271

CHAPTER 16
Molecular and Biotechnological Approaches in Plant
Disease Management

K.N. Chandrashekara*
Indian Institute of Horticulture Research,
Hessaraghatta, Bangalore - 560 089, Karnataka (India)
Shilpa Ramachandra Raju
Advanced Centre of Plant Biotechnology,
UAS campus, GKVK, Bangalore - 560 054, Karnataka (India)
[Link]
Vivekananda Parvatiya Krishi Anusandhan Sansthan, Almora - 263 601, Uttarakhand (India)
*
E-mail:knchandu1@[Link]

INTRODUCTION

A lot of agricultural crops are being threatened by a wide variety of biotic stresses by lowering of
vegetable and fruit quality leading to wipe out entire harvests. About 42% of the world’s total agricultural
crop is destroyed yearly by diseases and pests. Farmers often must contend with more than one pest or
disease and new pesticide-resistant pathogenic strains attacking the same crop. However, crop losses can
be minimized and specific treatments can be tailored to combat specific pathogens if plant diseases are
correctly diagnosed and identified early. These need-based treatments also translate to economic and
environmental gains. The traditional method of identifying plant pathogens is through visual examination.
This is often possible only after major damage has already been done to the crop, so treatments will be of
limited or no use. Pathogens capable of causing systemic infections on their host plants are usually transmitted
through vegetative propagation from infected mother plants to the young plants. If all the seedlings or
planting materials are infected, there may be severe loss of the crop after planting. Infected seedlings or
planting materials can easily be transported to new areas, resulting in the invasion and establishment of new
diseases. In areas where the pathogens already exist, infected seedlings or planting materials serve as
inoculums sources of pathogens that can be transmitted by insects and other vectors, causing outbreaks of
disease. Because these pathogens are always located inside the cells and systemically spread throughout
the plant, the diseases they cause are often very difficult to control by conventional measures such as
pesticide sprays. Experience over several decades tells us that the most effective way of dealing with this
problem is to produce pathogen-free seedlings or planting materials.

To save plants from irreparable damage by pathogens, farmers have to be able to identify an infection
even before it becomes visible. Is this possible? What happens when pathogens attack a plant? An attack by
272 Eco-friendly Innovative Approaches in Plant Disease Management

disease-causing organisms generates a complex immune response in a plant, resulting in the production of
disease-specific proteins involved in plant defense and in limiting the spread of infection. Pathogens also
produce proteins and toxins to facilitate their infection, before disease symptoms appear. These molecules
play vital role in the development of plant diagnostic kits.

Advances in molecular biology, plant pathology and biotechnology have made the development of
such kits possible. These kits are designed to detect plant diseases early, either by identifying the presence
of the pathogen in the plant (by testing for the presence of pathogen DNA) or the molecules (proteins)
produced by either the pathogen or the plant during infection. These techniques require minimal processing
time and are more accurate in identifying pathogens. And while some require laboratory equipment and
training, other procedures can be performed on site by a person with no special training. Using Real Time
PCR, it is possible not only to detect the presence or absence of the target pathogen, but also possible to
quantify the number of the pathogen in the sample. Enumerating the pathogen upon detection is crucial to
estimate the potential risks with respect to diseases development and provides a useful basis for diseases
management decisions. Crops can be attacked by many pathogens which, in addition, often occur in complexes.
Therefore, many disease diagnostic applications require simultaneous detection and quantification of several
targets. Methodological limitations, however, are in many cases the reason for developing simplex or assays
designed for only few targets. The DNA Microarray technology, originally designed to study gene expression
and generate single nucleotide polymorphism (SNP) profiles, is currently a new and emerging pathogen
diagnostic technology, which in theory, offers a platform for unlimited multiplexing capability. It is viewed as
a technology that fundamentally alters molecular diagnostics. The fast growing databases generated by
genomics and biosystematics research provides unique opportunity for the design of more versatile, high-
throughput, sensitive and specific molecular assays which will address the major limitations of the current
technologies and benefit plant pathology.

Table 1: Common causes of disease and how they can be visualize

Cause Description How they Equipment Magnification Examples
reproduce required*
Fungi Grow as tiny Spores, Cell Microscope 20-250 Rusts, smuts,
threadlike filaments; division (occasionally leaf spots, white
large fruiting structures a hand lens) mold, powdery
may develop from these mildew, cankers,
filaments fungal wilt, apple
scab, wheat and
barley scab etc.
Bacteria Tiny single-celled Cell division Microscope 400-1,000 Bacterial blights,
organisms fire blight,
Canker, bacterial
wilt etc.
Viruses Very tiny rod-shaped Cause host to Electron 20,000-100,000 Tomato Leaf curl,
or spherical particles, manufacture microscope Papaya ring spot
composed of RNA/DNA virus virus, Wheat streak
with a protein coat mosaic, barley stripe
mosaic, potato
viruses,tobacco
mosaic, cucumber
mosaic etc.
Molecular and Biotechnological Approaches in Plant Disease Management 273

Phytoplasmas Very tiny organisms Division Electron 20,000-50,000 Aster yellows, little
without a cell wall, microscope leaf, big bud (purple
no definite shape top in potato and
tomato).
Nematodes Tiny Roundworms Eggs Microscope 1-60 Disease caused by
(naked eye for larger forms)
organism in India
*The number of times a pathogen must be magnified to be visible. For example, a pathogen 1/1,000 inch in size when
magnified 100 times would appear to be 1/10 inch in size; a pathogen 1/1,000,000 inch in size when magnified
100,000 times would appear to be 1/10 inch in size.

METHODS USED FOR IDENTIFICATION OF PLANT DISEASE

Control measures depend on proper identification of diseases and of the causal agents. Without
proper identification of the disease and the disease-causing agent, disease control measures can be a waste
of time and money and can lead to further plant losses. Proper disease diagnosis is therefore vital. There are
several methods used for the identification of disease causing organisms. First and foremost step in identifying
the disease causing organism is:
1. Check for the symptoms: Variation in symptoms in diseased plants may sometimes lead to improper
diagnosis.
2. Look for signs of disease causal agents: Signs of plant disease agents are the observable evidence
of the actual disease-causing agent. Signs may include the mycelia of a fungal agent, fungal spores,
and spore-producing bodies. Indications of insects causing problems may include the actual insect,
insect frass, mite webbing, and insect eggs. Signs are much more specific to disease-causing agents
than are symptoms and are extremely useful in the diagnosis of a disease and identification of the
agent causing the disease.
3. Identify plant part Affected: Are symptoms associated with specific plant parts? The symptoms
of some diseases are most commonly seen on specific plant parts and this observation can be
important in diagnosis.
4. Check distribution of symptoms: One of the first things that a diagnostician should note is how the
diseased plants are distributed over the affected area. Are they distributed uniformly across an area
or are they localized? Is there a definite pattern to the distribution?
5. Check for host specificity: Is the problem occurring in only one plant species or are different plant
species affected? If different plant species are affected, this suggests the possibility of a non-
infectious problem which could be related to cultural or environmental problems.

The techniques available have evolved significantly in the last few years to achieve rapid and reliable
detection of pathogens, extraction of the target from the sample being important for optimizing detection.
For viruses, sample preparation has been simplified by imprinting or squashing plant material or insect
vectors onto membranes. To improve the sensitivity of techniques for bacterial detection, a prior enrichment
step in liquid or solid medium is advised. Specific monoclonal and/or recombinant antibodies are available
for many plant pathogens and have contributed to the specificity of serological detection. Molecular
detection can be optimized through the automatic purification of nucleic acids from pathogens by columns
or robotics. New variants of PCR, such as simple or multiplex nested PCR in a single closed tube, co-
operative-PCR and real-time monitoring of amplicon or quantitative PCR, allow high sensitivity in the
detection of one or several pathogens in a single assay. The latest development in the analysis of nucleic
acids is micro-array technology, but it requires generic DNA/RNA extraction and pre-amplification
274 Eco-friendly Innovative Approaches in Plant Disease Management

methods to increase detection sensitivity. The advances in research that will result from the sequencing
of many plant pathogen genomes, especially now in the era of proteomics, represent a new source of
information for the future development of sensitive and specific detection techniques for these
microorganisms.

6. Detection specificity and sensitivity: Sensitivity and specificity are numeric measures of effectiveness
of a detection system (Peruski & Peruski, 2003; Malorny et al., 2003). Diagnostic specificity is defined
as a measure of the degree to which the method is affected by non target components present in a
sample, which may result in false positive responses. Diagnostic sensitivity is defined as a measure of
the degree to detect the target pathogen in the sample, which may result in false negative responses
(Malorny et al., 2003). Too low sensitivity often leads to false negatives. Thus, a high degree of diagnostic
accuracy is characterized by the ability to detect, true and precisely the target micro organism from a
sample without interference from non target components. The high degree of sensitivity of molecular
methods made pre-symptomatic detection and quantification of pathogens possible. PCR is a highly
sensitive technology. However, its sensitivity is greatly affected by the presence of inhibitors which
prevent or reduce amplification. A wide range of inhibitors are reported (Yang and Rothman, 2004).
Although their mode of action is not clear, these inhibitors are believed to interfere with the polymerase
activity for amplification of the target DNA. On the other hand, it is worth mentioning that the high
sensitivity of PCR also causes one of the limitations of PCR that is detection sensitivity exceeding
threshold levels or clinical significance and false positive results from slight DNA contamination (Yang
and Rothman, 2004). Hence, stringent conditions are necessary in conducting the assay and proper
negative controls must be included in the test. It is also recommended to have separate dedicated areas
for pre- and post PCR handling.

DIFFERENT TECHNIQUES USED IN MOLECULAR DIAGNOSIS

Polymerase Chain Reaction (PCR)

PCR is used in the diagnosis of plant diseases has become very common in laboratory practice. Its
advantages (speed, sensitivity, specificity) are far more important than its drawbacks (risk of contamination,
sensitivity to inhibitors, complexity, and cost) and several modifications to solve these problems have been
performed with success. In general, PCR, with all its variants, is currently a basic tool in diagnosis, alone or
preferentially in combination with other techniques.

Efficiency for detection of bacteria, fungi and viruses is based on the primer specificity. Its efficiency
is also related to many parameters such as polymerase type, buffer composition, stability, purity and
concentration of dNTPs, cycling parameters as well as the characteristics of the starting template. In
Addition, as above indicated, the quality of the nucleic acid to be amplified is critical. Because PCR can
achieve a relatively high sensitivity and good specificity hence, different types of PCRs are available viz:

Nested PCR

Sensitivity and specificity problems associated with conventional PCR and RT-PCR can be reduced
by using nested PCR-based methods, based on two consecutive rounds of amplification (Fig. 1). Usually, the
products of the first amplification are transferred to another tube before the nested PCR is carried out using
one or two internal primers (if one is used it is semi nested or more that 1 primer is used then it is nested
Molecular and Biotechnological Approaches in Plant Disease Management 275

PCR). By using high sensitivity, specificity, and reliability nested PCR. Qi et al, (2000) reported the physiological
races of Corynespora cassiicola causing Corynespora leaf fall disease of Hevea brasiliensis.

Fig. 1 : Pictorial representation of Nested PCR

Co-operational PCR

PCR concept based on the simultaneous action of four or three primers, has also been developed.
This technique named co-operational amplification (Co-PCR) can be performed easily in a simple reaction
increasing the sensitivity level and using ten times less reagent than in conventional PCR.

The technique was used successfully, both in metal block and capillary air thermal cyclers for the
detection of plant RNA viruses (Cherry leaf roll virus, Strawberry latent ringspot virus, Cucumber mosaic
virus, Plum pox virus and Citrus tristeza virus). The sensitivity observed is at least 100 times higher than
that achieved with RT-PCR and similar to nested RT-PCR. Colorimetric detection was coupled with this
methodology facilitating its introduction for routine indexing programs and for phytosanitary selection of
virus-free plant material.

Multiplex PCR

The simultaneous detection of two or more DNA or/and RNA targets can be afforded by duplex or
multiplex PCR in a single reaction with several specific primers included in the PCR cocktail. Multiplex PCR
is very useful in plant pathology because different bacteria or viruses frequently infect a single crop or host.
In multiplex assays where closely related templates such as pathogen strains are distinguished by amplifying
differing sequence, primers for a sequence common to all templates provide a positive control for amplification.

Multiplex nested PCR

Multiplex nested RT-PCR method in a single tube, combines the advantages of the multiplex PCR
with the sensitivity and reliability of the nested PCR, saving time and reagent costs because two reactions
are sequentially performed using a single reaction cocktail. In addition, it enables simultaneous detection of
RNA and DNA targets. The accurate design of compatible primers is necessary to avoid hairpins and
primer-dimer formation.
276 Eco-friendly Innovative Approaches in Plant Disease Management

RT-PCR

Is the “gold standard” molecular method used for the detection of plant viruses due to its high
sensitivity and specificity. As the majority of them are RNA viruses, an initial step of reverse transcription
that converts single strand RNA to cDNA is necessary for PCR-based molecular amplification (Fig. 2).
When PCR or RT-PCR is applied routinely for detection purposes, the sensitivity usually afforded tends to
be similar to ELISA or Hybridisation techniques.

Fig 2: Pictorial representation of Reverse transcriptase PCR

Loop-mediated Isothermal Amplification (LAMP)

Is type of isothermal amplification that it is being increasingly used in the diagnostic field offering
sensitivity and economic costs. The method requires a set of four specifically designed primers that recognize
six distinct sequences of the target and a DNA polymerase with strand displacement activity. The amplification
products are stem-loop DNA structures with several inverted repeats of the target and cauliflower-like
structures with multiple loops, yielding >500ug/ml. The LAMP reaction was enhanced by the addition of loop
primers, reducing time and increasing sensitivity (Figure 3). The amplification takes place at 60-65 C for 60
min. Although it was initially developed for DNA it can be adapted to amplify RNA (RT-LAMP).

Fig. 3: Loop-mediated Isothermal Amplification (LAMP)

Molecular and Biotechnological Approaches in Plant Disease Management 277

Random Amplified Polymorphic DNA (RAPD)

Is method based on the PCR reaction using short usually 10 nucleotide primers of arbitrary sequences.
Polymorphism of amplified fragments are causes by, base substitutions or deletions in the priming sites,
insertions that render priming sites too distant to support amplification or insertions or deletions that change
the size of the amplifies fragment. RAPD is an inexpensive yet powerful typing method for many bacterial
species and for detection of viral species.

Restriction Fragment Length Polymorphism (RFLP)

Is a difference in homologous DNA sequence that can be detected by the presence of fragments of
different lengths after digestion of the DNA samples in question with specific restriction endonucleases.
RFLP as a molecular marker is a specific to a single clone/restriction enzyme combination.

Fluorescence RT-PCR using Taqman technology

Two primers flank the sequence of interest and a third fluorescently labelled primer anneals between
them. As the flanking primers extend, the labelled primer is released and fluorescence occurs (Fig. 4A &
4B). The advantages of this method are that no post-reaction processing is required to detect and that it is
quantitative.

Fig. 4 A : Pictorial representation of Real-time PCR reaction

278 Eco-friendly Innovative Approaches in Plant Disease Management

Fig. 4 B : Pictorial representation of Real-time quantitative PCR

Competitive fluorescence PCR (CF–PCR)

This is a variation on the above technique. It is used to simultaneously differentiate between virus
strains and multiple virus infections. Several primer sets, each labelled with a different fluorescent marker
are added to the reaction mixture. Virus strains are differentiated with primers that differ only at the 3’ end,
complementary to a nucleotide position that is polymorphic between strains. Extension occurs only where
the 3’ nucleotide is complementary. Only primers that generate amplicons fluoresce and the wavelength
emitted identifies the primers that have been extended.

Microarray Technology

Since the development of microarray technology for gene expression studies new approaches are
extending their application to the detection of pathogens. Microarrays are generally composed of thousand
of specific probes spotted onto a solid surface (usually nylon or glass). Each probe is complementary to a
specific DNA sequence provides a signal that can be detected and analysed. Although there is great potential
for microarray technology in the diagnosis of plant diseases, the practical development of this application is
still in progress (Fig. 5).
Molecular and Biotechnological Approaches in Plant Disease Management 279

Fig. 5 : Pictorial representation of Microarray

Enzyme Linked Immuno-Sorbent Assay (ELISA)

Is a biochemical technique used as a diagnostic tool in medicine and plant pathology. In ELISA an
unknown amount of antigen is affixed to a surface, and then a specific antibody is washed over the surface
so that it can bind to the antigen. This antibody is linked to an enzyme, and in the final step a substance is
added that the enzyme can convert to some detectable signal. Thus in the case of fluorescence ELISA,
when light is shone upon the sample, any antigen/antibody complexes will fluoresce so that the amount of
antigen in the sample can be measured. Modified ELISA with molecular techniques are in use for detecting
pathogens when pathogen is low in concentration.

Immunocapture PCR (IC-PCR)

This combines capture of virus particles by antibodies with amplification by PCR. In this method,
the virus is adsorbed by the antibody bound to a surface, then removed by heating with a non-ionic surfactant
such as Triton X-100. The nucleic acids are then amplified using RT-PCR. This method is especially useful
in concentrating virus particles from plant species where virus titre is low or where compounds that inhibit
PCR are present.
280 Eco-friendly Innovative Approaches in Plant Disease Management

Quartz Crystal Microbalance (QCM) Immuno-sensors

In this novel technique for plant virus detection, a quartz crystal disk is coated with virus-specific
antibodies. Volt age is applied across the disk, making the disk warp slightly via a piezoelectric effect.
Adsorption of virus particles to the crystal surface changes its resonance oscillation frequency in a
concentration-dependent manner. It is therefore qualitative and quantitative.

Single Chain Variable Fragments (scFv)

Single chain antibody refers to highly variable domains of antibody (VH and VL) linked by a linker
peptide (Fig. 6).

Fig. 6 : Antibody gene used for phage display

The small size and lack of assembly requirements of these engineered antibodies make them particularly
suitable for developing diagnostic kits and molecular characterization through epitope mapping. These can be
generated de novo, in the absence of any animal system, using phage display libraries. Herein, the amplified
immunoglobulin genes are fused to the N-terminal of M13 capsid protein, g3p. The phage library is then
screened for its ability to bind to an antigen of interest, which is artificially bound to a solid support. The
phages screened by this panning procedure are further expanded and enriched and the scFv together with
the gene encoding it is selected in this manner (Fig. 7). This technique has been successfully attempted in
producing monoclonal antibodies against toxins, hormones, growth factors, chemical, protein, enzyme, viruses,
fungi and bacteria. Recent accomplishments in the protein engineering of antibodies offers alternatives to
classical hybridoma technology, to the technique of the humanization of murine antibodies by grafting murine
Molecular and Biotechnological Approaches in Plant Disease Management 281

CDR regions onto a human antibody framework regions or to PCR cloning of rearranged VH and VL genes
from hybridomas.

Fig. 7 : Enrichment of bound phage and selection

A tremendous impact on the development of antibody engineering technology had the display of
antibody fragments on the surface of filamentous phage fd or M13 that live on Escherichia coli. The
antibody phage display is accomplished by fusing the coding sequence of the antibody variable regions to the
gene encoding one of the phage coat proteins: pIII, pVI, pVII, pVIII, or pIX (Fig. 8). Expression of the
fusion product and its subsequent incorporation into the mature phage coat results in the antibody being
presented on the phage surface, while its genetic material resides within the phage particle.

Fig. 8 : Filamentous phage display

282 Eco-friendly Innovative Approaches in Plant Disease Management

The affinity of phage display-selected antibodies depends to a large extend on the size of the displayed
antibody repertoire. In principle three different types of libraries can be utilized: a) a library derived from an
immunized donor; b) a naive V-gene library; c) a synthetic antibody library. Phage display has made a
tremendous impact on the discovery of peptides binding to a variety of receptors and high-affinity antibodies,
and it has been widely used to select and engineer peptides and proteins. Recently, this technology has also
been used in the directed evolution of enzymes taking advantage of the larger libraries it could accommodate
and the direct linkage between the phenotype and genotype.

Comparing Traditional Method with Presently used Techniques

In Traditional Method:
1. Identification is time consuming.
2. Not always sensitive and specific enough.
3. Required skilled person to perform.
4. Not suited for analysis of a large number of samples.

Using Present Day Techniques:

1. Accuracy in detection has greatly improved.
2. PCR offers several advantages, because organisms do not need to be cultured before their detection.
3. It is highly sensitive.
4. It enables a single target molecule to be detected in a complex mixture and it is also rapid and
versatile.
5. Biological assays are simple; require minimal knowledge of the pathogen.

REQUIREMENTS FOR TECHNOLOGY IMPLEMENTATION IN

PRACTICE

Since its introduction in the mid-1980s, PCR has become a fundamental aspect of fungal molecular
diagnostics, and several technologies based on PCR have been developed since then (Lievens et al., 2005;
McCartney et al., 2003). However, although extensively used as a tool in (academic) research, the use of
PCR-based technologies in horticultural and agricultural practice is still limited (McCartney et al., 2003;
Schaad et al., 2003). Nevertheless, several areas would benefit from the commercial availability of such
detection assays. With the opening of the borders of many countries and increased free-trade agreements,
rapid testing for possible contamination with quarantine organisms is in high question. In addition, in order to
be able to take timely control measures, the question from commercial growers for rapid, affordable pathogen
detection assays is increasing. However, different requirements have to be met before new detection methods
are implemented in practice. These requirements can be separated into technical and economical demands.
Whereas the technical demands are absolutely required for the development of any successful diagnostic
method, the economical criteria are important guidelines for the development of a commercially attractive
assay.

Technical Demands

When developing a tool for plant pathogen diagnostic purposes, several technical aspects related to
Molecular and Biotechnological Approaches in Plant Disease Management 283

plant disease management on one hand, and regulatory issues on the other hand, are to be considered. These
aspects mainly concern specificity, sensitivity, and robustness. In addition, multiplexing and quantification are
increasingly becoming required features for a diagnostic assay.

Specificity: the ability to specifically detect the target pathogens is vital for all diagnostic applications. One
of the most important advantages that molecular-based detection techniques have over conventional diagnostic
methods is the power to, in principle, distinguish closely related organisms. For fungal molecular diagnostics,
ubiquitously conserved genes are frequently used as target genes. Closely related microbial species often
differ in a single (single-nucleotide polymorphism [SNP]) to a few bases in such genes. However, the high
degree of specificity of nucleic acid-based detection techniques, achieved through the use of PCR primers,
hybridization probes, or detector oligonucleotides, allows detecting such SNPs (Consolandi et al., 2001;
Livak, 1999; Papp et al., 2003). Since closely related pathogens might have a different host range or display
a completely different pathogenicity, this is an extremely important trait. Ultimately, to enhance specificity of
a diagnostic assay, a combination of multiple unique diagnostic regions can be exploited. Obviously, the
specificity of nucleic acid-based techniques is determined by the sequences that are targeted. In general,
there are two approaches to select target sequences. The first, and most common, strategy involves targeting
ubiquitously conserved known genes carrying sequence variation that can be exploited. Currently, the primary
target in fungal and bacterial molecular diagnostics is the nuclear ribosomal DNA (rDNA), which has been
extensively used in molecular phylogenetic studies and is, therefore well characterized (White et al., 1990;
Anonymous, 2006). A large amount of rDNA sequence data is available in public databases, which aids the
design of a diagnostic assay. These extensive sequence data allow comparison of sequences which permits,
in turn, determining diagnostic regions harbouring the required specificity. Apart from the discriminatory
potential, the high copy number of rDNA genes in any genome permits a highly sensitive detection. Fungal
rDNA occurs as a structured unit consisting of three ribosomal RNA subunit genes that are separated by
internal transcribed spacers (ITS). This ITS region is an area of particular importance to fungal diagnostics
since it contains alternating areas of high conservation and high variability. This variability allows classification
over a wide range of taxonomic levels (White et al., 1990; Berg et al., 2005; Falloon et al., 2006; Anonymous,
2006), sometimes even below the species level (Atkins et al., 2003). The second strategy to select target
sequences for detection of plant pathogens involves the screening of random parts of the genome to find
diagnostic sequences. This can be achieved by several techniques, including random amplified polymorphic
DNA (Williams et al., 1990; Wang et al., 1993; Birch et al., 1997; Ferreira et al., 2000) and amplified
fragment length polymorphism (Darrase et al., 1994; Vos et al., 1995; Manceau and Horvais, 1997;
Mkandawire et al., 2004) technology. Nevertheless, since the location of possible useful sequences in the
genome is a prior unknown, there often are few sequence data available for comparison to other organisms
in order to guarantee specificity. As a consequence, extensive screening is required to ensure specificity of
the potential marker.

Sensitivity: early detection of pathogens, e.g., before crops are infected or symptoms have developed, is
essential to prevent diseases, spread of the inoculum, and economic losses. Therefore, diagnostic procedures
should be highly sensitive. Prior to the introduction of nucleic acid amplification methods, in particular PCR,
nucleic acid-based diagnostics mainly involved the use of specific probes to report the presence of a certain
organism (Yao et al., 1991; Manceau et al., 2000; Fanelli et al., 2007). However, these methods often led
to “false negatives” because of too low sensitivity. Because of this, PCR has been introduced in most
molecular diagnostic assays in recent years, allowing detection of minute quantities of pathogen DNA.
However, high sensitivity also causes one of the potential pitfalls of PCR technology: the slightest carry-over
contamination can give rise to “false positive” results. Therefore, stringent conditions and controls are
necessary, such as guarding the reagents and samples for accidental DNA contamination via aerosols,
running negative controls simultaneously with the test samples, and having separate dedicated areas for pre-
and post-PCR handling (Kwok and Higushi, 1989). Recently, attempts are being made to assess the potential
use of relatively novel highly sensitive amplification techniques such as rolling circle amplification (Baner et
284 Eco-friendly Innovative Approaches in Plant Disease Management

al., 1998) for pathogen detection. However, compared with PCR this procedure is fairly complicated (Andras
et al., 2001) and relatively expensive. Therefore, it is important to realize what level of sensitivity is required
for a method to be used for plant pathogen detection. Techniques more sensitive than conventional PCR
amplification will probably not be required when assessing whether measures have to be taken to prevent
yield losses, as the threshold level that has to be crossed can easily be detected using PCR. In contrast,
sensitivity is very important for quarantine organisms for which a nil-tolerance is wanted. The developers of
the QCM technique claim that it is as sensitive but more rapid than ELISA, and economical. In the first
described use of QCM for plant viruses, as little as 1 ng of particles of Cymbidium mosaic virus and
Odontoglossum ringspot virus were detected in crude sap extracts (Eun et al., 2002). Thermal images
were obtained using a non-cooled focal plane array infrared camera (TH7102 MX, NEC, Tokyo, Japan).
The instrument operates in the waveband between 8 and 14 mm. The detector array has a geometric
resolution of 1.58m rad (320 X 240 pixels focal plane array and a 29 X 220 field of view lens with a minimum
focus distance of 0.3 m). The thermal resolution is 0.060C and accuracy of absolute temperature measurement
close to ± 20C. The recognition of variations in leaf temperature led to the combined use of infrared
thermography and visible imagery to identify plant stress (Leinonen and Jones, 2004). Thermal imagery was
applied to non-destructive monitoring of a plant’s physiological status in response to pathogen attack (Chaerle
et al., 2004). Furthermore, infrared thermography can be applied as a useful tool for pre-symptomatic
detection of downy mildew in cucumber (Manfred et al., 2008). In this example, the maximum temperature
difference (MTD) within a leaf turned out to be a suitable method to differentiate between infected and non-
infected leaf tissue (Lindenthal et al. 2005; Oerke et al., 2006).

Multiplexing: most current molecular diagnostic assays used in plant pathology target one specific pathogen.
However, because crops can be infected by numerous pathogens which are, in addition, often present in
plants as complexes, it is desirable to develop assays that can detect multiple pathogens simultaneously. The
first multiplex PCR-based strategies involved the use of multiple primer sets in the same reaction. Nevertheless,
the development of a reliable multiplex PCR, in order to resolve at least a few amplicons by gel electrophoresis,
is a significant technical challenge (Elnifro et al., 2000). For real-time PCR (Heid et al., 1996), the amplification
process is monitored on-line, meaning that the size difference of amplicons to discriminate them on gels is
not necessary. Nevertheless, in this case multiplexing is limited by the availability of dyes emitting fluorescence
at different wavelengths on one hand, and the monochromatic character of the energizing light source in
real-time PCR instruments on the other hand (Varga and James, 2006; Reid et al., 2006; Osman and
Rowhani, 2008). As a result, detection of more than a few pathogens per assay is currently not possible
using these strategies. In contrast, array hybridization technology offers the possibility to add a multiplex
aspect to PCR-based detection. In theory, DNA arrays, originally designed to study gene expression or to
generate SNP profiles, can be used to detect an unlimited amount of different organisms in parallel (Lévesque,
2001; Lievens et al., 2005; Martin et al., 2000). The virtually unlimited screening capability of DNA arrays,
coupled with PCR amplification, results in high levels of sensitivity, specificity, and throughput capacity
(Lievens et al., 2003). A pathogen detection array typically consists of many discretely located pathogen-
specific detector sequences that are immobilized on a solid support, such as a nylon filter or a glass slide, to
create a macroarray or a microarray, respectively. For signal amplification, in general the target DNA to be
tested is amplified using consensus primers that target a genomic region containing the pathogen-specific
sequences, and is labeled simultaneously or subsequently. In this way, it may thus be possible to differentiate
a large number of organisms using a single PCR, provided that sufficient discriminatory potential exists
within the region that is used. Subsequently, labeled amplicons are hybridized to the array under stringent
conditions. In plant pathology, this approach was applied for identifying oomycete, nematode, bacterial and
fungal DNA from pure cultures (Fessehaie et al., 2003; Lévesque, 2001, Lievens et al., 2003; Uehara et al.,
1999) as well as for the identification of a number of viruses (Boonham et al., 2002). Despite these studies,
for application in practice, identification of pathogens from pure cultures is not very relevant as, in the end;
pathogens preferably should be assessed directly from plant and soil samples. In recent studies, the utility of
this technology for the diagnosis of multiple pathogens in such environmental samples was shown (Lievens
Molecular and Biotechnological Approaches in Plant Disease Management 285

et al., 2003; Lievens et al., 2004; Nicolaisen et al., 2005). Ultimately, such multiplex approach should lead
to a comprehensive diagnostic kit that can detect all relevant pathogens of a specific crop.

Quantification: with respect to plant disease management, especially quantification of a pathogen upon its
detection and identification is an important aspect as it can be used to estimate potential risks regarding
disease development, spread of the inoculum, and economic losses. Apart from this potential, it provides the
information required to take appropriate management decisions. Currently, in plant pathology, real-time PCR
is the most reliable culture-independent technique to quantify the detected pathogen (Gachon et al., 2004;
Schaad and Frederick, 2002) as well as for quantification of disease progress (Brouwer et al, 2003). However,
to quantify more than a handful of plant pathogens in a single assay, real-time PCR instrumentation needs to
be adapted or other techniques should be pursued. DNA array technology offers the most suitable technology
for multiplex detection of plant pathogens. Therefore, implementation of a quantitative aspect to this technology
would be highly desirable. Two recent studies have demonstrated that such a trait can be achieved (Lievens
et al., 2005; Rudi et al., 2003).

Robustness: obviously, each diagnostic assay developed should be robust, meaning that the methodology is
highly reproducible. Therefore, the assay must be optimized and thoroughly tested against multiple targets
and taxonomically related organisms, preferably isolated from various hosts or cultivars and from different
geographical areas. There are, however, specific obstacles that can affect the robustness and reliability of
PCR-based diagnostic assays. PCR efficiency can be drastically reduced or even inhibited due to a variety
of naturally occurring compounds that are co-extracted with the nucleic acids, such as phenolic compounds,
humic acids, fulvic acids, and heavy metals. However, in many cases, these specific problems have been
circumvented by improved extraction methods (McCartney et al., 2003) or by the use of optimized extraction
kits (Lievens et al., 2005) by which highly purified DNA can be obtained from complex environmental
samples. To improve reliability, PCR efficiency can be monitored by spiking the DNA extract with a certain
amount of exogenous control DNA that can be amplified in the same reaction (Cubero et al., 2002) or in
parallel (Lievens et al., 2005).

Validation: in many cases, detection procedures are developed to be used in specific research areas. As a
consequence, these tools are generally only evaluated to work under the experimental conditions used. The
steps required to evaluate new detection techniques for their direct use in practice are, however, rarely
taken. New diagnostic procedures should be validated and standardized using worldwide ring tests before
entering the market. Factors involved in validation include (i) specificity, (ii) sensitivity, (iii) reproducibility,
(iv) accuracy of results, and (v) consistency and reliability of detection. The reliability of the test must be
demonstrated unequivocally in blind tests in several different laboratories and results should be interpreted
without any ambiguity. Preferably, the evaluation process must be monitored by an internationally recognized
organization that ensures suitable expertise for the crop(s) and pathogen(s) involved in evaluating the test.
As soon as new methods and reagents are validated, they can be officially recognized and recommended for
plant pathogen detection and eventually replace more conventional gold standards in specific control directives
(Martin et al., 2000; Stead, 1999).

Economical Demands

Apart from the technical criteria, several economical aspects have to be considered in the development
of reliable detection methods that can be used by diagnostic laboratories. These demands include short
diagnosis time and high throughput capability. In addition, it should be possible to perform the test with a
minimum of taxonomical expertise and at a minimum of cost. With regard to quarantine and export, legislation
speed is the most important factor. When it comes to routine diagnosis requested by growers especially, cost
is of high importance.
286 Eco-friendly Innovative Approaches in Plant Disease Management

Speed: the speed at which results can be obtained is a very important issue for any commercial diagnostic
tool, especially for the detection of pathogens of high-risk potential or when it comes to timely disease
management decisions. Whereas culture-based traditional techniques are often laborious and time-consuming,
and typically take days to weeks to complete, molecular detection techniques can generate accurate results
much faster. In general, most molecular analyses can be accurately performed within 1 or 2 days, which is
a considerable gain of time compared with the more conventional analyses.

High-throughput sample analysis: another requirement for commercial applicability is the possibility to
screen a large number of samples in a short period of time. Nowadays, when using molecular techniques,
comprehensive screening of samples is made possible because of recent developments in automated high
throughput DNA extraction systems and because of the introduction of 96- to 384-well plate PCR systems.
In addition, the development of DNA arrays for plant pathogen diagnosis has enabled screening of multiple
pathogens in a single assay, eliminating the need of performing several singleplex assays. However, when
many samples need to be processed using such multiplex assays in a short time, there is still a lack of high
sample throughput capacity. Nevertheless, it can be expected that this will be achieved in the near future by
the use of low-density arrays in a multiwell configuration.

Expertise: traditionally, the predominant techniques used to identify pathogens have relied upon morphological
criteria and require highly specialized taxonomical expertise, which may take extensive education plus years
of work in practice to acquire. However, since many pathogens are difficult to identify using morphological
criteria, these techniques often lead to incomplete or even wrong diagnoses. Therefore, companies/institutes
that provide diagnostic services are intensively searching for generic diagnostic tools that can be executed
relatively easily and interpreted by technicians with a general education in molecular biology.

Cost issues: cost is perhaps the most important consideration for routine pathogen testing since, relative to
human clinical diagnostics, the willingness to spend money on expensive plant disease diagnosis is limited.
This is caused by the fact that profit margins in agriculture and horticulture are often low as is the emotional
value of a crop. On the other hand, when it comes to regulatory issues and risk management of exotic
pathogens, other criteria like specificity, sensitivity, and speed are more important than cost. While nucleic
acid-based assays provide an excellent opportunity for rapid and precise detection, currently their success
largely depends on well-equipped laboratory facilities. Therefore, first of all, companies that provide diagnostic
services should compare the many advantages afforded by nucleic acid-based diagnostic assays to the cost
of establishing and maintaining a suitable laboratory environment. When large numbers of samples need to
be assessed for the presence of one specific pathogen (as in some quarantine testing programs), diagnostic
assays that can detect only single pathogens may be the most cost-effective. In contrast, when one has no
idea about the causal agent of a disease or when several pathogens need to be targeted simultaneously,
detection of multiple pathogens increases efficiency, reduces cost, and saves time. With regard to a grower,
it is the price that one has to pay that counts. In general, a multiplex assay, like a DNA array-based test, is
the most cost effective per sample because the use of a singleplex assay often requires multiple consecutive
analyses to determine and confirm the cause of a disease, hence increasing the price per sample analyzed.
Nevertheless, the price of such DNA array-based analysis is largely determined by the instruments used. In
general, there are two types of DNA arrays, membrane-based macroarrays and high-density microarrays
using a glass slide (Schena et al., 1996) or beads (Yang et al., 1998). Whereas macroarray-based detection
generally does not require specialized equipment, highly specialized instruments are needed for microarray
fabrication and reading. As a consequence, implementation of microarray technology for disease diagnostic
use in plant pathology is currently relatively expensive and therefore commercially unattractive. In addition,
macroarrays are generally more sensitive than microarrays since spots on a macroarray often contain
higher amounts of detector oligonucleotides (Cho and Tiedje, 2002), favouring the use of macroarrays.
Besides, high density is probably not a necessity for host-based diagnostic assays since the number of major
pathogens of a given crop is usually not more than 100.
Molecular and Biotechnological Approaches in Plant Disease Management 287

CONCLUSION

With even more advances in molecular biology and immunology, scientists and farmers alike will be
able to improve plant disease diagnosis. Efforts are already underway to produce better diagnostic kits to
detect pathogens in crops important to developing countries. Diagnostic kits are an investment: they may be
expensive, but the costs can be offset by gains, such as reduced crop losses and more environment-friendly
crop-management practices. Their development should be made a priority by both the public and private
sectors in developing countries. It is only through advancing our understanding the ways in which plants and
microbes interact, and through dissecting the signaling pathways and changes in gene expression in greater
detail, that we will be able to develop rational and integrated control programmes that maximize protection of
plants from disease and consumers from harmful toxins and chemical residues. Whether through marker
assisted breeding, improved molecular diagnostics or the other methods discussed, more research and an
enhanced understanding will result in the development of methods that can be applied in the future. New
problems will continue to arise. Perhaps new diseases will evolve or will spread to regions where they have
previously been absent, and new techniques for studying these pathogens will be developed as technologies
advance. The plant pathologist, who understands and keeps abreast of modern concepts about the nature of
plant disease and the ways in which they can be controlled will be a vital resource for the agricultural and
horticultural.

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