First report of "Candidatus Liberibacter solanacearum" in carrot in France

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 »

1 – State of Knowledge

2 – Action following alert in France 2012

3 – Perspectives

                                            2

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)

State of Knowledge

• 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

             Leaf purpling and scorching

State of Knowledge
                                 Symptoms on potato:

         Confusion with:
« Potato purple top » (Aster yellow
     Phytoplasma), Stolbur or
        psyllids damages

State of Knowledge
Symptoms on potato:

                      « Zebra chip »
                       symptoms :
                          brown
                      decoloration of
                          vesels

State of Knowledge
  Symptoms on tomato

                        Chlorosis, stunting

  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

State of Knowledge
                       Symptoms on carrot

Foliage wound with discoloration (yellow,
      bronze, purple), uncontrolled
development by the collar, stunting of the
     main root and root proliferation

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                   Bactericera trigonalis
       haplotype C (?)                      haplotype D (?)

       Scandinavia                Spain, Canaries Island

State of Knowledge

     Others way of transmission
• Potato : tubers from mother plant towards
  plantlet and son tubers

• Seed ?:not yet observed

Alert in France 2012
       Détection of Ca. L. solanacearum

• 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

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
      Détection of Ca. L. solanacearum
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

Alert in France 2012
     Confirmation of Ca. L. solanacearum
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

Alert in France 2012
       Confirmation of Ca. L. solanacearum

• 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)

Measures taken and to be taken
 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

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