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11111111111111111111111111111

FOR CLASS USE ONLY

ACHARYA N. G. RANGA AGRICULTURAL UNIVERISTY

PRACTICAL MANUAL
B.Sc., (Ag.)
Course No:PATH-171
INTRODUCTION TO PLANT PATHOGENS

COMPLIED BY
SRI.V.CHANDRA SEKHAR
ASSISTANT PROFESSOR
DEPARTMENT OF PLANT PAHTLOGY
AGRICULTURAL COLLEGE,NAIRA
SRIKAKULAM-532 185
And
DR.M.JOHNSON
ASSOCIATE PROFESSOR & HEAD
DEPARTMENT OF PLANT PATHOLOGY
AGRICULTURAL COLLEGE,MAHANANDI
KURNOOL -518 502

2012

Certificate

Certified that this is a bonafide record of practical work done by

Mr. / Ms. ________________________ID No: _______________in
B.Sc.,(Ag.)

degree

INTRODUCTION

TO

program
PLANT

Course

No:PATH-171

PATHOGENS

________semester,20____-20____.

Date:

Signature of course in-charge

during

Contents
Ex No.

Title of the Exercise

Page No.

Study of vegetative structures of sungi and their modifications

Study of reproductive (sexual and asexual) structures of fungi

Study of Pythium and Phytophthora

Study of Albugo

Study of downy mildew fungi-Sclerospora and

Peronosclerospora

Study of downy mildew fungiPseudoperonospora,Peronospora,Plasmopara and Bremia and

Zygomycetes fungi-Rhizopus

Study of Powdery mildew fungi-Oidium,Oidiopsis and

Ovulariopsis

Study of ascocarps of Erysiphe,Phyllactinia,Podosphaera and

Microsphaera

Study of rust fungi-Puccinia (different stages) ,Uromyces and

Hemileia

10

Study of smut fungi Sphacelotheca,Ustilago and

Tolyposporium-Study of Ganoderma

11

Study of imperfect fungi-Septooria,Colletotrichum and

Pestalotiopsis

12

Study of imperfect fungi-Aspergillus,Penicillium and Pyricularia

13

Study of imperfect fungiDreschlera,Helminthosporium,Alternaria,Cercospora and

Phaeoisariopsis

14

Study of imperfect fungi-Fusarium,Rhizoctonia and Sclerotium

15

Isolation of phytopathogenic bacteria (locally available diseased

plant material) and study of colony characteristics and Grams
staining

16

Demonstration of mechanical transmission of plant vuruses

4

Evaluation of class work performance

Ex No:

Date Title of exercise

Remarks Initials of
the teacher

1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16

1. STUDY OF VEGETATIVE STRUCTURES OF FUNGI AND THEIR

MODIFICATIONS

VEGETATIVE STRUCTURES OF FUNGI:

Fungi produce various types of vegetative structures which are chiefly concerned with nutrition
and growth. The vegetative body (thallus) of a typical fungus consists of filamentous
mycelium, which is made up of branching hyphae. The hyphae may be aseptate(coenocytic),
typical of the lower fungi or septate(multicellular)with cross partitions, characteristic of higher
The

fungi.

mycelium

is

endophytic(inside

plant

tissue) in vast majority of cases or ectophytic(on the surface of the host) in such
fungi

that

cause

powdery

mildews.

The

endophytic

mycelium

may

be

either

intercellular (between the cells) or intra cellular (with in the cells ) .Special
structures

known

as

are

haustoria(drinkers)

produced

by

inter

cellular

and

ectophytic mycelia and help in obtaining nourishment from the host cells. Some
parasitic fungi develop appressoria at the tip of hyphaeby which it attaches to the
host surface.
TERMINOLOGY:
Thallus: (pI. Thalli;Gr.Thanos=shoot) The somatic
phase

of

the

fungus.

It

is

the

body

of the fungus typically consisting of microscopic

threads or filaments that branch in all directions,
spreading over or with in substratum utilizedfor food.

Septate mycelium Ex:Rhizoctonia

Mycelium: (pl.mycelia;Gr.Mykes= mushroom,fungus).A

mass

of

constituting

hyphae

the body (thallus) of a fungus.

Hyphae:(pI.
transparent

hyphae;
tubular

Gr.hypha=web)A
structure

filled

with

thin
a
Hypha (filamentous unit) of Septate
mycelium

layer of protoplasm.
6

Septum:(pl.septa;
L.septum=partition)
A cross wall in a
hypha.
Septate

hypha:A

hyphae with more

or

less

regularly

occurring

cross

walls
(septa).eg:-

Drechslera.
Various types of septa in fungi

Aseptatehypha:(coenocytic-Gr.koinos=common+kytos

without

the

septa.

The

nuclei

are

embedded

in

hollow
cytoplasm

separated by cross walls. eg:-Rhizopus.

Coenocytic (Aseptate) mycelium in Rhizopussp.

vessel).A

hypha

without

being

EctophyticI Ectobiotic: (Gr.ektos

(Gr.ektos= outside+bios = life). A fungal mycelium living
li
on the
surface of the host.eg:-Oidium.
Endophytic/Endobiotic:(Gr.endo=inside+bios=l
endo=inside+bios=life). A fungal mycelium living
g within
w
the cells
of the host.eg:-Oidiopsis.

Ectophytic growth in powdery mildew fungi and development of various shapes of haustoria
Ex: Oidiumsp.

MODIFICATIONS OF VEGETATIVE STRUCTURES

Sometimes

special

forms

of

mycelium

are

formed

to

perform

special

functions such as a. obtaining nourishment b. overcoming unfavourable conditions

i.e., over overwintering, over summering) and c. reproduction. The following are the various
types of modifications of fungal mycelium.

Rhizoids:(Gr.rhiza=root +oeides=like). Rhizoids are slender root like branched

structures produced by some fungi which are useful for anchoring the thallus
1.

to the substratum and for obtaining nourishment .eg:-Rhizopus stolonifer.

Rhizoids

2.

Rhizoids in Rhizopus

Rhizomorphs:( Gr.rhiza=root + morphe =shape).A thick strand of somatic hyphae

in which the hyphae lose their individuality and form complex tissues, which are resistant to
adverse conditions and remain dormant until favourableconditions return. The structure of
growing tip of rhizomorph somewhat resembles that of a root tip, hence the name
rhizomorph.eg:-Armillariamellea.

Rhizomorphs

Rhizomorphs Ex:Basidiomycetes fungi

9

Ex: Armillariamellea

Sclerotium:(pl.sclerotia.Gr.skleron=hard) Sclerotium is a hard resting body

3.
formed

due

to

remain

dormant

aggregation
for

long

of

mycelium,

periods

of

resistant

time

and

to

unfavorable

germinate

on

the

conditions,
return

of

favorableconditions.eg:-Sclerotiumrolfsii and Rhizoctoniasolani.

Sclerotial bodies in Rhizoctoniasp.

Sclerotial bodies in Rhizoctoniasp.

4.

Stroma:(pl.stromata

Gr

.stroma-mattress),

Stromais a compact somatic structure much like a

mattress or a cushion on which or in which
fructifications (spores
usually

or fruiting bodies) are

formed.eg:-Clavicepspurpureaand

Colletotrichum. falcatum.

5. Haustorium:(pI. haustoriaL.hauster=drinker).
Haustorium is a special absorbing organ produced

Perithium(Sexual fruiting body)

embedded in StromaEx:Claviceps

on certain hyphae by parasitic fungi for obtainin nourishment by piercing into living cells of
the host.They may be knob like, elongated or branched like in shape. eg:-knob like-Albugo
candida.; elongated-Erysiphecichoracearum.

Development of knobbed haustoria

in host cell Ex:Albugo

Development of elongated and branched

haustoria in Erysiphe Sp.
10

6. Appressorium:(pl.appressoriaL.apprimere=to press against). A flattened, hyphal

pressing

organ

from

which

minute

infection

peg

usually

penetrates the epidermal cells of thehost.eg:-Pucciniagraminisf.sp. tritici.

Appressorium

11

grows

and

OBSERVATIONS AND RECORD

Observe the different vegetative structures of fungi and make sketches of all
the structures and label the parts.

12

Work done report

Ex No:
Date:
Title:

13

2. STUDY OF REPRODUCTIVE STRUCTURES OF FUNGI

(ASEXUAL AND SEXUAL)
Reproduction is the formation of a new individual. Sexual reproduction involves the
fusion of compatible gametes / gametangia. This lacks in asexual and vegetative
reproduction. The sexual state is termed as perfect state or teleomorphic state and
the asexual state as imperfect state or anamorphic state. Fungi reproduce chiefly
by means of spores,which are produced in three ways viz., asexual, sexual and
vegetative. Spores may develop on specialized hyphae, exogenously or may be
borne in special fruiting bodies or fructifications, endogenously. A fruiting body is any
fungal structure that contains either sexual or asexual spores. The fruiting bodies
are of great importance to fungi, not only in tiding over conditions of desiccation and
freezing, but have vital significance from the point of view of multiplication of
inoculum and maintenance. The various fruiting bodies have also been utilized as
taxonomic character for distinguishing the various groups of fungi.

ASEXUAL SPORES:
Asexual reproduction occurs usually under conditions that favour growth and
several generations may be produced in one season. Asexual spores are capable of
immediate germination and bringing about a rapid increase in numbers and spread
of the organism under favourable conditions.
The following are the various types of asexual spores produced by fungi.
1.Zoospore(Planospore):Motile

asexually

produced sporangiospore bearing flagella and

capable of moving in water. Sporangium containing
zoospore is calledzoosporangium.
Ex: Pythiumdebaryanum,
Phytophthorainfestans.
Zoospores

14

2. Sporangiospore(Aplanospore):Non
motile asexual spore found in a sac like
structure

is

called

sporangiospore.

Sporangia are produced at the tips of

special hyphae called sporangiophore
Ex: Rhizopus stotonifer.
3.Conidiospore(Conidia):Non motile
asexual, single or multicelledspore
Liberation of Sporangiospores fromRhizopus
sporangium

produced at the tips or sides of a

specialhypha

called
may

conidiophore.Theconidia
directly

on

the

arise

tips

of

conidiophore(eg.Drechslera,
Helminthosporium, Alternaria) or inside
specialisedfruiting
pycnidium,

bodies

acervulus,

such

as

sporodochium

and synnemata.

(a)

Pycnidium:Itis

an

asexual,

spherical or flask shaped fruiting body

lined inside with conidiophores and
producing

conidia.eg.Phomalingam,

Phomopsisvexans.

(b)

Acervulus:A

saucer

shaped,

asexual fruiting body with a mat of

hyphae

producing

conidia

on

short

conidiophores.
Eg:Colletotrichumfalcatum,Pestalotiapsis

Pycnidium

palmarum.

15

(c) Sporodochium:A cushion shaped

asexual

fruiting

body

consi
sisting

of

cluster of conidiophores woven together

on a mass of hyphae
Eg: Fusariumoxysporumf.sp. ciiceri

(d)

Synnemata:A

conidiophores
diophores

united

group
at

of

the

base

producing conidia at its

ts tip or along the
length of synnema, resembling a long
handled feather duster.
Eg:Graphiumulmi

4.

Chlamydospore:A thick
ck walled

vegetatively produced asexual resting

spore formed by the thickening
ckening of a cell
of a hyphal cell.
Eg:Fusariumoxysporum. f.sp. ciceri
ciceri.

5.

Arthrospore:An

resulting

from

the

asexual

spore

fragment
fragmentation

of

vegetative
hyphae. Eg :Erysiphaecichoracearum
cichoracearum.

6.

Blastospore:Spores

formed

by

budding.Budding is the produ

production of
small outgrowths (bud) from a parent cell
cell.
The

bud

increases

in
n

ssize

and

gets pinched off from the parent cell

cell.
Eg:Yeast-Saccharomyces cerevvisiae.

16

SEXUAL SPORES:
Sexual

spores

of

fungii

are

variously

known

as

oospores,,

zygospores,

ascospores
ospores and basidiospores depending o
on the manner of their
ir formation and the
class of fungi to which
ch they belong
belong. They are the results off fusion between two
gametes of opposite
te sex and funct
function as resting spores, thus helping the fungus
f
in
over-summering or over-winter
wintering. Sexual spores in fungi are less
ss varied
vari
and less
frequent compared to asexual spo
spores, which are produced in great abundancce.
The following are the important types of sexual spores produce
produced by fungi.
1. Oospore: A thick walled sexual spore
produced

by

the

union
on

morphologically

of

two

different

gametangiaEg.Pythium
infestans.
debaryanum,Phytophthorainfestans.
2. Zygospore:A
A thick walled sexual
resting spore produced by the fusion of
two

morphologically
ogically

similar

gametangiaEg.Rhizopusstolonifer
.Rhizopusstolonifer.
3.Ascospore:

sexually

produced

meiospore borne in an ascus.

Ascus:A
A sac like structure generally
containing eight ascosporesformed
formed after
karyogamy and meiosis.
Asci may be produced in a sexual fruiting
body called ascocarp or may arise
naked without any ascocarp.
Naked ascus:Ascus
Ascus arising naked
without any fruiting body.Eg. Taphrinadeformans
Taphrinadeformans.

17

Ascocarp:

sexual

fruiting

body

containing asci. There

here are four types of
ascocarps produced by fungi.
(a) Cleistothecium:An
An entirely closed
closed,
more or less spherical shaped ascocarp
and iss many times provided on its body
with appendages of various typ
pes, which
serve as organs of anchorage and help in
dissemination.
Eg.Erysiphecichoracearum.
(b) Perithecium:A closed, flask shaped
ascocarp with a pore at the top ,a true
ostiole through which ascospo
ascospores are
released.eg. Clavicepspurpurea.
a.
(c).Apothecium: An open cup or saucer
shaped ascocarp. Eg:Tuber
Tuber indicum
indicum.
(d)

Ascostromata:Astromaticascocarp
Astromaticascocarp

bearing asci directly in loculessor cavities

within stroma.eg. Eisinoefawcetti
Eisinoefawcetti.
Pseudothecium:
Anuniloculateascostromatais
is

called

pseudothecium or pseudoperithecium
thecium.
Eg:Venturiainaequalis
4.Basidiospore.. Sexual spore borne on
a

basidiumfollowing
ng

karyogam
karyogamy

meiosis.eg.Pucciniagraminis.f.sp
sp.

and
tritici,

Agaricus.bisporus.

18

Basidum with four basidiospores on sterigmata

Basidium.A club shaped structure on

which basidiospores are borne.
Basidiocarp.Sexual fruiting body that
bears basidia and basidiospores.It may
be thin, fleshy, spongy, leathery, corky or
of any other structure.
Eg.Agaricus,
Pleurotus, Volvariella.

Various shapes of basidiocarps

19

OBSERVATIONS AND RECORD

Observe the different vegetative structures of fungi and make sketches of all
the structures and label the parts.

20

Work done report

Ex No:
Date:
Title:

21

3. STUDY OF PYTHIUM AND PHYTOPHTHORA

Division: Eumycota
Sub-division: Mastigomycotina
Class: Oomycetes
Order: Peronosporales
Family: Pythiaceae
eg: Pythium
Habitat: Fungus is a facultative parasite and lives in soil on dead organic matter or

parasitically on young seedlings of crop plants.

Mycelium:

Well developed, branched, coenocytic, hyaline, intracellular mycelium

without haustoria.
Asexual

reproduction: Sporangia are large, globose(P.debaryanum) or

irregularly

lobed

position

on

(P.

somatic.

aphanidermatum)
hyphae.

formed

Zoospores

are

either

terminally

produced

in

or

intercalary

vesicle

which

emerge out of sporangium.

Sexual

reproduction:

Gametangial contact. Oospores are smooth, thick walled,

round, light brown and aplerotic.

Diseases: Damping-off of vegetable seedlings of solanaceous crops caused by

Pythium debaryanum orP. aphanidermatum.

Pythium debaryanum
22

OBSERVATIONS AND RECORD

1. Obtain the culture of P.debaryanum/P.aphanidermatum.

2. Examine the morphological features of the fungus under a microscope.
3. Look for the mycelium, sporangia and oospores and describe the
characteristics.
4. Make sketches of all the structures and label.
PHYTOPHTHORA

Division: Eumycota
Sub-division:Mastigomycotina
Class: Oomycetes
Order: Peronosporales
Family: Pythiaceae
eg: Phytophthora

Habitat: Fungus is a facultative parasite

and lives in soil on dead organic matter

(parasitically on potato tubers.)
Mycelium:

Well

developed,

branched,

coenocytic, hyaline, intercellular mycelium

with haustoria.
Asexual

lemon

reproduction:

shaped,thin

Sporangia

walled,

are

papillate,

formed terminally on sympodially branched

sporangiophore. Zoospores are produce

Sporangiphore with sporangia

in sporangium.
Sexual reproduction: Gametangial contact. Oospores are smooth, thick walled

round, dark brown and plerotic.

Diseases: Late blight of potato ( Phytophthorainfestans), Leaf blight and black shank of

tobacco( Phytophthoraparasitica var. nicotianae).

23

24

OBSERVATIONS AND RECORD

1. Obtain the culture of P.infestansor diseased specimens of leaf blight and

black shank of tobacco.
2. Examine the morphological features of the fungus under a microscope.
3. Look for the mycelium, sporangia and oospores and describe the
characteristics.
4. Make sketches of all the structures and label.

25

Work done report

Ex No:
Date:
Title:

26

4. STUDY OF ALBUGO
Division: Eumycota
Sub-division: Mastigomycotina
Class: Oomycetes
Order: Peronosporales
Family: Albuginaceae
Eg: Albugo

Habitat: Fungus is an obligate parasite on crucifers and lives in soil in the form of
oospores or parasitically on plants.
Mycelium:

hyaline,

Coenocytic,

endophytic,

intercellular

mycelium

with

knob

shapedhaustoria.
Asexual reproduction: Sporangiophores are unbranched, hyaline, clavate, bears
sporangia
walled,

in

sessile,

chains

in

hyaline

basipetal
and

succession.

germinate

by

Sporangia

zoospores.

are

spherical,

Zoospores

are

thin

hyaline,

biflagellate and kidney shaped.

Sexual reproduction: Gametangial contact. Oospores are round, thick walled, dark
brown and outer wall warty.
Diseases: White rust on mustard caused by Albugo candida

Electron microscopic view of Albugo candida

27

OBSERVATIONS AND RECORD

1.Take out thin sections of diseased leaves.
2.Examine the sections under the microscope.
3.Look for the palisade 'layer of sporangiophoreswith chains of sporangia
and describe the characteristics.
4.Make sketches of all the structures and label.

28

Work done report

Ex No:
Date:
Title:

29

5.STUDY OF DIFFERENT GENERA OF DOWNY MILDEW FUNGISCLEROSPORA ,PERONOSCLEROSPORA

Division: Eumycota
S.D:Mastigomycotina
Class: Oomycetes
Order: Peronosporales
Family: Peronosporaceae
eg:Sclerospora
Peronosclerospora

The

members

of

family

Peronosporaceae

cause

downy

mildew

diseases

in

several economically important crops. The name downy mildew is given because of
soft feathery growth observed on the lower side of affected foliage consisting of
sporangiphores and sporangia which comes out through stomata.

Habitat: Fungus lives in the soil in the form of oospores and all the species are
obligate parasites of vascular plants.
Mycelium: Mycelium is well developed, branched, hyaline, coenocytic, intercellular
and produce haustoria.
Asexual

reproduction:

branched

and

papillate,

germinate

Sporangiophores

determinate
by

type.

zoospores

are

Sporangia
or

in

well
are

some

developed,

deciduous,
members

characteristically

papillate
germinate

or

non-

by

germ

tube directly, thus behaving like conidia.

Sexual

reproduction:

Gametangial

contact.

Oospores

are

the

sexual

resting

spores, smooth, round, thick walled, light brown or dark brown in color and
germinate by germ tube.
Downy

mildew

genera

are

differentiated

based

on

sporangiophores and method of germination of sporangia.

30

the

branching

pattern

of

Distinguishing generic characteristics:

1. Sclerospora:
Sporangiophores

are

stout,

having

upright branches, bearing sporangia on

sterigmata. Sporangia are hyaline, ovoid,
smooth walled, papillate and germinate
by zoospores. Oospore is plerotic
Eg:Sclerosporagraminicola

downy

mildew of bajra

2. Peronosclerospora:
Fungus possess characteristics of both
Peronosporaand Sclerospora.
Sporangiophores are erect, short, stout,
widening

towards

upper

portion,

dichotomously branched 2-5 times at

apex bearing sporangia on sterigmata.
Sporangia are hyaline, elliptical or ovoid,
thin walled, non- papillate and germinate
by germ tube like Peronospora. Oospore
is plerotic type like Sclerospora.
Eg:Peronosclerosporasorghi- downy
mildew of jowar
P. philippinensis- downy mildew of maize.

31

Work done report

Ex No:
Date:
Title:

32

6.STUDY OF DIFFERENT GENERA OF DOWNY MILDEW FUNGIPSEUDOPERONOSPORA, PERONOSPORA, PLASMOPARA, AND

BREMIA ANDZYGOMYCETES FUNGI - RHIZOPUS

Division: Eumycota
S.D:Mastigomycotina
Class: Oomycetes Order:
Peronosporales
Family:PeronosporaceaeEg:
PseudoperonosporaPeronos
pora, Plasmopara,
Bremia

1. Pseudoperonospora:
Sporangiophores are branched at acute
angles

with curved,

blunt

tips,

bearing

sporangia on sterigmata. Sporangia are

ovoid,

greyish,

papillate

and

germinate

by zoospores.
eg.Pseudoperonosporacubensis-downy
mildew of cucurbits

2. Peronospora:
Sporangiophores are dichotomously branched
2-

times

at

acute

angles

and

tips

of branches are curved and pointed bearing

sporangia

on

sterigmata.

Sporangia

are

hyaline, ovoid, non- papillate and germinate by

germ tube.
Eg:Peronospora destructor- downy mildew
of onion

33

3. Plasmopara:
Sporangiophores

are

angles

main

to

the

branched
axis

at

at

right

regular

intervals bearing sporangia on sterigmata.

Sporangia are ovoid and germinate by
zoospores.
Eg:Plasmoparaviticola- downy mildew of
grapes

4.Bremia:
Sporangiophores

are

dichotomously

branched,tips of branches are expanded

to cup shaped apophysiswith four sterigmata
bearing

sporangia.

Sporangia

are

ovoid, papillate and germinate by zoospores.

Eg:Bremialactucae-downy mildew on lettuce

34

ZYGOMYCETES FUNGI-RHIZOPUS
Division: Eumycota
Sub- Division: Zygomycotina
Class: Zygomycetes
Order: Mucorales
Family: Mucoraceae
Eg:Rhizopus.

Rhizopus stoloniferis known as common bread mold and is a general

contaminant of several food materials. The fungus is mostly saprophytic but is a
weak parasite. It causes soft rot of potatoes during storage.

Rhizopus stolonifer
Mycelium:
The fungus produces abundant soft cottony mycelium. The mycelium is coenocytic, well
developed and is differentiated into rhizoids, stolons and sporangiophores.
Rhizoids/holdfast area cluster of brown, slender, branched root like structures
which arise from the lower side of stolons and penetrate into the substratum .These are
useful for anchoring the thallus into the substratum and for absorption of nutrients.
Stolons or runners are aerial hyphae which grow on the surface of substratum
horizontally and connect the two nodal points (the junction of stolon and rhizoid or the point
35

from which rhizoids are produced)

Sporangiophores are erect,

unbranched

hyphae

usually produced

in fascicles

(groups) only from the nodes during asexual reproduction. Each sporangiophore
bears a single sporangium at its tip. Sporangia are large, globose,manyspored
with a sterile structure called columella.
Asexual reproduction:
Asexual reproduction

is

through

non

motile

sporangiospores(aplanospores).

Sporangiospores are uninucleate, globose, brown, smooth walled, non motileand

are produced inside columellate sporangium. The sporangiospores are liberated by
rupture of sporangial wall as a black mass on collapsed columella. The spores
germinate under favourable conditions by germ tube that gives rise to mycelium.
Sexual reproduction:
The

fungus

is

heterothallic.

Sexual

reproduction

is

by

isogametangial

copulation. Zygospores are thick walled, dark, warty sexual resting spores that
develop in a zygosporangium formed as a result of fusion of gametangiaduring
sexual reproduction.

Zygomycetes fungi-Rhizopuslife cycle

36

OBSERVATIONS AND RECORD

1. Obtain the culture of Rhizopus stotonifer.

2. Examine the morphological features of the fungus under a microscope.
3. Look for the structures viz., coenocytic mycelium, sporangiophores,
rhizoids, stolons, sporangia, columella and sporangiospores.
4. Make sketches of all the structures observed and label.

37

Work done report

Ex No:
Date:
Title:

38

7. STUDY OF POWDERY MILDEW FUNGI OIDIUM, OIDIOPSIS AND

OVULARIOPSIS
Three types of conidial stages are recognised in powdery mildews.
1.Oidium:( syn: Acrosporium)
Majority

of

powdery

mildew

genera

have

Oidiumtype

conidia.

Mycelium

is

ectophytic, hyaline.Conidia develop from a flask shaped mother cell (spore mother
cell) formed on a short conidiophore. Conidia are barrel shaped with flat ends and
produced in chains. The conidia are also referred to as meristem arthrospores as
these are formed by fragmentation of hyphae. The perfect stages viz., Erysiphe,
Podosphaera,

Uncinula,

Sphaerothecaand

Microsphaeraproduce

Oidiumas

conidial stage.

Oidium
2.Oidiopsis:
Mycelium is endophytic. Conidiophores may be branched or unbranched, erect,
septate, hyaline and emerge through stomata. Conidia are produced singly and
cylindrical in shape. Conidia are of two types. a. blunt tip b. pointed tip.
Eg:Leveillulasp. produce Oidiopsisas conidial stage.

39

3. Ovulariopsis:
Mycelium

is

partly

ectophytic

and

partly

endophytic.The

conidiophores

are

hyaline, septate, unbranched and bear a single conidium. Conidia are rhomboid in
shape. In some species, the conidiphores are spiral in shape.
Eg:Phyllactiniasubspiralis.

40

Work done report

Ex No:
Date:
Title:

41

8. STUDY OF ASCOCARPS OF
ERYSIPHE,PHYLLACTINIA,UNCINULA,PODOSPHAERAAND
MICROSPHAERA

Division:Eumycota
Sub- division :Ascomycotina
Class: Plectomycetes
Order: Erysiphales
Sexual state

Asexual state

Erysiphe
Uncinula
Podosphaera
Sphaerotheca
Microsphaera

Oidium spp.

Leveillula'
Phyllactinia

Oidiopsis sp.
Ovulariopsis sp.

Powdery mildews are the most common, conspicuous and easily recognisable
plant diseases characterized by the appearance of white powdery superficial hyphae
on aerial parts of living plants with large, one celled conidia produced terminally on
an isolated aerial branched conidiophores with haustoria in the epidermal cells of
their hosts. These diseases are most commonly observed on the upper side of the
leaf but also affect the underside of the leaves.

Habitat: Obligate parasites of vascular plants.

Mycelium:Mostly ectophytic (superficial), endophytic(internal), or semi- endophytic,
42

branched, septate, hyaline with globoseor finger shaped haustoria.

Sexual reproduction :
Species are homothallic or heterothallic. Sexual reproduction is by gametangial
contact. Antheridia and ascogonia are sex organs.Fruiting body (ascocarp) is a
cleistothecium which is produced on superficial mycelium without stroma as a
result of gametangial contact. The cleistothecia are first white and finally black in
color when mature, globose, completely closed (non-ostiolate), with a wall made up of
pseudoparenchymatous tissue of several layers called peridium.
The cleistothecia are provided with characteristic appendages,varyconsiderably
in length and character. Asciare released by disintegration of peridialwall of
ascocarp. Asci are globose to pyriform, sessile, unitunicate. Ascospores are elliptical
or oval, hyaline, unicellular and eight in number in a ascus.
Types of cleistothecial appendages in powdery mildew fungi

1.Myceloid
flexible,
somatic

appendage:These
flaccid

and

hyphae.

are

resemble
Eg:Erysiphe,

Sphaerotheca, LeveilIula.

2.Circinoid

appendage:

(hooked)These are rigid with curled or

coiled
tips. egoUncinula.

3.Dichotomously

branched

tips:These are rigid, flattened with

dichotomously branched tips.
Eg: Podosphaera, Microsphaera.

4.Appendages with bulbous base

and pointed tip: These are rigid,
spear like with bulbous base and
pointed tip.
Eg;Phyllactinia.

43

Powdery mildew diseases

of

various

crops

are

caused

by

many

genera

of

fungi and these genera are distinguished from one another based on the type of
cleistothecial

appendage,

number

of

asciper

cleistothecium,

conidial

type

and

nature of mycelium. Since cleistothecia are encountered rarely in nature, the

identification of genera presents a problem. Hence, these fungi are identified by the
characteristics of conidia and conidiophores.
KEY FOR THE IDENTIFICATION OF POWDERY MILDEW GENERA:

1. Nature of mycelium
a. Ectophytic b. Endophytic

c. Semi- endophytic

2 .Number of asci per cleistothecium

a.One

b. Many

3. Type of conidial state

a. Oidiumb.Oidiopsisc. Ovulariopsis
44

4. Type of cleistothecial appendage

a. Myceloid
b. Dichotomously branched
c. Circinoid
d.Bulbous base with pointed tip

1. Ectophytic
a.One ascus per cleistothecium
Oidiumtype conidial state

Myceloid appendageeg: Sphaerothecapannosa- powderymildew on rose

Dichotomously branched appendage

eg:Podosphaeraleucotricha-Powdery mildew on apple

b. Several asci
Oidiumtype conidial state
Myceloid-eg:Erysiphecichoracearum-Powdery mildew on cucurbits / bhendi
Circinoid-eg.:Uncinulanecator-Powdey mildew on grapes
Dichotomously branched- eg:Microsphaeraalni -Powdery mildew on lilac
2. Endophytic
Many asci
Oidiopsistype conidial state
Myceloideg:Leveillulataurica -Powdery mildew on chillies
3. Semi- endophytic:
Many asci
Ovulariopsistype conidial state
Bulbous base with pointed tip- eg:PhyllactiniacoryleaPowdery mildew on mulberry

45

Work done report

Ex No:
Date:
Title:

46

9. STUDY OF RUST FUNGI PUCCINIA (DIFFERENT STAGES),

UROMYCES AND HEMILEIA

Division: Eumycota
Sub- Division: Basidiomycotina
Class: Teliomycetes
Order: Uredinales
Family: Pucciniaceae
Eg:
1. Puccinia
2. Uromyces
3 Hemileia
The members of the order uredinales are popularly called as rust fungi. The
word rust refers to the reddish brown colour of some of the species. It is the brick red
colour of the uredia which gives its name i.e. rust and infected field appear rusty. The
disease mostly occurs on leaves as pustules and may appear on both the surfaces
depending on the host. The characteristic feature of rust fungi is the presence of
stalked teliospores. They may be free from each other or variously united but never
in the form of layers. The important rust genera are Puccinia, Uromyces and
Hemileia.
PUCCINIA

Habitat:Biotrophs, heteroeciousmacrocyclic rusts.

Mycelium:Septate, branched, intercellular with haustoria.
The fungus produces 5 distinct stages in its life cycle in a regular sequence.
0 Stage:Spermagonium: Flask shaped, ostiolate, contain numerous spermatia (male sex
cells) and receptive hyphae (female sex organs). Spermatia - ovate, hyaline, unicellular,
uninucleate and haploid.

47

0 Stage: Spermagonium

I Stage: Aecia: Cup shaped, yellow with round to angular aeciospores in chains.

I Stage: Aecia

II Stage:UrediumSub epidermal,uredospores-repeating asexual spores. Oval,binucleate,

unicellular, brown, thick walled, spiny, stalked, diploid.

II Stage: Uredium

48

III Stage:TeliumTeliospores- sexual resting spores, two celled, spindle shaped, round or
pointed at apex, constriction at septum, dark brown, thick walled, smooth, stalked, bi
nucleate.

III Stage: Telium

IV

Stage:

Basidium:

Basidiospores-

in

number

produced

on

sterigmata,

uninucleate, haploid.
Eg:

1.

Pucciniagraminis

f.

sp.

tritici-

black

stem

rust

of

wheat

2.

Puccinia

penniseti- bajra rust 3. Pucciniapurpurea- sorghum rust 4. Pucciniaarachidis- groundnut

rust.

IV Stage: Basidium

49

UROMYCES
Habitat: Biotroph, autoeciousmacrocyclic rust.
Mycelium: Septate, branched, intercellular with
haustoria.
Spermagonium: Flask shaped, ostiolate with
numerous

spermatia

(male

sex

organ)

and receptive hyphae (female sex organ).

Aecium: Aecia- short, whitish, cup shaped.
Aeciospores- round or elliptical, yellow, warty.
Uredium: Uredia- round, yellowish brown, appear
50

on both the surfaces, sub epidermal, Uredospores- ovate, light brown, echinulate, 1 celled,
stalked.
Telium: Teliospores- dark brown, ovate, pedicillate, smooth walled, 1 celled, hyaline
with papillum at the apex.
Basidium: Basidiospores- 4 in number produced on sterigmata.
Eg:Uromycesphaseolitypica-bean rust (syn: U. appendiculatus)

HEMILEIA

Habitat: Biotroph, autoeciousmicrocyclic rust.

Mycelium: Mostly inter cellular with haustoria,
Spermagonium and Aecium: unknown
Uredium: Uredospores- reniform, looking like
an orange segment, convex side echinulate,
concave side smooth
Telium:

Teliospores-

also

called

as

leptospores(a spore which germinate without

any resting period), turnip shaped, hyaline, thin
walled, stalked, unicellular.
Basidium: Basidiospores 4 in number produced on
sterigmata.
Eg:.Hemileiavastatrix-coffee rust
OBSERVATIONS AND RECORD
1. Collect the available diseased material
2. Take out thin sections of the diseased leaves
3. Examine the sections under microscope, identify and describe the
morphological features of the pathogen.
4. Make sketches of all the structures observed and label.

51

Work done report

Ex No:
Date:
Title:

52

10. STUDY OF SMUT FUNGI SPHACELOTHECA,USTILAGOAND

TOLYPOSPORIUM:STUDY OF GANODERMA.
Division: Eumycota
Sub-division:
Basidiomycotina
Class: Teliomycetes
Order: Ustilaginales
Family: Ustilaginaceae
Eg:Sphacelotheca
Ustilago
Tolyposporium

The smut fungi refer to the characteristic dark brown to almost black soot like
powdery mass of teleutospores produced in sori on the affected plant parts. Majority
of the fungi attack ovaries, which instead of forming grains, become transformed into
sori containing full of teleutospores. Teleutospores or chlamydospores or smut
spores commonly called as resting spores are the chief means of survival of the
fungus in the absence of the host plant. The important characteristics of smut fungi
are i. facultative saprophytes il. Teleutospores are formed from intercalary cells of
mycelium iii. Basidiospores / sporidia are not borne on sterigmata. Mycelium is
dikaryotic, majority with inter cellular hyphae with haustoria and clamp connections,
but without dolipore septum.

Asexual spores :
Budding
posses

is
no

the
sex

common

method

organs.Plasmogamy

of

asexual

takes

place

reproduction.
by

fusion

of

The
two

members
compatible

basidiospores or branches of promycelium. Teleutospores formed from dikaryotic

mycelium, germinate and give rise to septate or aseptatepromycelium(basidium)
and produce indefinite number of sporidia either laterally and / or terminally. Sporidia
are monokaryotic and unable to infect host plant (except Ustilagomaydis). Infection
of the host is by means of dikaryotic mycelium.

53

Sexual spores :
Teleutosporesor

chlamydospores

are

the

sexual

resting

spores.

Teleutospores

are of taxonomic importance and characteristics of the spores are used as basis for
identification of genera.

1. Sphacelotheca:
Majority of the grains are replaced by an oval or cylindrical dirty grey spore
sac with small sorus. Sorusis enveloped in a peridium made up 'of fungal cells with a
hard, slender central columella made up of host tissue.
Habitat:Seed borne
Mycelium: Intercellular
Teleutospores:Dark brown in mass, olive brown singly, smooth walled, oval, often united in
loose balls which break up to individual spores when placed in water.
Promycelium / basidium: Septate,4celled, produce sporidialaterally and terminally.
Sporidia: Spindle shaped, single celled.

Eg.Sphacelothecasorghi- grain / covered I kernal/ short smut of jowar

54

2. USTILAGO
ation on infected tissue mostly on grains
ains. Sorus is
Fungus induces gall forma
covered by a membrane off hos
host tissue.
Habitat:Seed borne.
Mycelium: Intercellular
Teleutospores:Separate, sing
single celled, spherical, black, tuberculate
e wall.
wal
Promycelium:Septate, 4 celled
celled, produce sporidia laterally and terminall
erminally.
Sporidia:Ovate, single celled, hyaline
hyaline.
Eg:Ustilagomaydis- common ssmut of maize

3. Tolyposporium:
Only a few grains are transformed into smut sori.
Each sorus is surrounded by healthy
ealthy grains
grains. Sori
are long, cylindrical, slightly currved.
Habitat: Soil borne.
Mycelium: Intercellular

55

Teleutospores:Spores united in solid permanent spore balls, globose, brownish

genechinulate, 1 celled
Promycelium:Septate,

celled,

produce

sporidia

laterally

and

terminally.

Sporidia:Singles or in chains, spindle shaped.

EgTolyposporiumehrenbergii- long smut of sorghum
CHARACTERISTICS OF FAMILY GANODERMACEAE:
The fruiting body of the fungus is called bracket fungi which is formed laterally at the
base of affected plant as a leathery stalked fan shaped or bracket shaped or without
stalk, made up of trimitichyphal system, hymenophoreporoid. The bracket is tough,
leathery or woody in texture and size vary from 1 to 20 inches in diameter. The stalk is
cylindrical and brown to black in color.
The upper surface of the bracket is reddish brown in color and coated with a hard shiny
substance resembling sealing wax while the lower side is white or yellowish in color.
When

examined

with

lens

minute

holes

or

pits

are

seen

allover

the

undersurface.These are the openings of numeral hymenial tubes or pores which are
vertically oriented inside the fruiting body. Each basidium gives rise to 4 sterigmata,
each of which bears a basidiospore at its tip.
Basidiospores are colored, 2 layered and cystidia are absent in hymenium. Bracket
shaped basidiocarp, broadly and horizontally attached to the tree trunks by means of a
short stalk or stipe. Ganoderma differs from other bracket fungi in having much longer
span of spore release, extending upto 5 months.
Ex: Ganodermalucidum- Root rot and wilt of coconutand other palm trees and citrus.

Bracket fungi

56

OBSERVATIONS AND RECORD:

1.Collect the available diseased material
2.Take
and

out

powdery

examine

under

mass

containing

microscope

and

of the pathogen.
3.Make sketches of the structures and label.

57

smut

spores

describe

the

from

diseased

materia!

morphological

features

Work done report

Ex No:
Date:
Title:

58

11.STUDY OF DIFFERENT GENERA OF IMPERFECT FUNGI

SEPTORIA COLLETOTRICHUM AND PESTALATIOPSIS

Division: Eumycota
Sub- Division:
Deuteromycotina
Class 1 :Coelomycetes
Order 1: Sphaeropsidales
Family: Sphaeropsidaceae
eg:Septoria
Order 2. Melanconiales
Family: Melanconiaceae
eg. 1. Colletotrichum
2. Pestalotiapsis
Class 2. Hyphomycetes
Order: Moniliales
Family 1.Moniliaceae
eg1. Aspergillus
2.Penicillium
3.Pyricularia
Family 2.Dematiaceae
eg1.Alternaria
2.Drechslera/Bipolaris
3.Helminthosporium
4.Cercospora
5.Phaeoisariopsis
Family 3. Tuberculariaceae
eg 1. Fusarium
Class 3.Agonomycetes
Order: Agonomycetales
Family: Agonomycetaceae
eg:1. Rhizoctonia
2.Sclerotium

The imperfect fungi technically called "fungi imperfecti" comprise a large

group of organisms whose perfect stage (teleomorphic or sexual stage) spores are
absent, rare or unknown. These are mostly conidial stages( anamorphic or asexual
stage) of Sub- Division Ascomycotina or more rarely Sub- Division Basidiomycotina.
Whenever the sexual stage of an imperfect fungus is discovered it is transferred to its
systematic position among fungi in Sub- Division Ascomycotina or Basidiomvcotina.
59

Some of them are saprophytes,many are plant pathogens causing severe

losses to the agricultural crops. These fungi are differentiated on the basis of their
conidial characteristics and by method of formation of conidiophores bearing conidia.

General characteristics:
Themycelium is septate, freely branched _and well developed. The fungi
sporulate producing conidiophores on which conidia are produced except in some
members. The conidiophores are produced singly in bunches or in compact masses
inside characteristic fruiting bodies, suchaspycnidium, acervulus, sporodochiumand
synnemata.
The shape, colour.size and septation of conidia are highly variable. Some
members are characterized by the formation of sclerotial bodies which are a bunch of
sterile, thick walled, septatehyphal cells, united together in the form of a ball. These
bodies may help the fungus to survive adverse climatic and environmental conditions
and to germinate when favourable conditions prevail.

1. Septoria:
Pycnidia:Globose, papillate, ostiolate and
brown. Pycnidial wall is consisting of pale
.

brown cells.
Conldliogenous cells:Hyaline, broad and
round at base and narrow at tip or barrel
shaped or cylindrical.

Conidia:

Hyaline,

many

septate

and

filiform.
Eg:SeptoriaIycopersici- leaf spot of tomato

60

2.Pestalotiopsis:
Acervulus:Epidermal

or

subepidermal, composed of thin walled

polyhedral cells

Conidiophores: Short, branched.

Conidia: Five celled, pointed hyaline
end cells with 2 or more apical simple or
dichotomously branched appendages,
median cells brown, fusiform, ego
Pestalotiopsispalmarum~ grey blight on
coconut

3. Colletotrichum:
Acervulus:Subepidermal,

saucer

shaped, with dark setae at the edge or

among conidiophores, setae- long,
darkbrown,septate.
Conidiophores:

Simple,

elongate,

brown, aseptate
Conidia: Hyaline, 1 celled, falcate
(sickle shaped)
eg.Colletotrichum falcatum- red rot of

Colletotrichum

sugarcane, C. Iindemuthianum- bean

anthracnose.

61

Work done report

Ex No:
Date:
Title:

62

12.STUDY OF DIFFERENT GENERA OF IMPERFECT FUNGIASPERGILLUS, PENICILLIUM AND PYRICULARIA

1. Aspergillus :
Well known saprophyte, grown on all
types of substrate and also a weak
parasite and commonly called as
"weed of the laboratory".
Mycelium: Septate, branched, with
multinucleate cells.
Conidiophores: The hyphal cell that
gives rise to conidiophore is called
foot cell. Conidiophores arise singly
on somatic hyphae, long, erect, non
septate and bears at its tip a
spherical structure called vesicle,
which bears two layers of bottle

Aspergillus

shaped structures called sterigmata

or phialides on which conidia are
produced in chains. The sterigmata
of first layer (lowermost) are called
primary

sterigmata

and

the

second

layer

(uppermost)

layer

secondary sterigmata.
Conidia: Globose, one celled, multinucleate, thick, rough walled and black.
Eg:Aspergillusniger- collar rot of groundnut

63

are

called

2.Penicillium

The

characteristic

conidial

apparatus

technically

is

called

"penicillus"

(L. Penicillium=small brush)because it resembles a small brush.

Mycelium:Highly branched, septate.
Conidiophores: Arise from any cell of hyphae (not from foot cell), branch once or
twice about 2/3 of the way to the tip in a characteristic symmetric or asymmetric broom
like fashion.
The first generation branches are called primary branches or rammi, on which
whorls

of

second

generation

branches

called

metulae

are

produced.Each

metula ultimately bears bottled shaped phialides which bear conidia in chains in
basipetal succession.
Conidia: Globose, hyaline.
eg: Penicilliumnotatum - citrus blue mold.

Penicillium

64

3.Pyricularia

Mycelium:
branched,

Immersed
septate,

in

host

tissue,

or

slightly

hyaline

coloured.
Conidiophores: Emerges through stomata
or

epidermal

cells

or

cuticle,

simple,

erect, long, septate, hyaline.

Conidia: Hyaline, pyriform( pear shaped),
broader at the base, tapering towards
apex, usually 3 celled with a small basal
appendage.
eg ..Pyriculariaoryzae- rice blast

Conidia of Pyricularia

65

Work done report

Ex No:
Date:
Title:

66

13.STUDY OF IMPERFECT FUNGI

FUNGIDRECHSLERA,HELMINTHOSPORIUM, ALTERNARIA, CERCOSPORA
AND PHAEOISARIOPSIS

1.Drechslera:
Mycelium:

Immersed,

branched,

brown.
Emerge

septate,

Conidiophores:
through

septate,

stomata,

brown,

simple,

conidiasinglyat.

erect,

producing

Apexthrough
pexthrough

sma
small

pores,

sympodialgrowth(continuing
g growth from

point below apex and then forming a second

conidia onnew apex), geniculate ( knee joints).

Conidia of Drechslera

Conidia: Dark, several celled, pseudoseptate,

cylindrical, straight, germinating from any or all
cells.
Eg: Drechsleragraminea- stripe disease of
barley
2.Helminthosporium:
Mycelium:: Dark, often in substrate.
Conidiophores: Single or clustered, tall, erect,
brown and simple.
Conidia:: Develop laterally through pores
beneath

septa,

often

brown,

obclavate,

appear

in

pseudoseptate

whorls,
with

prominent basal scar.

Eg:Helminthosporiummaydis-- southern corn
leaf blight

Conidia of Helminthosporium

67

3.Alternaria:
Mycelium: Branched, septate, dark brown
Conidiophore: Dark, septate, mostly simple
bearing

simple

or

branched

chain of conidia
Conidia:Dark, typically with both transverse
and

longitudinal

septa

with

simple

or branched apical appendage, obclavate,

frequently

borne

acropetallyin

Conidia of Alternaria

chains or borne singly

EgAlternariapalandui-onion blight
4. Cercospora
Mycelium: Immersed in host tissue. branched,
septate1 pale brown
Conidiophores:Emerge in clusters through
stomata, brown, septate, simple or
rarely branched with knee joints, marking the
scars of fallen spores
Conidia: Terminal, arise singly from
conidiophore, hyaline, filiform, several
celled ( 4 - 12 septate), a scar at the base.

Conidia of Cercospora

Eg:Cercosporaarachidicola- tikka(early leaf

spot)on groundnut

68

5. Phaeoisariopsis
Mycelium:

Septate,

intercellular

with

branched haustoria, pale brown, immersed

entirelyinleaf tissue.
Conidiophores : Emerging throughruptured
epidermis

in

pale

clusters,

to

olivaceous brown, smooth, geniculate or not,

septate

or

aseptate,

simple

with

prominent conidial scars.

Conidia: Light colored, cylindrical, usually
straight or slightly curved, rounded at
ends,

base

shortly

tapered

Phaeisariopsis
with

conspicuous hilum, mostly 3-4septate.

Eg:Phaeoisariopsispersonata(syn:
Cercosporidiumpersonatum)tikka
(late leaf spot) on groundnut

69

Work done report

Ex No:
Date:
Title:

70

14. STUDY OF IMPERFECT FUNGI- FUSARIUM, RHIZOCTONIA AND

SCLEROTIUM

1. Fusarium:
Mycelium: Superficial, cottony in culture, septate,
hyaline,grouped into sporodochia.
Conidiophores: Slender, simple, stout, branched
irregularly bearing awhorl o phialides bearing
conidia.
Conidia:Hyaline, 2 types- macroconidia(several
celled,slightly curved orbent, pointed at both
ends,

sickle

shaped

with

foot

cell),microconidia(1or2 celled, ovoid, single or in

chains, hyaline) .
Chlamydospores: thick walled, terminal or inter
asexual

calary,

spore

formed

by

modification of hyphal cell

EgFusariumoxysporiumf.sp.ciceri- wilt of gram .

2.Rhizoctonia:

FUSARIUM macro and micro

Cconidia

No spores
Mycelium: Brown, branches arise at acute
angles,

hyphal

cells

barrel

shaped

and

long.

Sclerotia: Brown to black, variable in form

(globose,

oval

or

irregular),

loosely

formed connected by mycelial threads, hard,

frequently

small

(less

than

mm

71

Mycelium

diameter), no differentiation of sclerotial tissue.

EgRhizoctoniasolani- sheath blight of paddy

3. Sclerotium

Sclerotia

Spores lacking
Mycelium: White or light coloured,
Sclerotia: Brown to black, globose or irregular,
compact,bigger(more than 1mm diameter in
size), hard, tissues are differentiated into rind,
central cortex, medullae.

EgSclerotiumrolfsii- root rot of groundnut

Sclerotia

OBSERVATIONS
AND RECORD

1. Collect the available diseased material/fungal cultures

2. Take out thin sections/ scrapings of the diseased material
3. Examine the sections / scrapings/ cultures under the microscope,
identify and describe the morphological features of the fungus
4. Make sketches of at! the structures and label.

72

Work done report

Ex No:
Date:
Title:

73

15. ISOLATION OF PHYTOPATHOGENIC BACTERIA (LOCALLY

AVAILABLE DISEASED PLANT MATERIAL) AND STUDY OF COLONY
CHARACTERISTICS AND GRAM STAINING.
Isolation and identification of bacteria associated with diseased plant is important to
determine whether bacteria are involved in plant disease. The method normally used to
isolate phyto-pathogenic bacteria differs from that used for fungi. A suspension of bacteria
is prepared from the infected material and loopfuls of this are streaked onto nutrient agar
plates. The aim is to produce single colony that can be subcultures pure. Pure cultures are
absolutely essential for pathogenicity assays and characterizing the pathogen for
identification. The serial dilution method is used for isolating bacteria from diseased tissues
contaminated with other bacteria. After surface sterilization of sections of diseased tissues,
the sections are ground in small volumes of sterile water and then part of this homogenate
is diluted serially. Finally, plates containing nutrient agar are streaked with a loop dipped in
each of the different serial dilutions and single colonies of the pathogenic bacterium are
obtained from the higher dilutions thatstill contain bacteria.
Choice of Material:Selection of the diseased tissue is important because pathogenic
bacteria may occupy different locations in the plant. In isolation of bacteria, it is generally
better to use newly collected material. The earliest stages of symptom development should
be used. Old lesions and dead areas usually contain few pathogens and many
saprophytes. Necrotic diseases usually start with tiny, dark greenish, spots which are
excellent for isolations. Cankers and soft roots should either be at an early stage, or if, older
lesions only are available, the advancing edge must be used, where the disease is
spreading into healthy tissue. When crown gall is suspected in a woody plant a search must
be made for young galls on young green stems. With wilts and other vascular infections
small pieces of infected stem are usually good for isolation.
Preparation of material:Clan leaves and stems, carefully chosen and handled as
aseptically, can often be used without surface sterilization. Roots and parts contaminated
with soil should be gently washed with clean water as soon as possible after collection.
Medium:Nutrient agar is suitable for the isolation of most plant pathogens. The medium
used for isolationmust have a dry surface. If, water is present the bacteria move around and
a carpet of mixed growth results instead of the required single colony.
This exercise deals with the isolation of bacterium, Xanthomonasaxonopodispv. Citri casual agent of citrus canker.
74

Materials:
Citrus leaves infected by Xanthomonasaxonopodispv. Citri(fresh), nutrient agar medium,
surface sterliant(I % less toxic sodium hypochlorite), sterile razor blade, glass rod, sterile
water, sterile test tubes and Petridishes, sterile pipettes (I ml), inoculation loop.
Procedure:
Put the U.V. lamp of inoculation chamber for 5 mts.Wipe the table topwith rectified spirit.
Wash hands with rectified spirit and air dry. Lit the burner/spirit lamp.Arrange sterile petri
dishes near the burner.

1.Select a diseased citrus leaf infected by canker and cut out a small portion of the
diseased tissue from the advancing lesion using sterile razor blade in a drop of sterile water
and after several minutes, examine under microscope. If bacterial ooze is seen, proceed for
isolation.

2.Surface disinfect the cut portions by dipping in sodium hypochlorite solution for 60 sec.
and then immediately rinse three times with sterile water.
3. Immerse the disinfected cut portions in 1 ml of sterile water taken in a clean sterilized test
tube.
4.Crush the cut portions of the leaf with a sterile glass rod. Allow it to stand for 5 minutes to
allow the bacteria to diffuse out of the cut tissue and into the water.
"
5.Gently lift the lid of a petridishwith left hand and using inoculation loop transfer several
loopfulsof the bacterial suspension to sterile pertridishes (three) containing 1 ml of sterile
water and mix thoroughly.
6.Hold flask filled with sterile luke warm nutrient agar medium in the right hand and remove
cotton plug near the flame and pour about 20 ml of medium into each dish and mix
thoroughly by gentle rotation. Allow time for solidification of medium.
7. Incubate the dishes in an inverted position at 250C for 36 to 72 hours.
8.Observation: Observe the dishes for appearance of desired bacterial colonies. If colonies

75

appear, select consistently found and well isolated colonies of the pathogen, for sub
culturing and further studies.
9.Select the isolated colonies and streak on the surface of a solidified medium in a zig-zag
manner and incubate the dishes at 25)0C. Bacteria isolated from nature may be
contaminated with saprophytic species, hence, re streaking for isolation ensures a pure
culture.TransfersomeofthepurifiedcoloniestoNAslants and grow them for further use.

Follow the protocol in the above procedure and isolate the desired bacterial colonies from
the given diseasedmaterial and maintain pure culture in petri dishes and slants for further
study.

76

STUDY OF COLONY AND MORPHOLOGICAL CHARACTERISTICS AND

GRAM'S STAINING REACTION OF A BACTERIUM

The classical approach to bacterial identification as soon as it is isolated from

diseased plant material in pure from involves a combination of information from
morphological and cultural characteristics, gram staining reaction which would form the
basis for the selection of biochemical tests to be performed to identify of the unknown
bacterium. The purpose of this exercise is to study the morphological and cultural
characteristics and gram staining reaction of the bacterium isolated from the diseased plant
material.

A. Study of colony characteristics of bacterium

Note: the following colony characteristics of the pure cultures of the bacterium isolated from
the diseased plant material and record the observations in the form of a table.

a) Color
b) Form

: yellow, white
: Punctiform,circular, rhizoid,irregular,
filamentous.

c)

Margins

: entire, undulate, lobate, filamentous

d) Elevation : flat, raised, convex, pulvinate, umbonate

e) Surface
f) Density

: smooth, glistening, rough, wrinkled, dry,

powdery
: opaque, translucent

B. Study of morphological characteristics of bacterium

Procedure:
1.Take clean glass. slide, wash and dry
77

2.Prepare the smear of the bacterium culture

Take inoculation loop and sterilize using alcohol lamp/burner. Place a drop of clean water
on the slide and mix it with a small portion of culture isolated from diseased plantmaterial.
Spread the bacterial suspension thinly to over an area of about 10 paise coin with
inoculation loop. This thin layer of bacterial culture spread on a glass slid is called smear or
film.
3.Allow the smear on slide to dry in air/heat (by passing the slide smear over the flame for
2-3 times). The main purpose of drying the smear is to fix the bacterial cells on the slide.
This is called fixation.

4.Flood the smear with five drops of basic stain (Methylene blue - 60 sec or crystal violet 30 sec) and allow it to react for designated time.

5. Pour off the stain and wash the smear gently with slowly running tap water.

6. Blot dry the slide using blotting paper (do not wipe the slide).

Observation and results:

Examine the stained slide under microscope and record the shape and arrangement of
bacterial cells and make colored drawing of the cells.
Colony and morphological description of bacteria
Colony
characteristics

Morphological

Description

characteristics

A.Color

a. Shape
b. Arrangement:

B. Form
C. Margins
D. Elevation
E. Surface
F. Density

78

Description

GRAM STAINING OF BACTERIA:

Gram staining developed by Christian Gram in 1884 is one of the most important and
widely used differential staining techniques useful in identifying and classifying bacteria into
two groups viz., Gram +ve and Gram -Ve. In this process the fixed bacterial smear is
subjected to four different solutions.
a. Basic Stain (Primary Stain) : It is an initial stain. The colour is in the positive ion of
the stain and reactwith cellular elements which are acidic in nature. Eg. Methylene
blue, Crystal Violet.
b. Mordant: It is a substance that increases the affinity between the cell and dye and
helps in fixing the dye on cells eg.Iodine.
c. Decolouring agent :It is a substance that removes the dye from a stained
celleg.Ethanol (95% alcohol).
d. Counter stain: It is a basic dye of a different colour from the primary stain. The
purpose of the counter stain is to give the decolourised cells a colour different from
that of the primary stain.
Those organisms that are not readily decolourised retain the colour of the primary
stain. Those that are readily decolourised take the colour of the counter stain
eg.Safranin.
The first step of Gram staining consists of staining the cells with a basic stain followed by a
treatment of these stained cells with a mordant. The cells are then treated with a
decolourising agent. The cells that retain the basic stain following decolourisation are called
Gram +ve, and those that are decolourised are Gram -ve and. can be restained with a
counter stain of contrasting colour. Gram +veisthicker than Gram -ve.
Materials: Fresh solutions of Crystal Violet, Gram's Iodine, 95% ethyl alcohol, inoculation
loop, blotting paper, alcohol lamp/burner, clean slides, bacterial culture, immersion oil,
microscope.
Procedure:
1) Prepare thin smear of given bacterial culture on a clean glass slide.
2) Stain the smear with crystal violet for 30 sec.
79

3) Wash the slide with distilled water for a few seconds, using wash bottle.
4) Cover the smear with iodine solution and allow it to act for about 30 sec.
5) Carefully add ethyl alcohol drop by drop on the slide, until no more colorflows
from the smear (usually it takes 30 see),

STEPS IN GRAM STAINING:

I. Firstly, take sterile &clean glass slide.
2. Take a drop of water & prepare thin smear using inoculation loop (culture of age
<0.4 hr.) & air dry it.
3. Add crystal violet (primary stain) to the smear and keep it for I min &wash it with
distilled water.
4. Then add iodine &keep it for 1 min &wash with water.
5. Then wash the smear with alcohol (decolourizer) till color is lost.
6. Add safraninedye to smear. If red colour retained we can conclude bacteria as gram
vebacteria.
If purple colouris retained it is gram +vebacteria. '
Conclusion:In experiment done, with Xanthomonaspurple color is retained. So, a
bacterium is Gram -Ve.

80

Work done report

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Date:
Title:

81

16. DEMONESTRATION OF MECHANICAL TRANSMISSION OF PLANT VIRUSES.

Plant viruses are transmitted from plant to plant in a number of ways. Modes of
transmission include vegetative propagation, mechanically through sap, seed, pollen,
dodder and vectors like insects, nematodes and fungi.
A. Demonstration of mechanical transmission of plant viruses through sap
Mechanical transmission of plant viruses in nature by direct transfer of sap
through contact of one plant with another plant is uncommon. Sap carrying virus
from diseased plants is transmitted to healthy plants through leaf injuries caused due
to strong winds or by hands or tools during cultural operations. Of the important plant
viruses, tobacco mosaic virus and potato virus- x are transmitted through sap in the
field.
Materials required
Test

plants

infected

with

tobacco

mosaic

virus,

potted

healthy

test

plants,

conical flasks, blotting paper, cheese cloth, muslin cloth, abrasive ( a substance
employed to create micro wounds eg.carborundum (400- 600 mesh) or celite( 1 %
diatomaceous earth ), mortar and pestle, fine washed sand, swabs, distilled water,
pieces of card board, vesse! for heating and heater, test tubes with stand, distilled
water or phosphate buffer solution ( a solution which is added to stabilize the virus)
of

pH

7.0

(eg.

dihydrogen
orthophosphate

disodium

hydrogen

orthophosphate
-

K2HP04&

orthophosphate

NaH2P04(0.1M)or

potassium

Na

2 H

P04&

hydrogen

eg.dipotassium

dihydroqenorthophosphate

sodium

H2

PO4(

0.1 M ), wash bottles.

Procedure
1.Collect two young mosaic infected leaves of test plant, wash with tap water
and dry with blotting paper. Weigh the leaves.
2. Prepare the standard extract by grinding the leaves alonqwith finely washed sand using a
mortar

and

pestle.

(for

preparing

standard

extract,

add

distilled

water or phosphate buffer equal to the weight of the leaves in to the mortar
and qrind ).
82

3.After through grinding, pass the leaf pulp through muslin cloth to get the
filtered standard extract of the leaves.
4.Divide the extract into two lots. Heat one lot to boiling and cool. Inoculate
the leaves of healthy plants with both the lots separately.
5.Dust the leaves with carborundum lightly or add the celite powder to the sap
to create micro wounds.
6.Apply the sap gently by rubbing the leaves (supporting the leaves with a
piece of card board or palm) in one direction i.e., only from petiole to the
apex of the leaves with a cheese cloth or fingers or painter's brush dipped
in

sap.

The

virus

enters

the

leaf

cells

through

the

wounds

made

water

to

remove

the

by

the

abrasive.
7.After

inoculation,

wash

the

leaves

with

excessive

inoculum and other extraneous particles if any.

8.Label the plants giving date of inoculation and other details of the host.
9. Place the inoculated plants in a glass house providing optimum conditions
for plant growth for observation.

83

Observations and record

Note
usually

the

date

of

appeared

inoculation

within3-

and
days.

appearance
Observe

the

of

symptoms.

mosaic

Symptoms

symptoms

in

are

plants

inoculated with unboiled extract and absence of symptoms in plants inoculated with
boiled extract.
Precautions
1.Inoculum should be obtained from the younger leaves of the infected plants
as the younger leaves contain more concentration of the virus particles.
2.Mortar and pestle should be thoroughly cleaned. Washed and sterilized before
and after use to avoid contamination.
3.Too much of carborundumpowder should not be used as it may cause lethal
injury to the plants.
4.Inoculated

plants

should

be

washed

thoroughly

to

remove

excess

and other external particles.

5. While inoculation, the leaves should be rubbed gently.
EXERCISE
1. Demonstration of transmission of any given mosaic viral disease through sap
and observation of mosaic symptoms on the test plant.

84

inoculum

Work done report

Ex No:
Date:
Title:

85