ITS2 metabarcoding analysis complements lichen mycobiome diversity data

Lichen thalli harbor complex fungal communities (mycobiomes) of species with divergent trophic and ecological strategies. The complexity and diversity of lichen mycobiomes are still largely unknown, despite surveys combining culture-based methods and high-throughput sequencing (HTS). The results of such surveys are strongly influenced by the barcode locus chosen, its sensitivity in discriminating taxa, and the depth to which public sequence repositories cover the phylogenetic spectrum of fungi. Here, we use HTS of the internal transcribed spacer 2 (ITS2) to assess the taxonomic composition and diversity of a well-characterized, alpine rock lichen community that includes thalli symptomatically infected by lichenicolous fungi as well as asymptomatic thalli. Taxa belonging to the order Chaetothyriales are the major components of the observed lichen mycobiomes. We predict sequences representative of lichenicolous fungi characterized morphologically and assess their asymptomatic presence in lichen thalli. We demonstrated the limitations of metabarcoding in fungi and show how the estimation of species diversity widely differs when ITS1 or ITS2 are used as barcode, and particularly biases the detection of Basidiomycota. The complementary analysis of both ITS1 and ITS2 loci is therefore required to reliably estimate the diversity of lichen mycobiomes.     

Algal and Fungal Diversity in Antarctic Lichens

The composition of lichen ecosystems except mycobiont and photobiont has not been evaluated intensively. In addition, recent studies to identify algal genotypes have raised questions about the specific relationship between mycobiont and photobiont. In the current study, we analyzed algal and fungal community structures in lichen species from King George Island, Antarctica, by pyrosequencing of eukaryotic large subunit (LSU) and algal internal transcribed spacer (ITS) domains of the nuclear rRNA gene. The sequencing results of LSU and ITS regions indicated that each lichen thallus contained diverse algal species. The major algal operational taxonomic unit (OTU) defined at a 99% similarity cutoff of LSU sequences accounted for 78.7–100% of the total algal community in each sample. In several cases, the major OTUs defined by LSU sequences were represented by two closely related OTUs defined by 98% sequence similarity of ITS domain. The results of LSU sequences indicated that lichen‐associated fungi belonged to the Arthoniomycetes, Eurotiomycetes, Lecanoromycetes, Leotiomycetes, and Sordariomycetes of the Ascomycota, and Tremellomycetes and Cystobasidiomycetes of the Basidiomycota. The composition of major photobiont species and lichen‐associated fungal community were mostly related to the mycobiont species. The contribution of growth forms or substrates on composition of photobiont and lichen‐associated fungi was not evident.     

Molecular diversity and distribution of marine fungi across 130 European environmental samples

Environmental DNA and culture-based analyses have suggested that fungi are present in low diversity and in low abundance in many marine environments, especially in the upper water column. Here, we use a dual approach involving high-throughput diversity tag sequencing from both DNA and RNA templates and fluorescent cell counts to evaluate the diversity and relative abundance of fungi across marine samples taken from six European near-shore sites. We removed very rare fungal operational taxonomic units (OTUs) selecting only OTUs recovered from multiple samples for a detailed analysis. This approach identified a set of 71 fungal ‘OTU clusters'' that account for 66% of all the sequences assigned to the Fungi. Phylogenetic analyses demonstrated that this diversity includes a significant number of chytrid-like lineages that had not been previously described, indicating that the marine environment encompasses a number of zoosporic fungi that are new to taxonomic inventories. Using the sequence datasets, we identified cases where fungal OTUs were sampled across multiple geographical sites and between different sampling depths. This was especially clear in one relatively abundant and diverse phylogroup tentatively named Novel Chytrid-Like-Clade 1 (NCLC1). For comparison, a subset of the water column samples was also investigated using fluorescent microscopy to examine the abundance of eukaryotes with chitin cell walls. Comparisons of relative abundance of RNA-derived fungal tag sequences and chitin cell-wall counts demonstrate that fungi constitute a low fraction of the eukaryotic community in these water column samples. Taken together, these results demonstrate the phylogenetic position and environmental distribution of 71 lineages, improving our understanding of the diversity and abundance of fungi in marine environments.     

Investigating Microbial Eukaryotic Diversity from a Global Census: Insights from a Comparison of Pyrotag and Full-Length Sequences of 18S rRNA Genes

Next-generation DNA sequencing (NGS) approaches are rapidly surpassing Sanger sequencing for characterizing the diversity of natural microbial communities. Despite this rapid transition, few comparisons exist between Sanger sequences and the generally much shorter reads of NGS. Operational taxonomic units (OTUs) derived from full-length (Sanger sequencing) and pyrotag (454 sequencing of the V9 hypervariable region) sequences of 18S rRNA genes from 10 global samples were analyzed in order to compare the resulting protistan community structures and species richness. Pyrotag OTUs called at 98% sequence similarity yielded numbers of OTUs that were similar overall to those for full-length sequences when the latter were called at 97% similarity. Singleton OTUs strongly influenced estimates of species richness but not the higher-level taxonomic composition of the community. The pyrotag and full-length sequence data sets had slightly different taxonomic compositions of rhizarians, stramenopiles, cryptophytes, and haptophytes, but the two data sets had similarly high compositions of alveolates. Pyrotag-based OTUs were often derived from sequences that mapped to multiple full-length OTUs at 100% similarity. Thus, pyrotags sequenced from a single hypervariable region might not be appropriate for establishing protistan species-level OTUs. However, nonmetric multidimensional scaling plots constructed with the two data sets yielded similar clusters, indicating that beta diversity analysis results were similar for the Sanger and NGS sequences. Short pyrotag sequences can provide holistic assessments of protistan communities, although care must be taken in interpreting the results. The longer reads (>500 bp) that are now becoming available through NGS should provide powerful tools for assessing the diversity of microbial eukaryotic assemblages.     

高通量测序分析环保肥料增效剂对马铃薯根际土壤真菌多样性变化影响

【目的】高通量测序技术对研究环境样品中微生物群落组成具有很大的应用价值。土壤微生物群落结构和多样性及其变化在一定程度上反映了土壤的质量。旨在从微生物群落结构的角度阐述环保肥料增效剂对马铃薯根际土壤主要真菌类群结构的影响。【方法】通过高通量测序结果对比分析应用增效剂前后马铃薯根际真菌宏基因组ITS1区,并依据RDP中设置的分类阈值对序列进行物种分类。【结果】测序结果经过质量控制,共获得有效条带437 375条,依据97%的序列相似性做聚类分析,获得全部样品的可分类操作单元(OTUs)共633个。子囊菌的总体数量在所有样品中最多(相对丰度在56.95%-97.23%之间),且处理后呈增加趋势(HY除外),而担子菌门数量在处理后呈下降的趋势。基于真菌ITS1高通量测序结果获得的α指数显示,样品内部处理和对照之间真菌物种多样性有差别。基于真菌ITS1高通量测序获得的β指数显示,处理组与对照组的真菌多样性之间没有差别,这表明真菌多样性之间的差异更多地取决于样品采集地点。【结论】土壤特性是影响真菌种群的重要因素之一,环保肥料增效剂显著改善了土壤真菌的种群结构。     

Fungal palaeodiversity revealed using high‐throughput metabarcoding of ancient DNA from arctic permafrost

The taxonomic and ecological diversity of ancient fungal communities was assessed by combining next generation sequencing and metabarcoding of DNA preserved in permafrost. Twenty‐six sediment samples dated 16 000–32 000 radiocarbon years old from two localities in Siberia were analysed for fungal ITS. We detected 75 fungal OTUs from 21 orders representing three phyla, although rarefaction analyses suggested that the full diversity was not recovered despite generating an average of 6677 ± 3811 (mean ± SD) sequences per sample and that preservation bias likely has considerable effect on the recovered DNA. Most OTUs (75.4%) represented ascomycetes. Due to insufficient sequencing depth, DNA degradation and putative preservation biases in our samples, the recovered taxa probably do not represent the complete historic fungal community, and it is difficult to determine whether the fungal communities varied geographically or experienced a composition shift within the period of 16 000–32 000 bp . However, annotation of OTUs to functional ecological groups provided a wealth of information on the historic communities. About one‐third of the OTUs are presumed plant‐associates (pathogens, saprotrophs and endophytes) typical of graminoid‐ and forb‐rich habitats. We also detected putative insect pathogens, coprophiles and keratinophiles likely associated with ancient insect and herbivore faunas. The detection of putative insect pathogens, mycoparasites, aquatic fungi and endophytes broadens our previous knowledge of the diversity of fungi present in Beringian palaeoecosystems. A large group of putatively psychrophilic/psychrotolerant fungi was also detected, most likely representing a modern, metabolically active fungal community.     

Long‐read metabarcoding of the eukaryotic rDNA operon to phylogenetically and taxonomically resolve environmental diversity

High‐throughput DNA metabarcoding of amplicon sizes below 500 bp has revolutionized the analysis of environmental microbial diversity. However, these short regions contain limited phylogenetic signal, which makes it impractical to use environmental DNA in full phylogenetic inferences. This lesser phylogenetic resolution of short amplicons may be overcome by new long‐read sequencing technologies. To test this idea, we amplified soil DNA and used PacBio Circular Consensus Sequencing (CCS) to obtain an ~4500‐bp region spanning most of the eukaryotic small subunit (18S) and large subunit (28S) ribosomal DNA genes. We first treated the CCS reads with a novel curation workflow, generating 650 high‐quality operational taxonomic units (OTUs) containing the physically linked 18S and 28S regions. To assign taxonomy to these OTUs, we developed a phylogeny‐aware approach based on the 18S region that showed greater accuracy and sensitivity than similarity‐based methods. The taxonomically annotated OTUs were then combined with available 18S and 28S reference sequences to infer a well‐resolved phylogeny spanning all major groups of eukaryotes, allowing us to accurately derive the evolutionary origin of environmental diversity. A total of 1,019 sequences were included, of which a majority (58%) corresponded to the new long environmental OTUs. The long reads also allowed us to directly investigate the relationships among environmental sequences themselves, which represents a key advantage over the placement of short reads on a reference phylogeny. Together, our results show that long amplicons can be treated in a full phylogenetic framework to provide greater taxonomic resolution and a robust evolutionary perspective to environmental DNA.     

Patterns of fungal communities among and within decaying logs, revealed by 454 sequencing

Owing to previous methodological limitations, knowledge about the fine-scale distribution of fungal mycelia in decaying logs is limited. We investigated fungal communities in decaying Norway spruce logs at various spatial scales at two environmentally different locations in Sweden. On the basis of 454 pyrosequencing of the ITS2 region of rDNA, 1914 operational taxonomic units (OTUs) were detected in 353 samples. The communities differed significantly among logs, but the physical distance between logs was not found to have a significant effect on whether fungal communities had any resemblance to each other. Within a log, samples that were closer together generally had communities that showed more resemblance to each other than those that were further apart. OTUs characteristic for particular positions on the logs could be identified. In general, these OTUs did not overlap with the most abundant OTUs, and their ecological role was often unknown. Only a few OTUs were detected in the majority of logs, whereas numerous OTUs were rare and present in only one or a few logs. Wood-decaying Basidiomycetes were often represented by higher sequence reads in individual logs than Ascomycete OTUs, suggesting that Basidiomycete mycelia spread out more rapidly when established. OTU richness tended to increase with the decay stage of the sample; however, the known wood decayers were most abundant in less-decomposed samples. The fungi identified in the logs represented different ecological strategies. Our findings differ from previously published sporocarp studies, indicating that the highly abundant fruiting species may respond to environment in different ways than the rest of the fungal community.     

Differences in Soil Fungal Communities between European Beech (Fagus sylvatica L.) Dominated Forests Are Related to Soil and Understory Vegetation

Fungi are important members of soil microbial communities with a crucial role in biogeochemical processes. Although soil fungi are known to be highly diverse, little is known about factors influencing variations in their diversity and community structure among forests dominated by the same tree species but spread over different regions and under different managements. We analyzed the soil fungal diversity and community composition of managed and unmanaged European beech dominated forests located in three German regions, the Schwäbische Alb in Southwestern, the Hainich-Dün in Central and the Schorfheide Chorin in the Northeastern Germany, using internal transcribed spacer (ITS) rDNA pyrotag sequencing. Multiple sequence quality filtering followed by sequence data normalization revealed 1655 fungal operational taxonomic units. Further analysis based on 722 abundant fungal OTUs revealed the phylum Basidiomycota to be dominant (54%) and its community to comprise 71.4% of ectomycorrhizal taxa. Fungal community structure differed significantly (p≤0.001) among the three regions and was characterized by non-random fungal OTUs co-occurrence. Soil parameters, herbaceous understory vegetation, and litter cover affected fungal community structure. However, within each study region we found no difference in fungal community structure between management types. Our results also showed region specific significant correlation patterns between the dominant ectomycorrhizal fungal genera. This suggests that soil fungal communities are region-specific but nevertheless composed of functionally diverse and complementary taxa.     

Amplicon –Based Metagenomic Analysis of Mixed Fungal Samples Using Proton Release Amplicon Sequencing

Next generation sequencing technology has revolutionised microbiology by allowing concurrent analysis of whole microbial communities. Here we developed and verified similar methods for the analysis of fungal communities using a proton release sequencing platform with the ability to sequence reads of up to 400 bp in length at significant depth. This read length permits the sequencing of amplicons from commonly used fungal identification regions and thereby taxonomic classification. Using the 400 bp sequencing capability, we have sequenced amplicons from the ITS1, ITS2 and LSU fungal regions to a depth of approximately 700,000 raw reads per sample. Representative operational taxonomic units (OTUs) were chosen by the USEARCH algorithm, and identified taxonomically through nucleotide blast (BLASTn). Combination of this sequencing technology with the bioinformatics pipeline allowed species recognition in two controlled fungal spore populations containing members of known identity and concentration. Each species included within the two controlled populations was found to correspond to a representative OTU, and these OTUs were found to be highly accurate representations of true biological sequences. However, the absolute number of reads attributed to each OTU differed among species. The majority of species were represented by an OTU derived from all three genomic regions although in some cases, species were only represented in two of the regions due to the absence of conserved primer binding sites or due to sequence composition. It is apparent from our data that proton release sequencing technologies can deliver a qualitative assessment of the fungal members comprising a sample. The fact that some fungi cannot be amplified by specific “conserved” primer pairs confirms our recommendation that a multi-region approach be taken for other amplicon-based metagenomic studies.     

Substantial compositional turnover of fungal communities in an alpine ridge‐to‐snowbed gradient

The main gradient in vascular plant, bryophyte and lichen species composition in alpine areas, structured by the topographic gradient from wind‐exposed ridges to snowbeds, has been extensively studied. Tolerance to environmental stress, resulting from wind abrasion and desiccation towards windswept ridges or reduced growing season due to prolonged snow cover towards snowbeds, is an important ecological mechanism in this gradient. The extent to which belowground fungal communities are structured by the same topographic gradient and the eventual mechanisms involved are less well known. In this study, we analysed variation in fungal diversity and community composition associated with roots of the ectomycorrhizal plant Bistorta vivipara along the ridge‐to‐snowbed gradient. We collected root samples from fifty B. vivipara plants in ten plots in an alpine area in central Norway. The fungal communities were analysed using 454 pyrosequencing analyses of tag‐encoded ITS1 amplicons. A distinct gradient in the fungal community composition was found that coincided with variation from ridge to snowbeds. This gradient was paralleled by change in soil content of carbon, nitrogen and phosphorus. A large proportion (66%) of the detected 801 nonsingleton operational taxonomic units (OTUs) were ascomycetes, while basidiomycetes dominated quantitatively (i.e. with respect to number of reads). Numerous fungal OTUs, many with taxonomic affinity to Sebacinales, Cortinarius and Meliniomyces, showed distinct affinities either to ridge or to snowbed plots, indicating habitat specialization. The compositional turnover of fungal communities along the gradient was not paralleled by a gradient in species richness.     

Patterns of community composition and diversity in latent fungi of living Quercus serrata trunks across a range of oak wilt prevalence and climate variables in Japan

Given that forest dieback due to emerging pests is increasing under global warming, understanding the relationships between pests, climate, and forest biodiversity is an urgent priority. In Japan, mass attacks of an ambrosia beetle, vectoring a pathogenic fungus, cause oak wilt outbreaks in recent decades. Here, the associations of oak wilt and climate with wood-inhabiting fungal communities in apparently healthy Quercus serrata trunks were investigated using DNA metabarcoding in seven sites along a climatic gradient in Japan. Amplicon sequencing of the fungal internal transcribed spacer 1 region generated 1,339,958 sequence reads containing 879 fungal operational taxonomic units (OTUs) in 234 wood samples. OTU compositions were significantly different between sites with and without oak wilt. OTU richness increased with temperature and precipitation at sites where oak wilt was present, but this relationship was not observed at sites without oak wilt, possibly due to interaction between oak wilt and climate.     

Fungi ahoy! Diversity on marine wooden substrata in the high North

Marine fungi are severely understudied in the polar regions. We used molecularly identified cultures to study fungi inhabiting 50 intertidal and sea-floor logs along the North Norwegian coast. The aim was to explore the taxonomic and ecological diversity and to examine factors shaping the marine wood-inhabiting fungal communities. The 577 pure cultures analyzed clustered into 147 operational taxonomic units (OTUs) based on 97 % ITS sequence similarity. Ascomycota dominated, but OTUs belonging to Basidiomycota, Mucoromycotina and Chytridiomycota were also isolated. Nine OTUs could not be assigned to any fungal phylum. Almost half of the OTUs were considered non-marine. The western and eastern part of the Norwegian Barents Sea coast hosted different communities. Geography, substratum and site level variables contributed to shaping these communities. We characterized a previously overlooked fungal community in a poorly studied area, discovered high diversity and report many taxa for the first time from the marine environment.     

A Comparison of Methods for Clustering 16S rRNA Sequences into OTUs

Recent studies of 16S rRNA sequences through next-generation sequencing have revolutionized our understanding of the microbial community composition and structure. One common approach in using these data to explore the genetic diversity in a microbial community is to cluster the 16S rRNA sequences into Operational Taxonomic Units (OTUs) based on sequence similarities. The inferred OTUs can then be used to estimate species, diversity, composition, and richness. Although a number of methods have been developed and commonly used to cluster the sequences into OTUs, relatively little guidance is available on their relative performance and the choice of key parameters for each method. In this study, we conducted a comprehensive evaluation of ten existing OTU inference methods. We found that the appropriate dissimilarity value for defining distinct OTUs is not only related with a specific method but also related with the sample complexity. For data sets with low complexity, all the algorithms need a higher dissimilarity threshold to define OTUs. Some methods, such as, CROP and SLP, are more robust to the specific choice of the threshold than other methods, especially for shorter reads. For high-complexity data sets, hierarchical cluster methods need a more strict dissimilarity threshold to define OTUs because the commonly used dissimilarity threshold of 3% often leads to an under-estimation of the number of OTUs. In general, hierarchical clustering methods perform better at lower dissimilarity thresholds. Our results show that sequence abundance plays an important role in OTU inference. We conclude that care is needed to choose both a threshold for dissimilarity and abundance for OTU inference.     

PhylOTU: a high-throughput procedure quantifies microbial community diversity and resolves novel taxa from metagenomic data

Microbial diversity is typically characterized by clustering ribosomal RNA (SSU-rRNA) sequences into operational taxonomic units (OTUs). Targeted sequencing of environmental SSU-rRNA markers via PCR may fail to detect OTUs due to biases in priming and amplification. Analysis of shotgun sequenced environmental DNA, known as metagenomics, avoids amplification bias but generates fragmentary, non-overlapping sequence reads that cannot be clustered by existing OTU-finding methods. To circumvent these limitations, we developed PhylOTU, a computational workflow that identifies OTUs from metagenomic SSU-rRNA sequence data through the use of phylogenetic principles and probabilistic sequence profiles. Using simulated metagenomic data, we quantified the accuracy with which PhylOTU clusters reads into OTUs. Comparisons of PCR and shotgun sequenced SSU-rRNA markers derived from the global open ocean revealed that while PCR libraries identify more OTUs per sequenced residue, metagenomic libraries recover a greater taxonomic diversity of OTUs. In addition, we discover novel species, genera and families in the metagenomic libraries, including OTUs from phyla missed by analysis of PCR sequences. Taken together, these results suggest that PhylOTU enables characterization of part of the biosphere currently hidden from PCR-based surveys of diversity?     

Metabarcoding of fungal communities associated with bark beetles

Many species of fungi are closely allied with bark beetles, including many tree pathogens, but their species richness and patterns of distribution remain largely unknown. We established a protocol for metabarcoding of fungal communities directly from total genomic DNA extracted from individual beetles, showing that the ITS3/4 primer pair selectively amplifies the fungal ITS. Using three specimens of bark beetle from different species, we assess the fungal diversity associated with these specimens and the repeatability of these estimates in PCRs conducted with different primer tags. The combined replicates produced 727 fungal Operational Taxonomic Units (OTUs) for the specimen of Hylastes ater, 435 OTUs for Tomicus piniperda, and 294 OTUs for Trypodendron lineatum, while individual PCR reactions produced on average only 229, 54, and 31 OTUs for the three specimens, respectively. Yet, communities from PCR replicates were very similar in pairwise comparisons, in particular when considering species abundance, but differed greatly among the three beetle specimens. Different primer tags or the inclusion of amplicons in separate libraries did not impact the species composition. The ITS2 sequences were identified with the Lowest Common Ancestor approach and correspond to diverse lineages of fungi, including Ophiostomaceae and Leotiomycetes widely found to be tree pathogens. We conclude that Illumina MiSeq metabarcoding reliably captures fungal diversity associated with bark beetles, although numerous PCR replicates are recommended for an exhaustive sample. Direct PCR from beetle DNA extractions provides a rapid method for future surveys of fungal species diversity and their associations with bark beetles and environmental variables.     

Comparing diversity levels in environmental samples: DNA sequence capture and metabarcoding approaches using 18S and COI genes

Environmental DNA studies targeting multiple taxa using metabarcoding provide remarkable insights into levels of species diversity in any habitat. The main drawbacks are the presence of primer bias and difficulty in identifying rare species. We tested a DNA sequence‐capture method in parallel with the metabarcoding approach to reveal possible advantages of one method over the other. Both approaches were performed using the same eDNA samples and the same 18S and COI regions, followed by high throughput sequencing. Metabarcoded eDNA libraries were PCR amplified with one primer pair from 18S and COI genes. DNA sequence‐capture libraries were enriched with 3,639 baits targeting the same gene regions. We tested amplicon sequence variants (ASVs) and operational taxonomic units (OTUs) in silico approaches for both markers and methods, using for this purpose the metabarcoding data set. ASVs methods uncovered more species for the COI gene, whereas the opposite occurred for the 18S gene, suggesting that clustering reads into OTUs could bias diversity richness especially using 18S with relaxed thresholds. Additionally, metabarcoding and DNA sequence‐capture recovered 80%–90% of the control sample species. DNA sequence‐capture was 8x more expensive, nonetheless it identified 1.5x more species for COI and 13x more genera for 18S than metabarcoding. Both approaches offer reliable results, sharing ca. 40% species and 72% families and retrieve more taxa when nuclear and mitochondrial markers are combined. eDNA metabarcoding is quite well established and low‐cost, whereas DNA‐sequence capture for biodiversity assessment is still in its infancy, is more time‐consuming but provides more taxonomic assignments.     

Assessment of Microbial Richness in Pelagic Sediment of Andaman Sea by Bacterial Tag Encoded FLX Titanium Amplicon Pyrosequencing (bTEFAP)

Microbial diversity of 1,000 m deep pelagic sediment from off Coast of Andaman Sea was analyzed by a culture independent technique, bacterial tag encoded FLX titanium amplicon pyrosequencing. The hypervariable region of small subunit ribosomal rRNA gene covering V6–V9, was amplified from the metagenomic DNA and sequenced. We obtained 19,271 reads, of which 18,206 high quality sequences were subjected to diversity analysis. A total of 305 operational taxonomic units (OTUs) were obtained corresponding to the members of firmicutes, proteobacteria, plantomycetes, actinobacteria, chloroflexi, bacteroidetes, and verucomicrobium. Firmicutes was the predominant phylum, which was largely represented with the family bacillaceae. More than 44 % of sequence reads could not be classified up to the species level and more than 14 % of the reads could not be assigned to any genus. Thus, the data indicates the possibility for the presence of uncultivable or unidentified novel bacterial species. In addition, the community structure identified in this study significantly differs with other reports from marine sediments.     

Soil drying procedure affects the DNA quantification of <Emphasis Type="Italic">Lactarius vinosus</Emphasis> but does not change the fungal community composition

Drying soil samples before DNA extraction is commonly used for specific fungal DNA quantification and metabarcoding studies, but the impact of different drying procedures on both the specific fungal DNA quantity and the fungal community composition has not been analyzed. We tested three different drying procedures (freeze-drying, oven-drying, and room temperature) on 12 different soil samples to determine (a) the soil mycelium biomass of the ectomycorrhizal species Lactarius vinosus using qPCR with a specifically designed TaqMan® probe and (b) the fungal community composition and diversity using the PacBio® RS II sequencing platform. Mycelium biomass of L. vinosus was significantly greater in the freeze-dried soil samples than in samples dried at oven and room temperature. However, drying procedures had no effect on fungal community composition or on fungal diversity. In addition, there were no significant differences in the proportions of fungi according to their functional roles (moulds vs. mycorrhizal species) in response to drying procedures. Only six out of 1139 operational taxonomic units (OTUs) had increased their relative proportions after soil drying at room temperature, with five of these OTUs classified as mould or yeast species. However, the magnitude of these changes was small, with an overall increase in relative abundance of these OTUs of approximately 2 %. These results suggest that DNA degradation may occur especially after drying soil samples at room temperature, but affecting equally nearly all fungi and therefore causing no significant differences in diversity and community composition. Despite the minimal effects caused by the drying procedures at the fungal community composition, freeze-drying resulted in higher concentrations of L. vinosus DNA and prevented potential colonization from opportunistic species.     

A diverse bacterial community in an anoxic quinoline-degrading bioreactor determined by using pyrosequencing and clone library analysis

There is a concern of whether the structure and diversity of a microbial community can be effectively revealed by short-length pyrosequencing reads. In this study, we performed a microbial community analysis on a sample from a high-efficiency denitrifying quinoline-degrading bioreactor and compared the results generated by pyrosequencing with those generated by clone library technology. By both technologies, 16S rRNA gene analysis indicated that the bacteria in the sample were closely related to, for example, Proteobacteria, Actinobacteria, and Bacteroidetes. The sequences belonging to Rhodococcus were the most predominant, and Pseudomonas, Sphingomonas, Acidovorax, and Zoogloea were also abundant. Both methods revealed a similar overall bacterial community structure. However, the 622 pyrosequencing reads of the hypervariable V3 region of the 16S rRNA gene revealed much higher bacterial diversity than the 130 sequences from the full-length 16S rRNA gene clone library. The 92 operational taxonomic unit (OTUs) detected using pyrosequencing belonged to 45 families, whereas the 37 OTUs found in the clone library belonged to 25 families. Most sequences obtained from the clone library had equivalents in the pyrosequencing reads. However, 64 OTUs detected by pyrosequencing were not represented in the clone library. Our results demonstrate that pyrosequencing of the V3 region of the 16S rRNA gene is not only a powerful tool for discovering low-abundance bacterial populations but is also reliable for dissecting the bacterial community structure in a wastewater environment.