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

• 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

Banana production

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)

Radopholus similis damage

Cosmopolites sordidus damage

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

Endophytes

Endophytes are all organisms that, at some

time in their life cycle, live symptomlessly

within plant tissues

rootsouter corm

innercorm

pseudostem base

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)

Continued

• Induced resistance determined as the most likely

mode of action against R. similis (Athman 2006, PhD

Thesis, University of Pretoria)

BA

A= Inducer half: Endophyte-inoculated

B= Respondent half: Nematode

challenged

Split-root experiments

Nematode numbers were reduced

in respondent half, compared to

control

• On-farm testing of F. oxysporum endophytes for

nematode control is on-going

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

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

Overview of PhD Research

Development of marked isolates for plant

colonization studies

• Generated benomyl and chlorate resistant mutants

• Mutants were tested for growth on PDA and plant

colonization abilities, in comparison with wild-type

isolates

Accepted for publication in Biocontrol 2009 (DOI

10.1007/s10526-009-9221-1)

Colony morphology of a benomyl-resistant

mutant and wild-type isolate on PDA

V5w2Wild-type isolate

Mutant isolate

Root colonization by benomyl-resistant

mutant isolates of Emb2.4o and V5w2

0

10

20

30

40

50

60

70

80

90

100

Em

b2.4

o

BR 1

BR 2

BR 3

BR 8

V5w

2

BR 1

BR 4

BR 6

BR 8

% r

oo

t c

olo

niz

ati

on

Emb2.4o V5w2

Root colonization by chlorate-resistant

mutant isolates of Emb2.4o and V5w2

0

10

20

30

40

50

60

70

80

90

100

Em

b2.4

o

CHR 2

CHR 3

CHR 4

CHR 6

V5w

2

CHR 1

2

CHR 2

CHR 4

CHR 9

% r

oo

t c

olo

niz

ati

on

Emb2.4o V5w2

Study of biochemical changes induced

in banana following endophyte

inoculation and nematode challenge

A. Expression analysis of putative banana

defense genes using qRT-PCR

• 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)

Published in Molecular Plant Pathology 2007; 71: 149-157

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

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

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

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

Root &

rhizome

sampling

RNA

extraction

mRNA & cDNA

synthesis

Selective

amplification AFLP gel

V5w2 Emb2.40Zero hr

Polymorph

ic bands

sequenced

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)

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

0.00

0.10

0.20

0.30

0.40

0.50

0.60

0.70

0.80

ZeroHr E2DPI E33DPI ERS CRS

Exp

ressio

n r

ati

o

Nabusa

Kayinja

0.00

0.50

1.00

1.50

2.00

2.50

3.00

3.50

4.00

4.50

ZeroHr E2DPI E33DPI ERS CRS

Exp

ressio

n r

atio

Nabusa

Kayinja

ABC-transporter β-1,3-glucan synthase

Coronatine Insensitive 1 Lipoxygenase

0.00

0.01

0.02

0.03

0.04

0.05

0.06

0.07

0.08

0.09

0.10

ZeroHr E2DPI E33DPI ERS CRS

Exp

ressio

n r

atio

Nabusa

Kayinja

0.00

2.00

4.00

6.00

8.00

10.00

12.00

14.00

16.00

18.00

ZeroHr E2DPI E33DPI ERS CRS

Ex

pre

ss

ion

ra

tio

Nabusa

Kayinja

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 andlignin 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

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

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

POX activity (Comparisons by cultivar and time point)

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

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)

Dual inoculation of isolates Emb2.4o

and V5w2

Published in Biocontrol Science and Technology 2009; 19:

639-655

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

Root and Rhizome colonization 4 weeks after

inoculation

0

10

20

30

40

50

60

70

80

Con

trol

Emb2

.4o

V5w

2

Emb2

.4o +V5w

2

Con

trol

Emb2

.4o

V5w

2

Emb2

.4o +V5w

2

% C

olo

niz

ati

on

Saprophytic strains

V5w2

Emb2.4o

Roots

Rhizome

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

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

Nematode densities in roots 12 weeks post challenge

Means within a column followed by different letters are significantly

different (P ≤ 0.005 Turkey’s)

Weevil damage to the rhizome 12 weeks after

challenge

For each plant organ, bars followed by different letters are significantly

different at P ≤ 0.05 (Tukey’s)

0

10

20

30

40

50

60

70

80

90

Rhizome periphery Inner rhizome Outer rhizome

Dam

ag

e (

%)

Emb2.4o

V5w2

Dual

Control

ab a

b

a

abab

b

a

ab

a

b

a

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

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

Continued

• Other than direct up-regulation, F. oxysporumendophytes prime defense genes for up-regulation following R. similis challenge

• Dual inoculation of isolates Emb2.4o and V5w2 increases plant colonization and reduces R. similispopulations in the screenhouse

• The effect of dual inoculation of isolates Emb2.4o and V5w2 on plant growth and weevil damage needs further investigations

Acknowledgements

BMZ for Funding