PRACTICAL PLP 201

EQUIPMENT USED IN PLANT PATHOLOGY LABORTARY

For detection and characterization of the plant pathogens, several machines and equipment are
required in a lab. The equipment generally required for the studies on plant pathogens include:
Autoclave:
It is a machine that runs a physical method of sterilization by killing bacteria, viruses, and even
spores present in the material put inside of the vessel using steam under pressure. This is
mandatory for the authentic execution of virus detection and characterization procedure. The
principle is employed in an autoclave where the water boils at 121°C at the pressure of 15 psi or
775 mm of Hg. Working principle of an autoclave is given in the figure below.

Water bath:
It is laboratory equipment that is used for keeping the samples incubated for specific time at a
certain temperature. It has a tank filled with water that keeps the samples dipped in it allowing
the samples to bear the same temperature as we set for the water.
Orbital shaker:
Orbital shakers are designed to provide circular shaking motion to the samples or microbial
cultures. It helps in obtaining, bacterial cultures or general mixing required as a prerequisite of
several lab procedures.

Microscope
Microscope is a device which magnifies a microbial cell or a group of microbial cells to enable
the human eye to study its structure, morphology etc. It is an instrument used to see objects that
are too small to be seen by the naked eye. Microscopy is the science of investigating small
objects and structures using such an instrument.

Vortex:
Vortex mixer is relatively simple machine used to mix the vials and liquid suspension in a
smaller quantity.
PCR Machine:
PCR machine is generally called as thermo-cycler and is used for making copies of a desired
fragment of DNA.

Incubator:
Laboratory incubators provide a controlled, contaminant-free environment for safe, reliable work
with cell and tissue cultures by regulating conditions such as temperature, humidity, and CO2.
Microbiological incubators are used for the growth and storage of bacterial and fungal cultures.

Weighing balance:
It is a scale used in the laboratory for weighing the mass of the lab chemicals with accuracy.
PH meter:
It is an electronic device to measure hydrogen-ion activity (acidity or alkalinity) in solution. It’s
used to determine the pH of unknown solution as well as for setting of pH of various media and
testing biochemical activity of microorganisms. PH is expressed as a number from 0 to 14. The
number is an expression of the concentration of H ion in the solution. The optimum range of pH
for bacteria is 6.5 to 7.5 and 4-6 for fungi.

Spectrophotometer:
It is an instrument that works on the principle of estimation of capacity of a chemical to absorb
or reflect the light of a specific wavelength. It is used for estimation of the amount of the
chemical in a compound. It also is used for the estimating population of bacteria based on the
principle of turbidity determination. Turbidity is the cloudiness of the suspension, the more
turbid a suspension, less light will be transmitted it.

Micropipette:
Micropipettes are used to measure the lab chemicals in very small quantities less than a
microliter.
Centrifuge:
This machine is used to separate different components of a liquid. This machine uses the
centrifugal force to separate different phases of a mixture based on their density. This apparatus
rotate at high speed and separates substances as particle on the basis of mass and density by
means of centrifugal force.

Refrigerators:
The refrigerators are used to preserve the samples and chemicals for a longer period. The
temperature range available for storage of samples is as low as -86oC.
Micro Plate Reader:
A micro-plate reader is a laboratory instrument that is used to measure chemical, biological or
physical reactions, properties and analyses within the well of a micro-plate. In plant virology
Laboratory, the micro-plate reader is used to estimate the intensity of the color development as a
result of ELISA reaction.

Gel Documentation System:

Gel Doc systems are used for analysis of RNA/DNA in the molecular lab. The DNA/RNA is
stained with the ethidium bromide, which has a property to glow when illuminated with UV
radiation. Gel Doc systems are used to visualize and record the DNA or RNA in a sample.
 TECHNIQUES USED FOR THE DETECTION OF PLANT VIRUSES
Making a correct diagnosis is the basis of plant disease management and accurate disease loss
assessment. In case of plant viral infections, the application of chemicals and control measures is
not very effective once the infection has established. Therefore, making a correct diagnosis is the
primary step in managing the viral diseases in the plants. To carryout correct diagnosis, several
disease diagnostic techniques are implemented on the basis of the nature of disease and the
facilities available at the Plant virology lab. Detection techniques in case of plant viruses are
mainly divided into following major groups:
1. Electron microscopy
2. Serological techniques
3. Molecular techniques.
Electron Microscopy

The use of electron microscope is of ever-increasing importance in plant virology, and with the
passage of time. New techniques are being developed with the purpose of increasing our
knowledge of viruses. Implementation of electron microscopy, the virologist can: a) record
morphological characteristics (both external and internal) of the virus particles and their
subunits; b) make association between infectivity with specific particles; c) identify the presence
of contaminants in a virus suspension; d) make identification of existing and new viruses in a
diseased sample; e) determine the locus of virus replication and other mechanisms; f) observe
cytological changes or development of inclusion bodies.
Serological methods
Serological methods generally include the techniques that are based on the principle of antigen-
antibody detection system. Warm blood animals generally produce antibodies upon introduction
of specific antigens to their immune system. Serological techniques include, Enzyme-Linked
Immunosorbent Assay (Elisa), Tissue Blot Immunoassay (TBIA) and Quartz Crystal
Microbalance Immunosensors (QCMI).

ELISA:
ELISA is used for detection of the plant viruses in plant samples, seeds and insects. Generally,
ELISA is performed using a polystyrene plate that has the capability to adsorb protein molecules
or antibodies. Specific antibodies are produced against the plant viruses and that are allowed to
react with the antigen (the viral proteins) which result in color development in case of positive
reaction in an ELISA test. Intensity of the color developed at the end of the reaction refers to the
virus concentration in the sample. Advantages of ELISA include its capacity to test a lot of
samples simultaneously; its sensitivity and the process can be semi-automated.
Molecular Techniques:
As compared to serological methods, molecular methods are more sensitive, accurate and
authentic. These techniques can be used when the genetic information of the pathogen is
available. Molecular techniques are mainly based on nucleic acid-based detection. These
techniques include Polymerase chain reaction, Reverse transcription polymerase chain reaction,
Nested PCR and Multiplex PCR.

Polymerase chain reaction (PCR)

The PCR technique one of the most popular techniques being used now a day for the detection of
plant viruses in diagnostic labs. The PCR technique is based on the amplification (multiplication
into millions of identical copies) of a specific fragment of the DNA or RNA of the plant virus
with the help of specific primers (primers are defined as the short single strand of DNA used for
the initiation of DNA synthesis).

PCR technique is carried out in following steps

Denaturation: This is the initial step of the PCR in which the heat treatment (at about 95oC) the
target/template DNA causes the hydrogen bond to break and two strands of the DNA molecule
are separated.

Annealing: During this step, the DNA and primers are cooled down to 50oC to 60oC so that the
primers can attach to the template DNA and start making copies.

Extension: In the last step the temperature is increased raised to about 72 °C, and the DNA
polymerase begins adding nucleotides onto the ends of the annealed primers. At the end of the
cycle, which lasts about five minutes, the temperature is raised and the process begins again. The
number of copies doubles after each cycle. Usually 25 to 30 cycles produce a sufficient amount
of DNA.
 PREPARATION AND STERLIZATION OF NUTRIENT AGAR
Material
Flask, test tubes, cheese cloth, pH meter funnel, wire basket absorbent cotton, sterile petri plates,
glass rod.
INGREDIENTS
Beef extract 3g
Peptone 5g
Agar 15g
Distilled water 1 liter
Procedure
Dissolve 3 gram of beef extract and 5 gram of peptone in 1000 ml of distilled water by gentle
heating, stir constantly and cool to room temperature. Note the pH of the solution with the help
of pH meter. Adjust the pH to 7 by using 1.0 normal NaOH solution drop by drop. Add 15 gram
of agar and heat till agar is completely dissolved. Adjust the volume to 1000 ml by adding
distilled water. Filter through cheese cloth. Pour half of the medium into culture tubes, plugging
them with cotton plugs. Place the tubes in a basket; put them in the autoclave along with flask
containing the other half of the medium.

PREPARATION AND STERLIZATION OF POTATO DEXTROSE AGAR

Material
Flask, test tubes, cheese cloth, pH meter funnel, wire basket absorbent cotton, sterile petri plates,
glass rod.

INGREDIENTS
Peeled diced potato 250g
Dextrose 20g
Agar 20g
Distilled water 1 liter
Procedure
To prepare PDA, boiled 250g of peeled diced potato in 500ml of water in a pot and strain. Boil
20gm of agar in a flask in the remaining 500 ml of water, stir constantly till agar is completely
dissolved, and add 20 gm of dextrose. Mix the two lots, make the volume up-to 1000 ml and
strain through cheese cloth. With the help of funnel, pore half the medium in test tubes each up-
to 1/4th full and plug them with already prepared cotton plugs place the plugged tubes in a wire
basket and put it in the autoclave. Save the other half of the medium for petri plates (15-20
ml/plates).

ISOLATION OF NEMATODES FROM SOIL AND PLANT MATERIALS

Nematodes are cosmopolitan in distribution and they have adopted various mechanisms of
parasitism. Few species are ecto-parasites, much longer than the other plant parasitic nematodes
and have long stylet for penetration in to the plant roots, e.g. Longidorus, Paralongidorus,
Trichodorus, Hoplolaimus and Xinphinema spp. Other plant parasitic nematode enter into plant
roots of their hosts completely and lay eggs there, e.g. Meloidogyne, Heterodera spp. Few are
semi-endoparasites, e.g. Tylenchulus semipeneterans whereas few others are stem and seed
parasites, e.g. Anguina tritici, Ditylenchus and Aphelenchoides.

Materials:

Soil and plant roots, funnel with stand, sauce pan, one intact and other perforated plastic or
metallic tray, a set of sieves (20, 60, 100, 150, 200, 300, 350, and 400 mesh), gloves, plastic
buckets, plastic tubs, mug, glass beakers, muslin cloth, tissue paper, Petri dishes, watch glass.

A) Samples Collection of the nematodes infested samples: -

From the field crops and vegetables collect the soil and root samples gently up-to the depth of
15-30 cm and about 1kg soil along with the whole plant in polyethylene bags. For shrubs and
trees, dig down 3 to 4 feet and from the four direction of the site and obtain one composite
sample. Bring the samples to the Laboratory and store them in refrigerator at 5 oC.

B) Isolation of nematodes from soil and plant roots

i) Baermann Funnel Technique (1917): -

Close the tube of the glass or plastic funnel with a spring clip. Pour the water in the
funnel up to the level that the test sample should be submerged in the water. Wrap 250 gms
quantity of soil and plant roots (cut in to small pieces) in tissue paper or muslin cloth and
suspend it on glass or plastic funnel. Nematodes move in wet soil, fall into water and gather
behind the clip in the tube. After 12-24 hrs of incubation, collect some quantity of water in glass
beaker, take 10ml of the nematode suspension in a Petri dish and observe the nematodes under
stereoscopic microscope.

ii) Whitehead and Hemming Tray Method (1965): -

In this method two types of plastic or metallic trays are used adjusted in to one another.
The lower tray is intact while the upper tray is perforated. Spread the tissue paper or muslin cloth
on the upper tray and put 250 gms quantity of soil and plant roots (cut in to small pieces) on
tissue paper or muslin cloth. Add such quantity of water in the lower tray that it should wet the
samples present on the upper tray. Nematodes move in the wet soil, fall down into water and
gather in the water present in the lower tray. After 12-24 hrs of incubation, collect the water from
the lower tray in a glass beaker, take 10ml of the nematode suspension in a Petri dish or watch
glass and observe the nematodes under stereoscopic microscope.

C) Isolation of nematode from soil by Cobb’s Sieving Method (1918): -

Dip 250 or 500 gms of soil to 3-4 liters of water in a bucket and stir vigorously to release
nematode. Break the soil colds with hand. Filter the soil suspension through a muslin cloth to
avoid the organic matter entering into suspension. Stir again and allow the soil particles to settle
down to the bottom for one minute while leaving the nematodes in the water suspension. Decant
the suspension into plastic tub through a nest of sieves with various openings (100, 200, 300,
350, and 400 meshes). Collect the nematodes by back washing of the sieves in to different
beakers. In this way you have the nematodes of various sizes in the beakers. Return the filtered
water in to the bucket. Repeat the entire process. Finally note the nematode fauna under
stereoscopic microscope as mentioned above.