Rodoni, Brendan

Gene- and genome-based approaches were used to determine whether Vigna little leaf (ViLL) phytoplasma, which occurs in northern Australia, is a distinct ‘Candidatus Phytoplasma’ species. The ViLL 16S rRNA gene sequences exhibited the highest known similarity to species in the 16SrXXIX-A and 16SrIX-D subgroups, namely ‘Candidatus Phytoplasma omanense’ (98.03–98.10%) and ‘Candidatus Phytoplasma phoenicium’ (96.87–97.20%), respectively. A total of 48 single-copy orthologue genes were identified to be shared among the two draft ViLL phytoplasma genomes, 30 publicly available phytoplasma genomes, and one Acholeplasma laidlawii genome as the outgroup taxon. Phylogenomic assessments using the 48 shared single-copy orthologue genes supported that ViLL and ‘Ca. Phytoplasma phoenicium’ were closely related yet distinct species. The 16S rRNA gene sequence analysis and phylogenomic assessment indicate that ViLL phytoplasmas are a distinct taxon. As such, a novel species, ‘Candidatus Phytoplasma vignae’, is proposed. Strain BAWM-336 (genome accession number JAUZLI000000000) detected in Momordica charantia (bitter melon) serves as the reference strain of this species, with infected plant material deposited in the Victorian Plant Pathology Herbarium (VPRI) as VPRI 44369.

AbstractThis study is the first to investigate the presence and movement of the novel Liberibacter species ‘Candidatus Liberibacter brunswickensis’ (CLbr) in eggplant, Solanum melongena. The psyllid, Acizzia solanicola can transmit CLbr to eggplant and CLbr can be acquired by CLbr-negative A. solanicola individuals from CLbr-positive eggplants. In planta, CLbr can replicate, move and persist. Investigation into the early development of eggplants showed that CLbr titres had increased at the inoculation site at 14 days post inoculation access period (DPIAP). CLbr had become systemic in the majority of plants tested by 28 DPIAP. The highest bacterial titres were recorded at 35 DPIAP in all samples of the inoculated leaf, the roots, stems and the midrib and petiole samples of the newest leaf (the top leaf). This finding strongly suggests that CLbr movement in planta follows the source to sink relationship as previously described for ‘Ca. Liberibacter asiaticus’ (CLas) and ‘Ca. Liberibacter solanacearum’ (CLso). No symptoms consistent with Liberibacter-associated diseases were noted for plants colonised by CLbr during this study, consistent with the hypothesis that CLbr does not cause disease of eggplant during the early stages of host colonisation. In addition, no significant differences in biomass were found between eggplant colonised with CLbr, compared to those that were exposed to CLbr-negative A. solanicola, and to control plants.

In Australia, Stylosanthes little leaf (StLL) phytoplasma has been detected in Stylosanthes scabra Vogel, Arachis pintoi Krapov, Saccharum officinarum L., Carica papaya L., Medicago sativa L., and Solanum tuberosum L. The 16S rRNA gene sequence of StLL phytoplasma strains from S. scabra, C. papaya, S. officinarum and S. tuberosum were compared and share 99.93–100 % nucleotide sequence identity. Phylogenetic comparisons between the 16S rRNA genes of StLL phytoplasma and other ‘Candidatus Phytoplasma’ species indicate that StLL represents a distinct phytoplasma lineage. It shares its most recent known ancestry with ‘Ca. Phytoplasma luffae’ (16SrVIII-A), with which it has 97.17–97.25 % nucleotide identity. In silico RFLP analysis of the 16S rRNA amplicon using iPhyClassifier indicate that StLL phytoplasmas have a unique pattern (similarity coefficient below 0.85) that is most similar to that of ‘Ca. Phytoplasma luffae’. The unique in silico RFLP patterns were confirmed in vitro. Nucleotide sequences of genes that are more variable than the 16S rRNA gene, namely tuf (tu-elongation factor), secA (partial translocation gene), and the partial ribosomal protein (rp) gene operon (rps19-rpl22-rps3), produced phylogenetic trees with similar branching patterns to the 16S rRNA gene tree. Sequence comparisons between the StLL 16S rRNA spacer region confirmed previous reports of rrn interoperon sequence heterogeneity for StLL, where the spacer region of rrnB encodes a complete tRNA-Isoleucine gene and the rrnA spacer region does not. Together these results suggest that the Australian phytoplasma, StLL, is unique according to the International Organization for Mycoplasmology (IRPCM) recommendations. The novel taxon ‘Ca. Phytoplasma stylosanthis’ is proposed, with the most recent strain from a potato crop in Victoria, Australia, serving as the reference strain (deposited in the Victorian Plant Pathology Herbarium as VPRI 43683).

Summary A novel candidate species of the liberibacter genus, ‘ Candidatus Liberibacter brunswickensis’ ( CL br), was identified in the Australian eggplant psyllid, Acizzia solanicola . This is the first discovery of a species belonging to the liberibacter genus in Australia and the first report of a liberibacter species in the psyllid genus Acizzia . This new candidate liberibacter species has not been associated with plant disease, unlike other psyllid‐vectored species in the genus including ‘ Candidatus Liberibacter asiaticus ’ ( CL as), ‘ Candidatus Liberibacter africanus’ ( CL af) and ‘ Ca . Liberibacter solanacearum’ ( CL so). This study describes novel generic liberibacter genus primers, used to screen Australian psyllids for the presence of microflora that may confound diagnosis of exotic pathogens. CL br forms a unique clade in the liberibacter genus based on phylogenetic analysis of the 16S ribosomal ribonucleic acid ( rRNA ) region and multilocus sequence analysis ( MLSA ) of seven highly conserved genes, dnaG , gyrB , mutS , nusG , rplA , rpoB and tufB . The MLSA mapping approach described in this article was able to discriminate between two ‘ Ca . Liberibacter’ species within a metagenomic data set and represents a novel approach to detecting and differentiating unculturable species of liberibacter. Further, CL br can confound the Li et al . (2006) quantitative PCR ( qPCR ) diagnostic tests for CL as and CL af.