“GLOBAL GERMPLASM COLLECTIONS:
SURE BENEFITS WITHOUT SEEDBORNE DISEASES”
Maritza Cuervo I.
September 25, 2014

Outline
1. The context: global collections
2. Key concepts around plant quarantine; cooperation CIAT-ICA
3. Health controls in the lab for CIAT Programs
4. Research to improve health in germplasm exports: 3 case studies
5. Perspectives: the lab towards certification

Holdings in Germplasm at CIAT
Registered into the International Treaty
(Agreement signed with the governing body on October 16, 2006)
Crops
Taxa
( No.)
Accessions
(No.)
Beans (Phaseolus)
Cassava (Manihot)
Tropical Forages
37,794
6,643
23,140
67.577
33
Number
(No.)
1
1
1
45
734
Origin Country
(No.)
112
28
75
Germplasm materials conserved/ distributed as International Public Goods
Source: GRP – CIAT, 2014
812
The International Treaty on Plant
Genetic Resources for Food and
Agriculture

What do we distribute?
Distribution of germplasm by the GRP in 1973-2013
Forage: 88,835 samples
109 countries
Bean: 431,203 samples
105 countries
Cassava: 37,230 samples
80 countries
Total samples distributed : 557,268 Source: GRP – Dec. 2014

Germplasm Selection and Process Order
Search
List of germplasm
Add to Cart
Display order registration
Select purpose
Select
Database
Method of acceptance
Accept the Material
Transfer Agreement
No accept
stop Decision
Accept
If select
on SMTA
Send request
Send email about request
to receptor and to
CIAT
Database
Save order
Display
SMTA
Information
A.Hernandez, 2014

O.Rivera, 2014
Material distributed over the last ten years
Countries receveing
materials from GRP
Countries no
distribution

Some concepts
 What is Plant Health?
It is the science that deals with the prevention and cure of plant diseases.
 Disease or quarantine pest
One that may have potential or true economic importance in the area where the germplasm
is introduced; therefore it is officially controlled.
 Plant quarantine
Any activity aimed at preventing the introduction and / or spread of quarantine diseases to
ensure their official control.
 Quarantine rules
They are considered as a technical-legal mechanism to prevent the entry, establishment and
dissemination of quarantine plant pests and plant products. International standards for
phytosanitary measures are subject to periodic review and amendment.

Office of the Division of Plant Health (Section of Inspection and Quarantine) of
the Colombian Agricultural Institute (ICA), organization assigned to the
Department of Agriculture of Colombia.
ICA-CIAT Cooperation agreement: Letter of Understanding No. 9
The objectives of the program are:
 to prevent the spread of seed borne diseases and to minimize the risk of
accidentally introducing exotic pests and pathogens to Colombia.
 To inspect screenhouses and glasshouses where imported germplasm is
increased.
 To inspect field and greenhouses where the germplasm intended for
national/international export is increased.
 To test the seed health status of germplasm for conservation/distribution.

Germplasm Health Laboratory (GHL)
Purpose: to ensure that
the germplasm
distributed by GRP and
other CIAT projects is
free of diseases of
quarantine importance.

GHL approving accessions
Approved accesion beans
Approved tropical grasses and legumes
2010 2011 2012 2013 Until July
2014
80% 77% 76%
84%
92%
70% 72% 70% 74% 75%
 Trying to avoid exogenous
contamination.
 Periodic visits to the
multiplication fields.
 Collecting samples for
phytosanitary diagnostic in
the GHL.
 Training field workers on
visual detection of pathogens
and disease management.
 Implementation of cultural
practices (clean bags, cleaning
tools, etc.)
 Implementation of best
laboratory practices.

2500
2000
1500
1000
500
0
Germplasm Health Laboratory tested
2005 2006 2007 2008 2009 2010 2011 2012 2013 Until July
2014
909
572
691
1190
1602 1544
1400
776 812
2306
Other projects of CIAT

Developing research to improve the
health of germplasm collections
Management of the fungus complex caused by Ergot
in Brachiaria sp.
 Standardization of molecular diagnostic
methodologies for virus detection in cassava.
 Use of tablets in GHL operations.

Ergot Disease
This disease is caused by certain species
of Claviceps; it has been registered in
Brachiaria in Africa - Ethiopia, Kenya,
Malawi and Zimbabwe - in Australia and
India. In South America, this fungus has
been reported in Brazil and Colombia.
Ergot disease in the inflorescences of
Brachiaria spp. Caused by Sphacelia sp.
(asexual state of Claviceps sp.),
producing a complex composed of
various fungal quarantine fungi.

Cleaning of tools
Handpicking
Removing
impurities
Transportation in
cloth bags
Labeling
and sealing
Collecting in
paper bags
J.C. Ramírez, A. Ciprián, M.Cuervo, 2014

Pruning plot
Incineration
vegetative growth
Harvest time
Soil fungicides
Foliar fungicides
J.C. Ramírez, A. Ciprián, M.Cuervo, 2014

Ergot disease
No. Colony Forming Units
J.C. Ramírez, A. Ciprián, M.Cuervo, 2014
0
Accepted
Rejected
21
42
21
45
40
35
30
25
20
15
10
5
0
Primera evaluación Segunda evaluación
Number of accessions
Chemical control and cultural management
in production of Brachiaria spp
First evaluation Second evaluation
1 Evaluation 2 Evaluation

BENEFITS (1)
Reduced symptoms of the disease caused by Ergot
generating a decrease in the presence of fungal complex
in Brachiaria accessions.
 Decrease in the inoculum source of the causative agent of
Ergot in the fields of seed production of GRP.
 Increased acceptance of the phytosanitary condition of
the accessions belonging to the genus Brachiaria for
distribution and subsequent storage in the genebank.

Developing research to improve the
health of germplasm collections
Management of the fungus complex caused by Ergot
in Brachiaria sp.
1 2 3 4 5 6
 Standardization of molecular diagnostic
methodologies for virus detection in cassava.
 Use of tablets in GHL operations.

As in other vegetative propagated crops, our research has
revealed the common occurrence of mixed virus infections
in diseased cassava plants therefore the methodologies of
diagnosis had to be changed accordingly.
Photo by Camilo Oliveros

Unknown viruses
Viruses with known
sequence

The standardization and implementation of new
molecular diagnostic techniques for cassava virus
 Modification in the type of extraction, was changed to make
dsRNA extractions of total RNA extractions
 Amount of RNA used
(1.8-4 μg) RNA ng/μl
Total RNA
used for cDNA
≤ 100 a 300 18μl
301 a 500 10μl
500 ≤ 5μl
1 2 3 4 5 6
LINE
CONCENTRATION
ng/μ 260/280 260/230
1 319.2 2.07 2.32
2 425.5 2.08 2.37
3 576.9 2.08 2.39
4 363.7 2.07 2.33
5 509.9 2.08 2.39
6 345.0 2.07 2.30
 Amount of cDNA used
Dilution 1/10 Dilution 1/100 Dilution 1/1000 (+) (-) Bl

 Modification in the use of primers for the realization of the
cDNA
cDNA prepared with
primer F110
(reovirus); PCR with
primer specific for
Reovirus
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 cDNA prepared with
Random-primer; PCR
with primer specific
for Reovirus
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30

 Viruses have high mutation and recombination rates. Therefore diagnostic
MCOL2737-4
MCOL2737-37
MCOL2737-56
MCOL2737-33
SM3375-113
MCOL2737-35
HEL-4
MCOL2737-36
MCOL2737-36
CM546010
FSD23
FSD80
FSD5
60
94 50
CM5460-10
Outgroup -ToTV
82
52
66
98
0.2
2014
(Eastern plains)
1980
(North Coast)
FSD5B
FSD80B
178 MCOL1754
003 MON
109 AMZ9 2
Cauca1A
17 FSD 29
111 AMZ9
16 FSD 29
181 COL1692
Llano1A
Llano1B
75
13 AMZ 16
Amz16A
69 AMZ16
AMZ 9
126 FSD29/Sec
92 FSD29
115 AMZ9 2
130 FSD29/Sec
FSD29A
FSD29B
Outgroup-RRSV
100
97
100
83
87
85
0.1
1980
(North Coast)
1990
(Amazonas
And Cauca)
Torrado
Reovirus 2005
(Eastern plains)
Jimenez, Cuervo, Carvajal, Lozano, Cuellar, 2014
methodologies must be adjusted accordingly.
 This requires continued collection of virus isolates and its biological
characterization.
 The method, besides providing a quick detection, also makes possible the
study of sequence variability/virus evolution.

Progress in certification of the CIAT collection of Manihot
Accessions available (negative for all viruses) 2233
Unavailable accessions evaluated (with positive results for at least one of
218
the viruses)
Pending accessions to assess 4220
Virus
Positive
accessions %
Cassava frogsking virus (CSFV), Reovirus: 99 4.43
Cassava polero-like virus’ (CsPLV, LUTEO) 87 3.89
Cassava Torrado-like virus’ (CsTLV) 22 0.98
Cassava new alphaflexivirus’ (CsNAV, POTEX) 16 0.71
Cassava X Virus (CsXV) 9 0.40
Cassava common mosaic virus (CCMV) 6 0.26
Other viruses evaluated
Accessions
evaluated
Negative
accessions
African cassava mosaic virus (ACMV) 382 382
Cassava Vein Mosaic virus (CVMV) 1260 1260
Virus Accessions
Cassava frogsking virus (CsFSaV) 81
Cassava polero-like virus’ (CsPLV) 67
Cassava Torrado-like virus’ (CsTLV) 19
Cassava new alphaflexivirus’ (CsNAV) 14
Cassava X virus (CsXV) 6
Cassava common mosaic virus (CsCMV) 5
CsPLV + CsFSaV 15
CsPLV + CsTLV 2
CsPLV + CsFSaV + CsTLV 1
CsPLV + CsNAV 2
CsXV + CsFSaV 2
CsCMV + CsXV 1
 The low percentage of mixed infections as compared to single infections, suggests again that Secundina is
useful detecting mixed infections.
 The high number of single infections indicates a dramatic improvement in indexing by using RT-PCR as
compared to Secundina.
 The low porcentage of CsXV and CsCMV suggest that ELISA was efficient in detection.
M.Cuervo, A.Martinez, E. Aranzales, J.C. Ramirez,, Virology Team

The standardization and implementation of new
molecular diagnostic techniques for cassava virus
 In cassava plants affected by the frogskin disease and the common
mosaic disease, several viruses were identified that may contribute to the
development of the disease, it is very important to make the diagnosis for
all of them.
 Looking to the future, we must have the implementation of molecular
methodologies for all viruses of cassava, as in the case of CsCMV and
CsXV where the antisera are not applied anymore.
 Other pathogens that are reported infecting the crop must be included.
 We have to evaluate other indicator plants to replace Secundina.

BENEFITS (2)
 With this method we can detect up to 7 viruses from one single plant RNA
extraction which saves time and money.
 More sensitive and specific it is also recommended in the evaluation of virus
cleaning methods.
 The method also makes possible the detection of these viruses even in
asymptomatic infections/ before they are part of a disease complex.
 The molecular methods are more effective and reliable than the
symptomatology and the use of warning plants.
 It can make a diagnosis earlier without waiting for the plants to grow.
 There is neither leaks nor confusion for other environmental effects.

Developing research to improve the
health of germplasm collections
Management of the fungus complex caused by Ergot
in Brachiaria sp.
 Standardization of molecular diagnostic
methodologies for virus detection in cassava.
 Use of tablets in GHL operations.

Process before tablet Implementation
Receipt of beans and tropical forages seeds in GHL
Generate and print formats of materials to test
Fungi test Virus test Bacteria test
Register results of evaluations
Compiling and transcribing data to
electronic format
Sync data to
GRP database
A.Hernandez, D.F.Gonzalez2014

Process after tablet Implementation
Receipt of beans and tropical forages seeds
in Germplasm Health Laboratory
Web site
Central
Data Base
Web services
Synchronization
of active
WI FI
Access point
Upload data
of materials
to be
evaluated
Registry of evaluations
Fungi Test Bacteria Test Virus Test evaluations
A.Hernandez, D.F.Gonzalez2014

Benefits (3)
 Material traceability through the
identification of materials with barcode.
 Minimize errors in the data taking during the
evaluation.
 Optimizing data collection.
 Streamlining the migration of the results to
the database.
 Reduction in time of giving results.
 Reduction of steps doing the evaluations
(17 vs. 10 steps).
 Generating easy reports.

Perspectives
 We have very high standards to evaluate germplasmfor distribution; it
is a must.
 To strengthen the CIAT diagnostic system related to the international
rules (ISTA) and standards that improve the quality and efficiency of
the laboratory.
 To collaborate permanently with CIAT scientists and other
laboratories.
 To improve and maintain communication with ICA officers to facilitate
the import and export of CIAT germplasm material.
 To implement a program of biosecurity in the GHL.
 To manage that the GHL starts to be part of the ICA national diagnostic
laboratories network (resolution 003823 of 04 September, 2013); so
the results of the lab analyses will be official.
We go to a certified laboratory !

Julio Cesar Ramirez
Angelica Maria Martinez
Angela Hernandez
Luis Guillermo Santos
Ericson Aranzales
Dr. Daniel Debouck
Josefina Martinez
Isabel Natalia Salas
Camilo Oliveros-Comunicaciones
Team of GHL
Cassava virology team at CIAT:
Dr. Wilmer Cuellar
Dr. Monica Carvajal
Ivan Lozano
Jenyfer Jimenez
Ana Maria Leiva

Great team !!