VASANTRAO NAIK

MARATHWADA KRISHI VIDYAPEEH, PARBHANI

COLEGE OF AGRICULTURE, LATUR
DEPARMENT OF PLANT PATHOLOGY

PRACTICAL MANUAL
of
PATH-121
Fundamentals of Plant Pathology
For
B.Sc.(Hons) Agriculture Semester II

Name of Student : Mr./Miss.___________________________________

Registration No. : ____________________________________________

College of Agriculture: ____________________________________________

Academic Year : 20 - 20

Compiled and Edited by

Dr. Sunita .J. Magar

Department of Plant Pathology

College of Agriculture, Latur
 VASANTRAO NAIK
MARATHWADA KRISHI VIDYAPEEH, PARBHANI
COLEGE OF AGRICULTURE, LATUR
DEPARMENT OF PLANT PATHOLOGY

CERTIFICATE

This is to certify that, Mr./Miss. _________________________________

(Reg. No.________ ), a student of semester-II, B.Sc. Hons (Agri.) has

completed all the exercises successfully of PATH-121 entitled “Fundamentals

of Plant Pathology” during Summer semester of the academic year 201 -1 .

Place: Latur

Date : Course Teacher

[1]
 INDEX
Course No. : PATH-121 Course title: Fundamentals of Plant
Pathology
Semester : II (New) Credits : 3 (2+1)

Ex. Name of Exercise Page Date Sign.

No. No.
01 Acquaintance with various laboratory 3-8
equipments and microscopy
02 General study of different structure of 9-11
fungi
03 Study of symptoms of various plant 12-14
diseases.
04 Study of representative fungal genera 15-25
05 Staining and identification of plant 26-30
pathogenic bacteria
06 Study of phanerogamic plant parasites 31-32
07 Transmission of plant viruses 33-34
08 Study of morphological features and 35-38
identification of plant parasitic
nematodes.
09 Prepartion of media 39-43
10 Isolation and purification of fungi and 44-45
bacteria
11 Extraction of nematodes from soil 46-47
12 Koch’s postulates 46-49
13 Study of fungicides and their 50-59
formulations
14 Methods of pesticide application and 60-69
their safe use
15 Calculation of fungicide sprays 70-72
concentrations.
16 Collection and preservation of disease 73-75
specimen

Course Teacher

[2]
EXERCISE NO. 01
Acquaintance with various laboratory equipments and microscopy
A. ACQUAINTANCE WITH LABORATORY EQUIPMENTS
1. Hot Air Oven:

It was originally developed by Pasteur.

Principle:
 It is accomplished by thermal (heat) conduction.
 When electricity is passed through the heating coils, electrical energy is converted into
heat energy. The temperature is controlled by thermostat.
 The chamber is double walled insulator and fitted with electrical heater which keep the
heat and conserve the energy.

Uses:
 Dry heat is mainly used to sterilize glassware or other heat stable materials.
 The materials are wrapped in an aluminum foil and exposed to the following and time
ratios.

1700C 60min, 1600C for 120min, 1500C 150 min.

2. Autoclave:

It was invented by Charles Chamberland in 1879.

Principle:
Works on the principle of steam under pressure.
Sterilization is carried out under pressure at 1210C for 15 minutes.
Uses:
It is used for sterilizing solid and liquid media for microbial cultures.

3. Laminar Air Flow Cabinet/ Chamber:

Principle:
 The underlying principle of a laminar air flow hood is that a constant flow of HEPA
(High Efficiency Particulate Air Filter) filtered air at a rate of approximately 90linear
feet per minute physically sweeps the area and prevents the entry of contaminated air.
 The space between the HEPA filter and sterile product being prepared is referred as the
critical work surface.
 HEPA filter – remove 99.99% of all air which have the diameter of 0.3 mm or larger.
 The normal filter surface air speed of 0.45 m/s (90 fpm) ensures a continuous change of
air in the enclosed area and therefore ensures the required purity of the air.

Uses:
 The hood workspace is used to prevent the contamination of compounded sterile products
and parental preparations.

[3]
 It is used for maintaining aseptic or microorganism free environment for performing
various activities such as pouring of sterilized media in pertridishes, test tubes, isolation
and sub culturing of pathogens etc.

4. Spectrophotometer /Colorimeter
It was invented in 1940, by Arnold J. Beckman and his colleagues at National
Technologies Laboratories in USA.
Principle:

 The spectrophotometer works based on the principle of turbidity determination.

 Turbidity or Optical density is the cloudyness of the suspension.
 A spectrophotometer is an instrument that measures the amount of light absorbed by
substance.
 Spectrophotometry is a process using electromagnetic radiation (light) to measure an
unknown analyte concentration.
 By using a spectrophotometer, the amount of transmitted light decreases s the cell
population increases. The transmitted light is converted to electrical energy, and this is
indicated on galvanometer. The reading, called absorbance or optical density, indirectly
reflects the number of bacteria.
 Using a spectrophotometer to measure the optical density at 600 nm (OD 600) of a
bacterial culture to monitor bacterial growth has always been a general technique in
microbiology.

Uses:
It is technique used for counting population of bacteria.
5. Quebec Colony Counter:

It is an instrument used to count colonies of bacteria or other microorganisms growing

on an agar plate.
 In Quebec colony counter, there is a platform which is marked with cross ruling (small
squares).
 An illuminator is below the platform to illuminate the colonies and above the platform, a
magnifying lens is present which magnifies the colonies and helps in counting.

6. Bunsen burner:
This apparatus was invented by Roberts Bunsen, German Chemist in 1856. A small
laboratory burner of a vertical metal tube connected to a gas source and producing a very hot
frame from a mixture of gas and air let in through adjustable holes at the base.

Uses: It is used for sterilizing inoculation loops or inoculation needles before and after
transferring the microorganisms. Flaming the mouth of test tubes, conical flask and other
glasswares to prevent contamination.

7. pH meter:

pH meter was invented by Arnold O. Beckman for Testing Acidity.

A pH meter is an electronic device used for the measuring the pH (acidity or alkalinity) of a
liquid.
[4]
Uses:

It is used to determine the pH of solutions of unknown pH.

It is used for setting the pH of various media used for the cultivation and testing biochemical
activities of microorganisms.

Incubator:
 In 1857, Jean Louise Pual Denuce invented the first incubator which was used for birds
like chicken to hatch eggs for a very long time.
 In 1891, French doctor Alexander Loin invented the modern incubator.
 It is similar to hot air oven in construction and operation.

Principle:

It is based on the principle to provide controlled temperature and humidity for

organisms which grow at their maximum growth rate.
Uses the range of temperature varies from room temperature and humidity for
organisms which grow at their maximum growth rate.

Uses:

The range of temperature varies from room temperature to about 50-600C .

It is used for incubation of microorganisms at suitable temperature.

8. Inoculating needle:

The tools like inoculating needles and inoculating loops are used for sub culturing of
microorganisms.
These are made up of platinum wire or nichrome wire fixed into a metal red at one
end.
Inoculation loop – the end of the wire is bent in the form of loop. It is for bacterial isolation
and sub-culturing.
Inoculating needle- the end of the nichrome wire is blunt or curved like crescent. It is
generally used for fungal sub culturing.

9. Shaking Water Bath:

Uses:
These baths can be used effectively in molecular biology protocols (such as
hybridization), bacterial culturing, as well as in solubility and metabolism studies.
Cultivation of microorganisms in a broth medium.

[5]
B) ACQUAINTANCE WITH MICROSCOPE
MICROSCOPE
OBJECT: To get mastery of the use of microscope; this is the basic to a thorough
understanding of microbiology.
Principle: The lenses in the instrument are so adjusted that minute objects invisible to naked
eyes are magnified and made visible
The word microscope is derived from the Greek, micros (small) and skopeo (look at).
The microscope is of different kinds
Introduction:
Light microscopy is the stone of microbiology for it is through the microscope that
most scientists first become acquitted with microorganisms. Microbiologists use a variety of
microscopes, each with specific advantages and limitations. This exercise presents the
principles of microscopy. A microscope is an optical instrument consisting of the one or more
lenses in order to magnify images of minute objects. S are of two categories.
Light microscope:
Magnification is obtained by a system of optical lenses using light waves. It includes
Bright field ii) Dark field iii) Fluorescence iv) Phase contrast v) UV microscope.
Electron microscope:
A system of electromagnetic lenses and a short beam of electrons are used to obtain
magnification. It is of two types
Transmission electron microscope (TEM) ii) Scanning electron microscope (SEM).
Light microscope can be broadly grouped in two categories
Simple microscope: It consists of only one bi-convex lens along with a stage to keep the
specimen.
Compound microscope: It employs two separate lens system namely i) objective and ii)
ocular (eye piece).
Parts of Compound microscope:
1. Mechanical parts:
These are secondary but necessary for working of a microscope. A Base which is
house hoe, shaped supports the entire framework for all parts from the base a ‘pillar’ arises.
At the top of the pillar through an inclination joint arm or limb is attached. At the top of the
pillar, a stage with a central circular opening called stage aperture is fitted, with a stage clip
to fix the microscope slide. Beneath the stage, there is one stage called as ‘sub stage’ which
carries the condenser. At the top of the arm, a hollow cylindrical use of standard diameter is
attached in line with the stage aperture, called ‘body tube’. The body be moves up and down
by two separate arrangements called ‘coarse adjustments’ worked with pinion head and fine
adjustment worked with micrometer head. At the bottom of the body tube an arrangement
called as ‘revolving nose piece’ is present for scanning different objectives. At the top of the
body tube eye piece is fixed.
Optical parts:
It includes mirror, condenser, objective and ocular lenses. All the optical parts should be kept
in perfect optical axis

[6]
a) Objectives:
Usually 3 types of magnifying lenses i) low power objectives (10x) ii) high dry
objective (45 x) and iii) oil immersion objective (100x).
Eye- piece:
Mostly have standard dimensions and made with different power lenses (5 x, 10x,
15x, 20x). A compound microscope with a single eyepiece is said to be monocular, and one
with two eye pieces to be binocular.
c) Condenser:
Condenser the light waves into a pencil shaped cone thereby preventing the escape of
light waves. Also raising or lowering the condenser can control light intensity. To the
condenser, iris diaphragm is attached which help in regulating the light.
d) Mirror:
It is mounted on a frame and attached to the pillar in a manner that it can be focus in
three different directions. The mirror is made of a lens with one plane surface and another
concave surface. Plane surface is used, when the microscope is with condenser.
Procedure for using microscope:
Rotate the nose-piece and bring the low power objective (10x) in the line with the eye
piece i.e. in optical axis.
Adjust the concave mirror, so that the brightly illuminated m microscope. Field is
seen through the objective.
View through the ocular (s) open and close the iris diaphragm by the lower to get an
optimum- illumination.
Keep the slides with specimen side up over the hole in the centre of the stage with the
coarse adjustment bring the low power objective close to the slide and slowly raise it, until
the specimen comes into focus.
To get clear image focus with the fine adjustment. Bring the specimen of our interest
to the centre of the field the stage adjustment. Rotate the nose piece and bring the high power
(45x) in working position.
If the microscope is of parfocal type the objective comes into focus and adjusts with
fine adjustment to get clear image.
If the microscope is not parfocal, lower the objective close to the specimen and slowly
raise it with coarse adjustment until the specimen comes into focus.
Adjust with the fine adjustment to get a clear image.

[7]
Fig 1 : MICROSCOPE

[8]
EXERCISE NO. 02
MORPHOLOGY OF FUNGI
General Study of Different Structures of Fungi
Fungi are eukaryotic, achlorophyllus, uni or multicellular have a definite cell wall
makeup of either chitin or cellulose, intake food by means of absorption, reproduce by means
of production of sexual and asexual spores. The asexual reproduction proceeds by means of
production of vegetative spores. The sexual reproduction is by means of production of male
and female gametangia unite to form a zygote. The body of fungus generally refereed as
thallus. They are classified under Kingdom – Fungi. (Ainswarth, 1973).
Constituents required
Microscope, slides, cover slips, two pointed needles and wash bottles.
Procedure:
Take piece of bread, moistened it and kept in moist chamber. After 48 hrs thread like whitish
growth appears on the bread. Take a thread like growth on a clean glass slide, with the help of
pointed needles, spread the threads in the drop of water on slide. Place the cover slip gently
over the water drop so that no air bubbles are let inside. Observe first under low power and
then focus under high power of the microscope.
Observations:
Look for an individual thread known as Hypha. Many of such hypha constitutes a mycelium.
Observe, if cross wall are present in the hypha. The cross wall is called as a septum or septa.
The mycelium having septa is called septate mycelium. A mycelium without septa is called a
septate or non septate or coenocytic mycelium. Observe also for sporangiophore, spores,
rhizoides, sporangiole, stolon and columella etc.
Sporangiophore:
A specialized hyphae upright in growth produced from the mycelium, bearing sac like
spore fruit or structure is known as sporangiophore.
Sporangium: Sporangium is a sac like structure or spore fruit containing spores.
Sporangiospores or spores: sporangiospores or spores are the unit of reproduction, round to
oval, hyaline, unicellular and produced internally or endogenously.
Columella: The knob like structure at the end of sporangiophore over which the sporangium
is attached. Its an attachment between sporangium and sporangiophore.
Rhizoids: The root like structures or appendages of fungus is known as rhizoids. It has two
main functions is anchoring or to hold fast with main host and absorption of food material.
Sporangiole: A small sporangium without columella.
Stolon: The hypha which joints two rhizoids is known as stolon. Also observe a culture of
fungus provided and note the type of mycelium, conidiophores and conidia.
Septate mycelium: Hypha divided by cross wall (septa).
Conidium: Asexual spore borne on the hypha (conidiophore) is called as conidium.
Conidiphores: The hypha bearing conidium.
II VEGETATIVE STRUCTURS
Morphology of fungi includes both vegetative and reproduction structure of fungi. A fungus
body generally consists of thread or filaments. An individual thread is called as hyphae. A
group of hyphae is known as mycelium. Examine the suitable preparation and record the
following types of mycelium within fungi.
[9]
Non-septate or aseptate or coenocytic mycelium:
The mycelium whose hyphae have no cross walls or septa and which consist of number of
nuclei embedded in cytoplasm. So mycelium is multinucleate or uninucleate e.g. fungi
belonging to sub division Zygomycotina and Mastigomycotina.
Septate mycelium:
The hyphal cells of mycelium are divided into the compartment by crosswalls or septa such
mycelium ids called as septate mycelium. Individual cell may contain one or more nuclei
depending upon species. Hence mycelium may be uni or multi nucleated e.g. fungi belonging
subdivision Ascomycotina, Basidiomycotinba, deuteromycotina.
Ectophytic mycelium:
The hyphae in this case grown on external surface or on epidermal cells, produces special
sucking organ or structures called as haustoria e.g. powdery mildew of pea, grapes and
cucurbits etc.
Endophytic mycelium:
When the hyphae of the mycelium grown inside the epidermal layer of plant tissues, it is
called as Endophytic mycelium.
Endophytic mycelium is of following types:
Intercellular mycelium, Intracellular mycelium and vascular mycelium
Intercellular mycelium: when the mycelium is found to grow in between two cells of plant
tissue without penetrating the cells s and obtained nourishment by sending haustoria in the
cells, it is known as Intercellular mycelium. e.g. stem leaf and yellow rust of wheat, jawar
rust, bajara rust etc.
Intracellular mycelium: when the mycelium is found to grow within the cells of plant tissue
is called as as Intracellar mycelium. e.g. bajra smut, sugarcane smuts etc. vascular mycelium:
Vascular mycelium: when the mycelium is confined to or found to grow invascular tissue of
plant it is called as vascular mycelium. E.g. fungus causing wilt viz., tur wilt, cotton wilt etc.
III. MODIFICATION OF MYCELIUM
Sclerotium: (Pl. Sclerotia) (Skeron- hard). Sclerotia are the modified form of vegetative
mycelium, which form hardened compact mass of hyphae and act as resting body which is
resistant to unfavorable condition and may remain dormant for long periods and germinate
under favourable condition e.g. Sclerotium sp.
Rhizomorph: (Rhizo- root + morphe- shape)
The number of fungi produced thick cable like strand made up of hyphae, where hyphae has
lossed their individuality. These grow either in soil or on the trunks of trees. They can
withstand unfavorable condition. They are found in fungi belonging to sub-division
Basidiomycotina.
Stroma: (Pl. Stromata) It is compact mass of hyphae and appears as pseudo paranchymatous
tissue and contains fruiting body of fungus. e.g Ergot sclerotia.
Chlamydospore: these are the thickened or swollen cell of mycelium containing stored food
materials and may be formed terminally and or intercalary. These are the resting cells
withstand the unfavourable conditions and germinate during favorable condition e.g.
Fusarium spp.
Dormant mycelium: It is mycelium, which hibernate in the host tissue and to tide over
unfavourable conditions. It remains in a dormant condition for a part of its lifecycle and
[10]
became active when condition are favourable e.g. downy mildew of grape, koleroga of
arecanut and loose smut of wheat.
Gemmae (Pl. Gemma): These are the chlamydospore produce in lower fungi whose wall is
thinner. e.g. Saprolegnia spp.
Questions:
Draw neat label diagram of typical fungus
What is fungus? Give its position.
What is initial source of mould on the bread?
What are the conditions required for proper growth bread mould fungus?
Draw neat lable diagram of different fungal vegetative structures
What is the role of haustoria, chlamydospore and sclerotia in the lifecycle of fungi?

Fig.2. Typical Fungus

[11]
Fig. 3 : Vegetative structures of fungi

[12]
EXERCISE NO. 03

Study of Symptoms of various plant pathogens

A) SYMPTOMS OF PLANT DISEASES PRODUCED BY FUNGAL PLANT

PATHOGENS
SYMPTOMS: Symptoms are expressions of diseased conditions. They are expressed
internally as well as externally and help in general diagnosis. With the help of symptoms a
diseased plant can be identified from the healthy one. However, symptoms alone are not
helpful in ascertaining the exact nature of the disease. Similarly, symptoms may result from
different causes, unrelated to each other e.g. Chlorosis may due to downy mildew, viral
infection or deficiency like the fever in human being may be wound, typhoid or by malaria.
SIGN: Sing are the experimental or scientific evidences of the diseases and generally
confirmed by various diagnostic techniques. Signs help in accurate diagnosis of the diseases.
Sign are the actual appearance of the pathogen or its structures on the host or in the host as a
result of manifestation. E.g. presences of whitish growth on the leaves in downy mildew of
grape or jowar, bacterial ooze in ring disease of potato.
1. MOTTLING: Partial destruction of chlorophyll in interveinal area. e. g. Mottle leaf of
citrus.
2. STEM GALLS: e.g. white rust of crucifers, Loranthus on mango.
3. CLUB: e.g. club root of cabbage.
4. BLIGHT: There is a general and rapid destruction of plant parts like shoots, leaves,
blossoms, twigs etc. the dead organ turn as brown to black showing burnt appearance.
e.g. Early and late blight of potato, Bacterial blight of paddy.
5. SPOT: it is localized destruction of the tissue in more or less circular manner. It is usually
found on the leaves, and may develop on stem or fruit. The dead tissues which are in limited
area give shapes as angular round or circular surrounded by yellow purple red margin.
e.g. Eyespot of jowar, tikka of groundnut and angular leaf spot of cotton.
6. TAR SPOT AND STREAKS OR STRIPES: Necrotic area become typically tar stained
found in forest trees, palm, grasses and jawar. Streaks are elongation of necrosis. e.g.
bacterial streak of paddy.
7. BLAST: same as blight but spots are distinct and spindle shaped. E.g. blast of paddy.
8. DIE BACK: Dying of plant organ especially stem and branches from the tip downward.
E.g. die back of citrus.
9. EXUDATION: Secretion of sticky gum like substances due to diseases e.g. gummosis of
citrus.
10. ANTHRCNOSE: Destruction of collenchyma and cambium tissue, lesions are sunken in
centre with raised and prominent margin e.g. anthracnose of grape, chilli and bean.
11. BLACK HEART: Blackening of central portion observed in potato due to high
temperature and poor ventilation in storage e.g. black heart in potato.
12. SCAB: Destruction of epidermal tissues in the form of scab. Infection is deep seated e.g.
Scab of potato and apple.
13. SHOT HOLE: Decayed leaf tissues blown are away leaving holes perforations e.g. shot
hole of Ashok and Mango.

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14. SMUTS: The floral parts are usually, affected the ovaries destroyed and replaced by
forming sori e.g. smut of jawar, loose smut of wheat.
15. RUSTS: The pustules of spore usually breaking through the epidermis are seen on the
host. Pustules may be dusty or compact and white, yellow, brown, red or black in colour. e.g.
White rust of crucifers, leaf rust and stem rust of wheat.
16. ERGOT: Normal grains are replaced by sclerotia e.g. ergot of bajra.
17. GREEN EAR (DOWNY MILDEW): Flowers are converted into green and elongated
diseased structures e.g. green ear of bajra.
18. POWDERY MILDEW: Powdery growth consisting of mycelium and numerous conidia
is seen on the host surface e.g. powdery mildew of grapes.
19. MUMMIFICATION: These are observed in fruits. The screen of fruits become hard and
fruits gate shriveled such fruits are called as mummified fruits e.g. downy mildew of grapes.
20. WILTS: Wilting or drying of entire plant observed in adults plants. The leaf and other
succulent parts loose turgidity become flaccid and droop. Its typical vascular symptom due to
plugging of xylem vessel or toxic effect e.g. tur wilt, cotton wilt, pea wilt, gram wilt etc.
21. DAMPING OFF: Sudden wilting and collapse of seedling observed commonly in seed
beds. The stem near the soil is affected, becoming constricted and weak. e.g. Damping off of
seedlings like tobacco, tomato, chilli, cabbage etc.
22. PALLOR: Partial destruction of chlorophyll in the forms of streaks. There is unhealthy
appearance of plant due to deficiency or excess of water or lack of light or reduction in
chlorophyll content due to pathogenic organisms. e.g. bajra seedlings affected with downy
mildew.
23. ROTS: The term is applied in cases where affected tissues decays or rots. Infection of
parenchyma, pitch tissues and various parts. Rot imparts different colour reactions and
designated accordingly.
a) Dry rot: Decay of tissues, even after rotting may sometimes remain firm or hard e.g.
dry rot of potato and corn.
b) Soft rot: Decay of soft tissues, rotting accompanied by softening of the tissues e.g. soft
rot of lemon, mango, tomato, banana etc.
c) Red rot: Affected tissues become red in colour e.g. red rot of sugarcane.
d) Wet rot: In addition to softening, there is slimy oozing of liquid e.g. storage rot in
potato, citrus and other fruits, usually due to fungi.
e) Root rot: Destruction of parenchyma of underground stems e.g. Rhizoctonia root rot of
cotton, hallow stem of jawar.
Rots may be described sometimes according to plant part affected e.g. stem rot (Papaya),
collar rot (Groundnut), neck rot (Paddy), rhizome rot (Ginger). After they are described after
the discolouration produced on infection e.g. brown rots (Potato) black rot (Cabbage), red rot
(Sugarcane) etc.

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B) SYMPTOMS OF PLANT DISEASES PRODUCED BY BACTERIAL PLANT
PATHOGENS
1. TUMORS AND GALLS: Tumors are knot like structure or over growth of the host tissue.
It is bigger in size e.g. tumor caused by the infestation of bacteria like Agrobacterium
radiobacter. Galls are abnormal swelling or blisters or pimples/ knot formed on plant parts.
The bacteria induces formation of galls in plants by stimulating mature cells to resume
meristematic growth, gall are smaller in size than tumors.
2. HAIRY ROOT: Formation of numerous fine roots e. g. infestation of Agrobacterium
radiobacter var. rhizogenes.
3. WILTS: Wilting or dying of entire plant observed in adult plants. The leaves and other
succulent parts loose turgidity become flaccid and droop. It is typical vascular symptom due
to plugging of xylem vessel or toxic effect e.g. bacterial wilt of tomato.
4. BLIGHT: Here there is a general and rapid destruction of plant parts like shoots, leaves,
blossoms, twigs etc. the dead organ turn as brown to black showing burnt appearance e.g.
bacterial blight of paddy.
5. SOFT ROT: The term is applied in cases where affected tissue decay or rots. Infection of
parenchyma, pitch tissues and various parts. Rot imparts different colour reaction and are
designed accordingly. e.g. brown rot of potato, black rot of cabbage etc.
6. CANKERS: Deep seated infection due to destruction of woody tissues and cambium
tissues. Cankers are raised from epidermal surface of the tissue and are rough to touch. e.g.
citrus canker, guava canker etc.

[15]
C) SYMPTOMS OF PLANT DISEASES PRODUCED BY VIRAL PLANT
PATHOGENS

1. YELLOWING: It is known as chlorosis. There is complete destruction of chlorophyll.

When the colour becomes white it is known as etiolation of chlorophyll. These symptoms
usually caused by viruses e.g. yellowing of dolichus bean due to viral like infection.
2. MOSAIC: Mosaic caused by virus infection is highly infectious. It is due to partial loss of
chlorophyll or chlorosis in uneven patches. e. g. papaya mosaic, tomato mosaic, chilli mosaic
etc.
3. YELLOW MOSAIC: Light green and yellow patches are observed in the leaf lamina e.g.
yellow mosaic of beans.
4. YELLOW VEIN MOSAIC: Clearing of veins becoming yellow and leaf lamina
remaining green e.g. yellow vein mosaic of bhendi and hibiscus.
5. MOTTLING: Partial destruction of chlorophyll in interveinal area e.g. mottle leaf of
citrus.
6. BUNCHY TOP: Here the leaves are given out in the form of a bunch at the crown of the
plant e.g. bunchy top of banana.
7. LEAF CURL: It is the curling or destruction of the leaves. In leaves, an increase in the
cells either side of mid rib and stimulation in growth of the palisade cells and to a lesser
extent of cells of spongy parenchyma result in puckering and curling e.g. leaf curl of chilli
and tomato.
8. HAIRY ROOT AND SINDLE TUBER: The formation of spindle tuber of potato due to
infestation of potato spindle tuber virus.

[16]
EXERCISE NO. 04
Study of representative fungal genera

A. Study of Pythium, Phytophtora, Perenospora and Sclerospora and Albugo

I. Genus: Pythium
This is an aquatic organism responsible for causing the extremely destructive disease like
damping off. The organism can be isolated on the baits in the water
The causal organism: Mycelium hyaline, coenocytic; sporangia globose to oval, terminal or
intercalary on the somatic hyphae; oogonia, globose, terminal or intercalary; antheridia,
small, elongated or club shaped; oospores thick walled.
Materials required:
 Affected specimen or culture of the pathogen
 Microscope
 Glass slides
 Cover-slips
 Watch glasses
 Cotton blue
 Lacto phenol
 Razor blade
 Needles
 Hair brush
 Inoculating needle
Procedure
Note pale green colour of the toppled- over seedlings. Examine the basal portion of
the stem and notice brownish, water-soaked lesion and the rotting tissues. Cut sections of the
affected areas, stain and examine under the microscope. Note the killed host cells and
collapsed tissues. Locate inter- and intracellular hyphae; sporangia; oogonia; antheridia and
oospores.
The culture of the pathogen, gently lift the small portion of the growth of the pathogen
on the slide with the help of the needle and mount on the glass slide and examine under the
microscope.
Record:
Record the following observations
 Symptoms of the disease.
 Pathogen different structure and microscope sketch
Classification of the pathogen
II. Genus: Phytophtora
The genus is responsible for the widespread epidemic in Ireland during 1845. It
continues to be a major disease in the cool and humid regions. In India, the disease is
prevalent in the Nilgiri Hills, Bihar, and plains of North India.

[17]
The causal organism: Mycelium, coenocytic, sporangiophores branched with side branches
showing bulbous enlargements; sporangia, hyaline, lemon-shaped with a papilla at apex.
Sporangia germinate by a germ tube or by zoospore but never form vesicle.
Materials:
 Potato plants and tubers infected by Phytophtora infestans or Culture of the pathogen
 Microscope, glass slides, cover-slips
 Watch glasses
 Test tubes
 Needles
 Hair brush
 10 per cent KOH solution
 Lactophenol
 Burner
 Moist Chambers
 Refgigerator
 Incubator adjusted to 24 0 C
Procedure
Cut a small portion of the infected leaf. Boil in 3-4 ml. of KOH solution. Place on the
slide with lower epidermis facing up and examine under low power. Watch the
sporangiaphores emerging out of the stomata. Look for the attached sporangia. Scrape an
infected leaf. Mount the scrapped material in lactophenol and study the characteristic
branching of sporangiaphores and the typical shape of sporangia. Brush out sporangia from
the infected leaf, place one moist chamber each in, a refrigerator at 10 0 C. and in an
incubator at 24 0C. Observe the type of germination at both the temperature after intervals of
3 hrs.
Take the culture of the pathogen, gently lift the small portion of the growth of the
pathogen on slide the with help of the needle and mount on the glass slide and examine under
the microscope.
Record:
Record the following observations
 Symptoms of the disease.
 Pathogen different structure and microscope sketch.
Classification of the pathogen
III. Genus: Perenospora and Sclerospora
Procedure, observation and record:
Examine infected leaves and note the sowny growth on the under surface of the
leaves, which consist of prorop0hores bearing spores.
Mount some sporophores on a slide under a cover glass. Note the type of branching.
A dichotomous branching is typical of the genus Perenospora. In Sclerospora, the
sporangiophores comprises of a swollen hypha bearing several upright branching at the apex.
Examine oosporic materials and note the thich oospore wall.

[18]
Fig. 4 : Oomycetous Fungi

[19]
IV. Genus: Albugo
Albugo candita (Lev.) Kunze
The genus attacks the many species of Cruciferous. In cabbage, cauliflower and raddish seed
crops, it induces the hypertrophy and hyperplasia of braches and floral parts cause losses.
The causal organism: Mycelium coenocytic, haustoria, knob-shaped; sporangiophores, club-
shaped; sporangia, hyaline, globose, in chain; oogonium, globose, terminal or intercalary;
antheridium, clavate, paragynous, oospore, thick-walled.
Materials:
 Crucifer plant infected by Albugo candida or Culture of pathogen
 Microscope
 Glass slides
 Cover-slips
 Watch glasses
 Lactophenol
 Cotton blue
 Razor blade
 Needles
 Hair brush
Procedure:
Note the symptoms and scrape the pustules with powdery consistency, mount in
lactophenol and examine sporangia under low-power
Cut cross sections of the leaves and hypertrophied organs, stain and observe under the
microscope. Watch, intercellular mycelium, intercellular haustoria, short conidiophores
arising from the mycelium in a compact layer beneath the epidermis, chains of the sporangia
and the sexual organs and oospores.
Take the culture of the pathogen, gently lift the small portion of the growth of the
pathogen on the slide with the help of the needle and mount on the glass slide and examine
under the microscope.
Record:
Record the following observations
 Symptoms of the disease.
 Pathogen different structure and microscope sketch
 Classification of the pathogen

[20]
B. STUDY OF THE FUNGAL GENERA: Synchitrium, Physoderma,
Plasmodiophora and Spongospora

I. Genus: Synchitrium
General comments: the fungus produces endobiotic and holocarpic thallus. It may become
converted directly into a group or sorus of sporangia or may form a prosorus which later
gives rise to a sorus of sporangia. In the resting stage, the thallus becomes a resting
sporangium which function as sorus or prosorus.
Procedure, observation and record:
1. Examine potato tuber infected with S. endobioticum. Note the effect of infection on the
host.
2. Observe prepared slide of the sections of infected tubers. Search for young prosorus,
mature
prosorus, germinating prosorus, sorus of sporangia and resting sporangium.
3. Observe the size of nuclei in young, mature and germinating prosorus.
4. Note the relative thickness of the walls of the prosorus and the resting sporangium.
II. Genus: Physoderma
General comments: obligate parasite, thick walled resting sporangia (resting spore) appear
as brown dist ion infected parts. These originate from fragmentation of the endobiotic
rhizomycelium. Resting sporangia are smooth, brown and flattened at one side having a lid or
cap like structure.
Procedure, observation and record:
Examine infected corn leaves for sporangia of P. zeamaydis. Cut off a very small portion of
the infected part and boil for a few seconds in 10% KOH in a test tube. Mount and examine
for sporangial sori.

III. Genus: Plasmodiophora and Spongospora

General comments: predominantly obligate endoparasites, somatic structure a true
plasmodium, no fruiting body, spores being borne singly or in spore balls within the host
cells, the zoospores unequally biflagellate with both flagella whiplash type.
Procedure, observation and record:
1. Examine cabbage roots infected with P. brassicaceae and compare with normal roots.
2. Cut very thin sections of diseased rootlet and examine for resting spores.
3. Study a prepared, stained slide of an infected root and observe disorganization of vascular
tissues.
4. Compare infected and normal cells for size and contents. Find some infected cells
containing
plasmodia and some containing the mature spores.
5. Examine potato tubers infected with S. subterranean.
6. Study prepared slides showing sections through an infected portion of a potato tuber. Note
the effect of the parasite in the cells. Find the spore balls of the fungus.

[21]
Fig. 5 : Fungal Genera: Synchitrium, Physoderma,

[22]
C. Study of ascomycetoces fungi: Taphrina, Protomyces, Erysiphe, Claviceps
and Sclerotinia
I. Genus: Taphrina
General comments: the taphrinales are parasites on plants, in culture they resemble yeast and
reproduce by budding. In nature they grow intercellularly in the ghost forming binucleate
hyphae. This further breaks up into chlamydospores-like cells from which the asci naked on
the host surface, forming a hymenium.
Procedure, observation and record:
1. Collect infected turmeric leaves (T. maculans) and peach leaves (T. deformans)
2. Cut fine sections, mount them in lactophenol and observe asci.
3. Study prepared slides showing hymenium layer with asci at different stages of
development.
4. Observe the stalk cells, the asci and ascospores.
II. Genus: Protomyces
General comments: P. macrosporus causes stemgall of coriander. Mycelium intercellular
forming thick walled resting spores; spore sacs formed by emergence of cell membrane
through ruptured outer wall of resting spore; endospores formed after germination of the
resting spores; the resting spores smooth walled and formed intercalary on the mycelium.
Procedure, observation and record:
1. Cut cross section of the plant parts bearing galls (hypertrophed parts) and mount in
lactophenol.
2. Observe resting spores (chlamydospores) formed in the host tissues.
3. Place chlamydospores on glass slide and treat with 1% acidulated (H2SO4) or alkalined
(NaOH) for 15 minutes.
4. Observe microscopically condensation of protoplasm, rupture of the outer two wall layers,
emergence of endospores as an oval or spherical vesicle.
III. Genus: Erysiphe
General comments: Biotrophic parasite, mycelium ectophytic, ascomyta basically
cleistothecial, thick walled, dark – brown colored, with complex appendages, asci one or few
per ascoma, anamorph oidium like.
Procedure, observation and record:
1. Collect plants infected with Erysiphe and examine under a dissecting microscope. Note the
hyphae which form a white mat over the infected portions. Strip a portion of the epidermis
and mount in lactophenol. Study the hypae of the fungus.
2. Observe the erect conidiophores and the conidial chains under the microscope.
3. Mount same cleistothecia in lactophenol. Note the appendages characteristic of the genus
(myceloid)
4. Press on the cover glass with a mounted needle and watch the asci with ascospores break
out of the cleistothecia.
5. Study the ascigerous stages of as many genera as are made available to you note the
appendages particularly the tip and base of appendages and to the manner in which the tips
may be branched. This will enable you to distinguish the genera from each other.

[23]
Fig. 6 : Ascomycetoces fungi: Taphrina, Protomyces

[24]
IV. Genus: Claviceps
General comments: The clavicipitales are parasitic on plants, insects, or on other fungi.
They are easily distinguished by the character of their asci and ascospores. The ascus has a
thick cap, Perforated by central narrow canal through which needle shaped ascospores are
successively ejected.
Procedure, observation and record:
1. If pure culture of any Claviceps sp. is available, study colony characters and somatic
hyphae.
2. Prepare slides of mycelia mates showing layers of conidiophores. Observe the large
numbers of conidia produced. The conidioferous mycelia mats develop eventually into the
sclerotia.
3. Examine infected earheads and note the size, shape and colour of sclerotia. Cut cross-
sections of the sclerotia. Note the pseudoparenchymatous tissue composing the rind and
interior of the sclerotia.
4. Incubate few scelerotia as suitable temperature and moisture. Observe germinated sclerotia
bearing stroma. Cut longitudinal sections through a stroma and observe the position of
perithecia. Note the ostiole, the periphyses, the asci and ascospore.
IV. Genus: Sclerotinia
General comments: Ascomata (Apothecium) usually stalked, arising from a sclerotium or
from the stromatized host tissue, often brown asci with an apical ring, ascospores often
longitudinally symmetrical.
Procedure, observation and record:
1. If pure culture of any Claviceps sp. is available, study colony characters and somatic
hyphae.
2. Obtain pure culture and study somatic stages.
3. Scrap some of the grey powdery substance of infected plant parts and mount. Study
characters of conidiophores and conidia.
4. Sclerotia along with infected host tissues falls on the ground and survives. Under suitable
conditions they germinate and bear apothecium. Make a thin section of a portion of an
apothecium and observe hymenial layer, paraphyses, asci and ascospore.

[25]
`
Fig. 7 : Ascomycetoces fungi: Erysiphe, Claviceps and Sclerotinia

[26]
D. Study of smut fungi (Class: Teliomycetes, Order: Ustilaginales)
General comments: The ustilaginales are the smuts. They produce their basidiospores on a
promycelium which grows out of germinating teliospores.
In contrast to the rusts, the smuts-
1. Produce their teliospores from intercalary hyphal cells.
2. Bear their basidospores not on sterigmata but sessile.
3. Do not discharge their basidiospores violently, and
4. Produce no specialized sex organs.
Procedure, observations and record:
1) Family: Ustilaginaceae
I. Genus: Ustilago
The teliospores of the family produce a 3-septate (4-celled) promycelium from which,
the basidiospores are budded off laterally.
Examine plant parts infected with various Ustilago spp. and note the conversion of
host parts into black powder. Examine sori which are naked or covered by host tissues but no
columella.
Mount some smut spores and examine under microscope. Note the minute spines on
the surface of the spores, the thick walls, the colour and size of spores.
Make a hanging drop of some smut spores in distilled water and at the next laboeatory
period, study the germination of the spores.
II and III .Genera: Sphacelotheca and Tolyposporium:
Study the teliospores of as many species of the above genera as are available and contrast
with Ustilago. Note particularly the spore grouping which are characteristic of these genera
and the presence or absence of membrane around the smut balls. In Sphacelotheca, the sori
have columella composed of host tissues in the centre and enclosed by pseudomembrane of
the fungus tissue. In Tolyposporium the spores remain united into permanent balls and are not
easily separable. Promycelium produced is septate with lateral and terminal sporidia.
2) Family: Tilletiaceae
Genera: Tilletia and Neovossia
The teliospores produce a non-septate promycelium which bears a cluster of
basidiospores (sporidia) at the tip.
Examine grains of wheat infected with Telletia spp.
Study the teliospore and note their size, colour, thickness of wall and surface
markings.
Prepare hanging drops of teliospores in distilled water and examine at the next
laboratory period for germination

[27]
Fig. 8 : Smut fungi
[28]
 E. Study of rust fungi (Class: Teliomycetes, Order: Uredinales)
General comments: The Uredinales are the plant rusts. They are specialized parasites with a
very complex life-cycle. Basidiocarp lacking, teliospores grouped in sori or scattered within
host tissues. The basidial apparatus is represented by the teliospores. Before karyogamy, it is
called PROBASIDIUM and after it is HYPOBASIDIUM. The PROMYCELIUM is the
EPIBASIDIUM. The basidiospores are formed on short pointed sterigmata and forcibly
discharged.
Rust fungi are either MACROCYCLIC or MICROCYCLIC. Macrocyclic rust passes through
the following phases in the order given:
Stage 0 : Spermagonia bearing spermatia and receptive hyphae
Stage I : Aecia bearing aeciospores
Stage II : Uredia bearing uredospores
Stage III : Telia bearing teliospores
Stage IV : Promycelium bearing basidiospores
The teliospores which are considered to represent the perfect stage of the rusts may be free,
loosely arranged, or fused into a compound head, a crust or a column.
Procedure, observations and record:
Family: Pucciniaceae
The stalked teliospores constitute the characteristic feature. The genera are based on
teliospore morphology, aecial characteristics and life-cycle pattern.
I. Genus: Puccinia
P. graminis tritici – Black stem rust of wheat
P. recondita - Brown rust of wheat
P. striiformis - Yellow stripe rust of wheat
a) Study the Puccinia spp. as follows
 Examine infected barberry leaf under dissecting microscope. Note the tiny spermagonia
on the upper epidermis. Invert the leaf and observe cluster cups (aecia) pushing through
lower epidermis.(Stage I and Stage II).
 Study a mature aecium. Note the basal “ aeciospores mother cells’ from which thye chain
of aeciospores can be observed.
 Now examine infected wheat leaves and stems. Note the rusty colour of the pustules and
the pattern of their discolouration. Scrap the pustule gently and mount onto a slide in
lactophenol.
 Note the colour of the spores, their shape, method of attachment. Study the wall, its
minute spines and germspores. (Stage II, uredial).
 Cut cross section of infected plant parts bearing uredia. Observe origin of uredia and their
effects on the host tissues particularly the epidermis. Note the long stalks of the
uredospores.
 Observe infected leaves and stem of wheat bearing telial stage of the fungus. Note colour
and distribution of pustules. Study teliospores in a lactophenol mount and note their
colour. Shape, number of cells , stalk and thickness of the wall.

[29]
 Cut cross section of infected plant parts bearing telial stage and observe the manner in
which teliospores are formed and their position with reference to the host cells and to
each other.
 Study the teliospores of all the three rusts infecting wheat and note differences in shape
number of cells and their location in host tissues.

II. Genus: Uromyces III. Genus: Melampsora

Fig. 9 : Rust fungi

[30]
F. Study of some parasitic fungi from Deuteromycetous group
I. Genus: Colletotrichum
It is common fungus on a number of plant hosts causing ‘Anthracnose’. Short
conidiophores are produced in an acervulus. This fungus usually produces dark brown to
black setae in the acervulus.
Procedure, observations and record
1. Observe a culture of C. lindimuthianum under the power of a binocular dissecting
microscope.
2. Note the bristle-like, brown or black, setae in each acervulus.
3. Observe the colour of acervulus or a section through an acervulus formed on host part.
4. Study the setae and conidia.
5. Study conidiophores. They are simple, hyaline, aseptate and packed.
6. Compare and contrast with Gloeosporium.

II. Genus: Alternaria

Some species are parasitic causing leaf spot and blight of plants; others are saprobic. Study
the culture of Alternaria as well as infected plant parts.
Make slides using lactophenol and observe mycelial network which are slender, septate,
branched, hyaline initially, becoming brown at maturity.
Observe conidiophores which are large, dark brown, obclavate, bottle shaped with a broad
cup like base and narrow beak, multiseptate with number of tranverse (5-10) and a few
longitudinal septa. The conidia mostly formed in chain.
Bring infected leaves of Brassica sp. and observe development of concentric rings.
Make slides (though scraping and sections) and observe hyphae, conidiophores and conidia.
III. Genus: Helminthosporium
Several species cause diseases of cereal and grasses. Obtain diseased specimen and culture.
Study and observe the followings:
Develop temporary mounts using lactophenol and the culture.
Observe mycelial thallus which are branched, septate, light to dark brown.
The mycelium in host tissues are inter or intracellular.
Locate conidiophores with conidia and study conidial attachment, shape, colour and septation
of conidia. Conidiophores are erect, brown to dark brown, the conidia are multi-septate,
brown- dark brown, cylindrical to fusiform.
IV. Genus: Cercospora:
Parasitic causing, leaf spot or blotch of higher plants.
Collect infected diseased plants as well as culture of the fungus.
Make temporary mounts using lactophenol both from the diseased plants and culture.
Observe the characters of hyphae which are septate, branched, hyaline or colored,
intercellular.
Observe the character of conidiophores which are septate, branched or unbrached hyaline or
coloured.
The conidia are long, round at the base and tepering above, slightly curved having 4-5 septa.

[31]
Fig. 10: parasitic fungi from Deuteromycetous group

[32]
V. Genus: Fusarium
Facultative parasite causes damping off, root rot, soft roots, dry rot and wilt.
Study a culture of Fusarium which produces both macro-conidiaa and micro conidia.
Develop 3-4 slides from the fungal growth and observe macro-conidia which are large,
hyaline, fusoid, curved or crescent shaped.
Observe micro-conidia which are small, hyaline, elliptical, ovoid or curved, unicellular or
with one or two septa. They are abstracted from the tips of the simple or branched
conidiophores.
Mount a portion of mycelium growth and note the large number of chlamydospores which
may be round, oval, thick walled and formed singly or in chains, terminal or intercalary.
VI. Genus: Pyricularia
Make temporary mounts using lactophenol fro the cultures as well as diseased plant parts.
Observe conidiophores which are erect, simple, rarely branched, septate, hyline to light
coloured, ultimate cells sympodulae.
Study the characters of conidia. They are ellipsoid, or more often pyriform, broader and
truncated at the attachment point, tapering towards the distal end, mostly one to two septate
hyaline to light pigmented.
VII. Genus: Phoma and Pyllosticta
Collect infected plants and the cultures. Observe development of pycnidial bodies.
Develop temporary slides for microscopic observations.
Observe pycnidia which may be immersed or semi-emmersed, sometimes becoming
erumpent or a short beak pierces the epidermis, unilocular, lenticular to globose, separate or
aggregated occasionally confluent and thin walled.
Apply pressure to beak open pycnidia and observe conidiophores and conidia. The
conidiophores may be short, hyaline and smooth.
Observe conidia which are hyaline, aseptate or sometimes bicelledd, thin walled, ovoid,
ellipsoid, cylindrical, fusiform, pyriform or globose.
G. Study of fungi grouped as “Mycelia Sterilia”
General comments: there are some imperfect fungi which do not form even asexual spores
and are, therefore, sterile. But they do form ‘Scelrotia” which perpetuate and disseminate the
fungus. There are two genera: Rhizoctonia and Sclerotium which include several plant
pathogenic species. However, in the few instances in which perfect stage have been found,
these have to be “Basidiomycetes”
Procedure, observations and record:
1. Collect diseased specimen and the cultures.
2. Study the pigmentation and growth pattern.
3. Study and record the number, size, pattern and pigmentation of sclerotial bodies.
4. Study the mycelium of a culture of Rhizoctonia. Note the characteristic branches of the
hyphae. The branches arise at right angles from below the septa and show distinct
constriction at the point of origin.
5. Study the culture of S. rolfsii. Observe the thick strands of white mycelium and the small
globose, sclerotia typical of this fungus. Section a sclerotium and study the internal
structures.

[33]
EXERCISE NO. 05
Staining and Identification of Plant Pathogenic bacteria
STAINING
1) SIMPLE STAINING OF BACTERIA
Materials:
Bacterial culture, Ethylene blue or Carbol Fuchsin, Slides, Spirit lamp, Glass wash
bottle, Muslin cloth, Microscope, Slide holder etc.
Procedure:
1. Clean the slide with detergent powder to wash off greasy surface. Dry the slide in air.
2. Sterilize the slide over flame on both the slides.
3. Preparation of smear: A little drop of bacterial suspension is mixed in distilled water
and placed on the slide, spread it uniformly and thinly with a glass rod or needle to form a
very thin film of smear.
4. Drying: Allow the smear to dry in air only. Do not dry on flame. They form clusters on
flame.
5. Fixing: Warm the slide slightly by passing through the flame two to three times so that
the bacteria get fixed on the slide.
6. Staining: Place two or three drops of any simple stain over the smear and allow to react
for specific time. (Methylne blue 1 to 1 ½ minutes, Carbol fuchsin 5 to 10 seconds).
7. Washing: Wash the slide with gentle stream of water; wipe out the lower surface and dry
the upper surface in air.
8. Mounting: Observe under low power. Select a good spot. Bring it in the centre, then
adjust under high power (and oil immersion objective).
Composition of Methylene blue (aqueous 3 %)
Methylene blue : 0.30 g
Ethanol (95 %) : 30.0 ml
Distilled water : 100 ml
Dissolve methylene blue in ethanol and then mix in distilled water.
Class work:
1. Prepare a stained smear of Bacteria.
2. Record the shape and cell grouping of Bacteria.

2) GRAM’S STAINING OF BACTERIA

This is a type of differential staining which divides bacteria into two groups namely
(1) Gram +ve and (2) Gram –ve Bacteria
Principle:
The organisms are first treated with main stains and then subjected to decolouring
agent. Later on, they are counter stained with other dye. The organisms which retain the
colour of the main stain are called as “Gram positive”, whereas those loose the colour of the
main stain during decolourization and take up the colour of counter stain are called as a
“Gram negative”.
The stain was first used by Christien Gram in 1984 to demonstrate the proportion of
bacteria in the diseased tissue. There are different modifications of Gram staining using the
same principles.
[34]
Materials:
Glass slide, 24 hours fresh bacterial culture, Gram A and Gram B solution, Gram’s
Iodine solution, 50 % Acetone-Alcohol solution, Basic fuchsin; Spirit lamp, Glass rod, Wash
bottle, Muslin cloth, Slide holder, Microscope etc.
Procedure : Kopeloff and Beerman’s method :
1. Prepare a thin smear, dry in air and fix on flame.
2. Cover the smear with Gram “A” and Gram “B” solution in 3:1 proportion (6 drops of
Gram `A’ and 2 drops of Gram `B’). Allow it to react for 5 minutes.
3. Treat with Iodine solution (Mordant). This helps in fixing the colour of the main stain. It
is done by dipping the slide in a jar containing Iodine solution for 2 minutes.
4. Decolourization: Pass the slide, serially through three jars containing 50 % Acetone-
Alcohol solution.
5. Wash with water and dry in air.
6. Counter stain with fuchsin for 20 to 30 seconds.
7. Wash the slide in air and examine under microscope under high power.
Results:
If the bacteria retain colour of main stain and appear violet or blue, they are “Gram
+ve” and if they take up the colour of counter stain showing red colour they are “Gram –
ve”.
Definitions:
1. Mordant: It is a chemical agent which helps in fixing the colour of the main stain e.g.
Iodine solution.
2. Decolourising agent: It is a chemical which decolourises (removes) the colour of the main
stain and thus the colour is lost.
3. Counter stain: It is stain used for staining after the main stain is decolourised eg. Basic
fuchsin, Safranin.
Composition of Stains
S.N. Stain Composition

i) Main stain (A) Crystal or Gention violet 1.0 gm

Distilled water 100 ml

1.
ii) Gram (B) Sodium carbonate NaHCO3 1.0 g

Distilled water 20 ml

Counter stain Basic fuchsin 1.5 g

2.
Distilled water 100 ml

Mordant Iodine crystals 2g

3.
NaOH (N/10) 10 ml

Make volume up to 100 ml of NaOH

[35]
Examples of Gram +ve and Gram –ve Bacteria :
S.N. Gram + ve Bacteria Gram –ve Bacteria

1 Bacillus subtilis Escherichia coli

2 Bacillus anthracis Azotobacter chroococcum

3 Lactobacillus bulgaricus Rhizobium leguminosarum

4 Mycobacterium tuberculosis Xanthomonas campestris pv. citri

5 Streptomyces scabies X. campestris pv. malvacearum

6 Corynebacterum diphtheriae Ralstonia soalanacearum

7 Clostridium tentani Erwinia amylovora

8 Diplococcus pnemoniae ----

9 Mycobacterium lepreae ----

10 Streptococcus lactis ----

Class work:
1. Prepare stained smear from curd and record Gram reaction.
2. Draw a neat sketch of Gram +ve and Gram –ve Cells.

………..

[36]
3. IDENTIFICATION OF PLANT PATHOGENIC BACTERIA
When a unknown bacterium is isolated in laboratory in the pure form, it is usually
identified by various methods viz. morphological characters, arrangement of cell cultural
(growth) characteristics on agar and in broth; the gram stain and other staining reactions
absence or presence of motility and physiological and biochemical tests.
1. Morphological Characteristics:
Shape - Spherical (Coccus),short-rods,long rod (Bacillus), filaments,
commas, spirals (Spirllum)
Cell Arrangement - Bacillus: Monobacillus (single), Diplobacillus (Pair), Streptobacillus
(chain), Palisade, Trichome, Rosette.
Coccus: Monococcus (single), Diplococcus (Pair), Streptococcus
(chain), Tetrad (four cells), Sarcina (cube of eight cells),
Staphylococcus (irregular).
Capsules - Present or absent.
Gram stain - Positive or negative.
Spore stain - Non-spore former, spore former (central, sub terminal, terminal).
Buds or sheaths - Positive or negative.
Motility - Motile or non-motile.
2. Cultural Characteristics on the Agar plate
Nutrition - Autotroph, heterotrophy
Colonies - Golden, yellow, white, glistering
Temperature - Minimum, maximum, optimum.
Growth - (agar media) absent (0), slight (+), moderate (++), abundant (+++).
Form - Circular, irregular, rhizoid.
Margins - Entire, lobate, undulated, serrate, filamentous.
Elevation - Flat, raised, convex, umbonate.
Density - Opaque, translucent.
3. Growth on Broth Media:
Surface growth - Ring, pellicle, none.
Clouding - Slight, heavy, none.
Sediment - Abundant, scanty, none, granular, flaky or flocculent
4. Biochemical Characteristics / Tests:
Perform the following biochemical tests with the bacterial culture by inoculating it into
different media, slants and broths as given below:
Carbohydrates fermentation:
Phenol red glucose broth
Phenol red lactose broth
Phenol red sucrose broth
Catalase activity - Trypticase soy agar slant
Litmus milk reactions - Litmus milk
H2S production - SIM medium
Indole production - SIM medium
Nitrate reduction - tryptycase nitrate broth

[37]
 Methyl red test - MR-VP broth
Voges-Proskauer test - MR-VP broth
i. Citrate utilization - Simmon’s citrate agar slant
ii. Urease activity - Urea broth
iii. Strach hydrolysis - Strach agar plate
iv. Lipid hydrolysis - Tributyrin agar plate
v. Gelatin hydrolysis - Nutrient gelatin deep tube
vi. Oxidase test - Tryticase soy agar plates
Incubation all the inoculated deep tubes, slants at 25oC and 37oC for 24 to 72 hours.
Biochemical Characteristics / Tests:
Tests Results
Carbohydrate fermentation (a) Glucose Red to yellow
(b) Mannitol Red to yellow
(c) Lactose Red to yellow
(d) Sucrose Red to yellow
Lactose fermentation Purple to pink
Gas formation; separation of the curd or
development of cracks or fissures within the
curd.
Litmus milk reaction Litmus reaction Purple to white or milk coloured. Proteolysis;
deep purple band on the upper portion and
whey- like brownish translucent medium.
Alkaline reaction: Deep blue (unchanged).
Starch hydrolysis A clear zone surrounding the bacterial growth.
Gelatin hydrolysis Liquefied cultures following incubation.
Casein hydrolysis Clear area surrounding the bacterial growth.
Lipid hydrolysis Clear area surrounding the bacterial growth.
Hydrogen sulfide Black precipitate
production
Indole production Formation of red layer
Methyl-red test IMViC Presence of red layer
Voges-proskauer (VP) test Deep rose (pink) colour
Citrate utilization test Green to blue
Urease test Red to deep pink
Nitrate reduction test Red colour
Catalase activity Bubbles of free oxygen gas
Oxidase test Development of pink, then maroon and finally
black colouration on the bacterial colonies.
Observations:
1. Examine the trytycase soy agar plate and slants for the cultural characteristics and the
temperature at which the culture grew best.
2. Record observations and results for the various biochemical tests performed with the
culture in the form of a table as given below

[38]
EXERCISE- 06
Study of Phanerogamic Plant Parasites
General comments: Some flowering and / or seed producing plant parasitize fruit trees,
forest trees and plantation. Both roots and stem/branches are targeted plant organs. Cuscuta
(Amarbel, Dodder) and Striga (Witch weed) parasitize stem and root only partially.

Procedure, observation and record:

1. Collect the parasite from their ecosystem.

2. Make a rough sketch showing host parasite relationship as shown in the Fugues.
3. Study complete plant anatomy and every morphological details- stem (colour, length,
presence/absence of leaves, type of leaves, colour; flowers (colour, numbers). Pod
formation, seed colour numbers, germination, etc.
4. Cut T.S. of the part showing apparent host x parasite relationships.
5. Locate absorbing organs (haustoria).
6. Preserve (both wet and dry for lab. record)

Flowering Plant Parasites (Phanerogams)

Most of the diseases are caused by fungi bacteria and viruses. There are few seeds
plants called flowering parasites (Phanerogams) which are parasitic on living plants. Some of
these attack roots of the host, while some parasites on stem. Some are devoid of chlorophyll
and entirely dependent on their host for food supply, while other have chlorophyll and obtain
only mineral constituents of food from host by drawing nutrition and water they are called as
Holoparasites or complete or total parasite. They have haustoria as absorbing organs, which
are sent deep into the vascular bundle of the host to draw nutrients, water and minerals.

Classification of Flowering Plant Parasites:

There are two types of parasites.

1) Root Parasites:
i) Striga (Partial root parasite)
ii) Orobanche (Complete root parasite)
2) Stem Parasites:
i) Dodder (Cuscuta) (Complete stem parasite)
ii) Loranthus (Partial Stem parasite)

1. Root Parasites:
1. Total or Complete or Holoparasite:
Orobanche (Broom rape or Tokra)
It is annual flashy flowering plant growing to height of about 15-50 cm long, yellow
or brownish colour and covered by small thin scaly leaves. Flowers appears in the axil of
leaves are white or tubular. Fruits appears in the axil of leaves are white or tubular. Fruits are
capsule containing and seeds are very small, black in colour remain viable for several years.
The haustoria of parasite penetrates into the roots of hosts and draw its nourishment. The

[39]
Fig. 11 : Phanerogamic Plant Parasites
[40]
growth of the plant is retarded, may die some times. It attacks tobacco, tomato, brinjal,
cabbage, cauliflower.

2. Hemi Partial or Semi Root Parasite:

Striga (Witch Weed or Turfula or Talop)
Family -Scrophulariaceae
It is a small plant with bright green leaves grows upto height 20-60 cm leaves beers
chlorophylls and developed in clusters of 10-20 % host plant. They are obligate parasites
therefore, do not obtain all their nutrient from their host root. Flowers are pink in colour, seed
are very minute and produce in grate number 5000 to 100000 seeds plant per years. One
flower contain 1200-1500 seeds and remains viable upto 12-40 years. Dissemination takes
place with rain water, flood, wind and irrigation water. It cause yellowing and wilting of host
leaves. It attacks sugarcane jowar, Maize, cereals and millets.
2. Stem Parasites:
A. Total or Complete or Holoparasite:
Cuscuta or dodder (Amarvel, Lovevine)
Family -cuscutaceae
Genus – Cuscuta
It is non-chlorophyllous, leaf less parasitic seed plant.
It is yellow pink or orange in colour and attached to the host. They do not bear leaves
but bear minute function less scale leaves is produces flower and fruits. Flowers are white,
pink or yellowish in colour and found in clusters. Seed are form in capsules. A single plant
may be produce 3000 seeds.
The first appearances of parasites is noticed as thread like leaf less stem which devoid
of green pigment and twine around the stem or leaves of the host.When stem of parasitic
plant comes in contact with host, the minute root like organs. i.e. hausteria penetrates into the
host and absorbs. When the relationship of the host is firmly established, the dodder plant
looses the contact from soil.
These affect plant get weakened and yield poorly the seeds spread by animals, water
and implements and remain viable when condition are unfavorable.
It attacks berseem alfalfa, clover, flax, onion, potato, ornamental and hedge plants.

B. Partial, Semi or Hemi Stem Parasites:

Loranthus
Family- Loranthaceae
It is a partial parasite of tree trunks and branches with brown stem, dark green leaves but no
roots.
1. Stem is thick and flattened of the node; appear in clusters at the point of attack which can
be easily spotted on the trees.
2. At the point of attachment with the tree, it shows swellings or tumourous growth where the
haustoria are produced. It produces flowers which are long, tabular, greenish, white or red
colour and found in clusters. It produces fleshy berries with single seed.
3. The affected host plant becomes stunted in growth and dispersal of seed is mostly through
the birds and animals. It attacks mango, citrus, apple and guava

********

[41]
EXERCISE 07
Identification and transmission of plant Virus

General comments: Plant viruses are nucleoprotein, submicroscopic; multiply only within
living cells and causes diseases. The virus particle (Virion) is composed by NA and protein.
Plant viruses contain either RNA or DNA but not both.
Procedure, observation and record
Identification
The virus concerned is inoculated on plants grown under controlled conditions. The
symptoms that develop carefully characterized and observed. The record of the HOST
RANGE and type of symptoms help in identifying the viruses.
After studying host-range and symptoms, transmission of viruses is studied.
Serological methods such as ELISA or fluorescent antibody microscopy are done.
Next step is electron microscopy to exactly know the shape of virus.
Indicator plants are inoculated with the virus and characteristic symptoms are noted.
Transmission:
1. Mechanical Transmission:
Freshly collected infected plant organs/ tissues are thoroughly macerated in 0.1 M potassium
phosphate buffer solution at pH 7-7.5 using mortar and pastle.
The sap thus collected is passed through muslin cloth to remove plant tissues.
Susceptible host plant species already raised under glasshouse condition are dusted with fine
abrasive powder carborandum (300- 600 mesh).
Therefore, the sap is inoculated by gently rubbing the leaves using cheese cloth, glass spatula
or brush.
Incubate the plants under suitable (glasshouse) conditions and record the time and type of
symptoms that develop.
2. Insect Transmission:
Several insects like aphids, leaf hopper, plant hopper, white flies, mealy bugs, thrips, mite,
beetles and grasshoppers are known to transmit viruses from disease to healthy plants.
Among them, white flies (Bemissia tabaci) and aphis (Myzus persici) are important.
Objective: To study transmission of virus by an aphid
Procedure, observation and record:
Collect aphids from natural habitat and watch a few single aphids under a dissecting
microscope for the appearance of young.
Take newly born aphid (which will not be viruliferous) on the tip of small brush and transfer
to a host plant already grown in the glasshouse. The host should be a non- host to virus under
study.
Keep the plants under a large chimney with cheese cloth over the open ends, or under a box
with transparent top and cloth sides, to prevent escape of the aphid.
Allow aphids colony to develop for a week or so.
Remove about 100 aphids from the stock culture and allow them to starve for about an hour
in the Petri dish.
Now transfer about 80aphids to virus infected plants for infection feeding.

[42]
After one minutes of infection feeding, carefully remove 15 aphids and place one on each of
15 healthy (test) plants for test feeding.
After 5 minutes, repeat the operation by moving 15 more aphids from the virus source to
healthy plants.
Do the same after 15 and 60 minutes.
For control, allow non-viruliferous stock aphids to feed for 5 minutes, on healthy leaves and
then transfer them to 15 teat plants.
After leaving the aphids on the test for an hour or so, treat the plants with some proprietary
insecticide to kill the aphids.
In 7-10 days, some of the inoculated plants will show symptoms. Tabulate the data, using the
number of plants infected as the numerator and the number of plants exposed to aphids as the
denominator.

********

[43]
EXERCISE 08
Study of morphological Features and Identification of Plant Parasitic Nematodes

Identification of Some Common Genera of Plant Parasitic Nematodes:

1. To identify plant-parasitic nematodes from soil and root samples, the following procedure
is recommended.
2. Examine the nematode under the lower power (10mm) objective of your microscope and
estimate the approximate body length. It helps to know the diameter of the microscope
field. This can be measured exactly be use of a stage micrometer, or approximately by
looking at a millimeter scale. For many microscopes, the field of the 16 mm objective
with 10X wide-field oculars is about 2.0 mm. A nematode extending halfway across the
field would be 1.0 mm long; a nematode one - fourth of the field diameter would 0.50
mm long. And so on.
3. Using the medium power (4 mm) objective, observe the stylet. Plant parasitic nematodes
always have stylet. If the nematode has no stylet, the species under study is probably not
plant –parasitic.
4. Observe the length of stylet relative to the width of the lip region of the nematode. Short
stylets are no more than 2 or 3 times the width of the lip region, long stylet are more
than 5 times the width of the lip region, and stylet of intermediate length are between
these two.
5. Observe the shape of the oesophagus. Determine if this has a medium bulb, than if it has
posterior bulb. If the oesophagus has a posterior has a posterior bulb, it is usually easy to
see. If there is doubt, you can usually assume that the oesophageal glands are in a lobe
overlapping the intestine.
6. Locate the vulva. If this is near the middle of the body, the nematode has two ovaries. If it
is on the posterior one-fourth of the body, the nematode has only one ovary. Observe the
spicules of the male. If neither vulva nor spicules are present, the nematode is a larve.
Look at other specimens, if all are larvae, search for adults.
7. Observe the tail shape.
8. If you are in doubt as to any of the above characters in adult nematodes, study other
specimens. Very often a character which is obscure on one specimen will be clear in
another.

Common Genera of Plant Parasitic Nematodes: Root-Knot Nematode, Meloidogyne spp.

Systematic position:
Order - Tylenchida
Sub order - Tylenchina
Super family - Tylenchoidea
Family - Heteroderidae
Sub family - Meloidogyninae
Genus - Meloidogyne
Diagnosis
Female: swollen, sub spherical, sedentary, cuticle annulated. Stylet with well developed
basal knobs. Gonads didelphic prodelphic, elongated and convoluated. Eggs laid in gelatinous
[44]
matrix outside the nematode body. Annus and vulva terminal surrounded by particular striae
on the cuticle known as perineal pattern.
Male: long and vermiform, spear well developed, tail short without caudal alae.
Second stage juvenile: Small vermiform, stylet small with slight knobs. Tail elongated
coided without hyaline portion.
Third and fourth stage juvenile: Swollen with spike tail, without stylet, found inside the
roots.

Important species:
Meloidogyne incognita
M. javanica
M. arenaria
M. hapla

Reniform nematode, Rotylenchulus Reniformis

Systematic position:
Order - Tylenchida
Sub order - Tylenchina
Super family - Hololaimoidea
Family - Hoplolaimidae
Sub family - Hoplolaiminae
Genus - Rotylenchulus

Diagnosis
Mature Females: Kidney shaped, swollen, anterior portion inside the foot tissue. Stylet
strong, amphidelphic, tall short, peg like, eggs laid in gelatinous matrix.
Immature Females: Vermiform, “C” shaped, cephalic sclerotization and stylet well
developed. Opening of dorsal oesophageal gland much posterior in carpus region,
oesophageal glades overlapping, vulva in the middle of the body, amphidelphic. Tail
elongate, conoid.
Male: Stylet and oesophagus reduced, caudal alae rudimentary.
Second stage juvenile: similar to immature females but fir sex organs.
Third and fourth stage juvenile: with superimposed cuticle.

Root Lesion Nematode, Pratylenchus species

Systematic position:
Order - Tylenchida
Sub order - Tylenchina
Super family - Tylenchoidea
Family - Pratylenchidae
Sub family - Pratylenchinae
Genus - Pratylenchus

Diagnosis:
[45]
Female: Vermiform, liop region flat infrong with rounded outer margins, stylets 14-20.4 µm
long with ronded basal knobs. The oesophagum overlaps intestine venterally. Ovary single
directed anteriorly, vulva posterior. Tail cylindrical with smooth of crenate terminus.
Male: with smooth or crenate terminus. Stylet and oesophagus fully formed, tail elongate
conoid enveloped with caudal alae.

Important species:
Pratylenchus coffee
P. zeae
P. thornei

4. Spiral Nematode, Helicotylenchus species

Systematic position:
Order - Tylenchida
Sub order - Tylenchina
Super family - Tylenchoidea
Family - Hoplolaimidae
Sub family - Rotylenehoidinae
Genus - Helicotylenchus

Diagnosis:
Female: Body acute to “C” shaped when relaxed, annules district, lop region hemispherical,
slightly offset, stylet moderately long, dorsal oesophageal gland orifice typically located
more than one half stylets lengh posterior to stylet knob ovaries two, vulva posterior to
middle of body. Tail of females founded to nearly pointed, often with short projections on
ventral side of males, short and with bursa.
Male: similar to female except sexual dimorphism.

5. Cyst Nematode, Heterodera and Globodera spp.

Systematic position:
Order - Tylenchida
Sub order - Tylenchina
Super family - Tylenchoidea
Family - Heteroderidae
Sub family - Heteroderinae
Genus - Heterodera and Globodera

Diagnosis:
Female: Body ovoid to globes with protruding nech and vulva cone, white in colour, cuticle
with lace-like or zig-zag pattern. Stylet strong with well developed knobs; genital track
didelphic, prodelphic; eggs retain inside body and some may be laid in gelatinous matrix.
Male: Body long, vermiform with posterior twist. Stylet fairly stout with well formed knobs.
Tail short, caudal alae absent.

[46]
Second stage juvenile: vermiform, stylet fairly stout with well formed knobs. Oesophagus
overlapping intestine. Tail elongated, conoid and with hyaline portion.

Third and fourth stage juvenile: with stylet no spike like tail. Cyst- lemon shaped with a
vulval cone. Vulva surrounded by transparent thin walled area which breaks down on
maturity to form birth pore/ fenestra. Ambi or bifenestrate. Eggs/ juveniles retained inside
body.

Important species:

Sr.
Important species Distribution Host
No.
Cereal cyst nematode, North India Wheat and Barley
1
H. avenae
Pigeonpea cyst nematode, Wide spread Pigeonpea, mungbean,
2 H. avenae urdbean, cowpea,
sesame, clusterbean
3 Maize cyst nematode Wide spread Maize
Rice cyst nematode Kerala, Madhya pradesh, Rice and Banana
4
Orissa, West Bengal

6. Citrus Nematode, Tylenchulus semipenetrans

Systematic position:
Order - Tylenchida
Sub order - Tylenchina
Super family - Criconematoidea
Family - Tylenhulidae
Sub family - Tylenchulinae
Genus - Tylenchus
Diagnosis:
Female: small sized, neck long inside the root tissues, posterior portion swollen. Stylet small,
oesophagus criconematid type, excretory pore near middle of the body, post uterine sac
present.
Male: Stylet and oesophagus degenerated, caudal alae absent.
Second stage juveniles: Small vermiform, stylet short, oesophagus criconematid type,
excretory pore near mid body, tail conical.

********

[47]
EXERCISE NO. 09
Preparation of Media
Objective:
Plant pathogens such as fungi and bacteria can be cultivated in -vitro on various
Culture medium. Which can be either solid or liquid. Due to great varity of a nutrient
requirement of different pathogens, the composition of the media used for their cultivation
varies.
Culture Medium:
Nutrient or combination of nutrients used for the growth of microorganisms is called
as a culture medium.
Culture:
It is the growth of microorganisms on a medium. If a culture contains the growth of
only one type of microorganisms it is called as pure culture, on the other hand when it
contains growth of more than one type of microorganisms it is called as “Mixed” or
“Contaminated culture”.
The main elements required for bacterial growth are carbon and nitrogen with certain
amount of minerals and vitamins with water. Carbon is usually supplied as carbohydrates,
water supplies the hydrogen and nitrogen can be supplied in organic or inorganic form.
(Plant or animal tissue extract supplying organic nitrogen with carbon are usually more
favorable). In the laboratory Peptone is commonly employed in the form of organic nitrogen,
it also supplies carbon. Since different organisms require various nutrients in different
proportions, general media supplying all the essential nutrients are commonly used e.g.
Nutrient Agar for bacteria and P.D.A. (Potato Dextrose Agar) for Fungi.
The medium is prepared by mixing or dissolving nutrients in water that makes a liquid
medium. If a solidifying agent (Agar or gelatin) is added a liquefiable solid medium can be
prepared.
Requirements of a good culture medium:
1. It should supply required nutrients in proper proportion.
2. It should have a proper pH.
3. It should be free from all living organisms and unwanted materials.
4. It should have proper moisture content.
5. It should have desirable physical properties e.g. solid, liquid.
Nutrient substances commonly used to prepare media:
1. Beef extract:
It is an aqueous extract of beef tissues concentrated to a paste. It contains the water
soluble substances salts, enzyme etc.
2. Peptone:
It is the principle source of organic nitrogen prepared from the digestion of protein
materials such as meat, casein and gelatin.
3. Gelatin:
It is an incomplete protein compound prepared from animal bones, horns, hoofs etc.
It is used by certain bacteria producing gelatinase called gelatin liquefying organisms. It
melts at 37 oC and solidifies a 20 oC. The medium containing gelatin is transparent and water

[48]
of condensation is not formed on its surface. It is used @ 10 to 20 % in the medium for
solidification.
4. Agar-Agar:
It is a complex carbohydrate prepared from sea weed or marine algae. It is used as
solidifying agent in culture media @ 1.5 to 2 per cent concentration. It melts at 98 oC and
solidifies at 37oC. The medium is opaque when solid and covered by water of condensation.
It does not supply any nutrient to the organisms.
Classification of media:
1. According to consistency:
a. Liquid: Nutrient broth (NB, Richard’s medium)
b. Solid: Potato cylinders
c. Liquefiable solid: PDA, NA
d. Solidifiable liquid: Egg Albumin, Blood serum.
2. According to composition:
a. Synthetic media:
Exact chemical composition of ingredients is known e.g. Richard’s medium. Ashby’s
medium, Conn’s medium.
b. Non-synthetic media:
Exact chemical composition is not known e.g. P.D.A., N.A., Malt extract etc.
3. According to use:
a. General or common use: e.g. P.D.A.; N.B.; N.A.
b. Selective or special medium: Used for specific organisms. Ashby’s medium for
Azotobacter and Yeast Manitol Agar medium for Rhizobium.
4. Differential media:
These are used to detect the production of certain characteristic growth of the various
species of bacteria e.g. Eosine methylene blue agar for testing coli form bacteria.
Solid media are used for studying cultural characters, for maintenance and obtaining
pure cultures by streaking, planting etc. While liquid media are commonly used for large
scale multiplication of the organisms.
Comparison between Agar - agar and Gelatin:

S.N. Particulars Agar-agar Gelatin

1. Source Red algae from sea Animal tissues i.e. hoofs, bones and
weed ligaments.
2. Composition Complex Complex nitrogenous compound
carbohydrates
3. Quantity required 1.5 to 2.0 % 12 to 15 % or more
for solidification
4. Melting point 98oC 37oC
5. Solidifying point 37oC 20oC
6. Availability as a Does not supply Some bacteria can use it as a food
food for microbes any nutrient (Liquefaction of gelatin).

[49]
 Agar agar is preferred as a solidifying agent because less quantity is required. It
remains solid at room temperature and is not acted upon or used by micro-organisms. Gelatin
is used to study the liquefying ability of bacteria (Liquefaction)
A. For Fungi: For artificial culturing of fungus in laboratory, the general media used is
Potato Dextrose Agar (PDA) or Czapek-Dox Agar
1) Composition of P.D.A. (Potato (peeled) Dextrose Agar):
Materials : Composition
1. Potato (peeled) : 200 g
2. Dextrose : 20 g
3. Agar agar : 20 g
4. Water : 1000 ml
Method:
The boiled and filtered extract of potatoes is mixed with dextrose and agar agar,
dissolved by indirect heating (in boiling water). The medium is distributed in tubes or flasks,
plugged and sterilized in autoclave at 15 lbs, pressure for 15 minutes. Tubes are slanted
during solidification to form slants. This is general medium for fungi.
B. For Bacteria: NA is the common medium used for isolation of bacteria in laboratory, 1)
Composition of N.A. (Nutrient Agar):
Materials : Composition
1. Beef extract : 3g
2. Peptone : 5g
3. Agar agar : 20 g
4. Water : 1000 ml
Weigh the ingredients and dissolve in required quantity of water by gentle heating and
stirring. Cool and adjust the pH is to 7.0, filled in tube or flasks, plugged and sterilized in
autoclave at 15 lbs pressure for 20 minutes.
Preparation of common media:
1. Nutrient Broth:
Materials : Composition
1. Beef extract : 3g
2. Peptone : 5g
3. Water : 1000 ml
The ingredients are mixed in water with slight heating. The pH is adjusted to 7.0,
filled in tube or flasks, plugged and sterilized in autoclave at 15 lbs pressure for 15 minutes.
2. Nutrient Gelatin:
It is prepared by adding gelatin 12 to 15 % to nutrient broth. The tubes are not slanted
but stabs are prepared.
3. Potato cylinders:
Potato cylinders of required size are cut from healthy potatoes with help or cork borer.
One end is cut in slanting position with knife, place in roux tube with slant facing upwards
and moist cotton at bottom. It is then plugged and sterilized in Arnold Steam Sterilizer for 3
successive days (100 to 110oC for 30 minutes on each day).
Adjustment of pH of culture Medium:

[50]
 pH is the measurement of acidity or alkalinity of a solution or it is the hydrogen-ion
concentration of medium. Different organisms grow well in the culture medium having
specific pH, Bacteria require slightly alkaline medium (pH 7.0 to 7.5), while fungi require
slightly acidic media (pH 6.0 to 7.0).
pH range 0 to 7 : Acidic (Distilled water)
pH range 8 to 14 : Alkali
Buffers:
Buffers are the substances which prevent rapid change in pH of a medium. Due to the
activities and respiration of micro-organisms, the pH of a medium is changed and hence it is
prevented by buffer e.g. Peptone, Carbonate protein, phosphates etc.
Filling and Pluging:
After adjustment of pH, the medium is distributed in suitable containers (tubes or
flasks). Before making it free from unwanted organisms, non-absorbent cotton stopers
(plugs) are applied to the mouth which allow easy exchange of gases but prevent entry of
unwanted organisms.
A suitable size of cotton piece, thickened in the centre is inserted in the mouth by
forcep or glass rod or pencil. Alternatively the piece of suitable size is held tightly and then
inserted by hand.
Requirement of a good plug:
1. It should be tight enough to bear the weight of the container.
2. It should be “1 to 1 ½” inside the mouth.
3. It should be easily removable or replaceable.
Preparation of slants:
After preparing agar medium the ingredients are dissolved by heating on a flame or in
autoclave. Then the tubes are filled in with 1 ml of the medium in each. The tubes are
plugged and sterilized in autoclave at 15 lbs. pressure for 15 minutes. After sterilization the
tubes are kept in slanting position for solidification.
Class work:
1. Observe the material used in the preparation of culture media.
2. Draw neat sketches of cork borer, scalpel, slant, stab.
3. Prepare NA, PDA and Ashby’s Agar media.
4. Prepare slants and stabs.
********

[51]
EXERCISE NO. 10
Isolation and purification of fungi and bacteria

Object: To isolate bacteria and fungi and to prepare their pure culture.
Principles: The microorganisms are required to be grown in pure form. Developed from
single cell on culture media for their detail study of morphology, physiology and cultural
characters.
Isolation
It is technique of separating the organisms from the diseased host tissue and growing them on
artificial media for the purpose of further study or investigation.
Culture: It is the growth of microorganisms on culture medium.
Colony: Growth of microorganisms usually round in appearance on a solid medium resulting
from a single cell or group of similar cells.
Contamination: Growth of desired microorganism along with other undesired
microorganisms on culture medium.
Fungal Isolation:
I. Isolation from diseased host tissue:
1. Wash the diseased part with tap water to remove dirt and soil and cut the affected part in to
small convenient pieces of 2 to 3 mm size in such a way that half the portion of healthy tissue
must be present on bits.
2. Surface sterilization with sodium hypocloride (NaOCl) solution (1-2%) by immersing the
pieces for 2-5 minutes depending upon the type of tissue.
3. Rinse the pieces of the diseased bits in distilled sterilized water, for three or four times so
as to remove the traces of disinfectant i.e. Sodium hypochloride.
4. Place the pieces of bits on slants or petriplates with pre poured potato dextrose agar
medium.
5. Incubate the petriplates in inverted position at optimum temperature 25-300C for 7 days.
6. After incubation period there is growth non the diseased bits, transfer the growth of
organism in new slants. Purify the culture either hyphal tip isolation or by single spore
isolation method.
2. Bacterial Isolation:
I.Dilution and Plating from soil:
1. Prepare 5 to 7 water blank by pouring 9 ml sterile water in sterile test tube.
2. Prepare nutrient agar medium.
3. Lift loopful quantity of contaminated bacterial culture with inoculating needle under
aseptic conditions using Laminar Air Flow Cabinet.
4. Dispense the culture in first water blank and this will be 10-1 dilution.
5. Take one ml suspension from 1st water blank and pour in 2nd water blank and this will be
10-2 dilution. Like wise prepare 10-5 to 10-7 serial dilution.
6. Take 1 ml from 10-5 to 10-7 dilution each and pour in Petriplates.
7. Pour 15 ml nutrient agar medium in the plates containing bacterial suspensions.
8. Incubate the plates at 30OC for 3-4 days.
Observations:
1. Observe the colonies of desired bacteria in the plates and transfer on agar slants.
[52]
2. Isolation from diseased tissue:

1.Collect samplesto be infected with the bacteria.

2. Wash the diseased part with tap water to remove dirt and soil and dip them in sodium
hypo-cloride (NaOCl) solution (1-2%) by immersing the pieces for 2-5 minutes depending
upon the type of tissue.

3. Rinse the pieces of the diseased bits in distilled sterilized water, for three or four times so
as to remove the traces of disinfectant i.e. Sodium hypochloride. cut the affected part in to
small convenient pieces of 2 to 3 mm size with sterilize scalpel. The bacteria willooze out in
about 5 minutes
1 Streak Plate Method:
1. Prepare nutrient agar plates.
2. Lift loopful bacterial contaminated culture and dispense in 1 ml sterile water in test tubes
under aseptic conition using Laminar Air Flow Cabinet.
3. Dip the inoculating needle in bacterial suspension and streak on agar medium by making
zigzag or straight and cross lines in Prelates.
4. Incubate the plates at 30 oC for 3 to 4 days.
Observations: Select well isolated colony and transfer on nutrient agar slants
II. Pour Plate Method:
Materials:
Mixed culture suspension, nutrient agar medium in a test tube or sterile water blanks,
inoculation needle, Bunsen burner, sterile petri-plates .
Procedure:
1. Melt three cubes of nutrient agar medium(12-15ml) & cool them to 450C.
2. Transfer one loopful of the sample of mixed culture provided, to the first tube of melted
nutrient agar.
3. Mix the inoculums well by thumbing the tube with fore finger. (The success of the method
depends upon through mixing & even distribution of the cell.)
4. Transfer two loopful of mixed inoculum from the first tube to the second tube of melted
agar & mixed contents well as stated above.
5. Transfer similarly from the second tube to third tube of agar & mix thoroughly.
6. Pour each of the tube into separate petri-plates by rotating clockwise & anti-clockwise
direction to have uniform distribution.
7. Incubate the petri-plates at room temperature for 3-7 daya depending on the organism.

Observations:

8. Examine the relative size of the colonies on crowded & on sparsely colonized plate.
Well isolated colonies are larger, with distinct characterize the shape, color & texture
of the colonies developed. Pick up the single colony & transfer to NA slant tubes.

Purification of fungal culture:

1. Following two methods does the purification of the fungal culture.

[53]
I. Hyphal Tip Isolation method:
1. In this method initially the growing hyphal tip is selected and transferred in the slant to
obtain the pure culture
2. Grow the colony of the fungus on the PDA medium.
3. Select the single growing hyphal tip of the mycelium with the help of microscope and
mark it with glass marking pencil.
4. Cut the marked position with the help of cork borer and transfer it on the PDA slant
medium and incubate at the temperature 27 ± 10C and observe for pure growth of culture.
II. Single spore isolation method:
1. Prepare the spore suspention from the fungus in order vto have 106 spore/ml.
2. Add above 10 ml of spore suspention in 250 ml of liquefied potato dextrose agar medium
when tepm. Of medium is about 400c.
3. Add the medium in sterilized petriplates, allow to solidify the medium.
4. After the solidification of the medium locate the single nspore with the help of
microscopeand mark with a glass marking pencil.
5. Cut the marked portion with the help of cork borer and transfer it on PDA slant.
6. Incubate the slants at the temp. 27 ± 20C and observe for pure growth of culture.
Purification of Bacterial culture:
The cultures of bacteria are purified by following three methods.
A) Streaking method B) Spread plate method
1 Streak Plate Method:
1. Prepare nutrient agar plates.
2. Lift loopful bacterial contaminated culture and dispense in 1 ml sterile water in test tubes
under aseptic conition using Laminar Air Flow Cabinet.
3. Dip the inoculating needle in bacterial suspension and streak on agar medium by making
zigzag or straight and cross lines in Prelates.
4. Incubate the plates at 30 oC for 3 to 4 days.
Observations: Select well isolated colony and transfer on nutrient agar slants.
2 Spread Plate Method:
1. Prepare nutrient agar Petriplates.
2. Prepare suspension of contaminated bacterial culture in sterile water.
3. Deep ‘L’ shaped bent glass rod in suspension and spread on agar plates.
4. Incubate the plates at 30oC for 3-4 day.
Observations: Observe well isolated bacterial colonies and transfer on nutrient agar slants
********

[54]
EXERCISE 11
Extraction of Plant Parasitic Nematodes from soil and plant

A . Extraction of Plant Parasitic Nematodes from soil.

Cobb Sieving and Gravity Method: (Plate XV, Fig. 1-6)
1. Stir soil sample in a bucket withy water, Nematode become suspended in water
2. Pour the water through a coarse sieve into a second bucket. Nematodes pass through
sieve. Heavy soil particles remain in the first bucket. Large particles of debris are
caught by sieve.
3. Pour water from the second bucket through a fine sieve. Many small soil particles
pass through the sieve. Nematodes remain on sieve with small particles of debris.
4. Wash the fine sieve with a gentle stream of water to remove more fine particles.
5. Wash the nematodes from the inverted fine sieve a cup.
6. Pour the nematodes into a shallow dish for examination under the dissecting
microscope
Baermann Funnel Method
1. Nematode may be removed from the soil particles by use of the Baerman funnel or
bowl. Put the sample in a cup and secure a piece of cloth over the top of the cup with
a rubber band.
2. Invert the cup containing the sample in a small bowl of the water to extract the
nematodes. Active nematodes will pass though the cloth and be found in the bottom
of bowl.
3. Invert the cup in a Baermann funnel in a rack. Nematodes pass through the cloth and
collect in the rubber tube A). They can be removed by opening the pinch clamps (b)
to allow about 5 cc of water to flow out.
4. Extraction of Plant Parasitic Nematodes from Plant Tissues:
5. Place a small piece of plant tissue in a shallow dish with a little water.
6. Place the dish under the dissecting microscope and pick the tissue apart, using two
dissecting needles under the flow or medium magnification of the binocular
microscope.
Root knot nematodes:
1. If the tissue is suspected of containing Meloidogyne spp., look for egg masses
clinging to the outside of the root. These are white to brown and roughly
hemispherical. They can be easily detached and when picked apart they will be found
to contain eggs in all stages of development.
2. If the egg mass is placed on a slide and flattened by gentle pressure on the cover glass,
the eggs and larvae can be plainly seen under microscope. Look for females by
pulling the root apart with the dissecting needles, being careful not to puncture the
nematode in the galls.
Heterodera species:
1. Females which are attached to the root with only their necks embedded, are easily
seen and detached.
2. Staining and detection of root- knot nematodes from the plant tissue.

[55]
 3. For demonstration and study of the re relationship
lationship of nematodes to plants, it is often
desirable to stain nematode in plant tissue. The procedure is as follows.
Phenol ……………………… 20 ml
Lactic acid ………………….. 20 ml
Glycerine. .…………………… 40 ml
Distilled water……………….. 20ml
Add to this 5 cc of a solution made by dissolving one gram of acid fuchsin or cotton blue
(aniline blue) in 100 cc of water.
2) Free the material from soil and wash the root materials.
3) Heat the staining solution to boiling immerse the plant tissue for 11-3 min.
n. Both plant tissue
and nematodes will be stained in this hot solution.
4) Remove the material from the staining solution and wash off the excess stain with cold
water.
5) Store in a destaining solution (clear
ar lactophenol soltution without acid fuschain or cotton
blue added) until properly destained. In this solution, the plant tissues will lose its colour
slowly and turns somewhat trascluscent, leaving the stained nematodes clearly visible.
After teasing the female of Meloidogyne spp. From the 3plant tissue, observe these females
under the stereobinocular microscope for its characteristics. The female are pear shaped in
structure with beak at the anterior end and the posterior end is a bulbous structure.

Fig. 12 : Cobb Sieving Method Fig. 13 : Baermann Funnel Method

********

[56]
EXERCISE NO. 12
KOCH’S POSTULATES
Introduction:
The main aim of the Koch’s Postulates to determine or establish the host pathogen
relationship between host and a new micro-organism which is suspected to be associated
with the diseases.
Robert Koch studying with the Anthrax disease in cattle followed out certain
conditions to prove the host pathogen relationship. These principle latter recognized as the
Koch’s Postulates.
Koch’s Postulates
Association
The suspected pathogen must be found associated with the disease in all the diseased
plant examined.
Isolation
When the organism is associated with the disease, it is separated i.e. isolated from the
natural host grown under artificial conditions.
Inoculation
The pure culture of organism when inoculated into healthy host should produce
symptoms of the same disease which was caused naturally.
Re-isolation
The re-isolated organisms should be similar to the original organism inoculated. The
organism must be re-isolated from the inoculated plants and must be shown to be the same
pathogen as the original.
Example: Koch’s Postulates for bacterial pathogen.
Erwinia caratovora is the etiologic agent of soft rot of served plants. Erwinia
caratovora carrot system will be used to demonstrate Koch’s postulates.
Materials:
Infected carrot with E. caratovora, healthy carrots, Nutrient agar plates, Razor blade, Potato
peeler, Forceps, Alcohol, Sterile water, Disinfectant, Gram-stain reagents, Inoculating loop,
Spirit lamp.
Procedure:
1. Isolate the bacterial pathogen from the infected carrot.
2. Purify the bacterial culture if contaminated.
3. Prepare the bacterial smear from the pure culture and gram stain it.
4. Wash the healthy carrot well, peel them and allow to dry.
5. Surface sterilizes the peeled carrots with disinfectant.
6. Cut the carrot into slices (5 to 8mm thick) with alcohol dipped and flamed scalpel.
7. Using flamed forceps, transfer four carrot slices in sterile Petri-plates.
8. Inoculate centre of three slices each with a loopful of bacterial culture, the 4th carrot
slice should be kept as un-inoculated comparative control.
9. Saturate the filter paper with sterile water.
10. Incubate the plates at room temperature (250C) for 3-5 days or until soft rot appear.
11. Streak the inoculums from diseased carrot on nutrient agar plate.

[57]
 12. Incubate the inoculated plates in inverted positions for 45 hrs at room temperature.
13. Prepare the smear from nutrient agar culture.
14. Make a smear from diseased carrot.
15. Grams stain both the smears.

Observations:
Observe infected carrot and artificially inoculated carrot pieces for soft rot symptoms;
observe the gram stain preparation for morphology and gram reaction of all the three smears.
Results:
If the disease symptoms are produced on artificially inoculated carrot pieces and morphology
and gram stain reaction in the three smears are same, then the Koch’s postulates are proved.

********

[58]
EXERCISE 13
Study of fungicides and their formulations
A) To study different group of fungicides
FUNGICIDES
The word ‘fungicide’ originated from two latin words, viz., ‘fungus’ and ‘caedo’. The word
‘caedo’ means ‘to kill.’ Thus the fungicide is any agency/chemical which has the ability to
kill the fungus. According to this meaning, physical agents like ultra violet light and heat
should also be considered as fungicides. However, in common usage, the meaning is
restricted to chemicals only. Hence, fungicide is a chemical which is capable of killing fungi.
Fungistat
Some chemicals do not kill the fungal pathogens. But they simply arrest the growth of the
fungus temporarily. These chemicals are called fungistat and the phenomenon of temporarily
inhibiting the fungal growth is termed as fungistatis.
Antisporulant
Some other chemicals may inhibit the spore production without affecting the growth of
vegetative hypha and are called as ‘Antisporulant’. Eventhough, the antisporulant and
fungistatic compounds do not kill the fungi, they are included under the broad term fungicide
because by common usage, the fungicide has been defined as a chemical agent who has the
ability to reduce or prevent the damage caused to plants and their products. So, some of the
plant pathologists prefer the term ‘Fungitoxicant’ instead of fungicide.
Although chemicals have been used in the management of plant diseases caused by fungi,
bacteria, nematodes, viruses and other nutritional deficiencies, the use of chemicals in
controlling fungal diseases has been established than other diseases. Fungicides can be
broadly grouped based on their (i) mode of action (ii) general use and (iii) chemical
composition.
Protectant
As the name suggests, protectant fungicides are prophylactic in their behaviour. Fungicide
which is effective only if applied prior to fungal infection is called a protectant, eg., Zineb,
Sulphur.
Therapeutant
Fungicide which is capable of eradicating a fungus after it has caused infection and there by
curing the plant is called chemotherapeutant. e.g. Carboxin, Oxycarboxin antibiotics like
Aureofungin. Usually chemotherapeutant are systemic in their action and affect the deep-
seated infection.
Eradicant
Eradicant are those which remove pathogenic fungi from an infection court (area of the host
around a propagating unit of a fungus in which infection could possibly occur). eg. Organic
mercurials, lime sulphur, dodine etc. These chemicals eradicate the dormant or active
pathogen from the host. They can remain effective on or in the host for some time.
II. Based on general uses
The fungicides can also be classified based on the nature of their use in managing the
diseases.
1. Seed protectants: e.g. Captan, thiram, organomercurials, carbendazim, carboxin etc.

[59]
2. Soil fungicides (preplant): e.g. Bordeaux mixture, copper oxy chloride, Chloropicrin,
Formaldehyde, Vapam, etc.,
3. Soil fungicides: e.g. Bordeaux mixture, copper oxy (for growing plants) chloride, Capton,
PCNB, thiram etc.
4. Foliage and blossom protectants: e.g. Captan, ferbam, zineb, mancozeb, chlorothalonil
etc.
5. Fruit protectants: e.g. Captan, maneb, carbendazim, mancozeb etc.
6. Eradicants: e.g. Organomercurials, lime sulphur, etc.
7. Tree wound dressers: e.g. Bordeaux paste, Chaubattia paste, etc.
8. Antibiotics: e.g. Actidione, Griseofulvin, Streptomycin, Streptocycline, etc.,
9. General purpose spray and dust formulations.
III. Based on Chemical Composition
The chemical available for plant disease control runs into hundreds, however, all are not
equally safe, effective and popular. Major group of fungicides used include salts of toxic
metals and organic acids, organic compounds of sulphur and mercury, quinones and
heterocyclic nitrogenous compounds. Copper, mercury, zinc, tin and nickel are some of the
metals used as base for inorganic and organic fungicides. The non metal substances include,
sulphur, chlorine, phosphorous etc. The fungicides can be broadly grouped as follows and
discussed in detail.
I) Sulphur fungicides
Use of sulphur in plant disease control is probably the oldest one and can be classified
as inorganic sulphur and organic sulphur. Inorganic sulphur is used in the form of elemental
sulphur or as lime sulphur. Elemental sulphur can be either used as dust or wettable sulphur,
later being more widely used in plant disease control. Sulphur is best known for its
effectiveness against powdery mildew of many plants, but also effective against certain rusts,
leaf blights and fruit diseases. Sulphur fungicides emit sufficient vapour to prevent the
growth of the fungal spores at a distance from the area of deposition. This is an added
advantage in sulphur fungicides as compared to other fungitoxicants.
Organic compounds of sulphur are now widely used in these days. All these compounds,
called as ‘carbamate fungicides’, are derivatives of Dithiocarbamic acid, Dithiocarbamates
are broadly grouped into two, based on the mechanism of action.
i) Inorganic Sulphur:
1.Dust Formulations: Sulphur dust,
2. Lime Sulphur : It can be prepared by boiling 9 Kg or rock lime and 6.75 Kg of sulphur
in
225 litres of water
3. Wetable Sulphur : Cosan, Wetsulf, Microsul
ii). Organic Sulphur :
i.Dithiocarbamates
a. Monoalkyl Dithiocarbamates: e.g. Zineb(Zinc ethylene bisdithiocarbamate), Maneb ,
Mancozeb(Dithane M45, Indofil M45, Manzeb), Nabam, Vapam.
b.Dialkyl Dithiocarbamates : e. g.. Thiram, Ziram, Ferbam

[60]
II) Copper Fungicides
The fungicidal action of copper was mentioned as early as 1807 by Prevost against
wheat bunt disease (Tilletia caries), but its large scale use as a fungicide started in 1885 after
the discovery of Bordeaux mixture by Millardet in France. The mixture of copper sulphate
and lime was effective in controlling downy mildew of grapevine caused by Plasmopara
viticola and later, late blight of potato (Phytophthora infestans).
Some other copper sulphate preparations later developed were Borduaux paste, Burgandy
mixture and Cheshnut compound which are all very effectively used in the control of several
plant diseases. In addition some preparations of copper oxy chloride preparations arev also
mused. These are all insoluble copper compounds very successfully used in managing several
leaf diseases and seeding diseases in nursery.
1. Copper Sulphate Preparations
1. Bordeaux mixture: It is prepared by mixing copper sulphate and lime in water (to get 1%
mixture, mix 1 kg of CuSO4 and 1 kg of lime in 100 litres of water)
2. Bordeaux paste: It is prepared by mixing 1 kg of CuSO4 and 1 kg of lime in 10 litres of
water.
3. Burgundy mixture: It is prepared by mixing 1 kg of CuSO4 and 1 kg of Sodium
Carbonate in 100 litres of water.
4. Cheshnut compound: It is prepared by mixing 2 parts of copper sulphate and 11 parts of
Ammonium Carbonate.

2. Copper carbonate preparation

i Chaubattia Paste : This is prepared by mixing 800 g of copper carbonate and 800 g of Red
lead in 1 litre of linseed oil or lanolin

3. Cuprous oxide: Fungimar, Perenox, Copper Sandoz, Copper 4% dust, Perecot, Cuproxd,
Kirt i copper.

4. Copper oxy-chloride: Blitox, Cupramar 50% WP, Fytolan, Bilmix 4%, Micop D-06,
Micop w-50, Blue copper 50, Cupravit, Cobox, Cuprax, Mycop

III .Mercury Fungicides :

Mercury fungicides can be grouped as inorganic and organic mercury compounds.
Both the groups are highly fungitoxic and were extensively used as seed treatment chemicals
against seed borne diseases. Ignorance compounds show bactericidal property also. However,
due to their residual toxicity in soil and plants and their extreme toxicity nature to animal and
human beings, the use of mercury fungicides is beings discouraged. In most of the countries,
the use of mercury fungicides is banned andin countries like India, the use of mercury
fungicides is restricted only in seed treatment for certain crops. The list of diseases against
which mercury fungicides used are listed below:
I. Inorganic Mercury
1. Mercuric chloride: Cyclosan, M-C Turf fungicide
2. Mercurous chloride: Agallol, Aretan, Emisan, Ceresan wet (India)
II. Organomercurials
Methoxy ethyl mercury chloride

[61]
Phenyl mercury chloride : Ceresan Dry (India), Ceresol, Leytosan.
Ethyl Mercury Chloride : Ceresan
Tolyl mercury acetate: Ceresan
Agrosan GN.
IV.Heterocyclic Nitrogen Compounds :
Heterocyclic nitrogen compounds are mostly used as foliage and fruits protectants. Some
compounds are very effectively used as seed dressers. Some of the commonly used
fungicides are listed below.
1.Captan (Kittleson’s Killer) : Captan 50W, Captan 75 W, Esso Fungicide 406, Orthocide
406, Vancide 89, Deltan, Merpan, Hexacap.
2. Captafol : Foltaf, Difolaton, Difosan, Captaspor, Foleid, Sanspor.
3. Glyodin : Glyoxaliadine, Glyoxide, Glyodin, Glyoxide Dry, Glyodex 30% liquid and 70%
WP.
4.Folpet (Folpet) : Phartan, Acryptan, Phaltan, Folpan, Orthophaltan

V. Benzene compounds :
Many aromatic compounds have important anti-microbial properties and have been
developed as fungicides. Some important benzene compounds commonly used in plant
disease control are listed below: Common Name Trade Name
1. Quintozene (PCNB) : Brassicol, Terraclor, Tritisan 10%, 20%, 40% D and 75% WP,
PCNB 75% WP.
2. Dichloran : Botran 50% WP and 75% WP, Allisan.
3. Fenaminsosuplh : Dexon 5% G and 70% WP.
4. Dinocap : Karathane, Arathane, DNOPC, Mildex, Crotothane, Crotothane 25% WP,
Crotothane 48% Liq.
VI .Quinone Fungicides :
Quinone areresent naturally in plants and animals and they exhibit anti-microbial activity and
some compounds are successfully developed and used in the plant disease control. Quinones
are very effectively used for seed treatment and two commonly used fungicides are listed
below

1. Chloranil : Spergon Chloronil is mainly used as a seed protectant against smuts of barely
and sorghum and bunt of wheat.

2. Dichlone : Phygon, Phygon XL WP. Dichlone has been used widely as seed protectant.
This is also used as a foliage fungicide, particularly against apple scab and peach leaf curl

VII. Organo-Phosphorous fungicide Ediphenphos (Edifenphos) : Ediphenphos is

available as Hinosan 50% EC and 2% D. It has a specific action against Pyricularia oryzae
(Rice blast). It is also effective against Corticium sesakii and Cochliobolus miyabeanus in
rice.

Systemic Fungicides
Since the late 1960s there has been substantial development in systemic fungicides. Any
compound capable of being freely translocated after penetrating the plant is called systemic.
A systemic fungicide is defined as fungitoxic compound that controls a fungal pathogen

[62]
remote from the point of application, and that can be detected and identified. Thus, a systemic
fungicide could eradicate established infection and protect the new parts of the plant. Several
systemic fungicides have been used as seed dressing to eliminate seed infection. These
chemicals, however, have not been very successful in the cases of trees and shrubs. On the
basis of chemical structure, systemic fungicides can be classified as Benzimidazoles,
Thiophanates, Oxathilins and related compounds, pyrimidines, morpholines, organo-
phosphorus compounds and miscellaneous group.
I. Oxathilin and related compounds : Oxathalins were the earliest developed compounds.
This group of systemic fungicide is also called as carboxamides, carboxyluc acid anillides,
carboxaanillides or simply as anillides which are effective only against the fungi belong to
Basidiomycotina and Rhizoctonia solani. Some of the chemicals developed are (i) Carboxin
(DMOC: 5,6 - dithydra-2-methyl-1, 4-oxathin-3-carboxanillide) and (ii) Oxycarboxin
(DCMOD-2,3-dihydro-5-carboxanillido-6-methyl-1, 4 oxathilin-4, 4, dioxide). The diseases
controlled by these chemicals are listed below:
1. Carboxin : Vitavax 10% D, Vitavax 75% WP, Vitavax 34% liq. Vitaflow
2. Oxycarboxin: Plantvax 5G, Plantvax 5% liq. Plantvax 1.5 EC, 10% dust, 75 WP.
3.Pyracarbolid: Sicarol
II. Benzimidazoles : The chemicals of this group show a very broad spectrum activity
against a variety of fungi. However, they are not effective against bacteria as well as fungi
belongs to Mastigomycotina. Two types of fungicidal derivates of benzimidazoles are known.
The first type of derivates includes fungicides such as thiabendazole and fuberidazole. The
fungicidal moiety of the second type is methyl-2-benzimidazole carbamate (MBC). The
fungicides of this group may be simple MBC such as carbendazim or a complex from such as
benomyl, which transforms into MBC in plant system. Some of the important diseases
controlled by these compounds are shown below
1.Benomyl : Benlate 50 WP, Benomyl.
2. Carbendazim (MBC) : Bavistin 50 WP, MBC, Dersol, B.Sten 50, Zoom,
3. Thiabendazole (TBZ) : Tagstin, Agrozim, Jkenstin.
4.Fuberidazole : Thiabendazole, Mertect, Tecto, Storite. Voronit.

III. Thiophanates : These compounds represent a new group of systemic fungicides based
on thiourea. They are the derivatives of thioallophanic acid. These fungicides contain
aromatic nucleus which is converted into benzimidazole ring for their activity. Hence,
thiophanates are often classified under benzimidazole group and the biological activity of
thiophanates resembles of benomyl. Two compounds are developed under this group are
discussed
e.g. Topsin 50 WP, Cercobin 50 WP, Enovit. , Topsin-M70 WP, Cercobin-M 70 WP,
Envovit-methyl, Mildothane.
IV.Morpholines : It is an eradicant fungicide with systemic action, being absorbed through
foilage and roots to give some protective action. It controls powdery mildew diseases of
cereals, vegetables and ornamentals. It is highly effective against Mycosphaerala musicola,
Exobasidium vexans and rust diseases or cereals, pulses and coffee. 1.Tridemorph : Calixin
75 EC, Bardew, Beaco and 2. Dodemorph: BAS 238F

[63]
V. Pyrimidines, Pyridines, Piperidines and Imidazole : It is highly effective against rusts
and powdery mildew of a variety of crops. It is also used against Venturia and Monilinia on
fruits and Cereospora leafspots of groundnut and banana

1. Triadimefon : Bayleton, Amiral 2. Tria dimenol : Baytan.

3. Bitertanal : Baycor 4. Etridiazole: Terrazole 30% WP, Terrazole 95% WP, Terrazole
25%
EC, Koban, Pansol EG, Pansol 4% DP, Turban WP, Terracoat
Aaterra.
VI. Hydroxy Pyrinidines :
1. Ethirimol: Milliatem 80 WDP, Milcurb Super, Milgo . It is effective against
powdery mildew of cereals and other field crops. It is also effective against powdery mildews
of cucumber and ornamentals.

2. Dimethirimol : Milcurb. It is very effective against powdery mildews of

chrysanthemum and cucurbits.

VII. Furan derivatives : It is effective against yellow rust on wheat and barley (P.
striiformis) and brown rust on barley (P. hordei). It is also having direct fungitoxic activity
against Sclerotium rolfsli and Rhizoctonia. brown rust of wheat, black rusts of wheat, rye,
barley and oats and rust of fig. 1. Furcarbanil and 2. Cyclafuramid : Calirus.

VIII.Benzanilide derivative:
1. Mebenil :
2. Benodanil: Calirus.
IX. Organo phosphorous compound: It is used to control powdery mildews of cereals,
vegetables, fruits and ornamentals. It is used to control Pyricularia oryzae and sheath blight
of rice. Eg.1. Pyrazophos : Afugan, Curamil, WP, Missile EC.
2. Iprobenphos (IBP) : Kitazin 48% EC, Kitazin 17G, Kitazin 2% D.

X. Piperazine : It is effective against powdery mildew, scab and other diseases of fruits and
rust on ornamentals and cereals. It is also active against storage diseases of fruits

Eg.1. Triforine : Saprol-EG, Fungitex

XI. Phenol derivative : It is highly fungistatic to Rhizoctonia spp., moderately so to Pythium

spp. and poorly to Fusarium spp. It is used as a supplemental seed treatment for beans and
soyabeans to control seedling diseases.

1. Choloroneb : Demonsan 65 WP, Tersan SP, Turf fungicide. It is highly fungistatic to

Rhizoctonia spp., moderately so to Pythium spp. and poorly to Fusarium spp. It is used as a
supplemental seed treatment for beans and soyabean to control seedling diseases.

XII. Triazole compounds. : It is a systemic fungicide with preventive and urative properties.
It controls powdery mildew of various vegetables, fruits and ornamentals. It is very effective
against leaf spot, rust and powdery mildew of many crops.

[64]
 1. Triazbutyl: Indar
2. Tricyclazole : Beam, Bim
3. Pyroquilon :Fongorene 50 WP &5 G
4. Hexaconazole : Contaf 5 EC, ANVIL 5 EC
5. Propiconazole : Tilt 25 EC
6. Terbuconazole : Folicur 25 EC, Raxil 2 DS
7. Penconazole : Topas 10 EC
8. Difenoconazole : Sare 25% EC
9. Cyperocoanazole :SAN 619
10. Probenazole : Orizemate
11. Isoprothiolane: Fuji - 1
12. Pyroxychior : Dowco 269, Nurelle
13. 15.Cymoxanil : Curzate
XIII. Other systemic fungicides: It is a fungicide with systemic and contact action and
effective against damping-off, root rots, stem rots, and downy mildew of grapes and millets.

1. Metalaxyl : Apron 35 SD, Ridomil

2. Metalaxyl + Mancozeb : Ridomil MZ 72 WP (8% Metalaxyl + 64% Mancozeb)
3. Fosetyl AL (Aluminium - Tris-aluminium : Alliette 80 WP

Other Fungicides: Some of the fungicides which are not included in any of the groups
described earlier are listed below
1. Binapacryl : Morocide 50% WP and 40% EC, Acricide 50% WP and 40% EC, Endosan.
2. Chinomethionat (Oxythioquinox) : Morestan 25% WP, Morestan 2% D.
3. Chlorothalonil : Kavach 25% WP, Bravo, Daconil, Termil, Chlorothalonil 40 SC,
Safegaurd, Spektrum. Cyprex 65% WP, Guanidol, Melprex WP, Syllit
4. Dodine : Cyprex 65% WP, Guanidol, Melprex WP, Syllit.

[65]
B) To study fungicides formulations
The names by which fungicides are known can be rather confusing, as there as usually
several names referring to one substance. Firstly, the chemical names describing the structure
and composition of the chemicals may be indicated. In the case of complex organic
compounds, different chemical names may be used to describe the same compound according
to the usages in different countries. Secondly, the common name may be identical to the
chemical name with simple compounds, or it may be an abbreviated and simplified derivative
of the chemical name, when this is complex. Thirdly, the trade names under which different
formulations of the same compound and marketed by different companies vary widely.
However, all marketed fungicides should state clearly the common name of the fungicide and
the amount of active ingredient of it contained in the formulated product. Commercial
fungicides are formulated in various ways and most commonly available formulations are
Emulsifiable Concentrates (EC), Wettable Powders (WP), Dusts (D) etc.
Commercially available fungicides usually consist of a mixture of active ingredient (a.i.) and
other substances including diluents, wetting agents, stickers, emulsifiers, etc. Formulations
containing mixtures of different active ingredients (especially mixtures of protectant and
systemic fungicides) are also widely used nowadays. Different formulations incorporating the
same active ingredient may be used for distinct purposes like seed treatment, foilar
application etc.

Emulsifiable Concentrates (EC)

These are liquid formulations which can be diluted with water before application. The active
ingredient is dissolved in a solvent. The fungicides and solvents will opten not mix with
water, so an emulsifying agent or water dispersible oil is mixed. When these emulsifiable
concentrate is added to water, a milky mixture is formed which is a suspension of active
ingredient and emulsified solvent in the water.

Wettable Powders (WP)

Wettable powder is a very common formulation for most of the fungicides, which is used for
spray mixtures. The modern wettable powders are water-dispersible which have the quality to
wet easily and disperse well in water. They are also called as Water-Dispersible Powders
(WDP). The active ingredient is incorporated, usually at the rate of 30-80%, with a finely
ground inert dust (filler) such as Kaolin, a wetting agent and a suspending agent. The
commonly used suspending agents are sodium lignin sulphonate (Sulphite dye), methyl
celluloses, polyvinyl acetate and aluminium silicate. In addition, spreader-sticker is
sometimes desirable, especially on plants with glossy or waxy leaves. Agitation is generally
necessary to keep uniform suspension.
A highly developed type of water-dispersible powder is called as colloidal powder, which is
so finely divided that the individual particles will never sediment out. A typical colloidal
powder contains 5-50% active ingredient, non-ionic wetting agent (1-10% polyethylene oxide
condensate), thickening agent like carboxy methyl cellulose and a hydrophilic diluent
(carrier) such as bentonite.

[66]
Dusts (D)
Dust formulations usually contain 1-10% active ingredient for direct application in dry forms.
They are manufactured in such a way that they are light enough to be carried by a slight
breeze for a considerable distance. The finely divided particle of active ingredient is carried
on a carrier particle. The commonly used carriers (diluents) are attapulgite, kaolin, talc,
pyrophylite, diatomaceous earth, bentonite, calcium silicate, hydrated silica, calcium
carbonate, magnesium carbonate, gypsum, lime etc.

Granules (Pellets)
Pellets are the formulations of the fugicide with inert materials formed into particles about the
size of coarse sugar. The granules normally contain 3-10% of the active ingredient. Due to
their size, the granules do not drift but have limited application being confined to soil and
seed treatments. Granules have the advantage they can be measured in dry form more easily
and acurately than dusts or wettable powders.

Suspension or slurries
These are formulation in which a dry form of the active ingredient is mixed with a liquid.
Such formulations usually contain a high percentage of active ingredient similar to wettable
powders. They are mixed with water for final use and require agitation. These are mostly
used as seed dressers in seed processing companies.

Solutions
True solutions are formulations in which active ingredient or a combination of active
ingredients and a solvent is dissolved in water Solutions have the advantage of requiring no
agitation after formulation is added in water.
Nowadays, the manufacturers are concentrating to develop new formulations to increase the
efficacy of the chemicals. Some new formulations developed are: Soluble Liquid (SL),
Soluble Powder (SP), Water Soluble Concentrate (WSC), Suspension Concentrate (SC) and
Aqua Flow (AF).

Adjuvants
The fungicides can be commonly applied either as spraying or dusting. In spraying method,
the toxicant is made into a suspension in water. In order to increase the efficacy of the water
mixed sprays, certain substances like wetting agents, dispersing agents, spreaders, stickers,
etc. are added during the formulation of fungicides. These auxillary spray materials are also
called adjuvants, which are usually inert materials added to improve the physical
characteristics of the toxicant and its carrier. Most of the materials used are surface active
agents and therefore induce variation either in surface tension or interfacial tension. The
various adjuvants are grouped as follows.

[67]
Dispersing agents (Deflocculating agents)
These are the substances which keep fine particles away from each other to prevent
deflocculation. These materials, when added to formulations, ensure uniform suspension and
retard sedimentation of particles in the spray suspension. These are also called as
deflocculating agents. Eg. Gelatin, plant gums and milk products.

Emulsifying agents
Powders (WDP). The active ingredient is incorporated, usually at the rate of 30-80%, with a
finely ground inert dust (filler) such as Kaolin, a wetting agent and a suspending agent. The
commonly used suspending agents are sodium lignin sulphonate (Sulphite dye), methyl
celluloses, polyvinyl acetate and aluminium silicate. In addition, spreader-sticker is
sometimes desirable, especially on plants with glossy or waxy leaves. Agitation is generally
necessary to keep uniform suspension.
A highly developed type of water-dispersible powder is called as colloidal powder, which is
so finely divided that the individual particles will never sediment out. A typical colloidal
powder contains 5-50% active ingredient, non-ionic wetting agent (1-10% polyethylene oxide
condensate), thickening agent like carboxy methyl cellulose and a hydrophilic diluent
(carrier) such as bentonite.
Many surface active substances like soap, function as emulsifying agent, which retard the
settling out of droplets of waterimmiscible liquids like oils. This helps in uniform mixing of
substances in water suspensions.

Wetting agent (Wetters)

These are the materials which are added to ensure that there will be no layer of air between a
solid and a liquid as they reduce the surface tension of the particles. Wetting agents, when
added to aqueous fungicidal preparation, help in easy deposition on leaves. Eg. Polyethylene
oxide condensat, esters of fatty acids and flour.

Spreading agent (Spreaders)

Spreaders are the materials added to establish improved contact between the spray materials
and plant surface and thus ensuring a good coverage of fungicide. Wetting must precede
spreading and this is the only distinction between wetting and spreading. Spreaders also
reduce the surface tension and thus improve contact. Eg. Soap, flour, sulphated amines,
soapamines, mineral oils, glyceride oil, terpene oil, resinates and petroleum sulphonic acids.

Stickers (Adhesives)
The materials which are added to spray or dust to improve the adherence to plant surfaces are
called as stickers. They increase the tenacity of the fungicidal preparations, thus increasing
the residual action. Eg. Polyvinyl acetate, polybutanes, fish oil, linseed oil, milk
casein,gelatin, dextrines, polyethylene polysulphide, starch, gum arabic, hydrocarbon oils and
bentonite clays; Milk casein, gelatin also act as good spreading and wetting agents besides
acting as stickers.

[68]
Safeners
Chemical which reduces the phytotoxicity of another chemical is called safener. For example
copper sulphate is phytotoxic to plants, but with addition of lime its toxicity is reduced. Lime
is, therefore, a safener. Lime is used universally with chemicals to prevent the formation of,
or to neutralise arsenic, which is phytotoxic to plants. Glycerine oils are also used as safeners.
Toxicity levels of chemicals
The toxicity levels of the fungicidal Toxicity Level LD50 Value
formulations are based on the LD50 values, (mg/kg body weight)
LD50 means the concentration of the chemical
at which 50 percent of the test animals dead.
The toxicity levels of the chemicals in all
formulations as coloured triangle. Triangle
Colour
Oral Dermal

Red Extremely toxic 1-50 1-200

Yellow Highly toxic 51-500 201-2000

Blue Moderately toxic 501-5000 2001-20000

Slightly > 20000

Green > 5000
toxic(Least toxic)

********

[69]
EXERCISE 14
Methods of fungicides application and their safe use

A. Methods of pesticide application

General comments: Different methods are employed to apply fungicides to manage
diseases. This mainly depends on the location of inoculums and plant organs to be protected.
Procedure, observations and record:

1. SEED DRESSING
The seed treatment with fungicides is highly essential because a large number of
fungal pathogens are carried on or in the seed. In addition, when the seed is sown, it is also
vulnerable to attack by many common soil-borne pathogens, leading to either seed rot,
seeding mortality or produce diseases at a later stage. Seed treatment is probably the effective
and economic method of disease control and is being advocated as a regular practice in crop
protection against soil and seed-borne pathogens. Seed treatment is therapeutic when it kills
pathogens that infect embroys, cotyledons or endosperms under the seed coat, eradicative
when it kills pathogens that contaminate seed surfaces and protective when it prevents
penetration of soil-borne pathogens into the seedling

Using fungicides on seed is one of the most efficient and economical methods of
chemical disease control. On the basis of their tenacity and action, the seed dressing
chemicals may be grouped as (i) Seed disinfectant, which disinfect the seed but may not
remain active for a long period after the seed has been sown and (ii) Seed protectants, which
disinfect the seed surface and stick to the seed surface for sometime after the seed has been
sown, thus giving temporary protection to the young seedlings against soil-borne fungi. Now,
the systemic fungicides are impregnated into the seeds to eliminate the deep-seated infection
in the seeds. The seed dressing chemicals may be applied by (i) Dry treatment (ii) Wet
treatment and (iii) Slurry.
. (i) Dry Seed Treatment
In this method, the fungicide adheres in a fine from on the surface of the seeds. A
calculated quantity of fungicide is applied and mixed with seed using machinary specially
designed for the purpose. The fungicides may be treated with the seeds of small lots using
simple Rotary seed Dresser (Seed treating drum) or of large seed lots at seed processing
plants using Grain treating machines. Normally in field level, dry seed treatment is carried
out in dry rotary seed treating drums which ensure proper coating of the chemical on the
surface of seeds.
In addition, the dry dressing method is also used int pulses, cotton and oil seeds with
the antagonistic fungus like Trichoderma vitide by mixing the formulation at the rate of 4g/kg
of the seed. Eg. Dry seed treatment in paddy.
Mix a required amount of fungicide with required quantity of seeds in a seed treating
drum or polythene lined gunny bags, so as to provide uniform coating of the fungicide over
the seeds. Treat the seeds atleast 24 hours prior to soaking for sprouting. Any one of the
following chemical may be used for treatment at the rate of 2g/kg : Thiram or Captan or
Carboxin or Tricyclazole.

[70]
(ii) Wet seed treatment
This method involves preparing fungicide suspension in water, often at field rates and
then dipping the seeds or seedlings or propagative materials for a specified time. the seeds
cannot be stored and the treatment has to be done before sowing. This treatment is usually
applied for treating vegetatively propagative materials like cuttings, tubers, corms, setts
rhizomes, bulbs etc., which are not amenable to dry or slurry treatment.
a. Seed dip / Seed soaking
For certain crops, seed soaking is essential. Seeds treated by these methods have to be
properly dried after treatment. The fungicide adheres as a thin film over the seed surface
which gives protection against invasion by soil-borne pathogens.
Eg. Seed dip treatment in paddy.
Prepare the fungicidal solution by mixing any of the fungicides viz., carbendazim or
pyroquilon or tricyclazole at the rate of 2g/litre of water and soak the seeds in the solution for
2 hrs. Drain the solution and keep the seeds for sprouting.
Eg. Seed dip treatment in Wheat.
Prepare 0.2% of carboxin (2g/litre of water) and soak the seeds for 6 hours. Drain the
solution and dry the seeds properly before sowing. This effectively eliminates the loose smut
pathogen, Ustilago nuda tritici.
b. Seedling dip / root dip
The seedlings of vegetables and fruits are normally dipped in 0.25% copper
oxychloride or 0.1% carbendazin solution for 5 minutes to protect against seedling blight and
rots.
c. Rhizome dip
The rhizomes of cardamom, ginger and turmeric are treated with 0.1% emisan
solution for 20 minutes to eliminate rot causing pathogen present in the soil.
d. Sett dip / Sucker dip
The setts of sugarcane and tapioca are dipped in 0.1% emisan solution for 30 minutes.
The suckers of pine apple may also be treated by this method to protect from soil-borne
diseases.

II. SOIL TREATMENT

It is well known that soil harbours a large number of plant pathogens and the primary
sources of many plant pathogens happens to be in soil where dead organic matter supports
active or dormant stages ofpathogens. In addition, seed treatment does not afford sufficient
protection against seedling diseases and a treatment of soil around the seed is necessary to
protect them. Soil treatment is largely curativ in nature as it mainly aims at killing the
pathogens in soil and making the soil ‘safe’ for the growth of the plant.
Chemical treatments of the soil is comparatively simple, especially when the soil is
fallow as the chemical is volatile and disappears quickly either by volatilization or
decomposition. Soil treating chemicals should be non-injurious to the plants in the soil
adjacent to the area where treatment has been carried out because there may be standing crop

[71]
in adjacent fields. The soil treatment methods involving the use of chemicals are (i) Soil
drenching, (ii) broadcasting, (iii) furrow application, (iv) fumigation and (v) chemigation.
(i) Soil drenching
This method is followed for followed for controlling damping off and root rot
infections at the ground level. Requisite quantity of fungicide suspension is applied per unit
area so that the fungicide reaches to a depth of atleast 10-15 cm.
Eg. Emisan, PCNB, Carbendazim, Copper fungicides, etc.
(ii) Broadcasting
It is followed in granular fungicides wherein the pellets are broadcasted near the plant.
(iii) Furrow application It is done specifically in the control of some diseases where the
direct application of the fungicides on the plant surface results in phytotoxic. It is
specifically practiced in the control of powdery mildew of tobacco where the sulphur dust is
applied in the furrows.
(iv) Fumigation
Volatile toxicants (fumigants) such as methyl bromide, chloropicrin, formaldehyde
and vapam are the best chemical sterilants for soil to kill fungi and nematodes as they
penetrate the soil efficiently. Fumigations are normally done in nursery areas and in glass
houses. The fumigant is applied to the soil and covered by thin polythene sheets for 5-7 days
and removed. For example, Formaldehyde is applied at 400 ml/100 Sq. m. The treated soil
was irrigated and used 1 or 2 weeks later. Vapam is normally sprinkled on the soil surface
and covered. Volatile liquid fumigants are also injected to a depth of 15-20 cm, using sub-soil
injectors.
(v) Chemigation
In this method, the fungicides are directly mixed in the irrigation water. It is normally
adopted using sprinkler or drip irrigation system.
III. FOLIAR APPLICATION
A. Spraying
This is the most commonly followed method. Spraying of fungicides is done on
leaves, stems and fruits. Wettable powders are most commonly used for preparing spray
solutions. The most common diluent or carrier is water. The dispersion of the spray is usually
achieved by its passage under pressure through nozzle of the sprayer.
The amount of spray solution required for a hectare will depend on the nature of crops
to be treated. For trees and shrubs more amount of spray solution is required than in the case
of ground crops. Depending on the volume of fluid used for coverage, the sprays are
categorised into high volume, medium volume, low volume, very high volume and ultra low
volume.
For spraying either low volume or high volume is used. WP or EC are normally used
for sprays. In high volume sprays the mount of water used is 500- 1000 litres/ha. The drop
size varies from 0.5 to 3 mm. In low volume sprays the liquid quantity may vary from a litre
to 100 litres. Air blast equipments are generally used and the size of droplets vary from 15 to
40 µ.

[72]
 The different equipments used for spray application are: Foot-operated sprayer,
rocking sprayer, knapsack sprayer, motorised knapsack sprayer (Power sprayer), tractor
mounted sprayer, mist blower and aircraft or helicopter (aerial spray).
B. Dusting
Dusts are applied to all aerial parts of a plant as an alternative to spraying. Dry
powders are used for covering host surface. Generally, dusting is practicable in calm weather
and a better protective action is obtained if the dust is applied when the plant surface is wet
with dew or rain drops. The equipments employed for the dusting operation are: Bellow
duster, rotary duster, motorised knapsack duster and aircraft (aerial application).
IV. POST-HARVEST APPLICATION
Fruits and vegetables are largely damaged after harvest by fungi and bacteria. Many
chemicals have been used as spray or dip or fumigation. Post harvest fungicides are most
frequently applied as aqueous suspensions or solutions. Dip application has the advantage of
totally submerging the commodity so that maximum opportunity for penetration to the
infection sites. Systemic fungicides, particularly thiabendazole, benomyl, carbendazim,
metalaxyl, fosety-AI have been found to be very effective against storage diseases. In
addition, dithiocarbamates and antibiotics are also applied to control the post-harvest
diseases. Wrapping the harvested products with fungicide impregnated wax paper is the latest
method available.
V. PAINTING (SWABBING)
This is practiced normally in most of the ornamentals and fruit trees after pruning.
The fungicidal solution/paste is painted on the cut ends to prevent the entry of pathogens.
Sometimes, the swabbing is done after removing the diseased portion of the plants.
Eg. Swabbing of Bordeaux paste in stem bleeding disease of coconut.
VI. SPECIAL METHODS
1. Trunk Application / Trunk Injection
It is normally adopted in coconut gardens to control Thanjavur wilt caused by
Ganoderma lucidum.
In the infected plant, a downward hole is made to a depth of 3-4” at an angle of 450C
at the height of 3’ from the ground level with the help of an auger. The solution containing 2g
of Aureofungin soil and 1 g of copper sulphate in 100 ml of water is taken in a saline bottle
and the bottle is tied with the tree. The hose is inserted into the hole and the stopper is
adjusted to allow the solution in drops. After the treatment, the hole is covered with clay.
2. Root Feeding
Root feeding is also adopted for the control of Thanjavur wilt of coconut instead of
trunk application. The root region is exposed; actively growing young root is selected and
given a slanting cut at the tip. The root is inserted into a polythene bag containing 100 ml of
the fungicidal solution. The mouth of the bag is tied tightly with the root.
3. Pseudostem Injection This method is very effective in controlling the aphid vector
(Pentalonia nigronervosa) of bunchy top of bannana. The banana injector is used for
injecting the insecticide.

[73]
 Banana injector is nothing but an Aspee baby sprayer of 500 ml capacity. In which,
the nozzle is replaced by leurlock system and aspirator needle No. 16. The tip of the needle is
closed and two small holes are made in opposite direction. It is for free flow of fluid and the
lock system prevents the needle from dropping from the sprayer.
One ml of monocrotophos mixed with water at 1:4 ratio is injected into the
pseudostem of 3 months old crop and repeated twice at monthly intervals.
The same injector can also be used to kill the bunchy top infected plants by injecting 2
ml of 2, 4-D (Femoxone) mixed in water at 1:8 ratio.
4. Corn Injection
It is an effective method used to control Panama will of banana caused by Fusarium
oxysporum f. sp. cubense.
Capsule applicator is used for this purpose. It is nothing but an iron rod of 7 mm
thickness to which a handle is attached at one end. The length of the rod is 45 cm and an iron
plate is fixed at a distance of 7 cm from the tip.
The corm is exposed by removing the soil and a hole is made at 45) angle to a depth
of 5 cm. One or two gelatin capsules containing 50-60 mg of carbendazim is pushed in
slowly and covered with soil. Instead of capsule, 3 ml of 2% carbendazim solution can also
be injected into the hole. It is well known that soil harbours a large number of plant
pathogens and the primary sources of many plant pathogens happens to be in soil where dead
organic matter supports active or dormant stages ofpathogens. In addition, seed treatment
does not afford sufficient protection against seedling diseases and a treatment of soil around
the seed is necessary to protect them. Soil treatment is largely curativ in nature as it mainly
aims at killing the pathogens in soil and making the soil ‘safe’ for the growth of the plant.
Chemical treatments of the soil is comparatively simple, especially when the soil is
fallow as the chemical is volatile and disappears quickly either by volatilization or
decomposition. Soil treating chemicals should be non-injurious to the plants in the soil
adjacent to the area where treatment has been carried out because there may be standing crop
in adjacent fields. The soil treatment methods involving the use of chemicals are (i) Soil
drenching, (ii) broadcasting, (iii) furrow application, (iv) fumigation and (v) chemigation.
(i) Soil drenching
This method is followed for followed for controlling damping off and root rot
infections at the ground level. Requisite quantity of fungicide suspension is applied per unit
area so that the fungicide reaches to a depth of atleast 10-15 cm.
Eg. Emisan, PCNB, Carbendazim, Copper fungicides, etc.
(ii) Broadcasting
It is followed in granular fungicides wherein the pellets are broadcasted near the plant.
(iii) Furrow application It is done specifically in the control of some diseases where the
direct application of the fungicides on the plant surface results in phytotoxic. It is specifically
practiced in the control of powdery mildew of tobacco where the sulphur dust is applied in
the furrows.

[74]
(iv) Fumigation
Volatile toxicants (fumigants) such as methyl bromide, chloropicrin, formaldehyde
and vapam are the best chemical sterilants for soil to kill fungi and nematodes as they
penetrate the soil efficiently. Fumigations are normally done in nursery areas and in glass
houses. The fumigant is applied to the soil and covered by thin polythene sheets for 5-7 days
and removed. For example, Formaldehyde is applied at 400 ml/100 Sq. m. The treated soil
was irrigated and used 1 or 2 weeks later. Vapam is normally sprinkled on the soil surface
and covered. Volatile liquid fumigants are also injected to a depth of 15-20 cm, using sub-soil
injectors.
(v) Chemigation
In this method, the fungicides are directly mixed in the irrigation water. It is normally
adopted using sprinkler or drip irrigation system.
III. FOLIAR APPLICATION
A. Spraying
This is the most commonly followed method. Spraying of fungicides is done on
leaves, stems and fruits. Wettable powders are most commonly used for preparing spray
solutions. The most common diluent or carrier is water. The dispersion of the spray is usually
achieved by its passage under pressure through nozzle of the sprayer.
The amount of spray solution required for a hectare will depend on the nature of crops
to be treated. For trees and shrubs more amount of spray solution is required than in the case
of ground crops. Depending on the volume of fluid used for coverage, the sprays are
categorised into high volume, medium volume, low volume, very high volume and ultra low
volume.
The different equipments used for spray application are: Foot-operated sprayer,
rocking sprayer, knapsack sprayer, motorised knapsack sprayer (Power sprayer), tractor
mounted sprayer, mist blower and aircraft or helicopter (aerial spray).
B. Dusting
Dusts are applied to all aerial parts of a plant as an alternative to spraying. Dry
powders are used for covering host surface. Generally, dusting is practicable in calm weather
and a better protective action is obtained if the dust is applied when the plant surface is wet
with dew or rain drops. The equipments employed for the dusting operation are: Bellow
duster, rotary duster, motorised knapsack duster and aircraft (aerial application).
IV. POST-HARVEST APPLICATION
Fruits and vegetables are largely damaged after harvest by fungi and bacteria. Many
chemicals have been used as spray or dip or fumigation. Post harvest fungicides are most
frequently applied as aqueous suspensions or solutions. Dip application has the advantage of
totally submerging the commodity so that maximum opportunity for penetration to the
infection sites. Systemic fungicides, particularly thiabendazole, benomyl, carbendazim,
metalaxyl, fosety-AI have been found to be very effective against storage diseases. In
addition, dithiocarbamates and antibiotics are also applied to control the post-harvest
diseases. Wrapping the harvested products with fungicide impregnated wax paper is the latest
method available.

[75]
V. PAINTING (SWABBING)
This is practiced normally in most of the ornamentals and fruit trees after pruning.
The fungicidal solution/paste is painted on the cut ends to prevent the entry of pathogens.
Sometimes, the swabbing is done after removing the diseased portion of the plants.
Eg. Swabbing of Bordeaux paste in stem bleeding disease of coconut.
VI. SPECIAL METHODS
1. Trunk Application / Trunk Injection
It is normally adopted in coconut gardens to control Thanjavur wilt caused by
Ganoderma lucidum.
In the infected plant, a downward hole is made to a depth of 3-4” at an angle of 450C
at the height of 3’ from the ground level with the help of an auger. The solution containing 2g
of Aureofungin soil and 1 g of copper sulphate in 100 ml of water is taken in a saline bottle
and the bottle is tied with the tree. The hose is inserted into the hole and the stopper is
adjusted to allow the solution in drops. After the treatment, the hole is covered with clay.
2. Root Feeding
Root feeding is also adopted for the control of Thanjavur wilt of coconut instead of
trunk application. The root region is exposed; actively growing young root is selected and
given a slanting cut at the tip. The root is inserted into a polythene bag containing 100 ml of
the fungicidal solution. The mouth of the bag is tied tightly with the root.
3. Pseudostem Injection This method is very effective in controlling the aphid vector
(Pentalonia nigronervosa) of bunchy top of bannana. The banana injector is used for
injecting the insecticide.
Banana injector is nothing but an Aspee baby sprayer of 500 ml capacity. In which,
the nozzle is replaced by leurlock system and aspirator needle No. 16. The tip of the needle is
closed and two small holes are made in opposite direction. It is for free flow of fluid and the
lock system prevents the needle from dropping from the sprayer.
One ml of monocrotophos mixed with water at 1:4 ratio is injected into the
pseudostem of 3 months old crop and repeated twice at monthly intervals.
The same injector can also be used to kill the bunchy top infected plants by injecting 2
ml of 2, 4-D (Femoxone) mixed in water at 1:8 ratio.
4. Corn Injection
It is an effective method used to control Panama will of banana caused by Fusarium
oxysporum f. sp. cubense.
Capsule applicator is used for this purpose. It is nothing but an iron rod of 7 mm
thickness to which a handle is attached at one end. The length of the rod is 45 cm and an iron
plate is fixed at a distance of 7 cm from the tip.
The corm is exposed by removing the soil and a hole is made at 45) angle to a depth
of 5 cm. One or two gelatin capsules containing 50-60 mg of carbendazim is pushed in

[76]
slowly and covered with soil. Instead of capsule, 3 ml of 2% carbendazim solution can also
be injected into the hole.
4. Equipments:
Sprayers- Napsak, Backpak, High tech, Bucket sprayer, Neumatic sprayer, Foot sprayer,
Mist sprayer.
Duster- Puf dusterSeed dressers, Gun injectors.
Exercise and Record: Based on Availability, utility and feasibility the course incharge
should develop the following exercise to train the students.
Treat seeds of a crop with fungicides by slurry and dry methods and record per cent
germination, health of root and shoot systems by blotter paper method, towel paper method
and in pots. Also record disease incidence and organisms involved.
Study equipments, their parts, calibration and operation used in plants disease control.

********

[77]
B. Safe use of fungicides:
Pesticides being toxic to human and domestic animals should be handled with almost
care. The following precautions should be observed.

1. The pesticides should always be stored in their original containers and kept in locked
cupboard where they are out of reach of the children and domestic animals.
2. They should be always from food or feed stuff and medicine.
3. The instruction found on the labels should be carefully read and strictly followed.
4. Bags and containers of pesticides should be cut open with a separate knife intended for
such purposes.
5. The empty containers, after the use of the chemicals, should be destroyed and should not
be put into some other use.
6. While preparing the spray solutions bare hands should not be used for mixing the
chemical with water.
7. Inhaling pesticide spray or dusts, smoking, chewing, eating or drinking while mixing or
applying the chemicals should be avoided.
8. Spilling of pesticides on skin or clothing should as far as possible be avoided.
9. The cloth should be washed after each operation.
10. Particles or drops of pesticide, which may accidently get into eyes, should be flushed out
immediately with large volume of clean water.
11. It is preferable that protective clothing and devices are used while handling poisonous
chemicals to avoid exposure to spray or drifts.
12. Dusting or spraying should never be done against the wind and it is preferable to have
them done in cool and calm weather.
13. Sprayer nozzles should not be blown by mouth if get blocked while spraying. Washers
and other contaminated parts should be buried.
14. After handling pesticides hands, face and body should be washed and clothing changed.
15. Washing of equipment after used and container in or near the wells or streams should be
avoided.
16. Person engaged in the handling the pesticides should undergo regular medical checkup.
17. In case of any suspected poisoning due to insecticides the nearest physician should be
called immediately.

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[78]
EXERCISE 15
Calculation of fungicide spray concentration
Calculation for preparation of Fungicide solutions:
Procedure:
The concentration of the test chemical is expressed as parts per million (ppm). It is
computed by using formula-

X units of solute "Y" grams

=
1,000000 units of solvent 1,000000 ml water

Thus, X ppm in Y ml of solvent

X gram of chemical z gram

=
1,000000 ml water y ml

Example: Its is desired to prepare 500 ppm of a chemical in 100 ml of water

500 gm chemical z gram

=
1,000000 ml water 100 ml

Solving for z= 0.05 grams

Thus 0.05 g in 100 ml = 500 ppm. It will be correct if the chemical has 100% AI.
Most fungicides available are having different percentages of active ingredient. In such cases,
a correction factor is applied. Suppose that the chemical has 50% AI. It should be calculated
as:
100
=2
50
Thus, the grams of chemical needed to give specified concentrations by the first
formula must be multiplied by “2”.
Examples
Problem 1:
Calculate the cost and quantity of fungicide for spraying 12 hectares of fruit garden for
controlling the disease problem on the points given below:

1 Sprays to be taken 02
2 Rate of fungicide to be used 0.25%
3 Spray solution required per plant 4.5 liter per plant
4 Cost of fungicide Rs. 650 per liter
5 Plant population per hectare 250

Answer:
Total plant population in 12 hectare = 12 x 250 = 3000 plants
Per plant spray 4.5 liter, hence for 2 sprays = 4.5 x 2 = 9 liter

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Total water requirement for total plants = 9 liter x 3000 plants = 27000 liter
As the fungicidal rate is 0.25%, it means that 2.5 g fungicide per liter of water.
Hence total fungicide requiring for 27,000 lit water will be = 2.5 x 27,000 = 67,500 g = 67.5
kg.
Problem No. 2:
Calculate the cost and quantity of fungicide for spraying 10 hectares of fruit garden for
controlling the disease problem on the points given below:

1 Sprays to be taken 02
2 Rate of fungicide to be used 0.25%
3 Spray solution required per plant 5.0 liter per plant
4 Cost of fungicide Rs. 650 per liter
5 Plant population per hectare 250
Answer:
Total plant population in 10 hectare = 10 x 250 = 2500 plants
Per plant spray 4.5 liter, hence for 2 sprays = 5 x 2 =10 liter
Total water requirement for total plants = 10 liter x 2500 plants = 25,000 liter
As the fungicidal rate is 0.25%, it means that 2.5 g fungicide per liter of water.
Hence total fungicide requiring for 25,000 lit water will be = 2.5 x 25,000 = 62,500 g = 62.5
kg.
There for, Cost of fungicide = 62.5 kg x Rs 500 per kg = 31,250/-
Hence, cost of fungicide is Rs. 31,250/-
Problem No.3
Calculate the cost and quantity of streptocycline @ 250 ppm for spraying 5 ha orchard of
pomegranate on the basis of following points :
Water requirement for 1 ha area = 500 liter
Rate of streptocycline = Rs. 20 per 5 gm
Answer:
Streptocycline @ 250 ppm = In 1 liter 0.25 gm streptocycline is required for 1 ha area 500
liter water required for spraying
Therefore for 5 ha area = 500 x 5 = 2500 liter water is required
In 1 liter = 0.25 gm streptocycline
Therefore in 2500 liter = ?
2500 x 0.25
-------------- = 625 gm streptocycline is required
1
Rate of streptocycline : for 5 gm = Rs. 20/-
Therefore, for 625 gm = ?
625 x 20
------------- = Rs. 2500
5
Answer: quantity of streptocycline required for spraying 5 ha orchard is 625 gm
cost of streptocycline for spraying 5 ha orchard is Rs. 2500/-

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Problem No. 4
Calculate the cost and quantity of streptocycline @ 500 ppm + Copper oxychloride @ 0.25
% for spraying 10 ha orchard of pomegranate on the basis of following points.
Water requirement for 1 ha area = 500 liter
Rate of streptocycline = Rs. 20/- per 5 gm
Rate of copper oxychloride = Rs. 230/- per 500 gm
Answer:
Streptocycline @ 500 ppm = in 1 ltr 0.5 g streptocycline is required
For 1 ha area 500 ltr water is required for spraying
Therefore for 10 ha area = 500 x 10 = 5000 ltr water is required
In 1 ltr = 0.5 gm streptocycline is required
Therefore, in 5000 ltr = ?
5000 x 0.5
----------------- = 2500 g streptocycline is required.
1
Rate of streptocycline: for 5 gm = Rs. 20/-
Therefore for 2500 gm = ?

2500 x 20
---------------- = Rs. 10,000/-
5

Similarly, copper oxychloride @ 0.25% = in 1 ltr 2.5 g copper oxychloride is required

For 1 ha area 500 ltr water is required for spraying
Therefore for 10 ha area = 500 x 10 = 5000 ltr water is required
In 1 ltr = 2.5 g copper oxychloride is required
Therefore in 5000 ltr = ?
5000 x 2.5
--------------- = 12,500 g copper oxychloride is required
1
Rate of copper oxychloride : for 500 g = Rs. 230/-
Therefore, for 12,500 gm = ?
12,500 x 230
---------------- = Rs. 5750 /-
500
Answer:
Quantity of streptocycline required for spraying 10 ha orchard is 2500 gm
Cost of streptocycline for spraying 10 ha orchard is Rs. 10000/-
Quantity of copper oxychloride required for spraying 10 ha orchard is 12500 gm
Cost of copper oxychloride for spraying 10 ha orchard is Rs. 5750/-

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[81]
EXERCISE NO 16

Collection and Preservation of Disease Specimen

Objective:
Collection of plant disease specimens from the field
Preservation of disease specimen
The micro organism like fungi, bacteria, viruses and nematodes cause disease in field
and plantation crops the affected or diseased specimen are necessary to collect and preserve
for plant pathological and mycological studies. These pathogens produce different types of
symptoms on leaves, stems, fruits, flowers and roots. Disease specimens collected from the
field can be preserved in laboratory and herbarium for further studiers.
A) COLLECTION OF PLANT DISEASE SPECIMENS:
While collecting the disease specimen the following precaution s should be taken
Proper section of the specimen:
Specimen should be fresh showing clear symptoms of the disease, very old or dried specimen
should be avoided as far as possible because saprophytic growth may have been established
on such specimen. For collection of plant disease specimens, following material are required
Hand lens
Blotting papers or absorbant paper
Paper bags
Vesiculum
Knife
Note book
Specimen, field condition and other relevant information should be noted in the book.
Specimen should be kept intact in the paper bags or vasculum. Certain plant diseases are
prevalent during a particular season, hence such disease specimen should be collected during
that sseseon only. In general, the specimens, which are collected and preserved are useful in
plant pathological and mycological studies.
It is essential to collect adequate amount of affected samples because much of the disease
sample would be exhausted during investigation. It is also necessary to preserve the specimen
and its symptoms, so that it becomes easy for the investigator to process the sample.
Disease specimen (may be stem, roots, leaves, inflorescence etc.) collected should be free
from soil particles and plant debris. The root samples should be washed thoroughly to remove
the soil particles. Care must be taken to gently put the sample on blotting or absorbent papers
to remove excess water. These absorbent papers need to be replaced quite often (every day)
to render the specimen dry. It is essential to indicate the name of host (local as well as latin
name ), stage of crop, locality, soil type, date of collection, name of collector, name of owner
of the field, tillage operations, and spraying undertaken.
Procedure:
1. Visit to different field.
2. Collect the affected samples along with roots.
3. Place them in vesiculum or paper bags.
4. On vesiculum/paper bags containing disease specimen, write the information about name
of host, the stage of the plant growth, time and season of collection etc. the disease caused

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 by phycomycetes fungi are to be collected other hypomyctes can be collected during dry
and cool season.
B) PRESERVATION OF PLANT DISEASE SPECIMEN
1) Dry preservation
In this method moisture from the specimen is removed by drying this can be done by two
ways as given below.
Drying in open air:
Hard and woody tissue specimens like stems and roots are often dried in the open air
preferably under shade.
Drying and pressing
In this method the specimens are well wrapped in blotting papers, after removal of soil and
dust etc. which are then pressed with some weight. Weight should give to press the specimen
uniformly. Every day the specimen should be exposed to the fresh air and turn again and
pressed till the specimen get dried completely. This method is generally used for leaves,
flowers and young plants
After drying, the specimen are mounted in various ways, labeled properly and preserved in
the herbarium.
Mounting in the packets
The specimen are mounted on a paper as such that the specimen will be visible though the
window of the specimens packets. The information about the disease specimen should be
filled in or written on the bags and kept in the herbarium cases.
Mounting on hard board
Hard specimen like root and stem are mounted on hard board directly with the help of
collection tape or string.
Riker mounts
Special boxes having glass on one side and detachahable side from the back are used for
preservation. The specimen is kept on the glasses from inside and cotton wool is filled in the
box, so as the specimen shouldnot move from the right place. Bottom board is fixed with
screw or immovable metal stripes. These specimens are kept in herbarium with information
about the specimen or the information should be self explanatory.
2. Wet preservation
Certain chemicals or preservatives are used for preservation. The preservations/chemicals
commonly used are: Formaldehyde, ethyl alcohol, rikee and Riker fluid or solution. These
chemicals help in retaining colour of the specimen. These chemicals are grouped in two:
General
Selective preservation
General preservatives:
Formalin or Formaldehyde (5% solution): Available commercially as formaldehyde 40%
is mixed with 95 ml of water to make a good preservative of 5 %. All parts of plants are
preserved in this solution.
Ethyl alcohol: Generally hard and woody specimens after boiling in water are preserved in
10 % ethyl alcohol.
Rikee or Riker solution: used for hard specimens and its composition is :
Ethyl alcohol : 150 ml
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Formaldehyde 40% : 25 ml
Water : 1000 ml
B) Selective preservation:
Certain chemicals are used for bleaching and retaining colour of the specimen, copper acetate
solution is commonly used. Crystals of the chemicals are dissolved in 50 % acetate acid and
diluted for 4 times with water before use. The solution is boiled and specimen are deeped for
3 minutes. First color of the specimen will disappear and regained after 15 minutes.
Specimens are washed thoroughly.

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