First report of “Candidatus Liberibacter solanacearum” in carrot

in France

Marianne LOISEAU1, Soraya GARNIER2, Violaine Boirin3, Maryse MERIEAU4, Anne LEGUAY1, Isabelle RENAUDIN1, Jean-Philippe RENVOISE2, Pascal GENTIT1, Françoise POLIAKOFF1

(1) ANSES-LSV Angers, 7 rue Jean Dixméras, 49044 Angers cedex 01, France (2) ANSES-LSV Unité de Quarantaine, Site de Clermont-Ferrand, 6 rue Aimé Rudel Marmilhat, 63370 LEMPDES, France (3) DRAAF-SRAL Centre, Cité Administrative Coligny, 131 Rue du Faubourg Bannier, 45042 ORLEANS cedex 1, France (4) FREDON CENTRE, Cité de l'Agriculture, 13 avenue des Droits de l'Homme, 45921 ORLEANS CEDEX 9, France

« Candidatus Liberibacter solanacearum »

2

1 – State of Knowledge 2 – Action following alert in France 2012 3 – Perspectives

State of Knowledge

• Gram negative bacterium • Phloem limited bacterium • 4 different haplotypes (ribosomal protein genes) (Nelson et al., 2011, 2012). Haplotypes A et B of Solanaceae:

potato (Solanum tuberosum), tomato (Solanum lycopersicum), pepper (Capsicum annuum), eggplant (Solanum melongena), tomatillo (Physalis peruviana), tamarillo (Solanum betaceum), tobacco (Nicotiana tabacum), and several weeds in the family Solanaceae

Haplotypes C et D of Apiaceae: carrot (Daucus carota), celery (Apium graveolens)

• Not Quarantine pest at EU level (2000/29/CE) = non regulated pathogen • Since september 2012: EPPO List for « Ca. L. solanacearum » on Solanaceae (haplotypes A and B). • Observed in different european countries on carrot (haplotype C) (Finland, Sweden, Norway – canaries island and Spain (haplotype D)

State of Knowledge

Geographical location

State of Knowledge

Symptoms on potato: Yellowing, chlorosis, stunting, excessive branching of axillary

shoots

State of Knowledge

Leaf purpling and scorching

Confusion with: « Potato purple top » (Aster yellow

Phytoplasma), Stolbur or psyllids damages

Symptoms on potato:

State of Knowledge

« Zebra chip » symptoms :

brown decoloration of

vesels

State of Knowledge Symptoms on potato:

Chlorosis, stunting

Symptoms on tomato

State of Knowledge

Mottling of leaves Curling of midveins

State of Knowledge Symptoms on pepper

Chlorotic or pale green leaves – sharp tapering of leaf apex (spiky appearance) – leaf cupping and shortened internodes – flower abortion

Foliage wound with discoloration (yellow, bronze, purple), uncontrolled

development by the collar, stunting of the main root and root proliferation

Symptoms on carrot

State of Knowledge

• Transmission by Psyllids on Solanaceae Bactericera cockerelli for haplotypes A and B

Vector present only in America and NZ

State of Knowledge

• Transmission by Psyllids on Apiacaea

Trioza apicalis haplotype C (?)

Bactericera trigonalis haplotype D (?)

Spain, Canaries Island Scandinavia

State of Knowledge

• Potato : tubers from mother plant towards plantlet and son tubers

• Seed ?:not yet observed

Others way of transmission

State of Knowledge

• Carrot fields "seed-bearing" with symptoms like "Aster yellow phytoplasma“

• Psyllids attacks observed on one of the fields • Recent report of 'Ca L. solanacearum "on carrots in

Europe

Alert in France 2012

Détection of Ca. L. solanacearum

Détection of Ca. L. solanacearum

Materials and methods 16 plants of symptomatic carrots; 6 plants of asymptomatic carrots harvested in the same

field ; 4 seed lots collected on symptomatic plants.

• DNA extraction with CTAB • Phytoplasma : real time PCR (Christensen et al., 2004) • Liberibacter : real time PCR (Li et al., 2009)

Alert in France 2012

Results in summer 2012: • No phytoplasma detected in samples. • Ca. L. solanacearum:

- All symptomatic and asymptomatic carrot plants gave positive results with real time PCR for Ca. L. solanacearum ;

- No amplification on the first seed batch.

carrot

T+ LOD

Alert in France 2012

Détection of Ca. L. solanacearum

Results march 2013: • 3 seeds batch sorted

Lot Tegument* Seeds*

1 10/10 7/10

2 9/10 0/10

3 10/10 10/10

*Nb positive/ nb sub samples tested

Détection of Ca. L. solanacearum Alert in France 2012

Method: • Amplification with PCR OA2/OI2c to amplify a portion of

16S rRNA gene (Liefting et al., 2009; Jagoueix et al, 1994)

• Amplification with PCR CL514F/R to amplify rpIJ/rpIL

ribosomal protein gene (Crosslin et al, 2009); • Sequencing of amplicons

Confirmation of Ca. L. solanacearum Alert in France 2012

• With both PCR, positive results confirmed for all plants of

carrot. PCR OA2/OI2c : 16S rRNA amplicon (2 sequences)

showed 99% of nucleotide identity with isolates of “Ca. L. solanacearum” amplified in Finland from carrot (GU373049)

• PCR CL514F/R : rpIJ/rpIL amplicon (2 séquences) was 99% identical to sequence of the analogous rpIJ/rpIL “Ca. L. solanacearum” ribosomal protein gene from carrot in Spain(JX308305)

Confirmation of Ca. L. solanacearum Alert in France 2012

Eradication in contaminated fields Consignement of harvested seeds

Notification to European Plant Protection Organisation (EPPO)

A survey to implement: - Evaluate the real time (Li et al., 2009); - Develop haplotype methods; - Sequence the genome of isolates; - Search of genetic marqueurs Research in progress: - Looking at weeds, others cultivated plants (celery) - Identification of Psyllids present - Psyllid and seed transmission - Detection in Psyllid

Measures taken and to be taken

Conclusion

This is the first report of in carrot in France.

Gel electrophoresis after PCR using primer pair CL514 F/R

Thank you for your attention