International Journal of Systematic and Evolutionary Microbiology

Phytoplasmas are a diverse but phylogenetically coherent group of cell-wall-less bacteria affiliated with the classMollicutes. Due to difficulties in establishing axenic culture, phytoplasmas were assigned to a provisional genus, ‘CandidatusPhytoplasma’, and the genus was embraced within the orderAcholeplasmatales. However, phytoplasmas differ significantly from species of the genusAcholeplasmain their habitat specificities, modes of life, metabolic capabilities, genomic architectures, and phylogenetic positions. This communication describes the unique ecological, nutritional, biochemical, genomic and phylogenetic properties that distinguish phytoplasmas from species of the genusAcholeplasmaand all other taxa in the classMollicutes. Since such distinguishing properties of the phytoplasmas are not referable to the descriptions of the orderAcholeplasmatalesand of all other existing orders, namelyMycoplasmatales,EntomoplasmatalesandAnaeroplasmatales, this communication raises the question of whether ‘CandidatusPhytoplasma’ should be retained in the orderAcholeplasmatalesor whether a novel provisional order and family should be created to accommodate the genus ‘Ca.Phytoplasma’.

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.

A new Gram-staining-positive, non-sporulating, mesophilic, amino acid-degrading anaerobic bacterium, designated strain OTA 102T, was isolated from an anaerobic sequencing batch reactor treating wastewater from cooking tuna. The cells were curved rods (0.6–2.5×0.5 µm) and occurred singly or in pairs. The strain was motile by means of one lateral flagellum. Strain OTA 102T grew at temperatures between 30 and 45 °C (optimum 40 °C), between pH 6.0 and 8.4 (optimum pH 7.2) and NaCl concentrations between 1 and 5 % (optimum 2 %, w/v). Strain OTA 102T required yeast extract for growth. Serine, threonine, glycine, cysteine, citrate, fumarate, α-ketoglutarate and pyruvate were fermented. When co-cultured with Methanobacterium formicicum as the hydrogen scavenger, strain OTA 102T oxidized alanine, valine, leucine, isoleucine, aspartate, tyrosine, methionine, histidine and asparagine. The genomic DNA G+C content of strain OTA 102T was 41.7 mol%. The main fatty acid was iso-C15 : 0. Phylogenetic analysis of the 16S rRNA gene sequence indicated that strain OTA 102T was related to Aminobacterium colombiense and Aminobacterium mobile (95.5 and 95.2 % similarity, respectively), of the phylum Synergistetes . On the basis of phylogenetic, genetic and physiological characteristics, strain OTA 102T is proposed to represent a novel species of the genus Aminobacterium , Aminobacterium thunnarium sp. nov. The type strain is OTA 102T ( = DSM 27500T = JCM 19320T).

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).

A phylogenetically novel proteobacterium, strain Shr3T, was isolated from sand gravels collected from the eastern margin of the Sahara Desert. The isolation strategy targeted bacteria filterable through 0.2-µm-pore-size filters. Strain Shr3T was determined to be a Gram-negative, aerobic, non-motile, filamentous bacterium. Oxidase and catalase reactions were positive. Strain Shr3T showed growth on R2A medium, but poor or no growth on nutrient agar, trypticase soy agar and standard method agar. The major isoprenoid quinone was menaquinone-7. The dominant cellular fatty acids detected were C16 : 1ω5c and C16 : 0, and the primary hydroxy acid present was C12 : 0 3-OH. The DNA G+C content was 54.0 mol%. Phylogenetic analysis based on 16S rRNA gene sequences revealed that strain Shr3T was affiliated with an uncultivated lineage of the phylum Proteobacteria ; the nearest known type strain, with 83 % sequence similarity, was Desulfomicrobium orale DSM 12838T in the class Deltaproteobacteria . The isolate and closely related environmental clones formed a novel class-level clade in the phylum Proteobacteria with high bootstrap support (96–99 %). Based on these results, the novel class Oligoflexia classis nov. in the phylum Proteobacteria and the novel genus and species Oligoflexus tunisiensis gen. nov., sp. nov. are proposed for strain Shr3T, the first cultivated representative of the Oligoflexia. The type strain of Oligoflexus tunisiensis is Shr3T ( = JCM 16864T = NCIMB 14846T). We also propose the subordinate taxa Oligoflexales ord. nov. and Oligoflexaceae fam. nov. in the class Oligoflexia.

A Gram-stain-negative, elongated rod-shaped, motile by gliding, green-pigmented, aerobic bacterial strain, designated LY03T, was isolated from lake water in Xiamen, Fujian Province, China. Phylogenetic analysis based on 16S rRNA gene sequencing revealed that the isolate was a member of the genus Chitinimonas , which belongs to the family Burkholderiaceae . Strain LY03T was most closely related to Chitinimonas taiwanensis LMG 22011T (96.02 % 16S rRNA gene sequence similarity), followed by Chitinimonas koreensis KACC 11467T (94.85 %), and the three strains formed a distinct lineage from other strains in the phylogenetic analyses. Optimum conditions for growth were 37 °C, pH 7–9 and without NaCl. The major fatty acids were summed feature 3 (C16 : 1ω6c and/or C16 : 1ω7c), C16 : 0 and C10 : 0 3-OH. The DNA G+C content of strain LY03T was 63.6 mol% and the major respiratory quinone was ubiquinone-8 (Q-8). The polar lipids were found to consist of diphosphatidylglycerol, phosphatidylglycerol, phosphatidylethanolamine, unknown polar lipids and unidentified phospholipids. Differential phenotypic properties and phylogenetic distinctiveness distinguished strain LY03T from all other members of the genus Chitinimonas . On the basis of its morphology, physiology, fatty acid composition and 16S rRNA gene sequence data, strain LY03T represents a novel species of the genus Chitinimonas , for which the name Chitinimonas prasina sp. nov. is proposed. The type strain is LY03T ( = MCCC 1F01209T = KCTC 32574T).

A thermotolerant, Gram-strain-negative, non-spore-forming and strictly aerobic bacterium, designated GU51T, was isolated from Guhai hot spring in Jimsar county, Xinjiang province, north-west China. Each cell of strain GU51Tconsisted of an oval body and two symmetrical long (3–6 µm) prosthecae. The strain moved by polar flagellum. Oxidase and catalase were produced. Strain GU51Tgrew within the ranges of 37–65 °C (optimum 48–50 °C), 0.5–7.5 % (w/v) NaCl (optimum 2–3 %) and pH 6.0–9.0 (optimum pH 7.5). The major respiratory quinone detected was ubiquinone 10 (U-10) and the genomic DNA G+C content was 66.7±0.4 mol%. Major fatty acids (>5 %) were C16 : 0, C18 : 1ω7cand 11-methyl C18 : 1ω7c. The polar lipids consisted of diphosphatidylglycerol, five glycolipids, phosphatidylglycerol and an unknown phospholipid. Phylogenetic analysis showed the closest relatives of strain GU51Twere members of the genusParvularculawith 92.3 % 16S rRNA gene sequence similarity. On the basis of this polyphasic taxonomic characterization, it is suggested that strain GU51Trepresents a novel species of a new genus in the family ‘Parvularculaceae’, for which the nameAmphiplicatus metriothermophilusgen. nov., sp. nov. is proposed. The type strain of the type species is GU51T( = CGMCC 1.12710T = JCM 19779T).

A thermophilic, filamentous, heterotrophic bacterium, designated strain JAD2T, a member of an as-yet uncultivated lineage that is present and sometimes abundant in some hot springs worldwide, was isolated from sediment of Great Boiling Spring in Nevada, USA. Cells had a mean diameter of 0.3 µm and length of 4.0 µm, and formed filaments that typically ranged in length from 20 to 200 µm. Filaments were negative for the Gram stain reaction, spores were not formed and motility was not observed. The optimum temperature for growth was 72.5–75 °C, with a range of 67.5–75 °C, and the optimum pH for growth was 6.75, with a range of pH 6.5–7.75. Peptone, tryptone or yeast extract were able to support growth when supplemented with vitamins, but no growth was observed using a variety of defined organic substrates. Strain JAD2T was microaerophilic and facultatively anaerobic, with optimal growth at 1 % (v/v) O2 and an upper limit of 8 % O2. The major cellular fatty acids (>5 %) were C16 : 0, C19 : 0, C18 : 0, C20 : 0 and C19 : 1. The genomic DNA G+C content was 69.3 mol%. Phylogenetic and phylogenomic analyses using sequences of the 16S rRNA gene and other conserved genes placed JAD2T within the phylum Chloroflexi , but not within any existing class in this phylum. These results indicate that strain JAD2T is the first cultivated representative of a novel lineage within the phylum Chloroflexi , for which we propose the name Thermoflexus hugenholtzii gen. nov., sp. nov., within Thermoflexia classis nov., Thermoflexales ord. nov. and Thermoflexaceae fam. nov. The type strain of Thermoflexus hugenholtzii is JAD2T ( = JCM 19131T = CCTCC AB-2014030T).

In this study, the taxonomic position and group classification of the phytoplasma associated with a lethal yellowing-type disease (LYD) of coconut (Cocos nucifera L.) in Mozambique were addressed. Pairwise similarity values based on alignment of nearly full-length 16S rRNA gene sequences (1530 bp) revealed that the Mozambique coconut phytoplasma (LYDM) shared 100 % identity with a comparable sequence derived from a phytoplasma strain (LDN) responsible for Awka wilt disease of coconut in Nigeria, and shared 99.0–99.6 % identity with 16S rRNA gene sequences from strains associated with Cape St Paul wilt (CSPW) disease of coconut in Ghana and Côte d’Ivoire. Similarity scores further determined that the 16S rRNA gene of the LYDM phytoplasma shared <97.5 % sequence identity with all previously described members of ‘Candidatus Phytoplasma ’. The presence of unique regions in the 16S rRNA gene sequence distinguished the LYDM phytoplasma from all currently described members of ‘Candidatus Phytoplasma ’, justifying its recognition as the reference strain of a novel taxon, ‘Candidatus Phytoplasma palmicola’. Virtual RFLP profiles of the F2n/R2 portion (1251 bp) of the 16S rRNA gene and pattern similarity coefficients delineated coconut LYDM phytoplasma strains from Mozambique as novel members of established group 16SrXXII, subgroup A (16SrXXII-A). Similarity coefficients of 0.97 were obtained for comparisons between subgroup 16SrXXII-A strains and CSPW phytoplasmas from Ghana and Côte d’Ivoire. On this basis, the CSPW phytoplasma strains were designated members of a novel subgroup, 16SrXXII-B.

A catalase-positive, rod-shaped, non-proteolytic, non-motile, anaerobic bacterial strain, designated B086562T, was isolated from a blood culture of an 84-year-old male patient in Israel. According to 16S rRNA gene sequence phylogeny, this strain has no known close relatives among recognized bacteria but should be placed within the familyLachnospiraceae. The most closely related recognized bacteria were from the ‘Clostridium clostridioformegroup’:C. clostridioforme(92.4 %) andClostridium bolteae(92.3 %). The isolate produced butyrate, lactate, acetate and succinate as major metabolic end products. The major fatty acids were C16 : 0and C18 : 1cis9 DMA and the DNA G+C content was 46.0 mol%. On the basis of the phenotypic properties and phylogenetic distinctiveness, the blood isolate represents a novel species of a new genus in the familyLachnospiraceae, for which the nameEisenbergiella tayigen. nov., sp. nov. is proposed. The type strain ofEisenbergiella tayiis B086562T( = LMG 27400T = DSM 26961T = ATCC BAA-2558T).