Molecular Methods Used For

Detection
of Plant Pathogens
 DNA extraction
• It is a routine procedure used to isolate
DNA from the nucleus of cells.

• Scientists can buy ready-to-

use DNA extraction kits.

• However, they can be expensive to use

routinely, so many labs have their own
methods for DNA extraction
• Plant materials are among the most difficult for
high quality DNA extractions.

• The key is to properly prepare the tissues for

extraction.

• In most cases this involves the use of liquid

nitrogen flash freezing followed by grinding the
frozen tissue with a mortar and pestle at room
temperature to form a fine powder
Step 1. Breaking cells open to release
the DNA
• The cells in a sample are separated
from each other, often by grinding
and put into a solution
containing salt (buffer)

• A detergent (chloroform) is then

added.

• The detergent breaks down

the lipids . DNA is released as
these membranes are disrupted.
Step 2. Separating DNA from proteins
and other cellular debris

• To get a clean sample of DNA, it’s necessary to

remove as much of the cellular debris as
possible.

• Often a protease ( protein enzyme) is added to

degrade DNA-associated proteins and other
cellular proteins.
Step 3. Precipitating the DNA with an
alcohol

• Ice-cold alcohol (either ethanol or isopropanol)

is carefully added to the DNA sample- DNA is
soluble in water but insoluble in the presence
of salt and alcohol.

• Vortexed and centrifuged, supernatant is

poured out and a white precipitate becomes
visible.
 Step 4. Cleaning the DNA
• The DNA sample can now be further purified
to remove other cellular contaminants
(cleaned in ethanol and rinsed).

• It is then resuspended in a
slightly alkaline buffer and ready to use.
 What can this DNA be used for?
• Once extracted, DNA can be used for
molecular analyses
including PCR, electrophoresis, sequencing,
fingerprinting and cloning.
 Polymerase Chain Reaction
• PCR is a method by which a particular
segment of DNA can be specifically
replicated.

• Invented by Karry Mullis(1987)

• PCR is an ingenious new tool for

identification of plant pathogens.

• Primer +DNA template, buffer , water

 PCR principle
• The double stranded DNA is denatured to
separate into two individual strands

• Each strand is then allowed to hybridize with a

primer

• These three steps denaturation, renaturation and

synthesis are repeated again and again to
generate multiple forms of target DNA
(amplification)
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

• Purpose: can be used to amplify a sample of

DNA when there isn't enough to analyze as a
method of identifying a gene of interest, or to
test for disease
 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.
RT-PCR (Reverse transcriptase-polymerase
chain reaction)
• “Gold standard molecular method” for detection
of viruses.
• Highly sensitive technique for the detection and
quantitation of mRNA.
• Allows the use of RNA as a template.
• 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.
 IC-RT-PCR: (immunocapture)
• Use of high-affinity binding antibodies to selectively
capture the target organism prior to detection by
conventional PCR amplification

• Technique can be performed in plates that have already

been used through a complete ELISA assay.

• Offers advantage of testing by PCR only those samples

found negative in an initial ELISA screening, without
compromising the sensitivity of the PCR amplification
procedure.

• Very well suited for large scale detection of plant pathogens

 IC-RT-PCR cont’d
• It has been successfully used to detect Xanthomonas
axonopodis pv. dieffenbanchiae, the cause of bacterial
blight in vegetative propagating material

• Pseudomonas atrosepticum was detected in potato peel

extracts containing both Pseudomonas atrosepticum and its
close relative Pseudomonas carotovorum at low levels

• 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
IC-RT-PCR cont’d
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 requires an antiserum against the pathogen in
question.
 Bio-PCR
• The biological amplification (growth media) is used to
increase the pathogen numbers to detectable levels in
asymptomatic tissue while PCR is used to increase the
number of copies of target DNA

• 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.
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
• It is a modification of PCR that was designed
to improve sensitivity and specificity.

• Involves the use of two primer sets and two

successive PCR reactions.

• It involves the introduction of a second round

of amplication using the amplicon of the first
PCR reaction as template for the second.
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.
 Multiplex PCR
• The simultaneous detection of two or more DNA
or/and RNA targets in a single reaction with
several specific primers

• 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.
Advantages:
• No false negatives due to reaction
failure/contamination
• Saving time and reagent costs.

Limitations:
• The accurate design of compatible primers is
necessary
• Cross hybridization of multiple primers
 Real Time PCR
• Real Time PCR 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.

• PCR uses Ethidium Bromide and UV light to visualize

bands in the agarose gel medium, while Real Time
PCR uses fluorescent dyes to detect the product.
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.
 Gel electrophoresis
• Gel electrophoresis is a
laboratory method used to
separate mixtures of DNA,
RNA, or proteins according
to molecular size.

• The molecules to be
separated are pushed by an
electrical field through a gel
Pouring gel on casting trays with Sample combs,
loading (dye added)and running
Agarose gel electrophoresis (AGE)
• Agarose is a natural linear polymer extracted
from seaweed that forms a gel matrix by
hydrogen-bonding when heated in a buffer
and allowed to cool.
 Polyacrylamide gel electrophoresis
(PAGE)
• Polyacrylamide gels are chemically cross-
linked gels formed by the polymerization of
acrylamide with a cross-linking agent, usually
N,N- methylenebisacrylamide
 Visualizing the DNA

• After the electrophoresis has been

completed there are different
methods that may be used to make
the separated DNA species in the
gel visible to the human eye.

• Staining that binds to DNA to make

it more visible under UV light
i. Ethidium bromide staining (EBS)
ii. Silver staining (SS)
Coding the bands
 Molecular markers-(PCR Based)
• Restriction Fragment Length Polymorphism
(RFLP)

• Amplified Fragment Length Polymorphism (AFLP)

• Random Amplified Polymorphic DNA (RAPD)

• Simple Sequence Repeat (SSR)/microsatellites

Summary
Indirect methods (Remote sensing)
 Thermography
• Allows imaging the differences in surface
temperature of plant leaves and canopies.

• The emitted infrared radiation can be

captured by thermographic cameras and
color difference can be analyzed.

• Loss of water in plants would be affected by

phytopathogens - The resulting disease can
be monitored through imaging and the
amount of water transpired
 Thermography

• Promising tool to monitor the heterogeneity in

the infection of soil-borne pathogens

• Practical applicability for disease monitoring is

limited due to its high sensitivity to the change of
environmental conditions during measurements.

• Lacks the specificity towards diseases, and

therefore cannot be used to identify the type of
infection or distinguish between diseases that
produce similar thermographic patterns.
 Fluorescence Imaging

• Chlorophyll fluorescence is
measured on the leaves and
change in fluorescence
parameters can be used to
analyze pathogen infections

• Although fluorescence
measurement provides sensitive
detection of abnormalities in
photosynthesis, the practical
application of this technique in a
field setting is limited
 Gas Chromatography
• Involves the profiling of the volatile chemical
signature of the infected plants.

• The pathogen infections of plants could result in

the release of specific volatile organic compounds
(VOCs) that are highly indicative of the type of
stress experienced by plants

• VOCs are also produced when green leaf plants

are damaged pathogenically and mechanically.
• Profiling VOC - a means to identify the type and
nature of infection and be used for disease
diagnostics and confirmation
• GC often combined with mass spectrometry (GC-
MS) to identify unknown compounds in the
volatile sample
• Can provide more accurate information about the
plant disease due to its high specificity.
• Allows the detection of diseases at different
stages based on the quantitative information
collected from the VOC sample.
• Requires sampling of pre-collected VOC for a
longer time, which severely limits its on-field
application
 Detection of Plant Diseases Using
Portable Sensors
• Biosensor Based on Nanomaterials

• Antibody-Based Biosensors

• DNA/RNA-Based Affinity Biosensor

• Enzymatic Electrochemical Biosensors

• Bacteriophage-Based Biosensors
PLANT TISSUE CULTURE
 Application of tissue culture?
• Advantages?
• Disadvantages?
Thank you