# Several subspecies and sequence types are associated with the emergence of Xylella fastidiosa in natural settings in France

Nicolas Denancé, B. Legendre, Martial Briand, V. Olivier, C. de Boisseson, F.

Poliakoff, Marie Agnes Jacques

► To cite this version:

Nicolas Denancé, B. Legendre, Martial Briand, V. Olivier, C. de Boisseson, et al.. Several subspecies and sequence types are associated with the emergence of Xylella fastidiosa in natural settings in France. Plant Pathology, 2017, 66, 10.1111/ppa.12695. hal-01607254

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# Several subspecies and sequence types are associated with the emergence of *Xylella fastidiosa* in natural settings in France

N. Denancé<sup>ab†</sup>, B. Legendre<sup>b†</sup>, M. Briand<sup>a</sup>, V. Olivier<sup>b</sup>, C. de Boisseson<sup>c</sup>, F. Poliakoff<sup>b</sup> and M.-A. Jacques<sup>a*, d</sup>

<sup>a</sup>IRHS, INRA, AGROCAMPUS-Ouest, Université d'Angers, SFR4207 QUASAV, 42, rue Georges Morel, F-49071 Beaucouzé; <sup>b</sup>Anses Laboratoire de la santé des végétaux, F-49044 Angers Cedex 01; and <sup>c</sup>ANSES Ploufragan, Viral Genetics and Biosecurity, F-22440 Ploufragan, France

*Xylella fastidiosa* is a plant pathogenic bacterium emerging in Europe. In France its emergence has been demonstrated through interceptions of contaminated coffee plants and, in 2015, by a survey of natural settings. The first French focus of contamination was detected in 2015 in Corsica; since then, almost 300 foci have been found and nearly 30 plant species have been declared contaminated, with *Polygala myrtifolia* remaining the principal host, suffering from severe leaf scorch. This study reports on the diversity of *X. fastidiosa* identified in France in 2015. Multilocus sequence analysis/typing revealed the presence of mainly *X. fastidiosa* subsp. *multiplex* sequence types (STs) ST6 and ST7. A focus of *X. fastidiosa* subsp. *pauca* ST53 was identified in mainland France; one sample contaminated by *X. fastidiosa* subsp. *sandyi* ST76, one novel recombinant, and coinfections of different isolates in individual samples were also identified, but could not be confirmed by successive samplings, indicating limited or transient contamination. Koch's postulates were fulfilled for two isolates of *X. fastidiosa* subsp. *multiplex* on *P. myrtifolia*, one being ST6 and the other ST7. Comparative genomics of the genome sequences of three French isolates (one ST6 and two ST7) with available sequences revealed that, unlike the American Dixon strain, the French ST6 and ST7 strains are devoid of a plasmid encoding a complete type IV secretion system. Other differences regarding phage sequences were highlighted. Altogether, the results suggest that the emergence of *X. fastidiosa* in France is linked to several introduction events of diverse strains from different subspecies.

**Keywords:** coffee, comparative genomics, multilocus sequence typing, *Xylella fastidiosa* subsp. *multiplex*, *Xylella fastidiosa* subsp. *pauca*, *Polygala myrtifolia*

## Introduction

Originally confined to the Americas, *Xylella fastidiosa*, a bacterial plant pathogen, recently emerged in Asia and Europe. In 2013, this pathogen was reported in Taiwan on grapevine (Su *et al.*, 2013). The same year, severe leaf scorch of olive trees, associated with *X. fastidiosa*, was reported from Apulia, Italy (Saponari *et al.*, 2013). A year later, this pathogen was detected in Iran in grapevine and almond trees (Amanifar *et al.*, 2014), and, in 2015, in France in *Polygala myrtifolia* (https://gd.eppo.int/taxon/XYLEFA/distribution/FR). In 2016, Spain also declared a focus of *X. fastidiosa* subsp. *fastidiosa* in the Balearic Islands (http://www.mercacei.com/noticia/46381/actualidad/primer-positivo-oficial-de-xylella-fastidiosa-en-espana.html). In addition, several outbreaks occurred in confined places in Europe, such as in 2016 in Germany, where oleander and rosemary plants in a

nursery were found to be contaminated by *X. fastidiosa* subsp. *fastidiosa*; these outbreaks were eradicated or are under eradication (https://gd.eppo.int/taxon/XYLEFA/distribution). At the same time, *X. fastidiosa* has been detected in coffee plants originating from various countries in Latin America that were intercepted in Europe (Jacques *et al.*, 2016; Loconsole *et al.*, 2016; https://gd.eppo.int/taxon/XYLEFA/distribution).

*Xylella fastidiosa* is a genetically diverse species subdivided into six subspecies, each one being more or less specific to a particular host range and a native zone in the Americas. The four most frequently reported subspecies are (i) *X. fastidiosa* subsp. *fastidiosa*, which causes Pierce's disease in grapevine and which was also recovered from various trees and other perennials (Janse *et al.*, 2012) and (ii) *Xylella fastidiosa* subsp. *sandyi* causing oleander leaf scorch. These two subspecies are supposed to have been introduced into the USA from Central America (Nunney *et al.*, 2010; Yuan *et al.*, 2010). (iii) *Xylella fastidiosa* subsp. *multiplex* is associated with scorch diseases of a large range of trees. This subspecies is mostly found in temperate climates of

<footnote>*E-mail: marie-agnes.jacques@inra.fr</footnote>

<footnote>†These authors contributed equally to this work.</footnote>
North America and has been introduced into South America (Nunes *et al.*, 2003). (iv) *Xylella fastidiosa* subsp. *pauca* is mostly found in South America on *Citrus* spp. and *Coffea* spp. (Almeida *et al.*, 2008). However, strains from this subspecies recently emerged in olive trees in Italy, Argentina and Brazil (Elbeiano *et al.*, 2014; Haelterman *et al.*, 2015; Coletta-Filho *et al.*, 2016) and were also recovered from coffee and oleander in Central America and Mexico (Nunney *et al.*, 2014b; Jacques *et al.*, 2016; Loconsole *et al.*, 2016). Collectively, strains belonging to the species *X. fastidiosa* cause diseases on more than 350 plant species (European Food Safety Authority, 2016a), but the most economically important diseases occur on grapevine in California, citrus in Brazil, and olive trees in Italy (European Food Safety Authority, 2015).

*Xylella fastidiosa* is naturally dispersed over short distances by a large range of sap-feeding insects (European Food Safety Authority, 2015), but long-distance dispersal depends predominantly on the human-mediated movement of infected planting and propagating material. Importation of coffee plants from the suspected area of origin of the agent of Pierce's disease has been linked to the first known outbreak of Pierce's disease in the USA (Nunney *et al.*, 2010). Similarly, plum leaf scald is supposed to have been introduced in the 1930s in Brazil by contaminated plant material (Nunes *et al.*, 2003). It has been documented that recombination contributed more to the genetic diversity of *X. fastidiosa* than point mutation (Nunney *et al.*, 2012); several cases of intersubspecific recombination events associated with host shifts have been reported (Nunney *et al.*, 2012, 2014a,b). Thus, the risk associated with the mixing of previously isolated strains should be avoided as this could result in novel genetic combinations with new host ranges.

Detection and identification of *X. fastidiosa* is currently based mostly on PCR tests. Several tests were proposed to detect *X. fastidiosa* in plant material and, among them, the amplification of a small fragment (180 bp) of *rimR* (16S rRNA processing gene) using qPCR was shown to be specific and sensitive (Harper *et al.*, 2010). Consequently, this test is included in the reference method currently used in France (https://www.anses.fr/fr/system/files/ANSES_MA039_Xylellafas tidiosa_final.pdf). Once *X. fastidiosa* is detected in plant material, further identification of the isolates can be based on PCR tests, such as the one developed by Hernandez-Martinez *et al.* (2006) to differentiate strains from three *X. fastidiosa* subspecies, *fastidiosa*, *multiplex* and *sandyi*; another test was designed to specifically identify strains of *X. fastidiosa* subsp. *pauca* (Pooler & Hartung, 1995). However, the bacterium identification is most often based on multilocus sequence analysis (MLSA) and its derivative, multilocus sequence typing (MLST). These methods are used for taxonomical and epidemiological purposes, respectively. A dedicated scheme has been proposed for *X. fastidiosa* and is currently used (Yuan *et al.*, 2010; European Plant Protection Organization, 2016; Jacques *et al.*, 2016).

Fragments of seven housekeeping genes, *cysG*, *gltT*, *holC*, *leuA*, *malF*, *nuoL* and *petC*, are amplified, ending in a 4161 bp sequence of concatenated data, and subsequent analyses rely on phylogenetic methods and allele assignation (http://pubmlst.org/xfastidiosa/).

*Xylella fastidiosa* was first detected in *P. myrtifolia* shrubs in Corsica, France in July 2015 and later, in October 2015, in mainland France (http://www.corse-du-sud.gouv.fr/IMG/pdf/Reunion_Xylella_21-01-16.pdf). These ornamentals presented symptoms, showing severe leaf scorch. Large surveys were undertaken to evaluate the phytosanitary status of the territory and measures were carried out to eradicate outbreaks. Throughout the same year, plants of coffee were intercepted and analysed for suspected contamination by *X. fastidiosa*. The objectives of the present study were to identify and further characterize the isolates of *X. fastidiosa* associated with leaf scorch detected in 2015 in France, and to identify probable routes of introduction.

## Materials and methods

### Analysis of plant material

Plants suspected to be infected by *X. fastidiosa*, based on symptomatology, were sampled by various national and regional services. Samples were sent for analysis to the plant health laboratory of Anses at Angers, France and/or, from November 2015, to laboratories officially certified by the Ministry in charge of agriculture (Rural code for agriculture and fisheries, articles L202-1 and R202-8 to R202-21). Sample analysis was performed as described in https://www.anses.fr/fr/system/files/ANSES_MA039_Xylellafastidiosa_final.pdf. Briefly, surface-sterilized fragments (0.5–1 g) of petioles and midribs were ground in demineralized sterile water (5 mL g⁻¹). Total genomic DNA was extracted using a commercial kit (QuickPick SML Plant DNA; Bio-Nobile) and a robot (KingFisher mL; Thermo Fisher Scientific).

### Detection of *X. fastidiosa* in plant material

The first step of detection of *X. fastidiosa* was based on qPCR using *X. fastidiosa*-specific primers and probe (XF-F, XF-R and XF-P) and TaqMan Fast universal PCR Master Mix 2× (Applied Biosystems), following the method of Harper *et al.* (2010). In specific cases, isolation of bacterial strains was attempted. Aliquots of plant extracts were streaked on modified PWG medium (European Plant Protection Organization, 2016). Plates were incubated for up to 30 days at 28 °C.

### Bacterial identification, typing and genome analyses

The multiprimer PCR identification test (Hernandez-Martinez *et al.*, 2006) was performed using DNA previously extracted from novel foci of disease and/or novel host plants. In order to allocate strains to sequence types (STs), housekeeping genes (Yuan *et al.*, 2010) were amplified from extracted DNA following the protocol described in European Plant Protection Organization (2016), with the following modifications. GoTaq G2 polymerase (Promega) and primers at a final concentration of 0.5 µM were employed. The PCR mix (50 µL) was composed of 10 µL Green GoTaq Reaction Buffer (5×), 5 µL each primer
(5 µM), 5 µL dNTPs (2 mM), 0.7 µL GoTaq G2 polymerase (5 U µL⁻¹), 4 µL DNA and 20.3 µL ultrapure water. Additionally, the melting temperature was fixed at 60 °C and elongation time was reduced to 45 s. PCR product sequencing and sequence analyses were carried out as described by Jacques *et al.* (2016). The genomes of the first three strains isolated were sequenced using Illumina Mi-Seq technology (Plateforme Génomique de Nantes, IRT-UN, France). Phylogenetic analyses were performed on the dataset of concatenated sequences obtained in this work and available from previous analyses for a collection of strains representing the *X. fastidiosa* diversity as previously described (Jacques *et al.*, 2016). Briefly, sequence alignment was performed with the MUSCLE algorithm using the default parameters in GENEIOUS v. 8.0.5 (www.geneious.com), and a maximum likelihood (ML) tree was constructed with 1000 replicates for bootstrap values using MEGA v. 6.0 (www.meg software.net).

## Pathogenicity tests on *P. myrtifolia*

The pathogenicity of *X. fastidiosa* strains CFBP 8416 and CFBP 8418 was tested on 1.5-year-old plants of *P. myrtifolia* grown in a confined growth chamber at 24 °C with 16 h of daylight and at 20 °C during night, under 70% relative humidity. Plants were watered daily with water supplemented with 1.4 g L⁻¹ nitrogen:phosphorus:potassium fertilizer (16:8:32). Ten leaves on two different stems of nine plants were inoculated per strain by the needle puncture method. Five drops (10 µL each) of inoculum were placed on the leaf petiole and/or vein and punctured with a needle. After 13 weeks, symptoms were monitored and samples with symptoms were analysed. Samples were tested by qPCR, as previously described by Harper *et al.* (2010) and bacterial isolations carried out on PWG or B-CYE medium according to the method of Jacques *et al.* (2016). Plant inoculations were carried out under quarantine at IRHS, Centre INRA, Beaucouzé, France under the agreement no. 2013119-0002 from the Prefecture de la Région Pays de la Loire, France.

## Nucleotide sequence accession numbers

The genome sequences of *X. fastidiosa* strains CFBP 8416, CFBP 8417 and CFBP 8418 reported here have been deposited in GenBank under accession numbers LUYC00000000, LUYB00000000 and LUYA00000000, respectively.

## Results

### Interception of imported coffee plants infected by *X. fastidiosa* subsp. *sandyi* and subsp. *pauca*

Within the framework of the *X. fastidiosa* national survey and control plan, coffee plants were checked upon importation. In 14 different interceptions of coffee plant material, 21 out of 135 samples of coffee plants were found to be contaminated by *X. fastidiosa*. These plant materials were imported from various Latin American countries and were intercepted in several regions in France Provence-Alpes-Côte d'Azur, i.e. PACA, Pays de la Loire, Centre and Ile de France). Among the 21 infected samples, nine were successfully typed by MLST. *Xylella fastidiosa* subsp. *sandyi* ST72 was identified in two coffee plants sampled in the Ile de France and Pays

de la Loire regions. *Xylella fastidiosa* subsp. *sandyi* ST76 was identified in three coffee plants in Ile de France. *Xylella fastidiosa* subsp. *pauca* ST53 was detected in four samples of coffee plants intercepted in the Ile de France and Pays de la Loire regions. The strains from the remaining infected plants could not be assigned to sequence types because several housekeeping genes could not be amplified and/or the sequences could not be obtained. In some cases, partial data suggest a possible infection by *X. fastidiosa* subsp. *sandyi* ST72 or ST76 (data not shown).

### A large range of plants are infected by *X. fastidiosa* in France

Besides the imported infected coffee plants, the first contaminated plant in a natural setting was identified in France in July 2015. From 31 December 2015, the bacterium was found in 237 foci in Corsica and 10 foci in mainland France. Most contaminated samples were *P. myrtifolia* (84% of the positive plants), but *X. fastidiosa* was also found in plants belonging to 21 different species (Table 1). In 2015, the survey in these two areas represented a total of 5962 analysed plant samples, among which 528 samples (8.9%) were declared contaminated by *X. fastidiosa*; these samples originated mostly from southern Corsica. The most frequently positive species was *P. myrtifolia* (29.5% of the tested samples were positive); *Pelargonium* spp., *Cistus* spp., *Lavandula* spp. and *Spartium junceum* represented 11% of the positive samples. Most importantly, no *Vitis*, *Citrus* or *Olea europaea* were detected positive to *X. fastidiosa* despite 69 *Vitis* spp., 238 *Citrus* spp. and 504 *Olea* spp. plants from Corsica being analysed.

In addition, as part of the national survey plan, plants in natural and cropped settings were sampled based on suspected symptoms of scorching. One apple tree (*Malus domestica*) originating from the Ile de France region was found to be infected by *X. fastidiosa*. However, the contamination of this apple tree appeared transient, as subsequent samplings of the same tree maintained in containment conditions failed to reveal any contamination. One peach tree (*Prunus persica*) gave an undetermined result based on qPCR ($C_t$ between 35 and 40; https://www.anses.fr/fr/system/files/ANSES_MA039_Xylellafastidiosa_final.pdf) as did one *Quercus ilex* sampled in April 2015 in Corsica.

### Subspecies of *X. fastidiosa* identified in France

A majority of the contaminated samples from French natural settings (307 out of 432 *X. fastidiosa*-contaminated samples typed) were infected by bacteria belonging to *X. fastidiosa* subsp. *multiplex*. Indeed, these samples clustered in the same branch as the type strain (ATCC 35871) of *X. fastidiosa* subsp. *multiplex*. The node of this branch was strongly supported (84%) indicating its robustness (Fig. 1). A set of 205 bacterial samples was allocated to a cluster grouping with the Dixon strain and
<table><caption>**Table 1** List and frequency of plants contaminated by *Xylella fastidiosa* in France in 2015</caption><thead><tr><th>Genus (total), species</th><th>French region</th><th>No. of analysed samples</th><th>Positive samples (%)</th></tr></thead><tbody><tr><td>Acer (total)</td><td>Corsica</td><td>24</td><td>4.17</td></tr><tr><td>Acer pseudoplatanus</td><td>Corsica</td><td>4</td><td>25.00</td></tr><tr><td>Artemisia (total)</td><td>Corsica</td><td>4</td><td>25.00</td></tr><tr><td>Artemisia arborescens</td><td>Corsica</td><td>3</td><td>33.33</td></tr><tr><td>Asparagus (total)</td><td>Corsica</td><td>29</td><td>3.45</td></tr><tr><td>Asparagus acutifolius</td><td>Corsica</td><td>23</td><td>4.35</td></tr><tr><td>Cistus (total)</td><td>Corsica</td><td>66</td><td>19.70</td></tr><tr><td>Cistus monspeliensis</td><td>Corsica</td><td>49</td><td>22.45</td></tr><tr><td>Cistus salviifolius</td><td>Corsica</td><td>7</td><td>28.57</td></tr><tr><td>Coronilla (total)</td><td>Corsica</td><td>2</td><td>50.00</td></tr><tr><td>Coronilla valentina</td><td>Corsica</td><td>2</td><td>50.00</td></tr><tr><td>Cytisus (total)</td><td>Corsica</td><td>33</td><td>12.12</td></tr><tr><td>Cytisus racemosus</td><td>Corsica</td><td>8</td><td>25.00</td></tr><tr><td>Genista (total)</td><td>Corsica</td><td>15</td><td>6.67</td></tr><tr><td>Genista ephedroides</td><td>Corsica</td><td>1</td><td>100.00</td></tr><tr><td>Hebe (total)</td><td>Corsica</td><td>10</td><td>30.00</td></tr><tr><td>Lavandula (total)</td><td>Corsica</td><td>111</td><td>17.12</td></tr><tr><td>Lavandula × heterophylla</td><td>Corsica</td><td>5</td><td>80.00</td></tr><tr><td>Lavandula × intermedia</td><td>Corsica</td><td>3</td><td>66.67</td></tr><tr><td>Lavandula angustifolia</td><td>Corsica</td><td>26</td><td>23.08</td></tr><tr><td>Lavandula dentata</td><td>Corsica</td><td>19</td><td>5.26</td></tr><tr><td>Lavandula stoechas</td><td>Corsica</td><td>29</td><td>6.90</td></tr><tr><td>Malus (total)<sup>a</sup></td><td>Ile de France</td><td>1</td><td>100.00</td></tr><tr><td>Malus domestica<sup>a</sup></td><td>Ile de France</td><td>1</td><td>100.00</td></tr><tr><td>Metrosideros (total)</td><td>Corsica</td><td>17</td><td>5.88</td></tr><tr><td>Metrosideros excelsa</td><td>Corsica</td><td>4</td><td>25.00</td></tr><tr><td>Myrtus (total)</td><td>Corsica</td><td>154</td><td>1.95</td></tr><tr><td>Myrtus communis</td><td>Corsica</td><td>138</td><td>2.17</td></tr><tr><td>Pelargonium (total)</td><td>Corsica</td><td>106</td><td>16.04</td></tr><tr><td>Pelargonium graveolens</td><td>Corsica</td><td>17</td><td>29.41</td></tr><tr><td>Polygala (total)</td><td>Corsica</td><td>1186</td><td>35.75</td></tr><tr><td>Polygala × dalmaisiana</td><td>Corsica</td><td>1</td><td>100.00</td></tr><tr><td>Polygala myrtifolia</td><td>Corsica</td><td>1176</td><td>35.97</td></tr><tr><td>Polygala (total)</td><td>PACA</td><td>328</td><td>6.71</td></tr><tr><td>Polygala myrtifolia</td><td>PACA</td><td>261</td><td>1.15</td></tr><tr><td>Prunus (total)<sup>a</sup></td><td>Corsica</td><td>254</td><td>0.39</td></tr><tr><td>Prunus cerasifera</td><td>Corsica</td><td>5</td><td>20.00</td></tr><tr><td>Quercus (total)</td><td>Corsica</td><td>321</td><td>0.93</td></tr><tr><td>Quercus ilex<sup>a</sup></td><td>Corsica</td><td>1</td><td>100.00</td></tr><tr><td>Quercus suber</td><td>Corsica</td><td>88</td><td>2.27</td></tr><tr><td>Rosa (total)</td><td>Corsica</td><td>43</td><td>2.33</td></tr><tr><td>Rosa × floribunda</td><td>Corsica</td><td>1</td><td>100.00</td></tr><tr><td>Rosmarinus (total)</td><td>Corsica</td><td>265</td><td>0.75</td></tr><tr><td>Rosmarinus officinalis</td><td>Corsica</td><td>265</td><td>0.75</td></tr><tr><td>Spartium (total)</td><td>Corsica</td><td>40</td><td>22.50</td></tr><tr><td>Spartium junceum</td><td>Corsica</td><td>40</td><td>22.50</td></tr></tbody></table>

Positive results were based on qPCR (Harper *et al.*, 2010). The frequency of detection of *X. fastidiosa* in these plants is calculated upon the analysed samples. Bold characters indicate the description of a plant species as a novel host for *X. fastidiosa*.

the other 102 bacterial samples clustered with the Griffith-1 and M12 strains (Fig. 1); these two groups correspond to the MLSA types ST6 and ST7, respectively

<figure><img src="image_2.png" /><figcaption>**Figure 1** Taxonomic position of *Xylella fastidiosa* isolates from France in 2015. Maximum likelihood tree based on the concatenated partial sequences of *cysG*, *gltT*, *holC*, *leuA*, *malF*, *nuoL* and *petC*. Samples harvested in France appear in bold. Green stars indicate samples from Seine-et-Marne (PRU, *Prunus persica*) or Corsica (QUER, *Quercus ilex*). Black stars indicate intercepted coffee (COF, *Coffea* sp.) plants. Red stars indicate samples of *Polygala myrtifolia* (POL) identified from the survey of disease foci (other than those from subsp. *multiplex*). Blue star shows sample with recombinant strains. One tenth of the ST6 and ST7 strains are represented, keeping proportionality in the subspecies *multiplex*. Other codes correspond to reference strains. Bootstrap scores (1000 replicates) are displayed at each node.</figcaption></figure>

<footnote><sup>a</sup>One sample from each of *Malus domestica*, *Prunus persica* and *Quercus ilex* tested positive, but no subsequent samplings could be taken to confirm these results.</footnote>
<table><caption>**Table 2** Incidence and identification of isolates of *Xylella fastidiosa* contaminating plant samples in France in 2015</caption><thead><tr><th>Identification</th><th>No. of samples</th><th>Foci monitoring in Corsica and PACA</th><th>French territory survey</th><th>Interceptions of coffee plants</th></tr></thead><tbody><tr><td>ST6</td><td>205</td><td>205</td><td>0</td><td>0</td></tr><tr><td>ST7</td><td>102</td><td>102</td><td>0</td><td>0</td></tr><tr><td>ST53</td><td>10</td><td>4</td><td>2<sup>a</sup></td><td>4</td></tr><tr><td>ST72</td><td>2</td><td>0</td><td>0</td><td>2</td></tr><tr><td>ST76</td><td>4</td><td>1<sup>b</sup></td><td>0</td><td>3</td></tr><tr><td>ST79</td><td>1</td><td>1<sup>b</sup></td><td>0</td><td>0</td></tr><tr><td>Undetermined type</td><td>8</td><td>8</td><td>0</td><td>0</td></tr><tr><td>Incomplete typing</td><td>117</td><td>108</td><td>1</td><td>8</td></tr><tr><td>Not typed</td><td>103</td><td>99</td><td>0</td><td>4</td></tr></tbody></table>

PACA: Provence-Alpes-Côte d'Azur; ST: sequence type.

(Table 2). Patterns with either two bands (521 and 638 bp) or three bands (412, 521 and 638 bp), which are characteristic of *X. fastidiosa* subsp. *multiplex* (Fig. S1), were obtained using the multiprimer PCR identification assay on these samples (Hernandez-Martinez et al., 2006). However, no correlations could be made between the patterns obtained using the multiprimer PCR identification assay and the ST assignation. Out of 133 results, 67 samples showed that a three-band pattern corresponded to ST7 and a two-band pattern corresponded to ST6; however, this correlation was contradicted in 66 samples. Furthermore, unclear patterns with additional bands were obtained for strains assigned by MLSA to the *X. fastidiosa* subsp. *multiplex*, leading to difficulty in using this method for identification of isolates. In consequence this test was no longer used.

Four samples of *P. myrtifolia* from one unique focus near Menton, PACA region, were found to be

contaminated by *X. fastidiosa* subsp. *pauca* ST53 (Table 2). This focus of disease was eradicated before the ST was assigned and in consequence no attempts to isolate the strain were performed. No *X. fastidiosa* was detected in further sampling in this vicinity. One sample of *P. myrtifolia* harvested in Corsica was infected by *X. fastidiosa* subsp. *sandyi* ST76 (Table 2). Again, the focus was eradicated before the ST was assigned and no confirmation of the presence of this ST could be made when new samples were taken in the same vicinity.

## One recombinant ST and coinfections in natural settings in France

One sample was found to be a recombinant ST between *X. fastidiosa* subsp. *multiplex* (ST6 or ST7) and *X. fastidiosa* subsp. *sandyi* (ST72 or ST76; Tables 2 & 3). For this novel ST (ST79) (*cysG_26*, *gltT_3*, *holC_3*, *leuA_3*, *malF_3*, *nuoL_3* and *petC_3*) a majority of alleles from subsp. *multiplex* were mixed with one allele from subsp. *sandyi*. In the phylogenetic tree (Fig. 1), this sample branched within the subsp. *multiplex*.

Eight samples could not be typed without ambiguities and are considered as 'undetermined ST', with four different patterns. For these samples, the sequence analysis of at least one allele (*gltT*, *holC*, *nuoL* or *petC*) was not strictly conclusive. The DNA chromatograms were of good quality all along the reads except at several positions, where superimposed peaks of two nucleotides were observed (Table 3; Fig. S2). Interestingly, such ambiguous positions were usually discriminant between several alleles described in the pubmlst database. Because of these chromatogram overlaps at strategic positions, no allele could be assigned. These undetermined STs may result from the simultaneous presence of distinct *X. fastidiosa* strains in the same plants. The allele numbers indicate the concomitant presence in these samples of *X. fastidiosa* ST6, ST7, ST22, ST41 or ST76 and/or recombinants of these STs. Indeed, all four undetermined STs are suspected to possess alleles that can be found in these five STs (Table S1). As indicated above, one plant of *P. myrtifolia* was infected by ST76 in a geographical

<table><caption>**Table 3** Allele designations for seven genes of *Xylella fastidiosa* and sequence types (STs) determined from the concatenated dataset from contaminated samples recorded in France in 2015</caption><thead><tr><th>ST</th><th>cysG</th><th>gltT</th><th>holC</th><th>leuA</th><th>malF</th><th>nuoL</th><th>petC</th></tr></thead><tbody><tr><td>6</td><td>3</td><td>3</td><td>3</td><td>3</td><td>3</td><td>3</td><td>3</td></tr><tr><td>7</td><td>7</td><td>3</td><td>3</td><td>3</td><td>3</td><td>3</td><td>3</td></tr><tr><td>53</td><td>24</td><td>14</td><td>10</td><td>7</td><td>16</td><td>16</td><td>6</td></tr><tr><td>72</td><td>26</td><td>1</td><td>24</td><td>12</td><td>15</td><td>18</td><td>12</td></tr><tr><td>76</td><td>26</td><td>1</td><td>24</td><td>12</td><td>15</td><td>18</td><td>13</td></tr><tr><td>79</td><td>26</td><td>3</td><td>3</td><td>3</td><td>3</td><td>3</td><td>3</td></tr><tr><td>Undetermined#1</td><td>26</td><td>3</td><td>3</td><td>3</td><td>3</td><td>3</td><td>3/11/13</td></tr><tr><td>Undetermined#2</td><td>26</td><td>1/3/10</td><td>24</td><td>3</td><td>15</td><td>18</td><td>13</td></tr><tr><td>Undetermined#3</td><td>26</td><td>3</td><td>24</td><td>3</td><td>3</td><td>3/18</td><td>13</td></tr><tr><td>Undetermined#4</td><td>26</td><td>3</td><td>3/4/9/24</td><td>3</td><td>3</td><td>3/18</td><td>3/11/13</td></tr></tbody></table>

Allele numbers and STs are coded in agreement with the pubmlst website (http://pubmlst.org/xfastidiosa/). In five cases, allele number at one or two loci could not be assigned due to superimposition of nucleotides, in consequence the ST is called undetermined.

<footnote><sup>a</sup>These two samples, one from *Quercus ilex* and one from *Prunus persica*, were not detected positive based on the EPPO protocol for MLSA used by the French National Laboratory of Reference</footnote>

<footnote><sup>b</sup>These samples of *Polygala myrtifolia* were not detected positive based on the EPPO protocol for MLSA used by the French National Laboratory of Reference.</footnote>
area where *X. fastidiosa* subsp. *multiplex* ST6/ST7 were
preponderant. The presence of other STs was not
observed but cannot be formally excluded. Additional
evidence of sympatry came from the isolation of the two
*X. fastidiosa* strains ST6 and ST7 from one plant (*Cistus
monspeliensis*) collected in 2016 (data not shown). In the
same vicinity in 2015, the simultaneous presence of dis-
tinct *X. fastidiosa* strains in the same plants had been
observed. Taken together, these data clearly indicate that
several distinct populations are currently living in sympa-
try in France.

At least one gene fragment could not be properly
amplified and/or sequenced in 117 samples, resulting in
incomplete typings. The partial results obtained for these
samples indicated contamination by ST6, ST7 or recom-
binants between one of these two STs and ST76. In addi-
tion, some samples that had incomplete typings had been
classed as undetermined (C<sub>t</sub> values from 35 to 40) when
identifying them as *X. fastidiosa* by qPCR (Harper *et al.*,
2010). This was the case for samples from phoenix (*Phoenix* sp.) and fig tree (*Ficus carica*). Finally, a set of
103 samples could not be typed, mostly as a consequence
of unavailability of plant material or DNA of sufficient
quality/quantity after the first identification step.

Koch's postulates tested for CFBP 8416 and CFBP
8418

Among the typed samples collected from France, *X. fas-
tidiosa* subsp. *multiplex* ST6 and ST7 were identified
from 171 and 92 *P. myrtifolia* samples, respectively. The
strains CFBP 8416 (ST7) isolated from *P. myrtifolia*
(Propriano, France) and CFBP 8418 (ST6) isolated from
*S. junceum* (Alata, France) were shown to be pathogenic
on *P. myrtifolia*. Indeed, 13 weeks after inoculation, typ-
ical symptoms of leaf scorch were observed on *P. myrti-
folia* plants. Symptoms occurred on leaves at the tip of
the inoculated stems, as well as on stems distinct from
those inoculated, for two and six plants inoculated with
CFBP 8416 and CFBP 8418, respectively, indicating that
the pathogen moved throughout the plant. The pathogen
was detected in one and four plants inoculated with
CFBP 8416 and CFBP 8418, respectively, based on
qPCR tests that were largely positive, with C<sub>t</sub> values
ranging from 20.9 to 23.1 for CFBP 8418 and from 18.3
to 22.9 for CFBP 8416. In addition, CFBP 8418 was
reisolated and properly identified from one inoculated
plant. These results confirmed that *X. fastidiosa* subsp.
*multiplex* ST6 and ST7 were responsible for the leaf
scorch observed on *P. myrtifolia* in natural settings in
France.

Genome sequence analyses reveal differences among
French and USA *X. fastidiosa* subsp. *multiplex* ST6
strains linked to mobile genetic elements

The genome sequences of three strains were used to con-
firm the identity of the strains and to infer hypotheses
concerning the origin of the disease in France. Among

the 20 strains belonging to ST6 and ST7 that were iso-
lated from various hosts and locations, the genomes of
three (CFBP 8416 isolated from *P. myrtifolia*, CFBP
8417 and CFBP 8418 isolated from *S. junceum*) were
sequenced. The shotgun sequencing yielded 1 147 101
(CFBP 8416), 1 150 672 (CFBP 8417) and 1 177 029
(CFBP 8418) paired-end reads with insert sizes of c.
135 bp, corresponding to 125× coverage ending, after
assembling, in 128, 256 and 271 contigs (for CFBP
8416, CFBP 8417 and CFBP 8418, respectively) of
length between 251 and 304 552 bp (CFBP 8416), 250
and 229 226 bp (CFBP 8417), and 250 and 229 246 bp
(CFBP 8418; Table S2). Based on average nucleic identi-
ties (ANI) (Richter & Rosselló-Móra, 2009), these three
strains shared more than 99% in pairwise comparisons
with the five other available *X. fastidiosa* subsp. multi-
plex genome sequences (strains ATCC35871, Sy-Va,
M12, Griffin-1 and Dixon, genome sequences available
at http://www.ncbi.nlm.nih.gov/genome/?term=xylella)
and more than 95.88% with 14 other *X. fastidiosa* gen-
ome sequences representing the subspecies *fastidiosa*,
*morus*, *sandyi* and *pauca* (Table 4). These results con-
firmed without ambiguities that these strains isolated in
France belong to *X. fastidiosa* and, more precisely, to
the subspecies *multiplex*. While the genomes of the two
strains (CFBP 8417 and CFBP 8418) isolated from
*S. junceum* sampled at Alata, Corsica were nearly identi-
cal (Fig. 2; Table 4), the genome of CFBP 8416 (isolated
from *P. myrtifolia* at Propriano, Corsica, France) differed
from them. Pairwise identities between the CFBP 8416
genome sequence on the one hand and CFBP 8417,
CFBP 8418 genome sequences on the other hand were
nevertheless higher than 99.43%, highlighting a very
high proximity among the three subsp. *multiplex* strains.
This identity level is comparable to the ones found
among Dixon, Griffin-1 and M12 strains (Table 4).

The comparisons of the genome sequences indicated
that the CFBP 8417, CFBP 8418 and Dixon strains were
mostly identical or highly similar (Table 4; Fig. 2). How-
ever, they differed by a large fragment, which was pre-
sent in the Dixon strain, but absent from CFBP 8417
and CFBP 8418 as well as from CFBP 8416, Griffin-1
and M12 genomes (Fig. 2). This fragment corresponds to
the sequence of an entire plasmid called pXF-RIV5 in
the American *X. fastidiosa* subsp. *multiplex* Riv5 strain
isolated from *Prunus cerasifera* (Rogers & Stenger,
2012). CFBP 8416 was closely related to Griffin-1 and
M12 (identities between 99.42% and 99.59%; Table 4;
Fig. 2). A high percentage (40.36%) of differences
(BLASTN) corresponded to mismatch or gaps at one or the
other end of contigs and could be associated with errors
of sequencing and assembling methodologies. However,
one difference between these strains concerned phage ele-
ments with a total length of 20 kb in Griffin-1 and M12
that are absent from CFBP 8416. These phage elements
presented high homologies (93% identity on 3013 bp) to
part of the temperate phage Xfas53, although this phage
has a genome of 36.7 kb and contains 45 coding
sequences (Summer *et al.*, 2010).
<table><caption>**Table 4** Average nucleic acid identities based on BLASTN searches for pairwise comparisons among *Xylella fastidiosa* subsp. *multiplex* strains and representatives of other subspecies</caption><thead><tr><th>Subspecies</th><th>Strain</th><th>ATCC<br>35871</th><th>Sy-VA</th><th>M12</th><th>Griffin-1</th><th>CFBP<br>8416</th><th>Dixon</th><th>CFBP<br>8417</th><th>CFBP<br>8418</th></tr></thead><tbody><tr><td rowspan="7">multiplex</td><td>ATCC 35871</td><td>—</td><td>99.45</td><td>99.39</td><td>99.40</td><td>99.55</td><td>99.43</td><td>99.68</td><td>99.63</td></tr><tr><td>Sy-VA</td><td>99.38</td><td>—</td><td>99.31</td><td>99.31</td><td>99.17</td><td>99.36</td><td>99.36</td><td>99.36</td></tr><tr><td>M12</td><td>99.31</td><td>—</td><td>99.35</td><td>99.95</td><td>99.58</td><td>99.68</td><td>99.69</td><td>99.69</td></tr><tr><td>Griffin-1</td><td>99.39</td><td>—</td><td>99.37</td><td>99.98</td><td>99.59</td><td>99.69</td><td>99.69</td><td>99.68</td></tr><tr><td>CFBP 8416</td><td>99.17</td><td>99.08</td><td>99.44</td><td>99.42</td><td>—</td><td>99.45</td><td>99.51</td><td>99.48</td></tr><tr><td>Dixon</td><td>99.33</td><td>99.27</td><td>99.58</td><td>99.57</td><td>99.47</td><td>—</td><td>99.92</td><td>99.92</td></tr><tr><td>CFBP 8417</td><td>99.23</td><td>99.22</td><td>99.49</td><td>99.45</td><td>99.45</td><td>99.95</td><td>—</td><td>99.98</td></tr><tr><td rowspan="2">morus</td><td>CFBP 8418</td><td>99.21</td><td>99.19</td><td>99.46</td><td>99.42</td><td>99.43</td><td>99.97</td><td>99.96</td><td>—</td></tr><tr><td>Mul-MD</td><td>98.19</td><td>98.10</td><td>97.73</td><td>97.78</td><td>97.72</td><td>97.76</td><td>97.80</td><td>97.83</td></tr><tr><td rowspan="2">sandyi</td><td>Mul0034</td><td>98.06</td><td>97.95</td><td>97.61</td><td>97.65</td><td>97.60</td><td>97.64</td><td>97.72</td><td>97.71</td></tr><tr><td>Ann-1</td><td>97.41</td><td>97.31</td><td>97.12</td><td>97.16</td><td>97.07</td><td>97.16</td><td>97.22</td><td>97.25</td></tr><tr><td rowspan="6">fastidiosa</td><td>CFBP 8073</td><td>97.39</td><td>97.32</td><td>97.13</td><td>97.17</td><td>97.16</td><td>97.16</td><td>97.19</td><td>97.17</td></tr><tr><td>Temecula1</td><td>97.68</td><td>97.58</td><td>97.35</td><td>97.40</td><td>97.44</td><td>97.36</td><td>97.56</td><td>97.57</td></tr><tr><td>M23</td><td>97.69</td><td>97.56</td><td>97.35</td><td>97.36</td><td>97.59</td><td>97.42</td><td>97.78</td><td>97.66</td></tr><tr><td>GB514</td><td>97.65</td><td>97.58</td><td>97.40</td><td>97.43</td><td>97.28</td><td>97.42</td><td>97.45</td><td>97.48</td></tr><tr><td>EB92.1</td><td>97.63</td><td>97.59</td><td>97.33</td><td>97.33</td><td>97.20</td><td>97.26</td><td>97.32</td><td>97.31</td></tr><tr><td>ATCC 35879</td><td>97.64</td><td>97.51</td><td>97.29</td><td>97.36</td><td>97.34</td><td>97.3</td><td>97.68</td><td>97.67</td></tr><tr><td rowspan="5">pauca</td><td>9a5c</td><td>96.17</td><td>96.12</td><td>96.07</td><td>96.13</td><td>96.03</td><td>96.04</td><td>96.14</td><td>96.11</td></tr><tr><td>6c</td><td>96.24</td><td>96.16</td><td>96.17</td><td>96.18</td><td>96.15</td><td>96.13</td><td>96.17</td><td>96.16</td></tr><tr><td>32</td><td>96.09</td><td>96.0</td><td>95.89</td><td>95.93</td><td>95.96</td><td>95.89</td><td>95.94</td><td>95.95</td></tr><tr><td>CFBP 8072</td><td>96.19</td><td>96.06</td><td>96.09</td><td>96.08</td><td>95.99</td><td>96.06</td><td>96.10</td><td>96.11</td></tr><tr><td>CoDiRO</td><td>96.28</td><td>96.25</td><td>96.19</td><td>96.20</td><td>96.06</td><td>96.19</td><td>96.76</td><td>96.86</td></tr></tbody></table>

Strains sequenced in the present study are shown in bold.

Discussion

*Xylella fastidiosa* is an emerging pathogen in Europe. *Xylella fastidiosa* was first detected in Apulia, Italy in October 2013. However, olive quick decline syndrome, the disease associated with *X. fastidiosa* in Apulia, appeared earlier, around 2008 (Martelli *et al.*, 2016). Based on MLST data, only one strain, the so-called CoDiRO strain (*X. fastidiosa* subsp. *pauca* ST53), is so far present in this area (European Food Safety Authority, 2016b). Emergence of *X. fastidiosa* in France was recently reported (European Plant Protection Organization, 2015) but the date(s) of introduction of the pathogen(s) remains to be specified. To identify potential origin and pathways of introduction(s) the positive samples from France in 2015 were typed. MLST is the most widely used genotyping technique for assessing the global epidemiology of pathogenic bacteria and it was proven to be efficient in the case of *X. fastidiosa* to reveal possible invasion routes (Nunney *et al.*, 2010, 2014b; Yuan *et al.*, 2010). MLST of DNA from a pure bacterial culture is traditionally used to assign an isolate to one of the known subspecies. Here, it was used directly with DNA extracted from plant material. A modification of the initial protocol (Yuan *et al.*, 2010; European Plant Protection Organization, 2016) was used to obtain a better amplification rate. This modified protocol led to 100% of amplification of the seven genes used for typing a set of 11 samples, whereas only 39% success rate was obtained with the European Plant Protection Organization protocol (data not shown).

The diversity of plants infected by *X. fastidiosa* in natural settings in France is large, but the frequency among species is highly variable. Frequency of infection should, however, be taken cautiously as the values are biased by the sampling frequency, which is unrelated to the species distribution in those natural settings. Most host plants of *X. fastidiosa* subsp. *multiplex* ST6 and ST7 are ornamentals and only a few trees were found to be infected. French and European regulations concerning *X. fastidiosa* make the eradication of contaminated foci mandatory (Directive 2000/29/EC, 2000; Commission Implementing Decision (EU) 2015/789, 2015). As a consequence, when *X. fastidiosa* was detected in samples, the plants were eradicated, as were plants with symptoms from the same species in the focus. Therefore, it was not possible to sample again to refine the diagnosis.

A large majority of the samples were contaminated by *X. fastidiosa* subsp. *multiplex*. This subspecies is supposed to be native to North America (USA) and to evolve slowly as a consequence of being adapted to life in temperate environments with a short growing season and few generations per year (Nunney *et al.*, 2010). The host range of this subspecies is large and it includes USA native and non-native trees, such as almond, elm, oak, olive, peach, pecan, pigeon grape, plum, red-bud, sweetgum and sycamore (European Food Safety Authority, 2015). Genetic heterogeneity was found in this subspecies, with 34 STs so far described (http://pubmlst.org/xfastidiosa/ downloaded 8 August 2016). Eight of these STs were shown to arise from intersubspecific homologous recombination (IHR) of alleles of *cysG*, *holC* and,
<figure><img src="image_3.png" /><figcaption>**Figure 2** Pairwise comparison of genome sequences of French and American *Xylella fastidiosa* strains. Genomic sequences of French (CFBP 8416, CFBP 8417 and CFP 8418) and American (M12, Griffin-1 and Dixon) strains were cut in 1 kb-length fragments. Conservation of each fragment of a query genome was searched in the others using the TBLASTN algorithm. The CGVIEW analytic tool was used to represent the results, with the order of strains given from the external (ext) to the internal (int) circles. The colour scale indicates the level of identity. White stripes indicate fragments from the query genome that are absent in the others. Pairwise comparisons of (a) CFBP 8416 (ST7) strain isolated from *Polygala myrtifolia* versus CFBP 8417 and CFBP 8418 (*Spartium junceum*, ST6); and comparisons of the French strains with the American strains (b), Dixon (ST6, *Prunus dulcis*); (c), Griffin-1 (ST7, *Quercus rubra*) and (d), M12 (ST7, *P. dulcis*).</figcaption></figure>

less frequently, *leuA*, whereas the STs 6 and 7 identified in France were considered to be non-IHR (Nunney *et al.*, 2013). The recombinant ST identified in the present study has not been previously described and appears to

have mixed alleles from *X. fastidiosa* subsp. *sandyi* ST72 or ST76 and *X. fastidiosa* subsp. *multiplex* ST6 or ST7. *Xylella fastidiosa* subsp. *sandyi* ST72 and ST76 have not so far been recorded in the USA and *X. fastidiosa* subsp.
*multiplex* ST6 or ST7 have not been recorded in Central America. Because these STs do not seem to be sympatric in the Americas, it is probable that the recombination events took place elsewhere. *Xylella fastidiosa* subsp. *sandyi* ST72 and ST76 have been detected in coffee plants intercepted in Italy (Loconsole *et al.*, 2016) and in France (this study). *Xylella fastidiosa* subsp. *multiplex* has been recorded in France, but not so far in Italy, indicating that the recombination probably occurred in France. The presence of *X. fastidiosa* subsp. *sandyi* outdoors in Corsica or PACA was recorded in the present survey in one sample among nearly 6000 samples analysed. Altogether this suggests that co-occurrence of both subspecies in France should be rare, rendering the occurrence of recombination even more rare.

The diversity of *X. fastidiosa* identified in this study included (i) *X. fastidiosa* subsp. *multiplex* ST6 and ST7, and (ii) a recombinant from these STs and *X. fastidiosa* subsp. *sandyi* ST72 or ST76. In addition to these individuals, probable mixing of strains was reported within eight samples, corresponding to four patterns of undetermined STs. In this study, recombination and coinfection were discriminated on the basis of the superposition or not of peaks at positions that are useful in allele differentiation. When nucleotides could not be properly assigned in a sequence, the sequence was considered to originate from more than one allele and so indicated a coinfection. Thus, MLST indicated either coinfection by *X. fastidiosa* subsp. *multiplex* and *X. fastidiosa* subsp. *sandyi* ST76, or a recombinant of both. Occurrence of coinfections with variants of the same pathogen has been described for several bacteria including phytoplasmas (Darrasse *et al.*, 2013; Wei *et al.*, 2016). As for *X. fastidiosa*, evidence of sympatry was previously reported for strains M12 (subsp. *multiplex*) and M23 (subsp. *fastidiosa*), which were isolated from the same almond orchard in Kern County, CA, USA (Chen *et al.*, 2005). In addition, the comparison of seven housekeeping gene sequences of M12 and M23 revealed several marks of recombination events (Nunney *et al.*, 2010). The results presented here indicate that distinct subspecies of *X. fastidiosa* are currently living in the same area and contribute to the generation of intersubspecific recombinants, increasing the genetic diversity of the pathogen.

A set of 109 samples from French natural settings could not be typed because of failure to amplify at least one gene. This incapacity to properly type all samples is a consequence of the direct (non-culture) MLST approach used. As mentioned earlier, MLST was carried out directly on DNA extracted from infected plants and not on purified bacterial isolates. Typing directly from host material has been reported in a few other studies (Arvand *et al.*, 2010; Griffiths *et al.*, 2010; Agampodi *et al.*, 2013). The limitations of direct MLST are linked to low bacterial load in samples preventing their typing and mixed infections that result in ambiguous STs. In the present study, MLST typing of the contaminated samples from France in 2015 indicated that ST6 and ST7 largely dominate over minor STs. Undetermined and incomplete

typings tentatively suggest a similar dominance of ST6 and ST7. These two major STs are well known in the USA where they were first recorded. ST6 was detected in almond trees and, so far, ST7 strains have been reported in almond trees, red oak, olive trees and black sage (http://pubmlst.org/xfastidiosa/). These two STs differ by only one nucleotide variation at the *cysG* locus, leading to the substitution of a guanine (in ST6) by an adenine (in ST7). Each of these STs clusters with strains that were isolated in France from various hosts and locations. Most of the plants infected in France were not previously known as hosts in the Americas and so far the host ranges found in France and in the USA are only partially overlapping. This clearly illustrates a well-known risk of host-shift linked to the introduction of a pathogen into a novel area (Parker & Guilbert, 2004).

Based on the analysis of the partial sequences of seven housekeeping genes, the French strains CFBP 8416 (ST6), CFBP 8417 and CFBP 8418 (ST7) were found to be closely related to the American strains Dixon (ST6), Griffin-1 and M12 (ST7). The Dixon and M12 strains were isolated from *Prunus dulcis* in the USA (Bhat-tacharyya *et al.*, 2002; Chen *et al.*, 2010), whereas the Griffin-1 strain was isolated in the USA from *Quercus rubra* (Chen *et al.*, 2013). The sequencing of the genomes of the French strains in the present study has allowed pairwise comparisons with the publicly available genomes of the American relatives. First, one fragment, showing high homology with the temperate phage Xfas53 (Summer *et al.*, 2010) is missing in CFBP 8416 isolated in France. This phage, isolated from *X. fastidiosa* subsp. *fastidiosa* strain 53 (host: *Vitis vinifera*), results from the recombination between a widespread family of *X. fastidiosa* P2-related prophage elements and a podophage distantly related to phage P22 (Summer *et al.*, 2010). Sequences related to Xfas53 are widespread among *X. fastidiosa* strains and have been reported in strains from the subspecies *fastidiosa*, *sandyi*, *multiplex* and *pauca* (Summer *et al.*, 2010; Jacques *et al.*, 2016).

The major difference found between the ST6 strains from France and the Dixon strain is the absence of an entire plasmid, which is also missing in the ST7 strain. This c. 38 kb plasmid has been analysed in detail in the *X. fastidiosa* subsp. *multiplex* strain Riv5 (Rogers & Stenger, 2012). It carries genes encoding a complete type IV secretion system involved in conjugation and DNA transfer among bacteria. A nearly identical plasmid was found in *X. fastidiosa* subsp. *fastidiosa* strain M23. The presence of an almost identical plasmid in strains belonging to different subspecies is considered as indicative of a recent horizontal gene transfer. It has been hypothesized that this plasmid could have transferred after the introduction of *X. fastidiosa* subsp. *fastidiosa* in the USA, after c. 1880 (Rogers & Stenger, 2012). Finding *X. fastidiosa* subsp. *multiplex* strains that are genetically closely related based on chromosome comparisons, but differ by the absence of this plasmid, indicates that the French strains may have (i) lost the plasmid since they have diverged from their American relatives, (ii) never
had the plasmid and originated from a ST6 strain depleted of this plasmid, or (iii) hosted the plasmid but lost it during *in vitro* isolation and subsequent cultures. This last hypothesis is highly unlikely as PCR performed with 10 pairs of primers designed from the pXF-Riv5 plasmid sequence (NC_020121) did not amplify the corresponding regions in the extracted DNA of six infected *P. myrtifolia* samples or strains from Corsica (data not shown). In addition, the entire plasmid sequence was conserved in other genomes of *X. fastidiosa* strains from the strain collection (data not shown). No other reports of ST6 lacking this plasmid have been made and, therefore, the first hypothesis is most probable.

To conclude, most French samples in the present investigation were contaminated by *X. fastidiosa* subsp. multiplex ST6 and ST7 isolates. In addition, *X. fastidiosa* subsp. pauca ST53 and *X. fastidiosa* subsp. sandyi ST76 were found in some samples and one recombinant isolate was also detected. Indications of coinfection by different strains were hypothesized based on complex indeterminate typings and were confirmed by the co-isolation of isolates of two different STs from one sample in 2016. Genomic analyses indicated that French strains are very closely related to the Dixon, Griffin-1 and M12 strains, without being identical. One striking difference with the Dixon strain is the absence of an entire plasmid encoding a type IV secretion system. The presence of various STs in *P. myrtifolia*, the identification of some of these STs in intercepted contaminated coffee plants, the existence of coinfections and of recombination indicate that *X. fastidiosa* must have been introduced several times via diverse contaminated plant material in France. The next step will be to pursue the analysis of ST6 and ST7 strains using other sets of markers to allow more detailed investigation at various evolutionary scales of the dispersal of the isolates.

## Acknowledgements

N.D. was funded by regional funds provided by Objectif Végétal. The work carried out at INRA has received funding from the European Union's Horizon 2020 research and innovation program under grant agreement 635646, POnTE (Pest Organisms Threatening Europe). The present work reflects only the authors' view and the EU funding agency is not responsible for any use that may be made of the information it contains. The authors greatly acknowledge SRAL, FREDON, DSF, DDSCPP for sampling in Corsica and PACA; Dimitri Molusson, Sandrine Paillard, Christèle Dousset, Antoine Sainte-Luce, Virginie Juteau, David Agud-Miguel, Christelle François, Carène Rivoal and Corinne Audusseau, LSV-ANSES at Angers, for sample analyses, bacterial isolations and identifications; Pauline de Jerphanion for quantitative data on hosts; Yannick Blanchard and Fabrice Touzain, ANSES at Ploufragan, for participating in library construction and handling strains for genome sequencing; Jérôme Gouzy and Sébastien Carrère, CATI-BBRIC, for genome sequence assembly and

annotation. The authors also thank CIRM-CFBP (French Collection for Plant-associated Bacteria: http://www6.inra.

fr/cirm_eng/CFBP-Plant-Associated-Bacteria) for strain conservation and are most grateful to the Biogenouest Genomics core facility for its technical support. The authors declare no conflicts of interest.

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## Supporting Information

Additional Supporting Information may be found in the online version of this article at the publisher's web-site.

**Figure S1.** Electrophoresis of products from multiprimer PCR identification tests (Hernandez-Martinez *et al.*, 2006) carried out on *Xylella fastidiosa*-contaminated samples from France in 2015 and strains representing the subspecies *multiplex* (CFBP 8070), *fastidiosa* (CFBP 7970), *pauca* (CFBP 8072) and *sandyi* (CFBP 8077) of *X. fastidiosa*. Lanes 2 and 3 show the typical patterns obtained from samples, lanes 1 and 8 are 1 kb ladder; lane 9 indicates negative control (ultrapure water test). Amplicon sizes are, from highest to lowest: 638, 521 and 412 bp.

**Figure S2.** Examples of alignment of the partial sequence of *petC* obtained from one sample of *Polygala myrtifolia* infected by *Xylella fastidiosa* with other *petC* alleles (numbers 3, 11 and 13) of *X. fastidiosa* referenced in the pubmlst database. Chromatograms are of good quality (one single peak at each position), except in several positions for which an overlap of two peaks appears, leading to an undetermined nucleotide 'N'. These positions are discriminant for four *petC* alleles.

**Table S1.** Tracing the putative origin of ambiguous alleles in the genomes of strains of *Xylella fastidiosa*.

**Table S2.** Main characteristics of genome sequences from three *Xylella fastidiosa* isolates isolated in France.