Napier grass stunt and head smut diagnostics Yaima Arocha and John Lucas Presented at the ASARECA/ILRI Workshop on Mitigat...
Rothamsted objectives <ul><li>Identify and characterise Napier stunt (NGS) phytoplasmas </li></ul><ul><li>Validate probe f...
NAPIER GRASS  STUNT
Etiology of NGS <ul><li>Phytoplasmas from 16SrIII and 16SrXI, associated with NGS in Ethiopia and Kenya respectively. </li...
Detection of NGS phytoplasmas <ul><li>Nested PCR with universal 16S ribosomal RNA primers </li></ul><ul><li>Further amplif...
Cynodon dactylon  L. Medicago sativa  L. <ul><li>Leptodelphax dymas  Fennah </li></ul><ul><li>Exitianus  sp. </li></ul><ul...
 
Napier Head Smut Disease Napier smut (NHS) due to infection by  Ustilago kamerunensis .
Smut Symptoms <ul><li>Smut-infected stems are much thinner and shorter than normal </li></ul><ul><li>with leaves reduced i...
Provision of isolates <ul><li>Collecting and Culturing Smut </li></ul>
Source of isolates <ul><li>15 cultures provide by Dr Margaret Mulaa (Kari Kenya). </li></ul><ul><li>Smut infected inflores...
Isolate purification <ul><li>Three morphologically similar isolates recovered from mixed Kenya cultures (putative  U. kame...
Ustilago cynodontis From smutted  Cynodon , Ethiopia, 2007 Putative  U. kamerunensis From Kenya, 2007 Cultural morphology 1
Mixture of budding and filamentous cells characteristic of smut fungus Cultural morphology 2 . Microscopic characters
Molecular Identification of  Ustilago   kamerunensis PCR amplifications from  U. kamerunensis  (2-4, 6-8, 10-12) and  U. c...
Hybridization signals from PCR products from Kenyan (UK) isolates with the   -tubulin probe.
NHS-infected Napier source plants (Kenya)
Uniformly smutted inflorescences of field-infected Napier plants
Distorted inflorescence
NG-1 month- post-inoculated plants
<ul><li>Plant infection tests (Koch’s Postulates) </li></ul><ul><li>Uninfected Napier grass cuttings were maintained in th...
PCR amplicons from inoculated plants using   -tubulin primers U U cynodontis  reference  U. kamerunensis Plant  Control s...
<ul><li>Outputs from the project  (Rothamsted) </li></ul><ul><li>Diagnostic primers for NGS and NHS </li></ul><ul><li>Sequ...
Draft diagnostic lab manual Edition 3
<ul><li>Conclusions </li></ul><ul><li>The phytoplasma associated with NG stunt in Ethiopia was molecularly characterized a...
<ul><li>Acknowledgements </li></ul><ul><li>Prof. Phil Jones, Rothamsted Research, UK </li></ul><ul><li>Colleagues at ILRI,...
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Napier grass stunt and head smut diagnostics

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A presentation prepared by Yaima Arocha and John Lucas for the ASARECA/ILRI Workshop on Mitigating the Impact of Napier Grass Smut and Stunt Diseases, Addis Ababa, June 2-3, 2010.

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Napier grass stunt and head smut diagnostics

  1. 1. Napier grass stunt and head smut diagnostics Yaima Arocha and John Lucas Presented at the ASARECA/ILRI Workshop on Mitigating the Impact of Napier Grass Smut and Stunt Diseases, Addis Ababa, June 2-3, 2010
  2. 2. Rothamsted objectives <ul><li>Identify and characterise Napier stunt (NGS) phytoplasmas </li></ul><ul><li>Validate probe for NGS detection and evaluation of resistance </li></ul><ul><li>Develop and validate probe for Napier smut (NHS) for detection and evaluation of resistance </li></ul><ul><li>Produce a training manual for molecular diagnostic use </li></ul><ul><li>Develop web page for project </li></ul>
  3. 3. NAPIER GRASS STUNT
  4. 4. Etiology of NGS <ul><li>Phytoplasmas from 16SrIII and 16SrXI, associated with NGS in Ethiopia and Kenya respectively. </li></ul>
  5. 5. Detection of NGS phytoplasmas <ul><li>Nested PCR with universal 16S ribosomal RNA primers </li></ul><ul><li>Further amplification with primers fU5/rU3 gives amplicon of 880 bp </li></ul><ul><li>RFLP to further characterise phytoplasma group using restriction enzymes eg. RsaI, AluI, and HaeIII </li></ul><ul><li>Fragments separated by electrophoresis </li></ul><ul><li>Specific probes for groups 16SrIII and 16SrXI from cloned fU5/rU3 PCR products </li></ul><ul><li>Non-radioactive nucleic acid hybridisation (nrNAH) detection </li></ul>
  6. 6. Cynodon dactylon L. Medicago sativa L. <ul><li>Leptodelphax dymas Fennah </li></ul><ul><li>Exitianus sp. </li></ul><ul><li>Molecular characterization of phytoplasma associated with NGS (NGS-E, subgroup 16SrIII-A) </li></ul><ul><li>Exitianus sp. and L. dymas , potential vectors </li></ul><ul><li>C. dactylon and M. sativa , alternative hosts </li></ul>Accepted for publication (full paper) in J. Phytopathology , 2008 16SrIII-A 16SrXI 16SrXIV 16SrXIV 16SrVII 16SrVI 16SrV 16SrVIII 16SrIV 16SrX 16SrI 16SrXII 16SrXIII 16SrII (DQ305977) (DQ3058983) (DQ305982) (DQ305980) (DQ305979) (DQ305978)
  7. 8. Napier Head Smut Disease Napier smut (NHS) due to infection by Ustilago kamerunensis .
  8. 9. Smut Symptoms <ul><li>Smut-infected stems are much thinner and shorter than normal </li></ul><ul><li>with leaves reduced in number and size, sometimes distorted </li></ul><ul><li>Severely infected stems have shortened internodes, increased aerial tillering slow regrowth after cutting </li></ul>
  9. 10. Provision of isolates <ul><li>Collecting and Culturing Smut </li></ul>
  10. 11. Source of isolates <ul><li>15 cultures provide by Dr Margaret Mulaa (Kari Kenya). </li></ul><ul><li>Smut infected inflorescence ( Cynodon dactylon ) from Ethiopia </li></ul>
  11. 12. Isolate purification <ul><li>Three morphologically similar isolates recovered from mixed Kenya cultures (putative U. kamerunensis ) on YPD antibiotic amended agar </li></ul><ul><li>Infected Cynodon grass gave a uniform colony morphology (putative U. cynodontis ) </li></ul>
  12. 13. Ustilago cynodontis From smutted Cynodon , Ethiopia, 2007 Putative U. kamerunensis From Kenya, 2007 Cultural morphology 1
  13. 14. Mixture of budding and filamentous cells characteristic of smut fungus Cultural morphology 2 . Microscopic characters
  14. 15. Molecular Identification of Ustilago kamerunensis PCR amplifications from U. kamerunensis (2-4, 6-8, 10-12) and U. cynodontis (5, 9, 13) with primers targeting the  -tubulin gene (F1/R2, 2-5; and F2/R1, 10-13) and ITS region (ITS1/4, 6-9). Lanes 14-16. Positive controls group 16SrIII and 16SrXI (16). More data can be obtained from authors 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17
  15. 16. Hybridization signals from PCR products from Kenyan (UK) isolates with the  -tubulin probe.
  16. 17. NHS-infected Napier source plants (Kenya)
  17. 18. Uniformly smutted inflorescences of field-infected Napier plants
  18. 19. Distorted inflorescence
  19. 20. NG-1 month- post-inoculated plants
  20. 21. <ul><li>Plant infection tests (Koch’s Postulates) </li></ul><ul><li>Uninfected Napier grass cuttings were maintained in the glasshouse </li></ul><ul><li>Spore suspensions harvested from infected inflorescences were mixed with SDW and Tween 20 detergent. </li></ul><ul><li>Four plants inoculated with suspension on cut emerging leaves. </li></ul><ul><li>A further four plants exposed to inoculum by application to the soil around emerging shoots. </li></ul><ul><li>One month after inoculation tissue samples were taken from inoculated plants and PCR amplification done with  -tubulin primers for Ustilago sp. </li></ul><ul><li>All plants tested positive with 860 bp amplicons with high sequence identity to U. kamerunensis . </li></ul><ul><li>Symptomatic inflorescences not observed. </li></ul>
  21. 22. PCR amplicons from inoculated plants using  -tubulin primers U U cynodontis reference U. kamerunensis Plant Control samples
  22. 23. <ul><li>Outputs from the project (Rothamsted) </li></ul><ul><li>Diagnostic primers for NGS and NHS </li></ul><ul><li>Sequences lodged in Genbank (first available for U. kamerunensis ) </li></ul><ul><li>Assay formats tested (but further optimisation required) </li></ul><ul><li>Laboratory manual for diagnostic protocols </li></ul><ul><li>Publications (One published on NGS and vector(s), second on NHS identification in draft form) </li></ul><ul><li>Networking. Project team and new boundary partners in UK and Canada? </li></ul>
  23. 24. Draft diagnostic lab manual Edition 3
  24. 25. <ul><li>Conclusions </li></ul><ul><li>The phytoplasma associated with NG stunt in Ethiopia was molecularly characterized as a member of subgroup 16SrIII-A </li></ul><ul><li>Potential leafhopper vectors and alternative plant hosts were identified for the NG stunt phytoplasma </li></ul><ul><li>Ustilago kamerunensis was morphologically and molecularly identified, using  -tubulin and ITS regions, closely related to U. trichophora and U. davisii </li></ul><ul><li>A non-radioactive nucleic acid hybridization assay was standardized for the detection of U. kamerunensis from cultures and PCR products, and its optimization for detecting directly from Napier leaves is in progress </li></ul><ul><li>Transmission tests on 1 month post-inoculated Napier plants, showed PCR amplifications for Ustilago with  -tubulin and ITS primers. </li></ul>
  25. 26. <ul><li>Acknowledgements </li></ul><ul><li>Prof. Phil Jones, Rothamsted Research, UK </li></ul><ul><li>Colleagues at ILRI, Ethiopia, and the project team for all their help </li></ul><ul><li>Dr Mike Wilson, National Museum, Cardiff, UK </li></ul><ul><li>Colleagues at Rothamsted Research, especially Bart Fraaije, Tim Bean and Mark Wilkinson. </li></ul><ul><li>ASARECA , GPC and BBSRC for financial support </li></ul>

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