Molecular diagnostic Techniques for Detection of Plant Pathogens

Molecular Methods Used For Detection

of Plant Pathogens • Polymerase Chain Reaction

• Molecular hybridization

• Molecular markers

• Nucleic acid sequence/Probes

• Micro-arrays

PCR is an in vitro method of nucleic acid

synthesis by which a particular segment of

DNA can be specifically replicated.

Invented by Karry Mullis(1987)

PCR is an ingenious new tool for molecular

biology for identification of plant pathogens.

Polymerase Chain Reaction

PCR Principle:

The double stranded DNA of interest is denatured to

separate into two individual strands each strand is then allowed

to hybridize with a primer (renaturation). The primer template

duplex is used for DNA synthesis (the enzyme DNA

polymerase). These three steps denaturation, renaturation and

synthesis are repeated again and again to generate multiple

forms of target DNA.

Steps in PCR: I Denaturation of DNA: Primers will only attach to end subsequently elongate the

Developing nucleic acid chain on a template of single stranded (ss) DNA

Thus if the target sequences is double stranded (ds)DNA, the strand need to be split apart from each other to produce ssDNA. This is performed by heating the material to 90-96◦C a process termed as denaturation.

The step is of 4 min in the first cycle of PCR but of only 2 min in subsequent cycles.

II Annealing of primers:

The primers are then attached to the ends of the segment to be amplified this process is called annealing.

This takes place at a temperature range of 37-50◦C.

III Polymerization:

After the primers are attached they “kick-start the polymerization which then elongates the developing nucleic acid chain between them.

Bases are successfully added on to the developing new nucleic acid chain according to the sequence of the segment, which is being detected, producing a new chain consisting of a complementary sequence to that of the target segment.

IV Amplification:

The new ds DNA need to be split apart again to yield two ss DNA strands by reheating the mixture to 95˚C i.e. denaturation, and the cycle is repeated.

Each cycle resulting in a logarithmic increase in the amount of DNA which is amplified.

Thus, within 20 cycles a million fold amplification of the starting amount of nucleic acid can be achieved.

The cyclical variation of temperature of the reaction is carried out by an automated thermal cycler.

V Detection of target DNA sequence:

• Amplification of DNA can be readily

demonstrated by conventional DNA detection

techniques such as nucleic acid hybridization

or by agarose gel electrophoresis.

Advantages:

The major advantage of PCR is sensitivity (one molecule of nucleic acid within 1,00,000 cells).

It can detect infections at an early stage.

Useful in detection of non-replicating virus.

PCR tests are more rapid (result within 24-48 hours).

Disadvantages:

False positive results due to contamination from operator, residual matter in testing utensils or air contamination can result in false positive reaction.

Reagents used are still very expensive.

Useful only for those pathogens for which primers have been specifically designed.

Application of PCR technique in plant pathology:

Diagnosis and quantification of diseases

Study of pathogen mating type

Production of virus free material.

Field surveys to assess the incidence and geographic distribution of plant pathogens

Domestic and international plant quarantine programmes.

• .

Detection of mixed virus infections

Analysis of virus distribution in different plant tissues.

Identification of alternative host plant

Germplasm screening programmes.

Evaluation of levels of resistance of cultivars.

Determination of virus

PCR – DDGE (Denaturing Gradient Gel Electrophoresis):-

This method is mainly applied for the

analysis of the genetic diversity of microbial

communities without the need of any prior

knowledge of the species.

(Muyzer, 1999; Portillo et al., 2011)

This method is however time-consuming, poorly reproduci-

ble and provides relative information about the abundance

of detected species. Eg. Phytophthora

Techniques: RT-PCR

“Gold standard molecular method” for detection of viruses.

An RT-PCR (Reverse transcriptase-polymerase chain reaction) is a highly sensitive technique for the detection and quantitation of mRNA (messenger RNA).

The technique consists of two parts: 1) The synthesis of cDNA (complementary DNA) from

RNA by reverse transcription (RT)

2) The amplification of a specific cDNA by PCR.

Schematic diagram of RT-PCR procedure

Many viroids like Apple dimple fruit viroid (ADFVd),Pear blister canker viroid (PBCVd), Peach latent mosaic viroid (PLMVd) have been detected by this method (Shamloul et al.,2002).It also applied in detection of viruses like CLRV, ACLSV, Apple mosaic virus and TomRV.

The sensitivity afforded tends to be similar to ELISA or hybridization techniques (Olmos et al., 2005)

IC-RT-PCR:• Very well suited for large scale detection of plant pathogens

• Exploiting high-affinity binding antibodies to selectively capture the

target organism prior to detection by conventional PCR

amplification (Mulholland,2005).

• Technique can be performed in plates that have already been used

through a complete ELISA assay.

• This offers the advantage of testing by PCR only those samples

found negative in an initial ELISA screening, without compromising

the sensitivity of the PCR amplification procedure.

It has been successfully used to detect Xanthomonas axonopodis pv. dieffenbanchiae, the cause of bacterial blight of anthurium,in vegetative propagating material (Khoodoo et al.,2005).

Similarly, Pseudomonas atrosepticum was detected by IC-PCR in potato peel extracts containing both Pseudomonas atrosepticum and its close relative Pseudomonas carotovorum at low levels (Van der Wolf et al.,1996).

It was possible to enumerate Pseudomonas atrosepticum to as low as 100 cells/ml in contrast to conventional PCR, in which 105 cells/ml was enumerated only after extract had been diluted 100 times to reduce the effect of inhibitors (Van der Wolf et al.,1996).

Advantages:

It provides an efficient way to remove interfering plant substances.

IC-RT-PCR allows the processing of large volume of plant extract.

Drawback:• It required an antiserum against the virus in question.

Bio-PCR BIO-PCR involves an enrichment technique prior

to extraction and amplification of DNA of the target bacterium (Schaad et al.,1995).

The intent of the method is to achieve target bacterium and suppress the growth of non target bacterium.

Normally the success of the method is wholly dependent on the availability of a reliable selective medium.

Application:

• Detection of Pseudomonas savastanoi pv. savastanoi by PCR was enhanced by pre-enrichment in either non selective King’s B medium or on a semi-selective medium design for Pseudomonas savastanoi pv. savastanoi by PCR (Penyalver et al;2000).

• Song et al. (2004) describe the development of nutrient enrichment medium for Acidovorax avenae subsp. avenae on rice seeds.

Advantages:

• BIO-PCR increases the sensitivity of detection.

• It avoids the possibility of detecting dead bacterial cells.

Limitations:• Quantification of bacterial population cannot be

readily done.

• If selective medium is lacking sensitivity of detection is lacking.

Nested PCR

Sensitivity and specificity problems associated with conventional PCR and RT-PCR can be reduced by using nested PCR-based methods.

It involves the introduction of a second round of amplication using the amplicon of the first PCR reaction as template for the second.

Some authors proposed single-tube nested-PCR protocols for the bacteria E. amylovora (Llop et al., 2000), for Pseudomonas savastanoi pv. savastanoi (Bertolini et al., 2003), and some viruses (Yourno, 1992).

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Bertolini et al., (2003) developed a nested PCR test for Pseudomonas savastanoi pv. savastanoi using external primers that amplified at 620C and internal primers that amplified at 500C.

A simple device based on the use of a compartmentalized Eppendorf tube, which enables RT reaction and nested PCR to be carried out in a single tube and in one-manipulation, has also been described for detection of Citrus tristeza virus (CTV) (Olmos et al., 1999 and 2003).

Coupling nested-PCR variants with squashed or printed samples on paper membranes has allowed the detection of RNA targets from several viruses in plant material and in individual insect vectors (Cambra et al., 2006a; Moreno et al., 2007).

Advantages:

• Sensitivity is increased by two orders of magnitude reaching about 102 bacterial cells/ml of extract.

Limitations:• Need to accurately establish the ratio between

external and internal primers • Use of limiting amounts of external primers to

avoid interference during the second amplification.

Nested PCR

Sensitivity and specificity problems associated with conventional PCR and RT-PCR can be reduced by using nested PCR-based methods.

It involves the introduction of a second round of amplication using the amplicon of the first PCR reaction as template for the second.

some authors proposed single-tube nested-PCR protocols for the bacteria E. amylovora (Llop et al., 2000), for Pseudomonas savastanoi pv. savastanoi (Bertolini et al., 2003), and some viruses (Yourno, 1992).

Co-operational PCR

It is based on the simultaneous action of four or three primers.

It can be performed easily in a simple reaction increasing the sensitivity level.

The sensitivity observed in virus detection is at least 1000 times higher than that achieved with RT-PCR and is similar to that of nested RT-PCR.

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For the detection of plant RNA viruses, such as CTV, Cucumber mosaic virus (CMV), Cherry leaf roll virus (CLRV) and Strawberry latent ringspot virus (SLRSV) (Olmos et al., 2002).

For the bacterium R. solanacearum in water (Caruso et al.,2003)

There are also some protocols for phytoplasmas as “Ca Phytoplasma mali”, “Ca Phytoplasma prunorum” and “Ca Phytoplasma pyri” (Bertolini et al., 2007).

Advantages:

• Co-PCR requires only one reaction, minimizing manipulation and reducing risk of contamination.

• Ten times less reagent required than in conventional PCR

Limitations:• The small volume of reagents could increase

susceptibility to inhibitors, requiring a previous RNA extraction to reach a good sensitivity in detection (Olmos et al., 2002).

Multiplex PCR

This methodology has demonstrated to be a valuable tool for detection and identification purposes (Lopez et al, 2006)

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

In this case the choice of primers for amplification depends upon the desired type or detection

Two successful examples are the simultaneous detection of

the six major characterized viruses affecting olive trees:CMV,

Arabis mosaic virus (ArMV), Olive latent virus-1 and

Olive latent virus-2 (Bertolini et al.,2001)

Nine grapevine viruses (ArMV, grapevine fanleaf virus,

grapevine virus A, grapevine virus B, rupestris stem pitting-

associated virus, grapevine fleck virus, grapevine leafroll-

associated virus-1, -2 and -3)(Gambino andGrinbaudo,2006).

Scheematic representation of multipex PCR

Multiplex nested PCR Combines the advantages of the multiplex PCR with the

sensitivity and reliability of the nested PCR.

It enables simultaneous detection of RNA and DNA targets.

The sensitivity achieved for the bacterium P. savastanoi pv. savastanoi by multiplex nested RT-PCR (1 cell/ml) was demonstrated to be 100-fold more sensitive than conventional PCR (Bertolini et al., 2003).

This technology performed in a single tube for specific detection of CMV, ArMV, and the bacterium P. savastanoi pv. savastanoi in olive plant material using 20 compatible primers (Bertolini et al., 2003).

Advantages:

• Saving time and reagent costs.

Limitations:• The accurate design of compatible primers is

necessary to avoid hairpins and primer-dimer formation.

Real Time PCR

An advancement of m-PCR is the development of Real Time PCR.

Real Time PCR is a quantitative procedure, which helps to detect, accumulation of PCR products during the PCR reaction.

PCR products can be monitored using either fluorescent DNA intercalating dyes such as SYBR Green I, or sequence specific probe based assays using TaqMan probes.

Taqman R

ABI prism 7900 continuously measures PCR product accumulation using Taqman probe

Sequence of probe is homologous to an internal target sequence

Probe intact

Energy transfer between fluoroaphore

Fluorescent emission is quenched

Probe cleaved by 5’ nuclease activity of Taq polymerase (extension phase)

Increase in emission intensity is measured

Real Time PCR

• For detection of Clavibacter michiganensis subsp. sepedonicus in potato tubers (Van Beckhoven et al., 2002),

• • Ralstonia solanacearum (Weller et al., 2000; Ozakman and Schaad,

2003),

• Erwinia amylovora, the causal agent of fire blight. (Salm and Geider, 2004),

• Ca. Liberibacter asiaticum (Li et al., 2006),

• X. fastidiosa (Bextine et al., 2005),

• X. fragariae (Weller et al., 2007)

Advantages:

It allows the monitoring of reaction while it is in course, thus avoiding the risk of contamination.

Rapid on-side diagnosis of quarantine pathogen.

It offers faster and more accurate detection assays.

Require fewer reagent and allows additional studies to be performed during detection.

Magnetic capture – hybridisation (MCH) PCR :-

Magnetic beads are coated with biotinilated oligonucleotide that is specific of a DNA region of the fungus of interest.

Hybridisation takes place between the fungal DNA and magnetic beads-oligomer and the conjugate is recovery separated from inhibitory compounds.

After the magnetic capture-hybridisation, PCR amplification was carried out using species-specific primers.

Langrell & Barbara, (2001) used this method to detect Nectria galligena in apple and pear trees.

Scheematic Representation of Magnetic capture – hybridisation (MCH) PCR

Nucleic Acid Hybridization

The Basic Process of Binding a Single Strand of Nucleic Acid (DNA or RNA) to Its Complementary Strand Is Called Nucleic Acid Hybridization.

First utilized in plant pathology to detect Potato spindle tuber viroid (Owens and Diener, 1981)

Principle:

• Denaturation of the ds DNA can be achieved by exposure to high temperature or alkaline pH. Dissociated strands of DNA can be immobilized on a solid phase support, such as latex, magnetic beads, microtitre plates ,nitrocellulose or nylon based membrane, and then hybridized with single strand, labeled nucleic acid (usually DNA) probe.

• The probes will hybridized only with the denatured strand of complementary nucleic acid.

a) Dot-Blot Hybridization Assay

samples of denatured nucleic acid from healthy and infected plants are directly spotted on to a prewetted, solid matrics such as a nitrocellulose or nylon based membrane.

Nucleic acids are then firmly immolised on a solid support by baking the membrane in an oven for 2hr. at 80◦C.

To block the remaining Free DNA binding sites, the membrane is incubated several minutes in a sealed plastic bag with prehybridisation solution.

Continued….

After removing the prehybridisation solution, the hybridization solution containing the denatured specific DNA probe is added to the plastic bag containing the sample membrane, and incubate for several hours to allow hybridization between the probe and the target nucleic acid.

The membrane is washed several times to remove the unbound DNA probe following hybridization.

Hybridization between the target nucleic acid immobilized on the membrane and the DNA probe is detected either by autoradiography on X-ray film, or by a calorimetric reaction.

Continued….

The sensitivity of dot-blot hybridization may equal or exceed that of ELISA (Polston et al., 1989).

This technique has been employed for Apple mosaic virus(ApMV), Prunus necrotic ringspot virus (PNRSV), Prune dwarf virus (PDV), and Apple chlorotic leaf spot virus (ACLSV) (Pallás et al., 1998).

Probe presentNo probe

Nucleic Acid Hybridization

b) Fluorescence in situ hybridization

(FISH) combines microscopical observation of bacteria and the specificity of hybridization (Volkhard et al., 2000).

It is dependent on the hybridisation of DNA probes to species-specific regions of bacterial ribosomes.

There is a high affinity and selectivity of DNAprobes because FISH takes place under very stringent hybridisation conditions.

In practice, FISH can reach a relatively low sensitivity levels in some cases, even though has been employed in some recent works (Ercolini et al.2006).

3. Molecular markers:

This technique makes use of restriction enzymes to fragment DNA, which is then separated by agarose gel electrophoresis.

Each restriction enzyme recognizes a specific nucleotide sequence and cuts the DNA specifically every time the sequence occurs.

The fragmented DNA is separated by gel electrophoresis, and the banding pattern is made visible by staining (ethidium bromide) or by autoradiography.

When mitochondrial DNA (mtDNA) from different species are examined, differences in the size and number of restriction fragments can be detected.

These differences can result from inserts or deletions between existing restriction-enzyme sites or mutations that destroy or create restriction sites.

a) Restriction fragment length polymorphisms:

b) RAPD Markers:

RAPDs are DNA fragments amplified by the PCR using short synthetic primers of random sequence.

These oligonucleiotides serve as both forward and raverse primer, and are usually able to amplify fragments from 1-10 genomic sites simultaneously.

Amplified fragments are separated by agarose gel electrophoresis, and polymorphism are detected.

These polymorphisms are considered to be primarily due to variation in the primer annealing sites, but they can also be generated by length differences in the amplified sequence between primer annealing sites.

Some of specific DNA fragments detected in a profile may be cut out of the gel and sequenced to obtain a SCAR (Sequence-characterized ampli- fied region), into which specific primers can be designed for a more precise PCR detection.

SCAR primers have been used to specifically identify Phytophthora cactorum (Causin et al., 2005), Fusarium subglutinans (Zaccaro et al., 2007) and Guignardia citricarpa (Stringari et al., 2009)

RAPD also useful for the analysis of the genetic diversity among populations.

Ex. Fusarium spp. ( Arici & Koc 2010) and pathotypes of Elsinoë spp. (Hyun et al., 2009).

The RAPD technique is rapid, inexpensive and does not require any prior knowledge of the DNA sequence of the target organism. Results obtained from RAPD profiles are easy to interpret because they are based on amplification or non amplification of specific DNA sequences.

Results obtained from RAPD profiles are easy to interpret because they are based on amplification or non amplification of specific DNA sequences.

RAPD analyses can be carried out on large numbers of isolates without the need for abundant quantities of high-quality DNA (Nayaka et al., 2011).

Continued….

Scheematic Representation of RAPD

4. Nucleic Acid Sequence /probes:

a) Nucleic Acid Sequence Based Amplification (NASBA):

It can be used to detect RNA targets and based on exponential amplification of single stranded RNA molecules by simultaneous activities of reverse transcriptase and polymerase enzymes (Kievits et al., 1991).

The reaction requires the use of three enzymes: i) Avian myeloblastosis virus reverse transcriptase( AMV-RT) for reverse transcription and to obtain double stranded cDNA,

ii) RNase H to hydrolyze the RNA fragment of the hybrid molecule DNA- RNA

iii) T7 RNA polymerase to produce a large amount of anti-sense, single strand RNA transcripts corresponding to the original RNA

target

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This technology has been applied for detecting plant viruses such as Apple stem pitting virus (Klerks et al., 2001)

PPV (Olmos et al., 2007a)

Potato virus Y, ArMV and the bacteria C. michiganensis subsp. sepedonicus and R. solanacearum (Szemes and Schoen, 2003).

The sensitivity of this method has proven similar to that obtained by real-time RT-PCR when applied to PPV detection (Olmos et al., 2007a).

b) Loop-mediated isothermal amplification (LAMP):

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.

The amplification takes place at 60-65ºC for 60 min.

Although it was initially developed for DNA it can be adapted to amplify RNA (RT-LAMP) (Fukuta et al., 2003).

The method has only been applied to the detection of some plant viruses such as PPV, with a sensitivity level similar to that obtained by real-time PCR (Varga and James, 2006).

5. Microarray technology:

Microarrays are generally composed of thousands of specific probes spotted onto a solid surface (usually nylon or glass).

Each probe is complementary to a specific DNA sequence (genes, ribosomal DNA) and hybridisation with the labeled complementary sequence provides a signal that can be detected and analysed.

Until now, the microarray technology focuses its use in multiplex format of similar or very different pathogens, taking advantage of the number of probes that can be employed in one chip (Pasquini et al., 2008).

continued…

Another type of microarray under development is called the nanochip (Sosnowski et al., 1997)

Based on an electronically addressable electrode array that provides direct electric field control over the transport of charged molecules to selected micro locations and concentration over an immobilized substrate

Future Perspectives:

Information resulting from detection by improved molecular methods could be used to optimize diseasecontrol through more rational decisions about the choice and use of control measures.

Besides optimization of PCR and real-time PCR protocols, the advances in microarray, microchip or biochip technology will allow to test simultaneously, the prospect of a wide variety of pathogenic microorganisms, and the potential of this tool will open new fields of studies in plant pathology.

Since cultivated plants can be affected by diseases caused by many types of organismsa method able to detect several pathogens simultaneously would be ideal for testing plant material, especially for quarantine pathogens.

Protocols based on PCR have already been developed for the most important pathogens and they should be optimized soon, looking for multiplex detection, trying to simplify the RNA or DNA extraction without decreasing the robustness of the methods

Conclusion: Nucleic acid-based methods are sensitive, specific and allow

genetic relationships to be determined.

In plant pathology compared to traditional methods, PCR offers several advantages, because, organisms do not need to be cultured before their detection.

It affords high sensitivity at least theoretically, enabling a single target molecule to be detected in a complex mixture.

It is also rapid and versatile.

In fact, the different variants of PCR, have increased the accuracy of detection and diagnosis, and opened new insights into our knowledge of the ecology and population dynamics of many pathogens.

Providing a valuable tool for basic and applied studies in plant pathology.

Thank you!