Publications

3810

“Candidatus Liberibacter europaeus” (Leu) is one of six currently known Liberibacter species. It is known primarily from pear and related species across Europe, and from Scotch broom and its associated psyllids in New Zealand (introduced from Britain). The psyllids were introduced to New Zealand as a biocontrol agent for broom and it is thought the bacterium may have been introduced as an endosymbiont of the psyllids. No symptoms in apple or pear trees have been reported, but mild symptoms can occur in broom. 16S and 16S–23S intergenic spacer region DNA sequences of this species are available from GenBank. Analysis of these sequences and associated ecological descriptions indicates the occurrence of two haplotypes, LeuA and LeuB, defined not only genetically but also by geographic range as well as by plant/psyllid host species composition. Liberibacter species are new to science and haplotype identifications enable historical accounts of relationships and spread to be generated.

“CandidatusNeoehrlichia mikurensis” is the tick-borne agent of neoehrlichiosis, an infectious disease that primarily affects immunocompromised patients. So far, the genetic variability of “Ca. Neoehrlichia” has been studied only by comparing 16S rRNA genes andgroELoperon sequences. We describe the development and use of a multilocus sequence analysis (MLSA) protocol to characterize the genetic diversity of clinical “Ca. Neoehrlichia” strains in Europe and their relatedness to other species within theAnaplasmataceaefamily. Six genes were selected:ftsZ,clpB,gatB,lipA,groEL, and 16S rRNA. Each MLSA locus was amplified by real-time PCR, and the PCR products were sequenced. Phylogenetic trees of MLSA locus relatedness were constructed from aligned sequences. Blood samples from 12 patients with confirmed “Ca. Neoehrlichia” infection from Sweden (n= 9), the Czech Republic (n= 2), and Germany (n= 1) were analyzed with the MLSA protocol. Three of the Swedish strains exhibited identicallipAsequences, while thelipAsequences of the strains from the other nine patients were identical to each other. One of the Czech strains had one differing nucleotide in theclpBsequence from the sequences of the other 11 strains. All 12 strains had identical sequences for the genes 16S rRNA,ftsZ,gatB, andgroEL. According to the MLSA, among theAnaplasmataceae, “Ca. Neoehrlichia” is most closely related toEhrlichia ruminantium, less so toAnaplasma phagocytophilum, and least toWolbachiaendosymbionts. To conclude, three sequence types of infectious “Ca. Neoehrlichia” were identified: one in the west of Sweden, one in the Czech Republic, and one spread throughout Europe.

Zebra chip disease of potato is caused by the bacterial pathogen ‘Candidatus Liberibacter solanacearum’ and is a growing concern for commercial potato production in several countries in North and Central America and New Zealand. ‘Ca. L. solanacearum’ is vectored by the potato psyllid Bactericera cockerelli, which transmits the pathogen to several cultivated and wild solanaceaous host plants. Silverleaf nightshade (SLN), Solanum elaeagnifolium, is a common weed in the Lower Rio Grande Valley of Texas and a host for both the potato psyllid and ‘Ca. L. solanacearum’. SLN plants were successfully inoculated with ‘Ca. L. solanacearum’ under laboratory conditions. Retention studies demonstrated that ‘Ca. L. solanacearum’-infected SLN planted in the field in January 2013, concurrent with commercial potato planting, retained the pathogen under field conditions throughout the year despite extensive dieback during summer. The presence of ‘Ca. L. solanacearum’ was confirmed in leaves, roots, and stolons of SLN plants collected the following year using polymerase chain reaction. Acquisition assays using B. cockerelli adults also revealed that SLN retained the pathogen. Transmission studies determined that B. cockerelli can acquire ‘Ca. L. solanacearum’ within a 2-week acquisition access period on ‘Ca. L. solanacearum’-infected SLN and subsequently transmit the pathogen to potato. These results demonstrate that SLN plants can serve as a reservoir for ‘Ca. L. solanacearum’, providing a source of inoculum for B. cockerelli adults colonizing potato the next season. The presence of SLN plants all year round in the LRGV makes the weed an epidemiologically important host. These findings underscore the importance of eradicating or managing SLN plants growing in the vicinity of potato fields to prevent spread of ‘Ca. L. solanacearum’ and damage caused by zebra chip.

‘Candidatus Liberibacter solanacearum’ contains two solanaceous crop-infecting haplotypes, A and B. Two haplotype A draft genomes were assembled and compared with ZC1 (haplotype B), revealing inversion and relocation genomic rearrangements, numerous single-nucleotide polymorphisms, and differences in phage-related regions. Differences in prophage location and sequence were seen both within and between haplotype comparisons. OrthoMCL and BLAST analyses identified 46 putative coding sequences present in haplotype A that were not present in haplotype B. Thirty-eight of these loci were not found in sequences from other Liberibacter spp. Quantitative polymerase chain reaction (qPCR) assays designed to amplify sequences from 15 of these loci were screened against a panel of ‘Ca. L. solanacearum’-positive samples to investigate genetic diversity. Seven of the assays demonstrated within-haplotype diversity; five failed to amplify loci in at least one haplotype A sample while three assays produced amplicons from some haplotype B samples. Eight of the loci assays showed consistent A–B differentiation. Differences in genome arrangements, prophage, and qPCR results suggesting locus diversity within the haplotypes provide more evidence for genetic complexity in this emerging bacterial species.