Research is being carried out for detecting plant diseases in earliest and easiest way.

2. Current and Prospective
Methods for Plant
Disease Detection
Speaker : Shivani
PhD 2nd Year
48020
Doctoral Seminar-I
on

3. OUTLINE
Introduction
Serological based detection methods
Nucleic acid based detection methods
Optical sensors based detection methods
Digital imaging based detection methods
Biosensors based detection methods
3
Case studies
Conclusion
Future Prospects

4. Importance
 Effective crop management and regulatory programmes
 Determination of cause, epidemiology and distribution of
diseases
 Providing suitable plant protection measures
 Resolving the components of complex diseases
 Seed certification and quarantine
 Studying taxonomic and evolutionary relationship of plant
pathogens
 Detection and identification of new pathogens
 Assessment of disease incidence and yield loss in field
4

5. Implications
 Risk of movement of pathogens and their vectors from one
country to another
 Preventing outbreaks and potentially devastating crop diseases
 Identifying variability of the organism
 To detect pathogens before symptom development
 To screen large number of samples accurately, reliably, quickly
with greater sensitivity
 To find the ways that provide additional information
Improvements
5

6. Current diagnostic methods for plant disease detection
 Use of recombinant antibodies
 Serological tests
• R-ELISA
• Lateral flow devices
 Micro-arrays (Bio-chips)
 Nucleic acid based tests
• Real-time PCR
• Nested and Multiplex PCR
 Digital Consulting System or imaging
 FISH
 Flow Cytometry
 Thermography
 Hyperspectral techniques
 Gas Chromatography
6

7. 7
Serological Based detection Techniques

8.  Polyclonal antibodies (Pabs)
 Demerits:
Cross-reactions to contaminants
Lack of sustainable source of Abs
Lack of specificity
 Monoclonal antibodies (Mabs)
 Superior to Pabs as can provide constant supply of specific antibodies
 Expensive to produce and maintain
 Recombinant antibodies
 Phage expressing complete Ab variable domain
 Commercial available libraries can be screened with plant pathogen to
identify clones
 Clones formed are used as a source of Abs for a diagnostic system e.g.
Potato leaf roll virus, Tomato spotted wilt virus, Alternaria alternata,
Ralstonia solanacearum, Phytophthora infestans, Cucumber mosaic virus
(Zienglar and Torrance, 2002)
Advantages
 No need to immunize any animal
 Monovalency
 Rapid, sensitive and low cost
 Potential to be used for a wide range of plant pathogens
8

9. 9

10. ELISA as diagnostic tools
Viruses
Tomato spotted wilt virus , Citrus tristeza virus, Groundnut viruses, Rice tungro
virus, Rice grassy stunt virus, Maize steak virus, Banana bunchy top , Papaya ring
spot , Sugarcane mosaic , Maize dwarf, Poty viruses, Tospo viruses, All potato
viruses, Tobra virus, Tobacco ring spot virus, Alfa-alfa mosaic virus
Fungi
Phytophthora infestans, Pythium spp., Rhizoctonia spp, Septoria tritici, Septoria
nodorum, Colletotrichum spp., Monilinia fructicola, Botrytis cinerea, Sclerotinia
spp., P. graminis, Fusarium spp.
Bacteria
Ralstonia solanacearum, Clavibacter michiganensis sub sp. sepidonicus and
michiganensis, Erwinia carotovora, Erwinia chrysanthemi, Xanthomonas
campestris pv. oryzae
Nematodes
Globodera pallida, G.rostochiensis, Meloidogyne spp.
Others
Spiroplasma citri
10

11. Pros and cons of using ELISA
Advantages
 Simple, Specific, accurate and quick
 Little expertise required
 Detection of pathogens with variable or latent symptoms
Disadvantages
 Not effective for viroids
 High initial cost
11

12. LATERAL FLOWDEVICES
12

13. Limitations
 Ideal for viruses and
bacteria (symptomatic
material)
 Limited use for fungi
(difficulty to achieve
species specificity)
 Life stage specific
13

14. Nucleic acid Based detection Techniques

15. Polymerase Chain Reaction (PCR)
 Most important technique in molecular biology
 Amplifies specific DNA sequence exponentially,
by chain reaction
15

16.  Classical PCR has not been
adopted by most plant disease
regulatory and diagnostic labs
Why ?
 Give results of PCR test in real
time
 As a diagnostic technology, it
is remarkably:
• Rapid
• Sensitive
• Specific
Real time or quantitative PCR
16

17.  Detection and quantification of
a fluorescent transmitter
during the process of
amplification
 Increase in the fluorescent
signal is directly proportional
to the quantity of amplicon
produced during the reaction
 Hybridization of fluorescently
labeled oligonucletide probe
sequences
 Specific region within the
target amplicon
 Amplification using traditional
forward and reverse PCR
primers
• Probe hydrolysis
(Taqman probe)
17

18. Typical Taqman Applications
18

19. Advantages of Real
Time PCR
 Time saving (no gel-
running)
 Closed-tube system
(reduced contamination risk)
 Easy multiplexing (internal
controls)
 Generic assays
 Increased sensitivity
 Quantitation
19

20. Real time PCR assays in diagnostics
 R. solanacearum, Agrobacterium, Xylella fastidiosa, C.
sepedonicus, Erwinia, Acidovorax avenae subsp. citrulli,
Pierce’s disease
 Tilletia indica, Phakospora pachyrhizi, Phytophthora
infestans, P. citricola, Diaporthe phaseolorum,Phomosis
longicola, Helmintosporium solani, Pyrenophora
graminea
 Tomato spotted wilt virus, Sugarcane yellow leaf virus,
Potato mop-top virus,Tobacco rattle virus, Cymbidium
mosaic potyvirus, odontoglossum ringspot tobamo virus
 Potato cyst nematode
(Schaad & Frederick, 2002, Schaad et al., 2003
20

21.  PCR can be used in combination
with pathogen isolation on agar
media
 Using a combine viable
enrichment (growth media) with
an enzymatic amplification (PCR)
 Target bacterium can be enriched
in liquid or solid media and
detected extremely low levels in
seeds and other propagative
materials
• Increased sensitivity
• Elimination of PCR inhibition
• Detection of viable cell only
BIO-PCR
21
Extracting a sample
Plating a sample onto agar media
Incubating for 15-72 h
Washing plates and
centrifugation
1 or 10 µL for direct PCR

22. Nested PCR
A second round of PCR is performed with
amplified DNA as template and primers internal to
first round primers
Multiplex PCR
Allows the simultaneous and senstivity detection
of different DNA or RNA targets in a single
reaction
Multiplex nested PCR
combines both multiplex and nested PCR
22

23. 23
Single site template PCR Multiple site template PCR

24. Nested PCR
24
First

25. Nested PCR in plant pathology
 Pythium ultimum, Plasmodiophora brassicae, Verticillium dahliae
 Clavibacter michiganensis subsp. sepedonicus, Xanthomonas axonopodis pv.
manihotis ,Erwinia amylovora, Pseudomonas savastanoi pv. Savastanoi, X.
fastidiosa
 Cucumber mosaic virus, Cherry leaf roll virus, Strawberry latent ring spot
virus, Arabis mosaic virus
(Pabla et al., 2000; Edson et al., 2002 ;Ojeda & Verdier, 2005)
Limitations
 False positive reaction due to contamination
 Cost of test is high
 Additional cost of reverse transcriptase for cDNA in case of
plant viruses
25

26. Micro-arrays (Bio-chip)
 Array:orderly arrangement
of samples
 Medium for matching
known and unknown NA
samples
 Generated by depositing a
few nanolitres of DNA on
a solid support
 Developed for high
throughout measurement
expression patterns of
thousands of genes
 Principle: Base pairing of
complementary sequences
by hybridization
26

27. 27

28. Loop mediated isothermal amplification
Loop-mediated
Refers to the loop structures formed when the LAMP
primers amplify their target DNA sequences
Isothermal
The reaction takes place at single temperature (63-
67oC
Amplification
The highest efficient polymerase enzyme used amplifies
the very small amount of target in the sample, generating
millions of the copies of the sequence
LAMP
28
LAMP is used in rapid diagnosis of viral and bacterial plant pathogens

29. 29

30. 30

31. PCR based techniques
Genomic assay Targeted taxa References
PCR Agrobacterium tumefaciens
Candidatus Liberobacter, E.
carotovora
Tospo &Citrus Tristeza virus
Tilletia indica
Cubero et al 2002, Ahlawat et
al., 2004, Dorasse et al ., 2003
Okuda et al., 2000,
Ramachandran et al., 2002
Frederick et al., 2000
Co-PCR R. solanacearum Caruso et al., 2003
Multiplex nested
PCR
Pseudomonas savastanoi pv.
savastanoi Begomo virus
Bertolini et al., 2003
Potter et al, 2003
Multiplex PCR Xiphinema index
Globodera rostochiensis, G.
pallida
Cucumovirus, Nepovirus
Necrovirus, Olea virus
Wang et al., 2002
Mulholand et al., 2001
Bertolini et al., 2001
Nested PCR X. axonopodis pv. citri, P.
savastanoi pv. savastanoi
Hartung et al. (1996)
(Bertolini et al., 2003)
31

32. 32
FISH (Fluorescence In Situ Hybridization)

33. Applied for bacterial detection in combination with microscopy and
hybridization of DNA probes and target gene from plant samples
Due to the presence of pathogen specific rRNA sequences in plants
Advantage of high sensitivity (103 CFU/mL)
Could also be used to detect fungi and viruses and other
endosymbiotic bacteria that infect the plant
33
This technique could also be used to detect unculturable or yet-to-be
cultured organisms to investigate complex microbiome

34. Indian Scenario
DAS- ELISA
DAC-ELISA
DIBA
PNC- ELISA
R-PAGE
Lily carla virus, Carnation
mottle virus, Bean yellow
mosaic, Groundnut bud
necrosis virus, CMV,
Banana streak virus, Water
melon mosaic virus, Stripe
teniuvirus in Fodder,
Viroids, TSV
Bhaik et al 2004, Singh et
al 2004, Roy et al 2005,
Raja & Jain, 2006,Bhat et
al 2004, Bhadramurthy et
al 2005, Biswas &Verma
2005, Narayana 2004,
Suryanarayana et al 2004
TEM (leaf dip
serology) ISEM
Watermelon mosaic virus,
Indian citrus ringspot virus
Biswas &Verma 2005
Hoa & Ahlawat 2004
RT-PCR Groundnut bud necrosis
virus, Indian citrus ringspot
virus, Banana streak virus,
Citrus yellow mosaic virus
in P. citri,
Raja & Jain 2006, Hoa &
Ahlawat 2004, Raman et
al 2004, Saxena et al 2005
Singh et al 2004
PCR (ITS) R. solanacearum ,
C.gloesporioides & falcatum
U.scitaminea
Kumar & Anandaraj
2006, Pandey & Pandey
2006, Naik & Gaikwad
2005
PCR (16s rRNA) Grassy shoot of sugarcane Srivastava et al 2004
Nested PCR GSD and yellow leaf
syndrome of sugarcane
Rao et al 2004
PCR-RFLP RTV,X. axonopodis pv.
malvacearum, CLCuV
Niazi et al 2005,
Chakraborthy et al 2004,
Microsatelite
markers
Fusarium spp. Prasad et al 2004
34

35. Optical sensors based detection
Techniques

36. FCM (Flow cytometry)
Flow cytometry (FCM) is a 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
FCM is used for rapid
identification of cells while cells
pass through an electronic detection
apparatus in a liquid stream
(Chitarra et al., 2003) 36

37. Thermography
Allows imaging the differences in surface temperature of plant leaves
and canopies
Emitted infrared radiation can be captured by thermographic cameras
and color difference can be analyzed
Also a promising tool to monitor the heterogenity in the infection of
soilborne pathogens
The practical applicability of thermography for disease monitoring is
limited due to its high sensitivity to the change of environmental
conditions during measurement
(Chaerle, et al., 2007)
37

38. Fluorescence imaging
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
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
(Kuchenberg, et al., 2009)
38
The practical application of this technique in a field setting is limited

39. Hyperspectral techniques
Used to obtain useful information
about the plant health over a wide
range of spectrum between 350 and
2500 nm
Highly robust and it provides a
rapid analysis of the imaging data
Magnaporthe grisea infection of
rice, Phytophthora infestans
infection of tomato and Venturia
inaequalis infection of apple trees
have been identified and reported
using hyperspectral imaging
techniques
(Kobayashi, et al., 2007)
39

40. 40
How does hyperspectral imaging work
• Hyperspectral imaging deals with the
imaging of narrow spectral bands over
a continuous spectral range, and
produces the spectra of all pixels in the
scene
• Hyperspectral sensors collect
information as a set of “images”
• These images are then combined and
formed into three-dimensional
hyperspectral data cube for processing
and analysis

41. Gas chromatography
A completely different non-
optical indirect method for plant
disease detection involves the
profiling of the volatile chemical
signature of the infected plants
An infection by Phytophthora
cactorum, the fungus that causes
crown rot diseases in strawberries,
results in the release of p-
ethylguaiacol and p-ethylphenol
as characteristic VOCs from the
infected portion of the strawberry
plant/fruit (Fang et al., 2014)
41

42. The volatile signature of plants could be analyzed using gas-
chromatography (GC) technique to analyze the presence of the
specific VOC that is indicative of a particular disease
It also allows the detection of diseases at different stages based on
the quantitative information collected from the VOC sample
42

43. VOCs emitted from whole, intact tomato plants or
detached leaves, and biotic stress causing agents
responsible for increase in VOC emissions
43

44. 44
Digital imaging based dtection techniques

45. Digital imaging system
 Distant analysis can be used
in first line diagnostic
 Can save a lot of time
 Better and faster
communication between
inspectors and specialists
 Sharing of knowledge
 Developing central database
 Always final identification
by diagnostic specialist
45

46. Imaging techniques Use
Magnetic resonance
microscopy (MRM)
Pine wilt disease (Utsuzawa et al.,
2005 )
Atomic force
microscopy (AFM)
Tobacco mosaic viruses
X-Ray
Spectroscopy
Mass spectrometry
based proteomics
Rhizoctonia solani, Fusarium
graminearum, and Ustilago maydis,
Uromyces appendiculatus (Padliya
and Copper, 2006)
46

47. Ideal diagnostic strategy
 High detectability
 Specificity
 Sensitivity
 Accuracy supported with simplicity
 Amenable to automation
 Low cost
Issues related to diagnostics
 Cost
 Specificity
 Sensitivity
47

48. 48
Biosensors based detection Techniques

49. 49
How does biosensors work?
Analyte of
interest
Biorecepter Transducer Signal
Detection of plant diseases using portable sensors
Antibody
Enzymes
Phages
Oligonucleotides
Optical
Electrochemi
cal
Mechanical
Cantilever
Luminescence
Fluorescence
UV-VS
Magnetic
Resonance

50. Biosensor platforms based on nanomaterials
Affinity biosensors
Antibody-based biosensors
DNA/RNA-based affinity biosensor
Enzymatic electrochemical biosensors
Bacteriophage-based biosensors
50
Types of biosensors
(Singh et al., 2010)

51. Comparison of affinity biosensors, enzymatic biosensors,
nanomaterials modified sensors and ELISA
51

52. List of commercially available sensors for plant fungal detection
Manufacturer Product Principle Pathogens
BIOREBA Agristrip Lateral flow immunochromatography Spongospora subterranea
Pocket Diagnostic Pocket Diagnostic Kits Lateral flow assay Phytophthora
Neogen Corporation Alert Test Kits Lateral flow assay Pythium, Phytophthora,
Rhizoctonia
Agdia Immunostrip Lateral flow immunochromatography Pythium, Phytophthora
La Chandra Bioscience Pocket Diagnostic test kits Lateral flow technology Pythium, Phytophthora,
Rhizoctonia solani, Botrytis
Cepheid Smart Cycler Real Time PCR Aspergillus, Phalospora sp.
Fera Portable PCR RT PCR Phytophthora ramorum
Ahram Biosystems Palm PCR RT PCR Aspergillus
Electronic Sensors
Technology
zNose 4200, 4300,7100 Electronic nose (E-nose) Oidium neolycopersici
Sensigent Cyranose 320 E-nose Botrytis cineria, Colletotrichum
gleosporioides, Alternaria sp.
Sensitive Technologies
Limited
Bloodbound ST214 E-nose Oidium neolycopersici
52

53. 53
Case studies

54. 54

55. 55

56. Multispectral imaging system (MSIS) developed for sunlight-induced fluorescence
imaging of outdoor plants
56
*EMCCD: Electron multiplying charge coupled device
Non-destructive method for detection and
classification of CMD infection based on
far:far-red chlorophyll fluorescence image ratio
Study was carried out in 14 varieties of
potted cassava plant with a MSIS consisting of
an EMCCD camera
Sunlight induced chl fluorescence (SICF)
images of plants were recorded using the MSIS
at different wavelength
The red: far red ratio was computed and
correlated with the laser induced chlorophyll
fluorescence (LICF) detetermined by point
monitoring, chl content and net photosynthetic
rate

57. Image recorded from a typical irrigated cassava plant : (a) a colour photo, (b)
monochrome image recorded at 687 nm, (c) monochrome image recorded at 760 nm,
and (d) ratio image (F687:F760) produced from (b) and (C), with colour ramp as
indicated by the legend
57

58. Scatter plot diagram of (a) net photosynthesis rate (Pn) and (b) total leaf
chl for different varieties of cassava plants
58

59. Scatter plot diagram of the red:far red fluorescence intensity ratio (a) F687:F760 ratio
from SICF imaging measurement and (b) F685:F735 ratio from point monitoring of
LICF. The cut-off lines for discrimination are drawn at the mean ratio value of the
adjoining groups
59

60. 60

61. 61

62. 62

63. Conclusions
 Validation of diagnostic test
 Integration of methods
 Sanitary and phyto-sanitary standards
 Seed certification programs and epidemiological studies
intended to monitor the distribution and spread of pathogens
 Modified PCR is adaptable to any experimental objectives
 Molecular techniques can reduce the time required for assay
and increase the sensitivity of assay, allowing detection of
pathogen before symptom expression
 Biosensor have new analytical tools this form allows for a
rapid response, is ideal for the plant disease detection
63

64. 64
Fabrication of test strips on ELISA will be an innovative strategy
Affinity sensors , although extensively studied, still remain in the
laboratory in many cases
Thermography being non-specific and susceptible to ambient
environment, therefore are less suitable for on-field crop disease
detection
It is also promising that the enzymatic and affinity sensors can be
fabricated for multiple pathogen detection rather for single pathogens
Future Prospects