Barcoding of Bacteria

In this work package the work focussed on the Q-species within the genera Ralstonia, Xylella, Clavibacter and Xanthomonas.

Objectives

  • To maintain and expand existing collection with relevant bacterial isolates (collection strains and recent field isolates).
  • To unravel the taxonomic and phylogenetic identity of quarantine bacteria in support of EU regulation.
  • To collect barcode data including genetic variation, inclusiveness and exclusiveness for the selected scope of Q-bacteria.
  • To generate barcode sequences from a selected set of regions and for a selected set of relevant quarantine bacteria.
  • To develop reliable routine DNA extraction protocols.
  • To develop robust primers for PCR amplification of the barcode gene regions in the different taxa.

Tasks

  • Task 4.1  Selection of target organisms and composition of the relevant working collection
  • Task 4.2  Selection of barcode target regions
  • Task 4.3  DNA isolation and DNA bank
  • Task 4.4  Generation of Barcode sequences, evaluation and deposit in QBOL database
  • Task 4.5  Generation of protocols for generation and evaluation of bacterial barcodes

Deliverables

  • D 4.1 List of selected bacterial Q-organisms (Month 6)
  • D 4.2 List of barcode regions for selected bacterial Q-organisms (Month 12)
  • D 4.3 Protocols for amplification of 4 barcode regions (Month 18)
  • D 4.4 Three protocols for DNA extraction and DNA bank (Month 20)
  • D 4.5 At least 3000 Barcode sequences of selected regions for the bacterial Q-organisms selected (Month 34)
  • D 4.6 A correct delineation of the Q-bacteria belonging to Xanthomonas, Clavibacter, Xylella fastidiosa and Ralstonia solanacearum in support of decision making on regulated bacterial pests (Month 34)
  • D 4.7 At least 3 scientific papers on taxonomic aspects of Xanthomonas and/or Clavibacter and/or Ralstonia and/or Xylella are submitted (Month 36).
  • D 4.8 Detailed contingency plan for WP4 (Month 6)

Results

In this work package the work focussed on the Q-species within the genera Ralstonia, Xylella, Clavibacter and Xanthomonas. Most of the Q-species within Xanthomonas on the EU Directive and EPPO list are on the pathovar level, which makes it difficult to select the barcoding gene as pathovars do not necessarily form a single taxonomical group.

Strains were retrieved from official collections, with a substantial amount being contributed from the BCCM-LMG collection hosted at LM-UGent. Especially for the xanthomonads, other official and working collections were contacted in regions of the world where the pathogens recently occurred.

DNA extraction procedures were evaluated and final protocols were written. Several genes were evaluated for their performance as  a barcode region. Finally a decision scheme was published to lead the end-user through the identification process. This scheme clearly shows when to use which barcoding genes.

Extensive sequencing also confirmed the taxonomic position of most of the target Q bacteria (within Ralstonia, Xylella, Clavibacter michiganensis and the Xanthomonas species) and also supports recent proposals for the taxonomic division of the Ralstonia solanacearum complex. On the other hand, our research also revealed that some Q-pathogens are represented by heterogeneous strains (e.g. X. axonopodis pvs. dieffenbachiae,  phaseoli and allii). Their classification under the same Q-pathovar name is questionable and needs further investigation by sequencing more genomic domains and performing host range experiments on plants. Within Clavibacter, the three Q- C. michiganensis subspecies are identified by the gyrB-based barcode. Also many non-pathogenic strains of the species (look-a-likes) were included in the study.

In total, the QBOL working collection increased to 1,008 strains and 3,667 sequences have been generated. Based on these sequences and on other strain characteristics (such as host, geographic origin and symptom type) a subset of reference strains has been identified for the end-users. The barcodes have been deposited in the Q-bank bacteria database and the strains are stored and available from the certified public service culture collections BCCM-LMG (BE), NCPPB (UK), and CFBP (FR). Under the initiative of ILVO, these culture collections collaborate on a reference collection of plant health-affecting bacteria.