Management of damping off disease (pythium aphanidermatum) in african nightshade using bacillus subtilis and trichoderma asperellum

1. Horticultural Innovation and Learning for Improved
Nutrition and Livelihood in East Africa
MANAGEMENT OF DAMPING OFF DISEASE (Pythium
aphanidermatum) IN AFRICAN NIGHTSHADE USING Bacillus
subtilis AND Trichoderma asperellum
AG31-2355/2014
Juma Patrick
Supervisors
Prof. Losenge Turoop and Dr. Lucy K. Murungi

2. Outline
 Introduction
 Efficacy of B. subtilis and T. asperellum against damping off disease
 Effect of B. subtilis and T. asperellum on growth and yield of African Nightshade
 Effect of Ultra-Violet radiation and temperature on the shelf life of B. subtilis and T.
asperellum
 Conclusion
 Acknowledgement
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3. INTRODUCTION
• African Nightshade is widely
produced and consumed in various
parts of Kenya.
• African Nightshade is most promising
AIVs and are likely substitute cash
crops.
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• This crop is plagued by a large number of fungal
diseases and insect pests (Mbugua et al., 2008).
• The yield is dismal 3 tones/ha compared to
potential yields of about 30 tones ha-1
11000
16000
21000
26000
31000
2012 2013 2014 2015
Production
(MT)
Years
Source: AFA 2015

4. Alternative crop management practices
• BCA can provide
mitigation
towards diseases
• BCA can also
improve plant
growth and yield
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Manipulation of crops
Pathogenic soil
biota
Crop production
Soil fertility &
health
Biological control of pest and disease
Beneficial soil biota

5. Objectives
Specific objectives
To evaluate efficacy of the Bacillus subtilis and Trichoderma asperellum against damping off disease
(pythium aphanidermatum) in African Nightshade
To determine the effect of the Bacillus subtilis and Trichoderma asperellum on growth and yield of
African Nightshade
To evaluate the effect of Ultra-Violet radiation and temperature on the shelf life of B. subtilis and T.
asperellum
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General objective
To evaluate the biological control of damping off in African Nightshade and shelf life of the
biological control agents as influenced by environmental factors

6. Bacillus subtilis and Trichoderma asperellum against
damping off disease
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OBJECTIVE ONE

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100% seedling loss in vegetables
Damping off disease
Pre-emergence Post-emergence

8. In Kenya, control of damping off diseases is mainly
through prophylactic application of synthetic fungicides.
Over use of synthetic chemical fungicides results to
Environmental Toxicity and development of Resistance
Biological control strategies against damping off disease
can provide an substitute to chemical fungicides.
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Management of damping off disease

9. Materials and Methods
• Biological control agents obtained from Real IPM
Company (K) Ltd.
• African Nightshade seeds var. Olevolosi, Abuku1 and
Abuku2 from World vegetable Centre and JKUAT.
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Bacillus Trichoderma

10. Seed coating
Media
preparation
Preparation of
inoculum
Initiation of
inoculum
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Activity 1: Efficacy of BCA against damping off
Data collection and analysisPlanting and crop management

11. 3/4/2019 juma 11
Layout of the experiment

12. Activity 2:Root colonization experiment
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Harvesting of roots
0.1 grams of roots
Grinding of roots
Addition of 0.1g sand
serial dilution
(1:10000)
Plating on PDA and NA
Inoculation with 0.5 ml
Bac Bac.Tric Trich Con
Roots at 6th
week
serial dilution and plating

13. The damping off disease incidence and root colonization data was subjected to
analysis of variance (ANOVA) using the Generalised Linear Models.
 Means comparison by Tukey at p≤0.05 level significance.
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Data analysis

14. Fig 1: Effect of biological control agents against damping off
BS-B. subtilis TA- T. asperellum C- Control
BCA application significantly
reduced incidence of damping
off disease irrespective of the
African Nightshade variety.
Single and combined
application of BCA
successfully suppressed the
incidence of damping off
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15. Root colonization
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Figure 2: Root colonization of three African Nightshade varieties
BS-B. subtilis TA- T. asperellum C- Control
 The root colonization was
significantly different in the three
African varieties (P ≤0.001)
 Colonization of African Nightshade
roots by either B. subtilis or T.
asperellum in either single or
combined application were
significantly (P = 0.001) different
from the control

16. Conclusion
• B. subtilis and T. asperellum have the ability to manage damping in African
Nightshade as single or combined seed coating.
• Application of B. subtilis and T. asperellum through seed coating resulted in
effective colonization the roots of African nightshades
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17. Effect of Trichoderma asperellum and Bacillus subtilis
on growth and yield of African Nightshade
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Objective two

18. Introduction
African Nightshade
vegetable is one of the
crops that require huge
amount of nutrients during
production.
Some soil borne
microorganisms promote
plant growth.
• BCA promote growth
through enhancing direct
and indirect mechanisms
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19. Materials and Methods
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Seed coating
1g of seeds:2g of BCA
Planting media preparation
3:2 of red soil and sand
Potting in 3kg pots
Planting and management
Watering and fertilizer application
6 weeks after
planting
16 weeks after
planting
Seed
coating
Planting
media

20. Materials and Methods
• Destructive harvesting was carried out twice, at 6th and 16th weeks after sowing and leaf
fresh weight was determined as the crop yield.
• Plant parts were partitioned into below and above-ground parts and oven dried at 70°C
for 72 hours.
• Leaf area was determined using a leaf area meter (LI-COR Li-3000, Lincoln, NE, USA)
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21. Data analysis
The relative growth rate, specific leaf area and leaf area ratio, Leaf weight fraction, Unit leaf rate
were estimated using software developed by Hunt et al. (2002) utilizing two harvesting intervals.
The growth parameters and yield were then subjected to the analysis of variance (ANOVA) using
the Generalised Linear Models and Means comparison by tukey at p≤0.05 level significance
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22. Results
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23. Table 1: Effect BCA on plant growth parameters
Treatment
Relative growth
rate
(g week -1)
Unit leaf rate
(g cm-2 week-1)
Leaf area ratio
(cm2 g1)
Leaf weight
fraction
Specific leaf area
(cm2 g1)
BacAbuku 1 0.32 ± 0.04 (0.08) 0.02 ± 0.01 (0.02) 28.74 ± 12.69 (26.00) 0.48± 0.02 (0.03) 101.86 ± 73.71 (151.03)
BacAbuku 2 0.29 ± 0.06 (0.14) 0.04 ± 0.01 (0.03) 8.65 ± 4.84 (9.91) 0.28 ± 0.12 (0.24) 35.59 ± 23.96 (49.09)
BacOlevolosi 0.31 ± 0.07 (0.14) 0.06 ± 0.05 (0.11) 17.93 ± 12.71 (46.53) 0.45 ± 0.24 (0.49) 68.95 ± 53.19 (149.97)
TrichAbuku 1 0.33 ± 0.06 (0.13) 0.05 ± 0.04 (0.08) 19.75 ± 4.92 (71.56) 0.27 ± 0.19 (0.58) 93.6 1± 61.31 (124.63)
TrichAbuku2 0.34 ± 0.06 (0.11) 0.06 ± 0.02 (0.04) 31.04 ± 3.33 (3.79) 0.13 ± 0.13 (0.33) 202.14 ± 81.71 (67.43)
TrichOlevolosi 0.28 ± 0.06 (0.13) 0.02 ± 0.02 (0.04) 18.45 ± 9.73 (19.93) 0.19 ± 0.12 (0.25) 131.74 ± 68.66 (140.69)
TrichBac Abuku 1 0.29 ± 0.06 (0.13) 0.05 ± 0.02 (0.03) 18.73 ± 4.92 (71.56) 0.47 ± 0.02 (0.04) 66.89 ± 44.50 (214.33)
TrichBac Abuku 2 0.22 ± 0.05 (012) 0.03 ± 0.01 (0.03) 19.04 ± 12.96 (26.36) 0.36 ± 0.06 (0.03) 66.58 ± 57.74 (118.31)
TrichBacOlevolosi 0.27 ± 0.07 (0.15) 0.03 ± 0.01 (0.03) 17.21 ± 11.11 (22.77) 0.29 ± 0.12 (0.27) 92.86 ± 4.52 (11.72)
Con Abuku1 0.16 ± 0.05 (0.10) 0.01 ± 0.01 (0.42) 11.16 ± 5.24 (10.73) 0.21 ± 0.15 (0.21) 45.67 ± 31.37 (64.14)
Con Abuku2 0.23 ± 0.06 (0.13) 0.03 ± 0.01 (0.54) 9.73 ± 4.97 (10.20) 0.15 ± 0.15 (0.01) 66.97 ± 50.84 (104.18)
Con Olevolosi 0.18 ± 0.09 (0.19) 0.01 ± 0.05 (0.10) 9.39 ± 7.32 (5.01) 0.14 ± 0.10 (0.20) 61.69 ± 42.93 (87.97)
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The unit leaf rate (ULR) and Leaf weight fraction (LWF) were significantly different among the
treatments in all the three varieties.

24. Fig 3:Effect of B. subtilis and T. asperellum on African Nightshade
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• Application of the biological control
agents resulted to a significant (P ≤
0.028) increase in yields among the
varieties.
• Furthermore, treatment with biological
control agents showed significant
(P ≤ 0.003) interaction with variety.
African Nightshade yield(g)

25. Conclusion
The application of B. subtilis and T. asperellum as seed coating can efficiently
increase growth and yield of African Nightshade.
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26. Output of objective one and two
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27. 3/4/2019 Juma 27
Effect of Ultra-Violet radiation and temperature on the
shelf life of B. subtilis and T. asperellum
Objective three

28. Introduction
• Food source, temperature, moisture and
time affect microorganism activities.
• Effect of Ultra-Violet (UV) B and A on
BCA not well established.
• This study therefore evaluated the shelf
life of B. subtilis and T. asperellum coated
on the African Nightshades seeds.
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29. Materials and Methods
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30. Activity 1:Effect of UV Irradiation on Shelf life
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Coated seeds Irradiation with UV light at 365
and 302 nm
Serial dilution Incubation and CFU counts

31. Activity 2:Effect of storage temperature on Shelf life
Coating
• Coating of seeds with B. subtilis or T. asperellum at rate of 1 gram of BCA to 2
grams of seeds
storage
• Coated seeds packed into 0.1 grams and stored at 5, 10, 15, 20 and 25 ℃
evaluation
• Viability of the spores was evaluated
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32. Data Analysis
The data on colony forming units (CFU) was collected for the two studies, Log
transformed before subjecting to ANOVA using the Generalised Linear Models. Means
compared using turkey at P < 0.05.
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33. Results
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34. Fig 6: Effect of (UV-A) 365 nm of T. asperellum (T) and B. subtilis (B)
Exposing the T. asperellum and B. subtilis coated seeds to UV-A (365 nm) for
upto100 minutes did not cause a significant reduction on CFU recovered on the
coated seeds (p<0.999 and p<0.336 respectively)
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y = -0.0029x + 7.68
R² = 0.1589
7.2
7.4
7.6
7.8
8
8.2
0 20 40 60 80 100 120
Log(CFU/0.1gseeds)
irradiation time (minutes)
T y = -0.0029x + 7.5365
R² = 0.3914
7.2
7.4
7.6
7.8
8
8.2
0 20 40 60 80 100 120
Log(CFU/0.1gseeds)
irradiation time (minutes)
B

35. Fig 6: Effect of (UV-B) 302 nm of T. asperellum (A) and B. subtilis (B)
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y = -0.056x + 7.3568
R² = 0.9265
0
1
2
3
4
5
6
7
8
0 20 40 60 80 100 120
Log(CFU/0.1gseeds)
irradiation time (minutes)
B
y = -0.0559x + 7.1081
R² = 0.9363
0
1
2
3
4
5
6
7
8
0 20 40 60 80 100 120
irradiation time (minutes)
T
Exposing T. asperellum and B. subtilis coated seeds to UV- B (302 nm) radiation for more than
20 minutes significantly reduced T. asperellum and B. subtilis colony forming units

36. Fig 4: Effect of temperature on the survival of the seed coating with T. asperellum A-5, B-10 C-15 E-25
However, storage of the T. asperellum at temperature higher than 10 ℃ resulted in a
significant decline of the viable CFU
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y = -0.2312x + 6.8594
R² = 0.9211
0
2
4
6
8
0 2 4 6 8 10 12 14 16
Log(colonyforming
units/0.1gseeds)
Time (weeks)
C
y = -0.1584x + 7.0226
R² = 0.8741
0
2
4
6
8
0 2 4 6 8 10 12 14 16
Log(colonyformingunits
/0.1gseeds)
Time (weeks)
B
y = -0.0188x + 7.4822
R² = 0.162
0
2
4
6
8
0 2 4 6 8 10 12 14 16
Log(colonyformingunits
/0.1gseeds)
Time (weeks)
A
y = -0.2385x + 6.585
R² = 0.8774
0
2
4
6
8
0 2 4 6 8 10 12 14 16
Log(colonyforming
units/0.1gseeds)
Time (weeks)
D

37. Fig 5:Effect of temperature on the survival of the B. subtilis
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Storage of seeds coated with B. subtilis for sixteen weeks at a temperature of 5°C and 10 °C
did not significantly reduce the CFU recovered on the treated seeds
y = -0.034x + 7.9162
R² = 0.2352
0
2
4
6
8
0 2 4 6 8 10 12 14 16
colonyformingunits/
0.1gseeds
Time (weeks)
A y = -0.0627x + 7.307
R² = 0.3692
3
4
5
6
7
8
0 2 4 6 8 10 12 14 16
colonyformingunits/
0.1gseeds
Time (weeks)
B
y = -0.1959x + 7.1296
R² = 0.7982
0
2
4
6
8
0 2 4 6 8 10 12 14 16
colonyformingunits/0.1g
seeds
Time (weeks)
C y = -0.2526x + 7.1429
R² = 0.9496
0
2
4
6
8
0 2 4 6 8 10 12 14 16
colonyformingunits/0.1g
seeds
Time (weeks)
D

38. Conclusion
• The survival of T. asperellum and B. subtilis on the seed coat during storage
can be enhanced by keeping the BCA at temperature below 15 ℃.
• irradiation of T. asperellum and B. subtilis with Ultra-Violet at 302 nm BCA
storability
• Radiation of T. asperellum and B. subtilis using 365 nm Ultra-Violet does
not affect spore survival
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39. Output Shelf life of BCA
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40. GENERAL CONCLUSION
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• Application of Bacillus subtilis and Trichoderma asperellum enhances African Nightshade
survival against damping off disease.
• Seed coating with B. subtilis and T. asperellum singly and in combination have the ability to
promote growth and yields of African Nightshade
• Temperature above 15 ℃ and UV radiation 302 nm affect the storability of the biological
control agents.

41. ACKNOWLEDGEMENTS
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42. Conference attended
• Efficacy of Bacillus subtilis and Trichoderma asperilum against damping off disease in Africa
nightshade and Ethiopian kale.- Tropentag conference held in Berlin Humbolt university) 2015
• Effect of application methods of biological control agents against Pythium aphanidermatum causing
Damping off disease in Ethiopian kales- conference held in JKUAT 2015
• Efficacy of Bacillus subtilis and Trichoderma asperilum against damping off disease in Africa
nightshade and Ethiopian kale – KALRO conference 2015
• UV Radiation and Wet Seed affects Shelf life of Trichoderma asperellum used for bio-control of soil
borne Fungal Pathogens- HAK conference 2016 held in Chuka
• Seed Treatment with Trichoderma asperellum and Bacillus subtilis to mitigate Abiotic, and
Physiological Stresses in Germinating Seeds and Seedlings of African nightshade held in JKUAT 2017
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THANK YOU