cassava brown streak virus studies
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Cassava brown streak virus
studies
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CBSD
Background
Research
Other work
Future perspectives
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CBSD
Background
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Background CBSD1890s, 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,Zambia2008, Angola? Burundi?
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Potyvirus genome strategy e.g. TEV
For pathogen-mediated strategies for virus resistance (PTGS) sequence data are needed
5’ VPg poly(A) 3’
0 10 KbgRNA
MP HC-Pro Pro Rep
?
Pol CP
Polyprotein
* **
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Field symptoms of CBSD
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10 days later
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CBSD
ResearchSurvey
Detection/symptoms
Symptom reliability
Transmission
Lab/screenhouse/
field
Virus preps
Sampling strategies
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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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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
200bp
RT-PCR using CBSV specific CP gene primers
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Diagnostic parameter Coast. Zone
(N = 27)
Lake Zone
(N = 36)
Southern
Zone (N = 28)
Mean
(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.
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TEV
0.1 changes
911
986
1000
1000
999
1000
CVYV
SPMMV
5508
DQ837302DQ837303DQ837304
AF52AF53AY40AY41AY42 AY97
40 sequencesKYCBSV
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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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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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1
9
45
32
6
7 8
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CBSV-Transmission experiments
3
4 5 6
1A B 2
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Sap transmission of CBSV
-10
0
10
20
30
40
50
60
3 4 5 6 7 8 9 10 16 24Weeks after inoculation
% C
BS
V-i
nfe
cte
d (
RT
-PC
R)
Buffer A Buffer B
Figure 1. Relationship between inoculation buffers and time to CBSV
detection
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Grafting experiments
0
20
40
60
80
100
120
140
1 2 3 4 5 6 7
Weeks after grafting
% C
BS
V in
cide
nce
RT
-PC
R
Infected scion Infected rootstock
BA
Rootstock
Scion
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Results
54 % (13/24 plants) inoculated with BufferB and 29 % (7/24 plants) inoculated withBuffer A tested positive to CBSV.
Grafting techniques had 100 % transmissionefficiency.
CBSV-free scions onto CBSV-infectedrootstocks was the most efficienttransmission technique
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Ctd
CBSV was not transmitted throughinfected root debris although 17 %(4.5/27 plants) test plants on thedebris-soil mixture produced foliarchloroses 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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Conclusions
CBSV may be transmitted through sap, grafting,cutting tools and leaf harvesting.
Graft inoculation with an infected rootstock isthe most efficient way to transmit the virus.
Transmission through CBSV-infected root debrisseems remote from the study.
For the first time, it was demonstrated thatagronomical practices have potential tohasten the spread of CBSV which may lead tonew epidemics.
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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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Relationship btn CBSV & type of root symptoms
1.1300
25.27
10.64
0
3.13
02.13
31.91
02.131.13
5.5
17.01
0
5
10
15
20
25
30
35
Positive Positive Negative Positive Negative
Brown mass
necrosis (%)
Chalkish necrosis (%) Necrotic specks (%)
Root symptoms & RT-PCR test
CB
SD
in
cid
en
ce
(%
)
Lake zone Coastal zone Southern zone
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0 20 40 60 80 100
Symptomatic &
positive
Symptomatic &
negative
Symptomless &
positive
Symptomless &
negative
Te
st
sa
mp
le
% of total
Relationship between CBSV infections and symptoms on samples
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14.81
6.17
9.17
6.066.17
0
16.05
1.2
8.64
2.12.8
1.2
12.34
2.47
6.59
2.01
1.02 1.2
0
2
4
6
8
10
12
14
16
18
Positive Negative Positive Negative Positive Negative
Chlorotic blotches (%) Chlorotic spots (%) Vein chlorosis (%)
Symptom type & RT-PCR test
CB
SD
in
cid
en
ce (
%)
Lake zone Coastal zone Southern zone
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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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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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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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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 conditionsUnknown relationship between infection with CBSV and symptom expressionUnknown 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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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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Y = 0.0906x + 1.2857
R2 = 0.7821
Y = 0.05x + 1.1333
R2 = 0.6172
0
1
2
3
4
5
6
7
0 10 20 30 40 50 60
Days after exposure
CB
SD
fo
liar
severi
tyLow temperature High temperatureLinear (Low temperature) Linear (High temperature)
Effect of temperature on cv. Albert
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Y = 0.7683x - 8.3066
R2 = 0.9529
Y = 0.0253x - 0.1816
R2 = 0.635
0
10
20
30
40
50
60
70
80
0 20 40 60 80 100Days after exposure
No
. o
f d
ead
leaves (
mean
)Diseased &stressed Diseased &IrrigatedLinear (Diseased &stressed) Linear (Diseased &Irrigated)
Effect of moisture stress on cv. Albert
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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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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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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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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
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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)
Silica gel-blue
Tissue paper
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PUNCH
10-leaf sample
representing 10
plants
Punched sample grind directly in
‘fuge tube with tube pestle for
RNA extraction
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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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CBSD
Other work
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• 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-operativesGovernment 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
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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: [email protected]
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8
m
8
m
Growth
Room 1
Growth Room
2
Biolistic Laminar
+ Extraction Laminar FC
OfficeStore
Molecular Lab
Coats/Sho
es
Storage
Washing
AUTO-
CLAVE
Terrazzo Lab Bench
Screen
House
Soil Storage
Potting Bench Pot Storage
5.4m
4.3
5.4m 5.4m
6.0m
10.3m
1.53.9
ACACAC AC
Fridge D/Freezer
AC
Transfer
AC
8m
4m
Fig.1: Sketch of the Proposed Containment Laboratory at ARI Mikocheni
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Cassava transformation for CMD and CBSV resistance
RF funded project to MARI with IITA
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AB
C D
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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
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Agricultural Biosafety Scientific Advisory Committee (ABSAC) reviews facility and work 21st January, 2009!
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Breeding services
Assessing (scoring trials)-more quantified
Testing “new sources of resistance”
51
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Breeding for CBSD field resistance
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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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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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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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96/1089A infected by CMD at Kibaha
Whitefly infested plant No whiteflies
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CBSD
Background
Future perspectives
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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
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THANKS
IITA people National Partners
All students and workers
All Donors
Asante sana