Barcoding of Phytoplasmas

In this work package a prioritized list of all phytopathogenic phytoplasms relevant for the EU to be barcoded was established.

Objectives

To generate barcode sequences from a selected set of genetic regions and for a selected set of relevant/quarantine phytoplasms. To reach this aim we will:

  • Establish a prioritized list of all phytopathogenic phytoplasms relevant for the EU to be barcoded.
  • Maintain and expand existing collection with relevant phytoplasma isolates.
  • Produce data to be used as a decision basis for the choice of 3 phytoplasma barcode genes and regions.
  • Develop reliable DNA extraction procedures.
  • Develop robust primers for PCR amplification of the phytoplasma barcode gene regions
  • Collect barcode data including intra- and interspecies genetic variation on the phytoplasma barcode regions (3 regions).

Tasks

  • Task 7.1 Availability collections and taxonomic data from non-partners
  • Task 7.2 Maintenance and expansion of collection at UB and INRA
  • Task 7.3 Selection of targets
  • Task 7.4 Selection of barcode regions
  • Task 7.5 DNA extractions
  • Task 7.6 Generation of protocols for amplification of barcode regions
  • Task 7.7 Generation of Barcode sequences

Deliverables

  • D 7.1 Access to non-partners collections and taxonomic expertise and information for selected Q-organisms. Seven named non-partners will be contacted (Month 6)
  • D 7.2 Collection of all relevant phytoplasms from about 30 taxonomic groups (Month 6)
  • D 7.3 List of selected phytoplasma Q-organisms (Month 6)
  • D 7.4 List of barcode regions for selected phytoplasma Q-organisms (Month 9)
  • D 7.5 Protocols (3-5) for DNA extraction for selected phytoplasma Q-organisms (Month 9)
  • D 7.6 Three protocols for generic amplification of barcode regions (Month 12)
  • D 7.7 Barcode sequences of the selected phytoplasma Q-organisms (at least 420 DNA sequences) (Month 30)
  • D 7.8 Detailed contingency plan for WP7 (Month 6)

Results

In this work package a prioritized list of all phytopathogenic phytoplasms relevant for the EU to be barcoded was established. An existing collection of phytoplasmas in micropropagated plants was maintained and expanded with relevant phytoplasma isolates during the project period. Phytoplasmas cannot be cultured in vitro and thus they need to be maintained in planta which requires considerable work. Partner 8 (University of Bologna) currently has 140 strains in micropropagation which covers all Q-phytoplasmas except palm lethal yellowing which is only available as DNA.

Colleagues and collections were contacted worldwide for specific strains throughout the project, for instance after publication of interesting new phytoplasma strains. The COST0807 network was mainly used for obtaining new strains. As the list of ‘Candidatus Phytoplasma’ species is constantly expanding we are still trying to include these in the collection/database though our international contacts.

Several DNA extraction methods were evaluated in the project for their effectiveness to extract phytoplasma DNA from infected host material and two methods have been recommended that work for most plant species/phytoplasmas.

Phytoplasma barcode regions 16S, tuf and SecA were selected to be used as DNA barcodes. Tuf and SecA regions are 400-600 bp whereas the 16S region is app. 1.8 kb, however, also a smaller region of the 5’ end of 18S has been selected for quick identification of phytoplasmas.

Barcode data from the 3 regions have been collected during the project. Until now, more than 460 barcodes have been produced from approximately 200 phytoplasma strains. These barcodes enable good separation between phytoplasma groups and are thus ideal for identification of phytoplasmas, including quarantine organisms.

Thus, a very efficient, sensitive and specific method for phytoplasma identification has been developed using a generic PCR followed by barcode sequencing.