Practical Manual

MYCOLOGY
APP-501 3(2+1)

For
M.Sc. (Ag.) Plant Pathology

2023

Department of Plant Pathology

College of Agriculture

Rani Lakshmi Bai Central Agricultural University

Jhansi, Uttar Pradesh-284003
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 Practical manual

MYCOLOGY
APP-501 3 (2+1)

M.Sc. (Ag.) Plant Pathology

Dr. Prashant Jambhulkar

Dr. Anita Puyam

Department of Plant Pathology

College of Agriculture

Rani Lakshmi Bai Central Agricultural University

Jhansi, Uttar Pradesh-284003

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 CONTENT
Practic Topics Page
al No. No.
1. Detailed comparative study of different groups of fungi 1
2. Saccardoan classification and classification based on conidiogenesis 7
3. Collection of cultures and live specimens, preservation, culturing of 8
plant parasitic fungi
4. Preparation of Potato dextrose Agar media 10
5. Isolation of plant pathogens from Diseased Plant Tissues 11
6. Study of different types of mycelium, asexual spores and fruiting 12
bodies
7. Study of different types sexual spores and sexual fruiting bodies 14
8. Somatic and reproductory structures of Pythium and Phytophthora 16
9. Somatic and reproductory structures of downy mildew and Albugo 18
spp.
10. Zygomycetes: Sexual and asexual structures of Mucor, Rhizopus 21
spp. under Phylum Zygomycota
11. Identification of the plant pathogens under the Phylum 23
Basidiomycota
12. Difference between Uredinial and Telial Stage 25
13. Identification of the general characters of fungi under the Class: 28
Eurotiomycetes
14. Identification of the general characters of fungi under the Class: 30
Sordariomycetes
15. Identification of the general characters of fungi under the Class: 32
Dothideomycetes
16. Identification of the general characters of fungi under the Class: 34
Letiomycetes
17. Identification of the general characters of fungi under the Class 37
Taphrinomycetes
18. Application of Molecular Approaches and technologies for 39
identification of fungal pathogens

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PRACTICAL NO. 1
Detailed comparative study of different groups of fungi
Objective: To study the comparative differences in different groups of fungi

Outline classification of fungi

The following classification is adapted from the 9th and 10th editions of The Dictionary of the Fungi (Kirk et al.,
2001, 2008), but it has been amended to adopt the phylogenetic arrangement emerging from the AFTOL
(Assembling the Fungal Tree of Life) project funded by the US National Science Foundation (visit:
http://www.aftol.org/; Blackwell et al., 2006), as set out by Hibbett et al. (2007).
Presently, then, the true fungi which make up this monophyletic clade called kingdom Fungi comprises the seven
phyla:

Kingdom Fungi (8 Phyla) Kingdom Chromista (3 phyla) Kingdom Protozoa (1 Phylum,

3 classes)

Chytridiomycota (2 classes) Hyphochitriomycota (1 class) Protostelea

Neocallimastigomycota (1 class) Labrynthulomycota (1 class) Myxogastrea

Blastocladiomycota (1 class) Oomycota (1 class) Dictyostelia

Zygomycota (4 subphyla)

Ascomycota (3 subphyla: 15
classes)

Glomeromycota (1 class)

Microsporidia

Basidiomycota (3 subphyla; 16
classes)
Summarised classification of Kingdom Fungi
The most recently-published phylogenetic classification of Fungi (Hibbett et al., 2007)
Kingdom: Fungi
Phylum: Chytridiomycota
Water moulds that live as aquatic saprotrophs or parasites in fresh water and soils; a few are marine. Chytrids
produce motile asexual zoospores (with a single posterior flagellum, both a kinetosome and non-functional
centriole, nine flagellar props, and a microbody-lipid globule complex) in zoosporangia. Golgi apparatus with
stacked cisternae; nuclear envelope fenestrated at poles during mitosis. Thallus may be unicellular or
filamentous, and holocarpic (where all of the thallus is involved in formation of the sporangium) or eucarpic
(where only part of the thallus is converted into the fruiting body, monocentric, polycentric or filamentous. Sexual

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reproduction with zygotic meiosis where known; sometimes produce motile sexual zoogametes. Considered to
be the most ancestral group of fungi.
Class : Chytridiomycetes
Order: Chytridiales
Class: Monoblepharidomycetes
Order: Monoblepharidales; example genus: Monoblepharis
Phylum: Neocallimastigomycota
Thallus monocentric or polycentric; anaerobic, found in digestive system of larger herbivorous mammals and
possibly in other terrestrial and aquatic anaerobic environments; lacks mitochondria but contains
hydrogenosomes of mitochondrial origin; zoospores posteriorly unflagellate or polyflagellate, kinetosome present
but non-functional centriole absent, kinetosome-associated complex composed of a skirt, strut, spur and
circumflagellar ring, microtubules extend from spur and radiate around nucleus, forming a posterior fan, flagellar
props absent; nuclear envelope remains intact throughout mitosis.
Class: Neocallimastigomycetes
Order: Neocallimastigales; example genus: Neocallimastix
Phylum: Blastocladiomycota
Very like the chytrids, characteristically, the Blastocladiomycota have life cycles with what is described as a
sporic meiosis; that is, meiosis results in the production of haploid spores that can develop directly into a new,
but now haploid, individual.
Class: Blastocladiomycetes
Order: Blastocladiales
Phylum: Microsporidia
No subdivision of the group is proposed yet because of the lack of well-sampled multigene phylogenies within the
group. Microsporidia are unicellular parasites of animals and protists with highly reduced mitochondria.
Microsporidia may be a sister group of the rest of the Fungi, but this suggestion may have arisen from incomplete
sampling.
Phylum: Glomeromycota
Until recently, arbuscular mycorrhizal (AM) fungi have generally been classified in the Zygomycota (being placed
in the Order Glomales), but they do not form the zygospores characteristic of zygomycota, and all ‘glomalean’
fungi form mutualistic symbioses.
Class: Glomeromycetes
Order: Archaeosporales; example genera: Archaeospora, Geosiphon.
Order: Diversisporales; example genera: Acaulospora, Diversispora, Pacispora.
Order: Glomerales; example genus: Glomus.
Order: Paraglomerales; example genus: Paraglomus.

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Phylum: Ascomycota
This is the largest group of fungi, and the lifestyles adopted cover the complete range from saprotrophs, to
symbionts (notably lichens), and to parasites and pathogens (plant pathogens are particularly numerous, but
there are many important human pathogens in this group also). The Ascomycota are characterised by having
sexual spores (ascospores) formed endogenously within an ascus A layered hyphal wall with a thin relatively
electron-dense outer layer and a thicker electrontransparent inner layer also appears to be diagnostic.
Subphylum: Taphrinomycotina
Class: Taphrinomycetes
Order: Taphrinales
Class: Neolectomycetes
Order: Neolectales; example genus: Neolecta
Class: Pneumocystidomycetes
Order: Pneumocystidales; example genus: Pneumocystis.
Class: Schizosaccharomycetes
Order: Schizosaccharomycetales; example genus: Schizosaccharomyces.
Subphylum: Saccharomycotina
Class: Saccharomycetes
Order: Saccharomycetales;
Subphylum: Pezizomycotina
Class: Arthoniomycetes
Order: Arthoniales
Class: Dothideomycetes
Class: Eurotiomycetes
Class: Pezizomycetes
Class: Lichinomycetes
Class: Leotiomycetes
Class: Lecanoromycetes
Class: Laboulbeniomycetes
Class: Sordariomycetes
Phylum: Basidiomycota
Saprotrophic or parasitic on plants or insects; filamentous; hyphae septate, the septa typically inflated (dolipore)
and centrally perforated; mycelium of two types, primary (homokaryotic) of uninucleate cells, succeeded by
secondary (heterokaryotic), consisting of dikaryotic cells, this often bearing bridge-like clamp connections over
the septa; asexual reproduction by fragmentation, oidia (thin-walled, free, hyphal cells behaving as spores) or
conidia; sexual reproduction by fusion of hyphae with each other or with hyphal fragments or with germinating
spores (somatogamy), resulting in dikaryotic hyphae that eventually give rise to basidia, either singly on the
hyphae or in variously shaped basidiomata.

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Class: Pucciniomycetes
Order: Septobasidiales; example genera: Septobasidium, Auriculoscypha.
Order: Pachnocybales; example genus: Pachnocybe.
Order: Helicobasidiales; example genera: Helicobasidium, Tuberculina.
Order: Platygloeales; example genera: Platygloea, Eocronartium.
Order: Pucciniales; example genera: Puccinia, Uromyces.
Class: Cystobasidiomycetes
Order: Cystobasidiales; example genera: Cystobasidium, Occultifur, Rhodotorula.
Order: Erythrobasidiales; example genera: Erythrobasidium, Rhodotorula, Sporobolomyces, Bannoa.
Order: Naohideales; example genus: Naohidea.
Class: Agaricostilbomycetes
Order: Agaricostilbales; example genera: Agaricostilbum, Chionosphaera.
Order: Spiculogloeales; example genera: Mycogloea, Spiculogloea.
Class: Microbotryomycetes
Order: Heterogastridiales; example genus: Heterogastridium.
Order: Microbotryales; example genera: Microbotryum, Ustilentyloma.
Order: Leucosporidiales; example genera: Leucosporidiella, Leucosporidium, Mastigobasidium.
Order: Sporidiobolales; example genera: Sporidiobolus, Rhodosporidium, Rhodotorula.
Class: Atractiellomycetes
Order: Atractiellales; example genera: Atractiella, Saccoblastia, Helicogloea, Phleogena.
Class: Classiculomycetes
Order: Classiculales; example genera: Classicula, Jaculispora.
Class: Mixiomycetes
Order: Mixiales; example genus: Mixia.
Class: Cryptomycocolacomycetes
Order: Cryptomycocolacales; example genera: Cryptomycocolax, Colacosiphon
Subphylum: Ustilaginomycotina (equivalent to the traditional Ustilaginomycetes)
Class: Ustilaginomycetes
Order: Urocystales; example genera: Urocystis, Ustacystis, Doassansiopsis.
Order: Ustilaginales; example genera: Ustilago, Cintractia.
Class: Exobasidiomycetes
Subphylum: Agaricomycotina (equivalent to the traditional Hymenomycetes or Basidiomycetes)
Class: Tremellomycetes
Class: Dacrymycetes
Class: Agaricomycetes Subclass: Agaricomycetidae
Subclass: Phallomycetidae Order: Geastrales
Class: Wallemiomycetes

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Class: Entorrhizomycetes
Kingdom Chromista
Phylum Hyphochytriomycota
Order: Hyphochytriales; example genera: Hyphochytrium, Rhizidiomyces.
Phylum Oomycota
Order Leptomitales; example genera: Apodachlyella, Ducellieria, Leptolegniella, Leptomitus. Order
Myzocytiopsidales; example genus: Crypticola.
Order Olpidiopsidales; example genus: Olpidiopsis.
Order Peronosporales; example genera: Albugo, Peronospora, Bremia, Plasmopara.
Order Pythiales; example genera: Pythium, Phytophthora, Pythiogeton.
Order Rhipidiales; example genus: Rhipidium.
Order Salilagenidiales; example genus: Haliphthoros.
Order Saprolegniales; example genera: Leptolegnia, Achlya, Saprolegnia.
Order Sclerosporales; example genera: Sclerospora, Verrucalvus.
Order Anisolpidiales; example genus: Anisolpidium.
Order Lagenismatales; example genus: Lagenisma.
Order Rozellopsidales; example genera: Pseudosphaerita, Rozellopsis.
Order Haptoglossales; example genera: Haptoglossa, Lagena, Electrogella, Eurychasma, Pontisma,
Sirolpidium.
Kingdom Protozoa
Phylum Plasmodiophoromycota Obligate intracellular symbionts or parasites of plant, algal or fungal cells
living in freshwater or soil habitats
Class Plasmodiophoromycetes
Order: Plasmodiophorales; example genera: Plasmodiophora, Polymyxa.
Phylum Myxomycota Free-living unicellular or plasmodial amoeboid slime moulds
Class Dictyosteliomycetes
Class Myxomycetes
Class Protosteliomycetes
Phylum Acrasiomycota Amoeboid slime moulds; generally saprotrophic, found on a very wide range of
decaying plant material. A total of 14 species assigned to 6 genera
Class Acrasiomycetes
Order Acrasiales; example genera: Acrasis, Copromyxa, Guttulinopsis, Fonticula
Phylum Choanozoa Amoebidiales and Eccrinales
Class Mesomycetozoea
Order Amoebidiales
Order Eccrinales
Activity:

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1. Collect isolates of fungi belongs to each phylum and show distinguishing features.
2. Get the culture belongs to different phylum and record the cultural characteristics

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PRACTICAL NO. 2

Saccardoan classification and classification based on conidiogenesis

Objective: To study the different forms of conidia based on conidiogenesis based on Saccardoan classification
Fungi in the Ascomycetes and Basidiomycetes groups produce a type of asexual reproductive structure
known as conidia. Some fungi only produce these asexual propagules and have not been observed to have a
sexual state. These fungi were classified as Fungi Imperfecti or Deuteromycetes. With advances in molecular
sequencing, some of them is being integrated into the classification system based on their sexual reproductive
structures.
Despite not having a sexual state, mitosporic fungi play significant roles ecologically, medically, and
industrially. Therefore, it is important to identify and name them accurately. In the past, two classification
approaches have been used for these fungi, one based on the overall morphology of the conidia and
conidiomata, and the other based on conidial development. Recently, identification manuals have combined
these two approaches.
The Saccardoan System, developed in 1886, classified Fungi Imperfecti based on the spore
characteristics of pigmentation, septation, and form. The primary combinations of these characters were used to
classify the species of Fungi Imperfecti.

Activity: Examine the species of fungi provided (cultures and slides). Draw the conidia of each species under
the terms that best describe them (be sure to write the name of the species under your drawing.
A. Hyaloamerosporae – hyaline or brightly colored single-celled conidia.
B. Phaeoamerosporae – dark pigmented single-celled conidia.
C. Hyalodidymosporae – hyaline or brightly colored, two celled condia.
D. Phaeodidymosporae – dark pigmented, two celled conidia.
E. Hyalophragmosporae – hyaline or brightly colored, two to many septate conidia.
F. Phaeophragmosporae – dark pigmented, two to many septate conidia.
G. Hyalodictyosporae – hyaline or brightly colored, transversely and longitudinally septate conidia
H. Phaeodictyosporae – dark pigmented, transversely and longitudinally septate conidia.
I. Hyalo or Phaeoscolecosporae – hyaline, brightly colored, or dark pigmented, long, curved, often sigmoidal
conidia.
J. Hyalo or Phaeohelicosporae – hyaline, brightly colored, or dark pigmented, coiled conidia.
K. Hyalo or Phaeostaurosporae – hyaline, brightly colored, or dark pigmented, star shaped (arms radiating out
from a central point) conidia.

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PRACTICAL NO. 3

Collection of cultures and live specimens, preservation of plant parasitic fungi

Objectives: To collect and preserve plant disease samples

Preservation means killing or restricting the growth of an organism in or on the substrate on which it
grows. Preservation of disease materials (herbaria) on their natural substrates as dry specimens or wet
specimens is essential for conducting systematic mycological work and important taxonomic research on various
micro-organisms.
Materials required: Polythene bags, Newsprint paper • Hand saw, Trowel, Pruning shear, knife, Scissors • Hand
lens • Pencil, Ink markers • Vasculum, Plant press, Paper bags, Envelopes • Ice box • Manual
Specimens
A herbarium specimen may be a single sporocarp or a portion of it, dried culture, slide or the material on
its host or substrate (e.g. leaf, stem, bark, rock, soil, paper, cloth). The following two types of preservation
methods are used for diseased plant specimen:
1) Dry Preservation: It involves following steps:
a) Collection and drying: The sample should have distinctively visible symptoms. Dry the specimen in
layer of blotting sheets under sunlight or in hot air oven for few days.
b) Labelling and packaging: The material should be kept in good herbarium packets. This is attached to
a chart paper sheets. The two sides of packet are folded first, then bottom flap and finally top flap. The name of
pathogen, host, locality, date, name of scientist who identified the specimen, should be mentioned on the label.
c) Disinfection and storage: The specimen folders are fumigated with methyl bromide vapours in
fumigation chamber for 24-48 hrs before storage.
2) Wet Preservation
Washed fresh diseased specimens are put in a boiling mixture of 1 part of glacial acetic acid saturated
with normal copper acetate crystals and 4 parts of water till the green colour reappears and then kept preserved
in 5 per cent formalin in the glass jars.
All mounted or preserved specimens must be labeled with as much of the following information as far as
possible:
1. Host (name of the diseased plant)
2. Name of the disease Parasite (the name of the organism causing the disease)
3. Place where collected (nearest town and state is usually sufficient)
4. Date collected
5. Name of the collector
Size of the specimen: A specimen should ideally be 25–40 cm long and up to 26 cm wide, allowing it to fit on a
standard herbarium mounting sheet which measures 42 x 27 cm. This is also the approximate size of tabloid
newspapers. Plant parts that are too large for a single sheet may be cut into sections pressed on a series of

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sheets, for example a palm or cycad frond. Long and narrow specimens such as grasses and sedges can be
folded once, twice or even three times at the time of pressing. In this way a plant of up to 1.6 metres high may be
pressed onto a single sheet. For very small plants, a number of individuals may be placed on each sheet.
Activity 1: Prepare herbarium of Plant disease with all following details in it:
1. Host (name of the diseased plant)
2. Name of the disease Parasite (the name of the organism causing the disease)
3. Place where collected (nearest town and state is usually sufficient)
4. Date collected
5. Name of the collector
Activity 2: Collect disease sample and preserve in the glass bottle following wet preservation protocol.

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PRACTICAL NO. 4
Preparation of Potato dextrose Agar media
Objective: To prepare Potato dextrose Agar media
Materials required: For the preparation of potato dextrose agar medium the following ingredients in different
quantities are used
(i) Peeled potato slices - 200g
(ii) Dextrose - 20g
(iii) Agar- agar - 20g
(iv) Distilled water - 1000 ml
Method:
(1) Potato slices are cooked in 500 ml of water.
(2) Then filtered with the help of muslin cloth.
(3) Agar-agar is melted in 500 ml of water.
(4) Potato juice is added to the melted agar.
(5) Volume is made 1000 ml by adding required water.
(6) Again lit is filtered through muslin cloth.
(7) Dextrose is added in this mixture and shaken well.
(8) Medium is sterilized in an autoclave at 1.1kg/cm 2 pressure for 20 minutes at temperature of 121.6°C. Thus
the medium is ready for use.
Activity 1: Prepare one litre of Potato dextrose Agar medium. Note the materials required and quantity of the
components.

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PRACTICAL NO. 5
Isolation of plant pathogens from Diseased Plant Tissues
Objective: To isolate plant pathogens from Diseased Plant Tissues
Tissues sampled during the active stage of an infection are likely to have within them only the pathogen
responsible for the infection; the surfaces of such tissues, however, are usually contaminated with saprophytic
organisms. The steps of isolation of the pathogen have been given in the flowchart below:

Collection of Infected plant sample

Rinsing the samples in running tap water

Sodium hypochlorite
solution (0.5 to 1.0 Surface disinfestation
percent) or 0.1 %
mercuric chloride or
Cutting of samples of uniform size 0.5
cm across

Inoculation of the pieces into freshly

prepared media under sterilized condition

Incubation of the culture in BOD

chamber

Re-isolation after 7-10 days from the

culture to make pure culture

Fig. 1. Flowchart showing steps of isolation of pathogen from plant tissues

Activity 1. Isolate the pathogen from the given sample and note the different morphological features
observed under the microscope and identify it based on morphology and cultural characteristics.

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PRACTICAL NO. 6
Study of different types of mycelium, asexual spores and fruiting bodies
Objective: To study different types of mycelium, asexual spores and asexual fruiting bodies
Mycelium: Network of hyphae is called as mycelium. It may be aseptate or septate.
i. Aseptate Mycelium- When the hyphae are undivided by cross-walls (septa) it is known as septate
mycelium. This type of mycelium is found in lower fungi.

ii. Septate Mycelium- When the mycelium is divided by cross walls (septa) at certain intervals, it is known
as septate mycelium. In the septa (singular septum), there is a minute hole, which is known as “septal
pore.” This type of mycelium is found in higher fungi.

Fig. 2. Structure of fungal mycelium

Different Types of Asexual Spores: Asexual spores are those in which sex is not involved. Generally five
types of asexual spores are produced in fungi. They are as follows:-

1. Arthrospores (Oidia): Formed in chains

(basipetal) on short conidiophores, single celled,
barrel or drum shaped.
2. Chlamydospores: Formed singly or in chains,
which may be terminal or intercalary, provided
with an envelop (covering).
3. Blastospores: Spores formed by process of
budding, which are single celled, first formed in
chains but later separated from each other.
Fig. 3. Different types of fungal asexual
4. Conidia: Formed at the tip or side of the hypha spores
(Conidiophore), may be formed singly or in chains, quite
variable in shape, size, septation, colour and also in ornamentation.

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5. Zoospores: Pear or kidney shaped, single shaped, naked, motile (flagellate), produced in sporangium
(zoosporangium).
6. Aplanospores: Oval or spherical in shape, single celled, non-motile (aflagellate) and produced mostly in
elliptical spora ngium.
Asexual fruiting bodies:
1. Pycnidia: These are spherical or flask shaped structures
in which the conidia or produced. They have the natural
opening known as ostiole through which the conidia are
liberated. This type of structure is produced in order
Spaeropsidales of sub division Deuteromycotina.
2. Acervuli: These are mat or cushion shaped structure
formed below the cuticle or epidermis of the host. They
may be provided with sterile hair like structures known as
setae.
3. Sporodochia: These are the cushion-shaped structure on
which the conidiophores are produced.
Fig. 4 Different types of asexual fruiting bodies
4. Synnemata: In these structures the conidiophores are
grouped together at the base and free towards apex.

Activity: 1. Examine the representative slides of asexual fruiting bodies and asexual spores and note
down the characteristic features of the structures observed.
Activity: 2 Enlist the organisms against the particular asexual fruiting bodies produced by them.

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PRACTICAL NO. 7
Study of different types sexual spores and sexual fruiting bodies
Objective: To study different types sexual spores and sexual fruiting bodies
Different types of sexual spores

Following four types of sexual spores are formed in fungi, which are produced by various methods and they form
the bases for the classification of fungi in different sub-divisions.

1. Oospores: Mostly spherical in shape, formed in the oogonium, usually smooth walled. They are formed by
gametangial contact (oogamy), characteristics of phylum lliptic.

Fig. 5 Different types of sexual spores

2. Zygospores: Black in colour, rough-walled, warty in appearance and provided with suspensors. They are
formed by gametangial copulation (zygogamy), characteristics of sub-division Zygomycotina.

3. Ascospores: Produced in asci, definite in number (usually 8). They are formed by
spermatization/somatogamy, characteristics of sub-division Ascomycotina.

4. Basidiospores: Borne on the basidium, definite in number (usually 4). They are formed by
spermatization/somatogamy, characteristics of sub-division Basidiomycotina.

Different Types of Ascocarps:

1. Cleistothecia (-um): Spherical in shape, black in colour, hard in structure and without any natural opening.
Asci come out by tearing or breaking of the cleistothecium. Cleistothecia are also provided with appendages.

2. Perithecia (-um): Flask shaped with natural opening known as “ostiole”, some time having long neck. Asci
are produced in the perithecium at basal region. Paraphyses may also be present in between the asci.

3. Apothecia (-um): The ascocarp, which produces its asci in an open disc or cup shaped structure, is called
as apothecium. It is exposed and form the layer of asci in a “hymenium” among them paraphyses may also be
present.

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 Fig.6 Different types of Ascocarps

Activity: 1. Examine the representative slides of asexual fruiting bodies and asexual spores and note
down the characteristic features of the structures observed.
Activity: 2 Enlist the organisms against the particular asexual fruiting bodies produced by them.

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PRACTICAL NO. 8
Somatic and reproductory structures of Pythium and Phytophthora
Objective: To study the somatic and reproductive structures of Pythium and Phytophthora and their differences
1. Genus – Pythium (Damping off)
Systematic Position
Phylum - Oomycota
Class - Oomycetes
Order - Peronosporales
Family - Pythiaceae

Fig. 7 Sporangium and oospore of Pythium spp

Mycelium – Aseptate, branched, cottony white.

Sporangiophores – Different from vegetative hyphae, erect, simple and bearing sporangia singly.
Sporangia- Spherical or globose, sometimes filamentous or toruloid.
Ooospores – Thick walled, spherical, usually smooth and three layered and plerotic.
Important species- P. aphanidermatum, P. ultimum, P. graminicolum (damping off disease)
2. Genus – Phytophthora

Mycelium – Aseptate, coenocytic, branched.

Sporangiophores – of indeterminate growth, zig-zag, sympodially branched, noduate (with nodular swellings)
Important species- P. infestans (Late blight of potato).
Systematic Position
Sporangia- Single celled, lemon shaped and papillate.
Phylum – Oomycota
Ooospores – Spherical in shape, smooth walled and
Class – Oomycetes
aplerotic.
Order - Peronosporales
Family - Pythiaceae

Fig. 8 Sporangium and oospore of Phytophthora spp

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 Activity:

1. Examine the representative slides of Pythium and Phytophthora and note down the characteristic
features of the mycelium, sporangia and the spores. Draw a proper neat and clean pencil sketch diagram. Note
the location of the antheridium in relation to the oogonium. How do oospores differ from oogonia?
2. Isolation of Phytophthora from plant tissue and soil
Materials required:
Root, stem, or leaves from diseased plant
Soil sampling tools
Plastic bags
Scalpel with #10 blade
Petri Plates
Sieves
Magnetic strirrer
Balance, weigh boats
BOD incubator
V8 Media
Compound microscope
3. Observation of zoospores and their characteristics.
i) Zoospore motility: Sporangia in an aqueous preparation have been placed in water, chilled, and then warmed
to induce the formation of motile zoospores. Observe the release of zoospores with a dissecting microscope
ii) Attraction of zoospores to plant roots: Place the roots of a pea seedling into an aqueous suspension of
Pythium zoospores in a Petri dish, and let stand for 5-10 minutes. Observe the suspension with a dissecting
microscope for the accumulation of zoospores around the root zone of elongation.
iii) Diagram your observations from the prepared cultures and slides:
a) Sporangia and zoospores (include vesicles if observed)
b) Oogonia and antheridia
c) Oospores
4. List the differences in the characteristic features of Pythium and Phytophthora.
Conclusive Questions:
1. What fungal structure differentiates species of Pythium from species of Phytophthora? What is the location of
this structure and its role in the Pythium life cycle?
2. How can wet, poorly drained soil contribute to root rots caused by Pythium and Phytophthora?
3. There will be no sexual reproduction in Phytophthora infestans, if only one mating type present in the
pathogen population. However, both mating types have been found. If you suspected that both mating types
were present in your potato field:
a. Where would you look to determine if sexual reproduction was occurring?
b. What fungal structure would be evidence of sexual reproduction?

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PRACTICAL 9
Somatic and reproductory structures of downy mildews and Albugo spp.
Objective: To study Somatic and reproductory structures of downy mildews and Albugo spp.
1. Genus – Peronospora (Downy mildew)
Mycelium : Aseptate, coencocytic, branched,
Systematic Position
hyaline, endophytic and intercellular. Phylum – Oomycota
Conidia : Single celled, spherical or oval in Class – Oomycetes
shape and borne singly. Order - Peronosporales
Family - Peronosporaceae
Branching : Dichotomous at acute angles. Last (ultimate) branch is changed into the sterigmata.
: Long and pointed and bearing conidia singly.
Sterigmata
Ooospores : Spherical and reticulate in Peronospora parastitica (downy mildew of Crucifers).
: Arise from the stomatal openings. They are slender, long, 2/3 portion unbranched and
Conidiophores only 1/3 portion is branched.
Important species- Peronospora parastitica (downy mildew of Crucifers), P. tabacinia (downy mildew of
tobacco). P. pisi (downy mildew of pea).
Systematic Position

2. Genus – Sclerospora Phylum - Oomycota

Mycelium – Aseptate, coencocytic, branched, hyaline, Class - Oomycetes

endophytic and intercellular. Order - Peronosporales

Sporangiophores– Arise from the stomatal openings. Family - Peronosporaceae

They are short and broader towards apex.

Branching – Dichotomous or even trichotomous. Last branch is changed into the sterigmata.
Sterigmata – Short and swollen and bearing sporangia singly.
Sporangia- Borne singly, single celled and sometimes papillate also.
Ooospores – Irregular in appearance because the sporangial wall shrinks and touches the oosporic wall at
several places.
Important species is Sclerospora graminicola, which causes green ear disease of Bajra.
3. Genus: Plasmopara
Mycelium – Aseptate, coencocytic, branched, hyaline, endophytic and intercellular.
Sporangia: The sporangia were hyaline, oval shaped formed on right angle sporangiophore. Sporangiophore:
The sporangiophore were hyaline, straight or slightly curved
Branching – Right angle branched.
Sterigmata: Sterigmata are mostly trichotomous
Ooospores – large, 25–50 μm in diameter, thick-walled spherical
Important species: Plasmopara viticola

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4. Genus: Bremia
Mycelium – Aseptate, coencocytic, branched, hyaline, endophytic and intercellular. Sporangiophores
branched more or less like Peronospora but ends with a disc-like or saucer shaped, sterigmata bearing
structure
Important species is Bremia lectuca
5. Genus: Basidiophora
Sporophores unbranched, apex swollen and with short sterigmata bearing papillate sporangia germinating by
zoospores; oospores aplerotic

Peronospora spp Sclerospora spp Plasmopara spp

Bremia spp Basidiophora spp

Fig.9 Sporangiophore of different Genus causing downy mildews.

6. Identification of Genus – Albugo spp (White blister/rust)
Systematic Position
Mycelium – Aseptate, coencocytic, branched, hyaline, Phylum –Oomycota
intercellular with knob shaped haustoria. Class – Oomycetes
Sporangiophores– Club shaped (clavate), simple, forming Order - Albuginales
palisade layer below the epidermis, lateranl wall thickened Family - Albuginaceae
and laterally free, bearing sporangia in basipetal chains.
Sporangia- Single celled, globose and produced in chains in basipetal succession and attached with each other
with a gelatinous pad known as “disjuctor”.

22
Ooospores – Rough and warty in appearance and yellow in colour.
Important species – Albugo candida (white blister / white rust of crucifers).

Ooospore

Sporangia Disjunctior

Sporangiophore
Host mesophyll

Fig. 10 Sporangiophore of Albugo candida producing conidia in chains

Activity:
1. Prepare slides by taking any downy mildew infected plant sample (Mustard, cucurbits,
2. Note the observations and draw a neat and clean diagram of the characteristic features observed.
Host Genus Branching of the Sterigmata
sporangiophore

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PRACTICAL NO. 10
Zygomycetes: Sexual and asexual structures of Mucor, Rhizopus spp. under
Phylum Zygomycota
Objective: Sexual and asexual structures of Mucor, Rhizopus spp

1. Genus – Mucor (Bread mould)

Mycelium : Aseptate, branched, cottony white Systematic Position
Phylum – Zygomycota
without stolons and rhizoids.
Sporangiophores : Arise singly, simple, aseptate, Class – Zygomycetes

bearing sporangia singly. Order - Mucorales

Sporangia : Spherical or globose, smooth walled, Family - Mucoraceae

fragile, columellate and multi-spored

Columella : Central portion in the sporangium which is sterile and “Dome shaped”
Aplanospores : Oval or spherical in shape and single celled.
Zygospores- : Rough walled, black, warty in appearance and provided with “suspensors”.
Important species: M. mucedo, M. basiliformis.
Systematic Position
2. Genus – Rhizopus (Bread mould)
Phylum – Zygomycota
Characters of this genus are Mucor- like except the
Class – Zygomycetes
formation of stolons and rhizoids, sporangiophores arise
Order - Mucorales
in-groups from rhizoids.
Family - Mucoraceae
Important species: R. stolonifer

Fig. 11.Spore bearing structure of Rhizopus spp Fig. 12.Spore bearing structure of Mucor spp

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 Fig.13 Gametangial copulation in Zygomycota

Activity:
1. Observed Rhizopus and Mucor culture available in the Laboratory. Note down its characteristic features.

2. If 2 different strains (called + and – strains) are placed together on a culture medium (or in nature), the hypha
will grow towards each other and conjugation will occur. This produces a sexual spore called a zygospore—a
diploid sexual spore.
A. On 10X and 40X, identify hyphae, sporangia, and sporangiospores.
B. Differentiate between the sexual zygospores and the sporangiospores on the slides.
Conclusive questions:
1. What are the vegetative structures present in Rhizopus and Mucor?
2. What is the form of sexual reproduction in Rhizopus and Mucor?

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PRACTICAL NO. 11

Identification of the plant pathogens under the Phylum Basidiomycota

Objective: To study the structures of teliospores and sorus of different genus under the Phylum Basidiomycota
1. Genus – Sphacelotheca

Sorus : Conical or cylindrical covered with the peridium Phylum-Basidiomycota

and filled with black spore powder. Class – Teliomycetes
Columella : In the central portion of sorus, slender on Order- Ustilaginales
curved, made up of host tissues in S. sorghi Family – Ustilaginaceae
Teliospores: Round to shortly oval, dark brown in mass but
olive brown singly, smooth walled. Mass but olive brown singly, smooth walled.
Important spp. S. sorghi (Grain smut of Jowar), S. cruenta (Loose smut of jowar), S. reiliana (Head smut of
Jowar)
2. Genus – Tolyposporium Phylum– Basidiomycota

Sorus : Though formed in various parts of the host,

Class – Teliomycetes
is more common in the ovary.
Order- Ustilaginales
Teliospores : They are formed in the form of “spore balls”
Family – Tilletiaceae
which are covered by member of host origin.
Important species: T. penicillariae (smut of bajra),
T. ehrenbergii (long smut of jowar)

3. Genus- Tilletia
Phylum– Basidiomycota
The disease caused by Tilletia are called as “Bunt”
Class – Teliomycetes
Teliospores :Teliospores are large, 16-54 smooth, verrucose Order- Ustilaginales
Important species :T. caries & T. foetida (stinking smut or hill bunt) Family –Tilletiaceae

4. Genus – Neovossia
Grains partially or wholly converted into black powdery mass enclosed by membrane (N.indica).
Teliospores: Dark brown, spherical to oval with reticulations on the epispore which appear as curved spines.

Important species: N. indica (Karnal bunt of wheat), N. horrida (Bunt of rice)

5. Genus – Ustilago

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Sorus: The teliosorus without a peridium; the black dusty Phylum– Basidiomycota
teliospores are covered by a membrane of host origin. Class – Teliomycetes
Teliospores: Small globose to oval or lliptical less than 20 m in Order-Ustilaginales
diameter in most of the species the outer wall (episopore) is Family –Ustilaginaceae
minutely echinulate but sometimes smooth also (U. hordei).
Important species: U. segetum tritici (U. tritici)
U. nuda – (Loose smut of barley)
U. maydis (corn smut)
U. scitaminea (whip smut of sugar came)

Activity:
1. Examine various parts of plant infected with smut and note the smut balls on the above ground plant
parts. Illustrate.
2. Mount some smut spores (teliospores) in lactophenol and study under the oil immersion of objective.
The teliospores of the Ustilaginaceae produce a 3-septate (four-celled) promycelium from which the
basidiospores are formed laterally. The teliospores have minute spines on their surfaces. They are thick-
walled, and dark in color. Note the size of the spores. llustrate.

Teliospores of Rust Fungi

Uromyces Puccinia Melampsora

1. Teliospores are stalked 1. Teliospores are bicelled 1. Teliospores single celled,
2. They are single celled 2. They are stalked 2. They are sessile and cylindrical
3. Apex of teliospores is in shape
thickened 3. Form layer below the epidermis

Uromyces Puccinia Melampsora

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 PRACTICAL 12
Difference between Uredial and telial stage of rust fungi
Objective: To observe the structural difference in Uredial and telial stage of rust fungi

(A) Uredial Stage (B) Telial Stage

1. Epidermis ruptured Uredospore 1. Epidermis intact (unbroken)
2. Uredospores stalked 2. Teliospores sessile
3. Uredospores finely echinulate 3. They are single celled, cylindrical in shape
4. Capitate paraphyses also present 4. Teliospores form layer below epidermis

Uredospore
Uredospore Epidermis

Layer of Teliospores

Paraphysis
Stalk

Host cell

Uredial Stage Telial Stage

Uredial and Telial Stages of Melampsora

Activity

Materials needed:
Infected plant parts: Aboveground parts of corn plant infected with smut.
Infected wheat stems with the black stem rust.
Fresh basidiocarp of Agaricus sp., Pleurotus ostreatus.
Media and chemicals: MEA; Calcium sulfate (gypsum); Pasteurized medium clay loam or
peat / limestone mixture (calcium carbonate).
Plant and animal materials: Fresh Horse or chicken manure; Wheat or barley straw; Wheat
bran; Grain (e.g., rye, wheat, or sorghum).
Procedure:
1- Under the microscope examine prepared slides of wheat leaves infected with the rust. The

28
 Uredinia (Stage 2) can be seen on the upper epidermis.
Scrape a uredinium with a scalpel and mount some urediniospores onto a slide in lactophenol
cotton blue. Study the spores under the oil immersion objective. Study a prepared slide showing cross section of
a wheat stem bearing uredinia. Observe the origin of the uredinium and its effect on the host epidermis. Note the
long stalks of the urediniospores. Illustrate and record your observations.
1. If wheat straw bearing the teletial stage (Telia, Stage III) of the fungus is available you could observe the black
pustules from which the disease "black stem rust of wheat" takes its name. Teliospores can then be studied in a
lactophenol mount under the oil immersion objective; their color, number of cells in each, and thickness of the
wall could also be noted. Illustrate and record your observations.
2. Study prepared slides showing cross sections of a wheat stem bearing telia. Note how the teliospores are borne
in the telia and note their position in relation to the host cells and to each other. Illustrate and record your
observations.
3. Label the following diagram showing life cycle of Puccinia graminis

Conclusive Questions:
1. Explain how the rusts obtain nourishment from the host?
2. Define basidium; clamp connection; dolipore septum; teliobasidium; dikaryotic;

29
spermatiophore; spermogonium; autoecious rusts; heteroecious rusts.
3. How do long-cycle rusts differ from short-cycled rusts?
4. How do the rusts reproduce sexually?
5. The uredospores are considered to be the conidia of the rusts. Why?
6. What structures represent the basidia of the rusts? Explain.

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PRACTICAL NO. 13

General characters of fungi under the class Class: Eurotiomycetes under the
Phylum Ascomycota

Objectives: To identify the general characters of fungi under the class Class: Eurotiomycetes
1. Genus – Aspergillus (Black mould)
Mycelium – Well developed, branched, septate, hyaline and
submerged in the substratum. Systematic Position
Phylum –Ascomycota
Conidiophores– Arise from the “foot cell,” aseptate, simple,
Class –Eurotiomycetes
terminating into vesicle.
Order - Eurotiales
Sterigmata– Two rows of the sterigmata are formed on the vesicle. Primary sterigmata are flat. Secondary
Family - Trichocomaceae
sterigmata are bottle shaped.
Conidia- Borne on secondary sterigmata in long basipetal chains. They are globose, single celled, and
echinulate.
Systematic Position
Important species: A. niger, A. flavus, A. fumigatus Phylum –Ascomycota
Perfect Stage: Eurotium Class –Eurotiomycetes
Order - Eurotiales
Family - Trichocomaceae

Fig.16 Conidiophore bearing conidia of Fig.17 Conidiophore bearing conidia of

Penicillium spp Aspergillus spp

2 Genus – Penicillium (Blue / green mould)

Mycelium – Well developed, branched, septate, hyaline and
submerged in the substratum.

31
Conidiophores– Septate and branched without forming vesicle. Foot cells absent.
Sterigmata– Single row of sterigmata is formed. They are peg like.
Conidia- Borne on sterigmata in long basipetal chains. They are, single celled, globose to ovoid, smooth walled
and resemble as “glass beads”.
Important species: P. notatum, P. chrysogenum
Perfect Stage: Talaromyces
Activity 1. Observed Penicillium and Aspergillus culture available in the Laboratory. On 10X and 40X, identify
hyphae, conidia fruiting structures, and the asexual conidiospores. Draw neat and labeled diagram of the spores
bearing conidiophores.

Point of differences Aspergillus Penicillium

Root cell

Conidiophores

Vesicle

Sterigmata

Conidia

6. Perfect stage

Activity 2: Examine under the low and high power objectives prepared slides showing different types of
ascocarps: cleistothecia (c.s. in ascocarp of Erysiphe graminis; cleistothecia, whole mount, Uncinula sallics),
perithecia (median sec. in apple leaf infected with Venturia inequalis; Xylaria sp.), apothecia (l.s., apothecium,
Morchella sp.), ascostroma (c.s. stroma, Claviceps purpurea, causal agent of ergot of Gramineae). Illustrate it.

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PRACTICAL NO. 14

General characters of fungi under the Class: Sodariomycetes under the Phylum
Ascomycota

Objective: To study the different features of different genera under Class: Sodariomycetes

1. Genus – Fusarium
Mycelium: Septate, branched, pinkish brown in colour. Systematic Position
Phylum–Ascomycota
Sporodochia: Spherical, oval or ovate. Class –Sordariomycetes
Conidiophores: Short, aseptate or septate, usually simple Order- Hypocreales
may be branched also bearing conidia singly. Family- Nectriaceae
Conidia: Micrococonidia – usually single celled or bicelled
Macroconidia – many celled (2-7), sickle shaped and knotched at the base.
Chlamydospores- formed in mycelium and macroconidia.
Important species: F. oxysporum (wilt diseases), F. udum (wilt of pigeonpea).
Perfect Stage: Gibberella and Nectria.
2. Genus Claviceps (Ergot) Systematic Position

The genus Claviceps, causes the important disease Phylum – Ascomycota

“Ergot”particularly of the cereals and millets. Class –Sordariomycetes

Common species is C. purpurea (Ergot of rye). Order- Hypocreales

Mycelium- Septate and branched, destroying ovary tissues and Family – Clavicipitaceae

replacing it by cottony white mycelial mat forming conidiophores

bearing conidia at their tips.
Conidia –Minute, oval and single celled forming “Honey dew” stage (Nector like secretion).
Sclerotia –Black, hard and variable in shape and actually the ovaries being destroyed and replaced by sclerotia.
Perithecia-Flask-like, ostiolate.
Asci – Several in a perithecium, and are elongated, cylindrical in shape.
Ascospores- Formed 8 in number in each ascus, which are long and thread like.
Important sp. C. purpurae (Ergot of rye), C. microcephate
(Ergot of bajra)
3. Genus – Pyricularia
Systematic Position
Conidiophores: Straight, septate (with 2-4 septa), slender Phylum-Ascomycota
and thickened at the base. Class –Sordariomycetes
Conidia: Pyriform (pear shaped) to obclavate base rounded Order- Magnaporthales
tapering at the apex, 2- septate (three celled), slightly darkened. Family-Magnaporthaceae
One to many conidia may found on a single conidiophore.
Important spp. P. oryzae (blast of paddy)

33
Perfect stage – Magnaporthe oryzae
4. Genus – Colletotrichum Systematic Position
Mycelium: Septate, light brown, branched Phylum –Ascomycota
Acervuli: Cushion shaped and provided with sterile, hair like Class –Sodariomycetes
black structure setae on acervuli. Order - Glomerellales
Conidiophores: Short, aseptate and unbranched Family - Glomerellaceae
Conidia: Single celled, falcate, often with oil globule
Important species- F. calcatum (red rot of sugarcane), C. truncatum
(Anthracnose of pulses)
Perfect stage- Glomerella, Physalospora
Activity: Identify the organism based on the features provided below and write their characteristics:

A B

C D

34
PRACTICAL NO. 15

General identification characters of fungi under the Phylum Ascomycota Class:

Dothideomycetes
Objective: To study the different features of different genera under Class: Dothideomycetes
1. Genus – Helminthosporium
Systematic Position
Conidiophores: Straight or zig-zag having knee joints Phylum–Ascomycota
(geniculate). Class – Dothideomycetes
Order- Pleosporales
Conidia: Conidia are produced singly at the apex and at knee- Family-Pleosporaceae
joints of the conidiophores. They are cylindrical, multi-septate,
mostly with rounded ends.
Important spp. H. gramineum (stripe disease of barley) and H. oryzae (brown spot of paddy).
Perfect stage – Cochliobolus and Pyrenophora.
2. Genus – Alternaria Systematic Position
Phylum–Ascomycota
Conidiophores: Septate, simple or some times branched.
Class- Dothideomycetes
Conidia: Conidia borne usually in chains (acropetal). Order - Pleosporales
Sometimes solitary also. Conidia are provided with cross as well Family- Pleosporaceae
as longitudinal or oblique septa (muriform).
Conidia are also provided with beak, which may vary from very short
to very long according to species.
Important spp. A. solani (early blight of potato), A. brassicae (Alternaria blight of crucifers), A. triticina (Leaf
blight of wheat).
Perfect stage – Pleospora
3. Genus – Cercospora
Systematic Position
Conidiophores: Straight or zig-zag having knee joint Phylum–Ascomycota
(geniculate). Class –Dothideomycetes

Conidia: Conidia are produced singly at the apex and at Order- Capnodiales

knee joints of the conidiophores. Family- Mycosphaerellaceae

They are acicular, multi-septate, tip acute and base broad.

Important spp. C. personata and C. arachidicola, which cause tikka disease of groundnut.
Perfect stage - Mycosphaerella

35
Activity: Identify the organism based on the features provided below and write their characteristics

B
A

36
 PRACTICAL NO. 16

General identification characters of fungi under the Phylum Ascomycota Class:

Leotiomycetes
Objective: 1. To understand the different forms of Cleistothecia in different powdery mildew genera
2.To study the different identification features of Sclerotinia
I. Identification of powdery mildew genera, Class: Leotiomycetes based on Cleistothecia
1. Mycelium internal and Cleistothecium with several asci
i. Appendages with bulbous base: Phyllactinia, Anamorph: Ovulariopsis
ii. Appendages hypha-like: Leveillula, Anamorph: Oidiopsis
2. Mycelium superficial and Cleistothecium with several asci
i. Appendages hypha-like: Erysiphe
ii. Appendages dichotomously-branched: Microsphaera
iii. Appendages with coiled tips: Uncinula
iv. Anamorph: Oidium
v. Appendages branched, hypha-like Golvinomyces
3. Cleistothecium with single ascus
i. Appendages dichotomously branched tip: Podosphaera
ii. Hypha-like: Sphaerotheca
The diagram is indicated in fig. no.18
II. Genus – Sclerotinia
Systematic Position
Mycelium Septate and branched mostly white in colour.
Phylum – Ascomycota
Conidiophores long septate land branched.
Class – Leotiomycetes
Conidia- oval or lemon shaped, single celled and
Order- Helotiales
formed in chains.
Family – Sclerotiniaceae
Sclerotia Black, hard, variable in shape
Apothecia Long and stalked cup or disc shaped.
Asci Clavate, slightly thickened at apex, with paraphyses.
Ascospores 8 in number in each ascus, single celled round, or elliptical or elongated.

Important sp. S. sclerotiorum causing root rot and white rot disease.

37
Fig. 18 Different features of Cleistothecia produced by powdery mildew fungi

38
Activity: 1. Collect the powdery mildew disease sample from the crop cafeteria and prepare slides and observe
under the microscope. Note down the following characteristics and identify the genera based on morphological
observation. Draw a neat and label diagram of the features observed .
A B C
Mycelium

Asexual stage

Conidiophores

Conidia

Sexual stage

Cleistothecia

Appendages

Asci

Ascospores

Host

Genus

2. Examine permanent preparations of l.s. through the head of a sclerotium under low magnification and draw a
general view, and then under higher magnification draw a section showing a part of a perithecium. The surface
of the stroma head is covered with sunken perithecia (ascostroma). The inoperculate asci have a typical cap at
their apex that is penetrated by a pore 2- Study the preserved specimen of the sclerotia available. Illustrate and
record your observations.

39
PRACTICAL NO. 17

General identification characters of fungi under the Phylum Ascomycota Class

Taphrinomycetes
Objective: To understand the identifying characters of the genus Taphrina spp. and its life cycle

Systematic Position
1. Genus Taphrina (Leaf curl fungus)
Phylum – Ascomycota
Mainly the spp. of this genus cause the disease symptoms as leaf
Class –Taphrinomycetes
curl, puckering, pockets and witches broom. The most important
Order- Taphrinales
species is T. deformans, the cause of “Peach leaf curl”.
Family – Taphrinaceae
Mycelium –Composed of septate hyphae, consisting of typically
binucleate cells. There hyphae may be intercellular on sub-cuticular or may grow within the walls for the
epidermal cells.
Asci- Naked, (without forming any fruiting body (ascocarp), forming the layer of naked (Hymenium on the
epidermis of the host, and each ascus harving 8 ascospores).
Ascospores- Eight in number, mostly located at upper portion of asci, single celled, round or ovoid.
Important and species. T. deformans (Peach leaf curl)
T. pruni (plum pocket)

Fig. 19 Life cycle of Taphrina pruni

Conclusive Questions:

40
1. How does the binucleate condition arise in Taphrina deformans?
2. How does Taphrina deformans reproduce asexually?
3. How does Taphrina deformans overwinter?
4. Collect the sample of plant infected with Sclerotinia rot from the crop cafeteria and prepare slides and observe
under the microscope. Note down the important observations

41
PRACTICAL 18
Application of molecular approaches and techniques for identification of fungal
pathogens.
Objective: To identify fungal isolates through molecular characterization

Procedure

1. Set water bath at 60° C.

2. Preheat the extraction buffer to 60°C followed by addition of mycelial mate into be pre-heated
buffer, mixProperly at 10 min interval by moving the tubes.
3. Cool the samples to room temperature.
4. Add equal vol. of PCI(Phenol: chloroform: Isoamylalcohal) (25:24:1) to the sample.
5. Centrifuge at 12,500rpm for 20min at 25° C.
6. Spell out the supper aqueous phase and repeat the step no-4.
7. Add Isoamyl alcohol (ice cooled) in to the sample and incubate for Ihr at -20°C.
8. Precipitate the DNA by centrifuge at 5°C at 10000 rpm for 10 min
9. Add wash buffer followed by ethanol to the sample.
10. Allow ethanol to evaporate.
11. Dissolve DNA pellet in Nucleus free water & TE buffet.
12. Store at 4°C until use.

Basic PCR Protocol

1. Place a 96 well plate into the ice bucket as a holder for the 0.2 ml thin walled PCR tubes.
Allowing PCR reagents to be added into cold 0.2 ml thin walled PCR tubes will help prevent nuclease
activity and nonspecific priming.
2. Pipette the following PCR reagents in the following order into a 0.2 ml thin walled PCR tube:
Sterile Water, 10X PCR buffer, dNTPs, MgCl2, primers, and template DNA . Since experiments should
have at least a negative control, and possibly a positive control, it is beneficial to set up a Master Mix
in a 1.8 ml microcentrifuge tube (See explanation in Notes).
3. In a separate 0.2 ml thin walled PCR tubes add all the reagents with the exception of template
DNA for a negative control (increase the water to compensate for the missing volume). In addition,
another reaction (if reagents are available) should contain a positive control using template DNA and
or primers previously known to amplify under the same conditions as the experimental PCR tubes.

42
 4. Taq DNA polymerase is typically stored in a 50% glycerol solution and for complete dispersal in
the reaction mix requires gentle mixing of the PCR reagents by pipetting up and down at least 20
times. The micropipettor should be set to about half the reaction volume of the master mix when
mixing, and care should be taken to avoid introducing bubbles.
5. Put caps on the 0.2 ml thin walled PCR tubes and place them into the thermal cycler . Once the
lid to the thermal cycler is firmly closed start the program.
6. When the program has finished, the 0.2 ml thin walled PCR tubes may be removed and stored
at 4 °C. PCR products can be detected by loading aliquots of each reaction into wells of an agarose
gel then staining DNA that has migrated into the gel following electrophoresis with ethidium bromide.
If a PCR product is present, the ethidium bromide will intercalate between the bases of the DNA
strands, allowing bands to be visualized with a UV illuminator.

Activity: Extract DNA and quantity it. Check its quality on nanodrop. Send the sample for sequencing. Get the
sequence and prove the identity of the pathogen

43