Publications

4871

The Asian citrus psyllid, Diaphorina citri, transmits the putative causal agent of citrus greening disease, ‘ Candidatus Liberibacter asiaticus’. The transmission occurs in a propagative, circulative manner. Specific protein–protein interactions are required for the accomplishment of the transmission process. We employed the far-Western technique, also known as protein overlay assay, and mass spectrometry to identify D. citri proteins that specifically recognize membrane proteins of ‘ Ca. L. asiaticus’. Whole-body protein extracts of D. citri were separated by electrophoresis and blotted onto polyvinylidene difluoride membranes. The membranes were overlayed with sonicated leaf midrib extracts from ‘ Ca. L. asiaticus’-infected citrus plants. The protein complexes were detected using different antibodies against ‘ Ca. L. asiaticus’ membrane proteins and peptidoglycans associated with lipoproteins. By comparison, spots were extracted from a polyacrylamide gel for liquid chromatography-tandem mass spectrometry analysis. Identified proteins included ATP synthase alpha and beta subunits, actin, alpha- and beta-tubulins, transitional endoplasmic reticulum ATPase TER94, 78 kDa glucose-regulated protein, and arginine kinase. These findings emphasize the involvement of energy machinery and muscle proteins in the ‘ Ca. L. asiaticus’– D. citri interactions. Understanding the pathogen–vector interactions will lead to better-designed control strategies based on the interference with specific interactions and/or blocking transmission.

A phytoplasma associated with poinsettia has previously been reported in Korea, but the report primarily focused on stem flattening symptoms and was based solely on partial 16S ribosomal RNA (rRNA) gene sequence analysis. In this study, phytoplasma-infected poinsettias collected in Jinan and Jeonju, Korea were examined for diverse symptom expression and additional molecular characterization of the associated phytoplasma. All seven plants showed a free-branching phenotype. Symptom severity ranged from low freebranching, with larger leaves and long internodes, to severe free-branching, with shortened internodes and small, irregularly twisted leaves. Based on 16S rRNA gene sequence and iPhyClassifier analyses, the phytoplasma detected in this study was assigned to subgroup 16SrIII-A of the X-disease phytoplasma group (16SrIII). Although this assignment differs from the previous Korean report, which classified poinsettia-associated phytoplasmas subgroup 16SrIII-H, the present study interpreted the classification using an updated 16SrIII subgroup framework. Sequence analyses of more variable regions, including the 16S-23S intergenic spacer region, ribosomal protein (rp) operon, and secA gene, showed 99.46-99.55% nucleotide identity with ‘Candidatus Phytoplasma pruni’-related reference strains. Phylogenetic analyses showed that the poinsettia-associated phytoplasmas in Korea were closely related to the subgroup 16SrIII-A. These results expand the known symptom spectrum of phytoplasma-infected poinsettias and provide multilocus molecular characterization supporting the phylogenetic placement of poinsettia-associated phytoplasmas in Korea.

Periploca aphylla witches’ broom (PapWB) is a phytoplasma-associated disease first reported in south-western Iran in 2010. Recent observations showed that the disease is spreading to new areas. PapWB phytoplasmas, detected in symptomatic P. aphylla plants, exhibited identical nucleotide sequences in the 1.8 kb PCR amplicon encompassing the 16S rRNA gene, the 16S-23S spacer region and the 5′-end of the 23S rRNA gene. When compared with the reference strains of previously described ‘ Candidatus Phytoplasma’ species, the selected representative strain PapWB-S exhibited the highest 16S rRNA gene sequence identity (98.15%) with salt cedar witches’ broom (SCWB1) phytoplasma, the reference strain of ‘ Ca . P. tamaricis’. Moreover, the sequence identity values for the tufB, rplV-rpsC and secY genes of PapWB-S strain, when compared with those of reference strains of previously described ‘ Ca . Phytoplasma’ species, were below the thresholds adopted for describing new species. Phylogenetic analyses confirmed the close relationship of the strain PapWB-S with SCWB1 followed by other ‘ Ca . Phytoplasma’ species belonging to the ribosomal groups 16SrX, 16SrXX and 16SrXXXIII. Following the revised guidelines for describing ‘ Ca . Phytoplasma’ species, ‘ Ca . P. periplocae’ sp. nov., associated with PapWB disease, is proposed, and the 16S rRNA gene sequence (accessible at GenBank/NCBI accession number OQ819043) is its nomenclatural type.

A Gram-stain-negative, aerobic, capsule-forming strain, designated DAS-389 T , was isolated from soil collected in the Kogelberg Nature Reserve, Western Cape, South Africa. The strain formed small pink colonies, with cells that multiplied by binary division. Optimum growth was observed at 20 °C to 26 °C and pH 5.5 to 6.5, and it tolerated a NaCl concentration up to 1.5% (w/v). DAS-389 T is chemoorganotrophic, with growth substrate preferences for sugars and polysaccharides, including fructose and starch, respectively. The genome of DAS-389 T had a genome size of 4,136,282 bp with an average G+C content of 63.49 mol% and encodes for an assortment of enzymes, carbohydrate metabolic pathways and several genes involved in stress and survival responses. Major fatty acids (>5%) of strain DAS-389 T were iso-C15:0, iso-C17:0, summed feature 3 (C16:1 ω7c and/or C16:1 ω6c) and summed feature 9 (iso-C17:1 ω9c and/or C16:0 10-methyl). Comparative analysis of 16S rRNA gene sequences revealed that strain DAS-389 T shared the highest similarity with Alloacidobacterium dinghuense 4Y35 T (96.6%), followed by Acidipila rosea AP8 T (96.6%) and Paracidobacterium acidisoli 4G-K13T (96.3%). Phylogenetic analysis based on 16S rRNA gene sequences and the whole proteome placed DAS-389 T within a distinct clade, separate from recognized genera in the family Acidobacteriaceae . We therefore propose that strain DAS-389ᵀ represents a novel genus and species, Candidatus Dedyshia acidiphila gen. nov., sp. nov., within the family Acidobacteriaceae , order Terriglobia . The whole-genome sequence of strain DAS-389ᵀ [accession number: JBMGJV000000000 (PRJNA1221820) ] is designated as the nomenclatural type.