defence mechanisim in plant

1. NON-PCR BASED MOLECULAR
APPROACHES FOR DETECTION AND
IDENTIFICATION OF PLANT PATHOGENS
DOCTORAL SEMINAR II
PUJA PANDEY
41124
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2. INTRODUCTION
Plant Pathology
Molecular Plant
Pathology
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3. • Visual observations
• Cultural
• Microscopy
CONVENTIONAL
TECHNIQUES
3

4. VISUAL OBSERVATION :
SIGNS AND SYMPTOMS
Rot - Sclerotium rolfsii
Late blight of potato – Phytophthora infestans
Citrus canker
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5. Problem with biotic and abiotic factors ??????????
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6. CULTURAL : LABORATORY TECHNIQUES
Bacterial ooze
Cultures of Ralstonia solanacearum
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7. Fungal cultures…..
Alternaria culture Fusarium culture
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8. MICROSCOPY : An Aid To
The Eyes
Microscope
Stereoscopic
Microscope
Fluorescence
Microscope
Light
Microscope
Bright Field Dark Field
Phase
Contrast
Confocal
Electron
Microscope
Scanning
electron
Microscope
Transmission
electron
Microscope
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9. Types of Microscope
1) Stereoscopic microscope
Function: Visible light to illuminate the surface
of a sample (2000x ), without disrupting them
2) Compound microscope (light)
Function: Visible light to illuminate a thin
section of sample, cells and tissues
3) Confocal laser scanning fluorescence
microscope
Function: Thin ‘slices’ in a sample while
keeping sample intact;
Specifically at parts of a cell (such as
individual proteins) by labelling them with
fluorescence
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10. Scanning Electron Microscope
Function: Surface of objects at high resolution
(3D image - 500 000x )
Principle : Beam of electrons being knocked off
the surface of the sample and then picked up by a
detector .
Why we use SEM ???????????
The SEM probably gives the best depth of field
out of any microscope
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11. Transmission Electron Microscope
Function: very thin cross-section of an
object (cell), internal structure of objects,
high resolution (500 000x )
Principle : Electrons pass through the
sample and some are deflected and some
pass right through, and that forms our
image, which is focussed on objective
lens
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12. Microscopy Techniques Applied to the Study of Phytoplasma
Diseases: Traditional and Innovative Methods
Rita Musetti and Maria Augusta Favali
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13. Light Microscopy
 Dienes’stain was first developed
as a specific stain for animal
mycoplasma colonies .
 Phloem tissues of stems infected
by phytoplasmas stained dark
blue while xylem was turquoise
and cortex light blue.
Musetti and Favali, 2004
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14. Fluorescence Microscopy
DAPI staining of hand cut
sections of healthy
Antibody plus fluorochrome,
such as fluorescein
isothiocyanate (FITC) and
stained
 Phytoplasma-infected :
the fluorescent bright spots,
visible at phloem level
Musetti and Favali, 2004
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15. Transmission electron microscopy (TEM)
Phytoplasmas in the phloem
cells of Catharanthus roseus
Musetti and Favali, 2004
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Phytoplasmas in the phloem of
apple tissues

16. Immuno-electron microscopy (IEM) of thin sections
Phytoplasmas in phloem tissues of Catharanthus roseus L. embeded with primary
monoclonal antibody and gold Labelledsecondary antibody
Gold partcle (15 nm) few
particles are visible on
phytoplasma membrane
Gold partcle (5 nm) particles are
well distributed over the periphery
of the phytoplasmas
Musetti and Favali, 2004
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17. High resolution autoradiography
Phytoplasmas in phloem cells of white clover (Trifolium repens L.), after 3
hours labelling with thymidine-3H. The silver grains were seen on the
dividing phytoplasmas
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18. Staining Technique : For Histopathological Tests
• Staining is an auxiliary technique used in microscopy to
enhance contrast in the microscopic image
e.g. Crystal violet stains only Gram positive bacteria
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20. Histopathological analysis of infected tissues
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Valencia sweet
oranges infected
with
Colletotrichum
acutatum.
Valencia
sweet orange
fruits infected
with Guignardia
citricarpa.
Toluidine blue staining Toluidine blue plus safranin staining Marques et al., 2013

21. 21
Limitations Faced due to conventional techniques !!!!!!!
 Latent infection : eg. Potato ring rot
 Misleading infection : eg. Black lesions (Alternaria) and bacterial
blight of carrot (Xanthomonas)
 Co-infection : Alteration of symptoms

22. SEROLOGICAL METHODS – De Vorac
Antigen Antibody
Antigen Antibody
Positive
Result 22

23. Antigen
 A molecule usually a protein, when it is injected into a
warm blooded animal produces antibody (immune
response)
Antibody
 A molecule produced in a warm
blooded serum of animal in
response to the stimulus antigens
 Antibodies are immune system-
related proteins called
immunoglobulin
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24.  Variable region : composed of 110-
130 amino acids, give the antibody
its specificity for binding antigen.
 Variable region includes the ends of
the light and heavy chains.
 Constant region : determines the
mechanism used to destroy antigen.
Structure of Antibody
24

25. MONOCLONAL ANTIBODY
POLYCLONAL ANTIBODY
Composed of a variety of antibody
Have multiple epitopes
Antibody derived from a single
clone and specific for a single
epitope
Consist of single type of antibody
Produced by hybridoma technique
Small quantity of antigen is enough
for development
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26. Production of Mabs by Hybridoma Technique
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27. Antigen antibody based technique
Direct test
 Precipitation test
1. Tube precipitation
2. Ring precipitation
 Micro Precipitation test
 Agglutination tests
1. Chloroplast agglutination
2. Latex agglutination
 Gel diffusion test
 Immuno-electrophoresis
Indirect test
 ELISA test
1. Direct ELISA (DAS ELISA)
2. Indirect (DAC ELISA)
3. DIBA ELISA
4. Lateral flow device
 Immunofluorescence
 Immuno Sorbent Electron
Microsopy(ISEM)
 Flow cytometry
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28. Tube precipitation test
Widely used
Reactants diluted in 8.5g/l
NaCl, followed by
incubation at 37°C in water
bath
Observations :
If elongated virus particle -
floccular
If spherical virus particle -
granular
Precipitation test
28

29. Done on a micro-scale to
economize on antiserum
Drop of dilution mixture (antiserum
& virus suspension) are mixed at
bottom of a Petri plate
The precipitates produced are
observed with a microscope with
dark-ground illumination
Precipitation varies depending on
the ratio of concentration of antigen
and antibody
Micro precipitition test
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30. Latex agglutination
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31. Chloroplast agglutination
Crude fresh
leaf sap from
diseased plant
Antiserum
Chloroplast
fragments
clump
together
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32.  The reactants, antiserum and virus solution are placed in well cut
in the agar (containg 0.85% NaCl and 0.02% sodium azide) in
Petri plate
 Antibody and virus diffuse into the agar from the adjacent wells
 Where they meet precipitation zones in the form of white band
are formed
Gel diffusion test
Oservation :
a) Bands : Identical or closely related
b) Spurs : Distantly Related
c) Intersect : Unrelated 32

33. Radial or single diffusion
Double or Ouchterlony diffusion
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34. Immuno-electrophoresis
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35. ELISA : Enzyme- Linked Immuno-Sorbent Assay
ELISA was initially applied for plant viruses by M.F. Clark and
Adams (1976)
Sensitive, detects at concentration of 1-10 ng/ml
It involves an enzyme-mediated colour change reaction to detect
antibody binding
Degree of colour change, usually measured quantitatively in
spectrophotometer at 405 nm
Ward et al., 2003
35

36. DAS ELISA : Double antibody sandwich ELISA
 Direct ELISA
 First time describe by Clark and Adams in 1977
p-nitrophenyl
phosphate
p-nitrophenol Ward et al., 2003
36

37. DAC ELISA : direct antigen coating ELISA
Indirect ELISA
Stand for Easy to rapid assay
Ward et al., 2003 37

38. Combination of electron microscopy
and serology
First time described by Derrick in1973
Virus and antiserum are reacted
together
Antigen are trapped onto grid coated
with specific antiserum, negatively
stained (Uranyl acetate -1%) and the
result viewed in the EM
Immuno Sorbernt electron microscope (ISEM)
Tubular particle of beet
necrotic yellow vein virus
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39. Dot Immunoblotting Assay (DIBA) OR Dot ELISA
Substrate : Nitro tetrazolium
BCIP
39

40. Lateral flow technique
• The principles used for rapid lateral flow devices are primarily
those of ELISA
• Various types of filters are used as the solid support for the
initial binding reaction
A lateral flow device test kit developed by Central Science
Laboratory, U.K., permits detection of R. solanacearum in a 3-
minute
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42. Ouchterlonie’s double diffusion test.
I – Healthy cane
extract
II – Control blood
serum of rabbit
III, IV, V – Antigen
of host pathogen
A – Antibody
raised against host
pathogen
Lingayya and Naik, 2002
42

43. Detection of Colletotrichum falcatum infection in sugarcane
tissue by DAC - ELISA
Lingayya and Naik, 2002 43

44. TDA = 3 X standard deviation of
healthy sample + mean value for
healthy sample
44
FUNGUS

45. Advantage of ELISA
It is sensitive
Semiautomatic technique
Application against large number of sample
Reproducible
Qualitative & Quantitative
Suitable for automation high speed
No radiation hazards
45

46. Immunofluorescence
The intercellular location &
distribution of viruses
Globulins mixed with a
fluorescent dye (Fluorescein
isothiocyanate and Rhodamine
B)
Introduced into the infected
cells/tissue, with antigen and
antibody reaction, fluorescence
takes place
46

47. Flow cytometry
Cell suspensions are filtered to remove large particles then
stained with fluorochrome-labelled antibodies
Fluorescent markers for viability
Stains, such as propidium and
hexidium iodide for red fluorescent
staining of dead cells
Carboxy fluorescein diacetate and calcein
AM for green fluorescent staining of
viable cells can be used to differentiate
live from dead cells
Detection of C. Michiganensis subsp. Michiganensis in tomato seed extracts
Detection of X. Axonopodis pv. Dieffenbachiae, causal agent of anthurium blight (Alvarez
et.al., 1999)
Determine viability of R. Solanacearum in seed potatoes (van derwolf et.al., 2004)
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48. HYBRIDIZATION BASED METHODS
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50. Southern blotting
• Professor Sir Edwin Southern
developed this method in 1975
• This method Involves separation,
transfer and hybridization
• Detection of a specific DNA
sequence in DNA samples
• Combines agarose gel
electrophoresis for size
separation of DNA and
hybridization with probe
Professor Sir Edwin Southern
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52. Northern Blotting
 Northern blotting is a technique for detection of specific RNA
sequences
 Northern blotting was developed by James Alwine and George
Stark at Stanford University (1979)
 Electrophoresed RNA is blotted on membrane and hybridized
52

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54. Western blotting
• Western blotting (1981) is an immunoblotting technique which
rely on the specificity of binding between a protein of interest
and a probe (antibody raised against that particular protein) to
allow detection of the protein of interest in a mixture of many
other similar molecules.
• The SDS (Sodium dodecyl sulphate) page technique is a
prerequisite for western blotting .
54

55. Steps in western blotting
Detected
through
auto-
radiography
55

56. DNA Microarray
56

57. Microarrays for Rapid Identification of Plant
Viruses
Neil Boonham, Jenny Tomlinson,and Rick Mumford
Central Science Laboratory, Sand Hutton, York, YO41 1LZ,
United Kingdom
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58. Boonham et al., 2007
A schematic diagram detailing a simple approach to virus
detection using a microarray.
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59. A microarray designed to detect and
discriminate a range of small spherical
viruses. Eg. Broad bean wilt virus 2
Indicator host Chenopodium quinoa A small spherical virus was identified
using electron microscopy.
Boonham et al., 200759
Positive control spots
Detection of virus

60. Sensitivity comparison between ELISA and microarray
Boonham et al., 2003
Dilution
end
point
Histogram showing local
background fluorescence for the
1/1600 dilution of RNA,

61. BIOCHEMICAL METHODS
• FAME analysis
• BIOLOG
• Volatile compound
Biochemical
techniques
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62. What is a BIOLOG?
 First and only bacterial identification system to identify both gram positive
and gram negative bacteria with a single universal test kit.
Add cells
96 wells contains different carbon sources and other
test. If the cells are metabolically active, they reduce
the redox dye and a purple colour is formed in al the
positive well
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63. Fatty Acid Methyl Ester ( FAME ) analysis
 Change in the fatty acid profile represent a change in the
microbial population
63

64. Detection of Diseased Plants by Analysis of Volatile
Organic Compound Emission
R.M.C. Jansen, J. Wildt, I.F. Kappers,
H.J. Bouwmeester, J.W. Hofstee,
and E.J. van Henten
64

65. Emission of volatile organic compounds (VOCs)
from non-infected and Botrytis cinerea–infected
tomato plants.
Jansen et al., 2011 65
Damaged cell membranes
Local emission of several
lipoxygenase (LOX)
oxidative cleavage of C18fatty
acids
(oxygen and lipoxygenases )
Characterize diseases due to
release of VOCs

66. Surface Plasmon Resonance (SPR)
 The Surface Plasmon Resonance
(SPR) sensor is used for label free
detection and real-time monitoring
 Their simplicity and sensitivity make
biosensors an effective means of
disease diagnosis and monitoring
 In SPR technique, shift of the
resonance angle is observed, when
the intended species is captured by
the immobilized antibody on the
sensor surface
66
Is it the specific protein, the virus fragment or the virion itself?

67. Development of Surface Plasmon Resonance (SPR) Based Immuno-
Sensing System for Detection of Fungal Teliospores of Karnal Bunt
(Tilletia Indica), a Quarantined Disease of Wheat
Sadhna Singh1, Manoj Singh1, Gohar Taj1, Sanjay Gupta2 and Anil Kumar1*
1Department of Molecular Biology & Genetic Engineering, College of Basic Sciences
& Humanities, G. B. Pant University of Agriculture & Technology, Pantnagar,
Uttaranchal, India
2Department of Biotechnology, SBS PG Institute of Biomedical Sciences, Balawala,
Dehradun, Uttarakhand, India
Journal of Biosensors &
Bioelectronics
67

68. Experiment conducted : Interaction of teliosporic wall
antigen with the anti-teliosporic antibody immobilized on sensor
chip
 The interaction of antigen at a concentration of 80, 40, 20, 10, 5.0, 2.5,
1.25, 0.625, 0.312, 0.156, .078 and 0.039 ng/μl with immobilized antibody
on sensor chip was examined
Observation
• The responses increased in proportion to the concentration of teliosporic
antigen due to the change of the refractive index near the SPR sensor chip
68

69. SPR sensor response after the interaction of different concentrations of antigen over
the immobilized antibody (1: 500)
Singh et al., 2012
(a) 0.312
ng/μl
(b)1.25 ng/μl
(c) 5 ng/μl
(d) 20 ng/μl
(e) 80 ng/μl

70. Advantages of SPR
Major advantages :
• Rapid, real-time
• Non-labeling analysis
• Miniaturization for portable application
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72. Phytophthora and Pythium Test Kits : 0.5 per cent of a
plant roots are infected
Tests for Phytophthora, Pythium, and Rhizoctonia root and crown decay
fungi can be performed on-site by growers in about 10 minutes
(A) Collect and grind samples using abrasive pads
(B) Fold pads and insert them into the extraction solution
(C) Apply solutions to detector
(D) Examine detector dots for color change.
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73. Molecular methods for detection of plant
pathogens—What is the future?
 Strategies are needed on how to exploit deduced genomics and
proteomics supported by in silico analysis for establishing
rational disease control measures.
 The reliability of each specific on-the-spot diagnostic method
needs to be validated before results are used exclusively to
implement costly disease control strategies and/or regulatory
actions.
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