Presentation given by Dennis Ochola, Bioversity International, on the 'Effect of Water Stress & the Interaction between Fertilizer & Inoculum Concentration'. The presentation was given at the International Horticultural Congress 2014. Find out more at www.promusa.org Read more about Dennis Ochola here: http://www.bioversityinternational.org/about-us/who-we-are/staff-bios/single-details-bios/ochola-dennis/

1. Effect of Water Stress & the Interaction
between Fertilizer & Inoculum Concentration
Dennis Ochola, Research Associate, Bioversity
International
International Horticultural Congress 2014 - 19th August

2. Content
Relevance of banana
Key constraints of
banana
Banana Xanthomonas wilt (BXW)
Soil nutrient depletion
Drought induced stress
Results 1: Water stress x BXW
Development
Results 2: Fertilizer x Inoculum concentration
x BXW
Conclusion

3. Relevance of bananas
• Banana is a versatile
food and income crop for
millions of smallholders
in EC Africa.
• Uganda #2 producer of
approx. 10 million
tonnes. Annual per
capita consumption
exceeds 300 kg.
• Continuous & reliable
production = Food
security (no drought-induced
famine). Erosion
Technoserve
Piet van Asten, IITA

4. Key Constraints of Banana
• Xanthomonas wilt
• Banana weevil
• Black sigatoka
• Fusarium wilt
• pH
• Potassium
• Nitrogen
• Phosphorous
Pests & Diseases
Soil Nutrient
Depletion
• Drought stress
• Temperature spikes
• Floods
Climate Change

5. Banana Xanthomonas Wilt
(BXW)
• Bacteria - Xanthomonas
campestris pv.
muscearum
(1.8 – 2.0 μ x 0.7-0.9μ)
• Devastating disease in EC
Africa; US$ 50 Million
losses.
• 1967 first noted on Enset
in the Ethiopian
highlands.
• Spread: Insects +
contaminated farm tools +
diseased suckers.

6. Drought induced stress
• Banana water req. >25 mm per
week = 1300 mm per year
(very sensitive to water stress).
• Most growing regions
experience suboptimal rainfall.
• CSIRO 2050: 100 mm rainfall
decline + 2 oC temperature rise.
• Drought = Δ Pathogen
suitability + Δ Crop
vulnerability.
YIELDS MIGHT
DECREASE BY
10-15%

7. Soil nutrient depletion
• Banana grown on Ferralsols +
Acrisols of low inherent
fertility.
• Crop is a heavy feeder i.e. 200-
400 kg N ha-1 N and 240-480 kg
K ha-1.
• Fertilizers not widely used =
high prices + limited and poor
access.
• Low fertility = reduced vigor +
high susceptibility to diseases.

8. Effect of Water
stress on BXW
Development
Research Question
Does duration & timing of water stress increase
susceptibility to Xanthomonas wilt?

9. Materials & Methods
• EAHB cv. Mbwazirume (persistent bracts + neuter flowers).
• 3.5 mm water per day until robust root system + leaves
• Water stress treatments – withholding water
• SF, 2W, 4W, SF+3W, 2W+3W and 2W+4W.
• Stress determined with a conventional water filled tensiometer DIK-
8333 pF meter.
• Inoculum and inoculation
• Xcm isolated + grown on Cellobiose Cephalexin Agar (CCA) (Mwebaze
et al. 2006)
• Bacterial colonies suspended in distilled water + OD adjusted to 0.5
approx. 1 x 108 colony forming units (cfu)mL-1) (Ocimati et al. 2013)
• 1 mL of Xcm injected in the petiole with the insulin syringe Micro-Fine+
0.33 x 12.7 mm.

10. Materials & Methods
• Disease development and wilt index
• Six-point scale (0-5) modified from Winstead & Kelman (1952)
0 = no wilt; 1 = 1 wilted leaf; 2 = 2-3 wilted leaves; 3 = 4 wilted leaves; 4 = All
leaves wilted; 5 = Dead or collapsed
• Percentage wilt index = [(0a + 1b + 2c + 3d + 4e + 5f/5n) x 100]
(Ssekiwoko et al. 2006)
• Incidence = proportion of plants expressing symptoms.
• Area under disease progress curve (Jeger & Viljanen-Rollinson, 2001)
AUDiPC + AUPSiPC
• Statistical analysis
• GENSTAT 11 Edition (VSNI, UK)
• ANOVA
• Mean separation LSD (P < 0.05)

11. Stress measurements with a pF
Meter
1.9
2.1
Moderate stress
2.3 2.3
2.8 2.8
3
2.5
2
1.5
1
0.5
0
SF 2W 4W SF+3W 2W+3W 4W+3W
pF value
Water stress treatments
Extreme
stress
Stress
free

12. BXW Parameters: Incubation
period
16.7 cd
13.6 b
15.6 c
12 a
15.1 bc
11.7 a
20
15
10
5
0
Incubation period (dpi)
Water stress treatments

13. BXW Parameters: Incidence
2268 a 2147 a
2735 ab2734 ab 2899 b
3162 bc
3500
3000
2500
2000
1500
1000
500
0
AUDiPC
Water stress treatments

14. BXW Parameters: % severity index
2735 ab
2268 a2147 a
3162 c
2734 b2899 bc
1400
1200
1000
800
600
400
200
0
AUPSiPC
Water stress treatments

15. Discussion & Conclusion
• Water stressed plants are physiologically weak = more
vulnerable to BXW. Hence BXW to be hastened by both
duration + timing of water stress.
• Increased incidence due to prolonged water stress
durations = shorter period Xcm to colonize the
parenchymatous tissue.
• Scenario in which BXW may be exacerbated by
changing climate, with effects large enough affect
smallholder production esp. in South-western Uganda.
• Temperature + rainfall data in 25 years show large
departures from normal.

16. Discussion & Conclusion
• Climate change-mediated impacts on plant diseases
and efficacy their management + productivity +
sustainability of agricultural systems (Chakraborty et al. 2000)
• Shifts in rainfall may alter host-pathogen interaction +
temperature spikes lead to breakdown of resistance.
• *Difficult to discriminate stress-induced from Xcm-inducing
wilting of banana.
• Xcm-induced wilting is due to reduced water + uptake
uptake due to embolism of the xylem from copious EPS
production.

17. Fertilizer &
Inoculum
Concentration &
BXW Development
Research Question
Does improved plant nutrition contribute to
increased Xanthomonas wilt resistance?

18. Materials & Methods
• EAHB cv. Mbwazirume (persistent bracts + neuter flowers).
• 3.5 mm water per day until robust root system + leaves
• 3 Fertilizer treatments – 0, 125 & 250 kg/ha
• 4 Inoculum treatments – 1 x 104, 1 x 106, 1 x 108 & 1 x 1012 cfu/mL
• Inoculum and inoculation
• Xcm isolated + grown on Cellobiose Cephalexin Agar (CCA) (Mwebaze
et al. 2006)
• Bacterial colonies suspended in distilled water + OD adjusted to 0.5
approx. 1 x 108 colony forming units (cfu)mL-1) (Ocimati et al. 2013)
• 1 mL of Xcm injected in the petiole with the insulin syringe Micro-Fine+
0.33 x 12.7 mm.

19. Materials & Methods
• Disease development and wilt index
• Six-point scale (0-5) modified from Winstead & Kelman (1952)
0 = no wilt; 1 = 1 wilted leaf; 2 = 2-3 wilted leaves; 3 = 4 wilted leaves; 4 = All
leaves wilted; 5 = Dead or collapsed
• Percentage wilt index = [(0a + 1b + 2c + 3d + 4e + 5f/5n) x 100]
(Ssekiwoko et al. 2006)
• Incidence = proportion of plants expressing symptoms.
• Area under disease progress curve (Jeger & Viljanen-Rollinson, 2001)
AUDiPC + AUPSiPC
• Statistical analysis
• GENSTAT 11 Edition (VSNI, UK)
• ANOVA
• Mean separation LSD (P < 0.05)

20. Fertilizer + Banana Growth
Fertilizer
Plant
height
(cm)
Pseudostem
Girth (cm)
Leaf Area
(cm2)
0 kg npk 89.9 13.4 953
125 kg npk 122.2 15.9 1589
250 kg npk 137.3 17.4 1722
Mean 116.5 15.5 1421
LSD (p<0.05) ** ** **

21. Fertilizer + Inoculum on BXW
Incidence
p<0.01 p>0.05
0npk 125npk 250npk
105
100
95
90
85
80
75
4e 6e 8e 12e
Incidence (%)
Log10 cfu mL-1
120
100
80
60
40
20
0
4e 6e 8e 12e
0npk 125npk 250npk
Fertilizer Treatment

22. Fertilizer + Inoculum on BXW
Severity
80
70
60
50
40
30
20
10
0
p<0.01 p>0.05
0npk 125npk 250npk
4e 6e 8e 12e
Wilt Index (%)
Log10 cfu mL-1
80
70
60
50
40
30
20
10
0
4e 6e 8e 12e
0npk 125npk 250npk
Fertilizer Treatment

23. Mortality (%)
Inoculum
(cfu mL-1)
Fertilizer Treatment
Meanx
0 125 250
104 15 ab 7.5 a 5.1 a 9.2 a
106 82.4 de 60.3 cd 72.7 d 71.8 d
108 65.4 d 80.8 de 79.5 de 75.2 de
1012 85.5 e 59.3 cd 67.9 d 70.9 d
Meany 62.1 d 52 c 56.3 cd

24. Discussion & Conclusion
• Fertilizers boost physiology of plants hence have
potential of augmenting disease resistance.
• Majority of cultivated East African highland bananas
have no resistance to Xcm infection.
• Xcm concentration has an overriding effect on the
effectiveness of fertilizers for BXW management.
• Type 3 effector proteins like YopJ-like C55 cysteine protease suppress
innate defenses in banana by enhancing nutrient uptake by Xcm.
• Findings contradict Atim et al. (2013) whereby
application of exogenous N, K+ & Ca2+ reduced BXW
susceptibility.

25. Discussion
• Studies reveal that fertilizers modulate predisposition to
facultative pathogens like Xanthomonas, Alternaria & Fusarium.
• Slow disease build up in 104 cfu/mL inoculated plants is
reminiscent of latent infections.
• Xcm survives latently in underground parts of banana mat
(corm) for 1-2 years (Ocimati et al. 2013)
• Prolonged duration of Xcm latency is associated with recent
resurgence of disease in fields wherein it had been
contained.
• Muted effect of fertilizer suggests a weak interaction with Xcm
concentration.
• Exercise caution prior to endorsements against fertilizer use in
BXW management.

26. Thank you
www.bioversityinternational.org