Large-Scale Characterization of the Soil Microbiome in Ancient Tea Plantations Using High-Throughput 16S rRNA and Internal Transcribed Spacer Amplicon Sequencing

There is a special interaction between the environment, soil microorganisms, and tea plants, which constitute the ecosystem of tea plantations. Influenced by environmental factors and human management, the changes in soil microbial community affected the growth, quality, and yield of tea plants. However, little is known about the composition and structure of soil bacterial and fungal communities in 100-year-old tea plantations and the mechanisms by which they are affected. In this regard, we characterized the microbiome of tea plantation soils by considering the bacterial and fungal communities in 448 soil samples from 101 ancient tea plantations in eight counties of Lincang city, which is one of the tea domestication centers in the world. 16S and Internal Transcribed Spacer (ITS) rRNA high-throughput amplicon sequencing techniques were applied in this study. The results showed that the abundance, diversity, and composition of the bacterial and fungal communities have different sensitivity with varying pH, altitude, and latitude. pH and altitude affect soil microbial communities, and bacterial communities are more sensitive than fungi in terms of abundance and diversity to pH. The highest α-diversity of bacterial communities is shown in the pH 4.50–5.00 and 2,200-m group, and fungi peaked in the pH 5.00–5.50 and 900-m group. Because of environmental and geographical factors, all microbes are similarly changing, and further correlations showed that the composition and structure of bacterial communities are more sensitive than fungal communities, which were affected by latitude and altitude. In conclusion, the interference of anthropogenic activities plays a more important role in governing fungal community selection than environmental or geographical factors, whereas for the bacterial community, it is more selective to environment adaptation than to adaptation to human activities.

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Different Amplicon Targets for Sequencing-Based Studies of Fungal Diversity

Applied and Environmental Microbiology ◽

10.1128/aem.00905-17 ◽

2017 ◽

Vol 83 (17) ◽

Cited By ~ 40

Author(s):

Francesca De Filippis ◽

Manolo Laiola ◽

Giuseppe Blaiotta ◽

Danilo Ercolini

Keyword(s):

Microbial Ecology ◽

High Throughput ◽

Biological Samples ◽

Fungal Diversity ◽

High Throughput Sequencing ◽

Amplicon Sequencing ◽

Fungal Species ◽

Fungal Communities ◽

Its2 Region ◽

Mock Community

ABSTRACT Target-gene amplicon sequencing is the most exploited high-throughput sequencing application in microbial ecology. The targets are taxonomically relevant genes, with 16S rRNA being the gold standard for bacteria. As for fungi, the most commonly used target is the internal transcribed spacer (ITS). However, the uneven ITS length among species may promote preferential amplification and sequencing and incorrect estimation of their abundance. Therefore, the use of different targets is desirable. We evaluated the use of three different target amplicons for the characterization of fungal diversity. After an in silico primer evaluation, we compared three amplicons (the ITS1-ITS2 region [ITS1-2], 18S ribosomal small subunit RNA, and the D1/D2 domain of the 26S ribosomal large subunit RNA), using biological samples and a mock community of common fungal species. All three targets allowed for accurate identification of the species present. Nevertheless, high heterogeneity in ITS1-2 length was found, and this caused an overestimation of the abundance of species with a shorter ITS, while both 18S and 26S amplicons allowed for more reliable quantification. We demonstrated that ITS1-2 amplicon sequencing, although widely used, may lead to an incorrect evaluation of fungal communities, and efforts should be made to promote the use of different targets in sequencing-based microbial ecology studies. IMPORTANCE Amplicon-sequencing approaches for fungi may rely on different targets affecting the diversity and abundance of the fungal species. An increasing number of studies will address fungal diversity by high-throughput amplicon sequencing. The description of the communities must be accurate and reliable in order to draw useful insights and to address both ecological and biological questions. By analyzing a mock community and several biological samples, we demonstrate that using different amplicon targets may change the results of fungal microbiota analysis, and we highlight how a careful choice of the target is fundamental for a thorough description of the fungal communities.

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Application of a High-Throughput Amplicon Sequencing Method to Chart the Bacterial Communities that Are Associated with European Fermented Meats from Different Origins

Foods ◽

10.3390/foods9091247 ◽

2020 ◽

Vol 9 (9) ◽

pp. 1247

Author(s):

Emiel Van Reckem ◽

Christina Charmpi ◽

David Van der Veken ◽

Wim Borremans ◽

Luc De Vuyst ◽

...

Keyword(s):

Microbial Communities ◽

High Throughput ◽

Bacterial Communities ◽

Geographical Origin ◽

Salt Content ◽

Amplicon Sequencing ◽

Taxonomic Resolution ◽

Future Application ◽

Processing Conditions ◽

The Impact

Insight into the microbial species diversity of fermented meats is not only paramount to gain control over quality development, but also to better understand the link with processing technology and geographical origin. To study the composition of the microbial communities, the use of culture-independent methods is increasingly popular but often still suffers from drawbacks, such as a limited taxonomic resolution. This study aimed to apply a previously developed high-throughput amplicon sequencing (HTS) method targeting the 16S rRNA and tuf genes to characterize the bacterial communities in European fermented meats in greater detail. The data obtained broadened the view on the microbial communities that were associated with the various products examined, revealing the presence of previously underreported subdominant species. Moreover, the composition of these communities could be linked to the specificities of individual products, in particular pH, salt content, and geographical origin. In contrast, no clear links were found between the volatile organic compound profiles of the different products and the country of origin, distinct processing conditions, or microbial communities. Future application of the HTS method offers the potential to further unravel complex microbial communities in fermented meats, as well as to assess the impact of different processing conditions on microbial consortia.

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Micro-aggregation of a pristine grassland soil selects for bacterial and fungal communities and changes in nitrogen cycling potentials

10.1101/2021.10.13.464334 ◽

2021 ◽

Author(s):

Christoph Keuschnig ◽

Jean Martins ◽

Aline Navel ◽

Pascal Simonet ◽

Catherine Larose

Keyword(s):

Particle Size ◽

Nitrogen Cycling ◽

Bacterial Communities ◽

Soil Microbial Communities ◽

Microbial Interactions ◽

Fungal Communities ◽

Supernatant Fraction ◽

Soil Microbial ◽

Microbial Analysis

Microbial analysis at the micro scale of soil is essential to the overall understanding of microbial organization and interactions, and necessary for a better understanding of soil ecosystem functioning. While bacterial communities have been extensively described, little is known about the organization of fungal communities as well as functional potentials at scales relevant to microbial interactions. Fungal and bacterial communities and changes in nitrogen cycling potentials in the pristine Rothamsted Park Grass soil (bulk soil) as well as in its particle size sub-fractions (PSFs; > 250 µm, 250-63 µm, 63-20 µm, 20-2 µm, < 2 µm and supernatant) were studied. The potential for nitrogen reduction was found elevated in bigger aggregates. The relative abundance of Basidiomycota deceased with decreasing particle size, Ascomycota showed an increase and Mucoromycota became more prominent in particles less than 20 µm. Bacterial community structures changed below 20 µm at the scale where microbes operate.Strikingly, only members of two bacterial and one fungal phyla (Proteobacteria, Bacteroidota and Ascomycota, respectively) were washed-off the soil during fractionation and accumulated in the supernatant fraction where most of the detected bacterial genera (e.g., Pseudomonas, Massilia, Mucilaginibacter, Edaphobaculum, Duganella, Janthinobacterium and Variovorax) were previously associated with exopolysaccharide production and biofilm formation.Overall, the applied method shows potential to study soil microbial communities at micro scales which might be useful in studies focusing on the role of specific fungal taxa in soil structure formation as well as research on how and by whom biofilm-like structures are distributed and organized in soil.

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Improved Selection of Internal Transcribed Spacer-Specific Primers Enables Quantitative, Ultra-High-Throughput Profiling of Fungal Communities

Applied and Environmental Microbiology ◽

10.1128/aem.03870-12 ◽

2013 ◽

Vol 79 (8) ◽

pp. 2519-2526 ◽

Cited By ~ 212

Author(s):

Nicholas A. Bokulich ◽

David A. Mills

Keyword(s):

High Throughput ◽

Internal Transcribed Spacer ◽

High Throughput Sequencing ◽

Work Flow ◽

Fungal Communities ◽

Amplification Bias ◽

Sequencing Technologies ◽

Yeast Community ◽

Primer Sets ◽

Sequencing Platforms

ABSTRACTUltra-high-throughput sequencing (HTS) of fungal communities has been restricted by short read lengths and primer amplification bias, slowing the adoption of newer sequencing technologies to fungal community profiling. To address these issues, we evaluated the performance of several common internal transcribed spacer (ITS) primers and designed a novel primer set and work flow for simultaneous quantification and species-level interrogation of fungal consortia. Primer comparison and validation were predictedin silicoand by sequencing a “mock community” of mixed yeast species to explore the challenges of amplicon length and amplification bias for reconstructing defined yeast community structures. The amplicon size and distribution of this primer set are smaller than for all preexisting ITS primer sets, maximizing sequencing coverage of hypervariable ITS domains by very-short-amplicon, high-throughput sequencing platforms. This feature also enables the optional integration of quantitative PCR (qPCR) directly into the HTS preparatory work flow by substituting qPCR with these primers for standard PCR, yielding quantification of individual community members. The complete work flow described here, utilizing any of the qualified primer sets evaluated, can rapidly profile mixed fungal communities and capably reconstructed well-characterized beer and wine fermentation fungal communities.

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Differential Responses of Soil Bacterial and Fungal Community to Short-Term Crop Tree Management in a Larix gmelinii Plantation

Forests ◽

10.3390/f12101411 ◽

2021 ◽

Vol 12 (10) ◽

pp. 1411

Author(s):

Li Ji ◽

Jiangbo Yu ◽

Xingzhe Zhang ◽

Yue Liu ◽

Lixue Yang

Keyword(s):

Organic Carbon ◽

Bacterial Communities ◽

Dissolved Organic Nitrogen ◽

Organic Nitrogen ◽

Soil Fungi ◽

Fungal Communities ◽

Larix Gmelinii ◽

Short Term ◽

Soil Microbial ◽

Soil Bacterial

Crop tree management (CTM) is a widely applicable silviculture technology that is used to improve the performance of individual trees. However, only little information is available about the effects of the CTM regime on the soil microbial community structure. We conducted a study to explore the effects of short-term (five years) CTM on the soil bacterial and fungal diversity, community composition, and structure in the 0–10 cm soil layer in a Larix gmelinii (Rupr.) Kuzen. plantation. We set out to investigate the differential response of bacterial and fungal communities to variations in soil properties mediated by short-term CTM. Compared with the control plots, the soil microbial biomass carbon and microbial biomass nitrogen in CTM increased significantly by 64.2% and 32.3%, respectively. CTM significantly promoted the content of soil organic carbon, dissolved organic carbon, and nitrate nitrogen, and reduced the content of dissolved organic nitrogen. CTM changed the Shannon and Simpson indices of soil fungi to a remarkable extent but had little effect on the α diversity of bacterial communities. The bacterial β diversity was more sensitive to CTM than fungi. The relative abundance of Verrucomicrobiae (the dominant class of soil bacteria) in CTM was significantly increased by 78.2%, while the relative abundance of Agaricomycetes (dominant class for soil fungi) was reduced by 43.3%. We observed a significantly increased number of unique OTUs for soil fungi in the CTM plots. Redundancy analysis showed that dissolved organic carbon, soil moisture, and total phosphorus content significantly affected the composition of bacterial communities, while soil dissolved organic nitrogen, C/N, and total phosphorus drove the high variation in fungal community composition. Overall, our results emphasize the divergent response of soil bacterial and fungal communities in Larix gmelinii plantations to short-term CTM. We must pay more attention to the functional role of soil microbiota in future forest management.

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High-throughput amplicon sequencing-based analysis of active fungal communities inhabiting grapevine after hot-water treatments reveals unexpectedly high fungal diversity

Fungal Ecology ◽

10.1016/j.funeco.2018.07.011 ◽

2018 ◽

Vol 36 ◽

pp. 26-38 ◽

Cited By ~ 12

Author(s):

Ales Eichmeier ◽

Jakub Pečenka ◽

Eliska Peňázová ◽

Miroslav Baránek ◽

Santiago Català-García ◽

...

Keyword(s):

High Throughput ◽

Fungal Diversity ◽

Amplicon Sequencing ◽

Hot Water ◽

Fungal Communities ◽

Water Treatments

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Variations in soil nutrient dynamics and bacterial communities in long-term tea monoculture production systems

10.1101/2021.04.25.441296 ◽

2021 ◽

Author(s):

Heng Gui ◽

Lichao Fan ◽

Donghui Wang ◽

Peng Yan ◽

Xin Li ◽

...

Keyword(s):

Bacterial Communities ◽

Nutrient Dynamics ◽

Soil Microbial Communities ◽

Soil Nutrient ◽

Stand Age ◽

Soil Microbial ◽

Soil Bacterial Communities ◽

Tea Plantations ◽

Soil Bacterial

AbstractLong-term monoculture agriculture systems could lead to soil degradation and yield decline. The ways in which soil microbiotas interact with one another, particularly in response to long-term tea monoculture systems are currently unclear. In this study, through the comparison of three independent tea plantations across eastern China composed of varying stand ages (from 3 years to 90 years after conversion from forest), we found that long-term tea monoculture led to significant increases in soil total organic carbon (TOC) and microbial nitrogen (MBN). Additionally, the structure, function and co-occurrence network of soil microbial communities were investigated by pyrosequencing 16S rRNA genes. The pyrosequencing analysis revealed that structures and functions of soil bacterial communities were significantly affected by different stand ages of tea plantations, but sampling sites and land-use conversion (from forest to tea plantation) still outcompeted stand age to control the diversity and structure of soil bacterial communities. Further RDA analysis revealed that the C and N availability improvement in tea plantation soils led to variation of structure and function in soil microbial communities. Moreover, co-occurrence network analysis of soil bacterial communities also demonstrated that interactions among soil bacteria taxa were strengthened with the increasing stand age of respective tea stands. Overall, this study provides a comprehensive understanding of the impact of long-term monoculture stand age on soil nutrient dynamics and bacterial communities in tea production.

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Environmental Microbiota Drives Microbial Succession and Metabolic Profiles during Chinese Liquor Fermentation

Applied and Environmental Microbiology ◽

10.1128/aem.02369-17 ◽

2017 ◽

Vol 84 (4) ◽

Cited By ~ 26

Author(s):

Xueshan Wang ◽

Hai Du ◽

Yan Zhang ◽

Yan Xu

Keyword(s):

High Throughput ◽

Bacterial Communities ◽

High Throughput Sequencing ◽

Fungal Communities ◽

Metabolic Profiles ◽

Microbial Succession ◽

Chinese Liquor ◽

Content Type ◽

Target Analysis ◽

Metabolite Target Analysis

ABSTRACT Many microorganisms in the environment participate in the fermentation process of Chinese liquor. However, it is unknown to what extent the environmental microbiota influences fermentation. In this study, high-throughput sequencing combined with multiphasic metabolite target analysis was applied to study the microbial succession and metabolism changes during Chinese liquor fermentation from two environments (old and new workshops). SourceTracker was applied to evaluate the contribution of environmental microbiota to fermentation. Results showed that Daqu contributed 9.10 to 27.39% of bacterial communities and 61.06 to 80.00% of fungal communities to fermentation, whereas environments (outdoor ground, indoor ground, tools, and other unknown environments) contributed 62.61 to 90.90% of bacterial communities and 20.00 to 38.94% of fungal communities to fermentation. In the old workshop, six bacterial genera ( Lactobacillus [11.73% average relative abundance], Bacillus [20.78%], Pseudomonas [6.13%], Kroppenstedtia [10.99%], Weissella [16.64%], and Pantoea [3.40%]) and five fungal genera ( Pichia [55.10%], Candida [1.47%], Aspergillus [10.66%], Saccharomycopsis [22.11%], and Wickerhamomyces [3.35%]) were abundant at the beginning of fermentation. However, in the new workshop, the change of environmental microbiota decreased the abundances of Bacillus (5.74%), Weissella (6.64%), Pichia (33.91%), Aspergillus (7.08%), and Wickerhamomyces (0.12%), and increased the abundances of Pseudomonas (17.04%), Kroppenstedtia (13.31%), Pantoea (11.41%), Acinetobacter (3.02%), Candida (16.47%), and Kazachstania (1.31%). Meanwhile, in the new workshop, the changes of microbial community resulted in the increase of acetic acid, lactic acid, malic acid, and ethyl acetate, and the decrease of ethyl lactate during fermentation. This study showed that the environmental microbiota was an important source of fermentation microbiota and could drive both microbial succession and metabolic profiles during liquor fermentation. IMPORTANCE Traditional solid-state fermentation of foods and beverages is mainly carried out by complex microbial communities from raw materials, starters, and the processing environments. However, it is still unclear how the environmental microbiota influences the quality of fermented foods and beverages, especially for Chinese liquors. In this study, we utilized high-throughput sequencing, microbial source tracking, and multiphasic metabolite target analysis to analyze the origins of microbiota and the metabolic profiles during liquor fermentation. This study contributes to a deeper understanding of the role of environmental microbiota during fermentation.

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Fungal endophyte diversity in table grapes

Canadian Journal of Microbiology ◽

10.1139/cjm-2020-0293 ◽

2021 ◽

Vol 67 (1) ◽

pp. 29-36

Author(s):

Champa Wijekoon ◽

Zoe Quill

Keyword(s):

Internal Transcribed Spacer ◽

Fungal Endophytes ◽

Pathogenic Fungi ◽

Amplicon Sequencing ◽

Fungal Endophyte ◽

Table Grape ◽

Fungal Communities ◽

Table Grapes ◽

Pharmaceutical Applications ◽

Microbial Sources

Plant fungal endophytes are diverse microbial sources that reside inside plants. Grapes (Vitis vinifera) are rich in polyphenols that have beneficial health effects, and recent research has shown that fungal endophytes in grapes may contribute to the production of these polyphenols and may serve as biocontrol agents. In this study, we determined the fungal microbial endophyte diversity in North American table grapes found at a Winnipeg, Manitoba, market. The amplicon internal transcribed spacer (ITS) metagenomics approach was used to profile the fungal communities of the fruit endophyte microbiome of three table grape types. The data supported endophyte diversity in different table grapes, including possible bioactive, saprophytic, and pathogenic fungi. Culturable endophytes were isolated and identified by morphology and ITS amplicon sequencing. The majority of the isolated culturable strains included Alternaria spp. and Cladosporium spp. The results provided evidence of the existence of diverse fungal endophytes isolated and identified from the fruit of the table grapes. These fungal endophytes may have potential in agricultural, industrial, and pharmaceutical applications.

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Changes of microorganism composition in fresh and stored bee pollen from Southern Germany

Environmental Science and Pollution Research ◽

10.1007/s11356-021-13932-4 ◽

2021 ◽

Author(s):

Carolin Friedle ◽

Paul D’Alvise ◽

Karsten Schweikert ◽

Klaus Wallner ◽

Martin Hasselmann

Keyword(s):

Dietary Supplement ◽

Fungal Community ◽

Bacterial Communities ◽

Amplicon Sequencing ◽

Storage Conditions ◽

Fungal Communities ◽

Human Consumption ◽

Palynological Analysis ◽

Bee Pollen ◽

Plant Pollen

AbstractAnalysis of plant pollen can provide valuable insights into the existing spectrum of microorganisms in the environment. When harvesting bee-collected pollen as a dietary supplement for human consumption, timely preservation of the freshly collected pollen is fundamental for product quality. Environmental microorganisms contained in freshly collected pollen can lead to spoilage by degradation of pollen components. In this study, freshly collected bee pollen was sampled at different locations and stored under various storage conditions to examine the hypothesis that storage conditions may have an effect on the composition of microorganisms in pollen samples. The samples were analyzed using 16S and 18S amplicon sequencing and characterized by palynological analysis. Interestingly, the bacterial communities between pollen samples from different locations varied only slightly, whereas for fungal community compositions, this effect was substantially increased. Further, we noticed that fungal communities in pollen are particularly sensitive to storage conditions. The fungal genera proportion Cladosporium and Mycosphaerella decreased, while Zygosaccharomyces and Aspergillus increased during storage. Aspergillus and Zygosaccharomyces fractions increased during storage at 30 °C, which could negatively impact the pollen quality if it is used as a dietary supplement.

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