Manual of Seed Health Testing Methods

Scope and Purpose

Seed health testing is a tool which helps to assess seed-borne disease risk and is an integral part of any seed-borne disease control program. This manual provides descriptions of seed health testing methods that are considered by ISHI-Veg to be the reference for the vegetable seed industry. The methods are the basis for the position adopted by the vegetable seed sector in May 2006 titled “Guidelines for the Use of Seed Health Methods by the Vegetable Seed Industry”, which was revised in 2010.

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. Development of a seed health testing method requires consideration of a number of factors, including 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. The seed pathologists within ISHI-Veg bring together the necessary experience, expertise and data in developing a test method. In addition ISHI-Veg makes recommendations on sub-sample and sample sizes for each method.

Method Description

For each crop/pathogen combination, the key elements of the method are provided in a manner that is familiar to laboratory staff with ample experience in working with plant pathogens. It is expected that the principles of Good Laboratory Practice (http://ec.europa.eu/enterprise/pharmaceuticals/eudralex/vol-7/a/7ag4a.pdf) are being followed at all times.

The methods described 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 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 members. Data related to validation of components of the method as well as the peer review are stored in the ISHI database.

The descriptions 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.

Interpretation of Results

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 on tomato seeds 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 then the bioassay must be completed.

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.

Disclaimer

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

Note

Methods presented in this 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.

Bean - Pseudomonas savastanoi pv. phaseolicola (Adopted as an ISTA Rule)

Bean - Xanthomonas axonopodis pv. phaseoli (Adopted as an ISTA Rule)

Brassica - Phoma lingam

Brassica - Xanthomonas campestris pv. campestris (modified Feb. 2013) (Adopted as an ISTA Rule)

Brassica - X.c. pv. campestris (disinfested/disinfected seed) (modified Feb. 2013)

Carrot - Alternaria dauci (Adopted as an ISTA Rule)

Carrot - Alternaria radicina (Adopted as an ISTA Rule)

Carrot - Alternaria radicina

Carrot - Xanthomonas hortorum pv. carotae (Adopted as an ISTA Rule)

Celery - Septoria apiicola

Cucurbit - Acidovorax avenae subsp. citrulli (An NSHS Standard)

Cucurbit - Squash mosaic virus, Cucumber green mottle mosaic virus and Melon necrotic spot virus (Adopted as an ISTA Rule)