Detection and identification of a Candidatus Liberibacter species from ash tree infesting psyllids

Candidatus Liberibacter species cause severe, economically important diseases. All known species of these pathogens are putatively insect-transmitted, specifically by psyllids. Detection of Liberibacter in plants is complicated by its uneven distribution in host plants and largely unculturable nature. The death of black (Fraxinus nigra) and mancana (Fraxinus mandshurica) ash trees in Saskatchewan, Canada has been associated with infestation with the cottony ash psyllid (Psyllopsis discrepans). We hypothesized that the symptoms and death could be due to a psyllid-transmitted Liberibacter. We used a combination of conventional PCR and Sanger sequencing of the 16S rDNA for detection of Liberibacter, and the genes CO1 and Cyt-b to determine species of psyllids. The 16S sequencing generated two sequences, NTHA 5 (GenBank accession number MK942379) and NTHA 6 (GenBank accession number MK937570) that were 1058 and 1085 bp long. A BLAST search for homology showed 99-100% sequence similarity to a Candidatus Liberibacter solanacearum sequence (GenBank accession number: KX197200) isolated from the Nearctic psyllid (Bactericera maculipennis) of US provenance. CO1 and Cyt-b gene sequencing of our psyllids yielded sequence information confirming that they were P. discrepans from comparisons with sequences in GenBank and BOLD. Confirmatory sequence comparison with a reference sample from the United Kingdom was concurrent. These results provide the first evidence on the likely cause of ash dieback in Saskatchewan. Further, they suggest a relatively rare example of a Liberibacter adapting a new host plant.