www.iita.org

Update on Cassava Brown Streak Disease (CBSD) Status, prospects and implications for cassava projects

R4D Week, R&T MTP, 24 November09

J Legg

E Kanju

P Ntawuruhunga

DJ Kim

M Ferguson

MN Maruthi (NRI-UK)Lava Kumar

www.iita.org

The Viruses of Cassava in Africa

Geminiviridae: Begomovirus

•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

Indian cassava mosaic virus*

EACMV-Uganda (Recombinant virus)

www.iita.org

The Viruses of Cassava in Africa

Poorly studied viruses

Cassava Ivorian bacilliform virus (Badnavirus)

Cassava Kumi virus

Cassava ‘Q’ virus

Cassava common mosaic virus* (Potexvirus)

Emerging virus

Cassava brown streak virus

Potyviridae: Ipomovirus

www.iita.org

Cassava brown streak disease

www.iita.org

www.iita.org

www.iita.org

Cassava brown streak disease

CMD CBSD CMD+CBSD

www.iita.org

CBSD: Pre-2000

1936: First report in northern Tanzania (Storey).

1950: CBSD reported to be endemic on East African coast and Malawi.

1995: Based on ‘pin-wheel’ inclusions potyvirus suspected

(Harrison et al.).

1995: Resistance/tolerance to CBSD identified in ‘local’ cassava

cultivars in Tanzania. e.g. 'Nachinyaya'.

1998: High CBSD incidences (70%) along the northern

Mozambique coast reported (Hillocks et al.)

•Considered as ‘locally’ important problem

www.iita.org

2000: Causal agent of CBSD identified as a potyvirus of genera Ipomovirus

by a research group at Bristol University, UK (Monger et al.)

2001-02: CBSV coat protein gene sequenced and RT-PCR-based diagnostic

tool established for CBSV detection

2003: Successful transmission of CBSD achieved with whitefly, Bemisia

tabaci (Maruthi et al.)

2004: Emergence of CBSD in Uganda (Alicai et al.) and Lake Zone area of

Tanzania (Legg et al.), the first major outbreak at high altitudes

CBSD: In new millennium

• High incidence of CBSD in highlands

• Increase in research efforts

www.iita.org

CBSD in epidemic era!

2009

• CBSV full genome sequence published (Mbazibwa et al.)

• Occurrence of at least two strains of CBSV reported (Mbazibwa et al.)

• Development of virus-resistant transgenic lines in N. benthamiana (Danforth)

• Report of Spiraling whitefly as CBSV vector in Kenya (unpublished)

2007 on wards: major initiatives to combat CBSD

2008

-Extensive disease incidence surveys initiated by (IITA)

-Progress on resistance breeding and selections (IITA)

-Development of improved RT-PCR diagnostics (many labs)

-Development of monoclonal antibodies and ELISA (Winter et al.)

www.iita.org

CBSD

CBSD

Control

Viral genes

(RNAi)

Transgenic

resistance

Biochemical,

molecular &

Biological

Properties

CBSV isolation

CBSV characterization

Bioassays

Serological &

Nucleic-acid assays

Diagnostic toolsVirus isolatesQuarantine

Awarness

Training

Monitoring

Landraces and

hybrids

Vector biology

Virus survival and spread

Environmental factors

Germplasm screening

Disease Epidemiology

Resistant

Varieties

Towards sustainable CBSD Control

Plant BreedingConventional & MAS

Virology / Pathology / Extension / NPPOs

Breeding / Agronomy / Extension(Conventional / MAS / transgenics)

www.iita.org

CBSV: R4D status

Ham-1

Concept R4D stage at IITA

• Characterization Advance

• Genome sequencing Advance

• Diagnostics Advance

• Transmission Advance

• Epidemiology (host-virus interaction) Advance

• Conventional Resistance Advance

• Transgenics Developmental stage

• Marker-assisted breeding Inception

• Phytosanitation / awareness / training etc Adequate

www.iita.org

CBSV: unique virus

Source: S Winter, 2008

Ham-1

www.iita.org

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

100

79

61

70

100

100

100

100

0.1

UG

, Ken

, Mal

Tz,

Mo

z

92-9

5%

86-8

7%

96%

79-8

0%

70-7

1%

M

KeUg

Tz

NJ Tree of full-length CBSV genomes

CBSV inter-relationships

www.iita.org

FN434109 Uganda Ug-23

EU916829 Uganda (LWR2)

EU916828 Uganda (HMA9)

EU916830 Uganda (IGA8)

EU916827 Uganda_Nam (NTG10)

EU916832 Uganda-Busia (BSA4)

EU916831 Uganda-Busia (BSA2)

FJ185044 Uganda-2006

FN433932 Malawi 2007 (Chitipa)

FN433933 Malawi 2007 (Salima)

FN433930 Kenya 125 1999

EU916826 Tanzania (MLB9)

NC 012698 Tanzania MLB3

EU916825 Tanzania (MLB3)

FN433931 Kenya 54 1997 (Kilifi)

FN434436 Moza Mo-83

GQ329864 Tanzania

FN434437 Tanzania- Tan70

AY008440 Tanzania (type C)

AY007597 Tanzania TZC2

AY008441 Tanzania (type B)

FJ821794 Tanzania (KBH2)

FJ821795 Tanzania (KBH1)

AY008442 Tanzania (type A)

AF311053 Mozambique MZQ1

AF311052 Mozambique MZQ2

NC 006941 CVYV

97

94

99

75

95

65

98

100

100

99

71

62

97

87

100

87

57

99 91-1

00%

69-7

1%

91-1

00%

M

KeUg

Tz

NJ Tree of full-length CBSV coat protein gene

CBSV inter-relationships

www.iita.org

Symptom diversity

Source: MN Maruthi, NRI-UK

www.iita.org

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 (species complex!)

CBSV and CMBVs are complex, often necessitating multiple tests.

CBSD Diagnostics

www.iita.org

Lanes 1 to 4: CBSV infected samples

Lane 5: Healthy cassava

Lane 6: CMD infected cassava

Lane M: Molecular weight marker (100 kb ladder)

Multiplex PCR for CBSV & CMBV

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

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

CBSV-D1/D2 + CMB

Sap DNA & RNA

EACMV

ACMV

• Primers CBSV-S1/S2 and CBSV-L1/L2

resulted in expected product size in

multiplex assay with CMBV primers.

• But, CBSV-D1/D2 did not result in

amplification.

www.iita.org

Ten fold serial dilution of sap extract (1:20 w/v) up to 10-10 dilution (v/v).

•CBSV detected consistently in sap diluted up to 10-3.

•Sometimes detection obtained even at 10-6 dilution.

CBSV Sensitivity assay

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

M 1 2 3 4 5 6 7 8 9 10 11M 1 2 3 4 5 6 7 8 9 10 11

Lane1 = undiluted sap; Lane 2 = 1:10; Lane 3 = 1:100; Lane 4 = 1:1000 (v/v)

www.iita.org

CBSV Composite Assay

•Sap extracted from composite sample comprising of 9 healthy leaves and one

infected leaf

•Sap extract from 100 mg tissue (1:20 w/v) used as template for RT-PCR

•Sap extract (1:20 w/v) from composite sample in 10 fold serially diluted up

to 10-10 dilution. CBSV detected in sap diluted up to 10-6.

CBSV-L1/L2 + CMB

M 1 2 3 4 5 6 7 8 9 1 0 1 2 3 4 5 6 7 8 9 10 M

Sample 1 Sample 2

www.iita.org

• Adequate ‘basic’ knowledge and technologies are

available that can contribute to studies on CBSD

epidemiology and development of host resistance and

more.

www.iita.org

CBSD EpidemiologyJ Legg

www.iita.org

CBSV epidemiolgy/

transmission studies

• Components

– Macro epidemiology. Through use of historical and new surveillance data from East/Central Africa

– Micro CBSV epidemiology CBSV, B. tabaci population dynamics trials – Kibaha (Tz)

– B. tabaci control trials – Ukerewe (Tz) and Namulonge (Ug)

– B. tabaci transmission studies – Kibaha and NRI

– Alternative host and alternative vector studies

(J. Ndunguru, ARI Mikocheni, Tanzania)

www.iita.org

Whitefly

(Bemisia tabaci)

CBSV Vector(s) ?

Spiraling whitefly

(Aleurodicus dispersus)

www.iita.org

‘Micro’ CBSV Epidemiology/

B. tabaci population dynamics

• Planting Material. Visually CBSD-free cv. Kiroba from

relatively unaffected Mkuranga District

• Virus Testing. Leaves from all parent stems tested for

CBSV using RT-PCR. Only negatives used

• Planting. November. In four separate locations at Kibaha

isolated from other cassava fields

• Data Collected. Weekly records of CBSD incidence,

severity and whitefly adult abundance. Samples collected

www.iita.org

CBSV Epidemiology

5 4 3 2 1 Spreader

Experimental Layout

Prevailing Wind

Initially CBSD-free plots

www.iita.org

CBSD Disease Progress - Kibaha

0

10

20

30

40

50

60

70

80

90

100

12/2

9/20

08

1/12

/200

9

1/26

/200

9

2/9/

2009

2/23

/200

9

3/9/

2009

3/23

/200

9

4/6/

2009

4/20

/200

9

5/4/

2009

5/18

/200

9

6/1/

2009

6/15

/200

9

6/29

/200

9

7/13

/200

9

7/27

/200

9

8/10

/200

9

CB

SD

in

cid

en

ce

(%

)

0

5

10

15

20

25

30

New infection

Incidence

www.iita.org

CBSD, Whiteflies - Kibaha

www.iita.org

CBSD/whitefly relationship

0

2

4

6

8

10

12

14

16

1 2 3 4 5 6 7 8

Week

Wh

itefl

y a

bu

nd

an

ce

0

0.5

1

1.5

2

2.5

CB

SD

tra

nsfo

rmed

in

fecti

on

Whitefly abundance 3 wks previous

CBSD transformed infection

y = 0.048x – 0.018

(F = 13.3; r2 = 0.63; P < 0.001)

www.iita.org

Whitefly Control and

CBSV Epidemiology

• Experimental Locations. High CBSD pressure locations in Uganda

(Namulonge) and Tanzania (Ukerewe Island)

• Planting Material. Visually CBSD-free cvs. TMS I92/0057 and Njule

(Uganda) and TMS I92/0057 and Liongo Kwimba (Tanzania) collected from

CBSD-unaffected locations

• Planting. December 2008. Replicated trial at each site

• Treatments. Whitefly controlled in ‘whitefly-free’ plots through combined use

of Imidacloprid (soil drench) and Cypermethrin (foliar application)

• Virus Testing. Samples collected from 10 plants per plot for each site in May

(Tanzania) and June (Uganda)

• Data Collected. Monthly records of CBSD incidence, severity and whitefly

adult abundance

www.iita.org

Whitefly Control and CBSV

Epidemiology

Whitefly

treatment

Variety Plants CBSD

(%)

CBSV W’fly CMD

(%)

Uganda Treated I92/0057 163 53.4 - 3.4 6.8

Untreated I92/0057 165 95.8 - 127.6 25.5

Treated Njule 165 50.9 - 2.0 13.3

Untreated Njule 167 97.6 - 113.4 100

Tanzania Treated I92/0057 60 86.7 22.5 0.6 0

Untreated I92/0057 62 90.3 45 94.9 0

Treated Liongo 58 69 17.5 0.6 58.6

Untreated Liongo 48 100 67.5 87.2 97.9

www.iita.orgPhoto: G. Okao-Okuja

Whitefly-treated Njule Whitefly-infested Njule

0

0.1

0.2

0.3

0.4

0.5

0.6

1 2 3 4 5 6 7 8

Months after Planting

Ne

wly

Dis

ea

se

d P

rop

ort

ion

0

20

40

60

80

100

120

140

160

180

Wh

itefl

y a

bu

nd

an

ce

CBSD

CMD

Whiteflies

Cassava Virus Spread, Whitefly Abundancecv. Liongo Kwimba - Tanzania

Cassava Virus Spread, Whitefly Abundancecv. Njule - Uganda

www.iita.org

Whitefly Control and CBSV

Epidemiology - Observations

• Planting material. Can’t be 100% sure of CBSV-free

status. Have established isolated CBSD-free multiplication

sites in the Usambara Mountains, eastern Tanzania

• Diagnostics. Cheaper and more reliable methods

essential for further work

• Results. Suggest an association between CBSD and

whitefly populations, contrasting with CMD

• Transmission. Results suggest whitefly transmission

through a non-persistent mechanism

• Climate interaction. Appears that disease will only be

expressed when climatic conditions are favourable –

drought and high temperature

www.iita.org

Digital Early Warning Network

(DEWN) Piloting in Tanzania

August 2009 September 2009

www.iita.org

Smart Surveillance

Systems

Digital Forms

Digital Images Digital Maps

www.iita.org

Disease Management:

Host resistance

Edward Kanju and Pheneas Ntawuruhunga

www.iita.org

Disease Management:

Host resistance

• Training: technicians on cassava grafting to test highly promising

clones for resistance to CBSD

• Grafting trials: established using clones that have remained disease

symptom free

• Multiplication: promising breeding lines

• Technical backstopping: of NARS on field screening of cassava clones

for resistance/tolerance to major diseases and pests (through PVS)

• Regional exchange: improved germplasm (with KEPHIS)

www.iita.org

Germplasm that respond to end-users’ needs

Enabling Technologies

Traits

Vectors

Promoters

Selectable markers

Dry matter yield/Unit/Time;

Disease/pest resistance

Nutritional and end users quality

Stress (drought) tolerance

Cassava improvement goal :To develop varieties

which combine high and stable yield with good

quality characteristics for end users

The most effective and realistic approach to

reducing losses to CMD and CBSD is the use of

host-plant resistance or deployment of less-

susceptible cultivars

www.iita.org

Grafting Trial Tanzania

• Seven participants were trained in germplasm screening.

• The following clones/breeding lines were grafted at ARI Chambezi (Bagamoyo district) and ARI Naliendele (Mtwara district):– KBH 06/98

– KBH 02/066

– KBH 02/363

– KBH 01/110

– KBH 06/18

– KBH 06/12

• > 80% successful grafts

• The clones are being multiplied at ARI Chambezi (4 are tested on-farm)

www.iita.org

CBSD Disease

index

Group No. genotypes

[Set 1 and 2]

Percentage

(%)

0 Resistant 410 42.6

0 – 20 Moderately resistant 172 17.9

>20 – 57.5 Moderately susceptible 186 19.3

>57.5 – 150 Susceptible 118 12.3

>150 Highly susceptible 77 8.0

• Performance of 963 cassava genotypes under high CBSD pressure at

Namulonge (Uganda) for 3 years.

• From further screening in 2008, 15 best genotypes selected for

dissemination through GLCI project.

Promising clones

www.iita.org

Promising clones

Ref.# Clone Quantity Ref.# Clone Quantity

1 MM06/0135 8 8 MM06/0131 8

2 MM06/0124 4 9 MM06/0012 5

3 MM06/0076 2 10 MM06/0019 6

4 MM06/0139 5 11 MM06/045 3

5 MM06/0013 3 12 MM06/0112 4

6 MM06/0011 3 13 MM06/0023b 4

7 MM06/0024 5 14 MM06/0079 9

www.iita.org

• A second screening trial conducted from botanical seeds developed

at IITA-Tanzania (Amani sources).

• From this, 8 best clones have been identified from Mukono trials

which were identified and selected also by farmers.

CMD and CBSD resistant cassava

• In total 52 genotypes have been selected as best potential resistant

clones to both CBSD and CMD. These have been transferred to

KEPHIS for cleaning, and tissue culture for exchange with NARS.

www.iita.org

Seedlings are evaluated

for their reaction to

diseases and pests.

Clones selected are

advanced for screening

for further activities in

different agro-ecologies

Seedlings

Transplantation

Crosses produce

seeds

Cassava Breeding

Scheme

www.iita.org

CBSD tolerant clones for Burundi

360 seedlings from

Tanzanian CBSD

tolerant parents

www.iita.org

New Varieties released in Zanzibar

It takes 7 – 8 years to develop a new variety

www.iita.org

Dual resistant clones in Uganda?

Clone Pedigree

1. MM

06/0013

Kitumbua-OP

2. MM

06/0046

Kigoma Red-

OP

3. MM

06/0074

Kigoma Red-

OP

4. MM

06/0082

Kibaha-OP

5. MM

06/0083

Kibaha-OP

6. MM

06/0138

Kibaha-OP

7. MM

06/0139

Kibaha-OP

Eight clones have remained CMD and CBSD symptom-free under

high pressure for three seasons. They will be challenged by grafting

www.iita.org

‘Biotechnology Applications to Combat CBSD’

Agreement signed: Nov 5th 2009

Implementing agency: IITA

Partner institutes: NARO, ARI –Tanzania

Collaborating institutes: DDPSC, BecA, ILRI

Budget: Approx $2.4m over four years

Associated project: Cassava Genomics: bridging the gap between

sequence and breeding applications

www.iita.org

Research Objectives

1. Improve existing markers for one source of CBSD

resistance/tolerance (SNP development and fine

mapping in Namikonga)

2. Develop new markers and breeding resources for six

new sources of resistance/tolerance to CBSD

3. Utilize markers in Tanzania and Uganda to begin

breeding new CBSD-resistant cultivars suitable for

the region (exit strategy)

4. Test a transgenic approach to controlling CBSD and

move the most promising transgene into a popular

Ugandan cultivar (CFT and transformation of

Ugandan varieties)

www.iita.org

•Development of surveillance protocols

•Training in field surveillance

Training & Capacity

www.iita.org

Regional Training for the Disease Objective of GLCI

Cassava Viruses: Biology, Diagnostics and Management28 October – 6 November 2009, IITA, Dar es Salaam, Tanzania

www.iita.org

• Consolidation and coordination of existing efforts

• Upstream research to address recalcitrant issues (eg. Combine resistance to CMD, CBSD and whitefly)

• Framework for improved cassava germplasm

distribution to partners (MTA / sMTA / IP Issues)

Future priorities

www.iita.org

Thank you