In this study, we investigated the fungal community structure in different plant tissues and rhizosphere soils of G. conopsea sampled from four sites in Tabet through high-throughput sequencing to explore the relationship between endophytic fungi and plants from a deeper perspective, with the aim of exploring the role of endophytic fungi and laying the foundation for the development and utilization of endophytic fungal resources.
A total of 14,800 OTUs belonging to 4 phyla, 73 families, and 99 genera obtained from tissues and rhizosphere soils were analyzed. It was previously reported that mycorrhizal fungi associated with orchids were mainly Basidiomycota and Ascomycota (Sisti et al. 2019). In our study, Ascomycota (33.71%-86.38%) was the dominant phylum, with the highest relative abundance in all samples, followed by Basidiomycota (6.98%-58.30%), while Mortierellomycota had the lowest relative abundance and existed only in roots and soil. These findings were similar to previous studies by Lin et al. (2020), who explored the differences in fungal communities from the roots and soils of G. conopsea. We also found that the community abundances of endophytes and soil fungi were different at the four sampling sites. The abundance of Ascomycota in fruits, leaves, and stems at low altitudes was higher than that at high altitudes, while the abundance in roots and soils was not consistent. Interestingly, the abundance of Basidiomycota was the opposite, with higher abundance at higher altitudes.
Several studies have shown that endophytes are closely related to the growth of Orchidaceae plants, playing important roles in the germination of Orchidaceae seeds (Bidartondo and Read 2008; Rasmussen 2002; Rasmussen et al. 2015), protocorm formation (Vujanovic et al. 2000), and seedling growth (Hou and Guo 2009; Huang et al. 2018), such as providing nutrients (Bonnardeaux et al. 2007; Sathiyadash et al. 2012; Kuga et al. 2014), secreting phytohormones to promote plant development (Zhang et al. 1999; Shah et al. 2019), and improving resistance against phytopathogens (Favre-Godal et al. 2020). Previous reports have shown that endophytes, including Tulasnellaceae, Ceratobasidiaceae, and Serendipitaceae, separated from orchids were cocultured with orchid seeds and could promote seed germination and protocorm development (Chen et al. 2012; Herrera et al. 2016; Meng et al. 2019). Gao et al (2020) isolated three kinds of endophytes from G. conopsea and observed that only the family Ceratobasidiaceae could promote protocorm formation and seedling growth. In this research, Ceratobasidiaceae was detected at a higher relative abundance in roots than in the other samples. The relative abundance of the species from the four collection sites was different, with the highest abundance at the FM site (14.19%). Fungi belonging to Serendipitaceae were detected only in soils but not in plant tissues. Lin et al (2020) showed that there were differences in the community diversity of endophytes at different stages of plant development. We used plant tissues of G. conopsea at the fruit stage as the study material and failed to find Tulasnellaceae, which might explain its absence among the classified fungi.
Plant hormones, including GA, IAA, and abscisic acid, are produced by some of the endophytic fungi that have been separated from medicinal orchids (Zhang et al. 1999). The literature has shown that the secretion of phytohormones by endophytic fungi belonging to the genera Fusarium (Vujanovic et al. 2000; Tsavkelova et al. 2008; Bell et al. 2020), Gibberella (Silva et al. 2018), Leptosphaerulina (Favre-Godal et al. 2020), Trichoderma (Zhang et al. 2016), Cladosporium (Favre-Godal et al. 2020), Phialophora (Berthelot et al. 2016), Aspergillus (Yuan et al. 2018), and Cadophora (Berthelot et al. 2016) might explain why endophytes promote seed germination and plant growth. In the current study, Cladosporium and Cadophora were the dominant species, with different degrees of richness in different tissue samples. Among them, Cladosporium was the dominant genus in fruits, accounting for a relative abundance of 10.11%-21.96%, and the abundance in stems was less than 5%; this genus was barely detected in other samples. These results imply that this species plays an important role in fruit development of G. conopsea. The abundance of Cadophora was the highest in roots, and this genus was confirmed by Berthelot et al (2016) to enhance plant growth. Ceratobasidium also had the highest abundance in roots. Ercole et al (2015) observed mycorrhizal fungi such as Ceratobasidium with diverse communities in the roots and protocorms of photoautotrophic orchids. N uptake from inorganic sources and transport to the protocorm by the genus Ceratobasidium were revealed by Kuga et al. (2014). However, the abundances of Phialophora and Aspergillus were only 1.22%-5.25% in roots, but that of Gibberella was less than 5% and 1% in stems and leaves, respectively, which might also contribute to the extinction of G. conopsea. The symbiotic relationship between orchid plants and endophytes exerts a significant influence on the growth and natural propagation of orchids. In other words, the distribution of fungi could affect key stages of orchids, such as seed germination, plant growth and survival (McCormick et al. 2018). Similar reports demonstrated that the diversity and abundance of endophytes affected the distribution and characteristics of plants, such as the size of populations and density of plants (McCormick et al. 2018; Favre-Godal et al. 2020). According to the results, one of the reasons why G. conopsea has become endangered in Tibet may be related to the low diversity and abundance of specific endophytic fungal communities that could promote plant growth and reproduction. Our results could provide a reference for the introduction of fungi into habitat soil to colonize roots to expand the population and protect the species.
According to the venn diagram analysis, the number of overlapping OTUs in roots and soils was 172. PCoA results showed that OTUs from roots and rhizosphere soils were clustered together. Compared with other plant tissues (stems, leaves, and fruits), the fungal community structures in roots and rhizosphere soils were more similar. The results implied that endophytes in roots might originate from rhizosphere soils. McCormick et al (2012) suggested that fungal community diversity was influenced by the soil environment. Supporting this view, Bell et al (2020) also showed that the diversity of endophytes in the roots of orchids was related to the soil environment. RDA indicated that the soil environment exerted a significant influence on the fungal community. In this study, the low abundance of fungal communities associated with plant growth promotion might be affected by the soil environment, which may be one of the reasons for the endangerment of G. conopsea in Tibet.
Endophytes can not only promote plant growth but also produce bioactive components (Sisti et al. 2019). Endophytic fungi from medicinal plants can synthesize many of the same or similar bioactive substances as their host plants (Yang et al. 2018). Relevant reports have shown that endophytes in medicinal plants have pharmacologically active compounds, such as antioxidants, antibacterial compounds, and tumor growth inhibitors (Sarsaiya et al. 2019), which Ma et al (2015) indicated was related to the diversity of endophytic fungal metabolites. All the sequences were classified into 73 families, among which Cladosporiaceae and Leptosphaeriaceae were the dominant flora. Fu et al (2020) showed that Leptosphaeriaceae reduced asthma severity. The highest abundance of Leptosphaeriaceae was found in fruits at low altitudes, with a relative abundance of 19.734%-19.89%, and this should be beneficial to the study and utilization of this dominant family. Ng et al (2013) declared that the genome of UM238, a dematiaceous fungus belonging to Herpotrichiellaceae, contained a variety of protective genes that helped the fungus survive in adverse environments. We also found that the abundance of Herpotrichiellaceae in roots was high, with the highest at the LD collection site; this family may aid G. conopsea adaptation to the environment of the Qinghai-Tibet Plateau, which is characterized by low temperature, strong UV ultraviolet light and low oxygen. Several species of Nectriaceae have previously been reported to have potential as biocontrol agents and biodegraders in industrial applications (Lombard et al. 2015; Ye et al. 2020). Similarly, the high abundance of these species in roots and rhizosphere soils in our results might make the plant more adaptable to the soil environment. In addition, secondary metabolites such as mycotoxins or antibiotics were secreted by several species belonging to the family Trichomeriaceae, which was described by Ye et al (2020). Trichomeriaceae were distributed in stems and leaves in our study. The analysis of the MetaCyc pathway with PICRUSt2 revealed several lipid metabolism pathways, such as fatty acid oxidation, CDP-diacylglycerol biosynthesis, stearate biosynthesis, phosphatidylglycerol biosynthesis, and monoacylglycerol metabolism. Interestingly, Mortierellaceae was distributed in roots and soils, with the highest abundance in roots. A large number of studies have shown that many species of Mortierellaceae are oleaginous fungi with great potential for yield of lipids and valuable fatty acids (Li et al. 2015; Gao et al. 2016). The MetaCyc pathway results also showed a high abundance of amino acids and carbohydrate metabolism. Amino acid metabolism mainly involved leucine, tyrosine, tryptophan, serine, valine, glycine, methionine, threonine, and arginine. Jiang et al (2018) isolated 9 kinds of amino acids from G. conopsea, which have high nutritional and medicinal value because they could maintain nitrogen balance in the body. Yu (2017) found that polysaccharides had the effect of delaying aging and alleviating fatigue through study of the pharmacological activity of polysaccharides in G. conopsea. Therefore, the metabolism of amino acids and carbohydrates in endophytes might be beneficial for improving the nutritional and medicinal value of G. conopsea. The natural propagation rate of G. conopsea is low, artificial cultivation of this species is difficult, and human destruction of habitat is increasing, which will cause the population to decrease. To avoid the decline of G. conopsea caused by the exploitation and utilization of the species for medicinal resources, effective medicinal ingredients can be obtained from microorganisms by mass culture in the future. Our study aimed to analyze the fungal community diversity and composition of G. conopsea, which may be valuable for screening fungi with biological activity.