Winning the race against virus threats to food crops in sub-Saharan Africa

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Winning the race against virus threats to food crops in sub-Saharan Africa:

What we do and how we do it!A review from August 2007

P Lava Kumar

Contract Review Seminar, 12 April 2010

www.iita.org

Outline

1. Introduction: The challenges

2. What we do and how we do it!• Clonal crops • Seed crops • Germplasm health and quarantine • Diagnostics • Capacity building

3. Future planStay fit and competitive

4. Conclusions

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1. The challenges

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0

5

10

15

20

25

30

35

Maize Sorghum Cassava Rice Wheat Musa Yams

12

34 5

6 7

Are

a, h

a (x

1 0

00,0

00)

1

2 34

4 76

0

20

40

60

80

100

120

140 Production, tonnes(x 1 000,000)

37%

16%15%

11%

8%

7%6%

cassava

maizeyam

musa

N = 327.2 million t76% IITA crops

• 10% increase in agriculture productivity in Africa is associatedwith 7.2% decrease in poverty (IFPRI 2004).

Food security and poverty reduction through agriculture development

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Virus diseases•Cause yield and quality loses

•Losses are often insidious. Frequently less conspicuous and go unnoticed or untreated.

•Reduction in growth•Reduction in vigor •Reduction in quality & market value•Reduction in transboundary trade •Costs of maintaining health

Direct and indirect losses:

• MSV: $180 million to $480 million at 5% annual incidence

• CMD: 42% yield reduction in EACMV-UG affected region

• CBSD: $100 million in 2003

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Effects of climate variability / change

• Distribution of pests and diseases

• Changes in geographical distribution of hosts and pathogens

• Altering crop yields and losses due to changes in efficacy of management strategies

Drivers of virus spread/evolution

Source: Nature Vol 438, No. 7066

Agriculture intensification • Raising population demand on food production• Rapid expansion in area• New crops & varieties, continuous cultivation (absence of breaks)

Effects of Global Warming

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Source:Wiki

Virus

Viruses are built to win?

• Intracellular pathogens, completely dependent on hosts.

• Difficult to eliminate them, without eliminating the host

• Do viruses are there to protect hosts from invasive plants?

• New paradigm - Viruses are evolutionary drivers

• For instance virus resistance in wild relatives / landraces and susceptibility of introduced species supports this thought. (new encounter diseases of introduced crops)

CMD in cassavaMSV in maizeCSSV in cocoaRosette of groundnut

• There is little choice for host and virus – either they adjust or both will perish

• Why is it important here?

•Viruses have mechanisms to negate preventive tactics

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• Diverse crops

• Diverse viruses

• Diverse vectors

• Diverse modes of virus spread &

• Diverse agro-ecologies

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Diversity in viruses

Types worked at IITA

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Leafhoppers Mealybugs

AphidsWhiteflies

ThripsBeetles

Diverse vectors and modes of dissemination

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Ecological diversity:Clonal and seed crops

Viruses of clonal crops –STATIC• Infected clones retain viruses

indefinitely (What goes in stays forever!).

• Increase in incidence incrementally Reduction depends on the replacement of infected stocks.

• In general viruses have narrow host range.

• Predictable annual situation.

Viruses of seed crops - DYNAMIC• Only seed-transmitted viruses are

retained and passed to next generation.

• Incidence depends on the vectors and virus sources (seed-borne / volunteer plants / alternative hosts). Conditions favoring insects favor high incidence

• In general, broad host range

• Unpredictable annual situation.

Different viruses – crops demands different tactics

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2. What we do?

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Develop disease control options, including resistant varieties

Develop serological and nucleic acid-based diagnostic tools

Ensure germplasm health safety and quarantine monitoring

Study virus-vector interactions and disease epidemiology

Characterize viruses(Biological and biochemical)

Knowledge and technology transfer to stakeholders

Fundamental and applied virology research for

mitigating the impact of virus diseases

What we do?1. Understand the foe

4. Disseminate the technologies

3. Establish technologies to prevent viruses (win over the virus)

2. Develop tools to monitor them

Strategic Objectives

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What we do?Ways to win the race

ExclusionQuarantine & Inspection

(From countries)Planting virus free material

PreventionCultivation of resistant

varieties Conventionally bread /

transgenics

Reduce sources of inoculumEliminate crop refuge,

alternate sources

Reduce impactCultivation of

tolerant varieties

Reduce spreadVector control

Physical barriersSeed testing

Avoidance by cultural methods

Field isolationPlant spacing / alternative

dates

Methods to reduce impact of virus

infections

All

All All

Breedin

g programs

Plant Health MonitoringVirus-free stocks

Breeding Programs

IPM

/ I

DM

(Cas

sava

& b

anan

a)

Not effective in SSA

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3. How do we do it?

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Inter-disciplinary approach

Vector biologyVirus survival and spread

Environmental factors

Biochemical, molecular & Biological Properties

BioassaysSerological &

Nucleic-acid assays

Viral genesHost R genes

Wild and Cultivated species

Quarantine

Virus Disease

Virus isolation

Virus characterization

Diagnostic tools

Germplasm screening for resistance

DISEASE MANAGEMENT

Disease Epidemiology

Transgenic resistance

Virus isolates

Resistant Varieties

Plant Breeding

Monitoring

Virology – Breeding – Biotechnology – Germplasm – Quarantine – Extension

Core businessCore business InterInter--disciplinary businessdisciplinary business

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Crop specific activities CassavaCassava mosaic begomoviruses & brown streak•Epidemiology•Virus diversity and diagnostics •Host resistance and seed systems •Whitefly control

YamYam potyvirus & badnavirus complex•Epidemiology•Virus diversity and diagnostics•Host plant resistance •Seed systems

BananaBanana bunchy top•Epidemiology•Investigations on management options

CocoaCocoa swollen shoot virus•Distribution and diversity•Seed systems

MaizeMaize streak virus•Host resistance

Cowpea & soybeanBean pod mottle virusBlackeye cowpea mosaic virusCowpea mottle virusCowpea mild mottle virusCowpea yellow mosaic virusCowpea aphid-borne mosaic virusCucumber mosaic virusSouthern bean mosaic virusCowpea chlorotic mottleSoybean mosaic virusTobacco ringspot virusTobacco streak virusSoybean begomoviruses•Host resistance•Diversity and distribution

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Generic activities

Plant health monitoring & quarantine•Virus indexing •Establishment of virus-free clonal and seed germplasm •Facilitation of germplasm distribution

Diagnostics •PCR and ELISA-based approaches •Viruses, fungi, bacteria and others•Mycotoxins

Capacity building•Graduate and post-graduates (MSc & PhD)•Training courses & workshops for groups•Methods manual •Public database•Supply of tools and materials

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How we do it!

Case Studies

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Cassava virology

1. CMD and CBSD diversity

5. Whitefly vector: dynamics, host interaction and control

2. Alternative hosts of CMD

3. Improve diagnostics

4. Virus-host interactions and host resistance

R Hanna, J Legg, G Melaku, E Kanju, P Ntawuruhunga & P Kulakow

•USAID-linkage grant, GLCI, IFAD, USAID•IITA Opportunity Grant, Travel Grant & Strategic Grant

OJ Alabi and RA Naidu (WSU, USA) SA Akinbade & Ope

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Caused by a complex of 7 species either alone or in mixed infection•African cassava mosaic virus (ACMV)•East African cassava mosaic virus (EACMV)•South African cassava mosaic virus (SACMV) •EACMV-Cameroon, EACMV-Malawi, EACMV-Kenya, EACMV-Zanzibar EACMV-Uganda (Recombinant virus)

Cassava mosaic disease

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CMG Distribution

01020304050607080

Nigeria

Ghana

08

Ghana

89Cam

eroon

08-09

Cote d'

Ivorie

-09Ben

in 07

-08Sier

ra Le

one 0

9Ang

ola 08

ACMVBothNoneEACMVUgV%

inci

denc

e

•Only ACMV was detected in samples in Mali and Niger.•EACMV-UG was detected in Angola and Cameroon.

• Surveys were conducted in 7 countries• Samples analyzed by differential PCR &

sequencing

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Tracking the spread of EACMV-UG

•As of 2005, Spread in 2.6 million sq. km causing an estimated loss of 47% in affected countries.

2009

2008

2009

•Spread into Cameroon in West-Central Africa •Spread into Angola in Southern Africa•Also reported from Burkina Faso and Togo in 2009

Kumar et al, 2008; Akinbade et al., 2010

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CMG DistributionConclusions

•ACMV is predominate, followed by mixed infection of ACMV and EACMV.•Other viruses found are EACMV and EACMCV, but not SCMV or other EACMV’s

Kumar et al., 2008; Akinbade et al., 2010

•EACMV-UG was detected only in Angola and Cameroon

•DNA-A segments of ACMV, EACMCV and EACMV-UG sequenced were 96-98% identical to previously reported sequences

• Evidence of EACMCV (recombinant species) in Ghana even in 1989, ten years before its discovery.

• Isolates have same age as the first ever sequences of ACMV published in 1980s.

• First ever recombinant ACMV found in in Angola

g i|148897747|| East Africa g i|221360434|East Afr ica g i|89330617|eEast Afr ican g i|89330671|e East African g i|7229288 | East African c g i|7229282 | East African c g i|148897740|| East Africa g i|89330555|East Afr ican g i|89330583|East Afr ican g i|89330942|East Afr ican g i|89330748| East African g i|89330963|East Afr ican g i|89330963 East Afr ican g i|3892569 |East Afr ican g i|7008113 |South African ACM V FN435277 G hana 1989 ACM V ICM V AY730035.2 SLCM V |NC 003861 EACM CV-T z1 AY795983 EACM CV-NG EU685323 EACM CV-Ivory AF259896 G hana-1989 - EACM CV

100

51

99

70

100

100

100

74

100

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Recombinant ACMV

•Two types of ACMV detected: Wild type and Recombinant ACMV

• Entire AV1 and AV2 (ca 1000 bp) in DNA-A segment in recombinant ACMV has high similarities with EACMV.

• Named as ACMV-ANG. Further studies required to assess its affect on pathogenicity.

ACMVX17095ACMVAJ427910

A16.3UGMld

ACMVICAF259894ACMVSvrAF126802ACMVMldAF126800ACMVTZAY795982EACMZVAJ717562EACMKVKEAJ717580SACMVNC003803EACMCVAF112354EACMCVNGEACMCVICEACMMVAJ006460EACMVKEAJ717542EACMVTZ

EACMVUG2SvrEACMVNC004674

A16.3RecACMV

EACMVUG2Mld Potential parent of AV1 and AV2

Potential parent of ACMV

Recombinant ACMV-ANG

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Alternative hosts to CMGV

ACMV+EACMVSenna occidentalisLeucana leucocephalaManihot glazioviiCombretum confertumGlycine max (soybean)

ACMV onlyRicinus communisAbelmoschus esculentus (Okra)*Centrosema pubescensSida cordifolia*

Okra

Centrosema Sida

Leucana

• ACMV and EACMCV has high homologies

• Indicates active migration between cassava and other hosts

• Risk of novel recombination's

Alabi et al., 2008*New record in 2009

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Cassava brown streak virusPotyviridae: Ipomovirus

• First recognized in 1920s.• Affecting 1.6 million people in Eastern Africa • Kenya, Uganda, Tanzania, Malawi and Mozambique• Suspected in DRC, Burundi and Rwanda

Prior to 2005

Post 2005

?

?

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CBSV Diversity

• About 50 partial coat protein (3’end) sequences generated at IITA.

• High diversity, two groups, but no evidence of geographic separation.

FJ687181 CBSV (GRL00508) pCP

FJ687175 CBSV (GRE05008) pCP


AY008440 CBSV (type C) CP

GQ329864 CBSV-Tz (full sequence) 200...



FN434437 CBSV-Tan 70 (full sequence) ...



FJ687164 CBSV (GRAO7208) pCP

FJ687197 CBSV (GRS00608) pCP



FJ687166 CBSV (GRE03108) pPC

FJ687173. CBSV (GRE04708) pCP

FJ687172 (GRE04608) pCP



FN434436 CBSV-Mo 83 (full sequence) 2...



FJ687202 CBSV (GRS05708) partial CP

FJ687201 CBSV (GRS05608) Partial CP



FN423417 CBSV-CP (Nampula-Mozambique ...




FJ687165 CBSV (GRA07308) pCP



AY007597 CBSV CP



FJ687203CBSV (GRS5908) partial CP





AY008442 CBSV (type A) CP

FJ821795 CBSV (KBH1) CP

FJ821794 CBSV (KBH2) CP

AF311053 CBSV

AF311052 CBSV



FN423418 CBSV-CP (Naliendele-2-Tanzan...

FN423416 CBSV-CP (Naliendele-1 Tanzan...

AY008441 CBSV (type B) CP

FN433930 CBSV Kenya 125 1999 (Kenya:K...

Sh1.1 U

Sh1.4 U

CBSV-TZ-Short-1

CBSV-TZ-Short-2

EU916832 CBSV (BSA4) CP

EU916830 CBSV (IGA8) CP

EU916827 CBSV (NTG10) CP

EU916829 CBSV (LWR2) CP

EU916828 CBSV (HMA9) CP

FN434109 CBSV-Ug 23 (full sequence) 2...

DQ837304 CBSV (WKS) pCP

DQ837303 CBSV (NAM) pCP

DQ837302 CBSV (MKN) p

Ten11.2 U

EU916831 CBSV (BSA2) CP

EU916825 CBSV (MLB3) CP

NC 012698

EU916826 CBSV (MLB9) CP

FN433932 CBSV-Ma 42 2007 (Malawi:Chit...

FN433933 CBSV Ma 43 2007 (Malawi:Salima)

FN433931 CBSV-Ke 54 1997 (Kenya:Kilifi)

CBSV-TZ-Long-1

Lg1.1 U

Lg1.3 U

CBSV-TZ-Long-2

95

92

89

87

7284

84

61

54

99

82

81

69

67

66

64

61

61

FN433933 CBSV Ma 43 2007 (Malawi:Salima) FN433932 CBSV-Ma 42 2007 (Malawi:Chit... FN434109 CBSV-Ug 23 (full sequence) 2... FN433930 CBSV Kenya 125 1999 (Kenya:K... FN433931 CBSV-Ke 54 1997 (Kenya:Kilifi) FJ185044. CBSV-Uganda (2006) NC 012698 CBSV isolate MLB3 full geno...

FN434437 CBSV-Tan 70 (full sequence) ... FN434436 CBSV-Mo 83 (full sequence) 2... GQ329864 CBSV-Tz (full sequence) 200... NC 006941 CVYV

1007961

70

100

100

100100

0.1

UG, K

en, M

alTz

, Moz

92-9

5%

86-8

7%96

%

79-8

0%

70-7

1%

M

KeUg

Tz

NJ Tree of full-length CBSV genomes

Sequences from Genebank

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Cassava mosaic begomoviruses (CMBVs) in SSA• African cassava mosaic• East African cassava mosaic• East African cassava mosaic Cameroon virus• East African cassava mosaic Zanzibar virus• East African cassava mosaic Malawi virus• East African cassava mosaic Kenya virus• East African cassava mosaic virus-Uganda • South African cassava mosaic virus • Indian cassava mosaic

Cassava brown streak virus•Sequence information points to divergent types (2 species and several strains?)

CBSV and CMBVs are complex and often necessitates multiple tests.

Improved diagnostics

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• Detects all CMBs reported in SSA, with exception of EACMZV, SACMV, ICMV, SLCMV

650 for EACMV20GGTTTGCAGAGAACTACATCEACMVRep/R

368 for ACMV17CAGCGGMAGTAAGTCMGACMVRep/R

19CRTCAATGACGTTGTACCACMBRep/F

Size (bp)Length (nts)Sequence (5’ to 3’)Primer

Simultaneous detection of ACMV and EACMV complex

Alabi et al., 2008

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Multiplex PCR for simultaneous detection of CMBV and CBSV

ACMVEACMCV

ACMV & EACMCV

Lanes 1 to 4: CBSV infected samplesLane 5: Healthy cassavaLane 6: CMD afffected cassavaLane 7: CBSD affected cassavaLane M: Molecular weight marker (100 kb ladder)

M 1 2 3 4 5 6 7 1 2 3 4 5 6 7 M 1 2 3 4 5 6 7 1 2 3 4 5 6 7 M

CBSV-S1/S2 + CMB CBSV-L1/L2 + CMB

Sap DNA & RNA Sap DNA&RNA

EACMV

ACMV

CBSV

TCTACCAACATTCGCTGCBSVcp-S2

GCAGGTAAGGCGTTTGTGCBSVcp-S1

CTACATTATTATCATCTCCCBSVcp-L2

CAGAATAGTGTTGCTGCAGGTAACBSVcp-L1

Sequence (5’ to 3’)Primer name

Kumar et al., 2009

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CBSD• Improvement in diagnostics – NASH and RT-PCR (GLCI)• Alternative hosts for CBSV (GLCI)• Impact of CBSV strains on host resistance (GLCI & USAID)• Understand the causation of root necrosis (GLCI)• CBSV evolution and within field and location diversity (USAID) • ELISA-based assays to CBSV (USAID / GLCI)• Protocol for the production of CBSV-free tissue culture material (GLCI)

Ongoing / future priorities

CMD and CBSD• Whitefly management programs as away to reduce disease incidence (IITA Strategic grant)

Generic• Monitoring programs to prevent the spread of CBSD and EACMV-UG spread CMGV and CBSV diversity and its impact on host resistance

• Development of dual resistant cassava varieties

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Maize virology

1. Host plant resistance to Maize streak virus

2. Mechanisms of resistance

A Menkir and S Hearne

•DTMA and Core funds

M Salaudeen, O Taiwo, A Razaq

3. High-throughput phenotyping for MSV

• MSV is restricted to only Africa

• The most damaging disease of maize in SSA

• Annual losses, $120 – 480 million

• Breeding for MSV resistance is integral in our programs

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6-7 DAS

9-10 DASInoculation with leaf hoppers

13-14 DASFirst symptoms

(most genotypes have 3-4 days incubation period)

30 to 50 DASSymptom scoring at weekly intervals

07

10

143036

4842

54

60

Days after sowing3

1Material selectionMaterial selection

Screening for MSV resistance

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High-throughput phenotyping for MSV

Disease evaluation 0 = No infection or escape 1 = Most resistant (less than 10% streaks) 2 = Resistant (10-25% streaks) 3 = Moderately resistant (25-50% streaks)4 = Susceptible (50-75% streaks)5 = Highly susceptible (>75% streaks)

Virus quantification in plants by ELISA

Test lineControl line

Rep # 1 Rep # 3Rep # 2 Rep # 4

S5 S4 S3 S2 S1

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Good recovery Susceptible

Good recovery

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Group C

0.01.02.03.04.05.06.0

1 2 3 4 5 6

Weeks

Seve

rity

Msv S27

MsvS16

MsvS15

Msv S28

MsvS13 MsvS21

MsvS23

Msv S25

Msv S29

Pool 6 control

No recover

Group A

0.0

1.0

2.0

3.0

4.0

5.0

1 2 3 4 5 6

Weeks

Seve

rity

scor

e MsvS10

MsvS09

MsvS18

MsvS08

Msv S26

MsvS07

MsvS12

High recovery

Group B

0.0

1.0

2.0

3.0

4.0

5.0

1 2 3 4 5 6

Weeks

scor

e

MsvS04

MsvS11

MsvS20

MsvS03

MsvS17

Msv S30

MsvS19

MsvS02

MsvS22

MsvS06

MsvS01

MsvS14

MsvS24 Moderate recovery

Performance of maize genotypes

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0.0

1.0

2.0

3.0

4.0

5.0

6.0

MX10 MX5 MX3 MX4

Genotype

scor

e

Week 1Week 2

Week 3week 4

week 5week 6

0.00

0.20

0.40

0.60

0.80

MX10 MX5 MX3 MX4

OD

vva

lues

Leaf 1

leaf 2

Leaf 3

Group A B C D

Virus concentration vs Severity score

After 6 weeks

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0.0

1.0

2.0

3.0

4.0

5.0

6.0

1 2 3 4 5 6

Group AGroup BGroup CPool 6 control

• No evidence of resistance to leafhopper feeding. • Incubation period is 3-4 days in genotypes evaluated so far• Most genotypes showed recover type resistance (reduction in chlorotic

streaks as well as virus concentration)• No indication of symptom remission. • No immunity

Recovery mechanism offers protection to MSV

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MSV resistance in S1 testcrosses

Salaudeen et al., 2010

• Field experiment for incidence, severity and agronomic performance• 6 S1 crosses were highly resistant to MSV and good agronomic performance.• Resistance is superior to what has been found in MSV transgenics

(Shepherd et al., 2007)

45.0 45.2

58.367.6

3.02.8

2.52.1 2.0 2.0

0.010.020.030.040.050.060.070.080.0

1WPI2WPI3WPI4WPI5WPI6WPI7WPI8WPI9WPITime (week)

Dis

ease

inci

denc

e (%

)

0

0.5

1

1.5

2

2.5

3

3.5

Dis

ease

sev

erity

IncidenceSeverity

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On-going and future plans

• Further characterization of promising lines and release to farmers

• Augment resistance to MSV in other backgrounds

• Identification of association markers for MSV (DTMA)

• Mechanisms of resistance

• Host-MSV interactions

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Yam virology

• Diversity and distribution of viruses infecting yams in West Africa

• Characterization of YBSD

• Development of diagnostic tools

• Symptomology and synergistic interactions

• Host resistance

• Evaluation of mapping population

• Virus-free seed systems

R Asiedu and A Sartie

•IFAD and Core funds

O Patricia, R Ronke, M Toually and S Asala,

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*Often detected in mixed infection; known to have synergistic effect on symptom expression **Virus-like disease of unknown etiology***Limited information on virus characters

Virus reported to infect Dioscorea yams in West Africa

High*Worldwide Yam mild mosaic virus(YMMV; Potyvirus)

HighWorldwideYam mosaic virus(YMV; Potyvirus)

Not known***Nigeria Dioscorea mottle virus (DMoV; Comovirus?)

Not known***TogoDioscorea ring mottle virus (DaRMV; Potyvirus)

High**Cote d’Ivory, Benin(?), The Caribbean

Yam internal brown spot virus (IBSV; Badnavirus*)

High*

Not known***

Worldwide

Benin

Dioscorea bacilliform viruses (many strains and species) (DBV; Badnavirus)

Dioscorea sansibarensis bacilliform virus (DsBV; Badnavirus)

High*WorldwideCucumber mosaic virus(CMV; Cucumovirus)

Disease importanceDistributionVirus

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• Diagnostic tools established for all major yam viruses.-Poly and monoclonal antibodies for Yam mosaic virus, Yam mild mosaic virus, Yam badna viruses available.

-Specific and generic primers for PCR/RT-PCR based diagnostics.

-Methods for virus detection in tubers established.

Yam virus diagnostics


• Uneven distribution of viruses in tubers

• Variation in symptom expression in plants germinated from the same tuber.

• Evidence of synergistic interaction between YMV and YMMV.

Yam virus diagnostics & symptomatology

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Evaluation of mapping population

Tolerant (Recommended)Y, BYNo information TDa98/ 0116620

Susceptible Y, BY, BSusceptible to anthracnose TDa92- 219

Tolerant Recommended Y, BY, BSusceptible to anthracnose TDa95- 31018

Tolerant (RecommendedY, BY, BSusceptible to anthracnose TDa95/ 0032817

Moderately Tolerant (Promising) Y, BYResistant to anthracnose TDa87/ 0109116

Moderately Tolerant (Promising) Y, BY,BResistant to anthracnose TDa85/ 0025015

Moderately Tolerant (Promising) Y, BYNo information TDr 98/ 0131714

Moderate Y, BYNo informationTDr 99/ 0278913

-ngYNo informationTDr 95/ 01932 12

Susceptible Y, BYNo informationTDr 95-12711

Susceptible Y, BYSusceptible to YMVTDr 226110

Moderately Tolerant (Promising) Y, B-Susceptible to YMVTDr36619

Susceptible Y, BYSusceptible to YMVTDr 7478

Susceptible Y, BYSusceptible to YMVTDr 95/ 185317

Moderately Tolerant (Promising) YYHighly susceptible to YMVTDr 93-326

Susceptible Y, BYHighly susceptible to YMVTDr 97/ 007775

--YResistant to YMVTDr 89/ 026654

Susceptible YYResistant to YMVTDr 16403

-ngYResistant to YMVTDr 16212

-ngYResistant to YMVTDr 93-21

PlantTuber

Following evaluationVirus indexing

Genotype details*Accession name

Sl. No.

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Evaluation of mapping population

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B

©Lava - 09

• A virus-like disease of unknown etiology recognized in Cote d’Ivory in early 1980s, and in Benin in 2008.

• Symptoms are similar to the internal brown spot disease (IBSD) first reported from the Caribbean in mid 1970s.

• Loss in tuber quality.

• This can emerge as a serious threat.

Yam brown spot disease (YBSD)(Potential CBSD of yam?)


Studies on IBSD etiology

1

2

3

4

5

1

2

3

1 2 3 4 5

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• Wide spread in Cote d’Ivoire

• Known for about 40 to 60 years

• Bètè-bètè is highly susceptible

Studies on IBSD etiology

2.12.52.83.03.060105178

5 (base)

4 (median-

base)3

(middle)2 (apico-median)

1 (Top)

Percent symp*

No. symp*

No. observed

Distribution and severity of necrotic symptoms in tuber sections (mean)*Tubers

1 2 3 4 5

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Phenotyping yams for virus resistance

•D. rotundata accessions were evaluated in 2008-09 season.

•987 accessions in total (3488 plants)

Score:1 = highly resistant (no visible symptoms)2 = resistant (mild mottling/mosaic on few leaves)3 = moderately resistant (mild mottling/mosaic on most leaves)4 = susceptible (severe mottling / mosaic on most leaves)5 = highly susceptible (severe mosaic/mottling, stunting and distortion)

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Phenotyping yams for virus resistance

Resistant 7%(Score 2)

Moderately resistant 83%(Score 3)

Susceptible 5%(Score 4)

Highly susceptible 5% (Score 5)

N = 987

•There is no immunity or very high resistance in these germplasm.

Score:1 = highly resistant (no visible symptoms)2 = resistant (mild mottling/mosaic on few leaves)3 = moderately resistant (mild mottling/mosaic on most leaves)4 = susceptible (severe mottling / mosaic on most leaves)5 = highly susceptible (severe mosaic/mottling, stunting and distortion)

www.iita.org

Next steps

• Determine the diversity of viruses in West Africa and improve diagnostic tools

• Understand the etiology of YBSD

• Symptomology and yield losses

• Evaluation of mapping population for YMV

• Virus-free seed systems

www.iita.org

Soybean virology

•TL2 and Core funds

H Tefera, C Fatokun,

T Imbor, R Adesida and P Ogunsanya

• Baseline studies to assess the impact of virus diseases on soybean

• Evaluation of improved varieties and breeding lines against widespread/economically important viruses.

• Monitor seed stocks and eliminate virus contaminated seed.

www.iita.org

Baseline studies in Nigeria

Begomovirus

Begomovirus

Begomovirus

Potyvirus

Nepovirus

Sobemovirus

Carlavirus

Comovirus

Cucumovirus

Comovirus

Carmovirus

Potyvirus

Potyvirus

Comovirus

Genus

Whiteflies CMDCassava mosaic virus**14

WhitefliesSbMMVSoybean mild mottle*13

WhitefliesSbCBVSoybean chlorotic blotch* 12

AphidsSMVSoybean mosaic 11

NematodesTSVTobacco streak 10

BeatlesSBMVSouthern bean mosaic9

Whiteflies CPMMVCowpea mild mottle8

BeatlesCpSMVCowpea severe mosaic7

AphidsCMVCucumber mosaic6

BeatlesCYMVCowpea yellow mosaic5

Beatles CMeVCowpea mottle 4

AphidsCABMVCowpea aphid-borne mosaic3

AphidsBICMVBlack eye cowpea mosaic 2

BeatlesBPMVBean pod mottle1

Vectors AbbreviationVirus

*New viruses; **New report Ezeri et al., 2009

www.iita.org

www.iita.org

4

2.5

4.533.2

4.24.1

2.7

3.93.4

3.33.4

3.3 3.7

3.8

www.iita.org

0

20

40

60

80

100

120

Benue

Taraba

Kogi

Oyo FCTNas

saraw

aKats

ina Kano

Kadun

aAda

mawa

Niger

Borno

Kwarapla

teau

Bauch

i

State

% in

fect

ion

Virus incidence in various states

•Virus incidence exceed 50% in 13 of the 15 states (87%) surveyed

www.iita.org

Relative abundance of viruses in Nigeria

0 20 40 60 80 100 120

CPMMV

CABMV

CMV

CYMV

CMeV

SBMV

BPMV

TSV

CpSMV

SMV

BICMV

CpSMVV

irus

Proportion

Imbor et al., 2010

www.iita.org

Relative abundance of viruses in latent infections

10099

94

21.51.50.7

0 20 40 60 80 100

viru

sCMeV

BPMVCABMV

CYMVTSV

CMVCPMMV

Total = 166

% infection

www.iita.org

1CPMMVBauchi

4CPMMV, CABMV, CMV, BPMVPlateauMid-Altitude, Sud/D. Sav

2CPMMV, CMVKwara

1CPMMVBorno

1CPMMVNiger

1CPMMVAdamawaSud/ South G.Sav

3CPMMV, CMV, BPMVKaduna

3CPMMV, CABMV, CMV Kano

1CPMMVKatsinaSud/Sahel/N.G.Sav

4CPMMV, CMV, BPMV,SMVNassarawa

7CPMMV, CMV, BPMV, CMeV, TSV, CpSMV,SMVFCT

2CPMMV, CMVOyo

1CPMMVKogi

7CPMMV, CMV, CYMV, BPMV, CMeV, TSV,SMVTaraba

9CPMMV,CABMV,CMV,CYMV, SBMV, BPMV, CMeV, TSV, SMVBenueDerived Sav

TotalViruses presentStateAgro-ecological zone

Distribution of viruses in Nigeria

www.iita.org

New whitefly-transmitted begomoviruses in soybean

[Legumoviruses]

•Soybean chlorotic blotch virus (SbCBV)

•Soybean mild mottle virus (SbMMV)

•Begomoviruses (legumogroup)

•Novel types within features of both new world and old world begomoviruses

Alabi et al., 2010

www.iita.org

SbCBV2647 bp

BC1(1219…2310)

CRB

BV1(373…1158)

CRA

SbCBV2708 bp

AV1(260…1021)

AC5(701…995)

AC3(1018…1431)

AC2(1163…1579)

AC1(1479…2567)

AC4(2117…2410)

SbMMV2768 bp

C4(2198…25)

IR

V2(1…507)

V1(299…1072)

C3(1069…1473)C2

(1187…1624)

C1(1563…2612) First monopartite legumovirus

First bipartite legumovirusesthat lack AV2 gene;

Novel features

www.iita.org

CoTSV DQ875869SiYMYuV DQ875873SiGMV AF0841AbMV X15984ToMHV Y14875ToMoTV AF012301ToMoV AY965901BDMV M88180ToLCSinV AJ508783CdTV AF101478SiGMCRV X99551SiGMHV Y11098SiYVV Y11100-1CLCrV AF480941DesLDV DQ875871PYMPV Y15033PYMV D00941BGMV M88687TGMV K02030SiMMV AJ5573ToRMV AF291706SiMoV AJ5574ToYSV DQ336351BGYMV AF173556MaYMFV AY044136MaMPRV AY044134DiYMoV AF170101MeMV AY965899TYMLCV AY508994PHYVV AY044163RhGMV DQ356429CabLCuJV DQ178609CabLCuV U65530PepGMV AY928517RhGMSV DQ6673BCaMV AF110190EuMV DQ3189CuLCrV AF327559MCLCuV AF4790SLCV M182SMLCV AF421553ToCMoV AF491306CoGMV DQ641689CoYVV AY727904SLCCNV AF509742SLCPHV AB085794LYMV AF5097ToLCGV AY190291ToLCNDV DQ169057ICMV Z24759SLCMV AJ579308ClCMV DQ641693PepLCIV AB2678TYLCKaV DQ169055TYLCTHV X63016HgYMV AJ627905MYMV AJ867554MYMIV AY049771KuMV DQ641691RhYMV FM208848ACMV X17096SbCBV GQ472986 SbCBV GQ472988 WmCSV AJ2653EACMCV AF112355EACMV AJ704949EACMZV AJ704942EACMKV AJ704965SACMV AF155807BSCTV U02311

100

100

100

100

66100

100

98100

100

100

100

100

100

56

100

100

100

99

95

99

99

97

100

100

100

100

100

100

100

97

97

99

68

5576

99

82

82

80

55

99

67

79

100

97

85

70

99

61

89

55

61

73

56

Old

Wor

ldN

ew W

orld

AYVV AJ558120SbCLV AB050781AYVHuV DQ866124TbLCYnV AJ512761ToLCMYV AF327436StaLCuV AJ810156EpYVV AB007990VeYVV AM182232PepLCV AF134484TYLCCNV AJ319675ToLCTWV DQ866125ToLCVV DQ641705AYVSLV AF314144ChiLCV DQ6759ToLCBDV AF188481AEV AJ437618CYVMV AJ507777PaLCuV Y15934ToLCNDV DQ169056ICMV Z24758SLCMV AJ579307CLCuBV AY7050BYVMV AF241479OYVMV AJ0021CLCuGV AF155064CLCUGV EU024120HoLCrV AY036009ACMV X17095TLCuKV EU350585TbLCZV AF350330ToCSV AF261885ToYLCrV AY502935ChaYMV AJ223191OYLCrV EU024119ToLCMLV AY502936ToLCArV DQ519575TYLCMalV AF271234TYLCMLV FM212660EACMCV AF112354EACMV AJ717542EACMMV AJ0060SACMV AF155806EACMKV AJ717580EACMZV AJ717562CPGMV AF029217SbCBV GQ472985SbCBV GQ472987SbMMV GQ472984DoYMV AY309241KuMV DQ641690RhYMV FM208848MYMIV AY049772HgYMV AJ627904MYMV AJ421642CoYVV AY727903CoGMV DQ641688DiYMoV AF1168PepGMV AY928516SLCV M183BGMV M88686BDMV M88179CdTV AF101476ToMoV AY965900SiGMV AF049336SPLCESV EF6741SPLCGV AF326775SPLCCaV EF6742SPLCLaV EF67IYVV AJ132548SPLCV AJ586885BSCTV U02311

100

10061

6899

100

100

100

91100

95100

100

100

8160

100

100

97

100

96

97

100

100

91

100

100

96

100

92

100

66

100

99

57

66

85

75

100

85

86

99

95

7786

99

98

75

66

6981

88

5993

56

76

75

92

80

61

10055

86

Asia

India

Africa

Americas

Sweet potato New

Wor

ldO

ld W

orld

Jute ‘Leg

umov

irus

’

Afr

ica

Asi

a

www.iita.org

Search for resistance to CPMMV

TGX1987-8F

TGX1987-62FTGX1895-50F

TGX1987-57FTGX1951- 3FTGX1895-33F

TGX1987-43FTGX1950-7FTGX1889-12F

TGX1987-14FTGX1949-7FTGX1876-4E

TGX1987-10FTGX1945-1FTGX1871-12F

TGX1972-1FTGX1937-1FTGX1869-31E

TGX1971-1FTGX1935-3FTGX1844-4E

TGX1965-7FTGX1932-1FTGX1844-18E

TGX1963-3FTGX1910-14FTGX1835-10E

TGX1961-1FTGX1908-8FTGX1830-20E

TGX1956-1FTGX1904-6FTGX1740-2F

TGX1955-4FTGX1904-4FTGX1485-ID

TGX1954-1FTGX1903-3FTGX1440-1E

TGX1951-4ETGX1903-1FTGX 1448-2E

•47 elite lines evaluated in screenhouse•No immunity, variation in susceptibility•Activity in progress

www.iita.org

Soybean reaction to CPMMV

0

50

100

150

200

250

1 3 5 7 9 11 13 15 17 19 21 23 25 27 29 31 33 35 37 39 41 43

Accessions(8 plants per accession)

No.

of s

eeds

Susceptible check

Control

www.iita.org

•Virus-like disease of unknown etiology.

•Extreme reduction in leaf lamina, stunting of plant and thickening of stems.

•African soybean dwarf?

Disease of unknown etiology

www.iita.org

New soybean disease in Southern Africa

•Incidence of soybean phyllody was high in Mozambique (15-25%)

Virus disease incidence was very low in Southern Africa•Low incidence •CPMMV and SMV detected in few fields

www.iita.org

•Evaluation of soybean genotypes for resistance to abundant viruses (CPMMV, SMV, CMV, BPMV, CABMV)

•Characterization of soybean phyllody

•Diagnostics for common soybean viruses

•Characterization of new soybean infecting viruses

Next steps

www.iita.org

Cowpea virology

Focus:

• Evaluation of germplasm (landraces / improved varieties) for resistance to viruses

[adding value to drought and strigaresistance; earliness etc.]

• Breeding for multiple virus resistance

• Establishment of virus-free cowpea seed stocks

C Fatokun, B Ousman

K Ogunsola, R Adesida, P OgunsanyaCore and TL2

www.iita.org

AphidsCucumovirusCMVCucumber mosaic3

CMMV

SBMV

CPMV

CMeV

CABMV

BlCMV

Abbreviation

WhitefliesCarlavirusCowpea mild mottle 7

BeetlesSobemovirusSouthern bean mosaic6

BeetlesComovirusCowpea mosaic5

BeetlesCarmovirusCowpea mottle4

AphidsPotyvirusCowpea aphid-borne mosaic2

AphidsPotyvirusBlackeye cowpea mosaic 1

VectorGenusVirus

Viruses of minor importance (sporadic incidence):Bean pod mottle virus; Peanut mottle; Sunn-hemp mosaic; Cowpea golden mosaic; Cowpea severe mosaic virus

Important viruses of cowpea in West Africa

*All these viruses are seed transmitted

www.iita.org

Distribution of cowpea infecting viruses in West Africa in 2008

www.iita.org

Cowpea virus diversity and distribution

1978 samples from 166 sites from 5 countries (2008-09)

CP

MV

CP

MV0

5

10

15

20

25

30

35

40

45

CA

bMV

BlC

MV

SB

MV

CM

VC

PM

VC

MeV

CP

MM

VM

ixed

infe

ct

CA

bMV

BlC

MV

SB

MV

CM

V

CM

eVC

PM

MV

Mix

ed in

fec

CA

bMV

BlC

MV

SB

MV

CM

V

CM

eVC

PM

MV

Mix

ed in

fect

Nigeria (N=833)

Benin and Mali(N=899)

Ghana and Togo(N=639)

%In

fect

ion

• Present situation is not significantly different from a decade ago.• Virus equation remains same: CABMV > BlCMV > CPMV > SBMV > CMoV > CMV

www.iita.org

Cowpea viruses in southern Africa

• Severe incidence in Mozambique

• Detected: BlCMV, CpSMV, CMV, CABMV, SBMV

• Evidence of new strains / species

www.iita.org

Screening elite lines for virus resistance

www.iita.org

RMRHSIT98K-205-8 Striga.

MRMRSIT99K-573-2-1 Striga

MRMRHSIT98K-128-3 Medium

MRMRRIT00K-1207 Striga

MRRHSIT99K-7-21-2-2 Dual

MRRHSIT03K-324-9 Early

RRRIT99K-573-1-1 Striga

MRMRSIT99K-1060 Early

MRSMRIT97K-819-118 Striga

RMRMRIT00K-901-5 Early

MRRRIT04K-405-5 Dual

RMRSIT98K-506-1 Early

MRRHSIT98K-311-8-2 Dual

MRSSIT99K-377-1 Early

RHSRIT99K-1122 Early

MRHSHSIT99K-216-24-2 Dual

RRRIT98K-133-1-1 Early

MRSSIT98K-692 Striga

CPMMVCABMVCMeV

*Genotype response to:

Genotype#

Best performing genotypes are selected for further analysis

www.iita.org

Breeding for multiple virus resistant cowpea

• Genotypes evaluated singly and also in combinations

[to account multiple disease resistance / breakdown of resistance due to synergistic impact]

• Eight elite lines selected for breeding.

• Screenhouse evaluation : BlCMV, CMV, CMeV, CYMV, SBMV and CABMV

[Virus load + severity score + effect on agronomic traits]

• The best line (multiple resistance) is being used for breeding

SusceptibleResistantSusceptibleSusceptibleResistantSusceptibleSusceptibleSusceptible

BICMV, SBMV CMV

BICMV, CMVSBMV, CMVCMVBICMV, CMVBICMV, SBMV, CMVBICMV, SBMV, CMV

BICMV, SBMV, CMVSBMVBICMVBICMV, SBMVSBMV

IT98K-133-1-1IT98K-1092-1IT97K-1069-6IT98K-503-1IT97K-1042-3IT04K-405-5IT99K-1060IT99K-573-1-1

12345678

RemarksVirus susceptibility*Virus resistanceAccessions

www.iita.org

IT99K-1060 (Early)Scuceptible

www.iita.org

19.010.517.011.015.516.016.015.5IT99K-573-1-1

20.05.513.514.513.513.514.515.0IT99K-1060

18.010.514.58.010.09.514.511.5IT04K-405-5

13.012.012.512.512.011.512.513.05. IT97K-1042-3

14.511.011.510.510.010.08.511.0IT98K-503-1

15.09.013.513.012.011.513.012.0IT97K-1069-6

10.59.59.07.510.010.010.09.0IT98K-1092-1

13.57.09.08.58.57.08.09.5IT98K-133-1-1

CONTROLBICMV+

SBMV+CMVSBMV+

CMVBICMV+

CMVBICMV+

SBMVCMVSBMVBICMVAccession

100 seed weight in grams

IT98K-133-1-1 IT98K-503-1 IT98K-1092-1

www.iita.org

1. BlCMV2. SBMV3. CMV

4. BlCMV+SBMV5. BlCMV+CMV6. SBMV+CMV7. BlCMV+SBMV+CMV

0

20

40

60

80

100

120

0

1

2

3

4

5

6SeverityIncidence (%)

Seve

rity

Inci

den

ce (

%)

IT98K-133-1-1 (early)Highly susceptible

IT98K-10921 (Striga)Resistant

IT99K-1060 EarlyHighly susceptible

1 2 3 4 5 6 7 1 2 3 4 5 6 7 1 2 3 4 5 6 7

Ogunsolo et al., 2010

www.iita.org

0.0

20.0

40.0

60.0

80.0

100.0

120.0

1 2 3 4 5 6 7 80.0

1.0

2.0

3.0

4.0

5.0

6.0Incidence Severity

SeverityIn

cide

nce

(%)

Genotype

CMV+SBMV+BlCMV

1 = No visible symptom = Resistant R2 = Mild symptom = moderately resistant (MR)3 = Moderate symptoms on few leaves = moderately susceptible (MS)4 = Symptoms on all leaves = susceptible (S) 5 = Severe symptoms on all leaves = highly susceptible (HS)6 = Very severe symptoms on many leaves, necrosis and plant death (HS)

www.iita.org

0100Buffer Control

0 (BICMV), 2 (SBMV), 4 (CMV)100BICMV+SBMV+CMV

6 (SBMV); 24 (CMV)98SBMV+CMV

10 (CMV)96BICMV+CMV

0100BICMV+SBMV

8 (CMV)100CMV

0100SBMV

0100BICMV

Seed transmission (%)% GerminationSeed from plants infected with

Cowpea # IT98K-133-1-1 (susceptible)

CMV facilitating SBMV seed-transmission (synergistic effect)

Rate of seed transmission in mixed infections

www.iita.org

100 seed wt (g)

02468

1012141618

1 3 5 7 9 11 13 15 17 19 21 23 25 27 29 31 33 35 37 39 41 43

Accessions

100

seed

wt (

g)

Susceptible check

Control

www.iita.org

•Evaluation of cowpea genotypes and genetics of resistance

•Characterization of new cowpea viruses occurring in Southern Africa

Next steps

www.iita.org

Focus• Studies on banana bunchy top disease –

virus distribution, diagnostics and management

• Diagnostics for Musa indexing

Musa virology

R Hanna, D Dumet, M Branco*, J Lorenzen, F Beed

Core funds and USAID

www.iita.org

Emerging virusBanana bunchy top virus

•Type species in the genus, Babuvirus (family, Nanoviridae)

•Transmitted by the banana aphid, Pentalonia nigronervosa, persistent circulative manner.

•Occurs in Africa, South and Southeast Asia, Australia and South Pacific

•Virus in Southeast Asia is different from South Asia

Banana aphid BBTV-South pacific group BBTV-Asian group

www.iita.org

•BBTV is amongst the list of top 100 of invasive species.

•Difficult to control and eradicate.

www.iita.org

Banana aphid

Vector banana bunchy top virus(+ other viruses)

Direct damage – reduced plant growth

www.iita.org

•Severe BBTV outbreaks in Malawi, Mozambique and Zambia.

•What is the cause for the recent surge of BBTV in SSA?

BBTV - not a new foe in SSA

•The virus or vector, introduction/evolution of a more virulent forms?

•Changes in cultural practices or the environment, including climate change effect, causing this spread?

Reproduced from “Foure and Manser (1982) Fruits Vol 37, 410.

•Earliest reports of BBTV are from Kisangani of DRC and Gabon

www.iita.org

Before 1960s

Since 1980s

Since 1990s

Since 2004

BBTV in SSABBTV in SSAPresent

Present 2009

www.iita.org

•Surveys were also conducted in Nigeria, Benin and Ghana, but There is no evidence of BBTV in these countries.

BBTV SurveyBBTV Survey

•Roving survey in major and minor banana growing areas.

•Focus of BBTV and banana aphids.

•Leaf samples collected from symptomatic and asymptomatic plants for virus analysis.

•Interviews with farmers

www.iita.org

BBTV DetectionBBTV Detection

BBTV specific(240 bp)

Internal Control [BRep-1] (400 bp)

Multiplex PCR with internal control primer

www.iita.org

3

62

34 31

68

310

18 2027

01020304050607080

Sites surveyed Sites with BBTV

Angola Cameroon Gabon DRC MalawiN

umbe

r of s

ites

Number of surveyed sites with BBTV

•Survey conducted in 198 sites in 5 countries.

•BBTV detected in 39.4% sites surveyed

N=7

6%

33%

44%

19%

05

101520253035404550

Angola Cameroon Gabon DRC Malawi

43%

N=520

N=224

N=295

N=107 22.7%N=1159

Perc

ent i

nfec

tion

Percent samples positive to BBTV

Total

•BBTV detected in 22.7% of the samples tested.

www.iita.org

• BBTV is well established in DRC, Gabon, and Congo Republic, and Northern Angola, Central Malawi.

• Widespread appearance since 1994.

• Most farmers are familiar with symptoms. SIDA (AIDS) or Witches Broom.

• Symptoms are less severe on most of the varieties, but severe on Cavendish Williams and Poyo.

SummarySummary

www.iita.org

• Intra and inter-village distribution of planting material seems to be main cause for widespread distribution.

• Isolated farms are free of BBTV.

• Aphids are omnipresent.

• Symptoms are less severe on landraces, but very severe in Cavendish and Poyo.

• BBTV + Sigatoka seems to be lethal on plantains. Elimination of source plants.

• Multiplication sites are the major sources for virus spread.

SummarySummary

Kumar and Hanna 2009

www.iita.org

•Nkumba village•Symptoms are not apparent, but BBTV positive; •Original source brought in 1997

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•Nkunga•Symptoms are not apparent, BBTV not detected•No new planting material for over 50 years.

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•Mampakasa (Inside Luki reserve, Boma, DRC)

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Needle in the banana stack!Production despite infection

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Movement of planting material

First outbreakFirst outbreak

Banana rich

Banana rich

Banana rich

BBTV spread mainly through infected suckers

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•BBTV is widespread in Central and Southern Africa. Widespread occurrence since 1994

•Severe disease expression in Cavendish, but local varieties, despite infection can tolerate (suppressed symptoms) the disease.

•Role of aphid transmission is significant in most places.

•Human movement of planting material seems to be the main reason for widespread distribution.

•Infected plants are the potential sources for new spread.

•Risk of spread is high in the routes of traditional exchange of planting material.

•Important to protect the source sites.

Summary from field surveys

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• Coat protein (DNA-S) and replicase (DNA-R) gene sequences of 10 BBTV isolates from Cameroon, Gabon, DRC, Malawi and Angola determined.

DNA-R(1)1111 nts

Geneology of BBTV

•Pair-wise comparisons of coat protein sequences (nucleotides / amino acids)

DNA-S(3)1075 nts

286aa

175aa•Very high sequence similarities 98-100% sequence identity

Kumar et al., 2009

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Coat protein-based geneology

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Replicase-gene based geneology

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•High sequence similarity between the BBTV isolates suggest a common origin.

•BBTV in SSA aligns with BBTV isolates from South Pacific group.

•There is no evidence of any unusual features in virus.

•Severe incidence and spread seems to be due to

-Increase in cultivation of most susceptible varieties, such as Cavendish

-Planting of infected suckers

-Aphids vector contributing to the secondary spread.

Conclusions from molecular work

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Exclusion & PreventionControl of material movement

Awareness campaigns Increased vigilance

Routine surveillanceField isolations

1. Reduce sources of inoculum-Eliminate crop refuges

3. Reduce impactReplace infected matsCultivate tolerant varieties

2. Reduce spreadVector controlPhysical barriers Seed testing

4. Avoidance by cultural methodsField isolation (buffer zone)Plant spacing

BBTV control in SSA

Regions/countries

free of BBTV

BBTV-affected

regions/countries

Curative Preventive

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International Workshop on BBTV and BXW:Meeting the Challenges of Emerging Disease Threats to Banana

and Strategies for Raising Awareness, Surveillance and Management of these Diseases in sub-Saharan Africa

24 – 28 August 2009, Arusha, Tanzania

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Priority setting and declaration to combat

emerging banana diseases

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Quarantine Monitoring & Production and Virus-free planting material

M Ayodele and D Dumet

Patricia and RonkeCore funds, Crop Trust and GPG2

Focus:•Virus indexing•Ensuring plant health and safe exchange of germplasm

www.iita.org

•Movement of germplasm involves a risk of virus and pathogen spread.

•Seed poses less risk than the clonal material

Less risk-Scope for phytosanitation -Only few viruses can transmitted through seed

High risk-Phytosanitation is difficult-All the pathogens spread through clonal material

Germplasm exchange and risks

Mode of formal exchange: • Cassava: Stems, in vitro plants, true seed• Yam: Tubers, in vitro plants and true seed• Musa: in vitro plants and true seeds• Maize, soybean and cowpea: Seeds

www.iita.org

• Exchange in the form of in vitro cultures or seed.

• International guidelines for production and certification of virus-free planting material.

Steps to minimize the risk – 1Production of healthy planting material

•In vitro cultures produced from meristem-tips

•Virus indexing of in vitro materials

•Production of virus-free true seed

•Task performed mainly by breeders / GRC / tissue culture specialists, etc

• No guidelines for pathogenic fungi / bacteria as they are eliminated during sterilization procedures.

•Distribute virus-free material

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Steps to minimize the risk – 2Inspection of planting material

• Phytosanitary / quarantine measures to prevent the spread of regulated pests

Requires• Knowledge on pathogens• Availability of diagnostic tools• Liaison with national quarantine agencies

Pests

Regulated Not regulated

Quarantine pests Regulated non-quarantine pests

• Pathogen matters, irrespective of disease causing potential

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Germplasm Health Unit

• Facilitate exchange of ‘pest-free’ germplasm

• Ensuring compliance with national and international regulations

• Facilitates-Inspection import and export material-Phytosanitation-Import and export permits -Establish certification schemes

Health Testing(For pathogens under containment facilities)

Release of pathogen

free-materialGermplasm

Indexing for insect pests, fungi and bacteria: Visual inspection, blotter test, and sedimentation assays (Optional seed treatment / fumigation)

Indexing for viruses: Grow-out tests, evaluation for

target virus by ELISA, PCR or EM

Import / Export

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Germplasm import

procedure NAQS may put specific conditions depending on the crop species/ material and country of origin.

Submit request to NAQS for import permit

Submit to exporter

Consignment received at IITA

Process germplasm (seed/ in vitro material / vegetative

propagules) as per the standard procedure

Certified consignments Uncertified consignments

Mandatory health testing •Material held in post-entry quarantine facility•Visual inspection•Biochemical analysis for quarantine pathogens

Release ‘clean’ material to requisitioner

Grow-out in post-entry quarantine isolation

Weekly inspections

Importer at IITA (GRC, breeders, etc.)

Routed throughGermplasm Health Unit

Permit issued

Importer to sign MTA as applicable; Exporter to comply with the importer’s phytosanitary needs. Communicate any difficulties to requester. A solution will be worked out to get the material.

Material that meets importing country standards

Material that do not satisfy importing country standards

Depends on the NAQS advise

Depends on the NAQS advise

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www.iita.org

Germplasm export procedure

Material owner(GRC, Breeding Units, etc.)

Submit request to GHU with necessary details*

*Requirements:-Accession details-Geographic origin of the material (site of production) -Year of propagation/production-Previous indexing history (if any)-Import permit from the recipient-Any other instructions

Indexing at GHUfor fungal and bacterial

pathogens, as per the IITA standard procedures, as well as importing country requirements.

Pest-free material selected for dispatch

NAQS certificate confirming pest-free status

Indexing in Virology Unitfor viral pathogens as per the IITA standard procedures, as well as importing country requirements.

Feedback results to GHU

Pack and dispatch pest-free material

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•Regulated non-quarantine pests of Musa

Pratylenchus coffeaeRoot lesion nematode

Radopholus similisBurrowing nematodes

Cucumber mosaic virusBanana mosaic

Banana streak virusBanana streak

Mycosphaerella musicolaMycosphaerella fijiensis

Sigatoka (yellow)Sigatoka (black)

Fusarium oxysporum f. sp. cubenseRaces 1 and 2

Fusarium wilt

PathogenPathogenDiseaseDisease

Banana mild mosaic virus Banana virus - X

Musa as example for monitoring and germplasm exchange procedures

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Quarantine pests of Musa

-++-Banana bract mosaic virus

Bract mosaic

9

-+-+Pratylenchus goodeyiRoot lesion nematode

4

---+Xanthomonascapestris pv. musacearum

Bacterial wilt

2

--++Mycosphaerellaeumusae

Eumusaeleaf spot

3

-+++Banana bunchy top virus

Bunchy top1

10

8

7

6

5

--+-Abaca mosaic virusMosaic

-+--Ralstonia sp.Blood

-+--Ralstoniasolanacearum Race 2

Moko

-++-Guignardia musaeFreckle

-++-Fusarium oxysporumf. sp. cubense Tropical Race 4

Fusarium wilt

Latin Latin AmericaAmerica

Australia & Australia & South PacificSouth Pacific

AsiaAsiaAfricaAfricaPathogenDisease

Concerns for exportConcern for im

port

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IPCC / IAPSC guidelines for Musagermplasm exchange

Vegetative (in vitro) material:• Import permit and phytosanitary certificate

• Compulsory treatment of consignment before shipment into Africa.

• Declaration certifying the absence of virus, bacteria, and nematodes (particular those quarantine/regulated pests.

• Exchange only virus tested, virus-free materials. • Musa hybrids with episomal BSV or BSV DNA integrated can be

exchanged without restrictions.

• Musa seed: Seed sanitation and in vitro plants derived from meristemsof regenerated plants

Country specific guidelines can differ from IPPC and IAPSC guidelines

www.iita.org

---++Nematodes

+

+

+

+

-

-

-

-

-

Steriletissue culture

++ 1++Cucumber mosaic virus

++ 1++Banana streak virus

-+ 1++Bract mosaic virus

-+1++Banana bunchy top virus

-+++BXW

-+++Ralstonia sp.

-+-+Guignardia musae

-+-+MycosphaerellaMusicola, M. fijiensis, M. eumusae

--++Fusarium oxysporumRaces 1, 2 and 4

seedLeaves Corms Suckers Pathogen

1Material access to insect vectors or propagation by tissue culture can perpetuate the virus from leaf source

Decrease in risk

Spread of pests through various Musa plant material

Inte

rnat

iona

l exc

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e

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International distribution

Depends on NPPO regulations:•Material can be supplied to end-users for further propagation and distribution.

or•Plant in ‘quarantine plots’ for certification by local agencies and release to end-users.

Certified Clones that meets the importer NPPO guidelines are released

Distribution of germplasm

In-country distribution

Follow established regulations.

•This could include certification of clones by authorized labs/agencies present in-country

Or

•Distribute directly

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Next steps

• Simplification of procedures• Integrated database for monitoring and traceability • Capacity development in other stations• Low-cost monitoring tools

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Establishing virus-free germplasm

D DumetR Adesida, O Patricia and O Taiwo

GPG2Crop Trust

www.iita.org

1. Blackeye cowpea mosaic (worldwide)2. Cowpea aphid-borne mosaic (worldwide)3. Cowpea mottle (West Africa)4. Cowpea mosaic (Cuba, Kenya, Suriname, USA)5. Cucumber mosaic (Worldwide)6. Southern bean mosaic (Africa, America)7. Cowpea mild mottle (worldwide)8. Bean pod mottle virus (Nigeria, USA)9. Peanut mottle (USA, Nigeria, India)10. Sunn-hemp mosaic (Africa, India, USA)

Seed transmissible cowpea viruses in West Africa

Establishing virus free seed crop germplasm:Cowpea as example

1. Adzuki bean mosaic (Korea and Japan)2. Blackgram mottle (India) 3. Cowpea green vein-banding (Latin America)4. Cowpea severe mosaic (Americas)*5. Cowpea Moroccan aphid borne mosaic (Morocco and South Africa) 6. Cowpea ring spot (Iran)7. Mungbean and urdbean mosaic 1 (India)8. Mungbean and urdbean mosaic 2 (India)9. Tomato black ring virus (Europe and India)* 10. Urdbean leaf crinkle (India)

Seed transmissible cowpea viruses elsewhere

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Seed collected for storage & distribution

Cowpea germplasm multiplied at IITA-Ibadan, Nigeria

Target viruses:-Bean pod mottle virus (BPMV; Comovirus)-Blackeye cowpea mosaic virus (BICMV; Potyvirus) -Cowpea mottle virus (CPMoM; Carmovirus) -Cowpea mild mottle virus (CPMMV; Carlavirus)-Cowpea yellow mosaic virus (CYMV; Comovirus)-Cowpea aphid-borne mosaic virus (CABMV; Potyvirus) -Cucumber mosaic virus (CMV; Cucumovirus) -Southern bean mosaic virus (SBMV; Sobemovirus)

• Endemic in West & Central Africa• Known to be transmitted through

cowpea seeds

Establishment of virus-free cowpea germplasm in the IITA gene bank

Seed planting in insect-proof screenhouse

Visual inspection of germinated plants

Symptomatic plants

Asymptomatic plants

Uproot & incinerate Virus indexing

by ELISA*Virus positive

Virus Negative

www.iita.org

Frequency of virus infection in cowpea and bambara accessions conserved in the IITA Gene bank

0102030405060708090

100

174 158 334 127 209 235 79 201 236 227 116 242 54 143 177 120 38

Batch

Perc

ent i

nfec

ted

acce

ssio

ns

Number of accessions

www.iita.org

Frequency of virus infected cowpea plants within cowpea germplasm

05

101520253035404550

Number of plants batches

Perc

ent i

nfec

ted

plan

ts

79 201 236 177 227 116 209 235 25 18 18

536

299284

8

3179

3685

1522

1566

1783 14

6 63 39

No. of accessions

www.iita.org

0

20

40

60

80

100

1 8 15 22 29 36 43 50 57 64 71 78 85 92 99 106 113 120 127 134

Cowpea accessions [N = 139]

% in

fect

ed p

lan

ts

• Accessions with infected plants (1% to 99%) = 119 (87%)• Accessions without infected plants = 19 (13%)• Accessions with 100% infected plants = 1

• Total accessions = 139• Total plants = 2315• Plants with symptoms = 13• Plants positive to virus = 743 (32%)• Plants negative to virus = 1572 (68%)• Virus detected = CABMV, BlCMV, CMV, BPMV

Different procedure to salvage virus-free germplasm

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Plants infected through seed-borne infection produce mild or no symptoms

Plant infected in field

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Seed from health plants are harvested for storage / distribution

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Conservation & distribution

•Cassava germplasm multiplied at IITA-Ibadan, Nigeria

Target viruses:-CMDs and CBSV

Cassava Clones from meristem culture

Sample from tissue culture plant

Virus indexing by multiplex Immunocapture PCR*

Virus positiveVirus Negative

Discard / incinerate

1337

0

200

400

600

800

1000

1200

1400

138 38

Total ACMVEACMV

EACMV

Establishing virus free clonal crop germplasm:cassava as example

• In vitro cleaning procedures• Virus testing and selection of

material for conservation

www.iita.org

Next steps

• Continue replacing virus infected stocks in the GRC

• Establish robust and cost-effective virus indexing procedures for virus indexing of yam

• Harmonize indexing systems across locations

• Develop database for archiving GHU data

• Revise GHU in house GHU procedures for accelerated processing of samples and documents

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Molecular diagnostics for fungal and bacterial pathogens

Bacterial pustule 4X. axonopodis pv glycineaSoybean

bacterial blight 2X. campestris pv vignicolaCowpea

Ear rots 12F. verticillioides

Grey leaf spot 135C. zeae-maydisMaize

Cassava anthracnose 86C. gloeosporioides f. sp manihotisCassava

Anthracnose 37C. gloeosporioides Penz.Yam

DiseaseTotal isolates collectedPathogenHost

Priority fungal and bacterial diseases

R Bandopadhyay and M Ayodele K Sharma

Focus• Development of molecular diagnostics for fungal, bacterial and

other pathogens• Determine the diversity in pathogen populations• Establish on-line database and diagnostics basket

Core fundsDutch APOService function

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Living collection/DNA extraction

DNA bank (Cryo-storage system –80°C)

Conserved gene amplification

Analysis e.g. Genotyping / sequencing

Primers for Diagnostics

Data reposition (DNA Barcode)

Diagnostics: Our approach

Diagnostic basket

www.iita.orgAnthracnose symptom on Dioscorea leaf Anthracnose symptom on cassava stem

GLS symptom on Maize leaf Severe GLS symptom on Maize leaf

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Shared and species-specific SNP display in Histone sequences10 20 30 40 50 60 70 80

. . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . |

YA08-8 AC G TC C G A A - GC AGC T T GC T T C C A - GGC C GC C C GC A AG AGC GC C C C C TC C AC C GG AGG T G T C A AG A AGC C C C AC C GC T ACYA08-1 . . . . . . . C . - . . . . . . C . . C . . . . - . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . T . . . . . T . . .YA08-2 . . . . . . . C . - . . . . . . C . . C . . . . - . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . T . . . . . T . . .YA08-3 . . . . . . . C . - . . . . . . C . . C . . . . - . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . T . . . . . T . . .YA08-4 . . . . . . . C . - . . . . . . C . . C . . . . - . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . T . . . . . T . . .YA08-5 . . . . . . . C . - . . . . . . . . . . . . . . A . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . T . . . . . . . . .YA08-6 . . . . . . . C . - . . . . . . . . . . . . . . A . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . T . . . . . . . . .YA08-7 . . . . . . . C . - . . . . . . C . . C . . . . - . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . T . . . . . T . . .YA08-9 . . . . . . . C . - . . . . . . . . . . . . . . A . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . T . . . . . . . . .YA08-10 . . . . . . . . . - . . . . . . . . . . . . . . - . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .CA08-1 . . TC . GC . . - . . . . . . . . . . . . . . - . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .CA08-2 . . TC . GC . . - . . . . . . . . . . . . . . - . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .CA08-3 . . TC . GC . . - . . . . . . . . . . . . . . - . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .YA08-11 . . . . . . . . . - . . . . . . . . . . . . . . - . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .YA08-20 . . . . . . . . . - . . . . . . . . . . . . . . - . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .YA08-21 . . . . . . . . . - . . . . . . . . . . . . . . - . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .YA08-24 . . . . . . . . . - . . . . . . . . . . . . . . - . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .YA08-35 . . . . . . . C . - . . . . . . . . . . . . . . A . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . T . . . . . . . . .YA08-36 . . . . . . . C . - . . . . . . . . . . . . . . A . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . T . . . . . . . . .CA09-6 - T C C . . . C . A . . . . . . . . . C . . . . A . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . T . . . . . . . . .CA09-16 - T C C . . . C . A . . . . . . . . . C . . . . A . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . T . . . . . . . . .CA09-22 - T C C . . . C . A . . . . . . . . . C . . . . A . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . T . . . . . . . . .CA09-53 - T C C . . . C . A . . . . . . . . . C . . . . A . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . T . . . . . . . . .YA09-83 - . C C . . . T . A . . . . . . . . . . . . . . A . . . . . . . . . . . . . . . . . . . . . . . . . . . T . . . . . . . . . . . . . . . . . T . . . . . . . . .YA09-84 - . C C . . . T . A . . . . . . . . . . . . . . A . . . . . . . . . . . . . . . . . . . . . . . . . . . T . . . . . . . . . . . . . . . . . T . . . . . . . . .YA09-85 - . C C . . . T . A . . . . . . . . . . . . . . A . . . . . . . . . . . . . . . . . . . . . . . . . . . T . . . . . . . . . . . . . . . . . T . . . . . . . . .

110 120 130 140 150 160 170 180. . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . |

YA08-8 C C G TG AG A TC C G TC GC T AC C AG A AG T C C AC C G AGC T T C T G A T C C GC A AGC TC C C C T TC C AGC GC C T GG T A AG T C - - - C C AYA08-1 . . . . . . . . . T . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G . . . A A A . T GYA08-2 . . . . . . . . . T . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G . . . A A A . T GYA08-3 . . . . . . . . . T . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G . . . A A A . T GYA08-4 . . . . . . . . . T . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G . . . A A A . T GYA08-5 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . - - - . . .YA08-6 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . - - - . . .YA08-7 . . . . . . . . . T . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G . . . A A A . T GYA08-9 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . - - - . . .YA08-10 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . - - - . . .CA08-1 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . - - - . . .CA08-2 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . - - - . . .CA08-3 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . - - - . . .YA08-11 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . - - - . . .YA08-20 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . - - - . . .YA08-21 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . - - - . . .YA08-24 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . - - - . . .YA08-35 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . - - - . . .YA08-36 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . - - - . . .CA09-6 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . C . - - - . . .CA09-16 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . C . - - - . . .CA09-22 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . C . - - - . . .CA09-53 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . C . - - - . . .YA09-83 . . . . . . . . . . . . . . . . . . . . . . . . . . . G . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . T . . . . . . . . AG - - - . . GYA09-84 . . . . . . . . . . . . . . . . . . . . . . . . . . . G . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . T . . . . . . . . AG - - - . . GYA09-85 . . . . . . . . . . . . . . . . . . . . . . . . . . . G . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . T . . . . . . . . AG - - - . . G

www.iita.org

Case study C. gloeosporioides

NJ Tree of based on ITS, Actin and Histone sequences

YA08-10

YA08-24

YA08-8

YA08-11

YA08-20

YA08-21

CA08-3

CA08-1

CA08-2

YA08-35

YA08-36

YA08-6

YA08-5

YA08-9

CA09-6

CA09-53

CA09-16

CA09-22

YA09-83

CA09-84

YA09-85

YA08-7

YA08-4

YA08-1

YA08-2

YA08-3

C. beticola

100

67

65

98

94

68

99

9198

95 94

83

64

Gro

up 1

Gro

up 2

Gro

up 2

Gro

up 1

YA08-5

YA08-6

YA08-36

YA08-9

YA08-35

AY188934C.gloeosporioides (SA)











EU520060C.coffeanum (China)

AY188927C.gloeosporioides(SA)

YA08-20

EU732732 C. camelliae (China)

CA08-3




CA08-1



YA08-10


YA08-24

CA08-2

CA09-6

CA09-53



YA08-8


YA08-11


AY376540C.kahawae (SA)

FJ172290C.fragariae (USA)

CA09-22


CA09-16




DQ003095C.musae (USA)

YA08-21

EU520091C.ampelinum (China)

YA09-83

YA09-84

EF016299 C.capsici (China)

EU400155C.brassicae (Canada)

FJ545227C.coccodes (USA)

EU000060C.linicola (USA)

DQ195690C.caudatum (HongKong)

FJ459919 C.orbiculare (China)

EU400136C.lindemuthianum (Canada)

YA08-1

YA08-3

YA08-2

YA08-4

YA08-7

AY898260 G. moniliformis

98

98

82

9182

63

99

61

59

63

65

Sharma et al., 2010

www.iita.org

Case study on GLS

AY840484 C.apiiItaly

AY840479 C.apii Germany

AY840486 C.apiiGermany

AY840487 C.apiiGermany

AY840490 C.beticola Romania

AY840494 C.beticolaItaly

AY752171 C.beticola New Zealand

AY840496 C.beticola Germnay

AY840481 C.apiiHungary

AY840495 C.beticolaIran


AY752168 C.beticola Netherlands

AY840480 C.apiiAustria

AY840492 C.beticola Germany


DQ185083 C.sp.South Africa

CZ10-46 (SRD 3231) Cercospora



CZM10-101 (BPF 332) Cercospora

CZM10-59 (BRD 1321) Cercospora


CZM10-4 (SAM 112) Cercospora

CZ10-129 (CSS 231) Cercospora

CZ09-2 Cercospora

CZ10-85 (UQ 3122) Cercospora

CZM09-1 Cercospora

CZM10-90 (BPF 1332) Cercospora

CZM10-75 (BRD 3411) Cercospora

DQ185085 C.zeae-maydis USA

DQ185090 C.zeae-maydisUSA








EU569212| C.zeina-SA CMW25454

DQ185093 C.zeina South Africa


DQ185094 C.zeina USA


EU569217C.zeina-Zambia CMW25445

EU569210.1| C.zeina-SA CMW25462



EU569218| C.zeina-Zambia CMW25467

EU569216|C.zeina-Zimbabwe CMW25442

100

98

100

62

99

77

82

GLS

SA

GLS

US

AG

LS N

iger

ia

•GLC agent in Nigeria is different from Southern African and USA

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DNA bank

DNA bank established and presently 306 type isolates of various pathogens are held in the ‘DNA bank’.

4X. axonopodis pv glycinea

2X. campestris pv vignicola

2 (Banana tissue culture contaminants)Enterobacter

19F. subglutinans

9F. oxysporum

12F. verticillioides

135C. zea-maydis

123C. gloeosporioides

Type isolate held in DNA bankPathogen

www.iita.org

M Y C Co S M B Mg O F

650500

B

3000

10000

A

Tuber Seed Leaf Fungi

rDNA amplification

DNA

2.141.89379.8CZM10-50

0.851.88151.96CZM10-46

0.181.2818.16CZM10-45

1.741.94299.06CZM10-30

1.431.89838.54CZM10-18

1.481.9862.33CZM10-12

0.741.371176.24CZM10-1

1.251.92766.98Maize

0.741.691581.92Soybean

0.551.61956.86Cowpea

A260/230A260/280concentrationSpecies

DNA Quality with High through put DNA protocol for leaves, seeds, tubers and fungi

0.4 ±0.41.7 ±0.21860.9 ±548.9Fungi

1.0 ±0.11.8 ±0.1851.9 ±28.3Okara

0.3 ±0.31.9 ±0.0917.4 ±30.0Mango

0.7 ±0.91.9 ±0.14417.0 ±334.3Banana

0.9 ±0.11.8 ±0.1870.3 ±32.4Maize

0.6 ±0.51.6 ±0.43896.6 ±522.1Soybean

0.7 ±0.61.7 ±0.24538.4 ±232.7Cowpea

0.4 ±0.31.7 ± 0.2506.5 ±36.2Cassava

0.5 ±0.41.8 ±0.01147.9 ±117.0Yam

A260/230A260/280concentration (ng/µl)

Species

High throughput DNA extraction protocol

www.iita.org

A

500650

M S1 S2 S3 S4 S5

E

M 1 M 1

F

400300

M CL CS CT

D

1 2 3 4 5 6 M

500650

G

ITS BSV

ACMV

ITS Specific

C. gloeosporioides

AC

MV

det

ectio

n fro

m F

TA c

ard

MS

V d

etec

tion

from

M

aize

leaf

Banana BSV

Versatile sample preparation protocol for PCR-based diagnosis

Sharma et al., 2010

www.iita.org

• ELISA have been proven to be easy for adoption in developing countries.

Low-cost aflatoxin kit

• Further modifications underway to reduce the cost and toxicity

Afla-ELISATM is a simple and low-cost test for quantitative estimation of

aflatoxins developed at IITA.

Kumar et al., 2008

www.iita.org

Quantification of aflatoxins in different commodities

0.000

0.200

0.400

0.600

0.800

1.000

1.200

1.400

1.600

1.800

1 2 3 4 5 6 7 8 9

Log AfB1(ng/ml)

A40

5nm

millet

sorghum

ground nut

cassava

rice

maize

Buffer

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Afla-ELISA

Trade Mark Registration

TM

Next step: Product registration

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• Training programs in surveillance and virus disease diagnosis.

Capacity building

• Strengthening physical capacity of one laboratory per country;

www.iita.org

Capacity building

Regional Training for the Disease Objective of GLCICassava Viruses: Biology, Diagnostics and Management

28 October – 6 November 2009, Tanzania

www.iita.org

Outreach

Workshop in the 3rd Annual Nigerian Mycotoxin Awareness and Study Network (NMASN) Conference, 29 April 2008,Standards Organization of Nigeria (SON), Lagos

www.iita.org

Outreach

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• Accounting all constraints in the farmers fields is necessary tosustain yield gains achieved by controlling pests/diseases/abiotic stress

Climate Change

Biotic stresses Abiotic stresses

Habitat change

Conclusions

Our R4D approach is competitive to out compete viruses

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Staff / Consultants• Sharma • Patricia• Taiwo• Ronke• Wale• Jimo• Opy• Ayodele

Ex-staff/students• Segun• Razaq• Ezeri• Tom• Oby• Buki

Students• Femi• Time• Ogunsola • Salaudeen• Mary K• Toually M• Asala• Edward

GIS-Unit

Acknowledgements

Winning the race against virus threats to food crops in sub-Saharan Africa

Technology

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