APPENDIX A Preparation of fungal media Potato Dextrose

APPENDIX A

Preparation of fungal media

Potato Dextrose Agar (PDA) per liter

Potato 200 g

Glucose 20 g

Agar 15 g

Distill water 1000 ml

Adjust pH 7

Media Preparation Potato Dextrose Agar

To prepare Potato Dextrose Agar (PDA) media:

1) Take 200 g of potatoes and cut small pieces after peeling. Boil the pieces

of potato in 500 ml water. After the potatoes are soft filter with miracloth

(any strainer, or cloth). Remove the pieces and take the nutrient broth. Add

20 g of dextrose (sugar) and 15 g of Agar.

2) Boil and make up the volume 1000 ml by distilled water.

3) Stir using magnetic plate.

4) Autoclave for 25 minutes.

5) Under sterilized laminar flow hood, let the media cool down then pour

into sterilized Petri dishes. Let the plates solidify.

6) Store plates in refrigerator.

87

APPENDIX B

Chitosan stock solution

1. Preparation of stock chitosan solution

Each chitosan concentration solutions tested in this study was prepared by

diluting 10 g/l chitosan in 1 M acetic acid stock solution and adjusting the pH

to 6.25 with 2N NaOH.

Appendix table 1 Chitosan each concentration ratio for working volume 300 ml

Treatments Chitosan

concentration (g/l) 10 g/l Chitosan solution (ml)

Distilled

water (ml)

0

1

2

3

4

5

6

7

8

Control

0.5

1.0

1.5

2.0

4.0

6.0

8.0

10.0

0

15

30

45

60

120

180

240

300

0

285

270

255

240

180

120

60

0

88

2. Preparation of PDA media with chitosan To prepare media of 250 ml of each chitosan concentration in PDA, stock

chitosan at 20 g/l was mixed with potato water as shown in Appendix table 2

Appendix table 2 Ratio of PDA media and chitosan solution

Chitosan

concentration (g/l)

Potato water

(ml)

Stock 20 g/l Chitosan

solution (ml)

0.5 185 66.25

2 175 75

6 150 100

10 125 125

1. Mixing between stock solution 1 and stock solution 2 at chitosan

concentration 0.5 g/l along with Appendix table 2 total volume 250 ml.

2. Another concentration was mixing with Appendix table 2 above.

89

APPENDIX C

Buffer preparation

1. Sodium acetate Buffer

Stock solutions

A: 0.2 M solution of acetic acid (11.55 ml in 1000 ml)

B: 0.2 M solution of sodium acetate (16.4 g of C2H3O2Na or 27.2 ml

of C2H3O2Na 3 •H2O in 1000 ml)

X ml of A + Y ml of B, diluted to a total of 100 ml:

X Y pH

46.3 3.7 3.6

44.0 6.0 3.8

41.0 9.0 4.0

36.8 13.2 4.2

30.5 19.5 4.4

25.5 24.5 4.6

14.8 35.2 5.0

10.5 39.5 5.2

8.8 41.2 5.4

4.8 45.2 5.6

2. Borate buffer

A: Sodium borate 3.8140 g in H2O 1000 ml.

B: Boric acid 0.6183 g in H2O 1000 ml.

pH 8.2-10.2 at 10 mM.

Adjust pH by NaOH 1N

90

APPENDIX D

Enzyme activity determination

1. β - 1, 3 - glucosanase activity

Enzyme activity was assayed by the laminarin-dinitrosalicylic acid method

(Saikia et al., 2005). The reaction mixture consisted of 62.5 μl of 4% (w/v) laminarin

and 62.5 μl of enzyme solution. Prepare the reaction mixture for measure enzyme

activity as in Appendix table 3.

Appendix table 3 Reaction mixture for determination of enzyme activity

Reaction Reaction mixture

Enzyme solution (μl) Substrate (μl) Acetate buffer (μl)

- - 125

62.5 62.5 -

62.5 - 62.5

Control

Enzyme Substrate (ES)

Enzyme Control (EC)

Substrate Control (SC) - 62.5 62.5

Reducing sugar quantity (mg/ml) in reaction can be calculated from net

absorbance (∆E) by calibration to the standard curve of glucose:

Net reducing sugar (∆E) = OD (ES-EC-SC)

Example for β-1, 3-glucosanase activity calculation;

Net reducing sugar = X mg

MW of glucose = 180 g = 1 mole

Quantity = X × 103/180 μ mole

Incubated reaction mixture = 10 min

1 min will resulted reducing sugar = X × 103 μ mole/min

180 x 10

Using enzyme volume = 0.0625 ml

91

So enzyme activity = X × 103 Unit/ml

180x10x0.0625

“One unit of enzyme activity is defined as the amount of enzyme, which

produced one μmol of reducing sugar per minute under standard assay condition”.

Appendix table 4 Absorbance at 500 nm of various concentration of glucose

Glucose (mg) Absorbance (500 nm)

0 0

200 0.0576

400 0.1610

600 0.2673

800 0.3553

1000 0.4413

y = 0.0004xR2 = 0.9922

00.050.1

0.150.2

0.250.3

0.350.4

0.450.5

0 200 400 600 800 1000 1200

glucose (ug/ml)

Abs

orba

nce

500

nm

Appendix figure 1 Calibration curve of glucose concentration versus absorbance at

500 nm

92

2. Chitosanase activity

Enzyme activity was assayed by estimating the amount of the reducing end of

sugar using the dinitrosalicylic acid (DNS) method (Miller, 1959).The reaction

mixture consisted of 0.3 ml of 0.4% (w/v) chitosan and 0.3 ml of enzyme solution.

Prepare the reaction mixture for measure enzyme activity as in Appendix table 5.


Reaction

Reaction mixture

Enzyme solution (ml) Substrate (ml) Acetate buffer (ml)

- - 0.6

0.3 0.3 -

0.3 - 0.3

Control

Enzyme Substrate (ES)

Enzyme Control (EC)

Substrate Control (SC) - 0.3 0.3

Reducing sugar quantity (mg/ml) in reaction can be calculated from net

absorbance (∆E) by calibration to the standard curve of N-acetylglucosamine:

Net reducing sugar (∆E) = OD (ES-EC-SC)

Example for chitosanase activity calculation;

Net reducing sugar = X mg

MW of N-acetylglucosamine = 221.21 g = 1 mole

Quantity = 221.21 μ mole


1 min will resulted reducing sugar = X μ mole/min

221.21x30


93

So enzyme activity = X Unit/ml

221.21x30x0.3

“One unit of enzyme activity is defined as the amount of enzyme, which

produced one μmol of reducing sugar per minute under standard assay condition”.

Appendix table 6 Absorbance at 540 nm of various concentration of

N-acetylglucosamine

N-acetlyglucosamine Absorbance (540 nm)

0 0

0.02 0.009

0.04 0.025

0.06 0.041

0.08 0.051

0.1 0.067

0.12 0.084

y = 0.6767xR2 = 0.9931

0

0.01

0.02

0.03

0.04

0.05

0.06

0.07

0.08

0.09

0 0.02 0.04 0.06 0.08 0.1 0.12 0.14

concentration (mg/ml)

OD

at 5

40 n

m

Appendix figure 2 Calibration curve of N-acetylglucosamine concentration versus

absorbance at 540 nm

94

3. Phenylalanine ammonia-lyase (PAL) activity

Enzyme activity was assayed following the method of Saunders and McClure

(1975). The reaction mixture consisted of 0.5 ml of 60 mM L-Phe and 0.5 ml of

enzyme solution. Prepare the reaction mixture for measure enzyme activity as in

Appendix table 7.


Reaction

Reaction mixture

Enzyme solution (ml) Substrate (ml) Acetate buffer (ml)

- - 1

0.5 0.5 -

0.5 - 0.5

Control

Enzyme Subatrate (ES)

Enzyme Control (EC)

Subatrate Control (SC) - 0.5 0.5

In reaction can be calculated from net absorbance (∆E) by calibration to the standard

curve of cinnamic acid:

Net absorbance (∆E) = OD (ES-EC-SC)

Example for PAL activity calculation;

Net absorbance = X mg

MW of cinnamic acid = 148.17 g = 1 mole

Quantity = X × 103 μ mole

148.17


1 min will resulted reducing sugar = X × 103 μ mole/min

95

148.17/60


So enzyme activity = X × 103 mUnit/ml

148.17x60x0.5

“One unit of enzyme activity is defined as the amount of enzyme, which produced one

μmol of reducing sugar per minute under standard assay condition”.

Appendix table 8 Absorbance at 280 nm of various concentration of cinnamic acid

Cinnamic acid (μmole/l) Absorbance (500nm)

0 0 25 0.6102 50 1.1256 75 1.614 100 2.2016 125 2.5146

150 3.0012

Appendix figure 3 Calibration curve of cinnamic acid concentration versus absorbance at 280 nm

APPENDIX E Raw data

Appendix table 9 Seed germination rate of rice cv. Luang San Pah Tawng at 10 days.

Seed germination rate (%)1±SD2 Time (day) Control 0.5 g/l 1.0g/l 1.5g/l 2g/l 4g/l 6g/l 8g/l 10g/l 2 Days 11.33±2.3a 13.0 ±3.5a 15±3.46c 13.0 ± 2.3a 15.66 ±3.7ab 17.33±3.0abc 22.0±4.0c 21± 4.3bc 16.66 ±1.1abc 3 Days 18 ± 0a 25.33±3.5ab 19.66±3.2ab 24 ± 8ab 27±3.0ab 28 ± 2.6b 24.66±1.5ab 21.66±7.5ab 18 ± 1.0a 4 Days 67 ± 1.1bc 68.66±6.1bc 53±7.3a 62.33±6.0ab 69.66±6.1bc 66 ±4.3abc 60 ± 4.3ab 79±0.5c 60 ±2.5ab 5 Days 79.33±6.6a 70.66±4.9ab 59±7.5a 77.33 ± 1.5b 79.33 ±4.0b 76 ±9.1b 78.66±6.5b 83.6±66b 79.66 ± 3.4b 6 Days 87.66±1.1c 75.66 ±1.5b 59.66 ± 6.0a 81.66±4.0bc 80± 4bc 85 ± 2.0bc 85.66±1.5bc 89.33 ± 2c 84.33 ±3.0bc 7 Days 88.6 ± 0.5c 77.33 ± 1.7b 62.33 ± 6.4a 82±2.6bc 82.33 ±2.5bc 85.33±2.0bc 87.33±3.0bc 89.33±1.5c 87 ± 1bc 8 Days 89.33±0.5bc 79.33 ± 2.6b 63±9.0a 82.3± 3.6bc 82.6 ±5.0d 89.33±4.5bc 89.33±5.5bc 90.66±1.5c 85.33 ±1.1bc 9 Days 89.66±1.1bcd 82.66 ±2.0b 75.66±9.8a 87.3 ± 2.6bc 83.66 ±3.5bcd 91.66±5.8cd 94± 2.0d 96.33±0.5d 88.33 ±1.1bcd 10 Days 89.66±1.1bcd 82.66 ±2.0b 75.66±10.2a 87.3bc±4.3c 83.66±3.5bcd 91.66±5.8cd 94± 2.0d 96.33±0.5d 88.33 ±1.1bcd

1 The experiment in this study was carried out in three replication of each 100 seeds. 2 Standards error a, b, c, d within a row are significantly different, (P<0.05)

97

Appendix table 10 Stem height of rice cv. Luang San Pah Tawng at 10 days. Stem height (cm)1±SD2 Time (day) Control 0.5 g/l 1.0g/l 1.5g/l 2g/l 4g/l 6g/l 8g/l 10g/l 2 Days 1.32±0.4ab 1.31±0.3ab 2.18 ± 0.8c 0.86 ±0.4a 0.86 ± 0.2a 1.57±0.5abc 1.55±0.3abc 1.86±0.6bc 1.67±0.3abc 3 Days 1.55±0.8ab 1.52±0.2ab 2.47±1.2b 0.88± 0.1a 1.14 ±0.1a 2.5 ± 0.6b 1.64± 0.8ab 1.96±0.5ab 1.87±0.4ab 4 Days 1.92±0.2a 2.10±0.1ab 2.90±0.8bc 1.96±0.05ab 1.88± 0.0a 2.55 ± 0.1bc 1.89±0.37a 2.04±0.2ab 2.±0.2ab 5 Days 3.69±0.3ab 4.59±0.7bc 4.67 ± 0.6c 3.77± .2abc 3.87±0.08abc 3.26 ±0.2a 3.70± 0.7ab 4.46±0.3bc 3.16±0.9a 6 Days 5.24±0.2a 5.78±0.3abc 6.51 ± 0.7c 5.36 ± 0.4a 6.22 ± 0.6bc 5.04 ±0.0a 5.80±0.77abc 5.41±0.4ab 5.14±0.3a 7 Days 6.82±0.5a 7.76±0.3ab 8.89±1.2b 7.33 ± 0.3a 8 ± 0.1ab 7.21±0.6a 6.97 ± 0.8a 7.38 ± 0.3a 7.49±0.5a 8 Days 8.28±0.2a 9.12±0.3abc 14.29±0.5d 9.70±0.5bcd 10.13±0.3cd 9.51±0.4bcd 9.02 ± 1.0ab 9.81±0.1bcd 8.79 ±0.4ab 9 Days 11.30±0.5a 12.56±0.4abc 14.95±0.9e 13.5±0.5cde 13.68±0.1de 12.02±0.3ab 12.34±0.8abc 13.05±0.5bcde 11.97±0.4ab 10 Days 14.48±0.3a 14.91±0.4abc 16.19±0.8c 15.95±0.8bc 16.24±0.4c 14.75±0.3ab 15.25±1.4abc 16.17±0.9c 15.47±0.4abc

1 The experiment in this study was carried out in three replication of each 100 seeds. 2 Standards error a, b, c, d, e within a row are significantly different, (P<0.05)

98

Appendix figure 4 Physiology of root length of rice cultivar Luang San Pah Tawng at 5 days

(A) (B)

(C) (D)

(E)

Appendix figure 5 Physiological of rice seedling cv. Luang San Pah Tawng at

chitosan concentration 10g/l (A), 8 g/l (B), 6 g/l (C), 4 g/l (D), control (E) at 20 days.

100

APPENDIX F

Statistical analysis

Appendix table 11 The data β - 1, 3 - glucanase enzyme activities during 7 days 1 Day

Chitosan concentration (g/l) Rep.1 Rep.2 Control 1.1022 0.3556

0.5 0.2311 0.8000 2 2.0267 1.7067 6 1.5111 1.1200 10 1.1733 2.0800

3 Days


0.5 1.1377 2.2222 2 4.3377 4.8888 6 2.773 3.5022 10 9.28 9.1022

5 Days


0.5 2.3467 3.0222 2 2.0444 1.3511 6 3.2000 2.0088 10 2.5688 4.6570

7 Days


0.5 3.4488 4.1066 2 1.1733 3.0755 6 1.7244 3.6622 10 1.7955 3.6800

101

Appendix table 12 The results from analysis β - 1,3 - glucanase on 1st day SS df MS Sig. Enzyme activity 3.988 2 1.61E-05 0.006 Error 0.979 8 0.122 Total 26.955 15

R Squared=0.833 (Adjusted R squared=0.655) CV%=32.23% Appendix table 13 The results from analysis β - 1,3 - glucanase on 3rd day SS df MS Sig. Enzyme activity 127.343 4 3.18E+01 0.000 Error 0.478 8 5.98E-02 Total 359.285 15

R Squared=0.996 (Adjusted R squared=0.933) CV%=31.40% Appendix table 14 The results from analysis β - 1,3 - glucanase on 5th day SS df MS Sig. Enzyme activity 6.316 4 1.58E+00 0.050 Error 3.291 8 4.11E-01 Total 106 15

R Squared=0.779 (Adjusted R squared=0.613) CV%=26.08% Appendix table 15 The results from analysis β - 1,3 - glucanase on 7th day SS df MS Sig.

Enzyme activity 4.291 4 1.07E+00 0.490 Error 2.223 8 2.78E-01 Total 128.855 15

R Squared=0.779 (Adjusted R squared=0.613) CV%=34.16%

102

Appendix table 16 The data chitosanase enzyme activities during 7 days 1 Day

Chitosan concentration (g/l) Rep.1 Rep.2 Control 0 0

0.5 0.0029 0.0155 2 0.063 0.0103 6 0.0207 0.05121 10 0.0556 0.0668

3 Days


0.5 0.1439 0.0467 2 0.2672 0.0987 6 0.1603 0.0051 10 0.0846 0.0742

5 Days


0.5 0.0378 0.0029 2 0.0482 0.0267 6 0.1120 0.0400 10 0.0014 0.0879

7 Days


0.5 0.0348 0.0155 2 0.0519 0.0356 6 0.0467 0.1291 10 0.0118 0.0170

103

Appendix table 17 The results from analysis chitosanase on 1st day SS df MS Sig. Enzyme activity 7.13E-03 4 1.18E-03 0.009 Error 2.00E-03 8 2.50E-04 Total 2.14E-02 15

R Squared=0.862 (Adjusted R squared=0.759) CV%=31.14% Appendix table 18 The results from analysis chitosanase on 3rd day SS df MS Sig. Enzyme activity 127.343 4 3.19E+01 0.000 Error 0.478 8 5.98E+01 Total 359.285 15

R Squared=0.996 (Adjusted R squared=0.993) CV%=33.32% Appendix table 19 The results from analysis chitosanase on 5th day SS df MS Sig. Enzyme activity 6.316 4 1.58E+00 0.050 Error 3.291 8 4.11E-01 Total 106 15

R Squared=0.660 (Adjusted R squared=0.405) CV%=36.13% Appendix table 20 The results from analysis chitosanase on 7th day SS df MS Sig. Enzyme activity 9.48E-03 4 2.37E-03 0.021 Error 3.49E-03 8 4.37E-04 Total 4.18E-02 15


104

Appendix table 21 The data PAL enzyme activities during 7 days 1 Day


0.5 0.3478 0.306 2 0.5503 0.4993 6 0.0185 0.0034 10 0.0865 0.1623

3 Days


0.5 0.5194 0.2767 2 0.9909 0.8239 6 0.0834 0.0448 10 0.2411 0.3555

5 Days


0.5 0.4776 0.4792 2 0.6245 0.5457 6 0.3972 0.405 10 0.221 0.388

7 Days


0.5 0.3308 0.354 2 0.4034 0.5364 6 0.66 0.6848 10 1.331 1.0929

105

Appendix table 22 The results from analysis PAL on 1st day SS df MS Sig. Enzyme activity 0.477 4 1.19E-01 0.000 Error 5.11E-03 8 6.38E-04 Total 1.286 15

R Squared=0.989 (Adjusted R squared=0.981) CV%=26.34% Appendix table 23 The results from analysis PAL on 3rd day SS df MS Sig. Enzyme activity 1.139 4 2.85E-01 0.000 Error 3.75E-02 8 4.69E-03 Total 3.753 15

R Squared=0.969 (Adjusted R squared=0.945) CV%=24.43% Appendix table 24 The results from analysis PAL on 5th day SS df MS Sig. Enzyme activity 0.128 4 3.20E-02 0.001 Error 1.80E-02 8 2.25E-03 Total 3.127 15

R Squared=0.877 (Adjusted R squared=0.785) CV%=29.87% Appendix table 25 The results from analysis PAL on 7th day SS df MS Sig. Enzyme activity 1.937 4 4.84E-01 0.000 Error 4.03E-02 8 5.03E-03 Total 6.902 15

R Squared=0.980 (Adjusted R squared=0.964) CV%=21.04% Appendix table 26 The data effect chitosan on mycelia growth

106

Isolation Chitosan concentration 0.5 g/l Rep.1 Rep.2 Rep.3

KLSPT 1 41.11 55.55 44.44 KLSPT 2 16.67 20 20 KLSPT 3 7.14 0 28.57 KLSPT 4 24.53 20 10 KLSPT 5 16.67 13.98 16.48

Isolation Chitosan concentration 2 g/l Rep.1 Rep.2 Rep.3

KLSPT 1 86.67 83.33 83.33 KLSPT 2 14 18.75 11.43 KLSPT 3 21.42 21.42 21.42 KLSPT 4 16.98 27.27 16 KLSPT 5 31.25 24.73 29.67


KLSPT 1 78.89 78.89 78.89 KLSPT 2 44.44 48 40 KLSPT 3 21.46 14.29 35.71 KLSPT 4 18.86 18.18 20 KLSPT 5 58.33 41.93 40.65


KLSPT 1 100 100 100 KLSPT 2 61.11 54 60 KLSPT 3 28.57 35.71 14.29 KLSPT 4 50.94 47.27 50 KLSPT 5 54.16 50.33 51.64

107

Appendix table 27 The result from analysis effect of chitosan on mycelia growth of unknown 1

SS df MS Sig. Mycelia growth 4453.728 3 1.48E+03 0.000 Error 1.01E+02 6 1.69E+01 Total 76821.11 12

R Squared=0.978 (Adjusted R squared=0.959) CV%=32.25% Appendix table 28 The result from analysis effect of chitosan on mycelia growth of

A.niger SS df MS Sig. Mycelia growth 3889.862 3 1.30E+03 0.000 Error 8.55E+01 6 1.43E+01 Total 17885.44 12


Aspergillus sp. SS df MS Sig. Mycelia growth 353.109 3 1.18E+02 0.505 Error 8.07E+02 6 1.35E+02 Total 6479.267 12


A.flavus SS df MS Sig. Mycelia growth 2072.85 3 6.91E+02 0.001 Error 1.54E+02 6 2.57E+01 Total 10805.24 12


unknown 2 SS df MS Sig.

108

Mycelia growth 2534.352 3 8.45E+02 0.000 Error 1.13E+02 6 1.88E+01 Total 18159.4 12

R Squared=0.959 (Adjusted R squared=0.925) CV%=31.11% Appendix table 32 The data effect of chitosan on spore germination A.niger

Chitosan concentration (g/l) Rep.1 Rep.2 Rep.3 Control 100 100 100

0.5 65 78 61 2 25 30 21 6 23 13 16 10 6 10 21

Aspergillus sp.


0.5 55 65 79 2 65 45 53 6 9 10 18 10 8 11 9

A.flavus


0.5 100 100 100 2 35 40 25 6 73 66 71 10 64 59 59

Appendix table 33 The result from analysis effect of chitosan on spore germination

of A.niger

109

SS df MS Sig.

spore germination 16835.067 4 4.21E+03 0.000

Error 2.33E+02 8 2.91E+01

Total 45932.0000 15 R Squared=0.986 (Adjusted R squared=0.976) CV%=35.31% Appendix table 34 The result from analysis effect of chitosan on spore germination

of Aspergillus sp. SS df MS Sig.


Error 4.60E+02 8 5.75E+01

Total 53321 15 R Squared=0.975 (Adjusted R squared=0.977) CV%=33.43% Appendix table 35 The result from analysis effect of chitosan on spore germination

of A.flavus SS df MS Sig.


Error 1.30E+02 8 1.63E+01

Total 89234.000 15 R Squared=0.992 (Adjusted R squared=0.985) CV%=31.22%

Appendix table 36 The data effect fungal on seed germination in Petri dish (in vitro) 7 days Time (Days)

Control A.niger Aspergillus sp. A.flavus

Rep.1 Rep.2 Rep.3 Rep.1 Rep.2 Rep.3 Rep.1 Rep.2 Rep.3 Rep.1 Rep.2 Rep.3

1 day 16 17 18 39 40 42 37 38 40 38 37 36

2 days 20 20 20 45 43 42 40 41 44 39 41 40

3 days 50 50 50 75 76 79 73 72 75 65 66 69

4 days 72 73 75 85 85 85 85 86 88 77 78 80

5 days 85 85 85 92 93 95 95 95 95 82 83 85

6 days 87 88 90 93 96 92 95 95 95 84 86 85

7 days 88 87 90 93 95 92 95 95 95 86 85 84

111

Appendix table 37 The result from analysis effect fungal on seed germination in Petri dish (in vitro) 1st day

SS df MS Sig. seed germination 1062.333 3 3.54E+02 0.000 Error 8.667 6 1.444 Total 14276.000 12

R Squared=0.992 (Adjusted R squared=0.985) CV%=26.88% Appendix table 38 The result from analysis effect fungal on seed germination in Petri

dish (in vitro) 2nd day SS df MS Sig. seed germination 1072.917 3 3.58E+02 0.000 Error 14.833 6 2.472 Total 16857 12


dish (in vitro) 3rd day SS df MS Sig.

seed germination 189.583 3 6.32E+01 0.000 Error 11.167 6 1.861 Total 98303 12


dish (in vitro) 4th day SS df MS Sig. seed germination 330.250 3 1.10E+02 0.000 Error 3.500 6 0.583 Total 78591.000 12

R Squared=0.990 (Adjusted R squared=0.981)

112

CV%=25.15% Appendix table 41 The result from analysis effect fungal on seed germination in Petri

dish (in vitro) 5th day SS df MS Sig. seed germination 195.667 3 6.52E+01 0.001 Error 15.333 6 2.556 Total 98494 12


dish (in vitro) 6th day SS df MS Sig.

seed germination 189.583 3 6.32E+01 0.000

Error 11.167 6 1.861 Total 98303 12


dish (in vitro) 7th day SS df MS Sig. seed germination 189.583 3 6.32E+01 0.000 Error 11.167 6 1.861 Total 98303 12


113

Appendix table 44 The data effect fungal on seed germination in Petri dish (in vivo) 7 days

Time (Days)

Control A.niger Aspergillus sp. A.flavus

Rep.1 Rep.2 Rep.3 Rep.1 Rep.2 Rep.3 Rep.1 Rep.2 Rep.3 Rep.1 Rep.2 Rep.3

1 day 42 43 45 51 51 52 80 79 81 61 60 63

2 days 66 67 69 65 64 64 91 93 96 86 84 83

3 days 72 73 75 74 75 75 91 96 93 91 92 94

4 days 75 76 79 80 78 78 96 91 93 92 91 94

5 days 79 75 76 81 81 82 96 93 91 91 92 94

6 days 79 76 75 81 82 81 93 96 91 92 91 94

7 days 79 75 76 81 81 82 96 93 91 94 92 91

114

Appendix table 45 The result from analysis effect fungal on seed germination in trial

culture (in vivo) 1st days

SS df MS Sig. seed germination 2241.667 3 7.47E+02 0.000 Error 3.333 6 0.556 Total 44258.000 12

R Squared=0.999 (Adjusted R squared=0.997) CV%=29.18% Appendix table 45 The result from analysis effect fungal on seed germination in trial

culture (in vivo) 2nd days SS df MS Sig. seed germination 1656.917 3 5.52E+05 0.000 Error 1.833 6 0.306 Total 73899 12


culture (in vivo) 3rd days SS df MS Sig. seed germination 1034.250 3 3.45E+05 0.000 Error 9.500 6 1.583 Total 84879.000 12


culture (in vivo) 4th days SS df MS Sig. seed germination 711.000 3 2.37E+02 0.000 Error 10.006 6 1.667 Total 87766.000 12


115

Appendix table 45 The result from analysis effect fungal on seed germination in trial

culture (in vivo) 5th days SS df MS Sig. seed germination 608.25 3 2.03E+02 0.000 Error 3.5 6 0.583 Total 89203 12






116

CURRICULUM VITAE

Name Ms. Chularat Chainan

Date of birth 8 December 1982

Education Nawamindrachutid Payap School, 1995 - 2001

Mea Fah Luang University, 2001 - 2004

E-mail [email protected]

Documents

APPENDIX A Preparation of fungal media Potato Dextrose