Community Research and Development Information Service - CORDIS

FP7

TESTA Report Summary

Project reference: 311875
Funded under: FP7-KBBE

Final Report Summary - TESTA (Seed health: development of seed treatment methods, evidence for seed transmission and assessment of seed health.)

Executive Summary:
An increasing world population is putting pressure on food supplies and on the environment. It is therefore crucial to maximise the efficiency of production from the land that is suitable for agriculture and horticulture. High quality seed is the foundation of high quality crops producing high yields and therefore maximising food and feed production. Plant seed is produced and traded across the globe and can carry and spread diseases and pests very efficiently to key production areas. Regulatory and quality controls are in place to reduce the risk of this but these must be supported by up-to-date underpinning methodologies for risk assessment, sampling and detection of pests and pathogens in seed lots and disinfection treatments.

The project developed novel approaches to assessing the risk of seed transmission by determining the relevant threshold inoculum levels and routes for infection for a range of important pathogens and pests. This work will add to the scientific literature underpinning seed health decisions. Methods developed in the project will also provide protocols which can be used in future seed transmission studies. The revision of the Annotated List of Seed-borne Diseases will provide an invaluable resource for all regulators, seed producers and laboratories.

Testing seed routinely is an important approach to maintaining seed health and to preventing the introduction of new pests and diseases and the unchecked spread of indigenous diseases and pests. However, testing methods rely on the robustness of sampling approaches. The project developed statistical approaches to improve the accuracy of seed sampling, especially for small lots or lots where the level of inoculum is likely to be low and the seed lots are likely to be large. The sampling approaches will be assessed by ISTA and made available to the National Plant Health services and the industry.

The development of validated and harmonised methods and tools for early detection is key to treatment and control. The awareness of the need for validation of protocols has increased rapidly over the last few years. Stricter regulations and quality requirements have resulted in increasing knowledge of and requirements for validation studies. TESTA has contributed to this awareness through dissemination of the work plans, teaching a validation course, presentations at meetings for different groups of stakeholders, and publication of the validation reports. 13 protocols were successfully validated and will be made available for use by stakeholders through inclusion in ISTA and EPPO protocols, by publication in peer-reviewed journals, publication on the TESTA website or TESTA-partner website, publication of instruction videos, presentations at national and international meetings, such as ISTA-meeting, EPPO/TESTA-meeting and ISHI-Veg meetings, and protocols are available upon request. Potential users such as NPPOs, inspection services, and industry can implement these protocols for reliable detection of these quarantine and quality pathogens.

The development of generic methods for seed testing will make testing quicker, cheaper and more accurate. These and other methods were validated within the project so that they can be immediately used by the Inspection Laboratories at the end of the project. The project also investigated the use of non-destructive methods for testing seed - these methods will allow the industry to save money currently wasted in destroyed seed.

Novel detection and diagnostic technologies were evaluated for their future role in seed testing - these included Luminex multiplex technology and Next and third generation sequencing technologies.

The ability to treat seed with suitable disinfectants would mean a huge saving in money for seed companies due to current losses of contaminated seed and also provide extra protection for the EU against disease. The project investigated and assessed the usefulness of a range of novel methods for disinfection of seed.
Project Context and Objectives:
A rapidly increasing world population is putting increasing pressure on world food supplies. The updated UN report (2015) reports that the world population continues to rise, at a rate of about 1.18% per year, or approximately an additional 83 million people annually (http://esa.un.org/unpd/wpp/Publications/Files/Key_Findings_WPP_2015.pdf). If the population continues to increase at the current rate there could be 11 billion people in the world by 2100. The interaction of climate change with population growth is also increasing the challenges for food production across the globe (UNEP report on the State of the World population 2009: https://www.unfpa.org/sites/default/files/pub-pdf/state_of_world_population_2009.pdf). The FAO report on Food Insecurity (2015) recognised that economic growth and investment in agriculture remains critical to sustainable long-term food security.

In addition, increased global trade can spread crop and plant diseases and pests more quickly than science can develop methods for their control. Concern for the environment also limits access to the use of chemical controls such as pesticides for example the EC Pesticide Authorisations Directive 91/414/EEC, the EC legislation on Maximum Residue Levels (MRLs) and a new European Commission initiative on the "Sustainable Use of Plant Protection Products".

High quality seed is the foundation of high quality crops producing high yields. Plant seed is produced and traded across the globe (see Figure 1) and can carry and spread diseases and pests very efficiently to key production areas.

Regulatory and quality controls are in place to reduce the risk of this but these must be supported by up-to-date underpinning methodologies for risk assessment, sampling and detection of pests and pathogens in seed lots and disinfection treatments.

The TESTA project was designed to provide the next generation of methods to ensure high quality seed for European farmers.

PROJECT OBJECTIVES

• Enhance the understanding of the biology of seed transmission by developing novel methods based on labelled microorganisms and microscopy.

• Establish a comprehensive web-based database as a global resource, detailing all known pests and diseases of crop plants transmitted by seed.

• Develop novel methods for assessing levels of seed transmission and their relevance to disease levels in crops in support of risk assessment.

• Improve generic sampling methodologies for seed lots to generate representative samples for testing.

• Establish generic platforms for testing methods for seed.

• Assess the potential for using innovative methods for testing seed.

• Assess the potential for using non-destructive testing methods for seed.

• Validate practical methods for immediate use in regulatory control laboratories.

• Develop novel disinfection methodologies for seed.

• Develop protocols for assessing the proficiency of disinfection methods.

• Disseminate results to National policy stakeholders, testing laboratories and inspection services via websites, workshops, scientific papers and conferences.

CURRENT EU REGULATORY AND QUALITY CONTROLS FOR SEED

In practice in the European Union (EU), if you want to market certified seed of agricultural and vegetable species covered by the legislation, you can only do so if the species/variety is on a national list and has been officially certified by the Member State (MS authority), and you are registered to market such seed. You can market Standard seed in the case of vegetables and ornamentals where it is under the control of the seed supplier only. Seed certification is a quality assurance process which ensures seed meets specific standards - relating to the crop and seed. These standards are clearly defined in the seed marketing regulations. Certified seed must be marketed in correctly packed, sealed and labelled packages. The aim of the scheme is to ensure high-quality crop and seed production. Seed that is imported into the EU must also have an equivalent certificate, for example under the Organisation for Economic Co-operation and Development (OECD) scheme.

There are various EU Marketing Directives that apply to true seeds and these impose quality standards regarding indigenous pests that affect quality. Their aim is to provide minimum harmonised standards that allow free trade throughout the single market. Growers and suppliers mainly operate these directives with official monitoring and supervision by the National Plant Protection Organization (NPPO). Growers are obliged in the legislation to inform the NPPO of any high levels of pests and then the latter will provide advice and if necessary enforce measures in order to reduce this to an acceptable level. Member states (MS) are also permitted their own additional voluntary certification schemes under these directives and these have tighter health standards than the directive minimum and stricter growing conditions, though they vary with the MS, and with official inspections and checks. Directives for vegetable and other seeds have some minimum requirements for seed-borne pathogens to “be at the lowest possible level” and in some cases tolerances are applied.

The primary regulation covering plant health in the EU is the “EU Plant Health Directive 2000/29/EC of 8 May 2000 on protective measures against the introduction into the Community of organisms harmful to plants or plant products and against their spread within the Community” (EU 2000). This EU Council directive covers the MS of the EU and is implemented by all MS in their national legislation. The directive specifically lists harmful organisms, (otherwise known as pests), which are not known to occur within the EU and whose introduction are prohibited, or other pests occurring in some or all MS but which are under regulatory control in planting material or other products. There are around 250 listed harmful organisms which according to the directive definition are considered to mean: “any species, strain or biotype of plant, animal or pathogenic agent injurious to plants or plant products”.

The implementation of the controls for the production of healthy seed are supported by the MS NPPOs, official seed testing laboratories, the seed industry and the supporting organisations such as the International Seed Testing Association (ISTA), the European Plant Protection Organisation (EPPO) and the International Seed Federation (ISF-ISHI). ISTA is responsible for the development of standard procedures for sampling and testing seeds and to promote uniform application of these procedures for evaluation of seeds moving in international trade. EPPO promotes the protection of the European and Mediterranean regions from non-indigenous pests by producing standards for diagnostics and plant protection products. The ISF provides guidelines for the seed industry, particularly on diagnostic methods for vegetable seeds through its VEG working group, but also on effective seed treatments.

Increasing awareness of the impact of seed-borne pathogens and pests on crop yields has led to pressure for the development of new techniques supporting the above strategies for control of contamination in seed. Successful and cost-effective implementation of these controls demands that the supporting sampling and detection methodologies are available to allow detection of contamination of seed at the required levels and disinfection methodologies to reduce levels of contamination to acceptable levels without affecting the quality and germination of the seed concerned.

As the number of pests and diseases implicated in seed transmission is large it is important that the methods are as generic and cheap as possible. To support the above developments it is also important to have the underpinning scientific knowledge on the biology of seed transmission of pathogens and pests to support decisions.

HOW THE PROJECT SUPPORTS THE PRODUCTION OF QUALITY SEED FOR USE IN EU AGRICULTURE

Outcomes from the project provide the toolbox supporting the NPPOs, official seed testing laboratories, the seed industry and the supporting organisations such as ISTA, EPPO and ISF-ISHI.

WP1 was designed to provide information on the number and type of pests and diseases of seed, mechanisms of seed transmission from crop to seed and seed to crop and tools for determining the transmission rates of seed borne plant pathogenic agents from seed to crops.

WP2 addressed the optimization of sampling plans to provide sufficiently low limits of detection for pests at minimum cost with objectives: optimising seed lot sampling and defining procedures for a range of model host/pathogen combinations through characterisation of infected seed lots and evidence based modelling and making recommendations for seed sampling plans.

WP3 developed testing methods for seed with the objectives of:
1. Developing the most efficient DNA/RNA extraction procedures to be used on seeds.
2. Developing uniform real-time PCR assays for seeds infected with pathogens.
3. Developing a protocol for generic multiplex detection to be used for multiple pathogens simultaneously in one sample.
4. Investigating the usefulness of Next Generation Sequence Technology for diagnostic purposes.
5. Developing a non-destructive detection method for pathogens on seeds.

WP4 developed novel methods for disinfection of seeds. The WP will also developed novel methods to assess the efficacy of disinfection.

In WP5 we validated these methods and other methods for practical applications. The inclusion of members of the ISTA Seed Health Committee, EPPO as a partner and representatives of ISF-ISHI ensured that the results from the project were tailored to their needs.

Through WP6 the project disseminated results to National policy stakeholders, testing laboratories and inspection services via the website, training courses/workshops, scientific papers and presentations at conferences.

SELECTION OF TARGET CROPS AND DISEASES

With a limited budget and a large number of crops and pests listed on the current Plant Health Directive it was important to choose a target list for the project. Crops targeted in the project were selected as those of economic importance to the EU and as far as possible to be representative of the variety of plant species, crops and geographical areas in the EU, 10 of those selected are in the top 30 most economically important crops (see Figure 2). Pests and pathogens of these crops were selected as far as possible to represent the groups of organisms i.e. fungi, bacteria, viruses, viroids and pests and the different types of seed transmission.
Project Results:
ADVANCES IN THE UNDERSTANDING OF THE BIOLOGY OF SEED TRANSMISSION USING NOVEL METHODS BASED ON LABELLED MICROORGANISMS AND MICROSCOPY

Transmission to and by seed is one of the most efficient means of dispersion for most plant-pathogens. Seed-borne bacterial plant pathogens are of particular concern because strategies for the management of bacterial diseases are very limited given the rare and antiquated chemical options available. Sanitary control of seeds is the main method currently used to control seed-transmitted bacterial disease. Sanitary control includes seed testing and treatments (chemical and physical) of infected seeds. The efficiency of the seed testing methods relies in part in the efficiency of the detection method that is used. In turn, the efficiency of the detection method depends on the location of the bacteria in the seed, which is linked to the pathway used by bacteria to reach the seed. Three pathways for seed contamination have been reported. i) Seed can be internally contaminated via the host xylem, as occurs for virus transmission, some fungi and other bacteria. This usually results in the invasion of seed through the hilum. ii) Seed may also become infested via the pistil, where bacteria move from the stigma through the stylar tissues down to the embryo. Finally, iii) an external and/or sub-tegumental contamination of seed occurs via fruit as a consequence of contact of the seed with fruit tissue showing symptoms, or at harvest and during threshing with residues carrying large bacterial populations (Maude, 1996). The aims of this part of the project were to monitor transmission of bacterial pathogen to seeds of their hosts using microscopy and gfp-tagged bacteria. A panel of plant pathogenic bacteria of economic concern were gfp-tagged including the agents of bacterial canker and wilt of tomato (Clavibacter michiganensis subsp. Michiganensis, Cmm), the agent of bacterial spot of tomato (Xanthomonas spp.), the agent of bactrerial fruit blotch of watermelon (Acidovorax citrulli, Acit), the agents of common bacterial blight (CBB) of bean (Xanthomonas spp.), and the agent of black vein of brassicas (Xanthomonas campestris pv. campestris, Xcc).

GFP labelled Acit was seen to progress from the penetration point, along the vascular system, to the seed. Such progression was very effective, if Acit entered through the immature fruits (pericarp), leading to seed contamination. GFP mutants of Cmm were inoculated into tomato plants through stem and/or fruilet lesions. The pathogen was seen to progress along the xylem vessels to the seed: such progression was very effective in both cases. Therefore, labelling bacteria with GFP proved to be a very efficient method to prove invasion pathways up to the seed.

Sets of microscopic examinations of plant material contaminated with the tagged strains of the CBB agents indicated that whatever the contamination pathway of the seed (flower or xylem vessels), bacteria are localized in the tracheid bar. This tracheid bar is specific to legumes seeds and it is the vascular supply of the seed. Bacteria were also localized in the parenchyma, which is adjacent to this tracheid bar. The surface of the testa and of the cotyledons were also colonized by CBB agent cells. No observations of gfp-tagged cells could be made in mature and dry seeds and after germination in plantlets. In seeds, bacterial cells certainly present a reduced metabolism, which limits emission of fluorescence and in consequence detection of the tagged cells. At germination time, the limited number of bacterial multiplication sites could be the explanation for the absence of detection.

Multiple spray-inoculations of flowers of Rapid Cycling Brassica plants with a GFP-tagged strain of Xcc resulted in contamination of flowers and petals, and subsequently in a systemic and symptomatic infection of siliques as shown by dilution plating and epifluorescence stereomicroscopy. Highly infected seed lots were harvested from flower-inoculated plants; ca. 7% of the lots were externally infected and 2% remained positive after a warm water treatment, indicating internal infections. Using confocal laser scanning microscopy, Xcc could be observed inside some of the seeds in endosperm and embryo. This also indicates that flower inoculations can result in internal seed infections. Flower infections may occur if contaminated insects visit flowers during pollination. We showed that bumble bees can transmit Xcc from infected to non-infected plants resulting in symptomatic siliques and infected seeds. Xcc could persist for 23 days in a colony of bumble bees. Seeds may also become infected via the vascular tissues, if after leaf-infection Xcc is translocated via stem, peduncle, pedicel, silique and funicle into the seed. We showed that after inoculation of the vascular tissues of peduncle or silique, Xcc can move through the xylem vessels into the septum from where it can infect seed via the funicle or via contact of externally infected septum tissues with the developing seed coat. We conclude that contamination of flowers is a very efficient way to derive seed, but that also leaf infections can potentially result in internal seed infections. This knowledge is of importance in the selection of Brassica seed production areas.

In conclusion, whatever the pathosystem under study, internal contaminations of seeds by pathogenic bacteria were observed following vascular and floral pathway. This indicates that early and late contaminations of crops can lead to internal seed contaminations, which are difficult to remove with seed treatments. Gfp-labelling of bacterial cells was found to be an interesting option to follow seed colonization.

The ascomycetes F. graminearum and F. cumorum are major causal agents of Fusarium Head Blight (FHB) and can infect many important grain cereals, such as wheat (Triticum aestivum), barley (Hordeum vulgare), rye (Secale cerealis), oats (Avena sativa). Seeds infected with GFP labelled F. graminearum and F. culmorum were monitored using the fluorescence detection systems including the PathoScreen platform. From these experiments it became clear that in spring wheat fusarium infection of seeds can result in seedling blight and with high frequency results in infected plantlets. In many cases these infected plants show only minor phenotypic differences. In these infected young plants Fusarium can be found in the shoot, roots and the remainder of the seed. Fusarium remains present and viable in the lower leaf parts until heading and flowering. However, we could not find evidence that Fusarium can grow internally from seedlings to the wheat head. We cut the stems and inspected these for hyphae that would be readily visualized because of the GFP label. However, the nodes seem to be an efficient barrier for such as progressive systemic Fusarium infection. Nevertheless, we expect that the sporulation found on the colonized lower leaves could result in infection of the wheat flowers later in the season.

REVISED DATABASE, DETAILING ALL KNOWN PESTS AND DISEASES OF CROP PLANTS TRANSMITTED BY SEED

The book “An Annotated List of Seed-borne Diseases” was first published in 1958 by the Commonwealth Mycological Institute in collaboration with the International Seed Testing Association (ISTA). The last time the book was updated was inn 1990 by M.J. Richardson producing the 4th edition, which was published by ISTA. The Annotated List includes information about all kinds of pathogens (fungi, bacteria, viruses and nematodes), as well as some physiological disorders which affect seed or seed assessment. This information is cross-referenced in 3 indices (Host common names; Host scientific names with families; Pathogens and other disease agents with nomenclature authorities, synonyms, references to descriptions and distribution maps). The list contains short synopses of relevant literature references. This publication is very useful for research and testing laboratories, as well as for both seed and plant pathologists, since important information can be obtained quickly together with relevant literature citations. However, it is out-of-date and was becoming misleading rather than helpful.

The search for new literature on seed-borne and seed transmitted diseases of 59 crops/hosts listed below was completed. Where new literature was found, it was saved on an online database (refworks), reviewed by a second person, uploaded onto Google drive and recorded on an Excel sheet. This was then uploaded onto the online ISTA “Annotated list of seed-borne diseases” database. This annotated list includes diseases of over 400 hosts. Furthermore, almost all the old articles on the ISTA database for 84 crops were sourced by Fera in England, collated at the University of Pretoria in South Africa and are now available for further review. The unpublished papers that could not be sourced will be removed from the online ISTA database after evaluation. It must be noted that the ISTA website is not currently available to researchers, the industry or public as all the uploaded new references and sourced old references still need to be reviewed before the database on the website can go “live”.

1. Almond
2. Apples
3. Black Wattle
4. Chestnuts
5. Chick-pea
6. Coffee
7. Cotton
8. Custard apple
9. Cypress
10. Elder
11. Elms
12. Endive
13. Eucalyptus
14. False cypress
15. White cedar
16. Fescue
17. Firs
18. Ginkgo
19. Ginseng
20. Gypsophila
21. Lemon/orange/citron
22. Lupins
23. Mahogany
24. Maple
25. Sycamore
26. Millet
27. Myrtle tree
28. Oaks
29. Papaya
30. Parsnip
31. Pearl millets
32. Bajra
33. Pears
34. Pistachio nut
35. Pine
36. Pomegranate
37. Poplar
38. Aspen
39. Potato
40. Red cedar
41. Red sandalwood
42. Rooibos tea
43. Rye
44. Ryegrasses
45. Safflower
46. Sage
47. Sesame
48. Gingilly
49. Till
50. Siberian fir
51. Soybean
52. Spruces
53. Strawberries
54. Sycamore
55. Triticale
56. Walnuts
57. White mustard
58. White rocket
59. Wild rye/lime grass

A total of 136 new seed-borne pathogens were recorded and 50 new seed transmitted pathogens. The definition for seed-borne and seed transmitted diseases that was used was according to that of the IPCC:

Seed-borne pest: A pest that can be carried by the seed externally or internally and may or may not be transmitted to resultant plants causing their infestation.

Seed transmitted pest: A seed-borne pest that is transmitted via seeds to resultant plants causing their infestation.

NOVEL METHODS FOR ASSESSING LEVELS OF SEED TRANSMISSION AND THEIR RELEVANCE TO DISEASE LEVELS IN CROPS IN SUPPORT OF RISK ASSESSMENT

An approach using the area under the disease progress curve (AUDPC) is a useful quantitative summary of disease intensity over time, for comparison across years, or locations, or management tactics of disease severity. The AUDPC is frequently used to combine multiple observations of disease progress into a single value: such values, once compared, may give a strong indication on differences of disease levels among experimental plots.

The calculation of AUDPC (Area Under the Disease Progression Curve) was used to assess disease quantity in growing crops. Such index has, then, been used to correlate seed contamination to disease levels, as observed in the field. For all pathosystems studied (Cmm/tomato, Xv/tomato, Xe/pepper, Acit/watermelon) AUDPC had a positive correlation with initial seed contamination rate. On the contrary, initial seed contamination rate was not correlated with disease observed in the field. Agro-environmental conditions are far more important than initial seed contamination rate for disease development: therefore, we observed that low or very low seed contamination rate could lead to an epidemic, in case of conducive conditions, and high seed contamination rate will lead to no disease, if environmental conditions were not favourable.

Naturally contaminated seed lots and contaminated seed lots that were produced in greenhouses following mother plant inoculations were used to determine the bacterial transmission rates from seed to four days- old seedlings and seven days-old plantlets. This was monitored for a panel of pathogens and plants: tomato, bean cabbage, and radish, faced to various Xanthomonas spp., Cmm, and Pseudomonas syringae pv. tomato. Analyses of four days- old seedlings, which have germinated in germination boxes give access to the potential of contamination by contaminated seeds with a bacterial load higher than 103 cfu/g of seed. This methods is highly favorable to bacterial multiplication and overestimate what we saw while analyzing the first leaves of seven days-old plantlets, which were grown in soil. The bacterial transmission to the first leaves of plantlets appeared to be a more constrained step for all bacteria that were tested. The analyses of primary leaves of seven days-old plantlets could give access to the number of potential epidemic foci.

IMPROVED GENERIC SAMPLING METHODOLOGIES FOR SEED LOTS TO GENERATE REPRESENTATIVE SAMPLES FOR TESTING

Most agricultural and vegetable seed for sowing is controlled through national or international certification schemes. Seed is processed in to seed lots. A seed lot is a specified amount of seed that has a unique identity. Seed can be contaminated or infected with seed transmitted pathogens (diseases or pests); there is therefore a risk that pathogens not present in a country or area can be introduced through movement of seed from one country or area to another. Some important seed transmitted pathogens occur in seed at very low frequencies and have low infection/contamination levels for economic loss or have a zero acceptance as quarantine organisms. Sampling of the seed lot to determine the presence or level of a pathogen is therefore very important.

There is concern from national plant protection organisations and some members of the seed trade that current sampling procedures described in the International Seed Testing Associations ‘International rules for seed testing’ or in the International Plant Protection Convention’s standard ‘Methodologies for the sampling of a consignment’ may not detect pathogens at low frequencies in seed lots. Many factors affect the distribution of a pathogen in a seed lot including seed production site, spatial variation of infection in field, physical characteristics of infected seed, seed processing methods, mixing of seed from different sites of production. There are very few studies on the distribution of seed transmitted pathogens in seed lots to confirm or challenge concerns on a sampling plans ability to detect pathogens at low frequencies.

To test these concerns the project work focused on four seed transmitted pathogens:

• A nematode in field bean, Ditylenchus species (Ditylenchus gigas and Ditylenchus dipsaci);
• Fungi in wheat, Fusarium species (Fusarium graminearum and Fusarium poae);
• Tilletia caries also a fungus in wheat; and
• Xanthomonas campestris pv. campestris (Xcc) a bacterium in brassica seeds,

where their presence in a seed lot was likely to be low and where their distribution between different parts of the seed lot was expected to be variable. Relevant statistical models were reviewed and those most likely to be useful for seed lots were used to develop experimental sampling plans depending on available knowledge of each pathogen and detection method. The statistical models were tested using data collected from 23 seed lots in this project and data from six wheat lots, infected with Tilletia caries, previously sampled in a project conducted by the UK Home Grown Cereals Authority.

Based on the results from the studies of the four organisms a model for the detection of pests and pathogens was developed. It is a simple model, which can be written as a single simple equation that provides an estimate of the limit of detection associated with sampling and testing for pests and pathogens in seed lots and includes the effects of:

• Variation in the level of pests or pathogens between different parts of the seed lot.
• The size of the working sample.
• Small scale clustering of the pest or pathogen.
• The recovery and limit of detection of the method used to detect the presence of the pest or pathogen.

Detailed sampling plans are available for each of the four organisms which have been studied. Each of the plans provides a detailed technical description of sampling and a range of test plans with an associated limit of detection: the method by which the limit of detection was estimated and the source of the information used to estimate the limit of detection. The aim was to provide sampling plans with sufficient practical information to allow them to be applied reliably by stakeholders, providing a reliable estimate of the likely limit of detection that is associated with the plan and to allow stakeholders to select an appropriate plan for their requirements objectively. For example Figure 3 shows 10 plans which are based on different numbers of primary samples, working sample size and test method that are all estimated provide a limit of detection of 10 nematodes/100g field beans using the methods described in this workpackage. Stakeholders can use the tools to estimate the limits of detection of a number of plans and then choose to use the plan the can be most easily applied.

Guidance is also provided that could be used by stakeholders to design their own studies on variation in lots.

Also, some additional consideration was given to the problem of “small seed lots”. The problem with these lots is that, often, the aim of sampling is to provide assurance that, say, fewer than 1 in 10 000 seeds is infected. Using the usual sampling and testing techniques we need to examine at least 30 000 seeds to provide this level of assurance. This is sometimes impossible or too expensive for high value seeds. Two potential ways of dealing with sampling of small lots were described: a scenario where normal seed processing has been shown to spread pathogens among seed if they are present, which allows the use of smaller samples. The second scenario was based on the concept of the infection unit, which is the smallest number of infected seeds that must be present if any infected seeds are present. The common feature of both of these approaches is that they rely on having some reliable information about the way that the seed lot has been produced.

Ten scenarios were presented that aimed to describe to stakeholders what type of information can be used and applied in the model to provide a sampling plan that meets their requirements. Four were based on the large formal studies (described above) where a relatively large number of samples had been analysed to provide information about the distribution of pests and pathogens.

Six other scenarios were based on less formal, more rapid assessments.

• PSTVD (viroid) in tomato seeds
• Dytilenchus dipsaci (nematode)in alfalfa
• Phoma lingam (fungus) in cabbage
• Clavibacter michiganensis subsp michiganensis (bacterium) in tomato seeds
• Xanthomonas axonopodis pv phaseoli (bacterium) in field beans
• Acidovorax citrulli (bacterium) in watermelon

For a number of these scenarios the information of the effect of testing was gained from results generated by TESTA’s work package concerned with validating practical seed testing methods for immediate use in regulatory control laboratories.

For “large lot” scenario estimates of the following parameters were presented:

Variation in the level of infected seeds or pest or pathogen between different locations in the lot (Expressed as an estimated relative standard deviation between prevalence at each location)

Small scale (Between-seed) variation in the prevalence of pest or pathogen (Expressed as a relative standard deviation between prevalence in replicate samples of known size)

Limit of detection of the analytical test method, or probability of detecting a single pest or pathogen (Expressed as a quantity of pest of pathogen with an associated probability of detection)

For small-lot scenarios estimates of the “infection unit size” and analytical performance were provided.

To enable stakeholders to get estimates of the relation between the prevalence of a pest or pathogen in a lot, the amount of variation in the prevalence of the pest or pathogen and the probability that a particular sampling plan would detect the presence of the pest or pathogen software is provided in excel. This software is of particular use in scenarios where there is little information on which to base estimates of the amount of variation in the prevalence of the pest or pathogen because they allow users to describe the amount of variation either as a statistical measure (between-location relative standard deviation) or the more intuitive “hot-spot size”.

In addition, plans were produced using the methods and tools, and information discussed that met targets for the detection of pests and pathogens described in test method, pathogen standards and validation reports available from example the International Seed Testing Association and the European Plant Protection Organisation.

Further dissemination of this work is ongoing to encourage international acceptance of the described methods to produce reliable sampling plans that meet stakeholder requirements.

GENERIC PLATFORMS FOR TESTING METHODS FOR SEED

Seed-borne pathogens pose a serious threat to modern agricultural cropping systems, as they can be disseminated to many geographical regions around the world. With trends of increasing global seed production and trade, seed-health testing including detection is an important quality control step to prevent the introduction and spread of these harmful pathogens. The following methods are used nowadays for detection:

Fungi: Blotter test: freeze, incubate, examine based upon morphology
Bacteria: Dilute plating, (semi-)selective media, confirmation by PCR and/or immunofluorescence, pathogenicity assay
Viruses: ELISA, indicator plants, RT-PCR and real-time RT-PCR
Viroids: RT-PAGE, real time RT-PCR

ISHI (International Seed Health Initiative), ISTA (International Seed Testing Association) and EPPO (European Plant Protection Organization) have developed, validated and published procedures within their seeds health committees and diagnostic panels. All of these methods are based on detecting a single pathogen and many methods have not been validated. Within TESTA we developed and validated new generic approaches for multiple pathogens. We compared different sample treatments to optimize nucleic acid extractions (soaking, crushing, shaking, macerating, ...), different DNA/RNA extraction kits and developed a more generic approach for DNA/RNA extraction using internal controls.

Protocols for DNA/RNA extraction

DNA/RNA extraction is an essential part of most molecular methods. Extraction of nucleic acid is a significant challenge in any routine diagnostic testing based on molecular biology procedures, because the robustness of any subsequent steps often rely on the quality of extracted nucleic acids. Several EU funded projects addressed this issue, however these did not include seeds.

For seeds many methods have been described but they are versatile in nature. Current limitations of DNA/RNA extraction for seeds:

- Treatments and/or coating of seeds with fungicides, dyes, pelleting materials, etc.
- Too many different extraction technologies are used, dependent on where the pathogen is: on or in the seeds and also the plant seeds vary dramatically in size and composition.
- Internal controls are often not used to screen for inhibitors.

Protocols were tested and the most effective ones have been validated within WP5 on seeds of tomato, cereals, cabbage and cucurbits for use in testing laboratories.

Real-Time PCR

One of the preferred methods for detection at the moment is real-time (TaqMan or SYBR Green) PCR. For many seed transmitted plant pathogens (bacteria, viroids, viruses and fungi) (real-time) PCR assays are already available or sequences are available from different sequence databases. However, these methods often use different PCR conditions, different enzymes and are often not well validated and implemented. Within this WP we developed generic methods for detection of pathogens in seeds (tomato, cereals, cabbage and cucurbits) using real-time PCR so that detection can be performed using standard conditions. For each of the crops the most optimal method was then validated and implemented in WP5.

Luminex

In an ideal situation one would like to detect all different pathogens (viruses, viroids, bacteria and fungi) simultanously in a DNA/RNA extract (multiplex detection). Within TESTA one strategy was tested for Generic and Multiplex Detection: Luminex. The Luminex platform consists of many different colored beads on which specific oligos are coupled. Individual beads are identified for colour and attachment of a specific amplicon to the oligo. The Luminex technology was investigated for simultaneous detection of viruses, viroids, and bacteria within different seeds. The target candidates were the bacteria Cmm and the viral pathogens Potato spindle tuber viroid (PSTVd) and Pepino mosaic virus (PepMV). Generic amplification was performed on the DNA/RNA extracted. The main focus was on the robustness of the amplification process under variable conditions. Enzymes, buffers and amplification conditions (temperature, hold times, etc.) were tested. A multiplex method for pospiviroids, including PSTVd, was optimized and tested on infected tomato seed lot. For PepMV there are several strains. Based on sequence difference of the entire genome a multiplex method was developed to identify which strain of PepMV was in a sample. Clavibacter michiganensis consists of several subspecies and also look-alikes. Based upon sequence difference in the RecA and GyrB genes a multiplex method was developed to identify to which subspecies a Clavibacter strain belongs or that it is a look-alike.

POTENTIAL FOR USING INNOVATIVE METHODS FOR TESTING SEED

Within the last few years many new sequence technologies have been developed (Next Generation and Third Generation Sequence Technology) to determine DNA/RNA sequences in a short time period and with high amounts of sequence data for low costs. Within TESTA, different applications were studied:

- Metagenomics of fungal species in cereal seeds
- Sequencing of DNA extracts from tomato seeds
- Whole genome sequencing of six bacterial and two fungal species

Depending on the NGS application different platforms can be used: Illumina MiSeq/HiSeq, Roche 454 and PacBio.

Metagenomics of fungal species in cereal seeds were successfully analysed using 454 sequence technology. NGS is able to detect a huge range of fungi in seeds and this is done semi-quantitatively (the output from NGS is relative abundances of species). The sensitivity depends on the depth of the sequencing. NGS is a valuable supplement to other techniques such as Videometer Lab or qPCR as it shows a more detailed picture of fungal species present in grain. This is valuable for example to support validation of these methods (e.g. Videometer Lab). In contrast to qPCR, NGS is a non-targeted method and should, in principle, detect all fungal species in a sample.

Sequencing of DNA and RNA extracts from tomato seeds showed good performance using Illumina HiSeq sequence technology. NGS using the Illumina HiSeq 2500 platform was able to detect the different spiked pathogens in DNA extracts of healthy tomato seed lots spiked with bacterial pathogens. In contaminated tomato seed lots NGS data showed the presence of several bacteria (Clavibacter michiganensis, Xanthomonas vesicatoria, X. gardneri, Pseudomonas syringae pv tomato). NGS data were obtained from several bacterial pathogens and PepMV with good coverage. Only PSTVd had a low coverage probably due to the small genome size(359 nts). More analyses will be performed to investigate the relevance of these results. NGS is able to detect different pathogens in RNA extracts of PepMV/PSTVd contaminated tomato seed lots. NGS data were also obtained from pure bacterial isolates which were used in the spike. They were used in de novo assembly to generate contigs which could be used in reference assembly. Once a good reference assembly is available a good read mapping can be performed.

Whole genome sequencing of six bacterial and two fungal species showed very good results obtained with Illumina Hiseq / MiSeq and PacBio sequence technology. A very high coverage with many reads were obtained. PacBio resulted in fewer contigs with much longer reads.

NGS using PacBio platform is able to produce long reads. These allow for a very good assembly with large contigs and generic features expected for F. oxysporum (11 corechromosomes + several LS chromosomes). These latter are lineage specific, that vary strongly in number and in sequence between different formae specialis. On the other hand it is very important to check the quality and quantity of the DNA used for PacBiosequencing is very critical. The purity of isolates is of eminent importance to have smooth assemblies. It becomes clear that more and more fungal strains are infected, contaminated or symbiotic with other micro-organisms. This complicates the assembly of raw PacBio reads, since most assembly programs have difficulty assembling sequences that vary in abundance.

High coverage was obtained with Illumina MiSeq sequencing of two Fusarium oxysporumstrains (F. oxysporum f.sp raphani isolate Fr 13-03 and F. oxysporum f.sp basilici isolate Fob 009) 27.15x and 18.98x respectively. Many SNPs were obtained for the two strains. Combined with the PacBio data of the two strains, genome annotation should be more effective and can lead to unravel the forma speciales concept.

POTENTIAL FOR USING NON-DESTRUCTIVE TESTING METHODS FOR SEED

For all molecular detection methods the seeds need to be destroyed since RNA or DNA needs to be extracted. Non-destructive detection methods would be a valuable tool to detect if seed lots contain pathogens. A non-destructive methodology based on a technology produced by the company Videometer was tested on cereal seeds to investigate if seed lots of cereals infected with different pathogens (different Fusarium species, Tilletia sp) can be discriminated from non-infected seeds. The Videometer technology is based upon reflectance of light. A number of different seed lots were tested using this technology to assess whether it was capable of selecting infected seeds lots from non-infected seed lots.

A non-destructive testing system with a feeder and conveyor that can handle samples of 500 grams of barley corresponding to more than 10.000 kernels was developed (Figure 4). To obtain efficient handling, kernels are only scanned on one side using multispectral reflectance imaging. Reproducibility studies show that this sample presentation enables selection of infected seeds lots from non-infected seed lots for Fusarium spp. There is only little gain in including multispectral fluorescence imaging and since this will increase the acquisition time significantly is has been decided to only use multispectral reflectance imaging. In conclusion, a non-destructive protocol for detection of Fusarium spp. in barley has been established and tested under real industrial conditions by a lab technician. It screens 10.000-15.000 kernels in app. 20 minutes with a reproducibility standard deviation over time and presentation of app. 10 infected kernels out of 10.000-15.000 i.e. less than 1 per 1000.

For Tilletia spp. on wheat a statistical model for 90 mm petri dish presentation has been established. This takes app. 200 wheat kernels presented in a single layer and the analysis time is 30 seconds including handling of the petri dish. The same imaging technology as for Microdochium spp. is used with the addition of multispectral fluorescence. Results suggest that Tilletia infection is detected even in samples with low degree of infection. This protocol will need further validation.

VALIDATED PRACTICAL METHODS FOR IMMEDIATE USE IN REGULATORY CONTROL LABORATORIES

With the increasing drive for quality, accredited and validated methods have become more important. For newly developed assays it is necessary to objectively determine whether the new assay meets the requirements dictated in advance. Validation of detection methods will make the results from research and development available for use by the regulatory control laboratories.

Within the TESTA project, thirteen methods were validated according to the EPPO Standard PM 7/98 (2) (EPPO, 2014), which describes guidelines for laboratories preparing for accreditation according to the ISO/IEC standard 17025. Whether the test is fit for its intended use was determined using several performance characteristics. For each test analytical sensitivity, analytical specificity, repeatability, reproducibility and if appropriate selectivity and trueness were determined.

In total thirteen protocols were validated. The protocols and validation reports will be disseminated in different ways. Three protocols are submitted to ISTA, four are being discussed within EPPO for inclusion in EPPO protocols and others will be submitted to peer-reviewed journals. Table 1 summarises the state of play with the validated methods.

The validation report for the assay for detection of Acit in melon and watermelon by direct seed wash Taqman PCR was the first to be finished and has since been discussed in EPPO Panel meetings for inclusion in the newly-written Acidovorax citrulli protocol. The validation report has been uploaded to the EPPO database and the protocol has been sent out for country consultation. When accepted, the protocol will be published in the EPPO Bulletin.

Most of the assays validated were developed outside of the TESTA project but could not be applied in practical use within a quality system in regulatory control laboratories because they had not been validated. We chose to validate these assays to releave this log-jam and support the testing laboratories in the immediate short term. The assay for detection of Cmm however was developed within the TESTA project. It will provide an assay based on another detection principle than the current standards.The new detection assay is a Taqman PCR on seed extract that can be used as a pre-screening method for the dilution plating assay that is currently described in the ISTA- and EPPO-protocols. The protocols for D. dipsaci and D. gigas, for Xcc and for P. lingam have been submitted to ISTA for inclusion in the current ISTA-rules.

The assay for the detection of pospiviroids in seeds of tomato and the molecular assay to distinguish between infectious and non-infectious Cucumber green mottle mosaic virus (CGMMV) on seeds of cucurbit crops were validated. The validated methods for pospiviroids are a RT Taqman for detection of seven pospiviroids in tomato seeds. Although the pathways for transmission are not proven, more pospiviroids are regulated and therefore a broader assay was developed based on the assay for PSTVd and Tomato chlorotic dwarf viroid (TCDVd) as described by Bakker et al. (2015). For CGMMV and other viruses there is a need for distinction between infectious and non-infectious CGMMV on seeds of important cucurbit crops. This protocol was developed and validated within TESTA and will be submitted to a peer-reviewed journal.

Four of the tests developed and validated in the framework of TESTA, Acit, D. dipsaci and D. gigas, Cmm, and pospiviroids, will be used to initiate a revision or preparation of new EPPO diagnostic protocols. The EPPO Secretariat is collaborating with TESTA partners (NAKT, UNIMORE, and GEVES) for the preparation of these drafts, based on the outcomes available so far.

The four generic extraction methods and real-time PCR assays developed in the TESTA project were validated. The validated methods include A) generic assay for bacteria and viruses in seeds of tomato, B) generic assay for the detection of Fusarium graminearum and Tilletia caries on cereal seeds, C) generic assay for the detection of Pseudomonas syringae pv. maculicola and Xcc on cabbage and radish seeds and D) an assay based on manual DNA extraction for the detection of Acit on cucurbit seeds.

NOVEL DISINFECTION METHODOLOGIES FOR SEED

Seed treatments have been used on a commercial basis for decades. Certain limitations and environmental disadvantages which have been associated with the use of chemicals as well as the uncertainties about the future availability of fungicides for minor uses called for the development of alternative methods for seed treatment. Compared to cereals, far less testing of alternative seed treatments has been done with vegetables.

TESTA WP4 aimed to develop physical and biological disinfection methods which are effective, do not affect germination rate and are suitable for industrial processes for conventional and organic production and for smaller crops of niche value. In fact, the ability to treat seed with suitable disinfectants would mean a huge saving in money for seed companies due to current losses of contaminated seed and also provide extra protection for the EU against disease. The project investigated the use of a range of novel methods for disinfection of seed, including the use of physical methods such as hot water, microorganisms ( bacteria, yeasts,etc) and fungal antagonists, plant and fungal extracts.

Table 2 gives an overview of the seed disinfection methods tested and validated within the project. In conclusion, the following methods may be recommended:

- For the pathosystem Fusarium fujikuroi/rice the treatment with aerated steam presented a pathogen control statistically similar to the chemical reference. Treatments with essential oils applied by fumigation allowed a germination rate of the rice seeds statistically similar to that from the seeds treated with the chemical reference. As the same time, seed fumigation with essential oils from savory and thyme presented results on pathogen control that were comparable to that from the chemical reference.

- For the pathosystem Alternaria spp./rocket and Alternaria spp./basil the treatment with aerated steam was the most effective among the physical methods tested.

- For Alternaria spp. on basil, carrot, and rocket the treatments with essential oils applied by fumigation were most effective against the pathogen and reached the control level provided by the chemical treatment.

- Seed treatments with steam thermotherapy at higher modalities (>condition1) can be approved as an official seed sanitation method for the control of D. dipsaci associated with alfalfa seed lots.

- Hot water at 50 °C for 30 minutes sufficiently controlled black rot disease caused by Xanthomonas campestris pv. campestris in rape plants. Its use can be recommended as a seed treatment for brassica vegetable production, among others for organic farming.

- Seed treatment with essential oils from savory and thyme significantly reduced incidence and severity of Fusarium wilts and crown galls caused by Fusarium oxysporum on basil, lettuce, and rocket seeds.

PROTOCOLS FOR ASSESSING THE PROFICIENCY OF DISINFECTION METHODS

Several performance protocols can be used to assess the efficacy of the disinfection methods and most of them are based on germination tests, tests on blotters and agar media, and efficacy tests on pots. TESTA project identified the best protocols for the following 16 seed-pathogen combinations (pathosystems):

• Alternaria spp. / Basil
• Alternaria spp. / Carrot
• Alternaria spp. / Rocket
• Fusarium fujikuroi / Rice
• Fusarium oxysporum / Basil
• Fusarium oxysporum / Lettuce
• Fusarium oxysporum / Rocket
• Dithylenchus dipsaci / Alfalfa
• Acidovorax avenae subsp. citrulli / Cucurbits
• Xanthomonas vesicatoria / Pepper
• Xanthomonas vesicatoria / Tomato
• Xanthomonas campestris pv. campestris / Brassica oleracea
• Alternaria / Brassica napus
• Xanthomonas campestris pv. campestris / Brassica napus
• Fusarium / Coriander
• Alternaria / Coriander

ISTA rules have been followed, when available, for performance protocols.

Germination tests on treated seeds were established for all pathosystems. For pathogens detection blotter tests and agar plate methods have been included. Tests on seedlings, seedling grow-out evaluation in glasshouse, and inoculation tests on leaves were chosen as pot tests. In addition, during the project implementation, it has been considered a priority to assess also seeds vigour when evaluating the proficiency of disinfection methods, thus vigour tests have been included as well in the performance protocols.

The results, included in the Deliverable 4.1. Performance protocols for the disinfection methods (D4.1.) are summarized in Table 3.

In addition, microbiological and molecular procedures for assessing the presence of Xanthomonas campestris pv. Campestris (Xcc) in cabbage seeds lot, including discrimination between living and dead organisms, have been developed (Deliverable 4.2. Microbiological and molecular procedures for Xanthomonas campestris campestris). The pathogen causes black leg in cabbage and is seed transmitted. Since Xcc is an important quality-determining organism, Xcc-contaminated batches of cabbage seeds are disinfected. Consequently, the distinction between living and dead Xcc in these batches is of great importance. This distinction is difficult and requires microbiological methods on highly selective media. Therefore we also developed a molecular assay based on DNA. Molecular techniques can be very selective and increasingly used, but usually do not distinguish between viable and non-viable organisms. Since dead Xcc cells may still contain DNA and thus DNA based methods will detect the presence of Xcc even when these cell are no longer viable. To resolve this issue we developed an assay based on a PMA treatment in combination with a TaqMan real-time PCR which detect specifically living cells with uncompromised cell membrane. As the required limit of detection could be a potential problem the test was also performed in combination with an Xcc enrichment strategy to reach a lower limit of detection.
Potential Impact:
WIDER SOCIETAL IMPLICATIONS AND SOCIO-ECONOMIC IMPACT OF SEED HEALTH TO THE EU

In 2008, the agricultural and food sector accounted for 17 million jobs (7.6% of total employment) and for 3.5% of total Gross Value Added in the EU-27. EU agriculture has a share of 18% in world food exports, worth €76 billion. Food demand is expected to increase by 50% but currently, a quarter of the world’s crops are lost to pests. In the longer term, the results of this work will help to ensure that European agriculture remains productive and delivers quality products that meet the expectations of consumers and the food chain. In addition, with imports of produce into the EU expanding it is important to maintain production in the EU to balance the economy and maintain self-reliance, for example, currently vegetables are produced in the EU on more than 400.000 hectares. The EU imports approximately 12.5 million metric tons of fresh fruit and vegetables with a value of 11 billion Euro. On the other hand, the EU exports account for 5 million metric tons with a value of 4 billion Euro (https://www.euroseeds.eu/vegetables).

The seed production sector in Europe is also a key component of the economy. The EU is the largest exporter of seed worldwide and the largest seed market worldwide. The seed production sector itself is of high value to the EU economy with a turnover of approximately 6.5–7billion euros, involving approx.1000 companies, of which the vast majority are SMEs (http://ec.europa.eu/food/plant/plant_propagation_material/legislation/review_eu_rules/docs/breeding_and_seed_prod.pdf). As an example, the estimated value of certified seed for sowing of cereals and pulses in the EU28 is estimated at 2.5 billion Euro. The value of entire commodity production of cereals and pulses in the EU28 is estimated at approximately 40 billion Euro (https://www.euroseeds.eu/system/files/publications/files/esa_16.0117_scp_factsheet.pdf.

It is therefore important to maintain a competitive, innovative and diverse seed sector.

POTENTIAL IMPACT OF PROJECT RESULTS

The knowledge generated by the project will have a significant direct economic impact on European agriculture by avoiding production losses and through substantial savings in controlling diseases. The project improved the knowledge of seed transmission, testing methods and control methods for a wide range of crops and pests relevant to EU agriculture and horticulture (see Table 4).

Gaining more detailed knowledge of the seed transmission mechanisms allows us to design control strategies and improve targeting of seed testing. The ISTA Annotated List includes information about all kinds of pathogens (fungi, bacteria, viruses and nematodes), as well as some physiological disorders, and has been used for many years as the key document for seed testing laboratories across the world. It had become obvious that it was out of date and was causing many problems with seed trade. The TESTA project revised a selection of the key crop entries ( about 25% of the total entries) for this list and has removed many doubtful entries. This will improve the efficiency of testing for seed types relevant to the EU.

Experimental work carried out during the project has extended the current knowledge of mechanisms of seed trasmission of pests and diseases. For example, internal contaminations of seeds by pathogenic bacteria were observed following vascular and floral pathway. This indicates that early and late contaminations of crops can lead to internal seed contaminations, which are difficult to remove with seed treatments.

A useful quantitative summary of disease intensity over time (AUDPC), for comparison across years, or locations, or management tactics of disease severity, was developed and can be used to compare results across field trials from different years and countries. This will allow experimental results to be compared between different groups.

Additionally, rapid methods for detection will prevent delays and other unnecessary costs to trade to the subsequent economic benefit of EU. Sampling methods are also key to the robust detection of pests.

There are differing views on interpretation of risk associated with seed-transmitted pathogens creating import restrictions on some countries of production. An internationally disseminated and accepted method for designing sampling plans may reduce the scope for disagreement about, at least, some aspects of the risk assessment of lots for seed pathogens. It is hoped that the methods and tools described will provide national plant protection organizations and the seed trade methods for defining sampling plans that are produced based on the best information available and to design their own studies on variation in lots where required.

The development of validated and harmonised methods and tools for early detection is key to treatment and control. The awareness of the need for validation of protocols is increasing rapidly over the last few years. Stricter regulations and quality requirements have resulted in increasing knowledge on and requirements for validation studies. TESTA work package 5 has contributed to this awareness through dissemination of the work plans, teaching a validation course, presentations at meetings for different groups of stakeholders, and publication of the validation reports.

13 protocols were successfully validated. These protocols have been and will be made available for use by stakeholders. The protocols will be disseminated through inclusion in ISTA and EPPO protocols, by publication in peer-reviewed journals, publication on the TESTA website or TESTA-partner website, publication of instruction videos, presentations at national and international meetings, such as ISTA-meeting, EPPO/TESTA-meeting and ISHI-Veg meetings, and protocols are available upon request. Potential users such as NPPOs, inspection services, and industry can implement these protocols for reliable detection of these quarantine and quality pathogens.

Results of the work will significantly enhance the capacity of plant health authorities and farmers to manage the disease and prevent further economic losses in crops.

ISHI (International Seed Health Initiative), ISTA (International Seed Testing Association) and EPPO (European Plant Protection Organization) have developed, validated and published procedures within their seeds health committees and diagnostic panels. All of these methods are based on detecting a single pathogen and many methods have not been validated. Looking to the future, within TESTA we developed and validated new generic approaches for multiple pathogens. We compared different sample treatments to optimize nucleic acid extractions and developed a more generic approach for DNA/RNA extraction using internal controls. Multi target detection strategies were developed using Taqman PCR and Luminex approaches- these will streamline testing for a range of target crops of interest to the EU.

Novel methods for non-targetted detection and identification of pests were also developed and assessed for suitability for use in seed testing laboratories e.g. NGS strategies. In future, these could allow the detection and identification of all pests associated with a seed lot and may reveal risks from new pathogens.

Non-destructive testing for use with valuable seed lots was investigated using a technology developed by Videometer. A protocol for detection of Fusarium spp. in barley has been established and tested under real industrial conditions (Figure 4). It was able to screen large numbers of seed lots reliably. The same imaging technology could also detect Tilletia infection even in samples with low degree of infection.

Valuable seed that has become contaminated can be used if suitable decontamination treatments are available. However, recently the number of chemicals available for use in seed decontamination has decreased markedly. In this project, a range of novel treatments were tested for efficacy against seed transmitted pests. Some of these proved suitable for future use, for example, steam thermotherapy could be approved as an official seed sanitation method for the control of D. dipsaci associated with alfalfa seed lots, seed treatment with essential oils significantly reduced incidence and severity of Fusarium wilts and crown galls of leafy vegetables and herbs and hot water treatment controlled black rot disease in rape plants and could be recommended as a seed treatment for brassica vegetable production under organic farming.

MAIN DISSEMINATION ACTIVITIES

7TH ISTA Seed Health Symposium (June 2014)

TESTA scientists attended the seventh International Seed Testing Association (ISTA) Seed Health Symposium held in Edinburgh from 12th-14th of June 2014. The meeting attracted 94 participants from 27 countries and was held in the National Museum of Scotland, in the heart of the city. The symposium was a valuable, unique meeting, bringing together industry, government and international organisations to discuss all aspects of seed transmitted diseases.

The three day symposium was split into five sessions. A total of 29 oral presentations were given by presenters from 12 different countries. Françoise Petter, from the European and Mediterranean Plant Protection Organization (EPPO) and Ruud Scheffer from the International Seed Federation were invited to give the opening presentations on Day 1 and Day 2 of the symposium. TESTA scientists gave 8 oral and 8 poster presentations during the conference.

Training workshops

Six training workshops were completed during the TESTA project. Some pictures from the workshops are included in the attached document of Figures and Tables.

1: Summer school on seed disinfection strategies:
The Centre of Competence Agroinnova, University of Torino, organized the TESTA summer school 2014 on “Seed Disinfection Strategies”, held in Grugliasco, Torino (Italy), between July 21-25, 2014. The course has been accredited (5 University courses credits) by the PhD Programme Agricultural, Forest and Food Science (Doctoral School of Sciences and Innovative Technologies, University of Torino) and it has been organized with the patronage of EXPO Milano 2015.

The TESTA summer school aimed to give the trainees deeper knowledge and expertise on sampling methods according to international standards (i.e. ISPM) and according the current legislation on regulated pathogens, seed disinfection strategies, diagnostics and new methods for detecting seed transmitted pathogens.

The course was attended by 9 participants from research and private sector from Italy, Spain and the Netherlands. The participants took part in a comprehensive scientific programme, including oral presentations, a technical excursion, practical laboratory exercises dealing with seed-borne pathogens biology, epidemiology and diagnostics, seed disinfection strategies and methods and a final proficiency test (Figure 5).

2. TESTA course on seed-borne diseases of arable crops
Twelve delegates attended a two day course at NIAB at the end of January 2015, exploring the significance of seed-borne diseases in northern European arable crops and learning about the activities of the TESTA project on some of these organisms (Figure 6). Delegates were from a range of backgrounds in either the seed trade or seed-borne disease research laboratories. Most, but not all, were already involved in seed health testing, but were not familiar with every disease, and wanted more background on the biology of the organisms, their importance, and the role of seed treatments and treatment thresholds in managing the diseases.

A wide range of pathogens was covered in presentations, but also in a number of “hands-on” laboratory sessions with identification and assessment exercises. Some molecular techniques were also demonstrated. Revised testing procedures under development in the TESTA project were discussed, focusing on the freezer blotter for Phoma lingam, and a new sieving method for Ditylenchus gigas in faba bean seed.

Some unusual seed-borne diseases, seen relatively rarely in routine testing, were available for examination, such as flag smut from rye, and Cochliobolus foot rot on barley seeds.

3. Training course on seed sampling
A four days training course on seed sampling for phytosanitary analyses has been organised by UNIMORE in March 2015, using different premises made available by seed companies in Italy (Figure 7). The course was attended by several international specialists involved in planning and organisation of seed sampling strategies. Most important seed crops have been taken into consideration during the practical activity (cereals, pulses, vegetables). Several topics have been discussed: proper tools and equipment for sampling (Figure 8), sampling methods for different seeds, sample size suitable form phytosanitary analyses, pathogen distribution in seed lots and its relationship to robustness of ophytosanitary analyses, statistical approach in developing sampling strategies. The trainees showed maximum interest during the whole course and had chances for confrontation among them on strategies in place in respective Countries (Figure 9).

4. Workshop on validation and quality monitoring
Validation is of increasing importance in the agricultural and horticultural sector. It is an important tool to ensure quality of laboratory assays.

Naktuinbouw organised a workshop on validation for researchers and technicians in the plant sector (Figure 10). The course examined the term validation and the value of validation studies for quality assurance. The theory was explained using the Dutch validation guideline and the EPPO Validation guideline PM7/98 (2). Practical examples were discussed to illustrate methods for determining performance characteristics.

Quality monitoring is an important aspect in plant health laboratories. The course discussed first, second and third line controls, such as method for monitoring positive and negative controls, blind samples and comparative tests.

The course aimed to provide answers to questions like: how can I guarantee the quality of my analyses, how do I assure continuous quality and how do I minimize risks in a health laboratory?

5. and 6. Workshops on Detection of tomato pathogens by molecular methods and Detection of Ditylenchus dipsaci on alfalfa and faba bean and Phoma lingam on brassicas
The final TESTA training workshops were held in association with the TESTA-EPPO conference in December 2015. Two days of workshop were organized by DLO and GEVES in Beaucouzé France, to inform and train people on methods set up during the TESTA project (Figure 11). The lectures and practicals were focused on molecular detection of tomato diseases, detection of Ditylenchus dipsaci and D. gigas by a filtration method and confirmation by PCR and detection of Phoma lingam on media and blotters. 47 people were registered for these workshops who appreciated the diversity of the techniques presented and the visit of the laboratory of phytopathology of GEVES.

ISHI-Veg meeting

A meeting with the industry especially those involved with vegetable seeds was arranged in Lyon in Sept 2014 by The International Seed Federation (ISF). A detailed discussion was held on progress on diagnostic methods, disinfection methods and the annotated list of seed transmitted and seed borne diseases (Blue book). Actions were agreed to follow up from this meeting, particularly to align our pest/ pathogen list with the ISF initiative. Also, they were particularly concerned with the practical issue of sampling small high value seed lots as mentioned in the WP2 report and follow up meetings were held to discuss this at the ISTA meeting in Edinburgh. The representatives from Incotec and Monsanto said they would be available for supporting some our activities in the disinfection method work area and with this aim, there was a further joint meeting with them in The Netherlands in Wageningen.

TESTA-EPPO Conference

TESTA scientists attended the TESTA-EPPO conference on Diagnostics for Plant Pests held in Angers, France from 30th November to 2nd December 2015. The meeting attracted over 100 participants from 21 countries. The conference was the latest in the series of EPPO conferences on new methods of diagnosis in plant protection. Most participants were experts in the field of diagnosis of plant pests and representatives of private companies were also present.

During the conference, there were two sessions dedicated to disseminating the results from the TESTA project (Figure 12). Seventeen presentations provided information on Sampling, Seed transmission, Diagnostic methods, Diagnostic method validation and Seed treatment. Representatives from EPPO and ISTA provided their conclusions on the project achievements and conference participants also provided feedback. The presentations and conclusions are available from the conference website: http://archives.eppo.int/MEETINGS/2015_conferences/testa.htm

Publications

From the project, there have been a total of 16 peer-reviewed publications plus 16 proceedings from conferences published. In addition two theses have been submitted and a book chapter is in press.

Oral and poster presentations

Project outputs have been presented at a number of national, European and international fora. A total of 50 oral and 14 poster presentations have been recorded from the conferences.

Newsletters

Three annual newsletters were published via the TESTA website and circulated to the mailing list to inform stakeholders about the progress and events of the project.

Website

The TESTA website (www.seedtesta.eu) was created at the beginning of the project and has been maintained throughout to provide project news and information on both project and other relevant events.
List of Websites:
www.seedtesta.eu
Project co-ordinator: Christine Henry, Fera Science Limited
Email: testa@fera.co.uk

Contact

Henry, Christine
Tel.: +441904 462329
E-mail
Record Number: 188016 / Last updated on: 2016-08-18