Agricultural crops are threatened by a wide variety of plant diseases and pests that can damage crops and lower yields. Correct diagnosis of plant diseases is important so that treatments can be tailored to specific pathogens. There are indirect and direct methods used for diagnosis. Indirect methods include environmental reviews and microscopic examination, while direct methods use molecular techniques like antibody-based methods (e.g. ELISA, polyclonal antibodies) and nucleic acid-based methods (e.g. PCR, DNA microarrays) to identify causal agents. DNA microarrays allow screening for multiple pathogens simultaneously. Correct diagnosis is key to minimizing crop losses from diseases and pests.

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Analytical Techniques in Plant Pathology
Agriculture crops are threatened by a wide variety of plant disease and pest. They can damage
crops, lower fruits and vegetable quality and wipe out entire harvest. Agricultural crop is yearly
destroyed by pest and disease 42%. Farmers often must with more than one pest or disease
pathogenic strains attacking the same crop and new pesticide resistant. Crops loses can be
minimized and specific treatments can be tailored to combat specific pathogen if plant disease are
correctly diagnosing and identified. Diagnoses of plant disease is to identify the disease nature and
to determine the causal agent whether living or non-living. The symptoms which appear on plants
may be because of Biotic factors A biotic factor. There are two types of methods.
1. Indirect Method
2. Direct Method
Indirect Method
1. Become acquainted with the environmental Conditions surrounding the plant
2. Review of agricultural operations
3. Examination of the plant sample microscopic examination
 Soil analysis and measurement of pH
 hypothesis Koch
 Biochemical methods
 microscopic examination
 serological methods
 the use of methods relies on DNA The methods used to identify the symptoms
are
Direct Method
Various molecular techniques are used are as follows
1. Antibody based Method
2. Nucleic Acid Base Method

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Antibody Based Methods
Serological methods are most widely used in diagnostic system in plants which are
1. ELISA
2. USE of polyclonal Antibody
3. Use of monoclonal Antibody
4. Lateral Flow Techniques
Polyclonal Antibody
Antibodies secreted by different B-cell lineages within a body. They are a collection of
immunoglobin molecules that react against a specific antigen, each identifying a different epitope.
Part of pathogen is used as antigen. Purity of antigen is crucial. Purified antigen is injected into
the animal with subsequent booster injections. Resultant antiserum contains a population of
antibodies that react with different determinants of antigen which is used for detection
Example
Polyclonal antibodies are used for diagnosis of seed transmitted plant pathogenic bacterium
Xanthomonas campesteris. Healthy seeds are treated with pure suspension X. campesteris and
shaken for 5 minutes at 125 rpm and incubated in room temperature for 2.5h. The suspension was
centrifuged at 11000 rpm for 5mins and re suspended in saline. This was used to inject the animal
to produce polyclonal antibodies which was later filtered and used for diagnosis of seeds. 10,000
seeds were selected per plot and were added into 0.85% saline. The seed extract was treated with
fluorescent tagged polyclonal antibodies incubated for 30 mints at room temperature in dark. Then
the suspension is centrifuged and the cells are suspended in saline. antibodies that react with
different determinants of antigen which is used for detection.
Monoclonal antibodies
An antibody produced by a single clone of cells or cell line and consisting of identical antibody
molecules. They are produced and are commercially available against a wide variety of viruses.
For example, the identification of Botrytis cinerea in grape juice and discrimination of cereal eye-
spot and other stem based diseases of wheat.

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Lateral flow technique
It is an immune chromatographic technique including both ELISA and chromatography. This
method is suitable for field use by crop inspectors and doesn’t require sophisticated equipment.
Nucleic acid based methods
The aim of nucleic acid based technique is based on sequences of the pathogen such as DNA for
bacteria and fungi; RNA for most viruses which can be used for further uses like diagnosis and
research.
1. PCR
2. RAPD
3. DNA Microarray
RAPD
In RAPD analysis short (10mer) primers chosen at random are used in PCRreactions. Differences
between template sequences will result in different patterns of bands on a gel. The method is
applied to previously uncharacterized fungi and used to distinguish between species or between
isolates within species.
DNA microarray technique
A DNA microarray (also commonly known as DNA chip or biochip) is a collection of microscopic
DNA spots attached to a solid surface. This method can also be used as a diagnostic tool. Increase
in DNA sequencing of plant pathogens increased the potential to develop diagnostic microarrays
that can be used to screen for the presence of multiple pathogens in one assay. In this method
known DNA sequences are arrayed on the solid surface. Universal primers available were used to
amplify ITS regions and the DNA sequences are labelled and used to probe array to identify the
organism present.

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Reference
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