Candidatus liberibacterafricanus in non-rutaceous alternate host species from South Africa

Abstract

Non-rutaceous plant species, potentially hosts to ‘Candidatus Liberibacter africanus’ (Laf) sensu lato, were sampled throughout the Cape Floristic Region from the Fynbos and Succulent Karoo biomes in South Africa, and tested for the presence of the insect-transmitted bacterial pathogen associated with ‘Citrus Greening disease’ (CG) in Citrus species (Rutaceae). Laf is considered a persistent problem to the production of citrus in South Africa as fruits produced from CG infected citrus are smaller in size, lopsided or misshaped and have a characteristic bitter taste. The information on the potential host range in indigenous and other plant species in South Africa is limited. In the current study three surveys were carried out during September 2017 (spring), January 2018 (summer), and August 2018 (winter) in the natural vegetation in Robertson, Worcester, Slanghoek, Vredendal, Lutzville and Klawer. Potential psyllid vectors were collected with vacuum sampling from approximately 20 randomly selected plant samples per plant species at each site. Branches and flowers, when available, of the same plants were collected for morphological identification. Leaf and petiole samples of 989 plant specimens, representing 19 plant families and 42 species, were collected. No typical galls induced on leaves by psyllid nymphs were observed on the plants. Psyllids were only collected from two Roepera foetida (Zygophyllaceae) plants. Of the 989 plant specimens of alternate host species tested for the presence of Liberibacters by real-time polymerase chain reaction (real-time PCR) assays 142 yielded a Ct value below the selected positive/negative threshold of 31 following the Liberibacter ‘Universal’ real-time PCR assay. Conventional PCR tests, including the amplification of the 16S rRNA, omp and rplJ genes of Laf, were conducted on these 142 plant specimens. Seven of these yielded very faint bands after gel electrophoresis analysis of the 16S rRNA conventional PCR test. Sanger sequencing of these suggested they were non-target amplicons. Therefore, none of the 42 plant species tested positive for ‘Ca. Liberibacter africanus’, the causal agent for CG in South African citrus. As a number of Atriplex semibaccata plants had yielded real-time PCR values <31 and yielded some amplicons with the 16S rRNA PCR, a sample with a DNA concentration above 250 ng/μl was selected for next-generation sequence (NGS) analysis using an Illumina HiSeq 2000 platform at Life Sequencing (Spain) to attempt to identify the presence of a potentially divergent Liberibacter. NGS data indicated that the bacterial entity amplified by the Liberibacter ‘Universal’ real-time PCR was not Liberibacter spp., but an, as yet, unidentified member of the Gammaproteobacteria. This bacterium may also be present in a number of other Atriplex samples tested during this study, including A. lindleyi and A. nummularia, which had also yielded amplicons in the real-time PCR assays. It may also be present in some of the other plant species testing positive in the real-time PCR test. The data generated from this study, and from the studies done in conjunction with this one, will be used for biological and epidemiological studies and the development of management strategies.