Characterizing fungal communities in medicinal and edible Cassiae Semen using high-throughput sequencing

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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Uncovering fungal community composition in natural habitat of Ophiocordyceps sinensis using high-throughput sequencing and culture-dependent approaches

10.21203/rs.3.rs-28393/v1 ◽

2020 ◽

Author(s):

Chuanbo Zhang ◽

Chao-Hui Ren ◽

Yan-Li Wang ◽

Qi-Qi Wang ◽

Yun-Sheng Wang ◽

...

Keyword(s):

Community Structure ◽

High Throughput ◽

Fungal Community ◽

High Throughput Sequencing ◽

Fungal Communities ◽

Bioactive Metabolites ◽

Fungal Community Structure ◽

Ophiocordyceps Sinensis ◽

Culturable Fungi ◽

Culture Dependent

Abstract Background The fungal communities inhabiting natural Ophiocordyceps sinensis play critical ecological roles in alpine meadow ecosystem, contribute to infect host insect, influence the occurrence of O. sinensis, and are repertoire of potential novel metabolites discovery. However, a comprehensive understanding of fungal communities of O. sinensis remain elusive. Therefore, the present study aimed to unravel fungal communities of natural O. sinensis using combination of high-throughput sequencing and culture-dependent approach. Results A total of 280,519 high-quality sequences, belonging to 5 fungal phyla, 15 classes, 41 orders, 79 families, 112 genera, and 352 putative operational taxonomic units (OTUs) were obtained from natural O. sinensis using high-throughput sequencing. Among of which, 43 genera were identified in external mycelial cortices (EMC), Ophiocordyceps, Sebacinia, Archaeorhizomyces were predominant genera with the abundance of 95.86%, 1.14%, 0.85%, respectively. Total 66 genera were identified from soil microhabitat, Inocybe, Archaeorhizomyces, Unclassified Thelephoraceae, Tomentella, Thelephora, Sebacina, Unclassified Ascomycota, Unclassified Fungi were predominant genera with an average abundance of 53.32%, 8.69%, 8.12%, 8.12%, 7.21%, 4.6%, 3.08% and 3.05%, respectively. The fungal communities in external mycelial cortices (EMC) were significantly distinct from the soil microhabitat (Soil). Meanwhile, seven culture media that benefit for the growth of O. sinensis were used to isolate culturable fungi at 16 °C, resulted in 77 fungal strains isolated for rDNA ITS sequence analysis, belonging to 33 genera, including Ophiocordyceps, Trichoderma, Cytospora, Truncatella, Dactylonectria, Isaria, Cephalosporium, Fusarium, Cosmospora, Paecilomyces, etc.. Among all culturable fungi, Mortierella and Trichoderma were predominant genera of total isolates. Conclusions The significantly distinction and overlap in fungal community structure between two approaches highlight that integration of approaches would generate more information than either of them. Our finding is the first investigation of fungal community structure of natural O. sinensis by two approachs, provide new insight into O. sinensis associated fungi, and support that microbiota of O. sinensis is an untapped source for novel bioactive metabolites discovery.

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Uncovering fungal community composition in natural habitat of Ophiocordyceps sinensis using high-throughput sequencing and culture-dependent approaches

10.21203/rs.3.rs-28393/v4 ◽

2020 ◽

Author(s):

Chuanbo Zhang ◽

Chao-Hui Ren ◽

Yan-Li Wang ◽

Qi-Qi Wang ◽

Yun-Sheng Wang ◽

...

Keyword(s):

Community Structure ◽

High Throughput ◽

Fungal Community ◽

High Throughput Sequencing ◽

Fungal Communities ◽

Comprehensive Understanding ◽

Fungal Community Structure ◽

Ophiocordyceps Sinensis ◽

Culturable Fungi ◽

Culture Dependent

Abstract Background: The fungal communities inhabiting natural Ophiocordyceps sinensis play critical ecological roles in alpine meadow ecosystem, contribute to infect host insect, influence the occurrence of O. sinensis, and are repertoire of potential novel metabolites discovery. However, a comprehensive understanding of fungal communities of O. sinensis remain elusive. Therefore, the present study aimed to unravel fungal communities of natural O. sinensis using combination of high-throughput sequencing and culture-dependent approaches. Results: A total of 280,519 high-quality sequences, belonging to 5 fungal phyla, 15 classes, 41 orders, 79 families, 112 genera, and 352 putative operational taxonomic units (OTUs) were obtained from natural O. sinensis using high-throughput sequencing. Among of which, 43 genera were identified in external mycelial cortices, Ophiocordyceps, Sebacinia, Archaeorhizomyces were predominant genera with the abundance of 95.86%, 1.14%, 0.85%, respectively. A total of 66 genera were identified from soil microhabitat, Inocybe, Archaeorhizomyces, unclassified Thelephoraceae, Tomentella, Thelephora, Sebacina, unclassified Ascomycota, unclassified Fungi were predominant genera with an average abundance of 53.32%, 8.69%, 8.12%, 8.12%, 7.21%, 4.6%, 3.08% and 3.05%, respectively. The fungal communities in external mycelial cortices were significantly distinct from the soil microhabitat. Meanwhile, seven types of culture media were used to isolate culturable fungi at 16°C, resulted in 77 fungal strains isolated by rDNA ITS sequence analysis, belonging to 33 genera, including Ophiocordyceps, Trichoderma, Cytospora, Truncatella, Dactylonectria, Isaria, Cephalosporium, Fusarium, Cosmospora and Paecilomyces, etc.. Among all culturable fungi, Mortierella and Trichoderma were predominant genera. Conclusions: The significantly differences and overlap in fungal community structure between two approaches highlight that the integration of high-throughput sequencing and culture-dependent approaches would generate more information. Our result reveal a comprehensive understanding of fungal community structure of natural O. sinensis, provide new insight into O. sinensis associated fungi, and support that microbiota of natural O. sinensis is an untapped source for novel bioactive metabolites discovery.

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Uncovering fungal community composition in natural habitat of Ophiocordyceps sinensis using high-throughput sequencing and culture-dependent approaches

10.21203/rs.3.rs-28393/v2 ◽

2020 ◽

Author(s):

Chuanbo Zhang ◽

Chao-Hui Ren ◽

Yan-Li Wang ◽

Qi-Qi Wang ◽

Yun-Sheng Wang ◽

...

Keyword(s):

Community Structure ◽

High Throughput ◽

Fungal Community ◽

High Throughput Sequencing ◽

Fungal Communities ◽

Comprehensive Understanding ◽

Fungal Community Structure ◽

Ophiocordyceps Sinensis ◽

Culturable Fungi ◽

Culture Dependent

Abstract Background: The fungal communities inhabiting natural Ophiocordyceps sinensis play critical ecological roles in alpine meadow ecosystem, contribute to infect host insect, influence the occurrence of O. sinensis, and are repertoire of potential novel metabolites discovery. However, a comprehensive understanding of fungal communities of O. sinensis remain elusive. Therefore, the present study aimed to unravel fungal communities of natural O. sinensis using combination of high-throughput sequencing and culture-dependent approaches. Results: A total of 280,519 high-quality sequences, belonging to 5 fungal phyla, 15 classes, 41 orders, 79 families, 112 genera, and 352 putative operational taxonomic units (OTUs) were obtained from natural O. sinensis using high-throughput sequencing. Among of which, 43 genera were identified in external mycelial cortices (EMC), Ophiocordyceps, Sebacinia, Archaeorhizomyces were predominant genera with the abundance of 95.86%, 1.14%, 0.85%, respectively. A total of 66 genera were identified from soil microhabitat (Soil), Inocybe, Archaeorhizomyces, unclassified Thelephoraceae, Tomentella, Thelephora, Sebacina, unclassified Ascomycota, unclassified Fungi were predominant genera with an average abundance of 53.32%, 8.69%, 8.12%, 8.12%, 7.21%, 4.6%, 3.08% and 3.05%, respectively. The fungal communities in external mycelial cortices were significantly distinct from the soil microhabitat. Meanwhile, seven types of culture media were used to isolate culturable fungi at 16°C, resulted in 77 fungal strains isolated by rDNA ITS sequence analysis, belonging to 33 genera, including Ophiocordyceps, Trichoderma, Cytospora, Truncatella, Dactylonectria, Isaria, Cephalosporium, Fusarium, Cosmospora and Paecilomyces, etc.. Among all culturable fungi, Mortierella and Trichoderma were predominant genera. Conclusions: The significantly differences and overlap in fungal community structure between two approaches highlight that the integration of high-throughput sequencing and culture-dependent approaches would generate more information. Our result reveal a comprehensive understanding of fungal community structure of natural O. sinensis, provide new insight into O. sinensis associated fungi, and support that microbiota of natural O. sinensis is an untapped source for novel bioactive metabolites discovery.

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Fungal communities in Nelumbinis semen characterized by high-throughput sequencing

International Journal of Food Microbiology ◽

10.1016/j.ijfoodmicro.2021.109428 ◽

2021 ◽

pp. 109428

Author(s):

Nan Long ◽

Jinxin Liu ◽

Xiaofang Liao ◽

Boyu Jia ◽

Jiali Liu ◽

...

Keyword(s):

High Throughput ◽

High Throughput Sequencing ◽

Fungal Communities ◽

Nelumbinis Semen

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Multi-element fingerprinting and high throughput sequencing identify multiple elements that affect fungal communities inQuercus macrocarpafoliage

Plant Signaling & Behavior ◽

10.4161/psb.5.9.12681 ◽

2010 ◽

Vol 5 (9) ◽

pp. 1157-1161 ◽

Cited By ~ 3

Author(s):

Ari Jumpponen ◽

Karen Keating ◽

Gary Gadbury ◽

Kenneth L. Jones ◽

J. David Mattox

Keyword(s):

High Throughput ◽

High Throughput Sequencing ◽

Fungal Communities

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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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High-throughput sequencing-based analysis of endogenetic fungal communities inhabiting the Chinese Cordyceps reveals unexpectedly high fungal diversity

Scientific Reports ◽

10.1038/srep33437 ◽

2016 ◽

Vol 6 (1) ◽

Cited By ~ 14

Author(s):

Fei Xia ◽

Xin Chen ◽

Meng-Yuan Guo ◽

Xiao-Hui Bai ◽

Yan Liu ◽

...

Keyword(s):

High Throughput ◽

Fungal Diversity ◽

High Throughput Sequencing ◽

Fungal Communities

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The Fungal Communities and Flavor Profiles in Different Types of High-Temperature Daqu as Revealed by High-Throughput Sequencing and Electronic Senses

Frontiers in Microbiology ◽

10.3389/fmicb.2021.784651 ◽

2021 ◽

Vol 12 ◽

Author(s):

Wenchao Cai ◽

Yu’ang Xue ◽

Yurong Wang ◽

Wenping Wang ◽

Na Shu ◽

...

Keyword(s):

High Temperature ◽

Nitrogen Oxides ◽

Organic Compounds ◽

High Throughput ◽

High Throughput Sequencing ◽

Electronic Tongue ◽

Illumina Miseq ◽

Fungal Communities ◽

Significant Difference ◽

Distinct Character

Polymicrobial co-fermentation is among the distinct character of high-temperature Daqu. However, fungal communities in the three types of high-temperature Daqu, namely, white high-temperature Daqu, black high-temperature Daqu, and yellow high-temperature Daqu, are yet to be characterized. In this study, the fungal diversity, taste, and aroma profiles in the three types of high-temperature Daqu were investigated by Illumina MiSeq high-throughput sequencing, electronic tongue, and electronic nose, respectively. Ascomycota and Basidiomycota were detected as the absolute dominant fungal phylum in all types of high-temperature Daqu samples, whereas Thermomyces, Thermoascus, Aspergillus, Rasamsonia, Byssochlamys, and Trichomonascus were identified as the dominant fungal genera. The fungal communities of the three types of high-temperature Daqu differed significantly (p < 0.05), and Thermomyces, Thermoascus, and Monascus could serve as the biomarkers in white high-temperature Daqu, black high-temperature Daqu, and yellow high-temperature Daqu, respectively. The three types of high-temperature Daqu had an extremely significant difference (p < 0.01) in flavor: white high-temperature Daqu was characterized by sourness, bitterness, astringency, richness, methane, alcohols, ketones, nitrogen oxides, and sulfur organic compounds; black high-temperature Daqu was characterized by aftertaste-A, aftertaste-B, methane-aliph, hydrogen, and aromatic compounds; and yellow high-temperature Daqu was characterized by saltiness, umami, methane, alcohols, ketones, nitrogen oxides, and sulfur organic compounds. The fungal communities in the three types of high-temperature Daqu were significantly correlated with taste but not with aroma, and the aroma of high-temperature Daqu was mainly influenced by the dominant fungal genera including Trichomonascus, Aspergillus, Thermoascus, and Thermomyces. The result of the present study enriched and refined our knowledge of high-temperature Daqu, which had positive implications for the development of traditional brewing technique.

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