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

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 and 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 and 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 soil microhabitat. Meanwhile, seven types of culture media were used to isolate culturable fungi at 16 °C, resulted in 77 fungal strains identified 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.


Background
The caterpillar fungus Ophiocordyceps sinensis parasitizes the larvae of Thitarodes that belongs to family Hepialidae (Lepidoptera) form the fungus-caterpillar complex (Chinese Cordyceps) [1]. O. sinensis mainly inhabits the alpine meadow ecosystem of Qinghai-Tibetan Plateau, Sichuan, Yunnan and Gansu Provinces in China, also distributes in certain areas of Bhutan, Nepal and India [2]. As the well-known traditional Chinese medicinal herb, O. sinensis has been widely used for centuries in Asia, exhibit beneficial properties and remedy for a variety of chronic disorders, including asthma, cancer, sexual dysfunction, obesity, type 2 diabetes and fatigue, etc., presumably, by modulating the composition of gut microbiota [3][4][5][6]. O. sinensis harbour a variety of bioactive metabolites, e.g., cordycepin, cordycepic acid, polysaccharides, ergosterol, mannitol and macrolides, etc.. It may be considered as key factors in the coevolution of O. sinensis and its host, coevolution of symbiotic and associated microbiota. The huge world market demand and price of O. sinensis continue to increase because of its pharmacological application, obligate parasitism and special dependence on ecological environments, which led to overexploitation, severely endangering numerous natural populations of O. sinensis towards its extinction and damage of its habitats. Artificial cultivation of O. sinensis has been successful, but commercial cultivation is difficult to perform because the cryptic factors triggering the development and maturation of O. sinensis remains obscure [7]. Currently, the soil originated from habitats of natural O. sinensis is indispensable in later stages of artificial cultivation. Therefore, illuminating mysterious drive factor in the occurrence and maturation of O. sinensis, enhancing the yield and discovery of its alternative substitutes, have already become hot topics.
Generally, O. sinensis is exclusively endemic to alpine meadow ecosystem of the Qinghai-Tibetan Plateau and its adjacent high-altitude area in China, which is characterized by low soil temperature, fragile plant community structure and extremely sensitive to climate change. Natural O. sinensis and its surrounding soil construct a microecosystem, which represent the reservoir of microbial species. Uncovering the fungal community structure of natural O. sinensis are significant in understanding the occurrence of O. sinensis and artificial cultivation. Recently, a wide variety of fungal species have been discovered from natural O. sinensis using culturedependent approach, including O. sinensis, Chrysosporium sinense, Paecilomyces hepiali, Tolypocladium sinense and Paecilomyces sinensis, etc., all of which are excellent potential sources of novel bioactive metabolites for drug discovery [8,9]. Except the caterpillar fungus O. sinensis, all the others are symbiotic or associated fungi [10,11]. Molecular evidence demonstrates the existence of both O. sinensis and P. hepiali DNA in the caterpillars and fruiting bodies of O. sinensis.
Recently, O. sinensis is confronted with reduction of output and geographical distribution shrink, which might result from microbial community disturbance in habitat environment by over-exploitation, global warming, heavy grazing and habitat deterioration, etc. [12]. Significantly, a variety of fungal species isolated from natural O. sinensis were applied in clinical treatment because of its health benefits. More than 200 novel bioactive metabolites have been isolated from these associated fungi (refered to as Cordyceps-colonizing fungi) [13,14]. Particularly, the mycelia of P. hepiali obtained by solid state fermentation or submerged liquid fermentation produce a large repertoire of bioactive metabolite similar to natural O. sinensis, also exhibit beneficial properties, including antioxidant, antifatigue, anti-aging, anti-diabetic, anti-inflammatory and anti-depressant-like effects [15,16]. Currently, P. hepiali has been widely used as an alternative to natural O. sinensis, because of its high productivity and similar health effects. These different fungal species produce similar bioactive metabolites, presumably, due to horizontal transfer of secondary metabolic biosynthetic gene cluster in microbiota of natural O. sinensis. Meanwhile, more fungal species originating from natural O. sinensis need to be discovered for developing alternative products, and applied in clinical practice.
Natural O. sinensis associated microbiota play vital roles in nutrient cycling, establishment of infection cycle of insect hosts, growth and maturation of sclerotia and fruiting body (stroma). Within the last decade, progressive studies have been devoted to isolate and identify fungal species from habitats of O. sinensis by culture-dependent approach. A total of 572 isolates have been obtained from different parts of O. sinensis (fruiting body, sclerotia and external mycelial cortices) at 25°C, belonging to 37 fungal genera, Ascomycota, Basidiomycota and Zygomycota were the predominant fungal phyla, which have provided fundamental insights into fungal community of native O. sinensis on the Qinghai-Tibetan Plateau [8]. However, only a small fraction of microbial community were culturable using traditional culture dependent approach [17,18]. Previously, 490 fungal clones were identified from the whole-community DNA using PCR-single-strand conformation polymorphism (PCR-SSCP) method, 266 of these clones were selected for sequencing and assigned to 21 genera [9]. Three main phyla, including Ascomycota, Basidiomycota, Zygomycota and a notable numbers of unclassified fungi, especially, in the sclerotia and external mycelial cortices were detected by high-throughput 454 sequencing technology [19].
Currently, Illumina Miseq high-throughput sequencing has been the most promising approach to explore microbial community composition and dynamic across special habitat, agriculture, traditional solid-state fermentation, deep-sea sediments and Chinese liquor starter, etc., due to its cost-effective, more sequences per sample, especially the capability of less abundant species mining [20][21][22][23][24][25]. However, the data obtained from samples by this approach were determined by the quality of microbial DNA extracted from samples, PCR amplification efficiency, quality of 16S rRNA or ITS regions library and other uncertain factors, which easily lead to offset or error of microbial diversity. In addition, due to short length of Miseq sequencing, the OTUs could be identified to the genus level.
Recently, O. sinensis associated fungi are increasingly attracting the attention as potential health benefits agent. The relationship between O. sinensis and its microbiota also represents one of the most evolutionarily ancient examples of symbiosis in ecosystem. The microbial community of unfertilized eggs from Thitarodes were analyzed via 16S rRNA and ITS sequencing, revealed that 348 bactetial genera belong to 26 phyla, and 289 fungal genera, mainly including Aureobasidium, Candida and Cryptococcus, etc., belonging to 5 phyla, namely, Ascomycota, Basidiomycota, Chytridiomycota, Glomeromycota and Zygomycota [26]. However, microbial communities of natural O. sinensis, ecological functions and metabolic potential remain largely unexplored. Here, the present study aimed to comprehensively uncover fungal communities in external mycelial cortices and soil microhabitat. The samples derived from different sites distributed across Golog Tibetan Autonomous Prefecture of Qinghai-Tibetan Plateau, were investigated using high-throughput sequencing and various types of culture media. These results would provide a comprehensive understanding of complex microbial ecosystem around natural O. sinensis, be beneficial to overcome the obstacle in artificial culture of O. sinensis.

Quality analysis of sequencing data
A total of 280,519 high quality fungal sequences were generated from six samples (external mycelial cortices: JM-1, JM-2, JM-3, soil microhabitat: 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 (Fig. 1). The total number of ITS reads obtained from all six samples, after filtering chimeric sequences and mismatches, were clustered into 352 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.

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 (Fig. 3). 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 (Fig. 4). 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 (Fig. 5 6).

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 culturedependent approach. Firstly, fungal isolates were classified according to the characteristics of the colonies. The representative fungal isolates were identified by 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,   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 Xylariales, Rachicladosporium antarcticum, Sebacinaceae, unclassified Sebacinales and

Discussion
The caterpillar fungus O. sinensis infects larvae of its hosts by oral cavity, insect cuticles or spiracle. After entering the hemolymph of host insect, the fungus grows as a hyphal body and threadlike hyphae, and converts it into caterpillar-shaped sclerotium. In the following year, mid-April to early June, the fruiting body bud growing on the head of sclerotium, emerging above the soil surface and forming a stalked fruiting body. Finally, ascospores were erupted from the mature fruiting body of O. sinensis, scatter in top soils, gradually infiltrate deeper into the soil, begin a new infection cycle. The dynamic changes and diversity of O. sinensis associated fungi in soil regulate the relationship of fungus-larvae, influence the occurence and geographical distribution of O. sinensis [27]. In Qinghai-Tibet Plateau, fungi community also are key components of the fragile ecosystems, perform critical ecological functions in biogeochemical cycles by decomposing organic matter and recycling nutrients. Uncovering fungi community composition of natural O. sinensis have great significance for better elucidating the interaction between microbiome and O. sinensis, the occurence and morphological variation of O. sinensis. However, it still lacked comprehensive investigation because of limitation of single approach. In present study, 5 fungal phyla, 112 genera, and 352 OTUs were identifed using Illumina MiSeq sequencing, a total of 43 fungal genera were discovered in external mycelial cortices, Ophiocordyceps, Sebacina and Archaeorhizomyces were predominant genera accounted for 95.86, 1.14, 0.85%. A total of 66 fungal genera were discovered from soil microhabitat, Inocybe, Archaeorhizomyces, unclassified Thelephoraceae, Tomentella, Thelephora, Sebacina, unclassified Ascomycota and 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. Previous study revealed that Ophiocordyceps was overwhelmingly dominant in the fruiting bodies and external mycelial cortices of the Chinese Cordyceps, which is consistent with our data. However, in soil samples, Ophiocordyceps, Verticillium, Pseudallescheria, Candida, Ilyonectria, Neonectria and Fusarium were the dominant genera [19]. Another previous study based on HiSeq sequencing of ITS genes of soil samples derived from the site that had a high density of Thitarodes larvae and Chinese Cordyceps at Shergyla Mountain revealed that Archaeorhizomyces, Hyphodiscus, Beutheromyces, Pezoloma, Venturia, Geogiossum, Clavulinopsis, Cotylidia, Rammanopsis, Peltigera and Cavana had high relative abundance [28]. Previous studies are inconsistent with each other, and also differ from our results, presumably, due to geographical origin, sampling processing and limitations of high-throughput sequencing, etc., which indicated that the O. sinensis associated microbial communities needs further study. In present study, 33 genera were isolated and identified using culture-dependent approach with different types of midia incubated at 16°C, which mimic the true growth temperature of natural O. sinensis. Mortierella and Trichoderma were predominant culturable fungal genera. The data obtained from previous study showed that Penicillium chrysogenum is the predominant fungal species from fruiting body, and Pseudogymnoascus roseus is the dominant fungal species from sclerotia and external mycelial cortices using PDA incubated at 20°C [8]. In our study, Pseudogymnoascus sp., was isolated from external mycelial cortices, but was not a predominant fungal species. There are significant differences in distinct studies, presumably, owing to different culture temperatures, media, sampling process and geographical origin, etc., which reflect the complexity of microbial communities from natural O. sinensis.
Compared with culture dependent approach which had limitations because of the selectivity of media and culture conditions, high-throughput sequencing was considered to provide better resolution of microbial community composition, but, it still showed biases. Each approach tend to capture different microbial community fractions, result in only a fraction of the taxa recovered did overlap, which also highlighting the complementarity of two approaches [29,30]. Therefore, it is believed that the coordination of culture-dependent and independent approaches would provide promising avenue to illuminate microbial community assembly in natural O. sinensis. The idea was supported by our results obtained from two approaches. Using high-throughput sequencing, many ectomycorrhizal fungi, Orchidaceae and lichen symbiotic fungi were unexpectedly discovered in external mycelial cortices. Although rarefaction curves combined with the estimated coverage values indicated that overwhelming majority of fungal species were revealed from the natural O. sinensis. There were only 2 fungal species obtained from culture dependent approach were detected by high-throughput sequencing, i.e., Tetracladium sp. and Lecythophora sp.. Significantly, several entomopathogenic fungi and Trichoderma sp. were isolated frequently using culture-dependent approach. Various environmental factors, including terrain, vegetation characteristics, altitude, climate change and anthropogenic disturbance, etc., strongly regulate soil properties, soil temperature and moisture, which alter the microclimate, then shape microbial communities composition of soil microhabitat [31]. The alpine meadow ecosystem of the Qinghai-Tibetan Plateau that suitable for the occurrence of O. sinensis, are the most fragile and sensitive to environmental factors change. The dynamic alterations of microbial community in natural habitat O. sinensis response to seasonal temperature fluctuation, are also need further study. Furthermore, soil microbial communities strongly influence the yield and occurrence of natural O. sinensis, because of the infection of host Thitarodes larvae mainly occurs in soils [28]. The suitable habitat of O. sinensis and host insects  [28]. Sebacina, a mycorrhizal fungus frequently isolated from Orchidaceae and plant, promote growth, enhance stress resistance and seed germination, was in high abundance in external mycelial cortices [33]. Archaeorhizomyces, a widespread fungal class with a dominant presence in many soil environments, increase plant bioactive components, enhance stress resistance and inhibit the occurrence of plant diseases by different mechanisms, also was high relative abundance genus in external mycelial cortices. Trichoderma spp. are proposed as major plant growth-promoting fungi in soils, significantly alter the rhizosphere soil chemistry, regulate microbial communities and improve grassland biomass [34]. Trichoderma species were frequently isolated from external mycelial cortices by culture-dependent approach, and also have been identified by highthroughput sequencing. Accumulated evidence and our data support the hypothesis that the caterpillar fungus O. sinensis parasitize the larvae of Thitarodes to form larva shape sclerotia, produce diverse secondary metabolites or organic ingredients, which could alter soil characteristic surrounding O. sinensis, influence the microbial community composition, form a complex hostmicrobiota microecosystem. The total protein content, pharmacological effect and bioactive components of natural O. sinensis are much higher than artificially cultivated O. sinensis, which indicate that shaping symbiotic and associated microbial species are significantly important in the growth and development of O. sinensis and formation of perithecial fruiting bodies. Presumably, these associated fungi might be the real producer of certain pharmacologically bioactive components in natural O. sinensis. A case of Truffles (Tuber spp.), are well known for their enticing aromas partially emitted by microbes colonizing truffle fruiting bodies, their associated microbes related to stimulation of the growth of Tuber mycelium, inhibition of pathogenic fungi, the elaboration of the complex aroma of truffles [35]. The data presented here highlight the importance of several fungi in O. sinensis, namely Trichoderma sp., Archaeorhizomyces, Sebacina, insect pathogenic fungi and mycorrhizal fungi, etc., the synergistic effect of different fungi help to produce O. sinensis.
Some studies demonstrate that other microbial species could colonize inside sclerotia except the caterpillar fungus O. sinensis. Cordyceps related fungal species, namely, P. hepiali, T. sinensis, P. sinensis, Synnematium sinense and Cephalosporium dongchongxiacae, etc., most of which belong to entomopathogenic fungi, were isolated from fruiting body or sclerotia, which resulting in the debate of unique anamorph of O. sinensis lasted for decades. Here, aiming to explain this fact, based on our data and previous studies, we hypothesized that the larvae of Thitarodes sp., were infected by O. sinensis accompanied with degradation of insect cuticle and tissue, which drive the soil microhabitat around O. sinensis highly suitable for entomopathogenic fungi, improve the abundance of various types of entomopathogenic fungi and parasitize inside the sclerotia. Yet, there are significant variations of entomopathogenic fungi species in microbial community of O. sinensis derived from different geographical region. The present study, taking internal tissue from sterilized fruiting body and sclerotia as material, only the fungus O. sinensis was isolated using traditional cultivated approach. However, 5 fungal species were isolated from the sterilized sclerotia surface tissue. This result is consistent with some previous finding that total 2 unique OTUs were isolated from fruiting body, 4 OTUs from sclerotia, and 115 OTUs from mycelial cortices [9]. However, some previous data revealed that many microbial species identified from fruiting body and sclerotia, presumably, due to the fact that the soil attached to sample were not removed. Our research aimed to reveal the fungal diversity in microecosystem of O. sinensis, and to elucidate the role of fungal community composition in the occurrence of O. sinensis. Therefore, we selected the external mycelial cortices and soil microhabitat for study using coordination of culture-dependent and independent approaches.
The soil microhabitat and O. sinensis form a complex microecosystem, which harbour huge number of fungi, bacteria and actinomycetes. The interactions among soil microbial community might play a significant role in the infection and occurrence of O. sinensis. Bacterial diversity of natural O. sinensis was also investigated by highthroughput sequencing in our group. In external mycelial cortices, 1667 bacterial OTUs, belonging to 17 genera were identified, the predominant genera including others (48.92%), norank Acidobacteria (5.39%), RB41 (4.16%), Flavobacterium (3.15%), norank Nitrosomonadaceae (2.81%), Bradyrhizobium (2.66%), Nitrospira (2.42%) and norank Anaerolineaceae (2.01%), etc.. In soil microhabitat, 1676 bacterial OTUs, belonging to 18 genera also were identified, and 1623 bacterial OTUs were shared (unpublished data). The average relative abundance of several bacterial genera, i.e., Flavobacterium, Ferruginibacter and Pseudomonas were higher in external mycelial cortices, indicated that these bacterial species might enhance fungal mycelial growth and promoting the occurence of O. sinensis. Recent evidence revealed that Pseudomonas sp. P7014 can enhance mycelial growth and reduce harvesting time of Pleurotus eryngii [36]. However, no studies have yet reported the best growth promoting microbes for O. sinensis, and it would be a hot topics.
O. sinensis and its soil microhabitat represent the potential source to explore microbial biosynthetic diversity and evolutionarily ancient examples of symbiosis, for some of O. sinensis associated fungi produce similar bioactive metabolites [6,37,38]. In microecosystem, polyketide synthase and nonribosomal peptide synthetase, the large multidomain and multifunctional megaenzymes involved in biosynthesis of secondary metabolites, evolve rapidly through horizontal transfer from bacteria to fungi, particularly, between fungi, which contribute to produce vast numbers of novel metabolites [39]. Presumably, horizontal gene transfer frequently occur in the microbiota of natural O. sinensis to improve the genomic flexibility and produce structurally similar metabolites during long-term symbiosis and evolution.
Previous study confirmed that the host insects of O. sinensis from different geographic origin are complex, mostly in the genus Hepialus and to a lesser extent in the genera Hepialiscus, Forkalus and Bipectilus [40]. Furthermore, the fungus O. sinensis and its hosts have coevolved. To better clarify the structure of microbial community of O. sinensis, extensively collecting sampling across the geographical distribution of O. sinensis are necessary. However, sampling collection was affected by traffic, sample storage and shorter period of emergence of O. sinensis. Here, we aim to unravel fungal communities of natural O. sinensis using combination of high-throughput sequencing and culture-dependent approach. Therefore, O. sinensis samples were collected at five sites from Golog Tibetan Autonomous Prefecture of Qinghai Province, which is a suitable geographical distribution of O. sinensis with high quality and yield. Although the limited sampling sites covered by this study possibly result in decreasing of credibility of the present results, considerable previous studies support our data and hypothesis [19,41].
As well know, O. sinensis usually occur in the humusrich horizon of soils. Unfortunately, in present study, we did not determine the characteristics of the soil samples. Other study reveal that the pH values of habitat soils of natural O. sinensis range from 5.01 to 6.00, and no significant difference among different habitats. The soil microhabitat of O. sinensis contain a large amount of organic matters and nitrogen, which control the whole microbial community composition and affect the occurence of O. sinensis. Further studies are needed to confirm the relationship between soil properties and microbial community composition and the occurence of O. sinensis. In present study, we only divided the sample into external mycelial cortices and soil microhabitat. The dynamics of the hyphal bodies and it's density in different distance from O. sinensis larvae in soil microhabitat are worthing further study, which would providing novel insight to occurrence mechanism of O. sinensis and be benefit to overcome the obstacle in artificial culture of O. sinensis.

Conclusion
This study revealed that each approach tend to capture different microbial community fractions, the significantly differences and little overlap in fungal community of natural O. sinensis between two approaches highlight that the combination of high-throughput sequencing and culture-dependent approaches would generate more information. A comprehensive investigation of the fungal community structure in natural O. sinensis performed by two approaches indicated that microbial species, namely Trichoderma sp., Archaeorhizomyces, Sebacina, entomogenous fungi and some bacterial species, etc., play an important role in the occurence and maturation of O. sinensis. In a word, our result provide novel insights into the fungal community structure of natural O. sinensis, reveal the occurrence mechanism of O. sinensis, and would be beneficial to overcome the obstacle in artificial culture of O. sinensis.

Site description, sampling collection and processing
The size and shape of O. sinensis vary in different areas owing to the variety of infected Thitarodes insects, environmental factors, including plant species, soil characteristic and soil microbial community, etc.. Therefore, these O. sinensis samples were collected at five sites from shrubland and alpine meadow with altitude of 4000-5200 m, which distributes in the territory of Maqen County, Darlag County and Gade County, Golog Tibetan Autonomous Prefecture of Qinghai Province (97°54′-101°50' longitude, 32°31′-35°40′ latitude) during May 10-20, 2015. These regions are representative geographical distribution of the O. sinensis with high quality and density. Five repeats were performed by the fivepoint method at each site. During sampling, the naturally growing O. sinensis was taken as the center point, soil block with 15 cm diameter and depth of 20 cm were collected. The samples were transported to the laboratory within 24 h under controlled temperature (4°C). Processed samples were stored at 4°Cfor fungal isolation and at − 80°Cfor DNA extraction, respectively.
Firstly, plant residues and stones in samples were removed with a sieve. The samples derived from same site were divided into three subsamples. sinensis represent the part that infected fungus growing on the head of caterpillar-shaped sclerotium. Samples from external mycelial cortices and soil microhabitat were investigated using high-throughput sequencing and culture-dependent approaches, yet, fruiting body was investigated by culture-dependent approach because of little microbial species (Fig. 7).

DNA extraction, high-throughput sequencing
For fungal community description, total genomic DNA was extracted from each subsamples, i.e., external mycelial cortices and soil microhabitat using two approaches, respectively. To improve the efficiency of the microbial DNA extraction, the samples were crushed after freezing with liquid nitrogen in precooled mortar. The total genomic DNA extracted by two approaches were pooled for further step.
First approach. Total genomic DNA extraction from external mycelial cortices and soil microhabitat were performed using the MoBio PowerSoil DNA Isolation Kit (MoBio Laboratories, Carlsbad, CA, USA) according to manufacturer's instructions, respectively. The integrity of genomic DNA was checked by 1.0% agarose gel electrophoresis (1% w/v).
Secondary approach. Before DNA extraction, the samples were treated using TENP buffer (50 mM Tris, 20 mM EDTA, 100 mM NaCl, 0.01 g/ml Polyvinylpyrrolidone, pH 10) and PBS buffer (137 mmol/L NaCl, 2.7 mmol/L KCl, 10 mmol/L Na 2 HPO 4 , 2 mmol/L KH 2 PO 4 , pH 7.4) to remove humus. Then, the genomic DNA was extracted using the traditional method with minor modification. Briefly, For each 2 g samples, add 3 mL DNA extraction buffer, 20 μL proteinase K, 30 μL lysozyme and 20 μL lytic enzyme, mix by vortexing 30 min, add 0.5 mL 20% SDS, subsequently incubated for 2 h at 65°C. Solution was centrifuged at room temperature 9000 r/min for 15 min, the supernatant was transferred to a new centrifuge tube. Equal volume of saturated phenol: chloroform: isoamyl alcohol (25,24,1) were used to remove protein contamination, and isopropanol was used to precipitate DNA for 30 min, followed by centrifugation at 6000×g for 30 min. Supernatant was discarded, pellet was washed with 70% ethanol, centrifuged again. Subsequently, the supernatant was removed, air dried and finally resuspended in an appropriate amount of Tris-EDTA buffer (10 mM Tris, 1 mM EDTA, pH 8.0).
The concentration and purification of mixed genomic DNA extracted using the above two approaches were determined by NanoDrop 2000 UV-vis spectrophotometer (Thermo Scientific, Wilmington, USA), the fungal rDNA ITS1 regions were amplified from the mixed total genomic DNA with primers ITSIF (5′-CTTGGTCATT TAGAGGAAGTAA-3′) and ITS2R (5′-GCTGCGTTCT TCATCGATGC-3′) by the ABI GeneAmp® 9700 PCR System (Applied Biosystems, Waltham, MA, USA) [42]. The PCR reaction mixture (20 μL) contained 4 μL of 5× FastPfu Buffer, 2 μL of dNTPs (2.5 mM), 0.8 μL of each primer, 0.4 μL of FastPfu Polymerase, 10 ng of template DNA. The PCR procedures were 95°C for 3 min initial denaturation, 27 cycles of 30 s at 95°C, 30 s for annealing at 55°C, 45 s for elongation at 72°C, and a final extension at 72°C for 10 min. Triplicate PCR reactions were carried out for each sample, the products were mixed, and evaluated by 2% agarose gel electrophoresis. The mixed products were purified using the AxyPrep DNA Gel Extraction Kit (Axygen Biosciences, Union City, CA, USA) and quantified using QuantiFluor™-ST (Promega, USA). Purified amplicons were pooled in equimolar and paired-end sequenced (2 × 300) on an Illumina MiSeq platform (Illumina, San Diego, USA) according to the standard protocols by Majorbio Bio-Pharm Technology Co. Ltd. (Shanghai, China). Raw sequencing data obtained from this study were deposited at DDBJ/ENA/ GenBank under the accession numbers: PRJNA609607.

Processing of sequencing data
Raw FASTQ files were demultiplexed, quality-filtered by Trimmomatic, and merged by the following criteria: (i) The reads were truncated at any site receiving an average quality score < 20 over a 50 bp sliding window. (ii) Primers were exactly matched allowing 2 nucleotide mismatching, and reads containing ambiguous bases were removed. (iii) Sequences whose overlap longer than 10 bp were merged according to their overlap sequence. After quality control, the reads were clustered into OTUs according to the pipeline of QIIME commands based on 97% sequence similarity. Representative sequences of each OTU were blasted against the UNITE database to obtain taxonomy with a threshold value of 0.8. The α-diversity indices, including ACE index, Chao1 index, Shannon index, Simpson index, and Coverage were calculated using Mothur 1.30.1. Euclidean-based Weighted UniFrac distances were employed to determine the distance (β-diversity) between fungal communities in any pair of samples. Venn diagrams were generated to illustrate the proportion of shared and unique taxa between different samples. The hierarchical clustering graph was generated using the MeV software, version 4.9.0 and the hierarchical clustering method.
Principal coordinates analysis (PCoA) is a visualization method for studying variation in data, based on UniFrac metrics, and is useful for describing the species composition similarity between samples. In present study, PCoA was performed using the vegan package 2.0-10. All software used was in Mothur package.

Isolation and identification of culturable fungi
The culturable fungi were isolated from the external mycelial cortices and soil microhabitat, which were pooled from all samples derived from different sites by the dilution-plate approach. A total of 10 g samples were added to a triangular flask containing 90 mL of sterile water and glass beads, shaking at 180 r/min for 1 h at 16°C, to prepare a suspension with a concentration of 10 − 1 , then serially diluted with sterile water to a concentration of 10 − 2 -10 − 6 . A total of 200 μL of each dilution were plated with triplicates in seven types of media. All of which contain 100 μg/mL streptomycin, 100 μg/mL penicillin and 10 mg/L rose bengal. The Petri dishes were incubated at 16°C in dark, and the characteristic of each emerging fungal colony was observed daily, then transferred onto a new PDA plate, until axenic cultures were obtained.
Fruiting body and sclerotia of O. sinensis were rinsed with sterile water, surface-sterilized using 75% ethanol for 3 min, followed by 2.5% sodium hypochlorite (NaClO) for 25 min, rinsed twice in sterile distilled water, dried on sterile filter paper. Subsequently, the internal tissue blocks obtained from the fruiting body and sclerotia of sterilized O. sinensis, were cut into 1-2 mm slice, then placed on different media described above. The Petri dishes were incubated at 16°C in dark. While hyphae germinate and grow from the tissue slice, the tip of hyphae were picked to purify culturable fungal species.
Effectiveness of surface sterilization: the surfacesterilized samples were washed in sterile distilled water three times, soaked in 1.5 mL sterile water, and stirred for 1 min. A total of 100 μL suspension were inoculated onto modified PDA (I) medium plates at 16°C in dark. If no microbial growth occurred on the medium, the sterilization was considered complete.

Identification of culturable fungal isolates
The purified fungal isolates were categorized preliminarily based on colony characteristics, including colony morphology, texture, aerial hyphae, substrate hyphae, spore mass color, sporophore and spore chain morphology, distinctive reverse colony color and diffusible pigment, etc.. The representative fungal isolates with distinct morphology were identified using a combination of morphology characteristics and the ITS sequence. The extraction of genomic DNA were performed using CTAB methods as previously described [43].
The nearly full-length ITS region of the rDNA gene was amplified by the primers ITS1 (5′-TCCGTAGGTG AACCTGCGG-3′) and ITS4 (5′-TCCTCCGCTTATTG ATATGC-3′) [44]. The amplicons were purified using the ExoSAP-IT reagent (USB Corporation, Cleveland, OH) following manufacturer's instructions, ligated into pMD18-T (TaKaRa, Japan) and transformed into competent E.coli DH5a following the manufacturer's protocol. The positive transformants were sent to Shanghai Invitrogen Biotechnology Co. Ltd., for sequencing. The sequences of fungal rDNA ITS region were compared with that of the most closely-related fungal species (higher than 97% similarity) in the NCBI database using the BLAST program. The ITS sequence data were submitted and deposited in GenBank under accession numbers MT133906-MT134006.