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 samples from soil microhabitat is 200~340 bp, the average length is 307.02 bp. The high quality sequence length of the samples from external mycelial cortices 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).
Richness and diversity of fungal community in natural O. sinensis
In all six samples derived from external mycelial cortices and soil microhabitat, the coverage values close to 100%, and the rarefaction curves were asymptotic, indicating that overwhelming majority of the fungal species were covered (Figure.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. One hundred and seventy OTUs were derived from the external mycelial cortices, 291 OTUs were derived from the soil microhabitat, and 109 OTUs were shared.
To better understand the differences among the microbial 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 are 102.9±23.0, 105.1±21.9, respectively, the Chao1 and ACE indices from soil microhabitat are 241.4±56.0, 248.9±50.4, respectively, which indicate that the external mycelial cortices had lower species richness of the fungal community than soil microhabitat (Table.2). The Shannon and Simpson index of the external mycelial cortices were 0.28, 0.9166, the soil samples were 1.89, 0.3367, respectively. The Shannon index of external mycelial cortices was lower than soil microhabitat, in contrast, the Simpson index of external mycelial cortices was higher than soil microhabitat. The results indicate that taxonomic diversity of the fungal community in soil microhabitat are higher than external mycelial cortices.
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 and soil microhabitat belong to 5 fungal phyla, 15 Classes, 41 orders, 79 families, 112 genera, 352 OTUs. A total of 4 fungal phyla were identified in external mycelial cortices samples, including Ascomycota, Basidiomycota, Zygomycota, and unclassified fungi, the average abundance were 98.02%, 1.79%, 0.07%, 0.12%, respectively. Ascomycota was the predominant fungus in external mycelial cortices samples of natural O. sinensis. A total of 5 fungal phyla were identified in soil microhabitat 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, and Glomeromycota was not detected in external mycelial cortices. The proportions of Ascomycota in the external mycelial cortices of O. sinensis were significantly higher than soil microhabitat.
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. In total, 43 genera were discovered in external mycelial cortices, Ophiocordyceps, Sebacina and Archaeorhizomyces were predominant genera accounted for 95.86%, 1.14%, 0.85%, and the genus Ophiocordyceps was overwhelmingly dominant in external mycelial cortices. A total of 66 genera were discovered in soil microhabitat, 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. In total, 34 genera were shared, among of which, Sebacina, Archaeorhizomyces, Apodus, Tetracladium, Mortierella and Cistella had higher abundance in external mycelial cortices, 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, 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 and Trichoderma were unique to external mycelial cortices. A total of 32 genera were unique to soil microhabitat 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 and 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 genera in different samples, which can visually reveal that the fungal communities in the external mycelial cortices of natural O. sinensis were significantly different from soil microhabitat 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 and soil microhabitat (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 across different areas. Venn diagram analysis revealed that 61 OTUs were exclusive to external mycelial cortices, 182 OTUs were exclusive to soil microhabitat, and 109 OTUs were shared (Figure.6). Comparative analysis at the OTU level reveal that caterpillar fungus O. sinensis was overwhelmingly dominant in the external mycelial cortices, 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 and unclassified_g_Sebacina were predominant fungal species in soil microhabitat, with proportion of 53.03%, 8.69%, 8.12%, 8.09%, 7.21%, 3.08%%, 3.05%, 2.63%, 1.57%, respectively (Figure.7).
Diversity of cultivable fungi
The surface sterilization effect meet the requirements of this study. Culture-dependent approach provide better taxonomic resolution than high-throughput sequencing. The fungal isolates varied with the different types of media, a total of 77 fungi were isolated from external mycelial cortices and soil microhabitat using culture-dependent approach. Firstly, fungal isolates were classified according to the characteristics of the colonies. The representative fungal isolates were identified by internal transcribed spacer (ITS) region of the rDNA gene. All sequenced fungal isolates belong to 2 fungal phyla, 4 Classes, 9 orders, 21 families, 33 genera, Ascomycota and Zygomycota were predominant fungi, with proportion of 77.33%, 22.67%, respectively. A total of 33 fungal 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 and Mortierella, among of which, the predominant ones were Mortierella and Trichoderma (Table.3).
Comparison of culture-dependent and-independent approaches
Surprisingly, there are significant differences in fungal communities of external mycelial cortices and soil microhabitat between two approaches. In external mycelial cortices, many ectomycorrhizal fungi, Orchidaceae and lichen symbiotic fungi were discovered by high-throughput sequencing, including, Tetracladium maxilliforme, Pyronemataceae_sp., unclassified_o__Xylariales, Rachicladosporium antarcticum, Sebacinaceae, unclassified_o__Sebacinales and Fusidium_griseum, etc.. However, several entomopathogenic fungi and Trichoderma sp. fungi were isolated using culture-dependent approach, including Tolypocladium cylindrosporum, Beauveria bassiana, Lecanicillium sp, Paecilomyces farinosus, Trichoderma paraviridescens and Trichoderma viridescens, etc.. In soil microhabitat, many cold adapted yeast, lichen symbiotic fungi, endophyte fungi and phytopathogenic fungi were discovered by high-throughput sequencing, including, Cryptococcus terricola, Cryptococcus victoriae, Elasticomyces elasticus, Rhodotorula lamellibrachiae and Alatospora sp, etc.. Using culture-dependent approach, we isolated many fungal species from soil microhabitat, which belong to Mortierella sp., entomopathogenic fungi and endophyte fungi, e.g., Mortierella minutissima, Isaria farinosa, Isaria fumosorosea and Paraphaeosphaeria sporulosa, etc..