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Seed Health Testing: Doing Things Right

    Affiliations
    Authors and Affiliations
    • Gerrit A. Hiddink1
    • Roland Willmann2
    • Joyce H. C. Woudenberg3
    • Rose Souza-Richards3
    1. 1Seed Technology Research, ENZA Zaden Seed Operations BV, Enkhuizen, Noord-Holland, 1602DB, The Netherlands
    2. 2Operations Technology, BASF, Nunhem, Limburg, 6083AB, The Netherlands
    3. 3Seed Health, International Seed Federation, Nyon, Vaud, 1260, Switzerland

    Abstract

    Since seeds can be a route for pathogen introduction, they are routinely inspected and tested to prevent pest outbreaks and introduction into new territories. The need for high throughput, short lead times, and cost reduction has played an important role in the development and application of techniques in seed health testing. Examples are molecular and serological techniques, such as ELISA and PCR assays, which are commonly called indirect tests. After signal detection in ELISA or PCR assay a seed lot is a suspect lot that requires further investigation for a final conclusion about the health status of the seed lot, since these tests do not provide any information about pathogen viability or pathogenicity. The seed industry uses them as a prescreen to identify healthy seed lots and in combination with classical methods, commonly called direct tests, to confirm viability of the target pathogen and demonstrate its pathogenicity. However, outside industry, indirect tests are increasingly used to make a final decision on the health status of a seed lot. This has led to a growing number of seed lots being rejected when the risk of introducing a pathogen to importing countries may have been negligible. We propose that investments continue to be made in the development of high-throughput prescreening detection methods like HTS and PCR assays, but together with direct tests that enable accurate assessment of the risks involved when target pathogens are detected using indirect tests. Close collaboration between molecular scientists and classical phytopathologists is essential.

    Copyright © 2023 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

    The seed industry has a twofold responsibility when moving seeds internationally: to deliver healthy seeds to customers and to comply with (inter)national phytosanitary regulations. Seeds are the foundation for production of most crops, and seed health impacts food production in many ways. Healthy seeds, free from seed-transmitted pathogens, are a prerequisite for sustainable food production (Gitaitis and Walcott 2007; Strange and Scott 2005). Because seeds can be a part of the pathogen life cycle and seed contamination a means of survival and introduction into (new) areas (Gitaitis and Walcott 2007; ISPM 38 2017; Lessl et al. 2007), they are inspected routinely and tested to prevent disease outbreaks and introduction of pathogens into new territories. The International Seed Federation Regulated Pest List Initiative (ISF-RPLI) gives an extensive review for which crop pest combinations seeds can be a pathway for regulated pests (ISF regulated pest list database). To control the spread of seed-transmittable pathogens, seed health tests are used as a phytosanitary requirement by National Plant Protection Organizations (NPPOs) before allowing seeds to enter their territories.

    The efficient production of high-quality seeds in sufficient volume and in an economically feasible manner to support the demand for seeds frequently requires the production of seeds in areas other than the locations used for food production. Today, there is no country that could fully supply farmers with seeds of their choice solely from their own domestic seed production (ISF 2021). Seed companies produce and trial seeds in different countries to mitigate the risk of crop failures due to adverse weather and growing conditions that hamper the production of high-quality seeds and to exploit different seasonality across the hemispheres. Therefore, global movement of high-quality seeds is of utmost importance. To reduce the risks of spreading seedborne pathogens with seeds, the seed industry invests in the development and participation in healthy seed production systems, such as Good Seed and Plant Practices (GSPP), and in partnering with USDA-APHIS in the ReFreSH program (https://www.aphis.usda.gov/aphis/ourfocus/planthealth/refresh). The International Seed Federation (ISF) has guidelines for healthy vegetable seed production (ISF 2020) and works on the development of a systems approach to pest risk management (ISPM 14 2002). By finding optimal locations for seed production, and by using appropriate preventive hygiene measures, disease inspections, and pathogen control programs during seed production, the seed sector ensures the steady supply of healthy and high-quality seeds to farmers globally.

    Under the umbrella of the ISF, the International Seed Health Initiative (ISHI: https://worldseed.org/our-work/seed-health/) brings together seed companies, public sector institutions, and private laboratories to develop seed health tests for economically important, seed-transmissible pathogens, with a particular focus on vegetables. Today, its members account for 70 to 75% of vegetable seeds that are traded internationally. The initiative aims to secure the delivery of sufficiently healthy seeds to customers by developing methods for seed health testing that are recognized internationally as reference methods and accepted as industry standards (https://worldseed.org/our-work/seed-health/ishi-methods/). Through the use of these methods and its expertise, the initiative strives to assist seed companies in their risk management and to facilitate the international movement of seeds (ISF 2010). ISHI focuses on the development of seed health test methods for pathogens that have a proven record of being seed transmittable and are thus able to cause disease of the germinating seedling or plant grown from that seed. These are referred to as pathway-proven pests (Ranganathan et al. 2020). It is important to note that a pathogen being transmittable extends further compared with a pathogen being seedborne. In the latter case, an organism can be present on a specific seed but is not able to initiate disease from its presence on the developing seedling or plant arising from that seed (Baker and Smith 1966). This pathway of transmission from seed to seedling and successively to the plant, although very effective for some pathogens, is a relatively rare route of disease establishment that few pathogens are able to use effectively. For pathogens for which seeds can be a proven pathway, ISHI develops seed health test methods based on a set of requirements and best practices that the seed health methods that ISHI publishes need to meet (https://worldseed.org/our-work/seed-health/ishi-best-practices/).

    To reliably assess the health status of a seed lot, many factors, such as trained personnel, access to high-quality chemicals and consumables, maintained and calibrated equipment, and above all a representative seed sample from a fully homogenized seed lot, are crucial. When these prerequisites cannot be met, the result of a seed health test is not reliable. Although their impact is major, we will consider these factors out of scope for this paper and assume the tests are performed under optimal conditions for seed health testing. The need for high throughput, short lead times, and cost reduction has played an important role in the development and application of techniques in seed health testing that permit rapid analysis of a large number of samples at relatively low cost. Examples of such methods are serological and molecular assays, such as the enzyme linked immunosorbent assay (ELISA), polymerase chain reaction (PCR), and toward the future, high-throughput sequencing (HTS) assays. In general, bulk samples of seeds are incubated in buffer, ground, and visualized with a fluorescently labelled protein (ELISA), or nucleic acid is extracted, and the target DNA or RNA is amplified by a PCR assay. These techniques are commonly called indirect assays and differ in the interpretation of certain aspects of the results from more classical methods. They do not provide any information about pathogen viability or pathogenicity. A number of published tests have incorporated methods such as DNA-binding dyes like propidium monoazide (Chai et al. 2020; Temple et al. 2013) that enable differentiation of nucleic acid from viable versus nonviable bacterial pathogen propagules. Still, limitations concerning pathogenicity remain. ELISA detects proteins associated with a target pathogen. However, the protein detected might be a (partial) remnant of a nonviable or no longer infectious pathogen. Furthermore, an ELISA might be subject to cross-reactivity with nontarget proteins. An ELISA positive result is, therefore, a putative positive, whereas a negative result indicates a healthy or noninfected lot. Similarly, for molecular methods that detect the presence of nucleic acid of the target pathogen, a positive detection does not indicate whether it is just the nucleic acid fragment that is present or whether the corresponding propagule source is present. It therefore does not return information about whether the source of the nucleic acid is pathogenic. As DNA and RNA are quite stable in seeds, shown for DNA by, for example, Walters et al. (2006), who isolated fungal DNA out of 135-year-old seed, the genome of a pathogen might not even have been intact because PCR detects only small fragments out of the pathogen genome. Additionally, examples can be found of horizontal exchange of nucleic acids, which is frequently observed between bacterial species (Thomas and Nielsen 2005), as well as in RNA viruses (Liu et al. 2012; Sztuba-Solińska et al. 2011), and transferred genomic fragments can be detected. Factors other than target nucleic acid or protein detection could also trigger a false positive signal, which can be difficult to distinguish from the signal produced by the target. Despite this drawback, PCR assays and ELISA are very useful to identify noninfected seed lots. After signal detection in an ELISA or a PCR assay, a seed lot is a suspect lot that requires further investigation on the viability and pathogenicity of the detected organism. A final conclusion about the infection status of the seed lot and the corresponding risk of disease establishment is determined by a direct test showing the infectiousness (or viability) and pathogenicity of the pathogen.

    The more classical methods for detecting seed-transmitted pathogens are based on demonstrating the presence of a living organism in or on the seed. The results obtained with these methods, also called direct tests, allow for interpretation of the biological relevance of the seed health assay, confirmation of the presence of the target pathogen, and proof of the viability and pathogenicity. The assays generally consist of three steps: (i) isolation of the pathogen from seeds for culturable plant pathogens, expression of typical symptoms on the seedlings grown from infected seed, or development of fungal structures on the seed; (ii) detection and identification of the pathogen; and (iii) confirmation of the viability and pathogenicity of isolates of the pathogen by inoculation onto plants of the relevant species to fulfill Koch's postulates for culturable organisms. For nonculturable organisms, confirmation should come from a grow-out test, for which, after development of typical symptoms, the pathogen can be identified and (extracts of the host plant be) used as inoculum on healthy assay plants in a bioassay. By isolating the target pathogen (or detecting the target pathogen in the case of nonculturable organisms) and demonstrating viability and pathogenicity, these methods provide conclusions on the health status of a seed lot. In situations where seed sanitation is applied (repair treatments inactivating the pathogen, e.g., physical treatments such as hot water soaks [Temple et al. 2013], exposure to dry heat [Ling 2010], and chemical solutions such as peracetic acid soaks [Hopkins et al. 2003]) and evaluation of the effectiveness of the seed treatment is needed, only these direct methods are relevant due to the persistence of nucleic acids or proteins, respectively. At any time, seed treatments eliminate or reduce the risk of transmission of pathogens from seed to seedling and are therefore one of the mitigation measures the seed industry can use (Maude 1996). However, seed health testing after curative treatments is always a necessity. Examples of widely used direct tests include planting seeds under disease-conducive conditions (grow-out or sweatbox assays), spreading seeds or seed extracts on a medium known to stimulate growth of fungi or bacteria (plating or dilution plating assays), incubating seeds on moist blotter paper to promote fungal growth that is then examined microscopically (blotter tests), or applying plant tissue extracts (e.g., seed or leaf) suspected of being infected with a pathogen to “indicator” plants to assess the infectivity status of the tissue (bioassay).

    Despite the valuable information the direct tests provide, the seed industry has largely embraced the use of indirect tests because they permit rapid analysis of a large number of seed samples at relatively low cost. This is in contrast to the relatively high costs, the relatively longer duration, the dependency of mostly specific conditions to grow the pathogens or generate disease expression, and the corresponding need for technical expertise to be able to cope with the complexity of direct tests. Indirect tests are used primarily as a prescreening tool to identify healthy seed lots and in combination with direct tests to confirm the viability of the target pathogen(s) and assess the pathogenicity of the target organism detected. The general tendency is that indirect tests are used increasingly to make decisions about the health status of seed lots (Munkvold 2009, 2012). The perceived increased sensitivity of molecular testing methods has led to a growing number of seed lots being rejected from seed trade or movement, even when the risk of introducing the seedborne pathogens to importing countries might have been negligible (ISF 2022). An example is the testing for Tomato brown rugose fruit virus by RT-qPCR that detects merely RNA fragments versus testing by indicator plants detecting relevant amounts of infectious virus. The lack of a clear link between the positive results in PCR assays or ELISA and the confirmed presence of infectious target pathogens in the seed lot is a weakness of serological and molecular detection methods. In the case of HTS methods, the situation is even more complex as many target pathogens might be detected for which the risk of transmission and spread on seeds is unknown. If the trend of using indirect tests to make final decisions about the health status of seed lots continues, the prospects for the seed industry to be able to move seed internationally are uncertain and could have an impact on global food security. We propose that investments continue to be made in the development of high-throughput prescreening detection methods such as HTS and PCR assays but together with direct tests that enable accurate assessment of the risks involved when target pathogens are detected using indirect tests. To value results obtained with technologies such as PCR assays or HTS, and to understand the biological relevance of the results of these assays, close collaboration between molecular scientists and classical phytopathologists is essential. ISHI members strive to link these areas of expertise to provide information about presence of the viability of the suspect plant pathogen detected and the biological relevance of the test results through the seed health detection methods evaluated and validated as a foundation to the mission of ISHI.

    AUTHOR-RECOMMENDED INTERNET RESOURCE

    ISF regulated pest list database: https://pestlist.worldseed.org/public/pestlist.jsp

    The author(s) declare no conflict of interest.

    LITERATURE CITED

    The author(s) declare no conflict of interest.