1. ACP Project 1.3 International quarantine facility for the exchange of sugarcane germplasm among ACP countries Mid-Term Review 1 October 2012 MSIRI Réduit Mauritius

2. International quarantine facility for the exchange of sugarcane germplasm among ACP countries Implementing Institution: Mauritius Sugarcane Industry Research Institute Countries targeted: ACP sugar producing countries Duration of project: 4 years Cost: Euros 918 510 Implementing Institution: Mauritius Sugarcane Industry Research Institute Countries targeted: ACP sugar producing countries Duration of project: 4 years Cost: Euros 918 510

3. INTRODUCTION Objective: To enhance the productivity and efficiency of the sugarcane industry in ACP countries by promoting germplasm exchange, making their industries more responsive to the challenges in the global economy Purpose of the International quarantine To provide ACP countries with sugarcane plants free from detectable pathogens in order to safeguard their industry from potentially damaging diseases

4. Sugarcane diseases  Some 120 diseases recorded on sugarcane  Most important ones are those known to be transmitted by cuttings, referred as systemic diseases  Their most common mode of spread is through exchange of varieties between countries  28 systemic diseases are recorded  The risks represented by them, if introduced, can vary from minor to severe

5. How to minimize risk of accidental introduction of sugarcane diseases? Safe movement of sugarcane germplasm Quarantine of germplasm Effective testing (indexing) of material - use of molecular tools Movement of tissue culture plants

6. Sugarcane Quarantine in Mauritius Mauritius has been regularly importing germplasm since 1914 A National closed quarantine is in existence since 1946 The responsibility for the national quarantine has been vested on the MSIRI since 1953 Setting-up of an international quarantine station in Mauritius, which any member of the ACP countries can use to facilitate exchange of disease- free germplasm

7. International Quarantine Benefits: Minimizing the risks of introducing potentially devastating diseases into ACP countries Avoiding duplication of resources by establishing an international facility in one state Using up-to-date disease detection technologies and tissue culture to speed availability of germplasm

8. Main Project Components Renovation of an existing quarantine to meet biosecurity level 3 Setting up and equipping of a plant pathology Lab Renovation of a tissue culture facility and equipping Training of staff Training of ACP members quarantine officials

9. project 1.3 Activity 1.1- Existing glass houses renovation International quarantine facility designed to meet biosecurity level 3 Near completion GH for imported germplasm & tissue culture plants

10. Activity 1.2 – Setting up of a new Plant Pathology Lab Near completion

11. Activity 1.3 – Upgrading of an existing Tissue culture laboratory Completed Tissue culture Laboratory designed & equipped

12. project 1.3 Activity 2.1 Training in molecular diagnostic of diseases Major diseases not present in Mauritius: SCMV, SCSMV, Fiji disease, Downy mildew CIRAD provided training to staff in molecular detection of SCMV,SCSMV, and red leaf mottle virus

13. Activity 2.2 Diagnostic tools development

14. DEVELOPMENT OF DISEASE TESTING AND ELIMINATION PROCEDURE FOR POTENTIAL DISEASES Based on: Country of origin of germplasm Requirements of importing country Risk assessment analysis

15. Sugarcane Systemic Diseases UNKNOWN ETIOLOGY FUNGIVIRUSESBACTERIA Ramu stuntDowny mildewFiji leaf gallGumming Chlorotic streakDry top rotLeaf fleckLeaf scald Fusarium settMild mosaicMottles stripe Pineapple diseaseMosaicRatoon stunt Red rotRed leaf mottleRed stripe SmutStreakPHYTOPLASMA SclerophthoraStreak mosaicGrassy shoot WiltStriate mosaicGreen grassy shoot Yellow leafWhite leaf Leaf yellows

16. Detection of Xanthomonas albineans  Primers and Probe designed from the albicidin gene cluster sequence of X albilineans  Primers/Probes checked for specificity  Conventional PCR & Real-time PCR tests optimized Leaf scald disease

17. Xa specific fragment amplified by PCR No amplification from Xcv Highly specific as compared to ITS based primers Xcv isolates Water control Xa

18. Taqman ® Real-Time PCR for X albilineans detection Inclusion of probe - added specificity guarding against non-specific annealing of PCR primer pairs Amplification curve observed from all isolates of Xa tested Flat line for Xcv isolates & water control

19. Sugarcane Mosaic Application of RT-PCR test for detection of SCMV, SrMV, JgMV using Primer pair Oligo1n/2n 327 bp fragment amplified from poaceae potyviruses

20. Detection of Sugarcane yellow leaf virus  Multiplex RT-PCR optimized  Real-Time RT-PCR optimized  The virus displays high genetic diversity  Development of real-time RT-PCR tests for screening genotypes in progress Leaf yellows disease

21. Multiplex RT-PCR for CUB, BRA-PER & REU genotypes of SCYLV BRA-PER CUB REUMultiplex Water RT-Step Water PCR Step Primers: REU-F/CPR, CUB-F/R & BRA-PER F/R 589 bp 450 bp 360 bp

22. Real-time RT-PCR specific for REU genotypes REU genotypes amplified No amplification from water controls, disease free plantlet, and plant infected with genotype BRA-PER

23. Activity 2.3 Tissue culture for elimination of diseases

24. Tissue culture/elimination of SCYLV- optimized Month 0 Month 1 Callus formation 3 subcultures of callus 3 subcultures of callus Infected Months 2-4 Months 5-6 Regeneration Multiplication & Rooting Months 7-8 Diagnosis

25. Success rate of SCYLV elimination in vitro PeriodInfected varieties Successful regeneration Virus elimination % success for virus cleaning 1991-20011816 100 2001-200338292586 2003-20051211 100 2006-20081310 100 2008-201013 100 2011-2012866100

26. Activity 2011201220132014 1.1. Setting up of a glasshouse for imported germplasm 1.2 Setting up of a diagnostic lab 1.3 Upgrading of a tissue culture laboratory for disease elimination & multiplication of plantlets 1.4 International advice on setting up the quarantine 1.5 Certification of the facility 2.1 Training of staff 2.2 Development of disease testing 3.0 Reception of germplasm in quarantine 4.0 Training of Plant health officials of ACP countries International quarantine facility for the exchange of sugarcane germplasm among ACP countries

27. Expected Project Outputs Quarantine facility available for ACP countries to allow safe movement of germplasm Create awareness on diseases of quarantine importance Diagnostic protocols available to ACP countries Capacity building of plant health officials in ACP countries

28. Collaborators Dr Salem Saumtally Mr Sonalall Dhayan Mr Guy Triton Dr Asha Dookun-Saumtally Mr Nawshad Joomun Mr Miguel Antoine

29. Acknowledgements