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Article

Research article

J. Microbiol. Biotechnol. 2024; 34(6): 1249-1259

Published online June 28, 2024 https://doi.org/10.4014/jmb.2401.01009

Copyright © The Korean Society for Microbiology and Biotechnology.

Diverse Mycena Fungi and Their Potential for Gastrodia elata Germination

Xiao-Han Jin1,2†, Yu-Chuan Wang3†, Dong Li1,2, Yu Li1, Hai-Yan He4,5, and Han-Bo Zhang1*

1State Key Laboratory Conservation and Utilization of Bio-Resources in Yunnan, Kunming, P.R. China
2School of Ecology and Environmental Science, Yunnan University, Kunming, P.R. China
3Gastrodia Tuber Research Institute of Zhaotong, P.R. China
4The Agriculture and Life Sciences College, Zhaotong University, Zhaotong, P.R. China
5Yunnan Key Laboratory of Gastrodia elata and Fungus Symbiotic Biology, Zhaotong, P.R. China

Correspondence to:Han-Bo Zhang,       zhhb@ynu.edu.cn

These authors contributed equally to this work.

Received: January 10, 2024; Revised: April 17, 2024; Accepted: April 18, 2024

Abstract

It remains to be determined whether there is a geographical distribution pattern and phylogenetic signals for the Mycena strains with seed germination of the orchid plant Gastrodia elata. This study analyzed the community composition and phylogenetics of 72 Mycena strains associated with G. elata varieties (G. elata. f. glauca and G. elata. f. viridis) using multiple gene fragments (ITS+nLSU+SSU). We found that (1) these diverse Mycena phylogenetically belong to the Basidiospore amyloid group. (2) There is a phylogenetic signal of Mycena for germination of G. elata. Those strains phylogenetically close to M. abramsii, M. polygramma, and an unclassified Mycena had significantly higher germination rates than those to M. citrinomarginata. (3) The Mycena distribution depends on geographic site and G. elata variety. Both unclassified Mycena group 1 and the M. abramsii group were dominant for the two varieties of G. elata; in contrast, the M. citrinomarginata group was dominant in G. elata f. glauca but absent in G. elata f. viridis. Our results indicate that the community composition of numerous Mycena resources in the Zhaotong area varies by geographical location and G. elata variety. Importantly, our results also indicate that Mycena’s phylogenetic status is correlated with its germination rate.

Keywords: Gastrodia elata variety, Mycena, phylogenetic signal, geographical distribution, germination

Introduction

The fungi of the genus Mycena (Pers.) Roussel belong to the phylum Basidiomycota, class Agaricomycetes, order Agaricales, and family Mycenaceae. The genus is species-rich and cosmopolitan. Several monographs of Mycena from different continents have been published over the years [1-5]. According to the tenth edition of Ainsworth & Bisby’s Dictionary of the Fungi, Mycena is one of the most diverse genera in Agaricales, comprising more than 500 described species worldwide [5]. As of 2023, Index Fungorum lists 2412 records for this genus. Most species of Mycena are saprotrophic fungi that decompose litter and contribute to the forest ecosystem [6, 7]. However, some species have symbiotic or parasitic interactions with plants. For example, Mycena galopus forms beneficial associations with Vaccinium corymbosum [8]; M. citrinomarginata colonizes the roots of Festuca roemeri [9]; and Mycena citricolor infects coffee trees and causes American leaf spot disease [10].

The Orchidaceae family is exceptionally diverse and widespread [11], comprising over 750 genera and approximately 27000 species, and is the most species-rich, accounting for approximately 10% of the world’s flowering plants [12, 13]. Orchid seeds are very small (dust-like), lack endosperm and have limited nutrition for germination, so they depend on certain fungi to supply them with nutrients. These plants are known as “initially mycoheterotrophic” plants [14-17]. All orchids are at least initially mycoheterotrophic [18, 19] and depend entirely on orchid mycorrhizal fungi (OMFs) for seed germination and protocorm formation [20, 21]. Subsequently, most orchids develop green leaves and perform photosynthesis (partially mycoheterotrophic type), whereas more than 200 orchid species never photosynthesize and rely entirely on fungal nutrition for growth (fully mycoheterotrophic type) [11, 22].

Gastrodia elata Bl. belongs to the fully mycoheterotrophic type and has important medicinal and edible value. It is widely used in China, Korea and Japan [23]. Each of its capsules produces tens of thousands of seeds [24], but they lack nutrition and require symbiosis with Mycena fungi to germinate and form protocorms. Subsequent nutritional growth depends on symbiosis with another fungus, Armillaria mellea, to complete the whole life cycle [25]. Although related cultivation technology and practices have achieved success, natural G. elata populations are still facing threats and are rated vulnerable on the IUCN Red List (IUCN 2022).

Currently, it is generally believed that G. elata seeds can only germinate in symbiosis with Mycena [26]. In the 1980s, Xu and Guo [27] were the first to successfully induce the fruiting body of a fungal strain for G. elata seed germination, GSF-8104, which was identified as Mycena osmundicola. In the 1990s, Guo and Fan et al. isolated three strains from the roots of 45 orchid species that could symbiotically enhance the germination of G. elata seeds. These strains were subsequently identified and named Mycena orchidicola [28], Mycena anoectochila [29] and Mycena dendrobii [30]. Since then, it has been rare to report new Mycena strains with the capacity for G. elata germination because Mycena are weakly saprophytic and have slow growth and low culturability. Until 2020, there were new reports on G. elata seed germination fungi, where two strains belonging to Mycena citrinomarginata and Mycena purpureofusa could germinate G. elata seeds [31]. In 2022, it was demonstrated that a new Mycena species, Mycena subpiligera, discovered in subtropical regions of China, could promote G. elata seed germination [32]. These studies indicate that research on the germinating fungi of G. elata seeds has not been interrupted, but few strains have been obtained, especially in Zhaotong, China, the main production area of G. elata, with scarce germinating fungi resources.

Nonetheless, Mycena is a diverse genus of fungi in China, and 76 names of Mycena have been recorded, including 6 new species and 16 new records from China [33, 34]. Recently, Na [35] used the ITS+nLSU+SSU multigene sequencing method to reconstruct the phylogenetic status of Mycena on a large scale. The two main branches of the genus are characterized by starch-like and nonstarch-like spores, divided into 7 clades, involving 56 taxonomic units of 23 groups of Mycena. Currently, it is unclear whether there is a phylogenetic relationship between Mycena species and the seed germination of G. elata. Moreover, it also remains to be determined whether different strains within the same species vary in their germination efficiency for different G. elata varieties. There are two varieties of G. elata with different geographical distributions in Zhaotong city, the most important G. elata production area in China, namely, G. elata Bl. f. glauca and G. elata Bl. f. viridis (Fig. 1). G. elata f. glauca is mainly found in Zhaoyang District, Zhenxiong County, Yiliang County and Weixin County, among which the Xiaocaoba G. elata in Yiliang County is the most famous, known as the “cloud G. elata”. G. elata f. viridis is mainly found in Qiaojia County, Yanjin County, Daguan County and Yongshan County, among which the Shibanba G. elata in Qiaojia County is the most famous, known as the “green gold”. If only some species can induce the germination of a given G. elata variety, it is thus expected that the geographic distribution of Mycena species with germination potential can determine the geographic distribution of G. elata variety and conservation.

Figure 1. Morphology of G. elata. f. viridis and G. elata. f. glauca. G. elata. f. viridis is shown on the far left, and G. elata. f. glauca is shown on the right. Fig. a depicts the morphological characteristics of the upright stem of G. elata after bolting; Fig. b illustrates the morphological characteristics of the tubers of G. elata; Fig. c demonstrates the morphological characteristics of the flowers of G. elata. G. elata. f. glauca has a plant height of 1.5-2 meters or more; a gray‒brown stem with white longitudinal stripes (A) blue‒green flowers (B) an elliptical or ovoid tuber, up to 15 centimeters or longer, with a maximum weight of 0.8 kilograms and a water content of 60-70% (C). G. elata. f. viridis has a plant height of 1-1.5 meters; a pale blue‒green stem (A) pale blue‒green or white flowers, which are rare (B) and an elliptical or inverted cone-shaped tuber, with a maximum weight of 0.6 kilograms and a water content of approximately 70% (C) [73].

In this study, we obtained numerically diverse Mycena by using an in situ fungal baiting method with different varieties of G. elata in natural habitats in Zhaotong, China, and verified the germination rate of these strains on G. elata varieties. We also used multigene methods to examine the correlation between the phylogenetic status of Mycena and the germination rate of G. elata. We aimed to answer the following questions: (1) What is the diversity of Mycena species in natural habitats in Zhaotong?(2) Is there a phylogenetic signal of Mycena for the germination of G. elata? (3) Is there a geographic distribution of Mycena and host preference of Mycena for the G. elata variety?Answering these scientific questions has important theoretical implications for understanding the evolution of Mycena-G. elata symbiosis, as well as practical guidance for isolating and obtaining novel germination-promoting Mycena resources.

Materials and Methods

In situ Fungal Baiting

In July 2021, we performed an experiment for in situ fungal baiting in three main production areas of G. elata in Zhaotong, China, including Yiliang (YL), Lianfeng (LF), and Panhe (PH). The baiting site was selected in the natural habitat with the distribution of wild G. elata populations. To attract suitable fungi for germination, we mixed local dead Fagaceae leaves with seeds of two varieties of G. elata, G. elata f. glauca and G. elata f. viridis, and then loaded them into a nylon mesh bag with dimensions of 45 cm (length) × 30 cm (width) and a grid of 1 mm. Nylon bags were buried 3 cm below the ground surface, with a minimum spacing of 1 m between each bag. At least three biological replicates for each variety of seeds were buried in each sample plot. In October 2021, we retrieved the bags buried in YL, and in December 2021, we retrieved the bags buried in LF and PH. These samples were put in a foam box and brought back to the laboratory with an ice pack to maintain a low temperature. The seeds of G. elata f. glauca and G. elata f. viridis were purchased from a G. elata farmer in Zhaotong, China. G. elata tissue samples were harvested from the nylon bags in the laboratory and stored at 4°C until utilization.

Isolation, DNA Extraction, and PCR Amplification of Endophytic Fungi

Endophytic fungi were isolated from the G. elata protocorms collected from the seed baiting bags. The protocorms were extracted and surface-sterilized by soaking in a 0.5% sodium hypochlorite solution for 30 s and then soaking in a 75% ethanol solution for 30 s, rinsed three times with sterile water, and then dried with sterile filter paper. The protocorms were cut into small pieces of approximately 2 mm using a sterile dissecting knife. We placed small pieces of protocorms on different Petri dishes: potato dextrose agar (PDA, potato 200 g/L, dextrose 20 g/L, agar 16 g/L), 1/5 nutrient potato dextrose agar (1/5 PDA, potato 40 g/L, dextrose 4 g/L, agar 16 g/L), wheat bran potato dextrose agar (wheat bran 150 g/L, potato 100 g/L, dextrose 20 g/L, agar 10 g/L), fungal isolation medium (FIM, (Ca(NO3)2·4H2O 0.5 g/L, KH2PO4 0.2 g/L, KCl 0.1 g/L, MgSO4·7H2O 0.1 g/L, yeast extract 0.1 g/L, sucrose 5 g/L, agar 10 g/L) and potato sucrose malt agar (potato 200 g/L, sucrose 20 g/L, malt 10 g/L, agar 16 g/L). We incubated the plates in the dark at 25°C for 3-5 days. Then, we transferred the fungi that grew out from the protocorm pieces to fresh PDA plates until we obtained pure strains.

We extracted genomic DNA from fungal strains using the cetyltrimethylammonium bromide (CTAB) method [36]. The sample was treated with a CTAB buffer solution containing salts and other reagents. This solution breaks down cell membranes, releasing DNA. After heating to precipitate proteins, DNA is extracted by adding alcohol, which causes it to precipitate out of the solution. The precipitated DNA is then purified to remove impurities, resulting in a relatively pure DNA solution suitable for PCR and sequencing. We first sequenced the ITS genes of all isolated strains using primers ITS4 and ITS5 and following the PCR cycling conditions described by White et al.[37]. We then amplified the nLSU and SSU regions of the Mycena DNA using primers LR0R and LR7 and primers MS1 and MS2, following the PCR cycling conditions described by Hopple and Vilgalys [38] and Ward and Gray [39], respectively. We used a 50 μl system consisting of 25 μl of 2 × PCR Master Mix (Sangon, Shanghai, China), 1 μl of 10 μl of forward primer and reverse primer each, 1 μl of template DNA and 22 μl of sterile ddH2O. The PCR products were sequenced by Sanger sequencing at Shanghai Sangon Biotech Co., Ltd. (China).

Phylogenetic Analysis

We performed OTU division and species identification for all isolated strains using ITS gene sequencing results and then used the combination of ITS, nLSU, and SSU gene fragments to perform OTU division for Mycena. We summarized and checked the orientation of the sequences with SeqMan 7.0.0 (DNAstar 5.0) and aligned, trimmed and concatenated each gene sequence with BioEdit 7.0.4.1 [40]. We assigned the trimmed sequences to OTUs at the unique level and 99% sequence similarity level using Muthur 1.35.1 software [41] and performed taxonomic classification based on the UNITE database [41]. We selected five representative strains of Mycena at the 99% similarity level in the combination of ITS, nLSU, and SSU gene fragments to construct the multigene phylogenetic tree.

All gene nucleotide sequences for these five representative strains reported in this study were deposited at GenBank under the accession numbers OR759509–OR759513 for ITS, OR754290–OR754294 for nLSU and OR763347–OR763351 for SSU (Table S1).

In this study, we obtained a total of 72 strains and one commercial Mycena J3) × 3 genes = 219 sequences by sequencing three genes (ITS, nLSU, and SSU) for 72 strains and one commercial Mycena J3. We also downloaded 70 sequences from GenBank, comprising 41 ITS, 17 nLSU, and 12 SSU sequences. The reference sequence information is presented in Table S2. We constructed a phylogenetic tree for the abovementioned 289 Mycena sequences based on the combination of ITS, nLSU, and SSU gene fragments. We also constructed a phylogenetic tree for the 17 Mycena sequences from this study based on the same combination of gene fragments. We determined the best nucleotide substitution model for each gene sequence using ModelFinder in PhyloSuite v1.2.2 [42]. We inferred the phylogeny of Mycena using Bayesian inference (BI) and maximum likelihood (ML) methods with Xeromphalina campanella (Batsch) Kühner & Maire as the outgroup. The BI analysis was performed with MrBayes version 3.2.7 (Sweden) [43]. Markov chain Monte Carlo (MCMC) chains were run for one million generations, sampling every 100th generation until the topological convergence diagnostic was less than 0.01 [44]. ML analysis was performed on MEGA 11 [45] using the GTR+G model. The bootstrap value was set to 1000 in the ML analysis, and the bootstrap values on the nodes were used to assess the reliability of the phylogenetic tree. Topology support values greater than 80% bootstrap support (ML) and 0.9 Bayesian posterior probabilities (BPP) are shown at each branch node. The resulting tree was visualized in Figtree version 1.4.4 (UK).

Germination Experiment of Mycena for G. elata Seeds

Germination test in the lab. The germination experiment was set up as shown in Fig. S1. Preparation of fungus-treated leaves: Leaves of the Fagaceae family were collected in the wild (Xishan Forest Park, Kunming, China, March, 2022), dried and cut into 2 cm × 2 cm pieces. The leaf pieces were sterilized according to soil sterilization standards (sterilized 3 times at 121°C, each time for 120 min, with a 24-h interval between each sterilization), dried and placed flat on PDA. Fresh Mycena hyphae were inoculated around the Fagaceae leaf pieces and incubated until the leaves were fully colonized by the hyphae (15 days). The leaf pieces colonized by the hyphae were placed flat on water agar medium. Then, G. elata seeds were evenly distributed on the fungus-treated leaf pieces. We used the commercially utilized strain Mycena J3 as a positive control and sprinkled the seeds directly on water agar plates (Fig. S2A), PDA plates (Fig. S2B), and sterile Fagaceae leaves and placed them on water agar plates (Fig. S2C) as a negative control. The plates were incubated at 25°C in the dark. We monitored seed germination under a dissecting microscope using the rupture of the seed coat as the germination criterion (Fig. S2D and S2E). We measured the germination rate in the fourth week after sowing, randomly selected 5 fields (Fig. S2F and S2G) under a dissecting microscope to record the number of germinated seeds and the total number of seeds, and calculated the germination rate. Germination rate (%) = (number of germinated seeds/total number of seeds) × 100%.

Germination test in the wild. We chose two Mycena strains (YL10 and YL16) with high germination rates to perform a field experiment. The site with sandy loam soils was located in YL, the main production area of G. elata in Zhaotong. The fungal bag was prepared according to the production formula of a Mycena fungus bag (57%broad-leaved tree leaves, 20% sawdust, 20.45% bran, 1% sucrose, 0.3% potassium dihydrogen phosphate, 0.25%magnesium sulfate, 1% gypsum powder, 65% water content, natural pH) and inoculated after sterilization according to the soil sterilization standard. After one and a half months, the bag was fully colonized by Mycena mycelium. The fungal materials were mixed with G. elata seeds and loaded into mesh bags with dimensions of 45 cm (length) × 30 cm (width) and a grid of 1 mm. The mesh seed bags were buried 5 cm below the soil surface, with a minimum distance of 1 m between each bag. After five months, they were retrieved, and G. elata tissues were sorted out in the laboratory. We counted the number of G. elata tissues and calculated the dry weight of each G. elata tissue in each bag. We also used commercial Mycena J3 as a positive control to verify the germination effect in the wild.

Data Analysis

We used Shapiro‒Wilk and Kolmogorov‒Smirnov tests to assess the normality of the data and performed independent sample T tests, analysis of variance (ANOVA) and post hoc comparisons if the data were normally distributed or nonparametric analysis if not. In this experiment, we used an independent sample T test to compare the germination rate between G. elata f. glauca and G. elata f. viridis for each strain, as well as the germination rate between each tested strain and the control commercial Mycena J3. We used one-way analysis of variance and post hoc multiple comparison to analyze the number of G. elata tissues and dry weight of single G. elata tissue. Among them, based on the homogeneity of data variance, Duncan’s test (homogeneous variance) and Dunnett’s T3 test (heterogeneous variance) were selected in post hoc multiple comparisons. We used nonparametric analysis to compare the germination rate by location, G. elata varieties and OTU division. SPSS 22.0 software (SPSS Inc., USA) was used for the normality test, one-way analysis of variance, post hoc multiple comparison and nonparametric Mann‒Whitney U tests. GraphPad Prism 7 software (GraphPad-software Inc., USA) was used to draw charts of fungal composition, germination rate of each strain, germination rate by location, germination rate by G. elata varieties, germination rate by OTU division, number of G. elata tissues and dry weight of single G. elata tissue.

Results

Geographic and Host Patterns of the Endophytic Fungal Community

In total, we obtained 280 strains from G. elata tissues (Table 1). Based on their ITS sequences, these strains phylogenetically belong to 208 strains of Ascomycetes and 72 strains of Basidiomycetes. The most abundant fungi at the genus level were Ilyonectria, Cadophora, and Mycena, accounting for 41.43%, 28.21%, and 24.29% of the total strains, respectively. Geographically, the fungal community is distinctively distributed (Table 1). The most abundant genus at the YL site was Mycena, accounting for 90% of the total isolates. In contrast, the most abundant genus at the LF site was Ilyonectria, with 51.49% of the total strains, followed by Cadophora, with 35.15% occurrence; similarly, the dominant genera at the PH site were also Ilyonectria and Cadophora, with 41.38% and 27.59%occurrence, respectively. Meanwhile, these two sites also harbored 11.76% and 10.71% of Mycena, respectively. Two varieties, G. elata f. glauca and G. elata f. viridis, harbored similar fungal communities, with the most prevalent being Ilyonectria, accounting for 43.11% and 34.55% of the total sequence, respectively. The second and third dominant genera were Mycena and Cadophora, which accounted for 27.11% and 27.11% of the total sequence of G. elata f. glauca, respectively, and 32.73% and 12.73% of the total sequence of G. elata f. viridis, respectively (Table 1).

Table 1 . Geographic distribution and G. elata source isolation information for all strains..

Taxonomic groupPhylumYLLFPHGVTotalRelative abundance
1AcremoniumAscomycota0010110.36%
2CadophoraAscomycota071861187928.21%
3ExophialaAscomycota0010110.36%
4IlyonectriaAscomycota010412971911641.43%
5LachnumAscomycota0101010.36%
6MollisiaAscomycota1000110.36%
7MycenaBasidiomycota452306176824.29%
8OphiosphaerellaAscomycota0100110.36%
9ParaphaeosphaeriaAscomycota0100110.36%
10Unclassified MycenaceaeBasidiomycota0134041.43%
11Unclassified SordarialesAscomycota0020220.71%
12XenopolyscytalumAscomycota4000441.43%
13Unclassified XylarialesAscomycota0011010.36%
Total502022822555280100%

YL, LF, and PH are strains isolated from Yiliang, Lianfeng and Panhe, respectively. G and V are strains isolated from G. elata f. glauca and G. elata f. viridis, respectively. The leftmost column exhibits the relative abundance of each taxonomic group..



Phylogenetic Analysis of Multiple Genes in Mycena

Based on the ITS gene sequences, we identified 72 Mycena that were classified into 5 OTUs at the 99% similarity level. Phylogenetic analysis showed that OTU1 and OTU4 were closely related to M. citrinomarginata and M. polygramma, respectively. OTU2 and OTU3 were sister taxa to M. adnexa and M. abramsii. OTU5 formed a separate branch (Fig. S3). Therefore, we further constructed a phylogenetic tree of the genus Mycena based on the combination of ITS+nLSU+SSU gene fragments (Fig. 2). According to the BI method and ML method, these strains were divided into 7 clades to form two large groups, with stable support rates for each clade, namely, the Basidiospore amyloid group (Clade 1-Clade 6) and Basidiospore nonamyloid group (Clade 7). All of our 72 Mycena strains are clustered in Clade 1. Consistent with the phylogenetic tree of ITS genes, OTU1 and OTU4 were closely related to M. citrinomarginata and M. polygramma, respectively. OTU5 formed a separate branch. However, OTU3 was closely related to M. abramsii, while OTU2 was sister to M. adnexa, M. abramsii and OTU3.

Figure 2. Mycena phylogeny from ITS+nLSU+SSU multi-gene sequences analysis (BPP ≥ 0.95, Bootstrap≥75% on nodes, – otherwise). The sequences of this study are displayed in bold red font, followed by the symbol “-” and the OTU at the 99% similarity level in ITS+nLSU+SSU genes indicating the OTU represented by this sequence. The tree is rooted with Xerphalina campanella.

The most abundant species were OTU1 (M. citrinomarginata group) and OTU2 (unclassified Mycena group 1), each accounting for 31.94% of the total Mycena strains, followed by OTU3 (M. abramsii group), with an abundance of 29.17% (Table S3). We obtained 45 Mycena from the YL site, of which OTU1 (M. citrinomarginata group) and OTU3 (M. abramsii group) had abundances of 51.11% and 46.67%, respectively; 24 Mycena from the LF site, of which OTU2 (unclassified Mycena group1) was the dominant species, with an abundance of 95.83%; and only 3 Mycena from the PH site, all of which were OTU4 (M. polygramma group) (Fig. 3A, Table S3). Regarding the two varieties of G. elata, both OTU2 (unclassified Mycena group 1) and OTU3 (M. abramsii group) were dominant, with abundances of 32.31% and 26.15% in G. elata f. glauca and 57.14% and 28.57% in G. elata f. viridis, respectively. In contrast, OTU1 (M. citrinomarginata group) occurred at 35.38% in G. elata f. glauca but at zero in G. elata f. viridis (Fig. 3B, Table S3). Therefore, the Mycena distribution depends on the geographic site and G. elata variety.

Figure 3. The OTU relative abundance of Mycena strains occurring in geographic site (A) and G. elata variety (B). YL, Yiliang; LF, Lianfeng; PH, Panhe. G, G. elata f. glauca; V, G. elata f. viridis.

Germination Promotion for G. elata Seeds

We first tested the germination rate for G. elata of 17 Mycena strains belonging to five OTUs in the laboratory (Fig. S4). With the exception of YL15 (OTU5), all strains were able to promote G. elata germination. Ten strains had significantly higher germination rates for G. elata f. glauca and eight for G. elata f. viridis than the positive control (a commercial Mycena J3). There was a certain correlation between the phylogenetic status of Mycena and the germination rate (Fig. 4A). The germination rates of OTU2, OTU3, and OTU4 were significantly higher than that of OTU1. The germination rates of OTU3 and OTU4 were not significantly different, but both were significantly higher than that of OTU2 (Fig. 4B). The germination rates of the strains were significantly different among the three geographic locations (Fig. 4C). Most strains showed higher germination for G. elata f. viridis than for G. elata f. glauca (Fig. S4); however, the average germination rate was not significantly different between G. elata f. viridis and G. elata f. glauca (Fig. 4D).

Figure 4. The effect of different Mycena on the germination rate of G. elata seeds. (A) Phylogenetic tree of the ITS+nLSU+SSU genes in this study’s sequences and their correlation with the germination rates of G. elata f. glauca and G. elata f. viridis. J3 is the control commercial Mycena. The uppercase letters and numbers in the column where J3 is located represent the Mycena strain number. (B) Statistical comparison of germination rate by OTU. (C) Statistical comparison of germination rate by sample plot (YL, Yiliang; LF, Lianfeng; PH, Panhe). (D) Statistical comparison of germination rate by G. elata variety (G, G. elata f. glauca; V, G. elata f. viridis). Different lowercase letters indicate significant differences (P<0.05), and identical lowercase or ns indicate nonsignificant differences (P > 0.05). Data are the mean ± SEM, n ≥ 3.

In the field germination experiment (Fig. S5), an average of 3511 and 2103 protocorms were harvested for the YL10 and YL16 strains, respectively, whereas only 157 protocorms were harvested for the commercial Mycena J3. Although the dry biomass of a single protocorm was not different among the three tested strains, the number of harvested protocorms of YL10 was significantly higher than that of J3 (Fig. 5).

Figure 5. The number of germinated protocorms (A) and the dry weight of a single G. elata protocorm (B) of Mycena strains YL10, YL16 and commercial Mycena J3 in field experiments. Different letters indicate significant differences (P < 0.05, Fig. a for Duncan’s test and Fig. b for Dunnett’s T3 test). Data are the mean ± SEM, n ≥ 3.

Discussion

We obtained numerically diverse Mycena in natural habitats in Zhaotong, China. Moreover, most Mycena strains showed a high capacity for G. elata germination, which is why Zhaotong is the main production area of G. elata in China. The genus Mycena comprises many species that are small and similar in appearance, posing challenges for identification and classification. Microscopic features of their basidiocarps, such as spores, lamellar cystesia, pileipellis and stipitipellis, are usually used for taxonomic purposes [5]. Because it failed to induce basidiocarps for these Mycena strains in this study, we had to deduce their phylogenetic position using molecular sequencing data and related literature, and the species were placed in the nearest known taxa. The phylogenetic tree constructed based on ITS+nLUS+SSU multigene analysis is more complete than that constructed based on single-gene analysis. The single-gene trees support the topology of the multigene tree and distinguish well between the two main clades of Mycena, namely, the Basidiospore amyloid clade and the Basidiospore nonamyloid clade, which are further divided into 7 clades (Fig. S3). However, in the ITS tree, the group of YL10 in the Fragilipedes section is not clear. In the multigene tree, Clade 1, Clade 2, and Clade 3 are sisters to Clade 4, while in the ITS tree, Clade 1, Clade 2, and Clade 3 are sisters to Clade 5 and Clade 6. The support value of Clade 1 in the ITS tree was higher than that in the multigene tree. Multigene analysis has also helped to resolve the phylogenetic relationships of some complex species within Mycena, and selecting appropriate gene fragments can better elucidate the divergence of the phylogenetic framework within Mycena. When screening suitable fragments, some species showed severe peak overlap and heterozygosity in ITS sequencing, while nLSU and SSU showed high conservation and good results. The possible new species are strains LF42 and YL15, which form a single branch in both the multigene tree and the ITS tree. Our data suggested that this area is valuable for exploring novel Mycena that can induce the germination of G. elata seeds.

From a geographical perspective, the compositions of both the total fungal communities and the Mycena community were different among the three plots (YL, LF, and PH) (Fig. 2A and 3A). Similarly, some studies have shown that orchids have different mycorrhizal fungal communities in different habitats, and environmental conditions strongly affect their mycorrhizal fungal communities [46, 47]. For example, Neottia ovata has different mycorrhizal fungal compositions in grasslands and forests [48]. The same orchid species has different mycorrhizal fungal communities in different habitats [49-52]. A cross-continental scale comparison of the mycorrhizal fungal communities associated with Gymnadenia conopsea and Epipactis helleborine revealed significant shifts in the fungal community composition of both orchids between China and Europe, and the similarity of their mycorrhizal fungal communities decreased significantly with increasing geographic distance [53].

On the other hand, the germination rates of Mycena isolated from the PH, YL, and LF sites showed significant differences, which reflect the unique distribution pattern of certain Mycena species with variation in germination. All Mycena isolates from the G. elata protocorms belong to Clade 1, indicating that Mycena species in Clade 1 have a strong ability to germinate G. elata seeds, while other clades may lack or have a weaker ability. A study also suggested that seed germination of G. elata depends on a narrow group of Mycenaceae fungi [54]. All Mycena isolated from PH were OTU4 (M. polygramma group), and the LF plot contained 95.83% abundance of OTU2 (unclassified Mycena group 1). These Mycena strains have not been reported previously to promote the germination of G. elata seeds, indicating that the PH and LF sites have high-quality Mycena resources for G. elata germination. The YL plot may have a lower germination rate due to the presence of OTU1 (M. citrinomarginata group) with an abundance of 51.11%. OTU1, OTU3, and OTU4 were assigned to the M. citrinomarginata group, M. abramsii group, and M. polygram group, respectively. The germination rates of OTU2, OTU3, and OTU4 were significantly higher than that of OTU1. This result suggests that there is a correlation between the phylogenetic position of Mycena and germination rate. Similarly, a study using in vitro symbiosis between six Mycena fungi and G. elata seeds showed that the seed germination rates of KFRI1212 and KFRI2121 were 60.1% and 47.0%, respectively, while the germination rates of the other four Mycena species were below 3.5% [54]. Molecular identification and phylogenetic analysis indicated that these two fungi belong to the same branch. Five OrM fungi isolated from Anacamptis papilionacea were selected to test the efficiency of seed germination. Among them, four belonged to the Tulasnella calospora species complex and one belonged to Ceratobasidium. The results showed that the T. calospora species complex had a higher germination rate [55]. Overall, the Mycena resources in the Zhaotong area are rich, and many Mycena species have a significantly higher germination rate than the control commercial Mycena J3. In this study, two strains (YL10 and YL16) had more effective germination to produce protocorms than the control commercial Mycena J3 in a field experiment, suggesting that these two strains have some market application potential.

The symbiotic relationship between orchids and mycorrhizal fungi changes as the plant develops. In Cephalanthera damasonium and Cephalanthera longifolia, Piriformospora indica and Sebacina vermifera are the main fungal partners during the seed germination and seedling development stages, whereas T. calospora and Ceratobasidium sp. dominate in the adult stage [56]. Cyrtosia septentrionalis shows a similar pattern, with Physisporinus inducing the seed germination stage and Armillaria associating with the adult stage [57]. The fungal community associated with G. elata also changes with the developmental stages [58]. In this study, most of the G. elata tissues from YL were at the protocorm stage of new germination (Fig. S6A), while most of the G. elata tissues from LF and PH were at the protocorm stage of elongation (Fig. S6B). The newly germinated protocorms (Fig. S6A) had low fungal diversity and were predominantly colonized by the basidiomycete Mycena (90% abundance), supporting the hypothesis that G. elata requires Mycena symbionts for germination [26]. However, the elongated protocorms (Fig. S6B) exhibited higher fungal diversity and a shift in fungal dominance to the Ascomycetes Ilyonectria and Cadophora, with Mycena abundance decreasing to 11.88% and 10.71%, respectively.

Orchid mycorrhizal fungi are predominantly Basidiomycetes [59, 60], but Shefferson et al. [61] also detected Ascomycetes in Phialophora. The mycorrhizal fungi that facilitate seed germination and protocorm formation may not sustain the growth of seedlings [61]. Orchids may switch to other fungi that offer greater advantages in resource acquisition. For instance, the fully mycoheterotrophic G. elata changed its fungal symbiont from Mycena to Armillaria, which may allow it to access a larger carbon pool [62]. Instead of switching to the colonization of Armillaria, we found that G. elata increases the abundance of Ascomycetes during the elongation protocorm stage. One possible reason is to sacrifice functional superiority for survival [63, 64]. Under ecological constraints, plants must associate with any accessible fungal species, regardless of whether they are the optimal partners or not [65]. In the absence of Armillaria in the vicinity, we assumed that G. elata may replace its fungal symbionts after germination to reduce the risk of death by symbioting with some Ascomycetes (Table 1). Therefore, it is interesting to explore the role of these non-Mycena fungi in symbiosis with G. elata.

The interaction between orchids and mycorrhizal fungi is also influenced by the genetic background of the orchid host. Different orchid genera or species may symbiose with different fungal groups. For example, the dominant symbiotic fungal group of plants in the genus Goodyera is Ceratobasidium [66], while the dominant symbiotic fungal group of plants in the genus Arundina is Tulasnella [67]. The host orchid’s genetic background, which includes its phylogeny, ploidy level and genome composition, may influence the composition of its mycorrhizal fungal community [68-72]. Strains isolated from G. elata f. glauca and G. elata f. viridis were different in abundance (Table 1) and Mycena composition (Fig. 3B, Table S3). Moreover, the average germination rate for G. elata f. viridis was higher than that for G. elata f. glauca (Fig. 4D). These facts reflect that fungi may have different affinities for the genetic characteristics of the G. elata variety.

Conclusion

We used an in situ baiting method to characterize the symbiotic fungal community associated with different varieties of G. elata in natural habitats. Moreover, we also explored the correlation between the phylogenetic status of the symbiotic fungus Mycena and the G. elata germination rate. We found that (1) there are diverse Mycena in natural habitats in Zhaotong, China. Multigene analysis revealed that they phylogenetically belong to the Basidiospore amyloid group (Clade 1). (2) There is a phylogenetic signal of Mycena for germination of G. elata. Those strains phylogenetically close to M. abramsii (OTU3), M. polygramma (OTU4), and an unclassified Mycena (OTU2) had significantly higher germination rates than those to M. citrinomarginata (OTU1). (3) The Mycena distribution depends on geographic site and G. elata variety. The YL site harbors OTU1 (M. citrinomarginata group) and OTU3 (M. abramsii group); the LF site harbors mainly OTU2 (unclassified Mycena group 1); and the PH site harbors all strains of OTU4 (M. polygramma group). Regarding the two varieties of G. elata, both OTU2 (unclassified Mycena group 1) and OTU3 (M. abramsii group) were dominant; in contrast, OTU1 (M. citrinomarginata group) occurred dominantly in G. elata f. glauca but was absent in G. elata f. viridis. Our results indicate that the community composition of numerous Mycena resources in the Zhaotong area varies by geographical location and G. elata variety. Importantly, our results also indicate that Mycena’s phylogenetic status is correlated with its germination rate, which is important for understanding the geographic distribution and coevolution of Mycena-G. elata symbiosis in nature. Moreover, it is also valuable for practical guidance for isolating and obtaining novel germination-promoting Mycena resources.

Supplemental Materials

Acknowledgments

This work was supported by Open Research Program of State Key Laboratory for Conservation and Utilization of Bio-Resource in Yunnan (2022KF002), National Natural Science Foundation of China (31960288), Special Funds for Central Guidance of Local Scientific and Technological Development (202307AB110011) and Innovative Research Foundation for Graduate Students of Yunnan University (KC23235551). Special thanks to Cheng-Shuai Zou (a G. elata farmer in Zhaotong, China) for providing G. elata seeds for this experiment.

Author Contributions

Conceptualization: HBZ, XHJ, and YCW; methodology: XHJ, YCW, YL and HYH; investigation: XHJ, YCW, DL, YL and HYH; formal analysis: XHJ; validation: XHJ and DL; writing—original draft preparation: XHJ; writing—review and editing: HBZ; visualization: XHJ; supervision: HBZ; funding acquisition: HBZ, and YCW; project administration: HBZ and YCW; resources: HBZ; supervision: HBZ; data curation: XHJ.

Conflict of Interest

The authors have no financial conflicts of interest to declare.

Data Availability

The data underlying this article are available in the GenBank Nucleotide Database at https://www.ncbi.nlm.nih.gov, and can be accessed with accession number OR759509–OR759513 for ITS, OR754290–OR754294 for nLSU and OR763347–OR763351 for SSU.

Material Availability

The strain YL10 was deposited at the China General Microbiological Culture Collection Center (CGMCC), with the registration number CGMCC No. 40281.

Fig 1.

Figure 1.Morphology of G. elata. f. viridis and G. elata. f. glauca. G. elata. f. viridis is shown on the far left, and G. elata. f. glauca is shown on the right. Fig. a depicts the morphological characteristics of the upright stem of G. elata after bolting; Fig. b illustrates the morphological characteristics of the tubers of G. elata; Fig. c demonstrates the morphological characteristics of the flowers of G. elata. G. elata. f. glauca has a plant height of 1.5-2 meters or more; a gray‒brown stem with white longitudinal stripes (A) blue‒green flowers (B) an elliptical or ovoid tuber, up to 15 centimeters or longer, with a maximum weight of 0.8 kilograms and a water content of 60-70% (C). G. elata. f. viridis has a plant height of 1-1.5 meters; a pale blue‒green stem (A) pale blue‒green or white flowers, which are rare (B) and an elliptical or inverted cone-shaped tuber, with a maximum weight of 0.6 kilograms and a water content of approximately 70% (C) [73].
Journal of Microbiology and Biotechnology 2024; 34: 1249-1259https://doi.org/10.4014/jmb.2401.01009

Fig 2.

Figure 2.Mycena phylogeny from ITS+nLSU+SSU multi-gene sequences analysis (BPP ≥ 0.95, Bootstrap≥75% on nodes, – otherwise). The sequences of this study are displayed in bold red font, followed by the symbol “-” and the OTU at the 99% similarity level in ITS+nLSU+SSU genes indicating the OTU represented by this sequence. The tree is rooted with Xerphalina campanella.
Journal of Microbiology and Biotechnology 2024; 34: 1249-1259https://doi.org/10.4014/jmb.2401.01009

Fig 3.

Figure 3.The OTU relative abundance of Mycena strains occurring in geographic site (A) and G. elata variety (B). YL, Yiliang; LF, Lianfeng; PH, Panhe. G, G. elata f. glauca; V, G. elata f. viridis.
Journal of Microbiology and Biotechnology 2024; 34: 1249-1259https://doi.org/10.4014/jmb.2401.01009

Fig 4.

Figure 4.The effect of different Mycena on the germination rate of G. elata seeds. (A) Phylogenetic tree of the ITS+nLSU+SSU genes in this study’s sequences and their correlation with the germination rates of G. elata f. glauca and G. elata f. viridis. J3 is the control commercial Mycena. The uppercase letters and numbers in the column where J3 is located represent the Mycena strain number. (B) Statistical comparison of germination rate by OTU. (C) Statistical comparison of germination rate by sample plot (YL, Yiliang; LF, Lianfeng; PH, Panhe). (D) Statistical comparison of germination rate by G. elata variety (G, G. elata f. glauca; V, G. elata f. viridis). Different lowercase letters indicate significant differences (P<0.05), and identical lowercase or ns indicate nonsignificant differences (P > 0.05). Data are the mean ± SEM, n ≥ 3.
Journal of Microbiology and Biotechnology 2024; 34: 1249-1259https://doi.org/10.4014/jmb.2401.01009

Fig 5.

Figure 5.The number of germinated protocorms (A) and the dry weight of a single G. elata protocorm (B) of Mycena strains YL10, YL16 and commercial Mycena J3 in field experiments. Different letters indicate significant differences (P < 0.05, Fig. a for Duncan’s test and Fig. b for Dunnett’s T3 test). Data are the mean ± SEM, n ≥ 3.
Journal of Microbiology and Biotechnology 2024; 34: 1249-1259https://doi.org/10.4014/jmb.2401.01009

Table 1 . Geographic distribution and G. elata source isolation information for all strains..

Taxonomic groupPhylumYLLFPHGVTotalRelative abundance
1AcremoniumAscomycota0010110.36%
2CadophoraAscomycota071861187928.21%
3ExophialaAscomycota0010110.36%
4IlyonectriaAscomycota010412971911641.43%
5LachnumAscomycota0101010.36%
6MollisiaAscomycota1000110.36%
7MycenaBasidiomycota452306176824.29%
8OphiosphaerellaAscomycota0100110.36%
9ParaphaeosphaeriaAscomycota0100110.36%
10Unclassified MycenaceaeBasidiomycota0134041.43%
11Unclassified SordarialesAscomycota0020220.71%
12XenopolyscytalumAscomycota4000441.43%
13Unclassified XylarialesAscomycota0011010.36%
Total502022822555280100%

YL, LF, and PH are strains isolated from Yiliang, Lianfeng and Panhe, respectively. G and V are strains isolated from G. elata f. glauca and G. elata f. viridis, respectively. The leftmost column exhibits the relative abundance of each taxonomic group..


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