Fungal Sequences Research Articles

Microbiome function predicts amphibian chytridiomycosis disease dynamics

BackgroundThe fungal pathogenBatrachochytrium dendrobatidis (Bd) threatens amphibian biodiversity and ecosystem stability worldwide. Amphibian skin microbial community structure has been linked to the clinical outcome of Bd infections, yet its overall functional importance is poorly understood.MethodsMicrobiome taxonomic and functional profiles were assessed using high-throughput bacterial 16S rRNA and fungal ITS2 gene sequencing, bacterial shotgun metagenomics and skin mucosal metabolomics. We sampled 56 wild midwife toads (Alytes obstetricans) from montane populations exhibiting Bd epizootic or enzootic disease dynamics. In addition, to assess whether disease-specific microbiome profiles were linked to microbe-mediated protection or Bd-induced perturbation, we performed a laboratory Bd challenge experiment whereby 40 young adult A. obstetricans were exposed to Bd or a control sham infection. We measured temporal changes in the microbiome as well as functional profiles of Bd-exposed and control animals at peak infection.ResultsMicrobiome community structure and function differed in wild populations based on infection history and in experimental control versus Bd-exposed animals. Bd exposure in the laboratory resulted in dynamic changes in microbiome community structure and functional differences, with infection clearance in all but one infected animal. Sphingobacterium, Stenotrophomonas and an unclassified Commamonadaceae were associated with wild epizootic dynamics and also had reduced abundance in laboratory Bd-exposed animals that cleared infection, indicating a negative association with Bd resistance. This was further supported by microbe-metabolite integration which identified functionally relevant taxa driving disease outcome, of which Sphingobacterium and Bd were most influential in wild epizootic dynamics. The strong correlation between microbial taxonomic community composition and skin metabolome in the laboratory and field is inconsistent with microbial functional redundancy, indicating that differences in microbial taxonomy drive functional variation. Shotgun metagenomic analyses support these findings, with similar disease-associated patterns in beta diversity. Analysis of differentially abundant bacterial genes and pathways indicated that bacterial environmental sensing and Bd resource competition are likely to be important in driving infection outcomes.ConclusionsBd infection drives altered microbiome taxonomic and functional profiles across laboratory and field environments. Our application of multi-omics analyses in experimental and field settings robustly predicts Bd disease dynamics and identifies novel candidate biomarkers of infection.6u4HXENqNsd5f8SBedLBdGVideo

Multiple Fungi May Connect the Roots of an Orchid (Cypripedium reginae) and Ash (Fraxinus nigra) in Western Newfoundland.

Showy lady's slipper (Cypripedium reginae Walter, Orchidaceae) and black ash (Fraxinus nigra Marshall, Oleaceae) often co-occur in close proximity in fens in western Newfoundland, Canada. Metabarcoding of DNA extracted from root samples of both species following surface sterilization, and others without surface sterilization was used to determine if there were shared fungal endophytes in the roots of both species that could form a common mycorrhizal network between them. A wide variety of fungi were recovered from primers amplifying the nuclear ribosomal internal transcribed spacer region (ITS2). Sixty-six fungal sequences were shared by surface-sterilized roots of both orchid and ash, among them arbuscular mycorrhizal fungi (Claroideoglomus, Dominikia, Glomus and Rhizophagus), ectomycorrhizal fungi (Inocybe and Tomentella), the broad-host root endophyte Cadophora orchidicola, along with root pathogens (Dactylonectria, Ilyonectria, Pyricularia, and Xylomyces) and fungi of unknown function. There appear to be multiple fungi that could form a common mycorrhizal network between C. reginae and F. nigra, which might explain their frequent co-occurrence. Transfer of nutrients or carbon between the orchid and ash via one or more of the shared fungal endophytes remains to be demonstrated.

Humidity and low pH boost occurrence of Onygenales fungi in soil at global scale

Soils are important reservoirs for potential human pathogens and opportunistic fungi such as the dermatophyte or dimorphic fungi in the order Onygenales. In soils, these taxa are decomposers but many of them have the potential to cause respiratory and skin diseases in humans and, in some cases, systemic infections. Even so, the factors that determine the biogeography and ecology of order Onygenales remain largely undocumented. To address this knowledge gap, we surveyed members of Onygenales from topsoil fungal communities at 235 sites across six continents and provided a first global atlas. We retrieved 4.3% of the total fungal sequences (∼420 Onygenales) across nine biomes ranging from deserts to tropical forests. This work advances our knowledge on the ecology and global distribution of order Onygenales and suggests the hypothesis that wet and acid soils support the larger proportions of these fungi, while their richness is constrained by aridity.

Plant organ and sampling time point determine the taxonomic structure of microbial communities associated to apple plants in the orchard environment

Plant-associated microbial communities interact with their host and are important components of the biodiversity of natural and agro-ecosystems. Scarce knowledge is available on the establishment of plant microbiota in perennial woody plants. In this work the variability in bacterial and fungal communities in aboveground organs was analysed in leaves, bark, flowers and fruits on three apple cultivars (Gala, Fuji and Golden), in order to understand changes of the microbiota community structure from orchard planting to the first year of fruit production. Our results indicate that Proteobacteria, Bacterioidetes, Actinobacteria and Firmiculites were the dominant bacterial phyla across all samples. The majority of fungal sequences were assigned to Ascomycota and Basidiomycota. The bacterial genera Pseudomonas and Sphingomonas, and the fungal genera Aureobasidium and Filobasidium, represented a major component of the aboveground microbiota. Different parts of the apple plant harboured a specific microbiota and the effect of plant organ on the bacterial and fungal taxonomic structure exceeded the influence of sampling time and plant genotype. This work highlights the specificity of the microbiota associated with aboveground apple organs, changes of the microbiota composition during the plant development from orchard planting to the first year of fruit production, and the negligible effects of apple cultivar.

Fungal Diversity in Sediments From Deep-Sea Extreme Ecosystems: Insights Into Low- and High-Temperature Hydrothermal Vents, and an Oxygen Minimum Zone in the Southern Gulf of California, Mexico

Deep-sea sediments are vast microbial habitats that cover almost two-thirds of the planet’s surface. Particularly, sediments in hydrothermal vents and oxygen minimum zones (OMZ) represent emblematic, and poorly understood extreme niches that pose strong selective pressures to life, representing untapped reservoirs of a unique microbial diversity. Nonetheless, the mycobiota in these systems remains relatively unknown. Here, we explored fungal diversity and community structure in deep-sea sediments collected from low- and high-temperature vent systems (Pescadero Basin, Pescadero Transform Fault, and Alarcón Rise) and an OMZ (Alfonso Basin) in the southern Gulf of California, by using high-throughput Illumina sequencing of the ITS1 region. We identified 102 fungal amplicon sequence variants (ASVs), principally affiliated to the Ascomycota and Basidiomycota. Our results also evidenced a high proportion of widely distributed, uncultured phylotypes (e.g., unknown clades closely related to the Basidiomycota such asFungisp. 18 and 19 formerly reported from deep-sea), evidencing the copious occurrence of novel fungal lineages with limited distribution to deep-sea sediments. Overall, the largest number of ASVs was recovered from high-temperature vent systems, corroborating these as diversity hotspots. In addition, clustering patterns across samples and the FUNGuild analysis revealed characteristic assemblages in each ecosystem, which could be linked to site-specific processes. This study provides baseline data paving the way for a better understanding of deep-sea fungal diversity, evidencing its potential importance in ecosystem functioning.

Diversity and Ecological Guild Analysis of the Oil Palm Fungal Microbiome Across Root, Rhizosphere, and Soil Compartments.

The rhizosphere microbiome is a major determinant of plant health, which can interact with the host directly and indirectly to promote or suppress productivity. Oil palm is one of the world’s most important crops, constituting over a third of global vegetable oil production. Currently there is little understanding of the oil palm microbiome and its contribution to plant health and productivity, with existing knowledge based almost entirely on culture dependent studies. We investigated the diversity and composition of the oil palm fungal microbiome in the bulk soil, rhizosphere soil, and roots of 2-, 18-, and 35-year old plantations in Selangor, Malaysia. The fungal community showed substantial variation between the plantations, accounting for 19.7% of community composition, with compartment (root, rhizosphere soil, and bulk soil), and soil properties (pH, C, N, and P) contributing 6.5 and 7.2% of community variation, respectively. Rhizosphere soil and roots supported distinct communities compared to the bulk soil, with significant enrichment of Agaricomycetes, Glomeromycetes, and Lecanoromycetes in roots. Several putative plant pathogens were abundant in roots in all the plantations, including taxa related to Prospodicola mexicana and Pleurostoma sp. The mycorrhizal status and dependency of oil palm has yet to be established, and using 18S rRNA primers we found considerable between-site variation in Glomeromycotinian community composition, accounting for 31.2% of variation. There was evidence for the selection of Glomeromycotinian communities in oil palm roots in the older plantations but compartment had a weak effect on community composition, accounting for 3.9% of variation, while soil variables accounted for 9% of community variation. While diverse Mucoromycotinian fungi were detected, they showed very low abundance and diversity within roots compared to bulk soil, and were not closely related to taxa which have been linked to fine root endophyte mycorrhizal morphology. Many of the fungal sequences showed low similarity to established genera, indicating the presence of substantial novel diversity with significance for plant health within the oil palm microbiome.

Constructing Micro-Landscapes: Management and Selection Practices on Microbial Communities in a Traditional Fermented Beverage

Colonche is a traditional beverage produced in Mexico by the fermentation of fruits of several cacti species. In the Meridional Central Plateau region of Mexico, where this study was conducted, it is mainly produced with fruits of Opuntia streptacantha; there, the producers perform spontaneous fermentation and/or fermentations through inoculums. Several factors can change the microbial community structure and dynamics through the fermentation process, but little attention has been directed to evaluate what type and extent of change the human practices have over the microbial communities. This study aims to assess the microbiota under spontaneous and inoculated fermentation techniques, the microorganisms present in the inoculums and containers, and the changes of microbiota during the process of producing colonche with different techniques. We used next-generation sequencing of the V3-V4 regions of the 16S rRNA gene and the ITS2, to characterize bacterial and fungal diversity associated with the different fermentation techniques. We identified 701 bacterial and 203 fungal amplicon sequence variants (ASVs) belonging to 173 bacterial and 187 fungal genera. The alpha and beta diversity analysis confirmed that both types of fermentation practices displayed differences in richness, diversity, and community structure. Richness of bacteria in spontaneous fermentation (0D = 136 ± 0.433) was higher than in the inoculated samples (0D = 128 ± 0.929), while fungal richness in the inoculated samples (0D = 32 ± 0.539) was higher than in spontaneous samples (0D = 19 ± 0.917). We identified bacterial groups like Lactobacillus, Leuconostoc, Pediococcus and the Saccharomyces yeast shared in ferments managed with different practices; these organisms are commonly related to the quality of the fermentation process. We identified that clay pots, where spontaneous fermentation is carried out, have an outstanding diversity of fungal and bacterial richness involved in fermentation, being valuable reservoirs of microorganisms for future fermentations. The inoculums displayed the lowest richness and diversity of bacterial and fungal communities suggesting unconscious selection on specific microbial consortia. The beta diversity analysis identified an overlap in microbial communities for both types of fermentation practices, which might reflect a shared composition of microorganisms occurring in the Opuntia streptacantha substrate. The variation in the spontaneous bacterial community is consistent with alpha diversity data, while fungal communities showed less differences among treatments, probably due to the high abundance and dominance of Saccharomyces. This information illustrates how traditional management guides selection and may drive changes in the microbial consortia to produce unique fermented beverages through specific fermentation practices. Although further studies are needed to analyze more specifically the advantages of each fermentation type over the quality of the product, our current analysis supports the role of traditional knowledge driving it and the relevance of plans for its conservation.

Global taxonomic and phylogenetic assembly of AM fungi.

Arbuscular mycorrhizal (AM) fungi are a ubiquitous group of plant symbionts, yet processes underlying their global assembly - in particular the roles of dispersal limitation and historical drivers - remain poorly understood. Because earlier studies have reported niche conservatism in AM fungi, we hypothesized that variation in taxonomic community composition (i.e.,unweighted by taxon relatedness) should resemble variation in phylogenetic community composition (i.e., weighted by taxon relatedness) which reflects ancestral adaptations to historical habitat gradients. Because of the presumed strong dispersal ability of AM fungi, we also anticipated that the large-scale structure of AM fungal communities would track environmental conditions without regional discontinuity. We used recently published AM fungal sequence data (small-subunit ribosomal RNA gene) from soil samples collected worldwide to reconstruct global patterns in taxonomic and phylogenetic community variation. The taxonomic structure of AM fungal communities was primarily driven by habitat conditions, with limited regional differentiation, and there were two well-supported clusters of communities - occurring in cold and warm conditions. Phylogenetic structure was driven by the same factors, though all relationships were markedly weaker. This suggests that niche conservatism with respect to habitat associations is weakly expressed in AM fungal communities. We conclude that the composition of AM fungal communities tracks major climatic and edaphic gradients, with the effects of dispersal limitation and historic factors considerably less apparent than those of climate and soil.

Seasonal airway microbiome and transcriptome interactions promote childhood asthma exacerbations

Seasonal variation in respiratory illnesses and exacerbations in pediatric populations with asthma is well described, though whether upper airway microbes play season-specific roles in these events is unknown. We hypothesized that nasal microbiota composition is seasonally dynamic and that discrete microbe-host interactions modify risk of asthma exacerbation in a season-specific manner. Repeated nasal samples from children with exacerbation-prone asthma collected during periods of respiratory health (baseline; n= 181 samples) or first captured respiratory illness (n= 97) across all seasons, underwent bacterial (16S ribosomal RNA gene) and fungal (internal transcribed spacer region 2) biomarker sequencing. Virus detection was performed by multiplex PCR. Paired nasal transcriptome data were examined for seasonal dynamics and integrative analyses. Upper airway bacterial and fungal microbiota and rhinovirus detection exhibited significant seasonal dynamics. In seasonally adjusted analysis, variation in both baseline and respiratory illness microbiota related to subsequent exacerbation. Specifically, in the fall, when respiratory illness and exacerbation events were most frequent, several Moraxella and Haemophilus members were enriched both in virus-positive respiratory illnesses and those that progressed to exacerbations. The abundance of 2 discrete bacterial networks, characteristically comprising either Streptococcus or Staphylococcus, exhibited opposing interactions with an exacerbation-associated SMAD3 nasal epithelial transcriptional module to significantly increase the odds of subsequent exacerbation (odds ratio= 14.7, 95% confidence interval= 1.50-144, P= .02; odds ratio= 39.17, 95% confidence interval= 2.44-626, P= .008, respectively). Upper airway microbiomes covary with season and with seasonal trends in respiratory illnesses and asthma exacerbations. Seasonally adjusted analyses reveal specific bacteria-host interactions that significantly increase risk of asthma exacerbation in these children.

Screening of endophytic fungi as potential antagonistic agents of Pyricularia oryzae and evaluation of their ability in producing hydrolytic enzymes

Abstract. Putri ND, Sulistyowati L, Aini LQ, Muhibuddin A, Trianti I. 2022. Screening of endophytic fungi as potential antagonistic agents of Pyricularia oryzae and evaluation of their ability in producing hydrolytic enzymes. Biodiversitas 23: 1048-1057. One of the most important phytopathogenic fungi is Pyricularia oryzae, as it is the causative agent of rice blast diseases, which is the most destructive and detrimental disease in rice. Biological control using endophytic fungi can be an alternative to control blast diseases. Endophytic fungi are fungi that colonize internal plant tissues without apparently damaging the host. The aim of this study was to isolate endophytic fungi that produce hydrolytic enzymes and to investigate their ability to suppress blast disease. In this study, fifty strains of endophytic fungi were successfully obtained from rice leaves. Three fungi with the highest inhibition against P. oryzae were identified as Trichoderma asperellum, Curvularia chiangmaiensis, and Fusarium solani by analyzing fungal ITS sequence. They can produce chitinase ranging from 1.43 to 151 ?g/mL and cellulase ranging from 1.83 to 4.09 ?g/mL, which were hydrolytic enzymes that account for the lysis of phytopathogens. This enzymatic activity can cause damage and degradation of cell walls. SEM results revealed that these endophytic fungi probably excreted wall lytic enzymes or antifungal substances, inflicting wrinkles and disintegrating P. oryzae mycelium.

FungalTraits vs. FUNGuild: Comparison of Ecological Functional Assignments of Leaf- and Needle-Associated Fungi Across 12 Temperate Tree Species

Recently, a new annotation tool “FungalTraits” was created based on the previous FUNGuild and FunFun databases, which has attracted high attention in the scientific community. These databases were widely used to gain more information from fungal sequencing datasets by assigning fungal functional traits. More than 1500 publications so far employed FUNGuild and the aim of this study is to compare this successful database with the recent FungalTraits database. Quality and quantity of the assignment by FUNGuild and FungalTraits to a fungal internal transcribed spacer (ITS)–based amplicon sequencing dataset on amplicon sequence variants (ASVs) were addressed. Sequencing dataset was derived from leaves and needles of 12 temperate broadleaved and coniferous tree species. We found that FungalTraits assigned more functional traits than FUNGuild, and especially the coverage of saprotrophs, plant pathogens, and endophytes was higher while lichenized fungi revealed similar findings. Moreover, ASVs derived from leaves and needles of each tree species were better assigned to all available fungal traits as well as to saprotrophs by FungalTraits compared to FUNGuild in particular for broadleaved tree species. Assigned ASV richness as well as fungal functional community composition was higher and more diverse after analyses with FungalTraits compared to FUNGuild. Moreover, datasets of both databases showed similar effect of environmental factors for saprotrophs but for endophytes, unidentical patterns of significant corresponding factors were obtained. As a conclusion, FungalTraits is superior to FUNGuild in assigning a higher quantity and quality of ASVs as well as a higher frequency of significant correlations with environmental factors.

The curse of the uncultured fungus.

The international DNA sequence databases abound in fungal sequences not annotated beyond the kingdom level, typically bearing names such as “uncultured fungus”. These sequences beget low-resolution mycological results and invite further deposition of similarly poorly annotated entries. What do these sequences represent? This study uses a 767,918-sequence corpus of public full-length fungal ITS sequences to estimate what proportion of the 95,055 “uncultured fungus” sequences that represent truly unidentifiable fungal taxa – and what proportion of them that would have been straightforward to annotate to some more meaningful taxonomic level at the time of sequence deposition. Our results suggest that more than 70% of these sequences would have been trivial to identify to at least the order/family level at the time of sequence deposition, hinting that factors other than poor availability of relevant reference sequences explain the low-resolution names. We speculate that researchers’ perceived lack of time and lack of insight into the ramifications of this problem are the main explanations for the low-resolution names. We were surprised to find that more than a fifth of these sequences seem to have been deposited by mycologists rather than researchers unfamiliar with the consequences of poorly annotated fungal sequences in molecular repositories. The proportion of these needlessly poorly annotated sequences does not decline over time, suggesting that this problem must not be left unchecked.

A long-read amplicon approach to scaling up the metabarcoding of lichen herbarium specimens.

Reference sequence databases are critical to the accurate detection and identification of fungi in the environment. As repositories of large numbers of well-curated specimens, herbaria and fungal culture collections have the material resources to generate sequence data for large number of taxa, and could therefore allow filling taxonomic gaps often present in reference sequence databases. Financial resources to do that are however often lacking, so that recent efforts have focused on decreasing sequencing cost by increasing the number of multiplexed samples per sequencing run while maintaining high sequence quality. Following a previous study that aimed at decreasing sequencing cost for lichen specimens by generating fungal ITS barcodes for 96 specimens using PacBio amplicon sequencing, we present a method that further decreases lichen specimen metabarcoding costs. A total of 384 mixed DNA extracts obtained from lichen herbarium specimens, mostly from the four genera Buellia, Catillaria, Endocarpon and Parmotrema, were used to generate new fungal ITS sequences using a Sequel I sequencing platform and the PacBio M13 barcoded primers. The average success rate across all taxa was high (86.5%), with particularly high rates for the crustose saxicolous taxa (Buellia, Catillaria and others; 93.3%) and the terricolous squamulose taxa (Endocarpon and others; 96.5%). On the other hand, the success rate for the foliose genus Parmotrema was lower (60.4%). With this taxon sampling, greater specimen age did not appear to impact sequencing success. In fact, the 1966–1980 collection date category showed the highest success rate (97.3%). Compared to the previous study, the abundance-based sequence denoising method showed some limitations, but the cost of generating ITS barcodes was further decreased thanks to the higher multiplexing level. In addition to contributing new ITS barcodes for specimens of four interesting lichen genera, this study further highlights the potential and challenges of using new sequencing technologies on collection specimens to generate DNA sequences for reference databases.

Ectomycorrhizal Fungi Dominated the Root and Rhizosphere Microbial Communities of Two Willow Cultivars Grown for Six-Years in a Mixed-Contaminated Environment.

There is a growing interest in plant microbiome’s engineering to optimize desired functions such as improved phytoremediation. This study is aimed at examining the microbial communities inhabiting the roots and rhizospheres of two Salix miyabeana cultivars that had been grown in a short-rotation intensive culture (SRIC) system for six years in a soil contaminated with the discharge from a petrochemical factory. DNA was extracted from roots and rhizospheric soils, and fungal ITS and bacterial and archaeal 16S rDNA regions were amplified and sequenced using Illumina MiSeq technology. Cultivars ‘SX61’ and ‘SX64’ were found to harbor a similar diversity of fungal, bacterial, and archaeal amplicon sequence variants (ASVs). As expected, a greater microbial diversity was found in the rhizosphere biotope than in the roots of both cultivars, except for cultivar ‘SX64’, where a similar fungal diversity was observed in both biotopes. However, we found that microbial community structures were cultivar- and biotope-specific. Although the implication of some identified taxa for plant adaptability and biomass production capacity remains to be explored, this study provides valuable and useful information regarding microbes that could potentially favor the implantation and phytoremediation efficiency of Salix miyabeana in mixed contamination sites in similar climatic environments.

Fresh Rumen Liquid Inoculant Enhances the Rumen Microbial Community Establishment in Pre-weaned Dairy Calves.

The development of the functional rumen in calves involves a complex interplay between the host and host-related microbiome. Attempts to modulate rumen microbial community establishment may therefore have an impact on weaning success, calf health, and animal performance later in life. In this experiment, we aimed to elucidate how rumen liquid inoculum from an adult cow, provided to calves during the pre-weaning period, influences the establishment of rumen bacterial, archaeal, fungal, and ciliate protozoan communities in monozygotic twin calves (n = 6 pairs). The calves were divided into treatment (T-group) and control (C-group) groups, where the T-group received fresh rumen liquid as an oral inoculum during a 2–8-week period. The C-group was not inoculated. The rumen microbial community composition was determined using bacterial and archaeal 16S ribosomal RNA (rRNA) gene, protozoal 18S rRNA gene, and fungal ITS1 region amplicon sequencing. Animal weight gain and feed intake were monitored throughout the experiment. The T-group tended to have a higher concentrate intake (Treatment: p < 0.08) and had a significantly higher weekly weight gain (Treatment: p < 0.05), but no significant difference in volatile fatty acid concentrations between the groups was observed. In the T-group, the inoculum stimulated the earlier establishment of mature rumen-related bacterial taxa, affecting significant differences between the groups until 6 weeks of age. The inoculum also increased the archaeal operational taxonomic unit (OTU) diversity (Treatment: p < 0.05) but did not affect the archaeal quantity. Archaeal communities differed significantly between groups until week 4 (p = 0.02). Due to the inoculum, ciliate protozoa were detected in the T-group in week 2, while the C-group remained defaunated until 6 weeks of age. In week 8, Eremoplastron dilobum was the dominant ciliate protozoa in the C-group and Isotricha sp. in the T-group, respectively. The Shannon diversity of rumen anaerobic fungi reduced with age (Week: p < 0.01), and community establishment was influenced by a change of diet and potential interaction with other rumen microorganisms. Our results indicate that an adult cow rumen liquid inoculum enhanced the maturation of bacterial and archaeal communities in pre-weaning calves’ rumen, whereas its effect on eukaryotic communities was less clear and requires further investigation.

Customization of a DADA2-based pipeline for fungal internal transcribed spacer 1 (ITS1) amplicon data sets.

Identification and analysis of fungal communities commonly rely on internal transcribed spacer–based (ITS-based) amplicon sequencing. There is no gold standard used to infer and classify fungal constituents since methodologies have been adapted from analyses of bacterial communities. To achieve high-resolution inference of fungal constituents, we customized a DADA2-based pipeline using a mix of 11 medically relevant fungi. While DADA2 allowed the discrimination of ITS1 sequences differing by single nucleotides, quality filtering, sequencing bias, and database selection were identified as key variables determining the accuracy of sample inference. Due to species-specific differences in sequencing quality, default filtering settings removed most reads that originated from Aspergillus species, Saccharomyces cerevisiae, and Candida glabrata. By fine-tuning the quality filtering process, we achieved an improved representation of the fungal communities. By adapting a wobble nucleotide in the ITS1 forward primer region, we further increased the yield of S. cerevisiae and C. glabrata sequences. Finally, we showed that a BLAST-based algorithm based on the UNITE+INSD or the NCBI NT database achieved a higher reliability in species-level taxonomic annotation compared with the naive Bayesian classifier implemented in DADA2. These steps optimized a robust fungal ITS1 sequencing pipeline that, in most instances, enabled species-level assignment of community members.

The effect of vineyard reclamation on soil properties and microbial communities in desertified land in Hongsibu, Ningxia

Desertified land reclamation for the purposes of viticulture can profoundly affect the properties of the underlying soil and the microbial communities therein. Herein, we assessed the effects of such reclamation of non-productive desert land on the soil microbial communities associated with the resultant vineyards, and to identify key soil properties related to these changes. Soil was collected from natural desert land (DL) and from different reclaimed vineyard types: Cabernet Sauvignon (CS), Merlot (M), Chardonnay (C), and Italian Riesling (IR). High-throughput sequencing was used to assess microbial community diversity and composition in these samples. Significant differences in soil organic carbon (SOC), total nitrogen, available nitrogen, available phosphorus, and pH were detected when comparing soil from DL and reclaimed lands. CS, M, C, and IR soils exhibited higher relative Actinobacteria, Proteobacteria, and Ascomycota abundance, while DL soil exhibited higher relative Acidobacteria and Mortierellomycota abundance. In total, 165 and 55 bacterial and fungal amplicon sequence variants (ASVs) were shared across land use types. Following reclamation, soil bacteria ASVs in CS, M, C, and IR soils rose to 6149, 6483, 10648, and 9821, respectively. Biomarkers of these different land use types were successfully identified via an LDA Effect Size (LEfSe) approach, revealing a correlation between the vineyard soil microorganisms at the genus level by network analysis, while key soil properties including pH, nitrogen, and SOC were found to be associated with these changes in microbial community structural composition following reclamation. As such, our data indicate that viticulture in desertified regions can enhance soil properties and microbial diversity, thereby supporting the soil quality of reclaimed land.

Endolichenic Fungal Community Analysis by Pure Culture Isolation and Metabarcoding: A Case Study of Parmotrema tinctorum

Lichen is a symbiotic mutualism of mycobiont and photobiont that harbors diverse organisms including endolichenic fungi (ELF). Despite the taxonomic and ecological significance of ELF, no comparative investigation of an ELF community involving isolation of a pure culture and high-throughput sequencing has been conducted. Thus, we analyzed the ELF community in Parmotrema tinctorum by culture and metabarcoding. Alpha diversity of the ELF community was notably greater in metabarcoding than in culture-based analysis. Taxonomic proportions of the ELF community estimated by metabarcoding and by culture analyses showed remarkable differences: Sordariomycetes was the most dominant fungal class in culture-based analysis, while Dothideomycetes was the most abundant in metabarcoding analysis. Thirty-seven operational taxonomic units (OTUs) were commonly observed by culture- and metabarcoding-based analyses but relative abundances differed: most of common OTUs were underrepresented in metabarcoding. The ELF community differed in lichen segments and thalli in metabarcoding analysis. Dissimilarity of ELF community intra lichen thallus increased with thallus segment distance; inter-thallus ELF community dissimilarity was significantly greater than intra-thallus ELF community dissimilarity. Finally, we tested how many fungal sequence reads would be needed to ELF diversity with relationship assays between numbers of lichen segments and saturation patterns of OTU richness and sample coverage. At least 6000 sequence reads per lichen thallus were sufficient for prediction of overall ELF community diversity and 50,000 reads per thallus were enough to observe rare taxa of ELF.

Differences of the Nasal Microbiome and Mycobiome by Clinical Characteristics of COPD Patients.

While studies suggest that the lung microbiome may influence risk of chronic obstructive pulmonary disease (COPD) exacerbations, little is known about the relationship between the nasal biome and clinical characteristics of COPD patients. We sampled the nasal lining fluid by nasosorption of both nares of 20 people with moderate-to-severe COPD. All 40 samples, plus 4 negative controls, underwent DNA extraction, and 16SV4 ribosomal RNA (rRNA) (bacterial) and ribosomal internal transcribed spacer 2 (ITS2) (fungal) sequencing. We measured the proportion of variance (R2) in beta diversity explained by clinical factors, including age, sex, body mass index (BMI), COPD treatment, disease severity (forced expiratory volume in 1 second [FEV1], symptom/exacerbation frequency), peripheral eosinophil level (≥150 versus <150 cells/µL) and season of sampling, with the PERMANOVA test on the Bray-Curtis dissimilarities, accounting for within-person correlation of samples. We assessed the relative abundance of microbial features in the nasal community and their associations with clinical characteristics using the Microbiome Multivariable Association with Linear Models (MaAsLin2) package. The most abundant nasal fluid bacterial taxa were Corynebacterium, Staphylococcus, Streptococcus, Moraxella, and Dolosigranulum, and fungal taxa were Malassezia, Candida, Malasseziales, Cladosporium and Aspergillus. Bacterial microbiome composition was associated with short-acting muscarinic antagonist use (R2 11.8%, p=0.002), sex (R2 8.3%, p=0.044), nasal steroid use (R2 7.7%, p=0.064), and higher eosinophil level (R2 7.6%, p=0.084). Mycobiome composition was associated with higher eosinophil level (R2 14.4%, p=0.004) and low FEV1 (R2 7.5%, p=0.071). No specific bacterium or fungus differed significantly in relative abundance by clinical characteristics in the multivariate per-feature analysis. The taxonomical composition of the nasal biome is heterogeneous in COPD patients and may be explained in part by clinical characteristics.

Analysis of microbial diversity in the root of Astragalus mongholicus.

The dry root of Astragalus mongholicus has therapeutic effects such as tonifing the middle - jiao, replenishing qi, solidifing the surface, promoting diuresis, dispelling sepsis outwards and nourishing muscle. There are some slices having black spots after slicing the root of astragalus. The diversity of endophytic fungi between slices with black spots and normal slices was analysed in this paper. The endophytic fungal sequences obtained by high-throughput sequencing were 298,044 and 297,396, and the 116 OTU subsets obtained after clustering belonged to 3 phyla, 9 classes, 22 orders, 38 families and 46 genera. The dominant classes were Eurotiomycetes and Leotiomycetes. The dominant order is Eurotiales and Helotiales. The dominant families are Helotiales_fam_Incertae_sedis and Aspergillaceae. The dominant genera are Cadophora and Aspergillus. There are some peculiar fungal flora in both normal slices and spotted slices. The study on endophytic fungi diversity of astragalus slices will provide some help for drug development of this plant.