategies, packaging,
reagent storage, temperature maintenance
during field operations, and detector technology (37). Although impediments to resolving
outstanding issues are not to be minimized,
significant progress is being made on an ongoing basis. A recent example of progress is the
stabilization of PCR reagents by their encapsulation in paraffin and subsequent integration
into a self-contained polycarbonate PCR chip
(63). Several research groups have developed
prototypes of handheld devices that illustrate
the practicality of DNA amplification and
characterization away from laboratory settings.
For example, a prototype handheld PCR-based
device containing a disposable cassette that
performs sample lysis, nucleic acid isolation,
DNA amplification by PCR, and amplicon
labeling and detection was developed for pointof-care pathogen detection (23). The cassette
integrated all the components necessary for
the reaction, including liquid storage and
pumping, solid-phase nucleic acid isolation,
dry storage of PCR reagents and their release,
temperature cycling, and lateral flow amplicon
detection. Subsequently, a disposable cassette
with innovative thermally activated valves was
developed for integrated detection of microbial
and viral targets (72). Another group developed
a prototype instrument in which a three-layer
microfluidic chip with a flexible membrane was
used for pumping and valving blood samples
to deliver them into a microchip chamber for
DNA amplification (78). Probably with some
modification in sample processing, ideas developed for blood or human tissue testing could
be directly usable for agricultural applications.
It would be advantageous to automate
the diagnostic process by incorporation of a
sample processing bioseparator directly on a
microfluidic platform. Microchannel-based
fluidics and its newer relative, digital microfluidics, hold promise for more sophisticated
devices to manipulate reagents and samples
(1). Extraction of complex samples using
microbeads either by immunological binding
or hybridization of DNA and integration into
a multifunctional microfluidic device is being
explored on several fronts. As in LFDs and
Luminex technology, the manipulation of
antigen-carrying magnetic beads and separation of them from supernatant suspensions
are extremely sensitive and selective extraction
strategies. Reducing bead size to nano-formats
optimizes analyte capture even further (42).
Already a multifunctional microfluidic device
has been developed that integrates a bioseparator using antibody-coated magnetic beads, a
micropump, a micromixer, and a microreaction
chamber for interrogating leukocytes for single
nucleotide polymorphisms (67). In another
approach to the problem of sample processing,
solid phase extraction of nucleic acid using a
nanoporous aluminum oxide membrane was
used for samples suspected of containing target
bacteria and/or viruses (64). The nucleic acids
in the lysate bind to the membrane, are washed,
and are then eluted directly into a PCR reaction
chamber. Although such applications are still
far removed from diagnostic field application
in agricultural settings, they illustrate the
technology being developed that potentially
can be integrated into on-the-spot diagnostic
tools.
Is it realistic to expect nucleic acid amplification technologies to become suitable for
on-the-spot diagnosis of plant diseases and
detection or identification of plant pathogens?
The answer seems to be a resounding yes. But
it is likely neither to be conventional PCR nor
any of its real-time variants. The immediate
future is more likely to lie with one of the
isothermal amplification strategies. Although
PCR has been a mainstay in pathogen detection, its need for rate-limiting temperature
cycling makes it less useful than isothermal
methods in terms of simplicity and speed.
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NASBA: nucleic acid
sequence-based
amplification
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Transcription-based amplification methods,
such as NASBA (nucleic acid sequence-based
amplification), requiring three enzymes to
sustain the reaction (25), have been simplified
recently (106). Yet other methods involve the
use of a strand displacement polymerase to
drive self-sustained sequence replication (131).
The LAMP technique utilizes a strand displacement strategy and is one of the most prominent
of these methods explored to date (91).
LAMP assays have been developed for all the
major classes of plant pathogenic microorganisms, including viroids, viruses, phytoplasma,
bacteria, fungi, and nematodes. The method
as first described by Notomi et al. (91) makes
use of four specially designed primers, a pair
of outer primers and a pair of inner primers,
which together recognize six distinct sites
flanking the amplified DNA sequence. In
place of heat denaturation of double-stranded
DNA as in PCR, the LAMP reaction relies
on a DNA polymerase with particularly high
strand displacement activity to remove newly
amplified strands. Because of the hybrid design
of the inner primers, amplified DNA structures
take on a loop configuration at one or both
ends of the elongated strands, which in turn
serve as stem-loop structured templates for
further displacement DNA synthesis. The final
amplified product consists of a mixture of stemloop DNA strands with various stem lengths
and structures with multiple loops. The rate
of DNA amplification can be accelerated, and
specificity increased, by additional loop primers
designed to hybridize to the loop structure and
uniquely amplify the stem-loop strands (84).
The LAMP assay has captured the imagination of many in the world of diagnostics because
its specific advantages make it suitable for adoption in on-the-spot testing devices (122). It can
be done in an essentially equipment-free environment. Only a water bath or heating block is
needed to maintain temperature in the range
of 60–68◦ C. LAMP assays are particularly
specific because hybridization to at least six
sites is required for the reaction to proceed and,
as with other DNA amplification methods,
De Boer
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López
the LAMP assay has exquisite sensitivity. A
detection level as low as 2 pg μl−1 of template DNA was achieved to detect latent
stage wheat stripe rust infections (52), and
E. amylovora could be detected at a sensitivity
of 102 –103 CFU per apple or pear flower (117).
Moreover, the rapid accumulation of multiple
amplification products yielding >500 μg ml−1
in 30–60 min is a significant advantage for
detection of end products (84, 121). Methods
for end-product analysis include direct visualization under ultraviolet light by addition
of DNA-intercalating fluorescent dyes and, in
visible light using other colorimetric procedures (45), measurement of turbidity caused by
accumulation of insoluble salts from reaction
of metal ions with the pyrophosphate ion (121),
hybridization of single-stranded amplicons to
DNA microarrays (85), and detection with
electrochemical sensors (3). LAMP assays can
also be configured in real-time quantitative
formats (7).
Another attractive feature of the LAMP
method for on-site testing is its tolerance to
template contaminants (58). In fact, for LAMP
assays such as the reverse transcription-LAMP
(RT-LAMP) test for peach latent mosaic viroid,
the sample template simply consists of traces
of plant sap eluted from a toothpick pricked
into a symptomatic leaf (13), and for potato
spindle tuber viroid the template consists of
plant sap picked up on insect pins that have
been stabbed into infected tubers 3–10 times
(124). Similarly, RT-LAMP assays for Tomato
yellow leaf curl virus and Melon yellow spot virus
use toothpicks for sample template acquisition
(107, 114). A commercial LAMP test kit even
recommends sampling by toothpicks for detecting Tomato yellow curl leaf virus in tomato (89). In
some instances, an undenatured template could
be amplified directly by LAMP, even further
simplifying the process (86). One of the downsides of LAMP assays is the complexity of the
primers required to drive the amplification reaction, but the burden of primer selection and
design is largely mitigated by readily available
software (http://primerexplorer.jp/e/).
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LAMP is not the only isothermal procedure
with potential for point-of-care and on-thespot detection. Another strategy involves linear
amplification with a nicking enzyme to cleave
off short oligonucleotides that are unstable
and dissociate readily from the complementary
strand to serve as new primer template (61,
127). Prototype assays exploiting NEAR (nicking enzyme amplification reaction) technology
could detect less than 50 genome copies in
5–10 min, showed little inhibitory effects
from sample template contaminants, and were
compatible with multiple readout options, including fluorescence detection and lateral flow
strips housed in a self-contained device (110).
Commercial exploitation of NEAR technology for plant-associated applications including
pathogen detection is in progress (http://www.
envirologix.com/artman/publish/article_
314.shtml).
ICAN (isothermal and chimeric primerinitiated amplification of nucleic acids) is another novel isothermic DNA amplification
strategy that has been investigated for rapid and
simple diagnostic purposes (82). ICAN is based
on a pair of 5 -DNA-RNA-3 chimeric primers,
a thermostable RNaseH, and a strand displacing DNA polymerase that drive two reactions—
a multipriming and a template-switching reaction (125). The ICAN strategy has been used
in a rapid and sensitive test for the detection
of citrus HLB disease and is particularly useful for pathogen targets with limited sequence
data (126). Sequence data for only two primer
regions is required, but the speed of the test,
specificity of amplification, and isothermic requirements make it a better candidate for testing field samples than PCR.
Array Technologies
Array technology refers to reverse dot blot
assays in which assorted DNA probes, rather
than sample DNA, are bound to a fixed matrix,
such as a nylon membrane or microscope
slides for microarrays, in a highly regular
pattern, allowing samples to be characterized
based on the probes to which they hybridize.
Detection of hybridization signals is achieved
by one of the various reporting strategies, such
as labeling target DNA with an identifiable
moiety. Additional options and specifics of
array development have been described (83).
The potential of array technology for plant
disease diagnosis and pathogen identification
lies in its marvelous multiplexing capabilities
(11, 12, 70). DNA macro- or microarrays have
the potential to simultaneously detect many
pathogens at one time and thus are suitable
techniques for high-throughput detection and
identification, particularly because they can
discriminate single nucleotide polymorphisms
and so distinguish among genomically similar
pathogens that may have very different host
specificity or virulence characteristics (69). Because the number of pathogen probes that can
be placed on a single array is almost limitless, it
can answer questions of “what is this?” and can
identify unknowns without the need for prior
knowledge. To date, DNA arrays have been
used to identify pathogens in commodities
such as potato, tomato, and apple (40, 68,
108), as well as to distinguish among strains
of regulated bacterial phytopathogens, differentiate species of Fusarium and Pythium, and
discriminate genotypes of Plum pox virus (95,
96, 115, 134). If concentration of target DNA
is high, direct hybridization of DNA extracts
without amplification may be practicable and
certainly desirable to avoid amplification bias
and dependence on primer specificity. Amplification is usually required, however, to enrich
the relative concentration of target DNA in the
sample and is facilitated by directing the test to
commonly present genes, such as the internal
spacer region of the ribosomal operon (40).
The potential of DNA array technology for
on-the-spot plant disease diagnosis has clearly
not yet been realized, and the future of planar
arrays for extensive application in plant pathology has been questioned on account of their
cost and limited capacity for sample throughput (17). However, innovative and inventive improvements in array technology are ongoing
www.annualreviews.org • Plant Pathogen Monitoring
NEAR: nicking
enzyme amplification
reaction
ICAN: isothermal
and chimeric
primer-initiated
amplification of
nucleic acids
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and bode well for the future. Alternate formats,
such as bead arrays, circumvent some of the limitations of planar arrays yet utilize the enormous
breadth of array technology. But even planar arrays are getting better by enhancing hybridization efficiency with longer dimeric probes (90)
or robustness and specificity by using cleavable
padlock probes (113) that require perfect target
hybridization to initiate a cascade of reactions
leading to visualization of positive responses.
Sensitive detection of positive responses can be
achieved by various strategies, including those
that do not require specific reporter molecules,
thereby setting the stage for the development of
highly specific and sensitive detection of unamplified pathogen DNA. One study on electrochemical biosensors used Agrobacterium tumefaciens DNA as the target for self-complementary
probes bound directly to electrode surfaces.
The probes behaved like molecular beacons, in
that hybridization of target DNA disrupted the
self-hybridization, causing it to undergo a conformational change, which resulted in diminished electron transfer efficiency, allowing direct measurement of hybridization as a drop in
peak redox current (56).
Arrays are not limited to those based on
DNA hybridization but can also be based on
protein interactions. Experimental work, for
instance, is being done on an electrochemical
immunosensor utilizing nanoparticles functionalized by targeted antibodies that when
immobilized onto a conductometric transducer
form an extremely sensitive biosensor for
bacterial and viral detection (42). However, to
format arrays, either in macro- or microconfiguration, into practical, robust devices usable
by nontechnical persons in field environments
remains a significant challenge. A particular
challenge is to simplify all of the functions
of testing devices to allow production at a
cost compatible with the cost savings realized
through knowledge attained by their use.
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Other Potential Technologies
The unprecedented taxonomic power of DNA
barcoding is very much applicable to the rapid
210
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and accurate identification of plant pathogens.
In DNA barcoding, short genome sequences,
such as those of the internal transcribed spacer
cistron, β-tubulin A gene, or the cytochrome
c oxidase 1 gene (CO1), are determined
for unknown samples and compared with
database sequences to establish the identity
of organisms (109). CO1 has been especially
useful for the identification of insect pests,
algae, and higher plants but has also already
been applied for the identification of fungal
plant pathogens (39, 109). A desire for more
rapid, efficient, and accurate identification
of plant pathogens has been the driver for
several consortiums coordinating development
of DNA barcode databases, such as Quarantine Barcoding of Life (http://www.qbol.
wur.nl), Plant Pathogen Barcode (http://www.
plantpathogenbarcode.org), and Plant Associated and Environmental Microbes Database
(http://genome.ppws.vt.edu/cgi-bin/MLST/
home.pl) (4). However, problems with taxonomic reliability and insufficient annotation
of fungal sequences in public DNA repositories may ultimately limit the usefulness of
sequence-based fungal identification (88).
Next-generation sequencing methods for
identifying microorganisms present in microbiomes of diseased plant tissue, as is being done
for other microbially complex milieus such as
endophyte communities in plant roots (80), are
becoming an increasingly realistic possibility.
Next-generation sequencing has already been
applied in plant virology to detect the presence
of unknown viruses by metagenomic analysis
of the entire plant genome along with any infecting viral genomes (2, 112). This approach is
heavily dependent on software discriminating
between host plant and viral sequences, along
with extensive databases to confirm virus identities. A concern with sequence-based identification is the erroneous and incomplete data
that tend to plague large reference databases
on which the procedures are entirely dependent
(59).
When prior knowledge of specific agents of
disease is entirely lacking, it may be beneficial
to detect and identify all the pathogens that are
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present in a sample. In such cases, DNA amplification methods with broad specificities may be
applicable to obtain information-rich mixtures
of amplicons analyzable by a universal biosensor
approach. Mass spectrometry can unambiguously measure the composition of nucleotides
in PCR amplicons, providing sufficient information to identify microorganisms to the
species level (29). Electrospray ionization-mass
spectrometry has already been used to analyze
the molecular mass of broad-range PCR products with a sufficiently high degree of precision
to unambiguously derive base composition
(99). Although the base order is not determined
by this method, organisms can be accurately
identified by matching base composition to
an appropriate database. The rapidity of the
method allows analysis of many PCR products
of protein-coding and housekeeping genes for
increased accuracy of diagnostic results. Mass
spectrometry measuring mass/charge ratio
fingerprints of proteins can also be used to
identify microorganisms rapidly in pure culture
(http://www.bdal.com/products.html). Single colonies of bacteria were reliably identified
in minutes using a procedure known as matrixassisted laser desorption ionization–time of
flight mass spectrometry (101).
CONCLUSION
Presently, highly specific methods can be developed rapidly for detecting and identifying plant
pathogens whenever a serious phytosanitary issue arises. Implementation of new technologies for routine use, however, has been delayed
by technical challenges and the cost factor. A
particular challenge has been the development
of simple, accurate, low-cost tests that growers
and producers can use routinely to improve production practices. Although no single method
can be used universally for plant disease diagnosis, the range of possible technologies is increasing, and in the near future there may be a choice
of rapid methods. Recent plant pathology textbooks (e.g., 105) already include LFDs or immunostrips as diagnostic tools because their use
is rapidly expanding, and low-cost multiplex
molecular-based testing methods may be available soon. It will be necessary to determine diagnostic parameters, such as sensitivity, specificity, and error rates, to validate new detection
and diagnostic methods. With the advent of
better and cheaper detection and identification
tools, the impact of increased testing, earlier
detection, and knowledge gained about disease
prevalence will need to be carefully assessed to
maximize their benefit (93, 129).
SUMMARY POINTS
1. Grower-friendly pathogen monitoring methods include on-the-spot or in-the-field testing devices as well as readily accessible laboratory services.
2. In order to be grower-friendly, pathogen monitoring methods must be cost-effective,
simple to use, robust, and rapid as well as have a long shelf life and appropriate specificity,
whereas the need for sensitivity depends on the purpose of the application.
3. In addition to growers and producers, rapid and easy-to-use pathogen detection methods
are also needed by regulatory agencies, exporters, importers, extension agents, and first
responders to crop-directed bioterrorist activity.
4. Pathogen monitoring methods are already being used in the agricultural industry, particularly in the fruit tree, citrus, and potato industries in which crops are propagated from
vegetative plant parts.
5. Lateral flow testing devices represent the type of grower-friendly method currently
widely available, whereas other technologies, such as Luminex and portable PCR machines, are available to growers only to a limited extent and are largely laboratory based.
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6. A lot of research is being devoted to the development of on-the-spot diagnostic devices using DNA amplification strategies, such as the isothermal LAMP method, and
multiplexing strategies, such as macro- and microarray technologies.
7. New technologies involving genomic barcoding, next-generation sequencing, biosensors, and mass spectrometry are also being explored for their application as diagnostic
and pathogen identification tools.
FUTURE ISSUES
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1. Increased usage of handheld devices for rapid diagnosis of plant diseases and identification of plant pathogens will require the development of additional pathogen-specific
primers and/or antibodies to address the many pathogens for which tools are currently
unavailable.
2. Accurate and reliable gene sequence databases need to be developed for plant-associated
microorganisms to support sequence-based diagnostic and identification schemes at an
international level.
3. New quantitative and multiplex formats for LFDs, biosensor technologies, and mass
spectrometric methods should be further explored to increase the accuracy and speed of
plant pathogen detection.
4. The reliability of each specific on-the-spot diagnostic method needs to be validated
before results are used exclusively to implement costly disease control strategies and/or
regulatory actions.
5. Sampling schemes need to be devised and laboratory-based assays formatted to provide
accurate data on pathogen or disease incidence, rather than only their presence or absence,
in a cost-effective manner.
6. With increased potential for determining presence of pathogens, economic thresholds
need to be established to effectively minimize pathogen dispersal without creating unnecessary obstacles to international trade in agricultural products.
DISCLOSURE STATEMENT
The authors are not aware of any affiliations, memberships, funding, or financial holdings that
might be perceived as affecting the objectivity of this review.
ACKNOWLEDGMENTS
The authors are grateful to M. Rott, M. Cambra, and A. Vicent for critical reading and valuable
suggestions to improve the manuscript. M.M. Lopez thanks the Spanish projects AGL200805723-C02-01/AGR and RTA2011-00140-C03 for funds.
LITERATURE CITED
1. Abdelgawad M, Wheeler AR. 2009. The digital revolution: a new paradigm for microfluidics. Adv. Mater.
21:920–25
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Contents
Annual Review of
Phytopathology
Volume 50, 2012
An Ideal Job
Kurt J. Leonard p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 1
Arthur Kelman: Tribute and Remembrance
Luis Sequeira p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p15
Stagonospora nodorum: From Pathology to Genomics
and Host Resistance
Richard P. Oliver, Timothy L. Friesen, Justin D. Faris, and Peter S. Solomon p p p p p p p p p p23
Apple Replant Disease: Role of Microbial Ecology
in Cause and Control
Mark Mazzola and Luisa M. Manici p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p45
Pathogenomics of the Ralstonia solanacearum Species Complex
Stéphane Genin and Timothy P. Denny p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p67
The Genomics of Obligate (and Nonobligate) Biotrophs
Pietro D. Spanu p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p91
Genome-Enabled Perspectives on the Composition, Evolution, and
Expression of Virulence Determinants in Bacterial Plant Pathogens
Magdalen Lindeberg p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 111
Suppressive Composts: Microbial Ecology Links Between Abiotic
Environments and Healthy Plants
Yitzhak Hadar and Kalliope K. Papadopoulou p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 133
Plant Defense Compounds: Systems Approaches to Metabolic Analysis
Daniel J. Kliebenstein p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 155
Role of Nematode Peptides and Other Small Molecules
in Plant Parasitism
Melissa G. Mitchum, Xiaohong Wang, Jianying Wang, and Eric L. Davis p p p p p p p p p p p p 175
New Grower-Friendly Methods for Plant Pathogen Monitoring
Solke H. De Boer and Marı́a M. López p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 197
Somatic Hybridization in the Uredinales
Robert F. Park and Colin R. Wellings p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 219
v
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Interrelationships of Food Safety and Plant Pathology: The Life Cycle
of Human Pathogens on Plants
Jeri D. Barak and Brenda K. Schroeder p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 241
Plant Immunity to Necrotrophs
Tesfaye Mengiste p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 267
Mechanisms and Evolution of Virulence in Oomycetes
Rays H.Y. Jiang and Brett M. Tyler p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 295
Variation and Selection of Quantitative Traits in Plant Pathogens
Christian Lannou p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 319
Annu. Rev. Phytopathol. 2012.50:197-218. Downloaded from www.annualreviews.org
by UNIVERSITY OF FLORIDA - Smathers Library on 01/16/13. For personal use only.
Gall Midges (Hessian Flies) as Plant Pathogens
Jeff J. Stuart, Ming-Shun Chen, Richard Shukle, and Marion O. Harris p p p p p p p p p p p p p p 339
Phytophthora Beyond Agriculture
Everett M. Hansen, Paul W. Reeser, and Wendy Sutton p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 359
Landscape Epidemiology of Emerging Infectious Diseases
in Natural and Human-Altered Ecosystems
Ross K. Meentemeyer, Sarah E. Haas, and Tomáš Václavı́k p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 379
Diversity and Natural Functions of Antibiotics Produced by Beneficial
and Plant Pathogenic Bacteria
Jos M. Raaijmakers and Mark Mazzola p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 403
The Role of Secretion Systems and Small Molecules in Soft-Rot
Enterobacteriaceae Pathogenicity
Amy Charkowski, Carlos Blanco, Guy Condemine, Dominique Expert, Thierry Franza,
Christopher Hayes, Nicole Hugouvieux-Cotte-Pattat, Emilia López Solanilla,
David Low, Lucy Moleleki, Minna Pirhonen, Andrew Pitman, Nicole Perna,
Sylvie Reverchon, Pablo Rodrı́guez Palenzuela, Michael San Francisco, Ian Toth,
Shinji Tsuyumu, Jacquie van der Waals, Jan van der Wolf,
Frédérique Van Gijsegem, Ching-Hong Yang, and Iris Yedidia p p p p p p p p p p p p p p p p p p p p p p 425
Receptor Kinase Signaling Pathways in Plant-Microbe Interactions
Meritxell Antolı́n-Llovera, Martina K. Ried, Andreas Binder,
and Martin Parniske p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 451
Fire Blight: Applied Genomic Insights of the
Pathogen and Host
Mickael Malnoy, Stefan Martens, John L. Norelli, Marie-Anne Barny,
George W. Sundin, Theo H.M. Smits, and Brion Duffy p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 475
Errata
An online log of corrections to Annual Review of Phytopathology articles may be found at
http://phyto.annualreviews.org/
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Contents