ACP Project 1.3

International quarantine facility for the exchange of sugarcane

germplasm among ACP countries

Mid-Term Review1 October 2012

MSIRIRéduit

Mauritius

International quarantine facility for the exchange of sugarcane germplasm among ACP countries

Implementing Institution: Mauritius Sugarcane Industry Research InstituteCountries targeted: ACP sugar producing countriesDuration of project: 4 yearsCost: Euros 918 510

INTRODUCTIONObjective: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 MauritiusMauritius has been regularly importing germplasm since 1914

A National closed quarantine is in existence since1946

The responsibility for the national quarantine has been vested on the MSIRI since 1953

Setting-up of an internationalquarantine 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

1 2 3 4 5 6 7 8 9 10 11 12 13

352 bp

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 mildewCIRAD 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 DiseasesUNKNOWN ETIOLOGY

FUNGI VIRUSES BACTERIA

Ramu stunt Downy mildew Fiji leaf gall Gumming

Chlorotic streak Dry top rot Leaf fleck Leaf scald

Fusarium sett Mild mosaic Mottles stripe

Pineapple disease Mosaic Ratoon stunt

Red rot Red leaf mottle Red stripe

Smut Streak PHYTOPLASMA

Sclerophthora Streak mosaic Grassy shoot

Wilt Striate mosaic Green grassy shoot

Yellow leaf White 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

isol

ates

Xcv

isola

tes

Wat

er c

ontr

ol

XaXa

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 CU

B REU

M

ultip

lex

Wat

er R

T-St

ep

Wat

er P

CR S

tep

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- optimizedMonth 0

Month 1Callus formation

3 subculturesof callus

Infected

Months 2-4

Months 5-6 RegenerationMultiplication & Rooting

Months 7-8

Diagnosis

Success rate of SCYLV elimination in vitro

Period Infected varieties

Successful regeneration

Virus elimination

% success for virus cleaning

1991-2001 18 16 16 1002001-2003 38 29 25 862003-2005 12 11 11 1002006-2008 13 10 10 1002008-2010 13 13 13 1002011-2012 8 6 6 100

Activity 2011 2012 2013 2014

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 plantlets1.4 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