ISOLATION AND TESTING

FOR THE DISEASE-REDUCING EFFECTS

AGAINST RICE BACTERIAL LEAF BLIGHT
OF SOIL BACTERIA FROM CHỢ GẠO, TIỀN GIANG
Châu Hoàng Trung Tín

Thesis Supervisors
Nguyễn Đắc Khoa, PhD.
Trần Kim Thoa, MSc.
Rice bacterial leaf blight (BB)

Disease symptoms Xanthomonas oryzae pv. oryzae (Xoo)

 Control of BB

IRRI

Chemical control Resistant cultivars

Controlled by antagonistic bacteria

 Objectives
Overall objective: Isolating soil bacteria with the
disease-reducing effects against rice bacterial leaf blight
Specific objectives:
1. Isolating at least 35 isolates from soil samples
2. Selecting at least 8 antagonistic isolates
3. Testing effects of antagonistic isolates on seed germination
and development
4. Testing for disease-reducing effects of at least 1 isolate
under greenhouse conditions
 Thesis outline

Soil samples collection Isolation Test for antagonistic effects

Test for effects on

seed germination and development
Test for disease-reducing effects
 Test for antagonistic effects

Bacterial
isolate

Xoo

Dual-culture test
Culture without H2O
bacterium
Xoo
NB

Well diffusion assays

 Test for effects on
seed germination and development

109 108 107

Culturing bacteria
in 48 hrs

Bacterial suspension
Soaking seeds in 2 hrs

Measuring the shoot

and root lengths of at
3, 5 and 7 DAP
 Test for disease-reducing effects
under greenhouse conditions
Cell density levels (109, 108, 107 CFU/ml)

Soil drenching Seed soaking Foliar spraying

at 7, 14, 21 DBI

Measuring at 5, 10, 15 DAI Inoculating at 45 DAS

 Results and discussion

• Isolation bacteria from soil samples

43 isolates from 4 soil samples

 Dual-culture test

Inhibition Inhibition
No. Isolates No. Isolates
zones (mm) zones (mm)
Inhibition zone
1 CG13 13.60 9 CG29 8.30
CG4
2 CG7 11.00 10 CG39 7.70
3 CG4 10.60 11 CG43 6.80
4 CG21 9.70 12 CG6 5.20
5 CG28 9.70 13 CG40 4.70
6 CG15 9.20 14 CG31 4.50
7 CG12 9.20 15 CG17 4.30
8 CG9 9.10 16 CG34 3.30
 Well diffusion test

Inhibition zones (mm)

No. Isolates
2 days 4 days 6 days 8 days
1 CG6 11.50 16.30 12.50 11.00
2 CG9 06.50 09.00 09.20 10.80
NB CG6
3 CG40 04.40 06.30 08.20 08.80
4 CG43 06.80 06.90 06.10 05.80 CG9 H2 O
5 CG29 04.50 07.50 05.40 05.10
6 CG34 04.80 05.50 05.00 04.90
7 CG4 04.20 10.20 03.80 03.50
8 CG12 06.30 04.50 03.50 02.80
 Selection criteria

1. Cultures with antagonistic effects showed in all culturing time

points
2. Inhibition zones were larger than 9 mm
 Well diffusion test

Inhibition zones (mm)

No. Isolates
2 days 4 days 6 days 8 days
1 CG6 11.50 16.30 12.50 11.00
2 CG9 06.50 09.00 09.20 10.80 NB CG6
3 CG40 04.40 06.30 08.20 08.80
4 CG43 06.80 06.90 06.10 05.80 CG9 H2 O

5 CG29 04.50 07.50 05.40 05.10

6 CG34 04.80 05.50 05.00 04.90
7 CG4 04.20 10.20 03.80 03.50
8 CG12 06.30 04.50 03.50 02.80
 Shoot lengths
Cell density Shoot lengths (mm)
Isolates levels
(CFU/ml) 3 DAP 5 DAP 7 DAP

109 16.57 bc 44.03 b 64.07 bcd

CG6 108 14.80 b 42.93 b 64.57 cd

107 17.63 c 44.16 b 66.63 d

109 15.17 b 41.87 b 63.17 bc

CG9 108 16.33 abc 40.00 b 61.67 ab

107 15.30 abc 44.27 b 65.93 cd

Control 8.67 a 33.07 a 59.93 a

 Root lengths
Cell density Root lengths (mm)
Isolates levels
(CFU/ml) 3 DAP 5 DAP 7 DAP
109 36.70 ab 80.13 a 113.20 a

CG6 108 34.70 ab 78.57 a 115.27 a

107 33.50 ab 70.23 a 102.13 a

109 39.23 b 81.20 a 118.43 a

CG9 108 31.67 ab 61.17 a 93.20 a

107 34.50 ab 70.07 a 111.03 a

Control 26.30 a 60.37 a 099.80 a

 Germination index

Germination index
Isolates
109 108 107
CG2-16 19430.0 b 15670.0 a 17543.3 ab
CG1-9 17723.3 ab 17976.7 ab 16870.0 ab
Control 16336.7 a

did not give any adverse effects on seed germination and

development
CG6 and CG9
promoted the growth of rice shoots

CG6 and CG9 were selected for the greenhouse experiments.

 Lesion lengths - Soil drenching

Cell density Lesion lengths (mm)

Bacterial
levels
Isolates 5 DAI 10 DAI 15 DAI
(CFU/ml)
109 18.47 i-m 156.13 f-I 293.07 efg

CG6 108 2.73 a 107.40 a-d 235.27 a-d

107 10.80 c-f 101.80 ab 218.73 a
109 09.33 cde 131.20 a-f 269.67 a-f

CG9 108 17.67 g-l 143.80 e-I 281.47 d-g

107 3.20 ab 98.40 a 230.87 a-d

Control 18.27 h-m 143.27 e-i 292.47 efg

 Lesion lengths - Seed soaking
Cell density Lesion lengths (mm)
Bacterial Isolates levels
(CFU/ml) 5 DAI 10 DAI 15 DAI
109 21.47 ijk 163.93 d-g 291.33 efg

CG6 108 17.87 d-j 150.73 d-g 298.13 efg

107 15.20 b-h 138.86 a-e 274.20 b-g
109 11.53 a-d 168.33 efg 306.00 fg

CG9 108 16.67 c-h 142.67 c-g 296.27 efg

107 27.00 ka- 175.47 ga- 246.87 a-e

Control 18.27 e-j 143.27 c-g 292.40 efg

 Lesion lengths - Foliar spraying
at 7 days before inoculation
Cell density Lesion lengths (mm)
Isolates levels
(CFU/ml) 5 DAI 10 DAI 15 DAI
109 7.53 a-d 98.87 a 249.53 a-e

CG6 108 14.47 e-k 132.53 e-h 327.60 gc-

107 17.07 f-l 157.93 b-f 275.87 c-g
109 12.40 d-I 130.20 a-e 264.27 a-f

CG9 108 24.20 mn 154.20 e-h 291.73 efg

107 14.47 e-k 134.73 b-f 244.33 a-e

Control 18.27 h-m 143.27 d-h 292.4 efg

 Lesion lengths - Foliar spraying
at 14 days before inoculation
Cell density Lesion lengths (mm)
Isolates levels
(CFU/ml) 5 DAI 10 DAI 15 DAI
109 11.73 c-g 146.00 e-I 279.00 c-g

CG6 108 13.67 c-I 174.06 hi 292.80 efg

107 9.06 a-d 106.33 abc 222.85 ab
109 15.60 d-k 140.47 d-h 226.80 abc

CG9 108 19.60 i-l 159.53 f-I 281.07 d-g

107 19.93 i-l 167.93 ghi 296.27 efg

Control 18.27 g-l 143.27 e-i 292.47 efg

 Lesion lengths - Foliar spraying
at 21 days before inoculation
Cell density Lesion lengths (mm)
Isolates levels
(CFU/ml) 5 DAI 10 DAI 15 DAI
109 14.20 e-j 143.13 e-I 274.73 b-g

CG6 108 05.00 abc 112.26 a-e 262.26 a-f

107 21.40 lmn 149.06 f-I 305.40 fg
109 15.07 e-l 133.67 b-g 293.47 efg

CG9 108 21.00 k-n 158.00 f-I 306.33 fg

107 11.87 d-I 107.20 a-d 288.60 efg

Control 18.27 h-m 143.27 e-i 292.40 efg

 Effective treatments

Application methods 5 DAI 10 DAI 15 DAI

Soil drenching CG6 (107), CG9 (107) CG6 (107), CG9 (107) CG6 (107), CG9 (107)
CG6 (108) CG6 (108) CG6 (108)
CG9 (109)

Seed soaking CG9 (109)

Foliar spraying 7 DBI CG6 (109) CG6 (109)

Foliar spraying 14 DBI CG6 (107) CG6 (107) CG6 (107)

CG9 (109)

Foliar spraying 21 DBI CG6 (108) CG9 (107)

 Selection criteria

1. High disease-reducing effects

2. Durability
3. Low cell density levels
Application methods 5 DAI 10 DAI 15 DAI
Soil drenching CG6 (107), CG9 (107) CG6 (107), CG9 (107) CG6 (107), CG9 (107)
CG6 (108) CG6 (108) CG6 (108)
CG9 (109)

Seed soaking CG9 (109)

Foliar spraying 7 DBI CG6 (109) CG6 (109)

Foliar spraying 14 DBI CG6 (107) CG6 (107) CG6 (107)

CG9 (109)

Foliar spraying 21 DBI CG6 (108) CG9 (107)

 Conclusions

Work packages Specific objectives Results

Bacterial isolation 35 isolates 43 isolates

Test for antagonistic effects 8 isolates 8 isolates

Test for disease-reducing

effects under greenhouse 1 isolate 2 isolates
conditions
 Perspectives

• Identification of two isolates CG6 and CG9

• Test for disease-reducing effects under field conditions
• Isolation of antagonistic bacterial strains from different
locations
Acknowledgement