Detection of plant Pathogens

ON SITE DETECTION OF PLANT
PATHOGENS USING ADVANCE
PORTABLE TOOLS
Presented By:
SANJAY KUMAR
Ph.D Plant Pathology
Punjab Agricultural University, Ludhiana

Content
 Why detection ?
 Disease detection tools
 Biosensors
 Application based detection

Why detection ?..
• Overcome outbreak of disease
• To reduce yield losses

CONVENTIONAL
TECHNIQUES
•Visual observations
•Cultural
• Foldscope

VISUAL OBSERVATION : SIGNS AND SYMPTOMS
Citrus canker
Rot - Sclerotium rolfsii Late blight of potato – Phytophthora infestans

Problem with biotic and abiotic factors ???????

Cultures of Ralstonia solanacearum
Bacterial ooze
CULTURAL : LABORATORY TECHNIQUES

Alternaria culture
Fusarium culture
Fungal cultures…..

Microscopy
Fusarium wilt Fusarium oxysporum
Wheat rust Urediospore and teliospore

FOLDSCOPE
Origami based print fold paper- microscope that we can build in
20 minutes
The foldscope costs less than a doller, weights 8 gm and fits in
your pockets
Invented by Manu Prakash and Jim Cybulski

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

Indirect techniques are non-invasive methods to identify plant
diseases, not through the direct identification of the pathogen, but by
detecting the impact of the pathogen on the physiological plant
response.
a) Thermography
b) Fluorescence Imaging
c) Hyperspectral Techniques
d) Gas Chromatography
Indirect Detection Methods

THERMOGRAPHY
Thermography allows the visualization of differences in
surface temperature by detecting emitted infrared radiation
[long-wave infrared (8–14 mm)].
Airborne sensors are suitable for the detection of field
patches that are diseased with soilborne pathogens (Hillnutter
et al 2011) or in later stages of the diseases (Mahlein et al
2012).

Berdugo et al 2014
Leaves inoculated with S. fuliginea had higher
MTD compared to the virus treatments and
the untreated control

FLUORESCENCE IMAGING
The chlorophyll fluorescence imaging can be an effective tool in monitoring leaf
diseases (Lenk et al 2007).
Chaerle et al (2007) used blue-green fluorescence to evaluate the effectiveness of this
technique in observing the development of tobacco mosaic virus (TMV) infection in
tobacco plants.
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

Csefalvay et al 2009
Heterogeneous distribution of FV/FM and ΦPSII in artificially inoculated leaves was
associated with the presence of the developing mycelium 3 days before the occurrence of
visible symptoms and 5 days before the release of spores.

HYPERSPECTRAL IMAGING
Hyperspectral imaging can be used to
obtain useful information about the
plant health over a wide range of
spectrum between 350 and 2500 nm.
Bravo et al (2003) investigated the
application of visible-NIR
hyperspectral imaging for the early
detection of yellow rust disease
(Puccinia striiformis) in winter wheat.
Also useful for the assessment of
mycotoxin producing pathogens in
maize (Del Fiore et al 2010).
Anne-Katrin Mahlein 2015

Trimble UX5
Key Features
 Apply imagery for crop scouting
to detect pests, weeds, mineral
deficiencies, and other potential
problems in agriculture
 Capture 180 acres (73 hectares)
of imagery at one inch (2.5 cm)
resolution in a single flight
 Operate in crosswinds up to 37
mph (60 kph)

Examples of plant diseases assessed by imaging techniques
Mahlein 2016

GAS CHROMATOGRAPHY
Plant disease detection involves the profiling of the volatile compounds
Profiling of VOC used as a means to identify the type and nature of infection

Jansen et al 2011
Emission of volatile organic compounds (VOCs)
from non-infected and Botrytis cinerea–infected
tomato plants.

BIOSENSORS FOR PLANT PATHOGEN
DETECTION

Pathogen biosensing strategies are based on biological recognition using different
receptors such as antibodies, DNA probe, phage,
Singh et al 2013

Antibody based biosensors
Electrochemical Immunosensors - based on label-free technologies
and enzymatic label based voltammetric approaches on mercury, gold
and carbon electrodes
1. Voltammetric detection based on the use of enzymes
2. Label-free electrochemical impedance spectroscopy (EIS)- based
detection
3. Label-free quartz crystal microbalance-based approaches

ECEIA sensor using gold nanoparticles as carriers of enzyme-labeled
antibodies for signal amplification, applied for Pantoea stewartii pv.
stewartii (PSS).
Zhao et al. (2014)

Optical Immunosensors
The principle of this test is based on antibody-antigen specific interaction.
The first LFIA for plant pathogen detection was designed to detect Tobacco mosaic
virus (Tsuda et al 1992).
Lateral flow microarrays

Fluorescent approaches
Charlermroj et al (2013)
Microsphere sandwich immunoassay technology based on fluorescence- loaded
magnetic microsphere and fluorophore-antibodies has been applied for detecting
multiple analytes such as biomarkers, food and plant pathogens

Quantum Dots
Fluorescence resonance energy transfer (FRET) mechanism, which
describes energy transfer between two light-reactive molecules.
Many QD-FRET-based sensors have been developed for phytoplasma
disease detection such as the witches’ broom disease of lime caused by
Candidatus aurantifolia (Rad et al 2012).
Rhizomania, which is the most destructive disease in sugar beet, is
caused by beet necrotic yellow vein virus (BNYVV) was successfully
reported to be detected by QD-FRET-based sensor (Safarpour et al
2012)

Surface Plasmon Resonance (SPR) systems

Dot Immunoblotting Assay (DIBA) OR Dot ELISA
ImmunoBlot Kit for Xanthomonas
campestris (Xc)

Loop Mediated Isothermal Amplification
(LAMP)
 LAMP was reported for the
first time by Notomi et al (2000)
for the detection of the hepatitis B
virus.
The fast results and the high
specificity of the technique for
pathogen detection (Yan et al
2017).

Real Time Isothermal Amplication

60,000 photographs
Covers 30 crops in India
60 crops worldwide
200 crop diseases.

Early detection of old, new and emerging infectious plant disease plays critical
role in plant disease management.
Most of reported biosensors for plant disease detection are still for use at lab level,
it is expected that more portable devices will emerge in the future being a strong
support for an efficient diagnostic.
The spectroscopic and imaging technology could be integrated with an
autonomous agricultural vehicle for reliable and real-time plant disease detection
to achieve superior plant disease control and management

Detection of plant Pathogens

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