Greening disease of citrus in South Africa is associated with ‘Candidatus Liberibacter africanus’
(Laf), a phloem-limited bacterium vectored by the sap-sucking insect Trioza erytreae (Triozidae).
Despite the implementation of control strategies, this disease remains problematic, suggesting
the existence of reservoir hosts to Laf. The current study aimed to identify such hosts. Samples
from 234 trees of Clausena anisata, 289 trees of Vepris lanceolata and 231 trees of Zanthoxylum
capense were collected throughout the natural distribution of these trees in South Africa. Total
DNA was extracted from samples and tested for the presence of liberibacters by a generic
Liberibacter TaqMan real-time PCR assay. Liberibacters present in positive samples were
characterized by amplifying and sequencing rplJ, omp and 16S rRNA gene regions. The identity of
tree host species from which liberibacter sequences were obtained was verified by sequencing
host rbcL genes. Of the trees tested, 33 specimens of Clausena, 17 specimens of Vepris and 10
specimens of Zanthoxylum tested positive for liberibacter. None of the samples contained typical
citrus-infecting Laf sequences. Phylogenetic analysis of 16S rRNA gene sequences indicated
that the liberibacters obtained from Vepris and Clausena had 16S rRNA gene sequences
identical to that of ‘Candidatus Liberibacter africanus subsp. capensis’ (LafC), whereas those
from Zanthoxylum species grouped separately. Phylogenetic analysis of the rplJ and omp gene
regions revealed unique clusters for liberibacters associated with each tree species. We propose
the following names for these novel liberibacters: ‘Candidatus Liberibacter africanus subsp.
clausenae’ (LafCl), ‘Candidatus Liberibacter africanus subsp. vepridis’ (LafV) and ‘Candidatus
Liberibacter africanus subsp. zanthoxyli’ (LafZ). This study did not find any natural hosts of Laf
associated with greening of citrus. While native citrus relatives were shown to be infected with
Laf-related liberibacters, nucleotide sequence data suggest that these are not alternative sources
of Laf to citrus orchards, per se.