Analysis Of Fungal Communities Research Articles

The Endophytic Microbiome of Wild Grapevines Vitis amurensis Rupr. and Vitis coignetiae Pulliat Growing in the Russian Far East.

Many grape endophytic microorganisms exhibit high potential for suppressing the development of grape diseases and stimulating grapevine growth and fitness, as well as beneficial properties of the crop. The microbiome of wild grapevines is a promising source of biocontrol agents, which can be beneficial for domesticated grapevines. Using next-generation sequencing (NGS) and classical microbiology techniques, we performed an analysis of bacterial and fungal endophytic communities of wild grapevines Vitis amurensis Rupr. and Vitis coignetiae Pulliat growing in the Russian Far East. According to the NGS analysis, 24 and 18 bacterial taxa from the class level were present in V. amurensis and V. coignetiae grapevines, respectively. Gammaproteobacteria (35%) was the predominant class of endophytic bacteria in V. amurensis and Alphaproteobacteria (46%) in V. coignetiae. Three taxa, namely Sphingomonas, Methylobacterium, and Hymenobacter, were the most common bacterial genera for V. amurensis and V. coignetiae. Metagenomic analysis showed the presence of 23 and 22 fungi and fungus-like taxa of class level in V. amurensis and V. coignetiae, respectively. The predominant fungal classes were Dothideomycetes (61-65%) and Tremellomycetes (10-11%), while Cladosporium and Aureobasidium were the most common fungal genera in V. amurensis and V. coignetiae, respectively. A comparative analysis of the endophytic communities of V. amurensis and V. coignetiae with the previously reported endophytic communities of V. vinifera revealed that the bacterial biodiversity of V. amurensis and V. coignetiae was similar in alpha diversity to V. vinifera's bacterial biodiversity. The fungal alpha diversity of V. amurensis and V. coignetiae was statistically different from that of V. vinifera. The beta diversity analysis of bacterial and fungal endophytes showed that samples of V. vinifera formed separate clusters, while V. amurensis samples formed a separate cluster including V. coignetiae samples. The data revealed that the endophytic community of bacteria and fungi from wild V. amurensis was richer than that from V. coignetiae grapes and cultivated V. vinifera grapes. Therefore, the data obtained in this work could be of high value in the search for potentially useful microorganisms for viticulture.

Analysis and comparison of fungal communities in wheat Qus and Lin‐fan yeast starters used as traditional starter cultures of Shaoxing Huangjiu

SummaryThe wheat Qu and Lin‐fan yeast starter are two vital components of Huangjiu brewing, the microbiome composition of which can have a major impact on the end product's quality. This investigation aimed to investigate and compare the fungal diversity of eight starters, including four wheat Qus and four Lin‐fan yeast starters collected from different manufacturers of Shaoxing Huangjiu using high‐throughput sequencing (HTS) technology. Significant variation was observed in the fungal community composition among different starter samples. Fusarium, Epicoccum, Aspergillus and Alternaria were the most predominant fungal genera in wheat Qus. In contrast, Lin‐fan yeast starters possessed relatively simple fungal communities and were predominated by Saccharomyces and Saccharomycopsis. LEfSe analysis showed that nine genus‐level fungal taxa could be retrieved to differentiate the two starter types. Furthermore, the complicated association among the dominant fungi in traditional starters was revealed by utilising Spearman's correlation‐based network analysis. A total of 42 positive and 28 negative correlations were identified respectively. Ultimately, this investigation described the fungal communities in wheat Qus and Lin‐fan yeast starters and will aid in screening fermentation strains, which can remarkably enhance the quality of Shaoxing Huangjiu.

Impact of Root Rot Disease of Zanthoxylum armatum on Rhizosphere Soil Microbes and Screening of Antagonistic Bacteria

Zanthoxylum armatum, a significant forest plant in southwestern China, is crucial for preserving soil and water resources. However, the presence of root rot disease has led to plant death, impacting the pepper sector. Effective control measures for this disease are still lacking. Rhizosphere microorganisms play a vital role in plant health by inhibiting plant pathogens and inducing plant resistance. This research aimed to isolate and characterize the pathogen responsible for root rot disease in Z. armatum. Comparative analysis of fungal and bacterial communities in the rhizosphere soil of healthy and diseased plants revealed Fusarium solani as the pathogenic fungus causing root rot disease. Diseased plants had a higher occurrence of Fusarium spp., while disease-free plants had a higher abundance of ecologically beneficial microbial communities that could potentially serve as biocontrol agents. Three bacterial strains (Bacillus subtilis, Bacillus amyloliquefaciens, and Bacillus siamensis) were identified as effective biocontrol agents, inhibiting the growth of the pathogenic fungus F. solani both in vivo and in vitro. This study deepens our understanding of the rhizosphere soil microbial community differences between diseased and healthy Z. armatum, providing potential biocontrol bacteria to enhance plant resistance against root rot disease.

A combined microscopy and single-cell sequencing approach reveals the ecology, morphology, and phylogeny of uncultured lineages of zoosporic fungi.

Environmental DNA analyses of fungal communities typically reveal a much larger diversity than can be ascribed to known species. Much of this hidden diversity lies within undescribed fungal lineages, especially the early diverging fungi (EDF). Although these EDF often represent new lineages even at the phylum level, they have never been cultured, making their morphology and ecology uncertain. One of the methods to characterize these uncultured fungi is a single-cell DNA sequencing approach. In this study, we established a large data set of single-cell sequences of EDF by manually isolating and photographing parasitic fungi on various hosts such as algae, protists, and micro-invertebrates, combined with subsequent long-read sequencing of the ribosomal DNA locus (rDNA). We successfully obtained rDNA sequences of 127 parasitic fungal cells, which clustered into 71 phylogenetic lineages belonging to seven phylum-level clades of EDF: Blastocladiomycota, Chytridiomycota, Aphelidiomycota, Rozellomycota, and three unknown phylum-level clades. Most of our single cells yielded novel sequences distinguished from both described taxa and existing metabarcoding data, indicating an expansive and hidden diversity of parasitic taxa of EDF. We also revealed an unexpected diversity of endobiotic Olpidium-like chytrids and hyper-parasitic lineages. Overall, by combining photographs of parasitic fungi with phylogenetic analyses, we were able to better understand the ecological function and morphology of many of the branches on the fungal tree of life known only from DNA sequences. IMPORTANCE Much of the diversity of microbes from natural habitats, such as soil and freshwater, comprise species and lineages that have never been isolated into pure culture. In part, this stems from a bias of culturing in favor of saprotrophic microbes over the myriad symbiotic ones that include parasitic and mutualistic relationships with other taxa. In the present study, we aimed to shed light on the ecological function and morphology of the many undescribed lineages of aquatic fungi by individually isolating and sequencing molecular barcodes from 127 cells of host-associated fungi using single-cell sequencing. By adding these sequences and their photographs into the fungal tree, we were able to understand the morphology of reproductive and vegetative structures of these novel fungi and to provide a hypothesized ecological function for them. These individual host-fungal cells revealed themselves to be complex environments despite their small size; numerous samples were hyper-parasitized with other zoosporic fungal lineages such as Rozellomycota.

Trichoderma viride F7 improves peanut performance while remedying cadmium-contaminated soil with microplastics

Microplastics (MPs) and cadmium (Cd) in the soil have serious negative impacts on plant growth. As a bioremediator, Trichoderma viride may modulate the impacts of MPs or/and Cd on plant growth and heavy metal uptake. In this study, T. viride F7 (F7) was used to treat Cd-contaminated soil with or without polylactic acid MPs (PLA-MPs) to investigate its effects on peanut growth, Cd uptake efficiency, and soil properties. The results showed that F7 increased the soil pH, dissolved organic carbon (DOC), cation exchange capacity (CEC), microbial nitrogen (MBN), and microbial carbon (MBC), which resulted in a 2.10–19.83% increase in peanut biomass and mitigated the negative effects of PLA and Cd on peanut growth. F7 reduced the soil effective-state Cd and plant Cd content by 16.68–34.20% and 5.37–25.84%, respectively. Regression path analysis showed that F7 could improve peanut performance by altering soil properties and reducing peanut Cd concentrations in PLA-MP- and Cd-contaminated soils. Soil fungal community analysis indicated that the microbial community was altered via F7 inoculation, which antagonized Fusarium and promoted peanut growth. This study reveals the mechanism of F7 in mitigating Cd contamination in soil in the presence of MPs, which provides new ideas for managing complex contaminated soil with MP–heavy metals and improving soil health.

Simultaneous Analysis of Bacterial and Fungal Communities in Oral Samples from Intubated Patients in Intensive Care Unit.

Intubated patients in intensive care units (ICUs) too frequently contract ventilator-associated pneumonia or Candida infections. Oropharyngeal microbes are believed to play an important etiologic role. This study was undertaken to determine whether next-generation sequencing (NGS) can be used to simultaneously analyze bacterial and fungal communities. Buccal samples were collected from intubated ICU patients. Primers targeting the V1-V2 region of bacterial 16S rRNA and the internal transcribed spacer 2 (ITS2) region of fungal 18S rRNA were used. V1-V2, ITS2, or mixed V1-V2/ITS2 primers were used to prepare an NGS library. Bacterial and fungal relative abundances were comparable for V1-V2, ITS2, or mixed V1-V2/ITS2 primers, respectively. A standard microbial community was used to adjust the relative abundances to theoretical abundance, and NGS and RT-PCR-adjusted relative abundances showed a high correlation. Using mixed V1-V2/ITS2 primers, bacterial and fungal abundances were simultaneously determined. The constructed microbiome network revealed novel interkingdom and intrakingdom interactions, and the simultaneous detection of bacterial and fungal communities using mixed V1-V2/ITS2 primers enabled analysis across two kingdoms. This study provides a novel approach to simultaneously determining bacterial and fungal communities using mixed V1-V2/ITS2 primers.

Yeast enrichment facilitated lipid removal and bioconversion by black soldier fly larvae in the food waste treatment

Food waste can be converted into insectile fatty acids (FAs) by the larvae of black soldier fly (BSFL), Hermetia illucens, for use in the feed sector or as a source of biodiesel. However, waste oil was less decomposed than carbohydrate or protein in frass due to the limitation of larval lipid metabolism. In this study, 10 yeast strains were screened, corresponding to six species, to examine their capacity of improving lipid transformation performance by BSFL. The species of Candida lipolytica was superior to the other five species, which exhibited significantly higher lipid reduction rate (95.0–97.1 %) than the control (88.7 %), and the larval FA yields achieved 82.3–115.5 % of the food waste FA matters, suggesting that BSFL not only transformed waste oil but also biosynthesized FAs from waste carbohydrate and other substances. Further, the CL2 strain of Candida lipolytica was examined for treating food waste containing high lipid content (16–32 %). The lipid removal rate was found improved from 21.4 to 42.3 % (control) to 80.5–93.3% in the waste containing 20–32 % lipid. The upper limit of lipid content that could be endured by BSFL was ≈16 %, and the CL2-enrichment elevated the upper limit to ≈24 %. Fungal community analysis indicated that Candida spp. accounted for the lipid removal improvement. The Candida spp. CL2 strain may facilitate the lipid reduction and transformation by BSFL through microbial catabolizing and assimilation of waste FAs. Altogether, this study suggests that yeast enrichment is feasible in improving lipid transformation by BSFL especially for food waste exhibiting high lipid content.

Diversity of Fungal Communities on Diseased and Healthy Cinnamomum burmannii Fruits and Antibacterial Activity of Secondary Metabolites.

The composition and structure of fungal communities on healthy and diseased fruits of Cinnamomum burmannii (Nees and Nees) Blume were characterized, with evaluation of the antibacterial activity of secondary metabolites from culturable fungi following the first identification of secondary metabolites in the fungus Medicopsis romeroi (Esf-14; GenBank accession number OK242756). These results are significant for understanding the functional variation in bioactivity in fungal communities and developing a broader range of bioactive resources. High-throughput sequencing results indicated that the fungal community in diseased fruit differed from that in healthy fruit at the phylum, class, order, or genus level, with significant differences in the species and relative abundance of the dominant flora. A total of 49 (healthy fruit) and 122 (diseased fruit) artificially cultivable endophytic fungi were isolated, and 41 different strains (11 from healthy fruit and 30 from diseased fruit) were successfully identified by morphological and molecular biological analyses, which were classified into 8 groups and 23 genera by phylogenetic tree analysis, with Pleosporales, Glomerellales, and Hypocreales being the dominant groups at the order level and Colletotrichum being the dominant group at the genus level. The results of the antibacterial assay demonstrated that the secondary metabolites of all strains had different degrees of antibacterial activity, while the secondary metabolites of endophytic fungi from diseased fruit were generally stronger than those of fungi from healthy fruit, with the active secondary metabolites dominated by small and moderately polar compounds. Combined analysis of fungal communities, phylogenetic tree analysis, and bioactivity analysis of culturable strains revealed strong antibacterial activity of both upregulated and downregulated flora in diseased fruit. Five compounds, including two new (5,6-dimethoxy-[1',1:4,1″-terphenyl]-2-ol [compound 1] and 5-(methoxycarbonyl)-2-methylbenzo[d][1,3]dioxole-2-carboxylic acid [compound 2]) and three known compounds (3,7-dihydroxy-1,9-dimethyldibenzofuran [compound 3], methyl 3-hydroxybenzoate [compound 4], and uracil [compound 5]), were isolated and identified for the first time from the endophytic fungus Medicopsis romeroi. In general, the diversity of fungal communities on diseased fruit was lower than that on healthy fruits, while the antibacterial activity of artificially cultured endophytic fungi on diseased fruits was generally stronger than that on healthy fruits, suggesting excellent promise for the development of secondary metabolites from active strains on diseased fruit as antibacterial agents. IMPORTANCE Powdery fruit disease is a notorious disease of Cinnamomum burmannii that causes severe loss in fruit production. Studies on the function of endophytic fungal communities in healthy plant tissues are not new, while little is known about the functional changes of fungal communities in disease-causing plant tissues. Our results demonstrate that fungal communities in diseased fruits differ from those in healthy fruits at the level of phylum, class, order, or genus, with significant differences in the species and relative abundance of dominant groups. Endophytic fungi in diseased fruits appeared to produce secondary metabolites with stronger antibacterial properties, although the community diversity was not as varied as that in healthy fruits. In addition, secondary metabolites of the Medicopsis romeroi strain from diseased fruits were identified for the first time. These results have important implications for understanding the functional variation of bioactivity in fungal communities and for developing a broader resource of bioactivity.

Fungal community analysis of hot spots in bulk maize under different storage conditions

Maize is one of the main cereals used for food and livestock feed. During storage, maize is easily infected by fungi to form mildew or cause hot spots, resulting in maize quality loss and potential safety hazards. We collected maize samples from normal spots and hot spots in three warehouses with different storage conditions (i.e., normal storage, N2-controlled atmosphere storage, and phosphine fumigation treatment storage) and used high-throughput sequencing and traditional culturing methods to investigate the fungal communities inducing hot spots. The results showed that there were large differences in the fungal communities on the maize within the three storage warehouses, and the community compositions of the hot spots and normal spots were different within the same warehouse. The storage conditions were highly associated with the fungal community alpha and beta diversities. Aspergillus penicillioides, A. vitricola, and A. chevalieri were the dominant fungal species inducing hot spots. Furthermore, the grain temperature and moisture content were significantly correlated with fungal community composition in hot spots and normal spots. These data are useful for grain depot managers evaluating the potential risks of fungi-induced hot spots and for optimizing storage conditions to reduce the risks of hot spots during maize storage.

DNA metabarcoding analysis of fungal community on surface of four root herbs

ObjectiveAngelicae Sinensis Radix (ASR, Danggui in Chinese), Cistanches Herba (CH, Roucongrong in Chinese), Ginseng Radix et Rhizoma (PG, Renshen in Chinese), and Panacis Quinquefolii Radix (PQ, Xiyangshen in Chinese), widely used as medicine and dietary supplement around the world, are susceptible to fungal and mycotoxin contamination. In this study, we aim to analyze their fungal community by DNA metabarcoding. MethodsA total of 12 root samples were collected from three main production areas in China. The samples were divided into four groups based on herb species, including ASR, CH, PG, and PQ groups. The fungal community on the surface of four root groups was investigated through DNA metabarcoding via targeting the internal transcribed spacer 2 region (ITS2). ResultsAll the 12 samples were detected with fungal contamination. Rhizopus (13.04%−74.03%), Aspergillus (1.76%−23.92%), and Fusarium (0.26%−15.27%) were the predominant genera. Ten important fungi were identified at the species level, including two potential toxigenic fungi (Penicillium citrinum and P. oxalicum) and eight human pathogenic fungi (Alternaria infectoria, Candida sake, Hyphopichia burtonii, Malassezia globosa, M. restricta, Rhizopus arrhizus, Rhodotorula mucilaginosa, and Ochroconis tshawytschae). Fungal community in ASR and CH groups was significantly different from other groups, while fungal community in PG and PQ groups was relatively similar. ConclusionDNA metabarcoding revealed the fungal community in four important root herbs. This study provided an important reference for preventing root herbs against fungal and mycotoxin contamination.

Effect of natural weed and Siratro cover crop on soil fungal diversity in a banana cropping system in southwestern China.

Natural weed cover and a legume cover crop were examined to determine if they could impact soil fungal diversity as an indicator of soil quality in banana production. Banana in Yunnan Province, China, was grown under three treatments: conventional tillage (bare soil), natural weed cover (primarily goosegrass (Eleusine indica (L.) Gaerth)), or a cover crop (Siratro (Macroptilium atropurpureum (DC.) Urb.)). Analysis of the soil fungal communities between 2017 and 2020 was done by Illumina Miseq high-throughput sequencing. Most significant effects were in the intercropping area for the treatments, whereas it was rarely observed in the furrow planted with banana. Based on the Shannon and Simpson diversity indices, soil fungal diversity in the intercropping area significantly decreased following planting banana in 2017 with all three treatments. However, both the Shannon and Simpson diversity indices showed that there were significant increases in fungal soil diversity in 2019 and 2020 with natural weed cover or Siratro compared to bare soil. At the end of the experiment, significant increases in fungal genera with Siratro compared to bare soil were observed with Mortierella, Acremonium, Plectophaerella, Metarhizium and Acrocalymma, and significant decreases were observed with Fusicolla, Myrothecium, Exserohilum, Micropsalliota and Nigrospora. Siratro resulted in higher stability of the soil fungal microbiome by increasing the modularity and the proportion of negative co-occurrences compared to bare soil. For fungal guilds, Siratro significantly increased saprotrophs_symbiotrophs in 2019 and 2020 and significantly decreased pathogens_saprotrophs in 2020 compared to bare soil. Using Siratro as a cover crop in the intercropping area of banana helped maintain soil fungal diversity, which would be beneficial for soil health with more symbiotrophs and less pathogens in the soil. However, further research is needed to determine the long-term impact of weed or Siratro cover crop on the fungal soil ecosystem and growth of banana.

The root-associated Fusarium isolated based on fungal community analysis improves phytoremediation efficiency of Ricinus communis L. in multi metal-contaminated soils

Phytoremediation is a widely accepted bioremediation method of treating heavy metal contaminated soils. Nevertheless, the remediation efficiency in multi-metal contaminated soils is still unsatisfactory attributable to susceptibility to different metals. To isolate root-associated fungi for improving phytoremediation efficiency in multi-metal contaminated soils, the fungal flora in root endosphere, rhizoplane, rhizosphere of Ricinus communis L. in heavy metal contaminated soils and non-heavy metal contaminated soils were compared by ITS amplicon sequencing, and then the critical fungal strains were isolated and inoculated into host plants to improve phytoremediation efficiency in Cd, Pb, and Zn-contaminated soils. The fungal ITS amplicon sequencing analysis indicated that the fungal community in root endosphere was more susceptible to heavy metals than those in rhizoplane and rhizosphere soils and Fusarium dominated the endophytic fungal community of R. communis L. roots under heavy metal stress. Three endophytic strains (Fusarium sp. F2, Fusarium sp. F8, and Fusarium sp. F14) isolated from Ricinus communis L. roots showed high resistances to multi-metals and possessed growth-promoting characteristics. Biomass and metal extraction amount of R. communis L. with Fusarium sp. F2, Fusarium sp. F8, and Fusarium sp. F14 inoculation in Cd-, Pb- and Zn-contaminated soils were significantly higher than those without the inoculation. The results suggested that fungal community analysis-guided isolation could be employed to obtain desired root-associated fungi for enhancing phytoremediation of multi-metal contaminated soils.

Diversity of Microbial Communities, PAHs, and Metals in Road and Leaf Dust of Functional Zones of Moscow and Murmansk

The impact of geographical factors, functional zoning, and biotope type on the diversity of microbial communities and chemical components in the dust of urban ecosystems was studied. Comprehensive analyses of bacterial and fungal communities, polycyclic aromatic hydrocarbons (PAHs), and metals in road and leaf dust in three urban zones of Murmansk and Moscow with contrasting anthropogenic load were conducted. We found that the structure of bacterial communities affected the functional zoning of the city, biotope type, and geographical components. Fungal communities were instead impacted only by biotope type. Our findings revealed that the structure of fungal communities was mostly impacted by PAHs whereas bacterial communities were sensitive to metals. Bacteria of the genus Sphingomonas in road and leaf dust as indicators of the ecological state of the urban ecosystems were proposed.

Porcine fungal mock community analyses: Implications for mycobiome investigations.

The gut microbiome is an integral partner in host health and plays a role in immune development, altered nutrition, and pathogen prevention. The mycobiome (fungal microbiome) is considered part of the rare biosphere but is still a critical component in health. Next generation sequencing has improved our understanding of fungi in the gut, but methodological challenges remain. Biases are introduced during DNA isolation, primer design and choice, polymerase selection, sequencing platform selection, and data analyses, as fungal reference databases are often incomplete or contain erroneous sequences. Here, we compared the accuracy of taxonomic identifications and abundances from mycobiome analyses which vary among three commonly selected target gene regions (18S, ITS1, or ITS2) and the reference database (UNITE - ITS1, ITS2 and SILVA - 18S). We analyze multiple communities including individual fungal isolates, a mixed mock community created from five common fungal isolates found in weanling piglet feces, a purchased commercial fungal mock community, and piglet fecal samples. In addition, we calculated gene copy numbers for the 18S, ITS1, and ITS2 regions of each of the five isolates from the piglet fecal mock community to determine whether copy number affects abundance estimates. Finally, we determined the abundance of taxa from several iterations of our in-house fecal community to assess the effects of community composition on taxon abundance. Overall, no marker-database combination consistently outperformed the others. Internal transcribed space markers were slightly superior to 18S in the identification of species in tested communities, but Lichtheimia corymbifera, a common member of piglet gut communities, was not amplified by ITS1 and ITS2 primers. Thus, ITS based abundance estimates of taxa in piglet mock communities were skewed while 18S marker profiles were more accurate. Kazachstania slooffiae displayed the most stable copy numbers (83-85) while L. corymbifera displayed significant variability (90-144) across gene regions. This study underscores the importance of preliminary studies to assess primer combinations and database choice for the mycobiome sample of interest and raises questions regarding the validity of fungal abundance estimates.

Soil amendment with cow dung modifies the soil nutrition and microbiota to reduce the ginseng replanting problem.

Ginseng is a profitable crop worldwide; however, the ginseng replanting problem (GRP) is a major threat to its production. Soil amendment is a non-chemical method that is gaining popularity for alleviating continuous cropping obstacles, such as GRP. However, the impact of soil amendment with either cow dung or canola on GRP reduction and the associated soil microbiota remains unclear. In the present study, we evaluated the effect of soil amendment with cow dung, canola seed powder, and without amendment (control), on the survival of ginseng seedling transplants, the soil bacterial and fungal communities, and their associated metabolic functions. The results showed that cow dung increased ginseng seedling survival rate by 100 percent and had a remarkable positive effect on ginseng plant growth compared to control, whereas canola did not. Cow dung improved soil nutritional status in terms of pH, electrical conductivity, , total carbon, total phosphorus, and available phosphorus. The amplicon sequencing results using Illumina MiSeq showed that canola had the strongest negative effect in reducing soil bacterial and fungal diversity. On the other hand, cow dung stimulated beneficial soil microbes, including Bacillus, Rhodanobacter, Streptomyces, and Chaetomium, while suppressing Acidobacteriota. Community-level physiological profiling analysis using Biolog Ecoplates containing 31 different carbon sources showed that cow dung soil had a different metabolic activity with higher utilization rates of carbohydrates and polymer carbon sources, mainly Tween 40 and beta-methyl-d-glucoside. These carbon sources were most highly associated with Bacillota. Furthermore, predicted ecological function analyses of bacterial and fungal communities showed that cow dung had a higher predicted function of fermentation and fewer functions related to plant pathogens and fungal parasites, signifying its potential to enhance soil suppressiveness. Co-occurrence network analysis based on random matrix theory (RMT) revealed that cow dung transformed the soil microbial network into a highly connected and complex network. This study is the first to report the alleviation of GRP using cow dung as a soil amendment, and the study contributes significantly to our understanding of how the soil microbiota and metabolic alterations via cow dung can aid in GRP alleviation.

Biochar drives humus formation during composting by regulating the specialized metabolic features of microbiome

Humus formation of composting is crucial for improving organic fertilizer quality. Biochar has been found to be a low-cost, eco-friendly and effective promoter for humus formation, while the mechanisms underlying the biochar-driven humus formation during composting remain unknown. The present study was aimed to explore the effect of biochar addition on humus formation by integrated analyses of fungal communities, functional genes and metabolic features during composting using the advanced techniques of fluorescence spectrometry, high throughput sequencing, metagenomics, and metabolomics. The efficiencies of fulvic acid and humic acid formation were increased by 17.4 % and 39.4 %, respectively, under biochar addition relative to the control at the mature phase. Biochar addition significantly increased temperature, lignocellulose degradation and the activities of urease, peroxidase, manganese peroxidase and lignin peroxidase, and altered the fungal community composition and diversity with the latter significantly correlated with humic acid content. The altered fungal community subsequently resulted in the stimulation of fungal function of Wood Saprotroph and expression of CAZyme genes at the thermophilic and mature phases. Ultimately, biochar addition significantly enhanced the Xenobiotics metabolism (Bisphenol metabolism and Quinone biosynthesis) and Amino acid metabolism (Tryptophan metabolism and Tyrosine metabolism), which were responsible for humus formation via producing metabolic compounds such as quinolines, phenolics and aromatic amino acids at the mature phase. Our findings suggest that biochar addition can effectively stimulate humus formation by selectively regulating fungal communities and metabolic features during composting, so that benefits the improvement of soil fertility and health upon the organic fertilizer application.

Analysis of Soil Fungal Community in Aged Apple Orchards in Luochuan County, Shaanxi Province

The Luochuan area is an important area for apple production in China. With the renewal and transformation of aged apple orchards, the occurrence of apple replant disease (ARD) was inevitable and has seriously affected the sustainable development of apples. Therefore, we randomly selected 14 soil samples from aged apple orchards in the Luochuan area to study the structural changes in the soil fungal community. The results showed that there were significant differences in the diversity of fungal communities between different aged apple orchards. The harmful fungi Gibberella, Fusarium, and Cryptococcus existed in 14 aged apple orchards in the Luochuan area, but their abundances were different in different aged apple orchards. A FUN Guild analysis showed that fungi were mainly present in the aged apple orchards in Luochuan in the saprotroph and pathotroph nutrition modes. Pathogenic fungi were widely present, which increased the risk of disease and seriously affected the growth and development of fruit trees. To sum up, there was a strong correlation between the ages of orchards and the unbalanced microbial community structure. Therefore, pathogenic fungi could be prevented and controlled during the renewal and transformation of aged orchards to reduce the impact of ARD on the apple industry.

Comparative analysis of fungal communities between herbicide-resistant and -susceptible Alopecurus aequalis.

Alopecurus aequalis is a grass species invading Chinese canola and wheat fields. An A. aequalis KMN-R population surviving mesosulfuron-methyl treatment with recommended rates was acquired from wheatland. Here, we aimed to confirm the resistance profiles of KMN-R to acetolactate synthetase (ALS) inhibiting herbicides and explore the possible resistance mechanisms to mesosulfuron-methyl in this weed population. The dose-response tests performed in our study were used to test the toxicity of A. aequalis to ALS-inhibiting herbicides. Sanger sequencing was used to analyze the ALS gene of mesosulfuron-methyl -resistant and -susceptible A. aequalis. RNA sequencing analysis was used to find candidate genes that may confer metabolic resistance to the mesosulfuron-methyl in resistant A. aequalis population. Mesosulfuron-methyl -resistant and -susceptible A. aequalis populations fungal composition was measured via Illumina MiSeq Sequencing. Dose-response results indicated that KMN-R population evolved resistance to mesosulfuron-methyl and other tested ALS-inhibiting herbicides. Known resistance-conferring Trp-574-Leu gene mutation in A. aequalis ALS was detected in the KMN-R population. Pretreatment with 4-chloro-7-nitrobenzoxadiazole reversed mesosulfuron-methyl resistance in KMN-R. Glutathione S-transferases (GST) gene GSTZ2 and GSTT3 were highly expressed in KMN-R population. In addition, we evaluated the alpha diversity in A. aequalis, centering on OTU abundance, equality, and multiplicity, and found that the fungal community composition had more unexplained variance between KMN-R and KMN-S A. aequalis. We also observed higher abundances of specific fungi in KMN-R A. aequalis. The results proved that resistance to mesosulfuron-methyl in A. aequalis KMN-R population is probably caused by target site- and non-target site-based relating GST and provided the basis for further research between fungal interaction and herbicide resistance.

Sample Preparation for Fungal Community Analysis by High-Throughput Sequencing of Barcode Amplicons.

Fungal species participate in vast numbers of processes in the landscape around us. However, their cryptic mycelial growth, inside various substrates and in highly diverse species assemblages, has been a major obstacle to thorough analysis of fungal communities, hampering exhaustive description of the fungal kingdom. Technological developments allowing rapid, high-throughput sequencing of mixed communities from many samples at once are currently having a tremendous impact in fungal community ecology. Universal DNA extraction followed by amplification and sequencing of fungal species-level barcodes such as the nuclear internal transcribed spacer (ITS) region now enables identification and relative quantification of fungal community members across well-replicated experimental settings.Here, we present the sample preparation procedure presently used in our laboratory for fungal community analysis by high-throughput sequencing of amplified ITS2 markers. We focus on the procedure optimized for studies of total fungal communities in humus-rich soils, wood, and litter. However, this procedure can be applied to other sample types and markers. We focus on the laboratory-based part of sample preparation, i.e., the procedure from the point where samples enter the laboratory until amplicons are submitted for sequencing. Our procedure comprises four main parts: (1) universal DNA extraction, (2) optimization of PCR conditions, (3) production of tagged ITS amplicons, and (4) preparation of the multiplexed amplicon pool to be sequenced. The presented procedure is independent of the specific high-throughput sequencing technology used, which makes it highly versatile.

Soil sample sizes for DNA extraction substantially affect the examination of microbial diversity and co-occurrence patterns but not abundance

As the cornerstone of most soil microbial diversity and abundance research, soil DNA extraction is inherently influenced by soil sample sizes, yet the effects remain far from fully understood. In this study, soils were collected from desert, meadow, forest, and cropland ecosystems to examine the effects of soil sample sizes (i.e., 0.01, 0.025, 0.25, 0.50, and 1.00 g) on prokaryotic and fungal community analysis. Soil microbial abundance and diversity were analyzed through real-time PCR and NovaSeq high-throughput sequencing, respectively. We found that soil sample sizes for DNA extraction generally did not affect microbial abundance determination. In contrast, microbial richness, community composition, and co-occurrence patterns varied with different soil sample sizes. The microbial richness and co-occurrence frequency obtained from 0.01 to 0.025 g of soils were lower than those determined based on 0.25–1.00 g of soils in most ecosystems. Microbial community composition was also affected by the soil sample sizes when they were 0.50 g or less. In particular, small soil sample sizes for DNA extraction (i.e., ≤ 0.025 g) revealed dramatic variations in microbial community profiles among soil microhabitats. Collectively, in most ecosystems, at least 0.25 g of soils should be used to determine the overall diversity of microbes. As for the soils with low microbial cellular density, DNA should be extracted from 0.50 g or even more soils. Thus, the analysis of soil microbial diversity and co-occurrence patterns rather than abundance are substantially affected by the soil sample sizes for DNA extraction, and the choice of optimum soil sample sizes depends on soil types and research purposes.