workshop on xylella fastidiosa: knowledge gaps … · workshop on xylella fastidiosa: knowledge...

EVENT REPORT

APPROVED: 31 May 2016 PUBLISHED: 20 June 2016

www.efsa.europa.eu/publications EFSA Supporting publication 2016:EN-1039

Workshop on Xylella fastidiosa: knowledge gaps and research priorities for the EU

European Food Safety Authority and European Commission Directorates General for Research and Innovation, Agriculture

and Rural Development, and Health and Food Safety

Abstract

EFSA in collaboration with the European Commission’s Directorates-General for Research and Innovation, Agriculture and Rural Development, and Health and Food Safety organised a workshop in

Brussels on 12-13 November 2015, to identify and analyse the uncertainties and knowledge gaps on Xylella fastidiosa and to discuss priorities for future research on this pathogen in the EU. More than

120 scientists and other experts from around the world met to propose and discuss research initiatives

that can foster scientific understanding of the X. fastidiosa diseases and help find solutions for their control. The workshop was structured through plenary sessions and four discussion breakout groups

dealing with knowledge gaps and research priorities on different research areas: 1) X. fastidiosa surveillance and detection; 2) vector identification, biology, epidemiology and control; 3) host plants:

host range , breeding, resistance and certification; and 4) pathogen biology, genetics, characterization

and control. This workshop also aimed to facilitate interaction among research groups to share previous experiences, establish new research collaborations, strengthen current collaborations among

European and non-European research organizations, and increase awareness about scientific work previously done by others. This publication reports on the presentations, discussions, and

recommendations made during the workshop.

© European Food Safety Authority, 2016

Key words: bacterial pathogen, certification, detection, host plants, surveillance, vectors control,

Xylella fastidiosa,

Question number: EFSA-Q-2015-00527

Correspondence: any enquires related to this output should be addressed to [email protected]

Xylella fastidiosa: knowledge gaps and research priorities for the EU

www.efsa.europa.eu/publications 2 EFSA Supporting publication 2016:EN-1039

Disclaimer: The views or positions expressed in this publication do not necessarily represent in legal

terms the official position of the European Food Safety Authority (EFSA). EFSA assumes no responsibility or liability for any errors or inaccuracies that may appear.

Acknowledgements: EFSA wishes to thank the workshop chairs and rapporteurs: Harry Arijs, Domenico Bosco, Claude Bragard, Helvécio Della Coletta-Filho, Marie-Agnes Jacques, Michael John

Jeger, Stephen Parnell, Stephan Winter; the EFSA staff members: Miren Andueza, Franck Berthe, Vanessa Descy, Ciro Gardi, Gabor Hollo, Viran Kertesz, Ioannis Koufakis, Svetla Kozelska, Gritta

Schrader, Giuseppe Stancanelli, Claudia Timanti, Sara Tramontini, Sybren Vos; the EU Commission

staff members Pasquale Di Rubbo, Marios Nektarios and Annette Schneegans.

Suggested citation: EFSA (European Food Safety Authority), 2016. Workshop on Xylella fastidiosa:

knowledge gaps and research priorities for the EU. EFSA supporting publication 2016:EN-1039. 74 pp.

© European Food Safety Authority, 2016



Summary

The plant pathogenic bacterium Xylella fastidiosa was detected in olive trees in Lecce province in

Apulia, Italy, in October 2013 (Saponari et al., 2013). The Apulian strain of X. fastidiosa is considered to be a genetic variant within the subspecies pauca and identical (based on a sequence of 7

housekeeping genes) to a variant infecting oleander in Costa Rica (Nunney et al., 2014; Loconsole et al., 2016). This was the first field outbreak of X. fastidiosa in the European Union (EU) (Cariddi et al.,

2014). Afterwards, since summer 2015, several outbreaks of X. fastidiosa belonging to a different

subspecies group (i.e., multiplex) were reported from woody ornamental plants in Corsica and southern mainland of France. X. fastidiosa is one of the most dangerous plant pathogens worldwide,

damaging major crops including fruit trees, grapevine and ornamentals. X. fastidiosa is a quarantine pest for the EU1 and emergency measures2 have been in place since its first outbreak in 2013.

In its Scientific Opinion published in January 2015, the Scientific Panel on Plant Health of the

European Food Safety Authority (EFSA) conducted a detailed assessment of the risks to plant health for the EU territory posed by X. fastidiosa, including the identification and evaluation of risk reduction

options, and recommended an intensification of research activities on the host range, epidemiology and control of the Apulian outbreak of X. fastidiosa (EFSA PLH Panel, 2015).

In May 2015, the European Parliament approved a non-legislative resolution demanding actions to halt the spread of the X. fastidiosa outbreak, including increased funding for research and increased

international networking. To respond to the X. fastidiosa emergency needs, the European Commission

(EC) has therefore reinforced research and innovation actions. A workshop entitled “Xylella fastidiosa: Options for its Control” was organised by the EC Directorate-General (DG) for Research and

Innovation (RTD) at Expo in Milan in July 2015, to discuss with key experts the role that research and innovation can play in tackling the control of this bacterium (Anonymous, 2015). Specific research

funding on X. fastidiosa topics have been made available by the EC within the EU framework

programme for research and innovation Horizon 2020: the project Pest Organisms Threatening Europe (POnTE), including a work package on X. fastidiosa, was funded in November 20153, and a dedicated

topic on X. fastidiosa was launched in the Sustainable Food Security 2016 call under Horizon 20204.

In September 2015, EFSA was tasked by the EC DG RTD to identify knowledge gaps and key priorities

for research on X. fastidiosa in the EU, including the organisation of a scientific workshop, and to

produce a periodical scientific report on the status of the biology, epidemiology and control of this pathogen.

Following this request, on 12-13 November 2015 in Brussels (BE), EFSA hosted a workshop entitled “Xylella fastidiosa: knowledge gaps and research priorities for the EU” in collaboration with the EC DG

for Research and Innovation (RTD), Agriculture and Rural Development (AGRI), and Health and Food Safety (SANTE). The goal of this workshop was to facilitate scientific discussions and analyse

knowledge gaps and priorities for EU research on X. fastidiosa. This report presents the outputs of the

discussions at this workshop. The workshop, attended by about 130 participants (including scientists, risk assessors, risk managers, and stakeholders) from 16 EU countries and four non-EU countries, was

structured through plenary sessions with presentations from keynote speakers and four discussion breakout sessions. Each breakout session focused on identification of knowledge gaps and research

priorities on a different area of research: surveillance and detection (breakout Group 1); vectors

identity, biology, epidemiology and control (breakout Group 2); plants: host range, breeding, resistance and certification (breakout Group 3); pathogen biology, genetics, characterization and

control (breakout Group 4). The role of cropping practices and farming systems was also part of the discussion in the different breakout sessions. Each breakout session was introduced by short

presentations from selected participants. All participants were expected to actively participate on identification and discussion of research topics. Abstracts of the plenary session and of the short

1 http://eur-lex.europa.eu/LexUriServ/LexUriServ.do?uri=CONSLEG:2000L0029:20090303:EN:PDF 2http://ec.europa.eu/food/plant/plant_health_biosecurity/legislation/emergency_measures/xylella-fastidiosa/index_en.htm 3 http://www.ponteproject.eu/ 4 Spotlight on critical outbreak of pests: the case of Xylella fastidiosa

http://ec.europa.eu/research/participants/portal/desktop/en/opportunities/h2020/topics/6071-sfs-09-2016.html

http://eur-lex.europa.eu/LexUriServ/LexUriServ.do?uri=CONSLEG:2000L0029:20090303:EN:PDF

http://ec.europa.eu/food/plant/plant_health_biosecurity/legislation/emergency_measures/xylella-fastidiosa/index_en.htm

http://www.ponteproject.eu/




presentations are available in the Appendix C of this report. Presentations of the plenary and breakout sessions and the recorded webstreaming of the plenary sessions can be downloaded from the

workshop page on EFSA website5.

In line with the conclusion of the previous workshop hosted by DG RTD in July 2015 (Anonymous, 2015), this workshop aimed to facilitate interaction among research groups to share previous

experiences, establish new research collaborations, strengthen current collaborations among European and non-European research organizations, and to increase awareness about scientific work previously

done elsewhere.

From the discussions in the breakout and plenary sessions, knowledge gaps and research priorities and opportunities were highlighted by the participants.

For surveillance and detection, it was stressed the importance of defining in advance why surveillance is needed. Already since the planning phase of a survey, there is a need to have defined a clear

survey purpose. Can research improve the identification of targets (pathogen, vector, host, and pathways) and locations for surveillance? What are the best methods and periods for surveillance?

Answers to these questions are likely to depend on improvement of the detection methods for the

pathogen, especially during the latent period of infection, and of the knowledge on the underlying vector behaviours. Advanced remote sensing techniques could help in delimiting potential infected

areas if survey resources are limited, however there could be a trade-off between size of area to be surveyed and sensitivity of detection methods. Research on survey design also can facilitate, by

collection and analysis of survey data, the establishment of accurate disease/vector incidence and

distribution maps, and a better understanding of epidemics and dispersal of X. fastidiosa. To facilitate research and enable data exchange and analysis across Member States, there is also a need for

harmonisation of survey methods and sharing of data.

For research on vectors, the need for an inventory of potential vector species in the EU, including

collection of biological and environmental data and geographical coordinates was highlighted, even more considering that vectors may not be pest species per se and hence their biology has been poorly

studied so far. Insect rearing methods, knowledge of insect reproductive biology, feeding behaviours,

population dynamics, host preference for feeding and reproduction, and the importance of studying the ecosystem as a whole were identified as key areas for research. This basic knowledge should then

be translated into studies of pathogen transmission by vectors, vector infectivity in the field, and X. fastidiosa population dynamics. This in turn links with surveillance and monitoring. As the vectors are

not general pests in their own right, there is also little experience with control methods, IPM, or on

their effects on biodiversity. A general need of epidemiological research was highlighted to support both the development of integrated pest and disease management strategies and the risk

management and containment measures to prevent spread.

For research on host plants, several questions were raised and discussed to identify knowledge gaps

and research priorities. Should research on X. fastidiosa host range and plant genetic resources for

breeding resistant varieties be general or specific? With newly-identified hosts, research may help to optimize certification schemes and procedures. Research on X. fastidiosa host range should be

conducted to provide lists of host plants and also to help understand outbreaks as it relates to sources of inoculum in cultivated and non-cultivated habitats. It was recommended to complete the EFSA

database on X. fastidiosa host plants by adding information on hosts of strains that have been characterized. There is need for research to test important EU crop varieties for susceptibility to

infection by known X. fastidiosa strains from Europe (Italy and France), but also to representative

strains from other clades that may invade Europe. For breeding of resistant or tolerant plant genotypes, the recommendation was to begin with olive trees by screening germplasm collections for

tolerance or resistance to X. fastidiosa. Screening of germplasm collections should focus initially on commercially relevant varieties. If resistance/tolerance is not found within varieties or immediate

lineages, then additional accessions should be tested. For certification of plant propagation material,

there is need for research on diagnostic tools and sanitation methods. Multidisciplinary agro-ecological research was identified as a tool to investigate solutions to keep infected plants alive and economically

viable in the outbreak areas where olives or other hosts contribute significantly to landscapes and old trees have a high cultural value.

5 http://www.efsa.europa.eu/en/events/event/151112a

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With regard to the pathogen X. fastidiosa, discussion focused on the priorities for the EU, given the long history of X. fastidiosa research in the Americas, the timescale envisaged, the need for

fundamental science, and socio-economic impact. In the short term, targets could be characterization

of isolates from the European outbreaks and availability of reference collections. In the mid to long term, biotic and abiotic interactions, host specificity, host response to infection, and histopathology in

new hosts were topics suggested and discussed. For research into pathogen control, there is need to address farmers’ expectations, especially in relation to physical approaches and methodologies of

application of different tools and their integration.

As overall recommendations, it was stressed the need to establish an open-field laboratory in olive orchards affected by X. fastidiosa in Salento, where experiments could be conducted to develop

strategies for disease and vector control, as well as studies on epidemiology and screening for tolerant or resistant genotypes. Best practices to optimise the research efforts, to promote interactions among

participants, and to avoid research duplications were discussed and the following general suggestions were made: need for systematic reading of literature before designing new experiments; need to

share information and data via free and centralized online databases, including ongoing research

programmes; and need to provide a forum for international research meetings on X. fastidiosa and its vectors.



Table of contents

Abstract ......................................................................................................................................... 1 Summary ....................................................................................................................................... 3 1. Introduction ........................................................................................................................ 8 2. Plenary session ................................................................................................................... 9 3. Breakout Group discussions ............................................................................................... 10

Breakout Group 1 – Surveillance and detection ................................................................... 10 3.1.

3.1.1. Structure of discussion ...................................................................................................... 10 3.1.2. Summary of the discussion ................................................................................................ 11

Breakout Group 2 – The Vectors: identity, biology, epidemiology and control ....................... 13 3.2.3.2.1. Biology and ecology of vectors ........................................................................................... 14 3.2.2. Vector species identification in European outbreaks and their role in disease epidemiology .... 14 3.2.3. Vector role in the spread of the pathogen to other areas ..................................................... 15 3.2.4. Vector management .......................................................................................................... 15 3.2.5. Innovative data collection on vectors and disease epidemiology ........................................... 16 3.2.6. How can EU scientists best ‘catch up’ with ongoing research elsewhere? How to avoid

repeating research already done? How to benefit from previous work and how to best interact

with ongoing research activities on the vectors?.................................................................. 16 Breakout Group 3 – The Plants: host range, breeding, resistance and certification ................ 16 3.3.

3.3.1. Host range ....................................................................................................................... 16 3.3.2. Germplasm genetic resources and breeding for resistance ................................................... 18 3.3.3. Certification ...................................................................................................................... 18 3.3.4. Multidisciplinary approach .................................................................................................. 18 3.3.5. Summary of the topics discussed in the breakout discussion session 3 ................................. 18

Breakout Group 4 – The Pathogen: biology, genetics, and control ........................................ 19 3.4.3.4.1. Strains characterisation and monitoring the xylem-associated microbial diversity .................. 21 3.4.2. Xylella fastidiosa and its interactions with the ecosystem ..................................................... 21 3.4.3. Control measures .............................................................................................................. 22 4. Concluding session ............................................................................................................ 22 Conclusions and recommendations ................................................................................................ 22 References ................................................................................................................................... 24 Abbreviations ............................................................................................................................... 25 Appendix A – Workshop programme ........................................................................................ 26 Appendix B – Briefing notes for breakout groups ....................................................................... 31 1. Breakout Group 1 - Surveillance and Detection ................................................................... 32

Introduction ...................................................................................................................... 32 1.1.

Discussion points .............................................................................................................. 32 1.2. Background documents ..................................................................................................... 33 1.3.

2. Breakout Group 2 | The Vectors: identity, biology, epidemiology and control ........................ 34 Introduction ...................................................................................................................... 34 2.1.

Discussion points .............................................................................................................. 34 2.2.

Background documents ..................................................................................................... 35 2.3.3. Breakout Group 3 | The Plants: host range, breeding, resistance and certification ................. 36

Introduction ...................................................................................................................... 36 3.1. Discussion points .............................................................................................................. 37 3.2.

Background documents ..................................................................................................... 37 3.3.

4. Breakout Group 4 - The pathogen: biology, genetics, control ............................................... 38 Introduction ...................................................................................................................... 38 4.1.

Discussion points .............................................................................................................. 39 4.2. Background documents ..................................................................................................... 40 4.3.

Appendix C – Abstract book ..................................................................................................... 41 1. Abstracts of Plenary session ............................................................................................... 41

Status and control of Xylella fastidiosa in the EU ................................................................. 41 1.1.

What is the risk of Xylella fastidiosa for Europe? ................................................................. 41 1.2. State of the art on biology and epidemiology of the Xylella fastidiosa outbreak in Apulia ....... 42 1.3.



Xylella fastidiosa outbreaks in France: identification of the causal agents and distribution of the 1.4.disease ............................................................................................................................. 43

State of the art on Xylella fastidiosa in North America ......................................................... 43 1.5.

Xylella fastidiosa in South America: Ecological basis for management ................................... 45 1.6. Research needs on X. fastidiosa: a perspective from the EU farmers .................................... 45 1.7.

Xylella fastidiosa from the Euphresco point of view ............................................................. 46 1.8. Openings for research on plant health and plant protection in Horizon 2020: Focus on Xylella 1.9.

fastidiosa .......................................................................................................................... 47 2. Abstracts of session 1. Surveillance and Detection .............................................................. 47

Surveillance methodology for Xylella fastidiosa.................................................................... 47 2.1.

How can remote sensing contribute to the surveillance of X. fastidiosa affected regions? ...... 48 2.2. Innovative tools in the early detection and surveillance of X. fastidiosa ................................ 49 2.3.

Detection of Xylella fastidiosa in the frame of surveillance activities in Austria ....................... 50 2.4. Electrochemical label-free sensing platform for rapid detection of X. fastidiosa ..................... 50 2.5.

Detection of Xylella fastidiosa in Coffea arabica ornamental plants ....................................... 51 2.6.

The sentinel nursery and plantation concept: an opportunity to respond to the X. fastidiosa 2.7.threat to Europe? .............................................................................................................. 52

Developing guidelines for a possible strategy for the surveillance of Xylella fastidiosa in Europe52 2.8. The EU Horizon 2020 POnTE project: the work plan on X. fastidiosa surveillance .................. 53 2.9.

3. Abstracts of session 2. The Vectors: identity, biology, epidemiology and control ................... 53 Overview of current knowledge on insect vectors/potential vectors of Xylella fastidiosa in 3.1.

Europe ............................................................................................................................. 53 How do vector numbers, movements, infectivity and transmission efficiency impact the 3.2.

incidence and spatial patterns of plant infection with X. fastidiosa? ...................................... 54 Ecological and behavioural traits that determine vector relevance ........................................ 55 3.3. Survey of potential vectors of Xylella fastidiosa in olives groves in southern Spain................. 56 3.4. The "vectors" aspects of the Belgian Xyleris research project ............................................... 56 3.5.

Options for control of meadow spittlebug (Philaenus spumarius, L.) in organic farming: 3.6.overview of available active substances .............................................................................. 57

Outline of EFSA outsourced project to collect data on biology and control of vectors of Xylella 3.7.fastidiosa .......................................................................................................................... 57

The EU Horizon 2020 POnTE project: the work plan on Xylella fastidiosa vectors .................. 58 3.8.

4. Abstracts of session 3. The Plants: host range, breeding, resistance and certification ............ 59 Lessons learned with CVC and X. fastidiosa subsp. pauca in Brazil: host range of bacterium, 4.1.

breeding for resistance and citrus nursery certification programme ...................................... 59 A comprehensive database on Xylella fastidiosa host plants ................................................. 59 4.2.

Rootstock Effects on Almond Leaf Scorch Disease Incidence and Severity ............................ 60 4.3.

Preliminary results of a pilot project on host range of X. fastidiosa CoDiRO strain and 4.4.evidences of resistance phenomena in the cv. Leccino upon the infection of X. fastidiosa ...... 60

The World Olive Collection of Córdoba ................................................................................ 61 4.5. Greek olive germplasm breeding and certification research priorities on X. fastidiosa............. 61 4.6.

The EU Horizon 2020 POnTE project: the work plan on host plants of X. fastidiosa ............... 62 4.7.5. Abstracts of session 4. The Pathogen: biology, genetics and control ..................................... 62

Can comparative genomics yield insights to control diseases caused by X. fastidiosa? ........... 62 5.1.

Genetic diversity of the CoDiRO strain and other EU intercepted isolates .............................. 63 5.2. Improved detection of X. fastidiosa and the potential of rapid whole genome sequencing for 5.3.

outbreak delineation and pathogenicity profiling ................................................................. 64 Antibacterial and plant defence elicitor peptides: novel tools against Xylella fastidiosa? ......... 64 5.4.

A biotechnological approach for Xylella fastidiosa targeting and population confusion ............ 65 5.5.

The EU Horizon 2020 POnTE project: the work plan on the pathogen “X. fastidiosa” ............. 66 5.6. Decoding the DSF quorum-sensing system in Xanthomonadaceae: lessons from 5.7.

Stenotrophomonas maltophilia ........................................................................................... 67 Appendix D – List of Workshop participants .............................................................................. 68 Appendix E – Workshop evaluation .......................................................................................... 74



1. Introduction

The plant pathogenic bacterium Xylella fastidiosa was detected in olive trees in Lecce province in

Apulia, Italy, in October 2013 (Saponari et al., 2013). The Apulian strain of X. fastidiosa is considered to be a genetic variant within the subspecies pauca and identical (based on a sequence of 7

housekeeping genes) to a variant infecting oleander in Costa Rica (Nunney et al., 2014; Loconsole et al., 2016). This was the first field outbreak of X. fastidiosa in the European Union (EU) (Cariddi et al.,

2014). Afterwards, since summer 2015, several outbreaks of X. fastidiosa belonging to a different

subspecies group (i.e., multiplex) were reported from woody ornamental plants in Corsica and southern mainland of France. X. fastidiosa is one of the most dangerous plant pathogens worldwide,

damaging major crops including fruit trees, grapevine and ornamentals. X. fastidiosa is a quarantine pest for the EU6 and emergency measures7 have been in place in the EU since its first outbreak in

2013.

In its Scientific Opinion published in January 2015, the Scientific Panel on Plant Health of the European Food Safety Authority (EFSA) conducted a detailed assessment of the risks to plant health

posed by X. fastidiosa for the EU territory, including the identification and evaluation of risk reduction options, and recommended an intensification of research activities on the host range, epidemiology

and control of the Apulian outbreak of X. fastidiosa (EFSA PLH Panel, 2015). In May 2015, the European Parliament approved a non-legislative resolution demanding actions to halt the spread of

the X. fastidiosa outbreak, including stepping up funding for research, and increasing international

networking. In order to respond to the X. fastidiosa emergency needs, the European Commission (EC) has therefore reinforced research and innovation actions. A workshop on “Xylella fastidiosa: Options

for its Control” was organised by the EC Directorate-General (DG) for Research and Innovation (RTD) at Expo in Milan in July 2015, to discuss with key experts the role that research and innovation can

play in tackling the control of this bacterium (Anonymous, 2015). Specific research funding on X. fastidiosa topics have been made available by the EC within the EU framework programme for research and innovation Horizon 2020: the project Pest Organisms Threatening Europe (POnTE),

including a work package on X. fastidiosa, was funded in November 20158, and a dedicated topic on X. fastidiosa was launched in the Sustainable Food Security 2016 call under Horizon 20209.

In September 2015, EFSA was tasked by the EC DG RTD to identify knowledge gaps and key priorities

for research on X. fastidiosa in the EU, including the organisation of a scientific workshop, and to produce a periodical scientific report on the state of the biology, epidemiology and control of this

pathogen. Following this request, on 12-13 November 2015 in Brussels (BE), EFSA hosted a workshop on “Xylella fastidiosa: knowledge gaps and research priorities for the EU” in collaboration with the EC

DG for Research and Innovation (RTD), Agriculture and Rural Development (AGRI), and Health and Food Safety (SANTE), aiming to provide a scientific environment to discuss and analyse knowledge

gaps and priorities for EU research on X. fastidiosa.

This report presents the outputs of the discussion at this workshop. The workshop, attended by ca. 130 participants (including scientists, risk assessors, risk managers and stakeholders) from 16 EU

countries and four extra-EU countries, , was structured through plenary sessions with presentations from keynote speakers and four discussion breakout groups. Each breakout session dealt with

knowledge gaps and research priorities on a different area of research: surveillance and detection

(breakout Group 1); vectors identity, biology, epidemiology and control (breakout Group 2); plants: host range, breeding, resistance and certification (breakout Group 3); pathogen biology, genetics,

typing and control (breakout Group 4). The role of cropping practices and farming systems was also part of the discussion in the different breakout sessions. Each breakout session was introduced by

short presentations from selected participants. All participants could actively take part in the identification and discussion of research topics. The abstracts of the plenary and of the short

presentations can be found in the Appendix C of this report. The presentations of the plenary and

6 http://eur-lex.europa.eu/LexUriServ/LexUriServ.do?uri=CONSLEG:2000L0029:20090303:EN:PDF 7 http://ec.europa.eu/food/plant/plant_health_biosecurity/legislation/emergency_measures/xylella-fastidiosa/index_en.htm 8 http://www.ponteproject.eu/ 9 Spotlight on critical outbreak of pests: the case of Xylella fastidiosa


http://eur-lex.europa.eu/LexUriServ/LexUriServ.do?uri=CONSLEG:2000L0029:20090303:EN:PDF

http://ec.europa.eu/food/plant/plant_health_biosecurity/legislation/emergency_measures/xylella-fastidiosa/index_en.htm





breakout sessions and the recorded webstreaming of the plenary sessions can be downloaded from the workshop page on EFSA website10.

In line with the conclusion of the previous workshop hosted by DG RTD in July 2015 (Anonymous,

2015), this workshop aimed to facilitate interaction among research groups to share previous experiences, establish new research collaborations, strengthen current collaborations among European

and non-European research organizations, and to increase awareness about scientific work previously done elsewhere.

2. Plenary session

The Plenary Session set the scene for identification of research gaps and priorities in the EU context for research initiatives on X. fastidiosa. The workshop opened with a welcoming address by Patrick

Kolar of the EC DG RTD, who emphasized in particular the opportunities for interaction among the participants in relation to the research agenda. Then, Pasquale di Rubbo of EC DG SANTE summarised

the regulatory framework for X. fastidiosa in the EU, in particular related to movement of plant

material, demarcated areas (affected and buffer zones), eradication and containment, and the need for research in support of regulation. Claude Bragard of the EFSA Plant Health Panel introduced the

audience to the EFSA risk assessment of X. fastidiosa for the EU, presenting the challenges not only to the olives but to EU agriculture more generally, and pointing out the unknown threat to the European

flora (including forests), the importance of databases and information on trade flows, and the extent to which climate could be a limiting factor for X. fastidiosa.

Following these speeches, a series of scientific and technical presentations were made by researchers

and stakeholders. A short summary of each presentation is given below.

Maria Saponari (Researcher, National Research Council of Bari, Italy) introduced the case of the

Apulian outbreak, with the most updated information concerning the list of host plants (including wild species) found infected by the Apulian strain of X. fastidiosa. The main primary host is considered to

be olive, without findings on weeds and wild flora, despite repeated surveys. Also, it was remarked

that, until then, X. fastidiosa was not found in the collected samples of grapevine or citrus plants in Apulia. The relevance of the role of Philaenus spumarius, the only confirmed vector in Apulia, in the

olive-to-olive transmission was indicated.

Charles Manceau (Director, Plant Health in ANSES, France) provided an update on the outbreaks of X. fastidiosa in France, the different plant species found to be infected, the identified strains of the

pathogen and the geographical locations of the findings. In those circumstances the main host identified was Polygala myrtifolia, followed (but with much lower numbers) by Spartium junceum,

Pelargonium graveolens, and Cytisus sp. The isolates of X. fastidiosa in Corsica and mainland France belong to the subspecies multiplex.

Alexander Purcell (Professor Emeritus, University of California, Berkeley, USA) and João Lopes (Professor, University of São Paulo, Piracicaba, Brazil) presented the state of the art concerning

knowledge on X. fastidiosa in California, USA, and São Paulo, Brazil, respectively. Purcell presented

the effects of low winter temperatures on X. fastidiosa survival to next year, in grapevines. The

effectiveness of physical (low temperatures) and chemical treatments against the pathogen, of vector control and of antagonistic microbes was also discussed. Lopes explained the relevance of considering

X. fastidiosa integrated within its pathosystem (pathogen/host plant/vector association) when dealing with the definition of the control strategy.

Giovanni Cantele (European farmers organisation Copa-Cogeca, Italy) indicated the research priorities

in the interest of EU farmers: identification and development of resistant or tolerant varieties, curative solutions, vector control by integrated pest management or biological control means, search for

alternative crops/renaturalization projects. He mentioned the promising method of heat treatment as a control option for planting material as well as the need for an integrated strategy based on

agronomic practices. Additionally, he pointed out the opportunity to take advantage of the infected

olive orchards in Salento as an open-field laboratory where experiments could be conducted to develop strategies for disease and vector control.

10 http://www.efsa.europa.eu/en/events/event/151112a

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Lastly, Baldissera Giovani (Coordinator, European ERA-Net network Euphresco) described the scope and structure of Euphresco highlighting how the research funded by this Network could help to

address the X. fastidiosa emergency. The Euphresco perspective on research needs stresses the

importance of coordination and complementarity of initiatives across Europe, but also the need for some level of focus. Among the ongoing and planned Euphresco research activities, a harmonized

protocol for monitoring and detection of X. fastidiosa will be funded and complementary research will be conducted on diagnostic tools for olives, grapevine, fruit trees, and ornamentals. Research gaps on

vector capacity and biology in plants should also be studied further.

3. Breakout Group discussions

Breakout Group 1 – Surveillance and detection 3.1.

This session was attended by 32 participants. Harry Arijs from EC DG SANTE acted as chair and

Stephen Parnell as rapporteur of the meeting. At the beginning of this breakout session, eight short presentations were given by participants covering different aspects of surveillance and research

needed to plan and execute effective surveys, including innovative and advanced methods for monitoring or detection. Stephen Parnell presented an overview of a methodology for surveillance,

particularly quantitative-based approaches, with a focus on X. fastidiosa. Pieter Beck presented a possible contribution of remote sensing technology to survey large areas for presence of disease,

particularly regarding early detection of disease symptoms. Anna Maria Donghia presented innovative

tools for early detection and surveillance of X. fastidiosa in the Apulian outbreak. Detection of X. fastidiosa in the frame of surveillance activities for Austria and the Netherlands was presented by

Richard Gottsberger and Maria Bergsma-Vlami, respectively. An electrochemical label-free sensing platform that could be used also for rapid, onsite detection of X. fastidiosa was presented by Alan

O’Riordan. Andrea Vannini presented a sentinel nursery concept and how it could provide an

opportunity to respond to the X. fastidiosa threat to Europe. Emilio Stefani discussed about guidelines for surveillance strategy for X. fastidiosa in Europe.

Surveillance for X. fastidiosa is crucial throughout the area of potential establishment in the EU. Effective surveillance can enhance control efforts by aiding swift identification of outbreaks and

provide data to improve our understanding of epidemiology and current geographical distribution of X. fastidiosa. Depending on the status of disease in a specific area, surveillance is required for early

detection of new outbreaks, monitoring and delimiting of individual outbreaks, and to map pest-free

areas.

This breakout group dealt with discussion of knowledge gaps and research priorities for surveillance

and detection of X. fastidiosa in Europe. Effective surveillance must be underpinned by research on host plant, pathogen and vector biology, rigorous statistical methods, risk-based modelling

approaches to target the deployment of sampling resources, and advanced diagnostic and detection

methods. Regarding the use of the survey data, the group highlighted the need for harmonisation of survey methods and sharing of survey data to facilitate research and enable inferences to be made

across the EU Member States.

3.1.1. Structure of discussion

The ensuing discussion was structured around three key discussion points relating to surveillance and

detection. The following questions were posed to the participants for discussion:

Why do we need surveillance and what are the related research needs? Identification of the

different purposes for surveillance and the research needs related to each.

What and where do we need to survey? Research needs to identify targets and locations for

surveillance.

How and when to perform surveillance? Research needs to identify best methods and

appropriate frequency and periods.

Following an open discussion around each of these discussion points, participants were invited to identify key research gaps for surveillance and detection. Ideas from the group were captured and the

participants individually assigned the identified gaps to categories relating to the three stages of



surveillance: (i) planning surveillance, (ii) conducting surveillance and (iii) using results of surveillance. The identified research gaps were further categorised by those that related to the pathogen, host

plants, vectors, or research gaps that spanned all three components of the pathosystem. Once

collected, the notes were discussed with the group to collate overlapping issues and to identify the most important research gaps. These are summarised in the following section and in Table 1.

3.1.2. Summary of the discussion

Planning surveillance – research needs to clearly define the purpose of the survey and

appropriate methodology

The group identified the need for a clear definition of survey purpose during the planning phase. The

range of surveillance purposes were discussed including for early detection in areas where X. fastidiosa is not yet known to occur, monitoring of outbreaks in affected areas, delimiting of outbreak

areas, and establishment of pest-free areas. How and what resources are utilised in surveillance will

depend on the specific purpose of the survey. In line with guidance provided in ISPM 611 broadly, surveys aimed at delimiting outbreaks should be targeted, whereas those aimed at monitoring the

incidence and distribution of X. fastidiosa should be random and representative of the population. It was noted that the purpose of the survey may be similar among regions, but conditions relating to the

pathogen, plants, and vectors necessitate that survey plans be tailored to each Member State. A key topic was the need for harmonisation of survey plans and information across Member States.

Approaches for understanding existing surveillance efforts amongst growers and the public and how

this could complement regulatory surveys were discussed.

Improved methods to identify spatial and temporal estimates of X. fastidiosa establishment and

spread across Member States was identified as important for targeted surveillance and necessitates the use of epidemic modelling, GIS (Geographic Information Systems) approaches, and better

collection of data on vector and host distribution. Methods to identify vector and host species, their

susceptibility, and accurate maps of distribution and density across potential areas of establishment in the EU are needed and should be utilised when planning surveillance. The utilisation of improved

information on long-distance pathogen dispersal by different dispersal mechanisms (e.g. vector flight, vector movement in trade material) also was identified as important. In addition to identifying the

most important invasion pathways, the areas/crops of most economic interest and those with most epidemiological significance is required.

There is a clear need for improved methods for early detection of X. fastidiosa in host plants. Methods

need to have high levels of sensitivity and specificity, low cost, and be available for rapid use. There is a need to better understand the length of the asymptomatic period of infections in hosts and select

detection methods appropriate to each asymptomatic period. These methods include those at a range of scales involving remote sensing, host inspection, and vector trapping. How to integrate and balance

resources across these different approaches should also be considered and understood in detail. For

example, remote sensing shows considerable potential for delimiting infected areas, particularly in case of scarce resources available for conducting surveys. However, there is a trade-off between the

size of a region that can be covered by the survey and sensitivity (i.e., cover a smaller amount of area with high sensitivity or larger amount of area with low sensitivity).

Conducting surveillance – Research needs to identify targets and locations for

surveillance.

Active surveillance programmes are expensive and resources are limited. Therefore, the group

identified the need to raise awareness among producers and the public when conducting surveillance to encourage alertness, compliance, and reporting. Partnering with the private sector should be

encouraged to facilitate and finance the surveillance process. The use of smartphone applications should be enhanced to enable reporting of symptoms by growers and the public, which can be further

investigated via serological and molecular methods. Interaction with other stakeholders and NPPOs is

also important to facilitate sharing and reporting of data.

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Validation of detection methods is very important for performing reliable surveillance; this could be done in third countries where X. fastidiosa is already present or in current affected areas in the EU.

The availability of standardised survey methods and protocols for X. fastidiosa is crucial and will

facilitate good practices such as recording and storage of negative data (locations that have been visited but no infection found), which is critical for understanding the distribution and incidence of X. fastidiosa in a particular area. The use and reporting of sample sizes (across multiple scales: number of locations visited, number of samples per location, number of samples per plant) should be noted.

In addition, sampling should be conducted to coincide with key stages of pathogen lifecycle and

temporal changes in symptom expression and X. fastidiosa population densities. The potential for using sentinel plants in nurseries for early detection was also discussed.

Use of surveillance results – Research needs to identify best methods and appropriate timing.

The use of survey results is inseparably intertwined with the way a survey has been conducted, reinforcing the need to consider the purpose of the survey and the eventual use of survey results

already at the planning stage. For example, results from targeted surveys biased toward highest risk

areas cannot readily be used to infer X. fastidiosa distribution and incidence in other areas. Therefore, there is need for research on survey design to facilitate the range of necessary uses of survey data,

including establishment of more accurate X. fastidiosa/vector incidence and distribution maps to improve understanding of epidemics and dispersal, inform pest risk assessment, and evaluate the

effectiveness of risk reduction measures. Survey data also can provide feedback to epidemic models

that enable analysis of surveillance data and control measures based on the current state of information. Surveys should also be designed to facilitate adaptation of survey methods as new

information emerges. Central to all of this is the harmonisation of methods and sharing of data to facilitate research and enable inferences to be made across Member States.

Table 1: Summary of the brainstorming session of the breakout session 1.

Planning surveillance

Plants - Identify host species and their susceptibility - Accurate maps of potential host distribution - Identify most important pathways - Identify areas/crops of most economic impact - Identify areas/crops of most epidemiological significance - Identify sentinel host species as canaries in mine shaft

Pathogen - Identify detection methods with high sensitivity, low cost and rapid result

- Differentiate surveys for areas with disease (i) known to be and (ii) not known to be present

- Understand length of asymptomatic period and impact on choice of detection method - Determine appropriate resolution of remote sensing method balanced with cost and

coverage - Identify high risk pathways and entry points

Vector - Understanding vector dispersal scale to inform surveillance

- How much survey resource to allocate to vector populations vs hosts? - Identify vector species and spatial distribution

All - Identifying the specific purpose(s) of each survey specific and tailor to each member

state - Harmonization of survey methods across member states - Country specific conditions and how to target survey for effectiveness - Understand existing surveillance effort from growers and public and how this

complements regulatory survey - How to determine spatial and temporal establishment and spread estimates to target

surveys? - How to optimise the deployment of multiple detection methods to maximise detections

Conducting surveillance

Plants - How to use remote sensing to identify host populations



- How to set up sentinel plants in nursery networks

Pathogen - Remote sensing to target survey effort to suspected locations - Methods to record negative data during survey (i.e. location of sites visited but no

disease found) - Validate survey methods such as remote sensing in endemic areas of the world before

application in EU Vector - Effective and standardized methods for vector trapping

- How to interpret detection in vector populations, i.e. what this means for source of infection and incidence

All - How to conduct awareness among growers/public and encourage compliance/reporting - Partnering with private sector to finance and conduct surveillance - Smart phone applications for growers/public to report suspected finds - Methods to adapt surveillance methods as new data/information become available

Use results of surveillance

Plants - How to use survey results to delimit size of buffer zones

Pathogen - How to determine thresholds to declare freedom disease - Develop maps of incidence across EU

Vector - Inform knowledge of vector population distribution

All - Harmonized methods across member states for comparison of results and

standardization of data recording - Normalize data acquisition to enable quantitative analysis - Perform retrospective analysis of existing survey data to address questions of X.

fastidiosa presence/absence, prevalence and spatial extent

Breakout Group 2 – The Vectors: identity, biology, epidemiology 3.2.and control

This session was attended by 28 participants. Giuseppe Stancanelli acted as chair and Domenico Bosco as rapporteur of the meeting. At the beginning of the breakout session, five short presentations

were given by participants that covered different aspects of insect vector

biology/ecology/behaviour/control. The main ongoing research projects on the topic were presented. Domenico Bosco presented an overview of current knowledge on insect vectors and potential vectors

of X. fastidiosa in Europe, as well as the work plan on X. fastidiosa vectors of the EU Horizon 2020 POnTE project. Alexander Purcell discussed how vector numbers, movements, infectivity, and

transmission efficiency can impact the incidence and spatial patterns of plant infection with X. fastidiosa. Joao Lopes presented the ecological and behavioural traits that determine vector relevance. Then, short presentations were given by participants on the present knowledge of potential vector

species in Spain (Alberto Fereres), the "vectors" aspects of the Belgian Xyleris research project (Jean-Claude Gregoire), options for control of Philaenus spumarius in organic farming (Vincenzo Verrastro),

and an outline of the EFSA outsourced project to collect data on biology and control of vectors of X. fastidiosa (Sabrina Bertin).

X. fastidiosa is exclusively transmitted by xylem fluid-feeding insects (order Hemiptera, sub-order

Auchenorrhyncha), belonging to three superfamilies: Cercopoidea (spittlebugs or froghoppers), Cicadoidea (cicadas), and Membracoidea (which includes a single xylem fluid-feeding subfamily, the

Cicadellinae, known as sharpshooters) (Redak et al., 2004). In general, vectors of X. fastidiosa are not considered direct pests unless present at very high population levels. The spittlebug species, Philaenus spumarius, is the only vector of X. fastidiosa identified so far in Europe (Saponari et al., 2014).

Vectors transmit X. fastidiosa in a persistent manner without the need for a latent period between acquisition and inoculation; acquired bacteria are limited to the foregut, which explains why nymphs

lose infectivity with moulting (Almeida and Purcell, 2006). All xylem fluid-feeding insects should be regarded as potential vectors of X. fastidiosa (Frazier, 1944; Purcell, 1989), but some species in

Europe are more likely candidates, owing to their wide geographical distribution, abundance, and host

plant range (for listing and review of European potential vectors, see pages 29-34 and Appendixes C



and D from EFSA PLH Panel, 2015a). There is a general lack of knowledge concerning all European putative vectors: their presence, abundance, role in the transmission and epidemic progress of X. fastidiosa, and on the control measures that can be applied to suppress vector populations. Therefore,

it is very difficult to assess the potential role of vectors in the spread and epidemics of X. fastidiosa in Europe.

The discussion points described in the briefing notes were adopted as a basis for the discussion with the goal of identifying knowledge gaps on vectors and research priorities needed to cover these gaps.

3.2.1. Biology and ecology of vectors

Jean-Claude Grégoire presented the Belgian Xyleris research project, which is in part dedicated to study the presence of possible insect vectors in Belgium, their life cycle and their capacity to transmit

the bacterium. Domenico Bosco presented the EU Horizon 2020 POnTE project, which focuses on X. fastidiosa strain CoDiRO vectors biology/ecology/phenology/epidemiology in the infected area of Italy

and in other potential areas of spread in the EU. Participants agreed on the need to establish an

inventory of the potential vector species in different geographic areas at different scales (environments/agro-ecosystems/crops). For this purpose, common survey protocols should be

established for the sampling methods and data records (see also breakout session 1). Also, reference specimen should be stored in vouchers and made available on demand for successive analyses (e.g.

species identification, genetic analysis, pathogen identification). Surveys should be carried out in all the Member States and in non-EU Mediterranean countries to obtain a comprehensive description of

vector species in the EU and in neighbouring countries. Therefore, the EU should support research to

fill in the gaps on data collection due to the lack of national funding support for surveys (see also breakout session 1). Development of rearing techniques for vector species is needed to provide

research material for experiments aimed at studying insect development, life cycle, reproductive and transmission biology, as well as to test the efficacy of control methods. Since the efficacy of the

available sampling methods has been questioned (namely yellow sticky traps, as not all potential

vectors are attracted by yellow, or by colours in the case of nocturnal species), sampling methods should be designed for each species. A consensus was achieved on the need of detailed studies on

vector reproductive biology and life history, as this knowledge is crucial to design effective and rational control programmes. To this purpose, presence and role of facultative endosymbionts such as

Wolbachia (Charlat et al., 2003) that disturb reproduction in some species should be investigated. Knowledge of the microbiome associated with the insect foregut is also of interest because microbes

co-localised with X. fastidiosa may interfere with phases of the transmission process (acquisition,

retention, and inoculation). Since transmission of X. fastidiosa is strictly dependent on xylem fluid-feeding, feeding behaviours of known vectors and potential vector species should be studied on

different host plants and for different life stages (nymphs and adults).

Since xylem fluid-feeding insects are polyphagous species, participants in this session recommended

that ecological studies on vectors should be targeted to the whole agro-ecosystem, not only to specific

crops. One of the main research needs is characterization of the xylem fluid-feeding fauna at different scales (landscape, agro-ecosystem, field crop), together with the description of the relative abundance

of vector species. Knowledge of insect population dynamics in different geographical areas, together with their host plant preferences for feeding and reproduction and their overwintering behaviour are

important for designing rational control programmes. Identification of non-host plants for vector

species should also be a research priority as these plant species may be used in the frame of a vegetation management programme to reduce vector populations in and around susceptible crops.

3.2.2. Vector species identification in European outbreaks and their role in disease epidemiology

To understand the spread of X. fastidiosa an accurate record of disease/infection progress (possibly

plant by plant) should be done in the infected areas. Since disease progression is not determined only by vector population densities, other factors driving epidemics should be clarified. Factors include

vector host plant preference, life cycle, feeding behaviour on different plants, and movement and dispersal behaviours. As for transmission of X. fastidiosa, besides controlled transmission experiments

in the laboratory, experiments with sentinel plants should be conducted in the field as they can help estimate infection pressure in a given environment as well as the seasonality of vector infectivity.



Also, identification of the sources of inoculum for vectors is imperative to understand disease epidemiology and prevent pathogen spread. There is an urgent need for vector transmission studies

with EU xylem fluid-feeding specialist insects, namely spittlebugs, which have been poorly studied in

the Americas. Since P. spumarius and other spittlebugs are relatively long-living insects (adults can live more than 6 months in warm Mediterranean areas), lifetime infectivity should be investigated. The

persistent association between insect vectors and X. fastidiosa is of high epidemiological importance and the time scale for presence of infective adults in nature has important consequences on control

programmes. Transmission capability of European sharpshooters and cicadas should also be

investigated. The cicada transmission competence should be ascertained because cicadas are very common and locally abundant in Mediterranean agro-ecosystems, including olive orchards.

Transmission experiments should be conducted considering both acquisition from and inoculation to different host plants to identify the main sources of inoculum and the most susceptible hosts.

3.2.3. Vector role in the spread of the pathogen to other areas

There is a lack of direct scientific data on the active and passive spread capacity of the vector P. spumarius. For the short-distance dispersal, techniques such as the mark release-recapture and the

flight mill can be applied to describe the active dispersal and flight activity of putative vectors. For long-distance dispersal, molecular markers can be developed to identify specific genotypes and their

long range spread. Preliminary observations proposed in the EFSA PLH Panel opinion (2015) suggest that spittlebugs can be transported by vehicles and can therefore cover long distances by human-

assisted dispersal. However, no experimental data on this aspect (e.g. survival on different means of

transportation, low and high lethal temperatures, survival time without feeding, etc.) are currently available and research aimed at filling these knowledge gaps would be useful.

3.2.4. Vector management

Development of innovative and effective control programmes for X. fastidiosa vectors relies heavily on

in-depth knowledge of vector ecology and biology (e.g. host plant preference for feeding of vector life

stages, host plant preference for egg laying, developmental time, longevity, prolificacy, response to temperature, and thermal requirements). According to Vincenzo Verrastro, these aspects are

particularly crucial in organic farming, which currently rely on a single product (citrus oil extract) for vector control in olive in Italy. Control programmes can integrate different measures such as soil and

vegetation management (e.g. soil tilling or weed control to reduce nymphal populations associated

with herbaceous hosts), application of insecticides, and augmentation of natural enemy populations (predators, parasitoids, pathogens). For these purposes, efficacy of soil and vegetation management

should be carefully examined in field experiments, sensitivity to insecticides (synthetic or natural) should be tested under controlled and field conditions, and vector natural enemies should be

identified, as they are not fully known and their efficacy in supressing insect populations has not been evaluated. Tools to disrupt insect reproduction should be investigated based on the evidence that

facultative endosymbionts like Wolbachia are present in field populations of P. spumarius (Lis et al.,

2015). Disruption of vector acquisition, retention, and inoculation of X. fastidiosa are likely to be a medium-long term goal of research on the microbiome associated with the vector foregut. With this

purpose, an EFSA outsourced project (RC/EFSA/ALPHA/2015/01) will start in January 2016 with the scope of collecting data on biology and control of vectors of X. fastidiosa. It will include a literature

review and field survey and will be concluded in two years.

Participants agreed that combining approaches for vector control is likely to be more successful than single approaches adopted individually, because there is not a control measure that alone can

suppress vector populations. According to Alexander Purcell’s presentation, a cumulative control approach means that mortalities caused by different control measures are accumulated (e.g. 60% of

the insects are killed by measure A, 40% of the insects surviving measure A are killed by measure B, 50% of the insects surviving measures A and B are killed by measure C, and so on) with a multiplier

effect provided by roguing of infected plants. Therefore, an integrated management programme could

be developed only if research provides data on the efficacy of a number of control measures against insect vectors of X. fastidiosa.



3.2.5. Innovative data collection on vectors and disease epidemiology

Participants suggested that analysis of disease progression through remote sensing-aerial image could

help data collection on pathogen spread and might enable inferences on the activity/mobility/dispersal

of vector insects (see also breakout session 1). In addition, it was suggested that an increased awareness of the problems caused by X. fastidiosa by all the stakeholders will help to prevent

pathogen spread and further economic damage. Moreover, the involvement of properly trained citizens may contribute in collecting data on vector presence and therefore in monitoring their

population density and distribution in a given area. It was also noted that the use of sticky traps

cannot fully substitute for visual observations, as the latter method provides more information on seasonal patterns than the former.

3.2.6. How can EU scientists best ‘catch up’ with ongoing research elsewhere? How to avoid repeating research already done? How to benefit from previous work and how to best interact with ongoing research activities on the vectors?

A final discussion was devoted to the best practices that might be applied to optimise all the research

efforts, promoting interactions among the different participants and avoiding research duplication. The

following suggestions arose from the discussion: i) systematic reading of literature before designing new experiments, ii) sharing information and data via free and centralized online databases, including

ongoing research programmes, and iii) provide a forum for international meetings on X. fastidiosa/vector research.

Breakout Group 3 – The Plants: host range, breeding, resistance 3.3.and certification

This session was attended by 27 participants. Stephan Winter acted as chair and Helvécio Della

Coletta-Filho as rapporteur of the meeting. At the beginning of the breakout session, six short presentations were given by participants covering different aspects of X. fastidiosa and its hosts. The

objective of this session was to discuss, identify, and rank according to their priority, the knowledge

and research gaps concerning the host range of X. fastidiosa. In particular the isolates causing CoDiRO in Apulia, breeding for resistance/ tolerance and, pathogen-free certification of plant

propagation material. Helvécio Della Coletta-Filho presented an overview of research for testing host range, breeding for resistance, and certification for X. fastidiosa subspecies pauca CVC strain in Brazil.

Ciro Gardi presented a comprehensive database on X. fastidiosa host plants developed by EFSA and

based on review of published literature and reports. Rodrigo Krugner presented research results on rootstock effects on almond leaf scorch disease incidence and severity in California, USA. Donato

Boscia presented preliminary results of an EFSA funded pilot project on host range of X. fastidiosa CoDiRO strain, evidences of resistance phenomena in the cv. Leccino upon infection with X. fastidiosa,

and the work plan on host plants of X. fastidiosa of the EU Horizon 2020 POnTE project. Angjelina Belaj introduced participants to the World Olive Germplasm Collection of Córdoba. Georgios Koubouris

presented the Greek olive germplasm breeding and certification research priorities on X. fastidiosa.

The talks initiated an open group discussion among participants to identify knowledge gaps and to rank research needs. The topics discussed and the research needs identified are summarized below.

3.3.1. Host range

The definition of the host plant concept was discussed. There was a consensus that, to be considered

as a X. fastidiosa host plant, bacteria must be able to multiply, move between xylem vessels, and

consequently systemically colonize the plant. Systemic invasion is independent from presence of symptoms since many endophytes move systemically in some plants and do not induce symptoms,

whereas in other hosts the same strain can cause disease. Questions were also addressed about the definition of primary and secondary hosts as sources of inoculum and their role in X. fastidiosa spread.

The role of secondary hosts, wild or cultivated plants found infected with X. fastidiosa, was discussed

and research needs expressed:

regarding the Olive Quick Decline (OQD) outbreak in Apulia, create a complete database of

hosts plants.



regarding secondary hosts, understanding the importance of wild hosts on X. fastidiosa

epidemics, i.e., if olive is the primary source of X. fastidiosa to wild plants or vice versa (host studies for the Apulian outbreak have been conducted within a pilot project funded by EFSA

funded12 and a more extensive and complete investigation of the host plants of the X. fastidiosa Apulian strain will be conducted within the H2020 POnTE project13).

concerning the impacts of X. fastidiosa on wild hosts, the spread of the pathogen among

those hosts and the general environmental impact of X. fastidiosa.

The importance of testing the susceptibility to X. fastidiosa infection of the most economically important EU crop species and plants was discussed and research gaps identified:

the need to test whether the most economically relevant plant species in the EU could sustain

infections with the isolate of X. fastidiosa subspecies pauca, CoDiRO strain;

the need to test if the most economically relevant plant species could be infected with the

isolate of X. fastidiosa subsp. multiplex found in Corsica;

the need to test the susceptibility of European host plants to X. fastidiosa isolates intercepted

in the EU, as this would provide data to reduce uncertainty of pest risk assessments.

However, the time span required to conduct these pathogenicity tests (possibly 2 to 3 years for the CoDiRO and Corsica isolates, and likely more than 6 years for other intercepted isolates) might make

such efforts unfeasible.

Although there is information available in the scientific literature on host specificity of some X. fastidiosa subspecies and strains, it was discussed and agreed that:

the whole genetic sequencing of X. fastidiosa isolates would be a significant element of the

host range definition and isolate characterization;

research should be intensified to understand the links between bacterial genetics, evolution,

and host specificity.

This would allow a definition of the host range for specific X. fastidiosa genotypes rather than an association of X. fastidiosa subspecies with a particular host.

Following, the creation of a global database of X. fastidiosa host plants by EFSA (EFSA, 2015; 2016), it

was suggested to add information on potential hosts for each subspecies and isolate. Participants agreed on the utility of a database also comprising images of specific/informative symptoms in

different plant species, information on disease progression, and seasonality to support early detection of X. fastidiosa in areas currently not invaded.

A very important discussion point was how to test host plants for X. fastidiosa. In particular, the advantages and disadvantages of each inoculation method (pinpricking – needle inoculation, grafting,

and insect transmission) were discussed. Pinpricking was considered to be effective in compatible

systems, but artificial. In the case of vector transmission, the effectiveness can be similar to the pinpricking inoculation method when using efficient vectors, but it largely depends on the host species

and the behaviour of the vector in relation to that plant species (i.e., during probing the insects can detect repellent compounds that can make them reject the plant and disperse). Hence, in case of

insect inoculation failure, it is difficult to discern whether it is due to the lack of susceptibility of the

test plant to X. fastidiosa or to the fact that the vectors are not able to transmit the bacteria to the plant.

There was consensus that needle inoculation is the easiest inoculation method and the standard method for greenhouse inoculations. Grafting and insect transmission studies are extremely important

and should be conducted if the necessary skills and facilities to apply these methodologies are available. A single standard inoculation protocol for all plant species was not advised since all host

plants and bacteria interactions would require specific adaptation of the methods used.

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More research would be needed to define the best methods (inoculum concentration, sub-culturing of bacterial colonies, number of treatments and repetitions, plant size, and monitoring duration) to test

for susceptibility in unknown or non-compatible hosts. Similarly, methods to determine whether

infections are chronic or transient need to be developed.

The influence of environmental conditions on X. fastidiosa-host susceptibility is known for some

pathosystems (e.g., Citrus Variegated Chlorosis and Coffee leaf scorch in Brazil), but not for Europe. It would however be highly significant to test host and disease development under different

environmental conditions (i.e. temperature) both under greenhouse and under field conditions.

3.3.2. Germplasm genetic resources and breeding for resistance

In Europe, the only information available on X. fastidiosa-olive interactions comes from observations

in Apulia. Hence, the most urgent research need was formulated as follows:

to determine whether resistance and/or tolerance to X. fastidiosa is present in germplasm

collections of Olea europaea and closely related plant species.

to screen the diversity of germplasm and to identify the most resistant genotypes and

cultivars.

However, given the large number of olive genotypes available in germplasm collections

(approximately 900 olive varieties in the World Olive Germplasm Collection of Córdoba), the question

was raised on how to select the genotypes to be tested. It was agreed that the most efficient procedure would be to test first the commercially relevant genotypes. A first screening should be done

with needle inoculation in plants propagated by cutting to test the ability of X. fastidiosa to infect those genotypes. Some participants remarked the importance of testing the susceptibility in both

greenhouse and field conditions. The experience of screening for pathogen resistance in olives (e.g.

resistance/tolerance to verticillium) from Spain could provide a useful guideline for the X. fastidiosa case. This screening for resistance should extend to other relevant crop species. Finally, the role of

traditional breeding for resistance was highlighted, which, although with only a long term perspective, was considered as a strategic target in sustainable and resilient crop production.

3.3.3. Certification

Certification of pathogen-free nurseries was identified as a crop management practice that should be covered by specific legislation. According to the participants of this session, different certification

requirements should be considered in different scenarios within the EU (i.e. geographic regions with and without outbreaks). The necessity of certification programmes in countries of origin (outside the

EU) where plant materials that are potential hosts of X. fastidiosa are produced also was discussed.

This was seen as the most effective measure to prevent new introductions of X. fastidiosa to the EU. However, pathogen detection would complement inspection, and thus research should be conducted

to improve speed and sensitivity of diagnostic methods, as well as on sanitation methods to eliminate X. fastidiosa from imported plant material, such as hot water treatment and thermotherapy.

3.3.4. Multidisciplinary approach

Multidisciplinary agro-ecology research was identified as a need to keep infected plants alive and economically viable in regions where olives or other host contribute significantly to landscapes and old

trees have a high cultural value (e.g. olives in Apulia). The research gaps identified include the role of pruning, fertilization, irrigation, use of bait plants, and vector control practices for enhancing plant

tolerance/resistance to X. fastidiosa infection.

The participants strongly agreed that suppression or reduction of X. fastidiosa inoculum must be done

in proximity to both pest-free areas and buffer zones.

3.3.5. Summary of the topics discussed in the breakout discussion session 3

The participants summarised the research needs for each of the four topics of the breakout discussion

session and discussed the priorities of the research lines. Results of the discussion are shown in table 2 below,



Table 2: Summary of the research needs and priority levels classified according to the four topics discussed in the breakout discussion session 3.

Host Plants Germplasm and resistance

Certification Multidisciplinary approach

High priority

Identification of host range for the isolates

already present in

Europe (CoDiRO and Corsica subsp.

multiplex)

Screening olive germplasms for

resistance

(especially varieties of

commercial interest)

On-site tests supporting

certification

Understanding the role of crop

management

practices on resistance and

tolerance

Host plants database Protocols for pathogen

detection and

sanitation in different species

Database of images with symptoms in

different hosts

Standard protocols for

detection of infection in different species

Medium

priority

Testing susceptibility of

present

subspecies/strains in EU (including

interceptions) and not present ones on

economically relevant crops

Screening other

relevant crop

species germplasms

Testing susceptibility of crop species in multiple

locations/environmental conditions

Understanding genetic basis of

resistance and tolerance

Low priority

To generate and

analyse data on trade/pathways of

plants for planting

Breakout Group 4 – The Pathogen: biology, genetics, and control 3.4.

This session was attended by 29 participants. Claude Bragard acted as chair and Marie-Agnes Jacques

as rapporteur of the meeting. At the beginning of the breakout session, Marie-Agnes Jacques gave an introductory presentation on comparative genomics and the insights it could provide to control

diseases caused by X. fastidiosa. Six short presentations were given by participants covering different

aspects of (i) the biology of X. fastidiosa, (ii) genome diversity and genetics, genome recombination, with particular focus on the CoDiRO strain and other EU intercepted isolates (Maria Saponari); (iii)

methods for genotyping of isolates and strains, including the potential of rapid whole genome sequencing for outbreak delineation and pathogenicity profiling for X. fastidiosa (Tanja Dreo) and (iv)



means for controlling the pathogen in the plant, including bactericidal substances such as antibacterial and plant defence elicitor peptides (Emilio Montesinos), resistance inducers, biotechnological

approaches (Pasquale Saldarelli and Xavier Daura), or cropping practices. The work plan on X. fastidiosa of the EU Horizon 2020 POnTE project was also presented (Blanca Landa). It has to be noted that, prior to this workshop, research needs to gain a better understanding of the bacterium

were already identified in the EFSA pest risk assessment on X. fastidiosa (EFSA Scientific Panel on Plant Health, 2015), such as: (i) genetic diversity of the pathogen and consequences in the field, (ii)

association of subspecies and strains with particular plant hosts and symptoms caused in the host, (iii)

association of strains and isolates with particular insect vectors, (iv) recombination between X. fastidiosa strains and its impact on host range, association with vectors, and disease severity, and (v)

cold susceptibility/tolerance of X. fastidiosa.

The discussion of this breakout group addressed the following questions:

How much should research focus on the development of genetic tools to study biology of X. fastidiosa?

Should all characterised strains be made available to researchers in publicly accessible culture

collections?

What not previously studied aspects of X. fastidiosa biology should be a research priority for

the EU?

Given the research done in the Americas aimed at controlling X. fastidiosa in the plant, what

tools for such control in the plant have potential for field application?

The discussion focused on which tools for the control of the bacterium in plants have a potential for

applications in the field, and which research should be repeated or prioritized in EU. As a first step, a set of issues that should be taken into account before the selection of methods or strategies for

control of the bacterium in planta were emphasised.

Participants stressed the time frame of the research to be done, with the need to specify short-, medium- and long-term targets. Applied basic research should certainly be conducted rapidly to

ascertain identification of strains that are isolated from intercepted plants, but also to control the bacteria in the zones where it has been established. Also, it was considered useful to anticipate the

needs and to propose research to deliver control methods based on in-depth knowledge of X. fastidiosa biology, which is currently lacking. Such research will deliver outputs and impacts within a

longer time frame.

X. fastidiosa is a new subject of research for most EU laboratories, but it has been studied for a long time in USA and Brazil. Hence, participants argued that the mid-long term research proposals should

position their contribution within the current knowledge on the pathogen, recognising the ongoing research, and indicating the added value of any collaboration with laboratories with long experience

with X. fastidiosa and associated vectors. The value of such collaborations is obvious. Additionally, the

interest to link also to the on-going extensive European research on Xanthomonas spp., another bacterial genus closely related to X. fastidiosa, would certainly be valuable.

The necessity to differentiate new research to be done from the implementation/duplication of research already published or disseminated, or the experimentation of on-going control strategies

already proposed in USA and Brazil was highlighted. The transfer of such research outputs requires first to verify that they would fit the specificity of potential diseases caused by X. fastidiosa that would

occur in the EU.

Integrated control approaches should be developed, not only for targeting the bacteria in olive trees and other host plants, but also allowing the use of all possible control means, including efficient

surveys and detections strategies (including bacterium eradication or curing possibilities), vector exclusion and control strategies altogether with the deployment of host plant resistance and the need

to minimize host plant stress. These approaches, if applied, could deal with targeting direct control of

the disease, which is different from the control of the bacterium alone.

Many participants stressed also the difficulty to target endophyte bacteria residing almost uniquely

either within the xylem vessels or in their insect vectors. Therefore, keeping note of the technical



methods that could help deliver control measures into xylem vessels, without impairing plant development, would certainly be useful.

The socioeconomic importance of X. fastidiosa diseases in crop plants is well recognized in the USA,

Brazil, and now in Italy. Not only yield losses are important, but also the impact of roguing infected plants in Southern Italy can be higher than the economic crop value, because olive trees represent a

cultural heritage, a symbol of local identity and a key component of the landscape. No information on the socioeconomic impact of the disease caused by X. fastidiosa is available concerning the recent

outbreaks in France, which so far affects only ornamentals. When implementing a specific control

method, it is important to study its social and cultural acceptability as well as its socioeconomic impact.

Finally, outside the scope of the discussion session, many participants stressed that the current regulations in the EU might not allow the development of certain strategies that are already

implemented or developed elsewhere. Such a consideration was raised for the transgenic approaches that are being investigated in the USA. Similarly, questions of biosafety might limit the possibility to

develop control strategies. For example, it was raised the risk of facilitating the occurrence of

recombination between attenuated strains that might be used over large areas for competing against X. fastidiosa. It was also stated that currently, within the EU, the use of antibiotic substances for the

control of bacterial plant pathogens is prohibited to avoid the emergence of resistances.

3.4.1. Strains characterisation and monitoring the xylem-associated microbial diversity

Key elements in studying epidemics are identification of inoculum sources and identification of potential routes of dissemination. This short term applied research already benefits from an efficient

and widely used genotyping scheme, such as the multilocus sequence typing (MLST) approach with the classification of isolates into sequence types (STs) (unique genotypes based on the seven loci

used in the MLST) that are recorded in a public MLST database (www.pubmlst.org). Several

alternative methods exist, with place for novel developments, partly as a consequence of the wider accessibility to the most recent sequencing technologies. While X. fastidiosa certainly coexists in xylem

vessels with other microbes, the structure and dynamics of the microbiome is yet poorly described, and its functioning is poorly known. To gain insights into microbiome functioning from the perspective

of developing novel control methods, assessing the dynamics of the structure of the whole xylem-

associated microbiome would benefit from comparisons between healthy and diseased plants.

X. fastidiosa takes its name from the difficulties to grow it in vitro. While it was mentioned that this

slow development could be an intrinsic property of this bacterium, there would be place to enhance cultivation methods as well as storage methods. It was also mentioned that inoculation methods may

fail to reproduce symptoms or to allow subsequent re-isolation of the bacterium from symptoms and hence failure to allow the fulfilment of Koch’s postulates. Proposals of alternative methods are

foreseen.

Due to the quarantine status of X. fastidiosa and its recent occurrence in the EU, the possibility of gathering its strains in European collections is very limited. Participants highlighted the need to

establish open collections storing not only bacterial strains (i.e. specimens) representing the natural worldwide diversity, but also their associated data such as genome sequence and metadata (including

place and year of isolation, host, phenotype). These collections should ensure the maintenance of the

genetic and virulence stabilities of the strains. Once again, it was emphasized that collaborations with non-EU research groups that have and maintain such collections would favour access to strains and to

efficient storage methodologies.

3.4.2. Xylella fastidiosa and its interactions with the ecosystem

X fastidiosa enters host plants by means of insect feeding and does not harbour any Type III

Secretion System, the role of which is to secrete effectors that interact with plant defence reactions. How does X. fastidiosa identify a plant as a host as it is directly injected into vessels? Does X. fastidiosa enter into a dialogue with the plant cells and, in case it does, with which cells? Answers to these questions could allow the development of control methods targeting directly or most certainly

indirectly the pathogen. The bacterium is known to be xylem-limited, nevertheless, thanks to novel



microscopy technologies and adequate bacterial cell tagging, an investigation of the distribution of the bacterium within the plant and the nature of the colonized tissues was considered as an option to

unveil the histopathology of diseases caused by X. fastidiosa. It has long been known that infections

become inactive during wintertime as an apparent consequence of low temperatures. What are the mechanisms involved in this phenomenon; and, more generally, what are the interactions between X. fastidiosa and its abiotic environment (temperature winter/summer) and biotic environment (plant, vector and microorganisms). These were questions raised by participants. A debate regarding the

ability to fulfil Koch’s postulates was raised and it was considered that molecular data provided by

research in the plant-bacteria interactions would provide the necessary insights to re-evaluate the need and methodology to fulfil Koch’s postulate in the case of new X. fastidiosa diseases.

3.4.3. Control measures

The mechanism of action of physical control measures

Besides the classical chemical control approaches, there is a need for better understanding of the

mechanisms explaining the effect of temperature on the bacterium in planta (cold temperature effect, hot water treatment effect in different hosts) and their mode of action, either direct or indirect on the

bacterium or based on the host plant defence mechanisms possibly activated.

Control strategies with the view of controlling the bacterium in the plant

Among control strategies in the plant, one of the most promising is certainly to improve the knowledge of the quorum sensing of X. fastidiosa and other Xanthomonadaceae. It was shown that X. fastidiosa modulates its activity in both the plant and the insect through its cell-cell sensor system.

Currently, transgenic grapevines for the control of X. fastidiosa are under trial in California based on this work. The direct use of the “diffusible factor” involved in this quorum sensing, or its secretion by

different microorganisms, possibly endophytes or virus vectors, could be foreseen.

Based on an improved knowledge of the endophyte microorganisms present in the plants affected by

X. fastidiosa, strategies could be developed by enhancing the presence of bacteria or other organisms

that might out-compete X. fastidiosa. Similarly, the use of bacteriophages is a possibility that was recently proposed, although the limitation of both the high specificity of the viruses and the possible

development of resistance should be foreseen, possibly through the use of cocktail of phages.

The possibility to deliver within the xylem molecules that could act as bactericide or bacteriostatic

agents or as plant defence stimulating compound (if a plant defence activity is proven to be effective against X. fastidiosa) was also proposed. Among such strategies, the possible use of synthetic

peptides has been highlighted.

4. Concluding session

In the final plenary session, the rapporteurs presented the results and discussions of the four

breakout sessions, followed by a questions and answers session and plenary discussion. Stephen

Parnell presented the outcome of the discussion of the breakout group session 1 on surveillance and detection. Domenico Bosco presented the outcome of the discussion of the breakout group session 2

on vectors identity, biology, epidemiology and control. Helvécio Della Coletta-Filho presented the outcome of the discussion of the breakout group session 3 on plant host range, breeding, resistance

and certification. Marie-Agnes Jacques presented the outcome of the discussion of the breakout group session 4 on pathogen biology, genetics and control. Annette Schneegans from EC DG AGRI presented

the new openings for research on plant health and plant protection in Horizon 2020, with focus on X. fastidiosa. Mike Jeger, Chair of the EFSA PLH Panel, made the conclusive remarks and closed the meeting.

Conclusions and recommendations

For surveillance and detection, the importance of defining in advance why surveillance is needed was

stressed. From the planning phase of a survey, there is a need to have defined a clear survey

purpose. Can research improve the identification of targets (pathogen, vector, host, and pathways) and locations for surveillance? What are the best methods and periods for surveillance? Answers to



these questions are likely to depend on improvement of the detection methods for the pathogen, especially during the asymptomatic period, and of the knowledge on the underlying vector behaviour.

Advanced remote sensing techniques can help in delimiting potential infected areas when survey

resources are limited, however there is a trade-off between coverage that can be achieved and sensitivity. Research on survey design can also result in accurate estimates of disease/vector

incidence and distribution by collection and analysis of survey data, and a better understanding of the epidemics and dispersal of X. fastidiosa. To facilitate research and enable data exchange and analysis

across MS, there is also a need for harmonisation of survey methods and sharing of data.

For research on vectors, the need for an inventory of potential vector species in the EU, including collection of biological and environmental data and geographical coordinates, was highlighted, in

particular considering that vectors may not be pest species per se and hence their biology has been poorly studied so far. Rearing methods for vectors, vector reproductive biology, feeding behaviours,

population dynamics, host preference for feeding and reproduction, and the importance of studying the ecosystem as a whole were identified as key areas for research. This basic knowledge should then

be translated into studies of X. fastidiosa transmission, seasonal vector infectivity, and disease

dynamics. This in turn links with surveillance and monitoring. As the vectors are not pests in their own right, there is also further experience to gain as regards the control methods, IPM, or on their effects

on biodiversity. A general need of epidemiological research was highlighted to support both the development of integrated pest and disease management strategies and the risk management and

containment measures to prevent spread.

For research on plants, several questions were raised and discussed to identify knowledge gaps and research priorities. For example, should research on host range, genetic resources, and resistance be

general or specific? With newly-identified hosts, research may help to optimize certification schemes and procedures. Host range should be considered not just to provide lists, but also to help understand

epidemics of subspecies and isolates of X. fastidiosa present, with the aim to find and/or confirm which plants species are the primary source of inoculum and what is the role of other crops and wild

plants in epidemics. It was recommended to complete the EFSA database on X. fastidiosa host plants

adding information on hosts of subspecies and isolates. There is a need for research to test important EU crops for their susceptibility to the isolates from the outbreaks in Italy and France, but also to the

other isolates found in intercepted plants at the EU borders. For selection and breeding of resistant or tolerant genotypes, the recommendation was to begin with olive trees by screening germplasm

collections to identify tolerant or resistant genotypes. While screening of germplasm could focus at

first on commercially relevant varieties, long term breeding programmes should also be established. For certification of plant propagation material, there is need for research on diagnostic tools and

sanitation methods. Multidisciplinary agro-ecological research was identified as a need to investigate solutions to keep infected plants alive and economically viable in the outbreak areas where olives or

other hosts contribute significantly to landscapes and old trees have a high cultural value.

With regard to the pathogen, discussion focused on identifying the priorities for the EU, given the long history of X. fastidiosa research in the Americas, the time scale envisaged, the need for fundamental

science, and socio-economic impact. In the short term, targets could be characterization of isolates from European outbreaks and availability of reference collections. In the mid- to long-term, biotic and

abiotic interactions, host specificity, host response to infection, and histopathology in new hosts were topics suggested and discussed. For research into pathogen control, there was a need to address

farmers’ expectations, especially in relation to physical approaches and the methodologies of

application of different tools and their integration.

As overall recommendations, it was stressed the opportunity to establish an open-field laboratory in

the olive orchards affected by X. fastidiosa in Salento, where experiments to develop strategies for disease and vector control could be conducted, as well as studies of epidemics and screening for

tolerant or resistant genotypes. Best practices to optimise the research efforts, to promote

interactions among the different actors and to avoid research duplications were discussed and the following general suggestions were made: the need for systematic reading of literature before

designing new experiments; the need to share information and data via free and centralized online databases, including ongoing research programmes; and the need to provide a forum for international

research meetings on X. fastidiosa and its vectors.



References

Almeida RPP and Purcell AH, 2006. Patterns of Xylella fastidiosa colonization on the precibarium of sharpshooter vectors relative to transmission to plants. Annals of the Entomological Society of

America, 99, 884–890.

Anonymous, 2015. Xylella fastidiosa: options for its control. Parallel Workshop at Conference "Health

Checks and Smart Treatments for Our Plants” EXPO Milan, 15th July 2015, © EU, 2015. ISBN 978-92-79-52732-6. doi: 10.2777/168991. Available online:

http://ec.europa.eu/research/conferences/2015/expo2015/pdf/presentations/xylella_final-

report_2015-expo.pdf#view=fit&pagemode=none

Cariddi C, Saponari M, Boscia D, De Stradis A, Loconsole G, Nigro F, Porcelli F, Potere O and Martelli

GP, 2014. Isolation of a Xylella fastidiosa strain infecting olive and oleander in Apulia, Italy. Journal of Plant Pathology, 96, 425–429.

Charlat S, Hurst GDD and Merçot H, 2003. Evolutionary consequences of Wolbachia infections.

TRENDS in Genetics, 19(4), 217–223.

EFSA (European Food Safety Authority), 2015. Categorisation of plants for planting, excluding seeds,

according to the risk of introduction of Xylella fastidiosa. EFSA Journal 2015;13(3):4061, 31 pp. doi:10.2903/j.efsa.2015.4061

EFSA (European Food Safety Authority), 2016. Scientific report on the update of a database of host plants of Xylella fastidiosa: 20 November 2015. EFSA Journal 2016;14(2):4378 [40 pp.] Excel

Database

EFSA PLH Panel (EFSA Panel on Plant Health), 2015. Scientific Opinion on the risks to plant health posed by Xylella fastidiosa in the EU territory, with the identification and evaluation of risk

reduction options. EFSA Journal 2015;13(1):3989, 262 pp., doi:10.2903/j.efsa.2015.3989

Frazier NW, 1944. Phylogenetic relationship of the nine known leaf-hopper vectors of Pierce’s disease

of grape. Phytopathology. 34, 1000–1001.

Lis A, Maryańska-Nadachowska A and Kajtoch Ł, 2015. Relations of Wolbachia infection with phylogeography of Philaenus spumarius (Hemiptera: Aphrophoridae) populations within and

beyond the Carpathian contact zone. Microbial Ecology, 70(2), 509–521.

Loconsole G, Saponari M, Boscia D, D’Attoma G, Morelli M, Martelli GP and Almeida RPP, 2016.

Intercepted isolates of Xylella fastidiosa in Europe reveal novel genetic diversity. European Journal

of Plant Pathology, ISSN: 0929-1873. DOI: 10.1007/s10658-016-0894-x.

Nunney L, Ortiz B, Russell SA, Ruiz Sa R and Stouthamer R, 2014. The complex biogeography of the

plant pathogen Xylella fastidiosa: genetic evidence of introductions and subspecific introgression in central America. PloS One, 9, e112463.

Purcell AH, 1989. Homopteran transmission of xylem-inhabiting bacteria. In: Advances in disease vector research, Vol. 6. Ed. Harris KF. Springer, New York, USA, 243–266.

Redak RA, Purcell AH, Lopes JRS, Blua MJ, Mizell III RF and Andersen PC, 2004. The biology of xylem

fluid-feeding insect vectors of Xylella fastidiosa and their relation to disease epidemiology. Annual Review of Entomology, 49, 243–270.

Saponari M, Boscia D, Nigro F and Martelli GP, 2013. Identification of DNA sequences related to Xylella fastidiosa in oleander, almond and olive trees exhibiting leaf scorch symptoms in Apulia (Southern

Italy). Journal of Plant Pathology, 95, 668.

Saponari M, Loconsole G, Cornara D, Yokomi RK, Stradis AD, Boscia D, Bosco D, Martelli GP, Krugner RC and Porcelli F, 2014. Infectivity and transmission of Xylella fastidiosa by Philaenus spumarius (Hemiptera: Aphrophoridae) in Apulia, Italy. Journal of Economic Entomology, 107, 1316–1319.

http://ec.europa.eu/research/conferences/2015/expo2015/pdf/presentations/xylella_final-report_2015-expo.pdf#view=fit&pagemode=none




Abbreviations

AGRI Agriculture and Rural Development

DG Directorate-General

EC

EFSA

EU

European Commission

European Food Safety Authority

European Union

IPM Integrated Pest Management

ISPM International Standards for Phytosanitary Measures

MS Member States

NPPO National Plant Protection Organization

OQD Olive Quick Decline

PLH Plant Health

RTD Research and Innovation

SANTE Health and Food Safety



Appendix A – Workshop programme

Workshop on Xylella fastidiosa: knowledge gaps and research priorities for the EU

12-13 November, Brussels

08.30-09.00 Registration of participants

1. PLENARY SESSION

Chair: Franck Berthe, Head of Animal and Plant Health Unit (EFSA)

OverallRapporteur: Michael John Jeger, Chair of the Plant Health Panel (EFSA)

09.00-09.15 Welcome and introduction to the event Patrick Kolar (EC, DG RTD)

09.15-09.30 Status and control of X. fastidiosa in the EU Pasquale Di Rubbo (EC, DG SANTE)

09.30-09.50 What is the risk of X. fastidiosa for Europe? Claude Bragard, Plant Health Panel (EFSA)

09.50-10.10 State of the art on biology and epidemiology of

the X. fastidiosa outbreak in Apulia Maria Saponari, CNR (Italy)

10.10-10.25 X. fastidiosa outbreak in France: identification of

the causal agent and distribution of the disease. Charles Manceau, ANSES (France)

10.25-10.35 Questions and answers

10.35-11.00 Coffee/Tea break

11.00-11.30 State of the art on X. fastidiosa in North America Alexander Purcell, University of California Berkeley (US)

11.30-12.00 Xylella fastidiosa in South America: ecological

basis for management João Lopes, University of São Paulo (Brazil )

12.00-12.10 Questions and answers

12.10-12.25 Research needs on X. fastidiosa: a perspective

from the EU farmers.

Giovanni Cantele, Copa Cogeca

12.25-12.40 Research needs on X. fastidiosa: an Euphresco

perspective

Baldissera Giovani, Euphresco coordinator (EPPO)

12.40-13.00 General discussion & Introduction to Discussion Groups

13.00-14.00 Lunch break



2. BREAKOUT SESSIONS ADRESSING SPECIFIC QUESTIONS

14.00-18.30

14.00-14.10

14.10-14.25

14.25-15.10

15.10-15.20

15.20-16.00

16.00-16.20

16.20-18.30

Session 1 | Surveillance and Detection

Chair: Harry Arijs

Rapporteur: Stephen Parnell

Scope of the session and overview of topics proposed by participants (EFSA)

Introduction to the session: surveillance methodology for X. fastidiosa (Stephen

Parnell, University of Salford, UK)

Short presentations from participants

How can remote sensing contribute to the surveillance of X. fastidiosa affected

regions? (Pieter Beck, European Commission – JRC)

Innovative tools in the early detection and surveillance of X. fastidiosa (Anna

Maria D’Onghia, Mediterranean Agronomic Institute, IT)

Detection of X. fastidiosa in the frame of surveillance activities in Austria

(Richard Gottsberger, AGES, Austria)

Electrochemical label-free sensing platform for rapid, onsite detection of X. fastidiosa (Alan O’Riordan, Tyndall National Institute, Ireland)

Detection of X. fastidiosa in Coffea arabica ornamental plants (Maria Bergsma-

Vlami, Netherlands National Authority)

The sentinel nursery concept as an opportunity to efficiently respond to the X. fastidiosa threat to Europe (Andrea Vannini, University of Tuscia, IT)

Developing guidelines for a possible strategy for the surveillance of X. fastidiosa

in Europe (Emilio Stefani, Università di Modena e Reggio Emilia, Italy)

The EU Horizon 2020 POnTE project: the work plan on X. fastidiosa surveillance (Maria M. Lopez)

Discussion

Coffee/Tea break

Discussion

14.00-18.30

14.00-14.10

14.10-14.20

14.20-14,30

14.30-14.45

14.45-15.00

Session 2 | The Vectors: identity, biology, epidemiology and control

Chair: Giuseppe Stancanelli

Rapporteur: Domenico Bosco


Overview of current knowledge on insect vectors/potential vectors of X. fastidiosa in Europe (Domenico Bosco, Università degli Studi di Torino, IT)

How do vector numbers, movements, infectivity and transmission efficiency impact the incidence and spatial patterns of plant infection with X. fastidiosa?

(Alexander Purcell, University of California, Berkeley, USA)

Ecological and behavioral traits that determine vector relevance (Joao Lopes, University of Sao Paulo, Brazil)


Present knowledge of potential vector species in Spain (Alberto Fereres, CSIC,



15.00-15.10

15.10-16.00

16.00-16.20

16.20-18.30

Madrid, Spain)

The "vectors" aspects of the Belgian Xyleris research project (Jean-Claude

Gregoire, Université Libre de Bruxelles, BE)

Outline of EFSA outsourced project to collect data on biology and control of

vectors of X. fastidiosa (Sabrina Bertini, Università degli Studi di Torino, IT)

The EU Horizon 2020 POnTE project: the work plan on X. fastidiosa vectors

(Domenico Bosco, Università degli Studi di Torino, IT)

Discussion

Coffee/Tea break

Discussion

14.00-18.30

14.00 - 14.10

14.10 - 14.25

14.25 – 15.05

15.05 - 15.15

15.15 – 16.00

16.00 - 16.30

16.30 - 18.30

Session 3 | The Plants: host range, breeding, resistance and

certification

Chair: Stephan Winter

Rapporteur: Helvécio DELLA Coletta-Filho


Testing host range, breeding for resistance and certification for X. fastidiosa subspecies pauca CVC strain (Helvécio Coletta-Filho, IAC / Centro de Citricultura,

Cordeiropolis, Brazil)


A comprehensive database on X. fastidiosa host plants (Ciro Gardi, EFSA)

Rootstock effects on almond leaf scorch disease incidence and severity (Rodrigo

Krugner, USDA – Agricultural Research Service, Parlier, USA)

Preliminary results of a pilot project on host range of X. fastidiosa CoDiRO strain

(Donato Boscia, CNR Institute for Sustainable Plant Protection, Bari, IT)

Evidences of resistance phenomena in the cv. Leccino upon the infection of X. fastidiosa (Donato Boscia, CNR Institute for Sustainable Plant Protection, Bari,

IT)

The World Olive Germplasm Collection of Córdoba (Raúl de la Rosa, IFAPA,

Junta de Andalucia, Córdoba, ES)

Greek olive germplasm and certification of olive plants (Georgios Koubouris,

Ellinikos Georgikos Organismos Dimitra, Chania, GR)

The EU Horizon 2020 POnTE project: the work plan on host Plants of X. fastidiosa

(Donato Boscia, CNR Institute for Sustainable Plant Protection, Bari, IT)

Discussion

Coffee/Tea break

Discussion



14.00-18.30

14.00-14.10

14.10-14.25

14.15-15.00

15.00-15.10

15.10-16.00

16.00-16.20

16.20-18.30

Session 4 | The Pathogen: biology, genetics, control

Chair: Claude Bragard

Rapporteur: Marie-Agnes Jacques


Can comparative genomics yield insights to control diseases caused by X. fastidiosa (Marie-Agnes Jacques, INRA, Beaucouze, FR)


Genetic diversity of the CoDiRO strain and other EU intercepted isolates (Maria

Saponari, Institute for Plant Protection, CNR, Bari, IT)

Improved detection of X. fastidiosa and the potential of rapid whole genome

sequencing for outbreak delineation and pathogenicity profiling (Tanja Dreo,

National Institute of Biology, SI)

Antibacterial and plant defence elicitor peptides. Novel tools against Xylella fastidiosa? (Emilio Montesinos, University of Girona, ES)

A biotechnological approach for Xylella fastidiosa targeting and population

confusion (Pasquale Saldarelli, CNR Institute for Sustainable Plant Protection, Bari, IT)

The EU Horizon 2020 POnTE project: the work plan on the X. fastidiosa Pathogen

(Blanca Landa, CSIC, Spain)

Discussion

Coffee/Tea break

Discussion

18.30-20.00 Networking cocktail



3. CONTINUATION OF THE BREAKOUT SESSIONS ADRESSING SPECIFIC QUESTIONS

08.30-10.00 Continuation of the breakout sessions including discussion on the

outcomes of the discussion groups and the production of reports to the plenary session

10.00-10.30 Coffee/Tea break

4. PLENARY SESSION

10.30-10.45

10.45-11.00

Report back from Session 1

Discussion

Stephen Parnell, University of Salsford (UK)

11.00-11.15

11.15-11.30


Discussion

Domenico Bosco, Università degli Studi, Torino (Italy)

11.30-11.45

11.45-12.00


Discussion

Helvécio Della Coletta-Filho, Instituto Agronômico – IAC (Brazil)

12.00-12.15

12.15-12.30


Discussion

Marie-Agnes Jacques, INRA (France)

12.30-12.45 Openings for research on plant health and plant protection in Horizon 2020: Focus on X. fastidiosa

Annette Schneegans (EC, DG AGRI)

12.45-13.15 Concluding remarks and end of meeting Michael John Jeger, Chair of the Plant Health Panel (EFSA)



Appendix B – Briefing notes for breakout groups

These briefing notes aim to provide participants with the relevant background information so as to be

prepared for an interactive exchange of views and expertise during the EFSA workshop “Xylella fastidiosa: knowledge gaps and research priorities for the EU”.

Background

The plant pathogenic bacterium Xylella fastidiosa was detected in olive trees in Lecce province in Apulia, Italy, in October 2013. The Apulian strain of X. fastidiosa is considered to be a genetic variant

within the subspecies pauca and identical (based on a sequence of 7 housekeeping genes) to a variant infecting oleander in Costa Rica. This was the first field outbreak in the European Union. X. fastidiosa is one of the most dangerous plant pathogens worldwide, damaging major crops including

fruit trees, grapevine and ornamentals. Emergency measures have been in place in the EU since the first outbreak of X. fastidiosa in the territory in 2013. Several outbreaks on woody ornamentals

caused by strains of X. fastidiosa subsp. multiplex, a subspecies different from the Apulian one, were then reported in summer and fall 2015 in Corsica and in the South of France.

In its Scientific Opinion published in January 2015, the EFSA’s Scientific Panel on Plant Health conducted a detailed assessment of the risks to plant health posed by X. fastidiosa for the EU,

including the identification and evaluation of risk reduction options, and recommended an

intensification of research activities on the host range, epidemiology and control of the Apulian outbreak of X. fastidiosa. Based on the knowledge acquired by this research, uncertainties could be

substantially reduced and a deeper assessment of the risk and of the mitigation measures could be conducted.

In May 2015 the European Parliament approved a non-legislative resolution demanding actions to halt

the spread of the X. fastidiosa outbreak, including stepping up funding for research, and increasing international networking. In order to respond to the X. fastidiosa emergency needs, the European

Commission (EC) has therefore reinforced research and innovation actions.

A workshop on “Xylella fastidiosa: Options for its Control” was organised at Expo in Milan in July

2015, in order to discuss with experts in the field having a wide range of expertise, the role that

research and innovation can play in tackling this bacterium, with the aim to provide a background document and ideas in preparation of the here presented wider audience workshop for identification

and discussion of knowledge gaps and research priorities on X. fastidiosa in the EU. Its specific conclusions will be considered below in these briefing notes in relation to the topics of each breakout

group.

Objective of this Workshop

This workshop, organised by EFSA in collaboration with the EC Directorates-General for Research and

Innovation, Agriculture and Rural Development, and Health and Food Safety, aims to provide a scientific environment to discuss and analyse knowledge gaps and priorities for EU research on X. fastidiosa. The workshop is structured through plenary sessions and four discussion breakout groups dealing with knowledge gaps and research priorities on: Surveillance and detection (breakout Group

1); Vectors identity, biology, epidemiology and control (breakout Group 2); Plants: host range,

breeding, resistance and certification (breakout Group 3); Pathogen biology, genetics, typing and control (breakout Group 4).

In line with the conclusion of the previous workshop held in July 2015, that the European research on X. fastidiosa should not reinvent the wheel, this workshop also aims to contribute to more interaction

with research groups abroad that already have experience in relevant topics, more collaboration among European research groups, and increased awareness of scientific work previously done by

others.

Organising committee

Miren Andueza, Vanessa Descy, Ciro Gardi, Virag Kertesz, Svetla Kozelska, Ioannis Koufakis, Gritta

Schrader, Giuseppe Stancanelli, Sara Tramontini, Sybren Vos



1. Breakout Group 1 - Surveillance and Detection

Introduction 1.1.

This breakout group deals with knowledge gaps and research priorities for surveillance and detection

of the bacterial pathogen X. fastidiosa in Europe. It is intended to discuss research needed to plan

and execute surveys, including innovative and advanced methods for monitoring or detection. It does not include the discussion of research needs on typing of isolates and strains, which is dealt with in

breakout group 4.

Surveillance and detection of X. fastidiosa throughout the area of potential establishment in the EU

are keys for early identification of further outbreaks and are a prerequisite to effective containment

and control of the bacterium and its vectors. According to the EFSA cooperation project PERSEUS (Bell et al., 2014), there is a need for clear and detailed documentation of the survey methods,

specific and appropriate sampling methods for the bacterium and its vectors, standardised detection and surveillance manuals and common reporting procedures.

Many plant species are reported as hosts of X. fastidiosa without expressing symptoms. In addition, in many host plants the development of symptoms can occur months or years after infection. This

makes early detection and surveys of X. fastidiosa difficult. The knowledge of the epidemiology, the

introduction pathways and the spread patterns in time and space is needed to support the planning of surveys for X. fastidiosa (EFSA PLH Panel, 2015a). However, to generate knowledge on the

epidemiology of X. fastidiosa in a given environment, reliable detection and monitoring programmes are necessary. Appropriate sampling strategies in the plants and in the landscape need to be

developed (Where does the infection occurs first in the plant? How are the bacteria distributed in the

plant spatially and seasonally? Which and how many plants should be sampled, when, and where? (Anonymous, 2015)). Should potential vector insects be sampled and tested for the presence of X. fastidiosa? Such approach is applied in surveys in Italy: adult vector insects could have chances to visit several different plants and become infected, thus they could represent “markers” for the

presence of X. fastidiosa in the environment when monitoring in a pest-free area; however the identification of an infected insect would not directly allow to identify an infected plant.

The benefits of remote sensing technology, including the analysis of standard images, or more

sophisticated multispectral images acquired from manned aircraft and satellites should be further investigated. Mapping of diseased plants based on the visual or automated analysis of remote sensing

data could inform the planning of in situ surveys (Anonymous, 2015; D’Onghia et al., 2014). Citizen science could also play a role in areas where the disease is not yet present, e.g. by an early

observation and reporting of typical symptoms in a given crop, however, as mentioned above, it is

limited by the occurrence of many asymptomatic hosts and the sometimes long incubation period between initial infection and symptoms expression. There are effective molecular tools for the

detection of the bacterium. The development of methodologies for field rapid detection can support surveys and reduce movement of samples from field to laboratory. Surveys and certification of

nurseries can significantly reduce the spread of bacteria by eliminating distribution of infected plants

to bacteria-free zones (Anonymous, 2015).

Discussion points 1.2.

Why do we need surveillance and what are the related research needs?

Elements for contingency planning

Delimitation of areas at risk

Identification of appropriate phytosanitary measures and timely implementation

Information about vectors of X. fastidiosa via surveillance and detection

What and where do we need to survey? Research needs to identify targets and locations

Identify areas of high disease risk where to target the surveys (e.g. high host density,

conducive environmental conditions, high vector density, high risk of entry, close to known positives etc.)



Specific pathways, commodities, host plants

Plants and potential vector insects or plants only?

European entry points (interceptions), field, greenhouses, uncultivated environment

How and when to perform surveillance? Research needs to identify best methods and appropriate timing

Detection and identification techniques – current and new, risks of misidentification

Standardised and harmonised surveillance and detection methodologies and reporting procedures

Protocols and guidelines for sharing real-time data, development of shared database(s)

Determination how much sampling effort is sufficient for detection

Appropriate timing for surveillance

Early detection, monitoring and delimiting surveys after an outbreak

How can EU scientists best ‘catch up’ with ongoing research elsewhere? How to avoid repeating

research already done, how to benefit from previous work and how to best interact with ongoing research activities on surveillance and detection?

Background documents 1.3.

Anonymous, 2015. Xylella fastidiosa: options for its control. Parallel Workshop at Conference "Health

Checks and Smart Treatments for Our Plants” EXPO Milan, 15th July 2015, © EU, 2015. ISBN 978-

92-79-52732-6.doi:10.2777/168991. Available online : http://ec.europa.eu/research/conferences/2015/expo2015/pdf/presentations/xylella_final-


Bell H, Wakefield M, Macarthur R, Stein J, Collins D, Hart A, Roques A, Augustin S, Yart A, Péré C,

Schrader G, Wendt C, Battisti A, Faccoli M, Marini L and Petrucco Toffolo E, 2014. Plant health

surveys for the EU territory: an analysis of data quality and methodologies and the resulting uncertainties for pest risk assessment. Supporting Publications 2014: EN-676. [212 pp.]. Available

online: http://www.efsa.europa.eu/en/supporting/pub/676e

EFSA PLH Panel (EFSA Panel on Plant Health), 2015a. Scientific Opinion on the risks to plant health

posed by Xylella fastidiosa in the EU territory, with the identification and evaluation of risk

reduction options. EFSA Journal 2015;13(1):3989, 262 pp. doi:10.2903/j.efsa.2015.3989. Available online at http://www.efsa.europa.eu/en/efsajournal/pub/3989

D'Onghia AM, Santoro F, Yaseen T, Djelouah K, Guario A, Percoco A and Valentini F, 2014. An innovative monitoring model of Xylella fastidiosa in Apulia Region, Italy. International Symposium

of the European Outbreak of Xylella fastidiosa in Olive, Gallipoli, Locorotondo, Italy (21-24 October 2014), 33 (abstract).



http://www.efsa.europa.eu/en/supporting/pub/676e

http://www.efsa.europa.eu/en/efsajournal/pub/3989



2. Breakout Group 2 | The Vectors: identity, biology, epidemiology and control

Introduction 2.1.

X. fastidiosa is exclusively transmitted by xylem sap feeding insects (order Hemiptera, sub-order

Auchenorrhyncha), belonging to the three superfamilies of Cercopoidea (spittlebugs or froghoppers), Cicadoidea (cicadas) and Membracoidea (which includes a single xylem fluid-feeding subfamily, the

Cicadellinae, known as sharpshooters) (Redak et al., 2004). These insects are generally not considered direct pests unless present at very high population levels. The spittlebug species Philaenus spumarius is the only vector of X. fastidiosa identified so far in Europe (Saponari et al., 2014). The

spittlebug Neophilaenus campestris was found positive for X. fastidiosa by PCR in Apulia, but its role as vector has not yet been demonstrated (Elbeaino et al., 2014). In the same study, also the phloem

sap feeder Euscelis lineolatus was found positive by PCR (Elbeaino et al., 2014), showing that some phloem sap feeders can also feed marginally to the xylem, although they are not expected to be

vectors (EFSA Pl, 2015a). Vectors transmit X. fastidiosa in a persistent manner, even though i) no

latent period is needed between acquisition and inoculation ii) bacteria do not colonize the insect body systemically and are limited to the foregut iii) nymphs lose infectivity with moulting (EFSA Plant

Health Panel, 2015a).

All xylem sap-feeding insects should be regarded as potential vectors of X. fastidiosa (Frazier, 1944;

Purcell, 1989), but some species in Europe are more likely candidates, owing to their wide geographical distribution, abundance and host plant range (for listing and review of European

potential vectors, see pages 29-34 and Appendixes C and D from EFSA PLH Panel, 2015a). Since few

studies have been conducted so far on the putative European native vectors (Lopes et al., 2014), it is very difficult to assess their potential role in the spread and epidemics of X. fastidiosa in Europe.

To demonstrate that an insect species is a vector, plant-to-plant transmission experiments needs to be conducted. As vector transmission efficiency is a parameter highly dependent on insect–plant–

pathogen associations, even a positive transmission to a given test plant does not necessarily imply

that the vector can transmit the pathogen to other host plant species, as vector species may have very different transmission efficiencies depending on host plant species, or even by feeding on

different parts of the same host plant (Lopes et al., 2010; Daugherty et al. 2011; for discussion on identification and testing of new vectors, see also pages 29-30 from EFSA PLH Panel, 2015). In

addition, a specific vector population might become infectious only at a given time of the year when

feeding on specific hosts, for example, thus for vector control it is important to understand the seasonality of their infectivity. There is also uncertainty on the distribution of various potential insect

vectors in Europe as well as their ecology and overwintering behaviour (see pages 112-113 from EFSA PLH Panel, 2015a).

Based on discussion at a previous workshop on “Xylella fastidiosa: options for its control” (Anonymous, 2015), there is a general lack of knowledge concerning all European putative vectors

role in the transmission and epidemics of X. fastidiosa. It is important to study their transmission

capacity and ecology (behaviour, seasonality, feeding habits, overwintering etc.), as well as their host plant preferences. Such information could be useful to understand for example how to interrupt their

life cycle and/or how to control their spread in the environment. It would be important for European research groups to develop systems to rear X. fastidiosa-free spittlebugs in laboratory colonies, which

could then be used for transmission experiments. Cicadas are very abundant in Europe and should

also be studied. Control measures to manage the vectors of X. fastidiosa should be developed both for conventional agriculture with integrated pest and disease management programmes and for low

input/organic farming.


What is the current knowledge on the biology and ecology of the potential European vectors of X. fastidiosa? What research should be conducted to improve our knowledge and preparedness?

How to identify vector species of X. fastidiosa in European outbreaks? How to study their role in

disease epidemiology?



How to study vectors role in the spread of the pathogen to other areas?

How to manage the vectors of X. fastidiosa in conventional agriculture, in integrated pest and disease management programmes and in low input/organic farming? What studies should be

conducted?

What data should be collected to support a more quantitative approach in risk assessment as well

as in vectors management?

How can EU scientists best ‘catch up’ with ongoing research elsewhere? How to avoid repeating research already done, how to benefit from previous work and how to best interact with ongoing

research activities on the vectors?


Anonymous (2015). Xylella fastidiosa: options for its control. Parallel Workshop at Conference "Health

Checks and Smart Treatments for Our Plants” EXPO Milan, 15th July 2015, © EU, 2015. ISBN 978-92-79-52732-6. doi: 10.2777/168991. Available online :


de Jong YSDM, 2013. Fauna Europaea version 2.6. Ed. de Jong YSDM. Web Service. Available online: http://www.faunaeur.org

Elbeaino T, Yaseen T, Valentini F, Ben Moussa IE, Mazzoni V and D’Onghia AM, 2014. Identification of

three potential insect vectors of Xylella fastidiosa in Southern Italy. Phytopathologia Mediterranea, 53(1), 126–130.

EFSA PLH Panel (EFSA Panel on Plant Health), 2015a. Scientific Opinion on the risks to plant health posed by Xylella fastidiosa in the EU territory, with the identification and evaluation of risk

reduction options. EFSA Journal 2015;13(1):3989, 262 pp. doi:10.2903/j.efsa.2015.3989. Available

online : http://www.efsa.europa.eu/en/efsajournal/pub/3989

Frazier NW, 1944. Phylogenetic relationship of the nine known leafhopper vectors of Pierce’s disease

of grape. Phytopathology, 34, 1000–1001.

Purcell AH, 1980. Almond leaf scorch – leafhopper (Homoptera, Cicadellidae) and spittlebug

(Homoptera, Cercopidae) vectors. Journal of Economic Entomology, 73, 834–838.

Purcell AH, 1989. Homopteran transmission of xylem-inhabiting bacteria. In: Advances in disease vector research, Vol. 6. Ed. Harris KF. Springer, New York, USA, 243–266.

Redak, R. A. et al. (2003). The biology of xylem fluid-feeding insect vectors of Xylella fastidiosa and their relation to disease epidemiology. Annu. Rev. Entomol. 2004. 49:243–70 doi:

10.1146/annurev.ento.49.061802.123403

Saponari M, Loconsole G, Cornara D, Yokomi RK, De Stradis A, Boscia D, Bosco D, Martelli GP,

Krugner R and Porcelli F, 2014. Infectivity and Transmission of Xylella fastidiosa by Philaenus spumarius (Hemiptera: Aphrophoridae) in Apulia, Italy. Journal of Economic Entomology 107(4), 1316-1319.



http://www.faunaeur.org/




3. Breakout Group 3 | The Plants: host range, breeding, resistance and certification

Introduction 3.1.

This breakout session deals with the research for identification of host range, plant breeding for

resistance or tolerance, as well as for certification of plant propagation material. Control of the pathogen in the plant by means of bactericidal substances or resistance inducers or cropping

practices is not included in this breakout session as it will be dealt in the breakout session 4.

X. fastidiosa has a very large host range. Based on currently available data, the host range comprises

plants in 69 families, 187 genera and more than 300 plant species (EFSA, 2015; EFSA PLH Panel,

2015a). However, not all of these plants express symptoms and are susceptible to disease and for some plant species, certain varieties have been reported showing symptoms whereas others remain

generally asymptomatic. In addition, not all host plant species are associated with each X. fastidiosa subspecies. There are indications of host specificity, however its mechanisms are not yet fully

understood. Overlap of host plants is possible (the same plant species susceptible to more than one

X. fastidiosa subspecies/strain), but also variability in sensitivity of different genotypes within the same host species has been shown. In fruit trees (e.g. vineyard, olive), there is further complexity,

related to the root-stock-graft complex. It is also important to further investigate the role of non-cultivated species (e.g. Hopkins and Alderz, 1988; Purcell and Saunders, 1999), considering their

potential role as reservoir of the disease in natural vegetation (EFSA PLH Panel, 2015a).

Each genotype of X. fastidiosa is different in terms of host range (whereas the general biology of the

bacterium will remain the same), therefore host range of a new genotype cannot be derived from

literature but studies need to be conducted. However, due to vector preference for host plants, there will be a difference between the artificial host range inferred from laboratory studies and the actual

host range determined by vectors for a given strain and region (Anonymous, 2015).

The current European outbreak provides the research opportunity to determine under natural

conditions which plants can or cannot host the specific strain. Investigation of naturally occurring

potential host plants (cultivated or not) requires however the testing of a large number of specimens of each plant species originating in zones where the disease is widely present, to ensure that the

results are statistically valid. Testing a limited number of specimens from areas where the disease is not widely present is certainly not conclusive. In addition, there is no indication that the distribution

of the bacterium is homogeneous in plants and that the density of bacteria is stable throughout the

year. As plants do not always show symptoms, analytical detection tools of sufficient quality are required. Thus, evaluating plant species under natural conditions is a difficult task that requires very-

well planned experiments or surveys and is time consuming. Studies in contained facilities may help (applying mechanical or insect-mediated inoculations of the bacterium to a range of plant specimens),

nevertheless, such analyses require special facilities, particularly in areas where the pathogen is absent or not widely distributed, and its success may be influenced by growing conditions (EFSA PLH

Panel, 2015a).

Breeding resistant or tolerant genotypes is very important for tackling the impact of this pathogen. The knowledge on the susceptibility of varieties of woody plants, fruit trees and perennials and the

identification of tolerant or resistant genotypes is crucial for growers in the outbreak area as well as for growers in the rest of Europe. This is particularly relevant for the identification of olive varieties

tolerant or resistant to X. fastidiosa CODiRO strain. Once identified, resistant genotypes could be used

in breeding programme aiming to develop resistant cultivars.

The trade of plants for plantings is considered the most important pathway for the introduction and

spread of X. fastidiosa into new areas. Certification and testing of plant propagation material is an effective option to reduce the risk of introduction and spread of X. fastidiosa along this pathway

(EFSA Panel on Plant Health, 2015a). It has been implemented effectively in Brazil for the control of Citrus Variegated Chlorosis in citrus nurseries production (Carvalho et al., 2002). Heat treatment has

proved effective for sanitation of dormant plant propagation material of Vitis sp. and pecan walnut

against X. fastidiosa, however studies on other woody species have not been conducted so far (EFSA PLH Panel, 2015b).




What is a “host plant” for X. fastidiosa?

How to identify the host range of a new strain of X. fastidiosa, or of a given strain in a new

geographical area? What is the difference between natural and experimental infection? What would be the best experimental plan to study the host range of a X. fastidiosa strain?

What is the role of plant breeding and cultivar/variety selection? How to screen the germplasm

collections of woody plants and tree species (e.g. by pin pricking inoculation of cultivated bacteria cells, or by grafting or by keeping young plants as ‘traps’ into a heavily infected areas)? Should

this screening be conducted on germplasm collections of those plant species reported to be susceptible to the X. fastidiosa strains occurring in Europe? Or also for main crops at risk by other

strains of X. fastidiosa absent from Europe?

The use of tolerant and resistant varieties has been modelled to lead to different epidemiological scenarios. Understanding the underlying mechanisms responsible for different types of resistance

may be helpful in breeding programmes.

How to develop and implement certification schemes for plant propagation material free of X. fastidiosa? What kind of tools for certification should research develop? Is there a need for further research on pathogen detection, plant propagation techniques or plants sanitation treatments?

How can EU scientists best ‘catch up’ with ongoing research elsewhere? How to avoid repeating

research already done, how to benefit from previous work and how to best interact with ongoing research activities on host range, plant breeding and certification of plant propagation material?


Anonymous (2015). Xylella fastidiosa: options for its control. Parallel Workshop at Conference "Health Checks and Smart Treatments for Our Plants” EXPO Milan, 15th July 2015, © EU, 2015. ISBN 978-

92-79-52732-6. doi: 10.2777/168991. Available online: http://ec.europa.eu/research/conferences/2015/expo2015/pdf/presentations/xylella_final-


Carvalho SA, Machado MA, Coletta Filho HD and Müller GW, 2002. Present status of the production of

citrus budwood and nursery trees free of graft and vector-transmissible diseases in São Paulo

State, Brazil. Abstract. Fifteenth IOCV Conference, 2002—Surveys and certification, 317–320.

EFSA (European Food Safety Authority), 2015. Categorisation of plants for planting, excluding seeds,

according to the risk of introduction of Xylella fastidiosa. EFSA Journal 2015;13(3):4061, 31 pp. doi:10.2903/j.efsa.2015.4061

EFSA PLH Panel (EFSA Panel on Plant Health), 2015a. Scientific Opinion on the risks to plant health

posed by Xylella fastidiosa in the EU territory, with the identification and evaluation of risk reduction options. EFSA Journal 2015;13(1):3989, 262 pp. doi:10.2903/j.efsa.2015.3989. Available

online at http://www.efsa.europa.eu/en/efsajournal/pub/3989

EFSA PLH Panel (EFSA Panel on Plant Health), 2015b. Scientific opinion on hot water treatment of

Vitis sp. for Xylella fastidiosa. EFSA Journal 2015;13(9):4225, 10 pp.

doi:10.2903/j.efsa.2015.4225. Available online: http://www.efsa.europa.eu/sites/default/files/scientific_output/files/main_documents/4225.pdf

Hopkins DL and Adlerz WC, 1988. Natural hosts of Xylella fastidiosa in Florida. Plant Disease, 72(5), 429-431.

Purcell AH and Saunders SR, 1999. Fate of Pierce's disease strains of Xylella fastidiosa in common riparian plants in California. Plant Disease, 83(9), 825-830.




http://www.efsa.europa.eu/sites/default/files/scientific_output/files/main_documents/4225.pdf



4. Breakout Group 4 - The pathogen: biology, genetics, control

Introduction 4.1.

This breakout session deals with the research for the biology, genetics, typing and means of control in

the plant of the plant pathogen X. fastidiosa, including bactericidal substances, resistance inducers or cropping practices. Detection methods for pathogen survey and cropping practices to control vectors

populations are not included in this breakout session as they will be respectively dealt with in the

breakout sessions 1 and 2.

X. fastidiosa is the causal agent of Pierce’s disease of grapevine, phony peach disease, plum leaf

scald, almond, elm, oak, American sycamore, mulberry and maple leaf scorch, and citrus variegated chlorosis disease, among other diseases. There are generally four accepted subspecies of X. fastidiosa — fastidiosa, pauca, multiplex and sandyi (Schaad et al., 2004; Schuenzel et al., 2005) - and a new

one – morus (Nunney et al., 2014) – has been proposed. In addition several strains have been identified which have not yet been allocated to a recognized entity. The multilocus sequence typing

(MLST) approach and the classification of isolates into sequence types (STs) (unique genotypes based on the seven loci used in the MLST) and the creation of the public MLST database (www.pubmlst.org)

have resulted in a robust taxonomy for X. fastidiosa and provided a tool to study evolution and host specificity (Almeida and Nunney, 2015; Scally et al., 2005; Yuan et al., 2010).

A horizontal research issue, in fact common among the topics of the four breakout sessions, is the

need of developing human capacity on culturing, maintaining and transfering X. fastidiosa in European plant health and research laboratories, as culturing this bacterium is still difficult. There is also a need

to develop tools for functional work, not only to identify functions, but also to disrupt them. Particularly with regard to research on bacterial biology it is important to capitalise on existing

international expertise on X. fastidiosa research, to establish cooperation, exploit maximum synergies

and avoid duplications (Anonymous, 2015).

The EFSA Scientific Panel on Plant Health (2015a) identified the following needs for research to gain a

better understanding of the bacterium:

the large but still only partly known genetic diversity of X. fastidiosa and its consequences in

the field need to be further studied;

the distribution of X. fastidiosa among various subspecies makes it difficult to predict the host

range and the association with vectors of any given strain, and the severity of the disease that strain can potentially cause;

need to know the extent to which the various subspecies of X. fastidiosa can be vulnerable to

cold temperatures and winter recovery;

recombination between X. fastidiosa strains should be further studied as it may greatly impact

the risks associated with X. fastidiosa in terms of host range, association with vectors and severity of the disease.

The latter point is of particular relevance as the Apulian and Corsica strains of X. fastidiosa reported in Europe are genetically different belonging to two different susbspecies with the associated risk of

recombination and creation of new genetic variants of the pathogen. Therefore there is a need to

closely monitor genetic changes or recombination arising within the populations of X. fastidiosa in different locations in Europe.

With regard to control of the pathogen in the plant, some research lines are on-going, but there is not

yet an effective control method of the pathogen applicable in the field. Control of X. fastidiosa in the Americas is therefore currently achieved by removing sources of inoculum, using healthy plant

propagation material and controlling the vector(s) (Anonymous, 2015).

http://www.pubmlst.org/




How much should research focus on the development of genetic tools to study biology of X. fastidiosa? Should generating a GFP-marked strain be a priority? Should specific isolates be

selected for biological research so that studies by independent groups can be compared?

X. fastidiosa taxonomy has improved significantly thanks to MLST. Should diagnostic guidelines be

updated incorporating newly accumulated knowledge on X. fastidiosa diversity? Should all characterised strains be made available to researchers in publicly accessible culture collections?

What aspects of X. fastidiosa biology should be a priority to study in the EU that has not already

been studied? Are findings on biology within one subspecies, for example, generally applicable to other subspecies or do they need to be repeated?

Given the research that has been already performed aimed at controlling/killing X. fastidiosa in the plant in the Americas, which tools for bacterium control in plant have a potential for applications in

the field and which researches should be reproduced or prioritized in the EU?

How can EU scientists best ‘catch up’ with ongoing research elsewhere? How to avoid repeating research already done, how to benefit from previous work and how to best interact with ongoing

research activities on the pathogen biology, genetics and control?




Almeida RPP, Pereira EF, Purcell AH and Lopes JRS, 2001. Multiplication and movement of a citrus strain of Xylella fastidiosa within sweet orange. Plant Disease, 85, 382–386.

Almeida RFP and Nunney L, 2015. How do plant diseases caused by Xylella fastidiosa emerge? Plant Disease 99, 1457-1467. http://dx.doi.org/10.1094/PDIS-02-15-0159-FE

Anonymous (2015). Xylella fastidiosa: options for its control. Parallel Workshop at Conference "Health Checks and Smart Treatments for Our Plants” EXPO Milan, 15th July 2015, © EU, 2015. ISBN 978-

92-79-52732-6. doi: 10.2777/168991. Available online :


EFSA PLH Panel (EFSA Panel on Plant Health), 2015a. Scientific Opinion on the risks to plant health posed by Xylella fastidiosa in the EU territory, with the identification and evaluation of risk

reduction options. EFSA Journal 2015;13(1):3989, 262 pp. doi:10.2903/j.efsa.2015.3989. Available

online at http://www.efsa.europa.eu/en/efsajournal/pub/3989

Hopkins D and Purcell A, 2002. Xylella fastidiosa: cause of Pierce’s disease of grapevine and other

emergent diseases. Plant Disease, 86, 1056–1066.

Meng Y, Li Y, Galvani CD, Hao G, Turner JN, Burr TJ and Hoch HC, 2005. Upstream migration of

Xylella fastidiosa via pilus-driven twitching motility. Journal of Bacteriology, 187, 5560–5567.

Nunney L, Schuenzel EL, Scally M, Bromley RE and Stouthamer R, 2014. Large-scale intersubspecific

recombination in the plant-pathogenic bacterium Xylella fastidiosa is associated with the host shift

to mulberry. Applied and Environmental Microbiology, 80(10), 3025–3033. http://doi.org/10.1128/AEM.04112-13.

Newman KL, Almeida RPP, Purcell AH and Lindow SE, 2003. Use of a green fluorescent strain for Analysis of Xylella fastidiosa colonization of Vitis vinifera. Applied and Environmental Microbiology,

69, 7319–7327.

Purcell AH and Saunders SR, 1999. Fate of Pierce’s disease strains of Xylella fastidiosa in common riparian plants in California. Plant Disease, 83, 825–830.

Schaad NW, Postnikova E, Lacy G, Fatmi M and Chang CJ, 2004. Xylella fastidiosa subspecies: X. fastidiosa subsp. [correction] fastidiosa [correction] subsp. nov, X. fastidiosa subsp. multiplex

subsp. nov, and X. fastidiosa subsp. pauca subsp. nov. Systematic and Applied Microbiology, 27,

290–300. Erratum in Systematic and Applied Microbiology, 27, 763.

Schuenzel EL, Scally M, Stouthamer R and Nunney L, 2005. A multigene phylogenetic study of clonal

diversity and divergence in North American strains of the plant pathogen Xylella fastidiosa. Applied and Environmental Microbiology, 71, 3832–3839.

Yuan X, Morano L, Bromley R, Spring-Pearson S, Stouthamer R and Nunney L, 2010. Multilocus sequence typing of Xylella fastidiosa causing Pierce's disease and oleander leaf scorch in the United

States. Phytopathol, 100, 601–611.

http://dx.doi.org/10.1094/PDIS-02-15-0159-FE






Appendix C – Abstract book

1. Abstracts of Plenary session

Status and control of Xylella fastidiosa in the EU 1.1.

Arijs, H. 1, P. Di Rubbo1, D. Simion1

1 European Commission, DG SANTE-Plant Health Unit, Brussels, Belgium

Xylella fastidiosa is regulated in the EU as quarantine organism under Council Directive 2000/29/EC ("plant health directive") on protective measures against the introduction into the Community of

organisms harmful to plants or plant products and against their spread within the Community. As such, the introduction of this organism into, and spread within all Member States, shall be banned.

The plant health directive provides Member States with the legal obligations to take, once the

organism is known to be present and irrespective of the symptoms, all necessary measures to eradicate it, or if that is impossible, inhibit its further spread.

Following the first outbreak of X. fastidiosa, subspecies pauca, notified by the Italian Authorities in the region of Apulia, in October 2013, preliminary EU emergency measures were taken in February 2014,

detailed in July 2014 and further strengthened in May 2015 with the aim to prevent the further spread of the bacterium within the EU.

Three audits were carried so far by the Commission's Food and Veterinary Office in Apulia, confirming

the limited implementation of the eradication/containment measures (e.g. removal of infected plants) and the further spreading of the bacterium out of the province of Lecce. No movement of specified

plants is so far authorised to be moved within and out of the demarcated areas established in Apulia.

In July 2015, French Authorities notified the first outbreak of X. fastidiosa, subspecies multiplex, in

Corsica. Numerous outbreaks have been reported since then in the area, including two outbreaks

recently reported in the PACA region (France mainland). Polygala myrtifolia is the main host plant, although numerous ornamental plants have been also confirmed to be infected. No positive cases

have been reported so far on Olea europea. Trace-back activities are ongoing to confirm the source of infection. EU emergency measures are in place.

Since January 2014, 6 imported consignments were intercepted from Honduras and Costa Rica at

import. Additional 41 EUROPHYT notifications were made in 2014/2015 based on trace-back activities carried out by Member States (AT, FR, DE, IT) and Switzerland on consignments already released in

the EU. Coffea plants, intended for planting, were the only plant species intercepted so far. 7 EUROPHYT notifications indicated the presence of X. fastidiosa, subspecies sandyi.

The revision of some elements of Decision 2015/789/EU, including the update of the list of regulated plant species, is foreseen by late 2015.

Lastly, survey activities were carried out across Member States during the 2015 growing season and

no further findings were reported. EU guidelines will be made available with the aim to harmonise survey activities across Member States.

What is the risk of Xylella fastidiosa for Europe? 1.2.

Bragard, C.1, 2, R. Almeida3, E. Czwienczek1, D. Bosco4, D. Caffier1,5, JC Grégoire1,6, G. Hollo1, S.

Parnell 1,7, G. Stancanelli1

1 EFSA, Plant health panel & staff, Parma, Italy 2 Université catholique de Louvain, Earth&Life Institute, Louvain-la-Neuve, Belgium 3 University of California, College of Natural Ressources, Berkeley, California UK 4 Universita degli Studi di Torino, Department of Agricultural, Forest and Food Science, Torino, Italy

http://eur-lex.europa.eu/legal-content/EN/TXT/?uri=CELEX:32015D0789



5 INRA, Paris, France 6 ULB, LUBIES, Bruxelles, Belgium 7 University of Salford, Ecosystems and Environment Research Center, Manchester, UK

E-mail: [email protected]

In 2013, the discovery of Xylella fastidiosa in Puglia, Italy, triggered major questions about the risk it represents for Europe. Despite the challenge of assessing the risk of a pathogen previously unreported

in the area and causing a “new disease”, the Plant health Panel of the European food safety authority published rapidly a statement on Xylella. This was followed by a comprehensive pest risk assessment

and an evaluation of risk reduction options, published in January 2015.

An exhaustive list of plant hosts was also compiled and published as a searchable electronic list.

Similarly, based on the assumption that all xylem-feeding insects are potential vectors, European

possible vectors have been listed. The different pathways for entry in Europe have also been outlined.

The probability of entry of the pathogen was rated very high with plants for planting and moderate

with infectious insect vectors carried with plant commodities or even travelling as stowaways. Establishment of the agent with new entries under current regulations was considered very likely.

Furthermore, the spread of X. fastidiosa from affected areas in Europe, already established or to be

potentially established in the future, was also rated as very likely. The consequences were considered to be major because yield losses and other damage would be high and require costly control

measures.

The recent detection of multiple outbreaks in both Corsica and Southern France stress the need to

carefully appraise continuously the risk at the whole European scale. Pending questions like a possible role of climate in limiting the spread, the contribution of human- and insect- associated spread or on

how to tackle with the worldwide and European plant traffic will be discussed, as well as the

knowledge gaps and research needs previously identified through the risk assessment proposed.

References:

EFSA, 2013. Statement of EFSA on host plants, entry and spread pathways and risk reduction options

for Xylella fastidiosa Wells et al. EFSA Journal 2013;11(11):3468 [50 pp.]

doi: 10.2903/j.efsa.2013.3468

EFSA, 2015. Categorisation of plants for planting, excluding seeds, according to the risk of

introduction of Xylella fastidiosa. EFSA Journal 2015;13(3):4061 [31 pp.] doi: 10.2903/j.efsa.2015.4061

EFSA PLH Panel, 2015. Scientific Opinion on the risks to plant health posed by Xylella fastidiosa in the

EU territory, with the identification and evaluation of risk reduction options. EFSA Journal 2015;13(1):3989 [262pp.] doi: 10.2903/j.efsa.2015.3989

State of the art on biology and epidemiology of the Xylella 1.3.fastidiosa outbreak in Apulia

Saponari, M.1

1 Istituto per la Protezione Sostenibile delle Piante, UOS Bari, CNR, Dipartimento di Scienze del Suolo, della Pianta e degli Alimenti, Università degli Studi di Bari Aldo Moro CRSFA Centro di Ricerca, Sperimentazione e Formazione Basile Caramia CIHEAM-Istituto Agronomico Mediterraneo di Bari


The geographic expansion of the olive quick decline epidemic in the Apulia region (southern Italy)

clearly revealed a strict relationship between the occurrence of diseased olive trees and the findings of Xylella fastidiosa infections; implying that the bacterium (X. fastidiosa strain CoDiRO) may have a

mailto:[email protected]




major role in the development of this emerging disease. Systematic surveys carried out over a 2-year period in the main outbreak area allowed the identification, besides olive as predominant susceptible

host, of more than 20 different susceptible host species. The majority consists of perennial trees and

ornamental shrubs found in gardens and backyards, and representing new hitherto host for the subspecies pauca, to which the CoDiRO strain belongs. Despite this large number of susceptible

species identified in the contaminated area, within the olive groves search for alternative hosts which may play a role in the epidemiology and spread of the bacterial infections in the Apulian olive groves,

failed to identify plant species which may act as relevant bacterial reservoir for the insect vectors. This

evidence coupled with the data showing that the infectivity of the indigenous ascertained insect vector (Philaenus spumarious) relates to the period when the adult population migrates on the olive canopy,

suggests that olive serves as an important source of inoculum for pathogen spread. Preliminary results of the artificial inoculations and exposure of experimental plants to infective P. spumarious support

field evidence on the host susceptibility and vector ability to acquire the bacterium from different host sources.

Xylella fastidiosa outbreaks in France: identification of the causal 1.4.agents and distribution of the disease

Manceau, C.1 and M.A. Jacques2

1 Anses - Plant Health Laboratory – 7 rue Jean Dixméras – 49 044 Angers, France 2 UMR1345 IRHS – EMERSYS - INRA - Centre Angers-Nantes, 42 rue Georges Morel – CS 60057 49071 Beaucouzé cedex, France


Xylella fastidiosa was recently intercepted several times in France on coffee plants imported from the

Americas. The first interception was carried out on four coffee plants (Coffea arabica and C. canephora) originating from Ecuador and Mexico. The strains isolated from C. arabica were identified

as X. fastidiosa subsp. pauca and those isolated from C. canephora as X. fastidiosa subsp. fastidiosa/sandyi. Other infected coffee plants originating from Costa-Rica which were imported via

The Netherlands, were intercepted in 2014 and 2015. All these outbreaks were eradicated. However,

schrubs of Polygala myrtifolia were unfortunately detected outdoor, contaminated by X. fastidiosa on July, the 22nd, 2015 in Propiano, South Corsica, France. The detection procedure was performed by

the Plant Health laboratory at Anses by real time-PCR as described in the official method published by Anses. A large survey has been conducted in Corsica and in Mainland France. By October the 15th,

2015, 330 samples out of more than 2,500 analysed samples were found contaminated by X. fastidiosa in Corsica. Most contaminated samples concerned Polygala myrtifolia (303) and X. fastidiosa

was found on surrounding plants belonging to Spartium, Cytisus, Pelargonium, Hebe, Lavandula,

Genista, Rosmarinus, Acer, Quercus, Asparagus and Artemisia. Recently, two outbreaks were observed in Nice and in Mandelieu la Napoule on the Azur Coast. All strains isolated in France were

identified as X. fastidiosa subsp. multiplex by MLSA/MLST analysis. Furthermore, the genome of three strains isolated from Polygala myrtifolia and Spartium junceum in Corsica, were sequenced. Their

analysis confirmed their identity to X. fastidiosa subsp. multiplex and the occurrence of two

genotypes, so far. These results indicate that the outbreaks observed in France are not an extension of the outbreak observed in Apulia in the south of Italia.

State of the art on Xylella fastidiosa in North America 1.5.

Purcell, A.1

1 Department of Environmental Science, Policy and Management University of California, Berkeley, CA 94720-3114






Most of what we know about the biology and control of the bacterium Xylella fastidiosa originated from research in the Americas. Research since the sequencing of the complete genome of X. fastidiosa in 2000 largely focused on molecular approaches, but field research continues to provide

the only management methods for control of some X. fastidiosa-caused diseases such as grape and citrus. The complex interactions of X. fastidiosa with host plants, insect vectors, environmental

factors, its own populations and those of other strains of X. fastidiosa, and other microbes drive the ecology (=epidemiology) of X. fastidiosa.

Host plant interactions. How do X. fastidiosa strains differ in colonizing various host species and how

do they cause disease? Still unknown. The X. fastidiosa-host interaction governs host resistance. Disease occurs only in “new encounters” of X. fastidiosa with some host plants that have no

evolutionary history of interactions with X. fastidiosa. The cell-to-cell signalling system of X. fastidiosa normally governs its behaviour within a plant host so as to not threaten the host’s survival and in turn

its own survival. This feature can be exploited to prevent disease by increasing cell signal concentration by spraying the signal, introducing antagonistic bacteria that have a similar signal, or

introducing genes into plants to have them produce the signal. The use of molecular markers over the

past 15 years have aided conventional breeding of grapevines resistant to Pierce’s disease (PD) strains of X. fastidiosa by using North American grape (Vitis) species as gene sources. Other strategies

involve introducing a variety of genes into plants to reduce the presence of X. fastidiosa or to impede its movements. Proceedings of Pierce’s disease symposia

(https://www.cdfa.ca.gov/pdcp/research.html) provide timely reports on currently funded research for

PD. Remember that many negative results are not published, but they are reported in these Proceedings.

Environmental factors. Freezing temperatures can eliminate X. fastidiosa from dormant grapes and almonds. This as yet unexplained feature seems to explain the geographic distribution of PD and other

X. fastidiosa-caused diseases in North America. Understanding the basis of this phenomenon can stimulate ideas for new therapies. Injections of antibiotics, copper, and other chemicals tested in

grape and almond to kill X. fastidiosa have not proven to be therapeutic, despite causing a temporary

lessening of symptoms, but searches for novel chemical therapies should continue. Scientific testing disproved early claims that applying some fertilizers and other soil-based chemicals cured PD.

Vectors. Any sucking insect that specializes in ingesting xylem sap should be considered a probable vector of X. fastidiosa. Only transmission tests using plants can identify which insects are vectors.

Ecological data are needed to identify the vectors important in the spread of X. fastidiosa; these will

vary with crop and location. So far, controlling vectors before they enter vineyards has been the most successful control method for PD in California but not in the south-eastern USA. Promptly removing

diseased trees was effective for reducing the spread of citrus variegated chlorosis disease in Brazil and is claimed to be important in controlling phony peach disease (both caused by X. fastidiosa) in the

southeastern United States, but so far this is not successful for PD.

Xylella strain interactions. A strain of X. fastidiosa that weakly colonizes grape without causing significant damage protects grapevines from PD in Florida. So far, this strain has not worked in

California. Quarantine restrictions make it difficult to study interactions among different strains of X. fastidiosa. Introductions of novel genetic diversity may lead to the emergence of new diseases.

Other microbes. Genomes of X. fastidiosa contain imbedded phage genomes and numerous signature sequences that indicate past attacks by phages. Recent reports indicate success in curing grapevines

with PD by applying mixtures of phages that attack X. fastidiosa. Phages may have important roles in

gene transfer and epidemiology of X. fastidiosa-caused diseases. X. fastidiosa phage biology is underexplored.

CONCLUSION. Fast access to the published record of successes and failures of X. fastidiosa research should be used to avoid “reinventing the wheel”. Urgency requires that we not ignore what others

have worked so hard to discover, but naïve investigators have made most of the major new

discoveries about the biology of X. fastidiosa. Every new setting or host for X. fastidiosa may have some unexpected properties.

https://www.cdfa.ca.gov/pdcp/research.html



Xylella fastidiosa in South America: Ecological basis for 1.6.management

Lopes, J.R.S.1

1ESALQ/University of São Paulo, Piracicaba, SP, Brazil.


The xylem-limited bacterium Xylella fastidiosa is the causal agent of important crop diseases in South

America, e.g. plum leaf scald (PLS), citrus variegated chlorosis (CVC) and coffee leaf scorch (CLS). Xylella fastidiosa pathosystems are usually complex, involving multiple host plants and vector species,

and there may be genetic and biological variation in the pathogen. An understanding of these disease components and interactions among them is basic for developing effective management strategies.

CVC and CLS are caused by biologically distinct sequence types (STs) within X. fastidiosa subsp.

pauca, whereas PLS is associated with X. fastidiosa subsp. multiplex and pauca. The known vectors are several species of xylem-fluid feeding leafhoppers (Hemiptera: Cicadellidae) of the subfamily

Cicadellinae (the ‘sharpshooters’), which are generally polyphagous and occur in different crops, but vary in geographic distribution, habitat and host preferences, and transmission efficiency. Various

weeds are known hosts of X. fastidiosa, but most of them are symptomless and their epidemiological role is not well understood. For CVC in São Paulo (SP) State, Brazil, both primary and secondary

spread occur, and citrus is the most important inoculum source. The climate influences disease

expression - CVC incidence and severity are higher in regions of SP with higher temperatures and longer dry season (water stress). CVC management includes control measures based on vector

control, pathogen exclusion (certification of nursery trees) and eradication (pruning or roguing of symptomatic trees), which have been successful in reducing disease incidence in SP over the last

decade. However, vector control is now heavily dependent on insecticides and the development of

alternative control methods is needed to make disease management sustainable. Research should focus on breeding for citrus resistance against the pathogen and bio-rational and behavioural methods

for vector control, based on fundamental knowledge on X. fastidiosa-plant-vector interactions.

Acknowledgments:

Research supported by FAPESP, Fundecitrus, FINEP and CNPq, Brazil.

Research needs on X. fastidiosa: a perspective from the EU farmers 1.7.

Cantele, G.1

1COPA-COGECA, Rue de Trèves 61, 1040 Bruxelles (BE)


It has been many months since the release of the first report in the European Union (EU) on Xylella fastidiosa, probably the most dangerous among the plant pathogens. This outbreak has affected in

Italy the whole province of Lecce, the southern part of the province of Brindisi and, it has recently been reported also in the province of Taranto. Since summer 2015 in France, another strain of X. fastidiosa, different from the Italian one, has been reported in several outbreaks in Corsica and on the

Mediterranean coast.

In the current scenario, the Italian territory can be considered as divided into two parts: the “Xylella

free” zone, recently established by the Italian Ministry of Agriculture and Forestry Policies, and the infected zone, which has a buffer zone in between to guarantee the pest freedom of the first zone.

Due to the activity of the Italian judiciary and in order to allow further investigations, any containment actions have been suspended so far, except for the implementation of voluntary good agricultural

practices by the farmers.

Meanwhile, according to the new reports released from France and Italy, the number of botanical species included on the list of plants which are susceptible to this pathogen has reached the number




of 359 species, a value that probably underestimates the gravity of the situation. This number well represents the idea of how practically impossible it is to eradicate the pathogen from the infected

areas and the idea of how much the containment strategies through roguing of diseased plants risk to

be ineffective.

Horizon 2020 certainly represents a great opportunity to improve the knowledge on this bacteria and

to start studying the strategies for coexistence and containment first, and for control then, but it is only the beginning of a journey in which the European research must be committed and strongly

supported for at least the next 10 years.

The objectives of the farmers from Italy, France, and generally from the Mediterranean EU countries, must converge on:

• Scientific validation of the cropping practices (the we apply everyday) and of the protocols for plant nutritional support that could allow the prevention of the spreading of the infection into the

“Xylella free” areas;

• Scientific validation of the guidelines for the control of the potential vector insects, that could

combine the achievement of the control about the presence of the vector insects with the need

that such measures do not cause a polluting environmental impact, to the benefit of the agricultural workers and all the citizens;

• Scientific validation of the cropping practices that could allow the slowdown of the infection, where the start of the desiccation symptoms is observed, in the infected areas where there is no

obligation to rogue the infected plants

• Development of protocols aimed at curing and removing the presence of the bacterium from diseased plants through the application of innovative technologies

• Identification of cultivars with forms of tolerance or resistance to the bacteria. This would possibility give great hope to maintain, already in short-medium term, the productive potential in

the most affected areas.

In order to achieve all this, it will be essential to be allowed to carry out researches under the most

extreme conditions, i.e. in the areas with the highest occurrence of infections where the ban to

replant susceptible species could represent a very severe limitation, particularly for the research of resistant cultivars.

Xylella fastidiosa from the Euphresco point of view 1.8.

Giovani, B.1

1 Euphresco coordinator (EPPO)


As a network of research programme owners and managers, Euphresco (European Phytosanitary

Research Coordination) is in front line for the coordination of national research activities, by supporting the exchange of knowledge and best practices on research programmes, by developing

common strategic research agenda and by financing transnational research projects.

Xylella fastidiosa was identified as one of the research priorities by a number of Euphresco members;

a plan for activities (research topic) was agreed and a research project should be launched by the spring 2016.

While the vectors of the disease are relatively well known in North- and South- America, this is not the

case for European countries; so far, the meadow spittlebug Philaenus spumarius (L.) (Hemiptera: Aphrophoridae) is the only species found in Salento that has been proved to be vector of X. fastidiosa

(Saponari et al., 2014). The project has the objective to produce rapid and reliable detection protocols for monitoring X. fastidiosa in insect vectors and in different plant species (symptomatic and

asymptomatic plant material), with emphasis on improving sampling techniques, protocols for




processing of samples (i.e., DNA extraction), and methods for isolating X. fastidiosa in culturing medium to fulfil quarantine and monitoring requirements.

Through Euphresco, the project will benefit from links with national and international research

projects and initiatives and complementarities will be pursued to ensure optimal use of the limited resources available. Researchers from North-America will provide valuable support to their European

fellows, as they will share their knowledge and expertise. The preferential relationships with National and Regional Plant Protection Organisations will contribute to the effective dissemination and use of

the project’s results.

Openings for research on plant health and plant protection in 1.9.Horizon 2020: Focus on Xylella fastidiosa

Schneegans, A.1

1European Commission, DG Agriculture and Rural Development

Email: [email protected]

Research on plant health and plant protection has been continuously supported throughout European

Framework Research Programmes. The current research programme Horizon 2020 is particularly well

placed to step up funding and scope of activities to deal with plant pests and diseases. With an increased focus on innovation Horizon 2020 allows to better link fundamental and applied research,

exchange of knowledge and testing of innovations. In addition to working on specific pests or diseases proposed research topics aim at increasing overall resilience of plants and production systems to biotic

stresses. Examples include research on beneficial plant-soil and plant-plant interactions in relation to plant health or the development of tools for integrated pest management. Research topics are

published in biannual work programmes14 and generally require that research consortia take well into

account the needs of the users of potential solutions. Involvement of farmers, advisors and plant health authorities is therefore particularly relevant. With regard to Xylella fastidiosa, the EU

Commission has provided funding for project POnTE to start work end of 2015 and will select a complementary project in the context of the 2016 evaluation exercise. Funding for the two projects

will amount to a total of about 10m€. Partners in these two major initiatives will be requested to work

in synergy to unravel many of the open questions with regard to the bacterium and its vectors. Results of their work are expected to enhance capacities of the farming and plant health sectors to

better prevent, control and manage disease outbreaks.

2. Abstracts of session 1. Surveillance and Detection

Surveillance methodology for Xylella fastidiosa 2.1.

Parnell, S.1, 2, N.J. Cunniffe3, C.A. Gilligan3, F. van den Bosch4.

1 University of Salford, School of Environment and Life Sciences, Manchester, UK

2 EFSA, Plant health panel & staff, Parma, Italy 3 University of Cambridge, Department of Plant Sciences, Cambridge, UK 4 Rothamsted Research, Computational and Systems Biology, Harpenden, UK


Surveillance for Xylella fastidiosa in the area of potential establishment in the EU is crucial for disease

containment and eradication. Surveillance is required for early detection of new outbreaks initiated by

14 For more information see Work Programme of Horizon 2020 Societal Challenge 2 "Food security, sustainable agriculture

and forestry, marine and maritime and inland water research and the bioeconomy":

http://ec.europa.eu/research/participants/portal/desktop/en/funding/reference_docs.html#h2020-work-programmes




human-mediated or natural dispersal of inoculum, to monitor shifts in the distribution and incidence of currently infected areas, and to establish the pest-free status of areas where X. fastidiosa is not yet

known to occur. Effective surveillance relies on well-defined survey objectives, utilisation of available

knowledge on vector, host and pathogen biology, and the application of efficient and sensitive detection technologies and sampling protocols. A range of ISPMs and reports are available as

guidance on surveillance for plant pests and which can be used to inform X. fastidiosa surveillance in the EU (Bell et al. 2014; FAO, 1997, EFSA 2014).

Knowing when, where, what and how to sample for an emerging disease such as X. fastidiosa is not a

trivial problem and involves the deployment of extensive survey resources over vast and complex host landscapes. We summarise some recent work looking at the use of epidemiological models to target

and optimise surveillance programmes to maximise the probability of success and minimise costs. The methods include risk-based survey methods to spatially-target survey resources in a landscape based

on available epidemiological information (Parnell et al. 2014) and methods to quantify the probability to detect an epidemic invading a new area given a certain surveillance programme is in place (Parnell

et al. 2015). Enhanced surveillance for X. fastidiosa will require modern approaches to survey which

account for the epidemiology of the pathosystem and which use the latest technologies for detection in hosts and vectors and information on their sensitivity and scope. Epidemiological modelling provides

a useful framework to incorporate this information for optimal and cost effective survey design.

References:

Bell H, Wakefield M, Macarthur R, Stein J, Collins D, Hart A, Roques A, Augustin S, Yart A, Péré C,

Schrader G, Wendt C, Battisti A, Faccoli M, Marini L and Petrucco Toffolo E, 2014. Plant health surveys for the EU territory: an analysis of data quality and methodologies and the resulting

uncertainties for pest risk assessment (PERSEUS). EFSA Supporting Publications 2014: EN-676. Available online at http://www.efsa.europa.eu/en/supporting/doc/676e.pdf.

EFSA PLH Panel, 2015. Scientific Opinion on the risks to plant health posed by Xylella fastidiosa in the EU territory, with the identification and evaluation of risk reduction options. EFSA Journal

2015;13(1):3989 [262pp.] doi: 10.2903/j.efsa.2015.3989

FAO (Food and Agriculture Organization of the United Nations), 1997. International standards for phytosanitary measures (ISPM) 06. Guidelines for surveillance. FAO, Rome. Available online:

https://www.ippc.int/id/ispms

Parnell S, Gottwald TR, Riley T and Van Den Bosch F, 2014. A generic risk‐based surveying method for

invading plant pathogens. Ecological Applications, 24(4), 779–790.

Parnell S, Gottwald TR, Cunniffe NJ, Alonso-Chavez V and van den Bosch F, 2015. Early detection surveillance for an emerging plant pathogen: a rule of thumb to predict prevalence at first

discovery. Proc. R. Soc. B 282 (1814), 20151478.

How can remote sensing contribute to the surveillance of X. 2.2.fastidiosa affected regions?

Beck, P.1

1 European Commission – JRC


Current remote sensing technology allows for the monitoring of plants, parcels, landscapes, and

regions, over a broad range of spatial resolutions (from centimetres to kilometres), and spectral

domains that include the visible and thermal wavelengths. Earth orbiting platforms, i.e. satellites, provide regular coverage of Earth’s surface at resolutions down to ca. 50 cm. This makes them well-

suited for land cover mapping, and for identifying areas that are at risk of X. fastidiosa infection because they have a high host plant density. Yet, more detailed agricultural land cover maps already

exist for individual Member States, and are better suited for this purpose. The latter maps are

produced from ortophotographs taken from manned aircraft. Manned aircraft, as well as remotely

https://www.ippc.int/id/ispms




piloted aerial systems (RPAS), offer the advantage over satellites that a) they can be equipped with imaging instruments that are better at estimating phenomena related to plant physiology and health,

such as evapotranspiration and fluorescence and b) that they can be deployed to acquire images with

resolutions down to a couple of cm; thermal infra-red images acquired from aircraft have been used to distinguish and map increasing levels of hydraulic stress in olive trees caused by Verticillium wilt.

Similarly, indices calculated from hyperspectral measurements of individual olive leaves, indicate that leaves infected by X. fastidiosa are spectrally distinct from uninfected ones. In short, while existing

land cover maps could greatly aid the planning of surveillance in the context of the X. fastidiosa

outbreak, the deployment of thermal or hyperspectral instrumentation on aircraft holds the greatest promise for the early detection of X. fastidiosa symptoms.

Innovative tools in the early detection and surveillance of X. 2.3.fastidiosa

D'Onghia, A.M.1

1 CIHEAM-Istituto Agronomico Mediterraneo di Bari (IAMB)


Xylella fastidiosa, the EU quarantine bacterium recently introduced in Italy (Apulia region), is highly associated to a severe disease affecting olive trees, the Olive Quick Decline Syndrome (OQDS). It may

seriously damage a number of host species, therefore its early detection and surveillance are of primary importance for a successful eradication/containment programme of the pathogen. To this aim

the CIHEAM-IAMB developed an innovative surveillance system and rapid diagnostic tools for the identification of infected olive trees at territorial level. This surveillance system, now operative in the

official monitoring of X. fastidiosa in Apulia region, combines in a central web server (XylWeb) data

from different components as: advanced tools of territorial analyses (e.g. photo-interpretation of high resolution aerial images in GIS environment of OQDS-suspected trees), information technology for

field data acquisition by smart devices (XylApp) and rapid diagnostic methods for in situ pathogen assessment in plant material (DTBIA, real time LAMP) and insects (real-time LAMP) that avoid the risk

of infection spread. In this system, pathogen detection in potential vectors (so called 'spy insects')

represents an effective preventive diagnostic tool that can reveal the presence of the bacterium before the symptoms appearance in host plants. Further research is needed to improve such a model,

e.g. the identification of the infection in asymptomatic trees and in host species other than olive through remote and proximal sensing methods, the development of a systematic sampling procedure

based on pathogen/vector spatial analyses, the enhancement of XylApp to extend its use to other European contexts, the improvement of in situ detection tools. Such a system has also a great

potential in facilitating the spread of information at different levels and the communication among all

actors.

References:

Ben Moussa IE, Valentini F, Lorusso D, Mazzoni V, Digiaro M, Varvaro L and D’Onghia AM, 2015.

Evaluation of 'Spy Insect” approach for monitoring Xylella fastidiosa in symptomless olive orchards in

the Salento peninsula (Southern Italy). In Proceedings of the 7th meeting of the IOBC/wprs WG 'Integrated Protection of Olive Crops' (Kalamata, Greece 2015).




Djelouah K, Frasheri D, Valentini F, D'Onghia AM and Digiaro M, 2014. Setting up of Direct Tissue Blot Immuno Assay (DTBIA) for the mass detection of Xylella fastidiosa in olive trees. Phytopathologia

Mediterranea, 53(3), 559–564.

D'Onghia AM, Santoro F, Yaseen T, Djelouah K, Guario A, Percoco A and Valentini F, 2014. An innovative monitoring model of Xylella fastidiosa in Apulia region, Italy. International Symposium of

the European Outbreak of Xylella fastidiosa in Olive (Gallipoli, Italy 2014), 33.

D'Onghia AM, Lacirignola C and Djelouah K, 2015. Contribution of CIHEAM-Bari for the early

surveillance of Xylella fastidiosa and its vectors on olive trees in Italy. CIHEAM publications Watch

letter, 33, 30–33.

Elbeaino T, Yaseen T, Valentini F, Ben Moussa IE, Mazzoni V and D’Onghia AM, 2014. Identification of

three potential vectors of Xylella fastidiosa in Puglia region (southern Italy). Phytopathologia Mediterranea, 53, 328–332.

Gualano S, Tarantino E, Santoro F, Valentini F, Dongiovanni N and D’Onghia AM, 2014. Analisi assistita da immagini aeree ad elevata risoluzione geometrica per il riconoscimento del CoDiRO

associato al batterio Xylella fastidiosa in Puglia. Atti Asita (Firenze 2014), 651–658.

Yaseen T, Drago S, Valentini F, Elbeaino F, Stampone G and D'Onghia AM, 2015. On-site detection of Xylella fastidiosa in olive trees (Olea europaea L.) and insects using the real-time loop-mediated

isothermal amplification method. Phytopathologia Mediterranea, 54, 1, temp17−25 DOI: 10.1460.

Detection of Xylella fastidiosa in the frame of surveillance activities 2.4.in Austria

Gottsberger, R.A.1

1 Austrian Agency for Health and Food Safety (AGES), Institute for Sustainable Plant Production, Dept. for Molecular Diagnostics of Plant Diseases, Spargelfeldstr. 191, 1220 Vienna


A detection and identification procedure for Xylella fastidiosa was established at the Austrian Agency for Health and Food Safety (AGES), to cover the requirements in the draft of the Decision

2014/497/EU; “Measures to prevent the introduction into and the spread of X. fastidiosa within the

Union” at national level. A scheme for the detection and identification of subspecies by molecular methods and sequencing was put in place and improvement in isolation procedures is being

implemented. In the frame of the surveillance activities to avoid an introduction, X. fastidiosa could be detected in ten out of 108 samples from different host plants recently tested. These plant samples

were sent in within the frame of the official monitoring, import samples and specific sampling of coffee plants in which X. fastidiosa were intercepted in some MS before. These 10 positive tested

samples consisted of big ornamental coffee plants which were traded to Austria in the EU internal

market. In addition there are plans in the near future to intensify the national surveillance activities including the testing of other host plants and potential vectors embedded in transnational research

activities.

Electrochemical label-free sensing platform for rapid detection of X. 2.5.fastidiosa

O’Riordan, A.1

1 Tyndall National Institute, Ireland


The large scale monitoring of X. fastidiosa (XL) is currently undertaken with ELISA kits and PCR

protocols. Although PCR sensitivity and specificity are sufficiently high, the procedures are time-





consuming, require expensive laboratory equipment and cannot be performed in the field because of the lack of convenient portable instruments. ELISA assays on the other hand have proven reliable and

cost effective. However, typical assay time for ELISA is 5 hours, and there are no electronic handheld

sensors commercially available for X. fastidiosa. This means rapid, on site detection of XL is currently not possible. We believe rapid, cost effective and onsite detection methods of XL (at locations with

high density and/or rapid turnaround of plants for planting such as at markets or points of entry) is essential for the promptness application of efficient control measures.

We have recently developed an electrochemical sensor and associated portable reader (including

communication protocols) for rapid, sensitive, multiplexed and on-site detection of pathogens. The platform was initially developed for animal health applications (Bovine Viral Diarrhoea- BVD), where

we successfully demonstrated label-free detection of both BVD antibodies and virus in serum samples. By modifying the biological capture molecules we believe our platform has tremendous potential for

cost effective, rapid (assay time <5min) and early detection of bacteria such as X. fastidiosa. The label-free sensing mechanism is versatile can be used on different approaches e.g., on serological (i.e.

detection of target antigen at the surface of the bacterium) or molecular detection (i.e. detection of

specific DNA strands after loop-mediated isothermal amplification –LAMP- based amplification). Our technology could therefore offer a promising alternative to the currently used ELISA for large scale

monitoring of the pathogen and be an invaluable tool in the fight against XL.

Detection of Xylella fastidiosa in Coffea arabica ornamental plants 2.6.

Bergsma-Vlami, M.1, J.L.J. van de Bilt1, N.N.A. Tjou-Tam-Sin1, M. Westenberg1

National Plant Protection Organization (NPPO), P.O. Box. 9102, 6700 HC Wageningen, the Netherlands Presenting author: M. Bergsma-Vlami


Xylella fastidiosa is a xylem-limited, Gram-negative bacterium restricted until recently to the Americas.

It causes significant economic losses in many agriculturally important plants, including citrus, coffee, grape and several different tree species, perennial crops and landscape plants (EFSA, 2015). Outside

the American continent was X. fastidiosa previously reported in Taiwan and Iran. Since recently, is the bacterium present in Europe and is responsible for the Olive Quick Decline Syndrome (OQDS) of olive

trees in southern Italy and for the decline of the ornamental plant Polygala myrtifolia in France.

As a preventive measure against the introduction of this harmful organism in the EU, emergency measures were adapted providing guidelines for the import and movement of particular host plants.

Following the Commission Implementing Decision 2014/87/EU, an official survey for the presence of X. fastidiosa on coffee plants was undertaken in 2014 in the Netherlands, based on import data from

the past few years. X. fastidiosa was presumptively diagnosed on ornamental Coffea arabica plants

showing a) mild leaf scorch symptoms, b) a range of “crespera”-like symptoms and c) no symptoms. Total genomic DNA was extracted from leaf veins and petioles of symptomatic and latent infected C. arabica plants with the QuickPick™ SML Plant DNA kit (Bio-Nobile, Finland), using a KingFisher isolation robot (Thermo Scientific, The Netherlands). Real-time PCR gave positive reactions (Harper et

al., 2010, erratum 2013) and application of a conventional PCR (Minsavage et al., 1994) confirmed the presumptive presence of X. fastidiosa in these samples. Sequence analysis of the resulting

approximately 700 bp amplicon of the RNA polymerase sigma 70 factor showed 100%-96% identity

with the homologous genomic regions of X. fastidiosa strain sequences present in GenBank. In total, three different sequences with 97-98% identity among each other (GenBank asscession no:

KP769842-KP769844) were acquired from the imported C. arabica plants.

References:

EFSA PLH Panel (EFSA Panel on Plant Health), 2015. Scientific Opinion on the risks to plant health

posed by Xylella fastidiosa in the EU territory, with the identification and evaluation of risk reduction options. EFSA Journal 2015;13(1):3989, 262 pp., doi: 10.2903/j.efsa.2015.3989.




Harper SJ, Ward LI and Clover GRG, 2010 (erratum 2013). Development of LAMP and Real-time PCR methods for the rapid detection of Xylella fastidiosa for quarantine and field applications.

Phytopathology, 100, 1282–1288.

Minsavage GV, Thompson CM, Hopkins DL, Leite RMVBC and Stall RE, 1994. Development of a polymerase chain reaction protocol for detection of Xylella fastidiosa in plant tissue.

Phytopathology, 84, 456–461.

The sentinel nursery and plantation concept: an opportunity to 2.7.respond to the X. fastidiosa threat to Europe?

Vannini, A.1, S. Woodward2, R. Eschen3

1 DIBAF-University of Tuscia – Viterbo (Italy); 2 University of Aberdeen (Scotland); 3 CABI (Switzerland)


The temporal trend of IAS invasion in Europe reflects the history of trading living plants between

Europe and other countries. In this context it is necessary to develop strategies to describe and study

new potential plant pathogens and evaluate the risk they might pose to European flora before their introduction to the old continent. The Sentinel plantation concept seems to properly respond to this

need. According to this hypothesis, living plant collections cultivated in an exotic environment are exposed to the inoculum pressure of the native pathogens providing information on their

susceptibility. In a different perspective, the Sentinel nursery concept provide a new model of pathway inspection by monitoring in the area of origin a selection of the most traded plants to Europe

for latent and symptomatic infections. These 2 strategies can be adopted to prevent the movement

and spread of Xylella fastidiosa from outside and throughout EU. The primary assumption is that known host range of X. fastidiosa (over 150 species) might not be exhaustive of the potential host

range. The first approach, based on susceptible hosts, aims to identify relevant EU species, selected based on agronomic (even olive varieties) and/or ecological relevance, to be exposed to X. fastidiosa

inoculum and vectors in areas where the epidemic is ongoing or where the pathogen is known to be

present. The second approach targets the pathway instead of the known hosts. Assuming that plant trade (horticulture and ornamentals) poses the main risk for X. fastidiosa spread, protocols should be

established for rapid inspection of the pathways to EU from areas where the epidemic is ongoing or where the pathogen is known to be present.

Developing guidelines for a possible strategy for the surveillance of 2.8.Xylella fastidiosa in Europe

Stefani, E.1

1 Department of Life Sciences, University of Modena and Reggio Emilia, Italy


According to the International Standards on Phytosanitary Measures (ISPM 5), a general surveillance is a process whereby information on particular pests, which are of concern for an area, is gathered

from many sources, wherever it is available and provided for use by the NPPO. Designing a specific survey for Xylella fastidiosa a justification is needed, in order to get approval by the NPPOs and in

order to give others specialists the chance to design an equivalent survey plan in another endangered

area. After defining the reason for a survey, i.e. early detection at Border Inspection Posts (BIPs) or monitor pest eradication, a robust strategy is chosen, aimed to get any useful information on the

pathogen and its vectors, helping decision making of the phytosanitary authorities. Harmonisation of plans and methods applied for surveys throughout the European Union will ensure the choice of

equivalent strategies, therefore preventing controversies related to X. fastidiosa introduction, spread





and possible establishment into new areas. The identification of sites to be surveyed is tightly connected with approval by NPPOs and should seek compliance of stakeholders and “understanding”

of citizens through a correct communication on risks posed by the pathogen. Due to the vast diversity

of EU regions, agricultural and natural areas, commercial networks, trade commodities, pathways, NPPOs and RPPOs organizations, pilot studies are urgently needed for analysing possible problems

encountered during planned survey activities and for a correct identification of nodes inside a general surveillance network. Mapping the relational network that spreads X. fastidiosa along trade flows

(extra et intra comunitatis) by surveys of BIPs, nursery sites, wholesale and retail markets, will greatly

help to identify hubs, where inspecting activity and phytosanitary checks should be implemented and intensified, and will assist in predicting risks and defining endangered areas. Therefore, NPPOs might

be timely alerted and, in case, are in a position to manage possible disease outbreaks.

The EU Horizon 2020 POnTE project: the work plan on X. fastidiosa 2.9.surveillance

López, M.M.1, J. Blasco1, J.A. Navas-Cortés2

1 Instituto Valenciano de Investigaciones Agrarias (IVIA). Moncada, Valencia, Spain 2 Instituto de Agricultura Sostenible (IAS). CSIC. Córdoba, Spain


The objectives of the work plan are the development of novel and high-throughput diagnostic

procedures for Xylella fastidiosa monitoring and surveillance programmes. Two Working Packages (WP4 and WP 6) and 17 partners are concerned. In WP 4 “Development and validation of rapid and

automated diagnostic assays for the sensitive and specific detection of the CoDiRO strain and all the X. fastidiosa subspecies” the production of new serological reagents, the design of new primers and

probes for improved and specific detection in host plants and vectors will be undertaken. Specifically,

High Resolution Melting (HRM) and TaqMan-based approaches will be used to target specific genomic regions of X. fastidiosa. Developing imprint –based extraction protocols (tissue spot and insect

squash) to capture the templates suitable either for serological or molecular assays, introducing automated steps for processing large number of samples and developing rapid tests based on

DNA:DNA hybridization using specific non-radioactive labelled oligoprobes will be also approached. The optimized procedures and reagents will be validated. Interlaboratory-tests or performance studies

will be organized among the different laboratories of the consortium and diagnostic parameters will be

calculated. In WP 6 ”Field and surveillance systems for disease monitoring” the objectives will be to determine the spatio-temporal dynamics of X. fastidiosa infections under natural field conditions, to

develop quantitative remote sensing methods for the early detection and monitoring of X. fastidiosa infection based on canopy-level high-resolution airborne hyperspectral and thermal imaging acquired

with aerial vehicles. They will be validated with visual inspection and handheld instruments for leaf-

level measurements, and validate the remote sensing devices with the end-user partners.

3. Abstracts of session 2. The Vectors: identity, biology, epidemiology and control

Overview of current knowledge on insect vectors/potential vectors 3.1.of Xylella fastidiosa in Europe

Bosco, D.1

1 Dipartimento di Scienze Agrarie, Forestali e Alimentari, Università degli Studi di Torino, Italy and Istituto di Protezione Sostenibile delle Piante, CNR, Torino, Italy


Xylella fastidiosa is a xylem-limited bacterium transmitted by xylem-sap feeding insects belonging to

the order Hemiptera, sub-order Cicadomorpha. Within xylem-sap feeders there is no species-




specificity for transmission and therefore all insects sharing this feeding behaviour are considered potential vectors.

Tens of vector species have been identified in North and South America, the original area of

distribution of X. fastidiosa, and the biology, ecology and pathogen-vector relationships of the main vector species have been characterized. Unfortunately, there is an important gap in the knowledge of

the European vector/potential vector species. We know there is a striking difference in the fauna of xylem-sap feeding insects between the New World and Europe. In particular, while in North and South

America there are many sharpshooters species, few species are present in Europe. Actually, out of

nine sharpshooter species recorded in the Fauna Europaea database only one, Cicadella viridis, is widespread and common, though mostly restricted to hygrophilous environments. On the contrary,

thirty six spittlebug species, are present in Europe and some of them are very common and widespread. Among these, the “meadow spittlebug” Philaenus spumarius, already identified as a

vector of X. fastidiosa, is very common and abundant in diverse ecosystems, and feeds on mono- and dicotyledonous grasses, on trees and shrubs. Until the recent introduction of X. fastidiosa in Europe,

very few spittlebugs were considered plant pests and therefore data on their biology, phenology,

ecology and control are missing. Among the European xylem-sap feeding insects, cicadas are represented by tens of species, represented mainly in the Mediterranean area. Some species, like

Cicada orni are common and locally abundant on olive trees.

The main priorities in X. fastidiosa vector studies in Europe are discussed.

How do vector numbers, movements, infectivity and transmission 3.2.efficiency impact the incidence and spatial patterns of plant infection with X. fastidiosa?

Purcell, A.1

1 Department of Environmental Science, Policy and Management University of California, Berkeley, CA 94720-3114


A simple probability model (Phytopathology 71(4): 429-435) for infection of a plant with Xylella fastidiosa transmitted by insect vectors is predicted on the numbers of infectious (capable of transmitting) vectors present and transmitting. Based on the Poisson distribution of zero (where 0 =

no transmission), the probability of transmission is Pnt = 1-e-niEt, where n = number of vector insects, i = infectivity (the fraction between 0 and 1 that transmit during a given time period), E = efficiency of

transmission (between 0 to 1), and t = time, which can be described an “average visit” to the plant by

a vector. Thus nt represents an “average vector visit”. The spatial distribution of nt is normally “clumpily” rather than randomly distributed, but can be estimated by adjusting the predicted

probability of randomly distributed infections or using another distribution equation. Both plant size and age are key factors because nt per plant increases with plant size and age, and varies with date

depending on seasonal abundance and behavior of vectors. E describes the fraction of infective

vectors that transmit per visit, varying with the vector species and plant species involved. The fraction of vectors that are infectious (i) is a function of the availability and degree of colonization of plants

already infected by X. fastidiosa. A graphic representation of Pnt as a function of nt at various values of iE illustrates the impact of i on transmission pressure. Mathematically i and n are of equivalent

value in predicting infections. This model shows that

1. Even very conservative (low) estimates of insect numbers, mobility, transmission efficiency

and infectivity predict substantial infection for large, long-lived plants.

2. The combination of simultaneously lowering i (removing diseased plants) and n (vector control) has a multiplier effect in reducing infection.

3. The decrease in the probability of infection with decreases in n, I, E, or t is nonlinear. Even large decreases in any of these inputs when infection probability is high may only reduce

infection likelihood slightly, even imperceptibly.




The diseases resulting from infection may be regulated by many subsequent factors such as temperature, plant resistance, and antagonistic microbes.

Figure 1: Probability of infection (Pnt) by n vectors after 1 days of different probabilities of single

vector transmission (iE). For equal numbers of vectors per plant (binomial distribution, solid line), Pnt = 1-(1-iE)nt . For vectors randomly distributed among plants (Poisson

distribution, broken line), Pnt = 1-e-niEt. (Figure adapted from Purcell AH, 1981. Vector preference and inoculation efficiency as components of varietal resistance to Pierce's

disease in European grapes. Phytopathology 71, 429-435).

Ecological and behavioural traits that determine vector relevance 3.3.

Lopes, J.R.S.1

1 ESALQ/University of São Paulo, Brazil.


Xylella fastidiosa pathosystems are usually complex, with multiple host plants and vector species, and

genetic variation in the pathogen. Determining what vector species are relevant for pathogen spread is basic to establish disease management strategies. Vector relevance depends not only on ability to

transmit the pathogen, but also on ecological and behavioural characteristics in consonance with other epidemiological components of the disease, such as source plants, pathogen dynamics in these hosts,

and favourable environmental conditions. Citrus variegates chlorosis (CVC), caused by X. fastidiosa

subsp. pauca in the State of São Paulo, Brazil, is an example of complex pathosystem, in which many vector and potential vector species exist, including around 20 species of sharpshooter leafhoppers

(Hemiptera: Cicadellidae: Cicadellinae) and spittlebugs (Hemiptera: Cercopidae). In transmission experiments, 13 out of 17 sharpshooters were able to transmit the pathogen to citrus. Transmission

efficiency is low, but variable among vector species. Some sharpshooter species are naturally infective

with the pathogen, showing that they visit inoculum sources. Because citrus is a major source of inoculum and secondary spread occurs in citrus orchards, vector species that frequently visit the tree

canopy and feed or reproduce on citrus are considered epidemiologically important. Among them, Acrogonia citrina, Bucephalogonia xanthophis, Dilobopterus costalimai and Oncometopia facialis, are

commonly found and reproduce on citrus during the rainy season, when X. fastidiosa infection and

multiplication are favored in citrus. They are also common in diverse habitats and host plants, including other crops susceptible to X. fastidiosa. Thus, they offer opportunities for X. fastidiosa to

colonize other hosts and build natural reservoirs outside the orchards Some sharpshooter vectors, e.g. Ferrariana trivittata, Plesiommata corniculata and Sonesimia grossa, are very abundant on the weedy

vegetation of citrus orchards, but much less frequent on citrus trees. Other vectors (A. virescens and Homalodisca ignorata) are associated with wild trees and restricted to citrus orchards adjacent to

woody vegetation. Although some weed species present in the ground cover of citrus orchards are

mailto:%[email protected]

mailto:%[email protected]



hosts of X. fastidiosa, their role as inoculum sources are still unclear. Feeding behavior can also impact vector competence. The blue-green sharpshooter, Graphocephala atropunctata, is the most

efficient vector of X. fastidiosa subsp. fastidiosa in grape in California. However, in alfalfa the green-

sharpshooter, Draeculacephala minerva transmits this pathogen more efficiently, probably because of its feeding preference for the basal portions of alfalfa stems, where the bacterium reach higher

populations.

Acknowledgments:

Research supported by FAPESP, Fundecitrus, FINEP and CNPq, Brazil.

Survey of potential vectors of Xylella fastidiosa in olives groves in 3.4.southern Spain

Fereres, A.1

1 Instituto de Ciencias Agrarias. Madrid. CSIC.


A new and destructive disease caused by Xylella fastidiosa was reported in olives in southern Italy in

2013. Because of climate similarities, other Mediterranean countries that grow olives in large areas

e.g. Spain, Greece and Portugal are at risk of pathogen introduction and spread if competent vectors are present in their olive orchards. The species Philaenus spumarius seems to be the most important

insect vector associated to the epidemics reported in Italy. However, other xylem sap-sucking auchenorrhyncans such as sharpshooters (Hemiptera: Cicadellidae: Cicadellinae), spittlebugs

(Hemiptera: Cercopoidea) and cicadas (Hemiptera: Cicadoidea) may also be involved.

The goal of our survey is to identify hemipteran insects associated to the major olive-growing regions

of Spain in order to investigate the presence of potential vectors of X. fastidiosa and assess risks for

disease establishment and spread.

The "vectors" aspects of the Belgian Xyleris research project 3.5.

Jean-Claude Grégoire1, Martine Maes2, Johan Van Vaerenbergh2 and Claude Bragard3

1 ULB - Université Libre de Bruxelles 2 ILVO - Institute for Agricultural and Fisheries Research, Merelbeke 3 UCL - Université Catholique de Louvain, Louvain-la-Neuve


The Xyleris research project, financed by the Belgian Federal Service Health, Food Chain Safety and Environment, will start in early 2016. It includes three components: a) assessing the disease

susceptibility of a set of plant species that could be at risk in Belgium and their suitability as sentinel plants for use here as well as in regions with disease outbreaks; b) Testing environmental factors such

as temperature and plant fertilization as possible inducers of disease; c) Assessing the presence of possible insect vectors in Belgium, their life cycle and their capacity to transmit the bacterium.

This presentation will develop the third component, which includes two parts.

1. A survey of potential vectors will be carried out, based on a systematic sampling protocol aimed at five of the species outlined as potential vectors in the EFSA Opinion and present in Belgium: Cercopis vulnerata; Cicadella viridis; Philaenus spumarius; Aphrophora alni; Aphrophora salicina. The project also includes a search for X. fastidiosa in these potential vectors, as well as an analysis of their

phenology and life history traits.

2. The transmission of X. fastidiosa to four model plant species (Prunus, Salix, Quercus and Vitis) by potential insect vector species common in Belgium will be tested under G2Q controlled conditions.

This will involve attempts at contaminating potential vectors using infected periwinkle and, later on,





attempts to contaminate model host plants using the artificially contaminated vector insects, as well as the detection and quantification of X. fastidiosa in the insect vectors. Another part of the project

will be to establish a “Sentinel nursery” in Puglia, with Belgian varieties of Prunus, Salix, Quercus and

Vitis.

Options for control of meadow spittlebug (Philaenus spumarius, L.) 3.6.in organic farming: overview of available active substances

Verrastro, V.1

1 CIHEAM – IAMB, Italy / IFOAM EU


The Xylella fastidiosa outbreaks on olive orchards in Salento Area and the consequence extraordinary

measures for her eradication/control are potentially dangerous also for a certain number of organic olive orchards. At the emanation of the first “Silletti Plan” (March 2015), no active substances were

authorised and registered in Italy organic agriculture against the vector Philaenus spumarius L. (Meadow spittlebug). The present work reports the active substances available in the market, duly

registered by the competent authorities and authorised for use in organic agriculture that potentially

can have a positive effect for the vector control and the setting up of an “organic strategy” for vector control in Salento Area giving priority to the application of preventive techniques, allowed substances

and any other technical means in compliance with EU, Italian and regional norms about organic agriculture. The strategy of organic olive growers is in full compliance with the organic production

method, with special regard to farmers with olive orchards directly interested and also to farms at drift risk, establish the necessity of vector control in correspondence of key stages of its

cycle/development, which are independent from the production cycle of the olive orchards and, finally

indicate actions whose efficacy could be easily monitored, allowing to collect on-field information, that can be used to better steer the actions to contain the bacteria in the years to come.

Outline of EFSA outsourced project to collect data on biology and 3.7.control of vectors of Xylella fastidiosa

Bertin, S.1,2

1 Dipartimento di Scienze Agrarie, Forestali e Alimentari, Università degli Studi di Torino, Italy 2 Istituto per la Protezione Sostenibile delle Piante, CNR, Torino, Italy


The EFSA project aims to improve the current knowledge on the biology and control of vectors and

potential vectors of Xylella fastidiosa in the EU, with emphasis on Philaenus spumarius. The project relies on literature and databases searches, and on two-year observations on the phenology of P. spumarius under field and semi-field conditions.

The literature search will include both peer-reviewed scientific publications and grey literature, and will provide summary files categorizing all the relevant information about vector biology and phenology.

The same searching method will be applied to evaluate the procedures suitable for vector control in conventional and organic farming agriculture, without geographical limitations. Good Agriculture

Practices and Integrated Pest Management programmes implemented in the UE Mediterranean countries will be identified and listed as well, in order to properly place the control measures against

X. fastidiosa vectors into the agriculture practices already applied for the protection of stone fruits,

citrus, grapevine and olive. The data will be gathered by contacting Ministry of agriculture, national phytosanitary services, IPM-related organizations, associations of producers and consumers, and

public and private organisations including cooperatives and NGO. Information will be obtained either by mailing the responsible officers (active approach) or by purposely-set questionnaires (passive

approach).





The field surveys will take place in four olive orchards in Salento area and Liguria region (Italy), representing different agro-climatic conditions. Host plants, time of egg hatching and first nymph

appearance, and emergence, abundance and persistence of adults on herbaceous plants and olive

canopies will be recorded for P. spumarius. The presence of other potential vector species will be also surveyed. More detailed data on P. spumarius phenology will be obtained in semi-field experiments

carried out in mesocosms, large cages equipped with data loggers for air temperature and humidity control, and hosting herbaceous plants together with potted young olives. All the field and semi-field

data will be analysed by means of proper statistical and simulation methods.

A final inventory table combining the data from literature, reports and field observations will be provided to EFSA as a tool for designing rational strategies of vector control.

The EU Horizon 2020 POnTE project: the work plan on Xylella 3.8.fastidiosa vectors

Porcelli, F.1 and D. Bosco2

1 Dipartimento di Scienze del Suolo, della Pianta e degli Alimenti, Università degli Studi di Bari Aldo Moro, Italy and Istituto di Protezione Sostenibile delle Piante, U.O. Bari, CNR, Italy 2 Dipartimento di Scienze Agrarie, Forestali e Alimentari, Università degli Studi di Torino, Italy and Istituto di Protezione Sostenibile delle Piante, Torino, CNR, Italy


Within the frame of the POnTE project on organisms threatening Europe, several activities are

scheduled with the aim of clarifying insect transmission and epidemiology of Xylella fastidiosa, with emphasis on the olive disease spread.

Main activities are devoted to the identification of the vector species, thus elucidating the role, the

biology/ecology/phenology of the most common xylem sap feeding insect populations in the X. fastidiosa contaminated area. The ability of putative vectors to acquire X. fastidiosa from infected olive

or other susceptible hosts and to transmit to olives or other susceptible hosts is under investigation.

Xylem-fluid feeders are collected with different methods and the presence of X. fastidiosa in the

insects determined by PCR assay. Insect species harbouring the bacterium are prioritized for X. fastidiosa transmission tests. Transmission experiments will be performed with xylem-feeding insects either field collected or reared in the laboratory under controlled conditions on experimental hosts. For

field-collected putative vectors, insects will be collected from contaminated groves and caged on receptor host plants (olives and periwinkles). Observations in the X. fastidiosa contaminated areas will

include collection of adult cicadas that will be caged on test plants following the same procedure. For controlled transmission experiments, X. fastidiosa-free insects (reared on X. fastidiosa-free host plants

or collected in X. fastidiosa-free areas) will be caged on infected olives, oleander or periwinkle and

then moved onto olive and periwinkle test plants. A mark–release–recapture study to measure dispersion capability of candidate vectors will be performed by treating adults with a protein marker,

followed by released in the field and re-capture with yellow sticky traps. ELISA will be used to identify marked insects re-captured at different distances from the release point. The xylem-sap-feeder

populations will also be investigated on different crops in infected and non-infected areas with high

risk of X. fastidiosa epidemics (Mediterranean area). Some activities also aim at providing preliminary indications for the management/suppression of vector populations, namely on the usefulness of the

vegetation management in suppressing nymph populations.




4. Abstracts of session 3. The Plants: host range, breeding, resistance and certification

Lessons learned with CVC and X. fastidiosa subsp. pauca in Brazil: 4.1.host range of bacterium, breeding for resistance and citrus nursery certification programme

Della Coletta Filho, H.1

Centro de Citricultura Sylvio Moreia – IAC, Cordeiropolis, SP, Brazil


The bacterium Xylella fastidiosa subsp. pauca causes problem for two economically important crops in

Brazil, coffee (Coffea sp.) and citrus (Citrus sp). But recently, others commercial plant species like the ornamental hibiscus (Hibiscus rosa-sinensis), plum (Prunus domestica), and olive (Olea europaea)

were also found as hosts of different strains of this bacterium. Even genetically close the X. fastidiosa pauca from coffee and citrus do not cause disease in its non-reciprocal host. Within the genus citrus

X. fastidiosa subsp. pauca causes the Citrus Variegated Chlorosis (CVC), a disease that affects all

commercial sweet orange varieties (C. sinensis) and is widely spread in Brazil resulting in significant economic impact in sweet orange production. Most of mandarin varieties (C. reticulata) and tangors

(C. sinensis x C. reticulata) are resistant which make them important sources of resistance to be explored in breeding programmes. In our citrus research center “Centro de Citricultura, IAC”, a

breeding programme using “Pera” sweet orange and tangor “Murcott” as parents was started in

1990’s producing CVC resistant hybrids that have been released to growers. In addition, for the CVC pathoystem, i. 13 different species of xylem-sap feeding sharpshooter leafhoppers (Hemiptera,

Cicadellidae) are known as vectors; ii. vertical transmission (rate of 20% efficiency) on budding even using buds taken from asymptomatic X. fastidiosa subsp. pauca infected branches; iii. latency period

of disease ranging from around 6 months to years, which seems to be directly correlated with warm temperature and water stress; iv. both primary and secondary forms of vector-borne bacterial

transmission are important for the disease spread in the field. All these information supported

scientific and technical decisions that made mandatory the Citrus Nursery Certification Programme (CNCP) in Sao Paulo State, Brazil, since January 2003. In general, this programme states the

requirements for the citrus nursery structure, propagation, growth and storage of vegetative materials free of X. fastidiosa and others pathogens with all the steps being conducted within vector-proof

screen-houses.

A comprehensive database on Xylella fastidiosa host plants 4.2.

Gardi, C.1, Czwienczek E. 1, Koufakis I. 1, Andueza M. 1, Hollo G. 1, Inverardi I. 1, Kertesz V. 1, Kozelska

S. 1, Mosbach-Schulz O. 1, Oszako T. 1, Pautasso M. 1, Tramontini S. 1, Vos S. 1, Stancanelli G. 1

1 European Food Safety Authority (EFSA), Animal and Plant Health (Alpha), via Carlo Magno, 1, 43100 Parma (Italy)


After the detection of Xylellla fastidiosa in Apulia in October 2013, EFSA was requested to produce a scientific opinion, supported by a database of the host plants for this pest.

A first review of the scientific literature was carried out during 2014 and more than 200 papers and

abstracts were collected. Data extracted allows identifying 312 host species, of X. fastidiosa, belonging to 75 plant families. In order to have a more comprehensive characterization of the host plants

several additional data were also extracted, such as the origin of the data (survey or experiment), the location and the geographical coordinates, the X. fastidiosa subspecies, the detection methods, the

presence of symptoms.





In 2015, based on a request of the European Commission, EFSA started the update of the database, by doing a second systematic literature review. This second review, using the same protocol and

searching criteria for the previous one, was realized using the Distiller SR platform. After removal of

duplicates, 337 paper were screened at first level (title/abstract screening), and 261 paper were retained for the second level screening (full text).

A first X. fastidiosa host plant database, in form of Excel file, has already made available (published on EFSA web site) and the updated version will be available at the beginning of 2016.

Rootstock Effects on Almond Leaf Scorch Disease Incidence and 4.3.Severity

Krugner R. 1 and C.A. Ledbetter1

1 USDA-ARS, San Joaquin Valley Agricultural Sciences Center, Parlier, California, U.S.A. 93648.


A five-year field study was conducted to evaluate effects of duration and exclusion of Xylella fastidiosa infections on young almond tree performance and their links to tree vigor. ‘Nemaguard’, ‘Okinawa’,

‘Nonpareil’, and Y119 were used as rootstocks for almond scion ‘Sonora’. Among X. fastidiosa-infected

trees there was significant etiological heterogeneity with 1) absence of leaf scorching symptoms in the presence of reduced growth, 2) presence of leaf scorching symptoms in the absence of reduced

growth, and 3) severe leaf scorching and reduced growth. Trunk cross sectional areas of X. fastidiosa-infected trees grafted on ‘Nemaguard’ and ‘Nonpareil’ rootstocks were significantly smaller than non-

infected trees, whereas trunk size of trees grafted on ‘Okinawa’ and Y119 was not affected by infection status. Severity of leaf scorching symptoms was highest on trees grafted on ‘Nonpareil’

rootstock, intermediate on ‘Okinawa’ and Y119, and lowest on ‘Nemaguard’. Xylella fastidiosa

infections and seasonal leaf scorching symptoms persisted on most inoculated trees throughout the study, except on trees grafted on ‘Nemaguard’ that manifested complete leaf scorching symptom

remission and apparent elimination of the pathogen after the second year. Results indicate that depending on rootstock type X. fastidiosa can affect trunk size in a relatively short period and/or

persist for years as trees grow.

Preliminary results of a pilot project on host range of X. fastidiosa 4.4.CoDiRO strain and evidences of resistance phenomena in the cv. Leccino upon the infection of X. fastidiosa

Boscia, D.1 and M. Saponari1

CNR - Institute for Sustainable Plant Protection, Unit of Bari


The “Pilot project on Xylella fastidiosa to reduce risk assessment uncertainties”

(NP/EFSA/ALPHA/2014/07) is presented, and its preliminary results are reported. The Project is focused on three objectives: 1) Evaluation of the host range of the X. fastidiosa CoDiRO strain; 2)

Development of an experimental research plan for a future comprehensive investigations on the full host range of the CoDiRO strain of X. fastidiosa; 3) Field observations and laboratory tests in support

of a pilot analysis of the spread patterns of X. fastidiosa in Apulia (with JRC and NERC). Preliminary

results, mainly based on artificial inoculations and exposure to natural infections of several plant species, indicate that Olive and Oleander appear to be the most susceptible hosts of the CoDiRO

strain, while in Grapes and Oak no systemic movement of the bacteria has been detected so far. In the case of Citrus, it seems that there is an initial establishment of the bacterial infection, however it

seems to remain confined as the plants grow. Artificial inoculations also showed different degrees of

susceptibility among Olive cultivars. These latter finding is also in agreement with the field observation of phenomena of resistance in the cv. Leccino upon the infection of X. fastidiosa. Clues to resistance





are i) the assessment of the bacterial titre, much lower in Leccino than in the severely affected cv. Ogliarola (unpublished), and ii) on the same direction go studies of transcriptome analysis, indicating

that the presence of X. fastidiosa induce a different response of gene expression in the two cultivars

which is different from the healthy plants (P. Saldarelli and A. Giampetruzzi, unpublished).

The World Olive Collection of Córdoba 4.5.

Belaj, A.1

1 IFAPA Centre “Alameda del Obispo”, Córdoba, Spain


Germplasm collections are basic tools for conservation, characterization and efficient use of olive

genetic resources. In this sense, the establishment in the 70s of the World Olive Collection of

Córdoba, Spain, represented the first international attempt of conservation and management of the olive germplasm. Nowadays, this Collection which belongs to the World Olive Germplasm Bank (CAP-

UCO.IFAPA) counts around 900 accessions from 25 countries, most of them from Mediterranean Basin. The olive cultivars maintained in the World Olive Collection have systematically been evaluated

for several pomological traits related to vigour, phenology, production, fruit and oil quality and for their resistance or susceptibility to Verticillium dahliae Kleb and other diseases and pests. All the

above mentioned evaluations have also revealed high levels of variability among the olive cultivars.

The results of such evaluations are very important to determine the most interesting cultivars for their use as potential parents in future crosses of the olive breeding programme as well as for the

establishment of comparative trials in different agro-climatic conditions. In addition, the olive cultivars could be a very useful source of diversity against new and adverse climatic changes as well as new

pest and diseases, like the case of Xylella fastidiosa. The activities carried out in the World Olive

Collection of Córdoba are fruit of collaboration between different research teams and scientific institutions.

Greek olive germplasm breeding and certification research 4.6.priorities on X. fastidiosa

Koubouris, G.C.1

1 Institute of Olive Tree, Subtropical Crops & Viticulture, Hellenic Agricultural Organization “Demeter”, Laboratory of Olive Cultivation, Agrokipion, 73100, Chania, Greece


Xylella fastidiosa has not been detected in Greece so far. The Ministry of Rural Development and Food coordinates preventive and survey actions. In parallel, immediate planning of research to cover

knowledge gaps is needed. Research to transfer know-how from Verticillium Wilt (Calderon et al., 2015, Remote Sens. 7, 5584-5610) to scan large areas and early detect Xylella-infected plants through

remote sensing (unmanned aerial vehicles) is considered as top priority. Additionally, research to develop low-cost, rapid and highly accurate e.g. molecular, chemical or spectral analysis of massive

samples of plants or insects is proposed. Finally, survey would be facilitated through the development

of a network of traps and automatization of monitoring of vector insect species and populations. The development of an efficient system for plant health testing of imported plants as well as the domestic

production of certified nursery material that is true to type and plant healthy, through the maintenance of mother plants in insect-proof facilities and implementation of traceability in plant

propagation is needed. For long term research planning, breeding for plant resistance through the

cooperation of International Olive Council network of germplasm banks, marker-assisted selection and pathogenicity tests would be suggested with caution to avoid spreading the disease in the countries

free from X. fastidiosa. Last but not least, research should focus on management of sustainable agro-ecosystems and identification of good agricultural practices for improved plant resilience, soil organic





matter management and biodiversity in a whole systems approach as applied in the case of the LIFE Oliveclima project.

The EU Horizon 2020 POnTE project: the work plan on host plants 4.7.of X. fastidiosa

Boscia D.1 and the POnTE-Xylella fastidiosa associate partners1,2,3,4

1 Consiglio Nazionale delle Ricerche (CNR), Istituto per la Protezione Sostenibile delle Piante, UOS Bari, Italy 2 Università degli Studi Aldo Moro (UNIBA) - Dipartimento di Scienze del Suolo, della Pianta e degli Alimenti, Italy 3 Institut National de la Recherche Agronomique (INRA), France 4 Agence Nationale de Securite Sanitaire de l'Alimentation, de l'Environnement et du Travail - Laboratoire de la Santé des Végétaux, France


In the framework of the POnTE research project, major efforts are devoted to gain knowledge about

the host range of the novel X. fastidiosa genotype infecting olive in southern Italy. Two Work

Packages (WP2 and WP 7), involving 4 different partners, will deal with the investigations on the hosts of X. fastidiosa. In WP 2 “Isolation, biology and pathogenicity of Xylella fastidiosa” the following

activities will be performed:

1. Artificial inoculations of a set of different olive cultivars and plant species (stone fruits, oleander,

citrus, grapes, Quercus spp. etc.), with the aim to understand a) the role of the CoDiRO strain in the OQDS; b) its pathogenicity on the currently ascertained natural hosts (almond, cherry,

oleander, etc.); c) its capability to colonize relevant crop and forest species (i.e. citrus, grapes,

Prunus spp., Quercus spp., etc.). Alongside with greenhouse pathogenicity tests, experiments will be conducted in selected experimental plots within the contaminated area;

2. Evaluation of weeds as alternative hosts; to this purpose a survey of the natural flora existing in selected olive groves (different soil conditions, different cultivation practices, etc.) will be

continued through a 2-year period, with the aim to understand as weeds can serve as source of

inoculum of the bacterium or alternative hosts for the vector populations.

In WP 7 “Signaling pathway, molecules and genes contributing to pest resistance in field” one task

(7c) will be focused on the screening of olive germplasm for X. fastidiosa resistance. This activity will include trials in open fields and experiments in confined conditions: i) an olive grove consisting of

different varieties which display different disease phenotypes has been already identified and will be used for this purpose; ii) an olive plot will be realized in the X. fastidiosa contaminated area, by

planting grafted olive plants belonging to at least 20 different varieties; iii) olive plants grown under

glasshouse condition and artificially inoculated with the bacterial suspension of by side-grafting infected cuttings. In all experiments plants will be inspected for symptoms and to quantitative

detection assays (qPCR). Differential gene expression analysis will be conducted on the most interesting varieties to determine the genetics and mechanisms of the tolerance/resistance.

More information and the latest updates on the research programme can be found at:

www.ponteproject.eu.

5. Abstracts of session 4. The Pathogen: biology, genetics and control

Can comparative genomics yield insights to control diseases caused 5.1.by X. fastidiosa?

Jacques, M.A.1

1 UMR1345 IRHS – EMERSYS - INRA - Centre Angers-Nantes, 42 rue Georges Morel – CS 60057, 49071 Beaucouzé cedex – France.





Xylella fastidiosa is a xylem-limited bacterium endemic to the Americas that has a broad host range

including grapevine, citrus, almond, and peach trees. The recent emergence of this phytopathogenic bacteria in Asia and Europe combined with its infection of new host species make X. fastidiosa a

serious threat for European agriculture. X. fastidiosa is a genetically diverse species whose members

are prone to recombination generating variants with novel pathogenic properties. Advances in genomics have increased our capacities to detect, type and track X. fastidiosa strains. Moreover

availability of genome sequences have provided insights into the mechanisms involved in interactions of X. fastidiosa with plants and insects. Nevertheless, control methods to fight this bacterial pathogen

are still limited and antiquated. Can the most recent next generation sequencing technologies and other omics methodologies help us to (i) improve the epidemiological survey of this pathogen, (ii) predict novel plant host(s), and ultimately (iii) avoid symptom development and X. fastidiosa

transmission by insect vectors? While the answers to these questions are undoubtedly positive, there are still obvious gaps in our knowledge on X. fastidiosa that restrict such developments. There are

mostly due to a yet unrecognized diversity of these bacteria and underrated extent of the worldwide current distribution of X. fastidiosa and its relatives.

Genetic diversity of the CoDiRO strain and other EU intercepted 5.2.isolates

Saponari, M.1, G. Loconsole1, A. Giampetruzzi1, M. Chiumenti1, D. Boscia1, P. Saldarelli1, G. D’Attoma1,2, M. Morelli1, G.P. Martelli2 and R.P.P. Almeida3

1 Istituto per la Protezione Sostenibile delle Piante, UOS Bari, CNR 2 Dipartimento di Scienze del Suolo, della Pianta e degli Alimenti, Università degli Studi di Bari Aldo Moro 3 Department of Environmental Science, Policy and Management, University of California, Berkeley


Due to the threat of X. fastidiosa to agricultural and environment, the European Union (EU) has implemented quarantine regulations and established inspections and controls at the ports of entry. As

a result, a large number of Xylella-infected stocks of propagating plant materials was intercepted,

mainly in The Netherlands, consisting of ornamental coffee plants (Coffea arabica) originating from Central America. Although some of the described coffee-infecting Xylella strains belong to X. fastidiosa

subspecies pauca, coffee plants are also susceptible to X. fastidiosa subsp. fastidiosa strains. Therefore, the importation of coffee plants from the Americas represents a threat to EU agriculture as

potential reservoir of genetically and biologically different X. fastidiosa strains. Similarly, the geographic expansion of the olive decline epidemic area of the Apulia region (southern Italy)

prompted investigations to identify new host plants. Here we report the interception of three novel

bacterial sequence types, based on multi-locus sequence typing, that cluster with different X. fastidiosa subspecies, illustrating the risk of the introduction of additional pathogen genetic diversity

into Europe. Conversely, in the epidemic area of Apulia, new foci as well as host plant species positive with X. fastidiosa were all infected with the same sequence type of this bacterium (ST53, or CoDiRO

strain). This work highlights the limited knowledge of X. fastidiosa phylogenetic and phenotypic

diversity, the risk of novel X. fastidiosa introductions via contaminated plant material, and corroborates other studies indicating that the Apulia epidemic emerged from a single introduction of

this pathogen into the region.

Such activities led to identify several infected coffee plants in the consignments originating from

Central America (Costa Rica and Honduras), and to detect several infected ornamental plants in

Corsica. Indeed, the rapid expansion of the infections in the epidemic area of the Apulia region (southern Italy) prompt investigations to determine the origin of the numerous foci now reported in

the area.





In this work, MLST analysis was used to determine the genetic relatedness of the isolates recently intercepted and isolated in the EU territory.

Sequence and phylogenetic analysis conducted on the different bacterial sources collected in the

epidemic area of Apulia (southern Italy) demonstrated they all share the same sequence type profile “ST53” as previously identified in the initial outbreak associated to the CoDiRO strain.

Similarly, the geographic expansion of the olive decline epidemic area of the Apulia region (southern Italy) prompted investigations to identify new host plants. Here we report the interception of three

novel bacterial sequence types, based on multi-locus sequence typing, that cluster with different X. fastidiosa subspecies, illustrating the risk of the introduction of additional pathogen genetic diversity into Europe. In the epidemic area of Apulia, new foci as well as host plant species positive with X. fastidiosa were all infected with the same sequence type of this bacterium (ST53, or CoDiRO strain). This work highlights the limited knowledge of X. fastidiosa phylogenetic and phenotypic diversity, the

risk of novel X. fastidiosa introductions via contaminated plant material, and corroborates other studies indicating that the Apulia epidemic emerged from a single introduction of this pathogen into

the region.

Improved detection of X. fastidiosa and the potential of rapid whole 5.3.genome sequencing for outbreak delineation and pathogenicity profiling

Dreo, T.1

1 National Institute of Biology (NIB), Department of Biotechnology and Systems Biology, Slovenia


Developing and optimizing methods and testing plants and vectors for X. fastidiosa are part of our official diagnostic activities since 2007. Real-time PCR is our method of choice testing grapevine,

oleander, olives, ornamental and other plants, and insects. Current activities include evaluation of

methods with the potential to address the issue of X. fastidiosa detection in low concentrations in particular in vectors using two different methods: (i) isothermal real-time LAMP, suitable for on-site

application and (ii) digital PCR, a sensitive method, resistant to inhibitors which is also suitable as a reference method in protocol harmonization, test performance studies and preparation of defined

reference material. The on-site LAMP was demonstrated to have 91% diagnostic sensitivity and

provides results in < 20 min which makes it a useful test for symptomatic samples where bacterial concentration is not a limiting factor. The digital PCR has been used for characterization of in-house

reference samples, normalization in comparative studies and as a detection method for low X. fastidiosa concentrations in difficult samples. Among further research gaps, rapid whole genome

sequencing is an important future priority. As for food disease outbreaks, X. fastidiosa outbreaks require rapid response. With the decreasing costs of sequencing microbial typing through sequencing

whole genomes is becoming a viable routine alternative to other techniques as demonstrated for

EHEC104 but necessitates highly optimized and standardised bioinformatics analysis. Another issue that is a mainstay of diagnostics is a question on the pathogenicity of a particular strain intercepted or

detected. Particularly with slow-growing bacteria that are difficult to isolate and maintain in pure culture, Koch’s postulates are far too time consuming to provide practical solutions. We hypothesise

that a molecular pathogenicity assay, based on gene expression analysis during initial contact

between X. fastidiosa and plant cells may be an alternative, faster option.

Antibacterial and plant defence elicitor peptides: novel tools 5.4.against Xylella fastidiosa?

Montesinos E.1

1 Institute of Food and Agricultural Technology-INTEA-CIDSAV, University of Girona, Spain





Antimicrobial peptides (AMPs) are the first barriers of innate immune defence in plants and animals

against infections, and are components of the antibiosis processes in microorganisms. Natural antimicrobial peptides offer great interest in human and veterinary disease control, food preservation

and plant protection, but their use is limited by several constrains. Synthetic antimicrobial peptides, based on natural structures or de novo designed, offer several advantages, because they can be

designed and produced by peptide chemistry or biotechnological approaches with optimized activity,

toxicity and biodegradability. Synthetic AMPs offer great possibilities as novel compounds for the control of bacterial and fungal plant diseases.

Libraries of near 300 small AMPs with linear, cyclic, 5-arylhistidine-containing peptides, cyclolipopeptides, peptidotriazole derivatives, and multivalent display structures have been prepared

by solid phase chemistry and combinatorial approaches, in collaboration with LIPPSO laboratory at the

University of Girona. Leads from these libraries have been developed and optimized against the main plant pathogenic bacteria causing quarantine diseases. Their minimal inhibitory concentrations are in

the range of 2.5 to 12.5 μM, and have minimized phytotoxicity, hemotoxicity, and mammal toxicity. Recently, some of the peptides confirmed their additional activity as plant defense elicitors in a model

plant, and novel multifunctional peptides have been developed (antibacterial and plant elicitor peptides).

Several of these peptides have been tested with success for disease control under greenhouse tests

and in some cases in field trials, against fireblight (Erwinia amylovora), bacterial blight of pear (Pseudomonas syringae pv. syringae), Prunus bacterial canker (Xanthomonas arboricola pv. pruni), and bacterial canker of kiwifruit (P. syringe pv. syringae). The efficacy was comparable to reference antibiotics, that are currently the most efficient compounds. Recently we have confirmed that some

peptides are active against phytoplasms, and that their delivery by endotherapy to wooden plants

increased significantly the efficacy of disease control.

It is expected that synthetic multifunctional antimicrobial peptides can be designed to interfere at

three levels with the biological cycle in Xylella fastidiosa diseases: pathogen (membrane disruption, inhibition of biofilm formation, interference with attachment, etc.), host plant (elicitation of defense

response), and vectors (insecticidal/pathogen transmission). In the case of the pathogen the design of these tools will be facilitated by the genome sequence of several strains, but it will be more difficult

due to the limited knowledge on the vectors and some plant hosts.

A biotechnological approach for Xylella fastidiosa targeting and 5.5.population confusion

Saldarelli, P.1

1 CNR - Istituto per la Protezione Sostenibile delle Piante UOS, Via Amendola 165/A 70126, Bari, Italy


Olive Quick Decline Syndrome is a new severe disease with which Xylella fastidiosa CoDiRO strain is

strongly associated. As in other Xylella-caused diseases, there are presently no effective treatments for affected plants, which show symptoms of leaf scorching and extensive die back. Several

therapeutic and prophylactic approaches are under experimentation to reduce bacterial movement

and multiplication or directly targeting X. fastidiosa cells for lysis. Pathogen confusion is a strategy in which grapevines expressing a X. fastidiosa Diffusible Signaling Factor (DSF) showed milder symptoms

of Pierce Disease (1). DSF accumulation causes X. fastidiosa to be less motile and more adhesive to surfaces with a consequent decrease of virulence in infected plants. A further approach directly

targets the bacterium by the expression of a protein chimera inducing lysis of X. fastidiosa outer membrane. Grapevines producing an elastase, cleaving a X. fastidiosa outer membrane protein, fused

to an antimicrobial peptide (AMP; 2) exhibit resistance to bacterial infection.





Both strategies, relying on transgenic expression, can be alternatively explored by transient and not stable expression mediated by plant viral vectors. The proposed approach uses engineered viral

vectors to induce expression of DSF or AMPs in X. fastidiosa - infected olives. Possible application in

olive and similar existing strategies in woody crops will be discussed.

References:

Dandekar AM, Gouran H, Ibáñez AM, Uratsu SL, Agüero CB, McFarland S, Borhani Y, Feldstein PA,

Bruening G, Nascimento R, Goulart LR, Pardington PE, Chaudhary A, Norvell M, Civerolo E and Gupta G, 2012. An engineered innate immune defense protects grapevines from Pierce disease. PNAS 109:

3721-3725.

Lindow S, Newman K, Chatterjee S, Baccari C, Iavarone AT and Ionescu M, 2014. Production of Xylella fastidiosa diffusible signal factor in transgenic grape causes pathogen confusion and reduction in

severity of Pierce’s disease. Phytopathology, 27, 244-254.

The EU Horizon 2020 POnTE project: the work plan on the pathogen 5.6.“X. fastidiosa”

Landa B.B.1 and the POnTE-Xylella fastidiosa associated partners1,2,3,4,5,6

1 Instituto de Agricultura Sostenible (IAS). Consejo Superior de Investgaciones Científicas (CSIC), Spain 2 Consiglio Nazionale delle Ricerche (CNR), Istituto per la Protezione Sostenibile delle Piante, UOS Bari, Italy 3 Università degli Studi Aldo Moro (UNIBA) - Dipartimento di Scienze del Suolo, della Pianta e degli Alimenti, Italy 4Institut National de la Recherche Agronomique (INRA), France 5 Agence Nationale de Securite Sanitaire de l'Alimentation, de l'Environnement et du Travail - Laboratoire de la Santé des Végétaux, France 6 Centro de Investigación en Biología Celular y Molecular, University of Costa Rica, Costa Rica


In the framework of the POnTE research project, major efforts are devoted to gain knowledge about

the virulence, the pathogenicity and the host range of the novel X. fastidiosa genotype infecting olive, and to obtain information on the genetic structure and diversity of the pathogen population in the

epidemic area. Two Work Packages (WP2 and WP 3), involving 6 different partners, will deal with the investigations on the biology, genomic and genetics of X. fastidiosa. In WP 2 “Isolation, biology and

pathogenicity of Xylella fastidiosa” the following activities will be performed: i) Establishment and implementation of the EU X. fastidiosa strain collections by performing a massive isolation campaign in diseased olive orchards and on potential hosts of X. fastidiosa identified in the area; ii) Pathogenicity tests: Artificial inoculations with the bacterial suspension will be conducted on a set of different olive cultivars and plant species (stone fruits, oleander, citrus, grapes, Quercus spp. etc.) under quarantine

conditions in confined facilities and in selected experimental field plots within the contaminated area;

iii) Studies on the host colonization: samples (collected from different branches and portions (root, suckers, etc.) of infected trees with different distribution and severity of symptoms of OQDS will be

used to determine the correlation amongst symptom development and X. fastidiosa plant colonization. In addition, grafting experiments will be performed to evaluate the movement of X. fastidiosa in olive

plants; iv) Evaluation of weeds as alternative hosts: Survey of the natural flora existing in selected olive groves will be performed to analyse the species present in the groves and their seasonality to

provide important elements for the management of the disease, as weeds can serve as source of

inoculum of the bacterium or alternative hosts for the vector populations. In WP 3 “Genotyping and genetic variability of Xylella fastidiosa” different techniques will be used including: i) Next generation sequencing (NGS) using Illumina or PacBio platforms, ii) MultiLocus Sequence Typing (MLST), iii) Multilocus sequence analysis of environmentally mediated genes (MLSA-E); iv) MultiLocus Variable number of tandem repeat Analysis (MLVA) or Short sequence repeats (SSRs), and v) Characterization



of the phage-related sequences. These techniques will allow generating the full length sequence of representative(s) isolate(s), clarifying phylogenetic relationships between closely related bacterial

strains with low genetic variability, and studying the population structure and their genetic diversity.

Furthermore, all of them will help in developing epidemiological and strain virulence studies as well as in the design of novel and high-throughput diagnostic procedures to detect X. fastidiosa.

More information and the latest updates on the research programme can be found at: www.ponteproject.eu.

Decoding the DSF quorum-sensing system in Xanthomonadaceae: 5.7.lessons from Stenotrophomonas maltophilia

Huedo, P.1, D. Yero1, S. Martínez-Servat1, O. Conchillo-Solé1, I. Gibert1, X. Daura1,2

1 Institute of Biotechnology and Biomedicine, Universitat Autònoma de Barcelona, Cerdanyola del Vallès (Barcelona), Spain 2 Catalan Institution for Research and Advanced Studies (ICREA), Barcelona, Spain.


Stenotrophomonas maltophilia are ubiquitous, worldwide distributed Gram-negative bacteria of the

Xanthomonadaceae family. S. maltophilia are common, often dominant members of the microbial communities found in plants, particularly in the rhizosphere but also in endophytic tissues, which are

their environmental reservoirs. In contrast to Xanthomonas and Xylella, no Stenotrophomonas species are known to be phytopathogenic. Instead some S. maltophilia strains have been shown to promote

plant growth and suppress plant pathogens.

Many pathogenic bacteria use cell-cell signalling systems involving the production and sensing of

diffusible signal molecules to regulate capacities such as biofilm formation, virulence and antibiotic

resistance as a response to cell density, a phenomenon known as quorum sensing (QS). These signals are often shared by different species, making QS a complex phenomenon with competing effects at

the community level. The Diffusible Signal Factor (DSF) QS system is common to different Xanthomonadaceae such as Xanthomonas campestris, S. maltophilia and Xylella fastidiosa, yet with

adaptive differences that raised, as we have shown, even within the same species.

Here, we will introduce our recent work on the DSF QS system of S. maltophilia (Huedo et al. 2014 and 2015) and briefly highlight its relation with the systems found in the phytopathogens X. campestris and X. fastidiosa. We will also introduce our strategy to identify virulence and resistance factors in S. maltophilia (Ferrer-Navarro et al., 2013; Yero et al.) and discuss its extension to X. fastidiosa.

References:

Huedo P, Yero D, Martínez-Servat S, Estibariz I, Planell R, Martínez P, Ruyra A, Roher N, Roca I, Vila J,

Daura X and Gibert I, 2014 Two different rpf clusters distributed among a population of

Stenotrophomonas maltophilia clinical strains display differential DSF production and virulence regulation. Journal of Bacteriology, 196, 2431–2442.

Huedo P, Yero D, Martínez-Servat S, Ruyra A, Roher N, Daura X and Gibert I, 2015. Decoding the genetic and functional diversity of the DSF Quorum-Sensing system in Stenotrophomonas maltophilia. Frontiers in Microbiology, 6, 761, 11pp.

Ferrer-Navarro M, Planell R, Yero D, Mongiardini E, Torrent G, Huedo P, Martínez P, Roher N, Mackenzie S, Gibert I and Daura X, 2013. Abundance of the Quorum-Sensing Factor Ax21 in Four

Strains of Stenotrophomonas maltophilia Correlates with Mortality Rate in a New Zebrafish Model of Infection. PLoS ONE, 8(6), e67207, 10pp

Yero et al. Enhanced antibiotic composition. EP15167740.8.





Appendix D – List of Workshop participants

Last name First name Affiliation Country

ANDUEZA Miren European Food Safety Authority (EFSA)

ANGELINI Martina European Parliament

ARAMPATZIS Christos Hellenic Ministry of Rural Development and

Food Greece

ARIJS Harry European Commission - DG SANTE

BAFORT Françoise Gembloux Agro-Bio Tech/ University of Liège Belgium

BALESTRA Giorgio University of Tuscia, Dep. DAFNE Italy

BAPTISTA Paula Polytechnic Institute of Bragança, School of Agriculture

Portugal

BATLLE Assumpció IRTA Spain

BECK Pieter European Commission - Joint Research Centre

BELAJ Angjelina Junta de Andalucia Spain

BERGSMA-

VLAMI Maria

National Plant Protection Organisation

(NPPO) The Netherlands

BERTACCINI Assunta Alma Mater Studiorum University of Bologna Italy

BERTHE Franck European Food Safety Authority (EFSA)

BERTIN Sabrina University of Torino - DISAFA Department Italy

BLEVE Gianluca Italian National Research Council, Institute of Science of Food Production

Italy

BOGLIOTTI Claudio CIHEAM - Mediterranean Agronomic Institute of Bari

Italy

BOSCIA Donato CNR Institute for Sustainable Plant Protection Italy

BOSCO Domenico Università degli Studi di Torino Italy

BRACHET Marie Lisa Centre technique interprofessionnel des fruits et légumes (CTIFL)

France

BRAGARD Claude Université catholique de Louvain (UCL) Belgium

BROUFAS Georgios Democritus University of Thrace Greece



BRUNEL Sarah Food and Agriculture Organization of the United Nations (FAO) – International Plant

Protection Convention (IPPC)

Italy

CALVITTI Maurizio ENEA-Italian National Agency for New Technologies, Energy and Sustanaible

Economic Development

Italy

CANDRESSE Thierry National Institute for Agricultural Research (INRA)

France

CANTELE Giovanni

Committee of Professional Agricultural

Organisations – General Committee for Agricultural Cooperation (COPA-COGECA)

CATARA Vittoria University of Catania Italy

CHATZIVASSILIOU

Elisavet Agricultural University of Athens - Plant Pathology Laboratory

Greece

CHROMY Zdenek Central Institute for Supervising and Testing

in Agriculture Czech Republic

CIAMPITTI Mariangela ERSAF Plant Protection Service Italy

COSTA joana University of Coimbra Portugal

COUTELIER Cecile Université catholique de Louvain (UCL) Belgium

CZWIENCZEK Ewelina Barbara

Bialystok University of Technology Poland

D'AMATO Rosa European Parliament

DAURA Xavier Institute of Biotechnology and Biomedicine –

Universitat Autònoma de Barcelona Bellaterra Spain

DE SANTIS Alessandra Confederazione italiana agricoltori Italy

DELLA

COLETTA-FILHO Helvécio IAC / Centro de Citricultura Brazil

DI RUBBO Pasquale European Commission – DG SANTE

D'ONGHIA Anna CIHEAM - Mediterranean Agronomic Institute of Bari

Italy

DREO Tanja National Institute of Biology Slovenia

FERERES Alberto Spanish Research Council (CSIC) Spain

FERGNANI Flavio European Food Safety Authority (EFSA)

FIRRAO Giuseppe Università di Udine Italy



FORMICA Lilia AGRITEST SRL Italy

GARDI Ciro European Food Safety Authority (EFSA)

GIOVANI Baldissera European and Mediterranean Plant Protection

Organization France

GOTTSBERGER Richard Austrian Agency for Health and Food Safety (AGES)

Austria

GOUMAS Dimitrios TEI of Crete - University of Applied Sciences Greece

GRECH Fiona Ministry for Sustainable Development, the Environment and Climate Change

Malta

GREGOIRE Jean-Claude Université Libre de Bruxelles (ULB) Belgium

GUERIN maxime Plante & Cité France

GUILLOT Gilles European Food Safety Authority (EFSA)

HARDY Tony Chair of the Scientific Committee (EFSA) United Kingdom

HERBEN Eveline Association of Dutch Flower Auctions - VBN Netherlands

HODGETTS Jennifer Food and Environment Research Agency (FERA)

United Kingdom

HOLLO Gabor European Food Safety Authority (EFSA)

IOANNIDOU Stavroula

Ministry of Rural Development and food -

Directorate of plant Health Production -

Dep.of Phytosanitary Control

Greece

IRIMIE Doru-

Leonard European Commission - DG RTD

JACQUES Marie-Agnès National Institute for Agricultural Research

(INRA) France

JAQUES Josep A. Universitat Jaume I Spain

JEGER Michael Imperial College United Kingdom

KAPSALIS Apostolos European Commission - DG SANTE

KERTESZ Virag European Food Safety Authority (EFSA)

KIEFFER Nadine

Ministère de l'Agriculture, de la Viticulture et

de la Protection des Consommateurs Administration des Services Techniques de

l'Agriculture

Luxembourg

KOLAR Patrik European Commission - DG RTD



KOUBOURIS Georgios Ellinikos Georgikos Organismos Dimitra, ELGO Dimitra

Greece

KOUFAKIS Ioannis European Food Safety Authority (EFSA)

KOZELSKA Svetla European Food Safety Authority (EFSA)

KRUGNER Rodrigo US Department of Agriculture - Agricultural Research Service

United States

KUZMANOVIC Nemanja University of Belgrade - Faculty of Agriculture Serbia

LANDA Blanca B. Spanish National Research Council (CSIC) Spain

LAVINA Amparo Institute of Agrifood Research and Technology (IRTA)

Spain

LOPES Joao University of Sao Paulo (USP) Brazil

LOPEZ María M. Instituto Valenciano de Investigaciones

Agrarias Spain

MACHADO Marcos Instituto Agronomico, Centro de Citricultura Brazil

MAES Martine Institute for Agricultural and Fisheries Research (ILVO)

Belgium

MANCEAU Charles French Agency for Food, Environmental and

Occupational Health & Safety (ANSES) France

MARKARIS Marios European Commission - DG RTD

MILELLA Luciana Regione Puglia Italy

MILLELIRI Isabelle Chambre Régionale d'agriculture de Corse France

MONTESINOS Emilio University of Girona Spain

MONTECCHIO Lucio Universirty of Padova Italy

MYLONA Panagiota European Commission - DG SANTE

NAVAJAS Maria National Institute for Agricultural Research (INRA)

France

NAVAS-CORTES Juan A. Spanish National Research Council - Institute

for Sustainable Agriculture Spain

OBRADOVIC Aleksa University of Belgrade, Faculty of Agriculture Serbia

OGRODOWCZYK Piotr Ministry of Agriculture and Rural

Development Poland

O'RIORDAN Alan Tyndall National Institute Ireland



OUSSET Didier

Consortium Européen Lubixyl_Xylella (Atelier

pour l'EUROPE, Université Marseille, CNRS, Université de BASILICATA

France

PAGES Josep M. European Nurserystock Association (ENA) Belgium

PARNELL Stephen University of Salford United Kingdom

PEETERS Luc BelOrta Belgium

PERCOCO Anna Regione Puglia Italy

PFEILSTETTER Ernst

Julius Kuehn-Institute, Federal Research

Centre for Cultivated Plants, Institute for National and International Plant Health

Germany

PILOTTI Massimo Plant Pathology Research Center (PAV), Consiglio per la Ricerca in Agricoltura e

l'analisi dell'Economia agraria (CREA)

Italy

PREDOIOU George Research Executive Agency (REA)

PURCELL Alexander UC Berkeley United States

SA PEREIRA Paula

INIAV - National Reference Laboratory for

Plant Health Pathology - Representative of Portugal GPP as X. fastidiosa expert

Portugal

SALDARELLI Pasquale Istituto per la Protezione Sostenibile delle Piante, Bari, Consiglio Nazionale delle

Ricerche

Italy

SAPONARI Maria Institute for Sustainable Plant Protection,

CNR, Bari Unit Italy

SCALIA Rosalinda European Commission – DG SANTE

SCHNEEGANS Annette European Commission – DG AGRI

SCHRADER Gritta European Food Safety Authority (EFSA)

SMITH Julian Food and Environment Research Agency (FERA)

United Kingdom

STANCANELLI Giuseppe European Food Safety Authority (EFSA)

STEFANI Emilio Università di Modena e Reggio Emilia Italy

TABONE Matthew European Commission – DG SANTE

TERRY SIMON European Food Safety Authority (EFSA)



TRAMONTINI Sara Valentina

European Food Safety Authority (EFSA)

TROTTA Luigi Regione Puglia Italy

TSAI Chi-Wei National Taiwan University Taiwan

TUFFEN Melanie Department for Environment, Food and Rural Affairs

United Kingdom

VALENTINI Franco CIHEAM - Mediterranean Agronomic Institute

of Bari Italy

VAN DER WERF Wopke Wageningen University Netherlands

VAN DER WOLF Jean Martin Wageningen University Netherlands

VANNINI Andrea University of Tuscia Italy

VAURY Charles Arysta Life Science United Kingdom

VELTMANS Jan Veltmans boomkwekerijen Netherlands

VERRASTRO Vincenzo CIHEAM –IAMB Mediterranean Agronomic

Institute - IFOAM EU

VERSTEIRT Veerle Avia-GIS Belgium

VERVERIDIS Filippos TEI of Crete - University of Applied Sciences Greece

VIVANI Laura Moverim sprl Belgium

VOS Sybren European Food Safety Authority (EFSA)

WINTER Stephan Leibniz-Institut DSMZ - Deutsche Sammlung

von Mikroorganismen und Zellkulturen GmbH Germany

WÖHNER Thomas Julius Kühn-Institut – Federal Research Centre for Cultivated Plants

Germany

XILOYANNIS Cristos University of Basilicata Italy



Appendix E – Workshop evaluation

An evaluation questionnaire was completed by delegates before the workshop closed. 41 questionnaires were received, indicating approximately a 30% response rate. Overall there was a 99%

positive feedback for logistics and administration issues and 97% positive feedback for the scientific

content.

Questions

Excellent Good Average Below

average Poor

% % % % %

Logistics and administration

Assistance before the event 68% 24% 2% 5% 0%

Travel management (if

applicable) 100% 0% 0% 0% 0%

Assistance during the event 73% 27% 0% 0% 0%

Quality of services offered 59% 41% 0% 0% 0%

Total 70% 29% 1% 0% 0%

Contents of the event

Content of the event 59% 41% 0% 0% 0%

Quality and relevance of the topics addressed

73% 24% 2% 0% 0%

Time allocated to questions and answers

56% 37% 7% 0% 0%

Networking opportunities 39% 51% 10% 0% 0%

Total 57% 38% 5% 0% 0%

Overall satisfaction level 63% 34% 3% 0% 0%

How would you rate your knowledge before and after the event?

Before the event 10% 54% 34% 2% 0%

After the event 41% 56% 2% 0% 0%

Difference 32% 2% -32% -2% 0%

workshop on xylella fastidiosa: knowledge gaps … · workshop on xylella fastidiosa: knowledge...

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