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RESULTS

Lower Yield of C. cicadae Under Natural Habitat Compared With Sterile Bottle

The proportion of pupae producing fruiting bodies was similar between CS and CB cultivation environments (92 ± 14% in CS and 95±9% in CB, Figure 2A), indicating that C. cicadae  can be cultivated in the soil of a natural forest habitat as in   glass bottle. However, the fresh weight, number, length and diameter of fruiting bodies in CS were all significantly lower than those in CB (p < 0.01, Figures 2B–E), and the relative yield in CS (534.52g/100 cicada) was also lower than that in CB (645.05g/100 cicada, Supplementary Table 2), indicating that the overall growth of C. cicadae in CB was greater than in CS.

FIGURE 2 | The effects of two cultivation environments on yield measurements in Cordyceps cicadae. (A) proportion of pupae producing fruiting bodies; (B) fresh weight of Cordyceps cicadae; (C) number of fruiting bodies; (D) length of fruiting bodies; (E) diameter of fruiting bodies. CB, cultivation in glass bottles; CS, cultivation in the soil of a natural habitat. **p < 0.01, according to non-parametric Mann–Whitney U-tests, error bars depict the standard deviation.

Contents of Metabolites in C. cicadae Differed Between Two Cultivation Environments

In total, 17 amino acids were detected in C. cicadae cultivated in both environments (Figure 3A). The total amino acid content of C. cicadae in CS (28.87  0.057%) was significantly higher than in CB (26.01 0.029%, p < 0.01, Figure 3B). The contents of aspartic acid, threonine, serine, glutamic acid, glycine, alanine, cysteine, valine, methionine, isoleucine, phenylalanine, histidine, lysine, arginine, and leucine of C. cicadae were significantly higher in CS than in CB (p < 0.01). Conversely, the proline content in CS (2.51  0.005%) was significantly lower than that in CB (3.04 0.015%, p < 0.01). Cordycepin and polysaccharide contents of C. cicadae were slightly lower in CS than in CB. Adenosine and mannitol contents were significantly lower in   CS than CB (p < 0.01, Figure  3B).  These  results  indicate that cultivating C. cicadae in natural habitats increases the production of nitrogen compounds (amino acids), but decreases that of carbon compounds (polysaccharides and mannitol), and nucleosides (cordycepin and adenosine).

FIGURE 3 | The effects of two cultivation environments on metabolites produced by Cordyceps cicadae. (A) content of 17 amino acids; (B) content of five metabolites. ASP, aspartic acid; THR, threonine; SER, serine; GLU, glutamic acid; GLY, glycine; ALA, alanine; CYS, cysteine; VAL, valine; MET, methionine; ILE, isoleucine; LEU, leucine; TYR, tyrosine; PHE, phenylalanine; HIS, histidine; LYS, lysine; ARG, arginine; PRO, proline; TAA, total amino acid; Cor, cordycepin; Ade, adenosine; Pol, polysaccharide; Man, mannitol. Error bars depict the standard deviation (*p < 0.05, **p < 0.01, according to Student’s t-tests).

Bacterial Communities in Sclerotia of C. cicadae Were Higher Richness When Cultivated in Natural Habitat Compared With Sterile Bottle

A total of 2,931,602 raw sequences were detected. After filtering, 1,465,801 effective reads were obtained, with 30,665–92,371 in each sample. The coverage index of samples (>0.99 for all samples, Supplementary Table 3) and the rarefaction curves (Supplementary Figure 1) indicated that the  sampling  depth of sequence data set in the current study was adequate for our analyses as the Shannon index flattens out after approximately 30,000 reads in each sample.

There was no significant difference in the Shannon diversity index of bacterial communities in the sclerotia of C. cicadae in each growth stage between the two environments, indicating that the bacterial community diversity was stable (Figure 4A). However, the Chao1 richness index in CS was higher than in CB; at week 2, the Chao1 richness index was significantly higher in SCS_2 than in SCB_2 (p < 0.05, Figure 4B). This result indicates bacterial richness is higher in C. cicadae cultivated in natural habitats than sterile bottle.

FIGURE 4 | Diversity index of bacterial communities in sclerotia of Cordyceps cicadae under the two cultivation environments (SCB, sclerotia of Cordyceps cicadae cultivating in glass bottles; SCS, sclerotia of Cordyceps cicadae cultivating in the soil of a natural habitat) at 1, 2, and 3 weeks (denoted by _1, _2, and _3, respectively) after inoculation. (A) Shannon index of diversity; (B) Chao1 index of richness. Different lowercase letters indicate significant difference (p < 0.05). Error bars depict the standard deviation.

Bacterial Community Composition in Sclerotia of C. cicadae Varied Between Cultivation Environments and Growth Stages

In total, 11 phyla with a relative abundance greater than 0.1% were detected (Figure 5A). Proteobacteria was the dominant phylum (31.69±22.74%–95.66±6.09%) across all growth stages in both environments. The Firmicutes represented the dominant phylum in SCB_1, and its abundance at this  time  point  in  week 1 (67.80    22.2%) was significantly higher than in week  2 or 3 under both cultivation environments (2.08 ±1.95%–7.93 ±1.27%, p < 0.01).

At the genus level (Figure 5B), the bacterial community composition varied between CB and CS and also across growth stages. In C. cicadae cultivated in sterile conditions: Enterococcus (37.05 ± 31.08%) and Serratia (26.08 ± 22.96%) were the dominant genera in SCB_1; Achromobacter (36.91 ± 15.24%) and Cedecea (33.18 ± 40.63%) were dominant in SCB_2; and Achromobacter (50.24 ± 46.77%) and Serratia (32.34 ± 56.01%) were dominant in SCB_3. In C. cicadae cultivated in natural habitats, Pseudomonas (61.80 ± 18.32%) and Achromobacter (80.75 ±15.18%) were the dominant genera in SCS_2 and SCS_3, respectively. The relative abundance of Achromobacter gradually increased from 0.02 ± 0.012%  to  80.75 ± 15.18%  with  the  growth of C. cicadae, which showed significantly different relative abundance among groups (p < 0.05). The relative abundance of Pseudomonas, Enterococcus, Ralstonia, Rhodococcus, and Amaricoccus were also significantly different among groups. Pseudomonas, Ralstonia, Rhodococcus,  and  Amaricoccus  had a significantly higher relative abundance in SCS_2 than  at  other stages in both environments (Rhodococcus, p <  0.05; other p < 0.01), while Enterococcus had significantly higher relative abundance in SCB_1 than at other stages in both environments (p < 0.05). Cedecea only had higher relative abundance (33.18 ± 23.46%) in SCB_2, but a low relative abundance (0.001 ± 0.00095%–1.09 ± 1.55%) at other stages   in both environments (Figure 5B). These data show that the bacterial community composition and dominant genera in the sclerotia of C. cicadae vary significantly between cultivation environments and growth stages.

FIGURE 5 | Bacterial community composition in sclerotia of Cordyceps cicadae at different growth stage in both cultivation environments (explanations for abbreviations in X axis are shown in Figure 4). (A) Phyla with relative abundance greater than 0.1%; (B) Top 30 most abundant genera (* and ** denotes significance between all growth stages in both environments of p < 0.05 and p < 0.01, respectively, according to ANOVA, error bars depict the standard deviation).

Predictive Functional Profiling of Bacterial Communities

The functions of bacterial communities were predicted by PICRUSt  to  determine  the  changes  in   relative   abundance of bacterial OTUs related to functions  in  samples  from different cultivation environments and time points (Figure 6). Bacteria OTUs related to metabolism had the highest relative abundance (62.80–66.33%) at the KEGG pathway Level 1, followed by environmental information processing (11.10– 14.06%), genetic information processing (6.04–12.14%), human diseases  (4.59–5.29%),  cellular  processes  (2.99–4.04%),  and organismal systems (1.71–2.36%).

At the KEGG pathway Level 2, bacteria OTUs with function related to carbohydrate metabolism (11.98–18.53%), global and overview maps (10.10–11.81%), Amino acid metabolism (7.47– 12.58%) in metabolism of Level 1 and membrane transport (6.55–10.11%) in environmental information processing of Level 1 showed higher  relative  abundance  than  other  functions.  The relative abundance of bacteria OTUs related to glycan biosynthesis and metabolism and carbohydrate metabolism were significantly lower in CS (1.26 ± 0.06% and 12.42 ± 0.31%, respectively) than in CB (1.57 ± 0.29% and 14.28 ± 2.04%, respectively; both p < 0.05). Conversely, in  CS  compared  with CB, the relative abundance of bacteria OTUs related to amino acid metabolism (12.08 ± 0.39% versus 10.44 ± 1.59%), xenobiotic biodegradation and metabolism (4.12 ± 0.50% versus 3.04 ± 1.02%),  metabolism  of  terpenoids  and  polyketides  (1.67 ± 0.03% versus 1.46 ± 0.15%), and biosynthesis of other secondary  metabolites  (1.60 ± 0.04%  versus  1.42  ± 0.13%)  were all significantly higher (all p < 0.05). These results suggested the relative abundance of bacteria OTUs with functions related to metabolism were significantly different between two cultivation environments, and further indicated the potential correlation between the changes of metabolite content and bacterial community composition in C. cicadae cultivated in different cultivation environments.

FIGURE 6 | Variation in KEGG metabolic pathways in functional bacterial communities of Cordyceps cicadae cultivated in different cultivation environments (SCB, sclerotia of Cordyceps cicadae cultivating in glass bottles; SCS, sclerotia of Cordyceps cicadae cultivating in the soil of a natural habitat) at 1, 2, and 3 weeks after inoculation (_1, _2, and _3, respectively). The final number in each sample label represents the replicate number of each sample (_1, _2, and _3, respectively). *+ denotes the relative abundance of bacteria related to the function is significantly higher in CS than that in CB, *- represents the relative abundance of bacteria related to the function significantly lower in CS than that in CB (p < 0.05, according to Student’s t-test).

The Relationship Between Bacterial Community and Metabolite

To further understand the relationship between bacterial communities and metabolites contents in cultivated C. cicadae, we calculated the Spearman’s rank correlation coefficient between the top 30 most abundant genera and the contents of 22 metabolites, including 17 amino acids (Figure 7). There was     a significant correlation between most bacterial genera and metabolite contents, especially those of amino acids.

Total amino acids, tyrosine, arginine, lysine, phenylalanine, valine, and leucine were all significantly positively correlated with Rhodococcus, Faecalibacterium, Enterobacter, Bacteroides, Candidatus Rhabdochlamydia, Brevundimonas, Amaricoccus, Ralstonia, and Bifidobacterium, but negatively correlated with Cosenzaea (all p < 0.05 at least). Pseudomonas, Burkholderia- Caballeronia-Paraburkholderia, and Acinetobacter were all significantly correlated with total amino acids, arginine, lysine, phenylalanine, valine and leucine (all p < 0.05 at least). Proline were significantly positively correlated with Serratia, Bacillus, and Enterococcus, but negatively correlated with Achromobacter and Faecalibacterium (all p < 0.05 at least). There was a significant positive correlation  between  polysaccharide  and  Cosenzaea  (p < 0.01). Cordycepin was significantly negatively correlated with numerous genera, including Rhodococcus, Faecalibacterium, Enterobacter, Bacteroides, Candidatus_Rhabdochlamydia, Brevundimonas, Amaricoccus, Ralstonia, Acinetobacter, Bifidobacterium, and Lactobacillus, but significantly positively correlated with Lactococcus (all p < 0.05 at least). Adenosine were significantly positively correlated with Cosenzaea (p < 0.01), but negatively correlated with Rhodococcus and Faecalibacterium (both p < 0.05). Mannitol were significantly positively correlated with Cosenzaea and Enterococcus, but significantly negatively correlated with Rhodococcus, Faecalibacterium, Enterobacter, and Brevundimonas (all p < 0.05 at least).

FIGURE 7 | Correlation of top 30 most abundant bacteria genera with metabolites in Cordyceps cicadae. The strength (Spearman’s ρ value) of correlations are shown here as different shades of red (positive correlation) and green (negative correlation). Explanations for abbreviations in X axis are shown in Figure 3. *0.01 < p ≤ 0.05; **0.001 < p ≤ 0.01; ***p ≤ 0.001.

DISCUSSION

This study confirmed that it is feasible to cultivate C. cicadae in the soil of a natural forest habitat, but the yield was lower than that of C. cicadae cultivated in sterile glass bottles. Our research also showed that different growth environments appear to affect the growth, bacterial community composition and metabolite content of C. cicadae, and bacterial community composition was correlated with metabolites content of C. cicadae.

Cultivating C. cicadae in natural habitats  could  help  to  meet consumer demand for natural products and reduce the exploitation of wild C. cicadae (Huang, 2008). However, in our study, the yield of C. cicadae cultivated in natural habitats was markedly reduced. Some research found that changes in growth environments can result in some resources being allocated to build a defense system to facilitate growth (Feng, 2008; Simon et al., 2010); it is therefore possible that, in the soil of their natural habitats, C. cicadae may also use resources to build a defense system to reduce the adverse effects  of  natural  competitors  and microorganisms on their growth. This would leave fewer resources allocated to growth of C. cicadae, which could have resulted in the reduced yield we observed in our study.

All organisms lived in complex microbial ecosystems and formed a close relationship with diverse microorganisms; changes in the growth environment can affect the microbial community associated with the host (Mueller et al., 2020). In this study, different growth environments were shown to affect bacterial community composition and dominant bacterial genera in the sclerotia of C. cicadae. Pseudomonas was significantly enriched in  sclerotia  of  C.  cicadae  cultivated  in  the  soil  of  a natural habitat. Researches showed that Pseudomonas was abundant in soil (Li et al., 2013), and Mou et al. (2021) found that Pseudomonas was the dominant  genus  in  the  sclerotia  and external mycelial cortices of wild C. cicadae. We thought therefore the Pseudomonas in C. cicadae cultivated in a natural habitat may originated from the soil; if this is the case, this indicates that C. cicadae may communicate with microorganisms in the soil within its natural habitat. Pseudomonas spp., regarded as broad-host-range entomopathogenic bacteria, are known to exhibit insecticidal activity toward certain agricultural pests (Chen W.J. et al., 2014). This could potentially benefit the growth of C. cicadae, for example by promoting the mummification process, as previously  observed  in  Chinese  Cordyceps  (Wu  et   al.,   2020).   In  addition,  in  this  study,  the sclerotia of C. cicadae cultivated in glass bottles was rich in Cedecea bacteria, but this genus had a very low relative abundance in sclerotia of C. cicadae cultivated in natural habitats. Previous researches isolated and  detected  Cedecea  in  C.  cicadae  (Qu  et al., 2019a,b); however, another study did not detect any Cedecea in sclerotia of C. cicadae (Mou et al., 2021), which indicated the abundance of Cedecea in C.  cicadae is unstable  and may be affected by the growth environment. Furthermore,  in our study, bacterial  community  composition  varied  with  the time point (and therefore growth stage) of C. cicadae, similar to previously  published  research  (Wu  et  al.,  2020).  In particular, the relative abundance of Enterococcus gradually decreased with the growth of C. cicadae, suggesting that I. cicadae injected into the pupae may have inhibited some bacterial genera.

In this study, we found that the changes in relative abundance of bacteria OTUs related to some functions were  consistent  with changes in metabolite content: for example, the relative abundance  of  bacteria  related  to  amino  acid  metabolism in C. cicadae in CS increased significantly than in CB, along with the content of total amino acids in this cultivation environment. The relative abundance of bacteria related to carbohydrate metabolism and glycan biosynthesis and metabolism in CS decreased significantly  than  in  CB,  along  with  the  content  of  carbon  compound  (mannitol  and  polysaccharides)  in  this cultivation environment. Researches showed culture conditions (Cheng et al., 2012; Lee et al., 2019), nutrient sources (Mao      et al., 2005; Chunyu et al., 2019), different strains (Kang et al., 2017), and  hosts  (He  Y.  et  al.,  2019)  can  affect  the  level  of metabolites, but these factors were consistent between two cultivation environment in this study. As a result, the main reason for different level of metabolite in two cultivation environment may be caused by the difference of microbial community composition and related functions (Figures 5, 6). Studies on food fermentation also showed that metabolite production related closely to bacterial community composition in the progress of fermentation (He and Chung, 2020; Zang et al., 2020). Some studies also reported that endophytic microbes in  Cordyceps spp. may affect the production of metabolites (Zhang et al., 2010; Qu et al., 2019a) and microbial co-culture can increase significantly the level of some metabolites (Akone et al., 2016; Yu et al., 2019). Therefore, further researches on determining which microbial species involve in the production of cordycepin, polysaccharide, adenosine, mannitol or amino acid and then using beneficially synthetic community or fungal-bacterial co- culture to improve the level of metabolites in C. cicadae are necessary. The amino acid content of edible mushrooms was known to be correlated  with  freshness  and  sweetness  (Mau  et al., 2001), which may explain why consumers prefer edible mushrooms grown in a natural habitat. In addition, the nitrogen compounds content of C. cicadae in natural soil habitats increased, while the carbon compounds decreased, which may suggest that C. cicadae may exchange carbon and nitrogen elements in the soil, similar function to Metarhizium robertsii (Behie et al., 2017; Barelli et al., 2019). We therefore speculated C. cicadae may exchange carbon and nitrogen elements in soil via endophytic bacteria to affect the content of carbon and nitrogen compounds in C. cicadae, which may potentially result in adding a branch to the carbon and nitrogen cycle in the ecosystem, entomogenous fungi C. cicadae is involved in carbon and nitrogen cycling in the soil.

Compared with the contents of metabolites in wild C. cicadae reported in other studies (Ge et al., 2007; Zhang et al., 2017),  the total amino acids, polysaccharides and mannitol contents of C. cicadae in our study were higher regardless of how they were cultivated. C. cicadae cultivated in a natural habitat could achieve microbial communication in soil and be favored by consumer,   it therefore be an alternative to the wild. Given the apparent close relationship between bacterial community composition and metabolites of C. cicadae, we will further isolate, test and screen microorganisms in future studies to identify which species may be beneficial to the growth and metabolite production of cultivated C. cicadae, so that the yield and metabolites contents could be improved by beneficial interaction between microorganisms.

CONCLUSION

Our study demonstrated for the first time  to  our  knowledge that cultivating C. cicadae in natural habitat soil is feasible, but further studies are required to improve yield. This cultivation method appeared to increase nitrogenous compound (amino acid) and decrease carbonaceous compound (mannitol and polysaccharides)  content,  possibly  through   the   interaction  of C. cicadae with microorganisms in the soil. Metabolite production was found to be closely related to the bacterial community during C. cicadae growth; C. cicadae may therefore also  participate  in  the  carbon  and  nitrogen  cycle  in natural ecosystems.

DATA AVAILABILITY STATEMENT

The datasets presented in this study can be found in online repositories. The names of the repository/repositories and accession number(s) can be found below: https://www.ncbi.nlm. nih.gov/, PRJNA698306.

AUTHOR CONTRIBUTIONS

ZZ designed and performed the experiments, analyzed the data, and wrote the manuscript. DM designed and performed the experiments and analyzed the data. LL, WZ, and LD collected the samples and performed the experiments. XZ designed the experiments and wrote the manuscript. All authors have read and approved the submission of the manuscript.

FUNDING

This work was supported by the National Natural Science Foundation of China (31860037), the Youth Science and Technology Talent Project of Guizhou Province ([2018]5781), the Science and Technology Plan Projects  of  Guizhou  Province (Qian Ke He Support [2019]2405), and (Qian Ke He Foundation [2020]1Z009).

ACKNOWLEDGMENTS

We thank Jiaxi Zhou, Chunbo Dong, Yeming Zhou, and  Jiaojiao Qu at Guizhou University for providing advice on data analysis and manuscript preparation. We also thank Guochun Fan,  Mingpeng  Qiu,  and  Jiaren  Zhang  for  help  with  part  of our experiment on cultivating C. cicadae in the soil of a natural forest habitat.

SUPPLEMENTARY MATERIAL

The Supplementary Material for this article can be  found  online at: https://www.frontiersin.org/articles/10.3389/fmicb. 2021.669785/full#supplementary-material

REFERENCES

Akone, S. H., Mándi, A., Kurtán, T., Hartmann, R., Lin, W. H., and Daletos, G. (2016). Inducing secondary metabolite production by the endophytic fungus Chaetomium sp. through fungal–bacterial co-culture and epigenetic modification. Tetrahedron 72, 6340–6347. doi: 10.1016/j.tet.2016.08.022

Barelli, L., Behie, S. W., and Bidochka, M. J. (2019). Availability of carbon and nitrogen in soil affects Metarhizium robertsii root colonization and transfer of insect-derived nitrogen. FEMS Microbiol. Ecol. 95:fiz144. doi: 10.1093/femsec/ fiz144

Behie, S. W., Moreira, C. C., Sementchoukova, I., Barelli, L., Zelisko, P. M.,   and Bidochka, M. J. (2017). Carbon translocation from a plant to an insect-pathogenic endophytic fungus. Nat. Commun. 8:14245. doi: 10.1038/ ncomms14245

Caporaso, J. G., Kuczynski, J., Stombaugh, J., Bittinger, K., Bushman, F. D., Costello, E. K., et al. (2010). QIIME allows analysis of high-throughput community sequencing data. Nat. Methods 7, 335–336. doi: 10.1038/nmeth.f. 303

Chen, W. J., Hsieh, F. C., Hsu, F. C., Tasy, Y. F., Liu, J. R., and Shih, M. C. (2014). Characterization of an insecticidal toxin and pathogenicity of Pseudomonas taiwanensis against insects. PLoS Pathog. 10:e1004288. doi: 10.1371/journal. ppat.1004288

Chen, Z. A., Li, Z. Z., and Chen, Y. P. (2014). Jin Chanhua. Beijing: Tranditional Chinese Medicine Classics Press.

Cheng, D. Q., Ding, Z. S., Lin, M. A., Pan, P. L., and Chen, Y. T. (2006). Isolation and fermentation culture of fungi from Cordyceps sobofifera. Zhong Yao Cai 2, 99–101.

Cheng, J. W., Wang, Y. B., He, L., Qian, H., Fu, L. Z., and Li, H. B. (2012). Optimization of fermentation process for the production of intracellular polysaccharide from Paecilomyces cicadae and the immuno-stimulating activity of intracellular polysaccharide. World J. Microbiol. Biotechnol. 28, 3293–3299. doi: 10.1007/s11274-012-1140-0

Chunyu, Y. J., Lu, Z. M., Luo, Z. S., Li, S. S., Li, H., Geng, Y., et al. (2019). Promotion of metabolite synthesis in Isaria cicadae, a dominant species in the cicada flower microbiota, by cicada pupae. J. Agric. Food Chem. 67, 8476–8484. doi: 10.1021/acs.jafc.9b02705

Edgar, R. C., Haas, B. J., Clemente, J. C., Quince, C., and Knight, R. (2011). UCHIME improves sensitivity and speed of chimera detection. Bioinformatics 27, 2194–2200. doi: 10.1093/bioinformatics/btr381

Feng, L. C. (2002). Study on Shanghai Tianma hill Cordyceps Sobolifera. J. Technol. 2, 125–127.

Feng, Y. (2008). Nitrogen allocation and partitioning in invasive and native Eupatorium species. Physiol. Plant. 132, 350–358. doi: 10.1111/j.1399-3054. 2007.01019.x

Ge, F., Xia, C. R., Li, C. R., Ding, T., Shao, Y., and Fan, M. Z. (2007). Analysis of the chemical compositions of Paecilomyces cicadae fermented mycelia and Cordyceps cicadae fruit body. Mycosystema 26, 68–75.

He, W., and Chung, H. Y. (2020). Exploring core functional microbiota related with flavor compounds involved in the fermentation of a natural fermented plain sufu (Chinese fermented soybean curd). Food Microbiol. 90:103408. doi: 10.1016/j.fm.2019.103408

He, W. W., He, L., and Li, W. Q. (2019). Research progress on chemical constituents and artificial culture of Cordyceps cicadae. Zhong Yao Cai. 42, 1691–1696.

He, Y., Zhang, W., Peng, F., Lu, R., Zhou, H., Bao, G., et al. (2019). Metabolomic  variation  in   wild   and   cultured   cordyceps   and   mycelia   of  Isaria  cicadae.  Biomed.  Chromatogr.  33:e4478.  doi:  10.1002/bmc. 4478

He, Y. Q., Zhang, W. C., Peng, F., Lu, R. L., Zhou, H., Bao, G. H., et al. (2019). Metabolomic variation in wild and cultured cordyceps and mycelia of Isaria cicadae. Biomed. Chromatogr. 33:e4478. doi: 10.1002/bmc.4478

Hsu, J. H., Jhou, B. Y., Yeh, S. H., Chen, Y. L., and Chen, C. C. (2015). Healthcare functions of Cordyceps cicadae. J. Nutrit. Food Sci. 5:100432. doi: 10.4172/2155- 9600.1000432

Hu, H. Y., Zou, X., Luo, L., Liu, A. Y., and Liang, Z. Q. (2009). Artificial culturing of Cordyceps cicadidae on living silkworm. Zhongguo Zhong Yao Za Zhi 34, 2140–2143.

Huang, W. (2008). Discussion on ecological wild cultivation of edible fungi. Edible Fungi China. 5:3334.

Kang, N., Lee, H. H., Park, I., and Seo, Y. S. (2017). Development of high cordycepin-producing Cordyceps militaris strains. Mycobiology 45, 31–38. doi: 10.5941/MYCO.2017.45.1.31

Ke,  B.-J.,  and  Lee,  C.-L.  (2016).  Investigation  on   the   fermentation  for high adenosine and N6-(2-hydroxyethyl)-adenosine (HEA) productions of Cordyceps cicadae. New Biotechnol. 33, S206–S207. doi: 10.1016/j.nbt.2016.06. 1433

Langille, M. G. I., Zaneveld, J., Caporaso, J. G., McDonald, D., Knights, D., Reyes,

J. A., et al. (2013). Predictive functional profiling of microbial communities using 16S rRNA marker gene sequences. Nat. Biotechnol. 31, 814–821. doi: 10.1038/nbt.2676

Latini, S., and Pedata, F. (2001). Adenosine in the central nervous system: release mechanisms and extracellular concentrations. J. Neurochem. 79, 463–484. doi: 10.1046/j.1471-4159.2001.00607.x

Lee, S. K., Lee, J. H., Kim, H. R., Chun, Y., Lee, J. H., Yoo, H. Y., et al. (2019). Improved cordycepin production by Cordyceps militaris KYL05 using casein hydrolysate in submerged conditions. Biomolecules 9:461. doi: 10.3390/ biom9090461

Li, L., Abu Al-Soud, W., Bergmark, L., Riber, L., Hansen, L. H., Magid, J., et al. (2013). Investigating the diversity of Pseudomonas spp. in soil using culture dependent and independent techniques. Curr. Microbiol. 67, 423–430. doi: 10. 1007/s00284-013-0382-x

Li, L., Zhang, T., Li, C., Xie, L., Li, N., Hou, T., et al. (2019). Potential therapeutic effects of Cordyceps cicadae and Paecilomyces cicadae on adenine-induced chronic renal failure in rats and their phytochemical analysis. Drug Des. Devel. Ther. 13, 103–117. doi: 10.2147/dddt.S180543

Lin, S., Liu, Z. Q., Xue, Y. P., Baker, P. J., Wu, H., Xu, F., et al. (2016). Biosynthetic pathway analysis for improving the cordycepin and cordycepic acid production in Hirsutella sinensis. Appl. Biochem. Biotechnol. 179, 633–649. doi: 10.1007/ s12010-016-2020-0

Liu, A. Y. (2012). Research and Application of Cordyceps cicadae Resources in China. Guiyang: Guizhou Science and Technology Publishing House.

Liu, S. Q., Wen, L., Xia, M., and Jiang, N. (2008). Determination of the active ingredient produced in the artificial cultivated Cordyceps sobolifera. J. Anhu. Agric. Sci. 36, 429–467.

Liu, T., Zhang, H., Shi, X., Ma, W., Zhang, Y., Liu, Z., et al. (2018). Illumina MiSeq sequencing investigation of Chanhua (Cordyceps cicadae Shing) fungal community structures in different regions. J. Trad. Chin. Med. Sci. 5, 206–212. doi: 10.1016/j.jtcms.2017.07.008

Lu, J., Gu, G., Hao, L., Jin, Z., and Wang, X. (2016). Characterization and in vitro antioxidant activity of a polysaccharide from Cordyceps sobolifera. J. Food Process. Preserv. 40, 447–452. doi: 10.1111/jfpp.12622 

Magoc, T., and Salzberg, S. L. (2011). FLASH: fast length adjustment of short reads to improve genome assemblies. Bioinformatics 27, 2957–2963. doi: 10.1093/ bioinformatics/btr507

Mao, X. B., Eksriwong, T., Chauvatcharin, S., and Zhong, J. J. (2005). Optimization of carbon source and carbon/nitrogen ratio for cordycepin production by submerged cultivation of medicinal mushroom Cordyceps militaris. Process Biochem. 40, 1667–1672. doi: 10.1016/j.procbio.2004.06.046

Mau, J. L., Lin, H. C., and Chen, C. C. (2001). Non-volatile components of several medicinal mushrooms. Food Res. Int. 34, 521–526. doi: 10.1016/s0963-9969(01) 00067-9

Mou, D., Zeng, Z. Y., Zhong, W. L., Zhou, J. X., Qu,  J.  J.,  and  Zou,  X.  (2021). Bacterial community structure and function of Cordyceps cicadae microecosystem in Guiyang. Act. Microbiol. Sin. doi: 10.13343/j.cnki.wsxb. 20200540 [Epub ahead of print],

Mueller, E. A., Wisnoski, N. I., Peralta, A. L., and Lennon, J. T. (2020). Microbial rescue effects: how microbiomes can save hosts from extinction. Funct. Ecol. 34, 2055–2064. doi: 10.1111/1365-2435.13493

Nakav,  S., Chaimovitz, C., Sufaro, Y., Lewis,  E. C., Shaked, G.,  Czeiger, D.,    et al. (2008). Anti-inflammatory preconditioning by agonists of adenosine A1 receptor. PLoS One 3:e2107. doi: 10.1371/journal.pone.0002107

Nxumalo, W., Elateeq, A. A., and Sun, Y. (2020). Can Cordyceps cicadae be used as an alternative to Cordyceps militaris and Cordyceps sinensis? – a review. J. Ethnopharmacol. 257:112879. doi: 10.1016/j.jep.2020.112879

Olatunji, O. J., Feng, Y., Olatunji, O. O., Tang, J., Ouyang, Z., and Su,  Z. (2016a). Cordycepin protects PC12 cells against 6-hydroxydopamine induced neurotoxicity via its antioxidant properties. Biomed. Pharmacother. 81, 7–14. doi: 10.1016/j.biopha.2016.03.009

Olatunji, O. J., Feng, Y., Olatunji, O. O., Tang, J., Ouyang, Z., Su, Z., et al. (2016b). Neuroprotective effects of adenosine isolated from Cordyceps cicadae against oxidative and ER stress damages induced by glutamate in PC12 cells. Environ. Toxicol. Pharmacol. 44, 53–61. doi: 10.1016/j.etap.2016.02.009

Qu, Q. S., Yang, F., Zhao, C. Y., and Shi, X. Y. (2019a). Analysis of the bacteria community in wild Cordyceps cicadae and its influence on the production of HEA and nucleosides in Cordyceps cicadae. J. Appl. Microbiol. 127, 1759–1767. doi: 10.1111/jam.14432

Qu, Q. S., Yang, F., Zhao, C. Y., Wang, J. R., Zeng, J. H., and Shi, X. Y. (2019b). Isolation and diversity of Cordyceps cicadae endophytic bacteria in Anhui and Zhejiang. Zhong Yao Cai. 42, 992–999. doi: 10.13863/j.issn1001-4454.2019.05. 009

Quast, C., Pruesse, E., Yilmaz, P., Gerken, J., Schweer, T., Yarza, P., et al. (2013). The SILVA ribosomal RNA gene database project: improved data processing and web-based tools. Nucleic Acids Res. 41, D590–D596. doi: 10.1093/nar/gks1219

San, W. J., Li, T. H., and Jiang, Z. D. (2010). Determination of cordyceps acid in Guangdong by colorimetry. Edible Fungi. 32, 73–74.

Simon, J., Gleadow, R. M., and Woodrow, I. E. (2010). Allocation of nitrogen    to chemical defence and plant functional traits is constrained by soil N. Tree. Physiol. 30, 1111–1117. doi: 10.1093/treephys/tpq049

Sun, Y. F., Kmonickova, E., Han, R. L., Zhou, W., Yang, K. B., Lu, H. F., et al. (2019). Comprehensive evaluation of wild Cordyceps cicadae from different geographical origins by TOPSIS method based on the macroscopic infrared spectroscopy (IR) fingerprint. Spectrochim. Acta A Mol. Biomol. Spectrosc. 214, 252–260. doi: 10.1016/j.saa.2019.02.031

Wang, Y., He, P., He, L., Huang, Q., Cheng, J.,  Li,  W.,  et  al.  (2019).  Structural elucidation, antioxidant and immunomodulatory activities of a novel hetero polysaccharide from cultured Paecilomyces cicadae (Miquel.) Samson. Carbohydr. Polym. 216, 270–281. doi: 10.1016/j.carbpol.2019.03.104

Wu, H., Rao, Z.-C., Cao, L., De Clercq, P., and Han, R.-C. (2020). Infection       of Ophiocordyceps sinensis fungus causes dramatic changes in the microbiota of its thitarodes host. Front. Microbiol. 11:577268. doi: 10.3389/fmicb.2020.57 7268

Xia, F., Liu, Y., Shen, G. R., Guo, L. X., and Zhou, X. W. (2015). Investigation and analysis of microbiological communities in natural Ophiocordyceps sinensis. Can. J. Microbiol. 61, 104–111. doi: 10.1139/cjm-2014-0610

Yu, M., Li, Y., Banakar, S. P., Liu, L., Shao, C., Li, Z., et al. (2019). New metabolites from the co-culture of marine-derived Actinomycete Streptomyces rochei MB037 and fungus Rhinocladiella similis 35. Front. Microbiol. 10:915. doi: 10.3389/fmicb.2019.00915

Zang, J., Xu, Y., Xia, W., Regenstein, J. M., Yu, D., Yang, F., et al. (2020). Correlations between microbiota succession and flavor formation during fermentation of Chinese low-salt fermented common carp (Cyprinus carpio L.) inoculated with mixed starter cultures. Food Microbiol. 90:103487. doi: 10.1016/ j.fm.2020.103487

Zeng, W. B., Chang, C. X., Li, J. P., Wang, Y. B., Dai, Y. D., and Yu, H. (2017). Micro-morphological variation pattern of Isaria cicadae. Acta  Microbiol.  Sin. 57, 350–362.

Zeng, W. B., Wang, Y. B., Yu, H., Dai, Y. D., and Chen, Z. H. (2015). Population distribution of Isaria cicadae and its habitat investigation. Edible Fungi China 34, 50–56.

Zeng, W. B., Yu, H., Ge, F., Yang, J. Y., Chen, Z. H., Wang, Y. B., et al. (2014). Distribution of nucleosides in populations of Cordyceps cicadae. Molecules 19, 6123–6141.

Zeng, Z. Y., Xu, Z. S., Mou, D., Xu, S. H., Du, F., and Zhou, J. X. (2019). Analyses on the structure and ecological function of fungal community in sclerotia, external mycelial layer covering ossified cicada epidermis and habitat soil of Isaria cicadae. Mycosystema 38, 1710–1723.

Zhang, H. M., Shi, X. S., Liu, T. F., Ma, W., Zhang, Y. C., and Shi, X. Y. (2017). Comparing research on contents of adenosine, cordycepin and ergosterol in Cordyceps cicadae. Glob. Trad. Chin. Med. 10, 297–301.

Zhang, H. X., Gao, X. H., Chen, W., and Huang, K. H. (2012). Levels of selected chemical components in Cordyceps sobolifera fruit bodies cultivated artificially and collected from the wild. Acta Edulis Fungi 19, 59–62.

Zhang, Q., Olatunji, O. J., Chen, H., Tola, A. J., and Oluwaniyi, O. O. (2018). Evaluation of the anti-diabetic activity of polysaccharide from Cordyceps cicadae in experimental diabetic rats. Chem. Biodivers. 15:e1800219. doi: 10. 1002/cbdv.201800219

Zhang, X., Hu, Q., and Weng, Q. (2019). Secondary  metabolites  (SMs)  of  Isaria cicadae and Isaria tenuipes. RSC Adv. 9, 172–184. doi: 10.1039/C8RA0 9039D

Zhang, Y., Xiao, X. J., Zhang, M. Y., Xiong, J., and Huang, Z. X. (2016). Key technique for artificial rearing of Cordyceps militaris. Guizhou Agric. Sci. 44, 65–68.

Zhang, Y., Zhang,  S.,  Wang,  M.,  Bai,  F.,  and  Liu,  X.  (2010).  High  diversity of the fungal community structure in naturally-occurring Ophiocordyceps sinensis. PLoS One 5:e15570. doi: 10.1371/journal.pone.00 15570

Zhou, J. X., Yu, L. F., Zhang, J., Zhang, X. M., Xue, Y., Liu, J., et al. (2020). Characterization of the core microbiome in tobacco leaves during aging. MicrobiologyOpen 9:e984. doi: 10.1002/mbo3.984

Conflict of Interest: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. 

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