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

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 Implementing Institution: Mauritius Sugarcane Industry Research Institute Countries targeted: ACP sugar producing countries Duration of project: 4 years Cost: Euros

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

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

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

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

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

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

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

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

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

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

Activity 2.2 Diagnostic tools development

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

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

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

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

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

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

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

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

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

Activity 2.3 Tissue culture for elimination of diseases

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

Success rate of SCYLV elimination in vitro PeriodInfected varieties Successful regeneration Virus elimination % success for virus cleaning

Activity 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

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

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

Acknowledgements