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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: mari@mari.or.tz

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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