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APPENDIX A

Stains and solutions for microscopic examination of fungi

1. India ink: was used to observing gelatinous appendages of ascospores in

some fungal species.

2. Lactophenol: was routinely used for observing fungi and preparing

semipermanant slides. The components: phenol, which will kill any living

organisms; lactic acid which preserves fungal structures.

Phenol (crystals) 10 g

Lactic acid 10 ml

Glycerol 10 ml

Distilled water 10 ml

Dissolve 10 g of phenol in 10 ml distilled water (do not heat). Then add 10 ml

glycerol and 10 ml lactic acid.

3. Lactophenol cotton blue (LPCB): widely used for staining and observing

fungi. The preparation has three components: phenol, which will kill any live

organisms; lactic acid which preserves fungal structures, and cotton blue

which stains the chitin in the fungal cell walls.

Phenol (crystals) 20 g

Lactic acid 16 ml

Glycerol 31 ml


ÅÔ¢ÊÔ·¸Ô ìÁËÒÇÔ·ÂÒÅÑÂàªÕÂ§ãËÁèCopyright by Chiang Mai UniversityA l l r i g h t s r e s e r v e d


276

Dissolve phenol in distilled water, then add lactic acid and glycerol. Add 0.05

g Poirrier's (cotton) blue or acid fuchsin.

4. Melzer's reagent: specifically for amyloid and dextrinoid reactions in spores,

asci, hymenial tissues. The staining result use for identification of ascomycete

fungi.

Chloral hydrate 100 g

Potassium iodide 5 g

Iodine 1.5 g


Agar medium

1. Corn meal agar (CMA)

Blend 50.0 g corn meal in 800 ml of distilled water. Leave overnight in the

refrigerator. Heat at 60 °C for one hour. Filter and make up volume to 1000

ml. Add 15.0 g agar and heat to dissolve, then autoclave for 15 minutes at 15

psi pressure-121 °C

2. Malt extract agar (MEA)

Agar 15.0 g

Glucose 20.0 g

Malt extract 20.0 g

Peptone 1.0 g



277

Add components to distilled water and bring volume to 1000 ml. Mix

thoroughly and gently heat and bring to boiling. Autoclave for 15 minutes at

15 psi pressure-121 °C. Pour into sterile Petri dishes.

3. Poly R-478 agar (Pointing, 1999) (per liter)

Glucose 20 g

KH2PO4 1 g

C4H12N2O6 0.5 g

MgSO4.7H2O 0.5 g

CaCl2.2H2O 0.01 g

Yeast extract 0.01 g

CuSO4.5H2O 0.01 g

Fe2(SO4)3 0.001 g

MnSO4.H2O 0.001 g

Poly R-478 0.02 g

Agar 15 g

pH 5.5

4. Potato dextrose agar (PDA)

Glucose 20.0 g

Agar 15.0 g

Potato, infusion form 1000 ml

Preparation of potato, infusion form:

Peel and dice potatoes. Add 300 g of potato to 500 ml of distilled water.

Gently heat and bring to boiling. Continue boiling for 30 minutes. Filter

through gauze. Bring volume of filtrate to 1000 ml.



278

Preparation of medium:

Add glucose and agar to 1000 ml of potato infusion. Mix, gently heat and

bring to boiling. Autoclave for 15 minutes at 15 psi pressure-121 °C.

5. Rose Bengal agar

Agar 15.0 g

Glucose 10.0 g

Malt extract agar 20.0 g

Rose Bengal 0.05 g

Chloramphenicol 10.0 ml

Preparation of chloramphenicol solution:

Add 0.10 g of chloramphenicol to distilled water and bring volume to 10.0 ml.

Mix thoroughly. Filter sterilize.

Preparation of medium:

Add components, except chloramphenicol solution, to distilled water and

bring volume to 990 ml. Mix thoroughly. Gently heat and bring to boiling.

Autoclave for 15 minutes at 15 psi pressure-121 °C. Cool to 45 °C.

Aseptically add sterile chloramphenicol solution. Mix thoroughly. Pour into

sterile Petri dishes.



APPENDIX B

Reagents for molecular techniques

1. 2× CTAB buffer (50 ml)

Cetyltrimethylammonium bromide (CTAB) 1.0 g (= 2%)

100 mM Tris-HCl (pH 8.0) 5.0 ml (= 100 mM)

5M NaCl 14.0 ml (= 1.4 M)

0.5 M EDTA 2.0 ml (= 20 mM)

2. Phenol-Chloroform: 25 parts phenol (saturated with extraction buffer), 24

parts chloroform, 1 part isoamyl alcohol, 0.1% (w/v) 8-hydroxyquinone. Store

refrigerated.

3. 10× TAE buffer (1000 ml)

Tris-base 48.4 g

Glacial Acetic Acid 10.9 g

EDTA (free acid f.w. 292.25) 2.92 g

Deionized water 1000 ml

4. TE buffer

10 M Tris-HCl (pH 7.4)

1 mM EDTA (pH 8.0)



APPENDIX C

Solutions for enzyme assay

1. 1.0 % Congo red

Congo red 10 g

NaNO3 0.5 g

Dissolve the components in 0.2 M K2HPO4. Prepare fresh before use.

2. DNS reagent

3,5-dinitrosalicylic acid 1.0 %

phenol 0.2 %

Na2SO3 0.05 %

NaOH 1.0 %

Dissolve 3,5-dinitrosalicylic acid and NaOH in 50 ml of distilled water, mix

thoroughly. Add phenol and Na2SO3. Adjust volume to 100 ml.

3. 40% Na-K tartrate

Dissolve Na-K tartrate 40 g in 100 ml of distilled water. Mix thoroughly.

4. 0.2 M K2HPO4

Dissolve 34 g of K2HPO4 in 1000 ml of distilled water. Mix thoroughly.



281

Broth medium

Liquid Basal Medium (LBM) (Pointing, 1999)

Carbon sources 1% (w/v)

KH2PO4 1 g/l

NH4NO3 0.1 g/l

MgSO4.7H2O 0.2 g/l

CaCl2.2H2O 0.01 g/l

Yeast extract 0.01 g/l

CuSO4.5H2O 0.001 g/l

MnSO4.H2O 0.001 g/l

Fe2(SO4)3 0.001 g/l

Calculation for enzyme activity

Calculation for cellulase activity

In 30 minutes of reaction, glucose was obtained = x µg

Glucose molecular weight = 180 = x / 180 µmol

.˙. In 1 minute, glucose was obtained = x / 180 / 30 µmol

Using crude enzyme volume = 0.5 ml

.˙. Cellulase activity = x / 180 / 30 / 0.5 Unit/ml



282

Calculation for xylanase activity

In 30 minutes of reaction, xylose was obtained = x µg

Xylose molecular weight = 150 = x / 150 µmol

.˙. In 1 minute, xylose was obtained = x / 150 / 30 µmol


.˙. Xylanase activity = x / 150 / 30 / 0.5 Unit/ml

Calculation for mannanase activity

In 30 minutes of reaction, mannose was obtained = x µg

Mannose molecular weight = 180.2 = x / 180.2 µmol

.˙. In 1 minute, mannose was obtained = x / 180.2 / 30 µmol


.˙. Mannanase activity = x / 180.2 / 30 / 0.5 Unit/ml

Calculation for polygalacturonase activity

In 30 minutes of reaction, D-galacturonic acid = x µg

was obtained

D-Galacturonic acid molecular weight = 212.2 = x / 212.2 µmol

.˙. In 1 minute, D-galacturonic acid was obtained = x / 212.2 / 30 µmol


.˙. Polygalacturonase activity = x / 212.2/ 30 / 0.5 Unit/ml



283

Calculation for laccase activity

Enzyme activity was calculated from the linear phase of the reaction using

molar absorbance of the oxidation product of DMP (ε=49,600 M-1cm-1). One unit of

laccase activity defined as the amount of enzyme that oxidize 1 µmole substrate per

minute under standard assay condition

The enzyme activities will be calculated by using Beer-Lambert’s law. In

mathematical terms:

A = εbC

A: absorbance of the sample at specific wavelength

ε: the adsorptivity coefficient or extinction coefficient of the

material at the wave length

b: the pathlength through the sample in cm

C: the concentration

Calculation

Net absorbance at 469 nm(A) = Y (min-1)

Pathlength of cuvette (b) = 1 (cm)

ε of DMP = 49600 (M-1cm-1)

A = εbC

C = A/εb

C = Y____(min-1) 49600 (M-1cm-1) 1 (cm) C = Y____ M/min 49600



284

C = Y____ mole/l/min 49600

C = (Y×106) µmole/ml/min 49.6

Using enzyme volume = 0.1 ml (dilution 10 times)

∴Laccase activity = (Y × 106 × 10) µmole/ml/min 49.6

= (Y × 107) Unit/ml 49.6



APPENDIX D

Standard curve

1. Standard curve of reducing sugar determination by DNS method (Miller, 1959)

Reagent

1. DNS solution

Dissolve 1 g of NaOH and 1 g of 3,5-dinitrosalicylic acid in 50 ml of

distilled water, add 0.2 g of phenol and 0.05 g of Na2SO3 in mixed solution

and adjust to a final volume of 100 ml.

2. 40% Na-K tartrate

Dissolve 40 g of Na-K tartrate in 100 ml of distilled water.

Procedure

1. Add 1 ml of reducing sugar sample and 2 ml of DNS solution to test tube

and using distilled water as control.

2. Vortex and boil for 15 minutes and place in cold water.

3. Add 1 ml of 40% Na-K tartrate to the test tube and vortex.

4. Record the absorbance at 550 nm using control as a blank. ÅÔ¢ÊÔ·¸Ô ìÁËÒÇÔ·ÂÒÅÑÂàªÕÂ§ãËÁèCopyright by Chiang Mai UniversityA l l r i g h t s r e s e r v e d


286

Table 1 Absorbance at 550 nm of various concentration of glucose, xylose, and

mannose by DNS method.

A550 Concentration of sugar (µg/ml) Glucose Xylose Mannose D-galacturonic â

0 0 0 0 0

100 0.099 0.101 0.111 0.127

200 0.264 0.357 0.275 0.395

300 0.441 0.765 0.516 0.661

400 0.676 1.091 0.752 0.923

500 0.911 1.508 0.984 1.232

600 1.141 1.818 1.177 1.469

700 1.375 2.166 1.533 1.676

800 1.675 2.461 1.728 1.871

900 1.900 2.718 1.981 2.235

1000 2.146 2.953 2.278 2.458

1100 2.338 3.111 2.430 -

1200 2.546 3.196 2.650 -

1300 2.670 3.252 2.788 - 1400 2.866 3.307 2.984 -



287

Figure 1 Satandard curve of glucose versus absorbance at 550 nm.

Figure 2 Satandard curve of xlucose versus absorbance at 550 nm.

y = 0.0021xR2 = 0.9900

0

0.5

1

1.5

2

2.5

3

3.5

0 200 400 600 800 1000 1200 1400 1600

Glucose concentration (ug/ml)

Abs

orba

nce

at 55

0 nm

y = 0.003xR2 = 0.9877

0

0.5

1

1.5

2

2.5

3

3.5

0 100 200 300 400 500 600 700 800 900 1000 1100

Xylose concentration (ug/ml)

Abs

orba

nce

at 55

0 nm



288

Figure 3 Satandard curve of mannose versus absorbance at 550 nm.

Figure 4 Satandard curve of D-galacturonic acid versus absorbance at 550 nm.

y = 0.0022xR2 = 0.9922

0

0.5

1

1.5

2

2.5

3

3.5

0 200 400 600 800 1000 1200 1400 1600

Mannose concentration (ug/ml)

Abs

orba

nce

at 55

0 nm

y = 0.0024xR2 = 0.9947

0

0.5

1

1.5

2

2.5

3

0 200 400 600 800 1000 1200

D-galacturonic acid concentration (ug/ml)

Abs

orba

nce

at 55

0 nm



289

2. Standard curve of bovine serum albumin (BSA)

Reagent

1. Reagent A: 0.1 M NaOH with 0.2% Na2CO3

Prepare 0.1 M NaOH by dissolve 1.0 g of NaOH in 250 ml of distilled

water. Dissolve 2.0 g of Na2CO3 in 80 ml of 0.1 M NaOH and adjust to a

final volume of 100 ml

2. Reagent B: 1.0% Na-K tartrate with 0.5% CuSO4.5H2O

Dissolve 1.0 g of Na-K tartrate in 80 ml of distilled water and add 0.5 g of

CuSO4.5H2O. Mix and adjust to a final volume of 100 ml by distilled

water.

3. Reagent C: Mix 50.0 ml of reagent A and 1.0 ml of reagent B (prepare

before use)

4. Reagent D: Folin-Ciocalteau reagent

Dilute Folin reagent with distilled water in 1:1

Procedure

1. Prepare a series of dilutions of 100-1000 µg/ml bovine serum albumin and

place 1 ml of each concentration to test tube.

2. Add 5.0 ml of reagent C and mix. Stand for 10 minutes at room

temperature.

3. Add 0.5 ml of reagent D and mix. Stand for 30 minutes at room

temperature.

4. Measure the absorbance at 750 nm using distilled water instead BSA as a

blank.



290

Table 2 Absorbance at 750 nm of various concentration of bovine serum albumin

(BSA) by Lowry method.

y = 0.0021xR2 = 0.9557

0

0.2

0.4

0.6

0.8

1

1.2

1.4

0 100 200 300 400 500 600 700

BSA concentration (ug/ml)

Abs

orba

nce

at 75

0 nm

Figure 5 Satandard curve of bovine serum albumin (BSA) versus absorbance at 750

nm

Concentration of BSA (µg/ml) A750 (average) 0 0

100 0.305

200 0.525

300 0.708

400 0.857

500 0.979

600 1.146

700 1.239

800 1.363

900 1.468

1000 1.534



APPENDIX E

Native PAGE

Reagent

1. 30% Acrylamide stock (30% (w/v) acrylamide, 0.8% (w/v) N,N΄-

methylenebisacrylamide)

Dissolve 60 g of acrylamide and 1.6 g of N,N΄-methylenebisacrylamide in 200

ml of distilled water. Filter the homogeneous solution through a 0.45 µm filter

membrane.

2. 1.5 M Tris-HCl (pH 8.0) (Resolving buffer)

Dissolve 18.5 g of Tris-Cl in distilled water and adjust to pH 8.0 with HCl and

add water to a final volume of 100 ml.

3. 0.05 M Tris-HCl (pH 6.8) (Stacking buffer)

Dissolve 6.0 g of Tris-Cl in distilled water and adjust to pH 6.8 with HCl and

add water to a final volume of 100 ml.

4. 0.9% ammoniumpersulfate

Dissolve 9 mg of ammoniumpersulfate in 1 ml of distilled water. Prepare fresh

daily.

5. TEMED (N,N,N΄,N΄-tetramethylenediamine)

6. Running buffer

Dissolve 3.0 g of Tris base and 14 g of glycine in 1000 ml of distilled water.



292

7. 5× Loading buffer

1 M Tris-HCl pH 6.8 0.6 ml

50% (v/v) glycerol 5.0 ml

10% (w/v) SDS 2.0 ml

Mercaptoethanol 0.5 ml

1% (w/v) bromophenol blue 1.0 ml

Distilled water 0.9 ml

Procedure

1. Clean the glass plates, spacers, combs and upper buffer reservoir of the gel

apparatus with detergent and completely dry.

2. Assemble the glass plate sandwich and the spacers.

3. Prepare the monomer solution for the resolving gel by combining all of the

reagents in Table 3.

4. Mix gently and quickly apply to gel cassette using autopipette to about 2

cm below the short plate.

5. Gently overlay with 1-5 mm of distilled water on top of the gel solution

and allow gel to polymerize for 1 hour.

6. Prepare stacking gel as recipe Table 3 on top of the resolving gel and insert

the well-forming comb into the solution.

7. Leave for polymerize about 30 minutes.



293

Table 3 Recipe for preparing 10% acrylamide gel for native PAGE.

Reagent Resolving gel (ml) Stacking gel (ml)

Resolving buffer (1.5 M Tris-HCl pH 8.0) 5.7

Stacking buffer (0.05 M Tris-HCl pH 6.8) 2.03

30% Acrylamide stock 3.3 0.6

0.9% Ammoniumpersulfate 1.0 0.3

Gently mix and degas for 30 second

TEMED 10 µl 4 µl

Gently mix and quickly apply to gel cassette

Sample preparation

1. Combine protein sample and 5X loading buffer (1:1) in an eppendorff tube.

2. Load 10 µl of sample in each well of gel

Electrophoresis

1. Place the gel into electrophoresis chamber and attach gel to electrode

assembly.

2. Add running buffer to inner and outer reservoir and remove the comb from

the gel carefully.

3. Load the prepared sample into the well in the stacking gel.

4. Attach the electrode plugs to power supply.

5. Turn on power supply to 200 Volt, 150 mA until the bromophenol blue

tracking dye front reaches the bottom of the gel.



CURRICULUM VITAE

Name Miss Itthayakorn Promputtha

Date of Birth September 6, 1978

Place of Birth Loei province, Thailand

Home Address 98 Moo 5 Ban Pong, Tambon Na-kham,

Amphur Muang, Loei, 42000, Thailand

E-mail Address [email protected]

Education Background B.Sc. in Medical Technology (Hons.),

Faculty of Associated Medical Science,

Chiang Mai University, Chiang Mai, Thailand,

May, 2001

Scholarship The Royal Golden Jubilee Ph.D. Program (2001-2006)

Publications

1. Promputtha, I., Lumyong, S., Vijaykrishna, D., McKenzie, E.H.C., Hyde,

K.D. and Jeewon, R. (2006). A phylogenetic evaluation of whether

endophytes become saprotrophs at host senescence. Microbial Ecology (In

press).

2. Promputtha, I., Jeewon, R., Lumyong, S., McKenzie, E.H.C. and Hyde, K.D.

(2005). Ribosomal DNA fingerprinting in the identification of non sporulating

endophytes from Magnolia liliifera (Magnoliaceae). Fungal Diversity 20:

167–186.



3. Promputtha, I., Lumyong, S., Lumyong, P., McKenzie, E.H.C. and Hyde,

K.D. (2005). A new species of Anthostomella on Magnolia liliifera from

northern Thailand. Mycotaxon 91: 413–418.

4. Promputtha, I.; Hyde, K.D.; Lumyong, P.; McKenzie, E.H.C.; Lumyong, S.

(2005). Fungi on Magnolia liliifera: Cheiromyces magnoliae sp. nov. from

dead branches. Nova Hedwigia 80: 527–532.


K.D. (2004). Fungal saprobes on dead leaves of Magnolia liliifera

(Magnoliaceae) in Thailand. Cryptogamie Mycologie 25: 315–321.


K.D. (2004). A new species of Pseudohalonectria from Thailand.

Cryptogamie Mycologie 25: 43–47.

7. Promputtha, I., Hyde, K.D., Lumyong, P., McKenzie, E.H.C. and Lumyong,

S. (2002). Dokmaia monthadangii gen. et sp. nov., a synnematous anamorphic

fungus on Manglietia garrettii. Sydowia 51: 99–103.


K.D. (2002). Fungal succession on senescent leaves of Manglietia garrettii in

Doi Suthep-Pui National Park, northern Thailand. Fungal Diversity 10: 89–

100.

295



Presentations in international conference

1. Promputtha, I., Hyde, K.D., Peberdy, J.F. and Lumyong, S. (2006).

Enzymatic activity of endophytic fungi on leaf decomposition. 8th

International Mycological Congress, 21-25 August 2006, Cairns Convention

Centre, Queensland, Australia (Poster presentation).

2. Promputtha, I., Jeewon, R., Hyde, K.D., Vijaykrishna. D., McKenzie, E.H.C.

and Lumyong, S. (2006). Do fungal endophytes of Magnolia liliifera become

saprobes at host senescence? RGJ-Ph.D. Congress VII, 20–22 April 2006,

Jontein Palmbeach Resort, Pattaya, Chonburi, Thailand (Oral presentation).

3. Promputtha, I., Lumyong, P., Hyde, K.D., Peberdy, J.F. and Lumyong, S.

(2005). The production pattern of carbohydrases during the decomposition of

Magnolia liliifera leaves. British Mycological Society Annual Scientific

Meeting, 5–8 September 2005, Hulme Hall, University of Manchester,

England (Oral presentation).


K.D. (2004). Diversity and succession of fungi on senescent leaves of

Meliosma simplicifolia (Sabiaceae). The IV Asia-Pacific Mycological

Congress and The IX International Marine and Freshwater Mycology

Symposium, 14–19 November 2004, Chiang Mai, Thailand (Poster

presentation).


K.D. (2002). Fungal succession on senescent leaves of Manglietia garrettii in

Doi Suthep-Pui National Park, northern Thailand. 3rd Asia-Pacific

Mycological Conference on Biodiversity and Biotechnology (AMC2002), 4–8

296



November 2002, Yunnan University, Kunming, China (Oral presentation).


K.D. (2002). Fungal saprobes on dead leaves of Manglietia garrettii in

Thailand. The 14th Annual Meeting of the Thai Society for Biotechnology

“BIOTECHNOLOGY FOR BETTER LIVING IN THE NEW ECONOMY”,

12–15 November 2002, Khonkaen University, Khonkaen, Thailand (Poster

presentation).


K.D. (2001). Saprobic Fungi on Magnolia garrettii. BioThailand 2001: From

Research to Market, 7–10 November 2001, Queen Sirikit National Convention

Center, Bangkok, Thailand (Poster presentation).

Workshops in field specialization

1. Workshop on “Unculturable Microbes: Molecular Techniques and

Biotechnology Application”. At National Center for Genetic Engineering and

Biotechnology (BIOTEC), National Science and Technology Development

Agency (NSTDA), and Ministry of Science and Technology (MOST). At

BIOTEC auditorium room, BIOTEC Building, Thailand Science Park,

Pathumthani, Thailand. 9–10 January 2006.

2. Workshop on “GBIF and EASIANET Proposed Collection/Names/Images

Digitization”. At Centre for Research in Fungal Diversity, Department of

Ecology and Biodiversity, The University of Hong Kong, Hong Kong SAR,

China. 14–19 March 2005.

297



3. Workshop on “Molecular Phylogenetics”. At Department of Ecology and

Biodiversity, The University of Hong Kong, Hong Kong SAR, China. 17–22

March 2004.

4. Workshop on “Denaturing Gradient Gel Electrophoresis”. At Department of

Biology, Faculty of Science, Chiang Mai University, Chiang Mai, Thailand.

4–6 May 2004.

5. Workshop on “Microbial Production of Surfactants and Their Applications”.

At Department of Biology, Faculty of Science, Chiang Mai University, Chiang

Mai, Thailand. 10–14 May 2004.

6. Workshop on “Mycology Taxonomy, Molecular Systematics and Using Key

Isolation and Preservation of Fungi”. At the Mushroom Research Centre,

Chiang Mai, Thailand. 7–27 July 2003

7. Workshop on “Fungal Diversity of Thailand: Towards a Checklist of Thai

Fungi, Fungi and Their Biotechnological Application”. At National Science

and Technology Development Agency (NSTDA), Bangkok, Thailand. 15–16

November 2001.

298



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