Characterization of Fungal Community Structure on a Weathered Pegmatitic Granite

This study exploited the contrasting major element chemistry of adjacent, physically separable crystals of framework and sheet silicates in a pegmatitic granite to investigate the mineralogical influences of fungal community structure on mineral surfaces. Large intact crystals of variably weathered muscovite, plagioclase, K-feldspar, and quartz were individually extracted, together with whole-rock granite. Environmental scanning electron microscopy (ESEM) revealed a diversity of fungal structures, with microcolonial fungi and fungal hyphae clearly visible on surfaces of all mineral types. Fungal automated ribosomal intergenic spacer analysis (FARISA) was used to generate a ribotype profile for each mineral sample and a randomization test revealed that ribotype profiles, or community fingerprints, differed between different mineral types. Canonical correspondence analysis (CCA) revealed that mineral chemistry affected individual fungal ribotypes, and strong relationships were found between certain ribotypes and particular chemical elements. This finding was further supported by analysis of variance (ANOVA) of the 16 most abundant ribotypes within the community. Significantly, individual ribotypes were largely restricted to single mineral types and ribotypes clustered strongly on the basis of mineral type. CCA also revealed that Al, Si, and Ca had a significant impact on fungal community structure within this system. These results show that fungal community structure was driven by the chemical composition of mineral substrates, indicating selective pressure by individual chemical elements on fungal populations in situ.     

NESTED AUTOMATED RIBOSOMAL INTERGENIC SPACER ANALYSIS: A RAPID AND ACCURATE METHOD FOR COMPARISON OF BACTERIAL COMMUNITY COMPOSITION

Nested automated ribosomal intergenic spacer analysis (ARISA) was used to examine the community structure of epilithic biofilms in freshwater streams experiencing different levels of human impact. This molecular fingerprinting technique generated reproducible profiles of bacterial community structure that varied significantly between stream sites. Nested ARISA was determined to be a cost-effective, high-throughput approach to assess bacterial community composition from very small sample volumes, requiring little sampling effort and without the need for taxonomic identification of individual organisms. In combination with multidimensional scaling, nested ARISA provides a rapid and sensitive method to carry out complex analyses of bacterial community structure.

PRACTICAL APPLICATIONS

Nested automated ribosomal intergenic spacer analysis (ARISA) provides a high-throughput molecular method with which to screen large numbers of environmental samples for differences in microbial community structure. This sensitive approach benefits assessments from small sample volumes or environments exhibiting reduced microbial biomass (both aquatic and terrestrial). Differences in bacterial community structure (obtained from ARISA profiles) could be used to characterize the impact of anthropogenic disturbance on freshwater systems, analogous to the current use of macroinvertebrate indicators of freshwater ecological health.     

Characterization of Bacterial Community Structure on a Weathered Pegmatitic Granite

This study exploited the contrasting major element chemistry of a pegmatitic granite to investigate mineralogical influences on bacterial community structure. Intact crystals of variably weathered muscovite, plagioclase, K-feldspar, and quartz were extracted, together with whole-rock granite. Environmental scanning electron microscopy revealed a diversity of bacterial structures, with rods and cocci clearly visible on surfaces of all mineral types. Bacterial automated ribosomal intergenic spacer analysis was used to generate a ribotype profile for each mineral. A randomization test revealed that community fingerprints differed between different mineral types, whereas canonical correspondence analysis (CCA) showed that mineral chemistry affected individual bacterial ribotypes. CCA also revealed that Al, Si, and Ca had a significant impact on bacterial community structure within the system, which contrasts with the finding within fungal communities that although Al and Si also had a significant impact, K rather than Ca was important. The bacterial populations associated with different minerals were different. Members of each of these populations were found almost exclusively on a single mineral type, as was previously reported for fungal populations. These results show that bacterial community structure was driven by the chemical composition of minerals, indicating selective pressure by individual chemical elements on bacterial populations in situ.     

Microbial community dynamics in a humic lake: differential persistence of common freshwater phylotypes

In an effort to better understand the factors contributing to patterns in freshwater bacterioplankton community composition and diversity, we coupled automated ribosomal intergenic spacer analysis (ARISA) to analysis of 16S ribosomal RNA (rRNA) gene sequences to follow the persistence patterns of 46 individual phylotypes over 3 years in Crystal Bog Lake. Additionally, we sought to identify linkages between the observed phylotype variations and known chemical and biological drivers. Sequencing of 16S rRNA genes obtained from the water column indicated the presence of phylotypes associated with the Actinobacteria, Bacteroidetes, Firmicutes, Proteobacteria, TM7 and Verrucomicrobia phyla, as well as phylotypes with unknown affiliation. Employment of the 16S rRNA gene/ARISA method revealed that specific phylotypes varied independently of the entire bacterial community dynamics. Actinobacteria, which were present on greater than 95% of sampling dates, did not share the large temporal variability of the other identified phyla. Examination of phylotype relative abundance patterns (inferred using ARISA fragment relative fluorescence) revealed a strong correlation between the dominant phytoplankton succession and the relative abundance patterns of the majority of individual phylotypes. Further analysis revealed covariation among unique phylotypes, which formed several distinct bacterial assemblages correlated with particular phytoplankton communities. These data indicate the existence of unique persistence patterns for different common freshwater phylotypes, which may be linked to the presence of dominant phytoplankton species.     

Spatial and temporal heterogeneity of the bacterial communities in stream epilithic biofilms

The spatial and temporal variability in bacterial communities within freshwater systems is poorly understood. The bacterial composition of stream epilithic biofilms across a range of different spatial and temporal scales both within and between streams and across the profile of individual stream rocks was characterised using a community DNA-fingerprinting technique (Automated Ribosomal Intergenic Spacer Analysis, ARISA). The differences in bacterial community structure between two different streams were found to be greater than the spatial variability within each stream site, and were larger than the weekly temporal variation measured over a 10-week study period. Greater variations in bacterial community profiles were detected on different faces of individual stream rocks than between whole rocks sampled within a 9-m stream section. Stream temperature was found to be the most important determinant of bacterial community variability using distance-based redundancy analysis (dbRDA) of ARISA data, which may have broad implications for riparian zone management and ecological change as a consequence of global warming. The combination of ARISA with multivariate statistical methods and ordination, such as multidimensional scaling (MDS), permutational manova and RDA, provided rapid and effective methods for quantifying and visualising variation in bacterial community structure, and to identify potential drivers of ecological change.     

Phospholipid Fatty Acid Composition and Heavy Metal Tolerance of Soil Microbial Communities along Two Heavy Metal-Polluted Gradients in Coniferous Forests

The effects of long-term heavy metal deposition on microbial community structure and the level of bacterial community tolerance were studied along two different gradients in Scandinavian coniferous forest soils. One was near the Harjavalta smelter in Finland, and one was at Ronnskar in Sweden. Phospholipid fatty acid (PLFA) analysis revealed a gradual change in soil microbial communities along both pollution gradients, and most of the individual PLFAs changed similarly to metal pollution at both sites. The relative quantities of the PLFAs br18:0, br17:0, i16:0, and i16:1 increased with increasing heavy metal concentration, while those of 20:4 and 18:2(omega)6, which is a predominant PLFA in many fungi, decreased. The fungal part of the microbial biomass was found to be more sensitive to heavy metals. This resulted in a decreased fungal/bacterial biomass ratio along the pollution gradient towards the smelters. The thymidine incorporation technique was used to study the heavy metal tolerance of the bacteria. The bacterial community at the Harjavalta smelter, exposed mainly to Cu deposition, exhibited an increased tolerance to Cu but not to Cd, Ni, and Zn. At the Ronnskar smelter the deposition consisting of a mixture of metals increased the bacterial community tolerance to all tested metals. Both the PLFA pattern and the bacterial community tolerance were affected at lower soil metal concentrations than were bacterial counts and bacterial activities. At Harjavalta the increased Cu tolerance of the bacteria and the change in the PLFA pattern of the microbial community were found at the same soil Cu concentrations. This indicated that the altered PLFA pattern was at least partly due to an altered, more metal-tolerant bacterial community. At Ronnskar, where the PLFA data varied more, a correlation between bacterial community tolerance and an altered PLFA pattern was found up to 10 to 15 km from the smelter. Farther away changes in the PLFA pattern could not be explained by an increased community tolerance to metals.     

Impact of catchment land use on bacterial communities within stream biofilms

The bacterial community structure in epilithic biofilms within 18 different streams was characterised using a community DNA fingerprinting technique (automated ribosomal intergenic spacer analysis—ARISA). Each stream has previously been described in terms of the dominant catchment land use, relative level of human disturbance and using a broad suite of water quality variables. Combination of ARISA with multivariate statistical analysis and ordination revealed that bacterial communities in streams located within rural catchments were significantly different to those within urban catchments. Broad-scale catchment land use described the largest component of the observed variation with no single water quality variable found to be a dominant determinant of the observed bacterial community variability, assessed using distance based redundancy analysis (dbRDA) of the ARISA data. This study highlights the potential of bacterial ARISA to provide a rapid and cost-effective approach to monitor the impact of catchment land use on aquatic ecosystems, such as the influence of encroaching urban development on the ecological health of rural streams.     

Effects of eutrophication, seasonality and macrofouling on the diversity of bacterial biofilms in equatorial coral reefs

Biofilms play an important role as a settlement cue for invertebrate larvae and significantly contribute to the nutrient turnover in aquatic ecosystems. Nevertheless, little is known about how biofilm community structure generally responds to environmental changes. This study aimed to identify patterns of bacterial dynamics in coral reef biofilms in response to associated macrofouling community structure, microhabitat (exposed vs. sheltered), seasonality, and eutrophication. Settlement tiles were deployed at four reefs along a cross-shelf eutrophication gradient and were exchanged every 4 months over 20 months. The fouling community composition on the tiles was recorded and the bacterial community structure was assessed with the community fingerprinting technique Automated Ribosomal Intergenic Spacer Analysis (ARISA). Bacterial operational taxonomic unit (OTU) number was higher on exposed tiles, where the fouling community was homogenous and algae-dominated, than in sheltered habitats, which were occupied by a variety of filter feeders. Furthermore, OTU number was also highest in eutrophied near-shore reefs, while seasonal variations in community structure were most pronounced in the oligotrophic mid-shelf reef. In contrast, the macrofouling community structure did not change significantly with seasons. Changes in bacterial community patterns were mostly affected by microhabitat, seasonal and anthropogenically derived changes in nutrient availability, and to a lesser extent by changes in the macrofouling community structure. Path analysis revealed a complex interplay of various environmental and biological factors explaining the spatial and temporal variations in bacterial biofilm communities under natural conditions.     

Bacterial community diversity assessment in municipal solid waste compost amended soil using DGGE and ARISA fingerprinting methods

Bacterial community structure and diversity of Tunisian agricultural soil treated with different amounts of municipal solid waste compost (MSWC) and other fertilizers were studied using DGGE and ARISA fingerprinting methods. Sequence analysis of dominant DGGE bands revealed the presence of three major clusters, Cytophaga/Flexibacter/Bacteroides (CFB) group, Proteobacteria and Acidobacteria group. Using ARISA profiles, dominant populations were assigned to low and high GC Gram positive bacteria, Cyanobacteria, Spirochetes and Cytophagales. The two methods revealed the absence of significant bacterial community shifts related to the different MSWC applications. Moreover, indigenous bacterial population of the used loam-clayey soil was observed to limit proliferation and survival of Proteobacteria, initially dominant in MSWC and farmyard manure. Effectiveness of the two methods for soil bacterial community studying was shown. While DGGE was more accurate for bacterial identification, ARISA was more practical for handling and rapid estimation of dominant bacteria.     

Seasonal influences on fungal community structure in unimproved and improved upland grassland soils

Seasonal and management influences on the fungal community structure of two upland grassland soils were investigated. An upland site containing both unimproved floristically diverse (U4a) and improved mesotrophic (MG7b) grassland types was selected. Samples from both grassland types were taken at five times in one year. Soil fungal community structure was assessed using fungal automated ribosomal intergenic spacer analysis (ARISA), a DNA-profiling approach. A grassland management regime was found to strongly affect fungal community structure, with fungal ARISA profiles from unimproved and improved grassland soils differing significantly. The number of fungal ribotypes found was higher in unimproved than improved grassland soils, providing evidence that improvement may reduce the suitability of upland soil as a habitat for specific groups of fungi. Seasonal influences on fungal community structure were also noted, with samples taken in autumn (October) more correlated with change in ribotype profiles than samples from other seasons. However, seasonal variation did not obscure the measurement of differences in the fungal community structure that were due to agricultural improvement, with canonical correspondence analysis indicating grassland type had a stronger influence on fungal profiles than did season.     

Comparison of two fingerprinting techniques, terminal restriction fragment length polymorphism and automated ribosomal intergenic spacer analysis, for determination of bacterial diversity in aquatic environments

We investigated bacterial diversity in different aquatic environments (including marine and lagoon sediments, coastal seawater, and groundwater), and we compared two fingerprinting techniques (terminal restriction fragment length polymorphism [T-RFLP] and automated ribosomal intergenic spacer analysis [ARISA]) which are currently utilized for estimating richness and community composition. Bacterial diversity ranged from 27 to 99 phylotypes (on average, 56) using the T-RFLP approach and from 62 to 101 genotypes (on average, 81) when the same samples were analyzed using ARISA. The total diversity encountered in all matrices analyzed was 144 phylotypes for T-RFLP and 200 genotypes for ARISA. Although the two techniques provided similar results in the analysis of community structure, bacterial richness and diversity estimates were significantly higher using ARISA. These findings suggest that ARISA is more effective than T-RFLP in detecting the presence of bacterial taxa accounting for <5% of total amplified product. ARISA enabled also distinction among aquatic bacterial isolates of Pseudomonas spp. which were indistinguishable using T-RFLP analysis. Overall, the results of this study show that ARISA is more accurate than T-RFLP analysis on the 16S rRNA gene for estimating the biodiversity of aquatic bacterial assemblages.     

Comparison of Two Fingerprinting Techniques, Terminal Restriction Fragment Length Polymorphism and Automated Ribosomal Intergenic Spacer Analysis, for Determination of Bacterial Diversity in Aquatic Environments

We investigated bacterial diversity in different aquatic environments (including marine and lagoon sediments, coastal seawater, and groundwater), and we compared two fingerprinting techniques (terminal restriction fragment length polymorphism [T-RFLP] and automated ribosomal intergenic spacer analysis [ARISA]) which are currently utilized for estimating richness and community composition. Bacterial diversity ranged from 27 to 99 phylotypes (on average, 56) using the T-RFLP approach and from 62 to 101 genotypes (on average, 81) when the same samples were analyzed using ARISA. The total diversity encountered in all matrices analyzed was 144 phylotypes for T-RFLP and 200 genotypes for ARISA. Although the two techniques provided similar results in the analysis of community structure, bacterial richness and diversity estimates were significantly higher using ARISA. These findings suggest that ARISA is more effective than T-RFLP in detecting the presence of bacterial taxa accounting for <5% of total amplified product. ARISA enabled also distinction among aquatic bacterial isolates of Pseudomonas spp. which were indistinguishable using T-RFLP analysis. Overall, the results of this study show that ARISA is more accurate than T-RFLP analysis on the 16S rRNA gene for estimating the biodiversity of aquatic bacterial assemblages.     

不同重金属污染水平下生物结皮及下层土壤细菌群落的差异性分析

为了探究生物结皮及下层土壤细菌群落多样性及结构在不同重金属污染水平下的差异及其影响因素,以宁夏东部工矿区和封育草地内广泛分布的两类生物结皮为研究对象,采用高通量测序技术,分析结皮层(A层)和结皮下层(B层)土壤细菌群落结构和多样性差异。采用相关分析、冗余分析探讨两个生境生物结皮各层次细菌群落与土壤环境因子的关系。结果表明：(1)相较于封育区,工矿区2类生物结皮对源自大气降尘的重金属元素具有更显著的表层富集作用,并表现出显著的种间差异：苔藓结皮>藻结皮。污染负荷指数评价结果显示：工矿区生物结皮各层次重金属污染程度更高,且均表现为A层污染程度高于B层。(2)两个生境的优势细菌门组成基本一致,均为放线菌门、变形菌门、酸杆菌门。受生物结皮类型、重金属污染程度的影响,细菌群落结构差异显著,主要表现为：工矿区藻结皮A、B层的α多样性指数均大于封育区,而苔藓结皮A、B层的细菌多样性指数高于封育区,丰富度指数低于封育区。主坐标分析结果显示,不同生境的生物结皮细菌群落结构差异明显,而同一生境的生物结皮细菌群落结构相似。重金属As、Pb、土壤黏粒含量和pH是影响生物结皮细菌群落结构的主要土壤环境因子...     

Comparison of methods for the extraction of DNA from stream epilithic biofilms

This study compares how different DNA extraction methods influence the quantity and quality of DNA yields from stream epilithic biofilms. Interpretations of bacterial community structure, using ARISA, revealed increased variability among samples processed using commercially-available kits, which also yielded lower DNA concentrations than other methods tested.     

Stream Macroinvertebrate Community Affected by Point‐Source Metal Pollution

The impacts of mining activities on aquatic biota have been documented in many stream ecosystems. In mining streams, point‐source heavy metal pollution often appears in the stream. We hypothesize that this pollution is toxic to macroinvertebrates owing to high concentrations of metals and therefore affects macroinvertebrate community structure. We investigated macroinvertebrate community structure in mountain streams, including heavy metal‐polluted sites and neutral‐pH streams, to determine the relationship between community structure and environmental factors such as low pH and heavy metal concentrations. Based on multidimensional scaling ordination, the macroinvertebrate community at heavy metal pollution sites was remarkably different from that at the other sites. Inductively coupled plasma mass spectrometry revealed high concentrations of aluminum and iron in surface water at the polluted sites. Macroinvertebrate community structure at the metal pollution sites was significantly different from that at other sites in the same stream and in neutral‐pH streams. Thus, point‐source metal pollution may reduce the density and diversity of in situ macroinvertebrates. (© 2007 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)     

Microbial community analysis of field-grown soybeans with different nodulation phenotypes

Microorganisms associated with the stems and roots of nonnodulated (Nod(-)), wild-type nodulated (Nod(+)), and hypernodulated (Nod(++)) soybeans [Glycine max (L.) Merril] were analyzed by ribosomal intergenic transcribed spacer analysis (RISA) and automated RISA (ARISA). RISA of stem samples detected no bands specific to the nodulation phenotype, whereas RISA of root samples revealed differential bands for the nodulation phenotypes. Pseudomonas fluorescens was exclusively associated with Nod(+) soybean roots. Fusarium solani was stably associated with nodulated (Nod(+) and Nod(++)) roots and less abundant in Nod(-) soybeans, whereas the abundance of basidiomycetes was just the opposite. The phylogenetic analyses suggested that these basidiomycetous fungi might represent a root-associated group in the Auriculariales. Principal-component analysis of the ARISA results showed that there was no clear relationship between nodulation phenotype and bacterial community structure in the stem. In contrast, both the bacterial and fungal community structures in the roots were related to nodulation phenotype. The principal-component analysis further suggested that bacterial community structure in roots could be classified into three groups according to the nodulation phenotype (Nod(-), Nod(+), or Nod(++)). The analysis of root samples indicated that the microbial community in Nod(-) soybeans was more similar to that in Nod(++) soybeans than to that in Nod(+) soybeans.     

Combined use of 16S ribosomal DNA and automated ribosomal intergenic spacer analysis to study the bacterial community in catfish ponds

AIMS: To apply culture-independent techniques to explore the bacterial community composition in catfish pond water. METHODS AND RESULTS: 16S rDNA libraries were constructed and sequenced from 15 pond water samples. Automated ribosomal intergenic spacer analysis (ARISA) was used to fingerprint each bacterial community. A broad diversity in bacterial species composition was found by 16S rDNA analysis. Alphaproteobacteria was the most represented class in all ponds, followed by Gammaproteobacteria and Gram-positive high G + C content bacteria. Uniqueness of bacterial communities from each individual pond was confirmed by ARISA. Catfish pathogens were detected sporadically. CONCLUSIONS: Bacterial communities in a catfish aquaculture setting can vary from pond to pond at one given point. No correlation could be made between bacteria composition and fish strain or between bacterial profile and the presence of catfish pathogens in a particular pond. SIGNIFICANCE AND IMPACT OF THE STUDY: This is the first report showing the composition of bacterial communities in catfish ponds. Fish health specialists and catfish aquaculture managers should be aware of the wide differences in bacterial communities between ponds and include this variable in fish husbandry practices.     

Comparison of primer sets for use in automated ribosomal intergenic spacer analysis of aquatic bacterial communities: an ecological perspective

Two primer sets for automated ribosomal intergenic spacer analysis (ARISA) were used to assess the bacterial community composition (BCC) in Lake Mendota, Wisconsin, over 3 years. Correspondence analysis revealed differences in community profiles generated by different primer sets, but overall ecological patterns were conserved in each case. ARISA is a powerful tool for evaluating BCC change through space and time, regardless of the specific primer set used.     

火电厂周边不同生物结皮细菌群落特征差异及其影响因素

为探究大气降尘重金属污染对矿区周边不同类型生物结皮细菌群落结构的影响,利用高通量测序技术分析位于宁东能源化工基地典型火电厂周边的3类生物结皮(藻结皮ZB、混生结皮HB、苔藓结皮TB)和对照(CK,裸土)的细菌丰度和群落结构,并探讨了影响细菌群落结构的环境因子。结果表明: 不同类型生物结皮的理化性质和重金属含量存在差异,且由于生物结皮对大气降尘重金属的富集作用造成各类结皮均达重度污染级别。在相对丰度排名前10的优势细菌门中,芽单胞菌门、蓝细菌门在不同类型生物结皮之间差异显著。细菌群落α多样性由高到低排序依次为CK>TB>HB>ZB。非度量多维排序(NMDS)结果显示,裸土细菌群落与其他3种生物结皮存在明显差异。相关性分析表明,生物结皮演替对细菌群落组成具有显著影响,细菌多样性和组成与pH、养分、重金属含量等密切相关。放线菌门、绿弯菌门相对丰度与pH值呈显著正相关关系,而与全氮(TN)、全磷(TP)、Pb、Zn、Cd均呈显著负相关关系;冗余分析结果表明,TN、pH、TP、有机碳(SOC)是影响3种生物结皮细菌群落α多样性以及一些优势菌群相对丰度的主要土壤环境因子,而重金属Pb、Zn、Cd是影响细菌群落结构的主要重金属元素,对细菌群落数量和多样性有抑制或刺激作用。说明pH、重金属和养分是影响结皮细菌群落组成的关键因子。总体而言,长期的重金属富集作用会对生物结皮的细菌多样性和群落组成产生影响。