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 918 510
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 since1946
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 diseasefree 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
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
Near completion
2
3
4
5
6
7
8
9
10
11
12
13
Activity 1.3 – Upgrading of an existing Tissue
culture laboratory
Tissue culture Laboratory designed &
equipped
Completed
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
Ramu stunt
FUNGI
Downy mildew
VIRUSES
BACTERIA
Fiji leaf gall
Gumming
Leaf fleck
Leaf scald
Fusarium sett
Mild mosaic
Mottles stripe
Pineapple disease
Mosaic
Ratoon stunt
Red rot
Red leaf mottle
Red stripe
Smut
Streak
Sclerophthora
Streak mosaic
Grassy shoot
Wilt
Striate mosaic
Green grassy shoot
Yellow leaf
White leaf
Chlorotic streak Dry top rot
PHYTOPLASMA
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
Water control
Xa
Xcv isolates
Xcv isolates
Xa specific fragment amplified by PCR
No amplification from Xcv
Highly specific as compared to ITS based primers
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
589 bp
450 bp
360 bp
Water PCR Step
Water RT-Step
REU
Multiplex
BRA-PER
CUB
Primers: REU-F/CPR, CUB-F/R & BRA-PER F/R
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
Infected
Month 0
Month 1
Callus formation
Months 2-4
3 subcultures
of callus
Months 7-8
Multiplication
& Rooting
Diagnosis
Months 5-6 Regeneration
Success rate of SCYLV elimination in vitro
Period
Infected
varieties
Successful
regeneration
Virus
elimination
% success for
virus cleaning
1991-2001
2001-2003
18
38
16
29
16
25
100
86
2003-2005
2006-2008
2008-2010
12
13
13
11
10
13
11
10
13
100
100
100
2011-2012
8
6
6
100
International quarantine facility for the exchange
of sugarcane germplasm among ACP countries
Activity
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
2011
2012
2013
2014
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