1. Master Seminar Presentation
Course Code- PLPATH- 591
Modern techniques for detection of plant pathogens
Presented by:
Megobi Punyü
Roll no: M-1607/22
Department of Plant Pathology
School of Agricultural Sciences
Medziphema, Nagaland
4. Process of identifying the presence of something hidden in an
object, which cannot be directly observed.
Detection
Ideal plant disease detection technique should be:
Specific
Sensitive
Accurate
Reliable
Fast
Ease of use
Cost-effective
Ability to detect pathogen in complex matrices
5. Objectives
Detect and identify new pathogens.
Determine the cause, epidemiology and distribution of disease.
Seed certification and quarantine.
Resolve the components of complex diseases.
Provide suitable plant protection measures.
Determine range of disease incidence and yield loss.
6. Serological based
Epitope: The part of an antigen that is recognized by antibodies.
Paratope: The part of an antibody that recognizes the antigen.
Antigen:
Any foreign substance / molecule which triggers an immune response in
the body and evokes the production of antibodies .
Antibody :
Glycoprotein secreted by immune cells in response of specific antigen.
7. Enzyme-linked Immunosorbent assay (ELISA)
Eva Engvall and Peter Perlman - 1971
It involves an enzyme-mediated color change reaction to detect
antibody binding
96 well Microtitre plate
A positive result is visualized when a colored product is
released by an enzyme-substrate reaction
Substrates: Para Nitrophenyl phosphate (PNPP), Hydrogen
peroxide
Enzymes: Horseradish peroxidase (HRP) and alkaline phosphatase
9. Immunofluorescence (IF)
Immunofluorescence is an antigen-antibody reaction where the antibodies are labeled with a
fluorescent dye and the antigen-antibody complex is visualized using ultra-violet (fluorescent)
microscope.
In this process dyes (Fluorochromes) are used which absorb ultraviolet rays and emit visible light.
This process is called fluorescence.
Fluorescent Dyes are of two type:
a. Fluorescein (green)
b. Rhodamine (red).
10. Direct immunofluorescence: In which the primary antibody is labeled with fluorescence dye.
Two types of immunofluorescence:
Indirect immunofluorescence: In which a secondary antibody labeled with fluorochrome is used to recognize a
primary antibody.
Fig: Direct and indirect immunofluorescence
11. Polymerase Chain Reaction (PCR)
A technique used to amplify specific DNA fragments
Detection of a PCR fragment with the expected size is used to
confirm the presence of the target pathogen.
Kary Mullis invented the PCR in 1983
Kary Mullis
Components of PCR:
DNA template: single or double stranded.
Thermostable DNA polymerase: Taq DNA polymerase
Primers: oligonucleotides
dNTPs : “building blocks” for new DNA strand
Buffer solution: example: Tris-HCL buffer
Divalent cations: Mg 2+
Thermocycler
Molecular based techniques
13. Types of PCR
RT-PCR (Reverse transcriptase): can be used on RNA targets.
Multiplex PCR: detection of multiple target pathogens.
Real Time / q PCR: Amplified DNA is measured in real time.
Nested PCR: increase sensitivity and specificity of DNA
amplification.
14. Micro arrays
Several synonyms of microarrays such as DNA chips, gene chips, DNA arrays, gene
arrays and biochips.
Microarrays are a collection of microscopic spots (< 200 µm ) of probe DNA (20-
5000
base pairs) attached to a glass or nylon slide.
Each probe represents a single gene which is complementary to a specific DNA
sequence (genes, ribosomal DNA) and hybridization with the labeled
complementary sequence provides a signal that can be detected and analyzed.
15. Fig: DNA Microarray
DNA microarray, protein microarray, chemical compound arrays,
carbohydrate arrays, cellular microarrays
Types:
16. Constant temperature (60-65°C).
Requires four to six primers.
LAMP products is detected by agarose gel electrophoresis, turbidity, fluorescent
DNA dyes, lateral flow devices.
Amplify nucleic acid at high specificity, sensitivity and speed.
Generation of stem looped (hairpin) structures during early stage of DNA synthesis.
Greek-: “isos” - equal, “therme”- heat
Rapid detection of viral and bacterial diseases.
17. Fluorescence In-situ hybridization(FISH)
Detecting and locating a specific DNA sequence on a chromosome.
It is a combination of Microscopy and Hybridization of DNA probes.
Gene probes
Attach
fluorescence
dye
Bind to target
sequence
within a cell
Emits
fluorescent
light
The high affinity and specificity of DNA probes provide high single-cell sensitivity in FISH, because the probe
will bind to each of the ribosomes in the sample.
Advantages:
Developed by M. L Pardue and J.G. Gall in 1969
The practical limit of detection lies in the range of around 103 cfu/ mL.
18. Fluorescence In-situ hybridization(FISH)
Detecting and locating a specific DNA sequence on a chromosome.
It is a combination of Microscopy and Hybridization of DNA probes.
Gene probes
Attach
fluorescence
dye
Bind to target
sequence
within a cell
Emits
fluorescent
light
Insufficient penetration, higher order structure of target or probe (e.g., three-dimensional rRNA, loop and
hairpin formation and rRNA-protein interactions), low rRNA content, photobleaching can give false negatives.
Limitation:
Developed by M. L Pardue and J.G. Gall in 1969
19. Flow cytometry
‘cyto’ : cell ‘metry’ : count / measurement
Laser-based optical technique widely used for cell counting and sorting,
biomarker detection and protein engineering
The technique uses an incident laser beam and measures the scattering and
fluorescence of the laser beam reflected from the sample.
Optical based techniques
Greek word “optikos “- of or having to do with sight.
Fluorescent dyes can be used to test the viability and metabolic state of microorganisms.
20. Flow Cytometry is used for rapid identification of cells while cells
pass through an electronic detection apparatus in a liquid stream.
Fig: Flow Cytometry
21. Thermography
A technique which observes changes in temperature of plant leaves and canopies due
to stomata transpiration.
Thermal imaging is useful for early detection of plant disease, particularly when the
disease directly affects transpiration rate, as it is shown that leaf temperature changes
with the change in transpiration rate.
22. Fluorescence Imaging
Fluorescence imaging - G.G. Stokes in 1852
Coined the word “fluorescence”.
The chlorophyll fluorescence is measured on the leaves as a
function of the incident light and the change in fluorescence
parameters can be used to analyze pathogen infections, based on
changes in the photosynthetic apparatus and photosynthetic
electron transport reactions (Bürling et al., 2011).
Fluorescent stains are called "fluorochromes”. e.g. - Acridine orange,
auramine O, fluorescent antibody (FA).
Temporal and spatial variations of chlorophyll fluorescence were analyzed
for precise detection of leaf rust and powdery mildew infections in wheat
leaves at 470 nm (Kuckenberg et al., 2009).
Fluorescence images (blue, green, red and infrared) of leaves.
23. Hyperspectral Techniques
“Huge numbers of continuous bands” are used for identification of a specimen.
Images are taken and spectral radiance assigned to each pixel, utilizing a range of
wavelengths across the electromagnetic spectrum (including visible and infrared
regions). Disease is detected by measuring the changes in reflectance resulting
from the biophysical and biochemical characteristic changes upon infection.
The range of spectrum is between 350 and 2500 nm.
Advantage:
Pixel-wise incorporation of a continuous spectral signature of hundreds of
wavelengths into a three-dimensional image of the object under inspection.
Highly robust and it provides a rapid analysis of the imaging data.
24. Gas chromatography
Plant infection
Release of
VOC(volatile
organic
compounds)
Gas
Chromatography
Technique
Detection of
Disease
Gas chromatography- the group of analytical separation techniques used to analyze
volatile substances in the gas phase.
Chromatography-M. Tswett (1901 )
Chromatography –separation of a mixture of chemical substances into its individual
components.
25. Gas chromatography
Plant infection
Release of
VOC(volatile
organic
compounds)
Gas
Chromatography
Technique
Detection of
Disease
Infected
Strawberry Plant
p-ethylguaiacol
and p-
ethylphenol
Gas
Chromatography
Technique
Phytophthora
cactorum
(crown rot
disease)
Example:
Gas chromatography- the group of analytical separation techniques used to analyze
volatile substances in the gas phase.
Chromatography-M. Tswett (1901 )
Chromatography –separation of a mixture of chemical substances into its individual
components.
27. Biosensor Platforms Based on Nanomaterials:
Greek word 'nano' means 'dwarf
The limit of detection (35nm) could be enhanced by the use of nanomaterial matrices as transducers.
Nanomaterials like Carbon Nanotubes, Fullerenes, Nano Chips, DNA Modified gold Particles,
Dendrimers and Quantum dots (QD) have been used for identification of plant disease.
Fluorescent silica nanoparticles (FSNPs) combined with antibody as a biomarker successfully
detected plant pathogens such as Xanthomonas axonopodis pv. vesicatoria that cause bacterial
spot diseases in Pepper and Tomato(Yao et al., 2009).
Gold nanoparticle-based biosensor has been used for detection of Karnal bunt disease in wheat
using surface plasmon resonance (SPR)(Singh et al., 2010)
Copper oxide (CuO) nanoparticles have been used in the detection of the Aspergillus niger fungi.
Quantum dots (QD) have also been used for biosensor construction for disease detection
such as the witches’ broom disease of lime (WBDL) caused by Candidatus Phytoplasma
aurantifolia (Rad et al., 2012).
28. Affinity biosensors
Devices in which biological molecules (receptor) binds with analyte/target molecules to form of a complex,
that is revealed by a transducer.
2 types:
1. Antibody-based Affinity sensors
Advantages: Fast detection, improved sensitivity, real-time analysis and potential for quantification.
Limitations: Exposure of a bacterial strain to environmental stress .
Antibodies require specific environment (pH, temperature, etc.) for the storage to prevent antibody
deterioration (Bryne et al., 2009).
Gold nanorods (AuNRs) functionalized by antibodies have been used to detect Cymbidium mosaic virus (CymMV)
or Odontoglossum ringspot virus (ORSV)
29. Affinity biosensors
2. DNA/RNA-Based Affinity Biosensor:
DNA-based biosensor enables early detection of diseases
before any visual symptoms appear.
This techniques use single stranded DNA (ssDNA) probes on electrodes with electro active indicators to
measure hybridization between probe DNA and the complementary DNA analyte.
Limitations : Requires the synthesis of specific DNA probe, amplification of DNA, high cost (DNA-based
molecular beacons) and unsuitability for real-time detection (DNA-based piezoelectric biosensor).
Eun and Wong (2000) detected two orchid viruses—Cymbidium mosaic virus (CymMV) and Odontoglossum
ringspot virus (ORSV) by this method.
30. Enzymatic
Electrochemical
Biosensors
High specificity of
the enzyme
Sensitivity of
electrochemical
transducers
Enzymatic electrodes have electrochemical probes with a thin layer of immobilized enzyme on their
surface. This enzyme provides the selectivity for the sensor and helps to catalyzes the formation of
the electro-active product for detection.
Many of the VOCs produced by infected crops are alcohols and aldehydes such as cis-3-hexen-1-ol
and trans-2-hexanal, which can be catalyzed by alcohol dehydrogenase enzymes. Accordingly, these
enzymes can be used for the development of biosensors for the detection of alcohol or aldehyde
based VOCs which are specific to the infection.
31. In addition to those specific volatile organic compounds, the common phytohormones such as
auxin, cytokinins and gibberellins which are indicative of plant health could also be deactivated by
oxidases.
Although enzyme-based biosensors usually provide high sensitivity and specificity for the
detection, stability of enzymes is of major concern. In addition, the enzyme catalysis varies with
factors such as temperature and pH which compromises the accuracy of the biosensor.
32. Bacteriophage-Based Biosensors (Phage therapy):
As a result of interaction between the bacteriophage and the target analyte, the impedance of charge transfer reactions at
the interface changes which is used as a signal for detection.
Detection and identification of Pseudomonas cannabina pv. alisalensis from cultures and diseased plant samples was
done by this method (Schofield et al., 2013).
Examples: Icosahedral bacteriophages (T4 and T7)
Filamentous bacteriophages (ie, M13).
33. Advantages:
Limitation:
•Bacteriophage as biosensors have high selectivity and comes at a low cost.
•Bacteriophage-based sensors are more thermostable which allows the detection in different temperature
ranges and longer shelf life.
•It can differentiate live and dead bacterial pathogens which decreases the false positive signals during
measurement.
•Bacteriophage-based sensor can only be used for detection of bacteria.
34. Conclusion
Plant pathogen detection can be through a variety of
techniques such as ELISA, PCR, immunofluorescence, thermal
imaging, biosensors, etc. based on different principles.
Researchers continue striving to find the most efficient and
ideal disease detection methods.
The use of remote sensing, artificial intelligence and
computers can complement the existing techniques.
There is need for constant research and innovation for the
best interest of the agricultural sector.
