Cassava brown streak virus         studies                         www.iita.org
CBSDBackgroundResearchOther workFuture perspectives                      www.iita.org
CBSDBackground               www.iita.org
Background CBSD1890s, Tanzania (Wahlberg)1930s, Tanzania (Storey)CBSD limited to coastal/low altitude areas2001, Causal or...
Potyvirus genome strategy e.g. TEV   0                                                              10 Kb         gRNA5’ V...
Field symptoms of CBSD                         www.iita.org
10 days later                www.iita.org
CBSDResearch    Survey    Detection/symptoms    Symptom reliability    Transmission    Lab/screenhouse/    field    Virus ...
1. Survey-CBSD and CBSV   further samples in TZ plus coastal   Kenya, Burundi and Rwanda     CBSD symptom analyses     CBS...
CBSV/CBSD assessmentsMajor surveys in Tanzania in 2005 and 2006Minor testing in coastal Kenya, Burundi and Rwanda in2007Fi...
www.iita.org
www.iita.org
Diagnostic parameter             Coast. Zone Lake Zone           Southern      Mean                                 (N = 2...
TEV              SPMMV                                  911                                                            AF5...
ConclusionsCBSD and CBSV found in all areas ofTanzania and KenyaIncidences and severity of CBSD had aninverse correlation ...
2. CBSV transmission experiments through   various inoculations to virus-free   material in controlled conditions with   T...
1   2   36   5       47   8   9                www.iita.org
CBSV-Transmission experiments    A   B                   31            24            5              6           www.iita.org
Sap transmission of CBSV                                          Buffer A                         Buffer B               ...
Grafting experiments                          140% CBSV incidence RT-PCR                          120                     ...
Results54 % (13/24 plants) inoculated with Buffer B and 29 % (7/24 plants) inoculated with Buffer A tested positive to CBS...
CtdCBSV was not transmitted through  infected root debris although 17 %  (4.5/27 plants) test plants on the  debris-soil m...
ConclusionsCBSV may be transmitted through sap, grafting,   cutting tools and leaf harvesting.Graft inoculation with an in...
3.Screen house experiment:Cuttings collected from plants with diverse CBSDsymptoms planted in potsSymptoms monitored for 1...
Relationship btn CBSV & type of root symptoms                                               Lake zone     Coastal zone    ...
Symptomless &                 negativeTest sample              Symptomless &                 positive              Symptom...
Lake zone          Coastal zone                     Southern zone                     18                              16.0...
Generation of virus –free cuttings• None of the cuttings from the  diseased plants germinated disease  free.• Only two pla...
ConclusionsCBSD symptoms are diverseCareful observation should be made to correctly diagnosethe disease.Newly established ...
Ctd.......Not all symptomless plants are free from CBSV.Detection of CBSV in symptomless plants complicates itsdiagnosis a...
5.Variability of Cassava Brown Streak DiseaseSymptoms and the Relationship Between VirusInfection and Symptoms Expression ...
6.Factors Affecting Disease Severity in Cassava brownstreak virus (Potyviridae; Ipomovirus) Infected PlantsSusceptibility ...
Low temperature                  High temperature                        7 Linear (Low temperature)         Linear (High t...
Diseased &stressed                  Diseased &Irrigated                            80       Linear (Diseased &stressed)   ...
ConclusionsCBSD affected cultivars tends to adapt to newenvironment.Foliar CBSD symptoms are more apparent than stemsympto...
7. Viral preparationsMaterials moved fromcoastal Kenya to KEPHIS-PQS Maguga in 2006-7 andkept under quarantineconditionsRe...
www.iita.org
Viral prep.Final density-centrifugationsediment fractions areanalysed by PAGE and RT-PCR -2007-8No success in cassava (2cu...
8.Sampling methods    TC free materials    CBSV materials    x cultivars x reps9.Parts of plant best for sampling     CBSV...
Take sample leaflet 2 orthree leaves from growingpoint of the plant             Preserve over             silica gel or   ...
10-leaf sample    representing 10    plants        Punched sample grind directly inPUNCH   ‘fuge tube with tube pestle for...
10-leaf sample                                     representing 10 plants       10 lobes from 10       leavesScissors     ...
CBSDOther work              www.iita.org
• Cassava genetic transformation for the  longevity of cassava virus resistance in  Tanzania• Mikocheni Agricultural Resea...
Application for Contained Laboratory and GreenhouseResearch on Cassava Transformation for the Longevity of          Cassav...
AC             AC       AC                                  AC                         5.4m8m8                            ...
Cassava transformation for CMD and             CBSV resistanceRF funded project to MARI with IITA                         ...
A        BC   D            www.iita.org
Cassava cultivars   No. of plantlets1    Ngwanzila                 492    Gago                      503    Konyu          ...
Agricultural Biosafety Scientific AdvisoryCommittee (ABSAC) reviews facility andwork 21st January, 2009!                  ...
Breeding servicesAssessing (scoring trials)-more quantifiedTesting “new sources of resistance”                            ...
Breeding for CBSD field resistance                              www.iita.org
New Sources of ResistanceTesting was done by grafting virus-free clones onto infected  rootstocks• CBSV:   – 96/1089A   – ...
I-96-1089A plant 3 with distinct leaf tertiary vein chlorosis to variousextents on two of the lower leaves 15/02/08       ...
Close up of the tertiary vein chlorosis on leaflet of plant 3 of I-96-1089A;arrows indicate tertiary leaflet veins 15/02/0...
96/1089A infected by CMD at Kibaha Whitefly infested plant   No whiteflies                                           www.i...
CBSDBackgroundFuture perspectives                      www.iita.org
CBSV infectious clone     replacement vectors     functionality of     genes     e.g. movement     HistologyCBSD epidemiol...
THANKSIITA people            National Partners                       All students and workers                       All Do...
Upcoming SlideShare
Loading in...5
×

cassava brown streak virus studies

1,722

Published on

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

Published in: Technology
0 Comments
0 Likes
Statistics
Notes
  • Be the first to comment

  • Be the first to like this

No Downloads
Views
Total Views
1,722
On Slideshare
0
From Embeds
0
Number of Embeds
1
Actions
Shares
0
Downloads
54
Comments
0
Likes
0
Embeds 0
No embeds

No notes for slide

cassava brown streak virus studies

  1. 1. Cassava brown streak virus studies www.iita.org
  2. 2. CBSDBackgroundResearchOther workFuture perspectives www.iita.org
  3. 3. CBSDBackground www.iita.org
  4. 4. Background CBSD1890s, Tanzania (Wahlberg)1930s, Tanzania (Storey)CBSD limited to coastal/low altitude areas2001, Causal organism- Cassava brown streak virus, ss + RNA (Monger et al.)Family;Potyviridae, Genus; Ipomovirus2005, Transmitted by Bemisia tabaci ca. 2%2005, Reports of the spread of disease/economic damage in high altitude areas-Uganda2006, Reports of symptoms in DRC,Zambia2008, Angola? Burundi? www.iita.org
  5. 5. Potyvirus genome strategy e.g. TEV 0 10 Kb gRNA5’ 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. 6. Field symptoms of CBSD www.iita.org
  7. 7. 10 days later www.iita.org
  8. 8. CBSDResearch Survey Detection/symptoms Symptom reliability Transmission Lab/screenhouse/ field Virus preps Sampling strategies www.iita.org
  9. 9. 1. Survey-CBSD and CBSV further samples in TZ plus coastal Kenya, Burundi and Rwanda CBSD symptom analyses CBSV sequence analyses www.iita.org
  10. 10. CBSV/CBSD assessmentsMajor surveys in Tanzania in 2005 and 2006Minor testing in coastal Kenya, Burundi and Rwanda in2007First surveys included evaluation of sampling storage for RT-PCR testsEvaluated the best type of tissue for sampling and tissue dilution tests for larger samplings by RT- PCR RT-PCR using CBSV specific CP gene primers200bp www.iita.org
  11. 11. www.iita.org
  12. 12. www.iita.org
  13. 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) 38.3Foliar CBSD severity 2.8 (3.9) 2.5 (2.8) 2.6 (3.3) 2.6Stem CBSD Incidence (%) 35.3 (87) 1.1 (20) 6.3 (56.7) 14.2Stem CBSD Severity 2.9 (4.5) 2.0 (2.0) 2.4 (2.6) 2.41Root CBSD Incidence (%) 57 (100) 18 (53) 33.1(90) 36.1Root CBSD severity 3.7 (5) 3.3 (3.6) 2.5 (3.5) 3.2Symptoms on leaf sample (%) 100 88.9 77.8 88.9CBSV positive by RT-PCR 78.6 72.2 70.4 73.7(%)Average crop age (months) 8 7 12 9B. tabaci per plant 2 24 1 9CMD incidence (foliar) (%) 32.2 35 24.4 30.5CMD severity 2.8 2.9 2.6 2.8The maximum values recorded for each parameter is given in brackets. www.iita.org
  14. 14. TEV SPMMV 911 AF52 AF53 986 AY40 AY41 1000 CVYV AY42 AY97 999 40 sequences 1000 KYCBSV DQ837302 1000 DQ837303 DQ8373040.1 changes 5508 www.iita.org
  15. 15. ConclusionsCBSD and CBSV found in all areas ofTanzania and KenyaIncidences and severity of CBSD had aninverse correlation with the altitudeSequence diversity is on the border ofspeciation limits for CBSV www.iita.org
  16. 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 www.iita.org
  17. 17. 1 2 36 5 47 8 9 www.iita.org
  18. 18. CBSV-Transmission experiments A B 31 24 5 6 www.iita.org
  19. 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 inoculationFigure 1. Relationship between inoculation buffers and time to CBSVdetection www.iita.org
  20. 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 RootstockA B www.iita.org
  21. 21. Results54 % (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 www.iita.org
  22. 22. CtdCBSV 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. www.iita.org
  23. 23. ConclusionsCBSV 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. 24. 3.Screen house experiment:Cuttings collected from plants with diverse CBSDsymptoms planted in potsSymptoms monitored for 12 months4.Field experiment:Representative cassava cultivars of the major types offoliar and root symptoms were identifiedIncluded Albert, Cheupe, Kibaha, Nachinyaya, Namikongaand AR 49/2.Grown in the 6 cv. x 3 reps. Total of 108 plants/cultivar atSRISymptoms monitored and recorded monthlyExperiment duration; 2 years. www.iita.org
  25. 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 www.iita.org
  26. 26. Symptomless & negativeTest sample Symptomless & positive Symptomatic & negative Symptomatic & positive 0 20 40 60 80 100 % of totalRelationship between CBSV infections and symptoms on samples www.iita.org
  27. 27. Lake zone Coastal zone Southern zone 18 16.05 16 14.81 14CBSD 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 www.iita.org
  28. 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 www.iita.org
  29. 29. ConclusionsCBSD symptoms are diverseCareful observation should be made to correctly diagnosethe disease.Newly established types of foliar and root symptoms ofCBSD may improve on the diagnosis.Relationship between CBSV infection & symptomexpression was established.The expression of CBSD foliar symptoms may notnecessarily indicate CBSV infection. www.iita.org
  30. 30. Ctd.......Not all symptomless plants are free from CBSV.Detection of CBSV in symptomless plants complicates itsdiagnosis and management.The expression of CBSD foliar symptoms may notnecessarily indicate CBSV infection. www.iita.org
  31. 31. 5.Variability of Cassava Brown Streak DiseaseSymptoms and the Relationship Between VirusInfection and Symptoms Expression in On-farmCassavaCBSD symptoms became complex and difficult tocomprehend under field conditionsUnknown relationship between infection with CBSV andsymptom expressionUnknown distribution of CBSV in various tissues ofinfected plantsFarmer belief that CBSD-free plants may be regeneratedfrom diseased plants www.iita.org
  32. 32. 6.Factors Affecting Disease Severity in Cassava brownstreak virus (Potyviridae; Ipomovirus) Infected PlantsSusceptibility of selected cultivars with time & ageField experiment at SRITemperature using a growth chamber www.iita.org
  33. 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 exposureEffect of temperature on cv. Albert www.iita.org
  34. 34. Diseased &stressed Diseased &Irrigated 80 Linear (Diseased &stressed) Linear (Diseased &Irrigated) 70No. 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 www.iita.org
  35. 35. ConclusionsCBSD affected cultivars tends to adapt to newenvironment.Foliar CBSD symptoms are more apparent than stemsymptoms.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 CBSDseverity. www.iita.org
  36. 36. 7. Viral preparationsMaterials moved fromcoastal Kenya to KEPHIS-PQS Maguga in 2006-7 andkept under quarantineconditionsResearch assistant trainedin purification methods atBeCA www.iita.org
  37. 37. www.iita.org
  38. 38. Viral prep.Final density-centrifugationsediment fractions areanalysed by PAGE and RT-PCR -2007-8No success in cassava (2cultivars) or threeherbaceous indicatorsKiribiti mwezi analysed induplicate looks promising inNicotiana benthamianaDec 08 www.iita.org
  39. 39. 8.Sampling methods TC free materials CBSV materials x cultivars x reps9.Parts of plant best for sampling CBSV infected plants at differentgrowth stages x cultivars x reps10. RNA extraction methods optimised www.iita.org
  40. 40. Take sample leaflet 2 orthree leaves from growingpoint of the plant Preserve over silica gel or between newspaper strips in sealed tubes (do not over-stuff) Tissue paper Silica gel-blue www.iita.org
  41. 41. 10-leaf sample representing 10 plants Punched sample grind directly inPUNCH ‘fuge tube with tube pestle for RNA extraction www.iita.org
  42. 42. 10-leaf sample representing 10 plants 10 lobes from 10 leavesScissors 10 strips from 10 lobes-grind in liq N2 for RNA sample www.iita.org
  43. 43. CBSDOther work www.iita.org
  44. 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 partnerTo be continued in 2009 by BMGates Foundation 44 www.iita.org
  45. 45. Application for Contained Laboratory and GreenhouseResearch 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 www.iita.org
  46. 46. AC AC AC AC 5.4m8m8 Store Officem8m Screen 3.9 1.5 House Growth Room 1 10.3m Growth Room 24m Biolistic Laminar + Extraction Laminar FC Potting Bench Pot Storage Transfer Soil Storage Fridge D/Freezer Storage Molecular Lab 6.0m Terrazzo Lab Bench4.3 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. 47. Cassava transformation for CMD and CBSV resistanceRF funded project to MARI with IITA www.iita.org
  48. 48. A BC D www.iita.org
  49. 49. Cassava cultivars No. of plantlets1 Ngwanzila 492 Gago 503 Konyu 204 Mwarusha 475 Lyongo Nyeupe 376 Pamba 167 Milundikachini 318 Kibandameno 2829 Konyu 3810 Marekani 3911 Rushura 2412 Pelagia 3013 Kitingisha 5214 Sagalatu 3315 Pikipiki 416 Karatasi 5817 Bukalasa 518 Mreteta 3219 Katakya 420 Mukandasa 3521 Kababi 622 Rangimbili 3623 Sungusungu 2224 Kibangili 3025 Kaitampunu 2526 Mkunungu 9 TOTAL 1014 www.iita.org
  50. 50. Agricultural Biosafety Scientific AdvisoryCommittee (ABSAC) reviews facility andwork 21st January, 2009! www.iita.org
  51. 51. Breeding servicesAssessing (scoring trials)-more quantifiedTesting “new sources of resistance” 51 www.iita.org
  52. 52. Breeding for CBSD field resistance www.iita.org
  53. 53. New Sources of ResistanceTesting 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) www.iita.org
  54. 54. I-96-1089A plant 3 with distinct leaf tertiary vein chlorosis to variousextents on two of the lower leaves 15/02/08 www.iita.org
  55. 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 www.iita.org
  56. 56. 96/1089A infected by CMD at Kibaha Whitefly infested plant No whiteflies www.iita.org
  57. 57. CBSDBackgroundFuture perspectives www.iita.org
  58. 58. CBSV infectious clone replacement vectors functionality of genes e.g. movement HistologyCBSD epidemiological trials CBSV-free trials/multilocational Herbaceous weeds surveyCBSV resistance field trials for transformed lines www.iita.org
  59. 59. THANKSIITA people National Partners All students and workers All Donors Asante sana www.iita.org
  1. A particular slide catching your eye?

    Clipping is a handy way to collect important slides you want to go back to later.

×