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Update on Cassava Brown Streak Disease (CBSD) Status, prospects and implications for cassava projects
1. Update on Cassava Brown Streak Disease (CBSD)
Status, prospects and implications for cassava projects
J Legg
E Kanju
P Ntawuruhunga
DJ Kim
M Ferguson
Lava Kumar MN Maruthi (NRI-UK)
R4D Week, R&T MTP, 24 November09
www.iita.org
2. 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)
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3. 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
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8. CBSD: Pre-2000
•Considered as ‘locally’ important problem
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.)
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9. CBSD: In new millennium
• High incidence of CBSD in highlands
• Increase in research efforts
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
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10. CBSD in epidemic era!
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.)
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)
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11. Towards sustainable CBSD Control
Breeding / Agronomy / Extension
(Conventional / MAS / transgenics)
Plant Breeding
Transgenic
CBSD resistance
Conventional & MAS
Viral genes Resistant
(RNAi) Varieties
CBSV isolation
Biochemical, Germplasm screening CBSD
molecular & CBSV characterization Landraces and Control
Biological hybrids
Properties
Diagnostic tools Disease Epidemiology Monitoring
Virus isolates
Quarantine
Bioassays Vector biology
Awarness
Serological & Virus survival and spread
Environmental factors Training
Nucleic-acid assays
Virology / Pathology / Extension / NPPOs
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17. CBSD Diagnostics
CBSV and CMBVs are complex, often necessitating multiple tests.
Cassava brown streak virus
•Sequence information points to divergent types (species complex!)
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
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18. Multiplex PCR for CBSV & CMBV
CBSV-S1/S2 + CMB CBSV-L1/L2 + CMB
Sap DNA & RNA Sap DNA&RNA
M1 2 3 4 5 6 7 1 2 34 5 6 7 M1 2 3 4 5 6 7 1 2 34 5 6 7 M
EACMV
Lanes 1 to 4: CBSV infected samples
ACMV Lane 5: Healthy cassava
Lane 6: CMD infected cassava
CBSV
Lane M: Molecular weight marker (100 kb ladder)
CBSV-D1/D2 + CMB
Sap DNA & RNA
• Primers CBSV-S1/S2 and CBSV-L1/L2
M1 2 3 4 5 6 7 1 2 3 4 5 6 7 M
resulted in expected product size in
EACMV multiplex assay with CMBV primers.
ACMV
• But, CBSV-D1/D2 did not result in
amplification.
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19. CBSV Sensitivity assay
CBSV-L1/L2 + CMB CBSV-S1/S2 + CMB
M 1 2 3 4 5 6 7 8 9 10 11 M 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)
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.
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20. 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
CBSV-L1/L2 + CMB
Sample 1 Sample 2
M 1 2 3 4 5 6 7 8 9 1 0 1 2 3 4 5 6 7 8 9 10 M
•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.
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21. • Adequate ‘basic’ knowledge and technologies are
available that can contribute to studies on CBSD
epidemiology and development of host resistance and
more.
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23. 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)
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25. ‘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
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30. 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
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35. 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
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36. Digital Early Warning Network
(DEWN) Piloting in Tanzania
August 2009 September 2009 www.iita.org
37. Smart Surveillance
Systems
Digital Forms
Digital Images Digital Maps
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38. Disease Management:
Host resistance
Edward Kanju and Pheneas Ntawuruhunga
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39. 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)
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40. Cassava improvement goal :To develop varieties
which combine high and stable yield with good
quality characteristics for end users
Dry matter yield/Unit/Time;
Disease/pest resistance
Traits Nutritional and end users quality
Stress (drought) tolerance
Vectors
Enabling Technologies Promoters
Selectable markers
Germplasm that respond to end-users’ needs
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
41. 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)
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42. Promising clones
• Performance of 963 cassava genotypes under high CBSD pressure at
Namulonge (Uganda) for 3 years.
CBSD Disease Group No. genotypes Percentage
index [Set 1 and 2] (%)
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
• From further screening in 2008, 15 best genotypes selected for
dissemination through GLCI project.
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44. CMD and CBSD resistant cassava
• 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.
• 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.
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45. Crosses produce
seeds
Cassava Breeding
Transplantation
Scheme
Seedlings are evaluated
for their reaction to
diseases and pests.
Clones selected are
advanced for screening
for further activities in
different agro-ecologies
Seedlings www.iita.org
46. CBSD tolerant clones for Burundi
360 seedlings from
Tanzanian CBSD
tolerant parents
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47. New Varieties released in Zanzibar
It takes 7 – 8 years to develop a new variety
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48. Dual resistant clones in Uganda?
Clone Pedigree
1. MM Kitumbua-OP
06/0013
2. MM Kigoma Red-
06/0046 OP
3. MM Kigoma Red-
06/0074 OP
4. MM Kibaha-OP
06/0082
5. MM Kibaha-OP
06/0083
6. MM Kibaha-OP
Eight clones have remained CMD and CBSD symptom-free under
06/0138
high pressure for three seasons. They will be challenged by grafting
7. MM Kibaha-OP www.iita.org
49. ‘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
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50. 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)
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52. Regional Training for the Disease Objective of GLCI
Cassava Viruses: Biology, Diagnostics and Management
28 October – 6 November 2009, IITA, Dar es Salaam, Tanzania
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53. Future priorities
• 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)
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