Quality analysis of sequencing data
A total of 280,519 high quality fungal sequences were generated from six samples (EMC: JM-1, JM-2, JM-3, Soil: TY-2, TY-3, TY-4) using Illumina Miseq sequencing. Each sample provided more than 32,568 fungal ITS sequences with an average length of 280.54 bp. The high quality sequence length of the soil microhabitat (Soil) is 200 ~ 340 bp, the average length is 307.02 bp. The high quality sequence length of external mycelial cortices (EMC) is 240 ~ 260 bp, and the average length is 254.06 bp, which is consistent with the prediction based on the primer site (Table 1).
Table 1
Quality analysis of sequences from different samples
Sample | Sequences | Bases(bp) | Average Length(bp) |
TY2 | 32568 | 10530892 | 323.35 |
TY 3 | 37531 | 11194578 | 298.28 |
TY 4 | 87493 | 16197280 | 299.42 |
JM 1 | 35546 | 9036787 | 254.23 |
JM 2 | 42817 | 10862727 | 253.70 |
JM 3 | 44564 | 11330766 | 254.25 |
Richness and diversity of fungal community in natural O. sinensis
In all six samples derived from external mycelial cortices (EMC) and soil microhabitat (Soil), the coverage values close to 100%, and the rarefaction curves were asymptotic, which indicating that overwhelming majority of the fungal species were covered (Fig. 2). The total number of ITS reads obtained from all six samples, after filtering chimeric sequences and mismatches, were clustered into 352 operational taxonomic units (OTUs) with at 97% similarity in nucleotide identity. 170 OTUs were derived from the external mycelial cortices (EMC), 291 OTUs were derived from the soil microhabitat (Soil), and 109 OTUs were shared between the external mycelial cortices (EMC) and soil microhabitat (Soil).
To better understand the differences among the communities, it is important to calculate the richness and diversity. The species richness of fungal communities can be demonstrated by Chao1 index and ACE index. Simpson index and Shannon index were used to analyze the diversity of fungal community, which demonstrate not only the species richness but the evenness of the species. In natural O. sinensis, the patterns of Chao1 and ACE are very similar to the OTUs numbers, the Chao1 and ACE indices from external mycelial cortices (EMC) are 102.9 ± 23.0, 105.1 ± 21.9, respectively, the Chao1 and ACE indices from soil microhabitat (Soil) are 241.4 ± 56.0, 248.9 ± 50.4, respectively, which indicate that the external mycelial cortices (EMC) had lower species richness of the fungal community than soil microhabitat (Soil) (Table 2). The Shannon and Simpson index of the external mycelial cortices (EMC) were 0.28, 0.9166, the soil samples were 1.89, 0.3367, respectively. The Shannon index of external mycelial cortices (EMC) was lower than soil microhabitat (Soil), in contrast, the Simpson index of external mycelial cortices (EMC) was higher than soil microhabitat (Soil). The results indicating that taxonomic diversity of the fungal community in soil microhabitat (Soil) are higher than external mycelial cortices.
Table 2
Richness and diversity of the fungal community from samples at 97% similarity.
Sample | OTU | ACE index | Chao1 index | Shannon index | Simpson index |
Soil | 291 | 248.9 ± 50.4a | 241.4 ± 56.0a | 1.89 ± 0.55a | 0.3367 ± 0.2104a |
EMC | 170 | 105.1 ± 21.9b | 102.9 ± 23.0b | 0.28 ± 0.14b | 0.9166 ± 0.0450b |
Note: values are means ± standard deviation, different letters (a,b) indicate significantly different (p < 0.05). |
Table 3
Phylogenetic affiliations of cultivable fungi isolated from natural O.sinensis.
Order | Isolates number | Closest identified relative | Accession number | Identity (%)% |
Ascomycota | TY105 | Valsa leucostoma | KF294008 | 99 |
| TY110 | Leptosphaeria sclerotioides strain IHBF 2251 | MF326616 | 100 |
| TY112 | Truncatella angustata strain SH9KPN | KT963797 | 100 |
| TY114 | Cladosporium sp. CLJ-7 | LC373150 | 100 |
| TY127 | Trichoderma polysporum isolate CTCCSJ-F-ZY40741 | KY750323 | 100 |
| TY128 | Trichoderma polysporum isolate TR3.3 | KX343127 | 100 |
| TY129 | Stagonosporopsis astragali strain AS1S2-1 | KP117286 | 100 |
| TY135 | Dactylonectria hordeicola isolate EFA 443 | MF440368 | 100 |
| TY137 | Trichocladium opacum | HF678530 | 100 |
| TY138 | Isaria farinosa strain IHBF 2244 | MF326609 | 100 |
| TY139 | Chalara sp. TMS-2011 | HQ630988 | 100 |
| TY141 | Leptodontidium sp. nc_besc_890f | HG936157 | 99 |
| TY147 | Preussia sp. AU_CryP01 | KC333159 | 99 |
| TY149 | Paraphaeosphaeria sporulosa isolate F08-02 | KX664338 | 99 |
| TY151 | Isaria fumosorosea isolate FFJC 27 | KF876832 | 99 |
| TY152 | Fusarium tricinctum strain WBS031 | KU350740 | 100 |
| TY154 | Cladosporium sp. strain 2–3 | KX378909 | 100 |
| TY156 | Paraphaeosphaeria sp. QTYC56 | KM103298 | 98 |
| TY162 | Cephalosporium sp. PF1_NA_8 | KT200264 | 97 |
| TY165 | Pseudogymnoascus sp. isolate UFMGCB 10326 | MG001401 | 100 |
| TY167 | Tetracladium sp. P_S3 | KP411581 | 99 |
| TY175 | Leptosphaeria sp. QLF95 | FJ025183 | 100 |
| TY182 | Paraphaeosphaeria sp. QTYC50 | KM103303 | 99 |
| TY185 | Cosmospora viridescens IMI 73377a | NR_154791 | 95 |
| TY187 | Paecilomyces hepiali | KX237743 | 99 |
| JM16 | Truncatella angustata strain C23RB1 | KT582088 | 100 |
| JM20 | Neonectria ramulariae isolate F744 | KM249079 | 99 |
| JM22 | Fusarium verticillioides strain NSH-5 | KX853851 | 100 |
| JM31 | Trichoderma paraviridescens isolate CTCCSJ-G-JK40841 | KY750503 | 100 |
| JM35 | Trichoderma polysporum isolate CTCCSJ-G-HB40843 | KY750506 | 99 |
| JM37 | Chaetosphaeronema achilleae MFLUCC 16–0476 | NR_153927 | 100 |
| JM42 | Tolypocladium cylindrosporum strain IHBF 2265 | MF326612 | 100 |
| JM45 | Neonectria candida | MG000969 | 100 |
| JM46 | Ilyonectria sp. strain P6011 | KT270205 | 100 |
| JM48 | Neosetophoma sp. strain P1802 | KT269074 | 99 |
| JM51 | Neonectria candida isolate VTN10Bs3 | KU588183 | 99 |
| JM57 | Microdochium sp. 5/97 − 31 | AM502258 | 100 |
| JM60 | Trichoderma viridescens voucher CTCCSJ-G-QT40323 | MF928756 | 100 |
| JM63 | Fusarium tricinctum isolate SBR01 | KX823410 | 99 |
| JM64 | Geomyces sp. AR-2009c | GU166479 | 100 |
| JM66 | Trichoderma nybergianum CBS 122500 | NR_134400 | 100 |
| JM74 | Leptosphaeria sp. QLF95 | FJ025183 | 97 |
| JM76 | Tetracladium sp. P_S3_F | KP411581 | 99 |
| JM78 | Cercophora sulphurella strain SMH2531 | AY587913 | 97 |
| JM84 | Cladosporium cladosporiodies isolate IGFRIWE10 | MF171065 | 100 |
| JM100 | Trichoderma polysporum isolate CTCCSJ-G-HB40843 | KY750506.1 | 99 |
| JM112 | Hypocrea pachybasioides strain T-50 | KC884807 | 100 |
| JM114 | Beauveria bassiana strain BLe-06 | JX149538 | 100 |
| JM117 | Paraphaeosphaeria sporulosa isolate F08-02 | KX664338 | 99 |
| JM125 | Helotiales sp. strain P2929 | KT270126 | 99 |
| JM126 | Tolypocladium cylindrosporum | AB208110 | 99 |
| JM129 | Helotiales sp. MKOTU39 | KP714632 | 100 |
| JM131 | Pseudogymnoascus sp. APA-2015 | KP902683 | 99 |
| JM132 | Cladosporium sp. CLJ-7 | LC373150 | 100 |
| JM137 | Paecilomyces farinosus strain RCEF446 | AF368797 | 99 |
| JM139 | Neonectria sp. CJL-2014 strain Rc-R-30 | KJ542219 | 100 |
| JM142 | Lecythophora sp. NG_p46 | HQ115712 | 99 |
| JM143 | Lecanicillium sp. strain UFSMQ06 | KX496884 | 100 |
| JM144 | Beauveria bassiana strain BLe-06 | JX149538 | 99 |
| ZG_1 | Ophiocordyceps sinensis isolate 1229 | KC184161 | 99 |
Zygomycota | TY100 | Mortierella minutissima strain JZ-26 | HQ637328 | 100 |
| TY101 | Mortierella sp. QLF53 | FJ025192 | 100 |
| TY103 | Mortierella sp. | HG935763 | 99 |
| TY104 | Mortierella alpina isolate QL-15 | MF939657 | 100 |
| TY113 | Mortierella sp. isolate UFMGCB 10336 | MG001402 | 100 |
| TY117 | Mortierella antarctica strain IHBF 2264 | MF326600 | 100 |
| JM1 | Mucor hiemalis isolate 349Jc14 | KU516636 | 100 |
| JM11 | Mortierella alpina isolate HG35 | KU523253 | 99 |
| JM15 | Mortierella sp. T_S4 | KP411583 | 100 |
| JM25 | Mortierella hyalina isolate FFJC 24 | KF876828 | 97 |
| JM30 | Mortierella elongata strain IHBF 2303 | MF326586 | 99 |
| JM85 | Mortierella sp. JZ-68 | HQ637326 | 100 |
| JM91 | Mortierella sp. GW20-2 | JQ670951 | 99 |
| JM99 | Mortierella sp. isolate N-4 | MF939652 | 99 |
| JM105 | Mortierella alpina | AB476411 | 99 |
| JM109 | Mortierella elongata strain PFY | JX155654 | 100 |
| JM113 | Mortierella sp. 02NH02 | JX270348 | 99 |
Note: TY indicates that the fungal isolated from soil microhabitat (Soil), JM indicates that the fungal isolated from external mycelial cortices (EMC). |
Fungal community structure analysis of O. sinensis using high-throughput sequencing
The fungal community structure of O. sinensis were evaluated at the phylum, class and genus levels. All high-quality sequences generated from external mycelial cortices (EMC) and soil microhabitat (Soil) belong to 5 fungal phyla, 15 Classes, 41 orders, 79 families, 112 genera, 352 OTUs. Total of 4 fungal phyla were identified in external mycelial cortices (EMC) samples, including Ascomycota, Basidiomycota, Zygomycota, and unclassified fungi, the average abundance were 97.97%, 1.83%, 0.07%, 0.12%, respectively. Ascomycota was the predominant fungus in external mycelial cortices (EMC) samples of nature O. sinensis. Total of 5 fungal phyla were identified in Soil samples, including Basidiomycota, Ascomycota, Zygomycota, Glomeromycota, and unclassified fungi, the abundance were 82.66%, 14.06%, 0.18%, 0.05% and 3.05%, respectively (Figure. 3A). Significantly, Basidiomycota and Ascomycotawas were the predominant fungi in soil microhabitat (Soil), and Glomeromycota was not detected in external mycelial cortices (EMC). The proportions of Ascomycota in the external mycelial cortices of O. sinensis were significantly higher than soil microhabitats.
The composition of fungal community were further analyzed at the genus level. Among 5 fungal phyla, a total of 112 genera were identified across all investigated samples. Total 43 genera were discovered in external mycelial cortices (EMC), Ophiocordyceps, Sebacina, Archaeorhizomyces were predominant genera accounted for 95.86%, 1.14%, 0.85%, and the genus Ophiocordyceps was overwhelmingly dominant in external mycelial cortices (EMC). Total 66 genera were discovered in soil microhabitat (Soil), Inocybe, Archaeorhizomyces, Unclassified Thelephoraceae, Tomentella, Thelephora, Sebacina, Unclassified Ascomycota, Unclassified Fungi were dominant genera with an average abundance of 53.32%, 8.69%, 8.12%, 8.12%, 7.21%, 4.6%, 3.08% and 3.05%, respectively.
Total 34 genera were shared between external mycelial cortices (EMC) and soil microhabitat (Soil), among of which, Sebacina, Archaeorhizomyces, Apodus, Tetracladium, Mortierella and Cistella had higher abundance in external mycelial cortices (EMC), accounted with 1.14%, 0.85%, 0.18%, 0.17%, 0.07%, 0.04%, respectively. However, Inocybe, Archaeorhizomyces, Tomentella, Thelephora, Sebacina and Geoglossum had higher abundance in soil microhabitat (Soil), accounted with 53.32%, 8.69%, 8.12%, 7.21%, 4.61%, 0.74%, respectively. The average abundance of other genera are lower than 0.01%.
In addition, a total of 9 genera, namely, Botrytis, Cladosporium, Coniochaeta, Hygrocybe, Lachnum, Laetisaria, Leucosporidiella, Ophiocordyceps, Trichoderma were unique to external mycelial cortices (EMC). Total 32 genera were unique to soil samples, Acremonium, Alternaria, Ambispora, Aspergillus, Basidiobolus, Cercophora, Clavaria, Clavulinopsis, Cotylidia, Cryptococcus, Elaphomyces, Elasticomyces, Entoloma, Glarea, Gloiocephala, Humicola, Hymenula, Lecythophora, Leptosphaeria, Minutisphaera, Monographella, Mrakia, Mycena, Neopeckia, Ophiosphaerella, Penicillium, Pluteus, Porotheleum, Rhodotorula, Russula, Scutellinia, Tarzetta. Among of which, Minutisphaera and Scutellinia have higher abundance of 0.29%, 0.02%, respectively, the others with an average abundance less than 0.01% (Figure. 3C).
Comparison of fungal communities from different samples in natural O. sinensis
Heatmap were performed based on the abundance information of each one from top 35 genus in different samples, which can visually revealed that the fungal communities in the external mycelial cortices (EMC) of nature O. sinensis were significantly different from the soil microhabitat (Soil) samples (Figure.4). The UniFrac-weighted Principal Coordinate Analysis (PCoA) showed that JM-1, JM-2, JM-3 were clustered together, TY-2, TY-3, TY-4 were separated on other side, which revealed that there are significant differences in fungal community structure between external mycelial cortices (EMC) with soil microhabitat (Soil) (Figure.5). Dispersed state was observed among the samples from soil microhabitat collected from different sites, which indicating that the fungal communities composition were highly varied significantly in soil microhabitat (Soil) across different areas. Venn diagram analysis revealed that 61 OTUs were exclusive to external mycelial cortices (EMC), 182 OTUs were exclusive to soil microhabitat (Soil), and 109 OTUs were shared in two samples (Figure.6). Comparative analysis at the OTU level reveal that caterpillar fungus O. sinensis was overwhelmingly dominant in the external mycelial cortices (EMC), with proportion of 95.85%. Other predominant fungal species including Archaeorhizomyces sp., Sebacina dimitica, Unclassified_p__Ascomycota, accounted with 0.85%, 0.86%, 0.24%, respectively. However, unclassified_g__Inocybe, Archaeorhizomyces sp., Unclassified_f__Thelephoraceae, Unclassified_g__Tomentella, Thelephora_sp., Unclassified_p__Ascomycota, Unclassified_k__Fungi, Sebacina dimitica, Unclassified_g_Sebacina were predominant fungal species in soil microhabitat (Soil), with proportion of 53.03%, 8.69%, 8.12%, 8.09%, 7.21%, 3.08%%, 3.05%, 2.63%, 1.57%, respectively (Figure.7).
Culture-dependent approaches can provide better taxonomic resolution than high-throughput sequencing. The surface sterilization effect meet the requirements of this study. The fungal isolates are varying with the different media, and a total of 77 fungi were isolated from external mycelial cortices (EMC) and soil microhabitat (Soil) using culture-dependent approaches. Firstly, fungal isolates were classified according to the characteristics of the colonies. Then, the representative fungal isolates were identified by rRNA gene ITS sequences. All sequencing fungal isolates belong to 2 fungal phylum, 4 Classes, 9 orders, 21 families, 33 genera, and Ascomycota, Zygomycota were predominant fungi, with proportion of 77.33%, 22.67%, respectively. Total of 33 genera, including Ophiocordyceps, Trichoderma, Cytospora, Truncatella, Dactylonectria, Isaria, Cephalosporium, Fusarium, Cosmospora, Paecilomyces, Tolypocladium, Cercophora, Beauveria, Neonectria, Microdochium, Coniochaeta, Lecanicillium, Ilyonectria, Stagonosporopsis, Cladosporium, Pleotrichocladium, Leptosphaeria, Chaetosphaeronema, Paraphaeosphaeria, Preussia, Neosetophoma, Leptodontidium, Chalara, Pseudogymnoascus, Geomyces, Tetracladium, Mucor, Mortierella. Among of 33 generic fungus identified, the predominant fungi observed were Mortierella and Trichoderma were the dominant fungi, respectively.
Surprisingly, there are significant difference in fungal communitiy of external mycelial cortices (EMC) and soil microhabitat (Soil) between two approaches. In external mycelial cortices (EMC), many ectomycorrhizal fungi, Orchidaceae or lichen symbiotic fungi were discovered by high-throughput sequencing, including, Tetracladium maxilliforme, Pyronemataceae_sp., unclassified_o__Xylariales, Rachicladosporium_antarcticum, Sebacinaceae, unclassified_o__Sebacinales, Fusidium_griseum, etc.. However, several entomopathogenic fungi and Trichoderma sp. fungi were isolated using culture-dependent approaches, including Tolypocladium cylindrosporum, Beauveria bassiana, Lecanicillium sp, Paecilomyces farinosus, Trichoderma paraviridescens, Trichoderma viridescens, etc.. In soil microhabitat (Soil), many cold adapted yeast, lichen symbiotic fungi, endophyte fungi or phytopathogenic fungi were discovered by high-throughput sequencing, including, Cryptococcus_terricola, Cryptococcus_victoriae, Elasticomyces_elasticus, Rhodotorula_lamellibrachiae, Alatospora_sp, etc.. Using culture-dependent approaches, we isolated many fungal species soil microhabitat (Soil) belong to Mortierella sp., entomopathogenic fungi and endophyte fungi, e.g., Mortierella minutissima, Isaria farinosa, Isaria fumosorosea, Paraphaeosphaeria sporulosa, etc..