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ISHI-Veg brings together seed companies, public sector institutions and private laboratories to develop detection methods for economically important seed-borne pathogens of vegetable crops.

The International Seed Health Initiative for Vegetables Crops (ISHI-Veg) was set up in 1993. Its members account for 70-75% of vegetable seed that is traded internationally. ISHI-Veg methods have a well-established track record in the seed industry and are recognized as reference methods by seed pathologists from testing laboratories of inspection services, private testing labs and the vegetable seed industry. In addition, some ISHI-Veg methods have been accepted as ISTA Rules and as Standards by the USDA-APHIS National Seed Health System (NSHS).

ISHI-Veg methods have been developed using the collective experience and expertise of its members. Developing a seed health testing method is complex and requires a consideration of disease dynamics. The epidemiology of a disease depends upon the pathogen infection levels within the seed, the climate in which the seed is grown, and the interactions of the host, pathogen and the environment.

Comprehensive studies that elucidate all these aspects are often not possible to find, as data may be scattered and sometimes inconclusive. Seed pathologists within ISHI-Veg bring together the necessary experience and data in developing a test method. In addition ISHI-Veg makes recommendations on sub-sample and sample sizes for each method.

Seed Health Testing

These seed health test methods are a tool for seed-borne disease risk management, which is itself a part of a company’s seed-borne disease control program. Plant disease control is achieved by reducing the progress of the disease and keeping disease development below a consequential level. ISHI-Veg methods are a reference for the vegetable seed industry, which adopted a position concerning their use in 2010 – see Guidelines for the Use of Seed Health Methods by the Vegetable Seed Industry.

Interpretation of seed health tests

The seed industry recognizes that even when tests are carried out correctly according to the protocol, differences can exist due to the expression or condition of the pathogen. Tests can show identical results in cases where the pathogen is viable but a deviating result if the pathogen is dead or inactivated.

Many countries active in seed health testing make use of modern laboratory techniques. A PCR may be easier to implement by a developing country than a traditional direct method that requires skilled pathologists. Indirect tests (e.g. serological techniques and DNA/RNA based techniques) will detect both dead/non-infectious and viable/infectious pathogens in contrast to direct tests (grow-outs and bioassays) that give final proof of infestation by a viable pathogen.

Diagnostic methods for seed health testing should have the right balance between preventing infected seed lots from being sold (as a consequence of a false negative test result) and preventing unjustified measures such as discarding seed lots (as a consequence of a false positive test result). There are, however, considerations in using such indirect techniques and ISF has taken a position on their use – see ISF Viewpoint on Indirect Seed Health Tests.

ISHI-Veg Manual

The ISHI-Veg Manual

The manual provides protocols for seed health testing methods that are considered by ISHI-Veg to be the standard for the vegetable seed industry.

ISHI-Veg methods are the outcome of one of the two processes or both:

  • A comparative test done by 6-8 company and public labs.
  • A peer review of the method description by ISHI-Veg members and/or external reviewers. The supporting data may be supplied by the laboratory responsible for development of the method and/or a validation test of certain components of the described method conducted by ISHI-Veg members.
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Method description and execution

Method Description

The descriptions in the ISHI-Veg seed health testing protocols include information on critical reagents and reference materials that were used in the test.

These methods serve as references for achieving uniformity in seed health testing and multiple methods may be used for detecting a target organism if they have been found to be equivalent in terms of the results they provide. For example, there are three fully separate methods for Alternaria radicina in carrot. Accepted alternatives for certain elements of the test method may also be indicated within the description.

Although these methods have been reviewed and tested over time by the industry, specific results may vary because standardized reference materials (or controls) are not readily available for seed health testing.

Information on verifying test performance and restrictions on use is provided in the section titled Sensitivity and Restrictions on Use or Restrictions on Use. A spiking control is often recommended to verify that the ability of the seed health test to detect the target organism (fungi, bacteria and viruses) is not inhibited by routine disinfestation/disinfection chemicals or the presence of other organisms such as fast growing saprophytes.

Method Execution

For each crop/pathogen combination, the key elements of the method are provided in a manner that should be sufficiently clear to laboratory staff with ample experience in working with plant pathogens. It is expected that the Principles of Good Laboratory Practice , as detailed in the OECD guidance document on the website of the European Commission, are followed at all times.

It is important to note that serological techniques and DNA/RNA based techniques will detect both dead/non-infectious and viable/infectious pathogens. In contrast, grow-outs and bioassays give final proof of infestation by a viable pathogen. Therefore, ELISA and bioassay for viruses for instance are not equivalent. The ELISA technique is considered an optional pre-screening method for the bioassay and the test can be terminated when a negative ELISA result is obtained. If a positive ELISA result is obtained this result should be considered preliminary and the bioassay must be completed for confirmation of the presence of pathogenic virus.

In all of the methods, results are more easily interpreted if a seed lot with a known infestation level is included as a positive check. Therefore, it is recommended that this be done each time a set of samples is tested.

Best Practices

To ensure accurate and reliable results ISHI-Veg has described best practices for molecular techniques, ELISA and dilution-plating assays used in routine seed health testing. It is strongly recommended following these best practices to ensure a valid test result.

Best Practices for Dilution Plating Assays in Seed Health Tests (2015)
Best Practices for ELISA Assays in Seed Health Tests (2015)
Best Practices for Molecular Techniques in Seed Health Tests (2014)

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Protocols

ISHI-Veg Protocols

Protocols presented in the ISHI-Veg Manual may be updated at any time. It is the responsibility of the user to see that the most recent version of a method is being applied.

Contact the ISF Secretariat if you wish to know more about the methods or validation reports.

Disclaimer

ISF cannot guarantee that the results obtained by laboratories that follow the protocol from the ISHI-Veg Manual of Detection Methods are accurate and representative. Many factors (e.g. personnel skills, lab conditions, quality of reagents, sampling methods etc.) can influence the results. Consequently, in case of any litigation ISF will not accept any liability with respect to the use of the methods in this manual.

Bean – Pseudomonas savastanoi pv. phaseolicola (ISTA Rule)
Bean – Xanthomonas axonopodis pv. phaseoli (ISTA Rule)
Brassica – Phoma lingam
Brassica – Xanthomonas campestris pv. campestris (untreated seed) (ISTA Rule)
Brassica – X.c. pv. campestris (disinfested/disinfected seed) (ISTA Rule)
Carrot – Alternaria dauci (ISTA Rule)
Carrot – Alternaria radicina (ISTA Rule)
Carrot – Alternaria radicina
Carrot – Xanthomonas hortorum pv. carotae (ISTA Rule)
Celery – Septoria apiicola
Cornsalad – Acidovorax valerianellae (ISTA Rule)
Cucurbit – Acidovorax avenae subsp. citrulli (NSHS Standard)
Cucurbit – Squash mosaic, Cucumber green mottle mosaic and Melon necrotic spot viruses (ISTA Rule)
Lettuce – Lettuce mosaic virus
Pea – Pea seed-borne mosaic virus and Pea early browning virus (ISTA Rule)
Pea – Pseudomonas syringae pv. pisi (ISTA Rule)
Pepper – Tobamoviruses
Pepper – Xanthomonas spp.
Spinach – Verticillium dahliae
Tomato – Clavibacter michiganensis subsp. michiganensis
Tomato – Pepino mosaic virus
Tomato – Tobamoviruses (ISTA Rule)
Tomato – Xanthomonas spp.