Farysizyma gen. nov., an anamorphic genus in the Ustilaginales to accommodate three novel epiphytic basidiomycetous yeast species from America, Europe and Asia

Among many isolates that resulted from four independent surveys of yeasts associated with plants in Brazil, the USA, Portugal and Taiwan, we have characterized eighteen basidiomycetous strains, two of which were conspecific with the type strain of Rhodotorula acheniorum, whereas the remaining sixteen isolates appeared not to correspond to any previously described species. Microsatellite-PCR fingerprinting with primers M13 and (GTG)5 confirmed that the latter strains formed three genetically distinct groups. Each group was considered to represent a distinct species based on nucleotide sequences of the D1/D2 domains of the 26S rRNA gene and the internal transcribed spacer (ITS) region. Phylogenetic analyses of sequence data placed the putative novel species in a clade with R. acheniorum and the dimorphic smut fungus Farysia chardoniana. A novel anamorphic genus, Farysizyma, is created to accommodate the three undescribed species, which were named Farysizyma itapuensis, Farysizyma setubalensis and Farysizyma taiwaniana. A new combination, Farysizyma acheniorum, is proposed for R. acheniorum, which may represent the yeast-phase anamorph of Farysia thuemenii.     

Tsukamurella spumae sp. nov., a novel actinomycete associated with foaming in activated sludge plants

A polyphasic taxonomic study was undertaken to establish the taxonomic position of six representative strains isolated from activated sewage sludge foam. The organisms were found to have chemical and morphological properties consistent with their assignment to the genus Tsukamurella. DNA:DNA relatedness studies showed that five out of the six isolates formed a distinct genomic species, the remaining strain was most closely associated with this taxon. The five isolates had a unique phenotypic profile that served to distinguish them from representatives of the validly described species of Tsukamurella. The combination of the genotypic and phenotypic data indicated that the five strains should be classified as a new species in the genus Tsukamurella. The name proposed for this taxon is Tsukamurella spumae, the type strain is N1171T (= DSM 44.113T = NCIMB 13947T). It was also shown that some of the reference strains were misclassified as Tsukamurella paurometabola.     

Discontinuous Genetic Variation Among Mesophilic Naegleria Isolates: Further Evidence that N. gruberi Is Not a Single Species

Naegleria isolates which are currently placed in the type species N. gruberi display great genetic, physiological and morphological heterogeneity. There are two possible interpretations of the nature of this species–that N. gruberi is a species complex or that it is a single continuously variable species. To distinguish between these alternatives, allelic states were determined for 33 loci in 74 new isolates selected to represent wide geographic sources and diverse temperature limits for growth. The results were compared with data for culture collection strains of N. gruberi and other species in the genus. The isolates formed a discontinuous series of clusters, separated by genetic distances similar to those separating the better-characterised taxa N. fowleri, N. lovaniensis, N. jadini, N. australiensis australiensis and N. australiensis italica . Culture collection strains assigned to N. gruberi fell into six distinct clusters, while other clusters were not represented by reference strains. The data are most consistent with the interpretation that N. gruberi is a group of several distinct species, each equivalent to the recently described species in the genus. Naegleria andersoni andersoni and N. andersoni jamiesoni also formed two distinct clusters, equivalent to species. Characteristic temperature limits for growth show that the mesophilic species are ecological as well as genetic entities.     

Discontinuous genetic variation among mesophilic Naegleria isolates: further evidence that N. gruberi is not a single species.

Naegleria isolates which are currently placed in the type species N. gruberi display great genetic, physiological and morphological heterogeneity. There are two possible interpretations of the nature of this species--that N. gruberi is a species complex or that it is a single continuously variable species. To distinguish between these alternatives, allelic states were determined for 33 loci in 74 new isolates selected to represent wide geographic sources and diverse temperature limits for growth. The results were compared with data for culture collection strains of N. gruberi and other species in the genus. The isolates formed a discontinuous series of clusters, separated by genetic distances similar to those separating the better-characterised taxa N. fowleri, N. lovaniensis, N. jadini, N. australiensis australiensis and N. australiensis italica. Culture collection strains assigned to N. gruberi fell into six distinct clusters, while other clusters were not represented by reference strains. The data are most consistent with the interpretation that N. gruberi is a group of several distinct species, each equivalent to the recently described species in the genus. Naegleria andersoni andersoni and N. andersoni jamiesoni also formed two distinct clusters, equivalent to species. Characteristics temperature limits for growth show that the mesophilic species are ecological as well as genetic entities.     

Phylogeny of Powellomycetaceae fam. nov. and description of Geranomyces variabilis gen. et comb. nov

The genus Powellomyces was described to accommodate two monocentric chytrid species from soil that develop exogenously and possess zoosporic ultrastructure similar to other members of the order Spizellomycetales. Despite Powellomyces-like chytrids being commonly observed in gross culture, the genus contained only two species. To determine diversity in this group I amassed 49 isolates of Powellomyces-like chytrids, including the cultures upon which species types were based and new isolates from pollen-baited water cultures of soils, plant detritus and manure. I sequenced portions of nucSSU and nucLSU rDNA regions and the EF-1α-like gene from each isolate to produce a molecular phylogeny. This phylogeny supports monophyly of spizellomycetalean chytrids with exogenous development and suggests that multiple distinct lineages exist within this group. This phylogeny, along with a reevaluation of the ultra-structural features of the two described species, supports the recognition of a new family, Powellomycetaceae, and genus, Geranomyces, which contains 31 isolates of G. variabilis comb. nov.     

Two new species of Rhynchosporium

Rhynchosporium consists of two species, R. secalis and R. orthosporum. Both are pathogens of grasses with R. secalis infecting a variety of Poaceae hosts and R. orthosporum infecting Dactylis glomerata. Phylogenetic analyses of multilocus DNA sequence data on R. secalis isolates originating from cultivated barley, rye, triticale and other grasses, including Agropyron spp., Bromus diandrus and Hordeum spp., resolved the monophyletic groups into three species according to their respective hosts. Host specificity according to phylogenetic lineages was confirmed with pathogenicity studies. Because R. secalis was described first on rye this name is retained for Rhynchosporium isolates infecting rye and triticale. Rhynchosporium isolates infecting cultivated barley and other Hordeum spp. and Bromus diandrus belong to a distinct species, R. commune. Similarly isolates infecting Agropyron spp. represent a distinct species of Rhynchosporium, namely R. agropyri. A PCR-RFLP assay was developed as a rapid tool for species identification of R. secalis and R. commune.     

Phylogenetic analysis of Cryptosporidium parasites based on the small-subunit rRNA gene locus

Biological data support the hypothesis that there are multiple species in the genus Cryptosporidium, but a recent analysis of the available genetic data suggested that there is insufficient evidence for species differentiation. In order to resolve the controversy in the taxonomy of this parasite genus, we characterized the small-subunit rRNA genes of Cryptosporidium parvum, Cryptosporidium baileyi, Cryptosporidium muris, and Cryptosporidium serpentis and performed a phylogenetic analysis of the genus Cryptosporidium. Our study revealed that the genus Cryptosporidium contains the phylogenetically distinct species C. parvum, C. muris, C. baileyi, and C. serpentis, which is consistent with the biological characteristics and host specificity data. The Cryptosporidium species formed two clades, with C. parvum and C. baileyi belonging to one clade and C. muris and C. serpentis belonging to the other clade. Within C. parvum, human genotype isolates and guinea pig isolates (known as Cryptosporidium wrairi) each differed from bovine genotype isolates by the nucleotide sequence in four regions. A C. muris isolate from cattle was also different from parasites isolated from a rock hyrax and a Bactrian camel. Minor differences were also detected between C. serpentis isolates from snakes and lizards. Based on the genetic information, a species- and strain-specific PCR-restriction fragment length polymorphism diagnostic tool was developed.     

Two new Ophiostoma species with Sporothrix anamorphs from Austria and Azerbaijan

The genus Ophiostoma includes numerous species of primarily insect-vectored, wood-staining fungi. Several anamorph genera that differ in their micronematous or macronematous conidiogenous cells have been associated with Ophiostoma species. Among the former group, Sporothrix is associated with many species and is characterized by conidiogenous cells that arise laterally or terminally from any place on the hyphae and produce nonseptate conidia on sympodially developing denticles. The purpose of this study was to characterize ophiostomatoid isolates with Sporothrix anamorphs recently collected in Austria and Azerbaijan. The isolates were characterized based on comparisons of rDNA and β-tubulin sequence data. Morphology, growth in culture, and sexual reproductive mode were also considered. Phylogenetic analyses of the combined sequence data showed that the isolates formed two distinct groups, one including isolates from Austria and the other isolates from Austria and Azerbaijan. Growth at 25 C and morphology revealed some differences between the two groups, and supported the view that they represent two new species, which we describe here as Ophiostoma fusiforme sp. nov. and Ophiostoma lunatum sp. nov. Both these groups phylogenetically were related to, but distinct from, Ophiostoma stenoceras.     

Leptographium piriforme sp. nov., from a taxonomically diverse collection of arthropods collected in an aspen-dominated forest in western Canada

During a survey of fungi associated with arthropods collected in a southern boreal mixed-wood forest in Alberta we obtained 29 isolates of a unique species of Leptographium. This species displayed a distinct combination of characteristics, including curved conidia on short-stipitate conidiophores, a secondary micronematous conidial state, stalked pear-shaped cells and an optimal growth rate at 35 C, and is described as Leptographium piriforme sp. nov. The isolates were most similar morphologically to L. crassivaginatum, but ITS sequence comparisons indicate that our isolates cannot be assigned to this or any other sequenced species in the genus. Initial observations on the pear-shaped cells in feeding experiments with Sancassania berlesei show that these structures may act as a nutritional incentive for visiting arthropods. Most arthropods carrying this new species were caught in traps baited with dung which, in light of its optimum growth temperature, suggests a coprophilous phase in the life cycle of this species. Additional isolates from woody species typical of the survey area might clarify whether Leptographium piriforme in its forest habitat occurs as a plant pathogen or saprobe.     

Salt adaptation in pseudomonads: characterization of glucosylglycerol-synthesizing isolates from brackish coastal waters and the rhizosphere

The compatible solute glucosylglycerol (GG) is widespread among cyanobacteria, but, until now, has been reported for only two species of heterotrophic bacteria. About 120 bacterial isolates from coastal regions of the Baltic Sea were screened by HPLC for their ability to synthesize GG. Positive isolates (26) were grouped by SDS-PAGE of whole-cell proteins and representative strains of each group were investigated by sequencing their 16S rRNA genes and phenotypic characterization. All GG-synthesizing isolates were shown to belong to the genus Pseudomonas (sensu stricto) and were assigned to 4 distinct groups, although none of the GG-synthesizing isolates could be unambiguously assigned to described species. The identity of GG was verified by 13C NMR analysis and enzymatic digestion with alpha- and beta-glucosidases. Besides GG, salt adapted cultures of the aquatic isolates accumulated the dipeptide N-acetylglutaminylglutamine amide (NAGGN) and glutamate. The accumulation of noncharged compatible solutes was also tested in previously identified pseudomonads isolated from the rhizosphere of oilseed rape and potato. The majority of these strains were fluorescent species of the genus Pseudomonas and accumulated trehalose and NAGGN when grown under salt stress conditions. However, rhizosphere isolates of Stenotrophomonas maltophilia synthesized GG and trehalose or only trehalose in a strain-dependent manner. These data indicate that the ability to synthesize GG is widely distributed among slightly or moderately halotolerant pseudomonads.     

Multilocus Sequence Analysis of the Marine Bacterial Genus Tenacibaculum Suggests Parallel Evolution of Fish Pathogenicity and Endemic Colonization of Aquaculture Systems

The genus Tenacibaculum, a member of the family Flavobacteriaceae, is an abundant component of marine bacterial ecosystems that also hosts several fish pathogens, some of which are of serious concern for marine aquaculture. Here, we applied multilocus sequence analysis (MLSA) to 114 representatives of most known species in the genus and of the worldwide diversity of the major fish pathogen Tenacibaculum maritimum. Recombination hampers precise phylogenetic reconstruction, but the data indicate intertwined environmental and pathogenic lineages, which suggests that pathogenicity evolved independently in several species. At lower phylogenetic levels recombination is also important, and the species T. maritimum constitutes a cohesive group of isolates. Importantly, the data reveal no trace of long-distance dissemination that could be linked to international fish movements. Instead, the high number of distinct genotypes suggests an endemic distribution of strains. The MLSA scheme and the data described in this study will help in monitoring Tenacibaculum infections in marine aquaculture; we show, for instance, that isolates from tenacibaculosis outbreaks in Norwegian salmon farms are related to T. dicentrarchi, a recently described species.     

Microtetraspora malaysiensis sp. nov., isolated from Malaysian primary dipterocarp forest soil

The taxonomic position of three actinomycete strains isolated from Malaysian soil was established by using a polyphasic approach. The isolates formed chains composed of four spores on the tip of sporophores branching from the aerial mycelium, and their chemotaxonomic properties were common to those of members of the family Streptosporangiaceae. These phenotypic properties as well as a phylogenetic analysis based on 16S rRNA gene sequences indicated that they should be classified in the genus Microtetraspora. The three isolates showed a unique pattern of cultural, physiological and biochemical properties that distinguished them from previously described species of the genus Microtetraspora. The isolates showed more than 72% DNA relatedness to each other, but only 58% or less relatedness to any previously described species. On the basis of the data presented, a new species of the genus Microtetraspora, Microtetraspora malaysiensis, is proposed. The type strain of the new species is strain H47-7(T) (=JCM 11278(T)=DSM 44579(T)).     

The ant-parasitic genus Rhipipalloidea Girault (Hymenoptera: Eucharitidae), with a description of a new species

The genus Rhipipalloidea Girault (1934 ), from the Australian region is revised. Rhipipalloidea gruberi Girault (1940 ) is synonymised with R. mira Girault 1934 . A new species, R. madangensis , is described from Papua New Guinea. This species is separated from R. mira from Australia by the distinct striation on the head, and ramose, 12-segmented antennae of the females. Males and a host for the genus are described for the first time.     

Photosynthetic bradyrhizobia from Aeschynomene spp. are specific to stem-nodulated species and form a separate 16S ribosomal DNA restriction fragment length polymorphism group.

We obtained nine bacterial isolates from root or collar nodules of the non-stem-nodulated Aeschynomene species A. elaphroxylon, A. uniflora, or A. schimperi and 69 root or stem nodule isolates from the stem-nodulated Aeschynomene species A. afraspera, A. ciliata, A. indica, A. nilotica, A. sensitiva, and A. tambacoundensis from various places in Senegal. These isolates, together with 45 previous isolates from various Aeschynomene species, were studied for host-specific nodulation within the genus Aeschynomene, also revisiting cross-inoculation groups described previously by D. Alazard (Appl. Environ. Microbiol. 50:732-734, 1985). The whole collection of Aeschynomene nodule isolates was screened for synthesis of photosynthetic pigments by spectrometry, high-pressure liquid chromatography, and thin-layer chromatography analyses. The presence of puf genes in photosynthetic Aeschynomene isolates was evidenced both by Southern hybridization with a Rhodobacter capsulatus photosynthetic gene probe and by DNA amplification with primers defined from photosynthetic genes. In addition, amplified 16S ribosomal DNA restriction analysis was performed on 45 Aeschynomene isolates, including strain BTAi1, and 19 reference strains from Bradyrhizobium japonicum, Bradyrhizobium elkanii, and other Bradyrhizobium sp. strains of uncertain taxonomic positions. The 16S rRNA gene sequence of the photosynthetic strain ORS278 (LMG 12187) was determined and compared to sequences from databases. Our main conclusion is that photosynthetic Aeschynomene nodule isolates share the ability to nodulate particular stem-nodulated species and form a separate subbranch on the Bradyrhizobium rRNA lineage, distinct from B. japonicum and B. elkanii.     

Comparative analysis of cyanobacteria in the rhizosphere and as endosymbionts of cycads in drought-affected soils

Does the diversity of cyanobacteria in the cycad rhizosphere relate to the cyanobiont species found in the coralloid roots of these ancient plants? The aim of this study was to identify the diversity of soil cyanobacteria occurring in the immediate vicinity of 22 colonized coralloid roots belonging to members of the cycad genera: Macrozamia, Lepidozamia, Bowenia and Cycas. The majority of coralloid roots were sampled at depths >?10?cm below the soil surface. A total of 32 cyanobacterial isolates were cultured and their 16S rRNA gene partially sequenced. Phylogenetic analysis revealed nine operational taxonomic units of soil cyanobacteria comprising 30 Nostoc spp., a Tolypothrix sp. and a Leptolyngbya sp. Microscopy indicated that all isolates were unialgal and confirmed their genus identity. Rhizospheric diversity was compared to existing data on cyanobionts isolated at the same time from the cycad coralloid root. The same isolate was present in both the cycad coralloid root and rhizosphere at only six sites. Phylogenetic evidence indicates that most rhizosphere isolates were distinct from root cyanobionts. This weak relationship between the soil cyanobacteria and cycad cyanobionts might indicate that changes in the soil community composition are due to environmental factors.     

Phylogenetic characterization of a new thermoacidophilic bacterium isolated from hot springs in Japan

Three strains of thermophilic-acidophilic bacteria isolated previously from different hot springs in Japan were characterized by molecular genetic methods. The strategy taken involved PCR amplification, sequencing and restriction pattern analysis of 16S rDNA, 16S-23S rDNA spacer polymorphism analysis and genomic DNA-DNA hybridization. A phylogenetic analysis based on 16S rDNA sequences showed that the new thermoacidophilic isolates formed a genetically coherent group at the species level and fell into a major cluster together with members of the genera Alicyclobacillus and Sulfobacillus with A. acidocaldarius and A. acidoterrestris as their closest relatives. The levels of binary sequence similarity between the isolates and the two Alicyclobacillus species were 97.6 to 97.9%, values considered low enough to warrant placement of the isolates in a distinct species of the genus Alicyclobacillus. The 16S rDNA restriction pattern analysis, but not 16S-23S rDNA spacer polymorphism analysis, was useful for differentiating the isolates from the established Alicyclobacillus species. DNA-DNA hybridization assays demonstrated a distinct phylogenetic position of our isolates as a genospecies within the genus Alicyclobacillus. On the basis of these results, the thermoacidophilic isolates should be classified into a new species of Alicyclobacillus. The results of this study suggest that this new genospecies of Alicyclobacillus is widely distributed in hot springs in Japan.     

Chitinophaga soli sp. nov. and Chitinophaga terrae sp. nov., isolated from soil of a ginseng field in Pocheon Province, Korea

Two novel strains of the Cytophaga-Flexibacter-Bacteroides (CFB) group, designated Gsoil 219" and Gsoil 2381, were isolated from soil of a ginseng field of Pocheon Province in Korea. Both strains were Gram-negative, aerobic, nonmotile, nonspore-forming, and rod-shaped. Phylogenetic analysis based on 16S rRNA gene sequences indicated that both isolates belong to the genus Chitinophaga but were clearly separated from established species of this genus. The sequence similarities between strain Gsoil 219T and type strains of the established species and between strain Gsoil 238T and type strains of the established species ranged from 91.4 to 94.7% and 91.6 to 94.2%, respectively. Phenotypic and chemotaxonomic data (major menaquinone, MK-7; major fatty acids, iso-C15:0 and C(16:1) omega5c; major hydroxy fatty acid, iso-C(17:0) 3-OH; major polyamine, homospermidine) supported the affiliation of both strains Gsoil 219T and Gsoil 238T to the genus Chitinophaga. Furthermore, the results of physiological and biochemical tests allowed genotypic and phenotypic differentiation of both strains from the other validated Chitinophaga species. Therefore, the two isolates represent two novel species, for which the name Chitinophaga soli sp. nov. (type strain, Gsoil 219T=KCTC 12650T=DSM 18093T) and Chitinophaga terrae sp. nov. (type strain, Gsoil 238T=KCTC 12651T=DSM 18078T) are proposed.     

Intraspecific Variation and Phylogenetic Relationships in the Genus Entamoeba as Revealed by Riboprinting

ABSTRACT. Eighty-seven isolates of amebae assigned to the genus Entamoeba have been studied by riboprinting (restriction enzyme polymorphism analysis of polymerase chain reaction amplified small subunit ribosomal RNA genes). Twenty-four distinct patterns were obtained, most of which corresponded to previously described species. In three species ( Entamoeba coli, Entamoeba gingivalis and Entamoeba moshkovskii ) intraspecific variation was detected that led to the grouping of isolates into 'ribodemes' (populations of amebae that share the same riboprint pattern). The riboprint data were used to estimate genetic distances among and within species for the construction of phylogenetic trees based on parsimony and distance analyses. The trees obtained with the two methods are largely congruent. In some cases the estimated distances between species were greater than the upper limit recommended for the fragment comigration method of analysis indicating unusually deep branches within this genus. However, it appears that those species producing cysts with eight nuclei, those producing cysts with one nucleus, and those producing cysts with four nuclei form morphologically based groups that are supported by the riboprint data. The oral parasite Entamoeba gingivalis , which does not encyst, clusters with the third group indicating secondary loss of this ability.     

Streptacidiphilus jiangxiensis sp. nov., a novel actinomycete isolated from acidic rhizosphere soil in China

The taxonomic position of three acidophilic actinomycetes isolated from acidic rhizosphere soil was established using a polyphasic approach. The morphological and chemical properties of the isolates were found to be consistent with their assignment to the genus Streptacidiphilus. Almost complete 16S rRNA gene sequences determined for the strains were aligned with corresponding sequences of representatives of the genera Kitasatospora, Streptacidiphilus and Streptomyces and phylogenetic trees inferred using three tree-making algorithms. The organisms formed a distinct subclade within the Streptacidiphilus 16S rRNA gene tree. They also shared nearly identical phenotypic profiles and rep-PCR fingerprint patterns that readily distinguished them from representatives of the established species of Streptacidiphilus. It is evident from the genotypic and phenotypic data that the three isolates form a new species in the genus Streptacidiphilus. The name proposed for this new species is Streptacidiphilus jiangxiensis, the type strain is isolate 33214T (= AS 4.1857T = JCM 12277T).     

Genetic diversity and phylogeny of rhizobia isolated from Caragana microphylla growing in desert soil in Ningxia, China

Rhizobia are soil bacteria with the capacity to induce nitrogen-fixing nodules on the roots or stems of legume plants. A total of 40 bacterial isolates from the root nodules of Caragana microphylla growing in desert soil in Ningxia, China, were analyzed for genetic diversity and phylogenetic position. These isolates were classified into 7 types of 16S ribosomal DNA (rDNA) using polymerase chain reaction-restriction fragment length polymorphism analysis. They were grouped into 4 clades, Rhizobium-Agrobacterium, Sinorhizobium, Phyllobacterium, and Bradyrhizobium, when the phylogenies of 16S rDNA, recA, and atpD genes were applied. Phylogenetic analysis showed that the tree generated from the 16S rDNA sequencing agreed with that produced from the recA and atpD genes. By analyzing phylogenetic relationship using the 3 loci, the isolates in the branches of Phyllobacterium and Sinorhizobium could be identified as P. brassicacearum and S. meliloti. The isolates in the branch of Rhizobium-Agrobacterium were the most abundant microsymbiont of C. microphylla and were designated R. leguminosarum, R. galegae, R. alamii, and A. tumefaciens. Two isolates with low sequence similarity to the known species of Bradyrhizobium might be novel species in this genus.