1. Hengodage Nirmalee Bhagya Wijayalath
Biological Control of Bacterial Wilt Disease in Tomato by Candida ethanolica and Enterobacter cloacae by
Hengodage Nirmalee Bhagya Wijayalath
Agricultural Systems and Engineering Field of Study
Examination Committee:
Dr. S. L. Ranamukhaarachchi (Chairman)
Dr. Oleg V. Shipin
Dr. Roland Cochard

2. Outline Introduction Objectives Methodology Results and Discussions
Summary
Conclusions
Future Recommendations

3. Introduction Bacterial wilt disease (BWD) Management practices
Biological control agents
Factors affecting the efficiency of biological control activities

4. Bacterial Wilt Disease (BWD)
Highly destructive
Can infect crops at any stage of growth
Causes heavy yield losses
Heavy economic losses for Solanaceae crops such as tomato,
chillie peppers, egg plants, tobacco,
Pesticides are very ineffective
Farmers apply high doses of pesticides, but cannot control
Pathogen is a bacteria (Ralstonia solanacearum) , which
has a wide range of hosts over 50 plant families
Pathogen persists in soil for long period
Difficult to control with normal cultural and pesticides

5. BWD
Tomato
Egg Plant
Chillies
Tobacco
Potato

6. Ralstonia solanacearum
R. Solanacearum in TZC medium
R. Solanacearum in SPA medium

7. Current Disease Management Methods
Soil fumigation
Chemical control
Field sanitation
Crop rotation
Soil amendments
Resistant varieties
Clean seeds and seedlings,
Modified cultural practices
Yet, control is unsuccessful.

8. Biological control agents
suppresses and control the growth and development
insect pests
pathogenic microorganisms
of crops
set of beneficial microorganisms
Bio control mechanisms
- antagonism
- parasitism
- antifungal compounds
- lytic enzymes
- competition for ferric iron,
- nutrients and colonization sites,
- systemic resistance in plants
- etc…
Biological control agents
- Pseudomonas fluorescens
- Tricoderma species
- Bacillus species
-Actinomycetes species
- Rhizobacteria species
- Candida ethanolica
- Enterobacter cloacae

9. Candida ethanolica
Enterobacter cloacae

10. sucrose as a carbon and energy mixed stands of bio control agents
Factors affecting the efficiency and stability of biological control activities
physical requirements
nutritional requirements
populations of other microorganisms
application method
time of application
combination of different bio control agents
sucrose as a carbon and energy
source
media pH
mixed stands of bio control agents

11. Objectives Overall objective
Determine the effect of sucrose as a supplementary carbon source and soil pH on the bio control ability of C. ethanolica , E. cloacae and their combination against R. solanacearum

12. to suppression of R. solanacearum
Specific objectives
In in vitro conditions
To identify the suitable levels of sucrose and media pH for optimum performances of
- C. ethanolica
- E. cloacae
- in combination of two antagonists
to suppression of R. solanacearum
In in vivo conditions
To determine the antagonistic ability of two antagonists and their combination under carbohydrate supplementation and optimum soil pH
to control BWD in tomato

13. Materials and Methods
Study location and duration
- Lab experiments in Agricultural Systems Laboratory- September to November in 2009
- Field experiments in the greenhouse of the Research Farm from November, 2009 to March in 2010

14. The overall study was comprised of the following steps:
In vitro multiplication of C. ethanolica , E. cloacae and R. solanacearum in SPA and SPB
In vitro evaluation of two antagonists alone and in combination at eleven concentrations of sucrose (1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6 (g/l)
In vitro evaluation of two antagonists alone and in combination under different pH (4,5,6,7,8)
In vivo evaluation of two antagonists individually and in combinations under optimum concentrations of sucrose and soil pH
against R. solanacearum

15. In vitro sucrose study
eleven sucrose concentrations 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6 (w/v)
were used to prepare the SPB medium
25 ml of each SPB added to test tubes
add one loop of antagonist alone and in combination
shacked hours at room temperature
centrifuged at 6000 rpm for 15 minutes
supernatant was added to perforated agar plates (SPA medium) contaminated with pathogen
incubated at C for 24 – 48 hours
mean distance of inhibition zone, were measured

16. In vitro pH study
media pH of modified SPB adjusted around 4,5,6,7, and 8
25 ml of each modified SPB added to test tubes
add one loop of antagonist alone and in combination
shacked hours at room temperature
centrifuged at 6000 rpm for 15 minutes
supernatant was added to perforated agar plates (SPA medium) contaminated with pathogen
incubated at C for 24 – 48 hours
mean distance of inhibition zone, were measured

17. Factorial experiment :
In vivo study
Factorial experiment :
Modified soil (5% sucrose /weight added and soil pH adjusted to )
A) Soil type
Normal soil (no sucrose added, no soil pH adjusted)
C. ethanolica
Control 1
with pathogen and without antagonists
B) Antagonist
E. cloacae
C. ethanolica + E. cloacae
Seed treatment
C) Antagonist inoculation method
Control 2
without both pathogen and antagonists
Drenching method
Root dipping method

18. Preparation of pathogen infected soil pots
Pots were filled with modified soil and normal soil
Initial microbial suspension of R. solanacearum in SPB (concentration of 109 CFU/ml)
15 ml of suspension was added to the soil /pot in three different times
20 days old tomato seedlings were transplanted
pots having BWD symptoms and dead plants were collected as contaminated pots
contaminated pots were used to setup the factorial experiment

19. Inoculation of tomato seeds and seedlings
Antagonist suspensions
-two antagonists
-in combination
(in SPB with 5% sucrose and pH)
Treated seeds
(by dipping for 12 hours)
Non treated seeds
Nursery management
(in seedling trays)
Modified soil Normal soil
(5% sucrose , pH)
Normal seedlings
Drenching method
Seed treatment
Root dipping method
Control

20. Crop establishment and management
Transplanting of tomato seedlings
Complete randomised design
Control pots
Drip irrigation
Fertilizing
Staking

21. Observations and collection of data
Disease severity , according to 5 point visual scale,
1 – no wilting; 2 – 1-25% wilting; 3 – 25-50% wilting;
4 – 50-75% wilting; 5 – % wilting
Plant height - cm
Tomato yield – g/plant
Dry weight ( dry biomass) – g/plant
Data analysis
Analysis of Variance (AOV) using SAS
Mean comparisons by Fisher’s protected least significant difference procedure and orthogonal contrast
Disease severity by ranking

22. Results and discussion
In vitro sucrose study
C. ethanolica + E. cloacae
E. cloacae

23. distance of inhibition zone between antagonist and pathogen as influenced by sucrose concentrations

24. In vitro pH study

25. distance of inhibition zone of three antagonist treatments at varying pH

26. Disease severity of tomato plants Severity according antagonist
In vivo study
Disease severity of tomato plants
Disease severity
1 – no wilting
2 – 1-25% wilting
3 – 25-50% wilting
4 – 50-75% wilting
5 – %
wilting
Severity according antagonist

27. Severity according inoculation method
Severity according to soil type
Severity according inoculation method

28. Plant height
Plant height according to soil type

29. Plant height according to antagonist
Plant height according to inoculation method

30. Tomato yield interaction between soil type and inoculation method
interaction between antagonists and inoculation method

31. Plant dry weight interaction between soil type and inoculation method
interaction between antagonists and inoculation method

32. Orthogonal comparison applied for plant height
Mean square
Mean plant height, cm
First comparison
Second comparison
Control 1 vs. other treatments 1
***
41.75
77.53
Control 1 vs. seed treatment
***
87.75
Control 1 vs. drenching method
***
96.23
Control 1 vs. root dip method ns
48.60
Control 1 vs. C. ethanolica
***
86.13
Control 1 vs. E. cloacae **
69.56
Control 1vs. in combination
***
76.90
Control 2 vs. other treatments *
94.00
Control 2 vs. seed treatment ns
Control 2 vs. drenching method
17.037ns
Control 2 vs. root dip method
***
Control 2 vs. C. ethanolica ns
Control 2 vs. E. cloacae **
Control 2vs. in combination *

33. Orthogonal comparison applied for yield
Mean square
Mean yield, g/plant
First comparison
Second comparison
Control 1 vs. other treatments 1
***
19.47
Control 1 vs. seed treatment
***
136.67
Control 1 vs. drenching method
***
170.28
Control 1 vs. root dip method ns
23.15
Control 1 vs. C. ethanolica
***
130.98
Control 1 vs. E. cloacae
***
80.35
Control 1vs. in combination
***
118.78
Control 2 vs. other treatments
***
174.47
Control 2 vs. seed treatment **
Control 2 vs. drenching method ns
Control 2 vs. root dip method
***
Control 2 vs. C. ethanolica **
Control 2 vs. E. cloacae
***
Control 2vs. in combination
***

34. Orthogonal comparison applied for plant dry weight
Mean square
Mean dry matter, g/plant
First comparison
Second comparison
Control 1 vs. other treatments 1
***
9.01
18.57
Control 1 vs. seed treatment
***
19.37
Control 1 vs. drenching method
***
25.75
Control 1 vs. root dip method ns
10.60
Control 1 vs. C. ethanolica
***
22.69
Control 1 vs. E. cloacae
***
15.13
Control 1vs. in combination
***
17.90
Control 2 vs. other treatments
***
33.54
Control 2 vs. seed treatment
61.589**
Control 2 vs. drenching method ns
Control 2 vs. root dip method
***
Control 2 vs. C. ethanolica
***
Control 2 vs. E. cloacae
***
Control 2vs. in combination
***

35. Conclusions In in vitro study
1) C. ethanolica, E. cloacae and combination of antagonists performed well at optimum sucrose concentrations of 5%, 4% and 5.5% (g/l) respectively against R. solanacearum
2) C. ethanolica, E. cloacae and their combination performed well in the pH range of 5-7 .

36. In in vivo study Conclusions…
1) All antagonist could suppress the R. solanacearum and the disease better when treated with sucrose concentration of 5% (g/l), and pH range of than in normal soil conditions
2) C. ethanolica with seeds treatment and drenching methods are more suitable to suppress the BWD and to enhance vegetative growth and yield of tomato in modified soil.

37. Future recommendations
evaluate the potential of antagonistic ability for field establishment, and efficient utilization of both antagonists and nutrient resources under varying environment conditions and cultural practices
develop a stable mixture of energy sources that can remain in soil for a considerable period and provide nourishment for the antagonist.
conduct further research with respect to bio control mechanisms of disease suppression by antagonists.
explore the findings of antagonists C. ethanolica , E. cloacae and their combination to suppression of R. solanacearum in other solanaceae crops such as potato, tobacco ,eggplant, etc….

38. Thank you