Probable dates and scenario of introduction of Xylella fastidiosa subsp. multiplex in

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 Porto- Samples Vecchio 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 (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