Crop Genebank Knowledge Base

Best practices

Best practices for safe transfer of forage grass germplasm

Contributors to this page: CIAT, Colombia (Maritza Cuervo, Cesar Medina, Jose Luis Ramirez, Socorro Balcazar, Josefina Martinez, Daniel Debouck); ILRI, Ethiopia (Jean Hanson, Janice Proud, Juvy Cantrell).

Flowchart 1.Germplasm management of Beans and Tropical Forages (Click to increase the size)

The agreement between the CIAT and the International Treaty on Plant Genetic Resources for Food and Agriculture implies of the Genetic Resources Program (GRP) the conservation on behalf of the countries of 65,505 materials for 745 plant species of bean, cassava and tropical pastures as the biological patrimony of 141 countries. This responsibility of conservation is associated with the distribution of samples of this patrimony (517,916 samples distributed to 136 countries to the date) according to a regulation defined by the countries inside the Agreement, and phytosanitary procedure established by the Colombian Agricultural Institute.

The job of the GRP is to safeguard the genetic diversity of beans, cassava, forages, and their wild relatives through a mix of conservation methods, both in situ (in a natural outdoor habitat) and ex situ (in the controlled environment of a gene bank). Among the GRP's activities are research to improve conservation methods (including ways to minimize risks to the collections); screening germplasm for diseases and certifying it; duplicating materials of the collection; collecting or otherwise acquiring novel materials; recording passport, characterization and evaluation data for accessions of the collections (See Flowchart 1).

Phytosanitary risks are associated with international movement of germplasm, especially the inadvertent transport of pathogens and pests of quarantine significance. To minimize the phytosanitary risks associated with exchange germplasm and to ensure that it is free from pests and pathogens of quarantine significance; CIAT has implemented regulatory measures and safeguards to complement quarantine guidelines. The process, wich is supervised by the the Agricultural Colombian Institute (ICA), includes the following activities:

Minimize the risk of accidental introduction of exotic pest and pathogens into Colombia;
Inspect plant and facilities in screenhouses and grenhouses where the imported germplasm is being incresased;
Inspect plants in fields and greenhouses where the germplasm intended for international export is produced; and
Determine the seed health status of germplasm for international export.

The responsibility over the areas dealing with animal and plant health, with regard to international trade, has been bestowed upon the Agricultural Colombian Institute, within regulatory decree 1840 of 1994 “for which (ICA) has the mission of preventing the risk of the entry, spread, and establishment of exotic diseases, those of national sanitary concern, and of chemical risks, and protecting the sanitary quality of animals, plants and products that are exported, to minimize losses in animal-plant production and contribute to the security in foodstuffs”.

ICA has established quarantine procedures to regulate the introduction of plant germplasm and the issuing of phytosanitary certificates that accompany exports. ICA has a Plant Quarantine Station at an altitude of 2600 m (4o 42’ N latitude and 74o 12’W longitude) near Mosquera (Cundinamarca), about 15 km west of Santafé de Bogotá. ICA provides the regulatory mechanism for germplasm exchange following guidelines of the International Plant Protection Convention (IPPC). The recommendations of the IPPC were adopted by Colombian Congress in 1981 and implemented under decrees 501 of 1989.

In order to facilitate the importation and exportation, the ICA has developed the SISPAP, which may accessed through the ICA webpage in which the parties interested in importing and exporting plant products may become aware of the following via internet.

In Colombia, additional guidelines to reduce phytosanitary risks are in place. These safeguards are implemented according to the geographic origin and economic importance of the plant species concerned, and characteristic of potential pathogens and pests. Currently, ICA has a plant quarantine agreement with CIAT establishing which guidelines and safeguards are updated to facilitate germplasm exchange according to national and international requirements. An agreement was signed in 1981 between CIAT and ICA for quarantine procedures to regulate the introductions of germplasm into Colombia. The agreement permits the transit of seed through customs and quarantine stations according to the level of potential risk of introducing pests and diseases no yet reported in Colombia. High risk areas include Africa, Asia, and some European countries. The agreement covers not only crops for wich CIAT has a mandate but also other crops of economic importance to Colombia.

After clearance, materials pass through a step of multiplication in greenhouse stage, and the harvested seeds are subsequently planted in isolated fields. During these two steps, a phytosanitary follow-up is carried out. Plants showing any symptom of fungal, bacterial or viral disease are destroyed.

CIAT facilities for germplasm health testing are designed as a multifunctional laboratory to test seeds and tissues for fungi, bacteria, viruses, and occasionally nematodes and insects (See table below). The purpose of the Germplasm Health Laboratory (GHL) is to ensure that the designated germplasm is kept under the international phytosanitary standards for each crop commodity, and also to ensure that the germplasm distributed by GRP is free of diseases of quarantine importance (listed in the table below).

The ICA Plant Quarantine Officer, stationed at CIAT, carries out field and greenhouses inspections and issues "ICA Phytosanitary Certificate" bases upon those inspections and results obtained by GHL. This document accompanies all out-going germplasm from Colombia (“decrees 1840 of 1994 articule 3”).

The germplasm leaving CIAT and the one used for exchange, conservation, and characterization in the GRP are multiplied in isolated fields under favorable ecological conditions with supervision by ICA quarantine officers. The GRP has seed multiplication sites in Popayan (Cauca) and Santander de Quilichao (Cauca). The harvested seeds are analyzed by GHL prior to shipment abroad or long-term conservation.

Seed health testing activities include:

Reception, registration, sampling and storage of incoming material.
Preparation of working samples for testing.
Analysis.

The seed health testing methods used at CIAT for beans and tropical pastures are summarized in The Handbook of Procedures of the Germplasm Health Laboratory (see also Flows charts 2, 3, 4, 5 below, for a quick reference).


Flowchart 2.Flow chart Germplasm Health Laboratory (Click to increase the size)	Flowchart 3. Flow chart Germplasm Heealth Laboratory for detection of fungi (Click to increase the size)

Flowchart 4. Flow chart Germplasm Health Laboratory for detection of bacteria (Click to increase the size)	Flowchart 5. Flow chart Germplasm Health Laboratory for detection of virus (Click to increase the size)

To detect pathogens of quarantine significance, the GHL uses the methodologies recommended by CIAT pathologists and virologists (see table below). When a recipient country has additional requirements, the GHL carries out additional tests wherever possible to comply with the specific quarantine regulations of the recipient country. The GHL realizes additional researches in the management and characterization of pathogens of quarantine importance and in the standardization of new methodologies of diagnosis that are more effective and sensitive.

ILRI carries out pathogen detection tests before each regeneration. Seeds are withdrawn from the genebank, scarified and germinated using appropriate conditions for the species. Seedlings are transferred to sterilized compost in seedling trays after germination and grown in a virus screened area until one to three leaf stage. Visual inspection is carried out and seedlings are batch tested in batches of 10 seedlings using TBIA for common grass viruses. Individual seedling tests are performed on any positive batches for confirmation. Clean seedlings are released to the field for regeneration.

If the seedlings are infected with seed borne pathogens and more original seeds are available for a second regeneration, the seedlings are destroyed by incineration. If no original seeds are available, the seedlings are transferred to large pots and retained in a virus screened area for seed production. Since virus transmission is rarely 100%, the seeds are harvested from infected plants and germinated. The seedlings are tested as above to identify clean seedlings for release to the field. If all seeds are infected, thermotherapy and meristem culture are used to eliminate the virus and obtain clean plantlets in in vitro culture. Rooted plantlets can be acclimatized in the greenhouse and these plants used as a source of clean seeds.

Plants in the field are regularly inspected for virus symptoms and samples taken for testing with either TBIA or ELISA for common grass viruses. Some grass species are maintained in the field genebank and also require regular inspection. Young leaves are sampled from Napier grass for diagnosis for phytoplasma associated with Napier grass stunt using NASH. Plants that test positive for virus diseases are rogued from the field.

Field inspection is also carried out for pathogenic fungi and insects and any infection is controlled through field management. Regeneration and field genebank plots are sprayed with fungicides or insecticides at early signs of infection. Regular cutting is done on grass plants in the field genebank to reduce fungal diseases.

ILRI uses a range of different methods to detect pathogens of forage grass germplasm. Click the forage grasses health table for specific species information about health diagnosis methods to detect some of these diseases. Detailed protocols were developed for the detection of:

Virus (ELISA and TBIA).
Phytoplasm (NASH).
Fungi (seed washing technique; blotter test; potato dextrose agar; oatmeal agar).

References and further reading

Cuervo MI, Balcazar MS, Ramirez JL, Medina CA, Debouck D. 2009. Manual de operaciones laboratorio sanidad de germoplasma – unidad de recursos genéticos. GRP, CIAT, Colômbia. 72 pp. Available here

Plant Biosecurity, Biosecurity Australia 2000. Viruses, Phytoplasmas and Spiroplasmas of Clonal Grasses and Their Diagnosis. Consultancy report. Available from: http://www.daff.gov.au/__data/assets/word_doc/0020/24761/consul_rpt_clonal.doc. Date accessed: 30 March 2010.