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Research article
Pyrosequencing and Taxonomic Composition of the Fungal Community from Soil of Tricholoma matsutake in Gyeongju
1Department of Life Sciences and Biotechnology, Kyungpook National University, Daegu 41566, Republic of Korea
2Department of Forest Environment, Gyeongsangbuk-do Forest Environment Research Institute, Gyeong-ju 38174, Republic of Korea
J. Microbiol. Biotechnol. 2021; 31(5): 686-695
Published May 28, 2021 https://doi.org/10.4014/jmb.2103.03021
Copyright © The Korean Society for Microbiology and Biotechnology.
Abstract
Keywords
Graphical Abstract

Introduction
Mycorrhizal fungi are an important group of fungi in the soil ecosystem. They have a mutualistic relationship with living plants, exchanging nutrients and some important compounds [15-19]. The major group of mycorrhizal fungi is the ectomycorrhizae (ECM) fungi group. ECM fungi, including
Next-generation sequencing (NGS) enables the rapid analysis of massive amounts of DNA sequences. It also permits the analysis of DNA sequences from microorganisms that are hard to cultivate [31-34]. Pyrosequencing is one of the sequencing approaches for NGS and has facilitated the study of mass and diverse microorganisms from soil samples. For this study, we used the Illumina MiSeq sequencing platform, which uses the sequences of ribosomal DNA as DNA barcodes to classify the soil fungi rapidly.
Here, we statistically analyzed the characteristics of soil fungal communities from two regions using metagenomic pyrosequencing with barcode to investigate diversity, species richness, and relative abundance or taxonomic composition of the communities in areas where
Materials and Methods
Collection of Soil Samples
We collected two soil samples each in pine forests in October 2017 in Gyeongju and in November 2017 in Yeongdeok, South Korea. Two samples were also collected in October 2018 and in February and June 2019 at Gyeongju. Immediately after harvesting the fruiting body of
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Table 1 . Sampling information of ten samples of the soil fungal community related to
T. matsutake .Gyeongju Yeongdeok October 2017 October 2018 February 2019 June 2019 November 2017 25~30 cm far from the fairy ring G-F1-1 G-F2-1 G-W-1 G-S-1 Y-F1-1 Under the fruiting body of T. matsutake G-F1-2 G-F2-2 G-W-2 G-S-2 Y-F1-2
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GPS coordinates of the sampling sites were as follows:
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• G-F1: 35°47'22.1" N, 129°14'06.9" E
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• Y-F1: 36°28'39.1" N, 129°22'12.0" E
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• G-F2, G-W, G-S: 35°47'20.0" N, 129°14'09.0" E
DNA Extraction and Pyrosequencing
Each 10 g of soil samples was used for isolation of metagenomic DNA using the FastDNA SPIN Kit for Soil (MP Biomedicals, USA) to conduct the pyrosequencing. Polymerase Chain Reaction (PCR) targeting the ITS2 region was used to amplify isolated chromosomal DNA. The sequences of the forward primer were 5′-AATGATACGGCGACCACCGAGATCTACAC-XXXXXXXX-TCGTCGGCAGCGTC-AGATGTGTATAAGAGACAG-GCATCGATGAAGAACGCAGC-3′. The sequences of the reverse primer were 5′- CAAGCAGAAGACGGC ATACGAGAT-XXXXXXXX-GTCTCGTGGGCTCGG-AGATGTGTATAAGAGACAGTCC-TCCGCTTATTGA TATGC-3′. “X” indicates that the barcode sequence differed between samples. A PTC-200 Peltier Thermal Cycler (MJ Research, USA) was used to conduct the PCR. For separation of PCR products, identical amounts of PCR products from each sample were used by agarose gel electrophoresis. The products of PCR were purified using the CleanPCR (CleanNA, Netherlands) and the quantification of the purified PCR products was performed with a Quant-iT PicoGreen dsDNA Assay Kit (Invitrogen, USA). PCR products longer than 300 bp were purified, and an Agilent 2100 Bioanalyzer (Agilent Technologies, USA) was used to analyze the base sequence of the DNA fragments [36]. In addition, the Illumina MiSeq sequencing platform (Illumina, USA) was used for the pyrosequencing analysis conducted by Chunlab Inc. (Korea), following the instructions supplied by the manufacturer [37]. Pyrosequencing reads data were submitted to the EMBL-EMI database (www.ebi.ac.uk) under accession number PRJEB42318 (primary) and ERP126157 (secondary).
Statistical Data Analysis and Taxonomic Identification
The pyrosequencing raw reads were processed by using the barcode sequences. The fusion primers with barcode and low-quality reads were trimmed and removed using Trimmomatic 0.32 [38]. We used CLcommunity software (Chunlab Inc.) for statistical analysis. To identify operational taxonomic units (OTUs) at 97% sequence similarity [39], the CD-HIT program was used. The Mothur platform [40, 41] was used to calculate the rarefaction curves and diversity indices. For taxonomic composition and relative abundance, random sample subsets were generated with the lowest number of reads. Conditionally,
Results and Discussion
Pyrosequencing Results and Statistical Data Analysis
For fungal communities, the total number of valid reads after preprocessing was 881,125 from all eight samples (Table 2). The number of valid reads of G-F2-2 was greatest, at 106,048, followed by G-S-2 at 102,565, G-F1-1 at 101,347, G-F1-2 at 94,695, G-S-1 at 94,346, G-F2-1 at 92,141, Y-F1-1 at 89,952, Y-F1-2 at 86,224, G-W-2 at 57,412, and G-W-1 at 56,395. Overall, there were a large enough number of reads to analyze the microbial communities. Obviously, with fewer reads in the winter samples, the overall number of surviving fungi was reduced. However, the spring samples showed that the number of reads increased again and the ecosystem of soil microbes recovered. In the sample from Yeongdeok, the number of valid reads was slightly lower than that from Gyeongju. The OTUs of Y-F1-1 were the highest, at 664, and those of Y-F1-2 were the lowest, at 124. These two samples showed the biggest difference, at 540. On the other hand, the G-S-1 and G-S-2 samples were 439 and 469, respectively, showing 30 different results. The OTUs of most Sample 2 groups were lower than those of Sample 1 groups. Naturally, the Shannon index, which is one of the most important diversity indices, was similar to the number of OTUs. For the most part, the number for Sample 2’s was lower. In particular, G-F2-1 and G-F2-2 showed a significantly large difference. The Shannon index measures species richness and evenness, which means that the higher the number, the greater the diversity of the community. Therefore, fungal diversity was low in the soil samples in which
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Table 2 . Pyrosequencing results and statistical analysis of the soil fungal community related to
T. matsutake .G-F1-1 G-F1-2 G-F2-1 G-F2-2 G-W-1 G-W-2 G-S-1 G-S-2 Y-F1-1 Y-F1-2 Number of Valid reads 101347 94695 92141 106048 56395 57412 94346 102565 89952 86224 OTUsa 347 203 578 211 554 230 439 469 664 124 Chao1b 348.68 204.38 596.83 217.52 561.17 238.12 447.52 475.43 665.60 124.58 Shannonc 2.90 2.05 3.69 0.97 3.54 1.25 2.61 2.65 4.01 1.70 Simpsond 0.11 0.30 0.06 0.64 0.08 0.58 0.19 0.28 0.04 0.29 Goods lib. Coveragee(%) 99.98 99.99 99.92 99.98 99.92 99.94 99.96 99.96 99.98 99.99 aOTUs: Operational Taxonomic Units
bChao1: Chao1 estimation for species richness
cShannon: Shannon index for species diversity, > 0, higher, more diverse
dSimpson: Simpson index for species diversity, 0 ~ 1, 1 = the simplest
eGoods Lib. Coverage: [1 - (number of singleton OTUs / number of total reads)] × 100
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Fig. 1.
Rarefaction curves for operational taxonomic units (OTUs) of fungi from all soil samples at Gyeongju and Yeongdeok. OTUs were clustered at 97% similarity with CD-HIT. This shows the rate of increase in the number of OTUs (species) with the number of acquired sequences. The curves ofT. matsutake -dominant soil samples were in low positions. This implies that the fungal diversity is low inT. matsutake -dominant soil samples (X-axis: the number of sequencing reads; Y-axis: the number of OTUs).
Comparison of Fungal Communities: Composition of T. matsutake
First, for analysis of taxonomic composition and relative abundance, random sample subsets were generated with the lowest number of reads: 56395 (G-W-1).
The composition of
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Table 3 . Composition (%) of
T. matsutake in each sample (using the sum of reads ofTricholoma matsutake ,Tricholoma matsutake_1 , andTricholoma matsutake_7 ).G-F1-1 G-F1-2 G-F2-1 G-F2-2 G-W-1 G-W-2 G-S-1 G-S-2 Y-F1-1 Y-F1-2 0.00 57.76 0.12 79.13 0.01 75.19 38.92 52.11 0.00 52.25
Comparison of Fungal Communities by Region
First, we conducted an analysis at the phylum level of the taxonomic composition of soil samples from G-F1-1, G-F1-2, G-F2-1, G-F2-2, Y-F1-1, and Y-F1-2. A total of five phyla were identified (>1%): Basidiomycota, Ascomycota, Mucoromycota, Fungi_p (phylum name unknown), and Mortierellomycota (Fig. 2). Including
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Fig. 2.
Taxonomic composition at the phylum level from G-F1-1, G-F1-2, G-F2-1, G-F2-2, Y-F1-1, and Y-F1- 2 samples. Samples of Gyeongju and Yeongdeok collected in autumn were compared. Fungal phyla with a relative abundance greater than 1% in at least one of the samples are shown and phyla less than 1% were shown as ETC. Basidiomycota was more abundant in Sample-2 (under fruiting bodies ofT. matsutake ) than Sample-1 (far from the fairy ring) regardless of regions and was significantly dominant in Y-F1-2 (G-F1: October 2017, G-F2: October 2018, and Y-F1: October 2017).
At the class level, a total of nine fungal classes found to represent more than 1% of all the classes were identified: Agaricomycetes, Umbelopsidomycetes, Eurotiomycetes, Leotiomycetes, Dothideomycetes, Sordariomycetes, Mortierellomycetes, Fungi_c (class name was unknown), and GS25 (unnamed). We focused on the classes of the relatively abundant phyla Basidiomycota, Ascomycota, Mucoromycota, and Mortierellomycota. In phylum Basidiomycota, we found Agaricomycetes in G-F1-1 (47.99%), G-F1-2 (65.95%), G-F2-1 (26.23%), G-F2-2 (80.12%), Y-F1-1 (46.39%), and Y-F1-2 (93.17%). Phylum Mucoromycota was represented by Umbelopsidomycetes: G-F1-1 (8.22%), G-F1-2 (14.82%), G-F2-1 (14.33%), G-F2-2 (10.60%), Y-F1-1 (11.63%), and Y-F1-2 (4.18%). Class Umbelopsidomycetes was the second most dominant class in all Sample 2’s after class Agaricomycetes, including
At the genus level, the top 10 most abundant genera were
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Table 4 . Gradient heatmap of the taxonomic composition (%) at the genus level of each sample for comparison by region (including
T. matsutake , _uc: unclassified, _g: the genus name was unknown).
Compared to the fungal community from Yeongdeok, Y-F1-1 shared
In our results,
However, in the
On the other hand, in our samples, there were several genera that were lower in Sample 2 than in Sample 1. The ratios of these genera were as follows:
For comparison of species comprising less than 1% of the community, only this number was compared with samples before normalization. Vaario and colleagues found that
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Table 5 . The number of reads for the common species in G-F1-2, G-F2-2, and Y-F1-2 (_uc: unclassified, _s: the species name was unknown).
Name G-F1-2 G-F2-2 Y-F1-2 Umbelopsis dimorpha 13420 11133 1095 Oidiodendron chlamydosporicum 450 347 325 Oidiodendron _uc642 759 26 Sagenomella diversispora 34 151 2 Cenococcum _uc4500 52 6 Sistotrema _uc2 15 10 Cladophialophora _uc872 7 115 Pezicula _uc128 7 85 Oidiodendron pilicola 657 160 8 Fungi_uc_s 47 8 7 Umbelopsis _uc231 10 379 Sagenomella _uc6 10 24 Tricholoma _uc230 2 125 Basidiomycota_uc_s 26 2 3
Comparison of Fungal Communities by Season
A total of seven distinct fungal phyla were found to represent more than 1% of the total phyla in the G-F2, G-W, and G-S samples (Fig. 3). From G-F2-2 to G-W-2 to G-S-2, the ratio of the phylum Basidiomycota gradually dropped from 82.84% to 77.01% to 60.28%, respectively. Additionally, the phylum Mucoromycota was reduced from 10.65% to 7.29% to 5.17%. In contrast, the level of phylum Ascomycota was increasingly higher at 6.37%, 14.70%, and 27.29%, respectively. Likewise, other phyla showed a small difference in ratio, including Fungi_p (0.03%, 0.37%, and 4.30%), Mortierellomycota (0.03%, 0.14%, and 1.72%), Rozellomycota (0.02%, 0.21%, and 0.52%), and Chytridiomycota (0.01%, 0.09%, and 0.17%).
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Fig. 3.
Taxonomic composition at the phylum level from the G-F2-1, G-F2-2, G-W-1, G-W-2, G-S-1, G-S-2 samples. Samples of only Gyeongju collected in autumn, winter, spring were compared. Fungal phyla with a relative abundance greater than 1% in at least one of the samples are shown and phyla less than 1% were shown as ETC. From G-F2-2 to G-W-2 to G-S-2, the ratio of the phylum Basidiomycota gradually dropped (G-F2: October 2018, G-W: February 2019, and GS: June 2019, -1: far from the fairy ring, -2: under fruiting bodies ofT. matsutake ).
A total of 11 fungal classes representing more than 1% of all the classes in each sample were identified: Agaricomycetes, Leotiomycetes, Eurotiomycetes, Umbelopsidomycetes, Dothideomycetes, Sordariomycetes, Fungi_c, GS25, Mortierellomycetes, Lecanoromycetes, and Rozellomycotina cls
At the genus level,
Park and his colleagues conducted in vitro transplantation of
In this study we analyzed the fungal community in the soil under fruiting bodies of
We got a high enough number of valid reads in each sample to analyze the fungal community. The valid reads of G-F2-2 were the highest, at 106,048, and those of G-W-1 were the fewest, at 56,395. The OTUs of most samples under fruiting bodies of
Basidiomycota was the dominant phylum in most samples. The classes in phylum Ascomycota had a tendency to show a smaller percentage in Sample 2 than in Sample 1.
With fewer reads from winter samples, the overall number was reduced, but in the spring, the number of samples and reads increased again. From fall to the following spring, the ratio of phyla Basidiomycota and Mucoromycota gradually dropped. In contrast, the phylum Ascomycota increased. A total of 11 fungal classes were found to represent more than 1% of the total classes in each sample. The classes Leotiomycetes, Eurotiomycetes, Sordariomycetes, Lecanoromycetes, and Mortierellomycetes increased, while Dothideomycetes and Umbelopsidomycetes decreased. The genus
This study provides a foundation for understanding the ecological relationships between
Acknowledgments
This study was supported by the Basic Science Research Program through the National Research Foundation of Korea (NRF), Ministry of Education (2016R1A6A1A05011910), and the Research Institute for Dok-do and Ulleung-do Island of Kyungpook National University.
Conflict of Interest
The authors have no financial conflicts of interest to declare.
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Article
Research article
J. Microbiol. Biotechnol. 2021; 31(5): 686-695
Published online May 28, 2021 https://doi.org/10.4014/jmb.2103.03021
Copyright © The Korean Society for Microbiology and Biotechnology.
Pyrosequencing and Taxonomic Composition of the Fungal Community from Soil of Tricholoma matsutake in Gyeongju
Minji Jeong1, Doo-Ho Choi1, Woo-Jae Cheon2, and Jong-Guk Kim1*
1Department of Life Sciences and Biotechnology, Kyungpook National University, Daegu 41566, Republic of Korea
2Department of Forest Environment, Gyeongsangbuk-do Forest Environment Research Institute, Gyeong-ju 38174, Republic of Korea
Correspondence to:Jong-Guk Kim, kimjg@knu.ac.kr
Abstract
Tricholoma matsutake is an ectomycorrhizal fungus that has a symbiotic relationship with the root of Pinus densiflora. Soil microbial communities greatly affect the growth of T. matsutake, however, few studies have examined the characteristics of these communities. In the present study, we analyzed soil fungal communities from Gyeongju and Yeongdeok using metagenomic pyrosequencing to investigate differences in fungal species diversity, richness, and taxonomic composition between the soil under T. matsutake fruiting bodies (Sample 2) and soil where the fairy ring of T. matsutake was no longer present (Sample 1). The same spot was investigated three times at intervals of four months to observe changes in the community. In the samples from Yeongdeok, the number of valid reads was lower than that at Gyeongju. The operational taxonomic units of most Sample 2 groups were less than those of Sample 1 groups, indicating that fungal diversity was low in the T. matsutakedominant soil. The soil under the T. matsutake fruiting bodies was dominated by more than 51% T. matsutake. From fall to the following spring, the ratio of T. matsutake decreased. Basidiomycota was the dominant phylum in most samples. G-F1-2, G-F2-2, and Y-F1-2 had the genera Tricholoma, Umbelopsis, Oidiodendron, Sagenomella, Cladophialophora, and Phialocephala in common. G-F1-1, G-F2-1, and Y-F1-1 had 10 genera including Umbelopsis and Sagenomella in common. From fall to the following spring, the amount of phyla Basidiomycota and Mucoromycota gradually decreased but that of phylum Ascomycota increased. We suggest that the genus Umbelopsis is positively related to T. matsutake.
Keywords: Fungal community, Tricholoma matsutake, metagenomics
Introduction
Mycorrhizal fungi are an important group of fungi in the soil ecosystem. They have a mutualistic relationship with living plants, exchanging nutrients and some important compounds [15-19]. The major group of mycorrhizal fungi is the ectomycorrhizae (ECM) fungi group. ECM fungi, including
Next-generation sequencing (NGS) enables the rapid analysis of massive amounts of DNA sequences. It also permits the analysis of DNA sequences from microorganisms that are hard to cultivate [31-34]. Pyrosequencing is one of the sequencing approaches for NGS and has facilitated the study of mass and diverse microorganisms from soil samples. For this study, we used the Illumina MiSeq sequencing platform, which uses the sequences of ribosomal DNA as DNA barcodes to classify the soil fungi rapidly.
Here, we statistically analyzed the characteristics of soil fungal communities from two regions using metagenomic pyrosequencing with barcode to investigate diversity, species richness, and relative abundance or taxonomic composition of the communities in areas where
Materials and Methods
Collection of Soil Samples
We collected two soil samples each in pine forests in October 2017 in Gyeongju and in November 2017 in Yeongdeok, South Korea. Two samples were also collected in October 2018 and in February and June 2019 at Gyeongju. Immediately after harvesting the fruiting body of
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Table 1 . Sampling information of ten samples of the soil fungal community related to
T. matsutake ..Gyeongju Yeongdeok October 2017 October 2018 February 2019 June 2019 November 2017 25~30 cm far from the fairy ring G-F1-1 G-F2-1 G-W-1 G-S-1 Y-F1-1 Under the fruiting body of T. matsutake G-F1-2 G-F2-2 G-W-2 G-S-2 Y-F1-2
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GPS coordinates of the sampling sites were as follows:
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• G-F1: 35°47'22.1" N, 129°14'06.9" E
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• Y-F1: 36°28'39.1" N, 129°22'12.0" E
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• G-F2, G-W, G-S: 35°47'20.0" N, 129°14'09.0" E
DNA Extraction and Pyrosequencing
Each 10 g of soil samples was used for isolation of metagenomic DNA using the FastDNA SPIN Kit for Soil (MP Biomedicals, USA) to conduct the pyrosequencing. Polymerase Chain Reaction (PCR) targeting the ITS2 region was used to amplify isolated chromosomal DNA. The sequences of the forward primer were 5′-AATGATACGGCGACCACCGAGATCTACAC-XXXXXXXX-TCGTCGGCAGCGTC-AGATGTGTATAAGAGACAG-GCATCGATGAAGAACGCAGC-3′. The sequences of the reverse primer were 5′- CAAGCAGAAGACGGC ATACGAGAT-XXXXXXXX-GTCTCGTGGGCTCGG-AGATGTGTATAAGAGACAGTCC-TCCGCTTATTGA TATGC-3′. “X” indicates that the barcode sequence differed between samples. A PTC-200 Peltier Thermal Cycler (MJ Research, USA) was used to conduct the PCR. For separation of PCR products, identical amounts of PCR products from each sample were used by agarose gel electrophoresis. The products of PCR were purified using the CleanPCR (CleanNA, Netherlands) and the quantification of the purified PCR products was performed with a Quant-iT PicoGreen dsDNA Assay Kit (Invitrogen, USA). PCR products longer than 300 bp were purified, and an Agilent 2100 Bioanalyzer (Agilent Technologies, USA) was used to analyze the base sequence of the DNA fragments [36]. In addition, the Illumina MiSeq sequencing platform (Illumina, USA) was used for the pyrosequencing analysis conducted by Chunlab Inc. (Korea), following the instructions supplied by the manufacturer [37]. Pyrosequencing reads data were submitted to the EMBL-EMI database (www.ebi.ac.uk) under accession number PRJEB42318 (primary) and ERP126157 (secondary).
Statistical Data Analysis and Taxonomic Identification
The pyrosequencing raw reads were processed by using the barcode sequences. The fusion primers with barcode and low-quality reads were trimmed and removed using Trimmomatic 0.32 [38]. We used CLcommunity software (Chunlab Inc.) for statistical analysis. To identify operational taxonomic units (OTUs) at 97% sequence similarity [39], the CD-HIT program was used. The Mothur platform [40, 41] was used to calculate the rarefaction curves and diversity indices. For taxonomic composition and relative abundance, random sample subsets were generated with the lowest number of reads. Conditionally,
Results and Discussion
Pyrosequencing Results and Statistical Data Analysis
For fungal communities, the total number of valid reads after preprocessing was 881,125 from all eight samples (Table 2). The number of valid reads of G-F2-2 was greatest, at 106,048, followed by G-S-2 at 102,565, G-F1-1 at 101,347, G-F1-2 at 94,695, G-S-1 at 94,346, G-F2-1 at 92,141, Y-F1-1 at 89,952, Y-F1-2 at 86,224, G-W-2 at 57,412, and G-W-1 at 56,395. Overall, there were a large enough number of reads to analyze the microbial communities. Obviously, with fewer reads in the winter samples, the overall number of surviving fungi was reduced. However, the spring samples showed that the number of reads increased again and the ecosystem of soil microbes recovered. In the sample from Yeongdeok, the number of valid reads was slightly lower than that from Gyeongju. The OTUs of Y-F1-1 were the highest, at 664, and those of Y-F1-2 were the lowest, at 124. These two samples showed the biggest difference, at 540. On the other hand, the G-S-1 and G-S-2 samples were 439 and 469, respectively, showing 30 different results. The OTUs of most Sample 2 groups were lower than those of Sample 1 groups. Naturally, the Shannon index, which is one of the most important diversity indices, was similar to the number of OTUs. For the most part, the number for Sample 2’s was lower. In particular, G-F2-1 and G-F2-2 showed a significantly large difference. The Shannon index measures species richness and evenness, which means that the higher the number, the greater the diversity of the community. Therefore, fungal diversity was low in the soil samples in which
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Table 2 . Pyrosequencing results and statistical analysis of the soil fungal community related to
T. matsutake ..G-F1-1 G-F1-2 G-F2-1 G-F2-2 G-W-1 G-W-2 G-S-1 G-S-2 Y-F1-1 Y-F1-2 Number of Valid reads 101347 94695 92141 106048 56395 57412 94346 102565 89952 86224 OTUsa 347 203 578 211 554 230 439 469 664 124 Chao1b 348.68 204.38 596.83 217.52 561.17 238.12 447.52 475.43 665.60 124.58 Shannonc 2.90 2.05 3.69 0.97 3.54 1.25 2.61 2.65 4.01 1.70 Simpsond 0.11 0.30 0.06 0.64 0.08 0.58 0.19 0.28 0.04 0.29 Goods lib. Coveragee(%) 99.98 99.99 99.92 99.98 99.92 99.94 99.96 99.96 99.98 99.99 aOTUs: Operational Taxonomic Units.
bChao1: Chao1 estimation for species richness.
cShannon: Shannon index for species diversity, > 0, higher, more diverse.
dSimpson: Simpson index for species diversity, 0 ~ 1, 1 = the simplest.
eGoods Lib. Coverage: [1 - (number of singleton OTUs / number of total reads)] × 100.
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Figure 1.
Rarefaction curves for operational taxonomic units (OTUs) of fungi from all soil samples at Gyeongju and Yeongdeok. OTUs were clustered at 97% similarity with CD-HIT. This shows the rate of increase in the number of OTUs (species) with the number of acquired sequences. The curves ofT. matsutake -dominant soil samples were in low positions. This implies that the fungal diversity is low inT. matsutake -dominant soil samples (X-axis: the number of sequencing reads; Y-axis: the number of OTUs).
Comparison of Fungal Communities: Composition of T. matsutake
First, for analysis of taxonomic composition and relative abundance, random sample subsets were generated with the lowest number of reads: 56395 (G-W-1).
The composition of
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Table 3 . Composition (%) of
T. matsutake in each sample (using the sum of reads ofTricholoma matsutake ,Tricholoma matsutake_1 , andTricholoma matsutake_7 )..G-F1-1 G-F1-2 G-F2-1 G-F2-2 G-W-1 G-W-2 G-S-1 G-S-2 Y-F1-1 Y-F1-2 0.00 57.76 0.12 79.13 0.01 75.19 38.92 52.11 0.00 52.25
Comparison of Fungal Communities by Region
First, we conducted an analysis at the phylum level of the taxonomic composition of soil samples from G-F1-1, G-F1-2, G-F2-1, G-F2-2, Y-F1-1, and Y-F1-2. A total of five phyla were identified (>1%): Basidiomycota, Ascomycota, Mucoromycota, Fungi_p (phylum name unknown), and Mortierellomycota (Fig. 2). Including
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Figure 2.
Taxonomic composition at the phylum level from G-F1-1, G-F1-2, G-F2-1, G-F2-2, Y-F1-1, and Y-F1- 2 samples. Samples of Gyeongju and Yeongdeok collected in autumn were compared. Fungal phyla with a relative abundance greater than 1% in at least one of the samples are shown and phyla less than 1% were shown as ETC. Basidiomycota was more abundant in Sample-2 (under fruiting bodies ofT. matsutake ) than Sample-1 (far from the fairy ring) regardless of regions and was significantly dominant in Y-F1-2 (G-F1: October 2017, G-F2: October 2018, and Y-F1: October 2017).
At the class level, a total of nine fungal classes found to represent more than 1% of all the classes were identified: Agaricomycetes, Umbelopsidomycetes, Eurotiomycetes, Leotiomycetes, Dothideomycetes, Sordariomycetes, Mortierellomycetes, Fungi_c (class name was unknown), and GS25 (unnamed). We focused on the classes of the relatively abundant phyla Basidiomycota, Ascomycota, Mucoromycota, and Mortierellomycota. In phylum Basidiomycota, we found Agaricomycetes in G-F1-1 (47.99%), G-F1-2 (65.95%), G-F2-1 (26.23%), G-F2-2 (80.12%), Y-F1-1 (46.39%), and Y-F1-2 (93.17%). Phylum Mucoromycota was represented by Umbelopsidomycetes: G-F1-1 (8.22%), G-F1-2 (14.82%), G-F2-1 (14.33%), G-F2-2 (10.60%), Y-F1-1 (11.63%), and Y-F1-2 (4.18%). Class Umbelopsidomycetes was the second most dominant class in all Sample 2’s after class Agaricomycetes, including
At the genus level, the top 10 most abundant genera were
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Table 4 . Gradient heatmap of the taxonomic composition (%) at the genus level of each sample for comparison by region (including
T. matsutake , _uc: unclassified, _g: the genus name was unknown)..
Compared to the fungal community from Yeongdeok, Y-F1-1 shared
In our results,
However, in the
On the other hand, in our samples, there were several genera that were lower in Sample 2 than in Sample 1. The ratios of these genera were as follows:
For comparison of species comprising less than 1% of the community, only this number was compared with samples before normalization. Vaario and colleagues found that
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Table 5 . The number of reads for the common species in G-F1-2, G-F2-2, and Y-F1-2 (_uc: unclassified, _s: the species name was unknown)..
Name G-F1-2 G-F2-2 Y-F1-2 Umbelopsis dimorpha 13420 11133 1095 Oidiodendron chlamydosporicum 450 347 325 Oidiodendron _uc642 759 26 Sagenomella diversispora 34 151 2 Cenococcum _uc4500 52 6 Sistotrema _uc2 15 10 Cladophialophora _uc872 7 115 Pezicula _uc128 7 85 Oidiodendron pilicola 657 160 8 Fungi_uc_s 47 8 7 Umbelopsis _uc231 10 379 Sagenomella _uc6 10 24 Tricholoma _uc230 2 125 Basidiomycota_uc_s 26 2 3
Comparison of Fungal Communities by Season
A total of seven distinct fungal phyla were found to represent more than 1% of the total phyla in the G-F2, G-W, and G-S samples (Fig. 3). From G-F2-2 to G-W-2 to G-S-2, the ratio of the phylum Basidiomycota gradually dropped from 82.84% to 77.01% to 60.28%, respectively. Additionally, the phylum Mucoromycota was reduced from 10.65% to 7.29% to 5.17%. In contrast, the level of phylum Ascomycota was increasingly higher at 6.37%, 14.70%, and 27.29%, respectively. Likewise, other phyla showed a small difference in ratio, including Fungi_p (0.03%, 0.37%, and 4.30%), Mortierellomycota (0.03%, 0.14%, and 1.72%), Rozellomycota (0.02%, 0.21%, and 0.52%), and Chytridiomycota (0.01%, 0.09%, and 0.17%).
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Figure 3.
Taxonomic composition at the phylum level from the G-F2-1, G-F2-2, G-W-1, G-W-2, G-S-1, G-S-2 samples. Samples of only Gyeongju collected in autumn, winter, spring were compared. Fungal phyla with a relative abundance greater than 1% in at least one of the samples are shown and phyla less than 1% were shown as ETC. From G-F2-2 to G-W-2 to G-S-2, the ratio of the phylum Basidiomycota gradually dropped (G-F2: October 2018, G-W: February 2019, and GS: June 2019, -1: far from the fairy ring, -2: under fruiting bodies ofT. matsutake ).
A total of 11 fungal classes representing more than 1% of all the classes in each sample were identified: Agaricomycetes, Leotiomycetes, Eurotiomycetes, Umbelopsidomycetes, Dothideomycetes, Sordariomycetes, Fungi_c, GS25, Mortierellomycetes, Lecanoromycetes, and Rozellomycotina cls
At the genus level,
Park and his colleagues conducted in vitro transplantation of
In this study we analyzed the fungal community in the soil under fruiting bodies of
We got a high enough number of valid reads in each sample to analyze the fungal community. The valid reads of G-F2-2 were the highest, at 106,048, and those of G-W-1 were the fewest, at 56,395. The OTUs of most samples under fruiting bodies of
Basidiomycota was the dominant phylum in most samples. The classes in phylum Ascomycota had a tendency to show a smaller percentage in Sample 2 than in Sample 1.
With fewer reads from winter samples, the overall number was reduced, but in the spring, the number of samples and reads increased again. From fall to the following spring, the ratio of phyla Basidiomycota and Mucoromycota gradually dropped. In contrast, the phylum Ascomycota increased. A total of 11 fungal classes were found to represent more than 1% of the total classes in each sample. The classes Leotiomycetes, Eurotiomycetes, Sordariomycetes, Lecanoromycetes, and Mortierellomycetes increased, while Dothideomycetes and Umbelopsidomycetes decreased. The genus
This study provides a foundation for understanding the ecological relationships between
Acknowledgments
This study was supported by the Basic Science Research Program through the National Research Foundation of Korea (NRF), Ministry of Education (2016R1A6A1A05011910), and the Research Institute for Dok-do and Ulleung-do Island of Kyungpook National University.
Conflict of Interest
The authors have no financial conflicts of interest to declare.
Fig 1.

Fig 2.

Fig 3.

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Table 1 . Sampling information of ten samples of the soil fungal community related to
T. matsutake ..Gyeongju Yeongdeok October 2017 October 2018 February 2019 June 2019 November 2017 25~30 cm far from the fairy ring G-F1-1 G-F2-1 G-W-1 G-S-1 Y-F1-1 Under the fruiting body of T. matsutake G-F1-2 G-F2-2 G-W-2 G-S-2 Y-F1-2
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Table 2 . Pyrosequencing results and statistical analysis of the soil fungal community related to
T. matsutake ..G-F1-1 G-F1-2 G-F2-1 G-F2-2 G-W-1 G-W-2 G-S-1 G-S-2 Y-F1-1 Y-F1-2 Number of Valid reads 101347 94695 92141 106048 56395 57412 94346 102565 89952 86224 OTUsa 347 203 578 211 554 230 439 469 664 124 Chao1b 348.68 204.38 596.83 217.52 561.17 238.12 447.52 475.43 665.60 124.58 Shannonc 2.90 2.05 3.69 0.97 3.54 1.25 2.61 2.65 4.01 1.70 Simpsond 0.11 0.30 0.06 0.64 0.08 0.58 0.19 0.28 0.04 0.29 Goods lib. Coveragee(%) 99.98 99.99 99.92 99.98 99.92 99.94 99.96 99.96 99.98 99.99 aOTUs: Operational Taxonomic Units.
bChao1: Chao1 estimation for species richness.
cShannon: Shannon index for species diversity, > 0, higher, more diverse.
dSimpson: Simpson index for species diversity, 0 ~ 1, 1 = the simplest.
eGoods Lib. Coverage: [1 - (number of singleton OTUs / number of total reads)] × 100.
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Table 3 . Composition (%) of
T. matsutake in each sample (using the sum of reads ofTricholoma matsutake ,Tricholoma matsutake_1 , andTricholoma matsutake_7 )..G-F1-1 G-F1-2 G-F2-1 G-F2-2 G-W-1 G-W-2 G-S-1 G-S-2 Y-F1-1 Y-F1-2 0.00 57.76 0.12 79.13 0.01 75.19 38.92 52.11 0.00 52.25
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Table 4 . Gradient heatmap of the taxonomic composition (%) at the genus level of each sample for comparison by region (including
T. matsutake , _uc: unclassified, _g: the genus name was unknown)..
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Table 5 . The number of reads for the common species in G-F1-2, G-F2-2, and Y-F1-2 (_uc: unclassified, _s: the species name was unknown)..
Name G-F1-2 G-F2-2 Y-F1-2 Umbelopsis dimorpha 13420 11133 1095 Oidiodendron chlamydosporicum 450 347 325 Oidiodendron _uc642 759 26 Sagenomella diversispora 34 151 2 Cenococcum _uc4500 52 6 Sistotrema _uc2 15 10 Cladophialophora _uc872 7 115 Pezicula _uc128 7 85 Oidiodendron pilicola 657 160 8 Fungi_uc_s 47 8 7 Umbelopsis _uc231 10 379 Sagenomella _uc6 10 24 Tricholoma _uc230 2 125 Basidiomycota_uc_s 26 2 3
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