Molecular investigation of genet distribution and genetic variation of Cortinarius rotundisporus in eastern Australian sclerophyll forests

The size and distribution of Cortinarius rotundisporus genets at three sclerophyll forest field sites in New South Wales, Australia, were estimated by using microsatellite-primed PCR (MS-PCR) of DNA extracted from sporocarp tissue. MS-PCR fingerprints generated with the primers (GTG)₅ and (GACA)₄ indicated that two to five genets were present at each site, with each site being characterized by a single large genet (9–30 m in diameter). Analysis of internal transcribed spacer (ITS)-RFLP patterns from individual sporocarps used in the study suggested that three distinct RFLP types were present in the sampled C. rotundisporus population. ITS sequence data indicate that the three RFLP types had less than 88.4% sequence identity to each other, strongly suggesting that C. rotundisporus is a complex of three species.     

Host effects on ectomycorrhizal fungal communities: insight from eight host species in mixed conifer-broadleaf forests

To advance our understanding of host effects on the community structure of ectomycorrhizal fungi (EMF), EMF communities were compared among different host species, genera and families in two mixed conifer-broadleaf forests in Japan. Using molecular identification methods we examined EMF root tips of eight coexisting species belonging to six genera (three families): Abies and Tsuga (Pinaceae), Betula and Carpinus (Betulaceae) and Fagus and Quercus (Fagaceae). In total, 205 EMF species were detected, and the total richness was estimated to exceed 300 species using major estimators. Of the 55 EMF species occurring three or more times, eight showed significantly biased host preference. A Mantel test showed a significantly negative correlation between EMF community similarity and host taxonomic distance. Detrended correspondence analysis separated EMF communities mainly by host taxonomic and successional status. Thus, EMF communities are similar on hosts with similar taxonomic and successional status. A significant proportion of EMF exhibited host specificity, which may contribute to the extremely diverse EMF community in conifer-broadleaf forests.     

ITS1 versus ITS2 as DNA metabarcodes for fungi

The nuclear ribosomal Internal Transcribed Spacer ITS region is widely used as a DNA metabarcoding marker to characterize the diversity and composition of fungal communities. In amplicon pyrosequencing studies of fungal diversity, one of the spacers ITS1 or ITS2 of the ITS region is normally used. In this methodological study we evaluate the usability of ITS1 vs. ITS2 as a DNA metabarcoding marker for fungi. We analyse three data sets: two comprising ITS1 and ITS2 sequences of known taxonomic affiliations and a third comprising ITS1 and ITS2 environmental amplicon pyrosequencing data. Clustering analyses of sequences with known taxonomy using the bioinformatics pipeline ClustEx revealed that a 97% similarity cut‐off represent a reasonable threshold for estimating the number of known species in the data sets for both ITS1 and ITS2. However, no single threshold value worked well for all fungi at the same time within the curated UNITE database, and we found that the Operational Taxonomic Unit (OTU) concept is not easily translated into the level of species because many species are distributed over several clusters. Clustering analyses of the 134 692 ITS1 and ITS2 pyrosequences using a 97% similarity cut‐off revealed a high similarity between the two data sets when it comes to taxonomic coverage. Although some groups are under‐ or unrepresented in the two data sets due to, e.g. primer mismatches, our results indicate that ITS1 and ITS2 to a large extent yield similar results when used as DNA metabarcodes for fungi.     

Pioneer dwarf willow may facilitate tree succession by providing late colonizers with compatible ectomycorrhizal fungi in a primary successional volcanic desert

To advance understanding of the contribution of ectomycorrhizal (ECM) fungi to tree successional processes, natural establishment patterns of secondary colonizing hosts and their ECM fungal communities were investigated with special reference to pioneer hosts. In the volcanic desert on Mount Fuji, Japan, vegetation is sparsely distributed, resembling islands in a sea of scoria. Of 509 vegetation islands in the research area, 161 contained Salix reinii (Salix), the first colonizing ECM host species. The spatial coincidence between secondary colonizing timber species and Salix was analysed, and ECM fungal communities were studied using molecular identification methods. I found 39 and 26 individuals of Betula ermanii and Larix kaempferi, respectively. Without exception, these individuals were all accompanied by Salix. The ECM fungal communities of these timber species showed high similarity to that of Salix and were dominated by generalists that were compatible with two or more plant families. In this desert, available ECM propagules are limited. Pioneer Salix may contribute to tree succession by providing adjacent late colonizers with compatible ECM fungal symbionts.     

Asterostroma species (Basidiomycota) from mangrove forests in Japan

A new homobasidiomycete, Asterostroma macrosporum, was found in mangrove forests of Iriomote Island, Japan. This species is morphologically characterized by having resupinate basidiomata, a monomitic (asterodimitic) hyphal system, simple septate generative hyphae, dextrinoid asterosetae, four sterigmate basidia and globose, tuberculate and amyloid basidiospores measuring 8.5–11 × 7.5–9 μm. It is similar to A. muscicola, but basidiospores in the latter are smaller (7–8 × 5.5–7 μm). Furthermore, phylogenetic analysis using internal transcribed spacer (ITS) region revealed that A. macrosporum is distinctly separated from A. muscicola. In Japan, A. muscicola is widely distributed in warm-temperate to subtropical regions, growing on a variety of broadleaved trees including mangroves, while A. macrosporum has been found only on mangroves.     

A simple method of genomic DNA extraction suitable for analysis of bulk fungal strains

Aims: A simple and rapid method (designated thermolysis) for extracting genomic DNA from bulk fungal strains was described. Methods and Results: In the thermolysis method, a few mycelia or yeast cells were first rinsed with pure water to remove potential PCR inhibitors and then incubated in a lysis buffer at 85°C to break down cell walls and membranes. This method was used to extract genomic DNA from large numbers of fungal strains (more than 92 species, 35 genera of three phyla) isolated from different sections of natural Ophiocordyceps sinensis specimens. Regions of interest from high as well as single‐copy number genes were successfully amplified from the extracted DNA samples. The DNA samples obtained by this method can be stored at ?20°C for over 1 year. Conclusions: The method was effective, easy and fast and allowed batch DNA extraction from multiple fungal isolates. Significance and Impact of Study: Use of the thermolysis method will allow researchers to obtain DNA from fungi quickly for use in molecular assays. This method requires only minute quantities of starting material and is suitable for diverse fungal species.     

辽宁省9种兰科植物根内与根际土壤中真菌群落结构的差异

兰科植物的生存及生长高度依赖其根中的共生真菌, 其中的菌根真菌更是对兰科植物的种子萌发与后续生长有着非常重要的作用, 研究兰科植物根中的真菌, 尤其是菌根真菌, 对兰科植物的保护有重要作用。该研究利用第二代测序技术, 对中国辽宁省境内的9种属于极小种群的兰科植物的根、根际土和根围土中的真菌群落和菌根真菌组成进行了研究。结果显示, 兰科植物根中的真菌群落和根际土、根围土中的真菌群落具有显著差异。兰科植物根中的总操作分类单元(OTU)数目远小于根际土和根围土中的总OTU数目。同时, 兰科植物根中菌根真菌的种类和丰度与根际土、根围土中菌根真菌的种类与丰度没有明显联系。FunGuild分析结果显示, 丛枝菌根真菌在根际土与根围土中的丰度非常高, 但在兰科植物的根中却数量极少。这些结果表明, 兰科植物根中的真菌群落与土壤中的真菌群落在一定程度上是相互独立的。     

广东湛江湾可培养丝状真菌分布状况的初步调查

通过一次航行对广东湛江湾19个站位的表层和底层海水进行采样。经过对样品的分离纯化,共获得253株丝状真菌菌株。通过测序获得了121个rDNA-ITS序列,并已提交给GenBank。经统计分析发现,湛江湾表层和底层海水真菌数量总体上表现为由湾内向湾口逐渐减少的水平分布格局;湛江湾表层丝状真菌的数量略多于底层,在海水表层和底层中真菌种类和数量的分布规律相似。根据形态特征及rDNA-ITS的Blast同源性分析,这些菌株分属于18个属和32个分类单元,其中包括7个海洋真菌新记录属。结果表明,湛江湾丝状真菌多样性Shannon指数达2.75,物种优势度变化范围为30.90%–0.02%。枝孢属Cladosporium的优势度最高,为湛江湾优势种群,其次是青霉属Penicillium、侧齿霉属Engyodontium、曲霉属Aspergillus、枝顶孢属Acremonium等。湛江湾表层和底层海水真菌群落的Jaccard相似系数为0.42。     

A fungal mock community control for amplicon sequencing experiments

Microbial ecology has been profoundly advanced by the ability to profile complex microbial communities by sequencing of marker genes amplified from environmental samples. However, inclusion of appropriate controls is vital to revealing the limitations and biases of this technique. “Mock community” samples, in which the composition and relative abundances of community members are known, are particularly valuable for guiding library preparation and data processing decisions. I generated a set of three mock communities using 19 different fungal taxa and demonstrate their utility by contrasting amplicon sequencing data obtained for the same communities under modifications to PCR conditions during library preparation. Increasing the number of PCR cycles elevated rates of chimera formation, and of errors in the final data set. Extension time during PCR had little impact on chimera formation, error rate or observed community structure. Polymerase fidelity impacted error rates significantly. Despite a high error rate, a master mix optimized to minimize amplification bias yielded profiles that were most similar to the true community structure. Bias against particular taxa differed among ITS1 vs. ITS2 loci. Preclustering nearly identical reads substantially reduced error rates, but did not improve similarity to the expected community structure. Inaccuracies in amplicon sequence‐based estimates of fungal community structure were associated with amplification bias and size selection processes, as well as variable culling rates among reads from different taxa. In some cases, the numerically dominant taxon was completely absent from final data sets, highlighting the need for further methodological improvements to avoid biased observations of community profiles.     

Biomass and compositional responses of ectomycorrhizal fungal hyphae to elevated CO2 and nitrogen fertilization

The extramatrical mycelia (EMM) of ectomycorrhizal fungi make up a large proportion of the microbial diversity and biomass in temperate forest soils. Thus, their response to elevated CO(2) can have large effects on plant nutrient acquisition and carbon movement through forests. Here, the effects of CO(2) and nitrogen (N) fertilization on EMM biomass and community structure in Pinus taeda forest plots were examined using sand-filled mesh bags buried in the field, the contents of which were analyzed by phospholipid fatty acid (PLFA) and DNA sequencing. A total of 2138 sequences comprising 295 taxa were recovered; most (83.5%) were from ectomycorrhizal fungal taxa. No biomass increase was detected in elevated CO(2) plots relative to control plots, but individual taxa responded to both CO(2) and N fertilization, four of the six most abundant taxa were less frequent in N-fertilized plots. Thelephoroid and athelioid taxa were both frequent and abundant as EMM, and thelephoroid richness was extremely high. Russula and Cortinariaceae taxa were less abundant and boletoid taxa were more abundant as EMM relative to ectomycorrhizas. The EMM community, sampled across seasons and years, was dynamic with a high degree of interspecific variation in response to CO(2) enrichment and N fertilization.     

Contrasting ectomycorrhizal fungal communities on the roots of co-occurring oaks (Quercus spp.) in a California woodland

Plant host species is considered an important factor influencing ectomycorrhizal (EM) communities. To gain insights into the role of host species in structuring EM communities, EM communities on sympatric oak (Quercus) species were compared in the Sierra Nevada foothills of California. Using molecular methods (polymerase chain reaction, cloning, restriction fragment length polymorphism and DNA sequencing), EM fungi on roots of deciduous Quercus douglasii and evergreen Quercus wislizeni trees were identified from 64 soil cores. The total EM species richness was 140, of which 40 taxa were detected on both oak hosts. Greater diversity and frequency of EM fungi with epigeous fruiting habit were found on Q. wislizeni, while taxa in the Ascomycota were more frequent and diverse on Q. douglasii. Using ordination, it was determined that both soil extractable phosphorus and oak host species explained a significant proportion of the variation in EM species distribution. These results indicate that plant host species can be an important factor influencing EM fungal community composition, even within congeneric trees.