agricultural microbiology

1. Molecular mechanisms of disease diagnosis
Presented by,
Jeeva Raj
Joseph
15KUSM6008
MSC.MB.

2. Diagnosis
• The act or process of identification of
the nature of an illness or other problem
by examination of the symptoms.
• Diagnosis helps in implementing right
control method.
• Diagnosis is important with the
increasing global trade of plants and
plant produce and associated risk of
movement of pathogen and their
vectors from one country to another.

3. Basic techniques
• Host range and symptomatology
• Morphology of the causal organism
• Selective media
• Biochemical markers like FAME
and protein analysis

4. Molecular techniques
• These techniques are more appropriate for pathogens that are difficult to
detect, identify or test for susceptibility with conventional methods
•Molecular methods are those used for identifying genetic variation within
pathogen population
• Various molecular techniques in use are as follows:
 Antibody based method
 Nucleic acid based methods

5. Antibody based methods
• Serology methods are most widely used in diagnostic systems in plant pathology
• In this method antibodies are raised in animals against specific antigens from the
pathogen which are purified and used for diagnostic purpose
• Different techniques are as follows:
 Use of polyclonal antibodies
 Use of monoclonal antibodies
 ELISA(Enzyme Linked Immuno
Sorbent Assay)
 Lateral flow technique

6. Polyclonal antibodies
• 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

7. 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 5minutes at 125rpm and incubated in room temperature for 2.5h.
• The suspension was centrifuged at 11000 rpm for 5mins and resuspended 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 mins at room temperature in dark.
•Then the suspension is centrifuged and the cells are suspended in saline.

8. example
•5μl of stained sample is placed on Neubauer counting chamber and read
with fluorescence microscope.
• They give information about sensitivity and specificity, not cell count.

9. 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.
•They are also produced against specific
oligosaccharide side chain of Erwinia
bacteria and phytoplasmas.
• For example, the identification of Botrytis
cinerea in grape juice and discrimination
of cereal eye-spot and other stem based
diseases of wheat

10. Enzyme linked immuno sorbent assay
• This technique is based on the principle of antigen and antibody interaction
with a detection system involving enzyme conjugated antibody.
• The activity of the enzyme is measured spectrophotometrically by adding an
appropriate substrate that results in colour change
DAS-ELISA
TAS-ELISA
PLATE TRAPPED ANTIGEN
DAS ELISA

11. Example
• A polyclonal antiserum specific to grapevine leafroll associated clestovirus-3
(GLRaV-3).The antigen was developed using a recombinant coat protein
expressed in E.coli. Specificity of antiserum against antigen GLRaV-3 is
done using Double Antibody Sandwich Enzyme-Linked Immunosorbent
Assay (DAS-ELISA).
• Pseudomonas solanacearum, a pathogen of potato, tomato and eggplant was
diagnosed by an antibody raised against the lipopolysaccharide of the
bacterium which made the diagnosis reliable and easy. DAS-ELISA technique
was used for this purpose.

12. Lateral flow technique
• It is an immunochromatographic
technique including both ELISA and
chromatography
• This method is suitable for field use
by crop inspectors and doesn’t
require sophisticated equipment.
• Application of sample
• Formation of Ab-Ag complex
• Flow of the complex
• Formation of test line
• Formation of control

13. 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
• Different methods are as follows:
 PCR based technique
 RAPD
 DNA microarray based technique

14. PCR based technique
• Polymerase chain reaction, PCR technology, is an efficient and cost-
effective way to copy or amplify small segments of DNA or RNA.
• PCR based methods are also developed for diagnostics because the
technique is rapid and can be used relatively easily in remote laboratories.
• The technique is fast and highly specific. It can be used to detect trace
amounts of fungal DNA from environment samples before symptoms
occur. It therefore allows the implementation of early disease control
methods.
• This technique utilizes specific oligonucleotide primers which are designed
based on nucleic acid sequences that are diagnostic for the pathogen.

15. PCR
• Primers developed are based on more specific sequences such as
The argk-tox gene of Pseudomonas syringae pv. phaseolicola which
encodes a gene involved in phaseolotoxin biosynthesis and can be used to
identify bacteria that posses this trait
DNA extracted from the leafhoppers that are potential vectors of
phytoplasmal diseases can be PCR amplified using phytoplasmal specific
primers to identify which species are true vectors.
• A number of techniques have been developed to improve the reliability,
efficiency and cost effectiveness of PCR based techinques such as multiplex
PCR kits capable of detecting more than one pathogen present in a particular
plant or soil sample .

16. • These multiplex systems are combination of primers that give different
size fragments for each of the species and it is further developed by
using primers that fluoresce at different wavelengths for different
pathogens.
•Kits are commercially available to unravel cereal stem-based complex of fungi
comprising Tapesia yellundae and T. acuformis (eye spot fungi), and
Microdochium nivale(snow mold of cereals).
PCR

17. PCR based diagnostic
technique
• Genomic DNA isolation
• Primers specific to the
pathogen
• PCR is run
• The amplified sample is run on
the gel along with the
positive control

18. Random amplified polymorphic Dna
• Random amplified polymorphic DNA (RAPD) is a powerful diagnostic tool that
can be used to distinguish genetic variation among different species, varieties,
form a biotype within population of plant pathogens
• RAPD is often suitable since it does not presume any knowledge of DNA
sequences of organism.
• In this technique, rather than selecting primers of known complementarities to
the pathogen, arbitrary sequences of about 10 nucleotides are used

19. RAPD
• In RAPD analysis short (10mer) primers chosen at random are used in
PCR reactions.
• 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.

21. 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.

22. DNA MICROARRAY
• Samples could be taken from soil, plant surfaces etc, and screened to determine
organisms present and these techniques could be further developed to establish the
efficacy of control methods in removal of specific species.