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Laboratory Manual: Fundamentals of Plant Pathology

This document provides guidelines for students in a plant pathology laboratory. It summarizes several key pieces of laboratory equipment and their uses, including microscopes for examining microbial cells, an autoclave for sterilizing materials, an incubator for culturing microbes at constant temperatures, and safety equipment like gloves and masks. Proper laboratory conduct, dress code, and safety procedures are also outlined to ensure a safe working environment.

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0% found this document useful (0 votes)

4K views

Laboratory Manual: Fundamentals of Plant Pathology

Uploaded by

Ch Dheeraj Rao

We take content rights seriously. If you suspect this is your content, claim it here.

Available Formats

Download as PDF, TXT or read online on Scribd

You are on page 1/ 63

LABORATORY MANUAL

Fundamentals of Plant Pathology

PTH-213

(For private circulation only)

Name of student ………………………

Registration no/Roll no ………………

Section and Group ……………………

School of Agriculture

Session-term: 2020-21 (Autumn Term)

LMPTH213 Page 1
General guidelines for the students

1. A Student is expected to maintain the decorum of the laboratory by maintaining

proper discipline.

2. Student is expected to be punctual in lab, should keenly perform the experiment allotted to
him without moving from one lab to another and even experimental set up should not be left
until it unavoidable.

3. Mobile phones are not allowed in the labs and should be kept in the bags in silent or
switch-off mode.

4. Keep the work area clear of all materials except those needed for your work. Extra
books, purses, bags etc. should be kept in the racks placed in the laboratories.

5. Clean up your work area before leaving.

Dress code:
1. Shorts and sandals should not be worn in the lab at any time. Shoes are
required when working in the laboratories.

2. Students must have lab coat, gloves and mask with them every time.

Compulsory things to be carried by the students in lab:

Lab coat, gloves, mask, calculator, butter paper, fractional weights and stationary items.

Safety Guidelines:
1. Do not use any equipment unless you are trained and approved as a user by
your supervisor.

2. Wear safety glasses when working with hazardous materials or use such
materials in fuming hood.

3. Wear gloves when using any hazardous or toxic agent.

4. If you have long hair or loose clothes, make sure it is tied back or confined.

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Table of Content

Sr. Title of Experiment Page No.

No.

1 To study various laboratory equipments and microscopy 4-11

2 To study collection and preservation of disease specimen 12-15

3 To study preparation of different culture media 16-20

4 To study Koch’s postulates for the isolation of plant pathogens 21-24

5 To study morphological structures of different fungal genera 25-28

6 To study staining and identification of plant pathogenic bacteria 29-32

7 To study transmission of plant viruses 33-35

8 To study of phanerogamic plant parasites 36-38

9 To study the morphological features and identification of plant parasitic 39-46

Nematodes

10 To study sampling and extraction of nematodes from soil and plant 47-54
material and preparation of nematode mounting

11 To study fungicidal formulations and their calculation 55-59

12 To study methods of pesticide application and their safe handling 60-63

LMPTH213 Page 3
Experiment 1

1. Experiment: To study various laboratory equipments and microscopy.

Equipment and Apparatus Required: Microscope, Autoclave, BOD, Laminar air flow
Centrifuge, Elisa Reader, Gel electrophoresis and PCR
Materials Required: Sterile Petri-dishes, sodium hypochlorite solution (1%), sterile water,
forceps, inoculation needle, burner/spirit lamp, spirit, incubator, antibiotic, Potato,
dextrose, Agar
2. Learning Objectives: Student will be able to understand laboratory techniques
to study Plant associated microbes
3. Theory/Principle/Background of the topic: These are basic equipment and tools to be
used in lab. It is very important to learn about their working. The students will observe the
following equipments and learn about their functioning. At the same time they will also learn
about their handling, and their use in preparation of artificial culture media for growth of
different pathogens. The autoclave is used to completely sterilize the media used in culturing
of microorganisms pure culture and remove all contaminations, for sterilizing glassware
another equipment is used called Hot air oven which produces very hot dry air, Incubator is
used for incubation (culturing of microbes) at a constant temp. Colony counter is an
electronic apparatus used to count the number of colonies on a Petri plate, inoculation
chamber is used to make most of the aseptic transfers, now-a-days laminar airflow system is
used as inoculation chamber, Water bath is used to maintain the temperature at desired level,
PH meter determines the pH of solutions of unknown pH as well as for setting of pH of
various media.
Equipment 1. Microscope and Microscopy
Microscope is a device, which can magnify a microbial cell or a group of microbial cells to
enable the human eye to study its structures, morphology etc. The optical microscope, often
referred to as the "light microscope", is a type of microscope which uses visible light and a
system of lenses to magnify images of small samples. Optical microscopes are the oldest and
simplest of microscopes. Microscopy is the use of a microscope or investigation by a
microscope.
i). Simple microscope: Consists of a simple lens system.
ii) Compound microscope: It consists of 2 or more lens systems- Depending
on source of illumination,

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A compound light microscope with a single eye-piece is called monocular; one with two eye-
pieces is said to be binocular. A compound light microscope with a single eye-piece is called
monocular; one with two eye-pieces is said to be binocular.

Microscopes that use a beam of electrons (instead of a beam of light) and electromagnets
(instead of glass lenses) for focusing are called electron microscopes. These microscopes
provide a higher magnification and are used for observing extremely small microorganisms
such as viruses.

Light microscopy
Brightfield microscopy
This is the commonly used type of microscope. In bright field microscopy the field of view is
brightly lit so that organisms and other structures are visible against it because of their
different densities. It is mainly used with stained preparations. Differential staining may be
used depending on the proper ties of different structures and organisms.
Darkfield microscopy
In darkfield microscopy the field of view is dark and the organisms are illuminated. A special
condenser is used which causes light to reflect from the specimen at an angle.
Phase-contrast microscopy
Phase-contrast microscopy allows the examination of live unstained organisms. For phase-
contrast microscopy, special condensers and objectives are used. These alter the phase
relationships of the light passing through the object and that passing around it.
Fluorescence microscopy
In fluorescence microscopy specimens are stained with fluorochromes/ fluorochrome
complexes. Light of high energy or short wavelengths (from halogen lamps or mercury
vapour lamps) is then used to excite molecules within the specimen or dye molecules attached
to it. These excited molecules emit light of different wavelengths, often of brilliant colours.

Equipment 2. Autoclave
It is an apparatus in which saturated steam under pressure affects sterilization called
autoclaving. The pressure increases boiling point of water and produces steam with a high
temperature. Cells are destroyed by high temp and not by the pressure. Most of the organisms
are killed at 121.6 °C and 151b pressure per sq. inch in 15 min. It is more efficient and
common instrument used to sterilize solids and liquid media for microbial culture. It is not
recommended for oils, powders, heat sensitive fluids and plastics. Autoclave is a double

LMPTH213 Page 5
walled cylindrical metallic vessel made of thick stainless steel copper, lid of which is opened
to receive the material to be sterilized. The lid is provided with pressure gauge noting the
pressure, steam clock for air exhaustion of the chamber. It is also provided with safety valve
to avoid explosion.
Equipment 3. Hot air oven
It is an electrically operated equipment with a thermostat (ambient Temp, to 300°C) used for
sterilizing glassware. An oven consists of an insulated cabinet, which is held at a constant
temp, by means of an electric thermostat. Some oven is also fitted with fan to keep hot air
uniformly circulated at constant temperature. For proper circulation of hot air, the shelves are
perforated. The scheduled temperature for sterilization with dry air is given in Table

Temperature (°C) Time in Minutes

120 480
150 150
160 120
170 60
180 20

Equipment 4. Incubator
It is used for incubation (culturing of microbes) at a constant temp. It is similar to an oven in
terms of construction and consists of an insulated cabinet fitted with a heating element at the
bottom. The temp, of the incubation is maintained at desired level by an automatic device
called thermostat. It is provided with double doors, made of glass so that the contents of
incubator maybe viewed without admitting outside air. Most incubators can be supplied by
placing a beaker of water in it to retard the dehydration of medium during growth of
microorganisms. Some incubators are provided with fluorescent light that can be used to
encourage sporulation.
Equipment 5. Colony counter
It is an electronic apparatus used to count the number of colonies on a Petri plate. A Petridis
fits into the recess in the platform. The colonies on plates are counted on an illuminated
screen, illuminated from beneath with a large magnifying lens which provides 1.5X
magnification. Some instruments are also fitted with electronic micro switch with pen and
counter.

LMPTH213 Page 6
Equipment 6. Inoculation chamber
Most of the aseptic transfers are made using inoculation chamber made of wood. Now-a-days
laminar air flow system is used as inoculation chamber. It is used for reducing danger of
infection while working with infective microorganisms and for preventing contamination of
sterile materials. It is a hood like structure having germicidal ultraviolet lamp and Bunsen
burner. It consists of mid table as working place onto which sterile air is pumped at uniform
velocity either in horizontal or vertical direction. It works on the principle of application of
high efficiency particulate air filters (HEPA)-or fiber glass filter which can retain all particles
including bacteria whose diameter is more than 5 microns.
Equipment 7. pH meter
It is used to determine the pH of solutions of unknown pH 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 for fungi it is 4-6. The
measure of pH with pH meter is done electrometrically. Measurement of pH depends upon
the" development of membrane potential by a glass electrodes. As an alternate, pH papers are
used to measure the pH of the medium.
Equipment 8. Water bath
It is an insulated metallic box fitted with an electric heating mechanism and a thermostat,
which maintains the temperature at desired level. There are racks for holding test tubes.
These are usually used for melting of media, testing enzymatic activities of various
microorganisms, widely test etc.
Equipment 9. Centrifuge
It is an apparatus that rotates at high speed and separates substances as particles on the basis
of mass and density by means of centrifugal force. The microbes are arrested from sediments
settled at the bottom of the tube after centrifugation. The centrifugal force is noted in rpm of
angular speed. A centrifuge consists of head which is rapidly revolving on upright motors.
Generally four metal caps are attached to the head for holding rubes or other container of the
material from which particulate matters to be separated. During centrifugation liquid
containing particulate matter is kept in the tubes, run at a particular speed and when
centrifugation is completed, the particulate matter gets settled at the bottom of the tubes. The
commonly used centrifuges are of low speed, high speed and ultracentrifuge with highest
speed limit of 5000 rpm, 18000 rpm, and 20,000 to 60,000 rpm, respectively. These are used
for separation of virus particles, bacterial cells, and fungal spores, separation of mixtures of

LMPTH213 Page 7
liquids varying in their density and concentrating microorganisms in various samples for
enzymatic and other studies.
Equipment 10. Electronic Balance
Various media components for culture media preparation and samples etc. are weighed on
an ordinary balance. Whenever precision is required an electronic monopan balance is
recommended. As most of the media ingredients are highly hygroscopic, the balance should
be cleaned immediately after use.
Equipment 11. Haemocytometer
The number of microorganisms present in a given liquid sample can be counted and
morphology of bacteria can be observed by direct cell count method using haemocytometer.
It is a special glass slide with a depression (0.1 mm - 0.02 mm deep) at the centre covering
an area of 1 mm, the area of 1 mm is further divided into 400 small squares. To get the
number of cells per ml of sample the following formula is used.The number of cells per ml
= average number of cells in a small square x 400 x 104 (factor) Following are some
tools and students are required to learn about them:
Equipment 12. Thermocycler PCR
The thermal cycler (also known as a thermo cycler, PCR machine or DNA amplifier) is a
laboratory apparatus most commonly used to amplify segments of DNA via the polymerase
chain reaction (PCR).Thermal cyclers may also be used in laboratories to facilitate other
temperature-sensitive reactions, including restriction enzyme digestion or rapid diagnostics.
The device has a thermal block with holes where tubes holding the reaction mixtures can be
inserted. The cycler then raises and lowers the temperature of the block in discrete, pre-
programmed steps.
Equipment 13. Agarose gel electrophoresis
Agarose gel electrophoresis is a method of gel electrophoresis used in biochemistry ,
molecular biology, and molecular plant pathology separate a mixed population of DNA or
proteins in a matrix of agarose. The proteins may be separated by charge and/or size
(isoelectric focusing agarose electrophoresis is essentially size independent), and the DNA
and RNA fragments by length. Bio molecules are separated by applying an electric field to
move the charged molecules through an agarose matrix, and the biomolecules are separated
by size in the agarose gel matrix. Agarose gels are easy to cast and are particularly suitable
for separating DNA of size range most often encountered in laboratories, which accounts for
the popularity of its use. The separated DNA may be viewed with stain, most commonly

LMPTH213 Page 8
under UV light, and the DNA fragments can be extracted from the gel with relative ease.
Most agarose gels used are between 0.7 - 2% dissolved in a suitable electrophoresis buffer.
TOOL 1. Inoculation loop or Inoculation needle
Used for aseptic transfer of culture. It consists of an insulted handle provided with screw
device at the tip which holds a heat resistant chrome or platinum wire approximately 3
inches long. The end of wire is bent to form a loop. Inoculation needle is similar to loop,
but the holder contains a straight piece of wire instead of a loop. They are sterilized by
flaming in the blue portion of burner flame until it is red. The loop is mainly used to
transfer culture of microorganism growing on liquid cultures. Inoculation needles are used
to transfer cultures of microorganisms growing on solid medium in form of colonies.
TOOL 2. Glass spreader
It is bent T or L shaped glass rod used for spreading of liquid culture and sample
on sterile agar plate
TOOL 3. Glass marking pen
All the culture material is labelled with the use of Glass marking pen.
TOOL 4. Petri-dish Cans
It is made of copper and used as container for keeping Petri-dishes. The Petri-dishes
in cans are sterilized in hot air oven at required temperature.
Glass wares to be used during lab
activities-1. 1. Test Tube:-
i) Test tubes of 18 x 150 mm size are used for preparation of broth, agar slants and agar
stabs, 25 x 150 mm size are used for preparation of dilution blank
ii) Screw caps tubes with round bottom of size 15 x 125 mm are used for
maintenance of culture.
2. Petri-dishes:-
Petri-dishes, a pair of circular glass containers named after Petri, are used for
the preparation of agar plates. The common size is 100 mm in diameter.
3. Pipettes, Flask and Beakers:
Different sizes of pipette and conical flask are used for preparation of dilutions and plating.
Generally pipette of 10 ml and 1 ml are used for sterile transfer of known volumes of liquid.
For preparation of dilutions conical flasks of 250, 500 and 1000 ml are used for preparation
of medium. The volume of media should not exceed 2/3 of the volume of flask. Beakers of
size 250, 500 and 1000 ml are used for preparation of medium.
4. Slides and cover slips:- Rectangular slides of 75 x 25 mm size made of glass with

LMPTH213 Page 9
polished edge are used for observation of microorganisms. Square or circular cover slips of
size18x18mm or 20 mm diameter are used for covering the specimen glass slide while
observing under high power objective of a microscope. The thickness of cover slip shouldn't
exceed 0.016 mm. Preparation of basic liquid Medium (broth) for routine Cultivation of
Bacteria. Bacteria are often cultivated in liquid broth (media lacking agar).
4. Outlines of the Procedure-
The student will observe the equipments mentioned above and learn their functioning.
5. Scope of the results: To learn about lab equipment is very essential as all the laboratory is
based on them. Media preparation should be according to the mentioned procedure. It is
required in almost all the experiments.
6. Results required: The students will identify the equipment and their handling
procedures.

7. Caution: All the given instructions to operate equipment and to use materials should be
followed. Handle the sample carefully to avoid any damage to its body parts during
collection and preservation.

Result & discussion:

Learning outcome

To be filled in by Faculty:
Sr. No. Parameter Marks Obtained Max Marks
1. Understanding of the student about 20
the procedure/apparatus
2. Observations and analysis 20
including learning outcome
3. Completion of experiment 10
Discipline and cleanliness
Signature of Faculty

LMPTH213 Page 11
Experiment 2

1. Experiment- To study collection and preservation of disease specimen

Equipment and Apparatus Required: Preservation jar
Materials Required: Disease sample, plastic bag, herbarium file

2. Learning Objectives: Student will be able to understand collection and

preservation of disease specimen.
3. Theory/Principle/Background of the topic: It is important to gather the best plant
samples possible and to record all pertinent background information for the diagnostician.
Diseased plants generally have been infected by one or more pathogenic microorganisms,
although they may also have an abiotic disease that does not involve a plant pathogen. Plants
affected often display a range of symptoms visual signs of the infection. Often, not all
symptoms of a particular disease will appear on any one plant within a diseased crop, and
more than one plant organ may be affected by a given disease.
4. Outlines of the Procedure-
Collecting plant samples
Following are general guidelines for collecting plant samples.
Examine the entire plant for symptoms
Examine all of the main plant organs for disease symptoms: roots, stems, leaves, and
blossoms. Collect samples from various plant organs as needed. Plants may suffer from more
than one disease simultaneously. Segregate different types of symptoms into different
samples.
Collect several plant specimens
A single plant sample may not be enough to allow a correct diagnosis of the problem; several
plant samples showing the range of symptoms may be needed. If possible, select samples
with various stages of disease development (early and late stages). Samples should be as
typical or representative of the overall problem as possible.
Do not collect dead plants or plant organs
Dead plants or plant organs may not be useful for diagnosis. Always select plant samples
from living tissues and focus your attention on plants or plant organs that are in early stages
of disease or are in the process of dying, and not already dead.
Collect the entire plant whenever possible
If the wrong part of a plant is submitted, a disease cannot be diagnosed with confidence. For
example, some leaf symptoms of disease (wilting, for example) are the result of damaged or

LMPTH213 Page 12
diseased roots that have rotted and are no longer functioning to support the plant; in such
cases, a correct diagnosis often depends on having a sample of the roots. Plants should be
carefully dug from the ground (not pulled out) so the root systems remain relatively intact. Be
sure that root samples are from the affected plants and not from adjacent weeds. If entire
plants cannot be sampled, submit photographs of affected plants where possible.
Provide as much background and related information as possible
Good information contributes to a better understanding of the problem. A complete
description of the problem and the crop’s history should accompany the sample. Give the
name of the plant submitted. Indicate when the problem appeared and when the sample was
taken. Specify all fertilizers and pesticides used. Examine the growing site carefully and note
the conditions. Make note of environmental conditions for the site such as elevation,
flooding, previous crop history, etc. Indicate any observable pattern of disease occurrence
(for example, in random patches, or uniformly throughout the crop).
Preserving plant samples
After collecting the samples, do not expose them to direct sunlight. Keep them cool and do
not allow them to dry out or cook. Place samples in plastic bags in the shade or in a cooler
until they are ready for delivery to the plant clinic. Leaves may be pressed between the pages
of a book or magazine or wrapped in tissue.
Packaging plant samples
It is important to package the samples properly to ensure they arrive in good condition at the
plant clinic. Following are general guidelines for handling and packaging plant samples.
Use plastic bags
For most samples including leaves, stems and roots, use plastic bags to prevent plant samples
from drying out during transport. However, fleshy fruits, vegetables, or tubers in stages of
decay should be wrapped individually in dry newspaper.
Do not add extra water
Do not add any water or moist paper towels. Moisture favors the growth of fungi and bacteria
that decay plant tissues, which can confuse or obscure the diagnosis of the pathogen.
Label the samples
Write your name, telephone number, and name of the plant or sample number on the plastic
bag, using marker or write the information on a piece of paper and insert in the bag.
Segregate plant tissue from soil
Keep soil off the foliage. Avoid contact of plant samples and information labels with any soil
that might be in the plastic bag.

LMPTH213 Page 13
Tie off the root ball
Place the root system and accompanying soil in a plastic bag and tie it securely to the lower
stem of the plant. This will prevent roots from drying out and will keep the soil secure. Place
a second plastic bag over the aboveground part of the plant, if possible, to prevent it from
drying out.
Submit samples as soon as possible
Decayed plant samples are useless for an accurate disease diagnosis. Always plan to have
samples arrive at the ADSC within one or two days of their collection, if possible, or take
steps to inhibit the deterioration or decay of samples (i.e., by refrigeration).
5. Scope of the results:
The student will observe the diseased plant samples in the field and learn their collection and
preservation procedure for further study. To learn about disease sample collection and
preservation is very essential as all the microbial study at microscopic level is based on them.
6. Results required:
Identification of proper disease sample on the basis of their symptoms and their
preservation for further microscopic study
7. Caution: Dead plants or plant organs may not be useful for diagnosis. Collect the entire
plant whenever possible. If entire plants cannot be sampled, submit photographs of affected
plants where possible. A complete description of the problem and the crop’s history
should accompany the sample. After collecting the samples, do not expose them to direct
sunlight. Use plastic bags to prevent plant samples from drying out during transport.

Suggested readings for students: Collecting Plant Disease and Insect Pest Samples for
Problem Diagnosis
Web links: https://www.ctahr.hawaii.edu/oc/freepubs/pdf/scm-14.pdf

LMPTH213 Page 14
Worksheet of the student
Date of Performance Registration Number:
Aim: Collection and preservation of pathological specimens and their maintenance.

Sr. Name of crop/plant Symptoms Pathogen

No.

Result & discussion:

Learning outcome

To be filled in by Faculty:
Sr. No. Parameter Marks Obtained Max Marks
1. Understanding of the student about 20
the disease symptoms
2. Observations and analysis 20
including learning outcome
3. Completion Of experiment 10
Discipline and cleanliness
Signature of Faculty

LMPTH213 Page 15
Experiment 3

1. Experiment: To study preparation of different culture media.

Equipment and Apparatus Required: Autoclave, BOD incubator, Laminar flow, hot air
oven
Materials Required: Potato, dextrose, agar agar powder, knife, muslin cloth, pan,
hot plate, distilled water
2. Learning Objectives: To study about the different methods of media preparation
for culturing different plant pathogenic and other micro-organisms.
3. Theory/Principle/Background of the topic: A wide range of media are used for growing
fungi. Most mycologists develop preferences for certain types of media based on experience
and peculiarities of the type of fungi that are routinely grown. Media will affect colony
morphology and color, whether particular structures are formed or not, and may affect
whether the fungus will even grow in culture. All fungi require several specific elements for
growth and reproduction. The requirements for growth are generally less stringent than for
sporulation, so it is often necessary to try several types of media when attempting to identify
a fungus in culture. Most fungi thrive on Potato Dextrose Agar (PDA), but this can be too
rich for many fungi, so that excessive mycelial growth is obtained at the expense of
sporulation.
4. Outlines of Procedure:

1. Potato Dextrose Agar (PDA)

Potato Tubers 200g
Dextrose 20g
Agar agar 20g
Distilled water 1000m l
2. Beef Peptone Agar
Beef Extract 3g
Peptone 10g
Agar agar 15g
Distilled water 1000m l
3. Czapek (Dox) Agar
Sodium nitrate 2g
Potassium phosphate 1g

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Potassium chloride 0.5g
Magnesium sulphate 0.5g
Ferrous sulphate 0.01g
Sucrose 30g
Agar agar 15g
Distilled water 1000m l
4. Nutrient Glucose Agar (NGA)
Beef Extract 3g
Peptone 5g
Glucose 2.5g
Agar agar 15g
Distilled water 1000m l
5. King’s Medium
Proteose peptone no. 3 (Difco) 20g
K2HPO4 1.5g
MgSO4.7H2O 1.5g
Agar agar 15g
Glycerol 15ml
Distilled water 1000m l
Media preparation:
Potato Dextrose Agar Media-
Materials: Peeled potatoes- 200g, Dextrose- 20 g, Agar- 20 g, Distilled water- 1L, beaker-
1L, 250 ml conical flasks, knife, muslin cloth, measuring cylinder, non-absorbent cotton,
pressure cooker.
Procedure-
i) Take 500 ml of distilled water in 1L beaker and add 200g of peeled and sliced potato
boil the potatoes till they become soft.
ii) Filter the contents of the beaker through muslin cloth and squeeze out all liquid
iii) Add the dextrose dissolved in water to this extract.
iv) Adjust the pH of medium to 6 to 6.5 using 0.1 N HCl or 0.1N NaOH as the ease maybe
v) Add the dissolved agar to dextrose potato extract and make the volume to 1lt and dispense
200ml each to 5 conical flask and plug with non-absorbent cotton. Sterilize the flasks at 15
Ibs pressure for 15 min in a pressure cook. Allow the pressure cooker to cool, "Remove the

LMPTH213 Page 17
conical flask and store at room temperature. Allow the flask to cool until the flask can be
held by hand.
vi) Prepare agar plate by pouring the media into Petridish quickly. Using aseptic
condition, allow the media in Petridish to solidify to produce the agar plate.
Preparation of Nutrient Agar Medium: Used for the maintenance and isolation of bacteria.
Material:
Peptone -5g, beef extract -3g, Agar -20g, distilled water-1lt, Petri-dish, 1lt beaker, 250 ml,
conical flasks, measuring cylinder, non-absorbent cotton, pressure cooker and hot plate.
Procedure
i) Dissolve the weighed amounts of peptone and beef extract into 500 ml of water.
ii) Heat and dissolve the chemicals and adjust the pH of medium to 7 by adding 0.1N
HCl or 0.IN NaOH.
iii) Weigh 20g agar and dissolve in 500 ml of distilled water in another beaker
iv) Mix the dissolved agar with chemical solution and make up the vol. to 1lt.
v) Dispense 200 ml each into 5 conical flasks.
vi) Plug the flask with non-absorbent cotton and sterilize at 15 Ibs pressure for 15 min in
a Pressure cooker.
vii) Allow the cooker to cool, remove the conical flask and store at room temperature.
viii) Allow the flask to sufficiently cool and prepare agar plates by pouring media
into Petri-dish under aseptic condition; allow the media with Petri-dish to solidify.
5. Scope of the results: To learn about lab equipment is very essential as all the laboratory is
based on them. Media preparation should be according to the mentioned procedure. It is
required in almost all the experiments.
6. Results required:
Preparation of media for the growth of different fungal and bacterial culture and its
identification.
7. Cautions:
i) Don’t pour the media over 2/3 of flask capacity.
ii) Cotton plug must be loose while autoclaving.
iii) Don't pour media to Petri-plate when the medium is too hot since it produce condensation of
water on underside of Petri plate lid and thus can fall on to agar surface and may lead to
iv) Pour medium quickly to avoid contamination by air-pores and close lid down as soon
as possible.

LMPTH213 Page 18
v) Perform the pouring of medium in inoculation chamber fitted with U. V. lamp with
filtered air.
vi) Pouring should be performed near the flame.

Result & discussion:

Learning outcome

LMPTH213 Page 20
Experiment 4

1. Experiment- To study Koch’s postulates for the isolation of plant pathogens.

Equipment and Apparatus Required: Autoclave, BOD incubator, Laminar flow and hot
air oven
Materials Required: Sterile Petri-dishes, PDA slants, sodium hypochlorite solution (1%),
sterile water, razor blade, forceps, inoculation needle, burner/spirit lamp, spirit, incubator,
infected plant material, susceptible healthy plants.
2. Learning Objectives: To study about the concept of Koch’s postulate and culturing
different plant pathogenic and other micro-organisms.
3. Theory/Principle/Background of the topic:
How would you prove that a particular organism was the cause of a plant disease? How could
you be sure you had found the right microorganism and not just confused it with another of
the millions of microorganisms that occur on a plant? This problem challenged scientists for
decades and it eventually led to "Koch's Postulates" as the accepted scientific method for
identifying the causal agent of a plant disease.
4. Outline procedure of Koch's Postulates
Three rules for experimental proof of the pathogenicity of an organism were presented in
1883 by the German bacteriologist, Robert Koch; a fourth was appended by E. F. Smith
(1905). Briefly, these rules state:
1. The suspected causal organism must be constantly associated with the disease.
2. The suspected causal organism must be isolated from an infected plant and grown
in pure culture.
3. When a healthy susceptible host is inoculated with the pathogen from pure
culture, symptoms of the original disease must develop.
4. The same pathogen must be re-isolated from plants infected under experimental
conditions. These rules of proof are often referred to as Koch's Postulates.
An example of the use of Koch's Postulates to study a disease of wheat leaves:
Steps to prove that the organism isolated from infected plant tissue caused the
original infection.
Isolation of pathogen from infected tissue.
1. Cut between 5 and 10 sections (1 cm) of infected leaf tissue
2. Surface-sterilize (SS) leaf tissue
3. Place SS leaf tissue in humidity chamber in plastic bag for 5 days

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4. Examine SS leaf tissue for fungal spores
5. Place a few spores in the centre of a petri dish containing agar medium
6. Allow to incubate at room temperature for 5 to 7 days
7. Examine culture under microscope to check that they are the spores of the pathogen
Inoculation of a susceptible host
1. Grow healthy wheat plants in soil for 3 weeks
2. Make inoculum with spores collected from pathogen on agar plate
3. Spray spore suspension on to leaves of wheat plants until run-off
4. Place large, clear plastic bag over plants to retain high humidity for 24 hours, and
place in the dark.
5. Remove plastic bag and leave at room temperature for 7 days in normal light/dark
conditions.
Re-isolation of pathogen from wheat plants
Follow steps 1 to 4 in isolation of the pathogen above.
Isolation of the fungal pathogens from diseased material
i) Isolation of the fungal pathogens from diseased material is made by surface sterilizing the
diseased area with surface sterilizing agents, removing a small portion of the infected tissue
(leaves, stems, fruits etc.) with a sterile scalpel.
ii) Plating it in a plate containing a nutrient medium. The most common method, for
isolating fungal pathogens from infected leaves as well as other plant parts involves cutting
several small sections 5-10 mm-square from the margin of the infected lesion to contain both
diseased and healthy looking tissue.
iii) These are placed in surface sterilizing agents solutions for about 15-30 seconds the
sections are taken out aseptically and blotted dry on clean, sterile paper towels or washed in
three changes of sterile water and are finally placed on the nutrient medium, usually three to
five petrdish.
iv) The pathogen will grow from the sections and the colonies of the pathogen are
sub cultured aseptically for further study
5. Scope of the results: To learn about isolation of plant pathogens, it is required in almost
all the infected plants for the confirmation of involvement of pathogens in disease
development.
6. Results required:
Application of Koch’s postulates for the confirmation of pathogenic activity of different
fungal, bacterial and viral pathogens.

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7. Cautions:
i) Pour medium quickly to avoid contamination by air-pores and close lid down as soon as
possible
ii). Perform the pouring of medium in inoculation chamber fitted with UV lamp with filtered
air.
iii). Pouring should be performed near the flame.

Suggested books for reading: Manual on diagnostic techniques in plant pathology By,
Bahadur, P., Singh, R.H., Jain, R.K., Aggarwal, R. and Jayaraman, J. 2000.
Web links
http://phytopath.ca/wp-content/uploads/2014/09/What-are-Koch.pdf

LMPTH213 Page 23
Worksheet of the student
Date of Performance Registration Number:
Aim: To isolate various plant pathogenic micro-organism through Koch Pastulates.
Sr. Name of crop Symptoms Pathogen
No.

Result & discussion:

Learning outcome

LMPTH213 Page 24
Experiment 5

1. Experiment- To study morphological structures of different fungal

genera Equipment and Apparatus Required: Light Microscope
Materials Required: Fresh and pure culture of fungi, infected plant material,
razor blade, forceps, slide, spirit, lactophenol cotton blue or aniline blue.

2. Learning Objectives: To study about the different structures of fungi with

example of some representative fungal genera.

3.Theory/Principle/Background of the topic:

The main body of most fungi is made up of fine, branching, usually colourless threads called
hyphae. Each fungus will have vast numbers of these hyphae, all intertwining to make up a
tangled web called the mycelium. The mycelium is generally too fine to be seen by the naked
eye, except where the hyphae are very closely packed together. But identification of different
fungi is not possible through their mycelium. Therefore identifying their fruiting bodies &
spores act as a tool of fungal identification. Hence, learning fungal morphology structures is
very important.
4. Outline of the procedure: This is a rapid and simple method for preparing a temporary
microscopic mount of a fungus without disturbing the arrangement of conidia and conidia
bearing hyphae, the conidiophores. Procedure as follows-
(a) Take a clean slide and place a drop of lactophenol cotton blue or aniline blue
in the centre of the slide.
(b) Hold the tape with sticky slide down, between the thumb and forefinger of each hand.
(c) Press firmly the centre of the sticky side of the tape onto the surface of the
fungus colony, where sporulation is visible.

(d) Pull away gently the tape from the colony and place it on the drop of lactophenol
cotton blue.

(e) Fold over the extended ends of the tape over the ends of the slide.

Some important fungal structure are as follows-Rhizopus-White to dark gray, non septate
mycelium with root like rhizoids;black columellate, sporangiophores in clusters.

Mucor- White to dark gray, non septate mycelium without rhizoids; singly columellate
sporangiophores.
Aspergillus- Greenish-blue, black or greencolonies; conidiophores arising from a foot-cell,
basipetal conidia on phialides on vesicle.
Penicillium: Greenish or blue green colonies; conidia in long chains on repeatedly branched
conidiophores resembling a brush like head.

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Fusarium: Woolly, white to pink; Sickle-shaped transversely septate macroconidia
produced in sporodochia.
Alternaria: Greyish green or black; transversely and longitudinally
septate (muriform), beaked conidia, in acropetal manner.

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5. Scope of the Result: The student will observe the morphological structure
of different micro organism and their distinguishing characteristic.
6. Result Required: Examine the slide under low power and high-power objectives of
the microscope for the fungus structure that aid the identification of culture.
7. Caution: Entry of air bubble should be avoided when cover slp is placed onto the
drop of lactophenol cotton blue.

Date of Performance Registration Number:

Aim: To study morphological structures of different fungal genera.

Sr. Name of Pathogen External Symptoms Internal Symptoms

No.

Result & discussion:

Learning Outcome:

To be filled in by Faculty:
Sr. No. Parameter Marks Obtained Max Marks
1. Understanding of the student about 20
the disease symptoms
2. Observations and analysis 20
including learning outcome
3. Completion of experiment 10
Discipline and cleanliness
Signature of Faculty

LMPTH213 Page 28
Experiment 6

1. Experiment- To study staining and identification of plant pathogenic bacteria.

Materials Required: Glass Slides, Iodine blue, crystal violet, Alcohol,
Safranin, Microscopes, stains, fresh bacterial culture

2. Learning Objectives: To learn the staining technique and identification

of plant pathogenic bacteria

3. Theory/Principle/Background of the topic: Bacteria have nearly the same refractive

index as water, therefore, when they are observed under a microscope they are opaque or
nearly invisible to the naked eye. Different types of staining methods are used to make the
cells and their internal structures more visible under the light microscope. Gram stain is
probably one of the most commonly used staining procedures used in the field of
microbiology. It is one of the differential stains that are used to characterize bacteria in one of
two groups: either gram positive bacteria or gram negative bacteria. Gram positive bacteria
will typically have a stronger affinity for crystal violet on applying gram's iodine than the
gram negative cell wall. Being a mordant, gram's iodine forms a complex with crystal violet
in the stain that has attached more tightly to the cell wall of gram positive bacteria than that
of the gram negative bacteria. Whereas the gram positive bacteria stain violet as a result of
the presence of a thick peptidoglycan layer in the walls of their cell, the gram negative
bacteria stain red, due to the thinner peptidoglycan layer in their cell wall (a thicker
peptidoglycan layer allows for the retention of the stain, but a thinner layer does not).

4. Outlines of Procedure:

1. Obtain a clean slide

2. Using aseptic technique, smear a loopful of bacterial sample from a pure culture
onto the center of the slide
3. Smear the sample in a circle on the slide, staying within the center 1/3 of the slide.
Particularly with the moistened sample from the agar slant, care must be taken to see
that the bacteria from the colony are separated from each other so they may be
observed Individually
4. Mark an “X” with a grease pencil to show the side of the slide with the bacteria.
5. The oil immersion lens will not focus of the bacterial cells if you have placed the slide
on the stage smear side down.

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6. Let the preparation on the slide dry on the slide dryers at top the Bacti-Cinerator. Then
hold the slide (smear-side away from the heat) near the mouth of the Bacti-Cinerator
for a few seconds, passing the slide in front of the chamber in sweeping motions to
avoid overheating
7. Let the slide cool
8. Put 2-3 drops of a stain on the smear and let it sit for 2 minutes. Use crystal violet (1
minute) or methylene blue (1 minute) or safranin (1-2 minutes). Rinse gently with tap
or distilled water.
9. Blot between sheets of bibulous paper. Place on microscope and observe using
all powers of your microscope, including oil immersion.

5. Scope of the results: To learn about the difference between gram negative and
gram positive bacteria.

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6. Result Required: Gram-negative bacteria will stain pink/red and Gram-
positive bacteria will stain blue/purple.
7. Caution: Handle the sample carefully to avoid any damage to its body parts during
staining of bacteria. A greater length of time for heat should be avoided the slide to
the breaking point.

Note-If you are struggling to remember the staining reagents used in this procedure
and their order you can remember this sentence “Come In And Stain” i.e. the order
is Crystal violet, Iodine, Alcohol/Acetone and the final one is Safranin.

Suggested readings for students: Bacterial Classification, Structure and Function By:
Frank Lowy

Web Links:

http://www.microscopemaster.com/gram-stain.html

https://homepages.wmich.edu/~rossbach/bios312/LabProcedures/Gram%20Stain%20Procedu
re.html

http://microbeonline.com/gram-staining-principle-procedure-results/

LMPTH213 Page 31
Worksheet of the student

Date of Performance Registration Number:

Aim: Staining and identification of plant pathogenic bacteria

Sr. Name of bacteria Symptoms Type of bacteria

No.

Result & discussion:

Learning Outcome:

To be filled in by Faculty:
Sr. No. Parameter Marks Obtained Max Marks
1. Understanding of the student about 20
the disease symptoms
2. Observations and analysis 20
including learning outcome
3. Completion of experiment 10
Discipline and cleanliness
Signature of Faculty

LMPTH213 Page 32
Experiment 7

1. Experiment- To study transmission of plant viruses

Equipment and Apparatus Required: Plant samples, petridish, foreceps
Materials Required: Record book, hand lance

2. Learning Objectives: Students will learn about the symptoms and types of
plant virus transmission
3. Theory/Principle/Background of the topic:
Viruses are distributed or transmitted from the infected plants to the healthy ones in various
ways in nature. Horizontal Transmission is by vectors, human pruning shears and tools, and
other direct, external contamination. Vertical Transmission occurs when a plant gets it from
its parent plant either through asexual propagation (cuttings) or in sexual reproduction via
infected seeds. As the plant viruses can not penetrate cuticle of their hosts and hence they can
enter into the host tissue through wounds only. The means of transmission are:
1. Mechanical transmission: The sap of the infected plant is manually transferred to the
healthy plants. It is the easiest method of experimental inoculation.
2. Graft transmission: In this practice, if either the scion (shoot portion) or stock (root
stock) is infected, the virus usually moves to the healthy partner which may later express
visible symptoms of disease.
3. Transmission through vectors
(i) Insects: Some insect species are the vector of plant viruses which can carry/ transmit
viruses from infected plants to the healthy plants e.g. aphid (potato virus Y, PLVR), white
flies (tobacco leaf curl), beetles (cowpea mosaic virus), mealy bugs (cacao mottle leaf),
thrips (tomato spotted wilt), lace bugs (sugar beet viruses), mites (sterility mosaic of arhar),
leaf hoppers (beet curly top, rice tungro etc.), plant-hoppers (maize mosaic, maize rough
dwarf), tree hopper (tomato pseudo curly top).
(ii) Nematodes: Five genera of nematodues viz., Xiphinema, Longidorus, Paralongidorus,
Trichodorus and Paratrichodorus can transmit plant viruses.
Fungi: Some species of fungi can also transmit viruses e.g. Olpiduim brassicae (tobacco
necrosis), O. cucurbitacearum (cucumber necrosis), Polymyxa graminis (oat mosaic, wheat
mosaic), P. betae (beet necrotic yellow vein) and Spongospora subterranea (potato mop top)
etc.

4. Dodder transmission: Many viruses can be transmitted through dodder (Cuscuta spp.).

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Dodder transmission is used in the laboratory to transfer viruses from the hosts.
5. Transmission through seeds and pollens: Seed coat (testa), embryo, and also pollens
of some plants can transmit viruses. e.g. alfalfa mosaic, barley stripe mosaic, bean common
mosaic, lettuce mosaic are transmitted by both seeds and pollens of Medicago sativa,
Hordeum vulgare, Phaseolus vulgaris and Lactuca sativa, respectively.

4. Outlines of Procedure:
1. Collect the disease sample
2. Observe under the microscope
5. Scope of Result: Student will learn about transmission of plant viruses.
6. Cautions: Sample should be fresh and handle it with carefully

Date of performance Registration no

Aim: To Study about the symptoms and method of transmission of plant viruses.
Observation Table:
Sr. Name of plant virus Mode of transmission and symptoms
No.

Result and Discuss.

Learning Outcomes:

LMPTH213 Page 35
Experiment 8

1. Experiment- To study of phanerogamic plant parasites.

Equipment and Apparatus Required: Microscope, foreceps.
Materials Required: Plant samples, petridish, foreceps

2. Learning Objectives: Student will learn about the Identifying

characters, reproduction and life cycle of phanerogamic plants.
3. Theory/Principle/Background of the topic:
Some flower and seed bearing higher plants (phanerogams) live parasitically on other living
plants and can cause important diseases on agricultural crops and also in forest trees. They
are classified in the following botanical families and genera,
1. Cuscutaceae (stem parasite): Genus: Cuscuta, the dodders
2. Viscaceae (stem parasites): Genus: Arceuthobium, the dwarf mistletoes of conifers
Genus: Phoradendron, the American true mistletoes of broad leaved
trees Genus: Viscum, the European tree mistletoes
Genus: Dendrophthoe, the giant mistletoes
3. Orobanchaceae (root parasite): Genus: Orobanche, the broomrapes
4. Scrophulariaceae (root parasite): Genus: Striga, the witchweeds
4. Identifying characters, reproduction and life cycle
1. Dodder (Love-vine, Amarbel): Dodder is slender, twining plant. The stem is tough,
succulent, threadlike, curling, leafless and bearing minute scales in place of leaves. Stem
is usually yellowish or orange in colour. They grow and climb in abundance on the wild
and cultivated plants. Their haustoria penetrate the stem or leave of the host plant and
absorb foodstuffs and water. Growth of the infected plants is suppressed and finally dies.
Tiny whitish flowers arise in clusters from the stem and they produce numerous grey to
reddish-brown seeds within few weeks of bloom. The seeds fall on the ground where they
either germinate immediately or remain dormant until next season. The seeds may be
spread to new areas by animals, water, equipments and by mixing with crop seeds.
2. Giant mistletoe (Loranthus) : Mistletoes are semi-parasites of thee-trunks and branches.
They have green leaves and many branches and hence grown like a small bush on the
host. They do not have true roots and hence develop haustoria to draw nutrients from the
vascular system of the host plant. Flowers are long, tubular, greenish white to red in
colour and bornein clusters. Seed is fleshy, contains one seed, sweet in taste and usually
eaten by birds and animals. The parasite is spread mostly through birds. Droppings of bird

LMPTH213 Page 36
containing seeds get deposited on the other trees, mainly at the junction of a branch and
main trunk. These seeds germinate, develop haustoria and established on the host plant.

3. Broomrape: Broomrape is a complete root parasite. It affects tobacco mostly and many
other Solanaceous and Cruciferous plants. The parasite has a stout, fleshy stem of 10-15
cm long. The stem is pale yellow or brownish red in colour and is covered by small, thin,
browny, scaly leaves. Flowers are white and tubular and appear in the axil of leaves.
Seeds are very small, black in colour and can remain viable in soil for several years. Roots
are haustoria-like, can penetrate the roots of the host for nutrition. The affected plants
become stunted and may die.
4. Witchweed: It is a semi root parasite but obligate in nature. Commonly affected plants by
this parasitic plant are sugarcane, cereals, maize and millets. Striga is a small plant with
bright green, slightly hairy stem and leaves. The weed grows 15-60 cm high in clusters.
Leaves are long and narrow in opposite pairs. Flowers are small, brick red or yellowish or
almost white with yellow centers, appear throughout the season. Tiny brown seeds are
produced in each capsule and thus a single plant can produce 50,000-500,000 seeds. The
seeds can remain viable for 12-40 years. Roots are watery white in colour without root
hairs and hence obtain nutrients by haustoria from the host plant. The life cycle from seed
germination to first seed setting on the developed plant needs 90-120 days.

4. Outlines of Procedure:
a. Sample is to be collected and brought into the laboratory.
b. By visual observation or under microscopic observation samples is to be
observed.
5. Scope of the result: Student will learn about higher plant parasities

Suggested readings for students: Introductory Plant Pathology, By. Dr. D.V. Singh,
Ex-Head and Emeritus Scientist, Division of Plant Pathology, Indian Agricultural
Research Institute, New Delhi-110012, (9-07- 2007)

Weblinks: http://nsdl.niscair.res.in/jspui/bitstream/123456789/658/1/Revised%20INTROD
UCTORY%20 PLANT%20PATH.pdf

LMPTH213 Page 37
Worksheet of the student

Date of Performance Registration Number:

Aim: To Study of phanerogamic plant parasites

Sr No. Name of phanerogamic plant parasites Symptoms

Result & discussion:

Learning outcome

LMPTH213 Page 38
Experiment-9

1. Experiment- To Study morphological features and identification of plant parasitic

nematodes.
Equipment and Apparatus required: Stereoscopic binocular microscope, compound
microscope
Materials Required: Record book, watch glass, nematode suspension and glass
slide, picking needle

2. Learning Objectives: Student will able to learn about the morphological characteristics of
the nematodes for identification of different type of nematode species
3. Theory/Principle/Background of the topic:
Nematodes are diverse metazoans with an estimated total number of a million species. They
are arguably the most numerous metazoans in soil and aquatic sediments. Nematodes are
generally free-living in marine, freshwater or soil environments, but a large number of
species are parasitic on different kinds of plants and animals. The parasitic species are of
considerable agricultural, clinical and veterinary importance as pests of plants and parasites
of man and livestock respectively. Plant nematodes are tiny worms usually 0.25 mm to 3 mm
long (1 / 100 " to 1 / 8 ") and cylindrical, tapering toward the head and tail. Females of a few
species lose their worm shape as they mature, becoming pear-, lemon- or kidney- shaped.
Plant parasitic nematodes possess all of the major organ systems of higher animals except
respiratory and circulatory systems. The body is covered by a transparent cuticle, which bears
surface marks helpful for identifying nematode species.

4. General Characteristics of Nematode-

As viewed under a microscope, the cuticle or body covering of a typical plant-parasitic

nematode is transparent allowing the viewing of internal systems. The major organs visible
are various components of the digestive system and of the reproductive system. At the
anterior end, all plant parasitic nematodes have a stylet (also called a spear), which can be
protruded and used to penetrate plant cells like a hypodermic needle. Nematodes which feed
on fungi and other sources of food can also possess stylets, so the structure is not diagnostic
for plant parasites. The esophagus is the area between the stylet and the start of the intestine.
The esophagus of plant parasitic nematodes will be variations of one of two major types.
Shown here is a tylenchoid esophagus consisting of three areas: the anterior procorpus, mid
region metacorpus or median bulb, and posterior basal glands or basal bulb. The median bulb
facilitates transfer of digestive enzymes produced in the basal glands into plant cells and of
plant products into the intestine. A two part esophagus is typical of dorylaimoid species and

LMPTH213 Page 39
consists of an anterior cylindrical part and an expanded glandular posterior part. Most of the
body cavity or pseudocoelom is filled with intestine and with components of the reproductive
system. The vulva is the opening of the female reproductive tract. The tube or tubes leading
from the vulva have been divided by morphologists based on observed functionality into
uterus, ovary, oviduct, seminal vesicle, etc. There are both female and male nematodes,
although not all species require the presence of males for reproduction, and in some species
males are rare. The tail of males may possess a number of structures such as spicules,
gubernaculum, bursa or caudal alae which facilitate transfer of sperm into the vulva. The
nervous system consists primarily of a nerve ring surrounding the esophagus with nerves
extending from it to various locations. A number of neurosecretory structures whose
functions are not well understood can be seen on the cuticle (sometimes only by very good
microscopists), including amphids, phasmids, and deirids. Depending on the species, the
excretory system consists of a simple excretory cell or a more extensive system of excretory
canals. The excretory pore is the portion of the system most easily visible. Nematodes do not
possess respiratory organs or a circulatory system, relying instead on diffusion through the
cuticle. Variations in morphological characteristics have been extensively utilized to
construct classification schemes for nematodes. These include variations in body shape,
stylets, esophagi, location of the vulva and numbers of ovaries, head shape and lip
configuration, amphids and phasmids, tail shapes, spicules, cuticlular ornamentation, etc.

Fig: Plant parasitic nematode stylet

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Fig: Gross morphology of a plant parasitic nematode

LMPTH213 Page 41
Fig: Male reproductive system: A- complete genital tract, B- leptoderan bursa,
C- peloderan bursa

Fig: Female reproductive system

LMPTH213 Page 42
5. Identification key for major plant-parasitic nematodes

Nematodes can be identified using several methods, including light microscopy, fatty acid
analysis, and PCR analysis. More specific methods (esterase and malate dehydrogenase
staining, host differentials, morphological studies, and fatty acid analysis) are used for
identifying Meloidogyne species and races.

Meloidogyne identification
It is commonly encountered in warmer regions and is seldom found in areas where average
monthly temperatures approach freezing. M. arenaria is the most morphologically and
cytologically variable of the Meloidogyne species. Two host races have been differentiated ,
one that reproduces on peanut (race 1), and race 2 which cannot. M. arenaria is a mitotically
parthenogenetic species with chromosome numbers that range from 30-50. Cuticle not
abnormally thick, annulated in all stages of the male and female.
Female. Sedentary, globose with projecting neck. No pre adult vermiform female stage.
Cuticle moderately thick; annulation forming finger-print like pattern around vulva and anus.
Labial disc dumb-bell shaped, not detached from labial sectors. Cephalic framework and
spear delicate. Excretory pore anterior to median oesophageal bulb, often only slightly
posterior to stylet base. Vulva and anus terminal. No cyst stage. Eggs not retained in body but
deposited in a gelatinous matrix (exception Meloidogyne spartinae where eggs ate deposited
individually without gelatinous matrix). Male. Labial area low, not set-off, irregularly
annulated. Lateral field with four lines.

Fig: Meloidogyne sp.; A. female, B. female anterior region, C. male, D. male tail, E.
second stage juvenile
Anguina:
Procorpus generally separated from the median bulb by a constriction. Median bulb with
or without valves; isthmus generally separated from the glandular bulb by a constriction;

LMPTH213 Page 43
esophageal glands enlarged, generally overlapping intestine. Oocytes in many rows;
columned uterus a long multinucleate tube. Mature females swollen. Form galls on above
ground parts of higher plants, generally grasses.

Radopholus: Secondary sexual dimorphism strongly marked. Two female genital branches
equally developed or, more rarely, posterior branch more or less reduced, degenerated and
nonfunctional. Female lip area low not offset. Oesophageal glands on line, with long dorsal
overlap of the intestine. Oesophago-intestinal valve not well developed. Tail long, tapering to
ternimus rounded or almost pointed; phasmids at mid-tail, or slightly anterior. Male lips high,
rounded, set-off; cephlic sclerotization weak; stylet reduced; basal knobs vestigial or absent;
oesophagus reduced. Gubernaculum slightly protruding from cloaca. Caudal alae sub-
terminal or, more rarely, terminal. Sperms more often rodlike.

Tylenchorhynchus: Lip region set off by constriction or continuous with contour. Phasmids
conspicuous, located well behind anal region. Spear usually strong with heavy basal knobs.
Basal bulb of esophagus connected to intestine by cardia. Vulva near middle of body. Ovaries
two, outstretched. Female tail conoid, blunt, usually 2 or more times anal body diameter.

Tylenchus
Small nemas rarely over 1.0 mm long. Tails of both sexes similar, elongate conoid to
filiform. Spear knobbed. Median bulb with valvular apparatus. Cardia present. Vulva well
posterior to middle of body. Anterioovary outstretched. Posterior uterine branch rudimentary.
Spermatheca a definite pouch in uterine branch. Bursa adanal, Phasmids not visible.

Xiphinema
Spear greatly attenuated with extensions bearing elongate, basal flanges. Spear guiding ring
deep in the pharynx. Basal part of esophagus 2-4 times as long as body width. Vulva
transverse with muscular labia. Ovaries 1 or 2. Tails hemispheroid to elongate cylindroid.
Males often unknown or rare.

6. Scope of Result: To learn about the morphological feature of important plant

parasitic nematode.
7. Caution: Morphological structure of different nematode should be clear
for proper identification.

Textbook on Introductory Plant Nematology by Raman K. Waliya and Harish K. Bajaj

Web links:
https://smartsite.ucdavis.edu/access/content/user/00002950/courses/slides/fromWWW/morp/
AMPHIDS.GIF

http://nematode.unl.edu/key/nemakey.htm

LMPTH213 Page 44
Worksheet of the student

Date of Performance Registration Number:

Aim: To Study the major plant parasitic nematode.

Identify the nematode species and draw the well labelled figure of the nematode species

LMPTH213 Page 45
Result & discussion:

Learning outcome

LMPTH213 Page 46
Experiment 10

1. Experiment- To study sampling and extraction of nematodes from soil and plant material
and preparation of nematode mounting.
Equipment and Apparatus Required: Stereoscopic Binocular microscope
Materials Required: Soil auger or hand hoe (Khurpa), polythene bag, aluminium foil
label, rubber band, note book, Beakers, pans, Petri plates, 20-, 60-, 300-mesh sieves, soil
samples, facial tissue papers, aluminium wire nets, glass funnels, funnel stand, rubber
tube, glass vials, Infected plant material, scissors, Petri plates, wire gauge, facial tissue
paper, warring blender, acid fuchsin stain, lactophenol etc.

2. Learning Objectives: Student will able to learn about method of collection of soil samples
and extraction methods of nematode from soil as well as from plant tissue to know the
nematode population present in the locality and to find out the best management practices for
controlling nematode population.
3. Theory/Principle/Background of the topic:
When making a sampling plan for estimating nematode numbers, biomass and species
composition, a number of practical and theoretical considerations must be observed. The
design of the sampling plan will partly depend on the purpose of the study, the required
accuracy, the time frame, as well as the costs involved. Besides, when making the plan,
knowledge about the variation in space and time of nematode populations, their biology, and
the influence of (a) biotic factors has to be considered, to arrive at a correct choice regarding
timing and depth of sampling, tools, number of samples and the sampling pattern. Extraction
is the easiest ways to isolate nematodes from their host material is by submerging the sample
in water and select the nematodes under a microscope. However, this is a tedious and
laborious job and it can only be done with very small samples
4. Outlines of Procedure:
Sampling Process:
1. Sampling from annual field crops
Leave about 1m peripheral area of the field.

Remove 2-3 cm upper layer of the soil with the help of a hand hoe.

Collect 50-100 cc soil along with feeder roots upto a depth of 15-20 cm (subsample).
Draw 10-20 such subsamples from one hectare area in a zigzag manner.

LMPTH213 Page 47
Put all the subsamples in the same polythene bag (Composite sample).

Put an aluminium foil label bearing the sample number in the polythene bag and tie
it with a rubber band.

Write the details (see labeling) separately in a note book.

2. Sampling from vegetable crops

Select 6 rows of a field (2 from the beginning, 2 from the middle and 2 from the far
end of the field).

Collect 8-10 subsamples upto a depth of 20-30 cm from each pair of rows in a
zigzag manner.

Put all the subsamples in the same polythene bag and label it.
3. Sampling from a tree
Collect 5 subsamples each from around the main stem and drip line of the tree by the
method described above.

The depth of the sampling will vary with the kind and age of the tree.

Put all the subsamples in the same polythene bag and label it.
4. Sampling from an orchard
Take 2 subsamples from one tree upto a depth of 30 to 60 cm (feeder
root zone) depending upon the age of the tree.

Collect subsamples from 10 trees randomly from one hectare area (Fig.1D).

Put all the subsamples in a polythene bag and label it.

LMPTH213 Page 48
Sampling patterns

Fig: Patterns of sample collection: A-from field crops, B- from vegetable crops, C- from
single tree, D- from orchard

Extraction:
Extraction from plant material
1. Direct extraction
technique Procedure
Wash the infected plant material thoroughly and chop it into small pieces of 1-2 cm.

Put this material in a Petri plate containing water.

The migratory semi endo/endoparasitic nematodes come out of the chopped

material into water by 24 hours and can be seen directly under stereomicroscope.

Alternatively the chopped material can be processed by modified

Baermann’s technique.

2. Warring blender technique

Procedure

LMPTH213 Page 49
Wash the roots under tap water to remove adhering soil particles.

Chop the roots to 1-2 cm pieces and transfer them to a blender containing about
100 ml water.

Operate the blender for 15 seconds.

Take out this material from the blender and put it on the modified Baermann’s funnel
assembly as described earlier.

3. Acid fuchsin staining technique

This technique is mainly used for detecting sedentary semi-endo and endoparasitic
nematodes. The stained but dead nematodes can be dissected out of the roots under a
stereomicroscope.
Extraction from soil and other substrates
1. Cobb’s decanting and
sieving technique Principle
The soil particles and nematodes settle at different rates due to differences in
their specific gravity.

Different sized nematodes are retained on sieves of different pore sizes.

Procedure

Take out the composite sample in a pan, mix it thoroughly and take 250
cc for processing.

Transfer 250 cc soil to a pan and add about one litre water, mix well breaking clods
and clumps.

Wait for 10-20 seconds and pass this soil suspension through a 20-mesh
sieve (pore size 840 micro meter), collecting the filterate in another pan.

Collect root bits present on the 20-mesh sieve in a beaker and discard the
remaining material.

Stir the suspension of second pan gently, wait for a few seconds and pour it through a
60-mesh sieve (pore size 250 micro meter) in 3rd pan. Collect the residue left over
60-mesh sieve in a beaker and label it as 60.

LMPTH213 Page 50
Pass the contents of 3rd pan through a 300-mesh sieve (pore size 53 micro
meter). Discard the suspension passed through the sieve.

Collect the residue left over 300-mesh sieve in a beaker and label as 300.

Examine the contents of beakers labelled as 60 for cyst nematodes directly under a
stereomicroscope.

Further process 300-mesh residue by Baermann funnel technique.

Fig: Metallic pans and sieves used in Cobb’s decanting and sieving te chnique

2. Baermann Funnel
Technique Principle
The active and motile nematodes pass through the tissue paper and get collected at the base
of rubber tube/glass vial due to movement and gravitational force whereas inert soil
particles/debris remain on the tissue paper.
Procedure
Process the soil sample by Cobb’s decanting and sieving technique as above.

Fill the Baermann funnel assembly with water and press the rubber tube gently to
remove the air bubbles. Baermann funnel assembly has a glass funnel with a piece of

LMPTH213 Page 51
rubber tube (10-20 inch long) bearing a glass vial (5 ml capacity) attached to its distal
end.

Transfer the sieved nematodes suspension (labelled as 300) to a moulded piece of

wire not covered with a double layered tissue paper and place it over the funnel.

Add water till it touches the lower surface of the wire net.

After 24-48 hours, remove the glass vial and observe the nematodes under a
stereoscopic binocular microscope.

Advantages
▪
Clear nematode suspension, free of debris etc. is obtained.
▪
The nematodes are collected in a small amount of water.
Disadvantages
▪
It is time consuming.
▪
Nematodes may lose their activity/viability due to lack of oxygen.
▪
Sedentary and slow moving nematodes cannot be extracted.

5. Result: Different types of nematodes will be identified.

6. Scope of the result: Student learn about the nematode extraction methods
7. Caution: Instrument should be washed properly. Generally sample of soil should
be collected from the rhizosphere region.

Suggested readings for students :Methods and techniques for nematology By. J.
Van Bezooijen Revised version 2006

Barker, K.R., Campbell, C.L. (1981). Sampling nematode populations. In: Plant
Parasitic Nematodes, Vol. III (B.M. Zuckerman, R.A. Rohde, eds.), pp. 451-474.
Academic Press, New York.

Web links: https://www.wageningenur.nl/upload_mm/4/e/3/f9618ac5-ac20-41e6-9cf1-

c556b15b9fa7_MethodsandTechniquesforNematology.pdf

LMPTH213 Page 52
Worksheet of the student

Date of Performance Registration Number:

Aim: To Study the collection of soil samples and extraction of plant parasitic nematodes

Extract the nematode from soil, root and draw the fig of typical plant parasitic nematodes
male and female

LMPTH213 Page 53
Result & discussion:

Learning outcome

LMPTH213 Page 54
Experiment 11

1. Experiment: To study fungicidal formulations and their calculation.

Equipments: Knapsack sprayer

Materials Required: fungicide, measuring cylinder, beaker, water, rod and Fogging
machine, hand glove
2. Learning objective: Student will learn about the different types of formulations and
calculation of insecticidal dosages for effective control of pest with less environmental effect.
3. Theory/Principle/Background of the topic: A fungicide formulation is a mixture of
chemicals which effectively controls a pathogen. Formulating a pesticide involves processing
it to improve its storage, handling, safety, application, or effectiveness. Fungicides are first-
discovered chemical compounds used by men to protect the plants from infections.
Fungicides are first-discovered chemical compounds used by men to protect the plants from
infections. Compounds used to protect the plant from causes of infections are divided into
fungistatic (inhibit the growth of the fungi) and fungicidal (kill the fungi). Based on the mode
of action of the pathogen fungicides can be preventive (protective). Fungicides can also be
contact – do not move to all parts of the plant, systemic – move through the plant upwards
and downwards and local-systemic – protect only the part where the spray is deposited,
mostly in the leaf so are therefore also called trans laminar. Fungicides can be used for seed
treatment, for fungal infection in soil (disinfection) and most widely spread use is for
treatment of upper parts of the plant (foliar use).
4. Pesticide formulations: Formulations are classified as solids or liquids on the basis of
their physical state in the container at the time of purchase. Solid formulation: Solid
formulations can be divided into two types: ready-to-use; and concentrates, which must be
mixed with water to be applied as a spray. Three of the solid formulations (dusts, granules,
and pellets) are ready-to-use, and three (wettable powders, dry flowables, and soluble
powders) are intended to be mixed with water. Liquid Formulations: Descriptions of four
common liquid formulations that are mixed with a carrier follow. The carrier generally will
be water, but in some instances labels may permit the use of crop oil, or some other light fuel
oil as a carrier. E.g. liquid flowables, emulsifiable concentrates, solutions etc. The various
types of formulations are-
Aerosols are liquids that contain the active ingredient in solution, packaged in a pressurized
container. “Bug bombs” contain a small amount of active ingredient mixed with a propellant

LMPTH213 Page 55
that forces the contents from thecan in a spray or mist. Never attempt to puncture or burn
aerosol cans because they may explode and produce shrapnel.

Baits (B) are composed of an edible substance or some other attractant mixed with a
poisonous active ingredient. The Bait either attracts pests or is placed in a location where the
pest animal will find it. The Pest must eat the bait to be killed. A major advantage is that baits
can be placed exactly where and only when needed, and can be removed after use.

Dusts (D) are ready to use as purchased without additional mixing. They Contain an active
ingredient plus a finely ground, inert substance such as talc, clay, nut hulls, or volcanic ash.
They are relatively expensive for the amount of active ingredient in the total formulation;
there are often problems with drift; they may be more irritating to the applicator than sprays;
often little active material reaches the target host and rain and wind easily remove dust
formulations from treated surfaces.

Emulsion: An emulsion is a mixture that occurs when one liquid is dispersed (as droplets) in
another liquid. Each liquid will retain its original identity and some degree of agitation
generally is required to keep the emulsion from separating.

Flowables (F) consist of finely ground solid particles suspended in a liquid carrier. The solid
in a flowable is similar to the active ingredient in a wettable powder, except that the solid is
formulated to stay in suspension in the liquid.

Fumigants (LG) are poisonous gases. Many fumigants are formulated as liquids under
pressure and become gases when released. Fumigants kill insects, weed seeds, nematodes,
rodents, fungi and other pests.

Granules and pellets (G) are dry, ready ‐to‐ Use materials normally containing from 2 to 15
Percent active ingredient. Since the particles are relatively heavy, granules do not normally
present a drift hazard and thus are safer to apply than most other formulations. Most are
prepared by applying the active ingredient as a liquid to a coarse, porous, solid material such
as clay or ground corn cobs. Granules are applied either directly to the soil, water or over
plants.

Sorption: In some cases it may be necessary or desirable to adhere a liquid active ingredient
onto a solid surface (e.g., a powder, dust, or granule). This process is called sorption.

LMPTH213 Page 56
Solution: A solution results when a substance (the solute) is dissolved in a liquid (the
solvent). The solute can be a solid or a liquid. The components of a true solution cannot be
mechanically separated. Once mixed, a true solution does not require agitation to keep its
various parts from settling.
Suspension: A suspension is a mixture of finely divided, solid particles dispersed in a liquid.
The solid particles do not dissolve in the liquid, and the mixture must be agitated to maintain
thorough distribution.
Water Dispersible granules are dry, granular materials designed to be mixed with water.
Upon contact with water, the granules disperse or break apart. The Resulting preparation has
all the characteristics of a flowable formulation or a finely dispersed wettable powder. The
Granules are easy to handle and are nearly dust free, which reduces their respiratory hazard.
Wettable Powders (WP) and soluble powders (SP) are dry, powdered formulations usually
containing from 25 To 80 Percent active ingredient. Wettable Powders are mixed with water
to produce suspensions, whereas soluble powders dissolve in water to form solutions. A
wetting agent is often added to keep suspended particles of wettable powders uniformly
dispersed. As a rule, wettable powders are safer to use on foliage and usually are not absorbed
through the skin as quickly as liquid formulations. They are generally easy to handle,
transport, store and mix and are relatively reasonable in cost.
5. Outlines of Procedure:
Fungicide calculations for recommended dose:
Formula: C1VI = C2V2
Where C1 = Concentration of commercial formulations in per cent or grams
V1 = Volume or amount of commercial formulation required in millilitre or grams
C2 = Desired concentration of spray fluid in per cent
V2 = Volume or amount of spray fluid required
Example:How much quantity of for the Carbendazim 50%SC control of plant disease and the
spray fluid recommended for spraying is 150 litres/ha.
Solution
Given C1 = 20%, V1 =?, C2 = 0.05%, V2 = 150 litres (1,50,000 ml)
Fromula- C1V1= C2V2
Given C1 = 20%, V1 =?, C2 = 0.05%, V2 = 150 litres (=150,000 ml)

6. Results: The calculated dose of fungicide will be generated by using the formulae.

LMPTH213 Page 57
7. Scope of Result: The accurate application of fungicide on the target pathogen.
8. Caution: Always wear the protecting clothes while spraying in the fields. Wash the
hands and clothes after spraying. Chemical formulations always keep away from
the reach of children

Date of Performance Registration Number:

Aim: To Study the pesticides and their formulation.
Sr. No. Name of pecticides Formulation/Recommended Dosage

Result & discussion:

Learning outcome

LMPTH213 Page 59
Experiment 12
1. Experiment- To study methods of pesticide application and their
safe handling. Equipment: Knapsack sprayer
Material Required: Fungicide, measuring cylinder
2. Learning Objective: The objective of the application of pesticide is to keep the pest under
check. The pest population has to be kept suppressed to minimum biological activities to
avoid economic loss of crop yields. The objective of pesticide application besides keeping the
pest population under check should also be to avoid pollution and damage to the non targets.

3. Theory/Principle/Background of the topic: Pesticides are toxic to both pests and

humans. Most pesticides will cause adverse effects if intentionally or accidentally ingested or
if they are in contact with the skin for a long time. Pesticide particles may be inhaled with the
air while they are being sprayed. An additional risk is the contamination of drinking-water,
food or soil. Special precautions must be taken during transport, storage and handling. Spray
equipment should be regularly cleaned and maintained to prevent leaks. People who work
with pesticides should receive proper training in their safe use.

4. Pesticides application procedure: To make an effective, safe, and efficient application,

read the label first. In addition, the equipment must be properly selected, operated, calibrated,
and maintained. To get the best result using the least amount of pesticide applicators need to
think through the application process carefully. Different application methods are appropriate
for different crop and pest types, but the method of application should always be consistent
with the label directions. Application methods include:

Band application: applying a pesticide in parallel strips or bands, such as between rows
of crops rather than uniformly over the entire field.
Basal application: directs herbicides to the lower portions of brush or small trees to control
vegetation.
Broadcast application: is the uniform application of a pesticide to an entire area or field.
Crack and crevice application: is the placement of small amounts of pesticide into
cracks and crevices in buildings, such as along baseboards and in cabinets, where insects
or other pests commonly hide or enter a structure.
Directed-spray application: specifically targets the pests to minimize pesticide contact
with non-target plants and animals.
Foliar application: directs pesticide to the leafy portions of a plant.

LMPTH213 Page 60
Rope-wick or wiper treatments: release pesticides onto a device that is wiped onto weeds taller
than the crop, or wiped selectively onto individual weeds in an ornamental planting bed. Soil
application: places pesticide directly on or in the soil rather than on a growing plant.

Soil incorporation: is the use of tillage, rainfall, or irrigation equipment to move the
pesticide into the soil.
Soil injection: is the application of a pesticide under pressure beneath the soil surface.
Space treatment: is the application of a pesticide in an enclosed area.
Spot treatment: is the application of a pesticide to small, distinct areas.
Tree injection: is the application of pesticides under the bark of trees.
5. Safety Systems
Storage and transport: Store pesticides in a place that can be locked and is not accessible to
unauthorized people or children; they should never be kept in a place where they might be
mistaken for food or drink. Keep them dry but away from fires and out of direct sunlight.

Disposal: Left-over insecticide suspension can be disposed of safely by pouring it into a

specially dug hole in the ground. Close the hole as soon as possible. Cardboard, paper and
cleaned plastic containers can be burned, where this is permitted, far away from houses and
sources of drinking-water.
General hygiene: Do not eat, drink or smoke while using insecticides. Keep food in tightly
closed boxes. Use suitable equipment for measuring out, mixing and transferring insecticides.
Do not stir liquids or scoop pesticide with bare hands. Wash the hands and face with soap and
water each time the pump has been refilled.

Protective clothing: Spray workers should wear overalls or shirts with long sleeves and
trousers, a broad-brimmed hat, a turban or other headgear and sturdy shoes or boots. The
mouth and nose should be covered with a simple device such as a disposable paper mask, a
surgical-type disposable or washable mask, or any clean piece of cotton.

Mixing: People who mix and pack insecticides in bags must take special precautions. In
addition to the protective clothing described above, it is recommended that gloves, an apron
and eye protection such as a face shield or goggles be worn.

Spraying: The discharge from the sprayer should be directed away from the body. Leaking
equipment should be repaired and the skin should be washed after any accidental
contamination. Persons and domestic animals must not remain indoors during spraying.

LMPTH213 Page 61
Rooms must not be sprayed if someone, e.g. a sick person, cannot be moved out. Cooking
utensils, food and drinking-water containers should be put outdoors before spraying.

Indications of pesticide poisoning

General: extreme weakness and fatigue.
Skin: irritation, burning sensation, excessive sweating, staining.
Eyes: itching, burning sensation, watering, difficult or blurred vision, narrowed or
widened pupils.
Digestive system: burning sensation in mouth and throat, excessive salivation,
nausea, vomiting, abdominal pain, diarrhoea.
Nervous system: headaches, dizziness, confusion, restlessness, muscle
twitching, staggering gait, slurred speech, fits, unconsciousness.
Respiratory system: cough, chest pain and tightness, difficulty with breathing, wheezing.

First-aid treatment

Give artificial respiration. If no insecticide has been swallowed, mouth-to-mouth

resuscitation may be given. If there is insecticide on the skin or in the eyes rinse the eyes with
large quantities of clean water for at least five minutes. Remove contaminated clothing from
the patient and remove the patient from the contaminated area. Do not induce vomiting unless
the patient has swallowed pesticide that is known to be highly toxic, and medical help is not
expected soon. Vomiting should be induced only if the patient is conscious.

6. Results: The application of fungicide will be done with proper care..

7. Scope of Result: The student will learn about the proper application of chemicals in the
field without any side effect.
8. Caution:. Always wear the protecting clothes while spraying in the fields. Wash the
hands and clothes after spraying.
Suggested readings for students:
Web links: https://en.wikipedia.org/wiki/Pesticide_application

http://www.who.int/water_sanitation_health/resources/vector385to397.pdf

https://www.unce.unr.edu/programs/sites/pesticide/files/pdf/GuidelinesForSafeUse.pdf
http://niphm.gov.in/Recruitments/PHE-ASO-Manual-22042013.pdf

LMPTH213 Page 62
Worksheet of the student

Date of Performance Registration Number:

Aim: To study the different pesticide and their application procedure.
Sr. Name of pesticides Application procedure
No.

Result & discussion:

Learning outcome

LMPTH213 Page 63

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Laboratory Manual: Fundamentals of Plant Pathology

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Laboratory Manual: Fundamentals of Plant Pathology

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LABORATORY MANUAL

Fundamentals of Plant Pathology

(For private circulation only)

Name of student ………………………

Registration no/Roll no ………………

Section and Group ……………………

Session-term: 2020-21 (Autumn Term)

1. A Student is expected to maintain the decorum of the laboratory by maintaining

5. Clean up your work area before leaving.

Compulsory things to be carried by the students in lab:

3. Wear gloves when using any hazardous or toxic agent.

Sr. Title of Experiment Page No.

1 To study various laboratory equipments and microscopy 4-11

2 To study collection and preservation of disease specimen 12-15

3 To study preparation of different culture media 16-20

4 To study Koch’s postulates for the isolation of plant pathogens 21-24

5 To study morphological structures of different fungal genera 25-28

6 To study staining and identification of plant pathogenic bacteria 29-32

7 To study transmission of plant viruses 33-35

8 To study of phanerogamic plant parasites 36-38

9 To study the morphological features and identification of plant parasitic 39-46

11 To study fungicidal formulations and their calculation 55-59

12 To study methods of pesticide application and their safe handling 60-63

1. Experiment: To study various laboratory equipments and microscopy.

Temperature (°C) Time in Minutes

Suggested readings for students: Laboratory Manual on Plant Pathology, D. K. Jha.

Result & discussion:

1. Experiment- To study collection and preservation of disease specimen

2. Learning Objectives: Student will be able to understand collection and

Sr. Name of crop/plant Symptoms Pathogen

Result & discussion:

1. Experiment: To study preparation of different culture media.

1. Potato Dextrose Agar (PDA)

Suggested readings for students:

Result & discussion:

1. Experiment- To study Koch’s postulates for the isolation of plant pathogens.

Result & discussion:

1. Experiment- To study morphological structures of different fungal

2. Learning Objectives: To study about the different structures of fungi with

3.Theory/Principle/Background of the topic:

Suggested readings for students: Introductory Mycology by Constantine J.

Date of Performance Registration Number:

Sr. Name of Pathogen External Symptoms Internal Symptoms

Result & discussion:

1. Experiment- To study staining and identification of plant pathogenic bacteria.

2. Learning Objectives: To learn the staining technique and identification

3. Theory/Principle/Background of the topic: Bacteria have nearly the same refractive

1. Obtain a clean slide

Date of Performance Registration Number:

Sr. Name of bacteria Symptoms Type of bacteria

Result & discussion:

1. Experiment- To study transmission of plant viruses

Suggested readings for students:

Date of performance Registration no

Result and Discuss.

1. Experiment- To study of phanerogamic plant parasites.

2. Learning Objectives: Student will learn about the Identifying

Date of Performance Registration Number:

Aim: To Study of phanerogamic plant parasites

Result & discussion:

1. Experiment- To Study morphological features and identification of plant parasitic

4. General Characteristics of Nematode-

As viewed under a microscope, the cuticle or body covering of a typical plant-parasitic

Fig: Plant parasitic nematode stylet

Fig: Female reproductive system

6. Scope of Result: To learn about the morphological feature of important plant

Suggested readings for students

Textbook on Introductory Plant Nematology by Raman K. Waliya and Harish K. Bajaj

Date of Performance Registration Number:

Aim: To Study the major plant parasitic nematode.

Write the details (see labeling) separately in a note book.

Put all the subsamples in a polythene bag and label it.

Put this material in a Petri plate containing water.

The migratory semi endo/endoparasitic nematodes come out of the chopped

Alternatively the chopped material can be processed by modified

2. Warring blender technique