Molecular characterization of Cylindrospermopsis raciborskii strains isolated from Portuguese freshwaters

Cylindrospermopsis raciborskii is a toxic bloom forming cyanobacteria that is a common component of the phytoplankton assemblage in temperate freshwaters, as well as in temperate climates. This species is of major concern in public health, due to its known ability to produce toxins, including cylindrospermopsin and paralytic shellfish poisoning toxin (PSP).In this study, M13 PCR fingerprinting, ERIC PCR fingerprinting and amplification of the internal transcribed spacer (ITS) region were used to characterize nine cultured strains of C. raciborskii, sourced from several freshwater lakes and rivers in Portugal, and two other Australian. Strains belonging to other taxa including Microcystis aeruginosa, Aphanizomenon spp., Planktothrix agardhii and Oscillatoria neglecta were also analysed to evaluate the taxonomical potential of the fingerprinting methods.Data obtained from genomic fingerprinting were used to perform hierarchical cluster analysis and demonstrated ability to differentiate strains at intra-specific level. However, the high level of variability prevents their use as an identification tool. ITS amplification displayed intra-specific polymorphism both in number and length of the obtained amplicons, but revealed itself as a good method for strain clustering. The unsuccessful amplification of peptide synthetase (PS) and polyketide synthase (PKS) genes pointed to the inability of Portuguese C. raciborskii strains to produce cylindrospermopsin. HPLC analysis further confirmed this lack of toxicity, since negative results were obtained for cylindrospermopsin and PSP toxins.     

Molecular phylogeny and phenotypic variability of clinical and environmental strains of Aspergillus flavus

Aspergillus flavus is the second most common cause of aspergillosis infection in immunocompromised patients and is responsible for the production of aflatoxins. Little is known about the population structure of A. flavus, although recent molecular and phenotypic data seem to demonstrate that different genetic lineages exist within this species. The aim of this study was to carry out a morphological, physiological, and molecular analysis of a set of clinical and environmental isolates to determine whether this variability is due to species divergence or intraspecific diversity, and to assess whether the clinical isolates form a separate group. The amdS and omtA genes were more phylogenetically informative than the other tested genes and their combined analysis inferred three main clades, with no clear distinction between clinical and environmental isolates. No important morphological and physiological differences were found between the members of the different clades, with the exception of the assimilation of d-glucosamine, which differentiates the members of the clade II from the others.     

On the systematics of some marine haploporids (Trematoda) with the description of a new species of Megasolena Linton, 1910

Megasolena mikra sp. nov. is described from the queen angelfish, Holacanthus ciliaris (Linnaeus), off Florida, USA. The new species can be differentiated from all other species of Megasolena Linton, 1910 except Megasolena littoralis Muñoz, George-Nascimento, and Bray, 2017 in possessing testes that are smaller in diameter than the ovary. The new species can be differentiated from M. littoralis in lacking tegumental spines and possessing oral sucker papillae. Molecular data are provided for two species each of Cadenatella Dollfus, 1946, Hapladena Linton, 1910, and Megasolena Linton, 1910. Bayesian inference analysis of concatenated internal transcribed spacer region-2 (ITS2) and partial 28S rDNA sequences of 50 haploporoids revealed 1) a monophyletic Atractotrematidae Yamaguti, 1939 sister to the rest of the haploporoids tested; 2) a paraphyletic Megasoleninae Manter, 1935 – if Hapladena is included; and 3) a monophyletic Cadenatellinae Gibson and Bray, 1982 sister to the ‘mugilid’ haploporids. The ‘mugilid’ haploporids formed a monophyletic clade consisting of the subfamilies Chalcinotrematinae Overstreet and Curran, 2005, Forticulcitinae Blasco-Costa, Balbuena, Kostadinova, and Olson, 2009, Haploporinae Nicoll, 1914, and Waretrematinae Srivastava, 1937. Based on our analysis we restrict the Megasoleninae to include Megasolena, Vitellibaculum Montgomery, 1957, and Metamegasolena Yamaguti, 1970, all of which have species with two testes. To accommodate the former megasolenine taxa with a single testis, we erect the Hapladeninae subf. nov. for species in Hapladena and tentatively, Myodera Montgomery, 1957. Our results further support that haploporoids had a common marine ancestor with two testes, and that members of the Haploporoidea Nicoll, 1914 underwent diversification following a shift from a primarily marine life history with eupercarian hosts to a more euryhaline one with diadromous hosts (namely mullet).     

我国南北大豆产区慢生大豆根瘤菌的遗传多样性和系统发育研究

利用16S rRNA基因RFLP、16S rRNA基因序列分析以及16S-23S rRNA IGS PCR RFLP技术对分离自我国南北大豆产区的慢生大豆根瘤菌进行了群体遗传多样性和系统发育研究。16S rRNA基因PCR RFLP分析以及16S rRNA基因序列分析结果表明:所有供试慢生大豆根瘤菌可分为B.japonicum和B.elkanii两个类群,其中属于B.japonicum的为优势种群,占供试菌株的91%,属于B.elkanii的仅占9%,多样性水平较低。16S-23S rRNA IGS PCRRFLP研究结果表明:属于B.japonicum的慢生根瘤菌具有较丰富的遗传多样性,在69%的相似性水平上可分为群Ⅰ和群Ⅱ两大类群。群I的菌株以分离自黑龙江和河北等北部区域的菌株为代表,群Ⅱ的菌株以分离自广西和江苏等南部地域的菌株为代表,反映出明显的地域特征。两群菌株在系统发育上均与USDA6、USDA110和USDA122等B.japonicum的模式或代表菌株有差异。     

Morphological and molecular characterization within 26 strains of the genus Cylindrospermum (Nostocaceae,Cyanobacteria), with descriptions of three new species

Twenty‐six strains morphologically identified as Cylindrospermum as well as the closely related taxon Cronbergia siamensis were examined microscopically as well as phylogenetically using sequence data for the 16S rRNA gene and the 16S‐23S internal transcribed spacer (ITS) region. Phylogenetic analysis of the 16S rRNA revealed three distinct clades. The clade we designate as Cylindrospermum sensu stricto contained all five of the foundational species, C. maius, C. stagnale, C. licheniforme, C. muscicola, and C. catenatum. In addition to these taxa, three species new to science in this clade were described: C. badium, C. moravicum, and C. pellucidum. Our evidence indicated that Cronbergia is a later synonym of Cylindrospermum. The phylogenetic position of Cylindrospermum within the Nostocaceae was not clearly resolved in our analyses. Cylindrospermum is unusual among cyanobacterial genera in that the morphological diversity appears to be more evident than sequence divergence. Taxa were clearly separable using morphology, but had very high percent similarity among ribosomal sequences. Given the high diversity we noted in this study, we conclude that there is likely much more diversity remaining to be described in this genus.     

Genomic and phenotypic heterogeneity of Acidithiobacillus spp. strains isolated from diverse habitats in China

The genetic variability among 32 Chinese Acidithiobacillus spp. environmental isolates and four reference strains representing three recognized species of the genus Acidithiobacillus was characterized by using a combination of molecular methods, namely restriction fragment length polymorphisms of PCR-amplified 16S rRNA genes and 16S-23S rRNA gene intergenic spacers, repetitive element PCR, arbitrarily primed PCR and 16S rRNA gene sequence analyses. 16S rRNA gene sequences revealed that all Acidithiobacillus spp. strains could be assigned to seven groups, three of which encompassed the Acidithiobacillus ferrooxidans strains from various parts of the world. A comparative analysis of the phylogenetic Group 1 and 2 was undertaken. Restriction fragment length polymorphism results allowed us to separate the 35 Acidithiobacillus strains into 15 different genotypes. An integrated phenotypic and genotypic analysis indicated that the distribution of A. ferrooxidans strains among the physiological groups were in agreement with their distribution among the genomic groups, and that no clear correlation was found between the genetic polymorphism of the Acidithiobacillus spp. strains and either the geographic location or type of habitats from which the strains were isolated. In addition, five unidentified sulfur-oxidizing isolates may represent one or two novel species of the genus Acidithiobacillus. The results showed that the Chinese Acidithiobacillus spp. isolates exhibited a high degree of genomic and phenotypic heterogeneity.     

Application of targeted metagenomics to explore abundance and diversity of CO2-fixing bacterial community using cbbL gene from the rhizosphere of Arachis hypogaea

Sequestration of CO₂ by autotrophic bacteria is a key process of biogeochemical carbon cycling in soil ecosystem. Rhizosphere is a rich niche of microbial activity and diversity, influenced by change in atmospheric CO₂. Structural changes in rhizosphere composition influence microbial communities and the nutrient cycling. In the present study, the bacterial diversity and population dynamics were established using cbbL and 16S rRNA gene targeted metagenomics approach from the rhizosphere of Arachis hypogaea. A total of 108 cbbL clones were obtained from the rhizospheric soil which revealed predominance of cbbL sequences affiliated to Rhizobium leguminosarum, Bradyrhizobium sp., Sinorhizobium meliloti, Ochrobactrum anthropi and a variety of uncultured cbbL harboring bacteria. The 16S rRNA gene clone library exhibited the dominance of Firmicutes (34.4%), Proteobacteria (18.3%), Actinobacteria (17.2%) and Bacteroidetes (16.1%). About 43% nucleotide sequences of 16S rRNA gene clone library were novel genera which showed < 95% homology with published sequences. Gene copy number of cbbL and 16S rRNA genes, determined by quantitative real‐time PCR (qRT PCR), was 9.38 ± 0.75 × 10⁷ and 5.43 ± 0.79 × 10⁸ (per g dry soil), respectively. The results exhibited bacterial community structure with high bacterial diversity and abundance of CO₂‐fixing bacteria, which can be explored further for their role in carbon cycling, sustainable agriculture and environment management.     

The rate and pattern of cladogenesis in microbes

Theories of macroevolution rarely have been extended to include microbes; however, because microbes represent the most ancient and diverse assemblage of organismal diversity, such oversight limits our understanding of evolutionary history. Our analysis of phylogenetic trees for microbes suggests that macroevolution may differ between prokaryotes and both micro- and macroeukaryotes (mainly plants and animals). Phylogenetic trees inferred for prokaryotes and some microbial eukaryotes conformed to expectations assuming a constant rate of cladogenesis over time and among lineages: nevertheless, microbial eukaryote trees exhibited more variation in rates of cladogenesis than prokaryote trees. We hypothesize that the contrast of macroevolutionary dynamics between prokaryotes and many eukaryotes is due, at least in part, to differences in the prevalence of lateral gene transfer (LGT) between the two groups. Inheritance is predominantly, if not wholly, vertical within eukaryotes, a feature that allows for the emergence and maintenance of heritable variation among lineages. By contrast, frequent LGT in prokaryotes may ameliorate heritable variation in rate of cladogenesis resulting from the emergence of key innovations; thus, the inferred difference in macroevolution might reflect exclusivity of key innovations in eukaryotes and their promiscuous nature in prokaryotes.     

Evaluation of four molecular typing methodologies as tools for determining taxonomy relations and for identifying species among Yersinia isolates

In the last few decades, molecular typing has become an important tool in taxonomic, phylogenetic and identification studies of numerous groups of bacteria, including the yersiniae. In this study, Enterobacterial Repetitive Intergenic Consensus PCR (ERIC-PCR), Pulsed-Field Gel Electrophoresis (PFGE), 16S rRNA gene sequencing and Multilocus Sequence Analysis (MLSA) were performed to determine the ability of these techniques to be used in taxonomy and identification of Yersinia strains. A total of 60 Yersinia strains were genotyped by ERIC-PCR and PFGE. Moreover, an in silico analysis was carried out for 16S rRNA gene sequencing and MLSA, using 68 and 49 Yersinia strains, respectively. A phylogenetic tree constructed from the ERIC-PCR, 16S rRNA gene sequencing and MLSA data grouped most of the Yersinia species into distinct species-specific clusters. In the PFGE assay these clusters were not observed. On this basis, ERIC-PCR, 16S rRNA gene sequencing and MLSA seem to be valuable techniques for use in taxonomic and identification studies of the genus Yersinia, whereas PFGE does not. Furthermore, ERIC-PCR has the advantage of being a cheaper, easier and faster assay than 16S rRNA gene sequencing or MLSA, and for these reasons can be considerate an alternative tool in taxonomic studies of yersiniae.     

Phylogenetic study of Geitlerinema and Microcystis (Cyanobacteria) using PC‐IGS and 16S–23S ITS as markers: investigation of horizontal gene transfer

Selection of genes that have not been horizontally transferred for prokaryote phylogenetic inferences is regarded as a challenging task. The markers internal transcribed spacer of ribosomal genes (16S–23S ITS) and phycocyanin intergenic spacer (PC‐IGS), based on the operons of ribosomal and phycocyanin genes respectively, are among the most used markers in cyanobacteria. The region of the ribosomal genes has been considered stable, whereas the phycocyanin operon may have undergone horizontal transfer. To investigate the occurrence of horizontal transfer of PC‐IGS, phylogenetic trees of Geitlerinema and Microcystis strains were generated using PC‐IGS and 16S–23S ITS and compared. Phylogenetic trees based on the two markers were mostly congruent for Geitlerinema and Microcystis, indicating a common evolutionary history among ribosomal and phycocyanin genes with no evidence for horizontal transfer of PC‐IGS. Thus, PC‐IGS is a suitable marker, along with 16S–23S ITS for phylogenetic studies of cyanobacteria.     

Presence of a novel 16S-23S rRNA gene intergenic spacer insert in Bradyrhizobium canariense strains

Seven slow-growing bacterial strains isolated from root nodules of yellow serradella (Ornithopus compressus) that originated from Asinara Island on North Western Sardinia in Italy were characterized by partial 16S rRNA gene and intergenic spacer (ITS) sequencing as well as amplified fragment length polymorphism (AFLP) genomic fingerprinting. The results indicated that the O. compressus isolates belong to the Bradyrhizobium canariense species. The analysis of ITS sequences divided the branch of B. canariense strains into two statistically separated groups (ITS clusters I and II). All the strains in ITS cluster I showed the presence of unique oligonucleotide insert TTAGAGACTTAGGTTTCTK. This insert was neither found in other described species of the family Rhizobiaceae nor in any other bacterial families and can be used as a natural and high selective genetic marker for ITS cluster I of B. canariense strains. ITS grouping of O. compressus isolates was supported by the unweighted pair group method with arithmetic averages cluster analysis of their AFLP patterns, suggesting that the strains of ITS cluster II were genetically closer to each other than to isolates from the ITS cluster I. A taxonomic importance is supposed of the revealed 19 bp ITS insert for an intraspecific division within high heterogeneous B. canariense species.     

Coral reef invertebrate microbiomes correlate with the presence of photosymbionts

Coral reefs provide habitat for an array of marine invertebrates that host symbiotic microbiomes. Photosynthetic symbionts including Symbiodinium dinoflagellates and diatoms potentially influence the diversity of their host-associated microbiomes by releasing carbon-containing photosynthates and other organic compounds that fuel microbial metabolism. Here we used 16S ribosomal RNA (rRNA) gene amplicon pyrosequencing to characterise the microbiomes of 11 common Great Barrier Reef marine invertebrate species that host photosynthetic symbionts and five taxa in which they are absent. The presence of photosynthetic symbionts influenced the composition but not the species richness, evenness and phylogenetic diversity of invertebrate-associated microbiomes. Invertebrates without photosynthetic symbionts were dominated by Alphaproteobacteria, whereas those hosting photosynthetic symbionts were dominated by Gammaproteobacteria. Interestingly, many microbial species from photosymbiont-bearing invertebrates, including Oceanospirillales spp., Alteromonas spp., Pseudomonas spp., Halomonas spp., are implicated in the metabolism of dimethylsulfoniopropionate (DMSP). DMSP is produced in high concentrations by photosynthetic dinoflagellates and is involved in climate regulation by facilitating cloud formation. Microbiomes correlated with host taxa and replicate individuals from most sampled species grouped in distance-based redundancy analysis of retrieved 16S rRNA gene sequences. This study highlights the complex nature of invertebrate holobionts and confirms the importance of photosynthetic symbionts in structuring marine invertebrate bacterial communities.     

Molecular phylogeny and biogeography of the narrow endemic Coelonema and affinitive Draba (Brassicaceae) based on two DNA regions

To clarify the relationship between two genera, Draba and the narrow genus Coelonema, endemic to the QilianMountains of the northeastern Qinghai-Tibet Plateau, phylogenetic analyses were conducted using nuclear ribosomal DNA ITS, and the chloroplast DNA trnL, from Coelonema draboides and 30 species of Draba representing eight sections, including 25 species of Chinese Draba, seven of which were endemic to the study region. The results unambiguously support several previously published proposals to unite Coelonema with Draba and accommodate C. draboides in the latter genus on the basis of morphological re-examination. Our molecular data presented here also provide evidence that these two genera should be combined as a monophyletic group with high support. In addition, it is estimated that Draba may have originated about 1.36–2.71 Mya, with C. draboides diverging from Draba about 0.15–0.31 Mya, based on the molecular calibration of ITS datasets. The assumed speciation and rapid expansion of these two genera is likely to have occurred in the eastern edge of the QilianMountains area according to molecular phylogeny and estimated divergence times, which correspond well with the known geological and paleobotanical histories of the Qinghai-Tibet Plateau.     

Raphidiopsis mediterranea Skuja represents non-heterocytous life-cycle stages of Cylindrospermopsis raciborskii (Woloszynska) Seenayya et Subba Raju in Lake Kastoria (Greece), its type locality: Evidence by morphological and phylogenetic analysis

The taxonomical relationship of Cylindrospermopsis raciborskii and Raphidiopsis mediterranea was studied by morphological and 16S rRNA gene diversity analyses of natural populations from Lake Kastoria, Greece. Samples were obtained during a bloom (23,830 trichomes mL⁻¹) in August 2003. A high diversity of apical cell, trichome, heterocyte and akinete morphology, trichome fragmentation and reproduction was observed. Trichomes were grouped into three dominant morphotypes: the typical and the non-heterocytous morphotype of C. raciborskii and the typical morphotype of R. mediterranea. A morphometric comparison of the dominant morphotypes showed significant differences in mean values of cell and trichome sizes despite the high overlap in the range of the respective size values. Additionally, two new morphotypes representing developmental stages of the species are described while a new mode of reproduction involving a structurally distinct reproductive cell is described for the first time in planktic Nostocales. A putative life-cycle, common for C. raciborskii and R. mediterranea is proposed revealing that trichome reproduction of R. mediterranea gives rise both to R. mediterranea and C. raciborskii non-heterocytous morphotypes. The phylogenetic analysis of partial 16S rRNA gene (ca. 920 bp) of the co-existing Cylindrospermopsis and Raphidiopsis morphotypes revealed only one phylotype which showed 99.54% similarity to R. mediterranea HB2 (China) and 99.19% similarity to C. raciborskii form 1 (Australia). We propose that all morphotypes comprised stages of the life cycle of C. raciborkii whereas R. mediterranea from Lake Kastoria (its type locality) represents non-heterocytous stages of Cylindrospermopsis complex life cycle.     

转mapk双链RNA干扰表达载体黄瓜对根际土壤细菌多样性的影响

随着RNA干扰技术的发展,通过植物表达病原物特异的dsRNA来防治植物病害的转基因作物越来越多.转根结线虫mapk双链RNA表达载体的黄瓜能够通过RNA干扰作用沉默线虫的mapk基因,对根结线虫具有良好的防治效果.为了评价该转基因黄瓜的安全性,明确mapk双链RNA干扰表达载体转基因黄瓜植株对根际土壤细菌多样性的影响,采用16S rRNA基因克隆文库方法对非转基因黄瓜和转基因黄瓜土壤细菌群落多样性进行分析.结果表明,非转基因黄瓜土壤细菌文库包含124个OTU(可操作分类单元),转基因黄瓜土壤细菌文库包含122个OTU.2个文库共同拥有的OTU为115个.2个文库都包含1 3个类群细菌,Acidobacteria、Actinobacteria、Armatimonadetes、Bacteroidetes、candidate division BRC1、Chloroflexi、Firmicutes、Gemmatimonadetes、Nitrospira、Planctomycetes、Proteobacteria、Verrucomicrobia、unclassified_Bacteria. 其中 Proteobacteria、Bacteroidetes 、Chloroflexi和Acidobacteria是优势菌群.其他细菌类群数量相对较少.在纲分类水平上,两个文库包含的细菌类群一致,且各类群细菌比例差异不大.在Acidobacteria门中,Acidobacteria_Gp6为优势菌群.在Bacteroidetes门中,Sphingobacteria纲细菌数量最多.在Chloroflexi门中,unclassified Chloroflexi细菌最多.在Proteobacteria门中,其中Betaproteobacteria纲的细菌数量最多.从多样性指数角度分析,两种土壤细菌群落的Shannon、Simpson和Chao值差异不大.总体来看,两种土壤细菌类群差异不显著,转基因黄瓜未对根际土壤细菌群落产生明显影响.     

Genotypic and phenotypic variation among Lysobacter capsici strains isolated from Rhizoctonia suppressive soils

Four Gram-negative bacterial strains, recovered from clay soils cultivated with different crops in the Netherland, were subjected to a polyphasic taxonomic study in order to clarify their taxonomic status. Comparative analysis of the 16S rRNA gene sequences revealed that they belong to the genus Lysobacter and to be highly related to the type strains of L. antibioticus DSM 2044^T, L. gummosus DSM 6980^T, and L. capsici DSM 19286^T, displaying 99.1–99.3%, 99.2–99.6% and 99.4–100% sequence similarities, respectively, to these species. The results of DNA–DNA hybridization studies unambigiously indicated that the four strains belonged to the species L. capsici. Nevertheless, DNA fingerprinting and phenotypic characterization indicated that there was a considerable diversification and niche differentiation among the strains belonging to L. capsici. The newly identified L. capsici strains strongly inhibit Rhizoctonia solani AG2 and originate from Rhizoctonia-suppressive soils where also populations of L. antibioticus and L. gummosus were present. This is the first report of the presence of combined populations of closely related Lysobacter spp. within agricultural soils.     

A bridge too far in naming species: a total evidence approach does not support recognition of four species in Desertifilum (Cyanobacteria)

A population of Desertifilum (Cyanobacteria, Oscillatoriales) from an oligotrophic desertic biotope was isolated and characterized using a polyphasic approach including molecular, morphological, and ecological information. The population was initially assumed to be a new species based on ecological and biogeographic separation from other existing species, however, phylogenetic analyses based on sequences of the 16S rRNA gene and 16S–23S ITS region, placed this strain clearly within the type species, Desertifilum tharense. Comparative analysis of morphology, 16S rRNA gene similarity, 16S–23S ITS secondary structure, and percent dissimilarity of the ITS regions for all characterized strains supports placing the six Desertifilum strains (designated as PD2001/TDC17, UAM‐C/S02, CHAB7200, NapGTcm17, IPPAS B‐1220, and PMC 872.14) into D. tharense. The recognition of Desertifilum salkalinema and Desertifilum dzianense is not supported, although our analysis does support continued recognition of Desertifilum fontinale. Pragmatic criteria for recognition of closely related species are proposed based on this study and others, and more rigorous review of future taxonomic papers is recommended.