Transcript

G20 MACS Transboundary and emerging pests: Xylella fastidiosa

Marie-Agnès Jacques1, Helvécio D. Coletta-Filho2, Lindsey Burbank3, Gerard Clover4, Sergey Elansky5,

Takashi Fujikawa6, Patrizia E. Ganci7, Aynur Karahan8, Rodrigo Krugner9, Stefania Loreti10, Abi

Marques11, Alessandra A. de Souza12, Mike Sutton-Croft13, Yuichiro Takai14, and Guan Wei15.

1 National Institute for Agricultural Research (INRA), France; 2 Centro de Citricultura Sylvio Moreira

(CCSM), Brazil; 3 US department of Agriculture (USDA), USA; 4 John Innes Centre, United Kingdom

5 Lomonosov Moscow State University, Russia; 6 National Agriculture and Food Research Organization

(NARO), Japan; 7 Directotate-General for Agriculture and Rural Development, European Commission; 8 Ministry of Agriculture and Forestry, Turkey; 9 US department of Agriculture (USDA), USA; 10 Council

for Agricultural Research and Economics (CREA), Italy; 11 Brazilian Agricultural Research Corporation

(EMBRAPA), Brazil; 12 Centro de Citricultura Sylvio Moreira(CCSM), Brazil; 13 Food and Rural Affairs

(Defra), United Kingdom; 14Ministry of Agriculture, Forestry and Fisheries (MAFF), Japan; and 15

Chinese Academy of Agricultural Sciences (CAAS), China.

November 27, 2019.

Table of contents General Introduction .............................................................................................................................. 2

1. Basic data on X. fastidiosa and its insect vectors .......................................................................... 2

1.1. General characteristics ........................................................................................................... 2

1.2. Distribution of X. fastidiosa .................................................................................................... 3

1.3. Ecology of X. fastidiosa and epidemiology of the diseases it causes .................................... 4

Host range ....................................................................................................................................... 4 Host range - X. fastidiosa subspecies or Sequence Type associations ............................................ 5

Transmission of X. fastidiosa by insect vectors ............................................................................... 6

Asymptomatic period ...................................................................................................................... 8

Short and long-range spread ........................................................................................................... 9

Entry pathways for X. fastidiosa .................................................................................................... 10

1.4. Impact of diseases due to X. fastidiosa ................................................................................. 11

2. Pest management technologies ................................................................................................... 14

2.1. Survey and sampling .............................................................................................................. 14

Plant material ................................................................................................................................ 14 Insect vectors ................................................................................................................................ 14

2.2. Diagnostic methods: detection and identification technologies targeting X. fastidiosa in

host plants or in insect vectors ......................................................................................................... 14

2.3. Border measures to avert introduction of infected plant material or insects ...................... 17

2.4. Measures for prevention and control ................................................................................... 18

Use of healthy plant material ........................................................................................................ 18

Curative measure on plant material .............................................................................................. 18

Vector control ................................................................................................................................ 18

Breeding resistant varieties to prevent pest damage ................................................................... 18

Genetically modified plants ........................................................................................................... 19 Agricultural practices ..................................................................................................................... 20

Biological control ........................................................................................................................... 20

Past attempts to eradicate X. fastidiosa ....................................................................................... 21

3. Challenges and future directions for international research collaboration ............................... 22

References ............................................................................................................................................. 26

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General Introduction

The 8th Meeting of G20 Agricultural Chief Scientists (G20-MACS) that was held in Japan (25-26 April 2019) identified Xylella fastidiosa as one of the transboundary plant pests that may pose a serious

threat to food security and the environment. It was recognized that collaboration and international

research is needed to implement effective action against this plant pathogen. A group of scientists (see

the list of Authors) from 10 participating members of G20 with relevant expertise in X. fastidiosa

and/or the diseases it causes, discussed biologically relevant items concerning the bacteria, its hosts,

its vectors, the diseases it causes and the control methods as suggested in the Draft Concept Note.

Then, challenges were identified for each of these items and future directions of research were

elaborated to solve these issues. Exchanges among discussion group members were made primarily

by e-mails, but also took place during the second European conference on X. fastidiosa (October 29-

31, 2019, France) for those members that attending it. This report presents the current state of the art concerning these items, the challenges and future directions for international collaborative research.

1. Basic data on X. fastidiosa and its insect vectors

1.1. General characteristics

Xylella fastidiosa is a plant-associated bacterium causing diseases of a wide host range of plant species

(EFSA, 2018a listed more than 560 species) including crops of high economic importance, species of

cultural/patrimonial importance, and plants from the landscape. The first description of a disease

caused by X. fastidiosa was made in 1892 by Newton Pierce, who reported on the California vine

disease, a disease now known as Pierce’s disease of grapevine. The bacterium was formally described

and named only in 1987, as a consequence of its fastidious nature (Wells et al., 1987). It was isolated

for the first time in 1978 from infected grapevine xylem tissues (Davis et al., 1978). The name of this

bacterium originates from the plant structure it infects (i.e., xylem vessels), and its fastidious nature,

meaning its slow growing behavior in vitro. Another important characteristic of this bacterium is that

it is transmitted from plant to plant by sap-sucking insects. This is the only natural means of dispersion of this bacterium.

X. fastidiosa is a rod shaped bacterium belonging to the class of gamma proteobacterium (Rapicavoli

et al., 2018). Its closest phylogenetic relatives are the plant associated bacteria belonging to the

Xanthomonas genus (Bern and Goldberg, 2005). In xylem vessels, X. fastidiosa aggregates and

produces a copious matrix forming biofilm that may plug the vessels. Symptoms resembling those of

water stress result from high populations of bacterial cells in xylem tissue as well as overproduction of

plant defense compounds such as pectins and tyloses, which are produced by the host plant in

response to infection (Sun et al., 2013). Embolisms (or air bubbles) eventually form and contribute to

plug affected xylem vessels, leading to reduced xylem function and water stress (Sabella et al., 2019). When prolonged, tissue available for photosynthesis is reduced and starch reserves are depleted,

resulting in leaf scorching and premature senescence. Symptom severity depends on climatic

conditions, physiological status of the plant, bacterial strain, host species and cultivar. Symptoms

commonly reported in the literature include marginal or apical leaf tissue necrosis, premature leaf

abscission, wilting of foliage, withering of branches, decrease in fruit production, decline in vigor,

stunting and/or reduced growth up to dwarfism, delayed bud break, dieback and eventually plant

death.

X. fastidiosa strains have been grouped into five subspecies, but only two of those, X. fastidiosa subsp. fastidiosa and X. fastidiosa subsp. multiplex, have valid names in the nomenclature (Schaad et al.,

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2004; Bull et al., 2012). X. fastidiosa subsp. pauca is widely recognized and has been formally described

(Schaad et al., 2004), but its name is not valid as the type strain has not been deposited in two

international collections. The two other subspecies that have been proposed (X. fastidiosa subsp.

sandyi and X. fastidiosa subsp. morus) cluster in X. fastidiosa subsp. fastidiosa according to several

genomics analyses (Marceletti and Scortichini, 2016; Denancé et al., 2019) and were not formally

described. Another subspecies (tashke) has been proposed (Randall et al., 2009), but no specimen nore

genomic information are available to support this description.

1.2. Distribution of X. fastidiosa

Briefly, X. fastidiosa is distributed nearly all over the Americas from Canada (British Columbia, Ontario,

Saskatchewan) up to La Rioja and Cordoba provinces in Argentina, present locally in Asia (Iran, Israel,

and Taiwan), and locally and in most cases supposedly transiently in Europe (France, Italy, Portugal,

and Spain) where strict control measures are in place with the aim to eradicate the pest or, where this

is no longer feasible, contain its further spread.

Figure 1. Distribution of X. fastidiosa on Oct 3, 2019. X. fastidiosa is present in states highlighted in

orange with a yellow dot, while X. fastidiosa presence is reported as transient in states highlighted in

orange with a purple dot (from EPPO website https://gd.eppo.int/taxon/XYLEFA/distribution).

The bacterium is endemic to the Americas. After the description of the Pierce’s disease of grapevine in

California in 1892, many other plant diseases such almond leaf scorch disease, alfalfa dwarf, and more

recently bacterial leaf scorch of blueberry have been reported in the USA. It was reported from South

America in the 1980s causing citrus variegated chlorosis and coffee leaf scorch disease. Presence of

X. fastidiosa has been also reported from Canada and Central America (EPPO website https://gd.eppo.int/taxon/XYLEFA/distribution), while it was reported from Taiwan in 2002 causing

Pierce’s disease of grapevine (Su et al., 2013). A related disease of Nashi pear trees in Taiwan was

described in 1993, but this disease is now known to be the result of infection by a different species,

X. taiwanensis, which is not known to occur elsewhere (Su et al., 2016). Efforts to eradicate

X. fastidiosa from Taiwan failed. Also in Asia, grapevines, almond, and later apricot trees were reported

infected by X. fastidiosa in Iran in 2014, and in almond in Israel in 2019. Pistachio leaf scorch disease

caused by X. fastidiosa was recently reported from Iran (2019). In Europe, X. fastidiosa was detected

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in Italy in Apulia (2013), France (2015), Spain (2016), and Portugal (2018). Cases of interceptions of

X. fastidiosa in controlled environments such as greenhouses and buildings were reported from

Germany (2016), Netherlands (2018), and Belgium (2018). The latter cases above recall interceptions

of introduced infected plant material that have been reported since 2012 in Europe (EFSA, 2019a; EPPO

website https://gd.eppo.int/taxon/XYLEFA/distribution).

Reports from other countries have not been confirmed. These are the cases for India (Jindal and

Sharma, 1987), Turkey (Güldür et al., 2005), Lebanon (Temsah et al., 2015), and China (Chu, 2001)

where reports are uncertain due to detection of the bacterial disease based solely on symptom expression, histological or serological tests.

Challenges on X. fastidiosa distribution

-To optimize the chances of eradication in case of emergence, early detection is essential as detailed in the revised version of the X. fastidiosa EU Pest Risk Assessment (EFSA, 2019a). Insect and plant

sentinel strategies could be of significant help to specific current distribution. However, conditions and

means of application of these sentinel strategies deserve more information and hence research

projects.

-Early detection is strongly linked to efficiency and performance of monitoring technologies (see

below).

-Year-round surveys are expensive and labor intense. Identification of periods of the season when the

pathogen and vectors are more likely to be detected, together with ad-hoc survey guidelines, could

improve efficiency and accuracy of programs designed to monitor new introductions or existing populations.

-Different strains and subspecies of X. fastidiosa may coexist within a region. Therefore, knowledge of

the identity and prevalence of each strains within a region could help control efforts.

1.3. Ecology of X. fastidiosa and epidemiology of the diseases it causes

Host range

X. fastidiosa host range contains 563 plant species belonging to 264 genera in 82 families (EFSA,

2018a), as determined using methods of detection ranging from laboratory diagnostic testing to solely

symptom expression. When considering records determined by at least two detection methods

(excluding only symptoms, microscopy and unspecified detection methods), 312 species in 152 genera

and 61 families have been reported. The most studied host plant genera, species, or hybrids regarding

X. fastidiosa infection are Vitis (n = 28, for a total of 226 reports), Citrus (n = 17 for a total of 183

reports), Prunus (n = 10 for a total of 61 reports), and Olea europaea (n = 1 species for a total of 13 reports). EFSA (2018a) provided a list of 46 plant species that were reported as non-hosts based on the

outcome of artificial inoculations that never resulted in infection under natural conditions.

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Figure 2: Xylella spp. host plant families – the most abundant in species (from EFSA, 2018a).

Blueberry (Vaccinium sp.) is an important host of X. fastidiosa in the southeastern USA. The disease

was first identified in 2006-2007 and the cause has been attributed (Harmon and Hopkins, 2009) to X.

fastidiosa subsp. multiplex and fastidiosa (Oliver et al., 2015). Bacterial leaf scorch of blueberry has

caused significant crop loss in the states of Georgia, Alabama, and Florida, and has been identified in

multiple other states. Vector is believed to be the glassy-winged sharpshooter, Homalodisca vitripennis

(Burbank et al., 2019). Research is less developed for this host than others such as grapevine but it is

worth considering for other regions of the world that cultivate blueberries and import plant material.

Host range - X. fastidiosa subspecies or Sequence Type associations

Some host plants are naturally hosts of strains belonging to several or multiple subspecies:

X. fastidiosa subspecies fastidiosa (f): 33 host species, but 14 are hosts of at least another subsp. X. fastidiosa subspecies multiplex (m): 117 host species, but 25 are hosts of at least another subsp.

X. fastidiosa subspecies pauca (p): 43 host species, but 14 are hosts of at least another subsp.

List of species that have been found naturally infected by several or multiple X. fastidiosa subspecies

(EFSA, 2018a Appendix B, classification E; when the data are based on only one report for one

subspecies it is indicated by ‘1 rep’):

X. fastidiosa f + X. fastidiosa m: Prunus dulcis, Cercis occidentalis, Cistus monspeliensis (X. fastidiosa f:

1 rep), Medicago sativa, and Sambucus sp. (1 rep).

X. fastidiosa f + X. fastidiosa p: Citrus sinensis, Coffea arabica, Nerium oleander X. fastidiosa f: 1 rep):

X. fastidiosa m + X. fastidiosa p: Acacia saligna (1 rep), Acacia sp (1 rep), Asparagus acutifolius (X. fastidiosa p: 1 rep), Cistus creticus (1 rep), Hebe sp. (X. fastidiosa p: 1 rep), Lavandula angustifolia

(X. fastidiosa p : 1 rep), Lavandula dentata (X. fastidiosa p : 1 rep), Lavandula stoechas (X. fastidiosa p:

1 rep), Myrtus communis (X. fastidiosa p: 1 rep), Olea europaea, Olea europaea subsp. sylvestris (

X. fastidiosa p: 1 rep), Periwinkle (common name) (1 rep), Prunus domestica (X. fastidiosa p: 1 rep),

Westringia fruticosa (X. fastidiosa m: 1 rep).

X. fastidiosa f + X. fastidiosa m + X. fastidiosa p: Polygala myrtifolia, Prunus avium (X. fastidiosa m: 1

rep), Prunus dulcis, Prunus persica (X. fastidiosa f and X. fastidiosa p: 1 rep), Rhamnus alaternus (1 rep),

Rosmarinus officinalis (X. fastidiosa f: 1 rep), Spartium junceum (X. fastidiosa p: 1 rep).

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Examples of frequently reported Sequence Types (STs) (EFSA, 2018a):

X. fastidiosa f ST1 has been recorded as naturally infecting 20 plant species (USA, Spain, Mexico)

X. fastidiosa m ST6/ST7: 45 plant species (France, Spain, USA); ST81: 11 plant species (Spain); ST8: 5

plant species (USA); ST9: 10 plant species (USA)

X. fastidiosa p: ST11: 3 plant species (Brazil), ST12 (2 plant species (Brazil), ST13: 2 plant species (Brazil),

ST53: 37 plant species (Italy, Brazil, Costa Rica, ST80: 7 plant species (Spain).

Challenges on host range determination

-How precisely can a host range be defined?

=> run experimental tests (i.e. potential host range), but then which parameter can reflect

susceptibility (development of symptoms, colonization/dispersal in the plant from point of inoculation,

….)

=> Follow naturalistic approach (large sampling in outbreaks, i.e. realized host range), but then how to distinguish transient from long-lived infection?

-What is the role of annual (vs perennial) host plants in epidemiology?

-Commensal vs. pathogen: is the outcome of the interaction between X. fastidiosa and the host plant

linked to their co-evolution?

-Establishing host range depends largely on performance of detection methods that are challenged by

the large range of plant species and potential inhibitors. The need of complementary methods, the

determination of the efficiency of DNA extraction methods and the limit of detection in various plant

species, the need of spiked controls are key elements that should be provided for detection methods

in the frame of interlaboratory performance tests.

-What are the key factors defining the host range of a given strain? They should result from a

combination of insect vector and bacterial strain determinants that remain to be identified. Increased

access to whole genome sequences would help to characterize bacterial genomic determinants, but

the interaction of the bacterium and the insect, and the insect behavior are other key determinants

that require considerable research efforts.

- An important consideration in host range is which hosts will have infections which are self-limiting

over time and which will have infections that rapidly spread? This can be dependent on vectors (ex.

very little plant-plant spread occurs in almond in California because it is not a preferred host of native

vectors), or it can be dependent on plant cultivars, general plant health, specific X. fastidiosa strains,

and climate (examples: rootstock effects, over winter recovery of grapevine, minor diseases such as X.

fastidiosa multiplex in olive in California). A challenge in inoculation experiments is evaluating longer

term disease progression for better understanding of which plant species and varieties are likely to

contribute to rapid spreading of disease.

Transmission of X. fastidiosa by insect vectors

Transmission of X. fastidiosa involves three distinct phases: acquisition of bacterial cells by the vector,

retention (and multiplication in the vector), and inoculation. X. fastidiosa is exclusively transmitted by

xylem sap-feeding insects belonging to the order Hemiptera, suborder Auchenorrhyncha and in three

families: Aphrophoridae (spittlebugs), Cicadellidae (leafhoppers and sharpshooters of the subfamily

Cicadellinae), and Cicadidae (Krugner et al., 2019). In the Americas, X. fastidiosa is transmitted mainly

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by sharpshooters. In Europe, spittlebugs are much more abundant and diverse than sharpshooters.

Recent outbreaks of Pierce’s disease in California have been linked to the introduction of the glassy-

winged sharpshooter in the 1990’s from southern states.

Some key features concerning X. fastidiosa transmission by insect vectors were recently reviewed by

Krugner et al (2019). Briefly, X. fastidiosa cannot be passed through eggs from an infected mother to

offspring, meaning that transmission lacks transovarial passage (Freitag, 1951). Nymphs and adults

must acquire X. fastidiosa from infected plants. Once acquired by adult vectors, X. fastidiosa is retained

and can be inoculated for life (Severin 1950; Turner and Pollard 1959). Transmission of X. fastidiosa

occurs via a non-circulative yet propagative mechanism (Hill and Purcell, 1995; Purcell and Finlay,

1979). Bacterial cells colonize, multiply, and form a biofilm only on the cuticle of the functional foregut

(Backus and Morgan, 2011; Purcell, 1979).

Studies indicate that transmission efficiency is not affected by insect gender (Krugner et al., 2012a;

Severin 1950), nor is there evidence for vector specificity (Redak et al., 2004). Turner and Pollard (1959)

hypothesized that any species in the subfamily Cicadellinae may be able to transmit X. fastidiosa.

Similarly, Philaenus spumarius L. (Hemiptera: Aphrophoridae) transmits at least X. fastidiosa sequence

types causing Pierce’s disease (Severin, 1950) and olive quick decline syndrome (Saponari et al., 2014).

While there is no specificity in the ability to transmit, the efficiency of transmission is expected to vary among vector species x bacterial genotype combinations.

Acquisition of X. fastidiosa occurs during ingestion and it can be temporarily held in suspension in the

insect foregut (Backus and Morgan, 2011). Once attached to the foregut, the bacteria undergo genetic

and morphological changes to form a biofilm (Killiny and Almeida, 2009). Inoculation of X. fastidiosa

to plants by sharpshooters is hypothesized to originate from the precibarium. Adult sharpshooters may

acquire and inoculate X. fastidiosa within a few hours after initiating feeding. Graphocephala

atropunctata and H. vitripennis carrying X. fastidiosa successfully inoculated bacteria into a host plant

within two hours of exposure to the plant (Almeida and Purcell, 2003; Severin, 1949). Because bacterial

suspension can be held in the foregut as the insect moves from one plant to another, and it has been proposed that ejection of such a suspension; termed the “flying syringe” hypothesis (Backus et al.,

2015); may explain the short latent period (about 1 to 2 h) of X. fastidiosa in vector foreguts. Very few

live bacterial cells in the vector’s foregut are required for transmission (Hill and Purcell, 1995).

Therefore, sensitive diagnostic tools are needed to detect the presence of X. fastidiosa cells in the

insect vectors. Enzyme-linked immunosorbent assay (ELISA) is not sensitive enough to detect

X. fastidiosa in the vector insects.

Challenges on insect transmission -The list of potential insect vectors and distribution has to be defined in each target area for

X. fastidiosa emergence.

-Transmission (i.e., pathogen acquisition, retention, and inoculation) is reputed not affected by

bacterial genotypes as reviewed for Homalodisca vitripennis by Krugner et al. (2019). Is this large

capacity of transmission true for any vector species and bacterial genotypes? This question would

beneficiate from research projects on mechanisms of interactions between foregut cells and X.

fastidiosa cells.

-Linking maps of insect and plant host distribution could help to determine at-risk area for surveillance,

but this is challenged by the large host range of X. fastidiosa.

-Reconstruction of plant-vector trophic networks might help to highlight at-risk area and certainly

deserves further research.

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-The performance of detection methods must be assessed in the light of the range of vector species

and optimized to deal with the low number of bacterial cells in insects.

-Eradication and containment strategies necessitate the determination of surveillance area around an

infected focus. Knowledge of flight distance in various contexts is important to define eradication area

or buffer zone.

-Determining the periods of the season when X. fastidiosa is most likely to be transmitted by insect

vectors would facilitate design of disease management strategies tailored to reduce pathogen spread.

- Development of differential control tools (mechanical, biological and chemical) under different

landscape scenarios (e.g. urban, semi-natural, agriculture).

Asymptomatic period

Symptoms caused by X. fastidiosa are often similar to those caused by water stress. Hence, these

symptoms are not specific or characteristic of any Xylella-diseases, and may be transient or completely

eliminated (Purcell, 2013, Krugner and Ledbetter 2016). The infection of some species causes rapid

death (Purcell and Saunders, 1999; Martelli et al., 2016). Early symptoms are not necessarily easy to

identify but could indicate X. fastidiosa infection. For example, delayed bud break of grapevine in the

beginning of the season can be used to identify infected vines several months before leaf scorching

symptoms are apparent and the pathogen can be detected by PCR (Feil and Purcell, 2003).

The asymptomatic period is the time from infection of a plant to expression of symptoms. This asymptomatic period is highly variable according to the plant species and age (generally shorter in

herbaceous than in woody hosts) and ranges from a few months (Lopes et al., 2005) to three years

(Krugner et al., 2019) after bacterial inoculation. The asymptomatic period of X. fastidiosa also varies

significantly for different hosts and pathogen subspecies combinations. For example, from a median

of up to 1 month in ornamental plants and up to 10 months in olive, for subsp. pauca. Temperature

has very significant effects on disease development as well as plant recovery from infection. Higher

temperatures during the growing season accelerate symptom development and bacterial population

levels and low winter temperatures impact disease reoccurrence. This has mostly been studied in

grapevine and almond (Feil and Purcell, 2001; Cao et al., 2010), suggesting that other host species should be investigated.

From a large literature review and parametric and non-parametric statistical analyses of the

quantitative results reported in peer-reviewed manuscripts (for methods and list of papers reviewed

see EFSA, 2019a), it appears that X. fastidiosa subsp. fastidiosa, X. fastidiosa subsp. multiplex, X.

fastidiosa subsp. pauca and X. fastidiosa subsp. morus showed a relatively rapid development of

symptoms in grapevines, almonds, ornamental species and mulberry, respectively following

mechanical inoculation of hosts. X. fastidiosa subsp. multiplex had a longer asymptomatic period in

shade trees (95% chance for symptoms in 941 days), and X. fastidiosa subsp. pauca had an even longer

asymptomatic period in both oranges and olives (95% chance for symptoms in 1,080 days and 1,354 days, respectively).

Little is known about colonization patterns within plant species. Nevertheless, available evidence

suggests that distribution within hosts are complex and depend on the plant species, bacterial

genotype, vector feeding behaviors, and abiotic factors.

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Challenges on asymptomatic period

-A number of factors such as host age, temperature and fertilization (quality and quantity) may impact

the asymptomatic period and the impact may vary with plant species-X. fastidiosa strain combination.

These factors may affect the reliability of experimental tests aiming at defining this asymptomatic

period and hence should be more precisely determined.

-Inoculation protocol(s) that limit caveats of artificial infection (including multiple infections, sampling

at a given time lapse after infection to ensure establishment of the inoculated strain, ….) are of interest

to evaluate more precisely the asymptomatic period.

-Resistant/tolerant cultivars can host X. fastidiosa populations asymptomatically. Are these resistance/tolerance traits stable in time or can they break? What are the epidemiological risks linked

to the low asymptomatic presence of X. fastidiosa in the resistant/tolerant cultivars?

-Asymptomatic period represents an epidemiological risk in natural environment as asymptomatic

infected plants can be reservoirs and serve as source of inoculum to insect vectors. Plant sentinel

strategy can help to detect discrete presence of X. fastidiosa. This sentinel strategy may also help

testing ecological theory. This strategy was recently reviewed (Eschen et al., 2019; Mansfield et al.,

2019).

-Modeling methods might help to identify the existence of such reservoirs (Soubeyrand et al., 2018)

and determine optimal distribution of sentinel plants in the environment.

-The asymptomatic period challenges the determination of key parameters for population genetics,

such as the generation time of X. fastidiosa that are essential to assess X. fastidiosa evolutionary

history and hence deserves research.

-How can infection clearing be differentiated from a cryptic infection (small population size)?

-Plant defenses against pathogen infection includes changes in the chemical composition of xylem sap

(terpenoids, etc…). Understanding such changes/differences related to X. fastidiosa infection could

help development of early-detection methods targeting widespread chemical compounds in plant

tissue in complement to X. fastidiosa DNA.

Short and long-range spread

The probability of spread was assessed for the update of the X. fastidiosa EU Pest Risk Assessment

(EFSA, 2019a). Two distinct modelling approaches were developed to capture the epidemiological

processes that occur at different spatial scales. Short-range spread is defined within an orchard and

long-range spread at a regional scale. The short-range spread model, which is a fully mechanistic

epidemiological model is mainly based on spread of X. fastidiosa in olive orchards in Apulia. Model

simulations showed that the application of highly effective insect vector control (nymphs and adults),

and reductions in the delay from infection to detection and from detection to implementation of control measures (e.g. removing plants) are the key factors for a successful local eradication.

Simulations also showed that with a smaller cut radius (50 m) it was possible to eradicate the pathogen

provided there was high efficacy of nymph and adult vector control. A cut radius of 100 m was more

efficient for eradication than a 50 m radius, but eradication could still fail if the vector was poorly

controlled and detection and instigation of control were too slow. The long-range spread model

describes the X. fastidiosa subsp. pauca spread in olive orchards in Italy. The model simulates spread

by coupling a generic epidemiological model with a dispersal-kernel model. The influence of changing

the epidemiological and landscape parameters were also assessed. Reducing buffer zone width in both

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containment and eradication scenarios increased the area infected, although the size of the zones

remains a final decision of risk managers.

Challenges on short/long range spread

-This is a challenging subject as the information is limited. The models that were recently developed

for this purpose (EFSA, 2019a) may be complemented by novel models and applied on larger sets of

data.

-Validation of the results of the models requires large experiments involving insect vectors,

X. fastidiosa strain(s), host plant(s), and environmental condition(s). Considering the quarantine status

of this pest, this is unfeasible in pest-free areas, and hence conditions should be organized for large

experiments being conducted in an infected area.

-The role of grafting operations, transmission through the use of contaminated tools, pruning

equipment and root self-grafting in short-range spread of X. fastidiosa needs to be determined. X. fastidiosa being a vascular pathogen, the apparent ineffectiveness of these transmission methods

justify more specific studies.

-Understanding vector movement and dispersal behaviors are key to understand pathogen spread.

Note that tracking disease symptoms only is not as informative because of long asymptomatic periods.

International collaborations could focus on development of new or improvement of current

techniques for tracking insect movement such as the protein marking technique (for review see Hagler,

2019).

Entry pathways for X. fastidiosa

EFSA (2015a) listed seven pathways of entry for X. fastidiosa. Plants for planting infected with

X. fastidiosa (including plants or plant material imported for research or breeding purposes) and

infectious insect vectors were considered the major pathways for entry. This was supported by data

on interceptions of infected plants and insects (Europhyt database). The other pathways, i.e. seeds,

fruits, cut flowers and ornamental foliage infected with X. fastidiosa were considered unlikely, while

the seventh one, detached wood, very unlikely. Uncertainty is high concerning some pathways, and

especially infected seeds. The only available data refer to citrus seeds (Coletta-Filho et al., 2014; Cordeiro et al., 2014; Hartung et al., 2014; Li et al., 2003) that were detected contaminated but no

transmission of the bacterium to the plantlets was observed. Coffee beans were reported as being

potentially infected (Crouzillat, pers. comm.). One report mentions that no seed-to-seedling

transmission was observed for olive seeds, but this analysis was based on limited number of individuals

(Altamurra et al., 2019).

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Challenges on entry pathways

-Investigate further the (in)effectiveness of the infected seed pathway with experiments on various

host seed material.

-Hot water bath treatments for propagative plant material have been tested and are currently recommended for export of grapevine material produced in infected area. Protocols need to be

determined for propagative plant species material.

-Low temperature treatments have not been tested, but may be of use for traded plant material

-Inspection and sampling are challenging due to a large consignments of plants (e.g. ornamentals) and

further modeling may be used to improve their efficacy.

-Live insect vectors unintentionally transported in plant products (e.g., fresh fruit, vegetables) could

serve as a source of bacterium for non-affected areas. Post-harvest protocols (temperature,

fumigation) developed specifically for vectors could help reduce the probability of invasions.

1.4. Impact of diseases due to X. fastidiosa

Impact of X. fastidiosa has been quantified or estimated in the case of six diseases that occur in the

USA and Brazil (this part has been mostly extracted from EFSA, 2015a).

Pierce’s disease: The disease is prevalent across the United States, from Florida to California and

grapevine production is considered to be economically unfeasible in the south-eastern USA (e.g.

Florida, Georgia) because of X. fastidiosa endemicity. Experimental vineyards are destroyed within

years of planting (Anas et al., 2008). Since the 1880s, Pierce’s disease has caused the decline of more

than 17,000 ha of vineyards in Southern California (Galvez et al., 2010). Grapevine production is

differentially affected within California, depending on vector species. In coastal California, vineyards

located near vector habitats such as riparian areas are affected by the blue-green sharpshooter,

Graphocephala atropunctata. In central California, vineyards and almond orchards located near weedy

alfalfa fields and irrigated pastures are affected by the green sharpshooter, Draeculocephala minerva.

In southern and central California, vineyards located near citrus orchards are affected by the glassy-winged sharpshooter, Homalodisca vitripennis. A recent study has estimated that Pierce’s disease costs

US$104 million per year to the grapevine industry in California (Alston et al., 2013; Tumber et al., 2014).

Without the control of H. vitripennis, which is ongoing, loss estimates for the California grapevine

industry would also increase. The pathogen threatens the country’s US$30 billion grape and wine

industries (Sanscartier et al., 2012).

Oleander leaf scorch: Its emergence in California in the 1990s was associated with high mortality of

plants used as decoration along highways. Oleander is a popular plant for landscaping along highways

because it is hardy and easy to care for; it is common in California because it can tolerate the extreme

high temperatures and dry climate found in the area. In 1997, CalTrans, the organisation responsible for the management of highways in California, estimated the economic impact of the loss of oleanders

along highways in the state at US$125 million, with additional cost needed for plant replacement

(Henry et al., 1997). In addition, motorways in southern California are now largely devoid of green

plants in central reservations.

Plum leaf scald (PLS) disease: PLS caused by X. fastidiosa subsp. multiplex, represents a limiting factor

for plum production in Brazil (Dalbo et al., 2018). Symptoms are characterized by leaf scorch and brown

rot but the disease causes low fruit quality affecting negatively the weight, firmness and size of the

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fruit (Kleina et al., 2018). The spread of PLS in Brazil is due to the presence of alternative hosts and

efficient and widespread vectors (sharpshooters) (Dalbo et al., 2016). In the state of Santa Catarina,

where the PLS disease caused damage to 90% of plum orchards from 1975 to 1982 (Ducroquet and

Mondi, 1997), an intensive breeding program was established using cultivar selections from Florida

(Dalbo et al., 2018). However, plum orchards usually have a short-lifespan and the most commonly

used strategy is to plant healthy material and spray insecticides for vector control (Dalbo et al., 2018).

It has to be considered that in Brazil the commercial orchards are based on Prunus salicina and its

hybrids while in the EU the most common species is Prunus domestica. In addition, vectors belong to

different families with different lifestyles (sharpshooters in Brazil and spittlebugs in EU).

Almond Leaf Scorch (ALS) disease: ALS was first noted in Southern California in the late 1950’s. In 1974,

it was reported from 14 different counties in California affecting 50% of the trees in some areas

(Sanborn et al., 1974). This disease is caused by X. fastidiosa subsp. multiplex and X. fastidiosa subsp.

fastidiosa is currently not a major problem for California almond growers, presumably due to

widespread use of a resistant rootstock (Krugner and Ledbetter, 2016). Areas affected have reported

yields of ALS affected trees reduced by 20% and 40% compared with unaffected trees for ‘Nonpareil’

and ‘Sonora’, respectively 9 of 183 ALS-affected trees died after 5 years (Sisterson et al. 2012).

Differences in disease incidence and severity were reported for almond varieties grown in California.

Citrus variegated chlorosis (CVC): From 1987 when the disease was observed the first time in São Paulo

to 2000, the disease affected 34% of the 200 million sweet orange trees. By 2005, the percentage had

increased to 43%, and CVC was present in all citrus growing regions of Brazil. From 1996 to 2005, the

percentage of trees with mild symptoms decreased from 16 to 6%, while the percentage of trees with

severe symptoms increased from 6 to 37%, indicating that trees with mild symptoms turned into trees

with severe symptoms. Yield losses of severely affected sweet orange trees can be as high as 60–80%.

In 1996, when CVC was still mild, 270 fruits were required to fill one box, while in 2006, when CVC was

more severe and fruits were smaller, 300 fruits were needed per box. The difference of 30 fruits per

box represents a loss of 10%, meaning that CVC decreases the number of boxes produced by 10% (Bové

and Ayres, 2007). From 2006 to 2008 in São Paulo state, Brazil the disease reduced the weight of fruits with symptoms by 50%. After 8 years of infection, 24% of difference between yield of trees from

healthy seedlings and trees from seedlings artificially inoculated with X. fastidiosa were recorded

(Goncalves et al. 2011).

For citrus plantations in a high inoculum area such as São Paulo, Brazil, considering percentage of small

fruits and percentage of estimated damage (PED), the varieties can be separated into three different

groups: highly susceptible (PED between 72 and 98%: Barão, Pêra, Lima, Rubi, Cadenera 17 and 51,

Berna, Valencia), susceptible (PED between 60 and 70%: Gardner, Pineapple, Sunstar, Folha Murcha,

Baianinha), mildly susceptible (Lue Gim Gong 43% PED and Westin 22% PED) (Laranjeira and Pompeu

Junior, 2002).

Leaf scorch and decline syndromes on broad-leaved tree species: X. fastidiosa has been reported to

cause great economic and aesthetic damage on broad-leaved trees, especially oak, elm, and sycamore

(Lashomb et al., 2002), although mulberry, red maple and other tree species may also be attacked

(Sinclair et al., 1987). Infected trees do not die immediately but tree life is shortened and the aesthetic

quality is reduced (Sherald and Kostka, 1992). In general, affected trees may decline to the point where

they must be removed (Hearon et al., 1980). In some New Jersey municipalities, leaf scorch was

reported to affect up to 35% of oaks planted as street trees and in landscapes (Lashomb et al., 2002;

Gould et al., 2004; Gould and Lashomb, 2005). Loss of value plus replacement costs for older trees

affected by this disease was estimated at $8,000 per tree (Gould and Lashomb, 2005). An analysis of economic impact of X. fastidiosa indicated that the affected communities in New Jersey would sustain,

and must plan for, losses ranging from US$0.7 to 1.6 million during the following 10 years (Gould et

al., 2004). In addition, it was noted that landowners and tree care professionals in these locations must

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plan for the loss of property values and high costs of replacement as shade trees in landscapes, wood

lots, and golf courses affected by leaf scorch decline and must be removed (Gould and Lashomb, 2005).

Other detailed and referenced impact concerning forest and urban trees are given in EFSA (2019a).

Concerning the EPPO region, the potential importance of X. fastidiosa infections has been evaluated

as major in the case of six plant entries (Table 1) and as minor for 111 entries (species or genus)

(https://gd.eppo.int/taxon/XYLEFA/hosts. This evaluation combines the biological importance of the

host plant for X. fastidiosa together with the economic importance of this plant for the EPPO region.

Table 1. List of hosts that severely affected by X. fastidiosa and are economically important for the

EPPO region (from https://gd.eppo.int/taxon/XYLEFA/hosts).

Furthermore, together with EFSA, the Commission’s Joint Research Centre (JRC) has estimated the

economic, social and environmental impact of X. fastidiosa in the Union territory in a full spread

scenario, taking into account the climatic suitability and potential establishment in the EU (Sanchez et

al, 2019). Data confirm that X. fastidiosa full spread could ultimately cost the EU over €5.5 billion per

year due to loss of production, with potential export losses of €0.7 billion per year. Moreover, it could

affect over 70% of the Union’s production value of olive trees older than 30 years, and 35% of the

younger trees. This could put nearly 300,000 jobs involved in olive trees, citrus, almonds and grapes

production at risk.

Challenges on impact

-Impact on production is the result of interactions between the host plant, the pathogen genotype,

the vectors and the abiotic environment. It will be affected by global changes. There is necessity for

modelling under diverse scenario.

-Evaluation of the potential impact in EU target area that are yet unaffected was done by EFSA using

the EKE process (EFSA, 2014) for grapevine, citrus and olive tree (EFSA, 2019a). These estimates were

based on various assumptions, which must be reassessed as new information becomes available.

-The behavior of tolerant/resistant varieties of grapevine and olive is known in some specific area, will

it last in different environments? What about the susceptibility of other host plants (e.g. varieties)?

-Uncertainties are large concerning the quantitative impact of X. fastidiosa on forest and urban trees,

especially concerning tree species that are absent in North America and native to other areas. Impact

should be modeled for these essential crop species and species of various landscapes.

-Qualitative and quantitative impacts of X. fastidiosa on biodiversity in natural environments has not

been documented in areas of long lasting occurrence (the Americas) and hence is difficult to predict in

introduced areas. This includes potential impact on native and endemic species and on genetic

resources.

-Socio-economic impact needs to be addressed on farming and rural systems, but also on landscape

use.

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2. Pest management technologies

2.1. Survey and sampling

Plant material

To survey for the presence of X. fastidiosa, multiple hosts have to be inspected and sampled over many different environments. Concerning plant material, guidelines are provided in EPPO diagnostic

standard PM 7/24 (EPPO, 2019) concerning both symptomatic and asymptomatic plant material in

order to maximise the probability of X. fastidiosa detection. These guidelines are based on recent

experimental data from the large European project XF-ACTORS and focused on a few plant species or

genera: O. europaea, N. oleander, P. myrtifolia, Lavandula spp., Prunus spp. and Coffea spp.

The timing of the optimal sampling period depends on the plant species, bacterial strain and

environmental factors. To maximize the likelihood of bacterial detection, sampling should be

performed during the period of active growth of the plant (Hopkins, 1981), which mostly corresponds

to late June up to autumn in Europe and North America.

Guidance on inspection is provided in PM 3/81 Inspection of consignments for Xylella fastidiosa (EPPO 2016a) and PM 3/82 Inspection of places of production for X. fastidiosa (EPPO, 2016b). ISPM 31 (FAO,

2016) provides useful information on the number of plants to be sampled.

Samples for analyses should be composed of material maximizing the number of xylem vessels, i.e.

small twigs, branches and cuttings with attached mature leaves.

On symptomatic plants, symptomatic samples should be collected preferably from a single plant and

consists of live organs representative of the symptoms seen on the plant(s). Depending on leaf size,

twigs with 10 to 25 leaves should be sampled.

For asymptomatic plants, the sample should be representative of the entire aerial part of the plant.

Depending on the host and plant size, the number of branches to be collected is at least 4 to 10, and

based on results obtained on olive trees in Italy, best results were obtained from samples of the mid-

upper parts of plant crown.

Insect vectors

Concerning insect vectors, adult vectors should preferably be collected with sweeping nets or

aspirators, as sticky traps are usually not as effective as active sampling for xylem feeders. To maximize

the likelihood of bacterial detection, sampling for insects should preferably be conducted from late spring until early autumn and should take into account insect behaviour in the area. X. fastidiosa being

persistent in adult vectors once acquired, detection in insects can indicate new spread before the

pathogen is detected in host plants (Moussa et al. 2016).

2.2. Diagnostic methods: detection and identification technologies targeting

X. fastidiosa in host plants or in insect vectors

The reference documents to be consulted concerning the diagnostic of diseases due to X. fastidiosa

are: the International Plant Protection Convention’s (IPPC) international standards on

phytosanitary measures (ISPM) 27 Diagnostic protocols for regulated pests, in particular DP 25: Xylella

fastidiosa, and ISPM 31 Methodologies for sampling of consignments (FAO, 2016, and 2018)

the European and Mediterranean Plant Protection Organization (EPPO) diagnostic protocol

for X. fastidiosa PM 7/24 (4) (EPPO, 2019),

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the pest survey card (EFSA et al., 2019)

the Guidelines for the prevention, eradication and containment of X. fastidiosa in olive-

growing areas (Catalano et al., 2019).

It is important to mention that isolation is not recommended as a screening test because X. fastidiosa

is very difficult to isolate. EPPO PM7/24 protocol recommends in X. fastidiosa -free area the use of two

screening tests based on different biological principles or targeting different parts of the genome. In

areas where X. fastidiosa is known to be present and in buffer zones, one positive test is sufficient to

consider that a sample is infected.

Because the detection threshold or limit of detection of molecular tests is lower than that of serological

tests, the use of molecular test is preferred for asymptomatic plant material, for insects, and for plant

material from X. fastidiosa-free area (EPPO, 2019). Considering the context of this report, only

molecular tests will be presented.

With X. fastidiosa forming biofilms, an additional ultrasonication step may help to disrupt the biofilms

and allow a better access of chemicals to bacterial cells to improve lysis (Dupas et al., 2019; Bergsma-

Vlami et al., 2017). For PCR-based detection methods, inhibitory compounds from plant material

(polyphenol content, polysaccharides) and insects can be removed by chemical extraction methods such as CTAB-DNA extraction protocol (Francis et al. 2006) or partial dissection of insects to utilize only

the head, where bacterial cells are located. Optimization of DNA extraction protocols is necessary

when dealing with the small numbers of bacterial cells found in insect mouthparts (Bextine et al. 2004).

For the detection of X. fastidiosa, several conventional and qPCR tests have been validated (EPPO,

2019). These includes the conventional PCR test developed by Minsavage et al. (1994) and the real-

time PCR tests described by Harper et al. (2010), Francis et al. (2006), Ouyanget al. (2013), Li et al.

(2013), and Bonants et al. (2019). The tests of Harper et al. (2010) can also be used in a LAMP version

(Yaseen et al., 2015). Another isothermal amplification test proposed as an AmplifyRP XRT kit has been

developed (Li et al., 2016).

Identification of X. fastidiosa and assignation of subspecies can be performed directly on DNA

extracted from plant material. The use of Multilocus Sequence Typing (MLST) analysis described by

Yuan et al. (2010) is recommended by EPPO (2019) to identify strains. To be used directly on DNA

extracted from plant material, slight modifications concerning Taq polymerase and primer

concentration, and melting temperature were applied to the original protocol (Denancé et al., 2019).

Being based on end-point PCR, MLST-based identification has a higher limit of detection than the qPCR

detection tests, also a nested version of the MLST-based identification has been proposed (Cesbron et

al., submitted). Sequencing of only two housekeeping genes is sufficient to assign a subspecies and

detect possible recombinants. Subspecies assignment may also be performed by subspecies-specific molecular markers on isolated strains (Pooler and Hartung 1995, Hernandez-Martinez et al. 2006) or

with multiplex qPCR test directly on extracted DNA from plant material (Dupas et al., 2019).

In agreement with the short-range spread model produced in the revised X. fastidiosa pest risk

assessment (EFSA, 2019a), the importance of early detection of new outbreaks is key to successful

control, demonstrating the importance of surveillance and detection capabilities. Further data on the

magnitude and pattern of long-range movements are needed due to their significant impact on success

of control. In consequence, the use of detection methods with low limit of detection has to be

promoted.

Because insects visit multiple plant species during life, they may acquire all genotypes of X. fastidiosa,

and the asymptomatic period in plant could be very long, insect sampling and testing may provide

useful results to complement plant material analysis (Cruaud et al., 2018; Stenger et al., 2019).

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Challenges on detection methods

-Interpretation of qPCR results may be complicated especially for surveying pest-free areas and when

considering asymptomatic plant material. Late positive (Ct> 35) signals are frequently observed.

Extensive data analyses are needed to further provide elements to interpret inconsistent results (eg

Ct> 35 present on 1-2 technical replicates out of 3, with 2 different real-time PCR methods) excluding

the hypothesis of contaminants, and document the phytosanitary risk associated to these results.

-While dealing with samples of diversified plant species or insects, the range of potential PCR-inhibitors

is large and may affect survey results. Some DNA extraction methods, such as CTAB-based protocols,

are more efficient than column-based one to remove these inhibitors, but are not automated and are time-consuming. Such DNA-extraction procedures have been compared in test-performance studies

in the frame of the Promode Euphresco project (https://zenodo.org/record/2656679#.XMqYG-gzbcs).

The use of spiked samples for each plant matrices is a good solution to detect the presence of potential

inhibitors, as is the use of an internal control. Harmonization of methods is essential for official survey

laboratories, but capacity to implement innovation is key in research laboratories. In this respect, in

2018, the EU established the European Reference laboratory (EURL) on bacteriology with the aim to

promote uniform and high standard practices across EU Member States in relation to the development

or use of the methods of analysis, test or diagnosis employed by the National Reference Laboratories

(NRLs) established by EU Member States. From August to October 2019, a proficiency test on X.

fastidiosa was carried out and the outcome of this exercise will serve as input for dissemination of

improved practices and for possible implementation of international diagnostic standards.

Implementation of innovative technologies to detect the pest that will be made in research

laboratories will serve as basis to improve EU-diagnosis by the EURL.

-Objectives and the performance of detection methods used in X. fastidiosa-infected or –free area

obviously has different purposes and challenges, the detection and identification strategies should be

chosen as a consequence.

-Novel technologies (remote sensing tools, Next-Generation Sequencings (NGS), for example) have

shown some promises for X. fastidiosa early detection (Bonants et al., 2019; Zarco-Tejada et al., 2018)

and more recent sequencing technologies, apparatus or imageries should be tested and performances described.

-Isolation of X. fastidiosa remains a difficult task. Can a better understanding of X. fastidiosa

metabolism helps to design improved isolation and in vitro growth procedures?

-Performance of sampling strategies would benefit from further testing. What are the tradeoffs

between sampling large areas for surveillance versus small areas for research (more samples per area

unit, but in a limited area)? That is, a survey conducted in multiple locations once versus fewer

locations but over longer periods. What information can we gather from both approaches?

Experiments could be conducted to determine the smallest number of samples per area that can give

a reliable estimate of vector population density or pathogen prevalence.

- Develop and determine the performance of survey activities in a statistically relevant manner to be able to confirm the X. fastidiosa-free pest status of areas with a certain level of confidence and a given

detection level.

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2.3. Border measures to avert introduction of infected plant material or insects

Major entry pathways for X. fastidiosa in X. fastidiosa-free area are i) plants for planting and ii) insect

vectors on their own and as hitchhikers (EFSA, 2018b). Hence, measures aiming at preventing entry at

borders shall deal with plant material and insects. Efficiency of measures aimed at preventing entry

can be limited by various biological and technical factors such as the asymptomatic period, the large

host range, the large volume of potentially infected plants traded, the presence of X. fastidiosa in some

plant material producing countries that are also exporting countries, and the ubiquitous presence of

potential insect vectors. Risk reducing options have been reviewed by EFSA (2015, 2018b, and 2019b).

The asymptomatic plant material may be infected with small population sizes that are at levels similar to the threshold for most sensitive detection methods, and hence may be undetected at entry tests.

Holding asymptomatic plant material from potential host species in quarantine is an option to be

considered for such types of asymptomatic materials. However, the asymptomatic period may be very

long for some combinations of plant species and bacterial genotype, and the duration is not known for

most combinations.

Entry trough trade of infected plant material can be limited through:

-prohibiting the import of plants for planting of X. fastidiosa host species from infected area. However,

the large host range of X. fastidiosa may restricts this measure to high-risk commodities.

-restricting the importation of plants for planting to pest-free areas or pest-free places of production, especially if associated with certification scheme (growing plants under exclusion conditions

associated to vector control in nurseries), and

-the thermotherapy of dormant propagative material, which has been recommended for grapevine

cuttings for planting.

The entry of insect vectors may be limited by insecticide treatment of traded plant material. This can

effectively reduce the likelihood of insect vectors being carried together with plant materials (see

below). The use of dormant plant material that has undergone thermotherapy has the advantage of

also limiting the introduction of exotic insect vector species, even those that lay eggs endophytically.

Sharpshooter and spittlebug vectors of X. fastidiosa are widely distributed, and a large number of

potential vectors have not been described yet, making the prohibition of this path a complicated

perspective. The most obvious solution to suppress vector populations and limit movement of insect

vectors is insecticide applications (see below).

With the aim to ensure consistency in the detection methods across EU and exporting third countries,

involvement in proficiency tests or training initiatives are under the scope of the EURL activities.

Challenges on border methods

-efficiency of border control methods is highly dependent on the efficacy of the survey, sampling and

testing. While detection methods with very good performance criteria are available, data on survey

and sampling protocols are still limited. Designing survey scheme and sampling strategies adapted for

asymptomatic periods with low bacterial numbers in samples are needed.

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2.4. Measures for prevention and control

Use of healthy plant material

The potential for X. fastidiosa to move in association with plants for planting is considered very likely,

hence reducing the risk of the entry through infected plant material is of major importance. The use

of healthy plant material means ensuring that the plant material has been produced in a pest-free area

or pest-free place of production, and/or using options to prevent or limit crop infestation by X.

fastidiosa. Concerning plant material originating from an area where X. fastidiosa is known to occur,

various options can be considered. These range from a strictly prohibiting importation of susceptible

plant material to the use of treatments to reduce X. fastidiosa /insect prevalence in shipments.

Concerning the vector pathway, measures should ensure that the plant material is free of infected

insects.

Curative measure on plant material

There is no curative measure against X. fastidiosa applicable in open field. The only curative measure

currently available has a restricted field of application as it has only been used for fully dormant

grapevine planting material. The method is hot water treatment (50◦C for 45 min; EFSA, 2015b).

Vector control

Considering the available information on transmission, vector ecology, and disease epidemiology,

management strategies should be established to reduce adult vector population density and to avoid

or mitigate both primary (from alternative hosts or between orchards) and secondary (tree-to-tree

spread within an orchard) spread in combination with other measures aimed at reducing the inoculum within the crop (e.g., planting healthy nursery trees and roguing of infected trees). For a review of the

available chemical, biological, and cultural control methods for olive refer to Bosco et al. (2019). For

control of sharpshooters in South and North America, refer to a recent review by Krugner et al. (2019).

Effective insecticides are available for conventional farming, but resistance rate in insect populations

to the widely used insecticides is increasing limiting their efficiency. Mass releases of egg parasitoids

of insect vectors have already encountered some success in California, but a key element remains to

identify parasitoid species capable of suppressing sharpshooter populations early in the season to

suppress the first annual generation (in spring months). Little is known about biological control agents

of vectors in Europe and South America. Control of insect vectors in Italy can also be achieved by

practices such as soil tilling in spring to suppress the population of nymphs on herbaceous plants found in olive orchards (Regione Puglia, 2016). A combination of physical practices and use of chemicals may

have additive effects.

Breeding resistant varieties to prevent pest damage

Various degrees of plant resistance, tolerance, and susceptibility have been observed in economically

relevant crops such as grape, citrus, almond, and olive. Genetic traits that confer resistance/tolerance

to Pierce’s disease have been identified and followed by conventional breeding programs to control

Pierce’s disease of grapevine. Tolerant/resistant cv were identified and characterized for citrus,

prunus, and olive tree. A resistance-associated locus, PdR1, was introgressed in Vitis vinifera and

cultivars combining agricultural and organoleptic characters were selected, tested and some are pre-

released (Walker and Tenscher, 2012; Walker et al., 2017). Breeding programs are ongoing to search

for alternative sources of resistance. Programs are ongoing to characterize susceptibility in Prunus

hybrids (Rogers and Ledbetter, 2015; Ledbetter and Lee, 2018), and search for resistant rootstocks

(Krugner et al., 2012b; Krugner and Ledbetter, 2016).

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All C. sinensis cultivars are susceptible to X. fastidiosa infection, except cv. Navelina ISA 315, which is

resistant (Fadel et al., 2014). Different levels of resistance/tolerance were observed in Citrus spp. and

hybrids, with mandarins (C. reticulata), tangors (C. sinensis × C. reticulata) and lemons (C. limon)

generally considered resistant (reviewed in EFSA, 2019b).

An extensive screening for olive tree resistance to X. fastidiosa was initiated in 2015 and progressively

extended in the following years under the auspices of the POnTE and XF-ACTORS H2020 programs. The

screening trials include hundreds of cultivars/accessions and feral olive seedlings that have been

mechanically inoculated under controlled greenhouse conditions and/or exposed to natural inoculum pressure in experimental plots in Apulia, Italy. Olive tree cv. Leccino has been identified as tolerant to

X. fastidiosa based on lower incidence, bacterial population, and symptom severity when compared to

cv. Ogliarola salentina, which is highly susceptible to X. fastidiosa p ST53 (Boscia et al., 2017). The

cultivar, FS17®, exhibits also no or mild development of symptoms and reduced prevalence of

X. fastidiosa. The study is ongoing.

Genetically modified plants

The gene rpfF encodes for a diffusible factor (DSF) involved in quorum sensing behaviour of

X. fastidiosa. This DSF up regulates factors required for biofilm formation and represses genes

encoding traits necessary for plant colonization (Newman et al., 2004). In a confusion approach to

control X. fastidiosa, genetically-modified (GM) grapevines producing DSF have been constructed and

tested in field conditions. Constitutive production of DSF in grapevines reduces movement and

systemic colonization by X. fastidiosa, which in turn translates into reduced disease symptoms (Lindow

et al. 2014). In DSF grapevines that remained symptomless, bacterial populations are highly aggregated. X. fastidiosa acquisition by insect vectors is then enhanced in DSF plants that do not show

symptoms and are more attractive to insect vectors that prefer to feed on healthy or asymptomatic

plants. In these plants, when insects acquire bacteria they acquire greater population size of

X. fastidiosa than on wild-type plants. Altogether, this leads to a lower but highly variable probability

of transmission from DSF plants than to control plants (Zeilinger et al., 2018). Hence, these GM plants

do not represent a satisfactory option for controlling X. fastidiosa in vineyards.

Hfx proteins (hemagglutinins) are involved in X. fastidiosa virulence and cell-to-cell aggregation

(Guilhabert and Kirkpatrick, 2005). Transgenic Hfx-expressing grapevines were shown to delay the

spread of X. fastidiosa but were unable to provide long-term protection against the bacterium (Gilchrist and Lincoln, 2016).

Polygalacturonase-inhibiting proteins (PGIP) are produced by plants to block cell wall-degrading

enzymes that are X. fastidiosa virulence factors (Esquerré-Tugayé et al., 2000). Dandekar et al. (2010)

selected a line with high PGIP activity (TS-50) that was evaluated in field studies during seven years as

rootstock and proved efficient to protect the scion from Pierce’s disease (Dandekar et al., 2017).

A chimeric transgene expressing an antimicrobial protein made of an elastase that recognizes and

cleaves MopB, a conserved outer membrane protein of X. fastidiosa f and a lytic peptide, which targets

conserved lipidmoieties and creates pores in the X. fastidiosa f outer membrane has been used to transform grape lines (Dandekar et al., 2012). These transgenic grapevines gave promising results and

novel constructs are being tested in the field (Dandekar et al., 2017). Other constructs, such as the

combination of transgenes, are currently being tested to provide longer-lasting and more robust

protection against X. fastidiosa in grapevines (Gilchrist et al., 2017)

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Agricultural practices

Impact of common agricultural practices, including pruning, irrigation and fertilization were evaluated

on transmission efficiency by insect vectors, symptom occurrence and/or X. fastidiosa population sizes.

Irrigation: Water stress impact the physiology of the host, its symptomatology, but also insect vector

behavior. McElrone et al. (2001) showed that bacterial leaf scorch symptoms of Virginia creeper

(Parthenocissus quinquefolia) were enhanced during drought stress in a greenhouse study. It was

suggested that maintaining plant vigor with regular watering can be used to sustain plants infected by X. fastidiosa, particularly during periods of water stress. In the same host, the whole shoot hydraulic

conductance caused by X. fastidiosa infection acts additively with the water limitation imposed by

drought stress (McElrone et al., 2003). Controlled deficit irrigation regimes in crops reduce vector

population density (Krugner et al. 2009), alter vector movement and dispersal behaviors (Krugner et

al. 2012c), and reduce duration of vector feeding behaviors associated with transmission of

X. fastidiosa (Krugner and Backus 2014). Further research showed that rates of X. fastidiosa

transmission to grapevines are affected by the intensity of plant water stress (Del Cid et al 2018).

Collectively, research indicates that moderate plant water stress enhances pathogen spread while

severe or no stress produce equivalent spread.

Fertilizer application: Zinc concentration levels in the plant may have a role in the establishment and

growth of X. fastidiosa (Navarrete and De La Fuente, 2015), but no practical treatment has yet been

proposed. N-Acetylcysteine (NAC) is an analogue of cysteine. It disrupts disulfide bonds in mucus.

Fertigation with NAC decreases both symptoms and bacterial growth rate in treated plants compared

to controls. Symptoms returned after treatment stopped in some of the treated plants. Although the

reported results showed that NAC had an antibacterial effect against X. fastidiosa (Muranaka et al.,

2013), it did not demonstrate that this measure provides a full control of the disease. Dentamet® (zinc,

copper and citric acid biocomplex) sprays may lead to a reduction in disease severity compared to

untreated trees, but the results did not demonstrate that Dentamet® provided a full control of the disease over the 3 years of the experiment (Scortichini et al., 2018).

Pruning/Roguing: Successful elimination of X. fastidiosa infection and disease symptoms by pruning

was reported from sweet oranges in Brazil (Amaral et al., 1994), but only in very specific conditions

and at the early stages of infection, before systemic infection occurred (Coletta-Filho & De Souza,

2014). Hopkins and Purcell (2002) reported that summer and autumn pruning of grapevines may

eliminate recent bacterial infections occurring on the outer canopy of grapevines (cane tips). Sisterson

and Stenger (2013) used modelling to identify roguing (removal of infected perennial trees) as a

potential management strategy for perennial crops. Results showed that successful application of

roguing depended on area-wide adoption of the approach (as opposed to few individual farms) and ability to quickly identify infected trees for removal.

Biotic environment and soil management: Nymphal populations of insect vectors thrive on herbaceous

hosts, removing these alternate hosts of insect vectors drastically limits their population (EFSA, 2016).

Removal of alternative host plants from riparian areas and citrus groves was proposed for coastal

vineyards in California (EFSA, 2015a).

Biological control

Reduction of disease severity was observed after spray applications of DSF homologues-containing

products, such as palmitoleic acid or macadamia oil soap 2 weeks before inoculation with X. fastidiosa

(Pierce’s disease strain), and monthly applications afterwards (Lindow et al., 2017).

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Application of the endophytic bacterium Paraburkholderia phytofirmans strain PsJN to control Pierce’s

disease shows promises (Baccari et al., 2019; Lindow et al., 2018), as was the case of the application

of an endophytic strain of Curtobacterium flaccumfaciens to control X. fastidiosa p ST53 in the model

plant Catharanthus roseus (Madagascar periwinkle) (Lacava et al.; 2007). Rolshausen et al. (2018)

explore the effect of a mixture of endophytes isolated from grapevine (two bacterial strains belonging

to Pseudomonas fluorescens and Achromobacter xyloxosidans, and one fungus, Cochliobolus sp.) to

control Pierce’s disease of grapevine. Here, again results are promising. However, none of these

studies demonstrated that the use of the biological control agents or mixtures could provide full

control of the disease, i.e. eliminating X. fastidiosa populations and leading to the absence of dissemination by vectors.

The use of bacteriophages is also a tempting strategy that has shown some promises in greenhouse

tests of grapevine protected by a cocktail of four bacteriophages (Ahern et al., 2014; Das et al., 2015).

Past attempts to eradicate X. fastidiosa

Eradication of X. fastidiosa by the complete removal of infected plants was attempted in Brazil to

eradicate citrus variegated chlorosis (Lopes et al., 2000; Machado et al., 2011) and in Taiwan to fight

Pierce’s disease of grapevine (Su et al., 2013), but both attempts proved unsuccessful. In Brazil, the

percentage of infected plants increased from 15.7 % in 1994 to 34 % in 1996 (Lopes et al., 2000).

Although , it has been reported that 40 % of the 200 million sweet orange plants in São Paulo state

were infected by X. fastidiosa (Almeida et al., 2014), more recently the disease incidence drop to

around 2%, in general (https://www.fundecitrus.com.br/pdf/levantamentos/levantamento-doencas-

2019.pdf). The use of X. fastidiosa free-plants to planting as a mandatory program started in 2003 whose plants represent 90% of all citrus plants in production in São Paulo State nowadays and the

minimization of vectors populations are pointed as the mainly factors for the well successful

management of CVC (Coletta-Filho, personal communication. In Taiwan, despite the removal of

thousands of infected grapevines since the first record of the disease in 2002 the disease is now

established (Su et al., 2013).

A list of factors influencing the success of eradication programs was established by Myers et al., 1998.

Among those factors, some factors linked to the biology of X. fastidiosa are difficult to meet. These are

for example i) the difficulty to early detect infection as a consequence of long asymptomatic infections,

ii) cost of efficient and sensitive detection methods that limit large scale monitoring, iii) large host range, and iv) dissemination by insect vectors.

Overall, considering the potential impact of X. fastidiosa, eradication remains the first option to

consider in case of recent introductions, while adapting the control strategy on a case by case situation.

Challenges related to prevention and control technologies

-Definitions of tolerance and resistance are still a matter of debate between scientists. Refining the

definition and providing extensive description of the situation (symptom characteristics, population,

sizes, dynamics, …) should help to clarify the debate.

-Efficiency of the eradication strategy in case of vector borne disease with a large host range is largely

dependent on a very rapid and efficient detection and identification of the initial introduction and

area-wide buy-in (adoption) from growers in the region (Sisterson and Stenger, 2013).

-Research on insecticides is required to determine what, when, where and how to apply products to

maximize mortality of the target pest while minimizing non-target effects.

Nov 27, 2019, G20-MACS Transboundary and emerging pests: Xylella fastidiosa discussion group report

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-When eradication is not feasible, containment status might be declared and accompanied by

measures to limit inoculum development. Removing inoculum sources is a key point of the

containment strategy. Public awareness is essential to ensure proper application of the strategy.

-For the design of a surveillance strategy, the critical early detection should be targeted in case of

emergence

-To prevent domestic introduction of X. fastidiosa infected materials, the reinforcement of public

awareness, measures at airport and ports are of critical importance.

-The adoption of regulatory measures and efficient detection measures at entry sites aimed at avoiding

X. fastidiosa introduction. Performance of detection measure is a challenging aspect to ensure

X. fastidiosa entry and establishment.

-Current vector control methods include insecticide application, release of natural enemies, and

cultural methods to suppress immature stages. Further investigations on classical, augmentative, and

conservational biological control could improve the impact of natural enemies on vector population.

-Insecticides are effective in suppressing vector populations, but development of insecticide resistance has been a concern. Identification of new insecticide chemistries to be included in a rotation program

could help slow down development of resistance.

-Public/grower resistance to eradication campaigns as has been seen in southern Italy

(https://www.oliveoiltimes.com/olive-oil-business/europe/charges-dropped-against-scientists-in-

lecce-xylella-case/67868)

3. Challenges and future directions for international research

collaboration

In the previous parts of the report, the presentation of the current situation regarding the key items

allowed to highlight challenges concerning basic knowledge required to improve methods to avoid

X. fastidiosa entry into yet uninfected area, limit its spread, and control it. In this last part of the

document, we attempted to identify future directions for international research collaboration that go

beyond those identified in 2016 during the workshop organized by EFSA on Xylella fastidiosa to identify

knowledge gaps and research priorities for the EU.

Still in need for improved survey and sampling methods: There is a need for improving survey and sampling strategies for accurate detection. Survey

should consider the wide potential host range in natural environments and crops, the irregular

distribution of X. fastidiosa in time along the seasons, while sampling should take into account

distribution in plant. The factors underlying the irregular distribution in time are still

unappreciated. A better knowledge of dynamics of X. fastidiosa population sizes within the

plant, along the seasons, and the years for several Xf × host combinations is hence required.

Designing survey scheme and sampling strategies adapted for asymptomatic i.e. lowly infected

material would improve confidence on tests at entry. Understanding of X. fastidiosa

interactions with the other actors of the ecosystem is still limited, and especially during the

asymptomatic period when population grows.

Innovative technologies such as remote sensing using different imageries has already shown

some promises for early detection on large orchards of olive trees. Application and adaptation

of this type of technologies remain a challeng for other species, other planting conditions, and

at a landscape scale.

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Modeling should help to design adapted protocols for sampling large consignments at entry,

considering the asymptomatic infection and large lot sizes.

Insect and plant sentinel strategies could be of significant help to determine current distribution

and to detect introductions. These are non-destructive surveillance methods and does not rely

on destructive sampling, at least in the case of sentinel insects. However, conditions and means

of application of these sentinel strategies deserve more information and hence research.

Indeed, parameters such as the species, the site for establishment of sentinels and their

surveillance, the sampling protocol and monitoring timeframe are critical for efficiency of these

strategies. Data obtained from such surveys could help elucidate prevalence of target

X. fastidiosa within areas affected by multiple bacterial genotypes. This approach could also

provide important information regarding acquisition efficiency of bacteria by vectors during

asymptomatic periods. Work can be performed in separate laboratories using regional vector

species.

Still in need for improved detection methods: Developing and improving detection protocols, but also rethinking detection methods to allow

tracking strains present at very small population sizes in plants is one of the key to gain

knowledge on asymptomatic colonization of plants and hence early detection of introductions,

but also differentiate outbreak with limited dispersal and epidemic development. This requires

large program to test complementary approaches including survey, sampling and detection per

se.

While a lot has been done on detection methods, there are still gaps as a consequence of the

large host range of the pest, the irregular and discontinuous distribution in plant complicating

sampling. Research projects should combine sound analysis of X. fastidiosa metabolism and

physiology to improve detection and isolation, by a better understanding of interactions during

host colonization. Still a lot of questions are pending concerning the performance of detection

methods that are challenged by the large range of plant species and potential inhibitors. The

need of complementary methods, the determination of the efficiency of DNA extraction

methods and the detection thresholds in various plant species, the need of spiked controls are

key elements that should be provided for detection methods in the frame of interlaboratory

performance tests.

Although challenging to implement for surveillance or large scale sampling, use of whole

genome sequencing bsed on Next Generation Sequencing (NGS) in detection and diagnostics

has the potential to give much better resolution to strain identification and outbreak tracking.

Future research should focus on development of rapid, inexpensive, and automated pipelines

for genome sequencing, assembly, and annotation to complement and extent existing PCR

based identification.

Assessing performance of detection tests on the range of potential insect vectors is essential,

as are establishments of potential vector occurrence and host plant distribution.

Mechanisms of interactions among X. fastidiosa and other actors of the

ecosystem to identify Achille’s heel. X. fastidiosa biology

X. fastidiosa is characterized by a fastidious behavior that translate slow multiplication rates both in

planta and in vitro. Fine characterization of the nutrient status of its environments (xylem fluids of

various hosts and foregut of insects) in terms of composition and elementary quantification in

association with a refine understanding of X. fastidiosa metabolic network will help to identify key

targets for improving in vitro growth conditions, but could also help to identify targets for a specific

control.

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The impact of environmental parameters (chemical parameters such as carbon and nitrogen sources,

physical parameters such as temperature, flow velocity) on X. fastidiosa physiology still requires

consolidated understanding to design ad-hoc control methods including agricultural practices.

X. fastidiosa – host plant interactions

The asymptomatic period challenges establishment of host list and parameters of population

genetics (generation time), has consequences on resistance-tolerance evaluation, and development

of inoculum reservoirs in the environment. Developing basic research program to highlight

mechanisms of interactions between the bacterium and the host during this asymptomatic period

would provide data to improve its monitoring, as will research on sentinel plants and insects.

Considering the various challenges related to host range determinants and the large access to

genome data in X. fastidiosa, a large collaborative program should be undertaken to identify

determinants associated to traits involved in pathogenicity (aggressiveness or amounts of symptoms

and virulence or host range) and their natural variation in X. fastidiosa populations. This will also

help to identify limitations of control methods targeting specific pathogenicity factor.

Understanding the molecular dialogue actors (virulence factors and defense mechanisms) is still an

emerging research field while identification of virulence factors of X. fastidiosa has been the subject

of research projects since decades. Improving control methods through targeted breeding should

beneficiate from a better understanding of this dialogue.

Impact of environmental conditions, and especially temperature, on X. fastidiosa physiology in

plants and in consequence survival, but also dispersal abilities requires a deep understanding to be

be transferred as control measures.

In planta biology of X. fastidiosa and vectors

While considerable recent knowledge has been acquired on X. fastidiosa, a lot still needs to be

addressed concerning insect vectors. Basic knowledge on flight distance, reproductive biology, plant-

vector trophic networks and mechanisms of interaction between insect (foregut cells) and bacterial

cells require sound developments.

Identification of periods of the season when the pathogen and vectors are more likely to be detected

could improve efficiency and accuracy of programs designed to monitor new introductions or

existing populations.

As host range depends partially on insect behavior, characterizing determinants of bacterium –

insect interactions, insect behavior and especially trophic networks are other key determinants that

require considerable research efforts. Understanding of insect movement and dispersal behaviors,

and feeding behaviors (e.g., number of plants and plant species visited) may help explain patterns

of pathogen spread. Research using mark-and-capture techniques may be used to investigate insect

mobility in the landscape.

Related to this, although a lot of work has been done to characterize virulence of different strains in

different host plants, very little is known regarding acquisition rates of the pathogen from a wider

range of host plants by a wider range of insect vectors. This would require extensive research efforts

focused on vectors from different areas, and acquisition experiments from different host plants

beyond just major crop species.

There has been intense interest in insect microbiome research. Very little is known about the role of

vector symbionts in insect developmental parameters and interaction with X. fastidiosa in insect

foreguts. While detection and identification of insect symbionts is becoming more common, future

research should start focusing more on describing the function of other organisms coexisting with

X. fastidiosa.

Interactions involving X. fastidiosa in its ecosystem

In planta but also in the foregut of insects, X. fastidiosa does not evolve in axenic environment, it

interacts with the other members of the microbiome. This is a nearly virgin field of research. But is

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has potentially profound impacts on potential biocontrol methods, and hence needs to be

deciphered.

How X. fastidiosa disperse? Research projects should allow a better understanding of X. fastidiosa dispersal through seeds,

if any, as data availability are yet more than limited. Note here that dealing with seed

transmission implies considering very large seed lots to be meaningful, as the unit of interest is

the orchard or field size. Assessing X. fastidiosa dispersal through seeds and infected

material/equipment is essential to limit short range spread.

Large experiments allowing to test for the various parameters involved in spread (plant factors,

insect determinants, and the environmental factors) should be run in infected area. The impact

of individual parameters can be tested in confined conditions that apply in non-infected

countries for X. fastidiosa experiments.

Assessing potential impact of X. fastidiosa introductions: Impact of X. fastidiosa on biodiversity including wild and endemic species, through large

naturalistic approaches, sentinel strategy and modeling should be assessed to estimate overall

impact and predict modifications of the environments.

Future research directed at quantifying impacts should include minor crops and ornamentals as

well as crops involved in major outbreaks (citrus, olive, grape).

Prevention and control technologies: The limited availability of curative measures and efficiency of prophylactic measures to control

X. fastidiosa infections and epidemics renders the development of research projects targeting

the various potential options highly important. As previously mentioned, the design of methods

aiming to cure infected plant materials have to be tested and are essential for trade of host

plant material. Similarly, improving plant tolerance or resistance to vectors and X. fastidiosa is

a key issue. An important limitation to these developments is that the efficiency of these

methods are certainly plant species specific and hence their development should be declined

per host species.

Applied research project should focus on the development of control methods targeting

specifically potential insect vectors in plant material to be trade and methods to clear bacterial

infection in trade plant material.

Concerning biological control, very encouraging preliminary data were obtained with a strain of

Paraburkholderia phytofirmans. Mechanism of activity should be deciphered and efficacy for

various combination of X. fastidiosa and host combination should be tested.

To breed for resistance, the genetic bases of such incompatible interactions should be identified

per targeted X. fastidiosa and host combination, but requires significant human and financial

support.

A better understanding of interactions involving X. fastidiosa in its natural environments, i.e.

with the xylem microbiome, insect microbiome, insect cells and plant defense system is

essential to improve plant defense or develop biocontrol methods to limit X. fastidiosa

multiplication and/or survival or prevent establishment. This can be part of a large international

collaborative research program considering the range of host plants, insect vectors and strain

to combine to have knowledge applicable in various conditions.

Interference with vector communication behaviours has received attention in the last few

years. While research on vector communication has focused on the glassy-winged sharpshooter

and Philaenus spumarius, nothing is known about communication signals of other vectors.

Knowledge of such behaviours may lead to development of novel vector control strategies, as

it was demonstrated for the glassy-winged sharpshooter (Krugner and Gordon, 2018).

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Risk managers need treatment solutions against vectors and pathogen concern chemical,

biological and mechanical tools (IPM).

Challenges with awareness and preparedness toward Xylella introductions and establishment

lie in reluctance to apply eradication procedures, including insecticide application. Humanities

and social sciences shall be brought in project to build on previous experiences to clarify

conditions for adoption of eradication/containment methods and consider the potential

unintentional consequences of such strategies.

Clarification of factors involved in “actual risk vis-a-vis perceived risk” of affected communities

should allow the design of targeted measures for specific targeted audiences (e.g. operators,

general public, etc.).

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