A sap alien: breaking news of Xylellafastidiosa, an emerging plant pathogen in France. Marie-Agnès Jacques

A sap alien: breaking news of Xylellafastidiosa, an emerging plant pathogen in France Marie-Agnès Jacques

American origin 6 subspecies >350 host plants fastidiosa sandyi morus multiplex pauca tashke undescribed subspecies Schaadet al., 2004; Randall et al., 2009; Nunneyet al., 2010, 2012, 2014a, 2014b

American origin 6 subspecies >350 host plants fastidiosa sandyi morus multiplex pauca tashke undescribed subspecies Schaadet al., 2004; Randall et al., 2009; Nunneyet al., 2010, 2012, 2014a, 2014b

France(Summer 2015) Recent emergences in Asia and Europe Italy(Fall 2013) Taiwan (Winter 2012) Iran (Summer 2014) Reports fromindia, Lebanon, Turkeyand China werenot confirmedor were invalidated. Saponariet al., 2013; Su et al., 2013; Amanifaret al. 2014; Jindal and Sharma, 1987; Chu 2001; Güldür et al., 2005; Habib, 2016.

2012-2015: Several interceptions of coffee plants infected by X. fastidiosa in France Ornamental plants: imported from Central America Production plants: voluntary-based survey on imported material from Latin America

Assignation of coffee isolates to X. fastidiosa subspecies Multiprimer PCR test (3 genes) Hernandez-Martinez et al., 2006 Control (+) fastidiosa multiplex pauca sandyi (-) MLSA (7 genes) fastidiosa fastidiosa sandyi multiplex pauca (-) sandyi morus MLSA: Partial sequences of 7 housekeeping genes: cysg, gltt, holc, leua, malf, nuol, petcas described in http://pubmlst.org/xfastidiosa/ Concatenated dataset; ML, 1000 bootstraps multiplex pauca = isolates from intercepted infected coffee plants Jacques et al., 2016; Unpublished data

New variants of coffee-infecting X. fastidiosa in France MLST scheme (7 genes; http://pubmlst.org/xfastidiosa/) Date Country Strain cysg gltt holc leua mal F nuol petc ST subsp 2012 Mexico CFBP 8073 29 1 1 9 10 19 1 75 2014 2015 2015 Costa Rica?? CFBP 8356 COF160 COF198.1 fastidiosa (sandyi) 26 1 24 12 15 18 13 76* sandyi 2015 Honduras COF167.2 26 1 24 12 15 18 12 72* sandyi 2012 2012 Ecuador Ecuador CFBP 8072 CFBP 8074 28 8 25 7 8 16 6 74 pauca 2015? COF1214.1 24 14 10 7 16 16 6 53* pauca Coffee plants trade Novel alleles and STs * Identical STs detected in France and Italy Giampettruzzi et al., 2015; Jacques et al., 2015; Loconsole et al., 2016; Unpublished data

Phylogenetic network of Xylella fastidiosa sequence types fastidiosa multiplex morus sandyi Intercepted isolates reveal additional genetic diversity Concatenated dataset Jacques et al., 2016; Unpublished data pauca

L affaire se Corse : X. fastidiosa outbreak in Corsica July 22 nd 2015 Symptomatic Polygala myrtifolia infected by X. fastidiosa were detected in Propriano Phytosanitary measures were applied And on Oct. 8 th, in Mainland France

Xf detection in plants SRAL, FREDON, DSF, DDSCPP Accredited labs French reference method: Anses-LSV MA039 version 01 https://www.anses.fr/fr/system/files/anses_ma039_xylellafastidiosa_final.pdf 1 sample= 5 to 100 petioles= 0.5 to 1 g 2 DNA extractions / sample AutomatedDNA extraction QuickPick Plant DNA kit (Bio-Nobile) and KingFisher ml 4 amplifications Real-time PCR Harper et al., 2010

Xf detection in plants Perfomance criteria of the method Performance criteria Pure strain cultures Inclusivity Exclusivity Matrix 100% (23 targeted strains representing the 4 major subsp.) 100% (29 non-targeted strains: Xylophilus, Xanthomonas, Liberibacter, sap saprophytes) Plant matrix ( 10² -10 5 bact./ml,15 samples) Orange tree + X. f. paucacfbp8072 Grappe + X. f. fastidiosacfbp7970 Olive tree + X. f.multiplex CFBP8173 Sensitivity 100% 94% 67% Specificity 100% 100% 100% Repetability 100% 96% 100% Reproductibility 98% Detection threshold (with detection probability of 100%) 10² bact./ml 10 3 bact./ml 10 5 bact./ml A performant method The presence of inhibitors (polyphenol, secondary metabolites, ) in some plant matrix (olive tree, oak) limits its sensitivity.

PACA: Provence, Alpes, Côte d azur 12 foci Situation in France - March 2016 Corsica 242 foci highestfocus: 530m Isolated from 2 plant species -Polygala myrtifolia -Spartium junceum foci buffer area sampled Isolated from 21 plant species http://www.corse-du-sud.gouv.fr/xylella-fastidiosa-une-menace-qui-demande-une-a1409.html http://www.alpes-maritimes.gouv.fr/politiques-publiques/securite-et-protection-de-la-population/xylella-fastidiosa/communiques

X. fastidiosacontaminatedplants in France Polygala myrtifolia Lavendula sp. Pelargonium graveolens Spartium junceum Cistus sp. Myrtus communis Hebe sp. Cytisus racemosus Metrosideros excelsa Acer pseudoplatanus Quercus suber Prunus cerasifera Rosa x floribunda Asparagus acutifolius Genista hephredoides Rosmarinus officinalis Total Nb analyzed samples Nb positive samples Nb negative samples % positive samples Acer(total) 22 22 1 20 4,5 % Asparagus(total) 32 32 1 31 3,1 % Cistus(total) 96 96 16 75 16,6 % Citrus(total) 264 260 0 259 0 % Cytisus racemosus 8 8 2 6 25 % Genista ephedroides 1 1 1 0 100 % Hebe(total) 11 11 3 8 27,3 % Lavandula(total) 138 138 23 110 16,7 % Metrosideros(total) 17 17 1 16 5,9 % Myrtus communis 166 165 3 158 1,8 % Olea europaea 548 548 0 548 0 % Pelargonium(total) 115 114 18 92 15,8 % Polygala myrtifolia 1284 1273 435 826 34,2 % Prunus(total) 243 240 1 238 0,4 % Quercus(total) 335 327 1 322 0,3 % Rosa(total) 44 44 1 43 2,3 % Rosmarinus(total) 286 286 2 284 0,7 % Spartium junceum 53 53 18 35 34 % Vitis(total) 80 80 0 79 0 % Autres 1246 1236 5 1214 0,4 % Total 4990 4952 533 4364 10,7 % 4,990 sampleson the 4 th of March 2016

Plants never found infected in France, so far # samples Olea europaea 548 Oleander spp. 111 Quercus spp. (other than Q. suber) 335 Citrus 264 14

Corsica/PACA regions: X. fastidiosa subsp. multiplex Multiprimer PCR test Control (+) MLSA7 HK genes Low bootstrap (63): 2 groups with high genetic promiscuity A Polygala myrtifolia 6 Cistus monspeliensis 2 Cystisus racemosus 1 Myrtus communis 1 Pelargonium sp. 1 Quercus suber 1 Rosa sp. 1 B 638bp 521bp 412bp A fastidiosa multiplex pauca sandyi (-) MLSA (7 genes) High bootstrap (96): subspecies multiplex B Polygala myrtifolia 22 Pelargonium sp. 9 Cistus sp. 4 Spartium junceum 3 Cystisus racemosus 2 Hebe sp. 2 Lavandula sp. 2 Artemisia arborencens1 Coronilla valentina 1 Metrosideros sp. 1 Prunus cerasifera 1 subsp. multiplex Hernandez-Martinez et al., Plant Dis (2006) No host specificity of the lineages >400 samples

Corsica/PACA strains share identities to US strains MLST(7 genes): 2 STs identical to subsp. multiplex US strains M12 (Prunus dulcis) Griffin-1 (Quercus rubra) ST cysg gltt holc leua malf nuol petc A 7 7 3 3 3 3 3 3 B 6 3 3 3 3 3 3 3 High genetic promiscuity: 1 SNP (G152A) at a single locus Occurrence of ST6 and ST7 isolates ST isolate country host species name year 6 ALS0003 USA almond Prunus dulcis 1990s 6 ALS0013 USA almond Prunus dulcis 2000s 6 ALS0015 USA almond Prunus dulcis 1994 6 ALS0073 USA almond Prunus dulcis 1994 6 ALS0091 USA almond Prunus dulcis 1994 6 ALS0092 USA almond Prunus dulcis 1994 7 OLV0029 USA olive Olea europaea 2003 7 SM0053 USA black sage Salvia mellifera 2004 7 SM0054 USA black sage Salvia mellifera 2004 7 SM0056 USA black sage Salvia mellifera 2004 7 ALS0206 USA almond Prunus dulcis. 7 ALS0299 USA almond Prunus dulcis 2003 7 ALS0361 USA almond Prunus dulcis. 7 OLV00471 USA olive Olea europaea 2011 Dixon ST6: Almond; USA ST7: Almond, black sage, olive, red oak; USA http://pubmlst.org/xfastidiosa/

Genome sequencing of 3 strains isolated in Corsica Isolation on PWG Illumina MiSeq: (Plateforme Génomique de Nantes, Biogenouest) CATI BBRIC analytic tools Strain Host, location ST Contigs CFBP 4616 CFBP 4617 CFBP 4618 Polygala myrtifolia, Propriano Spartium junceum, Alata Spartium junceum, Alata N50 (bp) Genome size (bp) GC% Genes Prot nc Genes 7 128 113,891 2,466,748 51.79 2697 2642 55 6 256 118,419 2,504,981 51.85 2618 2564 54 6 271 96,035 2,513,969 51.88 2681 2625 56 Data similar to those of other X. fastidiosa genomes publicly available

Average nucleotide identity (ANIb) multiplex morus sandyi fastidiosa pauca PLS 229 ATCC 35871 Sy-VA M12 Griffin-1 LSV CFBP 4677 4616 Dixon LSV CFBP 4678 4617 LSV CFBP 4679 4618 Mul-MD Mul0034 ANIb PLS 229 --- 83.64 83.56 83.64 83.65 83.59 83.59 83.61 83.62 83.47 83.41 83.44 83.49 83.44 83.41 83.40 83.46 83.51 83.40 83.77 83.77 83.80 83.77 83.77 ATCC 35871 83.89 --- 99.45 99.39 99.40 99.55 99.43 99.68 99.63 98.26 98.25 97.60 97.72 97.71 97.74 97.73 97.76 97.72 97.72 96.30 96.23 96.31 96.55 96.30 Sy-VA 83.70 99.38 --- 99.31 99.31 99.17 99.36 99.36 99.36 98.17 98.17 97.50 97.57 97.65 97.70 97.69 97.72 97.65 97.67 96.18 96.17 96.23 96.45 96.15 M12 83.74 99.31 99.35 --- 99.95 99.58 99.68 99.69 99.69 97.85 97.79 97.51 97.47 97.53 97.55 97.53 97.52 97.50 97.50 96.15 96.22 96.23 96.48 96.21 Griffin-1 83.77 99.39 99.37 99.98 --- 99.59 99.69 99.69 99.68 97.92 97.90 97.49 97.51 97.57 97.56 97.56 97.54 97.52 97.52 96.21 96.22 96.21 96.56 96.26 CFBP 4616 LSV 4677 83.65 99.17 99.08 99.44 99.42 --- 99.45 99.51 99.48 97.67 97.61 97.19 97.21 97.21 97.20 97.19 97.18 97.20 97.14 95.90 95.88 96.02 96.22 95.91 Dixon 83.59 99.33 99.27 99.58 99.57 99.47 --- 99.92 99.92 97.86 97.82 97.44 97.40 97.50 97.46 97.55 97.44 97.40 97.44 96.03 96.05 96.10 96.39 96.12 LSV 4678 83.64 99.23 99.22 99.49 99.45 99.45 99.95 --- 99.98 97.78 97.64 97.36 97.24 97.36 97.41 97.43 97.39 97.30 97.40 95.92 95.98 96.01 96.24 96.06 LSV 4679 83.57 99.21 99.19 99.46 99.42 99.43 99.97 99.96 --- 97.74 97.67 97.38 97.26 97.38 97.35 97.35 97.34 97.31 97.34 95.95 95.97 96.04 96.97 96.02 Mul-MD 83.51 98.19 98.10 97.73 97.78 97.72 97.76 97.80 97.83 --- 99.77 97.99 98.29 98.10 98.40 98.37 98.40 98.49 98.37 95.81 95.78 95.83 96.20 95.97 Mul0034 83.30 98.06 97.95 97.61 97.65 97.60 97.64 97.72 97.71 99.79 --- 97.89 98.22 97.97 98.33 98.29 98.39 98.42 98.31 95.67 95.65 95.74 96.08 95.80 Ann-1 83.38 97.41 97.31 97.12 97.16 97.07 97.16 97.22 97.25 97.89 97.82 --- 98.12 97.99 97.97 97.97 97.99 98.02 98.00 95.65 95.77 95.74 96.16 95.93 CFBP 8073 83.47 97.39 97.32 97.13 97.17 97.16 97.16 97.19 97.17 98.11 98.01 97.97 --- 98.34 98.75 98.72 98.74 98.84 98.78 95.65 95.76 95.77 96.14 95.84 CO33 83.36 97.39 97.26 97.10 97.17 97.12 97.11 97.13 97.13 97.76 97.68 97.72 98.26 --- 98.00 98.01 97.98 98.14 97.99 95.63 95.64 95.79 96.12 95.77 Temecula1 83.40 97.68 97.58 97.35 97.40 97.44 97.36 97.56 97.57 98.41 98.39 98.04 98.89 98.28 --- 99.90 99.91 99.71 99.77 95.82 95.80 95.90 96.23 95.92 M23 83.33 97.69 97.56 97.35 97.36 97.59 97.42 97.78 97.66 98.32 98.30 98.00 98.83 98.24 99.87 --- 99.83 99.64 99.77 95.79 95.78 95.87 96.19 95.93 GB514 83.44 97.65 97.58 97.40 97.43 97.28 97.42 97.45 97.48 98.43 98.39 98.09 98.82 98.37 99.93 99.89 --- 99.72 99.79 95.83 95.83 95.90 96.25 95.91 EB92.1 83.48 97.63 97.59 97.33 97.33 97.20 97.26 97.32 97.31 98.45 98.44 98.09 98.78 98.28 99.64 99.62 99.67 --- 99.68 95.74 95.73 95.78 96.15 95.97 ATCC 35879 83.29 97.64 97.51 97.29 97.36 97.34 97.30 97.68 97.67 98.30 98.32 98.05 98.83 98.21 99.76 99.79 99.75 99.69 --- 95.73 95.78 95.85 96.16 95.85 9a5c 83.73 96.17 96.12 96.07 96.13 96.03 96.04 96.14 96.11 95.85 95.76 95.83 95.95 95.87 95.84 95.86 95.84 95.85 95.85 --- 98.85 99.16 97.96 98.10 6c 83.91 96.24 96.16 96.17 96.18 96.15 96.13 96.17 96.16 95.83 95.82 95.90 95.95 95.92 95.85 95.82 95.84 95.86 95.83 98.86 --- 98.88 97.96 98.19 32 83.92 96.09 96.00 95.89 95.93 95.96 95.89 95.94 95.95 95.68 95.64 95.68 95.82 95.91 95.80 95.77 95.75 95.80 95.75 99.06 98.77 --- 97.86 97.98 CFBP 8072 83.72 96.19 96.06 96.09 96.08 95.99 96.06 96.10 96.11 95.96 95.92 96.01 96.02 96.11 96.02 95.99 96.09 96.07 96.01 97.74 97.67 97.86 --- 98.29 CoDiRO 83.95 96.28 96.25 96.19 96.20 96.06 96.19 96.76 96.86 96.11 96.05 96.05 96.19 96.20 96.05 96.07 96.05 96.15 96.05 98.20 98.25 98.25 98.64 --- CFBP 4617 CFBP 4618 Ann-1 CFBP 8073 CO33 Temecula1 M23 GB514 EB92.1 ATCC 35879 9a5c 6c 32 CFBP 8072 CoDiRO Corsica strains: >99% identity vs. X. fastidiosa subsp. multiplex.

CGview: Pairwise genome comparisons (1kb fragments) CFBP4617 ST6, Sjvs. -CFBP4618 ST6, Sj(ext) -CFBP4616 ST7, Pm (int) ST7 M12 (P. dulcis) CFBP 4616 (P. myrtifolia) ST6 BLAST hit = 100% identical BLAST hit >= 98% identical BLAST hit >= 96% identical BLAST hit >= 94% identical Dixon (P. dulcis) CFBP 4617, CFBP 4618 (S. junceum) Comparison among Corsica strains: - CFBP 4617 vs. CFBP 4618: nearly 100% identical. - CFBP 4617 vs. CFBP 4616: strong genetic promiscuity but lower identities. White stripes: specific fragments of CFBP 4617

CGview: Pairwise genome comparisons (1kb fragments) Dixon vs. -CFBP4618 ST6, Sj(ext) -CFBP4617 ST6, Sj -M12 ST7, Pd -CFBP4616 ST7 Pm -Griffin-1 ST7 Qr(int) ST7 ST6 M12 (P. dulcis) CFBP 4616 (P. myrtifolia) BLAST hit = 100% identical BLAST hit >= 98% identical BLAST hit >= 96% identical BLAST hit >= 94% identical Dixon (P. dulcis) CFBP 4617, CFBP 4618 (S. junceum) -CFBP 4617 and CFBP 4618: highest identities with the Dixon strain. White stripes: sequences of one plasmid from Dixon are missing in other strains No ORFs from pxf-riv5 in strains from Corsica, M12 and Griffin-1

Ongoing: 1. pathogenicity assays Aim:Getting knowledgeon Xfstrains from French outbreak hostrange and fulfilling Koch postulates Pays de la Loire region: Grapevine -Apple -Pear -Prunus Corsica region: Citrus - Olive - Polygala X. fastidiosa strains Subsp. fastidiosa, sandyi, multiplex, pauca US French Italian isolates Positive Controls: Coffee - Olive - Oleander

19 wpi Ongoing: 1. pathogenicity assays CoDiRO Nerium oleander CFBP 8072 Coffea arabica CoDiRO = ST53, first described for Coffee and Oleander isolates in Costa Rica Ongoing: Bacterial isolation and qpcr detection tests

Ongoing: 1. pathogenicity assays Aim:Getting knowledgeon Xfstrains from French outbreak hostrange and fulfilling Koch postulates Pays de la Loire region: Grapevine -Apple -Pear -Prunus Corsica region: Citrus - Olive - Polygala X. fastidiosa strains Subsp. fastidiosa, sandyi, multiplex, pauca US French Italian isolates Positive Controls: Coffee - Olive - Oleander Tibicina Corsica ssp. corsica Cicadetta fangoana Ongoing: 2. insect analyses Philaenus spumarius Cicada orni Ledra aurita Cicadella viridis Elaborate the reference method Aphrophora alni Aphrophora pectoralis Test insects sampled in Corsica last summer: Neophilaenus lineatus Lepyronia coleoptrata

Acknowledgments Sap Alien project Bruno Legendre Valérie Olivier Françoise Poliakoff Amandine Cunty Nicolas Denancé Martial Briand Sophie Cesbron Perrine Portier InEMteamfortestsonplants: Rémi Gardet Daniel Sochard Département SPE Muriel Bahut Maria Saponari Yannick Blanchard Claire de Boisséson Fabrice Touzaint Pauline de Jerphanion Dominique Crouzillat Stelly Mississipi Emmanuelle Morel