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Content Sr. No.

Title Page No.

1 Phylogenetic classification systems. 2-3 2 Extraction of fungal genomic DNA. 4-6 3 Isolation and culture of zoosporic fungi

by baiting Technique. 7

4 Slide culture technique of fungi. 8-9 5 Biocontrol potential of Trichoderma

against plant pathogenic fungi. 10-11

6 Production and estimation of citric acid from Aspergillus niger.

12

7 Production of Penicillin from Penicillium sp.

13-14

8 Determination of AM fungal diversity from soil and staining of AM colonized roots.

15-19

9 Isolation and culture of fungi from rhizosphere.

20

10 Various Media for culturing of Fungi. 21-22 11 Practical: To prepare temporary mounts

of the fungal specimen and fungal culture.

23

11 Use full links 24-25 12 Bibliography 26-28

2

1. Classification of fungi:

A higher-level phylogenetic classification of the Fungi by Hibbett et al. (2007)

Fig.: Phylogeny and classification of Ascomycota and Basidiomycota.

3

Kirk et al. (2008)

Kingdom Fungi

Phylum Ascomycota

Phylum Basidiomycota

Phylum Chytridiomycota

Phylum Glomeromycota

Phylum Microsporidia

Phylum Zygomycota

Kingdom Chromista, fungal phyla

Phylum Hyphochytriomycota

Phylum Labyrinthulomycota

Phylum Oomycota

Kingdom Protozoa, lineages with fungal phyla

Ramicristates (incl. classes Protostelia, Myxogastria and

Dictyostelia)

Heterolobosea (incl. order Acrasida)

Copromyxida (Copromyxa)

Fonticulida (Fonticula)

Plasmodiophorida

4

Practical: 2 Extraction of fungal genomic DNA: (Amir et al. 2015)

Extract the fruiting body/fungal culture (200 mg) in liquid

nitrogen. Homogenize the powder by adding 1 ml of lysis buffer

(100mM Tris HCL (pH-8.00); 50mM EDTA; 3% SDS). Centrifuge at

13000 rpm for 10 min. To the supernatant add equal volume of

phenol:chloroform:Isoamyl alcohol (25:24:1) and shake well. Again

centrifuge at 13000 rpm for 10 min. Separate the upper aqueous

layer in another tube and add equal volume of ethanol and keep at

20°C for 20 min. Again centrifuge it at 10,000 rpm for 10 min.

Remove supernatant and wash the pellet with 200ul 70 % ethanol.

Again centrifuge it at 10,000 rpm for 10 min. Remove the

supernatant and dry the pellet. After complete removal of ethanol,

dissolve the pellet in 20μl of 1X TE buffer. Add 2μL of RNase and

incubate it at 37°C for 30 min on dry bath. 2μL DNA will be run on to

0.8% agarose gel electrophoresis. Observe the gel under UV trans

illuminator system.

PCR amplification:

PCR master mix 12.5μL

ITS1 1μL

ITS4 1μL

Sterile dH2O 7.5μL

DNA template 3 μL

Total 25μL

5

PCR cycle conditions (For ITS1-ITS4):

Initial denaturation 95°C 5 min

Denaturation 95°C 1 min

30 cycles Annealing 58°C 45 sec

Extension 72°C 1 min

Final Extension 72°C 7 min

Hold 4°C Hold

Primer sequences for the DNA amplification:

ITS 1 Forward: 5' TCCGTAGGTGAACCTGCGG 3'

ITS 4 Reverse: 5' TCCTCCGCTTATTGATATGC 3'

Purification of PCR product:

Add 2 volumes of DNA binding buffer NT1 to the PCR product and

transfer it to the provided column.

Centrifuge at 13,000rpm for 1 min.

Discard the flow-through and add 600μL of NT3 wash buffer to

the column.

Centrifuge at 13,000 rpm for 1 min.

Discard the flow through and centrifuge again for 1 min

Place the column in sterile 1.5mL Eppendorf tube.

Add EB buffer or warm MilliQ water (20-30 μL) to the column and

incubate for 2-3 min.

Centrifuge at 13,000 rpm for 1 min 30 sec.

6

Sequencing of PCR product:

Sequencing will be done by Sanger sequencing method.

Identification by using NCBI:

The sequence will be aligned and compared by BLAST search in NCBI

(National Centre for Biotechnology Information) nucleotide

database.

Consensus sequences:

Make a consensus sequences by using software’s: Reverse

complement and Clustal omega.

Phylogenetic analysis by using MEGA 7 software:

Phylogenetic analysis will be carried out to establish the phylogenetic

placement of our collected isolates. Reference sequences will be

selected from the relevant literature and Genebank. Alignment will

be performed using CLUSTAL W and the phylogenetic analysis will be

performed by using the Neighbor-Joining method by Mega7 software

(Saitou and Nei 1987, Felsenstein 1985, Tamura and Nei 1993, Kumar

et al. 2016).

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Practical 3: Isolation and culture of zoosporic fungi by baiting

Technique.

The members of Saprolegniaceae are most ubiquitous in water

bodies.

Requirements:

Water samples in sterile bottles.

Baits (hemp, mustard seeds, grass leaves, house or drosophila flies,

termite, wings, cellophate, pollen grain of conifer, snake skin, white

human hair).

Sterile petri dishes, sterile distilled water, potato dextrose agar

medium, forceps, sterile tissue paper etc.

Procedure:

Bring the water sample from pond in beaker.

Add some bait in the water sample.

Incubate the water sample at 27°C for 3-6 days.

Grow good colonies on the baits.

The colonies are transferred to sterile culture dish, each

containing 25 ml of sterile tap water and 25 ml of sterile

distilled water.

If no pond are nearby then some soil ½ inches below the

surface can be collected and poured in beaker containing

autoclaved distilled water.

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Practical 4: Slide culture technique of fungi.

Requirements:

Petri dish, Slide, Coverglass, PDA medium, Glass rod, Glycerine etc.

Procedure:

Slide culture results in sporulation characteristics where

organism remains relatively unchanged.

Fungi growing on a block of agar are sandwiched between a

glass slide and a cover slip in a moist chamber (Figure 1).

Prepare Petri dish with agar suitable for organism to be grown;

use about 15 ml of agar per 100 mm dish. Cut solidified

medium rapidly into 1 cm squares using a sterile knife or

dissecting needle and a sterile glassrod.

Place a bent glass rod on a filter paper disc in the bottom of a

Petri dish, put a slide, cover and sterilize it.

Introduce sterile water sufficient enough to moisten the filter

paper (with 5% glycerine, if fogging occurs on slide).

Place agar block on slide, using aseptic condition throughout.

Inoculate the fungi on four sides of the agar block with spores

or mycelium.

Place a sterile cover slip centrally upon the agar block.

Incubate under temperature and light condition suitable to

sporulation of the organisms. (Add more sterile water if filter

paper dries).

Lift cover slip carefully and discard agar block.

Place drop of lactophenol on a clean slide and add the cover

slip with the fungus growth adhering to it.

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Figure 1: Diagram of a slide culture in petri-plates as moist

chamber.

Precautions:

Incubate the petri dishes in dark to discourage algal blooms.

Change the water frequently to avoid anaerobic condition and for

avoiding the growth of bacteria and protozoans.

Baits in petri plates should be shallowly covered with sterile

water.

Several baits may be used at a time to have a great diversity of

fungi.

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Practical 5: Study the Antagonistic effect of Trichoderma viride to

the Pathogenic Fungi by dual Culture Method. (Morton and

Straube, 1955).

Requirements:

PDA medium,

Trichoderma viride

Plant pathogen

Introduction:

Trichoderma species is a most common fungal biological control

agent that inhibits pathogens by cell wall degrading enzymes like

Chitinase and glucanase. They play major role in antagonistic

mechanism of Trichoderma sp. against wide range of fungal

pathogens.

Requirements:

PDA plates, Trichoderma culture, Fusarium oxysporum culture.

Procedure:

A mycelial disc (9mm diameter approx.) obtained from peripheral

region of 3-5 days old culture of tested fungi and T. viride will be

placed simultaneously on periphery, about 1 cm from the edges

of petri-plate (9mm dia.) at the opposite sides.

The petri-plate containing the PDA medium inoculated with

tested pathogen alone will serve as a control.

All plates are incubated at 28°C and measurement will be taken

after 5 days.

At the end of incubation period, radial growth will be measured.

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Observation:

The % inhibition growth of tested pathogens in presence of T. viride

will be calculated over control.

Per cent inhibition of the pathogen over control was calculated by

the formula given by Nene and Thapliyal (1982).

I (%) = (C-T)/ C x 100

I = Percent growth inhibition

C= Growth in control (monoculture)

T= Growth in treatment (dual culture)

Fusarium oxysporum

Trichoderma viride

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Practical 6: Production and estimation of citric acid from Aspergillus

niger (HailemariamFeleke, 2010).

Requirements:

PDA broth medium, Aspergillus niger strain, Spectrophotometer,

Unhydrous citric acid.

Procedure:

Citric acid estimation

The broth culture is filtered to separate mycelia, and the filtrate

will be used for estimation.

The citric acid estimation will be done by using

spectrophotometer at 420nm, after adding pyridine and acetic

anhydride.

For each 1ml of sample, 1.3 ml of pyridine and 5.7ml of acetic

anhydride will be added to develop color (Marrier and Boulet,

1958).

Citric acid standard will be prepared as follows: Anhydrous

citric will be used to prepare a stock solution (50mg/ml) which

is stable for at least one year at 0°C.

Standards prepared from the stock solution are stable for one

month at 00C. Using dilution rule, M1V1=M2V2.

To make 0.3mg/lm, 0.25mg/ml, 0.2gm/ml, 0.15gm/ml,

0.1mg/ml and 0.05gm/ml citrate solution, 60μl,50μl,

40μl,30μl,20μl and 10μl per 10ml distilled water will be used

respectively.

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Practical 7: Production of Penicillin from Penicillium sp.

Requirements: Penicillium culture, PDA broth medium, Brown sugar,

Nutrient Agar medium etc.

Procedure:

Culture the Penicillium sp.on PDA medium.

Grow the Penicillium on PDA broth medium (50 ml medium in 250

ml flask) with and without brown sugar (3gm/100 ml).

Incubate the flask at 25-280 C for 7-14 days.

Observation: Observe the flask for development of golden yellow

colour in the medium or in droplets on the mycelium indicating the

production of Penicillin.

Antibiotic sensitivity test.

Antibiotic sensitivity performs by agar diffusion method which

design to determine the growth inhibition of microorganism by

antibiotic. The resulting value is called minimal inhibitory

concentration (MIC) which is determine by measuring diameter of

growth inhibition zone surrounding the antibiotic disc.

Requirements: 28 hr. Nutrient Broth cultures of bacteria, culture

filtrate of Penicillium sp., Nutrient Agar plate (N.A.), filter disc, sterile

cotton, and solutions of Penicillin of known concentration.

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Procedure

Inoculate the Bacterial suspension culture on N.A. medium by

using sterile cotton.

Allow the agar surface to dry for 5 min.

Pick the sterile filter paper disc and dip it in the Penicillium

culture filtrate.

Place the disc on the agar surface in the plate.

Place the sterile disc on agar plate as a control.

Repeat the steps with other concentrations of Penicillin.

Incubate all the plate at 370C for 24-48 hrs.

Observations: Examine all the plates for zone of inhibition

surrounding the disc.

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Practical 8: Determination of AM fungal diversity from soil and

staining of roots for AM colonization.

Osmolyteaccumulation

0

1

2

3

4

5

C AM

Ton

/Ha.

Bulb Yield22%

AM fungi

P uptake

Abiotic stress Biotic stress

Key Functions

Plant growth

and yield

(Borde et al 2009)

(Fig.: Evelin et al 2009)

(Fig: Roy-Borde et al 2011)

(Fig.: M.J Pozo et al 2009)

Isolation of AM fungal spore by wet sieving and Decanting

methods: (Gerdemannand Nicolson 1963).

Requirements:

Rhizospheric soil sample, 500 ml beaker, sieves 710 µm, 250/µm, 75

µm, 45 µm, water, funnel, whatman filter paper.

Procedure:

Take 200ml water in 500 ml beaker.

Add 50 gm of rhizospheric soil and mix well until all soil aggregate

disperses and leaves a uniform suspension.

Allow the heavier particles to settle down.

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Decant most of the suspension through a 710µm sieve to remove

large organic matter.

Decant the suspension through 250µm, 75µm and 45µm sieves

consequently.

Collect all the residues on 45µm sieve.

Wash the residues with water and filter it by using whatman filter

paper to collect the spores.

Observation and results: Observe the AM fungal spore and identify

the genera based on color size, shape, shape of hyphae attachment

by referring Manual Schenck and Perez (1990).

Observations of intact spore

A: Spore colour; B: Spore diameter (for globose spore); C: Composite

spore wall thickness; D: Attachment present? Yes/No. If no, go to E,

E: Spore contents; F: Spore with mantle or other surface hyphae?

Yes/No. If no, go to G

1) Width of hyphae: 2) Colour of hyphae: 3) Hyphae sinous?

Yes/No. If no, go to G

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Observations on broken spores

A: Number of wall groups in the spore wall

B: Width of each wall group

C: Number of walls within each group

Diagrammatic representation of Isolation of AM fungal spore and

AM root colonization:

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Determination of AM root colonization: (Phillips and Hayman1970)

Requirements:

Test tube, forceps, water bath, KOH 10%, HCL 1N, 0.05% Trypan blue

in lactophenol, 50% glycerol-water (v/v) solution for de-staining and

storage of stained roots.

Procedure:

Wash the root in sterile water.

Cut the root pieces in to 1cm length.

Wash with water and Macerate the root in 10% KOH in water-

bath for 1 hr.

Acidified the root with 1N HCL.

Wash with sterile water and stain with 0.05 % trypan-blue in

lactophenol for 1hr.

50% glycerol-water (v/v) solution for de-staining and mount with

glycerol on slide and cover with coverslip.

Observation and result: Observe the AM colonization in roots by

observing vesicles and arbuscule, also calculate Percentage of AM

root colonization.

Percentage of AM root colonization: Giovannetti and Mosse (1980).

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Calculation of the percentage of the root length with mycorrhizal

colonization in a sample of 25 root segments (1 cm).

AM colonized roots

% AM colonization= ------------------------------------- X 100

Total No. of roots observed

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Practical 9: Isolation and culture of fungi from rhizosphere.

Requirements:

Freshly collected roots, Czapek-Dox agar medium, petriplate, 250 ml

flask, sterile distilled water, scrap bottle, sterile polythene bag and

autoclave.

Procedure:

Collect the rhizospheric soil and roots in bag.

Add 10 gm soil in 100ml sterile water and shake it for 15 min. on

shaker.

Prepare serial dilution 10-2 to 10-6.

Transfer 1ml of dilutions 10-2 to 10-6 to sterile petriplates.

Pour the melted sterile Czapek-Dox medium in petriplates.

Incubate the plate for 7 days at 25°C in an inverted position.

Results: Microbial counts in rhizospheric soil per g of the soil are

calculated by formula.

No. of fungal sp./g of soil= No. of colonies/ plate x dilution factor

------------------------------------------------

Dry wt. of soil taken

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Various Media for culturing of Fungi:

Potato Dextrose Agar

This media is used for cultivation of yeasts, fungi and moulds.

Potato (Peeled) - 200.0 gm

Dextrose - 20.0 gm

Agar - 15.0 gm

Distilled water - 1000.0 ml

pH - 5.6

Sabouraud Agar

Sabouraud agar is used for cultivation of yeasts and moulds.

Peptone - 10.0 gm

Dextrose - 40.0 gm

Agar - 15.0 gm

Distilled water - 1000 ml

pH - 5.6

Corn meal agar (CMA):

Agar - 15 gm

Corn-meal - 20gm

Distilled water - 1000ml

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Malt extract agar:

Malt Extract : 20gm

Agar : 15gm

Glucose : 20gm

Distilled water : 1000ml

Sucrose : 200gm

Adjust pH to about 6.8 with 1M NaOH

Oatmeal agar:

Porridge oats : 30gm

Distilled water 1 litre (Simmer for 2 hours, filter out solids through

muslin, make up to 1 litre)

Agar 15gm

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Practical: To prepare temporary mounts of the fungal specimen and

fungal culture.

Requirements:

Reagent: Lactophenol Cotton blue stain.

Equipments & Glassware: Clean glass slides, covers slips, ocular,

Bunsen-burner and transfer needles.

Theory/Principle:

Lactophenol cotton blue solution is used for fungal staining. It

contains lactic acid (20.0 ml), phenol (20 gm), glycerol (40 ml),

distilled water (20 ml) and aniline blue (0.05 gm). Glycerol gives

viscosity while phenol kills the fungal cells. Lactic acid is used for

viewing the colour. Cotton blue or aniline blue stains both the cell

wall and cytoplasm. Few drops of cotton blue stain are used for

staining purpose. After staining, the sample is observed at low, as

well as, at high power. Identifiable characteristics are noted and used

for identifying the fungal specimens.

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Useful links:

Clustal Omega

https://www.ebi.ac.uk/Tools/msa/clustalo/

Reverse complement

https://www.bioinformatics.org/sms/rev_comp.html

Finch TV

https://softfamous.com/finchtv/

Mycobank

http://www.mycobank.org/

indexfungorum

http://www.indexfungorum.org/names/names.asp

AFTOL classification project

http://www2.clarku.edu/faculty/dhibbett/AFTOL/AFTOL.htm

NCBI

https://www.ncbi.nlm.nih.gov/

American Type Culture Collection

https://www.atcc.org/

NFCCI-Agharkar Research Institute

http://nfcci.aripune.org/service.php

MTCC

https://mtccindia.res.in/

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NBAIM

http://nbaim.org.in/default.aspx

MCC-NCCS

Microbial Culture Collection, National Centre for Cell Science (NCCS) -

pune, India

NCIM

National Collection of Industrial Microorganisms (NCIM) - Pune, India

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Bibliogrpahy:

Aamir S, Sutar S, Singh SK and Baghela A. 2015. A rapid and efficient

method of fungal genomic DNA extraction, suitable for PCR

based molecular methods. Plant Pathology and Quarantine. 5:

74–81.

Felsenstein J. 1985. Confidence limits on phylogenies: An approach

using the bootstrap. Evolution. 39: 783–791.

Gerdemann JW and Nicolson TH. 1963. Spores of mycorrhizal

endogone species extracted from soil by wet-sieving and

decanting. Transactions of the British Mycological Society,

p.235-244.

Giovannetti M and Mosse B. 1980.An evaluation of techniques for

measuring vesicular arbuscular mycorrhizal infection in roots.

New Phytol. 84: 489–500.

Hibbett DS, Bindera M, Bischoff JF, Blackwell M, Cannon PF, Eriksson,

OE, Huhndorf S, James T, Kirk PM, Lücking R, Thorsten Lumbsch

H, Lutzonig F, Matheny PB, McLaughlin DJ, Powell MJ, Redhead

S, Schoch CL, Spatafora JW, Stalpers JL, Vilgalys R, Aime MC,

Aptroot A, Bauer R, Begerow D, Benny GL, Castlebury LA, Crous

PW, Dai YC, Gams W, Geiser DM, Griffith GW, Gueidan C,

Hawksworth DL, Hestmark G, Hosaka K, Humber RA, Hyde KD,

Ironside JE, Kõljalg U, Kurtzman CP, Larsson KH, Lichtwardt R,

Longcore J, Miądlikowska J, Miller A, Moncalvo JM, Mozley-

Standridge S, Oberwinkler F, Parmasto E, Reeb V, Rogers JD,

Roux C, Ryvarden L, Sampaio JP, Schüssler A, Sugiyama J, Thorn

RG, Tibell L, Untereiner WA, Walker C, Wang Z, Weir A, Weiss

M, White MM, Winka K, Yao YJ and Zhang N. 2007. A higher-

27

level phylogenetic classification of the Fungi. Mycological

Research. 111: 509–547.

Kirk P, Cannon PF, Minter DW and Stalpers JA. 2008. Ainsworth and

Bisby’s “Dictionary of the Fungi” (10th edition) CAB

International, Wallingford, UK, pp 771.

Kumar S, Stecher G and Tamura K. 2016. MEGA7: Molecular

Evolutionary Genetics Analysis version 7.0 for bigger datasets.

Mol Biol and Evol. 33: 1870–1874.

Morton DJ and Straube W. 1955. Antagonistic and stimulatory effect

of microorganism upon Sclerotium rolffsii. Phytopathology. 45:

417–420.

Nene YL and Thaplial PN. 1982. Fungicides in Plant Disease Control.

Oxford and IBH Publishing House, New Delhi, pp.163.

Phillips JM and Hayman, D.S. 1970. Improved procedure for cleaning

roots and staining parasitic and vesicular arbuscular

mycorrhizal fungi for rapid assessment of infection. Trans. Brit.

Mycol. Soc. 55, 158-160.

Saitou N and Nei M. 1987. The neighbor-joining method: A new

method for reconstructing phylogenetic trees. MolBiol and

Evol. 4: 406-425.

Schenck, N.C. and Perez, Y. (1990) Manual for Identification of

Vesicular Arbuscular Mycorrhizal Fungi. (INVAM). University of

Florida, Gainesville.

Stevens and Russell B. 1974. Mycology Guidebook. INSTITUTICN

Mycological Society of America, San Francisco, Calif. National

Science Foundation, Washington, D.C. pp. 719.

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Tamura K and Nei M. 1993. Estimation of the number of nucleotide

substitutions in the control region of mitochondrial DNA in

humans and chimpanzees. Mol Biol and Evol. 10: 512–526.

White TJ, Bruns T, Lee S and Taylor J. 1990. Amplification and direct

sequencing of fungal ribosomal RNA genes for phylogenetics.

In: Innis, M.A., Gelfand, D.H., Sninsky, J.J. & White, T.J. (Eds.)

PCR Protocols: a guide to method and applications. Academic

Press, San Diego, pp. 315–322.