Identification of plant pathogens on the basis of their morphology is a tedious, time consuming, difficult task, needs mycological expertise and may lead to wrong identification sometime. Thus, conventional phyto-pathological methods have limitations in developing quarantine methods, disease resistance breeding and disease management purposes. With the advent of genome analysis, it has become possible to accomplish the same task directly at the molecular level with the help of molecular markers. Molecular markers are nucleic acid segments that behave as landmarks for genome analysis and are based on naturally occurring genetic variabilities (usually termed polymorphisms). The evidences available clearly indicate that molecular markers are useful in separating morphologically similar species.

Molecular markers can be categorized in two groups according to the way they are generated i.e. Hybridization-based techniques and Amplification-based techniques. Hybridization-based techniques are based on the Watson-Crick complementary rules of base pairing and require the use of labeled nucleic acid molecules as hybridization probes. Probes can be short single-stranded nucleic acid segments (oligonucleotides) of synthetic origin or cloned DNA segments that bind to complementary nucleic acids, forming hybrid molecules. Eg.- Restriction fragment length polymorphic markers (RFLP). Amplification based techniques use oligonucleotides (DNA or RNA) to get the exponential accumulation of specific sequences from defined regions in a genome or transcriptome. PCR-based techniques have been used to amplify single targets such as ribosomal RNA (rRNA) genes, mitochondrial and chloroplast DNA sequences, and repetitive DNA.

The Polymerase Chain Reaction (PCR) is a powerful, extremely sensitive technique employed in the field of Molecular biology, Agriculture diagnostics, Forensic analysis and population genetics. It is based on the enzymatic amplification of DNA fragments that is flanked by oligonucleotide primer hybridizing to opposite strands of the target sequence. The PCR involves three basis steps which constitute a single cycle:

(i) Denaturation of the target DNA at 92-94^°C

(ii) Annealing of the primers to the template DNA.

(iii) Primer extension by addition of nucleotides to the 3’ end of the primers by the enzyme DNA polymerase.

As the number of PCR cycle increases, the amount of target DNA synthesized increases exponentially. Availability of thermostable DNA polymerase-Taq (from the bacteria Thermus aquaticus) has facilitated automation of the PCR. PCR is invented by Kary Mullis and this invention brought him a number of scientific awards, among them most important were the Japan Prize and the Nobel, both awarded to him in 1993.

Constituents of PCR reaction

Primer - The most essential requirement of PCR is the availability of short oligonucleotides called primers having sequence complementary to either ends of the target DNA segment. Primers are short strand of nucleic acid serves as starting point of DNA synthesis.

Template DNA - DNA segment to be synthesized in large amount. Genomic DNA or RNA is used as a template.

In general, CTAB method is used for isolation of genomic DNA from fungal plant pathogens. Liquid Nitrogen is used for grinding the mycelial mats, for effective disruption of the cell wall and cell membrane. 1M Tris buffer, 0.5 M EDTA (Ethylene diamine tetra) , 1M NaCl, 70% ethanol, 10% (w/v) Sodium dodecyl sulfate (SDS), 10% CTAB, 24:1 Chloroform Isoamyl solution, Isopropanol and TE buffer are different chemicals required for DNA isolation.

Template DNA should be pure i.e. without any contamination of RNA and protein. Ribonuclease enzyme and ProteinaseK are used (as per the protocol) for removal of RNA and protein contamination from the genomic DNA of target plant pathogen. Quantification of the purified genomic DNA should be done by checking the UV absorbance at 260 nm with the help of U.V. spectrophotometer. Take optical density (O.D.) of the sample at 280 nm to calculate the ratio OD260/OD280. This ratio gives the amount of RNA (or) protein in the preparation. A value of 1.8 is optimum for best DNA preparation.

Taq DNA Polymerase- Thermus aquaticus DNA polymerase (Taq DNA polymerase) is a thermostable enzyme that replicated DNA at 72-74^°C and remains functional even after incubation at 95 ⁰C. The enzyme has 5’-3’ polymerase activity and 3’-5’ exonuclease activity.

DNTP’s- The deoxynucleotidetriphosphates are dATP, dGTP, dCTP, dTTP (used as 10 mM each).

Assay Buffer (10X)- 10 X assay buffer for Taq polymerase enzyme. Assay buffer contains 10 mM Tris-HCl (pH 9.0), 15 mM MgCl2, 50 mM KCl and 0.01% gelatin.

Preparation of PCR mixture:
A master mix containing all the above mentioned components and no template DNA should be prepared in laminar airflow under sterile conditions for total number of PCR tubes to be used. This would reduce the pipetting errors. Then distribute the master mix in each tube (24 ul each) and finally add 1 ul of different DNA template in each tube. Gently mix and centrifuge the mixture for 10 sec. Annealing temperature should be standardized using T-gradient programme.

Analysis of the Amplification (PCR) Products:
For the analysis of PCR products/ amplicons, Agarose Gel Electrophoresis technique is used. In agarose gel electrophoresis, DNA and RNA can be separated on the basis of size by running the DNA through an agarose gel. Agarose gel of various thickness (1.0% or more, as per need) could be produced by dissolving appropriate amount of agarose in 0.5X TBE buffer. DNA loading dye and DNA samples are loaded in the gel. After electrophoresis, the gel is stained with ethidium bromide solution. After destaining in de-ionized water, the gel image could be viewed in U.V. transilluminator and stored in gel documentation system.

Significance of PCR
The central scientific fact that makes PCR so useful is that- The genetic material of each living organism-plant or animal, bacterium or virus-possesses sequences of its nucleotide building blocks (usually DNA, sometimes RNA) that are uniquely and specifically present only in its own species. These unique variations make it possible to trace genetic material back to its origin, identifying with precision at least what species of organism it came from, and often which particular member of that species.

Such an investigation requires, however, that enough of the DNA under study is available for analysis-which is where PCR comes in. PCR exploits the remarkable natural function of the enzymes known as polymerases. These enzymes are present in all living things, and their job is to copy genetic material (and also proofread and correct the copies). Sometimes referred to as "molecular photocopying," PCR can characterize, analyze, and synthesize any specific piece of DNA or RNA. It works even on extremely complicated mixtures, seeking out, identifying, and duplicating a particular bit of genetic material from blood, hair, or tissue specimens, from microbes, animals, or plants, some of them many thousands-or possibly even millions-of years old.

Application of PCR in Plant Pathology
PCR technology has become an essential research and diagnostic tool for improving knowledge regarding identification, characterization, detection and diagnosis of plant pathogens. PCR technology allows scientists to take a specimen of genetic material, even from just one cell, copy its genetic sequence over and over, and generate a test sample sufficient to detect the presence or absence of a specific virus, bacterium or any particular sequence of genetic material. Therefore, it is hard to exaggerate the impact of the polymerase chain reaction. PCR, the quick, easy method for generating unlimited copies of any fragment of DNA, is one of those scientific developments that actually deserve timeworn superlatives like "revolutionary" and "breakthrough."

Diagnosis and characterization of the pathogen
PCR utilises specific oligonucleotide primers, which are designed based on nucleic acid sequences that are diagnostic for the pathogen. These may be sequences identified by genomic sequencing or techniques such as DNA fingerprinting of pathogens. To improve the sensitivity of these techniques, it is sometimes an advantage to use nested PCR methods, in which the products from the initial PCR amplification are diluted and re-amplified with a second set of primers internal to the original primer set in the pathogen sequence. These conventional PCR-based techniques have proved to be useful for diagnosis of fungal, bacterial and phytoplasma-associated diseases with a number of good taxon-specific primers developed for example from the rRNA subunit genes. Because of the sequence variation between these regions in different isolates, it has sometimes been possible to identify restriction endonucleases that give different restriction patterns upon digestion of the PCR products depending upon the isolate.

Primers have also been developed based on more specific sequences such as the argk-tox gene of P. syringae pv. phaseolicola which encodes a gene involved in phaseolotoxin biosynthesis and can be used to identify bacteria that possess this trait, or the aflR gene of Aspergillus flavus which regulates aflatoxin production in these fungi. In addition, PCR-based techniques have proved useful for identifying the vectors for insect-transmitted diseases. For example, DNA extracted from leafhoppers that are potential vectors for phytoplasma diseases can be PCR amplified using phytoplasma-specific primers to identify which species are the true vectors.

Single-strand RNA viruses can be detected by modifying the PCR to include a reverse transcriptase step (RT-PCR). In this, the reverse transcriptase uses a viral specific primer to make a cDNA copy of the viral RNA which is then amplified using Taq polymerase through conventional PCR. RT-PCR has the potential for use in diagnostics of bacterial and fungal pathogens. Through identification of particular genes that are expressed during pathogen growth, or at specific stages of development, it may be possible to use RT-PCR to identify the developmental stage of the pathogen in infected material. It has also been possible to produce ‘multiplex’ PCR kits capable of detecting more than one pathogen present in a particular plant or soil sample. Kits are commercially available to unravel the cereal stem-based complex of fungi comprising Tapesia yallundae and T. acuformis (eyespot fungi), Fusarium culmorum, F. avenaceum, F. graminearum and F. poae (ear blights), and Microdochium nivale (snowmould of cereals).

References
1. Dickinson. M. 2005. Molecular Plant Pathology. BIOS Scientific Publishers, Taylor & Francis Group. London and New York.

2. Schaad, N. W. and Frederick, R. D. 2002. Real-time PCR and its application for rapid plant disease diagnostics. Can. J. Plant Pathol. 24: 250â€"258.

3. Gelsomino, G., Faedda, R., Rizza, C., Petrone, G. and Cacciola, S.O. 2011. New platforms for the diagnosis and identification of fungal and bacterial pathogens. In: Science against microbial pathogens: communicating current research and technological advances. (Ed.) A. Mendez Vilas. 622-630.


About Author / Additional Info:
I am a Scientist (Plant Pathology) at JNKVV, Regional Agricultural Research Station, Sagar, M.P.