Lecture 4

DIAGNOSIS AND
DETEcTION OF PLA
P L A N T D I S E A S s

Part A:
DLAGNOSIs BASED ON
Disease
diagnosis is very DISEASE niective strategies for scase
management. Without important for deve
developing effective

Crop disease
diagnosis is an art asdiagnosis, there Can
nanagement.

discase

ean be be no
involye
ves the
recognition of symptoms
process
well as
diugnostie

science
Science. a The
signs (which are not and
(which
outwardly observable) and are associuteu clnted
with
disease)

the use of scientific as

well
methods. Several requires intuitive judgment
as

incidence. disease
conventional diagnoSe to

techniques include visualtechniques are

These echniques tollowed are
symptoms and isolation
recognition
and
examination of crop
inspection and
and
Such techniques are
time-

consuming and may not be

genspathogens using
microscopy.
using n diagnostic assays
able to detect latent
4ole infections. Several
nave been
latent
developed for early and ranid diagnosis. These ne include immunoassays,

nucleic acid probe-based methods, and PCR-based techniques. The use of these
i n the diagnosis of fungal, bacterial. viral, viroid, and phytoplasma diseases is

described here.
Methods of diagnosis
n the tield of human medicine, the doctors invariably use the diagnostic tools

the ailment before starting a line of

(physical, chemical or serological) to be sure of
treatment. The symptoms provide the clues for possibilities, but conf+rmation comes

tests. In plant diseases, visual observations of the

only after performing diagnostic
infected plant/ plants parts continue to be the dominant method. Several sophisticated
Isolation and
being used for observation, which include microscopy.
techniques are
bio-chemical
identification of biotic agents
associated, besides serology, immunological,
and genome analysis.
and physiological analyses
observation (symptoms)
I. Visual
of host-pathogen interaction. The deviation
Symptoms are the visible expresSIOn
presence of pathogen structures form the
from normal
morphology coupled wi
basis the for preliminary diagnosis
osis.
characteristic symptoms ofsign,
fungi:
induced by parasitic
Symptoms
biotic and mesc esobiotic agents are identified primarily by
caused by
Diseases
on
host. The morphological
The morpholooie atures such as spores,
produced
and signs form
symptoms which ffer from
ditfer one
one
fungus species to another,
s, 72 fructifications,
or
sporophores,
26
 important part of diagnostic programme" esired structures of the fungus are not
readily visible on the infected host surlace, C parasite may be induced to sporulate by
proper incubation of the infected tissue.
Some pathogens produce characteristic symptoms that can be easilyrecognized
in the field. The
symptoms of fungus are DiigS, blast, mildews, rust, smuts, bunts, ergot
etc.

Symptoms induced by bacterial pathogens:

Plant pathogenie bacteria induce walcr soaked
lesions in the infected tissues at
the initial stages and these lesions turn necrOuc late.
Formation of bacterial ooze from
infected tissues is another distinguishing feature associated
with bacterial diseases. As
infection progresses, leaf spots, blights, scabs,
cankers, tumours, wilts, soft rots, etc.,
may be the
prominent symptoms.
Symptoms induced by phytoplasmas:
The phytoplasmas cause general stunting or dwarfing of affected plant parts or
whole plants. Chlorosis and reduced leaves are also frequently observed.
Phylody and
proliferation of floral tissues- Floral parts are transformed into green leaf-like
structures. Partial total
or
sterility of infected plants
may be commonly noted. These
symptoms are observed in plants infected by diseases such as aster yellows, little leaf of

brinjal, sesame phyllody and witche's broom disease of potato, peanut and grain
legumes. Proliferation of auxiliary buds and formation of a large number of thin shoots
are observed prominently in little of brinjal, rice
yellow dwarf and sugarcane grassy
shoot diseases. Reduction in leaf size and inter nodal length and tendency for the leaves
to stand out stiffly, giving a spike like appearance to the infected branches are the
distinguishing symptoms of sandal spike disease.
Symptoms induced by nematodes:
Nematodes infect both root and above ground portion of plants. Root symptoms
may be appear as root knots, root galls, root lesions, excessive root branching, injured
root tips and root rots when nematode infections are accompanied by parasitic or

saprophytic fungi and bacteria. These root symptoms are usually accompanied by non-
characteristic symptoms in the above ground parts of plants appearing primarily as
reduced growth, symptoms of nutrient deficiency, such as yellowing of foliage,
excessive wilting in hot or dry weather, reduced yield and poor quality of products.
Certain species of nematodes infect the above ground portions of plants. They cause

lesions and rots, twisting and distortion of stem and leaves and abnormal
galls, necrotic
development of floral parts. Certain nematodes attack grains of grasses forming galls
27
 full of
nematodes in
place of seed,
Symptoms induced by viruses
and
Methods of diagnosis viroids:
t r a n s m i s s i o n ,

mode of
of virus include s y m p t o m a t o l o E y ,

in gels.
host-range. particle Clude sy mobility

Because some of
morphology, antigenicity
genicity and
electrophoretic

and equipment
for
these
techniques require spec1a
confirmation, the symptoms and Cquire special
methodology

suffice
nary
for preliminary
frequently

diagnosis. Plant viruses cause distribution

rbution patteri
a
patterns

depending
on the host plant
variety
Cy of
of symp
symptoms,
host
species and different
unrelated viruses mav induce similar symptoms in the same

OS
may induce si exhibit
plant species. The viruses induce inoculated leaves, which
primary symptoms
* d r y symptoms on
"
on

chlorotic or necrotic local when the virus

becomes
lesion or vein clearing. Later
clearing. Latel
Or
necrosis and
systemic, secondary symptoms develoned as colour changes,
ua
abnormal growth form. Colour changes may vary from mosaic on leaves to colour
breaking in flowers. Various kinds of changes in size and shape of plant part may oOc
seen as leaf roll, leaf curl, enation, leaf crinkle, galls and tumours.
 Lecture 5: DIAGNOSIS AND DETECTION OF
PLANT DISEASES
Part B: Detection of Plant Diseases
Conventional plant-pathological techniques need high expertise for routine
identification. In the case of latent infections in vegetative planting materials, seeds, and
fruit, conventional methods may not be useful to diagnose infection.

Detection and identification of diseases in crops could be realized via both direct
and indirect methods. Direct detection of diseases includes molecular and serological
methods that could be used for high-throughput analysis when large numbers of samples
need to be analyzed. In these methods, the disease causing pathogens such as bacteria,
fungi and viruses are directly detected to provide accurate identification of the
disease/pathogen. On the other hand, Indirect detection methods identify the plant
diseases through various parameters such as morphological change, temperature change,
transpiration rate change and volatile organic compounds released by infected plants.

A. DIRECT DETECTION OF DISEASES

I.DIAGNOSIS BY IMMUNOLOGICAL TECHNIQUESs

provide a fast method of confirming visible symptoms
Immunodiagnostic assays
cannot be easily identified by other methods. They
as well detecting pathogens that
as
of pathogens.
pathogens and accurate identification
permit early detection of plant
only to certain pathogens or groups pathogens,
of
Because many fungicides are specific
treatment.
most appropriate
useful in the selection of the
immunodiagnosis will be
can be
as a sterile mycelium)
and
Viruses, bacteria, (especially those spreading
fungi
methods.
readily detected by these

antibodies specific to the particular

Immunoassays depend on the development of
to recognize
mammals, have the ability
animals, particularly
pathogen. Cells of living and carbohydrate
lipopolysaccharides,
binding sites
on proteins, glycoproteins, animal). Such
foreign to that
that are not present in their bodies (i.e., this leads
molecules
of the animal and
stimulate the immune system
known as antigens, binds
specifically recognizes and
molecules,
which
of specific antibodies, each of reveal the presence
to the production is to
immunoassay
antigen. The role of an
to its complementary

29
 ofspecifi complexes
thogen. The between the antibody
whichis es produced
antibodi to the
antigen, Ody and an antigen that
are unique

present on cell nin nan

ntibodies can recognize a animal body can recognize the
microbial

wall In other words, the

pathogen. In the plant pathogen
Ffound attached with them.
specifically withprinciple,
to the
Ben by recognizing the antigen specific

homolimmunoussays
the the fact that antibodies react
detect. Several
as described below.
ogous
techniqueshave been antigen.
1ascd o
are based on

"Ben, However, the reaction is not easy

to

develope to exploit this reaction in immunoassays

"Cveloped
Ia. Agglutination Test
This test can be
test, drops carried out
of
antigen and diluted in Slides
slides or
or in test
test tubes. In the slide agglutination
a
glassmicroscope slide. antiserum containing ant
rum containing antibodies are mixed together on

ambiguous). the test-tube

In Agglutination
aton is observed
obser by eye or microscope (if
test tubes,
and the agglutination
nation test,
test, the
the antigens
antigens are mixed with antibodies in
aggregation of antigensand antibodies is monitored with a binocular
microscope.
I b.
Precipitation Test
In this
test, aliquot dilutions of antien are layered over equal volumes of
antiserum diluted in normal serum in
capillary, or other small tubes. The test is
regarded
as
positive if there is precipitation at the interface. When the
the antibodies, the
antigens are layered over
antigens are precipitated out of solution by the
antiserum when
antigen and antibodies are related.
Ic. Immunoelectrophoresis
By this method, mixtures of antigens are
separated before immunodiffusion, A
narrow trough is cut in a layer of thin gci patallc o an electric current that passes close

to the antigens along the length of the gel. Eacn antigen moves in a separate wave at a

characteristic rate according to its distinct charge. As a result, proteins separate into
proteins
enarated suificiently,
have separated sufficiently, the
the
current is switched off and
bands. Once the
the trough cut
ch in the
tne gei.
gcl. Precipitin aarcs
antiserum is added to composed of complexes of
form where
the
the individual el

antibodies and antigens electrophoresced antigens have

reached.
ELISA
Id. Direct
Sandwich
ELISA method, 96-Wel
method 96-well
immunoplates are
sandwich
coated with the
In the
direct

(polyclonal
or areferably
preferably

30
mon
monoclonal antibody) and incubated
antibody
specific
 successively with the antigen containing sample followed by a second enzyme labeled
specific antibody that is directly conjugated with an enzyme. This leads to a colored
product in proportion to concentration of pathogen.
Ie. Double Antibody Sandwich ELISA
In the double antibody sandwich ELISA method, a specific capture antibody is

immobilized onto a solid surface, such as the wells ofa microtiter plate. The infected
plant tissue sample is added, and unbound material is washed away. Bound antigen is
detected by the addition of a detecting antibody that has been conjugated with an
enzyme, and unbound material is again washed away. The presence of the detectingg
antibody is determined through the addition of a substrate for the enzyme. The amount
of color that develops is proportional to the amount of antigen present in the sample, The

intensity of the color can be recorded by automated equipment.

I.f. Dot-blot ELISA

In this assay system, ELISA reactions are carriednitrocellulose membranes. A drop

out on

containing the specific monoclonal antibody is absorbed as a "dot" onto which a drop of the test sample is
later added and blotted.

I.g Immunofluorescence
Two methods of immunofluorescence are used to diagnose plant diseases. In. the direct

immunofluorescence method, specific antibodies bound to their target antigens are detected by using

second antibodies conjugated with fluorescent dyes such as fluorescein isothiocyanate (FITC) or
rhodamine isothiocyanate. Fluorescence, indicating the presence of the target antigen, is visualized
microscopically. The microscope should have a special device for fluorescence using ultraviolet light

(fluorescence microscopy).
I. h. Immunosorbent Electron Microscopy
This assay system is mostly used for the diagnosis of virus diseases. Electron microscope grids
coated with carbon strongly adsorb protein, and when they are floated on a drop of antiserum containing
antibodies to the pathogen, the antibodies become attached. The grids are then floated on a drop of the sap

virus particles) adsorbed to the antibodies

of an infected plant. After staining, the pathogen (particularly
can be seen under a transmission electron microscope.

I1. NUCLEIC ACID PROBE-BASED METHODS

Both DNA and RNA probes are used for crop disease diagnosis.
PCR-Based Methodology
technique to exponentially
ne polymerase chain reaction provides a nowerful and rapid

amplify specific DNA sequences by in vitro DNA synthesis. T hree

 essential
steps to a
oligonucl
leotide PCR
primers to theinclude
a thermostable
(1) Melting
denatured DNA the target DNA, (2) annealing two
sub-
sequent DNA meltine
rase, N.DNA strands, and (3) extending the primer via
specificity of the synthesis
synthesis sinee
base-pair to and method since thesynthesized DNA strands serve as targets for
defines derives r ethrec
thermostable Thermus each end ofthethe synthetic
es from
c steps
steps are repeated up to 50 times. The
sy oligonucleotide primers, which
oligonucleotide primers aquatints
strand cDNA, and (Taq)
the Target sequence to be amplified. PCR uses a
targe
DNA polymerase to synthesize D A from
strands of the
or
cloned
cloned
ned see.templatePrimers
sequences. DNA. The
The temp
template sequences. Primers
template D A
may be genomic, first-

replication templatesuch that synthesis

of the mers
DNA
are designed
des are anneal complementary
to to

The PCR region between theSvnthesis ine

initiated at each primer results in
involves three primers
deoxynucleotides,
overlaidwith mineral
distinct
buffer, and Taq steps governe
steps governed by temperature. DNA, primers,
oil. The polymerase are
POlymerase are combined in a microcentrifuge and
set of
short tube is placed in aa com
incubations at placed thermocycler programmed to repeat a
S

thermoey
DNA is
denatured predetermined temperatures. In the first step, the template
to temperatures.
Cseparate
h nthe
e
minutes. In the complementary
second step, the complementary stranas This is done at 95°C for 5
mixture is held at an
strands.

primers to hybridize to annealing temperature to allow uC

their
A PCR
complementary sequences. This is done at 55°C for 1 min.
primer
may comprise two
regions, a 3' (priming) region and a
region. The most important region in 5' (variabie)
determining the efficiency of annealing and
subsequent DNA synthesis during the PCR is the 3 region, which should be perfectly
complementary to the template sequence. The priming region should normally be 20 to
25 bases long. The Taq polymerase stabilizes these base-paired structures and initiates
DNA synthesis. In the last step, the reaction is heated to about 72°C for 1 to 5 minutes.

This process leads to a Taq polymerase<airecred DNA synthesis. The cycle is repeated

in a ycier for more than 20 times. In the

by keeping the reaction
tubes tnerd first

rise to a nowy SZd complement. Thus, the number of

cvcle each template gives
iss doubled.
doubled. Similarly, in each
e a c h subsequent cycle, the DNA
.

target region
copies of the dino to the target eion is almost doubled. About 20 cycles of

mnlification of
amplification the . target DNA. The PCR product is
of the
106-fold
produce
would onhoresis: The
The PCR
PCR product from a defined band can be
PCR electrophoresis:

agarose
gel DNA generated in
generated in a PCR can be re-amplified and used
a n a l y z e d by
DNA
The

from
agarosegel.
r e c o v e r e d

for sequencing.
32
 UORESCENCI IN-SITU HYBRIDIZATION
is fluorescence in-situ
Another type of molecular detection technique
hybridization (FISH)
W h i c h is applied for
bacterial detection in combination with

microscopy
to yOdization probesof DNA and target gene
from plant samples. Due

the presence of pathogen-specific ribosomal RNA (FRNA) sequences

in plants,

recognizing pecitic
this spe information by FISH can help detect the pathogen
infections in

" ddition to bacterial pathogens, FISH could also be used to detect fungi and

Viruses and other

IV. FLOw CYTOMETRY
endosymbiotic bacteria that infect the plant.
IOW
Cytometry (FCM) is
laser-based optical technique widely used for
a cell
a n d sorting, biomarker detection and protein engineering. FCM is usea Tor
rapid identification of
cells while cells pass
a
through an electronic detection apparatus in
lhquid stream. The
advantage of this technology is the capability for simultaneous
measurement of several
parameters. The technique uses an incident laser beam and
measures the
scattering and fluorescence of the laser beam reflected from the sample
B. INDIRECT
DETECTION METHODS
In addition to the direct
methods discussed above, indirect methods based on
plant stress
profiling and plant volatile profiling
have also been used for the
identification of biotic
and abiotic stresses as well as
pathogenic diseases in crops. In this
regard, new types of
optical sensors that detect biotic and abiotic stresses in plants have been .The developed
optical sensors provide detailed information based on different electromagnetic spectra
and thus, enable prediction of the plant health. Thermography, fluorescence imaging and
hyperspectral techniques are among the most favorable indirect methods for plant
disease detection.
1. Thermography
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.
2. Fluorescence Imaging
In this technique, the chlorophyll fuorescence is measured on the leaves as a

function of the incident light and the change in fluorescence parameters can be used to

apparatus and
analyze pathogen infections, based on changes in the photosynthetic
photosynthetic electron transport reactions

33
 3. Hyperspectral Techniques

health over a
Avperspectral imaging
wide can
beUsed to obtain uselul information about the plant
range of
is increasingly
being used forspectrum Detween 350 and 2500 nm. Hyperspectral imaging
scale agriculture. plant PHCnotyping und erop disease identification in large
phenotypin
4. Gas Chromatography
A
completely different
involves the
profiling of the non-optical
On-optical indirect
indirect method for plant disease detection
pathogen infections of volatile
echemical signature of the infected plants. The
chemica
plants could
Ould result in the
compounds (VOCs) that are relcase of specific volatile organic
the release
highly indicative
"gy indicative of
of the
the type
type of stress experienced by plants.
o
Detection of Plant
Diseases Using
A WIde
Portable Sensors
variety of sensors
deve loped and commercialized for various
have heen
applications including environmental monitoring and
medical diagnostics. Depending on
the operating principle of the sensor, the analytes could be detected using a sensor based
on electrical, chemical, electrochemical, optical, magnetic or vibrational signals. The
limit of detection could be enhanced by the use of nanomaterial matrices as transducers
and the specificity could be enhanced by the use of bio-recognition elements such as
DNA, antibody, enzymes etc.

Biosensor Platforms Based on Nanomaterials

Recent breakthroughs in nanotechnology enable the preparation of various

with rew technical hurdles. Nanoparticles display
nanoparticles and nanostructures
electronic and optical properics
and can be synthesized
using different types
fascinating
applications.
electronics and sensing
of materials for