SHC Committee report 2013, perspectives 2014

Valerie Grimault, Ilaria Alberti, Isabelle Serandat

Committee members SHC

1 Chair Valerie Grimault France 2 Vice-Chair Theresa A.S. Aveling South Africa 3 Member Ilaria Alberti Italy 4 Member Siham Asaad Syria 5 Member Rouke Bakker New Zealand

6 Member Harrie Koenraadt Netherlands 7 Member Ronit Kohen Israel 8 Member Gary Munkvold USA 9 Member Masatoshi Sato Japan

10 Member Karin Sperlingsson Sweden 11 Member Sandra Walker USA 12 Member Reyes Blanco Prieto Spain 13 Member Dorota Szopińska Poland 14 Member Mark Buimer Netherlands

Date - Presenter Name

èCall for new member for next congress

Rules development (Valerie Grimault)

• New methods submitted to vote June 2014: -2 methods validated by ISHI:

Date - Presenter Name

• Acidovorax/corn salad: grow out method + PCR

• Xanthomonas/brassicae: modification of media for method 7-019

-1 method validated by Euphresco: • Gibberella/Pinus: media + possible

confirmation by PCR (see following presentation)

Seed Health Testing Method 7-009: Gibberella circinata on Pinus taeda

and Pinus ellioti (Pine).

Method revision in collaboration with Euphresco Project

Ilaria Alberti (CRA-SCS, Italy)

SHC Committee report June 2014

Foto: I. Alberti, CRA-SCS (Italy) Foto: I. Alberti, CRA-SCS (Italy)

Gibberella circinata (anamorph Fusarium circinatum) former nomenclature Fusarium moniliforme var. subglutinans and F. subglutinans f.sp. pini.

Pitch canker of pine trees

Ilaria Alberti

Foto R. L. Anderson, USDA Forest Service, Usa

Foto D. Owen, California Department of Forestry and

Fire Protection, Usa

AREA

USA Forest

MEXICO Forest

HAITI Forest

SOUTH AFRICA Forest

JAPAN Forest

CHILE Forest

SPAIN Nurseries

FRANCE Nurseries

PORTUGAL Nurseries

ITALY Limited areas of the natural environment

SPAIN Limited areas of the natural environment

Ilaria Alberti

Seed Health Testing Method 7-009 (Chapter 7 of ISTA Rules)

Published in ISTA Handbook of Seed Health Testing in 1985 (Working sheet N. 56)

Incorporated into the newly revised Chapter 7 in 2002 from the 1999 edition of the ISTA Rules

Reviewed by ISTA-SHC in 2006 with the recommendation to accept for a further five years

Foto R. L. Anderson, USDA Forest Service, Usa

Ilaria Alberti

2011

ISTA SHC: revision of the method The EUPHRESCO project called “Gibcir-Diagseed”

Start Date: April the 1th, 2011 End Date: June the 30th, 2012

Ilaria Alberti

EUPHRESCO is a European Research Area Network (ERA-NET) project for research policy development and implementation in the field

of statutory and emerging plant pests, diseases and invasive species (but not: GMO's).

….. validated protocols and tests for the detection of the pathogen in seeds will be proposed to the ISTA in order to promote European standards within the international community.

Expected benefits

EUPHRESCO PROJECT

(Ring testing, 2011)

11 laboratories from 10 European Country

Three protocols evaluated (available 9 protocols from literature) Protocol 2: Isolation followed by morphological identification

Protocol 4: Isolation followed by conventional PCR

Protocol 9: IGS hydrolysis probe real-time PCR

For each protocol, the results obtained for the blinded samples were processed according to EN ISO 16140 to compute Accuracy, Specificity and Selectivity and their respective confidence interval

Foto R. L. Anderson, USDA Forest Service, Usa

All three protocols showed acceptable performance data

Final report and validation data were sent to SHC for scientific evaluation

SHC, during 2013 ISTA meeting, suggested to carry on the validation of Protocol 2 with PCR option of protocol 4. In the introduction of the method it has been suggested to introduce the references of protocol 9, validated by Euphresco

September 2013:

Euphresco filled Appendix 4 (Test Method Validation Application Form) and sent

to ISTA the Draft Test method sheet with the title: Detection of Gibberella

circinata in Pinus species and Pseudotsuga menziesii seed

Reviewed by 2 technical and 1 statistical reviewers.

SHC decided to propose it to Rules Proposals

Foto: I. Alberti, CRA-SCS (Italy)

ACKNOWLEDGEMENTS TO

Euphresco “Gibcir-Diagseed” Project Coordinator

Dr. Renaud Ioos

Rules development • New methods under development:

- SqMV/squash: grow out (ISHI), review of validation report done

- Botrytis/onion: media (ISHI), postponed - Leptosphaeria/brassicas: media and blotter,

pathogenicity tests and PCR confirmation under progress (TESTA, collaboration with ISHI), test plan under progress

- Fusarium/tomato: media and pathogenicity test and/or PCR for confirmation (ISTA, Israel), test plan under progress

- Fusarium/cereals (ISTA, Norway), test plan under progress

- Xanthomonas/rice (IRRI, Philippines), test plan under progress

ØBefore 2009: § No PT programme § No description of how to organize and analyze PTs è guidelines/standards to be defined è programme to be defined

Proficiency tests (Masatoshi Sato)

ØDecision: § Start to organize PTs: first PT in 2009, 7 completed yet § Use the experience of these PT to start defining

• Guidelines • Tools • Rating system • When ready integrate the PT Com programme in a more routine

basis § No influence of results on accreditation of labs yet

ØHow?: 3 seminars organized last 3 years, SHC members with chair of ISTA STATCOM

Proficiency tests (Masatoshi Sato)

• Organization, statistical analysis and rating of labs: - Seminar December 2013: Isabelle Serandat, Karin Sperlingsson,

Liana Politikou, Mark Buimer, Valerie Grimault, Jean-Louis Laffont

homogeneity test - seed health

Sample size 1000

Samples 1 2 3 4 5 6 7 8 9 10

infected seed nb. 10 11 10 9 12 11 9 13 12 10 Averagehealthy seed percentage 99 98.9 99 99.1 98.8 98.9 99.1 98.7 98.8 99 98.93

98.93

1.41

OK

AverageToleranceH valueHomogeneity check

- Web meeting May 2014: Isabelle Serandat, Mark Buimer, Valerie Grimault

- Guidelines defined and posted on ISTA website: http://www.seedtest.org/en/documents-_content---1--1177.html

- Homogeneity test: tool posted on ISTA website (still need to be validated by STATCOM)

Proficiency tests • Proposal for analysis for qualitative

and quantitative results - Tools developped by JL Laffont - Simulations done on former PT results in May

meeting. - Proposals for rules for rating defined, to be

discussed with JL Laffont and PTCOM.

Proficiency tests • 7-013 U.nuda/Hordeum vulgare: postponed

due to problem of heterogeneity in one seed lot

Date - Presenter Name

• 7-019 Xcc: results received, statistical analysis pending

• 7-006 Colletotrichum lindemuthianum and 7-005 Ascochyta: no contaminated seeds available

• 7-003 Botrytis sunflower: results received, statistical analysis under progress

• 7-016 Phomopsis: done (see presentation of Isabelle)

• 7-026 cucurbits viruses: planned • http://seedtest.org/en/shc-proficiency-

test-_content---1--1423.html

Status now and perspectives Ø Planning and execution depending on availablibity of

infected seed samples / possibility of producing artificially infected samples

Ø Conclusions are provided to laboratories showing if their performances are satisfactory

Ø Project ongoing to optimise evaluation and rating system

Ø Goal: § integrate PTCOM program on a routine basis § Provide to accredited and voluntary labs two Proficiency Test

rounds per year

ISTA Seed Health Example of proficiency test Phomopsis/Soybean PT 12-SH G.max

Isabelle Serandat

Proficiency test Phomopsis/Soybean PT 12-SH G.max • 2012: 22 laboratories

- 19 accredited - 3 non accredited

• Aim: verify ability of laboratories to detect Phomopsis complex on Glycine max seeds.

• Detection: ISTA method 7-016 - Direct plating on acidified potato dextrose agar (APDA) - Visual identification

Date - Presenter Name

Pre-test and sampling PT 12-SH G.max

• Pre-test ØDifficulty to find available contaminated seed lots ØCollaboration with ENSE who provided lots ØFour seed lots tested to select one healthy and one medium

contamination level • Healthy (lot A): 0% of Phomopsis complex • Medium (lot B): 16.75% of Phomopsis complex

• Samples ØAll samples sampled in the same time (homogeneity, stability, PT) according to the guidelines available on ISTA SHC webpage ØEach lab received a total of 5 coded samples

• 400 seeds per sample • 2 replicates of the healthy • 3 replicates of the medium contamination level

Extra samples /Analysis PT 12-SH G.max

• Homogeneity test Ø10 extra samples tested from each lot, after sampling and packaging

• Stability test Ø3 extra samples tested from each lot, after reception of all laboratories

proficiency test results • Scoring of results ØNumber of contaminated seeds counted automatic calculation of the

percentage • Statistical analysis ØHomogeneity and Stability tests: GEVES statistical software (based on H

test) available on ISTA SHC webpage (still to be validated by STATCOM) ØSamples: ISTA Boxplot tool (Jean Louis Laffont)

Homogeneity PT 12-SH G.max

• Homogeneity test results ØHealthy lot (A): no contaminated seeds (0% ), no positives. ØMedium lot (B): mean of 16.28% contaminated seeds

• Homogeneity of the lots confirmed • Results close to the pre-test

Stability PT 12-SH G.max

• Stability test results ØHealthy lot (A): no contaminated seeds (0% ), no positives ØMedium lot (B): mean of 14.58% of contaminated seeds

• Stability of the lots confirmed • Results are slightly lower but close to the pre-test

Results PT 12-SH G.max • Results of healthy lot (A) • 6 laboratories obtained false positive results

(4 out of 19 accredited labs and 2 out of 3 non accredited labs)

• Possible explanation: - Confusion between Diaporthe phaseolorum and Fusarium or

Arthrinium sp. - Cross contamination during manipulation (for low percentage)

0

1

2

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22

% o

f con

tam

inat

ed se

eds

laboratories

Healthy lot (A)

Arthrinium sp. Phomopsis sp.

• Results of medium lot (B) • Distribution of average results showed a small variability of

the results between laboratories. • 4 laboratories identified as outliers

- 1 under estimation (accredited) - 3 over estimations (2 accredited, 1 non accredited)

Confusion between Diaporthe phaseolorum and Fusarium or Arthrinium sp.

5

10

15

20

25

30

35

40

45

50

All labs(22)

% o

f con

tam

inat

ed se

eds

Medium contaminated lot

Results PT 12-SH G.max

Conclusion: ØPT highlighted a problem of identification for some

laboratories: False positives or over estimations ØISTA Seed Health Testing workshop (Poznan, Poland) in

September on several species including soybean ØResults will be used to optimise the rating system

Results PT 12-SH G.max

Handbook (Terry Aveling)

• 8 chapters under progress - 1 reviewed and ready for sending to ISTA for publication:

sampling

- 4 completed, to be reviewed for final comments

• General methods

• Fungi

• Bacteria

• Nematodes

- 3 to be completed

Annotated list (Terry Aveling)

• TESTA project - 5 postgraduate students

• reviewing papers cited in ISTA annotated list of seed borne diseases

• Finding new papers • Goal: update the list

- Collaboration with ISF pest list: • ISF focused on vegetables (published on their website) • TESTA project focuses on crops not covered by ISF: barley,

herbs… • Exchange info on new diseases identified in TESTA • TESTA: seed borne diseases è pest for which seed is a pathway • Still under discussion: how will the lists interact and be

available

Web page (Sandra Walker)

Workshops • Poznań, Poland

- 04.09.2014 - 7.09.2014 - Focused on diseases of economically

important crops in this part of the world (barley, cabbage, carrot, flax, pea, soybean)

- lectures of seed-borne diseases of these plants, hands-on practical work focused on recognizing fungal pathogens on seeds

- Presented by: Prof Theresa Aveling (South Africa), Prof Reyes Blanco (Spain), Prof Krystyna Tylkowska and Dr Dorota Szopinska (Poland), Ms Isabelle Serandat (France), and Ms Karin Sperlingsson (Sweden)

- Now fully booked

Workshops • BALAI BESAR WEST JAVA,

INDONESIA • 10 -14 November 2014 • Seed health training course • Lectures and Practical work on

- detection of fungi (blotter tests), bacteria (media and PCR), viruses (ELISA), nematodes (morphological identification)

- Rice, brassicas, pepper, tomato • Lectures on:

- Good Laboratory Practises in seed health laboratory

- Critical Control Point in seed health testing - Molecular techniques in seed health testing - Quality assurance in seed health testing

• Presented by: Valerie Grimault (France), Mark Buimer (Netherlands), Masatoshi Sato (Japan), Corinne Sarniguet (France), Bonny W. Soekarno (Indonesia)

Date - Presenter Name

Thank you for your attention