Different Decaying Wood Effects on Bacterial Diversity: Insights from Molecular Methods

Decaying wood is a novel key factor required for biodiversity and function of a forest, as it provides a good account of substrate and habitats for various organisms. Herein, the bacterial diversity in decaying wood of Betula platyphylla was discussed through high throughput sequencing. Our results showed that most of the obtained sequences belonged to the phyla Firmicutes, Proteobacteria, Bacteroidetes, Actinobacteria, Acidobacteria and Verrucomicrobia. Bacterial community compositions in samples with higher moisture content were obviously different than that with lower content, which could be reflected by richness estimators, diversity indices, and cluster and heatmap analysis. All three networks were non-random and possessed topological features of complex systems such as small-world and modularity features. However, these networks exhibited distinct topological features, indicating the potential ability of extensive cooperative and competitive interactions in the decayed wood microenvironments. Redundant analysis showed that most bacterial phyla were mainly distributed in higher-moisture trunks. The obtained data will increase the knowledge of the complex bacterial diversity associated with dead wood, and lay a foundation for the bioconversion technology of plant cell walls using bacteria.     

Molecular Diversity among Communities of Freshwater Microchlorophytes

Current hypotheses on the distribution of freshwater microchlorophytes lead to predictions of low diversity and wide environmental tolerances. Thus, the same few species should be found worldwide in many different habitats. However, these hypotheses are based on a morphospecies concept, which precludes the possibility of numerous cryptic species among these organisms. In this study, we examined the diversity of coccoid green microalgae and chlamydomonads (Chlorophyta) isolated from sites in Minnesota and North Dakota (USA) using techniques of 18S rDNA sequence analysis. Of 93 distinct 18S rDNA sequences identified from among 273 isolates examined by molecular techniques, all but four are new to science. The spatial distribution of organisms represented by these 18S rDNA sequences was not uniform, because some lakes and ponds yielded distinct 18S rDNA types not found at other sites. In addition, organisms generally considered to be cosmopolitan, such as Chlamydomonas reinhardtii and Chlorella vulgaris, were not found. These results challenge predictions of low species number and wide environmental tolerances among these eukaryotic microorganisms.     

微生物多样性的研究方法概况

介绍了进行微生物多样性研究多种方法,将其简要划分为三大部分：1．经典纯培养技术的改进方法;2．现代分子生物学技术;3．上述两种方法的联合使用;并重点阐述了这些方法的优缺点,展望了微生物多样性研究方法的发展前景。     

Diversity of Fungi, Bacteria, and Actinomycetes on Leaves Decomposing in a Stream

Although fungi, bacteria, and specific bacterial taxa, such as the actinomycetes, have been studied extensively in various habitats, few studies have examined them simultaneously, especially on decomposing leaves in streams. In this study, sugar maple and white oak leaves were incubated in a stream in northeastern Ohio for 181 days during which samples were collected at regular intervals. Following DNA extraction, PCR-denaturing gradient gel electrophoresis (DGGE) was performed using fungus-, bacterium-, and actinomycete-specific primers. In addition, fungal and bacterial biomass was estimated. Fungal biomass differed on different days but not between leaves of the two species and was always greater than bacterial biomass. There were significant differences in bacterial biomass through time and between leaf types on some days. Generally, on the basis of DGGE, few differences in community structure were found for different leaf types. However, the ribotype richness of fungi was significantly greater than those of the bacteria and actinomycetes, which were similar to each other. Ribotype richness decreased toward the end of the study for each group except bacteria. Lack of differences between the two leaf types suggests that the microorganisms colonizing the leaf biofilm were primarily generalists that could exploit the resources of the leaves of either species equally well. Thus, we conclude that factors, such as the ecological role of the taxa (generalists versus specialists), stage of decay, and time of exposure, appeared to be more important determinants of microbial community structure than leaf quality.     

Cultivation-Dependent and -Independent Approaches for Determining Bacterial Diversity in Heavy-Metal-Contaminated Soil

In recent years, culture-independent methods have been used in preference to traditional isolation techniques for microbial community analysis. However, it is questionable whether uncultured organisms from a given sample are important for determining the impact of anthropogenic stress on indigenous communities. To investigate this, soil samples were taken from a site with patchy metal contamination, and the bacterial community structure was assessed with a variety of approaches. There were small differences in microscopic epifluorescence bacterial counts. Denaturing gradient gel electrophoresis (DGGE) profiles of 16S rRNA gene fragments (16S-DGGE) amplified directly from soil samples were highly similar. A clone library generated from the most contaminated sample revealed a diverse bacterial community, which showed similarities to pristine soil communities from other studies. However, the proportion of bacteria from the soil samples that were culturable on standard plate-counting media varied between 0.08 and 2.2%, and these values correlated negatively with metal concentrations. The culturable communities from each sample were compared by 16S-DGGE of plate washes and by fatty acid profiling of individual isolates. Each approach indicated that there were considerable differences between the compositions of the culturable communities from each sample. DGGE bands from both culture-based and culture-independent approaches were sequenced and compared. These data indicated that metal contamination did not have a significant effect on the total genetic diversity present but affected physiological status, so that the number of bacteria capable of responding to laboratory culture and their taxonomic distribution were altered. Thus, it appears that plate counts may be a more appropriate method for determining the effect of heavy metals on soil bacteria than culture-independent approaches.     

Prospecting Environmental Mycobacteria: Combined Molecular Approaches Reveal Unprecedented Diversity

Background

Environmental mycobacteria (EM) include species commonly found in various terrestrial and aquatic environments, encompassing animal and human pathogens in addition to saprophytes. Approximately 150 EM species can be separated into fast and slow growers based on sequence and copy number differences of their 16S rRNA genes. Cultivation methods are not appropriate for diversity studies; few studies have investigated EM diversity in soil despite their importance as potential reservoirs of pathogens and their hypothesized role in masking or blocking M. bovis BCG vaccine.

Methods

We report here the development, optimization and validation of molecular assays targeting the 16S rRNA gene to assess diversity and prevalence of fast and slow growing EM in representative soils from semi tropical and temperate areas. New primer sets were designed also to target uniquely slow growing mycobacteria and used with PCR-DGGE, tag-encoded Titanium amplicon pyrosequencing and quantitative PCR.

Results

PCR-DGGE and pyrosequencing provided a consensus of EM diversity; for example, a high abundance of pyrosequencing reads and DGGE bands corresponded to M. moriokaense, M. colombiense and M. riyadhense. As expected pyrosequencing provided more comprehensive information; additional prevalent species included M. chlorophenolicum, M. neglectum, M. gordonae, M. aemonae. Prevalence of the total Mycobacterium genus in the soil samples ranged from 2.3×10⁷ to 2.7×10⁸ gene targets g⁻¹; slow growers prevalence from 2.9×10⁵ to 1.2×10⁷ cells g⁻¹.

Conclusions

This combined molecular approach enabled an unprecedented qualitative and quantitative assessment of EM across soil samples. Good concordance was found between methods and the bioinformatics analysis was validated by random resampling. Sequences from most pathogenic groups associated with slow growth were identified in extenso in all soils tested with a specific assay, allowing to unmask them from the Mycobacterium whole genus, in which, as minority members, they would have remained undetected.     

Comparison of Two Direct-Count Methods for Determining Metabolizing Bacteria in Freshwater

Planktonic bacteria collected from several freshwater environments and cultured bacteria were used to compare two methods for determining the numbers of metabolizing bacteria. The methods used were (i) reduction of 2-(ρ-iodophenyl)-3-(ρ-nitrophenyl)-5-phenyl tetrazolium chloride 2-(ρ-iodophenyl)-3-(ρ-nitrophenyl)-5-phenyl to tetrazolium chloride-formazan and (ii) elongation of cells by using yeast extract and nalidixic acid. No statistically significant difference was found between methods in determining metabolizing bacteria, although significant differences (P < 0.05) were found when comparing numbers of total bacteria. A combination of the two methods yielded significant changes, both positive and negative, in the numbers of metabolizing bacteria.     

Restoring Wetland Plant Diversity: A Comparison of Existing and Adaptive Approaches

Most restoration projects are not designed or assessed in ways that identify cause–effect relationships.When plants die, even detailed postmortem examinations cannot pinpoint causes; e.g., mortalities of 7% vs. 90% in two salt marsh transplantation projects were attributable to differences in hypersalinity and sedimentation, but other effects could not be ruled out. Adaptive restoration (the experimental testing of alternative approaches in restoration sites), however, can clarify cause–effect relationships, while simultaneously restoring plant diversity and informing future restoration efforts. Projects in Tijuana Estuary (California) and Greene Prairie (Wisconsin) demonstrate the approach: (1) A large field experiment at Tijuana Estuary showed that species-rich plantings of halophytes accelerated the development of ecosystem structure and function (over single-species plantings) while simultaneously vegetating an intertidal plain. The six-species assemblages produced more complex canopies and accumulated more biomass and nitrogen than singlespecies and unplanted plots. (2) Also at Tijuana Estuary, an experiment is testing the ability of tidal creek networks to accelerate revegetation and increase food-web support (via increased growth of plants, invertebrates, and fish) in an 8-ha project that simultaneously restored tidal flushing. (3) In Greene Prairie, the ability to establish 33 native species is being tested as replacements for an invasive grass (Phalaris arundinacea). In each case, the adaptive approach informs both the science and practice of restoration. Without experimentation, restorationists are hard-pressed to explain past mortality and to suggest better methods for restoring structure and function. Adaptive restoration can provide the knowledge required, especially when large projects are implemented as sequential modules with experiments that sequentially provide essential information.     

Succession of Fungi on Decaying Setaria glauca Beauv.: A Qualitative Analysis of the Mycoflora

The mycoflora recorded on the decaying shoots of Setaria glauca,from their early senescence onwards, has been analysed qualitatively.On the basis of time of appearance and sporing and durationperiods of fungi, three patterns of colonization have been recognized.The group I members, designated as primary colonists appearon shoots just after their senescence during August. This groupincludes six dematiaceous hyphomycetes and two sphaeropsidales.These are followed by group II members during the late rainyseason, and group III members that appear only for a short periodduring winter. The dominant forms of group II are, 15 dematiaceousand 3 tuberculariaceous hyphomycetes, 2 sphaeropsidales, and1 ascomycete, whereas those representing group III are, 27 dematiaceous,2 moniliaceous and I tuberculariaceous hyphomycetes, 5 sphaeropsidales,1 melanconiales, and 3 mycelia sterilia. These studies also showed that about 94 per cent of the totalmycoflora is constituted by the deuteromycetes of which 12 aresphaeropsidales, 1 melanconiales, 78 moniliales, and 3 myceliasterilia. The moniliales are represented by 3 species of moniliaceae,69 of dematiaceae, and 6 of tuberculariaceae. The phycomycetestage was absent, the three mucorales being discovered onlyby inoculation at an advanced stage of decay. The deuteromycetes,particularly dematiaceous hyphomycetes, have been found to playthe most important role in this phase of decomposition.     

Effects of Resources and Trophic Interactions on Freshwater Bacterioplankton Diversity

Abstract In a study of bacterioplankton in an oligotrophic lake in northern Wisconsin, a community fingerprinting technique, automated ribosomal intergenic spacer analysis (ARISA), was used to determine the effect of resources and trophic interactions on bacterioplankton diversity. Inorganic nitrogen and phosphorus (NP), carbon in the form of glucose (G) or dissolved organic matter extracted from peat (DOM), and carbon and NP in combination were added to two types of experimental systems. Ten-liter mesocosms contained all components of the original aquatic community except for large zooplankton. One-liter dilution cultures were prepared so that the effects of grazers and phytoplankton were removed. During a 3-day incubation, bacterial production showed the greatest response to the carbon plus NP treatment in both experimental systems, but bacterial diversity was strikingly different between them. In the mesocosms, the number of ARISA-PCR fragments averaged 41 per profile, whereas the dilution culture communities were highly reduced in complexity, dominated in most cases by a single PCR fragment. Further analysis of the mesocosm data suggested that whereas the NPDOM addition caused the greatest aggregate bacterial growth response, the addition of NP alone caused the largest shifts in community composition. These results suggest that the measurement of aggregate responses, such as bacterial production, alone in studies of freshwater bacterial communities may mask the effects of resources on bacterioplankton. Received: 24 January 2000; Accepted: 10 May 2000; Online Publication: 28 August 2000     

Comparing Morphological and Molecular Estimates of Species Diversity in the Freshwater Genus Synura (Stramenopiles): A Model for Understanding Diversity of Eukaryotic Microorganisms

We performed a comparison of molecular and morphological diversity in a freshwater colonial genus Synura (Chrysophyceae, Stramenopiles), using the island of Newfoundland (Canada) as a case study. We examined the morphological species diversity in collections from 79 localities, and compared these findings to diversity based on molecular characters for 150 strains isolated from the same sites. Of 27 species or species-level lineages identified, only one third was recorded by both molecular and morphological techniques, showing both approaches are complementary in estimating species diversity within this genus. Eight taxa, each representing young evolutionary lineages, were recovered only by sequencing of isolated colonies, whereas ten species were recovered only microscopically. Our complex investigation, involving both morphological and molecular examinations, indicates that our knowledge of Synura diversity is still poor, limited only to a few well-studied areas. We revealed considerable cryptic diversity within the core S. petersenii and S. leptorrhabda lineages. We further resolved the phylogenetic position of two previously described taxa, S. kristiansenii and S. petersenii f. praefracta, propose species-level status for S. petersenii f. praefracta, and describe three new species, S. vinlandica, S. fluviatilis, and S. cornuta. Our findings add to the growing body of literature detailing distribution patterns observed in the genus, ranging from cosmopolitan species, to highly restricted taxa, to species such as S. hibernica found along coastal regions on multiple continents. Finally, our study illustrates the usefulness of combining detailed morphological information with gene sequence data to examine species diversity within chrysophyte algae.     

Diversity of Aerobic Bacilli Analysis Using Molecular and Culture-Based Approaches in Debagh Hot Spring

This study reports the first attempt to describe aerobic bacilli communities in Debagh hot spring, from which 41 aerobic, thermophile, and halotolerant bacilli were isolated and selected based on morphological, physiological, and biochemical tests. 16S rDNA sequence analysis revealed that the recovered isolates belonged to four bacterial genera dominated by the genus Bacillus represented with species B. mojavensis (16), B. licheniformis (11), B. subtilis (2), B. atrophaeus (1), B.amyloliquifaciens (1), and B .pimulus (1). The genus Aeribacillus represented by the species A. pallidus (3), the genus Geobacillus represented by the species G. toebii (2), and the genus Hydrogenophilus represented by the species H. hirschii (4). While, MALDI-TOF analysis determined that isolates belonged to the genus Bacillus that contained B. licheniformis (12), B. mojavensis (6), B. subtilis (2), B. atrophaeus (1), and B. pumilus (1). Furthermore, the isolates exhibited high hydrolytic activity to casein, lecithin, tween 80, olive oil, and starch with 53.65%, 83.33%, 70.73%, 92.68%, and 56.09%, respectively. Among these isolates, 26.82% were able to hydrolyze all the substrates tested.     

Initial Colonization, Nutrient Supply, and Fungal Activity on Leaves Decaying in Streams

Aquatic hyphomycetes dominate leaf decomposition in streams, and their biomass is an important component in the diet of leaf-eating invertebrates. After 2 weeks of exposure in a first-order stream, maple leaf disks had low levels of fungal biomass and species diversity. Spore production by aquatic hyphomycetes also was low. Subsets of these disks were left in the stream for another 3 weeks or incubated in defined mineral solutions with one of three levels of nitrate and phosphate. Stream disks lost mass, increased ergosterol levels and spore production, and were colonized by additional fungal species. External N and P significantly stimulated mass loss, ergosterol accumulation, and spore production of laboratory disks. On disks incubated without added N and P, ergosterol levels declined while conidium production continued, suggesting conversion of existing hyphal biomass to propagules. In all other treatments, approximately equal amounts of newly synthesized biomass were invested in hyphae and conidia. Net yield (fungal biomass per leaf mass lost) varied between 1% (in the laboratory, without added N or P) and 31% (decay in stream). In most treatments, the three aquatic hyphomycete species that dominated spore production during the first 2 weeks in the stream also produced the largest numbers of conidia in the following 3 weeks. Principal-component analysis suggested two divergent trends from the initial fungal community established after 2 weeks in the stream. One culminated in the community of the second phase of stream exposure, and the other culminated in the laboratory treatment with the highest levels of N and P. The results suggest that fungal production in streams, and, by extension, production of invertebrates and higher tropic levels, is stimulated by inorganic N and P.     

The Diversity and Distribution of Fungi on Residential Surfaces

The predominant hypothesis regarding the composition of microbial assemblages in indoor environments is that fungal assemblages are structured by outdoor air with a moderate contribution by surface growth, whereas indoor bacterial assemblages represent a mixture of bacteria entered from outdoor air, shed by building inhabitants, and grown on surfaces. To test the fungal aspect of this hypothesis, we sampled fungi from three surface types likely to support growth and therefore possible contributors of fungi to indoor air: drains in kitchens and bathrooms, sills beneath condensation-prone windows, and skin of human inhabitants. Sampling was done in replicated units of a university-housing complex without reported mold problems, and sequences were analyzed using both QIIME and the new UPARSE approach to OTU-binning, to the same result. Surfaces demonstrated a mycological profile similar to that of outdoor air from the same locality, and assemblages clustered by surface type. “Weedy” genera typical of indoor air, such as Cladosporium and Cryptococcus, were abundant on sills, as were a diverse set of fungi of likely outdoor origin. Drains supported more depauperate assemblages than the other surfaces and contained thermotolerant genera such as Exophiala, Candida, and Fusarium. Most surprising was the composition detected on residents’ foreheads. In addition to harboring Malassezia, a known human commensal, skin also possessed a surprising richness of non-resident fungi, including plant pathogens such as ergot (Claviceps purperea). Overall, fungal richness across indoor surfaces was high, but based on known autecologies, most of these fungi were unlikely to be growing on surfaces. We conclude that while some endogenous fungal growth on typical household surfaces does occur, particularly on drains and skin, all residential surfaces appear – to varying degrees – to be passive collectors of airborne fungi of putative outdoor origin, a view of the origins of the indoor microbiome quite different from bacteria.     

利用培养和免培养方法研究红壤的真菌多样性

利用培养和免培养方法研究了一个云南红壤的真菌多样性。扩增的18S rRNA基因片段经过RsaⅠ、HinfⅠ、HaeⅢ三种限制性内切酶消化后,培养方法共获得16种限制性酶切长度多态类型（Restriction Fragment Length Polymorphism,RFLP）,而免培养方法仅获得了12种RFLP类型。每个代表类型经DNA测序及系统发育分析表明,免培养方法得到的真菌全部属于子囊菌门。优势物种与Aspergillus niger类似,占免培养真菌的40．9％。其次是Penicillium属和Paecilomyces属真菌,分别占免培养真菌的18％和17％。此外,有13个克隆仅和环境克隆具有较近的亲缘关系。培养方法获取的真菌包括Ascomycota门（11．5％）、Zygomycota门（86．5％）以及Basidiomycota门（1．9％）真菌,优势克隆来自Mortierella属,占全部培养克隆的55．8％,有21．2％的序列与Absidia glauca相似。两种方法观察到的真菌物种完全不同。     

Animal Adoption as Negotiated Order: A Comparison of Open Versus Traditional Shelter Approaches

ABSTRACT

Recently, the sheltering community has begun to reevaluate its adoption policies and the attitudes that shelter workers have towards adopters. Some shelters are now implementing what have been termed “open” adoptions as a way of increasing the number of animals adopted into good homes, moving away from more “traditional,” protective approaches. Based on in-depth interviews with, and observation of, the staff at two such shelters, this study examines how the adoption process is a negotiated order; namely, that workers in concert with each other and potential adopters figure out on a case-by-case basis how to interpret and implement formal adoption policies. Workers at both shelters similarly sorted potential adopters into various categories but relied on different strategies for influencing the outcome of the adoption process.     

Pigment Diversity in Freshwater Phytoplankton. I. A Comparison of Spectrophotometric and Paper Chromatographic Methods

Spectrophotometric and paper chromatographic analyses of the pigments in the phytoplankton were made from early spring till the end of summer in two small Dutch freshwater lakes. It was found that pigment diversity cannot be adequately estimated by MARGALEF'S pigment ratio nor by polychromatic spectrophotometric methods. The pigments detected with the paper chromatographic method were: chlorophyll-a, chlorophyll-b, chlorophyll-c, phaeophytin-a (traces), phaeophorbide-a, Mg-containing chlorophyll-derivatives, carotene, lutein, violaxanthin, neoxanthin (traces), fucoxanthin, diadinoxanthin, diatoxanthin (traces), peridinin and keto-carotenoids (traces). It is suggested to distinguish between a richness-component and an evenness-component of pigment diversity.     

The Freshwater Animal Diversity Assessment: an overview of the results

The geographic genetic structure of two common encrusting sponges, Hymeniacidon sinapium and Hymeniacidon flavia (family Halichondriidae), was investigated using two DNA markers, Internal Transcribed Spacers (ITS) of nuclear ribosomal DNA and NADH dehydrogenase subunit 5 (nad5) of mitochondrial DNA. In the ITS analyses, multiple sequence types were identified within each species. Geographic distribution patterns of sequence types showed higher diversity in the western than eastern areas in both species. However, intraspecific genetic diversity of the two species in Japan differed markedly. Hymeniacidon flavia had far more diverse sequence types, and several genetic differentiations between localities were detected. In contrast, H. sinapium had only four sequence types in Japan, and two Atlantic Hymeniacidon species had sequence types similar to this species. In comparison to ITS, nad5 showed very low genetic diversity in both species, with two haplotypes identified in each species. In H. flavia, frequency of haplotype changed gradually from north to south. In H. sinapium, one haplotype was predominant in most regions, and another haplotype was minor and distributed only in the Korean and Tsushima populations. Based on the unique distribution patterns of sequence types around Shikoku and Kyushu, geographical history and ocean currents were assumed to affect the generation of genetic structure. The geographic genetic structure of H. flavia suggests low dispersal ability of pelagic larvae, whereas higher larval dispersal ability and a far broader distribution range are suggested in H. sinapium. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users. Handling editor: C. Sturmbauer