Publications

4609

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.

Mycoplasma haemomuris is causative of infectious anaemia or splenomegaly in rodents. We examined the nucleotide sequences of the non-ribosomal genes, rnpB and dnaK, in strains of the species M. haemomuris detected in small field mice and black rats. rnpB nucleotide sequences in strains of the species M. haemomuris isolated from small field mice and black rats had only 89 % sequence similarity, suggesting their separation into two distinct subgroups. dnaK had a nucleotide sequence similarity of 84 % between the subgroups. These results support the classification of M. haemomuris into two genetically distinct subgroups. Here we propose the establishment of these subgroups as ‘Candidatus Mycoplasma haemomuris subsp. musculi’, detected in small field mice (Apodemus argenteus), and ‘Candidatus Mycoplasma haemomuris subsp. ratti’, detected in black rats (Rattus rattus).

The bacterium ‘Candidatus Liberibacter solanacearum’ is associated with zebra chip disease (ZC), a threat to potato production in North America and New Zealand. It is vectored by potato psyllids. Previous studies observed that ‘Ca. L. solanacearum’ infection causes potato tubers to undergo ZC-symptom-associated shifts in physiology, such as increased levels of amino acids, sugars, and phenolics. However, little is known about how ‘Ca. L. solanacearum’ infections caused by psyllid vector feeding may affect metabolism in potato foliage and stems. This study compared metabolism in potato plants fed upon by ‘Ca. L. solanacearum’-positive psyllids with potato plants not exposed to psyllids. Foliar levels of asparagine, aspartic acid, glutamine, fructose, glucose, sucrose, a ferulic acid derivative, and quinic acid were lower in ‘Ca. L. solanacearum’-inoculated than noninfected plants. However, foliar levels of proline, serine, four phenolic compounds, and most terpenoids were greater in ‘Ca. L. solanacearum’-inoculated than noninfected plants. Upper stem levels of asparagine and aspartic acid, upper and lower stem levels of ellagitannins and most monoterpenoids, and lower stem level of sesquiterpenoids were greater in ‘Ca. L. solanacearum’-inoculated than noninfected plants. These results suggest that many defense-related terpenoid compounds might increase in plants which had psyllids inoculate ‘Ca. L. solanacearum’. This could impact progression and spread of ZC.

‘Candidatus Liberibacter solanacearum’ has recently been reported to be associated with vegetative disorders and economic losses in carrot and celery crops in Spain. The bacterium is a carrot seedborne pathogen and it is transmitted by psyllid vector species. From 2011 to 2014 seasonal and occasional surveys in carrot, celery and potato plots were performed. The sticky plant method was used to monitor the arthropods that visited the plants. The collected arthropods were classified into Aphididae and Cicadellidae, and the superfamily Psylloidea was identified to the species level. The superfamily Psylloidea represented 35.45% of the total arthropods captured on celery in Villena and 99.1% on carrot in Tenerife (Canary Islands). The maximum flight of psyllid species was in summer, both in mainland Spain and the Canary Islands, reaching a peak of 570 specimens in August in Villena and 6,063 in July in Tenerife. The main identified psyllid species were as follows: Bactericera trigonica Hodkinson, B. tremblayi Wagner and B. nigricornis Förster. B. trigonica represented more than 99% of the psyllids captured in the Canary Islands and 75% and 38% in 2011 and 2012 in Villena, respectively. In addition, Trioza urticae Linnaeus, Bactericera sp., Ctenarytaina sp., Cacopsylla sp., Trioza sp. and Psylla sp. were captured. ‘Ca. L. solanacearum’ targets were detected by squash real-time PCR in 19.5% of the psyllids belonging to the different Bactericera species. This paper reports at least three new psyllid species that carry the bacterium and can be considered as potential vectors.

Significance TM7 is one of the most enigmatic bacterial phyla among the uncultivated candidate phyla referred to as “microbial dark matter,” and it has potential pathogenic associations. We revealed molecular insights into its uncultivability and pathogenicity, as well its unique epibiotic and parasitic lifestyle phases. These novel discoveries shed significant light on the biological, ecological, and medical importance of TM7, as well as providing useful information for culturing other TM7 and currently uncultivable bacteria that may evade standard cultivation approaches.