International Forest Quarantine Bodies

Eric Allen Natural Resources Canada Canadian Forest Service International Plant Protection Convention

Commission on Phytosanitary Measures

Standards Committee

Technical Panels Expert Working Groups RPPOs

International Forestry Quarantine Research Group Commission on Phytosanitary Measures

• Governing body of the International Plant Protection

Convention (IPPC)

• Members: National Plant Protection Organizations (179 countries) • International standard-setting body • Goal: prevent plant pests being spread through international trade International Plant Protection Convention

IPPC Technical Panels:

• Forest Quarantine (TPFQ) • Phytosanitary Treatments (TPPT) • Fruit Flies (TPFF) • Glossary Terms (TPGT) • Diagnostic Protocols (TPDP) Technical Panel on Forest Quarantine

Mandate: • address technical aspects of forest quarantine issues. • review relevant technical and scientific information to provide guidance to the IPPC Standards Committee as requested on development, amendment and revision of standards • ISPM 15 revision • work with other technical panels e.g. TPPT, TPDT International Forestry Quarantine Research Group

• Advisory body to the IPPC providing scientific analysis and review of global phytosanitary issues

• Identify and undertake collaborative scientific research aimed at high priority forestry quarantine questions International Forestry Quarantine Research Group

UPDATE OF EPPO ACTIVITIES IN FOREST QUARANTINE

Andrei D. ORLINSKI Scientific officer of EPPO

11th IFQRG meeting, Qingdao, China, 28 October – 1 November 2013 EPPO Region EPPO main activities on forest pests in 2012-2013 • EAB Agrilus planipennis • Cerambycids: ALB, CLB • New emerging pests and PRAs for Oemona hirta, Polygraphus proximus & Agrilus planipennis • Phytosanitary forest news • Forest pest interceptions • Safe use of biological control agents • Comments on new ISPMs EPPO standards PM 9 (NRCS) for EAB, ALB & CLB • PM 9 standard on EAB was under development since 2009, PM 9 standards on ALB & CLB - since 2010 • The aim is to provide guidance on measures at the detection of pests: clear-cut zones, detection and delimiting surveys, sampling, etc. • Difficulty: to get consensus • Standards re-drafted in 2012 • Adopted by the EPPO Council in September 2013 Pest Risk Analysis • Oemona hirta (LTB: Lemon Tree Borer, Cerambycidae, originating from New Zealand) was intercepted on Wisteria plants in the UK in 2010 • Attacks over 200 species of trees and shrubs from 81 families • PRA conducted in EPPO in June 2012 by experts from AT, GR, NL, NZ and UK • Recognized of quarantine significance • Pathway identified – plants for planting • Main measure identified – PFPP Pest Risk Analysis (2) • Polygraphus proximus (FFBB: Four- eyed Fir Bark Beetle, Curculionidae, Scolytinae, originating from the Far East) recently spread to Western Siberia and European Russia • Prefers fir both in the Far East and in newly invaded areas, but also may attack spruce and other coniferous; vectors Grosmannia aoshimae (initially described as Ophiostoma aoshimae) Pest Risk Analysis (3) • PRA conducted in EPPO in December 2012 by experts from BG, CZ, HU, IT, RU and SE • Recognized of quarantine significance for the EPPO region • Main pathways identified: WPM, wood with bark, particle wood and waste wood, isolated bark, plants for planting & Christmas trees of Coniferae • Main measures identified: PFA, bark freedom, treatments Pest Risk Analysis (4) • PRA for EAB • First conducted by EPPO Forestry Panel in January 2013 • PRM part of PRA revised following EPPO express-PRA scheme in January 2013 by experts from CA, DK, ES, NL, RU, UK and USA • HT 56°/30 min was not recognized efficient based on EFSA decision • Bark freedom was not recognized efficient • Chipping to 2,5cm size chips was not recognized efficient • Main measures identified: PFA, removal of bark & 2,5cm layer of sapwood, treatments Safe use of biological control agents (BCAs) • Joint EPPO/IOBC Panel on Biological Control Agents reanimated since 2008 • EPPO Standards of PM 6 series “Safe use of biological control”: - PM 6/1(1) First import of exotic biological control agents for research under contained conditions - PM 6/2(2) Import and release of non- indigenous biological control agents - PM 6/3(4) List of biological control agents widely used in the EPPO region (“Positive List”) Safe use of biological control agents (BCAs) • Targets both forest and agricultural pests • The “Positive List” is reviewed annually • Criteria: use in at least 5 EPPO countries for at least 5 years without records on negative non-target effects • Aim: to facilitate introduction and use of BCAs at the national levels • Concern only macroorganisms (Invertebrates: IBCAs) • Focus on safety of use, not on efficacy

Other EPPO activities on forest pests • Phytosanitary news (EPPO Reporting Service) on plant pests • Plant pest interceptions (collection of data received from NPPOs of EPPO countries) • Comments on new draft ISPMs (in 2013 the draft ISPM on international movement of wood was discussed at the regional EPPO/FAO/IPPC Workshop and at the EPPO Panel on CPM affairs) Thank you for your attention NAPPO Forestry Panel 2012-14 Key Progress

Shane Sela, Chair, NAPPO Forestry Panel IFQRG Meeting October 28 – November 1, 2013

© 2007 Her Majesty the Queen in Right of Canada (Canadian Food Inspection Agency), all rights reserved. Use without permission is prohibited. •Discussion paper regarding the applicability •of heat treatment for wood products HT standards variable from country to country; • Confusion on applicability to pest risk mitigation; • Panel proposed developing guidance to NPPOs; • Paper completed in August 2013; • Posted to NAPPO website and is a presented to IFQRG for review; • treatment; General structure of the paper: • History of heat as a wood ); Scientific basis for its effects on wood pests (including insects, fungi, • treated; nematodes and bacteria • H phytosanitary Industrial processes by which wood is heat approaches; ow heat treatment can be incorporated into systems

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•Workshop on the implementation of ISPM-

History of ongoing interceptions of pest and non compliant • - wood packaging reported by member countries Proposed joint Asia North American workshop which brings NPPOs and industry together to discuss successes • and areas for improvement; Panel met in Victoria, BC in March 2013 to develop a • - proposed agenda for the workshop; • S Agenda provided to APPPC NAPPO Steering Committee pecific timing and location of the workshop is yet to be • established Current proposal is to establish the workshop in Asia in early 2014.

(EAB) •Biological control of the emerald ash borer • Biocontrols EAB expanding in U.S. and Canada • are a critical tool in managing expansion; • Harmonization of management efforts more likely to slow spread; • Technical Advisory Group established to develop technical guidance; • T Guide nearing completion: • entomopathogenic biocontrol he general biology of EAB and available management options; • R biocontrol Guidance on the use of classical and agents;

• Fraxinusegulatory procedures guiding the use of in the three NAPPO countries; • biocontrol species at risk to EAB in Mexico; • biocontrol Research needs for EAB ; Steps to harmonize strategies in the three NAPPO countries and conclusions and recommendations.

2013-

• 14 Proposed work • Complete ISPM 15 workshop wood Develop• a regional standard on the potential use of systems approaches to manage pest risks associated with the movement of • Countries rely predominantly rely on treatments to manage pest risks; • phytosanitary Systems approaches may better manage pest risks • Situations where a single measure is insufficient or prohibitive to trade; • Could be applied to facilitate country to country trade or manage pests domestically • I Provide guidance on how integrated measures could be used: • nspections (at harvest, during production, prior to and or following export), • Prescribed production activities; • T Laboratory diagnostics; • areas; he application of treatments; • Surveillance Pest free

The work of IUFRO Unit 7.03.12 and its links to other initiatives on invasive alien species and international trade

Hugh Evans Forest Research, UK IUFRO Unit 7.03.12

Alien Invasive Species and International Trade • Established in 2005 • Coordinator – Kerry Britton, USA • Deputies – Kavile Veetil Sankaran (India) Hugh Evans, UK Kenji Fukuda, Japan • Aims: • To examine global forestry issues related to the unwanted international movement of alien invasive species, including fungi, insects, nematodes and plants • To share information and research needs • To develop data sharing agreements • To coordinate joint research and monitoring efforts • To encourage improvement and extension of Pest Risk Analysis techniques • To provide research support on mitigation measures • To interface with other IUFRO Units and other initiatives on global phytosanitary issues

2 IUFRO Unit 7.03.12 Alien Invasive Species and International Trade • Meetings: • 2006 - Jedlnia, Poland. Local organisers Tomasz Oszako, Leszek Orlikowski and Monika Małecka. Proceedings publication: http://www.forestry.gov.uk/fr/INFD-6YUGMQ • 2008 – Shepherdstown, USA. Local organisers Kerry Britton and USDA Forest Service colleagues. Presentations from the meeting: http://www.forestry.gov.uk/fr/INFD- 6YUGMQ • 2012 – Tokyo, Japan. Local organiser Kenji Fukuda. Report is on line http://www.iufro.org/science/divisions/division- 7/70000/70300/70312/activities/ • 2013 – Qingdao, China. Local organiser Jinghua Sun. Held last week! • Initiatives: • Position paper on Plants for Planting (P4P) Pathway by Kerry Britton, Marc Kenis (Switzerland) and Shiroma Sathyapala (New Zealand). Available for download - http://www.forestry.gov.uk/fr/INFD-6YUJRD

3 Links to previous and current initiatives concerned with invasive alien species

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Pathway Evaluation and pest Risk

Management In Transport (PERMIT)

WG2: Development Overall outlineof generic risk of the T4:Action Analysis & PERMIT COST mitigation development of generic measures to reduce Action risk mitigation measures pest movements Synergyalong: pathwaysjoint work with ganisation of EU and other groups n WG3: Analysis of T5: Education and

the level of dissemination of education and information for pest risk awareness of risks awareness and risk associated with reduction

different pathways

WG4: Identification T6: Gaps analysis and of research and definition of research and data gathering data requirements needs

5 PERMIT contributors

• Contributions from 26 COST Countries Austria, Belgium, Bulgaria, Croatia, Denmark, Finland, France, Germany, Greece, Hungary, Iceland, Ireland, Italy, FYR of Macedonia, Malta, The Netherlands, Poland, Portugal, Serbia, Slovak Republic, Slovenia, Spain, Sweden, Switzerland, Turkey, United Kingdom, • and 8 non-COST Countries Albania, Australia, Canada, China, New Zealand, Russia, South Africa, USA

6 PERMIT COST Action • Meetings of the Action: • November 2010 – Brussels, Belgium • May 2011 – Belgrade, Serbia • December 2011 – Warsaw, Poland • May 2012 – Alice Holt, UK • June 2012 sub group – Brussels, Belgium • September 2012 – Antalya, Turkey • December 2012 – Uppsala, Sweden • July 2013 – Training School, Sarajevo, Bosnia & Herzebovina • October 2013 – Workshop, Treviso, Italy • June 2014 – Training School, Warsaw, Poland • September 2014 – International Conference, Portugal • Short Term Scientific Missions • Funding to support Early Stage Researchers on short missions to COST countries or designated partners in non-COST countries 7 REPHRAME –Research Extending Plant Health Risk And Monitoring Evaluation • Full title: Development of improved methods for detection, control and eradication of pine wood nematode in support of EU Plant Health policy • Continuation of previous PHRAME project (http://www.forestresearch.gov.uk for PDF of the full report) • Formal start date: 1 March 2011 • Current end date: 28 February 2014 (but could be 30 November 2014 if extension is granted). • Small collaborative project involving 11 organisations in 8 countries (Austria, China, France, Germany, Norway, Portugal, Spain, United Kingdom) • International linkage with Canada, Japan and USA • Kick-off meeting 1-2 April, 2011 in Madeira

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Work Packages of the REPHRAME project and the leaders of each WP: WP 1 Management and coordination – Hugh Evans WP 2 Behaviour and dynamics of PWN in infested trees – Manuel Mota WP 3 Assessing phenology and dispersal capacities of PWN vectors – Edmundo Sousa WP 4 Development of new methods for monitoring and control of Monochamus spp and PWN – Juan Pajares WP 5 Determine risk of non-vector spread of PWN through various pathways to healthy forests – Thomas Schroeder WP 6 Host tree resistance to PWN and its vectors for future planting – Edmundo Sousa WP 7 Prediction of pine wilt expression across eco-climatic zones, taking account of latency – Christelle Robinet, Alain Roques and Philippe Castagnone WP 8 EU and international cooperation and collaboration – Manuel Mota WP 9 Synthesis and development of PWN Tool Kit for monitoring and management of PWN – Hugh Evans WP 10 Stakeholder Engagement & Dissemination – Hugh Evans

9 Thank you for your attention!

10 Management of pest risks associated with the international movement of wood (2006-029)

Shane Sela Member Technical Panel on Forest Quarantine

© 2007 Her Majesty the Queen in Right of Canada (Canadian Food Inspection Agency), all rights reserved. Use without permission is prohibited. Background

CPM- programme

2 (2007) added the topic to work SC approved specification 46 in May 2008 Technical Panel on Forest Quarantine (TPFQ) began drafting and presented draft ISPM to the Standards • Committee (SC) in April 2010 draft returned to TPFQ for further revision • Presented again to SC in November 2012 returned again for further drafting

SC revised draft in May 2013 and approved for member consultation in 2013 Specification 46

Wood in trade may pose a pest risk phytosanitary measures Standard to provide guidance on risk management using

• The standard should include guidance on: Treatments and tools to verify that treatments have been • applied;phytosanitary • certification criteria; • inspection information; • detection techniques for specific pest groups; the determination of pest status in an area;

General Considerations

Wood (with or without bark) moved in international trade may be a pathway for the introduction and spread of quarantine pests • Pest risk relate to the: • commodity type • presence or absence of bark • origin of wood commodity • intended use treatment applied

General Considerations commodities: Wood is commonly moved as one of following four •

• round wood • sawn wood • mechanically processed wood processed wood material

The standard describes the general pest risk profile for each wood commodity by indicating the major pest groups associated with each one

General Considerations

• phytosanitary The standard also describes: various options for measures for managing pest risk related to wood, including bark removal, treatments, andchipping and inspection • phytosanitary phytosanitary specific requirements such as verification of measures applied before harvest to import of wood consignments

Drafting Issues

The TPFQ drafted the standard addressing each of the tasks outlined in specification 46 It only provides guidance and NPPOs should conduct risk assessments to determine if a specific trade presents a pest risk In line with specification 46, the standard is intended to • cover only wood and associated bark bark as a commodity itself is not covered in the standard

Wood packaging material moving in international trade that has not been treated and marked in compliance with ISPM 15:2009 is covered in the standard Other relevant information

A specific section on intended use of wood commodity was developed The concept of contamination of treated wood was considered and addressed in the standard An appendix containing the world’s major forest pests of quarantine concern was considered not appropriate for this standard as potentially too large provided A number of references for such information was

ISPM 15 Treatment criteria

IFQRG-11, Qingdao, China 28 Oct - 2 Nov, 2013

Draft Appendix to ISPM 15:2009 (Wood packaging material in international trade): Submission of new treatments for inclusion in ISPM 15

The draft was submitted for IPPC Member Consultations in 2010 and was returned by the Standards Committee to the Technical Panel on Forest Quarantine (TPFQ) for revision

ISPM 15 Treatment criteria - IFQRG-11, Qingdao, China

Among the main difficulties during the 2010 Member Consultations were:

- the list of pest groups to be used for testing

- the statistical model used in the evaluation of the efficacy of submitted treatments (Probit-9)

ISPM 15 Treatment criteria - IFQRG-11, Qingdao, China

IFQRG-9 and IFQRG-10 brought the development of the ‘Cardiff Protocol’ as an alternative to Probit-9

TPFQ met in June, 2013 to revise the document

ISPM 15 Treatment criteria - IFQRG-11, Qingdao, China

Proposals agreed by the TPFQ are the following:

1. The title of the draft should be modified to read: Process for testing new treatments for ISPM 15 - to better reflect the content of the document

ISPM 15 Treatment criteria - IFQRG-11, Qingdao, China

2. The status of the document should be changed from ‘appendix’ to ‘annex’ - the described procedure should not be voluntary, but be a requirement under the IPPC

ISPM 15 Treatment criteria - IFQRG-11, Qingdao, China

3. The treatment testing process should follow a 4-step procedure:

Step 1: Screening

Step 2: Effect of physical parameters

Step 3: Validation of the effective treatment level

Step 4: Validation under operational conditions

ISPM 15 Treatment criteria - IFQRG-11, Qingdao, China Step 1: Screening (1)

Step 1 should:

- demonstrate that the treatment is lethal to all tested species at all life stages

- identify the treatment level effective against all tested species

- identify the pest species and its life stage most resistant to the treatment

ISPM 15 Treatment criteria - IFQRG-11, Qingdao, China Step 1: Screening (2)

Pests to be used for testing:

Bostrychidae Buprestidae Cerambycidae Scolytinae Siricidae

Heterobasidion sp.

Bursaphelenchus xylophilus

ISPM 15 Treatment criteria - IFQRG-11, Qingdao, China Step 1: Screening (3)

At least 1 species from each group should be used for testing

A minimum of 5 test units for every species should be used for testing

A test unit is:

- for insects - a single insect - for Heterobasidion sp. and PWN - a single piece of colonized wood

ISPM 15 Treatment criteria - IFQRG-11, Qingdao, China Step 2: Effect of physical parameters (1)

Step 2 should indicate:

- whether the effective treatment level identified in Step 1 is delivered throughout the profile of wood

- if not, then which other lethal treatment level is delivered throughout the profile of wood

ISPM 15 Treatment criteria - IFQRG-11, Qingdao, China Step 2: Effect of physical parameters (2)

In this step the pest species at its most resistant life stage as identified in Step 1 is exposed to the effective level of the treatment at different levels of physical parameters

ISPM 15 Treatment criteria - IFQRG-11, Qingdao, China Step 2: Effect of physical parameters (3)

The physical parameters may include:

- air and wood temperature - moisture content of wood - dimensions of wood - anatomy and density of wood - wood species - presence or absence of bark

The testing is performed in laboratory conditions

ISPM 15 Treatment criteria - IFQRG-11, Qingdao, China Step 3: Validation of the effective treatment level

Step 3 verifies the statistical reliability of the treatment under laboratory conditions

A minimum of 60 test units should be used

100 % treatment success should be achieved

ISPM 15 Treatment criteria - IFQRG-11, Qingdao, China Step 4: Validation under operational conditions (1)

Treatment should be validated on wood size and type normally used in the production of the wood packaging material

ISPM 15 Treatment criteria - IFQRG-11, Qingdao, China Step 4: Validation under operational conditions (2)

For Heterobasidion sp. and PWN at least 60 test units should be used

For insects, the minimum number of test units should be calculated using the formula provided by the ‘Cardiff Protocol’, which takes into account: - the biology of the pest - the pest relationship with wood packaging material - the international trading patterns

ISPM 15 Treatment criteria - IFQRG-11, Qingdao, China Step 4: Validation under operational conditions (3)

Treatment success should be achieved for all test units, which will indicate that the treatment is effective at statistical reliability of 0.95 at 95 % confidence level.

ISPM 15 Treatment criteria - IFQRG-11, Qingdao, China Future steps (1)

The draft will be submitted to the Standards Committee (SC) at its meeting in May, 2014 for consideration

The SC may decide to send the draft for Member Consultation in 2014 (July - November)

ISPM 15 Treatment criteria - IFQRG-11, Qingdao, China Future steps (2)

Country comments (2014) will be considered by the SC-7 in May, 2015, the draft will be adjusted and sent for Substantial Concerns Commenting Period (July - September)

Country comments will be considered by the SC in November, 2015 and the draft may be sent to the CPM for adoption in March 2016

ISPM 15 Treatment criteria - IFQRG-11, Qingdao, China Review of “Cardiff protocol”

Adnan Uzunovic

Location: IFQRG 11, Qingdao, China

Date: October 28- November 1st, 2013 IFQRG’s 3-Step approach in testing efficacy of a new treatment

§ Step 1:Small lab scale test-estimate the lethal dose of the most tolerant pest of quarantine importance that is found in wood packaging, and most challenging conditions (physical parameters)

§ Step 2:Larger lab test- replicate experiments at the estimated lethal dose to validate it for the most tolerant pest (demonstrates higher reliability)

§ Step 3: Simulated field test- confirm under simulated operational condition and provide statistical efficacy

Approved treatment will be scrutinized on ongoing base in subsequent real-life experience, and either kept, modified or withdrawn (e.g. discussions on HT tolerance of EAB)

Step 3 - Confirmatory Study Under Simulated Operational Conditions § Using the most tolerant pests under simulated operational conditions using wood samples similar in size to wood packaging material and infested to levels that reflect field conditions. Sample size can be discussed and perhaps be 60 (0.95) to 300 (0.99). § Pre test pest load can be estimated and used to calculate reliability figures following the treatment. When a sufficient amount of a pest is available (e.g. PWN) then the Probit 9 level will be easy to achieve and report but for more scarce pests the Probit level will be much less.

Still, there was a need to proscribe minimum amount of pest to be tested (with no survivors) to determine the required level of efficacy for step 3, that considers important factors along the biological pathway including host and commodity infestation, pest development and survival, population ecology effects related to invasion and persistence “The Cardiff Protocol”

A model to estimate the minimum test size for step 3 required for pests on wood packaging in international trade

Initially developed and presented by Mike Ormsby at:

IUFRO -7.03.1.2 (Alien invasive species and international trade), Tokyo, June 2012

Presented at and supported by IFQRG, in Cardiff, September 2012

“The Cardiff Protocol”

Recognises that there are two types of pests: § Those that infest at a relativelly low rate and can be counted as individuals (e.g. insects) § Those that infest at very high rate and cannot be counted as individuals (e.g. nematodes, fungi, bacteria…)

“Cardiff Protocol” model estimates treatment efficacy target that reflects:

§ The biology of the pest § The relationship between the pest and the wood packaging § The international trading patterns of wood packaging material

www.invasive.org “Cardiff Protocol” – biology of pest • The level of natural mortality of the pest from the time of treatment to reproductive fertility “Cardiff Protocol” –biology of pests

The founder population size (The number of individuals of the pest required to establish a new population) and level of natural mortality are combined to give the ‘Maximum Pest Limit’.

The Maximum Pest Limit or MPL is therefore the number of a pest infesting a unit of WPM that would be required to establish a new population in a new area

MPL =FP/Psurvival (By dividing the founder population size by the propability of a pest surviving to a mature breeding adult) “Cardiff Protocol”-relationship between pest and wood packaging

• INFESTATION RATIO: The maximum ratio of pallets likely to be infested by the pest

• INFESTATION RATE-The average number of the pest likely to be found in a unit of wood packaging.

“Cardiff Protocol” – trading patterns of wood packaging

• The maximum number of wood packaging units (e.g. pallets) found stacked or grouped together at a single location (e.g. a pallet recycling facility). “Cardiff Protocol” – Required Number of Dead Pests ”Cardiff Protocol” example for Southern pine beetle, Dendroctonus frontalis

• The infestation ratio = 75% (of pallets may be infested)

• The infestation rate = 60 larvae per pallet

• The maximum Pest Limit = 25 larvae (a Founder Population Size of 5 multiplied by a survival rate of 20%)

• The maximum number of units of WPM aggregated at a single location = 1000

‘”Cardiff Protocol” required pest mortality for Dendroctonus frontalis

= 0.75 (75%) x 60 x 1000 = 45,000 larvae

= 45,000 larvae ÷ 25 (MPL) = 1,800

‘”Cardiff Protocol” required pest mortality for Dendroctonus frontalis

Finally multipy by 3 to bring it to 95% level of confidence (Couey & Chew 1986)

1800 x 3 = 5400

Based on the Cardif protocol calculation we require experimental test size (no surivors) for the Step 3 of only 5,400 larvae. This is far less then 93,613 (probit nine) ‘”Cardiff Protocol” for pest with high rate infestation (nematodes, fungi…)

Infestation ration x Volume of trade MICL

MICL=Maximum infested commodity limit. It considders the number of infested commodity units (e.g. blocks) required to establish a population in a new area)

The required information for each high rate pest will neet to be collated and the test size calculated

Discussion points

§ Gathering and agreeing on data to establish sample size for different groups. § Pre calculated numbers for several examples from each of 7 pest groups mentioned in table 1 of step 1 § Should researchers calculate these? § Who will help and justify calculations? § Will TPPT or IPPC be available to help/advise in the process? § Sample size for fungi, nematodes? § The importance of individual’s variability and representativeness (age, origin…) Thank You Extra slides

Update from the IPPC Secretariat Standard Setting Group

Artur Shamilov (IPPC Secretariat) IFQRG-11, 28 October-2 November 2013 Qingdao, China

1

Agenda Item 8.1 Update on IPPC standards

2013 Member consultation

Draft ISPMs: · Management of phytosanitary risks in the international movement of wood (2006-029), Priority 1 · Preliminary draft: Minimizing pest movement by sea containers (2008-001), Priority 1 · Movement of growing media in association with plants for planting in international trade (2005-004), Priority 1 · Phytosanitary procedures for fruit fly (Tephritidae) management (2005-010), Priority 2 · Amendments to ISPM 5 (Glossary of phytosanitary terms) (1994- 001)

2013 Member consultation

Draft diagnostic protocols: · Draft Annex to ISPM 27:2006 – Potato spindle tuber viroid (2006- 022), Priority 1 · Draft Annex to ISPM 27:2006 – Xanthomonas citri subsp. citri (2004- 011), Priority 1

Draft phytosanitary treatment: Irradiation for Dysmicoccus Neobrevipes Beardsley, Planococcus Lilacinus (Cockerell) and Planococcus Minor (Maskell) (Hemiptera: Pseudococcidae) (2012-011), Priority 1

2013 Member consultation The following draft specifications have been submitted to member consultation: · Wood products and handicrafts made from raw wood (2008-008) · Revision of ISPM 4:1995 Requirements for the establishment of pest free areas (2009-002) · Revision of ISPM 8:1998 Determination of pest status in an area (2009-005)

Substantial Concerns Commenting Period for draft ISPMs

· Appendix to ISPM 12 on Electronic certification, information on standard XML schemes and exchange mechanisms (2006-003), Priority 1 · Establishment of fruit fly quarantine areas within a pest free area in the event of an outbreak (for inclusion as Annex 1 of ISPM 26) (2009-007), Priority 3 · Determination of host status of fruits and vegetables to fruit fly (Tephritidae) infestation (2006-031), Priority 1

2013 IPPC Secretariat Calls

• Call for topics • Call for experts (TPG, TPPT, EWGs) • Call for authors for diagnostic protocols • Call for participants in ECCT

Technical Panels • The Technical Panel for the Glossary of phytosanitary terms (TPG) met in Rome, Italy from 4 to 7 February 2013 • The Technical Panel on Diagnostic Protocols (TPDP) met in Paris, France from 24 to 28 June 2013. • The Technical Panel on Phytosanitary Treatments (TPPT) met in Fukuoka, Japan from 8 to 12 July 2013. • The Technical Panel on Forest Quarantine (TPFQ) met in Belem, Brazil from 10 to 14 June 2013. • Technical Panel on Pest Free Areas and Systems Approaches for Fruit Flies (TPFF) works virtually

Agenda item 8.2 The Commission on Phytosanitary Measures (CPM)

Status of ISPM 15 Implementation Two-part strategy to move forward was presented: • The first part of the strategy would cover five years in which efforts would be focused on registering the symbol in countries with FAO taking the lead. It suggested that allocating USD 70,000 per year from regular program funds for five years would be an investment while pursuing a longer term approach for maintaining and protecting the symbol into the future. •The second part of the strategy was aimed at maintaining the protection of the symbol and it was agreed to consider longer term options and approaches into the future.

Scientific session - review of phytosanitary security based on a Probit-9 treatment standard

The IPPC invited Experts: • Mr Robert Griffin • Mr Adnan Uzunovic

Relationship between ISPMs and standards created by other organizations

CPM-8 (2013) agreed that ISO standards are not mandatory for implementation of ISPMs and that in the phytosanitary area; ISPMs take precedence over ISO standards and encouraged contracting parties to take this into account.

Agenda item 8.2.1 Adopted Standards for Phytosanitary Measures (ISPMs)

CPM-8 (2013) adopted:

· Annex 4 (Pest risk analysis for plants as quarantine pests) and core text-consequential changes to ISPM 11:2004 (2005-001), noting that the title of ISPM 11 changed to Pest risk analysis for plants as quarantine pests and that the year of adoption of ISPM 11 changed to 2013. · Annex 1 (Approved treatments associated with wood packaging material) to ISPM 15:2009 (Regulation of wood packaging material in international trade) (2006-011), and consequential revision of Annex 2 (The mark and its application) to ISPM 15:2009 · The following ISPMs in the Russian language version: ISPMs 1 to 4, 6, 8 to 11, and 13 to 34, including Diagnostic Protocol 1 and Phytosanitary Treatments 1 to 14

Agenda item 8.3 TPPT

Topics for TPPT

• Irradiation treatments (2006-014) • Wood packaging material treatments (2006- 015) • Fruit fly treatments (2006-024) • Soil and growing media in association with plants treatments (2009-006) Review of wood packaging material treatments (2006-015)

The SC on request from the submitting NPPO, had removed following treatment from the List of topics for IPPC standards: Methyl isothiocyanate and sulfuryl fluoride (Ecotwin mixture) fumigation for Bursaphelenchus xylophilus, Coleoptera: Cerambycidae, and Coleoptera: Scolytinae of wood packaging material (2007-102)

Sulfuryl fluoride fumigation of wood packaging material (2007-101)

• In response to the 2010 TPPT request and 2010 TPPT meeting report, the submitter provided the results of research completed in 2011 and 2012 (Bonifacio et al. 2013). • It is plausible that the eggs stage of PWN (pinewood nematode, Bursaphelenchus xylophilus) are the most resistant life stage to sulfuryl fluoride fumigation, and would be controlled by extending the fumigation period to 48 hours as proposed. • The panel was concerned that the lower temperature treatment (15ºC to 20ºC) was not consistent with the life stage tolerance concept applied to the 20ºC treatment • It was agreed to consider this treatment at the next TPPT meeting, after consultation with PWN specialists, to complete the evaluation. Heat treatment of wood packaging material using dielectric heating (2007-114)

• This draft annex to ISPM 28:2007 was put on hold by the SC after member consultation in 2010 until member countries had gained some experience with use of dielectric heating. • It was noted that, once this Annex 1 to ISPM 15:2009 was adopted, the panel could again continue working on the heat treatment of wood packaging material using dielectric heating (2007-114). • SPMSince the I 28:2007 dielectric heating treatment schedule is for wood (not just wood packaging material), it is independent of ISPM 15:2009, and, therefore, the evaluation and approval process should proceed independently of the TPFQ, under the work plan of the TPPT. • The TPPT agreed to change the title to Heat treatment of wood using dielectric heating (2007-114). continue

• Since the Annex 1 to ISPM 15:2009 and the draft treatment schedule had been developed, new research has indicated that some of the limitations of the treatment stated in ISPM 15:2009 Annex 1 (e.g. limit the wood dimensions to 20 cm) may not be necessary or appropriate for a treatment schedule developed for ISPM 28:2007. • The TPFQ, at its meeting in June, suggested that the TPPT consider any discrepancy that might exist between the proposed ISPM 28:2007 DH treatment schedule and ISPM 15:2009 Annex 1. • It was a concern that additional research regarding radio waves would greatly broaden the application technology and the existing guidelines lacks such useful information. • The panel hold off evaluating the treatment based on the pending publication.

Update of Working TPPT criteria for treatment evaluation · saving voucher specimens during trials · placing the burden on the submitter to provide complete and accurate submission information · replicates with different load factor may be removed when calculated · age of colony · pest source · recording measurements and providing data on emergence for each replication · providing temperature data · including most tolerant life stage testing · accepting hot water dipping for most tolerant life stages · accepting artificial inoculation

Thank you! Scientific session: Eighth Session of the CPM Probit 9 as statistical method of establishing dose response

Adnan Uzunovic

Location: CPM 8, FAO, Rome, Itally

Date: April 11, 2013 Invited experts: § Robert L. Griffin – Security based on Probit 9 treatment standard • Report available: “Review of phytosanitary security based on probit 9 treatment standard” § Adnan Uzunovic – Evaluation criteria for phytosanitary treatments of wood packaging: alternative approach to probit nine

Introduction

§ 2013 IFQRG-11-12; 2013 IFQRG-11-14, 2013 IFQRG-11-17

§ TPPT requested in 2007 that TPFQ develops evaluation criteria for new treatments for ISPM-15, include list of test pests and required efficacy (“probit 9” considered as it has been arbitrary used as a standard in fruit flies quarantine research)

§ TPFQ asked International Forestry Quarantine Research Group (IFQRG) comprising of scientists, regulators, the industry representatives for help

§ IFQRG worked on and discussed evaluation criteria over several years

Introduction

§ ISPM No 28 – Phytosanitary treatments for regulated pests, does not specify required efficacy

§ ISPM No 15 (mentioned other possible treatments but provided no specific guidelines on efficacy data)

Introduction

§ In 2010 a draft appendix “Submission of new treatments for inclusion in ISPM 15” contained evaluation criteria for new treatments and was released for countries consultation (2013 IFQRG-11-14) • Included extensive list of target pests (including quarantine pests) and proscribed Probit 9 as a required level of efficacy • Criticised as too complex and too stringent

§ IFQRG reviewed the SC comments and worked on alternative approach especially to address the list of pests, and probit 9

Key points

§ Alternative treatments for wood packaging are urgently needed

§ Currently used methods (HT schedule adopted based on efficacy data for PWN and its vectors; Methyl Bromide has historically been accepted for variety of pests) are successfully used but did not go through that rigorous process

§ Wording in ISPM15 2009 changed to” practically eliminate” to significantly reduce risk of introduction and spread. Zero risk=zero trade

Key points

§ Probit 9 requirement and extensive list of pest including a quarantinable pests (ALB, EAB) is serious impediment and prevents new treatment development and adoption

§ Initially there were many discussions, approaches, confusion, requests and disagreements but during the process of negotiation, education and increased understanding, IFQRG in principal agreed on alternative approach in Sept 2011. Two peer-reviewed papers published. Haack et al, 2011. Seeking alternatives to probit 9 when developing treatments for wood packaging materials under ISPM No. 15 EPPO bulletin 41:39-45

Schortemeyer et al 2011. Appropriateness of Probit-9 in the Development of Quarantine Treatments for Timber and Timber Commodities J. Econ. Entomology, 104(3):717-731)

Wood packaging is a specific commodity

§ Could harbor many and diverse pests that can vary between countries and WPM

§ WPM go around the world repeatedly

§ Treatments (measures) must target multiple pests instead of discrete species or genus

Insects Fungi Others –Bark- inhabiting beetles (6,000 Scolitinae) –Rust fungi Mistletoes –Wood-inhabiting beetles (20,000 Cerambicids) –Decay fungi Nematodes (e.g. PWN) – Adelgids and scales –Canker fungi Mites, –Termites, Carpenter ants –Bluestain fungi Viruses, – Wood-inhabiting moths –Black yeasts Bacteria –Bark-inhabiting moths –Vascular wilt fungi – Wood wasps –Oomycetes 8

How to test efficacy of a treatment?

• Set up experiment, chose specimens and expose them to a treatment

§ Which organisms (stages) and how many do you need to test to represent all possible pests around the world? § Do you test specimen alone or infested wood as one unit? § Can you use naturally infested wood or lab inoculated? § Lab based tests versus scaled up field tests? § What replication is needed; what is acceptable statistics? § How to address the worse case scenarios: e.g most difficult to treat substrate type, effect of moisture content, frozen wood, with/without bark? § What if some organisms survive the treatment, what is acceptable? A Question of numbers/replication

§ If you use 10 insects (or 10 infested log units) and manage to kill all of them using a particular treatment, does that make a 100 % efficacy and can can you submit such data?

“Efficacy” versus “Reliability”

§ Efficacy (common word): The ability to produce a desired amount of an effect.

§ Reliability (statistical): The effectiveness of the treatment at a given confidence intervals; expressed as decimal portion of 1.0

§ Probability that the same result will be obtained again and again with repetition.

§ It addresses natural tolerance variability within test pest. A sample needs to represent the whole population

Common statistical formula for sample size and statistical reliability

Sample size necessary to n reliability demonstrate a given reliability 1 0.050000000 at 95% confidence level : 5 0.549280272 n=log(0.05)/log(pr) 10 0.741134449 15 0.818963727 20 0.860891659 25 0.887071855 30 0.904966147 35 0.917968364 40 0.927842475 45 0.935595711 50 0.941844921 59 0.95049239 60 0.951297087 100 0.97048695 299 0.990030853 2995 0.999000256 93613 0.999968 Two approaches to test reliability

n reliability 1 0.050000000 • BRUTE FORCE EXPERIMENTS - Involves exposing n number of 5 0.549280272 10 0.741134449 units to the selected dose and no units should have any 15 0.818963727 survivors 20 0.860891659 25 0.887071855 – 0.95% effective n=59 30 0.904966147 35 0.917968364 – 0.99% effective n=299 40 0.927842475 – 0.999% effective n=2995 45 0.935595711 50 0.941844921 – 0.999968% (Probit 9) (95% confidence) n=93,613 59 0.95049239 60 0.951297087 100 0.97048695 • DEVELOP MORTALITY CURVES – Dose Response Models 299 0.990030853 2995 0.999000256 – Develop a mortality curve (probability of death as a function of 93613 0.999968 temperature/exposure time that could be confidently used to determine the efficacy of any choice of thermal death time. – Done through extrapolation, so you need to accurately model the curve to achieve minimal variability within data – controlled lab experiments needed

‘Brute force’ has been done with fruit flies

§ Easy to rear in the lab, easy to put in a jar or collect fruits that are heavily infested, to treat and get probit 9 reliability. But what about wood pests?

14 Not easy with wood pests

§ There are potentially large diversity of pests to test

§ Scarce availability of key quarantine pests (ALB), impossible to find 93,613 individuals, § Rearing takes 6 months to over year, only 50 eggs produced by one female in lifetime; cost of rearing one ALB in the lab is $50, 4.7 million to rear and treat 93,613 individuals, in quarantine facility § Lack of separate units in some pests (e.g. pathogens)

§ Results from modeling give overestimated values 15

What is a Reasonable Target for reliability (efficacy)?

The short answer:

Whatever the trading partners and their scientific advisors are comfortable with, should be based on pest-risk assessment.

Countries and their advisors do not necessarily agree on this (179 NPPOs)

Robert Griffin – presentation

§ The meaning of probit nine § Criteria for requiring probit 9 efficacy § Statistical considerations § Research considerations § Policy considerations

Robert Griffin – key points

§ Explained background based on his USDA and IPPC work § Probit 9 mentioned as standard for fruit flies (1939); no references to suggest that Probit 9 was actually used as a standard by the USDA. Quarantine treatments (e.g. fumigations) developed and adopted (60s) used much lower sample size and less rigorous testing then are currently required by APHIS. The methodology was almost identical to that used for developing pesticide treatments where mortalities of 95% to 99% deemed adequate. § In 70s computers allowed Probit analysis possible to do § In last three decades the requirements for new treatments became so stringent requiring 3-5 years of research § Many publications questioned legitimacy of probit 9

Robert Griffin – key points § Criteria for requiring probit 9 efficacy (high risk situations) • The host is suitable • The infestation rate is high • The host is easily infested • Distribution of pest is highly clumped § Probit 9 is attractive as it provides a conservative level of security but does not provide consistency in all situations. § May result in substantial overkill, does not link to consistent policies; likely to violate the principle of “least restrictive measure” § Survivors are important. There is biological and other variables beyond this to consider likelihood of establishment. Criteria often not considered in light of PRA

Robert Griffin – key points § The use of probit nine as a standard for phytosanitary security is not based on any specific scientific data § Statistical problems when using and calculating probit § Probit treatment levels should be tailored for the specific commodity and pest of interest. § Allow provisional approval: When treatment is necessary to allow trade and when stopping the movement may cause major economic hardship it should be possible to use the best scientific evidence available to provide a treatment for a short term period. Later as new information and research progresses the treatment could be adjusted § Phytosanitary security can be achieved through several different methods: treatment, pest free areas, system approaches, combination of treatments…. § Probit nine should not be excersized as a standards in all quarantine situations

What then shall we do?

How to develop an acceptable approach and trust it enough to allow fast development and adoption of new treatments and support safe trade?

Quality systems used by structural engineers – Phase 1, 2 and 3

§ Used successfully for centuries to adopt a new component/structure (e.g. house, airplane, nuclear power plant, space shuttle….)

Phase 1

§ A committee of professional engineers identifies the most critical types of failures.

§ Proponents of new components asked to submit a engineering test results based on small lab testing

Phase 2

§ New component with promising small scale test results is required to be tested in SCALED –UP EXPERIMENT to demonstrate higher reliability.

• The scale depends on intended application and consequences of failure. At this stage testing can be cost prohibitive or impossible (e.g. new space shuttle component-no way to replicate in-situ conditions)

§ However the engineers and the engineering committee are willing to come up with a test and accept risk and engineering process and progress is not stopped just because there are risks

§ Successful large scale test results lead to PROVISIONAL APPROVAL of the component

Phase 3

§ Structures that suffer failure are subject to POST FAILURE EXAMINATION to determine the cause

§ If it is found that the new component was the cause-then it is BACK TO DRAWING BOARD.

§ The component is restricted for use in any more structures that could encounter that type of failure in service until the proponents demonstrate that can be used safely, Re-design the component, or redesign the structure

IFQRG’s had similar 3-Step approach

§ Step 1:Small lab scale test-estimate the lethal dose of the most tolerant pest of quarantine importance that is found in wood packaging

§ Step 2:Larger lab test- replicate experiments at the estimated lethal dose for the most tolerant pest to demonstrate higher reliability

§ Step 3: Simulated field test- confirm under simulated operational condition and provide statistical efficacy

Approved treatment will be scrutinized on ongoing base in subsequent real-life experience, and either kept, modified or withdrawn (e.g. discussions on HT tolerance of EAB)

Step 1 (lab): Estimating Lethal Dose of Most Tolerant Life Stage

§ Estimate treatment dose at which nearly all tested individuals will die using small sample size (5-10 experimental units each that can contain one or more target pests)

§ Use smallest wood samples as test units to ensure uniform dose delivery

§ How to address huge variety of possible pest and quarantine pest?

Step 1 (adjusted at IFQRG 2011) § Pre-screening for tolerance (low replication). Test one available species from 7 representative pest groups: • a reference-easy-to-rear insect (e.g. from Sitophilus, Oryzaephillus, Trogoderma or Ambrosia beetle genera), • Scolytinae (bark beetles) • Bostrychidae (horned powder post beetles) • Buprestidae (metallic wood boring beetles) • Cerambycide (large wood borers) • Pine wood nematode, • Decay fungus from Heterobasidion genus,

(2013 IFQRG-11-17) Other pests from the original list were dropped, after justifications were discussed (e.g. relevance to wood products and significantly reduced risk of pathway via wood packaging materials (e.g. Anobiidae, Lepidoptera: (Cossoid-Sessoid-Tortricoid assemblages, Siricidae, Fusarium circinatum, tree killing Phytophthora spp., deep penetrating blue stain fungi, canker fungi/chestnut blight, root rot fungi)

Step 2 (lab)

§ Replicated experiments (with no survivors) at the estimated lethal dose using the most tolerant pests determined during step 1. n reliability 1 0.050000000 § 5 0.549280272 Minimum sample size of 60 experimental units, which 10 0.741134449 15 0.818963727 achieves 0.95 statistical reliability at the 95% confidence 20 0.860891659 25 0.887071855 level. 30 0.904966147 35 0.917968364 40 0.927842475 45 0.935595711 § If possible test one or two doses above or below the 50 0.941844921 59 0.95049239 estimated lethal dose 60 0.951297087 100 0.97048695 299 0.990030853 § If there are survivors , increase dosage until no survivors 2995 0.999000256 93613 0.999968

Note: Scientifically and statistically this gives better confidence in the results then treating 93,613 individuals in a single unit (pseudoreplication, as in work with fruit flies). Although it “meets” probit nine requirement (killing 93,613 flies) this is really a reliability of only 0.05 (sample size =1) and not reliability of 0.999968

Step 3 - Confirmatory Study Under Simulated Operational Conditions § Using the most tolerant pests under simulated operational conditions using wood samples similar in size to wood packaging material and infested to levels that reflect field conditions. Sample size suggested to be 60 (0.95) to 300 (0.99). § Pre test pest load can be estimated and used to calculate reliability figures following the treatment. When a sufficient amount of a pest is available (e.g. PWN) then the Probit 9 level will be easy to achieve and report but for more scarce pests the Probit level will be much less.

“The Cardiff Protocol” IFQRG meeting in 2012 in Cardiff

§ Concept presented at IUFRO-allien species meeting in Japan and at IFQRG meeting in Cardiff by Mike Ormsby

“The Cardiff Protocol” IFQRG meeting in 2012 in Cardiff

The ‘Cardiff Protocol’ estimates a treatment efficacy target that reflects the biology of the pest, the pest relationship with wood packaging, and the trading patterns of wood packaging internationally. Concluding remarks

§ Raising bar too will preclude new treatments to emerge § Reliability levels need to be agreed and understood among those proscribing and accepting evaluation criteria for new tests § Trading countries need to be willing to accept proposed test protocol by a forum of knowledgeable individuals to manage risk. Forums such IFQRG provides a hub to secure an international agreement § Understanding of pathway analysis is important in order to comfortably accept agreed reliability IFQRG, TPFQ and TPPT are very close to agreeing on the final procedures

§ Proscribed efficacy tests will be possible to do without bearing huge cost

§ It will be scientifically sound and agreed among international group of scientists and regulators

§ It will give confidence that treatments adopted and its guidelines will “Significantly reduce the risk of introduction and spread of pests”

§ Following its adoption and adoption of new treatments it will allow necessary adjustment in real life situations

§ Treatments are just one measure as part of integrated approaches for safer and least restricted trade

Thank You Edited by: Thomas Schröder

Julius Kühn-Institute (JKI) Federal Research Centre for Cultivated Plants Institute for National and International Plant Health

Pine Wilt Disease Conference 2013 15th to 18th Oct. 2013 Braunschweig / Germany

Scientifi c Conference IUFRO unit 7.02.10 and FP7 EU-Research Project REPHRAME

- Abstracts -

Berichte aus dem Julius Kühn-Institut

Julius Kühn-Institut Bundesforschungsinstitut für Kulturpfl anzen

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Herausgeber / Editor Julius Kühn-Institut, Bundesforschungsinstitut für Kulturpfl anzen, Braunschweig, Deutschland Julius Kühn Institute, Federal Research Centre for Cultivated Plants, Braunschweig, Germany

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ISSN 1866-590X DOI 10.5073/berjki.2013.169.000

© Julius Kühn-Institut, Bundesforschungsinstitut für Kulturpfl anzen, 2013 Das Werk ist urheberrechtlich geschützt. Die dadurch begründeten Rechte, insbesondere die der Übersendung, des Nachdrucks, des Vortrages, der Entnahme von Abbildungen, der Funksendung, der Wiedergabe auf fotomechanischem oder ähnlichem Wege und der Speicherung in Datenverarbeitungsanlagen, bleiben, auch bei nur auszugsweiser Verwertung, vorbehalten. ©Julius Kühn-Institute, Federal Research Centre for Cultivated Plants, 2013 Copyrighted material. All rights reserved, especially the rights for conveyance, reprint, lecture, quotation of fi gures, radio transmission, photomechanical or similar reproduction and data storage, also for extracts.

IUFRO / REPHRAME International Conference on Pine Wilt Disease 2013

Worldwide the pine wood nematode (PWN), Bursaphelenchus xylophilus, is one of the severest quarantine pests mainly in coniferous stands. In the concerned countries severe damage was caused by the nematode induced pine wilt disease (PWD). Mainly the international trade of wood and wooden products led to an introduction of the pine wood nematode from its habitat in Northern America to Asia (Japan, China, Korea, Taiwan) and Europe (Portugal, Spain). Since then efforts were made in the infested areas to eradicate t he n ematode. N ot onl y i n t he i nfested c ountries but also i n m any other countries ne w r esearch a pproaches a nd c onception pl ans w ere pur sued i n t he pr evious years to manage the pine wilt disease. Since the last IUFRO Symposium in Nanjing/China in 2009, several expert groups in the whole world - among other things - worked intensively on the following topics: • Impact on the international trade as well as economic consequences in the infested areas including corresponding modeling of outbreak scenarios and pathways, • Pathway analysis a nd modeling/predicting of pine w ilt e xpression a cross e co- climatic zones taking account of latency , • Biology of Bursaphelenchus x ylophilus and ot her Bursaphelenchus species including their interaction with bacteria and fungi and their impact on host trees, • Diagnostic m ethods a imed t o a fast and r eliable de termination of P WN i n pur e culture and in plant tissue as well as in laboratory and under field conditions, • Examinations on the tree physiology and resistance characteristics of host trees, • PWN a nd ve ctor a ssociation, ve ctor di spersal c apacity, strategies f or ve ctor control, • Behavior and population dynamics in infested trees, • Non-vector transmission and treatment options for wood and wood products, • Management strategies for PWD. All r esearch approaches c ontribute t o e nhance procedures on t he e radication a nd t he management of the PWN resp. the PWD and thus to minimize the economical and the ecological impact on concerned forests. The aim of the symposium is to bundle the actual research progress and the management of the pine wood nematode and its vector beetles and to enhance the scientific exchange and thus to present the research results to a broad interested group of scientists, disease managers and decision makers. This symposium, organized by the Julius Kühn-Institut (JKI), Braunschwig, Germany, is a joint action of the International Union of Forest Research Organizations (IUFRO) unit 7.02.10 Pine Wilt Disease (PWD) and the group of the EU-research project REPHRAME „Development of improved methods for detection, control and eradication of pine wood nematode“ i n cooperation with the D eutsche P hytomedizinische G esellschaft - German Scientific Society for Plant Protection and Plant Health (DPG).

Braunschweig, Oktober 2013 Thomas Schröder

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SCIENTIFIC COMMITTEE

Thomas Schröder, Julius Kühn-Institut, Braunschweig, Germany

Christer Magnusson, Bioforsk, As, Norway

Isabel Leal, Pacific Forest Center, Victoria, Canada

Hugh Evans, Forest Research Wales, Aberystwyth, UK, REPHRAME, United Kingdom

Boguang Zhao, Nanjing Forestry University, Nanjing, China

ORGANIZING COMMITTEE

Thomas Schröder, Julius Kühn-Institut, Braunschweig, Germany

Falko Feldmann, Deutsche Phytomedizinische Gesellschaft e.V. (DPG), Braunschweig, Germany

Elke Vogt-Arndt, Julius Kühn-Institut, Braunschweig, Germany

Andrea Hopf, Julius Kühn-Institut, Braunschweig, Germany

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IUFRO / REPHRAME International Conference on Pine Wilt Disease 2013

Program

Tuesday, 15th Oct. 2013 Opening Ceremony

08:45 - 10:00 Registration; Forum of the Thünen-Institute

Welcome by the coordinator of IUFRO unit 10:00 - 10:15 T. Schröder 7.02.10 Pine Wilt Disease

Welcome and opening of the conference by the 10:15 - 10:35 G. Backhaus President of the Julius Kühn-Institut (JKI)

Welcome by the managing director of The 10:35 - 10:45 F. Feldmann German Scientific Society for Plant Protection and Plant Health

Welcome by the coordinator of the European 10:45 - 10:55 H. Evans Union Research Project "REPHRAME"

Introduction in Conference and organizing 10:55 - 11:05 T. Schröder issues

11:05 – 11:30 Group Photo and Coffee Break

Session 1: Insect Vector

Chairperson: Thomas Schröder

Sampling strategies for Monochamus sutor – a 11:30 – 11:50 Martin SCHROEDER potential vector of the pine wood nematode (103)

Assessing potential expansion of the Pine Wood Julien HARAN, nematode (Bursaphelenchus xylophilus) from the 11:50 – 12:10 Alain ROQUES, Géraldine ROUX spatial genetic structure of the vector (Monochamus galloprovincialis) (107)

Gonzalo ÁLVAREZ, Estela SÁNCHEZ, Effective traps for live trapping of PWN vectors 12:10 – 12:30 Iñaki ETXEBESTE, Diego GALLEGO, Monochamus spp. (134) Juan PAJARES

Effective attraction radius of pheromone traps for 12:30 – 12:50 Hervé JACTEL Monochamus galloprovincialis (172)

12:50 – 14:00 Lunch

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Continue Tuesday 15th Oct. 2013

Session 1: Insect Vector Chairperson: Juan Pajares

Iñaki ETXEBESTE, Gonzalo ÁLVAREZ, Monochamus galloprovincialis dispersal and the 14:00 – 14:20 Estela SÁNCHEZ, effective sampling area of operational traps (123) Juan PAJARES

Guillaume DAVID, Hervé JACTEL, Flight performances of Monochamus 14:20 – 14:40 Dominique PIOU, galloprovincialis, insect vector of the Pine Wood Pedro NAVES, nematode (149) Edmundo de SOUSA

Christelle ROBINET, Simulating the dispersal of Monochamus Guillaume DAVID, galloprovinciallis based on its flight mill 14:40 – 15:00 Dominique PIOU, Alain ROQUES, performance and testing several management Hervé JACTEL scenarios (110)

H. MAS, R. HERNÁNDEZ, Dispersal behavior and long distance flight 15:00 – 15:20 M. VILLAROYA, capacity of Monochamus galloprovincialis G. SÀNCHEZ (Olivier 1795) (170) et. al

Fabio CHINELLATO, Mauro SIMONATO, Smart-traps combined with molecular on-site Andrea BATTISTI, 15:20 – 15:40 Massimo FACCOLI, detection to monitor Monochamus spp. and Scott HARDWICK, associated pine wood nematode (152) Max SUCKLING

15:40 – 16:00 Coffee Break

Session 1: Insect Vector Chairperson: Hervé Jactel

E. Sanchez HUSILLOS, Physiological development of Monochamus Iñaki Etxebeste, 16:00 – 16:20 G. Alvarez BAZ, galloprovincialis immature adults through shoot Juan PAJARES feeding (133)

Celia K BOONE, Jean-C. GRÉGOIRE, Detection of exotic Monochamus spp. in Belgium - 16:20 – 16:40 Nick BERKVENS, Hans CASTEELS, testing the tools in the areas of origin (130) Nicole VIAENE

Preliminary results of potential vector species of Mehmet DAYI, 16:40 – 17:00 Süleyman AKBULUT Bursaphelenchus spp. (Nematoda:Parasitaphelenchidae) in Turkey (125)

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Continue Tuesday 15th Oct. 2013

Session 1: Insect Vector

Chairperson: Hervé Jactel

17:00 – 17:20 Final Discussion Insect Vector

17:30 Bus transfer to hotel

Guided Tour Braunschweig

19:00 Start from Tourist Information Address: Vor der Burg 1 (in front of the cathedral) Distance: 10 min from “Best Western Hotel”; 1 min from “Deutsches Haus"

Wednesday 16th October 2013 Field trip National Park Harz

07:30 Departure Hotel by bus

Field Trip National Park Harz Sabine MANÉ1 Christian LUX1 2 • Silvicultural concept in the National Park 09:00 Michael HABERMANN Melanie KIRCHHÖFER2 • Forest Protection • Game Management

12:00 – 13:30 Lunch at „Bavaria Alm“

1 Sabine MANÉ Continue Field Trip Christian LUX1 13:30 – 16:00 Michael HABERMANN • “Strolling-through-the-forest”-path! 2 Melanie KIRCHHÖFER . • continental raised bog

16:00 – 17:00 Bus transfer to city of Goslar

17:00 – 18:30 Guided tour in the world heritage site city of Goslar

18:30 – 22:00 Dinner in the Kennel of Goslar 1 Mangament Nationalpark Harz; http://www.nationalpark-harz.de/en/ 2 North-West-German Forest Research Center, Department Forest Protection (http://www.nw-fva.de/)

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Thursday 17th Oct. 2013 Session 2: Systematic and diagnostics Chairperson: Géraldine Roux

Validation of a real time PCR assay for the Anne-Marie CHAPPE, detection of Bursaphelenchus xylophilus in 09:00 – 09:20 Geraldine ANTHOINE targeted matrices in the framework of national survey (114)

Renske LANDEWEERT, Paul MOOIJMAN, Molecular detection of B. xylophilus in complex 09:20 -09:40 Winfried MULDER, Sven VAN DEN ELSEN, DNA backgrounds (112) Johannes HELDER,

Pine-Wood nematode assay and development of Ye, WEIMIN, real-time PCR for species identification in North 09:40 – 10:00 Robin M. GIBLIN-DAVIS Carolina department of agriculture & consumer services (159)

Isabel LEAL, Eric ALLEN, Jennifer ANEMA, Development of a reverse transcription loop- Brett FOORD, mediated isothermal amplification (RT-LAMP) 10:00 – 10:20 Caralyn REISLE, method to detect living pinewood nematode, Adnan UZUNOVIC, Bursaphelenchus xylophilus, in wood (100) Aniko VARGA, Delano JAMES

EUPHRESCO project - Bursaphelenchus 10:20 – 10:40 Geraldine ANTHOINE xylophilus, early detection methods (115)

10:40 – 11:00 Coffee Break

Session 3: PWN in international trade, pathways and phytosanitary treatments

Chairperson: Hugh Evans

Simone PROSPERO, Occurrence and distribution of Bursaphelenchus 11:00 – 11:20 Janina POLOMSKI, Daniel RIGLING species in Switzerland (119)

Luís FONSECA, José SANTOS, Hartmut NESTLER, Coniferous bark hot steam treatment for the 11:20 – 11:40 Joaquim VERDASCA, Rui OLIVEIRA, elimination of the pinewood nematode (146) Isabel ABRANTES, Clara SERRA

Andrea HOPF, Non vector spread of Bursaphlenchus xylophilus 11:40 – 12:00 Thomas SCHRÖDER via wood chips (155)

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Continue Thursday 17th Oct. 2013

Session 3: PWN in international trade, pathways and phytosanitary treatments

Chairperson: Hugh Evans

L. BONIFÁCIO, Complementary studies to validate the proposed M. L. INÁCIO, fumigation schedules of sulfuryl fluoride for 12:00 – 12:20 E. SOUSA, inclusion in ISPM No. 15 for the eradication of S. BUCKLEY pine wood nematode (Bursaphelenchus xylophilus) E. M. THOMS from wood packaging material (135)

Thomas SCHRÖDER, Efficacy of kiln drying as phytosanitary treatment 12:20 – 12:40 Johannes WELLING, C. Aukamp-TIMMRECK against wood borne nematodes (156)

12:40 – 13:40 Lunch

Session 4: PWN interactions with bacteria

Chairperson: Manuel Mota

O.A. KULINICH, E.N.ARBUZOVA, U.Sh. MAGOMEDOV, Recent Research on Pine Wilt Disease in Russia 13:40 – 14:00 N.I.KOZYREVA, E.S. MAZURIN, (116) M.S. KOLYCHIKHINA, A.Yu RYSS.

Jiajin TAN， Inoculation Effects of Pinus thunbergii with Hongye Qu， 14:00 – 14:20 Bursaphelenchus xylophilus and Two Bacterium ， Dejun HAO Strains of Bacillus firmus (141) Fengmao CHEN

Cláudia S. L. VICENTE, Yoriko IKUYO, Bursaphelenchus xylophilus and associated 14:20 – 14:40 Manuel MOTA, bacteria under oxidative stress conditions (104) Koichi HASEGAWA

Paula V. MORAIS, Diogo Neves PROENÇA, Gabriel PAIVA, Diversity and in vitro nematicidal activity of 14:40 – 15:00 Romeu FRANCISCO, Paula VERISSIMO, bacteria associated to pinewood nematode (161) Luís FONSECA Isabel M.O. ABRANTES

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Continue Thursday 17th Oct. 2013

Session 4: PWN interactions with bacteria

Chairperson: Manuel Mota

Cláudia S. L. VICENTE, F. X. NASCIMENTO, Margarida ESPADA, Natural bacterial communities associated with the 15:00 – 15:20 Pedro BARBOSA, pine sawyer beetle Monochamus galloprovincialis Koichi HASEGAWA, (105) Manuel MOTA, Solange OLIVEIRA

15:20 – 15:40 Final Discussion

15:40 – 16:00 Coffee break

Postersession and side events

Conference banquet

16:00 – 19:00 Poster session

16:00 – 17:00 Thomas SCHRÖDER IUFRO unit PWD management meeting

17:00 – 18:00 Geraldine ANTHOINE EUPHRESCO meeting

19:00 – 21:00 Conference banquet

21:10 Departure by bus to hotel

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Friday 18th Oct. 2013 Session 5: PWD management and contingency planning

Chairperson: Edmundo Sousa

Katsunori NAKAMURA, Noritoshi MAEHARA, Takuya AIKAWA, Yu ICHIHARA, Hajime KOSAKA, A research project to develop strategic action plan 09:00 – 09:20 Etsuko KAGAYA, in the pine-wilt-disease unaffected area in northern Hisahsi SUGITA, Japan (118) Takashi MASAKI, Koki KIMURA, Jun-ichi KON, Tomonori KANEKO

Xu FUYUAN, Study on the techniques of sustainable control of 09:20 - 09:40 Zheng HUAYING, pine wood nematode disease (Bursaphelenchus Xu MING xylophilus) (136)

P. NAVES, New Strategies for pine wilt disease (PWD) 09:40 – 10:00 M. VIEIRA, management in Portugal: preventive methods to E. SOUSA reduce the spread of the disease to new areas (158)

Session 6: PWN Biology, Population dynamics, Epidemiology, Modelling

Chairperson: Edmundo Sousa & Christer Magnusson

Inter-specific competition of Bursaphelenchus Marek TOMALAK, 10:00 – 10:20 Anna FILIPIAK xylophilus with native populations of B. mucronatus in pine (121)

Han ZHENGMIN, Interspecific hybridization between Ben AILING, 10:20 – 10:40 Guo YE, Bursaphelenchus xylophilus and Bursaphelenchus Cao DONGXIA mucronatus (102)

10:40 – 11:00 Coffee break

Tetsuro KATO, Akira KANEKO, Phenotypic and genotypic traits of recombinant 11:00 – 11:20 Ryoji SHINYA, inbred lines of pine wood nematode, Kazuyoshi FUTAI, Bursaphelenchus xylophilus (113) Yuko TAKEUCHI

Lihua ZHU, Pathogenicity, reproduction and survival of axenic 11:20 – 11:40 Lin HUANG, Bursaphelenchus xylophilus (128) Jianren YE

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Continue Friday 18th Oct. 2013

Session 6: PWN Biology, Population dynamics, Epidemiology, Modeling

Chairperson: Christer Magnusson

Rui-he GAO, Influence of pine wood nematode invasion on 11:40 – 12:00 Juan SHI, typical Masson pine ecosystem in Three Gorges You-qing LUO Reservoir Region of China (151)

Using an evapo-transpiration model to predict the Hannah GRUFFUDD, current and future range and severity of pine wilt 12:00 – 12:20 Hugh EVANS, Tom JENKINS disease caused by pine wood nematode, Bursaphelenchus xylophilus in Europe (124)

Ruifen HUANG, Cold-tolerance and adaption of Pine wood 12:20 – 12:40 Juan SHI, Youqing LUO nematode in China (153)

12:40 – 13:40 Lunch

Chairperson: Gernot Hoch & Thomas Schröder

Zheng HUAYING, A comparative proteomics analysis on resistant 13:40 – 14:00 Xu MING, provenance of Pinus massoniana inoculated with Xu FUYUAN Bursaphelenchus xylophilus (137)

Francisco LEISICO, Paula GOMES, Comparative transcriptomics to understand the Miguel PINHEIRO, 14:00 – 14:20 molecular basis of Bursaphelenchus xylophilus Luís FONSECA, Isabel ABRANTES, pathogenicity (154) Conceição EGAS

Lin HUANG, Minqi TIAN, The Function of Major Sperm Proteins (MSPs) in Xiuwen QIU, 14:20 – 14:40 reproduction of pine wood nematode, Yi ZHANG, Xiaoqin WU, Bursaphelenchus xylophilus (129) Jianren YE

14:40 – 15:00 Coffee break

Claudia SL VICENTE, Exploring the relation between virulence and Yoriko IKUYO, 15:00 – 15:20 oxidative stress response of Bursaphelenchus Manuel MOTA, Koichi HASEGAWA xylophilus and Bursaphelenchus mucronatus (157)

Final Discussion and conclusions 15:20 – 17:00 End of conference

17:00 Departure to hotel by bus

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Oral Presentations

Session 1:

Insect Vector

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Schroeder M, Sampling strategies for Monochamus sutor – a potential vector of the pine wood nematode. In: Schröder, T. (ed.), Pine Wilt Disease Conference 2013, pp. 12, Braunschweig, ISSN: 1866-590X

Sampling strategies for Monochamus sutor – a potential vector of the pine wood nematode

Schroeder M Swedish University of Agricultural Sciences, Department of Ecology, Box 7044, S – 750 07 Uppsala, Sweden Email: [email protected]

According to the European Commission Decision 2012/535 all EU countries are required to conduct detection and delimitation (if detected) surveys for the pine wood nematode (PWN). In more northern areas, where development of pine wilt disease is unlikely, the two most efficient survey methods for PWN is to sample dispersing Monochamus beetles by traps (baited with attractants) and wood substrates colonized by Monochamus (easily identified due t o characteristic l arval ga lleries). The ai m of t his pr oject was t o develop sampling strategies, based on these two approaches, for Monochamus sutor in Sweden. In addition, an identification key based on male genitalia was developed for the European Monochamus species ( Wallin et al . 2013). T rap c atches of M. s utor were com pared among three different stand types: fresh clear-cuts, old clear-cuts and pine stands. Catches were of the same magnitude on fresh and old clear-cuts, but 5 – 6 times higher on clear- cuts compared with in pine stands. Thus, clear-cuts should be used for trap locations if possible. Logging residues on c lear-cuts and t hinnings c onstitute t he major br eeding substrate for M. s utor in S weden. Thus, s urveys of average d ensities of colonized t ops and branches on clear-cuts and thinnings provide a method to plan the number of samples required for achieving a certain statistical significance at which PWN can be stated to be absent. The identification key provides a reliable method for discerning between M. sutor and M. galloprovincialis. In addition, no di fference between male genitalia of M. sartor and M. urussovi were found. Thus, we regard M. urussovi as a subspecies of M. sartor.

REFERENCES Wallin H; Schroeder M; Kvamme T (2013). A review of the European species of Monochamus Dejean, 1821 (Coleoptera, Cerambycidae) – with a description of the genitalia characters. Norwegian Journal of Entomology 60, 11-38.

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Haran J, Roques A, Roux G, Assessing potential expansion of the Pine Wood nematode (Bursaphelenchus xylophilus)from the spatial genetic structure of the vector (Monochamus galloprovincialis). In: Schröder, T. (ed.), Pine Wilt Disease Conference 2013, pp. 13, Braunschweig, ISSN: 1866-590X

Assessing potential expansion of the Pine Wood Nematode (Bursaphelenchus xylophilus) from the spatial genetic structure of the vector (Monochamus galloprovincialis)

Haran J, Roques A, Roux G INRA Orléans, URZF- 2163 Avenue de la Pomme de Pin 45160 ARDON, France Email: [email protected]

Monochamus galloprovincialis (Coleoptera, Cerambycidae) is the main factor involved in the natural s pread of t he P ine W ood Nematode (PWN), a s erious pest for pi ne forests. Since its introduction in Portugal, the PWN has rapidly expanded its range to a large part of t he c ountry a nd w ill pr obably k eep e xpanding t o t he r est of E urope. E stimation of dispersal abilities of M. galloprovincialis over various landscapes and across mountains is a ke y poi nt t o p redict t he i nvasion of t he P WN i n E urope, a nd w ill he lp t o s et u p management for this pest. Microsatellites a re hi ghly va riable genetic m arkers. Their pol ymorphism provides information on genetic structure of organisms at a broad scale (phylogeography), but also at loc al s cale ( migration of indi viduals). We de veloped a s et of 12 microsatellites loc i specific to M. galloprovincialis. First assessments conducted on s ix populations along a European North-South transect reveal a reduction of the genetic diversity northward, with a ma ximum of a llelic r ichness in Spain. This s eems c onsistent w ith post g lacial recolonization of M editerranean species as sociated with Pines t rees. We ha ve al so observed a significant differentiation between some Iberian populations. Our pe rspectives a re t o use t he m icrosatellites m arkers ( i) t o construct t he E uropean phylogeography of M. galloprovincialis, (ii) to estimate the effect of the Pyrenees and the landscape structure on dispersal abilities of this species, and finally, (iii) to look at the effect of PWN invasion on its genetic structure in Portugal.

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Alvarez, G.; Etxebeste, I.; Sánchez, E.; Gallego, D.; Pajares J.A. Effective traps for live trapping of PWN vectors Monochamus spp. In: Schröder, T. (ed.), Pine Wilt Disease Conference 2013, pp. 14-16, Braunschweig, ISSN: 1866-590X

Effective traps for live trapping of PWN vectors Monochamus spp.

Alvarez G, Etxebeste I; Sánchez E, Gallego D, Pajares JA

ABSTRACT

Pheromone-kairomone b lend r ecently de veloped ha s s hown a hi gh pow er t o a ttract Monochamus gal loprovincialis beetles to tr aps. T his lur e ma y b e us ed for e ffective monitoring and even mass trapping of pine wood nematode vectors. However, for this to be true, an effective trap for trapping these species is required. Monochamus beetles are very agile and easily escape from most of traps if they are not killed. However, keeping the captured beetles alive is a ke y feature for monitoring nematode loads in the beetles and for obtaining other valuable information on the beetle population. Several designs of traps were tested to determine their suitability in maximizing M. galloprovincialis caught beetles and in keeping them alive. These included different modifications on conventional multiple-funnel t raps a nd c ross-vane t raps, as o ne-way funnels, s lippery coated i nner surfaces, extended collector cups or wire screen bottoms. Experiments were carried out under di fferent f ield conditions i n S pain us ing randomized bl ock de signs. T raps were suspended f rom pol es 2m he ight a nd ba ited with t he ka iromone/pheromone bl end. Catches w ere s ampled w eekly dur ing M. gal loprovincialis flying pe riod. S ome experiments w ere r eplicated i n ot her c ountries. R esults of s everal ex perimental years showed that a slippery coat on t he trap doubled catches, whereas the slippery coat and a tight wire screen on the collection cups bottom avoided escape of trapped live adults and increased their survival. These results have led to commercial development of two models of efficient traps.

INTRODUCTION

Insects of t he genus Monochamus (Coleoptera: C erambycidae) i nclude s pecies t hat colonize recently dead or heavily damaged conifers by factors as drought and attack by other or ganisms. A lthough t hey h ave be en us ually considered a s s econdary p ests, t heir reported role as vectos of the pine wood nematode Bursaphelenchus xylophilus, has given them a main role in pest management. Since the introduction of the nematode in Europe, M. galloprovincialis has been confirmed as its only vector. A great deal of knowledge has been achieved in recent years on its chemical ecology leading to a pheromone-kairomone blend r ecently de veloped that has s hown a hi gh pow er t o attract Monochamus

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galloprovincialis beetles to traps. This lure may be used for effective monitoring and even mass trapping of pine wood nematode vectors. However, for this to be true, an effective trap for trapping these species is required. Monochamus beetles are very agile and easily escape from most of traps if they are not killed. However, keeping the captured beetles alive is a key feature for monitoring nematode loads in the beetles and for obtaining other valuable information on the beetle population.

MATERIAL AND METHODS

From 2010 to 2012, several designs of traps were tested in field assays to determine their suitability in maximizing M. galloprovincialis caught beetles and in keeping them alive. Assay in 2010 included multiple-funnel traps for 5 different treatments: 1) collecting cup provided with a small piece of DDVP (dimethyl 2,2-dichlorovinyl phosphate) insecticide strip ( Econex S . L., M urcia, S pain) t o ki ll t rapped be etles; 2) collecting c up w ithout modifications or insecticide; 3) collecting cup internally coated with slippery substance (Teflon); 4) bot h m ultiple-funnel t rap a nd c ollecting c up coated w ith T eflon a nd 5) collecting cup sheathed in a polystyrene cover and tight wire screen bottom with the aim of reduce internal temperature and improve insects survival. The assay carried out in 2011 compared the ef ficacy o f cat ching i nsects and mantaining t hem al ive of t hree di fferent designs of traps: conventional multiple-funnel trap, cross-vane trap and polytrap (a type of c ross-vane t rap). Finally i n 2012, effectiveness of ne w mul tiple-funnel t raps w ere compared with us ed m ultiple-funnel a nd c ross-vane t raps w ith the aim of eva luate t he suitability of t hese di fferent m odels a nd t he pos sible l oss of e fficacy of T eflon-coated traps t wo years af ter ha ving be en coated. All t hese e xperiments were carried out us ing randomized block designs. Traps were suspended from poles 2m height and baited with the ka iromone/pheromone bl end. C atches were s ampled w eekly during M. galloprovincialis flying period.

DISCUSSION

Comparison be tween conventional mul tiple-funnel t rap a nd t his s ame pr ovided w ith insecticide showed that exist a proportion of caught insects that escape from collecting cup. T he num ber of i nsects obt ained w ith collecting cup i nternally coated w as not different from t hat us ing conventional c ollecting c up, but c ollecting cup covered w ith polystyrene got better results, due to better conditions inside the collecting cup, reducing temperature and keeping insects quiet on t he background grid. However, the number of catches obt ained by both m ultiple-funnel t rap and c ollecting c up s lippery coated w as three times more than that by conventional multiple-funnel trap, suggesting that the key is not only to avoid escape of insects but maximize the number of attracted insects that fall into t he collecting c up. T he pr oportion of l iving i nsects w as not di fferent be tween treatments w ithout i nsecticide. C omparison be tween di fferent m odel of t raps i n 2011

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showed no differences be tween cr oss-vane t rap a nd m ultiple f unnel t rap, a lthough polytrap s howed clearly i nferior ( Figure 1) . F inally, 2012 e xperiments s howed no differences between old traps and new ones and confirmed no differences between cross- vane traps and multiple-funnel traps. Anyway, Teflón-coated traps have been not tested more than two years after being coated and long durability of Teflon coating under field conditions i s unknow n. The l oss of effectiveness dur ing t ime w ould ha ve t he doubl e effect of not only escape of insects but a decrease in the number of catches. These results have l ed to the com mercial de velopment of b oth t eflon-coated traps, ECONEX MULTIFUNNEL-12® and CROSSTRAP® (Econex S.L., Murcia, Spain), that are efficient enough to be recommended for monitoring and for mass trapping of M. galloprovincialis They are currently being used in Spain in the PWN eradication programs and live adult trapping is allowing the sampling for pine wood nematodes carried in the caught beetles within these programs.

Fig. 1) Different type of traps tested in 2011 field assay.

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Jactel H, Effective attraction radius of pheromone traps for Monochmaus galloprovincialis. In: Schröder, T. (ed.), Pine Wilt Disease Conference 2013, pp. 17, Braunschweig, ISSN: 1866-590X

Effective attraction radius of pheromone traps for Monochamus galloprovincialis

Jactel H INRA, UMR BIOGECO, Bordeaux, France [email protected]

Pheromone t rapping ha s be en considered a s a m eans t o m onitor p opulations of Monochamus galloprovincialis, the insect vector of the pine wood nematode (PWN) in southern Europe, since the identification and the synthesis of its aggregation pheromone, which s hows e xcellent bi ological a ctivity i n t he f ield. W ith t he de velopment of ve ry effective interception traps, pheromone trapping is also envisaged for PWN management through m ass-trapping. H owever little is know n about the pr actical de ployment o f pheromone t raps i n t he f ield, p articularly about opt imal de nsity of t raps i n t rapping networks. The concept o f Effective Attraction R adius (EAR, B yers et al. 1989), which represents the radius of a passive "sticky" sphere that would intercept the same number of flying ins ects a s the a ttractant, is o f pa rticular interest f or opt imizing t he de nsity of trapping ne twork. In t heory, c apture e fficiency i s e xpected t o reach an opt imum f or distance DOpt between traps equ al t o twice E AR. We de veloped a "quick and cheap" experimental m ethod t o e stimate E AR i n t he f ield, us ing p airs of ph eromone t raps a t increasing distance from each other. Plotting the mean capture of M. galloprovincialis per trap against the distance between two traps we obtained an asymptotic curve that levelled out at DOpt making it possible to estimate EAR.

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Etxebeste I, Álvarez G, Sánchez-Husillos E & Pajares J, Monochamus galloprovincialis dispersal and the effective sampling area of operational traps. In: Schröder, T. (ed.), Pine Wilt Disease Conference 2013, pp. 18-19, Braunschweig, ISSN: 1866-590X

Monochamus galloprovincialis dispersal and effective sampling area of operational traps

Etxebeste I, Álvarez G, Sánchez-Husillos E & Pajares J Sustainable Forest Management Research Institute, University of Valladolid – INIA 34004 Palencia, Spain Email: [email protected]

ABSTRACT

The a vailability of a n operational t rapping s ystem f or Monochamus g alloprovincialis (Olivier, Coleoptera: Cerambycidae), the European vector of the Pine Wood Nematode, Bursaphelenchus x ylophilus (Steiner & B uhrer), ha s a llowed the imp lementation of a reference t ool for m onitoring t he p resence and spread of t he P ine W ilt Disease as well developing m anagement m ethods ba sed on m ass-trapping of t he ve ctor. F urthermore, catch data gathered in traps can be potentially used in the study of M. galloprovincialis dispersal a nd t he de termination of a bsolute population de nsities. H owever, s uch applications require of t he parameterization of dispersal models and detection functions that are in turn used to estimate values such as diffusion rates or the effective sampling area of traps (Turchin, 1998). Two mark-release-recapture ex periments car ried out i n 2009 a nd 2010 a t two different pine stands in central-Spain were designed to describe such values. During the first trial, 174 unfed M. galloprovincialis imagoes were marked and released from the centre of a setup c onsisting of 28 t raps di stributed a long f our c oncentric r ings a t 5 0,100, 250 a nd 500m located on a natural Pinus pinaster stand (Figure 1A). A second study used a grid- based

Figure 1. Experimental de signs f ollowed dur ing Monochamus gal loprovincialis dispersal studies in 2009 (a) and 2010 (b).

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Figure 2. Proportions of recaptured Monochamus galloprovincialis [P(r)] at traps located a di verse d istances f rom t he r elease poi nt ( r) du ring f ield t rials i n 2009 a nd 2010 in Western Spain. The black line representes fitted linear regression for the estimated proportion of recaptures in relation to the common logarithm of the distance. design with 56 t raps that covered distances up t o 761 m , to track the movement of 353 individuals (Figure 1B). Up t o 35 and 29% of released be etles w ere r ecaptured dur ing 2009 and 2010 f ield experiments respectively. Values per each replicate during each experimental period were then modelled a nd t he r elationship be tween c apture pr obability a nd di stance t o r elease point c ould be s tudied unde r di fferent theoretical a nd empirical regression m odels (Turchin, 1998). On t he one h and, m odeling of m ean r ecaptures pe r di stance unde r a s imple di ffusion model f or t ime i ntegrated da ta s howed t hat a bout 50% of i ndividuals do not di spers beyond 40 m. On the other hand, empirical fitting of proportion of recaptures allowed to set the effective sampling area for traps at ca. 0.77ha, while the seasonal sampling range at which t he estimated proportion of t he catch would be z ero could be s et t o 570.44m (Figure 2; Ostrand & Anderbrant, 2003). The variations in the estimates when using other modelling approaches are discussed, as well a s the impl ications of t he study of M. gal loprovincialis dispersal on m anagement decissions and the mmonitoring of its population density. Keywords. Dispersal, Mark Release R ecapture, Population D ensity, B aited T raps, Sampling Range

REFERENCES Ostrand F; Anderbrant O (2003). From where are insects recruited? A new model to interpret catches of attractive traps. Agricultural and Forest Entomology 5, 163- 171. Turchin P (1998). Quantitative analysis of movement : measuring and modeling population redistribution in animals and plants. Sinauer Associates Inc: Sunderland, Massachusetts.

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David G, Hervé J, Piou D, Naves P, Sousa E, Flight performances of Monochamus galloprovincialis, insect vector of the Pine Wood nematode, In: Schröder, T. (ed.), Pine Wilt Disease Conference 2013, pp. 20, Braunschweig, ISSN: 1866-590X

Flight performances of Monochamus galloprovincialis, insect vector of the Pine Wood nematode

David G, Hervé J, Piou D, Naves P, Sousa E [email protected]

The P ine W ood N ematode ( PWN, Bursaphelenchus x ylophilus) is the mos t impor tant threat to pine pl antation forests in Europe s ince its int roduction from A sia a nd its establishment in pine f orests of P ortugal. It is currently s preading t owards S pain and France. The natural transmission from tree to tree is done by insect vectors of the genus Monochamus. H owever until now l ittle w as kno wn a bout t he f light c apacity of t hese vectors. To be tter eva luate t heir di spersal capacity under s tandardized c onditions, w e developed a utomatically r ecording f light m ills. W e f ound t hat M. ga lloprovincialis exhibits a wide array of flight capacities, with few beetles not flying at all, while others are abl e t o fly ov er several t ens of km when considering the cumulated flights through their life span. We also investigated the effect of several life traits on flight performances such as beetles’ gender, age, maturation status, body size and also the impact of nematode load. We will discuss the implication of these findings for the development of PWN risk management methods such as precautionary clearcuts.

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Robinet C, David G, Piou D, Roques A, Jactel H, Simulating the dispersal of Monochamus galloprovinciallis based on its flight mill performance and testing several management scenarios, In: Schröder, T. (ed.), Pine Wilt Disease Conference 2013, pp. 21, Braunschweig, ISSN: 1866-590X

Simulating the dispersal of Monochamus galloprovinciallis based on its flight mill performance and testing several management scenarios

Robinet C(1), David G(2), Piou D(2,3), Roques A(1), Jactel H(2) (1)INRA, UR633 Zoologie Forestière, F-45075 Orléans, France (2)INRA, UMR BIOGECO, F-33612 Cestas, France (3)DSF-MAAF, F-33612 Cestas, France Email: [email protected]

The potential spread of t he pi ne w ood ne matode, Bursaphelenchus x ylophilus, and its associated pine wilt disease are strongly associated with the flight capacity of the insect vector. Although some data were available from congeneric species in North America and Asia, t he f light pe rformances of t he E uropean vector, Monochamus ga lloprovinciallis, were largely unknown. They were assessed with flight mill experiments and used to fit a dispersal kernel. A stochastic individual-based model was then developed to simulate the trajectory o f a dults ove r one s eason. W ith t his di spersal m odel a nd a t ransmission function of the nematode, the area where the vector can transmit the pine wood nematode can be determined and several forest management scenarios to contain its spread can be tested. Although this dispersal model is still at an exploratory stage, and beetle’s dispersal capacity mi ght be ov erestimated with flight mill, this a pproach contributes to a be tter understanding of vector’s dispersal pattern and can be used to test the effects of several management scenarios. This dispersal model is a first step towards the development of a refined spread model of the nematode and the disease at larger spatial and temporal scales in Europe.

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Mas et al., Dispersal behavior and long distance flight capacity of Monochamus galloprovincialis (Olivier 1795), In: Schröder, T. (ed.), Pine Wilt Disease Conference 2013, pp. 22, Braunschweig, ISSN: 1866-590X

Dispersal behavior and long distance flight capacity of Monochamus galloprovincialis (Olivier 1795)

Mas H1, Hernández R2, Villaroya M2, Sánchez G3, Pérez-Laorga E1, González E 4, Ortiz A.2, Lencina J L5, Rovira J 6, Marco M 6, Pérez V2, Gil M2, Sánchez-García F J5, Bordón P1, Pastor C1, Biel M J1, Montagud L1, Gallego D5 1 Laboratori de Sanitat Forestal, Servei d’Ordenació i Gestió Forestal, Conselleria d’Infraestructures, Territori i Medi Ambient, Generalitat Valenciana. 2 Laboratorio de Sanidad Forestal en Mora de Rubielos, Servicio Provincial de Agricultura, Ganadería y Medio Ambiente, Departamento de Agr. Gan. y M.A., Gobierno de Aragón. 3 Servicio de Sanidad Forestal y Equilibrios Biológicos, Ministerio de Agricultura, Alimentación y Medio Ambiente. 4 SILCO S.L. 5 Departamento de Zoología y Antropología Física, Universidad de Murcia. 6 Conselleria d’Infraestructures, Territori i Medi Ambient, Generalitat Valenciana Email: [email protected]

ABSTRACT

It i s i ntended to evaluate t he capacity of l ong distance na tural s pread of Monochamus galloprovincialis (Olivier 1795) , ve ctor of t he pa thogen P ine W ood N ematode, Bursaphelenchus xylophilus (Steiner et Buhrer, 1934). Eight trials of trapping-marking-releasing-recapturing adult individuals have been held in different regions along the east of the Iberian Peninsula during 2009-2011. To catch the adults have been used three different types of traps: Lindgren funnel, Crosstrap and the Torre-LSF pr ototype, a ll of t hem ba ited w ith t he s pecific ka iromonal-pheromonal attractive of M. galloprovincialis. The traps were sited in the sampling areas at different distances from a central point where the insects have been released. Results show that released mature adults of M. galloprovincialis are able to achieve long- distance s pread, reaching m aximum values of 13600 m and 22100 m . These di stances, together with the high percentages of captures recorded above 3000 m (close to 2%) seem to show the low efficiency of quarantine belts (areas cleaned of possible host species) that are been used in the eradicating programmes and in the theoretical isolation of pest free areas.

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Chinellato F et al., Smart-traps combined with molecular on-site detection to monitor Monochamus spp. and associated pine wood nematode. In: Schröder, T. (ed.), Pine Wilt Disease Conference 2013, pp. 23-25, Braunschweig, ISSN: 1866-590X

Smart-traps combined with molecular on-site detection to monitor Monochamus spp. and associated pine wood nematode

Chinellato F.1, Simonato M.1, Battisti A.1, Faccoli M.1, Hardwick S.2, Suckling D.M.3 1 Department of Agronomy, Food, Natural resources, Animals and Environment, University of Padova, V.le dell’Università, 16 – 35020 Legnaro (PD), Italy. 2 AgResearch Limited, Lincoln, New Zealand. 3 The New Zealand Institute for Plant and Food Research Limited, Lincoln, New Zealand. Email: [email protected]

INTRODUCTION

The pi ne s awyer be etles M onochamus s pp. (Coleoptera C erambycidae) ar e t he m ain vectors of t he P ine W ood Nematode (PWN), Bursaphelenchus x ylophilus, t he a gent of pine w ilt disease i n v arious pa rts of t he w orld ( Mamiya 1983 ). In E urope, M . galloprovincialis (Olivier) gained importance as a vector after the finding of the PWN in Portugal i n 1999 ( Sousa e t al. 2001) . A n effective m onitoring m ethod b ased on e arly detection of bot h ve ctor i nsects a nd a ssociated ne matode i s ne eded i n or der t o a dopt appropriate phytosanitary measures (Rassati et al. 2012 and 2013).

MATERIALS AND METHODS

The pr esent s tudy s hows a n ew t echnology for t he r emote d etection of beetle catch combined with on-site molecular detection of both vector and nematode identity. A multi- funnel trap, baited with either specific or generic blend, and equipped with a specifically modified s ecurity camera (BioCam, M i5 S ecurity, Auckland, New Zealand), composed by a w ide-angle l ens, 1 or 3 MegaPixel s ensor, rechargeable ba ttery p ack and internal modem f or G eneral P acket R adio S ervice ( GPRS) c onnection w as us ed. T he i nterval between images taken by the camera can be programmed and saved in a Secure Digital (SD) m emory card. The images can be stored in the same SD card and simultaneously sent to a safe repository accessible through the web, from which they are downloadable. On the same repository it is possible to check the level of battery charge of each camera and the GPRS coverage as well. When a target beetle is detected, an on-site visit is planned, during which a fragment of the thorax is analyzed using a Loop Mediated Isothermal Amplification (LAMP) portable

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device (Genie II, Optigene, UK) to identify the trapped species of Monochamus spp. and to detect the PWN possibly vectored by the beetles. Currently, primers were deloped for the endemic M. galloprovincialis and M. sutor, and for the exotic M. alternatus and M. carolinensis. For the beetles identified as Monochamus, presence of PWN is also tested with the same device using slighty modified LAMP primers from Kikuchi et al. (2009), specific for the nematode ITS1 region. A positive control for the nematode is included in the test. The technique allows amplifying target DNA i n a f ew m inutes visualizing the results immediately.

RESULTS

Images obtained by cameras are definitely adequate to visually recognize large longhorn beetles s uch as Monochamus spp.. All the main morphological traits of the species are detectable ( Fig. 1) . T he s ystem w orks a lso und er s ub-optimal lig ht c onditions. LAMP primers de signed t o amplify t he ITS2 r egion of M. gal loprovincialis, M . sutor, M . alternatus and M. carolinensis show to be specific, giving a positive result only for these species a fter 10 -15 minutes a fter the te st s tart ( Fig. 2) . On t he ot her ha nd, no pos itive insects for PWN have been detected until now.

CONCLUSIONS

Both technologies are designed for quick and cheap on-site analyses, and can be used by non-expert staff with a short training. In case of positive samples, they must be taken to the l aboratory a nd analyzed m ore a ccurately w ith s tandard pr otocols f or of ficial confirmation.

REFERENCES Kikuchi T; Aikawa T; Oeda Y, Karim N, Kanzaki N (2009) A rapid and precise diagnostic method for detecting the pinewood nematode Bursaphelenchus xylophilus by Loop-Mediated Isothermal Amplification. Phytopathology 99, 1365-1369. Mamiya Y (1983). Pathology of the pine wilt desease caused by Bursaphelenchus xylophilus. Annual Review of Phytopathology 21, 201-220. Rassati D; Petrucco Toffolo E; Battisti A, Faccoli M (2012). Monitoring of the pine sawyer beetle Monochamus galloprovincialis by pheromone traps in Italy. Phytoparasitica 40, 329-336. Rassati D; Petrucco Toffolo E; Roques A; Battisti A; Faccoli M (2013) Trapping wood boring beetles in Italian ports: a pilot study. Journal of Pest Science DOI 10.1007/s10340-013-0499 Sousa E; Bravo MA; Pires J; Naves P; Penas AC; Bonifacio L; Mota MM (2001). Bursaphelenchus xylophilus (Nematoda: Aphelenchoididae) associated with Monochamus galloprovincialis (Coleoptera: Cerambycidae) in Portugal. Nematology 3, 89-91.

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Figure 1 Picture taken by 3M P t rap c amera. O ne i ndividual of Monochamus spp. i s clearly recognizable on t he l eft, together w ith s everal i ndividuals o f t he longhorn b eetle Acanthocinus gr iseus, o ne o f the w estern s eed b ug Leptoglossus occidentalis (above) and several small bark beetles (right).

Figure 2. Amplification profile f or t he L AMP assay car ried on Monochamus spp. a nd other cerambycid beetles. Positive curves are obtained in 10-15 minutes only for M. galloprovincialis, M. sutor, M. alternatus and M. carolinensis.

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Sánchez-Husillos E, Etxebeste I Álvarez-Baz G, Pajares J, Physiological development of Monochamus galloprovincialis immature adults through shoot feeding. In: Schröder, T. (ed.), Pine Wilt Disease Conference 2013, pp. 26-27, Braunschweig, ISSN: 1866-590X

Physiological development of Monochamus galloprovincialis immature adults through shoot feeding

Sánchez-Husillos E, Etxebeste Larrañaga I, Álvarez-Baz G, Pajares Alonso J Sustainable Forest Management Research Institute Sostenible, University of Valladolid- CIFOR-INIA. Avd. Madrid s/n, 34071 Palencia, Spain. Email: [email protected]

ABSTRACT

Adult shoot feeding is a vital feature of Monochamus life history playing a key role in the pine w ilt di sease inf ection cycle. A fter e mergence, adults mus t f eed on the phl oem o f healthy pine s hoots t horough a ll t heir l ife f or nut rition t o s ex m aturate, di sperse, reproduce a nd s urvive. F urthermore, i t i s know n t o be r equired b y s ome s pecies a s a necessary s tep for w ing m uscle d evelopment. D evelopment of s everal ph ysiological parameters a nd s exual maturation w as s tudied on f reshly e merged a dults of t he pi ne nematode vector M. galloprovincialis (Olivier, Col.: Cerambycidae) during one month of shoot feeding with the aim to gain knowledege on its dispersial behaviour. Gonadic development was assessed on adults of both sexes (n=24) at 0, 4, 8, 14, 18 and> 18 day age intervals. Genitalia dissections served to track morphological changes during gonadic maturation as well as the presence of eggs or oocytes. Sex maturation could be established to occur after feeding for 8-14 days in males and 16 days in females. Fat bodies of fed adults of both sexes (n=90) at previously mentioned age intevals were extracted as described by Anderbrandt (1988). M. galloprovincialis adults emerged with lipid content averaging 12.28% of their dry weight. This amount was decreasing during the f irst 4 da ys dow n t o 9.7% , a nd t hen i ncreasing t o pe ak 14 da ys a fter f eeding a t 13.68% of their dry weight (Figure 1).

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Figure 1:Histogram of mean fat content Figure 2:Histogram of mean toracic (%DW) muscle fraction (%DW)

Up to 60 insects used in lipid extraction had their wing segement extracted and macerated in K OH. T he dr y weight di fference be tween l ipid-less and diggested s egments, i. e. without m uscle, allowed t he determination of an approximated m uscle c ontent. R esults varied f rom 26.6% of d ry weight i n males and 20% i n females after emergence, to a maximum of 39% in males and 36% in females after 14-18 days of shoot feeding. Steady weight gain was recorded in fed adults through the first 16 days of feeding then before stabilizing. C onversely, weight l oss of un fed a dults m irrored gain w eight of f ed a dults (Figure 2). Finnaly, 29 individuals were fed with Pinus pinaster twigs until weight stabilization and 10 unf ed i nsects w ere k ept unt il de ath oc curred. S urvival of unf ed a dults a veraged 1 2 days and for a maximum of 20 days. Weight loss at the time of death of these beetles was 38% of dry weight. These results show that freshly emerged, unfed, Monochamus adults have fat content and wing muscles enough to undertake sustained dispersal flight. Key Words: Bursaphelenchus xylophilus, pine wood nematode, gonads, fat content, wing muscles.

ACKNOWLEDGEMENTS

This study was funded by the E.U. through the project REPHRAME (KBBE.2010.1.4-09)

REFERENCES Anderbrant O (1988) Survival of parent and brood adult bark beetles, Ips typographus, in relation to size, lipid content and re-emergence or emergence day. Physiological Entomology 13: 121-129.

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Boone CK, Grégoire J-C, Berkvens N, Casteels H, Viaene N, Detection of exoctic Monochamus spp. in Belgium – testing the tools in the area of origin, In: Schröder, T. (ed.), Pine Wilt Disease Conference 2013, pp. 28, Braunschweig, ISSN: 1866-590X

Detection of exoctic Monochamus spp. in Belgium – testing the tools in the area of origin

Boone CK1, Grégoire J-C1, Berkvens N2, Casteels H2, Viaene N2 1 Université Libre de Bruxelles, LUBIES, Avenue FD Roosevelt 50, B-1050 Brussels,Belgium 2 ILVO, Plant – Crop Protection, Burg. van Gansberghelaan 96, B-9820 Merelbeke, Belgium Email: [email protected]

ABSTRACT

In t he European Union, Commission decision 2012-535-EC on emergency measures t o prevent t he s pread w ithin t he U nion of Bursaphelenchus x ylophilus specifies t hat "Member States shall annually conduct surveys for Bursaphelenchus x ylophilus [...] on susceptible plants, susceptible wood and bar k and on t he vector, and de termine whether there is any evidence of the presence of PWN in their territory in areas in which PWN was previously not known to occur". Bursaphelenchus xylophilus has not been detected in Belgium to date, however, potential native B. xylophilus vectors, Monochamus galloprovincialis and M. s artor, have be en detected infrequently. To effectively monitor for potential vectors, both native and exotic, it is necessary to test the efficacy of traps and lures in both this country and the countries of or igin of pot ential vectors. In 2013, Belgium i nitiated a t wo-fold e xperimental programme aiming to: 1) monitor native Monochamus species susceptible t o vector t he pinewood nematode; and 2) implement surveillance for exotic species that could enter the country via wood pa ckaging m aterial. European t raps ( Econex C rosstrap) and l ures (Galloprotect Pack), which have been successfully tested in different EU countries, were deployed i n va rious l ocations t hroughout Belgium, i ncluding t en poi nts a djacent t o companies i mporting goods i n w ood pa ckaging m aterial. In pa rallel, to va lidate t he capacity of these traps and lures to capture exotic Monochamus species, traps were sent to several l ocations i n N orth A merica know n t o h arbour species t hat v ector P WN. In the United States, traps and lures w ere s ent t o Arkansas t argeting M. c aroliniensis and M. titillator, and Utah targeting M. scutellatus and M. clamator. In Canada, they were sent to New Brunswick targeting M. mutator and M. notatus, and British Columbia targeting M. obtusus. Here we present the first results of this control experiment.

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Dayi M, Akbulut S, Preliminary results of potential vector species of Bursaphelenchus spp. (Nematoda:Parasitaphelenchidae) in Turkey , In: Schröder, T. (ed.), Pine Wilt Disease Conference 2013, pp. 29, Braunschweig, ISSN: 1866-590X

Preliminary results of potential vector species of Bursaphelenchus spp. (Nematoda:Parasitaphelenchidae) in Turkey

Mehmet DAYI and Süleyman AKBULUT Düzce University, Faculty of Forestry, Konuralp Campus 81620, Düzce/TURKEY

ABSTRACT

The de tection of Bursaphelenchus x ylophilus in 1999 i n E urope p rompted m any European countries to carry out surveys to determine B. xylophilus and its insect vectors, and to prevent the pine wilt disease. As a result of these surveys, many Bursaphelenchus species isolated and reported from stressed, dying or newly dead conifer trees. In Turkey, several Bursaphelenchus species were found to be associated with dead or wilted conifer trees, but no records w ere av ailable about i nsect ve ctors of t hese Bursaphelenchus species. For t his pu rpose, s everal s tudies h ave been s tarted i n conifer forests i n t he Aegean and the Marmara regions of Turkey. In these studies, five trap t rees, free from Bursaphelenchus species, were selected. These trees were cut and laid down in the same place to attract possible insect vector of Bursaphelenchus species reported from previous studies in the s ame r egions. The t rap trees w ere kept i n the f ield between March and September to obtain oviposition of potential vector species in 2012 a nd 2013. The t rap trees w ere checked periodically f or i nsect a nd ne matode pr esence. T he w ood c hip samples w ere t aken from each trap trees and controlled for t he pr esence of Bursaphelenchus s pp. In t he l ab. When the samples w ere pos itive f or pr esence of Bursaphelenchus species, several log samples were taken from the trap trees. These logs were ke pt unde r c onstant c onditions ( 25±0C, 60 -70 % R H) du ring t he d evelopment of insects. Orthotomicus e rosus and Ips s exdentatus emerged from B. sexdentati isolated Pinus brutia logs, Monochamus galloprovincialis emerged from B. mucronatus isolated P. brutia logs, O. erosus and Acanthocinus griseus emerged from B. vallesianus isolated P. br utia logs a nd Pityokteines cur videns and Rhagium i nquisitor emerged from B. hellenicus isolated Abies cilicica logs.

Key words: Vector species, Bursaphelenchus spp., Conifer

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Session 2:

Systematics and Diagnostics

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Anthoine G, Chappé AM, Validation of a real time PCR assay for the detection of Bursaphelenchus xylophilus in targeted matrices in the framework of national survey. In: Schröder, T. (ed.), Pine Wilt Disease Conference 2013, pp. 31-32, Braunschweig, ISSN: 1866- 590X

Validation of a real time PCR assay for the detection of Bursaphelenchus xylophilus in targeted matrices in the framework of national survey

Anthoine G, Chappé A-M, Laboratoire de la santé des végétaux, Unité de nématologie, Domaine de la Motte, BP35329, F35653 Le Rheu cedex, France. Email: [email protected], [email protected]

Surveys on Bursaphelenchus x ylophilus need detection methods t hat ar e r eliable, sensitive, t hat allow hi gh t hroughput analysis and can be applied on a ny m atrix tested, such as wood material or insect for a direct detection. Many molecular tests, especially real time PCR tests, were recently published but none of them was fully validated to fulfill all the previous requirements. From a selection of three real time PCR assays, François et al. (2007), Leal et al. (2007), Cao et al . (2005), a v alidation pr ocess w as de signed ba sed on EPPO r ecommendation PM7/98 ( EPPO, 2010) and a pplied f or t he evaluation of t he f ollowing pe rformance criteria: sensitivity, specificity, repeatability, reproducibility or robustness. The three real time PCR assays tested proved to be very sensitive as they all detect one individual of B. xylophilus, even if the reaction profile is clearly different from one assay to another probably due to the target gene. They also are repeatable and reproducible. The specificity of the three tests is different especially when analysing wood: some tests gave f alse pos itive r esults w ith routine w ood s amples. With some te sts, f alse pos itives were obt ained from no n target B ursaphelenchus s pecies, especially i n case of l arge amount of DNA, with Ct values that could lead to confusion with B. xylophilus. Based on its performance, the real-time PCR assay developed by François et al. (2007) was considered as the m ost adapted for our routine use. So, this test was coupled with universal primers and p robe as i nternal control, t argeting 18S gene which i s present i n plant and insects cells (Ioos et al., 2009). The performance of the B. xylophilus specific assay was not affected by the addition of this universal primer set, whatever the sample, wood or i nsect.This du plex r eal t ime P CR e nabled t he de tection of one s ingle B. xylophilus.

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Consequently, t he de tection s cheme applied f or French na tional s urvey i ncludes a f irst step of s creening us ing real t ime P CR as say f rom F rançois et al. (2007) for wood and insect s amples. If a ny p ositive r esult i s obt ained f or w ood, i t w ould b e c onfirmed b y morphological analysis and complementary molecular approach on a compulsory basis. For insect sample, any positive detection would lead to further investigations on site.

REFERENCES Cao A.X; Liu X.Z; Zhu S.F. and Lu B.S. (2005). Detection of the pinewood nematode, Bursaphelenchus xylophilus, using a real-time polymerase chain reaction assay. Phytopathology 95, 566–571. EPPO (2010) EPPO standards PM 7/98. EPPO Bulletin 43, 105–118. François C; Castagnone C; Boonham N; Tomlinson J; Lawson R; Hockland S; Quill J; Vieira P; Mota M; & Castagnone-Sereno P (2007). Satellite DNA as a target for TaqMan real-time PCR detection of the pinewood nematode, Bursaphelenchus xylophilus. Molecular Plant Pathology 8, 803–809. Ioos R; Fourrier C; Iancu G and Gordon TR (2009). Sensitive detection of fusarium circinatum in Pine seed by Combining an Enrichment Procedure with a real-time Polymerase chain reaction using a Dual-Labeled Probe Chemistry. Phytopathology 2009 May; 99(5) : 582-90. Leal I; Green M; Allen E; Humble L; Rott M (2007). Application of a real-time PCR method for the detection of pine wood nematode, Bursaphelenchus xylophilus, in wood samples from lodgepole pine. In: Nematology, 9, Number 3, 351-362.

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Landewert R et al., Molecular detection of B. xylophilus in complex DNA backgrounds, In: Schröder, T. (ed.), Pine Wilt Disease Conference 2013, pp. 33-34, Braunschweig, ISSN: 1866-590X

Molecular detection of B. xylophilus in complex DNA backgrounds

Renske Landeweert1, Paul Mooijman2, Winfried Mulder1, Sven van den Elsen2, Johannes 2 Helder 1 ClearDetections, Binnenhaven 5, 6709 PD Wageningen, The Netherlands, [email protected] 2 Wageningen University, Laboratory of Nematology, Droevendaalsesteeg 1, 6708 PB Wageningen, The Netherlands

Dozens of Bursaphelenchus species are associated with pine trees (Pinus spp.), most of them being harmless, as they feed exclusively on fungi associated with stressed or dying pine trees. Bursaphelenchus xylophilus is exceptional as it is a facultative, mostly lethal parasite of vi tal pi ne t rees. M orphological i dentification of Bursaphelenchus species i s predominantly based on spicule characters and depends on the availability of adult males. Invariably, microscopic identification of B. xylophilus in nematode suspensions is a time- consuming specialist job, and this largely limits sample throughput. To c ontrol t he s preading of B. x ylophilus through E urope, f ast a nd hi gh t hroughput detection tests are required. DNA-based screening assays allow for the sensitive screening of l arge num bers of n ematode s amples of a ny ki nd, a nd do not depend on t he developmental s tage of the ne matodes unde r i nvestigation. S everal di agnostic m ethods for molecular detection of B. xylophilus have been published. Some of them are designed against a f ramework with representatives f rom m ost Bursaphelenchus groups ( e.g. Burgermeister et a l. 2009) but a re l aborious a s t hey include e nzymatic a mplicon digestion f ollowed b y gel-based fragment ana lysis. On t he ot her ha nd, a r elatively fast satellite DNA-based TaqMan assay h as been developed with verified contrast against a limited number (n=10) of congeneric species (Francois et al. 2007). On the basis of a framework of ~ 2,800 full length nematode SSU rDNA sequences (Van Megen et al . 2009), w e ha ve de veloped a ne w m olecular a ssay f or t he qua ntitative detection of B. xylophilus. Using this framework, we identified unique DNA motifs that enable ‘ blind’ i dentification of B. x ylophilus in c omplex D NA ba ckgrounds. T his B. xylophilus specific t est was developed using 35 S SU rDNA sequences of B. xylophilus and 113 S SU rDNA sequences of 44 non -target Bursaphelenchus species (including 13 sequences of B. mucronates). SYBR Green-based detection assays (similar to Rybarczyk et al. 2012) allow for reliable and cost-effective molecular screening of large numbers of

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nematode s amples i n a ny ( inspection) l aboratory. R esults w ill be pr esented on t he validation of t his ne w t est a nd i nclude a c omparison w ith t he pe rformance o f t he B. xylophilus test (Francois et al. 2007) from the EPPO Standard PM 7/4 (3).

REFERENCES Burgermeister et al. (2009) ITS-RFLP analysis, an efficient tool for differentiation of Bursaphelenchus species. Nematology, 11(5), 649-668. Francois et al. (2007) Satellite DNA as a target for TaqMan real-time PCR detection of the pinewood nematode, Bursaphelenchus xylophilus. Molecular Plant Pathology 8, 803-809. Rybarczyk-Mydlowska et al. (2012) Small Subunit Ribosomal DNA-Based Phylogenetic Analysis of Foliar Nematodes (Aphelenchoides spp.) and Their Quantitative Detection in Complex DNA Backgrounds. Phytopathology 102, 1153-1160. Van Megen et al. (2009) A phylogenetic tree of nematodes based on about 1200 full- length small subunit ribosomal DNA sequences. Nematology 11(6), 927-950.

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Ye W, Giblin-Davis R M, Pinewood Nematode Assay and Development of Real-Time PCR for Species Identification in North Carolina Department of Agriculture & Consumer Services, In: Schröder, T. (ed.), Pine Wilt Disease Conference 2013, pp. 35-38, Braunschweig, ISSN: 1866-590X

Pinewood Nematode Assay and Development of Real-Time PCR for Species Identification in North Carolina Department of Agriculture & Consumer Services

Weimin Ye1; Robin M. Giblin-Davis2 1 Nematode Assay Section, Agronomic Division, North Carolina Department of Agriculture & Consumer Services, Raleigh, NC 27607, USA Email: [email protected] 2 Fort Lauderdale Research and Education Center, University of Florida/IFAS, 3205 College Avenue, Davie, Florida 33314, USA. Email: [email protected]

Bursaphelenchus xylophilus, the pinewood nematode (PWN), is the causal agent of pine wilt di sease, one of the mos t da maging e merging p est pr oblems t o f orests a round t he world. PWN was introduced in Japan at the beginning of the 20 th century ( Yano 1913) and later in mainland China (Cheng et al. 1983), Taiwan (Chang & Lu 1996) and Korea (Yi et al. 1989), causing massive mortality of native pine trees. PWN was first recorded in E urope ( Portugal) i n 1999 ( Mota et al . 1999); l ater on t he P ortuguese i sland o f Madeira, 900 km SW of the European continent in 2010 (Fonseca et al. 2012); and more recently in three locations in Spain close to the Portuguese border (Robertson et al. 2011). The international spread of PWN occurs mainly through the movement of infested logs, untreated w ood pr oducts a nd w ood-packaging m aterial. It is na tive to North America where it causes relatively minor damage to native conifers but is labeled an EPPO-A-2 pest and a quarantine nematode for many countries outside of the United States because of i ts pot ential f or d estruction t o t heir na tive c onifers. E xports of w ood l ogs and commodities i nvolving s oftwood pa ckaging materials now r equire a la b test f or th e presence/absence of this regulated nematode species. The A gronomic Division of the N.C. Department of A griculture & C onsumer S ervices operates a hi gh-throughput and publ icly operated nematode assay l ab. R ecently, due t o more strict regulations on PWN, a large number of pine-wood samples were submitted to our lab (Table 1). In fiscal year 2013, 3,934 pine-wood samples were analyzed and 233 reports were generated f or U SDA/APHIS/PPQ i n c onnection w ith t he i ssuing o f phytosanitary certificates f or e xported pi ne-wood l ogs t o C hina; t his w orkload represented a more than six-fold increase over the previous year. Although in the first two

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months of fiscal year 2014, NCDA&CS assayed 1,139 samples for pinewood nematode— accounting for 55% of the sample total to date, July and August are during our non-busy season in receiving samples. PWN prevalence in pine-wood samples was 0.82%, 0.89% and 4.48% for fiscal year 2012, f iscal year 2013 and the first two months of fiscal year 2014, r espectively. T hese r esults i ndicate t he l ow pr esence of P WN i n e xported pi ne- wood logs in the USA and the importance of regulatory measures and laboratory testing 0.82% 0.89% 4.76% Identification of t hese s pecies us ing t raditional m orphology requires a hi gh l evel of expertise and can be very time-consuming and inconclusive. However, rapid and accurate identification of PWN is required in order to comply with quarantine regulations and to prevent its movement between countries. Molecular diagnosis is potentially simple, rapid, sensitive and reliable and can be used with high precision to determine t he presence of PWN i n w ood. We cha racterized the D NA s equences of t he ribosomal D NA s mall subunit, l arge s ubunit D 2/D3, i nternal t ranscribed s pacer and m itochondrial D NA cytochrome ox idase s ubunit one of a large c ollection of A phelenchid s pecies. T his allowed the de velopment of a r eal-time-PCR m ethod b y either s implex ( Figure 1) or duplex ( Figure 2) f or r apid a nd a ccurate i dentification of P WN t argeting the IT S-1. A total of 97 ne matode populations were used to evaluate the specificity and sensitivity of this a ssay, i ncluding 45 populations of B. x ylophilus and 36 popul ations of 21 ot her species of Bursaphelenchus, which b elong t o t he abietinus, cocophilus, eggersi, fungivorus, hofmanni, kevini, leoni, sexdentati and xylophilus groups and one unassigned group f rom a t otal of 13 gr oups i n t he genus Bursaphelenchus; 15 populations of Aphelenchoides b esseyi, A. f ragariae, A. spp. a nd Aphelenchus av enae; a nd one population of m ixed ne matode s pecies from a soil s ample. T his a ssay pr oved t o b e specific to B. xylophilus only and was sensitive to a single nematode specimen regardless of t he l ife s tages pr esent. T his a pproach pr ovides t he r apid s pecies i dentification necessary to comply with the zero-tolerance export regulations.

REFERENCES Chang R J; Lu S S (1996). Investigation of the occurrence of pine wilt disease and its naturally infected hosts in Fushan Botanical Garden. Taiwan Journal of Forest Science 11, 201207. Cheng H; Lin M; Li W; Fang Z (1983). The occurrence of a pine wilting disease caused by a nematode found in Nanjing. Forest Pest and Disease 4, 15. Fonseca L; Cardoso J M S; Lopes A; Pestana M; Abreu F; Nunes N; Mota M; Abrantes I (2012). The pinewood nematode, Bursaphelenchus xylophilus, in Madeira Island. Helminthologia 49, 96-103. Mota M M; Braasch H; Bravo M A; Penas A C; Burgermeister W; Metge K; Sousa E (1999). First report of Bursaphelenchus xylophilus in Portugal and in Europe. Nematology 1, 727-734.

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Robertson L; Cobacho Arcos S; Escuer M; Santiago Merino R; Esparrago G; Abelleira A; Navas A (2011). Incidence of the pinewood nematode Bursaphelenchus xylophlius Steiner & Buhrer, 1934 (Nickle, 1970) in Spain. Nematology 13, 655-757. Yano M (1913). Investigations on the cause of pine mortality in Nagasaki prefecture. Sanrin-Koho 4, 1-14. Yi C; Byun B; Park J; Yang S; Chang K (1989). First finding of the pine wood nematode, Bursaphelenchus xylophilus (Steiner & Buhrer) Nickle and its insect vector in Korea. Research Reports of the Forestry Research Institute (Seoul) 38, 141-149.

Table 1. Pinewood nematode assay summary in NCDA&CS

FY2012 FY2013 FY2014 (7/1/11- (7/1/12- (7/1/13- 6/30/12) 6/30/13) 8/31/13) Total Total pine-wood samples 613 3934 1139 5686 Total pine-wood reports 31 234 80 344 Positive PWN samples 5 35 51 91 Positive PWN reports 3 16 16 35

Yearly nematode samples 34129 35012 2090 71231

Yearly nematode reports 4606 4744 279 9629 Pine-wood-sample percentage 1.80% 11.29% 54.50% 7.98% Pine-wood-report percentage 0.67% 4.97% 28.67% 3.57% PWN prevalence in pine-wood samples 0.82% 0.89% 4.48% 1.60%

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Figure 1. Example of a real-time-PCR result for testing sample 2013-33423 by Pinewood- nematode -specific and nematode-universal primer/probes.

Figure 2. Duplex r eal-time-PCR r esult. A. A pos itive r esult w ith two sigmoid FAM (Pinewood-nematode-specific) a nd HEX ( nematode-universal) curves in a mplification plot. B. A negative result with one sigmoid HEX curve, and nonincreased FAM curve.

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Leal I, et al., Development of a reverse transcription loop-mediated isothermal amplification (RT-LAMP) method to detect living pinewood nematode, Bursaphelenchus xylophilus, in wood. In: Schröder, T. (ed.), Pine Wilt Disease Conference 2013, pp. 39-40, Braunschweig, ISSN: 1866-590X

Development of a reverse transcription loop- mediated isothermal amplification (RT- LAMP) method to detect living pinewood nematode, Bursaphelenchus xylophilus, in wood.

Leal I1, Allen E1, Anema J1, Reisle C1, Foord B1, Uzunovic A2, Varga A3, James D3 1 Natural Resources Canada, Canadian Forst Service, 506 W. Burnside Rd. Victoria, BC V8Z 1M5, Canada 2 FPInnovations, Forintek Division, 2665 East Mall, Vancouver, BC V6T 1W5, Canada 3 Sidney Laboratory, Centre for Plant Health, Canadian Food and Inspection Agency, 8801 East Saanich Rd., BC V8L 1H3, Canada Email:[email protected]

Current molecular techniques for the detection of PWN rely on t he presence of genomic DNA and thus cannot differentiate between living and dead PWN. The detection of dead nematodes c ould l ead t o unne cessary t rade di sruption. W e ha ve de veloped a r everse transcription l oop-mediated isothermal a mplification (RT-LAMP) assay, which specifically identifies living PWN in wood by detecting the presence of expansin mRNA as a viability marker. This diagnostic method was found to be more sensitive, faster, more cost-effective, and allows for simpler visual detection as compared to PCR. We chose an expansin g ene, be cause i t ha d be en s equenced f or bot h P WN a nd i ts cl osest r elated species, B. mucronatus, (Kikuchi et al., 2009), and because it contains an intron, which is present onl y i n g enomic D NA. In order for a n RT-LAMP me thod to be a r eliable indicator of vi ability, it w as important to ascertain that onl y cDNA transcribed f rom mRNA was amplified, and to eliminate the possibility that any genomic DNA (gDNA) could be amplified. We designed 6 LAMP primers that recognize 8 distinct regions in the target sequence (Notomi et al., 2000). One of the primers was positioned at an exon-exon junction of the expansin gene, so that genomic DNA could not be amplified. When testing gDNA samples and cDNA from different Bursaphelenchus species, we found exclusive amplification of c DNA f rom P WN. Positive s amples w ere de tected with HNB (hydroxynaphthol blue) by a change of colour from violet to blue (Goto et al., 2009). The sensitivity of the R T-LAMP diagnostic to de tect l iving P WN w as hi gher t han t hat obtained by a conventional PCR diagnostic method, and similar to a real time RT-PCR

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assay. We m odified a n R NA e xtraction pr otocol ( Chomczynski a nd S acchi, 1986) i n order to improve extraction quality from wood. We have optimized the RT-LAMP assay not onl y on ne matodes from pure isolate cultures, but also on s amples directly isolated from 4 g of P WN-infected w ood. This as say w as us ed to test t he pr esence/absence o f living PWN in wood that had been heat treated according to ISPM 15 (FAO 2009). From the r esults obt ained, w e f ound t hat a ll heat-treated w ood s amples w ere f ree o f l iving PWN, a nd t hus t he he at t reatment applied t o t hese w ood s amples was an effective treatment to kill PWN. We will be using this method to verify the efficacy of other wood treatment such as sulfuryl fluoride and phosphine. This method will help resolve disputes over the detection of PWN by clarifying whether any PWN present in wood is alive or dead. It can also be used to evaluate the efficiency of wood treatment procedures.

REFERENCES Kikuchi T; Li H; Karim N; Kennedy M; Moens M; Jones T (2009). Identification of putative expansin-like genes from the pine wood nematode Bursaphalenchus xylophilus, and evolution of the expansin gene family within the Nematoda. Nematology 11(3), 355-364. Notomi T; Okayama H; Masubuchi H; Yonekawa T; Watanabe K; Amino N; Hase T (2000). Loop-mediated isothermal amplification of DNA. Nucleic acids research, (12) e63 Goto, M., Honda, E., Ogura, A., Nomoto, A., & Hanaki, K.-I. (2009). Colorimetric detection of loop-mediated isothermal amplification reaction by using hydroxy naphthol blue. BioTechniques, 46(3), 167–72. Chomczynski P; Sacchi N (1986). Single-Step Method of RNA Isolation by Acid Guanidinium Thiocyanate-Phenol-Chloroform Extraction. Analytical Biochemistry 162, 156-159. FAO International Standards for Phytosanitary Measures. 2009. Guidelines for regulating wood packaging material in international trade. Publication No. 15. Food and Agriculture Organization, Rome, Italy.

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Anthoine G, EUPHRESCO BURSA project: early detection methods of the pinewood nematode, In: Schröder, T. (ed.), Pine Wilt Disease Conference 2013, pp. 41, Braunschweig, ISSN: 1866-590X

EUPHRESCO BURSA project: early detection methods of the pinewood nematode

Anthoine G Laboratoire de la santé des végétaux, Unité de nématologie, Domaine de la Motte, BP35329, F35653 Le Rheu cedex, France. Email : [email protected]

EUPHRESCO is an EU-funded ERA-NET (Coordination Action) which aims to increase cooperation a nd c oordination of na tional ph ytosanitary (statutory pl ant health) r esearch programmes at the EU level through networking of research funding activities. EUPHRESCO-1 w as f unded b y t he E U 6t h F ramework P rogramme ( FP6) f rom 2006 - 2010. EUPHRESCO-2 i s f unded f rom t he E U 7t h F ramework P rogramme (FP7) f rom 2011 - 2013. It is composed of 31 partners in 22 countries with 12 European Observer countries and 2 i nternational O bservers. Its pa rtners are l eading o rganisations i nvolved w ith funding phytosanitary research in Europe. EUPHRESCO a ims t o continue a s a s elf-sustainable l ong-term ne twork of E uropean phytosanitary research funders after 2013. The Eranet Euphresco initiates scientfic project on regulated or emerging pest. In this regard, a specific project is about to start on Bursaphelenchus xylophilus and early methods of detection. The project would aim at validating published detection methods that could be used on many matrices and that could avoid nematodes extraction as such. The PWD conference will be the best opportunity for a kick off meeting of this project including discussion with scientists and especially those involved in REPHRAME. This discussion would allow to avoid duplication of work.

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Session 3:

PWN in International Trade, Pathways and Phytosanitary Treatments

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Prospero S, Polomski J, Rigling D, Occurrence and distribution of Bursaphelenchus species in Switzerland, In: Schröder, T. (ed.), Pine Wilt Disease Conference 2013, pp. 43, Braunschweig, ISSN: 1866-590X

Occurrence and distribution of Bursaphelenchus species in Switzerland

Prospero S, Polomski J, Rigling D Swiss Federal Institute for Forest, Snow and Landscape Research WSL, CH-8903 Birmensdorf, Switzerland Email: [email protected]

In Switzerland, pine forests cover about 43000 hectares and are dominated by Scots pine (P. sylvestris). M ost o f t hese forests a re l ocated in t he Alps where t hey protect hum an infrastructures a gainst t he i mpacts of na tural ha zards. Here, we pr esent t he r esults of a nation-wide s urvey on t he oc currence of Bursaphelenchus species t hat was conduc ted between 2009 a nd 2011 in Swiss pine forests, including sites in proximity of r isk areas (e.g. airport and sawmills), and in pine bark and solid wood packing material. In the pine forests, a t otal of e ight Bursaphelenchus species w ere i dentified b y m orphological a nd molecular m ethods. The m ost f requent s pecies w ere B. v allesianus, f ollowed b y B. sexdentati, B. l eoni, B. e ggersi, a nd B. m ucronatus. B. bor ealis, B. pi nophilus, a nd B. polygraphi were only rarely found. Although most species can probably be considered as saprotrophs, B. vallesianus and B. mucronatus may be involved in pine dieback observed in some ar eas. Five of t hese ei ght Bursaphelenchus species ( including B. m ucronatus) were also isolated from symptomatic pines at risk areas. In the bark of the sampled trees Bursaphelenchus nematodes were practically absent. The quarantine species B. xylophilus was onl y d etected i n i mported pi ne b ark from P ortugal. T his s urvey shows t hat B. xylophilus is not pr esent i n pi ne forests i n S witzerland. However, t he recovery of t he closely related species B. mucronatus suggests that local climatic and ecologic conditions may be suitable for the establishment of B. xylophilus.

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Fonseca L et al., Coniferous bark hot steam treatment for the elimination of the pinewood nematode. In: Schröder, T. (ed.), Pine Wilt Disease Conference 2013, pp. 44-45, Braunschweig, ISSN: 1866-590X

Coniferous bark hot steam treatment for the elimination of the pinewood nematode

Fonseca L1, Santos J2, Nestler H3, Verdasca J4, Oliveira R5, Abrantes I1, Serra C6 1IMAR-CMA, Department of Life Sciences, University of Coimbra, 3004-517, Coimbra, Portugal 2LNEG, Estrada do Paço do Lumiar, 22, 1649-038 Lisboa, Portugal 3LEAL & SOARES S.A., Zona Industrial de Mira, Apartado 9, 3071-909 Mira, Portugal 4MADECA, Apartado 1, 2436-909 Caxarias, Portugal 5ALFARROXO TRADING, Parque Industrial Figueira da Foz, Lote 10, 3090 – 380 Gala, Figueira da Foz, Portugal 6DGAV, Avenida Afonso Costa 3, 1949-002 Lisboa, Portugal Email: [email protected]

ABSTRACT

In or der to develop an a rtificial h eat tr eatment to eliminate the pi newood ne matode (PWN), Bursaphelenchus x ylophilus, from c oniferous b ark, an industrial e quipment, based on hot steam was build up which enables continuous bark treatment for more than 30 m in w ith t emperatures a bove 80ºC . B iological a ssays w ere pe rformed us ing experimental units (bags) with Pinus pinaster bark and wood chips containing more than 100 000 P WN (˃60% third dispersal juvenile s tage). The bags were heat treated for 30 min a nd t he t emperature i nside m onitored b y t emperature pr obes. T he t otal num ber o f live nematodes was quantified immediately after treatment and after incubation (25ºC for 15 da ys) a nd i n bot h situations no ne matodes w ere de tected revealing ef ficacy in eliminating PWN from coniferous bark.

INTRODUCTION

The Food and Agriculture Organization of the United Nations, through the International Plant P rotection C onvention, a dopted t he I nternational S tandard f or P hytosanitary Measures N o. 15 ( ISPM N º 15) w hich de fines g uidelines f or t he pi newood ne matode (PWN), Bursaphelenchus xylophilus, elimination from wood products by heat treatment with a minimum temperature of 56ºC for 30 m in (FAO, 2009). Since November 2012, pine ba rk t hat i s t raded from P ortugal t o ot her c ountries ha s t o be heat treated by ho t steam. This treatment is being used in six Portuguese companies and enables continuous bark t reatment f or m ore t han 30 m in w ith t emperatures above 80ºC . The hot s team treatment is performed i n industrial equipment composed b y a bark feeder tank, a bark

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inlet, a s team i njection cha mber and a he at s torage cha mber m onitored by s everal temperature probes which permit real-time recording and storage of temperature data. In the present study, we have evaluated the efficacy of the hot steam treatment to eliminate PWN.

MATERIALS AND METHODS

In order to evaluate the efficacy of the hot steam treatment to eliminate PWN, biological assays w ere pe rformed using six experimental uni ts ( bags) with bark a nd w ood c hips, from P WN i nfected Pinus pi naster trees, containing m ore t han 100 00 0 P WN˃60% ( third dispersal juvenile stage) (Magnusson & Schröder 2009). Temperature probes were introduced in each bag. Then, the bags were placed into de hot steam equipment together with bark and heat treated for at leat 30 min. The number of nematodes, present in each bag was estimated before and after t he t reatment us ing t he t ray m ethod (Whitehead & Hemming 1965). After t he treatment, the bags w ere r ecovered. In t hree of t hem, t he number of live nematodes was quantified immediately and the other three were incubated at 25ºC f or 15 da ys to allow an y l ive ne matode pr esent t o breed and m aximise t he likelihood of de tection ( EPPO 2013) . A fter t he i ncubation pe riod, the num ber of l ive nematodes was also quantified.

RESULTS The t emperature i nside t he ba gs ex ceeded the recommended by t he ISPM N º15 being higher t han 80ºC . In b oth s ituations, after t reatment a nd a fter i ncubation pe riod, no nematodes were detected. The results confirmed that the continuous hot steam system go beyond the ISPM Nº15 and revealed effective in eliminating PWN from coniferous bark.

REFERENCES EPPO (2013). Diagnostics - Bursaphelenchus xylophilus. Bulletin OEPP/EPPO 43, 105- 118. FAO (2009). International standards for phytosanitary measures: guidelines for regulating wood packaging material in international trade. ISPM No. 15. Rome, Italy. Magnusson C, Schröder T (2009). Technical protocol for testing nematodes during treatment development. International Forestry Quarantine Research Group Meeting, September 2008, Rome, Italy. Whitehead AG, Hemming JR (1965). A comparison of some quantitative methods of extracting small vermiform nematodes from soil. Annals of Applied Biologists 55, 25–38.

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Hopf A, Schroeder T, Non vector spread of Bursaphelenchus xylophilus via wood chips. In: Schröder, T. (ed.), Pine Wilt Disease Conference 2013, pp. 46-47, Braunschweig, ISSN: 1866-590X.

Non vector spread of Bursaphelenchus xylophilus via wood chips

Hopf A, Schroeder T Julius Kühn-Institut, Institute for National and International Plant Health, Messeweg 11- 12, D-38104 Braunschweig, Germany Email: [email protected], [email protected]

ABSTRACT

The transmission of Bursaphelenchus xylophilus (PWN) to new host trees depends on its vector b eetles be longing t o the genus Monochamus. Nevertheless s ince t he first interception of PWN in wood chips in the European Union in 1984 di scussions on non vector tr ansmission of P WN f rom w ood c hips t o he althy t rees t akes place. C urrently increasing interest in importing wood chips from North America for energy pu rpose o r paper pr oduction can b e obs erved. W ithin t he E U research pr oject R EPHRAME w e investigated und er l aboratory conditions us ing Pinus sy lvestris saplings, whether P WN can spread from artificially infested Pinus sylvestris wood chips to pine trees.

MATERIALS AND METHODS

For this purpose pine logs (eight cm average diameter) were inoculated with PWN and incubated at 25 °C for 24 da ys. After stripping of the bark logs were processed to wood chips using a laboratory wood mill with a maximum size of 10 x 20 x 4 mm. At test start 100 g wood chips each were placed in three liter pots of 3-4 years old Pinus sylvestris saplings. Test temperatures were 15 °C and 25 °C respectively. Different combinations of tree conditions and wood chips were investigated. Except for the variant “healthy trees” the pines were either wounded on t he stem, the roots or were cut above the root collar. The wood chips were mixed in the soil or were placed on the soil with direct contact or in a distance to the stem. The control variant did not include wood chips. In total 12 t ree- wood c hip c ombinations w ere i nvestigated f or e ach t emperature. Four v ariants w ithout wood c hips but w ith di fferent t ree c onditions s erved a s c ontrols. D uring 12 w eeks t he trees were evaluated concerning their development of wilt symptoms. Six wilt classes (0 to 5 = no wilt symptoms to death of the tree) were used for assessing the physiological condition of the trees. In this time trees with more than 75 % wilting needles but still alive (wilt class 4) were sampled for nematode extraction using the modified Baermann funnel

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method. After 12 weeks al l r emaining t rees w ere al so sampled f or ne matodes irrespectively of their wilt class.

RESULTS

At 25 °C m ore s aplings de veloped hi gher w ilt c lasses a nd f ound t o b e P WN i nfested compared to the 15 °C variant. Stem injured pines with direct wood chip contact and root injured pines combined with wood chips in the soil at 25 °C showed the majority of trees with wilt class 4. PWN could be extracted from 47 of all 480 non-control trees. At 15 °C three trees in the variant with chips directly attached to stem wounded trees were affected by PWN. At 25 °C s even of 12 t ree-wood c hip-combinations w ith in total 44 trees showed PWN infestation.

CONCLUSIONS

The r esults of t he current i nvestigation i ndicate t he pos sibility of non v ector s pread of PWN w ith w ood c hips t o t rees unde r l aboratory c onditions. T he t emperature, t ree condition and wood chip location are influencing factors for this infestation pathway. The results ne ed t o be evaluated unde r out door conditions. F or P est R isk Assessment t he changing end-use of wood chips as well as the increasing amounts in international wood chip trade needs to be considered.

ACKNOWLEDGEMENTS

This study was funded by the European Union through the research project REPHRAME (KBBE.2010.1.4-09)

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Bonifácio L, Inácio M L, Sousa E, Buckley S, Thoms E M, Complementary studies to validate the proposed fumigation schedules of sulfuryl fluoride for inclusion in ISPM No. 15 for the eradication of Bursaphelenchus xylophilus from wood packaging material, In: Schröder, T. (ed.), Pine Wilt Disease Conference 2013, pp. 48-50, Braunschweig, ISSN: 1866-590X

Complementary studies to validate the proposed fumigation schedules of sulfuryl fluoride for inclusion in ISPM No. 15 for the eradication of Bursaphelenchus xylophilus from wood packaging material

Bonifácio L1 , Inácio M L1, Sousa E1, Buckley S2, Thoms E M3 1 INIAV –, Avenida da República, Quinta do Marquês 2780 - 159 Oeiras, Portugal Email: [email protected] 2 Dow AgroSciences S.A.S., ZAC du Font de l’Orme, Mougins, 06250, France Email :[email protected] 3 Dow AgroSciences LLC, 7257 NW 4th Blvd. No. 20, Gainesville, Florida, USA Email: [email protected]

INTRODUCTION

Sulfuryl f luoride ( SF), a br oad-spectrum f umigant, is unde r e valuation f or i nclusion i n standard ISPM N o. 15 - Guidelines f or R egulating W ood P ackaging M aterial i n International T rade. The T echnical P anel on Phytosanitary T reatments ( TPPT) ha s evaluated the ef ficacy d ata s ubmitted on SF ag ainst a r ange of i nsect s pecies and pine wood nematode (Bursaphelenchus xylophilus) [PWN] to support the fumigant inclusion in t he S tandard. F ollowing t heir e valuation, t he T PPT c onsidered t hat t here were no further requirements for insects; however, additional information on PWN was requested. Several e fficacy s tudies of S F on P WN were conducted and s ubmitted: (Dwinell et al. 2005); ( Flack et al . 2008) a nd (Bonifácio et a l. 2013) but a dditional information on efficacy w ithin 18 -29.9°C was r equested. A new s tudy w as c ompleted on PWN in Portugal in 2013 to validate a proposed treatment schedule for that temperature range.

MATERIALS AND METHODS

Boards of 45cm x 10cm x 5cm were prepared from PWN-contaminated pine trees (Pinus pinaster) f elled i n P ortugal, w here t his pe st i s now na turalized. T he boa rds w ere maintained i n i ncubation c hamber a t 25°C 75 % r h t o pr ovide opt imal c onditions f or reproduction o f P WN. B oards w ere t hen pl aced i n 1 m 3 chamber and f umigated with

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commercial grade ProFume® gas fumigant (99.8% sulfuryl fluoride, Dow AgroSciences, Indianpolis, IN U SA). A r ange of S F dos ages a nd t wo e xposure t imes w ere t ested a t 20oC as follows: 2,514- 4,263 g-h/m3 at 24 h exposure and 2,459-3,216 g-h/m3 at 48 h exposure. Each fumigation was monitored for temperature with a thermocouple and for SF concentration with an IR- specific m onitoring d evice. Once t arget ex posure t ime w as achi eved ( 24 or 48 h), chambers were aerated and wood boards placed in incubation chamber before counting PWN. R epresentative samples from both ends of boards were prepared by cutting them into wood cubes of ca. 1 cm3. Live PWN were immediately extracted by total immersion of t he w ood c ubes i n w ater for 48 h, then s ieving th e w ater thr ough a 38µm sieve to identify and count the nematodes under a microscope (Penas et al. 2002). Complementary genomic D NA ide ntification of e xtracted n ematodes w as pe rformed when no adult nematodes were found.

RESULTS AND CONCLUSION

The s tudy conditions c reated a de manding, w orst c ase scenario f or P WN i nfestation. Initial popul ations of P WN r anged f rom 237 t o 331 i ndividuals pe r gr am of w ood, exceeding 3 m illion individuals per treatment, and further increased in the controls after fumigation of the treated samples. Infested wood contained a high proportion (53-90%) of the J III juvenile dispersal stage. Wood moisture content was 43-61.4% before fumigation and decreased to 15.7-19.4% 21 days after fumigation. At 3 days after application, 100% control of the JIII juvenile dispersal stage was achieved with all SF dosages and exposure times. All dosages tested at 48 h exposure, compared to similar dosages at 24 h exposure, achieved be tter ne matode c ontrol; 99.971 -100% ve rsus 99.617 -99.998%, respectively.

Survivors f or 24 h exposure w ere m ainly young l arvae ( J2, J 3 stages), w hich would support the assumption that only eggs survived the SF treatment at this exposure. At 21 days after a pplication, ne matode c ontrol was 99.852 -99.999% at 24 h e xposure a nd 99.991-100% at 48 h exposure. 3 The dosage of 3,000 g-h/m in 48 h achieved 99.999% to 100% control at T3 and T21 and was s elected for fu migation a t 20°C -29.9°C. As a general r ule, S F f umigant dos age decreases as temperature increases. A previous study verified this observation on PWN at 20 and 25°C (Flack et al. 2008). New proposed SF fumigation schedules, using the SF schedules developed in 2010 (Bonifácio et al. 2013) for temperatures below 20°C (3,200 g-h/m3 with 24 h e xposure) a nd a bove 30°C ( 1,400 g -h/m3 with 24 h e xposure) a nd adding 3,000 g-h/m3 with 48 h exposure for 20ºC-29.9°C, were submitted to TPPT.

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REFERENCES Bonifácio LF; Sousa E; Naves P; Inácio ML; Henriques J; Mota M; Barbosa P; Drinkall MJ; Buckley S (2013). Efficacy of sulfuryl fluoride against the Pine Wood Nematode, Bursaphelenchus xylophilus (Nematoda: Aphelenchidae), in Pinus pinaster Aiton. boards. Journal of Pest Management. DOI 10.1002/ps.3507. Dwinell L D; Thoms E; Prabhakaran S (2005). Sulfuryl fluoride as a quarantine treatment for the pinewood nematode in unseasoned pine. Proceedings Annual International Research Conference on Methyl Bromide Alternatives and Emissions, pp. 68, 1-6, November 2005, San Diego. Flack E; Barak A; Messenger M; Thoms E (2008). Confirmation of Proposed Quarantine Fumigation CT Dosage Rates for the Control of Pine wood Nematode (Bursaphelenchus xylophilus) in Unseasoned Pine at 20oC and 25oC. Report submitted to the TPPT. Penas A C; Dias L S; Mota M M (2002). Precision and selection of extraction methods of aphelenchid nematodes from maritime pine wood, Pinus pinaster L. Journal of Nematology. 34, 62-65.

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Schröder T, Welling J, Aukamp-Timmreck C, Efficacy of kiln drying as phytosanitary treatment against wood borne nematodes. In: Schröder, T. (ed.), Pine Wilt Disease Conference 2013, pp. 51-52, Braunschweig, ISSN: 1866-590X

Efficacy of kiln drying as phytosanitary treatment against wood borne nematodes

Schröder T1, Welling J2, Aukamp-Timmreck C1 1 Julius Kühn-Institut, Institute for National and International Plant Health, Messeweg 11-12, D-38104 Braunschweig, Germany; 2 Thünen Institut of Wood Research, Leuschnerstraße 91c, D-21031 Hamburg, Germany [email protected], [email protected]

INTRODUCTION

Kiln drying (KD) often is considered equivalent to a phytosanitary treatment, because it is believed that the reduction of the wood moisture content (MC) will inhibt the growth of harmful organisms and kill them. KD with a wood moisture content reduction to less than 20 % can be a chieved using a wide r ange of p rocess pa rameters no rmally de fined in a drying schedule. In contrary to a phytosanitary measure the main aim of a KD process is moisture content reduction. Therefore, usually, a minimum drying temperature to be used is not de fined – e.g. t he E U qua rantine l egislation onl y r efers t o a n "appropriate time/temperature s chedule" (EU 2000 ). T herefore t he w ell know n l ethal t emperature (56°C for 30 minutes) is not in all cases reached in kiln drying operations. Within the E UPHRESCO mode l pr oject P EKID the inf luence of low te mperature K D treatments on t he s urvival of Bursaphelenchus x ylophilus and B. m ucronatus was investigated in comparison to KD tr eatments w hich included standard heat tr eatment conditions as described in ISPM No. 15 (FAO 2013).

MATERIALS AND METHODS

In a first step pre-trials concerning artificial infestion of pine wood with B. xylophilus and B. m ucronatus as w ell as l aboratory KD t reatments w ere conduc ted. In a s econd step pilot-scale KD treatments were carried out to investigate the efficacy of KD with respect to phytisanitary aspects. Freshly cut lo gs (Pinus sy lvestris mean di ameter 29 cm an d length 100 cm) were inoculated with B. mucronatus. After 59 days incubation time, 4 cm thick boards with a length of 100 cm and widths between 9 and 20 cm were sawn with a mobile band saw. Boards were stacked with 2.5 cm stickers to a final stack of 0.8 m x 1.0 m x 1.0 m ( WxHxL). Each drying/phytosanitary tr eatment w as c arried out in a s mall pilot-scale ki ln b y us ing t he following d rying pa rameters ( temperature ki ln ( Tair); equilibrium moisture content (EMC)), to reach the target wood moisture content (MCtg) of 20 %:

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1. Low temperature KD treatment: Tair = 35 °C and EMC = 13%,

2. KD treatment simulating conditions in a condensation kiln: T air = 35 °C and EMC = 13 % until fibre saturation point (FSP) is reached followed by Tair = 50 °C until MC = < 20 %,

3. Low temperature pre-drying plus ISPM 15 treatment: T air = 35 °C and EMC = 13 % until FSP is reached followed by Tair = 60 °C until 56 °C core temperature is reached for 30 minutes

4. KD treatment with parameters satisfying ISPM 15 requirments: Tair = 35 °C and EMC = 13 % until FSP is reached, Tair = 60 °C until MC =< 20 %

RESULTS

1. B. mucronatus survives a low temperature KD treatment (MC < 20 %) using a drying temperature of 35 °C. 2. B. mucronatus was effectively killed on Probit 9 level with a KD treatment (MC < 20 %) using a treatment temperature of 50 °C. 3. B. m ucronatus does not s urvive a K D t reatment ( MC < 20 % ) us ing a t reatment temperature of 60 °C. 4. B. mucronatus does not survive an ISPM 15 t reatment (56°C core temperature for 30 minutes, without drying the wood) using a treatment temperature of 60 °C.

CONCLUSIONS

A s tand a lone K D t reatment us ing dr ying pa rameters t hat do not i nclude t hreshold conditions (e.g. 56°C for 30 m inutes t hroughout t he whole wood profile [FAO 2013] ), which are lethal to harmful organisms such as B. xylophilus, are not suitable to be used as phytosanitary treatment. Import regulations referring to KD therefore need to specify the minimum te mperature a s w ell a s tr eatment time s to make s ure tha t ph ytosanitary requirements are met.

ACKNOWLEDGEMENTS

This investigation w as f inanzed bei the German F ederal M inistry o f Food, Agriculture and Consumer Protection under the framework of the EUPHRESCO Phytosanitary ERA- NET.

REFERENCES EU, 2000: Council 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 (in its current version) Official Journal L 169: 184pp. FAO, 2013: Regulation of Wood Pakaging in International Trade. International Standards for Phytosanitary Measures No. 15: 20 pp.

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Session 4:

PWN Interactions with Bacteria

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Kulinich et al., Recent Research on Pine Wilt Disease in Russia, In: Schröder, T. (ed.), Pine Wilt Disease Conference 2013, pp. 54-55, Braunschweig, ISSN: 1866-590X

Recent Research on Pine Wilt Disease in Russia

Kulinich O A , A rbuzova E N, M agomedov U S h, K ozyreva N I, Mazurin E S , Kolychikhina M S, Ryss A Yu All-Russian Centre for Plant Quarantine, Pogranichnay St., 32, Bykovo, Moscow region, 140150, Russia Email: [email protected]

In e arly 1990 w e i nitiated a survey in Russian conifer f orests to determine if the pine wood nematode (PWN), Bursaphelenchus xylophilus. occurred in Russia. Later, in 2010- 2011 l arger, m ore w idely-distributed surveys w ere c onducted f or t he P WN i n c onifer forests a nd i n s tored l umber i n e leven r egions of R ussia. B ased on t he r esults of t he surveys B. xylophilus has be en not f ound i n Russia, however, t he closely related nematode s pecies B. m ucronatus has be en f ound. S pecificially i n t he 3718 s amples analyzed B. mucronatus was found in 11.5% of the samples in 2010 and 5.6% in 2011. Inoclation experiments do ne in R ussia w ith B. m ucronatus isolates s howed that sometimes the se is olates ki lled pine ( Pinus sy lvestris) a nd l urch ( Larix olgensis) seedlings. Also recent r esearch done i n China and S outh K orea s howed that PWD of conifers can oc cur following i noculations done us ing a complex of pa thogenic ba cteria and the PWN of B.xylophilus that carried them (Zhao et al. 2009; Kwon et al., 2010; Wu et al. 2013). As well, it has been shown that B. mucronatus species carry such bacteria and perhaps some B. mucronatus populations can vector such pathogenic bacteria. During our 2010-2012 survey twenty six isolates of the wood-inhabiting nematode B. mucronatus were extracted and pr opagated in vitro to de termine, us ing s equencing techniques, t he identity of the associated symbiotic bacteria.. Twenty species of ba cteria b elonging to the families Enterobacteriacea, Xanthomonadaceae, Pseudomonadaceae, Burkholderiaceae, Rhizobiaceae, Nocardiaceae, Flavobacteriaceae, Bacillaceae, Paenibacillaceae were i solated from t he nematodes and identified a s be longing t o t he genera Achromobacter, B acillus, B urkholderia, Enterobacter, F lavobacterium, K lebsiella, P seudomonas, R ahnella, R hodococcus, Stenotrophomonas, P antoea, P aenibacillus, and Serratia (Fig 1) . T he m ost f requently encountered bacterium belonged to the genus Pseudomonas (44%). Five species of this genus were identified: P. lurida, P. brenneri, P. geniculata, P. fluorescens, Pseudomonas sp. T he ba cterium Pseudomonas f luorescens was i solated from ni ne B. mucronatus isolates f rom th e di fferent r egions. T oo ba cteria-associated s pecies w ere f ound on t he

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dauerlarva s tage of B. mucronatus nematodes w ere i solated from be etles i dentified as Monochamus urussovi Fisch. Four species of bacteria including Pseudomonas fluorescens species were isolated from the larva stage of a B. mucronatus isolate. According to the results of Chinese researchers, P. fluorescens is an essential species of the nematode-bacterial complex that induces PWD in the pine forests of southern China (Zhao, 2008) . It i s assumed that B. mucronatus nematodes and symbiotic ba cteria P. fluorescens can cause d eath of s ome R ussian pine f orests i n areas w here t he m ean air temperature during the summer months excedes 250C. The average monthly temperature in the Centre of European Russian in 2010 was 26.4 ° C in July and 25.5 ° C in August. Widespread de ath of Pinus sylvestris occurred t here a fter 2010. Bursaphelenchus mucronatus nematodes and t he s ymbiotic ba cterium Pseudomonas fluorescens were isolated from some dead trees. Local foresters b elieve t hat t he death of these t rees was caused by drought, but we do not exclude the possibility that that PWD played some role in the death of these trees..

REFERENCES Kwon Hyeok Ran; Gyung Ja Choi; Yong Ho Choi; Kyoung Soo Jang; Nack-Do Sung; Mun Seong Kang; Yilseong Moon; Seung Kyu Lee; Jin-Cheol Kim (2010). Suppression of pine wilt disease by an antibacterial agent, oxolinic acid. Pest Manag. Sci., 66, 6, 634-639. Wu X Q; Yuan W M; Tian X J; Fan B., Fang X; Ye J R.; Ding X L (2013). Specific and functional diversity of endophytic bacteria from pine wood nematode Bursaphelenchus xylophilus with different virulence. Int. J Biol Sci., 9, 1, 34-44. Zhao B G (2008). Bacteria carried by the pine wood nematode and their symbiotic relationship with nematode. In: Zhao B G, Futai K, Sutherland J R, Takeuchi Y. (eds.), “Pine Wilt Disease”, Tokyo. Springer, 264-274. Zhao B G; Lin F; Guo D; Li R.G; Li S N; Kulinich O; Ryss A (2009). Pathogenic roles of the bacteria carried by Bursaphelenchus mucronatus. Journal of Nematology, 41, 1, 11-16.

Fig 1. Symbiotic b acteria extracted from Bursaphelenchus mucronatus isolates f rom different regions of Russia

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Tan J J, Qu H Y, Hao D J, Chen F M, Inoculation effects of Pinus thunbergii with Bursaphelenchus xylophilus and two strains of Bacillus firmus. In: Schröder, T. (ed.), Pine Wilt Disease Conference 2013, pp. 56-57, Braunschweig, ISSN: 1866-590X

Inoculation Effects of Pinus thunbergii with Bursaphelenchus xylophilus and two strains of Bacillus firmus

Tan J J, Qu H Y, Hao D J, Chen F M College of Forest Resources and Environment, Nanjing Forestry University, Nanjing, China Email: [email protected]

ABSTRACT

To c larify t he r ole of b acterium i n t he pa thogenesis of pi ne wilt di sease, 3 years ol d Japanese black pine (Pinus thunbergii) were inoculated with disinfected Bursaphelenchus xylophilus (Bx) , bacterium is olate G D1 of Bacillus firmus (isolated f rom t he bod y of Bx)、GD2 of B. firmus (isolated from the healthy P. massoniana) and the mixture of the nematode and bacterium. The results showed that the pine seedlings were diseased when inoculated with the di sinfected B x and the mix ture of Bx and bacterium， while not diseased when inoculated with B. firmus singly. Disease de velopment of t he pi ne seedlings w as s lower af ter i noculated w ith Bx s ingly t han w ith t he m ixture of Bx a nd bacterium. The disease of pine seedlings was heavier when the inoculation concentration of ba cterium w as hi gher. A fter i noculated with B x s ingly a nd t he m ixture of Bx a nd bacterium, t he pi th of pi ne s eedlings br owned, t he pr ocess o f pi th br owning was from lower part to upper part of the inoculated maim stem, while the pith of pine seedlings was normal af ter inoculated with B. firmus singly and t he c ontrol. At ea rly s tage af ter inoculation with the mixture of Bx and B. firmus, the number of bacterium detected in the pine s eedlings was l arger. Therefore, it w as c oncluded that the tw o ba cterium s trains enhanced the disease development of pine wilt disease. Key words: Pinus thunbergii; Bursaphelenchus xylophilus; Bacterium; Bacillus firmus

INTRODUCTION

Up t o now , t he pa thogenic m echanism of pi ne w ilt di sease ke eps obs cure. T he r ole of bacterium i n pathogenesis of t he di sease i s s till unc lear ( Oku et al 1980; K awazu & Kaneko 1997; Zhao & Guo 2004; Zhu et al 2012).

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MATERIALS AND METHODS

3 years ol d J apanese black pi ne (P. thunbergii) were i noculated with disinfected Bursaphelenchus x ylophilus (Bx) , bacterium is olate G D1 of Bacillus firmus (isolated from the body of Bx)、GD2 of B. firmus (isolated from the healthy P. massoniana) and the mixture of the nematode and different concentration of bacterium by bark inoculation method. The i noculation num ber of B x a nd ba cterium w as 3500 , 2.3×105CFU, 2.3×106CFU and 2.3×107CFU per s eedling. At 5 da ys after i noculation, the num ber of bacterium in the inoculation main stem 3-4 cm above inoculation point was detected.

DISCUSSION

This inoculation expriment was conducted on 3 -year-old Japanese black pine seedlings, its r esults w ere s imilar to that on 1-2 years o ld excised br anch o f m asson pi ne ( P. massoniana) ( Tan 2001 ). T he ba cterium s train GD2 w as isolated from t he he althy P. massoniana , it belongs to pine endophytic bacterium. The research on t he relationship between pine endophytic bacterium and pine wilt disease is being carried out.

ACKNOWLEDGEMENTS

This r esearch was supported b y N ational N atural S cience F oundation of C hina ( No 31170606; 31170599).

REFERENCES Kawazu K; Kaneko N (1997). Asepsis of the pine wood nematode isolate OKD-3 causes it to lost its pathogenicity. Japanese Journal of Nematology 27(2), 76-80. Oku H; Shiraishi T; Ouchi S; Kurozumi S; Ohta H (1980). Pine wilt toxin, the metabolite of a bacterium associated with a nematode. Naturwissenschaften 67, 198-199. Tan J J (2001). Studies on relationship between Bacillus firmus and pine wood nematode disease (PhD thesis). South China Agricultural University: Guangzhou, China. Zhao B G; Guo D S (2004). Isolation and pathogenicity of a bacterium strain carried by pine wood nematode. Journal of Beijing Forestry University(Natural Sciences Edition) 26 (1), 125-128. Zhu L H; Ye J R; Sapna N; Xu X L; Wang Z L; Ji J Y (2012). Pathogenicity of aseptic Bursaphelenchus xylophilus. PloS One 7(5), 1-8

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Vicente C, Ikuyo Y, Mota M, Hasegawa K, Bursaphelenchus xylophilus and associated bacteria under oxidative stress conditions. In: Schröder, T. (ed.), Pine Wilt Disease Conference 2013, pp. 58-59, Braunschweig, ISSN: 1866-590X

Bursaphelenchus xylophilus and associated bacteria under oxidative stress conditions

Vicente C, Ikuyo Y, Mota M, Hasegawa K ICAAM - Instituto de Ciências Agrárias e Ambientais Mediterrânicas, Departamento de Biologia, Universidade de Évora, Núcleo da Mitra, Ap. 94, 7002-554 Évora, Portugal Email: [email protected]

Plant pa thogens ha ve e volved a m achinery o f a ntioxidant e nzymes a nd de toxyfying systems t o r educe t he plant ox idative bur st i mpact upon i nvasion, a nd a llow t heir successful c olonization. O ur study aimed to unde rstand t he c ontribution of Bursaphelenchus xylophilus-associated bacteria in interaction with the nematode, and as well i ndependently, und er oxidative s tress conditions in an attempt to mimick their behaviour in the oxidative burst conditions of the host tree in the early stages of pine wilt disease (PWD). Thus, we begin by examining the oxidative stress resistance of three B. xylophilus-associated bacteria (Serratia spp. LCN-4, LCN-16 and PWN-146) (Vicente et al., 2011 a nd 2012) , and a s w ell Escherichia c oli OP50 ( control s train), i n increasing concentrations of hydrogen peroxide (H2O2) ranging from 15 to 40 mM in a 24h-exposure period. W e could s ee t hat a ll Serratias were abl e t o tolerate t he s trong and prolonged

H2O2 conditions, in contrast with control E. coli. Following, we checked the mortality of two isolates of B. xylophilus (virulent Ka4 and avirulent C14-5) in absence and presence of associated-Serratia and control strain in the same stressful conditions. Without bacteria (surface s terilized nematode), Ka4 a nd C 14-5 presented s ignificant di fferences i n their

ability to tolerate H2O2, being Ka4 clearly more resistant than C14-5. With Serratia spp.,

both Ka4 and C14-5 were able to survive at all H2O2 concentrations tested, with mortality rates l ower t han 10 %. In the pr esence of the E. c oli OP50, mor tality percentage of avirulent C 14-5 was hi gher a nd c loser t o t he va lues obt ained i n ne matode a lone conditions, with no s tatistical di fferences be tween treatments. These r esults i ndicate a beneficial and potential helper e ffect towards B. x ylophilus, suggesting th at these associated Serratia spp. are able to express several antioxidant enzymes and detoxifying systems, which explain their high tolerance to H2O2-mediated stress. Next, we focused on

B. xylophilus catalase transcript levels to target H2O2. Two catalases were predicted in the B. xylophilus genome, BxyCTL-1 (BUX.s00579.159) and BxyCTL-2 (BUX.s01109.377), with a high protein similarity with other nematode catalases. Relative gene expression of catalase genes of B. xylophilus Ka4 and C14-5 in both absence and presence of Serratia

spp. PWN-146 were studied under stress conditions (24h-exposure to 15mM H2O2) and

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compared with non-stress condition. Bacterial effect w as t ransversal t o virulent and avirulent B. xylophilus. Relative gene expression of catalase genes of B. xylophilus show that, without ba cteria, t he vi rulent i solate K a4, f or bot h non -secreted Bxyctl-1 and secreted Bxyctl-2 genes, pr esented a 1.5 -fold di fference t o avirulent C 14-5. When i n interaction with bacteria ( Serratia spp. P WN-146), bot h vi rulent a nd a virulent B. xylophilus catalase l evels de creased to levels com parable t o normal con ditions w ithout oxidative stress, which is also in agreement with mortality test results.

Futher, we explored the bacterial interaction with B. xylophilus, namely the attachment to the nematode cuticle, an important characteristic in bacteria dissemination and that, to our knowledge, has not been studied before. We performed co-culturing of B. xylophilus and GFP-labelled bacteria in Botrytis cinerea plates. We observed that after 24-hour contact with Serratia spp. LCN-16, the density of nematode-attached bacteria was sparse, and no GFP f luorescence s ignal w as de tected from i nside t he ne matode. From t hese r esults, adhesion of these bacteria to the nematode surface and organs seems to be weak and non- specific. Previously, Shinya et al . (2010) have s hown, t hrough s canning e lectron microscopy (SEM), the presence of few bacteria on the nematode cuticle even after the nematode was vigorously w ashed. B. x ylophilus-associated bacteria ar e reported to be carried on t he ne matode’s s urface, a nd i n a verage 290 w ere counted on t he c uticle of PWN isolated from diseased trees (Zhao et al., 2003). If bacteria are not attached to the nematode surface, how can they be transported by B. xylophilus from and into a pine tree?

New insights into the nematode-bacteria interaction are given in this study. We report, for the f irst time , tha t B. x ylophilus associated ba cteria may assist the ne matode opportunistically in the disease, and that a virulent B. xylophilus isolate was able to better tolerate OS conditions than an avirulent isolate.

REFERENCES Vicente CSL, Nascimento F, Espada M, Mota M, Oliveira S (2011). Bacteria associated with the pinewood nematode Bursaphelenchus xylophilus collected in Portugal. Antonie van Leeuwenhoek Journal of Microbiology 100, 477–81. Vicente CSL, Nascimento F, Espada M, Barbosa P, Mota M, Glick BR, Oliveira S (2012). Characterization of bacteria associated with pinewood nematode Bursaphelenchus xylophilus. PloS one 7:e46661. Shinya R, Morisaka H, Takeuchi Y, Ueda M, Futai K (2010). Comparison of the surface coat proteins of the pine wood nematode appeared during host pine infection and in vitro culture by a proteomic approach. Phytopathology 100, 1289–97. Zhao BGZ, Ang HLW, An SFH, An ZMH (2003). Distribution and pathogenicity of bacteria species carried by Bursaphelenchus xylophilus in China. Nematology 5:899–906.

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Morais PV et al., Diversity and in vitro nematicidal activity of bacteria associated to pinewood nematode. In: Schröder, T. (ed.), Pine Wilt Disease Conference 2013, pp. 60-62, Braunschweig, ISSN: 1866-590X

Diversity and in vitro nematicidal activity of bacteria associated to pinewood nematode

Morais PV1,3, Proença DN1, Paiva G1, Francisco R1, Verissimo P2,3, Fonseca L1, Abrantes IMO1,3 1 IMAR-CMA, University of Coimbra, 3004-517 Coimbra, Portugal, 2 Center for Neuroscience and Cell Biology, University of Coimbra, 3004-504 Coimbra, Portugal, 3 Department of Life Sciences, FCTUC, University of Coimbra, 3004-517 Coimbra, Portugal Email: [email protected]

Bacteria h ave be en suggested to play a r ole on pine w ilt di sease s ince t hey h ave b een isolated associated with the pinewood nematode. The aim of this work was to evaluate the diversity o f t he n ematode as sociated bacteria and their pot ential r ole i n this di sease b y determining the ir in vitro nematicidal a ctivity. The ba cterial is olates, identified by 16S rRNA g ene s equence, belonged to the f amilies M icrobacteriaceae, Oxalobacteriaceae, Burkholderiaceae, Enterobacteriaceae, Pseudomonadaceae and Xanthomonadaceae. The most nematicidal strain, Serratia sp. A88copa13, produced proteases in the supernatant.

INTRODUCTION

Bacteria have been suggested to play a role on pine wilt disease (PWD) since they have been found associated with the pinewood nematode (PWN), Bursaphelenchus xylophilus. Therefore, PWN isolates from across the globe have been studied in order to understand whether these bacteria can produce toxins that could be involved in the development of PWD (Proença et al. 2010). The microbial community associated to PWN was accessed in nematodes from different recently affected areas in Portugal. The aim of this work was to evaluate the diversity of the nematode associated bacteria and their potential role in the PWD by determining their in vitro nematicidal activity.

MATERIALS AND METHODS

The mic robial c ommunity associated to PWN w as a ssessed isolating the s trains on t he track of ne matodes f rom i nfected Pinus pi naster trees, from af fected areas i n Portugal. The bacterial isolates were identified by 16S rRNA gene sequence. Phylogenetic analysis were pe rformed b y us ing A RB s oftware pa ckage a nd t ype s trains f rom i nternational databases. All isolates were screened for their ability to produce siderophores, lipases and

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proteases (Proença et al. 2010). Strains were tested against B. xylophilus to evaluate their nematicidal activity assessed as the percentage of dead nematodes when incubated with bacteria supernatant during 24 h at 26ºC.

RESULTS

Strains is olated be longed t o t he f amilies Microbacteriaceae, Oxalobacteriaceae, Burkholderiaceae, Enterobacteriaceae, Pseudomonadaceae and Xanthomonadaceae. Forty-seven s trains w ere t ested a nd 21 s trains pr oduced e xtracellular pr oducts w ith nematicidal a ctivity ( Figure 1) . The mos t ne maticidal s train, Serratia sp. A 88copa13, produced proteases i n the s upernatant. Biological as says revealed di fferences i n nematicidal activity of the proteases to different species of Bursaphelenchus.

100% Pseudomonas putida P.moorei 90% P.lutea P.gengeri 80% P.koreensis P. costantinii 70% Pseudomonas sp. Burkholderia sordidicola 60% B. fungorum 50% B. glathei Burkholderia sp. 40% Serratia marcescens S. plymuthica Rhanella aquatilis Percentage of bacterial isolates bacterial of Percentage 30% Yersinia intermedia 20% Pantoea cypripedii Pantoea sp. 10% Erwinia sp. Cronobacter sp. 0% Luiteibacter rizhinovicinus Curtobacterium pusillum

Nematotoxicity

Figure 1 — Biochemical pr operties and ne maticidal a bility of ba cteria a ssociated with Bursaphelenchus xylophilus. Most strains produced siderophores and lipases.

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CONCLUSIONS

In Portugal, strains belonging to the families Enterobacteriaceae and Pseudomonadaceae have be en isolated associated to the P WN a nd s ome ha ve pot ential to e liminate the nematode in v itro. In t his pr ocess, pr oteolytic e nzymes a nd l ipases, surfactants a nd possibly s iderophore, pr oduced b y t he ba cteria t o t he e xtracellular m edium, m ay b e involved, being the proteases (metalloproteinases and serine) the most relevant.

ACKNOWLEDGMENTS

Fundação para a C iência e a T ecnologia P roject P TDC/AGR-CFL/115373/2009. DNP: graduate f ellowship S FRH/BD/61311/2009. GP: f ellowship P TDC/AGR- CFL/115373/2009

REFERENCES Proença DN, Francisco R, Santos CV, Lopes A, Fonseca L, Abrantes IMO, Morais PV (2010). Diversity of bacteria associated with Bursaphelenchus xylophilus and other nematodes isolated from Pinus pinaster trees with pine wilt disease. PLoS One 5, e15191

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Vicente et al., Natural bacterial communities associated with the pine sawyer beetle Monochamus galloprovincialis. In: Schröder, T. (ed.), Pine Wilt Disease Conference 2013, pp. 63-64, Braunschweig, ISSN: 1866-590X

Natural bacterial communities associated with the pine sawyer beetle Monochamus galloprovincialis

Vicente C, Nascimento F, Espada M, Barbosa P, Hasegawa K, Mota M, Oliveira S ICAAM - Instituto de Ciências Agrárias e Ambientais Mediterrânicas, Departamento de Biologia, Universidade de Évora, Núcleo da Mitra, Ap. 94, 7002-554 Évora, Portugal Email: [email protected]

Most studies of cerambycids microbiota are related with gut-bacterial communities due to their importance in the insect’s biology and ecology. Essentially, the research in this field showed t hat i nsect m icrobial c ommunities a re li mited by specific ni che characteristics. The pr esent s tudy inv estigates the n atural b acterial c ommunities of Monochamus galloprovincialis collected from P ortuguese Pinus pi naster trees and Bursaphelenchus xylophilus-free, using a metagenomics approach. A total of 492 partial sequences (750- 1200bp) of 16S rRNA gene were considered in this study. The rarefaction curves analyses showed 18 OTUs (operational taxonomic units) at genus-species level (95-97% sequence similarity). Bacterial c ommunities a ssociated with M. gal loprovinciallis are m ainly composed by Proteobacteria (78.5%), followed by Firmicutes (20.8%) and Bacteroidetes (<1%). From the phylum Proteobacteria, three classes were present: γ-proteobacteria (87.9%), β-proteobacteria (11.6%), and α-proteobacteria (0.5%). T he most a bundant genera of Proteobacteria were Serratia (76.4%), followed by Janthinobacterium (11.6%), Rahnella (5.0%), Pseudomonas (3.6%), a nd Nevskia (2.1%). Among Firmicutes, the genera f ound were: Bacillus (95%), Paenibacillus (3%), Lactococcus (1%), and Lysinibacillus (1%). From t he ph ylum Bacteroidetes, representatives w ere i dentified as Sphingobacterium (n=1), Sediminibacterium (n=1) a nd unc ultured Bacteroidetes (n=2). Our results show a predominance of γ-proteobacteria i n M. gal loprovincialis, w hich might be int rinsically related with their feeding di et and habitat c haracteristics. Interestling, a high-density population of Serratia spp. was found in M. galloprovincialis. The pr esence of Serratia in insects is well documented, both as s ymbiont or pathogen, and its related with their fitness ability to resist antibacterial substances ingested by the insect, as well as the powerful enzymatic cocktail produced (chitinases, lecithinases, and proteinases) (Grimmont and Grimmont, 2006). Previous culture-dependent studies have also reported isolation of Serratia marcescens in M. al ternatus (Ma et al ., 2009) ,

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Aerobacter aerogenes and Bacillus cereus var. mycoides in M. scutellatus, M. notatus and M. marmorator (Soper and Olsen, 1963). Further studies are needed to understand their functional c ontribution t o t he ba cterial c ommunity s tructure of M. gal loprovincialis. In light of all knowledge r egarding ba cterial communities of B. xylophilus and t he results here presented, it is tempting to establish the hypothesis that perhaps the B. xylophilus can harbour ba cteria from t he i nsect. A n e xample i s t he pr edominance of Serratia in M. galloprovincialis and a lso B. x ylophilus (Vicente et al ., 2011) . V icente et al . ( 2012) described that some nematode-associated bacteria, including Serratia spp., were able to degrade c ellulose, an advantage i n t he a daptation a nd c olonization of w ood t issues (Harakava and Gabriel, 2003). Although with t his s tudy i t’s not pos sible t o establish a comparison between insect-vector a nd B. x ylophilus bacterial c ommunities, the r esults presented are us eful and e ncourage f uture w ork i n t his s ubject ( Vicente e t a l., 2013) . Understanding the role of bacteria transmission in the PWD complex will bring important knowledge for future prospects in the disease management and control.

REFERENCES Grimmont F; Grimmont PAD (2006). The Genus Serratia. In: The Prokaryotes - a Handbook on the biology of Bacteria: Ecophysiology, isolation, identification, Application. New York, NY: Springer Verlag. 219–244. Harakava R; Gabriel DW (2003). Genetic differences between two strains of Xylella fastidiosa revealed by suppression subtractive hybridization. Applied Environmental Microbiology 69: 1315–1319. Ma LJ; Zhang LQ; Lin HP; Mao SQ (2009). Investigation of pathogens of Monochamus alternatus in East China and virulence. Chinese Journal of Biology Control, 25:220-224. Soper RS; Olsen RE (1963) Survey of biota associated with Monochamus in Maine. Canadian Entomology 95: 83-95 Vicente CSL, Nascimento F, Espada M, Mota M & Oliveira S (2011) Bacteria associated with the pinewood nematode Bursaphelenchus xylophilus collected in Portugal. Antonie Van Leeuwenhoek Journal of Microbiology 100: 477–81. Vicente CSL; Nascimento FX; Espada M et al. (2012) Characterization of bacteria associated with pinewood nematode Bursaphelenchus xylophilus. PloS one 7: e46661. Vicente CSL; Nascimento FX; Espada M; Barbosa P; Hasegawa K; Mota M; Oliveira S (2013). Characterization of bacterial communities associated with the pine sawyer beetle Monochamus galloprovincialis, the insect vector of the pinewood nematode Bursaphelenchus xylophilus. FEMS Microbiology Letters (accepted).

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Session 5:

PWD Management and Contingency Planning

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Nakamura et al., A Research Project to Develop Strategic Action Plan in the Pine-wilt-disease Unaffected Area in Northern Japan. In: Schröder, T. (ed.), Pine Wilt Disease Conference 2013, pp. 66, Braunschweig, ISSN: 1866-590X

A Research Project to Develop Strategic Action Plan in the Pine-wilt-disease Unaffected Area in Northern Japan

Nakamura K, Maehara N, Aikawa T, Ichihara Y, Kosaka H, Kanzaki N, Kagaya E, Sugita H, Masaki T, Kimura K, Kon J, Kaneko T Tohoku Research Center, Forestry and Forest Products Research Institute, Nabeyashiki 92-25, Shimo-kuriyagawa, Morioka, Iwate 020-0123, Japan Email: [email protected]

Aomori is the northernmost prefecture in Honshu, the main island of Japan, that had not been affected by pine wilt disease (PWD). To develop an action plan to prevent from and be prepared for introduction of PWD into Aomori Prefecture, we tried to acquire critical information related to introduction, colonization and spread of the disease from the view point of pr esence/absence of t he i nsect ve ctors and c ompetitive substitution of Bursaphelenchus nematodes, a s w ell a s t he t olerance of t ree popul ations a nd f orest communities to the loss of pine trees resulted form the disease. Captures of adult Monochamus alternatus by attraction traps and genetical identification of the trapped adult using SSR markers indicated that accidental incoming of the vector insect oc curred at t he s outh-west bor der t o t he ne ighboring pr efecture h aving s everely damaged forests by PWD, but was effectively checked by the 2-km clear-cut zone of pine trees. According to the whole tree investigations for subcortical insects in 124 dying and newly dead pi ne t rees conducted i n va rious l ocations i n A omori, i t s eemed t hat ne ither M. alternatus nor B. mucronatus, as substitutive species for B. xylophilus, was distributed in the prefecture. The ol d-growth popul ation of Pinus de nsiflora did not c ompensate t he l oss of f orest canopy caus ed by P WD, and t he br oad-leaved trees mix ed in P. d ensiflora or P. thunbergii dominated forests could not substitute the status and functions of pine trees in the or iginal f orests. T hus w e c oncluded t hat t he l oss of pi ne t rees b y P WD e pidemic would bring about severe degradation of the forests in the area. On the ba sis of t he i rreplaceableness o f t he p ine s pecies i n the f orests, absence of effective vector and substitutive species of B. xylophilus, and limited entry route of the disease into the prefecture, we proposed regionally-specialized action plan against PWD in Aomori Prefecture.

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Xu, F et al., Study on the techniques of sustainable control of pine wood nematode disease (Bursaphelenchus xylophilus). In: Schröder, T. (ed.), Pine Wilt Disease Conference 2013, pp. 67, Braunschweig, ISSN: 1866-590X

Study on the techniques of sustainable control of pine wood nematode disease (Bursaphelenchus xylophilus)

Xu Fuyuan, Xu Ming, Zheng Huaying, Xie Chunxia, Liu Yunpeng, Gao Yue Forestry Academy of Jiangsu Province, 211153, dongshanqiao, Nanjing, China Email: [email protected]

ABSTRACT

Pine wood nematode (PWN) is a devastating global forest diseases and its spread is still increasing. According t o t he s ituation above w e carried out r esearch for more t han 30 years. By the results we found that the sustainable control of PWN was the effective way to protect our afforestation and ecological security. Both b y lab and field tests the tests results showed as following: 1. By use selected GD5, GX2 and GX3 3 Pinus massoniana provenances resistant to pine wood nematode (PWN), monitoring their resistibility and large area of afforestation, resistant provenances selection provide technical support for breeding resistant stand. 2. Comprehensive development and utilization of the resources of natural enemy to control Monochamus alternatus. The technique developed the mass raising 15 million head of Dastarcus helophoroides, Scleroderma guani annually, and the technique of combined D. helophoroides with S. guani releasing to control M. alternatus larva w ere s tudied. Parasite r ate to M. alternatus larva w ere m ore t han 74.1%. P ine mortality rate at the beginning of the 25% fell to below 0.3% which were killed by PWN, has significant control effect in the test field.

KEY WORDS: resistant provenances, the mass raise natural enemy, combined Dastarcus helophoroides with Scleroderma guani releasing, to control Monochamus alternatus larva, parasite rate, control effect

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Naves P, Vieira M, Sousa E, New Strategies for pine wilt disease (PWD) management in Portugal: preventive methods to reduce the spread of the disease to new areas. In: Schröder, T. (ed.), Pine Wilt Disease Conference 2013, pp. 68, Braunschweig, ISSN: 1866- 590X

New Strategies for pine wilt disease (PWD) management in Portugal: preventive methods to reduce the spread of the disease to new areas

Naves P, Vieira M, Sousa E Instituto Nacional de Investigação Agrária e Veterinária (INIAV, IP), Oeiras, Portugal Email: [email protected]

Despite t he i mportance of t he pi ne w ood ne matode Bursaphelenchus x ylophilus, t he casual agent of pine wilt disease, and its insect vector Monochamus galloprovincialis in Portugal, there ar e f ew available options t o control t hese o rganisms and t o prevent t he spread of the disease to new areas. Some new strategies were developed in Portugal: (i) Preventive trunk injection of Emamectin Benzoate (EB), (ii) Preventive dissemination of the di sease b y application of an insecticide ne t with a lpha-cypermethrin to w ood transport. Concerning th e f irst tr ial ( i), t runk-injections w ith EB were performed in a maritime pine (Pinus pinaster) forest in Portugal, testing three dose-rates: 0.032 g a.i./cm diameter at breast height (DBH), 0.064 g a.i./cm DBH and 0.128 g a.i./cm DBH, along with a n unt reated c ontrol pl ot. E B w as s uccessfully i njected a nd t ranslocated i n pi nes, resulting in low mor tality f or the inoc ulated t rees s everal mont hs after inoculation, contrasting w ith m uch hi gher m ortality of non -treated pines. Emamectin be nzoate w as successfully r ecovered i n br anches of t reated pi nes dur ing a p eriod of more t han t hree years. Concerning the s econd trial ( ii), two s tudies w ere p erformed with the a im o f studying the effectiveness and minimum exposure time of the insecticide-net to the vector and to test the net´s efficiency in relation to insects emerging from wood logs during a simulation of a truck transport. Results showed that exposure to the insecticide net proved fatal to M. galloprovincialis adults even at very short periods of contact with the net, of just 1 t o 5 m inutes. These t wo novel strategies to manage and control w ilt di sease in Europe offer new possibilities to prevent the spread of wilt disease natural and artificial spread of the disease by the vector.

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Session 6:

PWN Biology, Population Dynamics, Epidemiology and Modelling

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Tomalak M, Filipiak A, Inter-specific competition of Bursaphelenchus xylophilus with native populations of B. mucronatus in pine. In: Schröder, T. (ed.), Pine Wilt Disease Conference 2013, pp. 70-71, Braunschweig, ISSN: 1866-590X

Inter-specific competiton of Bursaphelenchus xylophilus with native populations of B. mucronatus in pine

Tomalak M, Filipiak A Institute of Plant Protection – National Research Inst., Węgorka 20, 60-318 Poznań, Poland Email: [email protected]

Both t he qua rantine pe st ne matode, Bursaphelenchus x ylophilus and na tive t o E urasia, unnharmful B. mucronatus are genetically closely related and present similar bionomics. They develop and reproduce in pine, can use the same insect vectors, and with contiuous colonization of ne w l ocalities b y B. x ylophilus, t he ove rlap of t heir geographic distribution continues to increase. The laboratory and field study in natural ecosystems of the F ar East r evealed a com petitive di splacement of t he na tive B. mucronatus by t he invasive B. xylophilus (Cheng et al., 2009), which could be attributed to faster population development i n the l ater s pecies (Futai 1980) . Considering t he obs erved g enetic a nd phenotypic variation among European populations of B. mucronatus we have undertaken a research on interactions between selected strains of B. xylophilus (both European and Asiatic) and a series of native Polish isolates of B. mucronatus during concurrent invasion and development in the same host.

MATERIALS AND METHODS

In our quarantine glasshouse study, conducted on 2-3-year old seedlings and 20-cm-long logs of P. silvestris, r eproduction of a single Chinese (Nanjing) and two P ortuguese (Mad25c and Pt67OL) strains of B. xylophilus, five geographically distant isolates of B. mucronatus collected i n P oland, a nd t he recently constructed m ulti-strain intra-specific hybrid of t he l ater s pecies ( MT-Rol-01) m arked w ith a R oller Bmrol-1(mt4) mutation, were com pared when reared separately or in two-species mixed popul ations. The seedlings w ere i noculated w ith a dos e of 2500 or 5000 ne matodes f or s ingle-species cultures, and of 2500 nematodes for each species in mixed populations. In logs the dose was reduced to 500 and 1000 nematodes, respectively. The nematodes were incubated for 1 m onth a t 20 oC. Then, t he w ood w as c hopped a nd s ubjected t o water e xtraction of nematodes. T he ne matodes w ere i dentified ba sed on t he s hape of f emale ta il te rminus. The pr oportions of ph enotypes pr esent i n t he of fspring w ere counted f or e ach species/strain variant. Selected populations were also subjected to molecular (ITS-RFLP) analysis to confirm the taxonomic status of the offspring.

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RESULTS AND DISCUSSION

Phenotypic examination o f the ne matode of fspring revealed tha t in s ingle-species populations of B. xylophilus the range of morphological variation of the female tail has increased w hen compared t o ne matodes o f t he pa rental popul ations, w hich w ere originally r eared in vitro, on Botritis c inerea. This w as pa rticularly o bvious i n t he Chinese s train ( Nanjing), w here i n 28 -42% i ndividuals t he t ail t erminus ha d a conical projection or a small mucro, compared to mostly broadly rounded terminus of in vitro- reared females. The shape of mucro was, however, distinctive from that in B. mucronatus. In B. mucronatus the female tail was similar in both the nematodes reared in vitro and in wood. In the o ffspring of t wo-species m ixed popul ations t he pr oportions of f emales w ith B. xylophilus- and B. m ucronatus-like ta il g enerally dr ifted to one of t he pa rental phenotypes. Interestingly, not only B. xylophilus but also B. mucronatus could dominate and contribute to significant reduction of the second species. Among five isolates of B. mucronatus examined i n l ogs, one ( Mdz-1) pr oved t o dom inate B. x ylophilus (Nanjing and P t67 O L) i n 73 a nd 63% of r eplicates, r espectively, w hile t he i solate M az-02 dominated these strains in almost 50% of replicates. B. xylophilus (Mad 25c) dominated in a ll e xperimental va riants, how ever, i n i ndividual s eedlings or l ogs t hese ne matodes could also be outperformed by local isolates of B. mucronatus. Insertion of the Roller mutation into the intra-specific hybrid population of B. mucronatus (MT-Rol-01) clearly simplified the process of phenotypic examination of the offspring in two-species m ixed c ultures. B y p roducing t he unique phe notype i n a ll de velopmental stages of B. mucronatus the Roller mutation s eems t o be a very handy marker for a ny intra- and inter-specific hybridization and controlled rearing of this nematode. In s imilar environmental c onditions t he r esults of t he i nter-specific competition in the host di ffered a mong s pecies / s train c ombinations and were app arently related with variation in innate cha racteristics of t he n ematode popul ations. We s peculate t hat t he observed phenomenon may have some retarding effect on early success of B. xylophilus in c olonization of ne w r egions w here native pop ulations of B. m ucronatus are pr esent. These observations need, however, further substantiation in field experiments.

REFERENCES Futai K (1980). Developmental rate and population growth of Bursaphelenchus lignicolus (Nematoda: A phelenchoididae) and B. m ucronatus. Applied E ntomology a nd Zoology 15, 115-122. Chen X-Y; Xie P-Z; Cheng F-X; Xu R-M; Xie B-Y (2009). Competitive displacement of the na tive s pecies Bursaphelenchus m ucronatus by an alien s pecies Bursaphelenchus xylophilus (Nematoda: Aphelenchida; Aphelenchoididae): a case of successful invasion. Biological Invasions 11, 205-213.

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Zhengmin H, Ye G, Ailing B, Dongxia C, Interspecific hybridization between Bursaphelenchus xylophilus and Bursaphelenchus mucronatus In: Schröder, T. (ed.), Pine Wilt Disease Conference 2013, pp. 72-73, Braunschweig, ISSN: 1866-590X

Interspecific hybridization between Bursaphelenchus xylophilus and Bursaphelenchus mucronatus

Han Zhengmin1, Guo Ye1, Ben Ailing2, Cao Dongxia1 1 Nanjing Forestry University，China 2 Nanjing Xiaozhuang Normal College, China

ABSTRACT:

Pine wood nematode (Bursaphelenchus xylophilus ) is the pathogen of pine wilt disease that c auses pi ne w ilt or de ath. Bursaphelenchus m ucronatus, be longing to s ame g enus with B. x ylophilus, was pr eviously considered as no -virulence or w eak pa thogenicity. Because B. mucronatus exists widely in the pine forests of Eurasia, there has been a lot of interest in the h ybridization between B. mucronatus and B. x ylophilus and many investigations c ould be f ound i n t he l iterature. H owever, t he i nvestigations w ere inconclusive a nd m any i ssues r emained unr esolved a nd un addressed. F or e xample, t he enhance pathogenicity of B. mucronatu and the indistinctive classification characteristic of the Bursaphelenchus mucronatus and B. xylophilus and so on. Gaining new insights on the i ssues w ill c ontribute not onl y t o Bursaphelenchus classification, but a lso to the quarantine and control of the pine wilt disease. We c onducted i ndoor h ybridization us ing t he n ematode isolates of B. xylophilus from China, Japan and the nematode isolates of B. mucronatus from China, Japan and France. Our obj ectives w ere to e xamine the tw o species in mating a bility, h ybrid offspring survival and fecundity. The s tudy r esults would provide experimental evidences on t he hybridization of the two species and insights on whether the two species could be merged. Our study has important implications for the classification of genus Bursaphelenchus and pine disease quarantine and control. Male and female adul ts of t he i solates of B. x ylophilus and B.mucronatus, were orthogonal or reverse cr ossed, with the intraspecific self mated and single female adult cultured as control, cultivated 7 days to observe the F1 generation. All the combinations were able t o cross and had the abi lity t o generate F 1 progeny, a lthough t he of fspring counts varied from tube to tube in a range of 17- 44. C ompared with intraspecific self mating, the hybrid of B. xylophilus and B.mucronatus produced a smaller number of F1 generation offspring. In the intraspecific mating, the number of offspring produced each

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tube i s a round 100. In t he i nterspecific m ating, however, t he num ber of F 1 generation offspring is around 50, only half of what is in the intraspecific mating. For the research of F2 generation nematodes production , each F1 generation of 10 larva was s elected and in Botrytis ci nerea slope, 2 5 cultured 7 da y, o bserved und er microscope , and r ecorded t he r esults. From t he results we can see t hat al l t he ℃ combination has F2 generation produced, just differ in nematodes total quantity, larva rate and nematodes vigor. In or der t o unde rstand t he r eproductive c apacity of h ybrid of fspring, w e obt ained the hybrid offspring of Chinese combinations (BxZJ×BmCHN). The result indicated that the hybrid Chinese combination could produce up to 22 generations. The average number of larvae i ncreased w hile the ave rage num ber of a dults de creased w ith a n i ncreasing generation. In addition, ne matode m ortality i ncreased. But s ex r atio w as s table f rom generation to generation. The ba ckcross out comes of di fferent c ross c ombinations w ith their pa rents were al so researched ,and the combination of F1 generation of female adults and their parents were mated. The r esult indi cated that a ll F 1 h ybrids could ba ckcross w ith t heir pa rents a nd produce offspring. Based on these study findings, we propose to merge B. xylophilus and B. mucronatus into one "species" and each be longs t o a " .pathogenic t ype" und er t he “species”.

Keywords: Bursaphelenchus x ylophilus, Bursaphelenchus m ucronatus, interspecific hybridization

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Kato T, Akira K, Ryoji S, Futai K, Takeuchi Y, Phenotypic and genotypic traits of recombinant inbred lines of pine wood nematode, Bursaphelenchus xylophilus. In: Schröder, T. (ed.), Pine Wilt Disease Conference 2013, pp. 74-78, Braunschweig, ISSN: 1866-590X

Phenotypic and genotypic traits of recombinant inbred lines of pine wood nematode, Bursaphelenchus xylophilus

Kato T, Akira K, Ryoji S, Futai K, Takeuchi Y Graduate School of Agriculture, Kyoto University, Kitashirakawa Oiwake-cho, Sakyo-ku, Kyoto 606-8502, Japan Email: [email protected]

ABSTRACT

Pine wood nematode, Bursaphelenchus xylophilus, exhibits a w ide range of intraspecific variation in several bi ological tr aits. Among t hem vi rulence ( degree of pathogenicity), reproductive ability and boarding ability on the vector beetle are important pathogenicity- related traits, although t heir m olecular basis has not been d etermined. In t his s tudy we generated a set of recombinant inbred lines (RILs) of B. xylophilus from two inbred lines, F7 and P9, which greatly differ in the degree of pathogenicity. In addition, we conducted bioassays to estimate above-mentioned three traits in the newly obtained 17 RILs and two parental i nbred l ines. A s a r esult, R ILs s howed va rious vi rulences a nd r eproductions along a continuum a nd two distinct tr ansmission a bilities. This indicates t hat vi rulence and r eproduction m ay b e qua ntitative, pol ygenic trait, while tr ansmission ability is a qualitative trait which is controlled by a single or few genes.

1. INTRODUCTION

Pine wilt is a disease of pine caused by the pine wood nematode, Bursaphelenchus xylophilus, transmitted by vector beetle of the genus Monochamus. In recent years, molecular biological apploach h ave be en v igorously c onducted f or c omprehension of di sease mechanism (e .g. Jones et al. 2008), although the pathogenic factor has not yet determined. In this study we applied a classical genetics to address this matter by using newly conducted recombinant inbred lines (RILs) of B. xylophilus.

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2. MATERIALS AND METHODS

2.1. Construction of RILs Two parental lines, a virulent inbred strain ‘P9’ and an avirulent inbred strain ‘F7’ of B. xylophilus (Shinya et al. 2012), served consecutive full-sib mating (brother-sister mating) to yield a set of R ILs. One unm ated vi rgin female of on e s train w as t ransferred i nto a breeding plate containing one adult male of the other strain (P9 female for F7 male, and vice versa) to let them cross. Unmated adult nematodes of F1 generation thus obtained served crossing to obtain the F2 generation. Each couple of nematodes of F2 generation was used as ancestral RIL for subsequent full-sib mating that was repeated 20 times.

2.2. SSR marker-based characterization of RILs Genomic da ta of B. x ylophilus (sequence d ata ver1.2.) do wnloaded f rom t he G eneDB website (http://www.genedb.org/Homepage) was used to find out the repeated sequences and for each SSR a unique pair of primers were designed. For genotyping a set of RILs, PCR am plification of candi date S SR m arkers w as car ried out using genomic D NA of them as template. PCR amplicons were then separated by electrophoresis and compared.

2.3. Reproductive ability of RILs on grey mould Bursaphelenchus xylophilus of 19 t est population including 17 R ILs and 2 progenitors, the virulent P9 and avirulent F7, was examined for reproduction. The food source fungus Botrytis cinerea was ini tially cultured on PDA medium in a P etri di sh. A ne matode suspension containing 100 individuals was inoculated on the fungal mat and incubated at 25ºC. Twelve days a fter i noculation ne matodes w ere e xtracted and c ounted unde r a stereomicroscope.

2.4. Estimation of Virulence of RILs against Japanese Black Pine Seedlings by Inoculation Test To determine the virulence of each of the RIL populations of B. xylophilus 3-month-old seedlings o f a hi ghly s usceptible J apanese bl ack pi ne, Pinus t hunbergii, s erved as experimental hos t pl ants to be c hallenged. After ma king a le ngthwise s lit tha t r eached cambium on main stem, nematode suspension containing 500 individuals was inoculated into t he i ncision. T wenty seedlings w ere cha llenged with each test popul ation a nd 20 other seedlings were inoculated with an equal volume of distilled water as control. This experiment was repeated 4 times. Seedlings inoculated were incubated for 2 months with weekly health checks.

2.5. Boarding Ability of RILs on Vector Beetle To create a culture vessel, barley and woodchip of Japanese red pine (Pinus densiflora) were added in this order to a glass tube and plugged. The tube was inoculated with the

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blue-stain fungus (Ophiostoma minus). After incubation, 100 B. xylophilus individuals of each test population was inoculated to the tube and incubated for another 2 weeks. Finally a l arva o f Monochamus a lternatus, obt sined f rom naturally-infected pine t rees, was introduced t o t he t ube, i ncubated a nd m onitored a t t he s ame hour e very da y. Eclosed beetle was taken from the tube and nematodes were extracted and counted both from the beetle and from the medium in the tube.

3. RESULTS AND DISCUSSION

3.1. Construction and SSR marker-based characterization of RILs Using two separate inbred lines of B. xylophilus, P9 and F7, a set of 17 RILs derived from 17 couples of F2 generation has been generated. Among them was eight RILs descended from F7 female and P9 male, and nine RILs descended from P9 female and F7 male. A search of the genomic da ta of B. xylophilus permitted 16 S SRs primer d esign in different s caffolds. These SSRs PCR-amplified with uni que primer pairs s howed pol ymorphism across t he two progenitors, i.e. P9 and F7, and they were therefore used in genotyping of RILs as SSR marker. As a result, 17 RILs showed unique genotype different from each other with high degree of homozygosity ranging from 0.88 to 1.00 (0.99 in average).

3.2. Reproductive Ability of RILs on Grey Mould Change i n t he num ber of B. x ylophilus grown on the fungus is sho wn i n Figure 1 . Reproductive ability shown by the newly obtained RILs were intermediate between those shown b y t he t wo pr ogenitors; t hat i s, no R IL s howed a s ignificantly l arger or a significantly s maller po pulation t han P 9 or F 7, r espectively. Thus t he re sultant R IL populations showed a continuously varying distribution of reproductive ability, which can be explained by quantitative inheritance controlled by polygene.

Figure 1. T he num ber of each RIL population grown on B. cinerea for 12 days.

3.3. Estimation of Virulence of RILs against Japanese Black Pine Seedlings by Inoculation Test Figure 2 shows increase of the number of dead P. thunbergii seedlings after inoculation of B. xylophilus. The s eedling m ortality, which indicates vi rulence of t he B. xylophilus isolate i noculated, w idely v aried, f rom 0% t o 58% . T he pr ogenitor F 7 c aused 1% mortality, while the other progenitor P9 caused 49% mortality. Distribution of mortality

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resulted from the 17 RIL populations varied along a continuum between those from the progenitors, w ith two exceptions. T his s uggests t hat vi rulence of B. xylophilus is a quantitative trait to which a combination of several genes contributes.

Figure 2. M ortality of pine s eedlings after inoc ulation with each of t he R IL populations

3.4. Boarding Ability of RILs on Vector Beetle Data obtained by coculture of B. xylophilus with its vector beetle is summarized in Figure 3. Three R ILs w ere om itted since no nematode of fspring w as ex tracted from ei ther of beetle body and medium in culture vessels. All RILs showed a largely similar fluctuation pattern in number o f t otal ne matodes recovered f rom t he b eetle bod y and m edium decreased w ith time. Multiple c omparisons de monstrated t hat t he pr ogenitor P 9 ha d a significantly hi gher v alue t han t he ot her pr ogenitor F 7. R ILs w ere di vided i nto t wo groups; one gave ex tremely l ow s cores i n the s imilar m anner t o F7, and the other generated a large number of boarded nematode that got aboard in the similar manner to P9. Thus the pattern of inheritance for boarding ability seems monogenic, suggesting that this trait is influenced by one or few genes with a major effect.

Figure 3. t he num ber of J iv ne matode boarding on M. al ternatus in each of RILs populations

4. ACKNOWLEDGEMENTS

This s tudy w as s upported b y KAKENHI (numbers 23248024, 25660121) a nd Inamori Foundation.

REFERENCES Shinya R; Takeuchi Y; Ichimura K; Takemoto S; Futai K (2012). Establishment of a set of inbred strains of the pine wood nematode, Bursaphelenchus xylophilus

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(Aphelenchida: Aphelenchoididae), and evidence of their varying levels of virulence. Appled Entomology and Zoology 47, 341-350. Jones JT; Moens M; Mota M; Li H; Kikuchi T (2008). Bursaphelenchus xylophilus: opportunities in comparative genomics and molecular host-parasite interactions. Molecular Plant Pathology 9, 357-368.

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Zhu l-H, Ye J-R, Huang L, Pathogenicity, reproduction and survival of axenic Bursaphelenchus xylophilus In: Schröder, T. (ed.), Pine Wilt Disease Conference 2013, pp. 79, Braunschweig, ISSN: 1866-590X

Pathogenicity, reproduction and survival of axenic Bursaphelenchus xylophilus

L.-H. Zhu, J.-R. Ye and L. Huang Institute of Forest Protection, College of Forest Resources and Environment, Nanjing Forestry University, Nanjing, 210037, P. R. China Jiangsu Key Laboratory for Prevention and Management of Invasive Species, Nanjing, 210037, P. R. China. [email protected]

Pine wilt disease (PWD) is the most serious tree epidemic which causes vast catastrophic damage t o pi ne forests. For a lon g time, the pi ne w ood nematode ( PWN), Bursaphelenchus x ylophilus was be lieved t o be the onl y p athogenic agent c ausing t he disease. More r ecently, it has be en h ypothesized t hat some bacteria as sociated with B. xylophilus may play a c rucial r ole on pi ne w ilt. T he r ole of nematodes and associated bacteria i n PWD development remains t o be f urther s tudied. Here, we f ocused on t he biology of axenic B. xylophilus. The pathogenicity of axenic B. xylophilus was tested by inoculating greenhouse-grown 4- year-old seedlings and 6 -month-old axenic microcuttings of P. densiflora with aseptic PWNs a nd non-aseptic P WNs. Seedlings w ere i noculated with 5,000 PWNs. Microcuttings were i noculated unde r a xenic conditions w ith 200 nematodes. After 20 days, the microcuttings inoculated with aseptic PWNs and non-aseptic PWNs wilted, and the wilting ratios were 90% and 80%, r espectively. The av erage num bers of recovered nematodes were (364 +355) and (66+52) per microcutting, respectively. Similarly, after 38 days, both of aseptic PWNs and non-aseptic PWNs wilted 80% of greenhouse-grown seedlings, with (34733 + 34162) and ( 25057 + 21410) nematodes per seedling, respectively. To compare the reproduction of aseptic PWNs and non-aseptic PWNs, 100 nematodes were transferred into a PDA plate with Botrytis cinerea and cultured at 25°C. One week later, the nematodes were isolated from the plate and aseptic and non-aseptic nematodes were counted. The results showed that there was no significant difference in the number between them. Furthermore, the survival of aseptic PWNs a nd non-aseptic PWNs under axenic conditions w as s tudied. F ive t housand PWNs we re maintained in flasks containing 2 ml sterile water and incubated at 25°C. After 36 days, the survival rate of non-aseptic PWNs was lower than 5%, on t he other hand, the survival rate of aseptic PWNs was about 50%. Based on our research, it c an be concluded t hat aseptic B. xylophilus does not lost its pathogenicity character. Also, it w as a mazing to s ee the m live lon ger tha n non-aseptic one under axenic condition.

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Gao RH , Shi J, Luo YQ. Influence of pine wood nematode invasion on typical Masson pine ecosystem in Three Gorges Reservoir Region of China. In: Schröder, T. (ed.), Pine Wilt Disease Conference 2013, pp. 80-81, Braunschweig, ISSN: 1866-590X

Influence of pine wood nematode invasion on typical Masson pine ecosystem in Three Gorges Reservoir Region of China

Rui-he Gao, Juan Shi, You-qing Luo Beijing Forestry University, No.35 Tsinghua East Rd, Haidian District, Beijing 100083. Peoples Republic of China Email: [email protected]

ABSTRACT:

Due t o complex terrain and bi ological di versity, t he Three Gorges Reservoir Region is becoming hot spot region for r esearches f rom C hina t o t he w orld. H owever i n r ecent years, the invasion of Bursaphelenchus xylophilus (pine wood nematode ,PWN) caused a devastating impact on M asson pine stand ecosystem and terrible effects on water quality of Yangtze River as well as the ecological safety of Three Gorges Dam.The purpose of this r esearch i s t o s erve f or pr otecting t he ecological safety of Three Gorges Dam and pine resources in Three Gorges Reservoir region.Based on “sample plots setting” and “the measurement of al l i ndividual t rees” m ethods, we ana lyzed the species composition , diversity changes and t he d ynamic changes o f structure a nd f unction o f M asson pi ne communities a fter a ttacked by P WN w ith different years（0year,1year,3year,5year and 7year） in 2012. R esults indicated, for the pine stand ecosystem infected by PWN, pure Masson pi ne f orest h ad e volved i nto coniferous a nd br oad-leaved mixed forest. Moreover, the Masson pine was ranked as the dominant species meanwhile some broad- leaved trees, such as Cinnamomum camphora, Quercus aliena, Quercus variabilis Blume and Loropetalum chinensis, were ranked as the subdominant species. As for the indicators that reflect healthy status of Masson pine’s structure and function, the healthy pine stand ecosystem was higher than infected pine one. With the increasing of infected years, each indicator showed a trend of decreasing. Through analyzing the relationship between pine wilt disease and stand structure in infected pine stand ecosystem, results indicated that the invasion of PWN had great influence on biological diversity of arbor, shrub and herb. In general, the relationship between species diversity indicators and infected years followed the “Mid-altitude bul ge” t heory. S pecifically, both one -year i nfected and seven-year infected Masson pine forest would have the decline of plant species diversity in certain degree. Various practices could be carried out to prevent the further spread of PWN, to

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improve the simple structure of Masson pine forest into a complex one for increasing the pine forest resistant ability in Three Gorges Reservoir region.

Key words: Three Gorges Reservoir region; pine wilt disease; plant community; Masson Pine; ecosystem

REFERENCES Dropkin VH; Foudin AS; Kondo E (1981). Pine wood nematode: a threat to US forest? Plant Disease 65,1022-1027. Gentry AH (1988). Changes in plant community diversity and floristic composition on environmental and geographical gradients. Annals of the Missouri Botanical Garden 75,1-34. Shi J; Luo YQ; Yan XS; Chen WP;Jiang P (2007). Effects of different disturbance ways on the diversity of pine forest invaded by pine wood nematode. Ecological Science 26, 289-292. Yoshimura A; Kawasaki K; Takasu F (1999). Modeling the spread of pine wilt disease caused by nematodes with pine sawyers as vector. Ecology 80,1691-1702. Zhang WL; Liu J; Wang JZ; Chen FQ (2010). Soil heterotrophic respiration and its temperature sensitivity in different-aged orange plantations in Three Gorges Reservoir area of China. Chinese Journal of Plant Ecology 34,1265-1273.

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Gruffudd H, Evans H F, Jenkins T, Using an evapo-transpiration model to predict the current and future range and severity of pine wilt disease caused by pine wood nematode, Bursaphelenchus xylophilus in Europe. In: Schröder, T. (ed.), Pine Wilt Disease Conference 2013, pp. 82-83, Braunschweig, ISSN: 1866-590X

Using an evapo-transpiration model to predict the current and future range and severity of pine wilt disease caused by pine wood nematode, Bursaphelenchus xylophilus in Europe

Gruffudd H, Evans H F, Jenkins T Forest Research in Wales, Edward Llwyd Building, Penglais Campus, Aberystwyth, Wales, UK, SY23 3DA Email: [email protected], [email protected], [email protected]

ABSTRACT

Pine Wilt Disease (PWD) is a x ylem restricting disease of pine trees, caused by the Pine Wood Nematode (PWN) Bursaphelenchus xylophilus. The nematode is carried from host tree t o hos t t ree b y v ector l onghorn beetles i n t he genus Monochamus. The i nteraction between the nematode and beetle is crucial in the establishment and spread of the disease. PWN, a na tive of N orth A merica w here i t doe s not ki ll pi ne t rees, ha s s pread internationally ki lling t rees i n J apan, C hina, K orea, T aiwan and, f rom 1999, P ortugal. Based on the locations where tree mortality has been recorded, it appears that pine trees growing in hot, dry conditions are more susceptible to the nematode, resulting in pine wilt disease. Results in the literature s how t hat t he g rowth a nd de velopment of P WN i s temperature-dependent a nd t hat t here i s a t emperature r ange, out side w hich ne matode development i s r estricted. In t his pa per w e describe t he E TPN m odel, a n e vapo- transpiration model (previously developed by Forest Research), which has been modified to incorporate the presence of PWN inside a tree, to predict the regions of Europe that are likely to succumb to pine wilt disease. ETPN acts independently of the vector; hence we predict t he l ikelihood of P WD, a ssuming t hat a t ree i n a p articular r egion has be en infested by the pine wood nematode.

We have run the ETPN model for various locations in Japan, where PWD has been killing pine trees for over a century. The results of the ETPN model are in good agreement with observations in Japan and provide strong validation for the model.

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We ha ve a lso c onsidered di fferent r egions i n E urope: P ortugal; w here PWD ha s be en found, France; where we would not currently expect to see PWD, but a region that might become s uitable un der future cl imates and Sweden; w here w e w ould not ex pect t o see PWD. We de monstrate how t he di fferent cl imates of t hese t hree r egions g ive ve ry different r esults. F inally, w e c onsider va rious c limate c hange s cenarios t o de monstrate how PWD is likely to affect different regions in Europe in the future, especially in areas where there might be a shift from nil to low likelihood of PWD to a higher likelihood of tree mortality.

ACKNOWLEDGEMENTS

This study was funded by the E.U. through the project REPHRAME (KBBE.2010.1.4-09)

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Huang R, Shi J, Luo Y, Cold-tolerance and adaption of Pine wood nematode in China. In: Schröder, T. (ed.), Pine Wilt Disease Conference 2013, pp. 84-85, Braunschweig, ISSN: 1866-590X

Cold-tolerance and adaption of Pine wood nematode in China

Rui-fen Huang, Juan Shi, You-qing Luo Beijing Forestry University, No.35 Tsinghua East Rd, Haidian District, Beijing 100083. Peoples Republic of China Email: [email protected]

ABSTRACT:

Bursaphelenchus x ylophilus (pine w ood n ematode, P WN) i s a ki nd of plants pa rasitic nematode w hich s urvival i n t ropical a nd t emperate z one, t emperature i s a n i mportant environmental factor affecting i ts spread areas. From 2012 t o 2013, w e collected PWN species from the different location which represent separately is the most north (Shannxi province), the most south(Guangdong province), and the middle part (Zhejiang and Hubei province) of distribution area of PWN in China to explore the influence of temperature on different geographical populations.Results showed that PWN are depressed by - 5℃ for 24 h, there exists difference in survival of the PWN among different regions .The survival rate of Shannxi, Zhejiang，Hubei and Guangdong strain are 39.10%, 37.48% ，48.51% and 29.21% separately. In addition, the survival of pine wood nematode after cultivation of 20 d a t 15 ℃ was ob viously hi gher t han t hat c ultivation of 20d a t 2 5℃. In general, there exists some cold tolerance and adaption ability of PWN in China, which improved the s urvival of P WN i n C hina, the de ep r eason t hat how l ow t emperature c ultivation improve the PWN survival would be further discussed in the future.

Key words: pine wood nematode，cold-tolerance，cultivation.

REFERENCES Chen C; Xie H; Xu CL（2008）. Cold tolerance abilities of Radopholus similis (in Chinese). Journal of Huazhong Agricultural University 27: 49-51. Dai SM; Cheng XY; Xiao QM(2006). Research progress in Nematode cold tolerance. Acta Ecologica Sinica 26: 3885-3890. Zhang JP; Cai X(2007). The Biological Effect of the Temperature on Bursaphelenchus xylophilus. Journal of Sichuan Forestry Science and Technology 28: 69-72.

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Farman Ali; David A. Wharton(2013). Cold tolerance abilities of two entomopathogenic nematodes, Steinernema feltiae and Heterorhabditis bacteriophora. Cryobiology 66: 24-29. Jagdale G B; Grewal P S (2003). Acclimation of entomopathogenic nematodes to novel temperatures: trehalose accumulation and the acquisition of the tolerance.Int . J. Parasitol 33: 145- 152.

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Huaying Z, Ming Xu, Fuyuan Xu, A comparative proteomics analysis on resistant provenance of Pinus massoniana inoculated with Bursaphelenchus xylophilus. In: Schröder, T. (ed.), Pine Wilt Disease Conference 2013, pp. 86, Braunschweig, ISSN: 1866-590X

A comparative proteomics analysis on resistant provenance of Pinus massoniana inoculated with Bursaphelenchus xylophilus

Zheng Huaying, Xu Ming, Xu Fuyuan Forestry Academy of Jiangsu Province, 211153, dongshanqiao, Nanjing, China Email: [email protected]

ABSTRACT:

Pine wilt disease caused by B. xylophilus, also known as the pine wood nematode (PWN), is the most devastating disease of pine trees. From different geographical provenance of

P. massoniana were inoculated with nematodes, test results selected the provenance GD5 which has strongly resistance to PWN. This article used resistant provenance GD5 as the

experimental material, and sensitive provenance SX1 as comparison. Total proteins were extracted by us ing 2-DE and MALDI-TOF/TOF technology from the provenances pine needles respectively. Differentially expressed proteins in the provenance before and two weeks a fter i noculated with PWN, were ana lyzed. At l ast, 89 differentially ex pression proteins were successfully identified by MALDI-TOF–TOF. The test result also fund that there w ere f ive pr oteins i nvolved in hydrogen peroxide s cavenging c apacity an d protecting the redox homeostasis system from damaged. Their up-regulation may be the

main cause of the provenance GD5 resistant to PWN. Keywords: Pinus massoniana, pine wilt disease, resistant provenance, proteomic, 2-DE, MALDI-TOF/TOF

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Egas et al. Comparative transcriptomics to understand the molecular basis of Bursaphelenchus xylophilus pathogenicity. In: Schröder, T. (ed.), Pine Wilt Disease Conference 2013, pp. 87-89, Braunschweig, ISSN: 1866-590X

Comparative transcriptomics to understand the molecular basis of Bursaphelenchus xylophilus pathogenicity

Egas C1, Leisico F2, Duarte T1, Gomes P1, Fonseca L3, Abrantes I3, Pinheiro M1. 1 Genoinseq, Biocant Park, 3060-197 Cantanhede, Portugal; 2 Department of Chemistry University of Aveiro, Campus de Santiago, 3810-193 Aveiro, PT 3 Department of Life Sciences, University of Coimbra, 3001-401 Coimbra, Portugal Email: [email protected]

Pinewood ne matode ( PWN) bi ology a nd e cology are s trictly associated t o pi ne w ilt disease (PWD) and have been extensively investigated. However, the disease molecular mechanism ha s not yet be en e stablished. Aiming to unravel the mechanism o f pathogenicity w e us ed a t ranscriptomics approach t o s tudy the gene e xpression of Bursaphelenchus x ylophilus and the cl osely r elated Bursaphelenchus m ucronatus. Furthermore, w e s equenced t he t ranscriptomes of B. x ylophilus males, females and dispersal j uveniles JIII. We t hen bui lt a t ranscriptomics pl atform t o c arry out e ducated searches on differential g ene e xpression t o hi ghlight t he m olecular basis of PWN pathogenicity or discover new targets with high interest for nematode control. The f ive t ranscriptomes w ere s equenced i n t he 454 pl atform ( Roche). Pyrosequencing generated on a verage 45 5,000 reads and 8,500 t ranscripts per t ranscriptome; more than 60% of t hese corresponded t o InterPro t erms ( Table 1) . Nucleotide a nd a minoacid sequences a nd c orresponding a nnotation w ere o rganized i n a w eb-based da tabase. The huge a mount of da ta generated r epresents a n important oppor tunity t o i ncrease t he available scientific knowledge on the nematode and to carry out comparative analysis.

Table 1. Summary of sequencing, a ssembly and a nnotation data. B. xylophilus and B. mucronatus mixed stages were collected from fungal cultures, while B. xylophilus males, females and JIII were collected from infected pines. Total RNA was isolated from each nematode isolate, and cDNA synthesized according to the SMART technology. T ranscript assembly a nd annotation were performed as described in Bettencourt (2010).

B. xylophilus B. mucronatus B. xylophilus (pine) (fungi) (fungi) male female JIII # Reads 647,641 465,256 407,835 227,307 531,049

# Transcripts 11,006 8,822 6,724 5,760 10,608 # Amino acid sequences 12,038 9,231 6,897 6,013 11,444 # Amino acid sequences 7,321 5,547 4,148 4,135 7,120 assigned to InterPro

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The pl atform w as qu eried f or t hree m ain c omparisons: B. x ylophilus versus B. mucronatus grown i n f ungi; PWN males versus f emales ve rsus J III grown on pi ne a nd PWN grown on fungi versus growth in pine. The transcriptomes were compared for genes exclusively present in each condition according to different annotation strategies such as KEGG a nd G ene O ntology, a nd a lso ba sed on sequences s imilarity us ing the C D-HIT program ( Huang 2010) . In addition, w e a lso s tudied t he gene expression of pot ential nematode parasitism effetors directly on the database using Myrna as described in Santos (2012). T he c omparisons i dentified m ore t han 30 g enes po tentially inv olved in PWD parasitism that are being experimentally validated by RT-PCR. Here, w e f ocus on t he c omparative gene e xpression of ox idative r esponse g enes, ubiquitination-related genes and a potential secreted purple acid phosphatase (Figure 1). The gene expression experimental validation indicated an overexpression in the oxidative response i n males and J III when c ompared t o f emales grown i n pi ne a nd a lso a n overexpression i n B. m ucronatus when c ompared t o B. x ylophilus grown on f ungi. Interestly, onl y B. x ylophilus males s howed a n ove rexpression of 4H PPD ( 4- hydroxyphenylpyruvate di oxygenase) and a n u nderexpression of H GD ( homogentisic acid oxidase), indicating that also homogentisate can be a potentially important nematode oxidative de toxification m echanism i n pi ne ( Martin and B atkoff, 1987; Arias-Barrau, 2004). Ubiquitination-related genes were overexpressed in females when compared to the other B. x ylophilus samples, suggesting a n i mportant r egulation of i nternal pr oteolytic activity, probably to reach normal homeostasis required for reproduction (Comyn, 2013).

The marked overexpression of secreted PAP (purple acid phosphatase) in females and JIII when compared to B. xylophilus grown in fungi and males grown in pine may represent a potential role in the nematode interaction with pine due to the relation of PAPs to a wide range of actions such as reactive oxygen species generation (Schenk, 2013).

Figure 1. Differential gene expression determined by RT-PCR. Gluthathione peroxidase (GPx), superoxide dismutase ( SOD), cat alase ( CAT) were s tudied i n t he five co nditions, while 4 -hydroxyphenylpyruvate dioxygenase (4HPPD), homogentisic acid oxidase (HGD), Ubiquitin-specific-processing protease 33 ( Usp 33), Ubiquitin-specific-processing protease 14 (Usp 14) and purple acid phosphatase (PAP) were studied in the four B. xylophilus samples. Data is presented under the form mean±SD (standard deviation). * indicates significative differential expression with p < 0.05 as determined with the REST software. The cell division control protein 42 was the endogenous control and B. xylophilus grown in fungi the control sample.

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Additional di fferentialy e xpressed g enes c orresponded t o pe ptidases a nd r espective inhibitors, c arbohydrate-active e nzymes, genes i nvolved i n ox idative de toxification, phenolic c ompound de gradation or hos t m imicking. T hese genes s howed higher expression levels for t he nematode grown i n pi ne, and al so differences be tween the 3 developmental stages. These results suggest differences between males, females and JIII while growing in pine and may elucidate the contribution of the different stages to the PWN pathogenicity. Results a nd di scussion of t hese g enes will be pr esented at th e meeting.

REFERENCES

Arias-Barrau E; Olivera ER; Luengo JM; Fernández C; Galán B; García JL; Díaz E; Miñambres B (2004). The homogentisate pathway: a central catabolic pathway involved in the degradation of L-phenylalanine, L-tyrosine, and 3- hydroxyphenylacetate in Pseudomonas putida. Journal of Bacteriology 186, 5062- 5077. Bettencourt R; Pinheiro M; Egas C; Gomes P; Afonso M; Shank T; Santo RS (2010). High-throughput sequencing and analysis of the gill tissue transcriptome from the deep-sea hydrothermal vent mussel Bathymodiolus azoricus. BMC genomics 11, 559. Comyn S; Chan G; Mayor T (2013). False start: Cotranslational protein ubiquitination and cytosolic protein quality control. Journal of Proteomics (0). Huang Y; Niu B; Gao Y; Li W (2010). CD-HIT Suite: a web server for clustering and comparing biological sequences. Bioinformatics 26(5), 680-682.

Martin Jr JP; Batkoff B (1987). Homogentisic acid autoxidation and oxygen radical generation: implications for the etiology of alkaptonuric arthritis. Free Radic Biol Med 3, 241–250. Santos CS; Pinheiro M; Silva AI; Egas C; Vasconcelos MW (2012). Searching for resistance genes to Bursaphelenchus xylophilus using high throughput screening. BMC Genomics 13, 599. Schenk G; Mitić N; Hanson GR; Comba P (2013). Purple acid phosphatase: A journey into the function and mechanism of a colorful enzyme. Coordination Chemistry Reviews 257(2), 473-482.

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Huang L et al. The Function of Major Sperm Proteins (MSPs) in reproduction of pine wood nematode. In: Schröder, T. (ed.), Pine Wilt Disease Conference 2013, pp. 90-92, Braunschweig, ISSN: 1866-590X

The Function of Major Sperm Proteins (MSPs) in reproduction of pine wood nematode

Lin Huang, Minqi Tian, Xiuwen Qiu, Yi Zhang, Xiaoqin Wu, Jianren Ye Institute of Forest Protection, College of Forest Resources and Environment, Nanjing Forestry University, Nanjing, 210037, P. R. China. Email:[email protected]

Abstract BxMSP1, B xMSP2 a nd B xMSP3 of Bursaphelenchus x ylophilus were c loned i n t his study. T he s enior s tructure of t hese pr oteins w as r ich of β sheets, w hich w as hi ghly conserved in MSP members of the nematode species. In situ hybridization showed that these genes were specifically expressed in the seminal vesicle tissue of the male adults. The reproduction ability of B. xylophilus decreased when the nematodes were soaked by the dsRNA of BxMSP1 and BxMSP2. qPCR analysis showed lower transcript abundance of t he t argeted m RNAs when t he nematodes were s oaked b y t he ds RNA of t he M SPs. These results indicated that BxMSP1 and BxMSP2 were required for reproduction of B. xylophilus.

1 Introduction The major s perm pr otein (MSP) i s a nematode specific protein. M SP ha s f irst b een identified in Caenorhabditis elegans. It is the most abundant protein present in nematode sperm, MSP is t he ke y player i n t he m otility m achinery of n ematodes t hat pr opels t he crawling movement of ne matode s perm in C. elegans. But t he f unction of M SP i n t he plant ne matode is still very limited known. In this paper, three MSPs were cloned form B. xylophilus and gene function were identified by RNAi method.

2 Materials and methods BxMSP1, B xMSP2 a nd BxMSP3 were c loned b y t he m ethods of t ranscriptomic sequencing and r apid a mplification of c DNA ends ( RACE). In s itu hybridization was used to locate the gene expressed tissue site. Nematodes were treated 48h by the dsRNA of BxMSP1, BxMSP2 and BxMSP3 respectively. Then these nematodes were fed on the Botrytis c inea to e valuate t he r eproduction ability. qP CR w as us ed t o d etect t he gene expressed level.

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3 Results 3.1 Gene cloning of BxMSP1, BxMSP2 and BxMSP3 Genomic s equence analysis i ndicated that BxMSP1, BxMSP2 and BxMSP3 contained a intron respectively. Mobile-Sperm domain was contained in these MSPs. These proteins was rich of β sheets, which was highly conserved in MSP members of the nematode species. 3.2 Tissue expression site of MSPs In s itu hybridization showed that thr ee MSPs were s pecifically expressed in the seminal ve sicle tis sue of the ma le a dults. There i s no h ybridization s ignal i n t he females and larvas. 3.3 RNAi of MSPs dsRNA of BxMSP1 and BxMSP2 significantly suppressed the reproduction ability of B. xylophilus, and significantly decreased the yield and the hatching rate of eggs. But dsRNA of BxMSP3 had no significantly effect on the nematode reproduction. 3.4 qPCR of gene expressed level of MSPs qPCR i ndicated t hat ds RNA of BxMSP1 a nd BxMSP2 s ignificantly d ecreased t he expression l evel of B xMSP1 a nd B xMSP2. B ut t he ds RNA of BxMSP3 ha d no significantly effect on the e xpression l evel of BxMSP3. T hese r esults i ndicated BxMSP1 a nd B xMSP2 ha d i mportant r oles of r egulating t he reproduction of B. xylophilus.

4 DISCUSSION

The pi ne wood nematode is a di sastrous pathogen of t he pi ne forests i n East Asia and European. But because of limited understanding of its pathogenic mechanism, there is no efficient measures to control this nematode. In t his study, BxMSP1 and BxMSP2 were required for reproduction of B. xylophilus. But how these genes regulate the pine wood nematode is still unknown. The interactional proteins and regulation network of BxMSP1 and BxMSP2 in the reproduction process need to be illuminated in the future. These work will help us to understand the molecular mechanism of nematode sperm development and reproduction. It is useful for screening the potential target gene for control this nematode.

Acknowlegement This research was supported by the National Natural Science Foundation of Chian (No. 31000303) and the Natural Science Foundation of Jiangsu Province (No. BK2010566).

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REFERENCES Ashcroft N R; Srayko M ; Kosinski M E; M ains PE; G olden A ( 1999). RNA-Mediated interference of a cdc25 ortholog in Caenorhabditis elegans results in defects in the embryonic c ortical me mbrane, meiosis and m itosis. Developmental B iology 206,15-32. Kosinski M; McDonald K; Schwartz J; Yamamoto I; Greenstein D (2005) . C. elegans sperm bud vesicles to deliver a me iotic ma turation signal to di stant ooc ytes. Development, 132: 3357-3369. Kikuchi T ; Cotton J A; Dalzell J J; Hasegawa K ; Kanzaki N ; et al (2011). Genomic insights i nto t he origin of parasitism in the emerging plant pathogen Bursaphelenchus x ylophilus. PLoS P athogens, 7(9): e 1002219. doi:10.1371/journal.ppat.1002219

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Vicente C et al., Exploring the relation between virulence and oxidative stress response of Bursaphelenchus xylophilus and Bursaphelenchus mucronatus. In: Schröder, T. (ed.), Pine Wilt Disease Conference 2013, pp. 93-94, Braunschweig, ISSN: 1866-590X

Exploring the relation between virulence and oxidative stress response of Bursaphelenchus xylophilus and Bursaphelenchus mucronatus

Vicente C, Ikuyo Y, Mota M, Hasegawa K ICAAM - Instituto de Ciências Agrárias e Ambientais Mediterrânicas, Departamento de Biologia, Universidade de Évora, Núcleo da Mitra, Ap. 94, 7002-554 Évora, Portugal Email: [email protected]

Tolerance to host-mediated oxidative stress (OS) conditions is an essential characteristic of pl ant-parasitic or ganisms. Susceptible Pinus thunbergii reacts t o Bursaphelenchus xylophilus invasion with a strong oxidative burst (Hirao et al., 2012), which may indicate that virulent B. xylophilus must possess an efficient antioxidant system to cope with these conditions. Previous s tudies ha ve s uggested t hat P RX ( 2-cysteine pe roxiredoxin), GST (glutathione S -transferase) and GAPDH, all l ocalized in t he s urface coa t, are pot ential scanvengers of B. xyophilus to plant reactive oxygen species (ROS) (Shinya et al., 2010; Li et al ., 2011) . M ore r ecently, 12 a nti-oxidant pr oteins w ere ide ntified in the B. xylophilus secreotome a fter pl ant e xtract s timuli, emphazing the ir imp ortance in the control of global oxidative stress of B. xylophilus (Shinya et al., 2013). In this work, our main concern was to study of OS tolerance of B. xylophilus isolates and B. mucronatus and t he r elation w ith t heir pa thogenicity ( virulence l evel) t o s usceptible pi ne s pecies. Previous results (Vicente et al., submitted) have al ready suggested a relation virulence- OS tolerance among B. xylophilus isolates virulent Ka4 and avirulent C14-5. So, firstly, three B. xylophilus isolates, Ka4 and T 4 (virulent) a nd C 14-5 ( avirulent), a nd one B. mucronatus (avirulent) were tested for OS tolerance using hydrogen peroxide as oxidative agent, in concentrations ranging from 0-40 mM H2O2. After 24h-exposure to this oxidant agent, nematode survival was checked. A clear difference between virulent and avirulent isolates w as r ecorded i n O S c onditions, e ven i n t he l owest H 2O2 concentration. T he virulent isolates (Ka4 and T4) presented lower mortality percentage in all concentrations than avirulent ones (C14-5 and B. mucronatus). Statistical differences between Ka4 and

T4 w ere a lso found unt il 30m M H 2O2 treatment, being K a4 the mos t r esistant is olate. Concerning a virulent i solates, m ortality p ercentage w as hi gher t han 90% i n a ll concentrations, with no s tatistical differences found between C14-5 and B. mucronatus. Next, w e a ssessed t ranscription l evels of 5 m ain a ntioxidant a nd de toxifying e nzymes during t he O S c onditions ( 15mM H 2O2, 24h -exposure), a nd c ompared w ith nor mal conditions (no stress applied) by qRT-PCR. The following enzyme genes were analysed:

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CTL ( catalases, Bxy-ctl-1 and Bxy-ctl-2), S OD ( superoxide di smutase, Bxy-sod-1, Bxy- sod-2 and Bxy-sod-3), GXP (glutathione peroxidase, Bxy-gxp-1, Bxy-gxp-2 and Bxy-gxp- 3), GST (glutathione S-transferase, Bxy-gst-1 and Bxy-gst-3) and PRDX (peroxiredoxin, Bxy-prdx-2). In the case of B. mucronatus, this analysis was not possible to conduct since no information is available about its genome. From the selected enzymes, only Bxy-ctl-1 and -ctl-2 were significantly upregulated (P<0.05) in virulent isolates Ka4 and T4. In the case o f C 14-5, onl y Bxy-ctl-2 was significantly downregulated (P<0.05) in comparison with nor mal c onditions. For S ODs and GPXs, there w ere no statistical di fferences between isolates, although we could assess t hat Bxy-sod-1 and -2 were ne arly 1-fold upregulated for T4; Bxy-sod-3, Bxy-gxp-2 and Bxy-gxp-3 for Ka4 and T4 were expressed at the same level than normal conditions; and that Bxy-sod-2 and -3, and Bxy-gxp-2 and -3 were downregulated for avirulent C14-5. Concerning the detoxifying enzymes GST and PRDX: Bxy-gst-1 of i solates K a4 and T4 were, r espectively, downregulated and unchanged unde r O S c onditions, a nd t hat e xpression of Bxy-prdx-2 for bot h vi rulent isolates was suppressed in stress conditions. In contrast, Bxy-gst-1 of avirulent C14-5 was upregulated i n O S c onditions a nd Bxy-prdx-2 remained una ltered. G ST-3 w as not detected in all isolates. Following, we will analyse gene expression of these enzymes in in vivo conditions for all B. xylophilus isolates to ascertain the global oxidative status of the nematode a s r esult of n atural ox idative s tress c onditions. W e w ere able c heck 100 % sequence similarity of coding sequences of CTLs, SODs and GXPs for Ka4, T4 and C14- 5, suggesting t hat i f di fferent enz ymatic act ivities ar e pr esented may be due t o posttranslational modifications. Based in these results, we hypothese a possible positive correlation between the level of OS tolerance and the level of virulence of B. xylophilus, which can be further investigated as a virulence marker.

REFERENCES Hirao T, Fukatsu E, Watanabe A. (2012) Characterization of resistance to pine wood nematode infection in Pinus thunbergii using suppression subtractive hybridization. BMC plant biology 12:13. Li Z, Liu X, Chu Y, Wang Y, Zhang Q, Zhou X (2011) Cloning and characterization of a 2-Cys peroxiredoxin in the pine wood nematode, Bursaphelenchus xylophilus, a putative genetic factor facilitating the infestation. International Journal of Biological Sciences 7:823-836. Shinya R, Morisaka H, Takeuchi Y, Ueda M, Futai K (2010) Comparison of the surface coat proteins of the pine wood nematode appeared during host pine infection and in vitro culture by a proteomic approach. Phytopathology 100, 1289–97. Shinya R, Morisaka H, Kikuchi T, Takeuchi Y, Ueda M, Futai K (2013) Secretome analysis of pine wood nematode Bursaphelenchus xylophilus reveals the tangled roots of parasitism and its potential for molecular mimicry. PloS One 8:e67377. Vicente C S L, Ikeyo Y, Mota M, Hasegawa K. Pinewood nematode-associated bacteria contribute to oxidative stress resistance of Bursaphelenchus xylophilus. BMC Microbiology submitted.

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Poster Presentations

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Sarniguet C et al., Bursaphelenchus xylophilus identification, from literature to routine analysis: how to make morphological analysis reliable. In: Schröder, T. (ed.), Pine Wilt Disease Conference 2013, pp. 96-97, Braunschweig, ISSN: 1866-590X

(101) Bursaphelenchus xylophilus identification, from literature to routine analysis: how to make morphological analysis reliable.

Sarniguet C, Buisson A, Anthoine G Laboratoire de la santé des végétaux, Unité de nématologie, Domaine de la Motte, BP35329, F-35653 Le Rheu cedex, France Email: [email protected]

Official surveys on Bursaphelenchus xylophilus are conducted in a compulsory manner since 2000 in European Union including in France. Considering the Portuguese situation and t he di spersal of Bursaphelenchus x ylophilus throughout i ts t erritory a nd recent occurrences in Spain, the reliability of sampling and analysis undertaken is a critical issue to early detect any further dispersal. The i mplementation of quality assurance f or of ficial ana lysis al so led to evaluate t he reliability of identification methods, based on morphology or on molecular principles. Published morphological identification keys to xylophilus group (Braasch et al. 2009) and to B. x ylophilus species l evels ( EPPO 2009) w ere co nsidered f or e valuation. T he ke ys were s ubmitted t o a p anel of s lides i ncluding B. x ylophilus individuals, m ales a nd females. Conclusions were drawn about the possibility to observe specific criteria, such as lateral lines number, shape of spicules, number of caudal papillae and presence of vulval flap f or t he g roup l evel ( Table 1) , a nd t he pos ition of e xcretory por e f or s pecies l evel (Table 2). Some criteria were shown not to be reliable for routine use: number of caudal papillae and excretory pore position. Consequently, reliable identification keys for B. xylophilus were designed (Table 3) and validated according to a standardized process and taking into account recommendations from EPPO protocol (EPPO 2010). Additional results are available in Sarniguet et al. (2013).

REFERENCES Braasch H; Burgermeister W; Gu J (2009). Revised intra-generic grouping of Bursaphelenchus Fuchs, 1937 (Nematoda: Aphelenchoididae). Journal of Nematode Morphology and Systematics 12 (1), 65-88.

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EPPO (2009). EPPO standards PM7/4 (2) Diagnostics. Bursaphelenchus xylophilus. Bulletin OEPP/EPPO bulletin 39, 344-353. EPPO (2010). EPPO standards PM7/98 (1) Diagnostics. Specifics requirements for laboratories preparing accreditation for plant pest diagnostic activity. Bulletin OEPP/EPPO bulletin 40, 5-22. Sarniguet C; Buisson A; Anthoine G (2013). Validation of morphological keys for identification of Bursaphelenchus xylophilus (Nematoda, Parasitaphelenchidae) to group and species level. Bulletion OEPP/EPPO bulletin 43 (2), 255-261.

Table 1. Results of ev aluation of the xylophilus group key according to Braasch et al. (2009): number of conform observation/total number of observations

Observed criteria Females Males

Lateral lines 23/60 41/60

Spicules NA 60/60

Caudal papillae NA 0/60

Vulval flap 46/60 NA

NA: not applicable

Table 2. Results o f e valuation of the E PPO ( 2009) B. x ylophilus identification key: number of times the criteria is observed/ total number of observations

Features to be observed Results

Excretory pore not observed 29/60

Excretory pore not at expected place 11/60

Position of the excretory pore conform to B. xylophilus 20/60

Table 3. Key de signed f or Bursaphelenchus x ylophilus species ide ntification from female individuals

1 Female with conical or slender tail with or without mucro not B. xylophilus

Female with sub-cylindrical tail ………………………… 2

2 Female with sub-cylindrical tail, rounded end without mucro B. xylophilus Female with sub-cylindrical tail with a terminal mucro not B. xylophilus or B. xylophilus mucronate form (1)

(1) molecular identification needed

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Akiba et al., Genetic diversity of the pinewood neamtode, Bursaphelencus xylophilus after 100 years of invasion in Japan. In: Schröder, T. (ed.), Pine Wilt Disease Conference 2013, pp. 98-99, Braunschweig, ISSN: 1866-590X

(106) Genetic diversity of the pinewood nematode, Bursaphelenchus xylophilus after 100 years of invasion in Japan

Akiba M, Kanzaki N, and Sahashi N Forestry and Forest Products reserarch Institute, Matsunosato 1, Tsukuba, Ibaraki, 305- 8687, Japan Email: [email protected]

The pinewood nematode Bursaphelenchus xylophilus is a pa thogen that causes pine wilt disease. T he n ematode is t hought t o ha ve be en i ntroduced i nto s outhwestern J apan (Nagasaki P refecture i n 1905 a nd H yogo P refecture i n 1921) from North A merica followed b y di spersion t hroughout J apan, except t o Hokkaido, t he northernmost i sland. However, details on t he origins of the invading nematodes and the routes of dispersal in Japan are unknown. We collected 223 nematode isolates from dead trees in affected areas covering almost all damaged areas in Japan and analyzed their nuclear ribosomal DNA (ITS1-5.8SRNA-ITS2) and m itochondrial D NA (cytochrome ox idase subunit I [C OI]) sequences. Three SNPs in the ITS1 region, 2 SNPs and 2 indels of two bases in the ITS2 region, and 6 haplotypes (R1–R6) were detected in the nuclear DNA sequence data. Five percent o f i solates s howed he terogeneity o f t wo ha plotypes. H aplotype di versity w as 0.625 ± 0.019 a nd nucleotide diversity was 0.0016 ± 0.0001 a mong all isolates. R1 and R2 were t he dom inant h aplotypes (38.2% and 4 7.2%, respectively) and only t hese t wo haplotypes oc curred i n nor theastern J apan, w here di sease e xpanded a fter t he 1970s . In southwestern Japan where the history of pine wilt disease is older, four other haplotypes (R3–6) w ere de tected. Twenty-eight va riable s ites w ere d etected within t he 658 -bp mitochondrial DNA sequence and the isolates separated into 11 ha plotypes (4 haplotype groups). Haplotype di versity w as 0.596 ± 0.032 and nucleotide di versity was 0.0109 ± 0.0005. T he dom inant haplotypes in all ar eas of J apan were C1a ( 60.1%) and C4a (18.8%). T he C 3 ha plotype w as d etected onl y o n O kinawa Island, pos sibly due t o t he founder e ffect of i ntroduced ne matodes. D ifferences i n bot h nuc lear a nd m itochondrial DNA in each haplotype may indicate different geographic origins of B. xylophilus, and therefore, m ultiple i nvasions of ne matodes f rom di fferent n ative l ocations. T he hi gher diversity of ha plotypes in s outhwestern J apan may r eflect mul tiple inva sions in that region f ollowed b y expansion of several ha plotypes to nor theastern J apan. T he ITS haplotype pattern did not correspond to that of COI. Considering the different inheritance

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modes of nuclear and mitochondrial DNA, hybridization between nematodes of different origins m ust ha ve oc curred after i ntroduction t o J apan. C omparison of J apanese haplotypes with sequences registered in public DNA databases showed that the R3 and R4 ITS h aplotypes and s ome C OI h aplotypes were uni que. A lmost a ll B. xylophilus isolates f rom P ortugal w ere consistent w ith t he R 2 ITS ha plotype a nd t he C 1a C OI haplotype, which were d ominant haplotypes in J apan. The diversity of B. xylophilus in Japan was higher than in Portugal where nematode invasion occurred in 1999. This study shows that multiple invasions are likely to have occurred in Japan, but the origins of the invading n ematodes a re unknow n. T o a ddress this g ap i n ou r know ledge, e xtensive sampling of nematodes in native locations (USA and Canada) and more detailed analysis using molecular markers with high resolution such as SSRs or SNPs are necessary.

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Kozlovsky M, Bursaphelenchus mucronatus as a cause of dying a secondary fir forests in Ukrainian Carpathians. In: Schröder, T. (ed.), Pine Wilt Disease Conference 2013, pp. 100, Braunschweig, ISSN: 1866-590X

(109) Bursaphelenchus mucronatus as a cause of dying a secondary fir forests in Ukrainian Carpathians

Mykola Kozlovsky Institute of ecology of the Carpathians NAS of Ukraine, Kozelnytska str., 4, 79026, Lviv, Ukraine Email: [email protected]

Natural range of the stem nematode Bursaphelenchus mucronatus (Mamya & Enda, 1979) in the Ukrainian Carpathians reach upper tree-line. In fir-wood belt (1000-1400 m above sea level) there were found some dead and dying fir trees with a few stem nematodes. No mass infection of wood was revealed. At lower altitude (400-900 m) in zone of beech forest formations the secondary fir woods are exposed noticeably more on infection (from 50% to 100% of inspected trees of age 40 – 100 years). Trees at the age 60-110 years are infected much more and often, compare to 40 years ol d f ir t rees. Stem ne matodes w ere found i n t rees o f di fferent he althiness categories – dead, dying as well as trees without signs of disease. A lot of trees are infected in severe parts of trunk. Entirely infected fir tree, from butt to top of crown, there was not found. Mostly, stem nematodes were found on upper part of crown and lower or middle part of trunk, that indicates frequentative infection of trees. In beech-forest belt the infectiousness of fir trees by stem nematodes counts in the range from dozens to 300 nematodes in 1 g of dry wood. Perhaps, stem nematodes expansion is stimulated here by warmer climate. On some trees were found stem nematodes in space between yellow top and green low part of crown; damages of roots by mushrooms and trunk by x ylophagous insects were not revealed. Therefore, we assumed that stem nematodes indeed causes dying of crown top of fir. Diameter of body of Bursaphelenchus mucronatus averages for female 20-24,6, for male - 25-30 µ ; m orphological pa rameters w ithin♀: L = 648,2±78,5; a = 30,2±2,4; b = 10,5±0,9; c = 16,2±1,2; V = 69,1±0,8%; St = 13,7±0,1. ♂: L = 714,2±35,9; a = 30,9±8,0; b = 11,0±0,7; c = 18,6±1,3; Sp = 24,3±0,6; St = 13,7±0,1. In our opinion, in zone of beech forest the stem nematodes are the one of the main reason of fir wood dying.

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Takefumi Ikeda, Keisuke Kobayashi, Shoji Naoe, Growth properties of pine trees died from pine wilt disease In: Schröder, T. (ed.), Pine Wilt Disease Conference 2013, pp. 101-102, Braunschweig, ISSN: 1866-590X

(111) Growth properties of pine trees died from pine wilt disease

Takefumi Ikeda1, Keisuke Kobayashi1 and Shoji Naoe2 1 Department of Forest Science, Kyoto Prefectural University, Sakyo, Kyoto 606-8522, Japan; 2 Department of Ecosystem Studies, The University of Tokyo, Bunkyo, Tokyo 113-8657, Japan; email: [email protected]

ABSTRACT

Are there any traits in appearance of dead trees in the early stage of massive pine death caused b y pi ne w ilt di sease? I n ot her w ords, what i s t he l andmark f or Monochamus species as a vector choosing pine trees for after-ripening? The study was conducted in P. thunbergii community of Amonohashidate, Kyoto, Japan. Pine trees with larger diameter were e asy to di e in comparison with those w ith smaller di ameter. Trees w ith larger diameter seem to have a larger tree crown. This might show that Monochamus alternatus can easily access t o pi ne t rees with a l arger crown and do not come flying to d eclined pine trees.

INTRODUCTION

What is the landmark to Monochamus species as a ve ctor choosing pine trees for after- ripening? Researchers on pine wilt disease almost certainly know thi s. It is important to know to which pine tree Monochamus comes flying in order to manage control programs. This s tudy t ried to evaluate a r elationship between tree growth properties s uch as t ree height and diameter at breast height (dbh) and pine death caused by pine wilt disease.

STUDY AREA

The study was conducted in P. thunbergii community of Amanohashidate, Kyoto, Japan (35°34’N, 135°11 ’E). Amanohashidate h as be en be st know n f or i ts c oastline of w hite sands in Japan. Death of pine tree is of crucial significant to its landscape maintenance. The pi ne c ommunity o f A manohashidate doe s not c onnect w ith the pine f orests of surrounding mountainous area and is on a flatland.

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TESTED TREES AND ANALYSIS

1. Tree height and dbh: 36 pine trees died in 2002 and 154 live pine trees surrounded dead pines were selected in September 2003. Relationship between dead or not in pine trees as objective variables, and height or dbh of pine trees as explaining variables was analyzed using t he Generalized Linear M ixed Model ( GLMM). Tree he ight a nd dbh ha ve be en measured in March 2002. 2. Annual ring analysis: 5 dead pine trees and 10 live pine trees selected among above pine trees were selected. Width of annual ring on increment core collected by an increment borer was measured.

RESULTS AND DISCUSSION

1. Tree height and dbh: There was significant correlation between death and dbh (p=0.06, RIV=0.78). T his s hows t hat pi ne t rees w ith larger di ameter w ere e asy t o di e i n comparison with those with smaller di ameter. M. alternatus just af ter em erging m akes choice of he althy pi ne t ree in r andom m anner a nd t hen f eeds younger s hoots ( Togashi 2006). M. alternatus can easily access to pine trees with a larger crown because of larger target. 2. Annual ring analysis: In both dead pines and live pines the width of annual ring in 2001 was narrower than that in 2000. This seems to be due to low rainfall in early summer of 2001. Dead pine trees did not suffer severe stresses as live pine trees. That’s not to say that M. alternatus did not target at declined trees for after- ripening.

CONCLUSION

M. alternatus can easily access to pine trees with a larger tree and do not come flying to declined pine trees.

REFERENCES Togashi K (2006) The life of Monochamus alternatus. In The fascinating lives of insects residing in tree trunks – An introduction to tree-boring insects -. eds Shibata E ; Togashi K, pp. 83-106, Tokai University Press, Hatano

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Hoch et al., Testing attractants for trapping Monochamus sartor and Monochamus sutor. In: Schröder, T. (ed.), Pine Wilt Disease Conference 2013, pp. 103-104, Braunschweig, ISSN: 1866-590X

(117) Testing attractants for trapping Monochamus sartor and Monochamus sutor

Hoch G1, Halbig P1,2, Menschhorn P1, Hall DR3, Krehan H1 1Department of Forest Protection, BFW – Federal Research and Training Center for Forests, Natural Hazards and Landscape, Vienna, Austria 2Department of Forest Entomology, Forest Pathology and Forest Protection, BOKU – University of Natural Resources and Life Sciences, Vienna, Austria 3Natural Resources Institute, University of Greenwich, United Kingdom Email: [email protected]

ABSTRACT

Trapping vectors i s on e i mportant m easure f or m onitoring a nd c ontrol of pi ne w ilt disease. Lures consisting of ba rk be etle phe romone c omponents a nd a Monochamus pheromone compound have been developed for Monochamus galloprovincialis, the main pine w ood ne matode v ector i n E urope. The Monochamus pheromone c ompound 2 - undecyloxy-1-ethanol has been shown to be attractive for other species in the genus, such as M. al ternatus. W e t ested t he r esponse o f M. sartor and M. s utor to l ures know n t o attract M. galloprovinicialis. These two species are important colonizers of weakened or freshly ki lled N orway spruce i n C entral E urope a nd ha ve t he pot ential t o be come important vectors should the pine wood nematode be introduced in this area.

The experiment was set up in a mountainous mixed spruce forest in a wilderness area in Lower Austria. No forest sanitation measures had been carried out following attacks of spruce by bark beetles as well after major damage by an avalanche allowing build-up of populations of M. sartor and M. sutor as well as other phloeo-xylophagous insects. Teflon coated 12-funnel traps (ECONEX, Spain) with three different combinations of attractants were de ployed i n f our randomized bl ocks. T he f ollowing l ures were t ested: ( 1) t he commercially ava ilable G alloprotect-2D ( SEDQ, S pain) c onsisting of 2 -undecyloxy-1- ethanol, i psenol, and 2 -methyl-3-buten-2-ol, (2) Galloprotect-2D plus α-pinene (SEDQ, Spain), and (3) Galloprotect-2D plus α-pinene plus a blend of smoke volatiles (produced in D .R.H.'s l aboratory at t he U niv. G reenwich). P ositions of l ures w ere r e-randomized every 10 days; the experiment lasted fom 10 July to 20 August 2012. Traps were emptied every 3 or 4 days.

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Traps baited with Galloprotect-2D caught 4.6 ± 0.6 female and 1.6 ± 0.3 male M. sartor on average per 10-day trapping period (Figure 1). Highest catches were attained when the host tree volatile α-pinene was added (6.4 ± 1.0 females and 3.3 ± 0.5 males); the increase in males was statistically significant. Further addition of smoke volatiles did not enhance captures. Due to lower M. sutor catch, no s ignificant differences in response to the lures

were established. In total, our traps caught 277 M. sartor females and 107 males as well as 68 M. sutor females and 45 males over the entire trapping period. Figure 1. N umbers of Monochamus sartor and M. sutor beetles caught per trap per 10-d period (means + SE, n = 16). G = Galloprotect-2D, GA = G plus α-pinene, GAS = G plus α-pinene pl us s moke vol atiles. Different le tters indicate s ignificant di fferences ( Mann- Whitney U tests (corrected α = 0.017) following up Kruskal-Wallis H tests).

Catches of M. sartor and M. sutor were significantly correlated with mean air temperature (Kendall's τ = 0.6 26 and τ = 0.657, respectively). No beetles w ere c aught w hen mean temperatures w ere be low 15° C. Traps c aught hi gh num bers of ot her phl oeo- or xylophagous i nsects, s uch a s ot her c erambycids a nd bupr estids ( total of 95 a nd 24 specimens). Most frequent s pecies w ere Acanthocinus gr iseus, Arhopalus r usticus, Spondylus buprestoides, and Leptura rubra. Moreover, 136 specimens of the bark beetle predator Thanasimus f ormicarius were cau ght du ring the tot al 40 -d t rapping p eriod. Generally, bycatch was highest in traps a dditionally ba ited with α-pinene. Woodwasps were only caught in traps containing this host tree volatile.

This e xperiment ga ve first ins ight int o flight a ctivity of tw o potential pi ne w ood nematode ve ctors i n m ountainous A ustria a nd t heir a ttraction t o vol atiles. T he r esults indicate that M. sartor and M. sutor respond to the pheromone compound 2-undecyloxy- 1-ethanol (monochamol). Attractants developed for M. galloprovincialis appear suitable for monitoring these potential pine wood nematode vectors.

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Nakamura et al., Inhabitation of the Pinewood Nematode and Its Vectors in the Tsunami-damaged Pinus thunbergii and P. densiflora trees. In: Schröder, T. (ed.), Pine Wilt Disease Conference 2013, pp. 105, Braunschweig, ISSN: 1866-590X

(120) Inhabitation of the Pinewood Nematode and Its Vectors in the Tsunami-damaged Pinus thunbergii and P. densiflora trees

Nakamura K, Aikawa T, Ichihara Y, Maehara N, Mizuta N Tohoku Research Center, Forestry and Forest Products Research Institute, Nabeyashiki 92-25, Shimo-kuriyagawa, Morioka, Iwate 020-0123, Japan Email: [email protected]

The tsunami (tidal wave) following the Great East Japan Earthquake on March 11, 2011, devastated vast ar eas of s eacoast f orest, mainly com posed of t he Japanese bl ack pine (Pinus t hunbergii), a nd red pi ne (P. densiflora). N eedle c hlorosis o ccurred not onl y in severely damaged trees but al so in t hose without c onspicuous external da mage. W e investigated presence/absence of Monochamus al ternatus and Bursaphelenchus xylophilus in t he pi ne t rees w ith di scolored f oliage i n t he t sunami-damaged seacoast forests, to evaluate the potential of the trees as the source of infection of pine wilt disease (PWD). B. xylophilus was rarely detected in the wood samples col lected from t he dead trees i n November 2011, except for the trees that was considered to have been latent infected and died a fter t he t sunami. P. t hunbergii trees grown a long t he s horeline w ere severely damaged by t he t sunami and most of t hem di ed pr omptly. In t he s tands behind the frontline f orests, small P. t hunbergii trees s uppressed by t he canopy trees and then flooded with sea water from tsunami tended to die shortly after the disaster. Those trees were ha rdly at tacked by M. al ternatus. In c ontrast, m ost of P. de nsiflora trees became declined after the tsunami a nd ke pt s tressed c ondition through the summer. S uch t rees were, resultingly, infested by M. alternatus, when there were PWD damaged trees from which adult sawyers emerge in the vicinity. Emerging a dults of M. al ternatus from t he i nfested trees in t he f ollowing year of ten carried B. xylophilus, though we could not detect it in those trees in the November survey. Since the P. densiflora trees seemed to be debilitated by sea water flooding, not because of B. xylophilus infection, it is unlikely that the trees harbored the nematode before M. alternatus adults' ovi position. T he ne matode was possibly transmitted to the ts unami- damaged trees w hen M. al ternatus adults la id t heir eggs ont o t hem. C onsequently, tsunami-induced damage in seacoast pi ne f orest m ay facilitate t he s pread of PWD epidemic w hen the forest i s c omposed of P. de nsiflora and PWD da maged t rees ha ve already been there.

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Kato T, Futai K, Takeuchi Y, Bacterial flora and its association with the pine wood nematode (Bursaphelenchus xylophilus). In: Schröder, T. (ed.), Pine Wilt Disease Conference 2013, pp. 106-109, Braunschweig, ISSN: 1866-590X

(126) Bacterial flora and its association with the pine wood nematode (Bursaphelenchus xylophilus)

Kato T, Futai K, Takeuchi Y Graduate School of Agriculture, Kyoto University, Kitashirakawa Oiwake-cho, Sakyo-ku, Kyoto 606-8502, Japan Email: [email protected]

ABSTRACT

In r ecent years a h ypothesis w as pr oposed t hat not pi ne w ood ne matode, but i ts accompanying bacteria is responsible for the symptom development in pine wilt disease. To ascertain this, we investigated the bacterial flora associated with the nematode and its possible roles in the disease by means of molecular biological techniques. As a result, the dominant bacterial species were different from those in past researches and none of them showed a significant pathogenicity against susceptible pine seedlings. On the other hand, one of the dominant species was frequently detected in seedlings inoculated with bacteria- free ne matodes and i n vector b eetle-associated samples, indicating its possible involvement in the disease.

1. INTRODUCTION

Pine w ilt di sease i s c aused b y t he pa thogen, pi ne w ood ne matode ( PWN; Bursaphelenchus xylophilus). Recently, it is suggested that PWN needs bacteria inhibiting its body surface in host infection to cause wilt symptoms (Han et al. 2003), although this hypothesis still remains a matter of debate. At this time we have no promising method for effectively controlling pine w ilt di sease, pa rtly be cause of l ack of de finitive caus al therapy, and it is necessary to identify the pathogenic factor(s) precisely. In this study, in order to clarify the significance of PWN-associated bacteria in this disease, we described the ba cterial f lora on t he P WN bod y a nd de termined t he pot ential pa thogenicity of isolated bacteria to the host pine trees.

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2. MATERIALS AND METHODS

2.1. Bacteria Accompanied by PWN in Naturally Infected Pine Stands We investigated the bacterial flora on t he body surface of PWN isolated from naturally- infected pine trees. Woody tissues were taken from dead pine trees during the infection season in 2011 a t two pine stands; one of Japanese black pine, Pinus thunbergii, located at A rid Land R esearch C enter, T ottori U niversity, T ottori, J apan, a nd t he ot her of Japanese r ed pine, Pinus densiflora, on M t. Ogura, Ukyo, K yoto, J apan. Samples were used for nematode extraction with Baermann funnels to obtain B. xylophilus, from which accompanying b acteria were i solated on a pl ate of R 2A m edium. All b acterial is olates served DNA extraction for species identification based on the nucleotide sequence of 16S rRNA.

2.2. Pathogenicity of Accompanying Bacteria To examine the interaction between host plant (pine) and PWN-accompanying bacteria, we t est t he pa thogenicity of t wo bacteria w hich w ere f requently i solated in experiment 2.1, namely, Serratia proteamaculans and Erwinia mallotivora. Seedlings of 4-month old P. thunbergii which were grown under axenic condition were challenged with 1) bacteria- free PWN (virulent isolate, Ka4), 2) 1) mixed with S. proteamaculans, 3) 1) mixed with E. m allotivora, 4) S. pr oteamaculans alone, 5) E. m allotivora alone, and 6) s terilized water as control and the symptom development was monitored.

2.3. Succession of Bacterial Flora on the Body Surface of PWN during Symptom Development Potted seedlings of P. thunbergii were artificially inoculated with bacteria-free cultured PWN ( virulent i solate, K a4) i n t he ope n air t o monitor t he t ransition of bacterial flora accompanied by PWN inside host plant. One, 2, 4, and 6 weeks from inoculation, woody tissues of t he s eedlings were s ampled t o s erve Baermann funnels e xtraction, ne matode thus obt ained were us ed as ba cterial s ource, and the cul tured bacterial mixture s erved molecular characterization by t-RFLP techniques with a set of universal primers specific to bacteria.

2.4. Interaction of Accompanying Bacteria and the Vector Beetle The result of experiment 2.3 showed that S. proteamaculans was frequently accompanied by PWN. Considering the past report that S. proteamaculans was symbiotic to a kind of beetles (Morales-Jimenez et al. 2009), we examined the interaction between this bacteria and the vector beetle of PWN. Sampling of dead pine log that harbored the beetle larvae were conducted at Mt. Ogura in January 2013. By chopping the log with an ax, larvae of Japanese pine sawyer beetle, Monochamus alternatus and its pupal chambers were taken. DNA samples extracted from the larvae gut and from bacterial bodies scraped from the chamber were us ed for m olecular ch aracterization by t -RFLP i n a s ame m anner as experiment 2.3.

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3. RESULTUS AND DISCUSSION

3.1. Bacteria Accompanied by PWN in Naturally Infected Pine Stands In the an alysis of ba cteria accom panied by P WN i solated from pi ne f orests na turally infected with pine wilt, neither Pseudomonas nor Bacillus were frequently detected and in especially Pseudomonas f luorescens was ne ver de tected (Figure 1 ), all w hich were suggested t o be a ssociated w ith pa thogenicity o f pi ne w ilt (Kawazu & Kaneko 1997; Zhao et al. 2003). Dominant species in both pine stands were Serratia proteamaculans and Erwinia mallotivora, which is consistent with the previous study in Portugal (Vicente et al. 2011).

Figure 1. Bacterial flora on the bo dy s urface o f p ine w ood nematode i solated from naturally infected pine stands in Japan

3.2. Pathogenicity of Accompanying Bacteria In pathogenicity test using axenic pine seedlings, PWN inoculation caused a significant mortality regardless of whether and which b acteria was inoculated (Figure 2). Also, no bacteria was detected in the seedlings killed by PWN inoculation. All these suggest that PWN-accompanying bacteria is not necessary for the pathogenicity of pine wilt disease.

Figure 2. Mortality of pine seedlings after inoculation with (1) bacteria-free PWN, (2) PWN mixed with S. proteamaculans, (3) PWN mixed with of E. mallotivora, (4) S. proteamaculans alone, (5) E. mallotivora alone, and (6) sterilized water is shown. Values are averaged number of de ad seedlings in two experiments (n=24 for each).

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3.3. Succession of Bacterial Flora on the Body Surface of PWN during Symptom Development Bacterial flora accompanied by PWN after inoculation into host pine was monitored by means of t-RFLP. In all samples a clear peak of 366 bp was detected, which was identical to t hat of S. pr oteamaculans. It s uggests tha t S. pr oteamaculans get i nfected i nto pi ne plant by any way to establish a specific relation with PWN.

3.4. Interaction of Accompanying Bacteria and the Vector Beetle Bacterial flora i n t he pupal c hamber a nd the g ut of v ector b eetle of P WN w as characterized by means of t-RFLP. In the pupal chamber, more species of bacteria were detected and t he dom inance of S. pr oteamaculans (detected a s a pe ak at 366 bp) w as lower than in the gut. Thus S. proteamaculans may be accompanied by the body surface of PWN which is infecting host plant, propagate inside the plant (on the surface of pupal chamber), and then preferentially colonize the beetle gut to encounter PWN.

4. CONCLUSION

Our r esults s trongly s uggest a l ack of pa thogenicity o f P WN-accompanying ba cteria, although it is highly possible that certain species of bacteria inhabit on t he body surface of PWN. Further studies need to determine the ecological significance of such bacterial species in association with pine wilt disease.

5. ACKNOWLEDGEMENTS

Authors thank Asahi Glass Foundation for financial support.

REFERENCES Han Z M; Hong Y D; Zhao B G (2003). A study on pathogenicity of bacteria carried by pine wood nematodes. Journal of Phytopathology 151, 683-689. Kawazu K; Kaneko N (1997). Asepsis of the pine wood nematode isolate OKD-3 causes it to lose its pathogenicity. Japanese Journal of Nematology 27, 76-80. Morales-Jimenez J; Zuniga G; Villa-Tanaca L; Hernandez-Rodriguez C (2009). Bacterial community and nitrogen fixation in the red turpentine beetle, Dendroctonus valens LeConte (Coleoptera: Curculionidae: Scolytinae). Microbial Ecology 58, 879-891. Vicente C S L; Nasciment F; Espada M; Mota M; Oliveira S (2011). Bacteria associated with the pinewood nematode, Bursaphelenchus xylophilus collected in Portugal. Antonie van Leeuwenhoek 100, 477-481. Zhao B G; Wang H L; Han S F; Han Z M (2003). Distribution and pathogenicity of bacteria species carried by Bursaphelenchus xylophilus in China. Nematology 5, 899-906.

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Akami et al., Embolism development observed with a compact MRI in Japanese black pine clones resistant to pine wilt disease. In: Schröder, T. (ed.), Pine Wilt Disease Conference 2013, pp. 110-111, Braunschweig, ISSN: 1866-590X

(127) Embolism development observed with a compact MRI in Japanese black pine clones resistant to pine wilt disease

Akami A, Kusumoto D, Hirao T, Watanabe A, Fukuda K The University of Tokyo, 5-1-5 Kashiwanoha, Kashiwa-shi, Chiba 277-8563, Japan Email: [email protected]

1. INTRODUCTION

The symptom development of pine wilt disease is divided into two stages, the early and advanced s tages. In t he early s tage, pi newood n ematodes (Bursaphelenchus xylophilus) mainly disperse in cortical and xylem resin canals, then induce parenchyma denaturation which cause x ylem e mbolism pa rtially. In t he a dvanced s tage, n ematode popul ation drastically increase, which coincides with cambial destruction and embolism development in the whole stem. Symptoms observed in resistant pines, which were selected in national projects, don’t progress to the advanced stage in general. However, their resistance shows great variation among families or clones, which have been ranked from grade 1 (low) to 5 (high). In t his s tudy, t o c larify t he s ymptom de velopment i n de tail, we ex amined embolism development in resistant pine clones inoculated with nematodes.

2. MATERIALS AND METHODS

Potted Japanese black pine ( Pinus t hunbergii) clones, two r esistant (Tosashimizu 63 (grade 4), Oita 8 (grade 1)) and t wo susceptible (Kashima 2, Futaba 1) varieties, were used in this study. Virulent (S10) or avirulent (C14-5) isolates of pinewood nematodes reared on t he f ungus ( Botrytis ci nerea) w ere i noculated into 1-year-old s tem of each seedling. Embolism development was observed in multi-cross-sectional slices taken with a compact MRI. At 16 positions along the stem at 1 cm intervals from 5 cm above to 10 cm below the inoculation point (0 cm), embolisms were monitored periodically.

3. RESULT

Xylem e mbolism onl y occurred a round i noculation site in a ll c lones i noculated with avirulent ne matodes. O n t he ot her ha nd, in t he seedlings i noculated w ith t he vi rulent nematodes, embolism occurred beyond the inoculation site even in the resistant clones,

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but it enlarged more moderately in the resistant clones than in the susceptible ones. The pattern of t he e mbolism de velopment i n t he highly resistant c lone ( Tosashimizu 63) spread from out er t o i nner x ylem a long ray t issues regardless of di stance f rom t he inoculation site, while in the low er resistant (Oita 8) and t he susceptible (Kashima 2, Futaba 1) clones, xylem embolism was mainly enlarged as a cluster from the inoculation site verticality and horizontally (Figure 1).

4. DISCUSSION

Avirulent nematodes did not induce embolisms in xylem except the inoculation sites even in susceptible c lones, while vi rulent is olate indu ced embolism in resistant c lones. This indicates that some num bers of vi rulent ne matodes can migrate i n xylem r esin canals even in highly resistant clones. However, in the highly resistant clone, nematodes did not cause a mass embolism around the inoculation site. This may suggest that proliferation of nematodes around the inoculation site was inihibited in these seedlings. In conclusion, restricted development of mass embolisms corresponded to the suppression of symptoms development to the advanced stage in resistant pine clones.

Figure 1. Embolism pattern in pine clones observed with a compact MRI

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Liu F Y, Chen F M, Xie L Y, Ye J R, Tan J J, Analysis of genetic diversity of Bursaphelenchus mucronatus and B. xylophilus isolates based on ISSR markers. In: Schröder, T. (ed.), Pine Wilt Disease Conference 2013, pp. 112-114, Braunschweig, ISSN: 1866-590X

(131) Analysis of genetic diversity of Bursaphelenchus mucronatus and B. xylophilus isolates based on ISSR markers

Liu F Y, Chen F M, Xie L Y, Ye J R, Tan J J College of Forest Resources and Environment, Nanjing Forestry University, Nanjing, China Email: [email protected]

ABSTRACT

Nine of 130 primers were collected by using PCR screening. The nine primers were used to amplify the genomic DNA of 11 Bursaphelenchus mucronatus parasitic groups from six provinces a nd of 10 B.xylophilus groups f rom 10 pr ovinces. A t otal of 1111 c lear strips were amplified with 1069 pol ymorphic strips, which reached a percentage as high as 96.2% . P rimer P TY1424 a mplified t he m aximum num ber of b ands, whereas pr imer PTY888 a mplified the l east ba nds. The r esult s howed that the g enetic s imilarity coefficient range of t he 21 ne matodes was from 0.4014085 t o 0.9436620. The DNA molecular dendrogram of these 21 groups were established through the un-weighted pair group method with arithmetic average (UPGMA) cluster analysis. The 11 B.mucronatus groups w ere g athered as one c ategory when the similarity coefficient v alue was 0.715 , whereas the 10 B.xylophilus groups were gathered as one when the coefficient was 0.755. However, the 21 groups divided into two species when the similarity coefficient was 0.52. No obvious difference was observed in the geographic relationship among the clustering results. The r esults of the i nter s imple s equence repeat (ISSR) markers c an be us ed to effectively di stinguish t he genetic r elationship be tween B.mucronatus and B.xylophilus groups. B ased on t he r esult of P CR, ISSR pr oduced hi gher po lymorphism on B.mucronatusas com pared with B.xylophilus groups. The reason m ay be t hat compared with B.xylophilus, B.mucronatus are na tive s pecies t hat ha ve undergone geographical isolation for a long time. Further study is required to explain whether the genetic diversity of B.mucronatus is related to their pathogenic differentiation. Key words: Bursaphelenchus mucronatus, B.xylophilus, genetic diversity, ISSR

INTRUDUCTION Recent research supported that B.mucronatus has pathogenicity to pine trees, especially to those with adversity stress(Chen et al 2010; Zhang et al 2002, 2004). In this study, ISSR

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was us ed t o explore the genetic di versity between di fferent B.mucronatus and B.xylophilus groups.

MATERIALS AND METHODS

Nematode sources: 11 B.mucronatus groups f rom 6 provinces a nd o f 10 B.xylophilus groups from 10 infected areas of China. PCR amplification primer: 100 primers (UBC800-UBC900) and 30 PTY primers. Detection of PCR products: QIAxcel was used to detect PCR products.

CONCLUSION AND DISCUSSION

11 B.mucronatus groups a nd 10 B.xylophilus groups w ere an alyzed b y using the ni ne primers of ISSR. The r esults obt ained 1111 a mplified D NA f ragments w ith the s ize ranging from 200 to 2000 bp (Fig.1). The band spectrum of these primers could clearly distinguish the geographical population of the 21 nematodes. Compared with B.xylophilus groups, B.mucronatus groups have m ore va rieties. Considering t he or igin of t hese t wo kinds of ne matode, B.mucronatus are widely di stributed i n E urope a nd A sia, w hereas B.xylophilus originated in North America. B.xylophilus began to spread i n Japan in the early 20th century, and to China in the 1980s. In China, B.mucronatus are native species, which have undergone geographical isolation for a long time. Such long isolation may be proven by their larger genetic variation. By contrast, B.xylophilus have spread to China for a short time, leading to higher genetic similarity between groups.

Fig.1 PCR-amplifiedfragments of 21isolated nematodes with primer PTY1425 1：marker；2-11:B.xylophilus groups，12-22: B. mucronatus groups

ACKNOWLEDGEMENT

The project was supported by the special research program for non-profit forestry of State Forestry Administration, P. R. China (201204501) and the National Science Foundation of China (31170599).

REFERENCES Chen FM; Shi YM; Wang SY;Tang J; Sun CY; Ye JR(2010). Pathogenicity of Different Isolates of BursaphelenchusmucronatustoPinustaiwanensis and P.thunbergii Seedlings. Scientia Silvae Sinicae 48,86-90.

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Zhang ZY; Lin MS; Yu BY(2004). Pathogenicity of Bursaphelenchus mucronatus on the seedings of black pine. Journal of Nanjing Agricultural University 27,46-50. Zhang ZY; Zhang KY; Lin MS; Luo HW; Xu FY(2002). Pathogenicity determination of Bursaphelenchus xylophilus isolates to Pine thunbergii. Journal of Nanjing Agricultural University 25,43-46.

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Magnusson C, Henriques J, Sousa E. Studies on non-vector transmission of Bursaphelenchus xylophilus on Pinus pinaster. In: Schröder, T. (ed.), Pine Wilt Disease Conference 2013, pp. 115, Braunschweig, ISSN: 1866-590X

(132) Studies on non-vector transmission of Bursaphelenchus xylophilus on Pinus pinaster.

Magnusson C1, Henriques J2, and Sousa E2 1 Norwegian Institute for Agricultural and Environmental Research (Bioforsk), Høgskoleveien 7, N-1430 Ås, Norway. 2 Instituto Nacional de Investigação Agrária e Veterinária, I.P. Av. da República, Quinta do Marquês, Edifício da ex-EFN. 2780-159 Oeiras – Portugal. Email: [email protected];

Since t he pi newood ne matode ( PWN), Buraphelenchus x ylophilus was r egulated as a quarantine pest questions have been raised concerning the possibility of nematode spread with means other than the vector beetles Monochamus spp. In the REPHRAME-project these concerns are addressed in Work Package 5. In Portugal experiments on non-vector transmission of PWN to maritime pine Pinus pinaster have been started. Studies on r oot transmission in the field are carried out in the Lisbon area and are located at Herdade da Comporta, Companhia das Lezirias and Mata da Machada. Here, PWN transmission from nematode-inoculated adult t rees (50 000 i nd/tree) t o covered undergrowth t rees will be studied. In an out door greenhouse f acility r oot t ransmission of P WN i s s tudied on 7 - years-old trees. The experiment includes 60 trees potted in pairs in 30 containers, where 1 tree in each pair is inoculated with 6 000 P WN and the spread of PWN from one tree to its neighbor is followed. Transmission of PWN from boards to trees is studied in the field. Infested boards ( n=5) a nd nematode-free boa rds (n= 5) will be tied to trees w ith intact bark, to trees with exposed cambium and to trees with exposed xylem. Transmission from infested c hips t o t rees will be studied i n t he o utdoor greenhouse f acility on 7 -year-old potted trees, with chips placed on t op of soil or in contact with roots. In both situations there will be a treatment with intact and a treatment with artificially wounded roots.

Keywords: Non-vector t ransmission, Bursaphelenchus x ylophilus, Pinus pi naster, Portugal.

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Berkvens N et al., Bursaphelenchus xylophilus does not occur in Belgium, but what about its vectors, the Monochamus spp.? In: Schröder, T. (ed.), Pine Wilt Disease Conference 2013, pp. 116-117, Braunschweig, ISSN: 1866-590X

(138) Bursaphelenchus xylophilus does not occur in Belgium, but what about its vectors, the Monochamus spp.?

Berkvens N 1, Casteels H 1, Damme N 1, Bighiu A1, Witters J 1, Grégoire J-C2, Boone C 2, Michelante D3, Viaene N1 1 ILVO, Plant – Crop Protection, Burg. van Gansberghelaan 96, B-9820 Merelbeke, Belgium 2 Université Libre de Bruxelles, LUBIES, Avenue FD Roosevelt 50, B-1050 Brussels,Belgium 3 FASFC, Food Safety Center, Kruidtuinlaan 55, B-1000 Brussels, Belgium Email: [email protected]

INTRODUCTION

It is unclear if pine wilt di sease (PWD) t hreatens the conifer fo rests of Belgium. Entry and presence of Bursaphelenchus xylophilus (PWN) are monitored by the Belgian NPPO (the Federal Agency for the Safety of the Food Chain, FASFC), but the nematode was never found. However, knowledge about the presence of its vector, Monochamus spp., in Belgium is lacking. Single specimens of M. galloprovincialis and M. sartor were reported in Belgium on 7 oc casions (Anonymous, 2013). The origin of these beetles (endemic or imported) is unknown. It is essential to gather more information about Monochamus spp. in Belgium to assess the risk of PWD.

1. MATERIALS AND METHODS

From 2000 unt ill t oday, F ASFC c ollected s uspicious s amples dur ing ph ytosanitary controls and national surveys in pine stands, public green areas, and logging and wood processing f acilities. Imported packaging m aterial s uspected of i nsect at tack were sampled a lso. A n a verage o f 170 w ood a nd ba rk s amples w ere a nalysed yearly a t t he Institute for Agricultural and Fisheries Research (ILVO). All procedures were according to the EPPO Standard PM 9/1(5) (EPPO 2012).

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1. RESULTS AND DISCUSSION

No specimen of B. xylophilus were detected in the 72 and 108 samples analysed in 2000 and 2001, respectively (De Wael et al. 2002), nor in the following years (Table 1). Only two living Monochamus individuals (adult and larva) were intercepted. Table 2. Results of phytosanitary controls and national surveys in Belgium for Monochamus spp. (packaging wood from import) and Bursaphelenchus xylophilus; n.d.: not determined

2004 2005 2006 2007 2008 2009 2010 2011 2012 Nematodes Total number of samples 106 90 123 239 251 213 200 178 143 (samples of imported materials) (25) (55) (51) (96) (96) Samples with B. xylophilus 0 0 0 0 0 0 0 0 0 Import with Laimaphelenchus spp. n.d. n.d. n.d. n.d. 1 0 0 4 1 Import with Aphelenchoides spp. n.d. n.d. n.d. n.d. 0 0 3 17 42 Import with other saprophytic nem. n.d. n.d. n.d. n.d. 16 49 43 68 45 Insects Total number of samples 13 2 9 1 11 4 7 6 8 Samples containing Monochamus spp. 0 0 0 0 0 1 1 0 0 Samples containing other longhorn spp. 2 0 0 0 1 1 0 0 0 Samples containing other insects 2 1 2 1 2 0 0 0 4

However, l ive s aprophytic n ematodes, i ncluding genera closely r elated to PWN (Aphelenchoides spp. and Laimaphelenchus spp.), as well as some live beetles belonging to the B ostrichidae and Cerambycidae ( Anoplophora gl abripennis, X ylotrechus r ufilius and Phoracantha s emipunctata) were detected on several oc casions on i mported materials.Their presence in imported wood and bark can indicate insufficient treatment of imported wood m aterial us ing h eat or fumigation (EPPO S tandards PM 10/6 and10/7). The c onsequences of an i ntroduction of P WN a re unc lear due t o a l ack of know ledge about the Monochamus spp. in Belgium. This essential information will be gathered in a three-year pr oject of ILVO and the Biological C ontrol a nd S patial E cology Lab (LUBIES), in cooperation with FASFC.

2. REFERENCES

Anonymous (2013). Saproxylic beetles from Belgium. projects.biodiversity.be/beetles. De W ael L; D e S utter N ; M oens M (2002). Diagnostic r esearch at t he D epartment o f Crop Protection in 2001 (Diagnostic centre for plants). IV Nematology: Survey of the pinewood nematode Bursaphelenchus xylophilus. Parasitica 58, 27-29. EPPO ( 2012). E PPO S tandard P M 9/ 1(5). Bursaphelenchus x ylophilus and i ts ve ctors: procedures for official control. Bulletin OEPP/EPPO Bulletin 42, 477–485.

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Li Sheng Nan et al., Construction of Engineering Bacterium Expressing Flagellin of Pseudomonas fluorescens and its Toxicity to Pinus thunbergii in Vivo. In: Schröder, T. (ed.), Pine Wilt Disease Conference 2013, pp. 118-119, Braunschweig, ISSN: 1866-590X

(139) Construction of Engineering Bacterium Expressing Flagellin of Pseudomonas fluorescens and its Toxicity to Pinus thunbergii in Vivo

Li Sheng Nan1, ZHAO Bo Guang2 , LI Rong Gui2 , GUO Dao Sen2 1 Department of Biology , Qingdao Univer sity , Qingdao 266071 , China ; 2 College of Forest Resources and Environment , Nanjing Forest ry University , Nanjing 210037 , China

A constitute and s ecreting expressing pl asmid p UC18ompA was constructed. The gene fliC encoding flagellin of Pseudomonas fluorescens Pf-5 was cloned into this plasmid to construct pUC18ompA-fliC. The plasmid was transformed into E. col i BL21 (DE3) to construct engineered bacteria.

1 supernatant of E. c oli BL21 ( DE3); 2 supernatant of the engineered bacteria ; 3 Proteins of E. c ol i BL21 ( DE3); 4 Proteins of the engineered bacteria; 5 Marks of the standard proteins Fig. 1 Western blotting of the proteins in the engineered bacteria

Bacterium-free seedlings of P inus t hunbergii were i noculated wit h a mixture of t he engineered bacteria and the aseptic pine wood nematodes (Bursaphelenchus xylophilus) to determine it s pathogenicity. The results of inoculation showed that inoculation with a mixture of engineered bacteria and aseptic pine wood nematodes also caused wilt of pine seedlings to some extent . T he important role of flagellin played in vivo in pathological process was further verified.

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Table 1. The wilting rate of the inoculated seedlings and re-isolation of the namatodes and bacteria

Bx: non-sterilized nematodes; ABx: Sterilized nematodes; E. coli: E. coli BL.21 (DE3); EB: the engineered bacteria; ABx+ E. coli: the mixture of sterilized nematodes and E. coli BL.21 (DE3); ABx+EB: the mixture of sterilized nematodes and the engineered bacteria; Pf: Pseudomonas fluorescens; ABx+Pf: the mixture of sterilized nematodes and Pseudomonas fluorescens: Ck: sterilized water control; “-”: neither bacteria no nematodes were re-iosolated.

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Cardoso JMS et al., Gene silencing in Bursaphelenchus xylophilus: knock down of a calponin gene and its effect on nematodes movement. In: Schröder, T. (ed.), Pine Wilt Disease Conference 2013, pp. 120-122, Braunschweig, ISSN: 1866-590X

(140) Gene silencing in Bursaphelenchus xylophilus: knock down of a calponin gene and its effect on nematodes movement

Cardoso J M S1, Fonseca L1, Gomes P2, Egas C2 and Abrantes I1 1 IMAR-CMA, Department of Life Sciences, University of Coimbra, 3004-517 Coimbra 2 Genoinseq, Next Generation Sequencing Unit, Biocant, 3060-197 Cantanhede; Portugal Email: [email protected]

INTRODUCTION

Post-transcriptional gene s ilencing b y R NA int erference ( RNAi) w as f irst di scribed in Caenorhabditis elegans (Fire et al . 1998) a nd oc curs when doubl e-stranded R NA (dsRNA) is recognized by an organism as foreign, trigering a chain of processes in which both ds RNA a nd i ts m RNA hom olog a re de graded p reventing t he s ynthesis of t he encoded protein. RNAi became an established experimental technique to investigate the function of different genes and its application and efficiency in the pinewood nematode, Bursaphelenchus xylophilus, function genomics has also been studied (Park et al. 2008; Cheng et al. 2010; Kang et al. 2011; Li et al. 2011; Ma et al. 2011; Kang et al. 2012; Wang et al . 2012 ). T here a re also some evidences t hat R NAi can be used to confer resistance t o engineered hos t pl ants t hat ex press ds RNA t o target and silence s pecific nematode genes (Lilley et al. 2012). New ways for the manegement of B. xylophilus are needed and gene silencing by RNAi is a potencial strategy. The calponin gene (unc-87), in C. elegans, is required to maintain the structure of myofilaments in muscle cells of the body wall (Kranewitter et al. 2001). In the present study, the knock down of the calponin homolog Bx-unc-87 was performed by RNAi to evaluate the role of this gene in B. xylophilus and the applicability of Bx-unc- 87 silencing as a control strategy for this nematode.

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MATERIALS AND METHODS

The knock down of the B. xylophilus calponin homolog gene was carried out by soaking the ne matodes i n a s olution c ontaining ds RNA of t he Bx-unc-87 gene dur ing 24 h. Afterwards, the phenotype of the nematodes was estimated by mobility and ne matodes reproduction. The relative Bx-unc-87 transcript a bundance, after ds RNA treatment, was assessed by R T-PCR with SybrGreen using t he A BI P RISM 7900 HT F ast S ystem

(Applied Biosystems) and the Comparative CT (ΔΔCT) method.

RESULTS

The dsRNA treated nematodes revealed some paralysis and uncoordinated movement in contrast t o t he regular and s inusoidal m ovement of t he non-treated n ematodes and reproduction was lower in treated nematodes. The reduction in the Bx-unc-87 transcript abundance conf irmed the ef fectiveness of Bx-unc-87 gene knock down. Further s tudies are being conducted in order to improve the efficiency of the silencing effect.

ACKNOWLEDGMENTS

This r esearch was pa rtially s upported by FEDER f unds t hrough the P rograma Operacional Factores de Competitividade (COMPETE) and national funds through FCT (Fundação para a C iência e a T ecnologia) und er t he pr oject FCOMP-01-0124-FEDER- 008794 (FCT PTDC/AGR-CFL/098916/2008). Cardoso JMS is funded by a post-doctoral fellowship SFRH/BPD/73724/2010 financed by QREN-POPH-Typology 4.1-co-financed by MES national funding and The European Social Fund.

REFERENCES Cheng XY, Dai SM, Xiao L, Xie BY (2010). Influence of cellulase gene knockdown by dsRNA interference on the development and reproduction of the pine wood nematode, Bursaphelenchus xylophilus. Nematology 12, 225-233. Kang JS, Koh YH, Moon YS, Lee SH (2012). Molecular properties of a venom allergen- like protein suggest a parasitic function in the pinewood nematode Bursaphelenchus xylophilus. International Journal for Parasitology 42, 63-70. Kang JS, Lee D-W, Koh YH, Lee SH (2011). A soluble acetylcholinesterase provides chemical defense against xenobiotics in the pinewood nematode. PLoS One 6, e19063. Kranewitter WJ, Ylanne J, Gimona M (2001). UNC-87 is an actin-bundling protein. Journal of Biological Chemistry 276, 6306-6312. Li X, Zhuo K, Luo M, Sun L, Liao J (2011). Molecular cloning and characterization of a calreticulin cDNA from the pinewood nematode Bursaphelenchus xylophilus. Experimental Parasitology 128, 121-126.

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Lilley CJ, Davies LJ, Urwin PE (2012). RNA interference in plant parasitic nematodes: a summary of the current status. Parasitology 139, 630-640. Ma HB, Lu Q, Liang J, Zhang XY (2011). Functional analysis of the cellulose gene of the pine wood nematode, Bursaphelenchus xylophilus, using RNA interference. Genetics and Molecular Research 10, 1931-1941. Park JE, Lee KY, Lee SJ, Oh WS, Jeong PY, Woo T, Kim CB, Paik YK, Koo HS (2008). The efficiency of RNA interference in Bursaphelenchus xylophilus. Molecules and Cells 26, 81-86. Wang X, Cheng X, Li Y, Zhang J, et al. (2012). Cloning arginine kinase gene and its RNAi in Bursaphelenchus xylophilus causing pine wilt disease. European Journal of Plant Pathology 134, 521-532.

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Vieira P et al., Comparative analysis of MspI satellite repeats of the pinewood nematode, Bursaphelenchus xylophilus, at different geographic scales. In: Schröder, T. (ed.), Pine Wilt Disease Conference 2013, pp. 123, Braunschweig, ISSN: 1866-590X

(142) Comparative analysis of MspI satellite repeats of the pinewood nematode, Bursaphelenchus xylophilus, at different geographic scales

Vieira P, Castagnone C, Mallez S, Espada M, Navas A, Mota M and Castagnone-Sereno P NemaLab/ICAAM, Instituto de Ciências Agrárias e Ambientais Mediterrânicas, Universidade de Évora, Núcleo da Mitra, Ap. 94, 7002-554 Évora, Portugal Email: [email protected]

The M spI s atellite D NA ( satDNA) f amily of the pi newood ne matode B ursaphelenchus xylophilus is herein analyzed in an attempt to understand the intraspecific variability at different geographic s cales. A t otal of 425 M spI m onomer uni ts, e ither PCR-amplified from isolates of local (Peninsula of Setúbal, Portugal) or worldwide origin, or retrieved from t he B . x ylophilus genome s equence, w ere characterized and compared. W hatever their origin, s liding window analysis of s equence variability patterns among m onomers revealed l ow, m oderate and hi ghly v ariant dom ains, indicating th at va riable le vels o f evolutionary constraint m ay act upon t he entire m onomers. The ph ylogenetic i nference based on t he di fferent sets of M spI s atDNA f amily f or t his s pecies s hows a br oad polymorphism of t he i ndividual m onomers, w hich w ere di stributed i nto f our m ain clusters. However, s uch clustering appeared i ndependent from t he geographic origin of the ne matodes, a nd c ould not di scriminate i solates or groups o f geographically c lose isolates. R ather, t he f ormation of di fferent ph ylogenetic g roups w ithin t his s atDNA family s uggests a n a pr iori e mbodying of a s et of di verging r epeats f rom a c ommon ancestor s atDNA l ibrary, which have b een differently amplified along t he evol utionary pathway o f t his s pecies. T he p resent w ork i mproves know ledge on t he e volutionary dynamics of satDNA at the intraspecific level, and provides new information on satDNA sequence variability among natural populations sampled at a local geographic scale.

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Abrantes I et al., Physiological responses to water stress and temperature on the pine wilt disease development in Pinus spp. In: Schröder, T. (ed.), Pine Wilt Disease Conference 2013, pp. 124-126, Braunschweig, ISSN: 1866-590X

(143) Physiological responses to water stress and temperature on the pine wilt disease development in Pinus spp.

Abrantes I 1, F onseca L1, Fernandes P2, Mendes A2, Colwell F2, Costa C 2, Máguas C2, Correia O2 1 IMAR-CMA, Department of Life Sciences, University of Coimbra, 3004-517, Coimbra, Portugal 2 Centre for Environmental Biology, Faculty of Sciences, University of Lisbon, 1749-016 Lisboa, Portugal Email: [email protected]

ABSTRACT

Four to five-years-old Pinus pinaster, P. pinea and P. radiata trees were inoculated with 6000 pinewood nematodes (PWN) and symptoms evolution, trees physiological responses and P WN popul ation de nsities w ere a ssessed under hi gh a nd l ow w ater a vailability conditions, at 25 a nd 30ºC. Pine wilt disease symptoms were observed in all infected P. pinaster and P. r adiata leading t o a de crease i n phot osynthetic a ctivity and water potential va lues, unde r the highest temperature a nd low w ater a vailability. P inus pi nea did not de velop s ymptoms a nd no s ignificant c hanges i n ph ysiological s tatus w ere detected. Nematodes were found in higher numbers, in high temperatures and low water availability, in P. pinaster followed by P. radiata and P. pinea.

INTRODUCTION

After e ntering i nto t he host t ree, the pinewood ne matodes (PWN), Bursaphelenchus xylophilus, multiply intensively and migrate throughout the plant. During this process, the blockage of xylem water conduction by tracheid embolisms causes high decrease in leaf water potential and cessation of photosynthesis (Kuroda 2008). To develop proper control methods, ba sed on e arly di agnosis of t he di sease, i s i mportant t o unde rstand t he physiological r esponses a nd i nternal changes of i nfected hos t s pecies. T he m ain objectives of this study were to understand the effect of water stress and temperature on PWN development and to evaluate the photosynthetic activity and water potential values of PWN infected P. pinaster, P. pinea and P. radiata.

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MATERIALS AND METHODS

A t otal of 120 four t o five-years-old trees (40 t rees/Pinus species) were gr own in a greenhouse under high and low water availability conditions at 25 a nd 30ºC. Trees were inoculated w ith 6000 PWN and trees i noculated with sterilized water w ere us ed as controls. Predawn xylem pressure potential of needles was measured using a S cholander pressure chamber and photosynthetic and transpiration rates of needles were taken, twice a week, using a portable infra-red gas analyzer (GFS-3000, Walz) equipped with a red led light source. Symptoms development was followed for 50 days and classified in six stages based on the wilting and consequent discoloration of the needles according to Proença et al. (2010). At the end of the experiment, the trees were cut and the final PWN population was estimated, in each tree, at the branches, trunk, roots and soil.

RESULTS

Pine w ilt di sease s ymptoms (PWD) were obs erved in all i nfected P. p inaster and P. radiata leading to a decrease in photosynthetic activity and water potential values, under the highest temperature a nd low w ater a vailability. Pinus pi nea did not de velop symptoms and no significant changes in physiological status were detected. Nematodes were found, in higher numbers, in the highest temperature and low water availability, in P. pinaster followed by P. radiata and P. pinea. In P. pinaster and P. radiata, nematodes were detected in all PWN inoculated trees, at the branches, trunk and roots while in P. pinea they were de tected onl y i n f our t rees, a t t he br anches a nd t runk. Pine s pecies reacted di fferently t o P WN, w ater s tress c onditions a nd t emperature, e nhancing t he development of the PWD, which may have implications on t he enlargement of infected area and on the shortening period of PWD development under climate change scenarios.

ACKNOWLEDGMENTS

This r esearch was pa rtially s upported by FEDER f unds t hrough the P rograma Operacional Factores de Competitividade (COMPETE) and national funds through FCT (Fundação para a Ciência e a Tecnologia) under the projects FCOMP-01-0124-FEDER- 008794 ( Refª. F CT P TDC/AGR-CFL/098916/2008) and FCT P TDC/AGR- CFL/098869/2008.

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REFERENCES Kuroda K (2008). Physiological incidences related to symptom development and wilting mechanism. In Pine Wilt Disease, eds B.G. Zhao, K. Futai, J.R. Sutherland &Y. Takeuchi, pp. 204-222. Springer: Tokio, Japan. Proença DN, Francisco R, Santos CV, Lopes A, Fonseca L, Abrantes IMO, Morais PV (2010). Diversity of bacteria associated with Bursaphelenchus xylophilus and other nematodes isolated from Pinus pinaster trees with pine wilt disease. PLoS ONE 5 e15191. doi:10.1371/journal.pone.0015191.

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Braasch H, Schönfeld U, Improved key to the species of the xylophilus group of the genus Bursaphelenchus Fuchs, 1937. In: Schröder, T. (ed.), Pine Wilt Disease Conference 2013, pp. 127-128, Braunschweig, ISSN: 1866-590X

(144) Improved key to the species of the xylophilus group of the genus Bursaphelenchus Fuchs, 1937

Helen Braasch1 and Ute Schönfeld2 1 Kantstrasse 5, D-14471 Potsdam, Germany, before: Federal Biological Research Centre for Agriculture and Forestry, Messeweg 11, D-38104 Braunschweig, Germany 2 State Office for Rural Development, Agriculture and Land Reallocation, Steinplatz 1, D-15806 Zossen, Germany

A c orrect de termination of t he pi ne w ood ne matode be comes m ore di fficult, t he m ore related or similar species are d escribed. In view of the fact that a further spread of the nematode s hould be c onsidered a nd c ontinuously ne w Bursaphelenchus species ar e detected, i dentification keys m ust b e a djusted accordingly. S ince t he publication of a dichotome m orphological ke y for s pecies of t he xylophilus group of t he ge nus Bursaphelenchus (Braasch, 2008), several new species of this group have been described. These species are B. macromucronatus Gu, Zheng, Braasch & Burgermeister, 2008; B. populi Tomalak & Filipiak, 2010; B. firmae Kanzaki, Maehara, Aikawa & Matsumoto, 2011; B. par aluxuriosae Gu, W ang, Braasch, B urgermeister & S chröder, 2012; B. koreanus Gu, W ang & Chen, 2013 a nd B. gi llanii Schönfeld, B raasch, Riedel & Gu, 2013. B ased on t he ke y published b y Braasch ( 2008) a nd t he r evised i ntrageneric grouping of Bursaphelenchus by B raasch et al . (2009), a n i mproved di chotome ke y including in the meantime newly described species is presented. The decision-making is supported by pictorial representation of important features. Two species found in aspen partially share characters with the xylophilus group and are not c onsidered i n t he k ey: B. trypophloei Tomalak & Filipiak 2011 a nd B. m asseyi Tomalak, W orrall & Filipiak, 2013, w hich c an easily b e s eparated f rom t he xylophilus group species by spicule morphology, having shorter condylus and rostrum. Characters us eful f or i dentification of xylophilus group species ar e f emale t ail s hape (cylindrical, s ubcylindrical or c onoid, round-tailed or di gitate, w ith or w ithout m ucro), shape and s ize of s picules, s everal m easurements a nd ratios ( a, c’) and pos ition of excretory pore with a certain variation of the last character.

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The r ecognition of t he ha rmful qua rantine pe st B. x ylophilus using m orphological characteristics in laboratories of the National Plant Protection Services is facilitated by an adapted and simplified key, w hich s eparates s pecies clearly distinguishable f rom B. xylophilus without s pecies ide ntification and includes onl y tw o very s imilar s pecies. Molecular methods are additionally advisable to confirm a morphological diagnosis.

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Fonseca L et al., Vacuum pressure impregnation for the elimination of the pinewood nematode from pine wood. In: Schröder, T. (ed.), Pine Wilt Disease Conference 2013, pp. 129-130, Braunschweig, ISSN: 1866-590X

(147) Vacuum pressure impregnation for the elimination of the pinewood nematode from pine wood

Fonseca L1, Santos A2, Amado S2, Pedroso M2, Abrantes I1 1 IMAR-CMA, Department of Life Sciences, University of Coimbra, 3004-517, Coimbra, Portugal 2 MTL – Madeiras Tratadas,2426-908 Monte Redondo, Leiria, Portugal Email: [email protected]

ABSTRACT

In order t o evaluate the efficiency of t he va cuum pr essure i mpregnation w ith chemical preservatives in the elimination of the pinewood nematode (PWN) from wood, naturally PWN infected Pinus pinaster sections were introduced into a wood treating autoclave and exposed t o the vacuum and pressure i mpregnation w ith t he w ood pr eservative TANALITH® E N B. A fter t reatment, w ood s ections w ere r emoved a nd l eft a t room temperature to dry and the total number of nematodes was quantified after incubation at 25ºC for 15 and 30 days. No nematodes were detected in the treated wood sections after the incubation periods. These preliminary results revealed a potential use of this process to eliminate PWN from wood.

INTRODUCTION

The i ntroduction of s everal i nvasive a lien pl ant pe sts i nto non -native a reas l ed t o t he development of appropriate phytosanitary measures against the introduction and spread of these species. Since wood material was recognized as one of the most important pathways for introductions of forest-related pests, the International Plant Protection Convention adopted the International Standard for Phytosanitary Measures No. 15, which serves as a guideline for the regulation of wood packaging material used in international trade (FAO 2009). The use of pinewood material for agricultural commodities and industrial items, has i ncreased significantly as global t rade has d eveloped over recent d ecades. This has resulted i n a n i ncreased risk of m ovement of no n-native pe st s pecies on w ood us ed i n international t ransport. T his i s t he c ase of t he pi newood ne matode ( PWN), Bursaphelenchus x ylophilus. In t his s tudy, w e have e valuated t he e fficacy of v acuum pressure impregnation with chemical preservatives to eliminate PWN from wood.

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MATERIALS AND METHODS

Pinewood nematode infected Pinus pinaster trees were felled and trunk sections were cut. Initial nematode population was estimated by cutting 5 c m segments of each end of the trunk sections. Nematodes were extracted using the tray method (Whitehead & Hemming 1965) a nd t he t otal P WN num ber w as estimated. W ood m oisture c ontent ( WMC) w as measured using a digital wood moisture meter. Trunk sections with less than 25 % WMC and containing m ore t han 100 000 P WN ( ›60% t hird di spersal j uvenile s tage) (Magnusson & S chröder 2009) were s elected and introduced i nto a wood t reating autoclave and exposed t o t he vacuum and pressure i mpregnation with t he w ood preservative T ANALITH® E N B pl us t he a dditive A C 3744 f ollowing t he s ubsequent treatment scheme: i) initial vacuum with -950 mbar for 25 min; ii) autoclave filling with the pr eservative s olution a t 12500 m bar f or 75 m in; i ii) e mptying of t he r emaining solution t o t he a utoclave s upport t ank a nd i v) f inal v acuum t o r emove t he excess preservative solution on the surface of the wood at -800 mbar for 8 min. After treatment, wood sections were removed and left at room temperature to dry and then, incubated at 25ºC for 15 a nd 30 days to allow any live nematode present to breed and maximise the likelihood of detection ( EPPO 2013). Nematodes extraction was p erformed by t he t ray method.

RESULTS

In the treated trunk sections, 100% nematode mortality was achieved in all post-treatment assessment i ncubation pe riods ( 15 a nd 30 da ys). These pr eliminary r esults r evealed a potential use of this process to eliminate PWN from wood.

REFERENCES EPPO (2013). Diagnostics - Bursaphelenchus xylophilus. Bulletin OEPP/EPPO 43, 105- 118. FAO (2009). International standards for phytosanitary measures: guidelines for regulating wood packaging material in international trade. ISPM No. 15. Rome, Italy. Magnusson C, Schröder T (2009). Technical protocol for testing nematodes during treatment development. International Forestry Quarantine Research Group Meeting, September 2008, Rome, Italy. Whitehead AG, Hemming JR (1965). A comparison of some quantitative methods of extracting small vermiform nematodes from soil. Annals of Applied Biologists 55, 25–38.

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Đođ N, Pernek M, Carletti B, Pine wood nematodes - as a factor of pine decline in Croatia. In: Schröder, T. (ed.), Pine Wilt Disease Conference 2013, pp. 131-132, Braunschweig, ISSN: 1866-590X

(150) Pine wood nematodes - as a factor of pine decline in Croatia

Đođ N, Pernek M, Carletti B Croatian Forest Research Institute, Cvjetno naselje 41, HR-10450 Jastrebarsko, Croatia [email protected] Croatian Forest Research Institute, Cvjetno naselje 41, HR-10450 Jastrebarsko, Croatia [email protected] C.R.A. - Research Centre for Agrobiology and Pedology, via di Lanciola 12/A-50125 Cascine del Riccio, Firenze, Italy [email protected]

In recent years significant decline of pine trees of different species, age, size and position in the forests of Northern Dalmatia (Croatia) has been recorded. Climatic extremes, especially drought, can be considered the basic adverse factor causing stress a nd ph ysiological w eakening o f pi ne t rees a nd s imultaneously i mproving t he conditions f or a ttacks of va rious t ypes of pe sts. A nalyzing s everal biotic f actors associated with climate extremes shows presents of Pine Processionary Moth, longhorn beetles, bark beetles, needle cast disease caused by fungus but also pine wood nematodes. So far it is not possible to determine the scope of impact of wood pathogenic nematodes in the chain of pine die back without further studies. 20 s amples of Pinus nigra, Pinus pinaster and Pinus halepensis were collected at 6 locations along the coast of Northern Dalmatia. The first results indicate the presents of several groups of nematodes that leave in w ood Aphelenchoides spp., Laimaphelenchus spp., Ditylenchus spp., s aprofitic nematodes Cephalobus spp., Rhabditis spp. a nd Plectus spp., n ematodes on i nsects i n order Neotylenchidae and Diplogasteride, as well as species of genus Bursaphelenchus: B. mucronatus, B. sexdentati, B. eggersi, B. minutus of witch two first ones are considers as pa thogenic. In a ddition, ne matode ve ctor Monochamus gal loprovincialis has be en determined w hich m ay pl ay an i mportant role i n pos sible oc currence of qu arantine species of Bursaphelenchus xylophilus. Keywords: pine de cline, climate extremes, bark beetles, pine w ood nematodes, Bursaphelenchus spp.

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REFERENCES Pernek M et al. (2012). The role of biotic factors on pine (Pinus spp) decline ni North Dalmatia. Šumarski list 7-8, 343-354. Pernek M; Matošević D; (2003). Karatenski štetočinja Bursaphelenchus xylophilus – vektorski odnos prema rodu Monochamus i opasnost za hrvatsko šumarstvo. Glasilo biljne zaštite 6, 278-383. Carletti B; (2008). Bursaphelenchus species with their natural vectors in Italy: distribution and essential diagnostic features. Redia XCI, 111-117. Braasch H; (2001). Bursaphelenchus species in conifers in Europe: distribution and morphological relationship. Bulletin OEPP/EPPO Bulletin 31, 127-142.

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Filipak A, Tomalak M, The use of PCR-HRM technique for detection of the quarantine nematode Bursaphelenchus xylophilus. In: Schröder, T. (ed.), Pine Wilt Disease Conference 2013, pp. 133-135, Braunschweig, ISSN: 1866-590X

(162) The use of PCR-HRM technique for detection of the quarantine nematode Bursaphelenchus xylophilus

Anna Filipiak, Marek Tomalak, Department of Biological Pest Control, Institute of Plant Protection – National Research Institute, ul. Wladyslawa Wegorka 20, 60-318 Poznan, Poland. Email: [email protected]

INTRODUCTION

High Resolution Melting Analysis (PCR-HRM) is a recently developed technique for fast, high-throughput pos t-PCR ana lysis of genetic m utations or va riance i n nucleic aci d sequences. It enables r esearchers t o detect r apidly and categorize genetic m utations, identify new genetic variants without sequencing, or determine the genetic variation in a population prior to sequencing (Pasay et al., 2008). We have examined this technique for detection and distinguishing of the quarantine pest, B. x ylophilus from the morphologically and genetically most similar ne matode B. mucronatus.

MATERIALS AND METHODS

Genomic DNA of B. x ylophilus (isolate C hina) a nd B. m ucronatus (isolate W ro-01) (about 100 nematodes) was isolated with QIAamp DNA Micro Kit (QIAGEN) according to t he pr otocol pr ovided b y t he m anufacturer. F or each s pecies t he i solation w as conducted separately. The s pecific p rimers were de signed f rom t he ITS-1 r egion (forward: 5’ - CGTGCAACGGTAAAGTCTGGGTTT-3’ and r everse 5 ’- AATCCTACGCTCGCCAGAACGAAT-3’) (Fig. 1). The PCR product was expected to be 112 bp i n length. The PCR-HRM assays were performed with the use of Rotor–Gene Q T hermocycler ( Qiagen). The obt ained d ata w as an alysed according t o t he manufacturer’s instructions. A ....|....| ....|....| ....|....| ....|....| ....|....| 160 170 180 190 200 BmWro-01 CGCACGGAAG CCGAGAGGTG ACCGTGCAAC GGTAAAGTCT GGGTTTCTAT Bx CGCATGGAAG CCGAGAGGCG ACCGTGCAAC GGTGAAGTCT GGGTTTCTAC

B ....|....| ....|....| ....|....| ....|....| ....|....| 260 270 280 290 300 BmWro-01 GCTGCTTGCC GATTCGTTCT GGCGAGCGTA GGATTGAAAA GCCCGAGCGG Bx GCTGCTTGCC GATTCGTTCT GGCGAGCGTA GGATTGAAAA GCCCGAGAGG

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Fig. 1. Diagrams indicating differences in sequence alignment of the forward primer (A) and reverse primer (B) between B. xylophilus and B. mucronatus.

RESULTS AND DISCUSSION

The study conducted with the designed primers allowed us to distinguish and identify two morphologically similar nematode species, i.e. B. xylophilus and B. mucronatus. Melting curve analysis of t he r eaction pr oducts s howed t he pr esence of a s ingle a mplification product for each of the performed reactions (Fig. 2). The graph shows mean values from four replicates of each reaction.

Fig. 2. Melting curves of PCR-HRM products: B. xylophilus and B. mucronatus. The vertical axis shows the fluorescence value (dF/dT), and the horizontal axis the value of the melting temperature (Tm) (°C).

The normalized DNA m elting curves obt ained in the HRM analysis di ffered from each other in denaturation temperature, as evidenced by a substantial shift of these curves in relation t o e ach ot her. This i ndicates t he di fferences b etween ex amined species i n the composition of nuc leotides w ithin t he amplified r egion o f t he genome. The a nalogous differences are also evident in the normalized differentiating graph (Fig. 3).

Fig. 3. Normalized differentiating graph of PCR-HRM products for: B. xylophilus and B. mucronatus.

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Conducted study confirmed the high efficiency of the designed primers to distinguish the quarantine nematode B. xylophilus from the most closely related B. mucronatus. Since the PCR-HRM reaction allows for detection of single changes in nucleotides in tested of PCR products, it seems to be a very promising method for supporting identification decisions in the case of closely related species and isolates which could give ambiguous results in the r eal-time P CR r eaction. C ompared t o t he other m olecular m ethods, P CR-HRM technique c an be m uch s impler a nd l ess expensive w ay t o i dentify t he qua rantine nematode B. xylophilus.

REFERENCES Pasay C, Arlian L, Morgan M, Vyszenski-Moher D, Rose A, Holt D, Walton S, McCarthy J. (2008). High-resolution melt analysis for the detection of a mutation associated with permethrin resistance in a population of scabies mites. Medical and Veterinary Entomology 22, 82–88.

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Knapič M et al., Modelling of potential spread of Pine Wood Nematode by natural means in Slovenia at present climate conditions and in light of predicted climate changes. In: Schröder, T. (ed.), Pine Wilt Disease Conference 2013, pp. 136-137, Braunschweig, ISSN: 1866-590X

(164) Modelling of potential spread of Pine Wood Nematode by natural means in Slovenia at present climate conditions and in light of predicted climate changes

Gerič Stare B, Knapič M, Ogrica N, Širca S, Urek G Agricultutral Institute of Slovenia [Hacquetova ulica 17, SI-1000 Ljubljana, Slovenia] Email: [email protected]

ABSTRACT

Pine w ood ne matode (PWN), Bursaphelenchus xy lophilus, threatens econom y and biodiversity of S lovenian f orest. A m odel w as de veloped t o s imulate na tural m eans of spread of PWN from three (3) potential entry points to Slovenia. The stochastic model of natural spread was developed on a 1 × 1 km grid. The model is based on real data, such as host t ree de nsity, s usceptibility of each c onifer s pecies t o P WN i nfestation, s uitable temperature for P WN development, and appearance of t he drought s tress. Modelling is performed on the assumption that Monochamus spp. vectors are present in all Pine tree growing areas and that t heir maximum annual spread is 3 km . Simulation results show that if no containment measures are applied, at present climate conditions PWN would be spread almost over the entire Pine forest-covered and temperature appropriate territory of Slovenia in average of a pproximately 200 years. The extent and speed of P WN s pread differ depending on entry point and climate conditions. The modeling was simulated also using a medium scenario of predicted climate changes for two periods: 2021–2050 and 2061–2090. A t pr esent environmental c onditions, 8.954.000 m 3 of Pinus spp. g rowing stock i s e ndangered. The mode l c learly d emonstrated that the s preading of P WN is relatively slow by the natural means. The human factor is the most critical for rapid PWN spreading.

INTRODUCTION

PWN represents a serious threat to susceptible conifer trees by causing Pine wilt disease. By na tural me ans the P WN is tr ansmitted by v ectors – beetles of genus Monochamus. The ma ximum annual distance of be etle m ovement i s up t o 3.3 km (Kobayashi et al ., 1984). On long distance PWN is dispersed by human activity which increases the speed

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of s pread c onsiderably. O n t he ot her ha nd i t i s di fficult t o pr edict s uch l ong di stance jumps (Robinet et al. 2011). We m odelled t he spread o f P WN i n S lovenia b y n atural means only, c onsidering different i nfluencing factors, such a s hos t t ree de nsity, susceptibility of each conifer species to PWN infestation, suitable temperature for PWN development, and appearance of the drought stress. International trade is the most likely pathway of P WN pot ential introduction so m odelling w as don e f or 3 di fferent ent ry points: sea port, airport and a saw-mill.

MATERIAL AND METHODS

Spread of P WN w as m odeled i n 1 x 1 km g rid w ith c ellular a utomata us ing r ule of extended M oor’s ne ighborhood. P robability f or establishment of P WN i n t he c ell w as defined with temperature and drought stress while speed of dispersal was determined with maximal a nnual di stance o f be etle m ovement ( 3 km ) a nd hos t t ree de nsity and susceptibility. Each factor was classified and their influence in the rule of PWN spread was defined. The drought stress in the model was defined as a point in time, when soil moisture d ecreases b elow 50% of pl ant av ailable w ater and last at l east f or t wo consecutive months. The climate changes were simulated with general circulation models (Bergant 2007). The modelling simulations were performed in 300 replicates.

RESULTS AND DISCUSSION

Spread of P WN b y na tural m eans i s r elatively s low. T he m odeling r esults s how t hat appropriate space for PWN would be infested in 200 years at present climate condition. The modeling was also simulated using a m edium scenario of predicted climate changes (Bergant, 2007) for t wo periods, 2021–2050 and 2061 –2090. At pr esent environmental conditions, 8.954.000 m 3 of Pinus spp. g rowing s tock i s e ndangered. W ith c limate changes endangered qu antities of growing s tock would increase t o 14.464.000 a nd 18.577.000 m3 for periods of 2021–2050 and 2061–2090, respectively.

REFERENCES Bergant K (2007). [Climate change projections for Slovenia]. Podnebne spremembe: vpliv na gozd in gozdarstvo. Studia forestalia Slovenica 130, 67-86. Kobayashi F; Yamane A; Ikeda T (1984). The Japanese Pine Sawyer Beetle as the Vector of Pine Wilt Disease. Annual Review of Entomology 29(1). 115-135. Robinet C; Opstal N; Baker R; Roques A (2011). Applying a spread model to identify the entry points from which the pine wood nematode, the vector of pine wilt disease, would spread most rapidly across Europe. Biological Invasions 13(12), 2981- 2995.

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Xu JR, Wu XQ, Fang X, et al., Analysis of Transcriptional Expression Variation of Pine Wood Nematode (Bursaphelenchus xylophilus) using EST-based Simple Sequence Repeats. In: Schröder, T. (ed.), Pine Wilt Disease Conference 2013, pp. 138-139, Braunschweig, ISSN: 1866-590X

(167) Analysis of Transcriptional Expression Variation of Pine Wood Nematode (Bursaphelenchus xylophilus) using EST- based Simple Sequence Repeats

Jun-Rong Xu, Xiao-Qin Wu, Xin Fang, Jian-Ren Ye, Yun Liu, Lu Xu, Yao Lu Jiangsu Key Laboratory for Prevention and Management of Invasive Species, College of Forest Resources and Environment, Nanjing Forestry University, Nanjing, China Email: [email protected]

ABSTRACT

EST-SSR marker was used to study the transcription variation of Pine Wood Nematode (PWN) Bursaphelenchus x ylophilus. The r esults imply that there is ve ry low transcriptional e xpression va riation l evel of B. xylophilus in China, a nd C hinese B. xylophilus were more likely to be introduced from Japan.

INTRODUCTION

Pine w ilt di sease was fi rst detected in Nanjing in 1982 a nd t hen i t has s pread t o 176 counties of 15 provinces (SFA 2013) in China. The ES T-SSR marker could reveal t he population genetics of individual species based on exon sequence. Thus, EST-SSR was used to identify t he di fferential ex pressed genes of B. x ylophilus from di fferent a reas aiming at addressing the relationship between various genetic population and spread route in B. xylophilus.

MATERIALS AND METHODS

138 B. xylophilus DNA samples assorted randomly and mixed equally to 3 DNA pooling samples, w hich f rom 59 infected areas i n 12 provinces of C hina. A nd 2 D NA pool ing samples consist of 6 DNA samples from Japan and 3 from America respectively and one B. mucronatus DNA pooling sample including 63 DNA samples from different areas of China. ESTs of B. xylophilus which 13357 from NCBI and 884 from our laboratory were preprocessed by E ST-trimmer, removed ve ctor by c ross-match, spliced by C AP 3, a nd searched S SR l oci b y MISA (Hu 2005 ). All t he E ST-SSRs w ere designed primers b y Primer 5.0, and pre-screened with one Chinese B. xylophlus DNA pooling for PCR, after

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that, re-screened with 6 D NA pool ings. The r esult of pr oducts d etected by QIAxcel automatic g el el ectrophoresis ana lysis s ystem (Wang 2009) , a nd N TSYS-pc 2.10e w as used for UPGMA cluster.

DISCUSSION

A total of 14241 ESTs of B. xylophilus were spliced, and 6945 Uingenes (non-redundant ESTs) w ere obtained. 2 37 U ingenes c ontained 265 S SR l oci, c omprising 76 t ypes of repeat m otif, w hich account f or 3.41% o f a ll U nigenes. T he a verage di stance b etween SSRs was 12.14 kb. In the SSRs, the tri-nucleotide repeat motifs were the most abundant (64.53%), followed by t etra-nucleotide r epeat nu cleotide m otifs (20.38%). Further, 189 primers were designed and synthesized based on the above motif types, then verified with 5 DNA pooling samples of B. xylophilus and one B. mucronatus DNA pooling sample for PCR. The r esult of pr oducts s howed t hat t he products of 130 pr imers a re c lear and effective i n B. xylophilus, and 120 pr imers are v ersatile in B. mucronatus. All of EST- SSRs showed no polymorphism in B. xylophilus from China, including 101 homozygous loci and 29 heterozygous loci, which indicate the SSRs developed from the exons have no obvious di fferentiation. However, there a re 8 pleomorphic EST-SSRs d eveloped i n B. xylophilus from America and Japan, and B. mucronatus from China respectively, which showed higher polymorphic c ontent in B. m ucronatus. Above all, transcriptional expression variation level of B. xylophilus is very low, after invading China 30 years. The result of UPGMA dendrogram supports the view that B. xylophilus was introduced into China from Japan.

ACKNOWLEDGEMENTS

This work was supported by the National Nature Science Fund of China (No.31270683), the P roject f or Natural S cience R esearch of J iangsu University i n China (No.11KJA220002), an innovation P lan f or G raduate S tudents i n J iangsu P rovince i n China, and a Project Founded by the Priority Academic Program Development of Jiangsu Higher Education Institutions (PAPD).

REFERENCES State Forest Administration (2013). Announcement of Pine Nematode Disease-affected areas in 2013 (No.2, 2013). www.forestry.gov.cn/portal/main/s/3600/content- 581164.html (17.01.2013) Hu SN (2005). Manual of data analysis of Gene expressed sequence tags. Zhejiang University Press: Zhejiang. Wang XW; Rinehart TA; Wadl PA; et al (2009). A new electrophoresis technique to separate microsatellite alleles. African Journal of Biotechnology 8, 2432-2436.

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Van Bruggen AS et al, The Dutch apporoach to Pine Wilt Disease (PWD): longhorn beetles and nematodes under surveillance. In: Schröder, T. (ed.), Pine Wilt Disease Conference 2013, pp. 140-141, Braunschweig, ISSN: 1866-590X

(171) The Dutch approach to Pine Wilt Disease (PWD): longhorn beetles and nematodes under surveillance

Van Bruggen AS, den Nijs LJMF, Loomans AJM Wageningen NPPO, National Reference Centre. Geertjesweg 15 6706 EA Wageningen, the Netherlands [email protected]

Climatic c onditions in the N etherlands ar e similar to those of the a rea of pine w ood nematode ( PWN) distribution i n N orth A merica. Environmental c onditions in N orth America are generally unsuitable for pine wilt expression (Evans et al. 1996). According to this study, below the summer isotherm of 20 degrees expression of pine wilt disease (PWD) is not expected. It is, therefore, presumed that infested trees in the Netherlands will not e xpress s ymptoms. The a bsence of s ymptoms m akes i t di fficult t o de sign a survey on b asis of e arly detection w ithout a nalysing a l arge num ber of s amples. We explored experiences and investigations of other European countries without conditions for PWD expression (Magnusson et al, 2007; Magnusson, 2009; Schröder et al, 2009), to clarify the possibilities for a survey strategy in the Netherlands. Since t he Netherlands ar e a m ajor importer and exporter of products, the establishment potential of PWN (Bursaphelenchus xylophilus) is foremost determined by the possible introduction of i nfested l onghorn be etles Monochamus in wood or w ood pa ckaging material ( WPM). Therefore, the N etherlands o pted for a s imilar s trategy as ot her European countries where P WD s ymptomes are not t o be expected. The m onitoring of PWN and Monochamus in the Netherlands is mainly based on s urveys at locations were wood a nd/or w ooden pr oducts ( bark, w ood c hips, l umber, pa ckaging), or iginating i n countries were PWN occurs, are imported or stored. Additionally surveys are carried out for the vector in the rest of the Netherlands.

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Each year, our survey plan for the Netherlands consists of 7 parts, which are as follows: 1. On high risk locations Monochamus pheromone traps are used at the site itself. When longhorn beetles are found, they are analysed for the presence of nematodes. Dead or dying trees at a distance of 50 to 100 m are inspected for beetle activity. In collaboration with the European Invertebrate Survey (EIS-NL) at 10 selected locations with 3 pheromone baited traps each, the presence of Monochamus populations is monitored in nearby forests or groups of host plants (beyond 100 m). 2. Random survey of forest areas for the presence of Bursaphelenchus species in dead or dying trees. 3. Monitoring programme of wood packaging material originating in countries where PWN is known to occur. 4. Sampling and testing of coniferous wood (including chips, particles, shavings, etc.) originating in countries where PWN is known to occur. 5. Sampling and testing of coniferous bark originating in Portugal. 6. Monitoring and sampling of potted plants with coniferous bark used as mulch coverage originating in Portugal. 7. Inspection and sampling of plants for planting of Chamaecyparis, Pinus and Juniperus originating in Japan and South Korea, with reference to Commission Decisions 2002/887/EC and 2002/499/EC.

Results of our surveys from the last five years will be presented. PWN, Bursaphelenchus xylophilus, has not been found in the Netherlands so far, although it has been recorded once f rom i mported w ood. In i mported b ark o nly B. f ungivorus, B. m inutus and B. sexdentati have be en f ound. Its ve ctor, Monochamus spp., ha s be en f ound i n i mported wood package material or other wooden products. Surveys performed in The Netherlands show that a small population of M. galloprovincialis is persistent in a small pine wood forest near the coast (3x5 km), whereas it is absent in all other parts of the Netherlands.

REFERENCES Evans, H., McNamara, D.G., Braasch, H., Chadoeuf, J., Magnusson, C., 1996. Pest Risk Analysis (PRA) for the territories of the European Union (as PRA area) on Bursaphelenchus xylophilus and its vectors in the genus Monochamus. Bulletin OEPP/EPPO Bulletin 26, 199-249. Magnusson, C., Thunes, K.H., Nyeggen, H., Overgaard, H., Rafoss, T., Haukeland, S., Brurberg, M.B., Rasmussen, I., Strandenaes, K-A, Okland, B., and Hammeraas, B., 2007. Surveillance of Pine Wood Nematode (PWN) Bursaphelenchus xylophilus Norwegian Surveys 2000-2006. Bioforsk Report Vol. 2 No. 104 2007. 27 pp

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