Research on CBSV and CBSD. Variability of Cassava Brown Streak Disease Symptoms and the Relationship Between Virus Infection and Symptoms Expression in On-farm Cassava.Factors Affecting Disease Severity in Cassava brown streak virus (Potyviridae; Ipomovirus) Infected Plants

1. Cassava brown streak virus
studies
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2. CBSD
Background
Research
Other work
Future perspectives
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3. CBSD
Background
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4. Background CBSD
1890s, Tanzania (Wahlberg)
1930s, Tanzania (Storey)
CBSD limited to coastal/low altitude areas
2001, Causal organism- Cassava brown streak virus, ss +
RNA (Monger et al.)
Family;Potyviridae, Genus; Ipomovirus
2005, Transmitted by Bemisia tabaci ca. 2%
2005, Reports of the spread of disease/economic
damage in high altitude areas-Uganda
2006, Reports of symptoms in DRC,Zambia
2008, Angola? Burundi?
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5. Potyvirus genome strategy e.g. TEV
0 10 Kb
gRNA
5’ VPg poly(A) 3’
Polyprotein
?
MP HC-Pro Pro Rep Pol CP
* * *
For pathogen-mediated strategies for virus resistance (PTGS) sequence
data are needed www.iita.org

6. Field symptoms of CBSD
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7. 10 days later
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8. CBSD
Research
Survey
Detection/symptoms
Symptom reliability
Transmission
Lab/screenhouse/
field
Virus preps
Sampling strategies
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9. 1. Survey-CBSD and CBSV
further samples in TZ plus coastal
Kenya, Burundi and Rwanda
CBSD symptom analyses
CBSV sequence analyses
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10. CBSV/CBSD assessments
Major surveys in Tanzania in 2005 and 2006
Minor testing in coastal Kenya, Burundi and Rwanda in
2007
First surveys included evaluation of sampling storage
for RT-PCR tests
Evaluated the best type of tissue for sampling and
tissue dilution tests for larger samplings by RT-
PCR
RT-PCR using CBSV specific CP gene primers
200bp
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11. www.iita.org

12. www.iita.org

13. Diagnostic parameter Coast. Zone Lake Zone Southern Mean
(N = 27) (N = 36) Zone (N = 28) (N=91)
Foliar CBSD Incidence (%) 61.8 (100) 26 (70) 27 (93.3) 38.3
Foliar CBSD severity 2.8 (3.9) 2.5 (2.8) 2.6 (3.3) 2.6
Stem CBSD Incidence (%) 35.3 (87) 1.1 (20) 6.3 (56.7) 14.2
Stem CBSD Severity 2.9 (4.5) 2.0 (2.0) 2.4 (2.6) 2.41
Root CBSD Incidence (%) 57 (100) 18 (53) 33.1(90) 36.1
Root CBSD severity 3.7 (5) 3.3 (3.6) 2.5 (3.5) 3.2
Symptoms on leaf sample (%) 100 88.9 77.8 88.9
CBSV positive by RT-PCR 78.6 72.2 70.4 73.7
(%)
Average crop age (months) 8 7 12 9
B. tabaci per plant 2 24 1 9
CMD incidence (foliar) (%) 32.2 35 24.4 30.5
CMD severity 2.8 2.9 2.6 2.8
The maximum values recorded for each parameter is given in brackets. www.iita.org

14. TEV
SPMMV
911
AF52
AF53
986
AY40
AY41
1000
CVYV AY42
AY97
999 40 sequences
1000 KYCBSV
DQ837302 1000
DQ837303
DQ837304
0.1 changes 5508
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15. Conclusions
CBSD and CBSV found in all areas of
Tanzania and Kenya
Incidences and severity of CBSD had an
inverse correlation with the altitude
Sequence diversity is on the border of
speciation limits for CBSV
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16. 2. CBSV transmission experiments through
various inoculations to virus-free
material in controlled conditions with
Tanzanian CBSV isolates
(TC and meristem culture)
Cassava indicators
Backed up with laboratory indexing
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17. 1 2 3
6 5 4
7 8 9
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18. CBSV-Transmission experiments
A B 3
1 2
4 5 6 www.iita.org

19. Sap transmission of CBSV
Buffer A Buffer B
60
50
% CBSV-infected (RT-PCR)
40
30
20
10
0
3 4 5 6 7 8 9 10 16 24
-10 Weeks after inoculation
Figure 1. Relationship between inoculation buffers and time to CBSV
detection www.iita.org

20. Grafting experiments
140
% CBSV incidence RT-PCR
120
100
80
60
40
20
0
1 2 3 4 5 6 7
Weeks after grafting
Infected scion Infected rootstock
Scion
Rootstock
A B www.iita.org

21. Results
54 % (13/24 plants) inoculated with Buffer
B and 29 % (7/24 plants) inoculated with
Buffer A tested positive to CBSV.
Grafting techniques had 100 % transmission
efficiency.
CBSV-free scions onto CBSV-infected
rootstocks was the most efficient
transmission technique
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22. Ctd
CBSV was not transmitted through
infected root debris although 17 %
(4.5/27 plants) test plants on the
debris-soil mixture produced foliar
chloroses in veins.
Cutting tools and leaf harvesting led to
(6.6/30) 22 % and (1/15 plants) 7 %
transmission efficiency respectively.
None of 180 seedlings from CBSV-
infected plants had detectible CBSV.
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23. Conclusions
CBSV may be transmitted through sap, grafting,
cutting tools and leaf harvesting.
Graft inoculation with an infected rootstock is
the most efficient way to transmit the virus.
Transmission through CBSV-infected root debris
seems remote from the study.
For the first time, it was demonstrated that
agronomical practices have potential to
hasten the spread of CBSV which may lead to
new epidemics. www.iita.org

24. 3.Screen house experiment:
Cuttings collected from plants with diverse CBSD
symptoms planted in pots
Symptoms monitored for 12 months
4.Field experiment:
Representative cassava cultivars of the major types of
foliar and root symptoms were identified
Included Albert, Cheupe, Kibaha, Nachinyaya, Namikonga
and AR 49/2.
Grown in the 6 cv. x 3 reps. Total of 108 plants/cultivar at
SRI
Symptoms monitored and recorded monthly
Experiment duration; 2 years.
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25. Relationship btn CBSV & type of root symptoms
Lake zone Coastal zone Southern zone
35
31.91
30
CBSD incidence (%)
25
25.27
20
17.01
15
10.64
10
5
5.5
2.13 3.13 2.13
1.13 1.13
0 0 0 0 0 0
Positive Positive Negative Positive Negative
Brown mass Chalkish necrosis (%) Necrotic specks (%)
necrosis (%)
Root symptoms & RT-PCR test
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26. Symptomless &
negative
Test sample
Symptomless &
positive
Symptomatic &
negative
Symptomatic &
positive
0 20 40 60 80 100
% of total
Relationship between CBSV infections and symptoms on samples
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27. Lake zone Coastal zone Southern zone
18
16.05
16 14.81
14
CBSD incidence (%)
12.34
12
10 9.17
8.64
8
6.59 6.17
6.17 6.06
6
4 2.8
2.47 2.1 2.01
2 1.2 1.02 1.2 1.2
0
0
Positive Negative Positive Negative Positive Negative
Chlorotic blotches (%) Chlorotic spots (%) Vein chlorosis (%)
Symptom type & RT-PCR test
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28. Generation of virus –free cuttings
• None of the cuttings from the
diseased plants germinated disease
free.
• Only two plants from one diseased
mother plant (score 2) became
disease free
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29. Conclusions
CBSD symptoms are diverse
Careful observation should be made to correctly diagnose
the disease.
Newly established types of foliar and root symptoms of
CBSD may improve on the diagnosis.
Relationship between CBSV infection & symptom
expression was established.
The expression of CBSD foliar symptoms may not
necessarily indicate CBSV infection.
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30. Ctd.......
Not all symptomless plants are free from CBSV.
Detection of CBSV in symptomless plants complicates its
diagnosis and management.
The expression of CBSD foliar symptoms may not
necessarily indicate CBSV infection.
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31. 5.Variability of Cassava Brown Streak Disease
Symptoms and the Relationship Between Virus
Infection and Symptoms Expression in On-farm
Cassava
CBSD symptoms became complex and difficult to
comprehend under field conditions
Unknown relationship between infection with CBSV and
symptom expression
Unknown distribution of CBSV in various tissues of
infected plants
Farmer belief that CBSD-free plants may be regenerated
from diseased plants
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32. 6.Factors Affecting Disease Severity in Cassava brown
streak virus (Potyviridae; Ipomovirus) Infected Plants
Susceptibility of selected cultivars with time & age
Field experiment at SRI
Temperature using a growth chamber
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33. Low temperature High temperature
7 Linear (Low temperature) Linear (High temperature)
6 Y = 0.0906x + 1.2857
R2 = 0.7821
CBSD foliar severity
5
4
3
Y = 0.05x + 1.1333
2 R2 = 0.6172
1
0
0 10 20 30 40 50 60
Days after exposure
Effect of temperature on cv. Albert www.iita.org

34. Diseased &stressed Diseased &Irrigated
80 Linear (Diseased &stressed) Linear (Diseased &Irrigated)
70
No. of dead leaves (mean)
Y = 0.7683x - 8.3066
60 R2 = 0.9529
50
40
30
20
Y = 0.0253x - 0.1816
10
R2 = 0.635
0
0 20 40 60 80 100
Days after exposure
Effect of moisture stress on cv. Albert
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35. Conclusions
CBSD affected cultivars tends to adapt to new
environment.
Foliar CBSD symptoms are more apparent than stem
symptoms.
CBSD incidence and severity levels are cultivar specific &
increases with plants age.
Low temperature is critical to the survival of CBSD-
affected plants.
Moisture stress is also a vital determinant of CBSD
severity.
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36. 7. Viral preparations
Materials moved from
coastal Kenya to KEPHIS-
PQS Maguga in 2006-7 and
kept under quarantine
conditions
Research assistant trained
in purification methods at
BeCA
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37. www.iita.org

38. Viral prep.
Final density-centrifugation
sediment fractions are
analysed by PAGE and RT-
PCR -2007-8
No success in cassava (2
cultivars) or three
herbaceous indicators
Kiribiti mwezi analysed in
duplicate looks promising in
Nicotiana benthamiana
Dec 08
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39. 8.Sampling methods
TC free materials
CBSV materials
x cultivars x reps
9.Parts of plant best for sampling
CBSV infected plants at different
growth stages
x cultivars x reps
10. RNA extraction methods optimised www.iita.org

40. Take sample leaflet 2 or
three leaves from growing
point of the plant
Preserve over
silica gel or
between
newspaper strips
in sealed tubes
(do not over-stuff)
Tissue paper
Silica gel-blue
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41. 10-leaf sample
representing 10
plants
Punched sample grind directly in
PUNCH ‘fuge tube with tube pestle for
RNA extraction
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42. 10-leaf sample
representing 10 plants
10 lobes from 10
leaves
Scissors 10 strips from 10 lobes-grind in liq N2 for RNA
sample
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43. CBSD
Other work
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44. • Cassava genetic transformation for the
longevity of cassava virus resistance in
Tanzania
• Mikocheni Agricultural Research Institute
(MARI) under
Ministry of Agriculture, Food Security and Co-operatives
Government of The United Republic of Tanzania
• The Rockefeller Foundation funded
• IITA as partners; IITA-capacity building in cassava
transformation and biosafety training plus increasing the
laboratory facilities (Ingelbrecht & Herron)
• Tropical Crops Research Institute (TPRI) –additional partner
To be continued in 2009 by BMGates Foundation
44
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45. Application for Contained Laboratory and Greenhouse
Research on Cassava Transformation for the Longevity of
Cassava Virus Resistance in Tanzania
Submitted for review to
Ministry of Agriculture Food Security and Cooperatives
November 2006
Applicant:
Dr JOSEPH NDUNGURU
MIKOCHENI AGRICULTURAL RESEARCH INSTITUTE
Box 6226, Dar es Salaam
Tel. 255-22-2700552
Fax: 255-22-2775549
Email: mari@mari.or.tz
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46. AC AC AC AC
5.4m
8m
8
Store Office
m
8
m
Screen
3.9 1.5
House
Growth
Room 1
10.3m
Growth Room
2
4m Biolistic Laminar
+ Extraction Laminar FC
Potting Bench Pot Storage
Transfer
Soil Storage Fridge D/Freezer
Storage
Molecular Lab
6.0m
Terrazzo Lab Bench
4.3 AUTO-
CLAVE
Coats/Sho
es Washing
AC AC
5.4m 5.4m
Fig.1: Sketch of the Proposed Containment Laboratory at ARI Mikocheni www.iita.org

47. Cassava transformation for CMD and
CBSV resistance
RF funded project to MARI with IITA
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48. A
B
C D
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49. Cassava cultivars No. of plantlets
1 Ngwanzila 49
2 Gago 50
3 Konyu 20
4 Mwarusha 47
5 Lyongo Nyeupe 37
6 Pamba 16
7 Milundikachini 31
8 Kibandameno 282
9 Konyu 38
10 Marekani 39
11 Rushura 24
12 Pelagia 30
13 Kitingisha 52
14 Sagalatu 33
15 Pikipiki 4
16 Karatasi 58
17 Bukalasa 5
18 Mreteta 32
19 Katakya 4
20 Mukandasa 35
21 Kababi 6
22 Rangimbili 36
23 Sungusungu 22
24 Kibangili 30
25 Kaitampunu 25
26 Mkunungu 9
TOTAL 1014 www.iita.org

50. Agricultural Biosafety Scientific Advisory
Committee (ABSAC) reviews facility and
work 21st January, 2009!
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51. Breeding services
Assessing (scoring trials)-more quantified
Testing “new sources of resistance”
51
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52. Breeding for CBSD field resistance
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53. New Sources of Resistance
Testing was done by grafting virus-free clones onto infected
rootstocks
• CBSV:
– 96/1089A
– TME 117
• CMV:
– 96/1089A (does not support neither virus
multiplication nor movement)
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54. I-96-1089A plant 3 with distinct leaf tertiary vein chlorosis to various
extents on two of the lower leaves 15/02/08
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55. Close up of the tertiary vein chlorosis on leaflet of plant 3 of I-96-1089A;
arrows indicate tertiary leaflet veins 15/02/08
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56. 96/1089A infected by CMD at Kibaha
Whitefly infested plant No whiteflies
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57. CBSD
Background
Future perspectives
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58. CBSV infectious clone
replacement vectors
functionality of
genes
e.g. movement
Histology
CBSD epidemiological trials
CBSV-free
trials/multilocational
Herbaceous weeds
survey
CBSV resistance
field trials for
transformed lines www.iita.org

59. THANKS
IITA people National Partners
All students and workers
All Donors
Asante sana
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