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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
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

•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.)
                                                                     www.iita.org
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




                                                                     www.iita.org
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)



                                                                    www.iita.org
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

                                                                                                 www.iita.org
CBSV: R4D status


Concept                                             R4D stage at IITA
•   Characterization                                Advance
•   Genome sequencing                               Advance
•   Diagnostics                                     Advance
•   Transmission                                    Advance
•   Epidemiology (host-virus interaction)           Advance
•   Conventional Resistance                         Advance
                                                                Ham-1
•   Transgenics                                     Developmental stage
•   Marker-assisted breeding                        Inception
•   Phytosanitation / awareness / training etc      Adequate




                                                                        www.iita.org
CBSV: unique virus




         Ham-1




        Source: S Winter, 2008




                      www.iita.org
CBSV inter-relationships

Ug    Ke

Tz
                       100 FN433933 CBSV Ma 43 2007 (Malawi:Salima)
                      79   FN433932 CBSV-Ma 42 2007 (Malawi:Chit...




                                                                                                           UG, Ken, Mal
                                                                            92-95%
                      61
                           FN434109 CBSV-Ug 23 (full sequence) 2...




                                                                                           86-87%
                     70
                           FN433930 CBSV Kenya 125 1999 (Kenya:K...
  M                 100




                                                                                                       70-71%
                           FN433931 CBSV-Ke 54 1997 (Kenya:Kilifi)
              100
                           FJ185044. CBSV-Uganda (2006)
                           NC 012698 CBSV isolate MLB3 full geno...
                                 FN434437 CBSV-Tan 70 (full sequence) ...




                                                                                              79-80%


                                                                                                              Tz, Moz
                                 FN434436 CBSV-Mo 83 (full sequence) 2...




                                                                                     96%
              100
                          100    GQ329864 CBSV-Tz (full sequence) 200...
                                 NC 006941 CVYV


0.1
           NJ Tree of full-length CBSV genomes
                                                                                           www.iita.org
CBSV inter-relationships
                                     62      FN434109 Uganda Ug-23
                               97            EU916829 Uganda (LWR2)
                         100                 EU916828 Uganda (HMA9)
                                             EU916830 Uganda (IGA8)
Ug   Ke             87
                                             EU916827 Uganda_Nam (NTG10)
                                     87
               57
                                             EU916832 Uganda-Busia (BSA4)




                                                                                91-100%
                                             EU916831 Uganda-Busia (BSA2)
Tz                                   71      FJ185044 Uganda-2006
                                             FN433932 Malawi 2007 (Chitipa)
          99                         99      FN433933 Malawi 2007 (Salima)
                                             FN433930 Kenya 125 1999




                                                                                             69-71%
                                             EU916826 Tanzania (MLB9)

                               100
                                             NC 012698 Tanzania MLB3
 M                                   100     EU916825 Tanzania (MLB3)
                                             FN433931 Kenya 54 1997 (Kilifi)
                                     65      FN434436 Moza Mo-83
                                             GQ329864 Tanzania
                                             FN434437 Tanzania- Tan70
                                             AY008440 Tanzania (type C)




                                                                               91-100%
                                     99
                                             AY007597 Tanzania TZC2
                    98
                                             AY008441 Tanzania (type B)
                                     94      FJ821794 Tanzania (KBH2)
                                             FJ821795 Tanzania (KBH1)
                         95                  AY008442 Tanzania (type A)
                                             AF311053 Mozambique MZQ1
                               75
                                     97      AF311052 Mozambique MZQ2
                                             NC 006941 CVYV

          NJ Tree of full-length CBSV coat protein gene                                   www.iita.org
Symptom diversity




Source: MN Maruthi, NRI-UK
                                         www.iita.org
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



                                                               www.iita.org
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.

                                                                                                      www.iita.org
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.



                                                                                 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

                               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.
                                                                           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 Epidemiology
      J 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
CBSV Vector(s) ?




Whitefly           Spiraling whitefly
(Bemisia tabaci)   (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


Experimental Layout




    5            4               3                2    1       Spreader
                      Initially CBSD-free plots




                                                      Prevailing Wind




                                                                        www.iita.org
CBSD incidence (%)
         12
            /    29
                    /2




                              0
                                  10
                                       20
                                            30
                                                   40
                                                        50
                                                             60
                                                                   70
                                                                                80
                                                                                                 90
                                                                                                      100
               1/ 008
                 12
                    /2
               1/ 009
                 26
                    /2
                       0
                2/ 09
                  9/
                     2
               2/ 009
                 23
                    /2
                       0
                3/ 09
                  9/
                     2
               3/ 009
                 23
                    /2
                       0
                4/ 09
                  6/
                     2
               4/ 009
                 20
                    /2
                       0
                5/ 09
                  4/
                     2
               5/ 009
                 18
                    /2
                       0
                6/ 09
                  1/
                     2
               6/ 009
                 15
                    /2
               6/ 009
                 29
                                                                        Incidence




                    /2
               7/ 009
                 13
                                                                                     New infection




                    /2
               7/ 009
                 27
                    /2
               8/ 009
                 10
                    /2
                       00
                          9
                              0
                                       5
                                              10
                                                        15
                                                                  20
                                                                                    25
                                                                                                      30
                                                                                                            CBSD Disease Progress - Kibaha




www.iita.org
CBSD, Whiteflies - Kibaha




                     www.iita.org
CBSD/whitefly relationship
                     16                                                                 2.5
                               Whitefly abundance 3 wks previous
                     14        CBSD transformed infection




                                                                                              CBSD transformed infection
                                                                                        2
                     12
Whitefly abundance




                          y = 0.048x – 0.018

                     10   (F = 13.3; r2 = 0.63; P < 0.001)                              1.5

                     8

                                                                                        1
                     6

                     4
                                                                                        0.5
                     2

                     0                                                                  0
                          1         2          3             4          5   6   7   8
                                                                 Week                   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     Variety   Plants   CBSD   CBSV   W’fly   CMD
           treatment                       (%)                    (%)
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.org
Whitefly-treated Njule   Whitefly-infested Njule




Photo: G. Okao-Okuja                               www.iita.org
Cassava Virus Spread, Whitefly Abundance
                                               cv. Liongo Kwimba - Tanzania

                            0.6                                                             180
                                       CBSD
                                       CMD                                                  160
                            0.5        Whiteflies
                                                                                            140
Newly Diseased Proportion




                            0.4                                                             120




                                                                                                  Whitefly abundance
                                                                                            100
                            0.3
                                                                                            80

                            0.2                                                             60

                                                                                            40
                            0.1
                                                                                            20

                             0                                                              0
                                   1       2        3        4         5        6   7   8
                                                        Months after Planting
Cassava Virus Spread, Whitefly Abundance
            cv. Njule - Uganda
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
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
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
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.



                                                                         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
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.




                                                                     www.iita.org
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
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             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
‘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
Training & Capacity




•Development of surveillance protocols

•Training in field surveillance

                                                       www.iita.org
Regional Training for the Disease Objective of GLCI
Cassava Viruses: Biology, Diagnostics and Management
      28 October – 6 November 2009, IITA, Dar es Salaam, Tanzania

                                                                    www.iita.org
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)




                                                      www.iita.org
Thank you




            www.iita.org

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  • 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) www.iita.org
  • 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 www.iita.org
  • 4. Cassava brown streak disease www.iita.org
  • 7. Cassava brown streak disease CMD CBSD CMD+CBSD www.iita.org
  • 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.) www.iita.org
  • 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 www.iita.org
  • 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) www.iita.org
  • 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 www.iita.org
  • 12. CBSV: R4D status Concept R4D stage at IITA • Characterization Advance • Genome sequencing Advance • Diagnostics Advance • Transmission Advance • Epidemiology (host-virus interaction) Advance • Conventional Resistance Advance Ham-1 • Transgenics Developmental stage • Marker-assisted breeding Inception • Phytosanitation / awareness / training etc Adequate www.iita.org
  • 13. CBSV: unique virus Ham-1 Source: S Winter, 2008 www.iita.org
  • 14. CBSV inter-relationships Ug Ke Tz 100 FN433933 CBSV Ma 43 2007 (Malawi:Salima) 79 FN433932 CBSV-Ma 42 2007 (Malawi:Chit... UG, Ken, Mal 92-95% 61 FN434109 CBSV-Ug 23 (full sequence) 2... 86-87% 70 FN433930 CBSV Kenya 125 1999 (Kenya:K... M 100 70-71% FN433931 CBSV-Ke 54 1997 (Kenya:Kilifi) 100 FJ185044. CBSV-Uganda (2006) NC 012698 CBSV isolate MLB3 full geno... FN434437 CBSV-Tan 70 (full sequence) ... 79-80% Tz, Moz FN434436 CBSV-Mo 83 (full sequence) 2... 96% 100 100 GQ329864 CBSV-Tz (full sequence) 200... NC 006941 CVYV 0.1 NJ Tree of full-length CBSV genomes www.iita.org
  • 15. CBSV inter-relationships 62 FN434109 Uganda Ug-23 97 EU916829 Uganda (LWR2) 100 EU916828 Uganda (HMA9) EU916830 Uganda (IGA8) Ug Ke 87 EU916827 Uganda_Nam (NTG10) 87 57 EU916832 Uganda-Busia (BSA4) 91-100% EU916831 Uganda-Busia (BSA2) Tz 71 FJ185044 Uganda-2006 FN433932 Malawi 2007 (Chitipa) 99 99 FN433933 Malawi 2007 (Salima) FN433930 Kenya 125 1999 69-71% EU916826 Tanzania (MLB9) 100 NC 012698 Tanzania MLB3 M 100 EU916825 Tanzania (MLB3) FN433931 Kenya 54 1997 (Kilifi) 65 FN434436 Moza Mo-83 GQ329864 Tanzania FN434437 Tanzania- Tan70 AY008440 Tanzania (type C) 91-100% 99 AY007597 Tanzania TZC2 98 AY008441 Tanzania (type B) 94 FJ821794 Tanzania (KBH2) FJ821795 Tanzania (KBH1) 95 AY008442 Tanzania (type A) AF311053 Mozambique MZQ1 75 97 AF311052 Mozambique MZQ2 NC 006941 CVYV NJ Tree of full-length CBSV coat protein gene www.iita.org
  • 16. Symptom diversity Source: MN Maruthi, NRI-UK www.iita.org
  • 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 www.iita.org
  • 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. www.iita.org
  • 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. www.iita.org
  • 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. www.iita.org
  • 21. • 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
  • 22. CBSD Epidemiology J Legg www.iita.org
  • 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) www.iita.org
  • 24. CBSV Vector(s) ? Whitefly Spiraling whitefly (Bemisia tabaci) (Aleurodicus dispersus) www.iita.org
  • 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 www.iita.org
  • 26. CBSV Epidemiology Experimental Layout 5 4 3 2 1 Spreader Initially CBSD-free plots Prevailing Wind www.iita.org
  • 27. CBSD incidence (%) 12 / 29 /2 0 10 20 30 40 50 60 70 80 90 100 1/ 008 12 /2 1/ 009 26 /2 0 2/ 09 9/ 2 2/ 009 23 /2 0 3/ 09 9/ 2 3/ 009 23 /2 0 4/ 09 6/ 2 4/ 009 20 /2 0 5/ 09 4/ 2 5/ 009 18 /2 0 6/ 09 1/ 2 6/ 009 15 /2 6/ 009 29 Incidence /2 7/ 009 13 New infection /2 7/ 009 27 /2 8/ 009 10 /2 00 9 0 5 10 15 20 25 30 CBSD Disease Progress - Kibaha www.iita.org
  • 28. CBSD, Whiteflies - Kibaha www.iita.org
  • 29. CBSD/whitefly relationship 16 2.5 Whitefly abundance 3 wks previous 14 CBSD transformed infection CBSD transformed infection 2 12 Whitefly abundance y = 0.048x – 0.018 10 (F = 13.3; r2 = 0.63; P < 0.001) 1.5 8 1 6 4 0.5 2 0 0 1 2 3 4 5 6 7 8 Week www.iita.org
  • 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 www.iita.org
  • 31. Whitefly Control and CBSV Epidemiology Whitefly Variety Plants CBSD CBSV W’fly CMD treatment (%) (%) 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.org
  • 32. Whitefly-treated Njule Whitefly-infested Njule Photo: G. Okao-Okuja www.iita.org
  • 33. Cassava Virus Spread, Whitefly Abundance cv. Liongo Kwimba - Tanzania 0.6 180 CBSD CMD 160 0.5 Whiteflies 140 Newly Diseased Proportion 0.4 120 Whitefly abundance 100 0.3 80 0.2 60 40 0.1 20 0 0 1 2 3 4 5 6 7 8 Months after Planting
  • 34. Cassava Virus Spread, Whitefly Abundance cv. Njule - Uganda
  • 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 www.iita.org
  • 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 www.iita.org
  • 38. Disease Management: Host resistance Edward Kanju and Pheneas Ntawuruhunga www.iita.org
  • 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) www.iita.org
  • 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) www.iita.org
  • 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. www.iita.org
  • 43. 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
  • 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. www.iita.org
  • 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 www.iita.org
  • 47. New Varieties released in Zanzibar It takes 7 – 8 years to develop a new variety www.iita.org
  • 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 www.iita.org
  • 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) www.iita.org
  • 51. Training & Capacity •Development of surveillance protocols •Training in field surveillance www.iita.org
  • 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 www.iita.org
  • 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) www.iita.org
  • 54. Thank you www.iita.org