several subspecies and sequence types are associated with the emergence of xylella fastidiosa in natural settings in france

Received Date : 04-Jan-2017 Accepted Article Revised Date : 02-Feb-2017 Accepted Date : 03-Feb-2017 Article type : Original Article Several subspecies and sequence types are associated with the emergence of Xylella fastidiosa in natural settings in France N DENANCE1,2§, B LEGENDRE2§, M BRIAND1, V OLIVIER2, C de BOISSESON3, F POLIAKOFF2, and M-A JACQUES1* § equal contribution IRHS, INRA, AGROCAMPUS-Ouest, Université d’Angers, SFR4207 QUASAV, 42, rue Georges Morel, 49071 Beaucouzé, France Anses Laboratoire de la santé des végétaux, F- 49044 Angers Cedex 01 ANSES Ploufragan, Viral Genetics and Biosecurity, F-22440 Ploufragan * Corresponding author: Marie-Agnès Jacques IRHS 42, rue Georges Morel, CS60057, 49071 Beaucouzé cedex, France, Tel: +33 41 22 57 07, Fax: +33 41 22 57 05, email: marie-agnes.jacques@inra.fr Running head: Emergence of X fastidiosa in France Keywords: Polygala myrtifolia, multiplex, pauca, coffee, MLST, comparative genomics This article has been accepted for publication and undergone full peer review but has not been through the copyediting, typesetting, pagination and proofreading process, which may lead to differences between this version and the Version of Record Please cite this article as doi: 10.1111/ppa.12695 This article is protected by copyright All rights reserved SUMMARY Accepted Article Xylella fastidiosa is a plant pathogenic bacterium emerging in Europe In France its emergence has been evidenced through interceptions of contaminated coffee plants and in 2015 by the survey of natural settings The first French contaminated focus was detected in 2015 in Corsica; then almost 300 foci and nearly 30 plant species were declared contaminated, with Polygala myrtifolia remaining the principal host suffering from severe leaf scorches We report on the diversity of X fastidiosa identified in France in 2015 Multilocus sequence analysis/typing revealed the presence of mainly X fastidiosa subsp multiplex ST6 and ST7 A focus of subspecies pauca ST53 was identified in mainland France; one sample contaminated by X fastidiosa subsp sandyi ST76, one novel recombinant, and co-infections of different isolates in individual samples were also identified, but could not be confirmed by successive samplings indicating limited or transient contaminations 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, our results suggest that the emergence of X fastidiosa in France is linked to several introduction events of diverse strains from different subspecies INTRODUCTION Originally confined to the Americas Xylella fastidiosa, a bacterial plant pathogen, recently emerged in Asia and Europe This pathogen was reported in Taiwan from grapevine in 2013 (Su et al., 2013) The same year, severe leaf scorches of olive trees were reported from This article is protected by copyright All rights reserved Apulia, Italy to be associated with X fastidiosa (Saponari et al., 2013) A year later, this Accepted Article pathogen was detected in Iran in grapevine and almond trees (Amanifar et al., 2014), and in 2015 in France from Polygala myrtifolia (https://gd.eppo.int/taxon/XYLEFA/distribution/FR) In 2016, Spain also declared a focus of X fastidiosa subsp fastidiosa in Baleares 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 as in 2016 in Germany where oleander and rosemary plants were detected contaminated in a nursery by X fastidiosa subsp fastidiosa; these outbreaks were eradicated or are under eradication (https://gd.eppo.int/taxon/XYLEFA/distribution) In 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) X fastidiosa is a genetically diverse species sub-divided 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) (ii) X 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) X fastidiosa subsp multiplex is associated with scorch diseases of a large range of trees This subspecies is mostly found in temperate climates of Northern America and has been introduced in South America (Nunes et al., 2003) (iv) X 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., This article is protected by copyright All rights reserved 2015; Coletta-Filho et al., 2016) and were also recovered from coffee and oleander in central Accepted Article 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) X 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 the 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 1930’s in Brazil by contaminated plant material (Nunes et al., 2003) It has been documented in several cases 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 documented (Nunney et al., 2012; 2014a; 2014b) In consequence, the risk associated with the mixing of strains that were previously isolated should be avoided as this could results in novel genetic combinations with yet undescribed host range Detection and identification of X fastidiosa is currently mostly based 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 (Harper et al., 2010) was shown to be specific and sensitive, and in consequence is included in the reference method in use in France (https://www.anses.fr/fr/system/files/ANSES_MA039_Xylella fastidiosa_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 This article is protected by copyright All rights reserved al (2006) to differentiate strains from three X fastidiosa subspecies, fastidiosa, multiplex, Accepted Article and sandyi, while 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 Multi Locus Sequence Analysis (MLSA), and its derivative the Multi Locus Sequence Typing (MLST) These methods are used for taxonomical and epidemiological purposes, respectively A dedicated scheme has been proposed for X fastidiosa and is of current use (Yuan et al., 2010; Jacques et al., 2016; European Plant Protection Organization, 2016) Fragments of seven housekeeping genes, cysG, gltT, holC, leuA, malF, nuoL and petC, are amplified, ending in a 4161bp sequence of concatenated data and subsequent analyses rely on phylogenetic methods and allele assignation (http://pubmlst.org/xfastidiosa/) X fastidiosa was first detected in Polygala myrtifolia shrubs in Corsica Island, France in July 2015 and later on in October 2015 in mainland France (http://www.corse-dusud.gouv.fr/IMG/pdf/Reunion_Xylella_21-01-16.pdf) These ornamentals were symptomatic and presented severe leaf scorches 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 scorches detected in 2015 in France and to identify probable routes of introduction MATERIAL AND METHODS Analyses of plant material Plants suspected to be contaminated by X fastidiosa based on symptomatology were sampled by various national and regional services and samples were sent for analysis to the plant health laboratory of Anses at Angers, France and/or from November 2015 to laboratories This article is protected by copyright All rights reserved officially certified by the Ministry in charge of agriculture (Rural code for agriculture and Accepted Article fisheries, articles L202-1 and R202-8 to R202-21) Sample analysis was run as described in https://www.anses.fr/fr/system/files/ANSES_MA039_Xylellafastidiosa_final.pdf Briefly, surface-sterilized fragments (0.5 to 1g) of petioles and midribs were ground in demineralized sterile water (5 mL/g) 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 A 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, Harper et al., 2010), and TaqMan® Fast universal PCR Master Mix 2X (Applied Biosystems) In specific cases isolation of bacterial strains was attempted Plant extract aliquots 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 run on the previously extracted DNAs representing novel foci and/or novel host plants Housekeeping genes (Yuan et al., 2010) were amplified from extracted DNAs following the protocol described in European Plant Protection Organization (2016), with modifications GoTaq G2 polymerase (Promega) and primers at a final concentration of 0.5 µM were employed The mix PCR (50µl) was composed 10µl Green GoTaq Reaction Buffer (5X), 5µl each primer (5µM), 5µl dNTPs (2mM), 0.7µl GoTaq G2 polymerase (5U/µl), 4µl DNA, and 20.3µl ultrapure water Additionally, the melting temperature was fixed at 60°C and elongation time was reduced to 45s PCR product sequencing, and sequence analyses were done as described (Jacques et al., 2016) The genomes of the three first strains that were isolated were This article is protected by copyright All rights reserved sequenced using the Illumina Mi-Seq technology (Plateforme Génomique de Nantes, IRT- Accepted Article UN, France) Genome sequencing, assembling, annotation, and analyses were run as previously described (Jacques et al., 2016) Pathogenicity tests on Polygala myrtifolia Pathogenicity of CFBP 8416 and CFBP 8418 strains was tested on 1.5 yr-old plants of Polygala myrtifolia grown in confined growth chamber at 24°C during 16h of daylight and at 20°C during night under 70% of relative humidity Plants were watered daily with water supplemented with 1.4 g/L 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 Notation of symptoms and analyses of symptomatic samples were monitored 13 weeks after inoculation Samples were tested as previously described based on qPCR test designed by Harper et al (2010) and isolations on PWG or B-CYE (Jacques et al., 2016) Plant inoculations were carried out under quarantine at IRHS, Centre INRA, Beaucouzé, France under the agreement N° 2013119-0002 from the Prefecture de la Région Pays de la Loire, France Nucleotide sequence accession numbers The CFBP 8416, CFBP 8417, and CFBP 8418 genome sequences reported here have been deposited in GenBank under accession numbers LUYC00000000, LUYB00000000, and LUYA00000000, respectively This article is protected by copyright All rights reserved RESULTS Accepted Article Interceptions of X fastidiosa subsp sandyi and subsp pauca through the importation of coffee plants Within the frame of the X fastidiosa national survey and control plan, coffee plants were controlled upon importation Twenty one out of 135 samples of coffee plants were detected contaminated by X fastidiosa representing 14 interceptions of coffee plant material These plants materials were imported from various Latin America countries and were intercepted in several regions in France (Ile de France, Pays de la Loire, Centre and Provence-Alpes-Côte d’Azur, i.e PACA) Among the 21 infected samples, nine were successfully typed by MLST X fastidiosa subsp sandyi ST72 was identified in two coffee plants sampled in Ile de France and in Pays de la Loire area X fastidiosa subsp sandyi ST76 was identified in three coffee plants in Ile-de-France X fastidiosa subsp pauca ST53 was detected from four samples of coffee plants intercepted in Ile-de-France and Pays de la Loire regions For the remaining infected intercepted plants, no sequence types could be assigned due to the impossibility to amplify and/or obtain several housekeeping gene sequences 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 is infected by X fastidiosa in France Beyond the intercepted infected coffee plants the first contaminated plant in natural settings was identified in France in July 2015 As of December 31 2015, the bacterium was found in 237 foci in Corsica and 10 foci in mainland France Most contaminated samples concerned Polygala myrtifolia (84% of the positive plants), but X fastidiosa was found in plants belonging to 21 different species of plants (Table 1) In 2015, the survey in these two areas represented a total of 5,962 plant samples that were analyzed among which 528 samples (8.9%) were declared contaminated by X fastidiosa Samples originated mostly from Southern Corsica The species P myrtifolia was the most frequently positive (29.5% of the This article is protected by copyright All rights reserved tested samples were positive); Pelargonium spp., Cistus spp., Lavandula spp., and Spartium Accepted Article junceum totalized 11% of the positive samples Most importantly, no Vitis, no Citrus, and no Olea europaea were detected positive to X fastidiosa while the sampling effort was important as 69 Vitis sp., 238 Citrus sp., and 504 Olea sp plants from Corsica were analyzed Moreover, 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 Ile de France region was detected contaminated by X fastidiosa However, the contamination of this apple tree appeared transient as subsequent samplings of the same tree maintained in containment condition failed to reveal any contamination One peach tree (Prunus persica) gave an undetermined result based on qPCR (Ct between 35 and 40, (https://www.anses.fr/fr/system/files/ANSES_MA039_Xylellafastidiosa_final.pdf) as was the case for one Quercus ilex sampled in April 2015 in Corsica X fastidiosa identified in France mostly belong to the subspecies multiplex, but also to the subspecies pauca A majority of the contaminated samples (307 samples out the 432 typed X fastidiosa contaminated samples from French natural settings) presented bacteria belonging to X fastidiosa subsp multiplex Indeed, these samples clustered in the same branch than the type strain (ATCC 35871) of X fastidiosa subsp multiplex The node of this branch was strongly supported (84%) indicating its robustness (Figure 1) A set of 205 bacterial samples was allocated to a cluster grouping with the Dixon strain and the other 102 bacterial samples clustered with the Griffin-1 and M12 strains (Figure 1) These two groups correspond to ST6 and ST7, respectively (Table 2) Patterns with either two bands (521 and 638 bp) or three bands (412, 521, and 638 bp) that are characteristic of X fastidiosa subsp multiplex (File S1 in Supporting Information) were obtained using the multiprimer PCR identification assay on these samples (Hernandez-Martinez et al., 2006) However, no correlations could be made This article is protected by copyright All rights reserved between the patterns obtained using the multiprimer PCR identification assay and the ST Accepted Article assignation Over a set of 133 cases, 67 correspondences between a three-band pattern and ST7 or a two-band pattern and ST6 were counted, but this was contradicted in 66 cases Additionally, unclear patterns with additional bands were obtained for strains assigned with the MLSA to the X fastidiosa subsp multiplex leading to a difficult use of this method for identification of the isolates In consequence this test was no longer used Additionally, four samples of P myrtifolia from one unique focus near Menton, PACA region, were detected contaminated by X fastidiosa subsp pauca ST53 (Table 2) This focus was eradicated before the ST assignation was made, and in consequence no attempts to isolate the strain were performed Further sampling in its vicinity did not lead to X fastidiosa detection One sample of P myrtifolia harvested in Corsica was found infected by X fastidiosa subsp sandyi ST76 (Table 2) Again the focus was eradicated before the ST assignation was made and no confirmation of the presence of this ST could be made when a new sampling was made in the vicinity of this first case One recombinant ST and co-infections were also evidenced for natural settings in France One sample presented a recombining ST between X fastidiosa subsp multiplex (ST6 or ST7) and X fastidiosa subsp sandyi (ST72 or ST76) (Tables and 3) For this novel ST ([STnew1, comment: this ST number will be updated as soon as it is provided by PubMlst] [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 (Figure 1), this sample branched within the subsp multiplex Eight samples could not be typed without ambiguities and are considered as “undetermined typing”, with four different cases For these samples, the sequence analysis of at least one allele (gltT, holC, nuoL, or petC) was not strictly conclusive The DNA chromatograms were This article is protected by copyright All rights reserved material in France The next step will be to pursue the analysis of ST6 and ST7 strains using Accepted Article other sets of markers for allowing finer investigations at various evolutionary scales to refine the dispersal of the isolates ACKNOWLEDGMENTS ND was funded by regional funds provided by Objectif Végétal The work that has been done at INRA was partly funded by H2020 project POnTE SEP-210177514 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 Franỗois, Carốne 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 We thank CIRM-CFBP (French Collection for Plant-associated Bacteria: http://www6.inra.fr/cirm_eng/CFBP-Plant-Associated-Bacteria) for strain conservation We are most grateful to the Biogenouest Genomics core facility for its technical support Authors declare no conflicts of interest This article is protected by copyright All rights reserved REFERENCES Accepted Article Agampodi SB, Moreno AC, Vinet JM, Matthias MA, 2013 Utility and Limitations of Direct Multi-Locus Sequence Typing on qPCR-Positive Blood to Determine Infecting Leptospira Strain American Journal of Tropical Medicine and Hygiene 88, 184-5 Almeida RPP, Nascimento FE, Chau J, Prado SS, Tsai CW, Lopes SA, Lopes JRS, 2008 Genetic structure and biology of Xylella fastidiosa causing disease in citrus and coffee in Brazil Applied and Environmental Microbiology 74, 3690-701 Amanifar N, Taghavi M, Izadpanah K, Babaei G, 2014 Isolation and pathogenicity of Xylella fastidiosa from grapevine and almond in Iran Phytopathologia Meditereranea 53, 318-27 Arvand M, Raoult D, Feil EJ, 2010 Multi-locus sequence typing of a geographically and temporally diverse sample of the 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Gonzalez CF, 2010 Genomic and biological analysis of phage Xfas53 and related prophages of Xylella fastidiosa Journal of Bacteriology 192, 179-90 Wei W, Davis W, Davis RE, Lee IM, Zhao Y, 2016 Development of molecular markers and a diagnostic tool for investigation of coinfections by and interactions between potato purple top and potato witches’-broom phytoplasmas in tomato Annals of Applied Biology 168, 133–41 Yuan X, Morano L, Bromley R, Spring-Pearson S, Stouthamer R, Nunney L, 2010 Multilocus sequence typing of Xylella fastidiosa causing Pierce's disease and oleander leaf scorch in the United States Phytopathology 100, 601-11 This article is protected by copyright All rights reserved SUPPORTING INFORMATION LEGENDS Accepted Article Supplemental File S1 Gel photograph showing the patterns obtained for X fastidiosa contaminated samples from France in 2015 in comparison to strains representing the subspecies multiplex (CFBP 8070), fastidiosa (CFBP 7970), pauca (CFBP 8072), and sandyi (CFBP 8077) of X fastidiosa with the identification tests described by Hernandez-Martinez et al (2006) Lanes and show the patterns with three or two bands that were obtained, lanes and are 1kb ladder; lane indicates negative control (ultrapure water test) Amplicon sizes are, from highest to lowest: 638bp, 521bp, 412bp Supplemental File Examples of alignment of the partial sequence of petC obtained from one infected sample of P myrtifolia, with other petC alleles (numbers 3, 11 and 13) referenced in the pubmlst database Chromatograms are of good quality (one single pick at each base), except in several positions for which an overlap of two picks appears, leading to an undetermined nucleotide ‘N’ These positions are discriminant for four petC alleles Supplemental File S3 Tracing the putative origin of ambiguous alleles Supplemental Fible S4 Main characteristics of genome sequences from three X fastidiosa isolates isolated in France TABLE LEGENDS Table List and frequency of plants contaminated by X fastidiosa in France in 2015 Positive results were based on qPCR (Harper et al., 2010) The frequency of detection of X fastidiosa in these plants is calculated upon the analyzed samples Bold characters indicate the description of a plant species as a novel host for X fastidiosa Table Characteristics of plant contaminations by X fastidiosa identified in France in 2015 This article is protected by copyright All rights reserved Table Allele designations for each gene and STs determined from the concatenated data Accepted Article set for contaminations recorded in France in 2015 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 Table Average nucleic identities based on blast for pairwise comparisons among X fastidiosa subsp multiplex strains and representatives of other subspecies FIGURE LEGENDS Figure Taxonomic position of X fastidiosa characterized in 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 indicates 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 foci survey (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 (1,000 replicates) are displayed at each node Figure Pairwise comparison of genome sequences of French and American Xylella fastidiosa strains Genomic sequence of French (CFBP 8416, CFBP 8417 and CFP 8418) and American (M12, Griffin-1, and Dixon) were cut in 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 This article is protected by copyright All rights reserved Pairwise comparisons of (A) CFBP 8416 (ST7) strain isolated from P myrtifolia versus Accepted Article CFBP 8417 and CFBP 8418 (S junceum, ST6); and comparisons of the French strains with the American strains as reference: (B), Dixon (ST6, P dulcis); (C), Griffin-1 (ST7, Q rubra) and (D), M12 (ST7, P dulcis) Table List and frequency of plants contaminated by X fastidiosa in France in 2015 Positive results were based on qPCR (Harper et al., 2010) The frequency of detection of X fastidiosa in these plants is calculated upon the analyzed samples Bold characters indicate the description of a plant species as a novel host for X fastidiosa Genus (total), speciesa Acer (total) Acer pseudoplatanus L Artemisia (total) Artemisia arborescens (Vaill.) L Asparagus (total) Asparagus acutifolius L Cistus (total) Cistus monspeliensis L Cistus salviifolius L Coronilla (total) Coronilla valentina Cytisus (total) Cytisus racemosus Hort.-Cf Marnock Genista (total) Genista ephedroides DC Hebe (total) Lavandula (total) Lavandula × heterophylla Viv Lavandula × intermedia Emeric ex Loisel Lavandula angustifolia Mill Lavandula dentata L Lavandula stoechas L Malus (total) b Malus domesticab Metrosideros (total) Metrosideros excelsa Sol ex Gaertn Myrtus (total) French Region Corsica Corsica Corsica Corsica Corsica Corsica Corsica Corsica Corsica Corsica Corsica Corsica Corsica Corsica Corsica Corsica Corsica Corsica Corsica Corsica Corsica Corsica Ile de France Ile de France Corsica Corsica Corsica This article is protected by copyright All rights reserved Nb of analyzed samples 24 4 29 23 66 49 2 33 15 10 111 26 19 29 1 17 154 % of positive samples 4.17 25.00 25.00 33.33 3.45 4.35 19.70 22.45 28.57 50.00 50.00 12.12 25.00 6.67 100.00 30.00 17.12 80.00 66.67 23.08 5.26 6.90 100.00 100.00 5.88 25.00 1.95 Accepted Article Myrtus communis L Pelargonium (total) Pelargonium graveolens L'Hér Polygala (total) Polygala × dalmaisiana Dazzler Polygala myrtifolia L Polygala (total) Polygala myrtifolia L Prunus (total) b Prunus cerasifera Ehrh Quercus (total) Quercus ilexb Quercus suber L Rosa (total) Rosa x floribunda Rosmarinus (total) Rosmarinus officinalis L Spartium (total) Spartium junceum L Corsica Corsica Corsica Corsica Corsica Corsica PACA PACA Corsica Corsica Corsica Corsica Corsica Corsica Corsica Corsica Corsica Corsica Corsica 138 106 17 1186 1176 328 261 254 321 88 43 265 265 40 40 2.17 16.04 29.41 35.75 100.00 35.97 6.71 1.15 0.39 20.00 0.93 100.00 2.27 2.33 100.00 0.75 0.75 22.50 22.50 a : when a species could be identified, specified data are given in following lines For instance, among 24 plants of the genus Acer that were analyzed, 4.17% were positive (1 plant) Among these 24 plants, were assigned to the A pseudoplatanus L species, from which 25% were positive (1 plant) b : one sample from a Malus domestica, a Prunus persica and a Quercus ilex were tested positive, but no subsequent samplings confirmed these results This article is protected by copyright All rights reserved Accepted Article Table Characteristics of plant contaminations by X fastidiosa identified in France in 2015 Identification Number of samples Foci monitoring in Corsica and PACA French territory survey Interceptions of coffee plants ST6 205 205 0 ST7 102 102 0 ST53 10 4 ST72 0 ST76 STnew1 1 0 Undetermined type 8 0 Incomplete typing 117 108 Not typed 103 99 This article is protected by copyright All rights reserved Table Allele designations for each gene and STs determined from the concatenated data Accepted Article set for contaminations recorded in France in 2015 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 ST cysG gltT holC leuA maIF nuoL petC 3 3 3 53 72 24 26 14 10 24 12 16 15 16 18 12 76 26 24 12 15 18 13 New1 Undetermined#1 Undetermined#2 Undetermined#3 Undetermined#4 26 26 26 26 26 3 1/3/10 3 3 24 24 3/4/9/24 3 3 3 15 3 3 18 3/18 3/18 3/11/13 13 13 3/11/13 This article is protected by copyright All rights reserved Table Average nucleic identities based on blast for pairwise comparisons among X Accepted Article fastidiosa subsp multiplex strains and representatives of other subspecies multiplex morus sandyi fastidiosa pauca ATCC Sy-VA 35871 M12 Grif fin-1 CFBP Dixon CFBP CFBP 8416 8417 8418 ATCC 35871 - 99.45 99.39 99.40 99.55 99.43 99.68 99.63 Sy-VA 99.38 - 99.31 99.31 99.17 99.36 99.36 99.36 M12 99.31 99.35 - 99.95 99.58 99.68 99.69 99.69 Griffin-1 99.39 99.37 99.98 - 99.59 99.69 99.69 99.68 CFBP 8416 99.17 99.08 99.44 99.42 - 99.45 99.51 99.48 Dixon 99.33 99.27 99.58 99.57 99.47 - 99.92 99.92 CFBP 8417 99.23 99.22 99.49 99.45 99.45 99.95 - 99.98 CFBP 8418 99.21 99.19 99.46 99.42 99.43 99.97 99.96 - Mul-MD 98.19 98.10 97.73 97.78 97.72 97.76 97.80 97.83 Mul0034 98.06 97.95 97.61 97.65 97.60 97.64 97.72 97.71 Ann-1 97.41 97.31 97.12 97.16 97.07 97.16 97.22 97.25 CFBP 8073 97.39 97.32 97.13 97.17 97.16 97.16 97.19 97.17 Temecula1 97.68 97.58 97.35 97.40 97.44 97.36 97.56 97.57 M23 97.69 97.56 97.35 97.36 97.59 97.42 97.78 97.66 GB514 97.65 97.58 97.40 97.43 97.28 97.42 97.45 97.48 EB92.1 97.63 97.59 97.33 97.33 97.20 97.26 97.32 97.31 ATCC 35879 97.64 97.51 97.29 97.36 97.34 97.3 97.68 97.67 9a5c 96.17 96.12 96.07 96.13 96.03 96.04 96.14 96.11 6c 96.24 96.16 96.17 96.18 96.15 96.13 96.17 96.16 32 96.09 96.0 95.89 95.93 95.96 95.89 95.94 95.95 CFBP 8072 96.19 96.06 96.09 96.08 95.99 96.06 96.10 96.11 CoDiRO 96.28 96.25 96.19 96.20 96.06 96.19 96.76 96.86 This article is protected by copyright All rights reserved Accepted Article This article is protected by copyright All rights reserved Accepted Article This article is protected by copyright All rights reserved