Banana production,Study of biochemical changes induced in banana following endophyte inoculation and nematode challenge

1. Plant defense responses against Radopholus
similis in East African Highland bananas
(EAHB-AAA) inoculated with endophytic
non-pathogenic Fusarium oxysporum
Pamela Paparu
Supervisors: Prof Altus Viljeon, Dr
Thomas Dubois and Dr Daniel Coyne
International Institute of Tropical Agriculture FABI,
University of Pretoria

2. Banana production
• 3rd most important fruit crop in the world
•Types grown: cooking, brewing and dessert
• Uganda has the highest consumption, with an estimated
250 kg per capita consumption annually
• World exports projected to reach15 million tons by 2010

3. Major banana production constraints
Biotic factors
• Pests such as banana weevil and plant parasitic
nematodes (most importantly Radopholus similis)
• Diseases such as Black sigatoka, Fusarium wilt,
Banana streak virus, banana bunchy top virus and
Banana bacterial wilt
Abiotic factors
• Loss of soil fertility due to poor soil management
• Socio-economic factors (land shortage, migrations)

4. Radopholus similis damage

5. Cosmopolites sordidus damage

6. Current control strategies for banana weevil
and the nematode
• Clean planting materials (tissue culture plants
and hot water-treated suckers)
• Chemical control
• Cultural practices e.g Field sanitation and
trapping of adult weevils
• Host plant resistance
• Biological control of weevils using Beauveria
bassiana and that of R. similis using Fusarium
oxysporum endophytes is being investigated

7. Endophytes
Endophytes are all organisms that, at some
time in their life cycle, live symptomlessly
within plant tissues
pseudostem base
inner
roots
corm outer corm

8. Endophyte research at IITA-Uganda
• A collection endophytic isolates with biocontrol
potential (From 1995)
• In vitro and in vivo efficacy tests against target pests
• Development of inoculation methods that result in high
colonization percentages (Paparu et al.2006-Journal
of Crop Improvement 16: 81-95)
• Root colonization patterns have also been determined
(Paparu et al. 2006-Annals of Applied Biology 149:1-8)

9. Continued
• Induced resistance determined as the most likely
mode of action against R. similis (Athman 2006, PhD
Thesis, University of Pretoria)
Split-root experiments
A= Inducer half: Endophyte-inoculated
B= Respondent half: Nematode
challenged
Nematode numbers were reduced
in respondent half, compared to
A B control
• On-farm testing of F. oxysporum endophytes for
nematode control is on-going

10. Induced resistance
• Refers to the activation of plant defense responses
by biotic or abiotic elicitors
• Resistance is expressed locally or systemically
Types of induced resistance
• Systemic acquired resistance (SAR)- activated
following exposure to pathogens or wounding;
signal molecule involved in its pathway is Salicylic
acid
• Induced systemic resistance (ISR)- resistance
induced in plants following root colonization by
rhizobacteria; signal molecule is Jasmonic acid

11. Gaps
• Development of markers to trace endophytes within
plant
• Need to determine biochemical changes in banana
following resistance induction
• Effect of dual inoculation of isolates on plant
colonization, growth and control of target pests

12. Overview of PhD Research

13. Development of marked isolates for plant
colonization studies
Accepted for publication in Biocontrol 2009 (DOI
10.1007/s10526-009-9221-1)
• Generated benomyl and chlorate resistant mutants
• Mutants were tested for growth on PDA and plant
colonization abilities, in comparison with wild-type
isolates

14. Colony morphology of a benomyl-resistant
mutant and wild-type isolate on PDA
V5w2
Wild-type isolate
Mutant isolate

15. Root colonization by benomyl-resistant
mutant isolates of Emb2.4o and V5w2
100
Emb2.4o V5w2
90
80
% root colonization
70
60
50
40
30
20
10
0
1
2
3
8
1
4
6
8
w2
o
.4
BR
BR
BR
BR
BR
BR
BR
BR
V5
b2
Em

16. Root colonization by chlorate-resistant
mutant isolates of Emb2.4o and V5w2
100
90
80
Emb2.4o V5w2
% root colonization
70
60
50
40
30
20
10
0
2
3
4
6
2
4
9
12
w2
o
.4
HR
HR
HR
HR
HR
HR
HR
V5
b2
HR
C
C
C
C
C
C
C
Em
C

17. Study of biochemical changes induced
in banana following endophyte
inoculation and nematode challenge

18. A. Expression analysis of putative banana
defense genes using qRT-PCR
Published in Molecular Plant Pathology 2007; 71: 149-157
• Genes screened included PR-1, Catalase,
Endochitinase, Peroxidase, PAL and Lectin
• These genes were up-regulated following
colonization of tolerant banana cvs by Fusarium
oxysporum f.sp. cubense (Foc)
(Van den Berg 2006, University of Pretoria;
Forsthy 2006, University of Queen’s Land)

19. Experimental design
• Banana cvs susceptible (Nabusa) and tolerant
(Kayinja) to R. similis
Treatments
1. Plants of both cvs not inoculated (control)
2. Plants of both cvs inoculated with isolate V5w2 and
sampled at 2 and 33 days after inoculation
3. Endophyte-inoculated plants of both cvs challenged
with R. similis and sampled 3 days after challenge
4. Control plants of both cvs challenged with
nematodes and sampled 3 days after challenge

20. Results
• Susceptible cv: genes not up-regulated after
endophyte inoculation or nematode challenge
• Tolerant cv: up-regulation of Catalase and PR-1 at
zero hr, 33 DPI and 3 DPNC

21. B. cDNA-AFLPs for identification of genes
up-regulated in banana following endophyte
colonization
Why cDNA-AFLPs?
• Allows detection of changes in gene expression
between samples
• Requires no prior sequence information

22. Summary of treatments and sampling time
Treatments Zero hr 2 DPI 7 DPI 30 DPI
Nabusa control +
Km-5 control +
Nabusa + V5w2 + + +
Nabusa + Emb2.4o + + +
Km-5 + V5w2 + + +
Km-5 + Emb2.4o + + +
DPI = Days Post Inoculation + = Sampling time

23. Root &
rhizome RNA mRNA & cDNA Selective
extraction synthesis amplification AFLP gel
sampling
Polymorph
ic bands
sequenced
Zero hr V5w2 Emb2.40

24. Results
• 50 differentially expressed fragments cloned and
assigned putative identities
• Fragments of interest included
 Lipoxygenase (associated with ISR and involved in
phenylpropanoid pathway)
 Glycolate oxidase (involved in oxidative burst)
 Coronatine insensitive 1(signal molecule essential for jasmonate
responses)
 Β-1,3-glucan synthase and cellulose synthase (cell wall
appositions)
 ABC transporter (implicated in transport of defense molecules)

25. Expression analysis of up-regulated genes
using qRT-PCR
• Expression studied in susceptible and tolerant
banana cvs following endophyte inoculation and
nematode challenge
• Genes included ABC transporter, COI 1, β-1,3-
glucan synthase and lipoxygenase

26. Nabusa Nabusa
18.00 0.80
Kayinja Kayinja
16.00 0.70
14.00
0.60
Expression ratio
Expression ratio
12.00
0.50
10.00
0.40
8.00
0.30
6.00
4.00 0.20
2.00 0.10
0.00 0.00
ZeroHr E2DPI E33DPI ERS CRS ZeroHr E2DPI E33DPI ERS CRS
ABC-transporter β-1,3-glucan synthase
4.50 Nabusa Nabusa
0.10
4.00 Kayinja 0.09 Kayinja
3.50 0.08
Expression ratio
Expression ratio
3.00 0.07
0.06
2.50
0.05
2.00
0.04
1.50 0.03
1.00 0.02
0.50 0.01
0.00 0.00
ZeroHr E2DPI E33DPI ERS CRS ZeroHr E2DPI E33DPI ERS CRS
Coronatine Insensitive 1 Lipoxygenase

27. C. Activities of phenylpropanoid pathway
enzymes following endophyte inoculation and
R. similis challenge
• PAL, POX and PPO are major enzymes in the
phenylpropanoid pathway (leading to phenolic and
lignin synthesis)
• Root protein extracts were assayed for PAL, POX
and PPO activity following endophyte inoculation
and nematode challenge
• Roots were also assessed for nematode population
density
Paper submitted to Nematology Journal

28. PAL activity (Comparisons by cultivar and time point)
Cultivar Treatment 7 DPI 7 DPNC 30 DPNC 60 DPNC
Nabusa Control
Nabusa Control + R.similis
Nabusa V5w2
Nabusa V5w2 + R.similis
Km-5 Control
Km-5 Control + R.similis
Km-5 V5w2
Km-5 V5w2 + R.similis

29. POX activity (Comparisons by cultivar and time point)
Cultivar Treatment 7 DPI 7 DPNC 30 DPNC 60 DPNC
Nabusa Control
Nabusa Control + R. similis
Nabusa V5w2
Nabusa V5w2 + R. similis
Km-5 Control
Km-5 Control + R. similis
Km-5 V5w2
Km-5 V5w2 + R. similis

30. PPO activity (Comparisons by cultivar and time point)
Cultivar Treatment 7 DPI 7 DPNC 30 DPNC 60 DPNC
Nabusa Control
Nabusa Control + R. similis
Nabusa V5w2
Nabusa V5w2 + R.similis
Km-5 Control
Km-5 Control + R. similis
Km-5 V5w2
Km-5 V5w2 + R.similis

31. R. similis population densities in roots of
challenged plants
Cultivar Treatment 7 DPNC 30 DPNC 60 DPNC
Nabusa Control + R. similis 0 83.3 a 6913.9 a
Nabusa V5w2 + R. similis
83.3 a 0 3915.1 b
Km-5 Control + R. similis 249.9 a 0 499.8 c
Km-5 V5w2 + R. similis 83.3 a 83.3 a 249.9 c
Means within a column followed by different letters are significantly
different (P ≤ 0.005 Turkey’s)

32. Dual inoculation of isolates Emb2.4o
and V5w2
Published in Biocontrol Science and Technology 2009; 19:
639-655

33. Effect of dual inoculations on plant
colonization, growth and pest control
Inoculation treatments
a. V5w2 (chlorate mutant)
b. Emb2.4o (benomyl mutant)
c. V5w2 + Emb2.4o
d. Control

34. Root and Rhizome colonization 4 weeks after
inoculation
80
Saprophytic strains
70
60
Roots V5w2
% Colonization
50 Emb2.4o
40
30 Rhizome
20
10
0
E m ol
E m ol
w2
w2
2
2
o
o
.4
.4
5w
5w
r
r
t
t
V5
V5
on
on
b2
b2
+V
+V
C
C
o
o
.4
.4
b2
b2
Em
Em

35. Effect of Dual inoculation on pest control
• Reduced nematode densities in plants inoculated with V5w2
alone and in those inoculated dually
• Reduced weevil damage to the rhizome

36. Nematode densities in roots 12 weeks post challenge
Inoculation treatment Total nematodes (x100)/100g root
Rep 1 Rep 2
Control 217.7 a 217.3 a
Emb2.4o 249.4 a 223.5 a
V5w2 55.9 b 218.1 a
Emb2.4o + V5w2 79.8 b 122.2 b
Means within a column followed by different letters are significantly
different (P ≤ 0.005 Turkey’s)

37. Weevil damage to the rhizome 12 weeks after
challenge
90 a a Emb2.4o
80
ab a ab a a V5w2
ab ab Dual
70 b Control
b b
60
Damage (%)
50
40
30
20
10
0
Rhizome periphery Inner rhizome Outer rhizome
For each plant organ, bars followed by different letters are significantly
different at P ≤ 0.05 (Tukey’s)

38. Effect of Dual inoculation on plant growth
• Dually inoculated plants challenged with R. similis
showed increased height and girth in one replicate
• Effect on growth neutral in replicate 2

39. Conclusions
• Chemically marked F. oxysporum endophytes are
stable mutants that can be effectively used to
determine actual plant colonization percentages
• Defense pathways activated following banana root
colonization by pathogenic and non-pathogenic F.
oxysporum are dissimilar
• There is significant evidence for the involvement of
JA-induced defense pathway in endophyte-
mediated defense responses against R. similis

40. Continued
• Other than direct up-regulation, F. oxysporum
endophytes prime defense genes for up-regulation
following R. similis challenge
• Dual inoculation of isolates Emb2.4o and V5w2
increases plant colonization and reduces R. similis
populations in the screenhouse
• The effect of dual inoculation of isolates Emb2.4o
and V5w2 on plant growth and weevil damage
needs further investigations

41. Acknowledgements
BMZ for Funding