G.P. Munkvold, Professor Iowa State University Seed Science Center Department of Plant Pathology & Microbiology

Transcription

1 Advances in Seed Health Testing for Ensuring Quality in Seed Certification and Phytosanitary Systems XX Congresso Brasileiro de Sementes 10 August, 2017 G.P. Munkvold, Professor Iowa State University Seed Science Center Department of Plant Pathology & Microbiology

2 Iowa State University Seed Science Center Seed Testing Laboratory Seed Pathology & Physiology research programs National Seed Health System administration Continuing education in seed testing & conditioning BIGMAP Risk Analysis Research & training Global Programs Graduate education: oncampus and on-line

3 Thank you! Dr. Nancy Gonzalez Dr. Silvina Arias Derrick Mayfield Mike Stulberg (USDA-APHIS) Dr. Charlie Block Jillian Lang (CO State Univ.) Dr. Jan Leach (CO State Univ.) Dr. Kirk Broders (CO State Univ.)

4 Outline Problems associated with seedborne pathogens Role of seed health testing in international trade International standards for seed health testing Role of seed health testing in domestic seed certification Development of new testing methods Examples

5 Risks associated with seedborne microorganisms Stand loss due to seed quality reduction Yield or quality loss due to disease outbreaks Direct transmission of disease Disease in subsequent crops Loss of seed marketability quality; phytosanitary; appearance Introduction of pathogen into new areas Locally or internationally Phytosanitary regulations

6 Growth and complexity in international seed movement Corn Soy Corn Cotton Soy Canola Cotton Sorghum Canola Sorghum Courtesy of Ric Dunkle, ASTA

7 Tomato Example Courtesy of Ric Dunkle, ASTA 1. Basic seed or breeder s seed production in Country A. 2. Foundation seed production in Country B. 3. Stock seed production in Country A. 4. Commercial seed production in Country C. 5. Shipment of commercial seed back to Country A for cleaning treating and packaging. 6. Shipment to Country D for distribution. 7. Shipment to final destination after sale (Country E).

8 Pathogen testing for quality and phytosanitary compliance: Melons BFB GSB C. lag M. phas CGMMV MNSV SqMV BFB GSB C. lag M phas CGMMV MNSV SqMV Pseudomonas sp. MRMV BFB GSB C. lag M. phas CGMMV MNSV SqMV (BFB) GSB (C. lag) M. phas MNSV SqMV Courtesy of Ric Dunkle, ASTA Disease Codes BFB: Acidovorax citrulli GSB: Didymella bryoniae C. lag: Colletrotrichum lagenarium M. phas: Macrophomina phaseolina CGMMV: Cucumber Green Mottle Mosaic Virus MNSV: Melon Necrotic Spot Virus MRMV: Melon Rugose Virus SqMV: Squash Mosaic Virus

9 What can go wrong? Phomopsis spp. on spinach seeds Spinach seed shipments from Europe held at U.S. border due to presence of Phomopsis spp. Unknown phytosanitary risk No established seed health test Pseudomonas on rye seeds Multiple rejections of rye seed shipments to S. Korea Positive tests for Pseudomonas syringae pv. atrofaciens Korean PCR method is nonspecific CGMMV on cucurbits Introduction into California No testing requirements

10 Increasingly complicated phytosanitary regulations regulators are requiring imports to be certified free of all known pathogens, regardless of whether that pathogen is already present

11 Pathogens regulated in seed ISF Pest List

12 International standards for seed IPPC - ISPM 38 May 2017 Describes phytosanitary measures NAPPO RSPM 36 March 2013 Appendix 1: Recommended seed testing methods movement

13 ISPM 38 Adopted by IPPC at CPM 12 (April, 2017) Next step: NPPOs have months to prepare to implement Training sessions and workshops are being planned ISF has developed a training manual and several ppts Major benefits: specific guidance on PRA, recognition of seed production practices that reduce phytosanitary risk, paves way for accreditation approaches Immediate impact: eliminate regulations where seed is not a pathway

14 ISPM 38 Importance of determining the nature of the seed pathway PRA guidance Is the plant a host? Is the pathogen seed transmitted, seed borne or a contaminant on the seed? ISPM 38 takes into account that different uses of seed pose different phytosanitary risks Recognition that many seed production practices reduce phytosanitary risk Recognition that, to facilitate export and re-export, phytosanitary measures need to be harmonized

15 Phytosanitary measures (ISPM 38) Pest freedom Pest free areas Pest free places of production Pest free production sites Areas of low pest prevalence Field inspection Backed-up by laboratory confirmation Crop treatment Visual inspection of seeds Testing of seeds Seed treatment System approaches - sets of integrated phytosanitary measures applied by seed producers and approved and monitored by an NPPO (eg., GSPP, ReFreSH) Post-entry quarantine Prohibition

16 Seed health testing method development and validation Numerous organizations are involved Int. Seed Testing Assoc. (ISTA) Int. Seed Federation (Int. Seed Health Initiative, ISHI) U.S. National Seed Health System (NSHS) Int. Plant Protection Conv. (IPPC) NAPPO, EPPO USDA-APHIS-PPQ Naktuinbouw Accreditation system (NAL) Individual NPPOs often go their own way!

17 Criteria for seed health testing method validation Specificity Inclusivity, Exclusivity How many strains must be tested? Sensitivity Lack of false negative results at desired limit of detection What is the appropriate limit of detection? Selectivity - Need to assess in seed extract matrices Repeatability & reproducibility Method gives consistent results within and between labs How many samples/labs are required? Statistical assessment of criteria How stringent? Other criteria robustness, precision, accuracy,

18 ISTA Seed Health Committee Responsible for: Establishing criteria for ISTA seed health testing methods Developing tools and guidelines for seed health testing method development and evaluation Evaluating proposed methods Making recommendations to ISTA membership for method approval 32 approved methods

19 ISTA Method Validation 1. Method selection and development. 2. Validation through comparative testing. 3. Review of comparative test results and preparation of a Method Validation Report. 4. Approval of validation status by the ISTA Seed Health Committee and preparation of a Rules Proposal for the method. 5. Final acceptance by the ISTA Membership which will allow publication of the validated method in the ISTA Rules. SHC Resources: Statistical tools Proficiency testing Workshops

20 ISTA Comparative test procedures Naturally or artificially contaminated seeds, 3 levels of contamination (clean, low, medium ) Pretests for contamination level, homogeneity, stability # of subsamples depends on contamination level & homogeneity (at least 3 subsamples per cont. level) Subsample size depends on desired detection limit Eight participating labs is the goal Statistical criteria must be met for: Sensitivity Specificity Accuracy Repeatability (accordance) Reproducibility (concordance)

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22 ISHI Method Development ISHI currently has 24 approved methods ISHI-Veg methods are the outcome of one of the two processes or both: A comparative test done by 6-8 company and public labs. A peer review of the method description by ISHI-Veg members and/or external reviewers. The supporting data may be supplied by the laboratory responsible for development of the method and/or a validation test of certain components of the described method conducted by ISHI-Veg members. ISHI Resources: Guidelines for use of seed health testing methods Best practices policies

23 National Seed Health System Responsible for: Establishing criteria for seed health testing methods for export certification from U.S. Overseeing accreditation of public & private labs to perform phytosanitary testing and inspections Evaluating proposed seed health testing methods 82 approved methods

24 NSHS Seed Health method evaluation Peer review process: Technical panels of experts in each disease formed Review of protocol & validation data Evaluation criteria defined by NSHS Sensitivity, specificity, repeatability, reliability Comparison with existing methods Technical Panel recommendation: Accept as Standard test Accept as Temporary Standard Test Not accepted Policies & Procedure Advisory Board vote on Technical Panel recommendation

25 International alignment-phytosanitary testing Alignment of methods NSHS, ISHI, ISTA Alignment of evaluation criteria 2016 international working group NSHS, ISTA, ISHI, EPPO, USDA-APHIS, NAL Assessment of procedures and criteria Goal to move toward harmonization, esp. ISHI and NSHS Report available

26 Seed certification System designed to ensure the availability of high quality seed of genetically distinct crop varieties Importance for publicly released varieties (traditionally field crops in the U.S.) Recent trend toward privatization Exports More stringent requirements in Europe & other countries

27 Seed certification Organization for Economic Cooperation and Development (OECD) OECD Seed Schemes International seed certification system Oversight by USDA for exports Association of Official Seed Certifying Agencies (AOSCA) US, Canada, S. America, Australia, NZ State Crop Improvement Associations

28 Seed certification stages Breeder seed Foundation seed Registered seed Certified seed Seed health testing Source: Copeland & McDonald, 2001

29 Seed certification requirements Documentation (pedigree, etc.) Production practices Field inspection Seed inspection Source: Copeland & McDonald, 2001

30 Seed health testing method advancements Traditional methods are still widely used and effective in many cases Visual inspection, microscopy Direct incubation methods for fungi Selective media for bacteria Grow-outs and bioassays Newer technologies have constraints Serological methods (ELISA) Nucleic-acid based methods (PCR)

31 International standard seed health tests for fungi and viruses Fungi Visual Blotter Agar ELISA PCR Gr.-out Indic. ISTA ISHI 3 2 NSHS Viruses & viroids Visual Blotter Agar ELISA PCR Gr.-out Indic. ISTA 2 1 ISHI 4 3 NSHS

32 Important considerations for nucleic-acid based methods Primers must be carefully designed and tested Extraction methods are often the main obstacle to accuracy Instrumentation needs are high Sensitivity and quantifiability are increasing, but information is lacking on relationship between test results and risk of seed transmission Viability of detected pathogens is uncertain Epidemiological information needed

33 Bacterial leaf streak of maize Xanthomonas vasicola pv. vasiculorum First officially reported in U.S. in 2016 Severe disease in irrigated crops Unknown if seed is a pathway for movement

34 Bacterial leaf streak of maize Primer design - CO State Univ., USDA-APHIS Partial or whole genome sequencing, phylogenetic analysis Lang et al., 2017; Stulberg et al., 2017

35 Bacterial leaf streak of maize Confirmation of primer specificity vs host range Lang et al., 2017

36 Pospiviroids in tomato & other Smallest known infectious agents Single Stranded RNA solanaceous crops RT-PCR or bioassays are the only detection options Pospiviroids - family of nine viroids Seed transmission risk is controversial Phytosanitary regulations E.g., Australia

37 Extraction methods Extraction from seeds is challenging Inhibitors / interference from seed matrix Throughput Magnetic bead technology Kingfisher Flex

38 Magnetic bead technology Can be used for generic DNA / RNA purification or Beads can be designed with specific probes Immuno-magnetic separation: Antibodycoated beads are used to capture pathogen or antigens in seed extract Magnetic capture hybridization: beads coated with nucleic-acid probes

39 PCR results with different extraction methods for F. oxysporum in bean seeds Fop seed infestation (%) Sousa et al., 2014 Crude Extract 0/9 0/9 0/9 0/9 0/9 Detection Frequency MCH MCH Qiagen (20 μl) e (40 μl) e (no MCH) 0/9 3/9 4/9 3/9 9/9 0/9 9/9 9/9 9/9 9/9 0/9 9/9 9/9 9/9 9/9

40 Instrumentation needs PCR-based method requirements can be an obstacle Isothermal amplification methods (e.g., LAMP) Can be done without thermal cycling (even in the field!) Complicated primer design Does not resolve extraction issues Peng et al., 2012

41 Loop mediated isothermal AMPlification Created in 2000 by Notomi et al. Requires 4-6 primers and DNA polymerase with strand displacement activity Forward inner Backward inner Forward outer Backward outer

42 Loop mediated isothermal AMPlification Created in 2000 by Notomi et al. Requires 4-6 primers and DNA polymerase with strand displacement activity Forward inner Backward inner Forward outer Backward outer

43 LAMP methods for seeds Late wilt of maize Gonzalez and Munkvold, 2016 Pospiviroids in tomatoes Gonzalez and Munkvold, 2017 Bacterial leaf streak of maize Stulberg et al., 2017 Other examples in the literature

44 Interpretation of nucleic-acid based seed health tests Viability of propagules Live/dead PCR assay Propidium monoazide (PMA) Blocks amplification of DNA from damaged cells PMA assay for bacterial leaf streak Stulberg et al., 2017

45 Interpretation of nucleic-acid based seed health tests Use of nucleic-acid based tests as initial screening Negative samples are considered clean Positive samples are processed for direct detection of pathogen Recovery of live propagules Bioassays Grow-out assays Use of nucleic-acid based tests as confirmation after recovery of live propagules

46 Bacterial leaf streak of maize Confirmation of identity of recovered colonies

47 Interpretation of nucleic-acid based seed health tests Relationship between low contamination levels and disease risk - Bacterial leaf streak of maize Stulberg et al., 2017 Arias et al., 2017

48 Important considerations for nucleic-acid based methods Primers must be carefully designed and tested Extraction methods are often the main obstacle to accuracy Instrumentation needs are high Sensitivity and quantifiability are increasing, but information is lacking on relationship between test results and risk of seed transmission Viability of detected pathogens is uncertain Epidemiological information needed

49 Common themes Need for robust seed health tests that: Capture the diversity of pathogenic strains Do not detect closely related non-target organisms/viruses Are connected with risk of disease occurrence Need for harmonization of testing methods To avoid costly re-testing and rejection of shipments Epidemiological studies connected with results of PCRbased seed health tests Seed transmission data for positive seedlots Establishing connection between genomic information and economic risk E.g., Diaporthe/Phomopsis on spinach seeds Dynamic pathogen populations that require continuous adaptations in detection and management

50 National Seed Health System

51 Iowa State University on-line Seed Technology & Business Graduate Program