2. 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.

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

4. 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

5. Overview of RYMV Diversity in Africa Criteria Serological tests 5 Serotypes W/C Africa Ser 1 to 3 E. Africa Ser 4 & 5 Nucleotide sequences W/C Africa S1 to S3 E. Africa S4 to S6

6. 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

7. 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)

8. 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

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

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

11. 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

12.  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 SEQUENCING OF CP GENE

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

14.  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. Serological (ELISA Tests) Results

15. DISTRIBUTION OF RYMV IN TZ Key S4 S5 S6

16. RT-PCR PRODUCT Amplified fragments of the expected size (720bp) were obtained No fragment was amplified for Health control plant (6) blank (7) & water (9) Lane M=maker 1=Ifakara 2=Lumemo 3=Hembenti 4=Dihombo5=Malinyi 6=H/Plant7 Blank 8=Ndungu 9=water 10=RNA11=cDNA 2 M1243567891011

17.  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 high diversity 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 CP SEQUENCE RESULTS

18. Identified Strains of RYMV in Tanzania

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

20. DNA-Divergence between Populations ParameterPopulations P1(S4-L. Malawi)P2(S4-L.Victoria) No of Sequences510 Polymorphic sites 2942 Total No of Mutation 2943 Nucleotide diversity (Pi) 0.0186 (1.9%)0.0176 (1.8%) Polymorphic P1 & Monomorphic P2 22 Polymorphic P2 & Monomorphic P1 36

21. DNA-Divergence between Populations ParametersPopulations P1 (S6 Tz)P2 (S4 Tz) No of Sequences5020 Polymorphic sites12079 Total No of Mutation 13682 Nucleotide diversity (Pi) 0.03925 (3.9%)0.04248 (4.2%) Polymorphic P1 & Monomorphic P2 101 Polymorphic P2 & Monomorphic P1 47

22. DNA-Divergence between Populations Parameters Populations P1 (S4 Tz)P2 (S6 Tz) P3 (EAM Strains) No of Sequences205060 Polymorphic sites79120182 Total No of Mutation 82136217 Nucleotide diversity (Pi) 0.04248 (4.2%) 0.03925 (3.9%) 0.06115 (6.1%) Polymo P1/ Mono P3 Mono P3/ Polymo P1 149 / 14 Polymo P2/ Mono P3 Mono P3/ Polymo P2 0 / 81

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

24. 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

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

26. ASANTE SANA THANK YOU MERCI BEAUCOUP