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Rice Yellow Mottle Virus in Tanzania: 1. Distribution and Genetic Diversity

Rice Yellow Mottle Virus in Tanzania: 1. Distribution and Genetic Diversity. 1 Kanyeka, Z.L., E.Sangu 1 , D. Fargette 2 , A. Pinel-Galzi 2 & H. Hebrard 2 1 Department of Botany , UDSM, P. O. Box 35060 Dar es Salaam, Tanzania. 2 IRD, BP 34394 Montpellier Cedex 5, France . INTRODUCTION.

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Rice Yellow Mottle Virus in Tanzania: 1. Distribution and Genetic Diversity

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  1. Rice Yellow Mottle Virus in Tanzania: 1.Distribution and Genetic Diversity 1Kanyeka, Z.L., E.Sangu1, D. Fargette2, A. Pinel-Galzi2 & H. Hebrard2 1Department of Botany, UDSM, P. O. Box 35060 Dar es Salaam, Tanzania. 2IRD, BP 34394 Montpellier Cedex 5, France.

  2. INTRODUCTION • Part 1: • Distribution and Genetic Diversity • Part 2: ( In Progress) • Resistance breaking isolates • More Surveys in TZ & MAL, MOZ. & ZAM

  3. INTROD. cont’d • RYMV is a major constraint of rice production in Africa • Tanzania is severely affected • Devastating disease epidemics, reported in all rice growing areas • Hot spots areas: Mbeya, Morogoro, Arusha, Lake Victoria Zone, ZNZ & Pemba

  4. Overview of RYMV Diversity in Africa

  5. WHY THIS RYMV STUDY? • DITRIBUTION & DIVERSITY UNDER-ESTIMATED IN TZ • Few isolates characterized • Number of strains identified • Strain Localization • Habitat Fragmentation • Diversity btn & w/n strains • New rice growing areas • Intensification of the rice cultivation systems • Eastern Arc Mountains proposed Center of Origin • EMERGENCE OF RESISTANCE-BREAKING ISOLATES • SPREADING PATHWAYS FROM ITS CENTER OF ORIGIN

  6. OBJECTIVES • Characterize the molecular variability of isolates • Identify and Determine the distribution of strains • Identify their phylogenic relationship with the known strains and isolates • Investigate resistance-breaking isolates (Pathogeny) • Propose possible spreading pathways from its Center of Origin (EAM)

  7. MATERIALS AND METHODS • Field Collection of isolates in TZ • Recovery of isolates on S-CV • Transport of samples to IRD-Laboratory, Montpellier , France • Molecular Analysis of isolates • Immunological typing-Elisa tests • Molecular Typing • Coat Protein(CP) Gene Sequencing

  8. Field trip in TZ Map 1. Field collection of RYMV isolates -May 2005.

  9. MOLECULAR CHARACTERIZATION OF ISOLATES • Immunological typing (ELISA Tests) • DAS-ELISA using polyclonal antibodies • (IgGs ant RYMV Mg diluted at 1/1000 with Carbonate buffer pH 9.6) • TAS-ELISA using monoclonal antibodies • MAb A, MAb M, MAb E, MAb D & MAb G • Molecular typing • Extraction of total RNA • Plant RNeasy Mini Kit • RT-PCR • Coat protein (CP) gene amplification • Primers Amerce M & III

  10. RT-PCR Temperature Conditions • Denaturation (5min at 94°C), • 30 cycles • Denaturation (3min at 94°C), • Annealing (1min at 58°C), • Elongation (1min at 72°C • Final extension (10min at 72°C) • Visualisation on 1% Ethidium bromide stained agarose gel • 720bp band expected

  11. SEQUENCING OF CP GENE • Sent to another istitute for sequencing • SEQUENCE ANALYSIS • Assembling (EditSeq) DNASTAR • Alignment CLASTAL V DNASTAR • Estimation of genetic distances (Distance matrix) (MegAlign) DANSTAR • PHYLOGENETIC ANALYSIS (PAUP) VERSION 4 (Swafford, 2000) • Phylogenetic relationship RYMV strain • DNA Polymorphism

  12. RESULTS AND DISCUSION • ELISA Tests • RT-PCR Product • CP Sequences • Phylogeny • DNA-DIVERGENCE • POLYMORPHISM W/N & BTN CLADES

  13. Serological (ELISA Tests) Results • Two Serological Groups: Ser4 & Ser5 (Monoclonal Tests ) • Several Variants of Ser4 & Ser5 • All Ser4 variants had negative rxn with MAb M • Three Ser5 variants had negative rxn with Mab A • Two Ser5 variants had no rxn with Mabs A & G • Serotype variants in the same site showed similar rxn with Mabs.

  14. DISTRIBUTION OF RYMV IN TZ Key S4 S5 S6

  15. RT-PCR PRODUCT M 1 2 3 4 5 6 7 8 9 10 11 • Amplified fragments of the expected size (720bp) were obtained • No fragment was amplified for Health control plant (6) blank (7) & water (9) 2 Lane M=maker 1=Ifakara 2=Lumemo 3=Hembenti 4=Dihombo 5=Malinyi 6=H/Plant 7 Blank 8=Ndungu 9=water 10=RNA 11=cDNA

  16. CP SEQUENCE RESULTS • Three strains identified; 9=S4, 1=S5 & 13= S6 • No new strains were found • First record of strain S4 occurrence & distribution in EAM- confirming the highdiversity and proposal of RYMV Center of Origin • Most rare strain was - S5 observed twice in same area also Ali 1999 • S4 is a highly diverse strain than S6 • High molecular diversity btn & w/n strains

  17. Identified Strains of RYMV in Tanzania

  18. Main Population Clades and Sub clades of RYMV isolates in TZ

  19. DNA-Divergence between Populations

  20. DNA-Divergence between Populations

  21. DNA-Divergence between Populations

  22. Phylogeography of RYMV in TZ C & WA LV S4 EAM LM S5 EAM EA S6 EAM

  23. Conclusions • No new RYM strain were found however, the study reconfirmed the high diversity of RYMV in TZ • Strain S4 isolates were recorded for the first time in the EAM Region and share the common ancestor with Lake Malawi group • Strain S5 isolates are rare and are confined only to the Kilombero Valley. • Strain S6 isolates are widely spread but restricted only to the EAM region • Reinforcement of the proposition that EAM is the center of origin of RYMV

  24. ACKNOWLEDGEMENT • UDSM-Sida/SAREC Project • Financial support • IRD-Montpellier, France • Facilitation of Laboratory analysis

  25. ASANTE SANA THANK YOU MERCI BEAUCOUP

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