- Volume 54, Issue 4, 2004

The taxonomic position of two Tsukamurella strains isolated from activated sludge foam was clarified. The organisms, isolates JC85 and N1176T, were found to have chemical and morphological properties typical of members of the genus Tsukamurella. DNA–DNA relatedness studies showed that the strains formed a distinct genomic species that was most closely related to Tsukamurella spumae. The two isolates also share a range of phenotypic properties that distinguishes them from representatives of all species of Tsukamurella with validly published names. It is evident from the data that the two organisms should be classified as a novel Tsukamurella species, Tsukamurella pseudospumae sp. nov. The type strain is N1176T (=DSM 44118T=NCIMB 13963T).

Apple proliferation (AP), pear decline (PD) and European stone fruit yellows (ESFY) are among the most economically important plant diseases that are caused by phytoplasmas. Phylogenetic analyses revealed that the 16S rDNA sequences of strains of each of these pathogens were identical or nearly identical. Differences between the three phytoplasmas ranged from 1·0 to 1·5 % of nucleotide positions and were thus below the recommended threshold of 2·5 % for assigning species rank to phytoplasmas under the provisional status ‘Candidatus’. However, supporting data for distinguishing the AP, PD and ESFY agents at the species level were obtained by examining other molecular markers, including the 16S–23S rDNA spacer region, protein-encoding genes and randomly cloned DNA fragments. The three phytoplasmas also differed in serological comparisons and showed clear differences in vector transmission and host-range specificity. From these results, it can be concluded that the AP, PD and ESFY phytoplasmas are coherent but discrete taxa that can be distinguished at the putative species level, for which the names ‘Candidatus Phytoplasma mali’, ‘Candidatus Phytoplasma pyri’ and ‘Candidatus Phytoplasma prunorum’, respectively, are proposed. Strains AP15R, PD1R and ESFY-G1R were selected as reference strains. Examination of available data on the peach yellow leaf roll (PYLR) phytoplasma, which clusters with the AP, PD and ESFY agents, confirmed previous results showing that it is related most closely to the PD pathogen. The two phytoplasmas share 99·6 % 16S rDNA sequence similarity. Significant differences were only observed in the sequence of a gene that encodes an immunodominant membrane protein. Until more information on this phytoplasma is available, it is proposed that the PYLR phytoplasma should be regarded as a subtype of ‘Candidatus Phytoplasma pyri’.

A novel moderately thermophilic bacterium, strain TH13T, was isolated from a hot spring in Kamchatka. It was found to be a Gram-positive, facultative aerobe; the straight, non-motile rods grew at 30–64 °C (optimum 54 °C). The isolate was positive for catalase and oxidase tests and reduced nitrate to nitrite, but was negative for H2S production and growth in more than 3 % NaCl (w/v). The isolate grew at pH 7–8, but not at pH values higher than 9. The DNA G+C content was 43·9 mol%. Phylogenetic analysis based on 16S rRNA gene sequencing indicated that strain TH13T was a member of the genus Anoxybacillus. DNA–DNA hybridization revealed a low relatedness (less than 30·2 %) between the isolate and its close phylogenetic neighbours Anoxybacillus pushchinoensis and Anoxybacillus flavithermus. On the basis of phenotypic characteristics, phylogenetic data and DNA–DNA hybridization data, it was concluded that the isolate merited classification as a novel species, for which the name Anoxybacillus voinovskiensis sp. nov. is proposed. The type strain of this species is TH13T (=NCIMB 13956T=JCM 12111T).

The trivial name ‘phytoplasma’ has been adopted to collectively name wall-less, non-helical prokaryotes that colonize plant phloem and insects, which were formerly known as mycoplasma-like organisms. Although phytoplasmas have not yet been cultivated in vitro, phylogenetic analyses based on various conserved genes have shown that they represent a distinct, monophyletic clade within the class Mollicutes. It is proposed here to accommodate phytoplasmas within the novel genus ‘Candidatus (Ca.) Phytoplasma’. Given the diversity within ‘Ca. Phytoplasma’, several subtaxa are needed to accommodate organisms that share <97·5 % similarity among their 16S rRNA gene sequences. This report describes the properties of ‘Ca. Phytoplasma’, a taxon that includes the species ‘Ca. Phytoplasma aurantifolia’ (the prokaryote associated with witches'-broom disease of small-fruited acid lime), ‘Ca. Phytoplasma australiense’ (associated with Australian grapevine yellows), ‘Ca. Phytoplasma fraxini’ (associated with ash yellows), ‘Ca. Phytoplasma japonicum’ (associated with Japanese hydrangea phyllody), ‘Ca. Phytoplasma brasiliense’ (associated with hibiscus witches'-broom in Brazil), ‘Ca. Phytoplasma castaneae’ (associated with chestnut witches'-broom in Korea), ‘Ca. Phytoplasma asteris' (associated with aster yellows), ‘Ca. Phytoplasma mali’ (associated with apple proliferation), ‘Ca. Phytoplasma phoenicium’ (associated with almond lethal disease), ‘Ca. Phytoplasma trifolii’ (associated with clover proliferation), ‘Ca. Phytoplasma cynodontis' (associated with Bermuda grass white leaf), ‘Ca. Phytoplasma ziziphi’ (associated with jujube witches'-broom), ‘Ca. Phytoplasma oryzae’ (associated with rice yellow dwarf) and six species-level taxa for which the Candidatus species designation has not yet been formally proposed (for the phytoplasmas associated with X-disease of peach, grapevine flavescence dorée, Central American coconut lethal yellows, Tanzanian lethal decline of coconut, Nigerian lethal decline of coconut and loofah witches'-broom, respectively). Additional species are needed to accommodate organisms that, despite their 16S rRNA gene sequence being >97·5 % similar to those of other ‘Ca. Phytoplasma’ species, are characterized by distinctive biological, phytopathological and genetic properties. These include ‘Ca. Phytoplasma pyri’ (associated with pear decline), ‘Ca. Phytoplasma prunorum’ (associated with European stone fruit yellows), ‘Ca. Phytoplasma spartii’ (associated with spartium witches'-broom), ‘Ca. Phytoplasma rhamni’ (associated with buckthorn witches'-broom), ‘Ca. Phytoplasma allocasuarinae’ (associated with allocasuarina yellows), ‘Ca. Phytoplasma ulmi’ (associated with elm yellows) and an additional taxon for the stolbur phytoplasma. Conversely, some organisms, despite their 16S rRNA gene sequence being <97·5 % similar to that of any other ‘Ca. Phytoplasma’ species, are not presently described as Candidatus species, due to their poor overall characterization.

The possibility that the strains included within the Mycobacterium avium complex (MAC), but not belonging either to M. avium or to Mycobacterium intracellulare, may be members of undescribed taxa, has already been questioned by several taxonomists. A very homogeneous cluster of 12 strains characterized by identical nucleotide sequences both in the 16S rDNA and in the 16S–23S internal transcribed spacer was investigated. Similar strains, previously reported in the literature, had been assigned either to the species M. intracellulare on the basis of the 16S rDNA similarity or to the group of MAC intermediates. However, several phenotypical and epidemiological characteristics seem to distinguish these strains from all other MAC organisms. The unique mycolic acid pattern obtained by HPLC is striking as it is characterized by two clusters of peaks, instead of the three presented by all other MAC organisms. All of the strains have been isolated from humans and all but one came from the respiratory tract of elderly people. The clinical significance of these strains, ascertained for seven patients, seems to suggest an unusually high virulence. The characteristics of all the strains reported in the literature, genotypically identical to the ones described here, seem to confirm our data, without reports of isolations from animals or the environment or, among humans, from AIDS patients. Therefore, an elevation of the MAC variant was proposed and characterized here, with the name Mycobacterium chimaera sp. nov.; this increases the number of species included in the M. avium complex. The type strain is FI-01069T (=CIP 107892T=DSM 44623T).

Strain XLG9A10.2T was isolated from an alkaline salt marsh soil in western China. 16S rRNA gene sequence analysis indicated that strain XLG9A10.2T constitutes a distinct lineage within the family Promicromonosporaceae, sharing 94·8–95·1 % gene similarity with members of the genus Promicromonospora and 94·4–95·7 % similarity with those of Xylanimonas and related genera. The general colony and cell morphology of strain XLG9A10.2T is similar to that of members of Promicromonospora, but differs from members of the genus Xylanimonas in forming a well-developed branching mycelium and production of coccoid spores. Strain XLG9A10.2T shows the peptidoglycan type A4α (l-lys←l-thr←d-Glu), contains glucose, mannose and galactose as whole cell sugars and has MK-9(H4) and MK-9(H6) as major menaquinones, while phospholipids are phosphatidylglycerol, diphosphatidylglycerol, phosphatidylinositol, three unidentified phospholipids and one unidentified glycolipid. The DNA base composition is 71·9 mol% G+C. On the basis of morphological, chemotaxonomic, metabolic and phylogenetic differences from other species of Promicromonosporaceae, a new genus and species, Myceligenerans xiligouense gen. nov., sp. nov., is proposed. The type strain is XLG9A10.2T (=DSM 15700T=CGMCC 1.3458T.)

A strictly aerobic, non-motile, rod-shaped actinomycete strain, named JC2055T, was isolated from a sediment sample of getbol, the tidal flat of Korea. 16S rDNA analysis revealed that the getbol isolate belonged to the genus Nocardioides with the highest sequence similarity to Nocardioides aquiterrae GW-9T (95·5 %). The major menaquinone was MK-8(H4) and the predominant cellular fatty acids were iso-16 : 0 and 17 : 1ω8c. Tuberculostearic acid was absent. The G+C content of DNA was 72 mol%. Based on the morphological, physiological, biochemical and chemotaxonomical data presented in this study, strain JC2055T can be readily differentiated from other validly named Nocardioides species. The name Nocardioides ganghwensis sp. nov. is proposed for the isolate. The type strain is JC2055T (=IMSNU 14028T=KCTC 9920T=JCM 12124T).

Three strains of a hitherto unknown, Gram-negative, anaerobic coccus were isolated from human samples. At the phenotypic level, the isolates displayed all the characteristics of bacteria belonging to the genus Veillonella. Sequence analysis revealed that the three strains shared >99·5 % similarity in 16S rDNA sequence and >98·4 % similarity in dnaK sequence. The three unknown strains formed a separate subclade that was clearly remote from Veillonella species of human and animal origin. Based on these results, the three strains were considered to represent a novel species within the genus Veillonella, for which the name Veillonella montpellierensis is proposed. The type strain of the species is ADV 281.99T (=CIP 107992T=CCUG 48299T).

A Gram-positive, motile, round to ellipsoidal, endospore-forming, rod-shaped bacterial strain, SF-57T, was isolated from a marine solar saltern in Korea. This organism grew between 4 and 39 °C, with optimum growth at 30 °C. Strain SF-57T grew in the presence of 0·5–15·0 % NaCl, with optimum growth at 2–3 % NaCl. The peptidoglycan type of strain SF-57T was A1α linked directly through l-Lys. In strain SF-57T, menaquinone-7 (MK-7) was the predominant isoprenoid quinone and anteiso-C15 : 0 was the major fatty acid. The DNA G+C content was 41·8 mol%. Phylogenetic analyses based on 16S rRNA gene sequences showed that strain SF-57T formed a coherent cluster with Marinibacillus marinus, with a bootstrap resampling value of 100 %. The level of 16S rRNA gene sequence similarity between strain SF-57T and M. marinus DSM 1297T was 98·9 %. The mean DNA–DNA relatedness level between strain SF-57T and the type strain of M. marinus was 20·6 %. Based on phenotypic properties, phylogenetic analyses and genomic data, strain SF-57T merits placement in the genus Marinibacillus as a representative of a novel species, for which the name Marinibacillus campisalis sp. nov. is proposed. The type strain is SF-57T (=KCCM 41644T=JCM 11810T).

An actinomycete strain, isolated from a Mata Atlântica soil sample, showing cellulolytic activity was subjected to polyphasic taxonomic characterization to determine its identity. Strain M7aT presented morphological and chemotaxonomic characteristics consistent with its assignment to the genus Streptomyces. Phylogenetic analysis of its 16S rDNA sequence revealed that the strain differed from described streptomycetes available in the public databases; the most closely related species was Streptomyces laceyi, with 98·4 % nucleotide similarity. It also differed from other cellulolytic strains in its phenotypic characteristics. It is therefore proposed that strain M7aT, a cellulolytic strain with biotechnological potential, represents a novel species, named Streptomyces drozdowiczii sp. nov. The type strain is M7aT (=CBMAI 0498T=CIP 107837T=NRRL B-24297T).

Two actinomycete strains, DSA1T and HD9T, were isolated from a potato tuber and soil from a potato-cultivating field in Jeju, Korea, respectively. A comprehensive 16S rDNA analysis revealed that the isolates belong to the genus Kribbella and share 97·7–98·6 % sequence similarity to Kribbella species. The strains also contained typical chemotaxonomic markers of the genus Kribbella: ll-diaminopimelic acid, alanine, glycine and glutamic acid in the cell wall peptidoglycan; mannose, glucose, galactose and ribose as whole cell sugars; diphosphatidylglycerol, phosphatidylcholine and phosphatidylinositol as characteristic phospholipids; and the major menaquinone MK-9 (H4). DNA–DNA hybridization experiments showed that the isolates represented two distinct genomic species. A number of phenotypic properties can be used to differentiate the two isolates from Kribbella species. On the basis of polyphasic evidence, two novel species are proposed: Kribbella solani sp. nov. for strain DSA1T (=KACC 20196T=JCM 12205T) and Kribbella jejuensis sp. nov. for strain HD9T (=KACC 20266T=JCM 12204T).

Clover proliferation phytoplasma (CPR) is designated as the reference strain for the CP phylogenetic group or subclade, on the basis of molecular analyses of genomic DNA, the 16S rRNA gene and the 16S–23S spacer region. Other strains related to CPR include alfalfa witches'-broom (AWB), brinjal little leaf (BLL), beet leafhopper-transmitted virescence (BLTV), Illinois elm yellows (ILEY), potato witches'-broom (PWB), potato yellows (PY), tomato big bud in California (TBBc) and phytoplasmas from Fragaria multicipita (FM). Phylogenetic analysis of the 16S rRNA gene sequences of BLL, CPR, FM and ILEY, together with sequences from 16 other phytoplasmas that belong to the ash yellows (AshY), jujube witches'-broom (JWB) and elm yellows (EY) groups that were available in GenBank, produced a tree on which these phytoplasmas clearly clustered as a discrete group. Three subgroups have been classified on the basis of sequence homology and the collective RFLP patterns of amplified 16S rRNA genes. AWB, BLTV, PWB and TBBc are assigned to taxonomic subgroup CP-A, FM belongs to subgroup CP-B and BLL and ILEY are assigned to subgroup CP-C. Genetic heterogeneity between different isolates of AWB, CPR and PWB has been observed from heteroduplex mobility assay analysis of amplified 16S rRNA genes and the 16S–23S spacer region. Two unique signature sequences that can be utilized to distinguish the CP group from others were present. On the basis of unique properties of the DNA from clover proliferation phytoplasma, the name ‘Candidatus Phytoplasma trifolii’ is proposed for the CP group.

Forty-eight bacterial strains were isolated at dairy farms from raw milk, the milking apparatus, green fodder or feed concentrate after a heat treatment of 30 min at 100 °C. In this way, spore-forming bacteria with a very high intrinsic heat resistance were selected for. The aerobic spore-forming isolates were subjected to a polyphasic taxonomical study, including repetitive element sequence-based PCR typing, whole-cell protein profiling, 16S rDNA sequence analysis, DNA–DNA hybridizations, DNA base composition, fatty acid analysis, and morphological and biochemical characteristics. A comparison of the REP- and (GTG)5-PCR and whole-cell protein SDS-PAGE profiles resulted in three clusters of similar strains. Analysis of the 16S rDNA sequences and DNA–DNA relatedness data showed that these clusters represented three novel species. The highest 16S rDNA similarity to a recognized species found for the three groups was around 94 % with Bacillus lentus and Bacillus sporothermodurans. Further phenotypic characterization supported the proposal of three novel species in the genus Bacillus, Bacillus farraginis, Bacillus fortis and Bacillus fordii. The respective type strains are R-6540T (=LMG 22081T=DSM 16013T), R-6514T (=LMG 22079T=DSM 16012T) and R-7190T (=LMG 22080T=DSM 16014T); their G+C DNA base contents are 43·7, 44·3 and 41·9 mol%, respectively. Although in variable amounts, a predominance of the branched fatty acids iso-C15 : 0 and anteiso-C15 : 0 was observed in all three novel species.

In studying the composition of the Lactobacillus flora of faeces from pigs fed different diets, isolates with notable differences in their 16S rRNA gene sequence compared to recognized species were found. Phenotypic characteristics together with 16S rRNA gene sequences revealed that the isolates represented a novel species belonging to the Lactobacillus mali subgroup of lactobacilli. The name Lactobacillus saerimneri sp. nov. is proposed (type strain GDA154T=LMG 22087T=DSM 16049T=CCUG 48462T).

Strain Sd/3T (=MTCC 4374T=DSM 15820T), an arsenic-resistant bacterium, was isolated from a sand sample obtained from an arsenic-contaminated aquifer in Chakdah district in West Bengal, India (23° 3′ N 88° 35′ E). The bacterium was Gram-positive, rod-shaped, non-motile, endospore-forming and yellowish-orange pigmented. It possessed all the characteristics that conform to the genus Bacillus, such as it had A4 β murein type (l-orn-d-Asp) peptidoglycan variant, MK-7 as the major menaquinone and iso-C15 : 0 and anteiso-C15 : 0 as the major fatty acids. Based on its chemotaxonomic and phylogenetic characteristics, strain Sd/3T was identified as a species of the genus Bacillus. It exhibited maximum similarity (95 %) at the 16S rRNA gene level with Bacillus cohnii; however, DNA–DNA similarity with B. cohnii was 60·7 %. Strain Sd/3T also exhibited a number of phenotypic differences from B. cohnii (DSM 6307T). These data suggest that Sd/3T represents a novel species of the genus Bacillus. The name Bacillus indicus sp. nov. is proposed.

The taxonomic position of a soil isolate, strain SA 233T, recovered from Saharan soil from Algeria was established using a polyphasic approach. This isolate has been previously reported to produce three novel dithiolopyrrolone antibiotics, and preliminary chemotaxonomic and morphological characteristics suggested that it was representative of a member of the genus Saccharothrix. Phylogenetic analysis of the strain from 16S rDNA sequences, along with a detailed analysis of morphological, chemotaxonomic and physiological characteristics, indicates that it belongs to the genus Saccharothrix and represents a novel species that is readily distinguished from all recognized Saccharothrix species. The name Saccharothrix algeriensis sp. nov. is proposed for the isolate, with type strain SA 233T (=NRRL B-24137T=DSM 44581T).

The recently described facultatively anaerobic Promicromonospora pachnodae is phylogenetically only moderately related to authentic members of Promicromonospora. P. pachnodae is closely related to Xylanibacterium ulmi and slightly less closely related to Xylanimonas cellulosilytica and Isoptericola variabilis (basonym Cellulosimicrobium variabile). Members of the different genera of Promicromonosporaceae have similar chemotaxonomic properties; they share the same peptidoglycan type (A4α) and have similar profiles of polar lipids, menaquinones, fatty acids and whole cell sugars. However, they differ from each other in the detailed amino acid composition of peptidoglycan, a taxonomically significant character that has previously been used in the delineation of actinobacterial genera. Recognized Promicromonospora species and Xylanibacterium ulmi exhibit the l-lys–l-ala–d-Glu type, Xylanimonas cellulosilytica and I. variabilis show the l-lys–d-Asp type, whereas P. pachnodae has the l-lys–l-ser–d-Glu type. This property, together with the distinct phylogenetic position of Promicromonospora pachnodae, suggests a novel genus for the xylanolytic organism Xylanimicrobium pachnodae (Cazemier et al. 2004) gen. nov., comb. nov.

Strains of Rhodotorula fujisanensis (Basidiomycota, Urediniomycetes, Microbotryomycetidae), including the type strain, are sexually compatible and produce clamped mycelium with teliospores. However, as teliospore germination had not been documented, the complete sexual cycle was not known. During the course of this work, the basidial stage of R. fujisanensis was characterized. In addition, mating studies employing isolates that were identified preliminarily as Rhodotorula nothofagi, a species that is related closely to R. fujisanensis, yielded mycelium with teliospores, which formed basidia and basidiospores. The new data were evaluated by using several criteria, including the available molecular phylogenetic framework for the Microbotryomycetidae. Curvibasidium gen. nov. is described here, to accommodate two teleomorphs: Curvibasidium cygneicollum sp. nov. (CBS 4551T), which is described as the sexual stage of R. fujisanensis, and Curvibasidium pallidicorallinum sp. nov. (CBS 9091T), which is related closely to R. nothofagi, but does not represent its sexual stage.

Among ascomycetous yeasts that were isolated from several nature reserve areas in China, three anamorphic strains isolated from soil (QL 5-5T) and fruit (QL 21-2T and SN 15-1T) were revealed, by conventional characterization and molecular phylogenetic analysis based on internal transcribed spacer and large subunit (26S) rRNA gene D1/D2 region sequencing, to represent three novel species in the genus Candida. Candida qinlingensis sp. nov. (type strain, QL 5-5T=AS 2.2524T=CBS 9768T) was related closely to a teleomorphic species, Williopsis pratensis. The close relatives of Candida diospyri sp. nov. (type strain, QL 21-2T=AS 2.2525T=CBS 9769T) are Candida friedrichii and Candida membranifaciens. Candida asparagi sp. nov. (type strain, SN 15-1T=AS 2.2526T=CBS 9770T) forms a clade with Candida fructus.