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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.

Three novel lineages of ‘Candidatus Liberibacter africanus’ associated with native rutaceous hosts of Trioza erytreae in South Africa