Global germplasm collections: sure benefits without seedborne diseases

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Information about Global germplasm collections: sure benefits without seedborne diseases

Published on September 25, 2014

Author: CIAT



The Genetic Resources Program is the germplasm bank of CIAT which conserves the collections of bean and tropical forage seeds, and the collection of cassava "in vitro" for a total of approximately 67,500 different accessions. The conservation of these collections allows the benefit of the distribution of germplasm of approximately 6,000 samples of genetic material per year, at national and international level. To minimize the risks associated with the movement of germplasm, especially the transport of pathogens of quarantine interest, it is required a process of laboratory tests certifying the plant quality. This process is carried out in the Germplasm Health Laboratory of the GRP, where also research is developed to improve the effectiveness of the detection, testing reliability and efficiency of operations.


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 View slide

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 View slide

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 !!

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