Probable dates and scenario of introduction of Xylella fastidiosa subsp. multiplex in France
Enora DUPAS
26 - 30 April 2021 Online
1 Background World distribution of Xylella fastidiosa
Legend : Before 2013 After 2013 subsp. fastidiosa subsp. sandyi subsp. morus subsp. multiplex subsp. pauca indeterminate
2 Background World distribution of Xylella fastidiosa
~2002
? Legend : ~1890 Before 2013 After 2013 ~1980 subsp. fastidiosa ~1930 subsp. sandyi subsp. morus subsp. multiplex subsp. pauca indeterminate
Nunes et al., 2003; Nunney et al., 2010, 2012, 2014; Yuan et al., 2010; Saponari et al., 2013, 2019; Su et al., 2013; Martelli, 2016; Denancé et al., 2017; Vanhove et al., 2020 3 Background European distribution of Xylella fastidiosa
France (2015)
Legend : subsp. fastidiosa subsp. multiplex subsp. pauca
Spain (2016)
ST6 ST7 ST53 ST87 ST7 ST76 Portugal (2018) ST6 Italy (2013) ST6 ST7 ST53 ST81
ST6 ST1 ST80 ST7 ST53 ST81
EPPO Global Database; Saponari et al., 2013, 2019; Denancé et al., 2017; Landa, 2017; Olmo et al., 2017; Marchi et al., 2018; Gomila et al., 2019; Cunty et al., 2020 4 Background European distribution of Xylella fastidiosa
Legend : subsp. fastidiosa subsp. multiplex subsp. pauca
~1985
~1998
ST6 ST7 ~1995 ST53 ST87 ST7 ST76 ST6 ST7 ST6 ~2008 ST53
ST81
ST6 ST1 ST80 ST7 ST53 ~2005 ST81
Martelli et al., 2016; Vanhove et al., 2019; Moralejo et al., 2020 5 Objective Define the probable dates and scenario of introduction of Xylella fastidiosa subsp. multiplex in France
6 Background French distribution of Xylella fastidiosa
M12 Griffin-1 Red Olive Geographical distribution of Xylella fastidiosa-infected samples Almond oak tree in France between 2015 and 2018 ST7
ST6
Dixon https://pubmlst.org/xfastidiosa/
France: 917 positive samples
Positive ST6: 51% Positive subsp multiplex Positive subsp pauca ST7: 49% Positive, unknown subsp Denancé et al., 2017
7 Background French distribution of Xylella fastidiosa
M12 Griffin-1 Red Olive Geographical distribution of Xylella fastidiosa-infected samples Almond oak tree in France between 2015 and 2018 ST7
ST6
Dixon https://pubmlst.org/xfastidiosa/
Positive 917 positive samples Positive subsp multiplex Positive subsp pauca 52 isolated strains Positive, unknown subsp Denancé et al., 2017
8 Background French distribution of Xylella fastidiosa
M12 Griffin-1 Red Olive Geographical distribution of Xylella fastidiosa-infected samples almond oak tree in France between 2015 and 2018 ST7 To trace the evolutionary history of X. fastidiosa subsp. multiplex in France ST6 Sequencing of the 52 French strains of X. fastidiosa subsp. multiplexDixon https://pubmlst.org/xfastidiosa/
Positive 917 positive samples Positive subsp multiplex Positive subsp pauca 52 isolated strains Positive, unknown subsp Denancé et al., 2017
9 Diversity of French X. fastidiosa subsp. multiplex strains Dating of the introduction-divergence
82 genomic sequences of X. fastidiosa subsp. multiplex
France USA ST6 : 23; ST7 : 29 ST6 : 1; ST7 : 3 Spain Italy others ST : 10 ST6 : 9; ST81 : 3 ST87 : 3
Brazil ST26 : 1
10 Diversity of French X. fastidiosa subsp. multiplex strains Dating of the introduction-divergence
82 genomic sequences of X. fastidiosa subsp. multiplex
Xfm genome ~2,5.106 pb France ST6 : 23; ST7 : 29 USA ST6 : 1; ST7 : 3 Spain Italy others ST : 10 ST6 : 9; ST81 : 3 ST87 : 3 Core genome 1 679 574 pb
Brazil ST26 : 1
13 818 SNP mutations
SNP: single nucleotide polymorphism 11 Diversity of French X. fastidiosa subsp. multiplex strains Dating of the introduction-divergence
: tip-dating analyses Date the nodes of the tree using the substitution rate and the date of isolation of the strains
Other ST
12 Diversity of French X. fastidiosa subsp. multiplex strains Dating of the introduction-divergence
: tip-dating analyses 2011 o 3, 2165 x 10-7 substitutions/site/year RAAR6Butte o subsp. pauca -> 7,62x10-7 substitutions/site/year (Vanhove et al., 2019) 1026 o subsp. fastidiosa -> 7,71x10-7 substitutions/site/year (Moralejo et al., 2020) 6,38x10-7 substitutions/site/year (Vanhove et al., 2020)
1971
755 M12 Griffin-1
1424
1987 Dixon
Other ST
13 Diversity of French X. fastidiosa subsp. multiplex strains Dating of the introduction-divergence
: tip-dating analyses 2011 o 3, 2165 x 10-7 substitutions/site/year RAAR6Butte o subsp. pauca -> 7,62x10-7 substitutions/site/year (Vanhove et al., 2019) 1026 o subsp. fastidiosa -> 7,71x10-7 substitutions/site/year (Moralejo et al., 2020) 6,38x10-7 substitutions/site/year (Vanhove et al., 2020)
o ST7 divergence between French and American strains : 1971 1971 (95% HPD: 1924-1993) 755 M12 Griffin-1
1424
o ST6 divergence between French and American strains : 1987 1987 (95% HPD: 1974-1994) Dixon
Other ST
HPD: Highest Posterior Density 14 M12 Griffin-1 Red Olive Geographical distribution of Xylella fastidiosa-infected samples almond oak tree in France between 2015 and 2018 ST7
ST6
Dixon https://pubmlst.org/xfastidiosa/ To trace the evolutionary history of X. fastidiosa subsp. multiplex in France To develop a MLVA scheme for direct typing of frozen samples
Positive 917 positif samples Positive subsp multiplex Positive subsp pauca 52 isolated strains Positive, unknown subsp Denancé et al., 2017
15 Diversity of French X. fastidiosa subsp. multiplex strains MLVA: Development of a new scheme
Repeat Nb repeats Strain A : attgctATTATTATTATTgccgta 4 Strain B : tgctagATTATTATTgtcagt 3 Strain C : tgaactATTATTATTATTATTgctacg 5 Strain D : gctgaaATTATTATTATTATTATTgctcga 6
1 3
5 3
2 5
MLVA profile Strain A : 1 - 3 - 2 - 5 Strain B : 3 - 2 - 5 - 3
16 Diversity of French X. fastidiosa subsp. multiplex strains MLVA: Development of a new scheme
Repeat Nb repeats Strain A : attgctATTATTATTATTgccgta 4 Strain B : tgctagATTATTATTgtcagt 3 Strain C : tgaactATTATTATTATTATTgctacg 5 o 13 VNTRs for Xfm typing Strain D : gctgaaATTATTATTATTATTATTgctcga 6 COSS1 o 9 from de Lin et al 2005 o 1 from Francisco et al 2017 GSSR7 o 3 newly designed ASSR12 ASSR19 1 3 ASSR11 VNTR58
OSSR16 3 5 ASSR16 GSSR4
2 5 VNTR40 OSSR19 VNTR37 MLVA profile ASSR9 Strain A : 1 - 3 - 2 - 5 Strain B : 3 - 2 - 5 - 3
17 Diversity of French X. fastidiosa subsp. multiplex strains MLVA: Development of a new scheme
1. Validation of the MLVA scheme in silico
10 Xfm genomes COSS1
GSSR7 ASSR12 and in vitro ASSR19 ASSR11 VNTR58 OSSR16 86 Xfm strains ASSR16 GSSR4
VNTR40 OSSR19 VNTR37 ASSR9 o 52 French strains (ST6, ST7) o 16 American strains(ST6, ST9, ST10, ST23, ST27, ST37, ST41, ST42, ST43, ST45, ST51) o 18 Spanish strains (ST6)
18 Diversity of French X. fastidiosa subsp. multiplex strains MLVA: Development of a new scheme
1. Validation of the MLVA scheme in silico 2. Direct typing
10 Xfm genomes COSS1
GSSR7 ASSR12 and in vitro ASSR19 ASSR11 VNTR58 Spiking of Polygala OSSR16 myrtifolia samples 86 Xfm strains ASSR16 and DNA extraction GSSR4
VNTR40 OSSR19 VNTR37 ASSR9 o 52 French strains (ST6, ST7) Comparison : o 16 American strains(ST6, ST9, 13 naturally infected ST10, ST23, ST27, ST37, ST41, Source: Gerbeaud Photo: E. Douzery. Source: Wikipedia samples ST42, ST43, ST45, ST51) VS o 18 Spanish strains (ST6) Their isolated strain
Source: pepinierebotanique.com Photo: de Salvator Source: wikipedia Reliable and robust method 19 Diversity of French X. fastidiosa subsp. multiplex strains MLVA : Characterization of French individuals
Selection criteria for the 534 samples 917 Available samples: Bacterial load: Cq ≤ 32 (>104 b/mL) 738 Corse + 179 PACA Maximum coverage of the French territory for each year Maximum diversity of plant species 534 selected samples : 383 Corse + 151 PACA Successful MLVA typing on 396 samples: 74% of tested samples 396 samples typed 43 % of French samples previously typed by MLST by MLVA: 283 Corse + 113 PACA
20 Diversity of French X. fastidiosa subsp. multiplex strains MLVA : Characterization of French individuals: 320 haplotypes ST6
ST6 : 184 samples 148 haplotypes 4 clonal complexes
Founder CC : 11 individuals from 3 plant species (Polygala myrtifolia, Lavandula, Calicotome)
ST7 : 212 samples 172 haplotypes 15 clonal complexes
Founder CC : 15 individuals from 3 plant species (Polygala myrtifolia, Genista, Helichrysum italicum)
1 locus CC 2 loci ST7 21 ST6 Year of isolation: Founder haplotype sampled over time
Haplotypes sampled in 2015 present at the end of genetic lineages
Xfm in France : a diverse population
1 locus CC 2 loci ST7 22 ST6 Year of isolation: Founder haplotype sampled over time
Haplotypes sampled in 2015 present at the end of genetic lineages
Xfm in France : a diverse population
1 locus CC 2 loci ST7 23 ST6 Year of isolation: Founder haplotype sampled over time
Haplotypes sampled in 2015 present at the end of genetic lineages
Xfm in France : a diverse population
1 locus CC 2 loci ST7 24 ST6 Year of isolation: Founder haplotype sampled over time
Haplotypes sampled in 2015 present at the end of genetic lineages
Xfm in France : a diverse population
1 locus CC 2 loci ST7 25 Diversity of French X. fastidiosa subsp. multiplex strains MLVA : Identification of introductions and dissemination routes ABC analyses of MLVA data
ST6 ST6 ST6 ST7 ST7 ST7 C1P1 C2 P2 C1P1 C2 P2 ABC groups
135 Corse + 2 PACA 19 Corse 110 Corse + 8 PACA 19 Corse 75 PACA 28 PACA
FST/RST : p-value < 0,05
C1P1: Corse1 et PACA1 ; C2: Corse 2 ; P2: PACA2 26 Diversity of French X. fastidiosa subsp. multiplex strains MLVA : Identification of introductions and dissemination routes
ST6 ST6 ST6 ST7 ST7 ST7 C1P1 C2 P2 C1P1 C2 P2 ABC group FST/RST : p-value < 0,05
1987 1971 ST6 ST7
Menton Nice Antibes Nice Saint Raphael Antibes Cannes
La Seyne-sur-mer ? Legend : ? C1P1 group Bastia Corbara Calvi Founders
Ota C2 group P2 group Casaglione
Ajaccio Ajaccio Porto- Samples Vecchio Propriano Porto- Strains Vecchio
Bonifacio
C1P1: Corse1 et PACA1 ; C2: Corse 2 ; P2: PACA2 27 Diversity of French X. fastidiosa subsp. multiplex strains MLVA : Identification of introductions and dissemination routes ABC analyses of MLVA data
1971 1987 ST6 ST7 t3
1 1 Step 1: t2 Step 1: Introduction P2 Introduction P2 t1 Menton Nice Antibes Nice t Saint Raphael Antibes USA C1P1 C2 P2 0 Cannes
La Seyne-sur-mer
Bastia Corbara Calvi Legend : C1P1 group Founders Ota
Casaglione C2 group
Ajaccio P2 group Ajaccio Porto- Vecchio Propriano Porto- Samples Vecchio Strains Bonifacio
28 Diversity of French X. fastidiosa subsp. multiplex strains MLVA : Identification of introductions and dissemination routes ABC analyses of MLVA data
1971 1987 ST6 ST7 t3
1 1 t2 Step 2: Step 2:
C1P1 differentiation t1 Menton C1P1 differentiation 2 Nice from P2 2 from P2 Antibes Nice t Saint Raphael Antibes USA C1P1 C2 P2 0 Cannes
La Seyne-sur-mer
Bastia Corbara Calvi Legend : C1P1 group Founders Ota
Casaglione C2 group Ajaccio 2 P2 group Ajaccio 2 Step 2: Porto- Step 2: C1P1 differentiation Vecchio Propriano Porto- C1P1 differentiation Vecchio from P2 Samples from P2 Strains Bonifacio
29 Diversity of French X. fastidiosa subsp. multiplex strains MLVA : Identification of introductions and dissemination routes ABC analyses of MLVA data
1971 1987 ST6 ST7 t3
1 1 Step 3: t2 Step 3: C2 differentiation C2 differentiation from P2 t1 Menton from P2 2 Nice 2 Antibes Nice t Saint Raphael Antibes USA C1P1 C2 P2 0 Cannes
La Seyne-sur-mer
Bastia Légende : Corbara Calvi Groupe C1P1 Fondateurs Ota
Casaglione Groupe C2 3 3 Groupe P2 Ajaccio 2 Ajaccio 2 Porto- Vecchio Propriano Porto- Échantillon Vecchio
Souches Bonifacio
30 Conclusion
ST6 1987 Hypothesis: introduction de X. fastidiosa subsp. multiplex ST6: -> from the USA Nice -> via myrtle leaf milkwort plant? Antibes
Bastia Calvi
Source: Gerbeaud
Ota myrtle leaf milkwort plant (Polygala myrtifolia)
Casaglione
Ajaccio Porto- Vecchio
31 Conclusion
ST6 1987
Nice Antibes
Bastia Calvi
Ota
Casaglione
Ajaccio Porto- Landa et al., 2020 Vecchio
32 Conclusion
ST6 1987
Nice Antibes
Bastia Calvi
Ota
Casaglione
Ajaccio Porto- Vecchio
33 Conclusion Hypothesis: introduction of X. fastidiosa subsp. multiplex ST6: ST6 1987 -> Entering trade of plants in Corsica (Chauvel et al., 2015) Nice -> Massive imports of alien plants (70s to 90s) Antibes (Jeanmonod & Natali, 1997) -> Ajaccio & Porto Vecchio commercial ports
Bastia Number of alien species introduced in Corsica Calvi Chauvel et al., 2015
Ota
Casaglione
Ajaccio Porto- Vecchio
Jeanmonod & Natali, 1997 1987 34 Conclusion
1971 Hypothesis: introduction de X. fastidiosa subsp. multiplex ST7: ST7 -> Scenario close to the ST6 one Menton -> Earlier introduction Nice Saint Raphael Antibes -> Wider initial spread Cannes
La Seyne-sur-mer
Corbara
Ajaccio
Propriano Porto- Vecchio
Bonifacio
35 Perspectives
X. fastidiosa subsp. multiplex What should we do next? French climate favorable for subsp. multiplex spread and survival
A threat for French environment? (Lousteau et al., 2020) Maintenance of the epidemiosurveillance Development of sentinel plant strategy as part of the monitoring plan?
Godefroid et al., 2019
36 Acknowledgements
Marie Agnès Jacques Sophie Cesbron Karine Durand Olivier Pruvost Martial Briand Virginie Ravigné Quentin Beaurepere Adrien Rieux Marina Blanc
Amandine Cunty Françoise Poliakoff Valérie Olivier Bruno Legendre Virginie Juteau Aurélie Forveille Muriel Bahut