The kingdom Fungi is estimated to include 1.5 million or more species, playing key roles as decomposers, mutualists, and parasites in every biome on the earth. To comprehensively understand the diversity and ecology of this huge kingdom, DNA barcoding targeting the internal transcribed spacer (ITS) region of the nuclear ribosomal repeat has been regarded as a prerequisite procedure. By extensively surveying ITS sequences in public databases, we designed new ITS primers with improved coverage across diverse taxonomic groups of fungi compared to existing primers. An in silico analysis based on public sequence databases indicated that the newly designed primers matched 99% of ascomycete and basidiomycete ITS taxa (species, subspecies or varieties), causing little taxonomic bias toward either fungal group. Two of the newly designed primers could inhibit the amplification of plant sequences and would enable the selective investigation of fungal communities in mycorrhizal associations, soil, and other types of environmental samples. Optimal PCR conditions for the primers were explored in an in vitro investigation. The new primers developed in this study will provide a basis for ecological studies on the diversity and community structures of fungi in the era of massive DNA sequencing.

In terrestrial ecosystems, plant roots are colonized by various clades of mycorrhizal and endophytic fungi. Focused on the root systems of an oak-dominated temperate forest in Japan, we used 454 pyrosequencing to explore how phylogenetically diverse fungi constitute an ecological community of multiple ecotypes. In total, 345 operational taxonomic units (OTUs) of fungi were found from 159 terminal-root samples from 12 plant species occurring in the forest. Due to the dominance of an oak species (Quercus serrata), diverse ectomycorrhizal clades such as Russula, Lactarius, Cortinarius, Tomentella, Amanita, Boletus, and Cenococcum were observed. Unexpectedly, the root-associated fungal community was dominated by root-endophytic ascomycetes in Helotiales, Chaetothyriales, and Rhytismatales. Overall, 55.3% of root samples were colonized by both the commonly observed ascomycetes and ectomycorrhizal fungi; 75.0% of the root samples of the dominant Q. serrata were so cocolonized. Overall, this study revealed that root-associated fungal communities of oak-dominated temperate forests were dominated not only by ectomycorrhizal fungi but also by diverse root endophytes and that potential ecological interactions between the two ecotypes may be important to understand the complex assembly processes of belowground fungal communities.

BackgroundMany local workers have been involved in rescue and reconstruction duties since the Great East Japan Earthquake (GEJE) on March 11, 2011. These workers continuously confront diverse stressors as both survivors and relief and reconstruction workers. However, little is known about the psychological sequelae among these workers. Thus, we assessed the prevalence of and personal/workplace risk factors for probable post-traumatic stress disorder (PTSD), probable depression, and high general psychological distress in this population.MethodsParticipants (N = 1294; overall response rate, 82.9%) were workers (firefighters, n = 327; local municipality workers, n = 610; hospital medical workers, n = 357) in coastal areas of Miyagi prefecture. The study was cross-sectional and conducted 14 months after the GEJE using a self-administered questionnaire which included the PTSD Checklist–Specific Version, the Patient Health Questionnaire-9, and the K6 scale. Significant risk factors from bivariate analysis, such as displacement, dead or missing family member(s), near-death experience, disaster related work, lack of communication, and lack of rest were considered potential factors in probable PTSD, probable depression, and high general psychological distress, and were entered into the multivariable logistic regression model.ResultsThe prevalence of probable PTSD, probable depression, and high general psychological distress was higher among municipality (6.6%, 15.9%, and 14.9%, respectively) and medical (6.6%, 14.3%, and 14.5%, respectively) workers than among firefighters (1.6%, 3.8%, and 2.6%, respectively). Lack of rest was associated with increased risk of PTSD and depression in municipality and medical workers; lack of communication was linked to increased PTSD risk in medical workers and depression in municipality and medical workers; and involvement in disaster-related work was associated with increased PTSD and depression risk in municipality workers.ConclusionsThe present results indicate that at 14 months after the GEJE, mental health consequences differed between occupations. High preparedness, early mental health interventions, and the return of ordinary working conditions might have contributed to the relative mental health resilience of the firefighters. Unlike the direct effects of disasters, workplace risk factors can be modified after disasters; thus, we should develop countermeasures to improve the working conditions of local disaster relief and reconstruction workers.

The use of environmental DNA (eDNA) methods for community analysis has recently been developed. High-throughput parallel DNA sequencing (HTS), called eDNA metabarcoding, has been increasingly used in eDNA studies to examine multiple species. However, eDNA metabarcoding methodology requires validation based on traditional methods in all natural ecosystems before a reliable method can be established. To date, relatively few studies have performed eDNA metabarcoding of fishes in aquatic environments where fish communities were intensively surveyed using multiple traditional methods. Here, we have compared fish communities’ data from eDNA metabarcoding with seven conventional multiple capture methods in 31 backwater lakes in Hokkaido, Japan. We found that capture and field surveys of fishes were often interrupted by macrophytes and muddy sediments in the 31 lakes. We sampled 1 L of the surface water and analyzed eDNA using HTS. We also surveyed the fish communities using seven different capture methods, including various types of nets and electrofishing. At some sites, we could not detect any eDNA, presumably because of the polymerase chain reaction (PCR) inhibition. We also detected the marine fish species as sewage-derived eDNA. Comparisons of eDNA metabarcoding and capture methods showed that the detected fish communities were similar between the two methods, with an overlap of 70%. Thus, our study suggests that to detect fish communities in backwater lakes, the performance of eDNA metabarcoding with the use of 1 L surface water sampling is similar to that of capturing methods. Therefore, eDNA metabarcoding can be used for fish community analysis but environmental factors that can cause PCR inhibition, should be considered in eDNA applications.

Recent advances in environmental DNA (eDNA) analysis using high‐throughput sequencing (HTS) provide a noninvasive way to evaluate the intraspecific genetic diversity of aquatic macroorganisms. However, erroneous sequences present in HTS data can result in false positive haplotypes; therefore, reliable strategies are necessary to eliminate such erroneous sequences when evaluating intraspecific genetic diversity using eDNA metabarcoding. In this study, we propose an approach combining denoising using amplicon sequence variant (ASV) method and the removal of haplotypes with low detection rates. A mixture of rearing water of Ayu (Plecoglossus altivelis altivelis) was used as an eDNA sample. In total, nine haplotypes of Ayu mitochondrial D‐loop region were contained in the sample and amplified by two‐step tailed PCR. The 15 PCR replicates indexed with different tags were prepared from the eDNA sample to compare the detection rates between true haplotypes and false positive haplotypes. All PCR replications were sequenced by HTS, and the total number of detected true haplotypes and false positive haplotypes was compared with and without denoising using the two types of ASV methods, Divisive Amplicon Denoising Algorithm 2 (DADA2) and UNOISE3. The use of both ASV methods considerably reduced the number of false positive haplotypes. Moreover, all true haplotypes were detected in all 15 PCR replicates, whereas false positive haplotypes had detection rates varying from 1/15 to 15/15. Thus, by removing haplotypes with lower detection rates than 15/15, the number of false positive haplotypes was further reduced. The approach proposed in this study successfully eliminated most of the false positive haplotypes in the HTS data obtained from eDNA samples, which allowed us to improve the detection accuracy for evaluating intraspecific genetic diversity using eDNA analysis.