Singer, Burton H.

Summary Citrus huanglongbing (HLB) is the most devastating citrus disease worldwide. ‘ Candidatus Liberibacter asiaticus’ (Las) is the most prevalent HLB causal agent that is yet to be cultured. Here, we analysed the flagellar genes of Las and Rhizobiaceae and observed two characteristics unique to the flagellar proteins of Las: (i) a shorter primary structure of the rod capping protein FlgJ than other Rhizobiaceae bacteria and (ii) Las contains only one flagellin‐encoding gene flaA (CLIBASIA_02090), whereas other Rhizobiaceae species carry at least three flagellin‐encoding genes. Only flgJ Atu but not flgJ Las restored the swimming motility of Agrobacterium tumefaciens flgJ mutant. Pull‐down assays demonstrated that FlgJ Las interacts with FlgB but not with FliE. Ectopic expression of flaA Las in A. tumefaciens mutants restored the swimming motility of ∆ flaA mutant and ∆ flaAD mutant, but not that of the null mutant ∆ flaABCD . No flagellum was observed for Las in citrus and dodder. The expression of flagellar genes was higher in psyllids than in planta . In addition, western blotting using flagellin‐specific antibody indicates that Las expresses flagellin protein in psyllids, but not in planta . The flagellar features of Las in planta suggest that Las movement in the phloem is not mediated by flagella. We also characterized the movement of Las after psyllid transmission into young flush. Our data support a model that Las remains inside young flush after psyllid transmission and before the flush matures. The delayed movement of Las out of young flush after psyllid transmission provides opportunities for targeted treatment of young flush for HLB control.

SUMMARYCandidatus Liberibacter spp. are Alphaproteobacteria associated with plants and psyllid vectors. Most cause plant diseases, including Ca Liberibacter asiaticus (Las) associated with citrus huanglongbing (HLB). Replacing HLB-infected by Las-free citrus trees results in fast re-infection despite psyllid control. To check if HLB could be soil-borne, we performed an insect-free greenhouse-experiment with 130 mandarin seedlings in two citrus-grove soils (A and B), non-autoclaved or autoclaved. Liberibacter-specific 16S-rDNA PCR primers to detect Las were used to search for Ca. Liberibacter spp. in mandarin leaves. Seven plants grown in non-autoclaved soil B showed HLB-like symptoms and tested positive after 2.5 and 8.5 months using three different primer systems: two based on the 16S-rDNA gene (primers HLBas/HLBr and OI2c/OI1) and one based on the rplA/rplJ gene (primers LAA2/LAJ5). DNA segments from these plants amplified by primers OI2c/OI1 were cloned and sequenced; they were 95.9 % similar to Las and 94.8% to Ca. Liberibacter africanus (Laf). The DNA product from Liberibacter-group specific PCR primers for the rplA/rplJ gene was 87.6% similar to that of Las and 78.2% of Laf. As the strain obtained originated from soil and was different from existing Ca. Liberibacter species, this strain may be a new species.