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Preliminary exploration on the ectomycorrhizal status of a wild edible Gomphus species from Southwest China
2023 Volume 64 Issue 2 Pages 83-95
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2023 Volume 64 Issue 2 Pages 83-95
A wild edible Gomphus species was discovered at local wild mushroom markets from May to November in Southwest China, where it was eaten for hundreds of years. However, litter information on the taxonomy is available. Whether Gomphus is a saprotrophic, parasitic, or ectomycorrhizal (ECM) fungus is unclear. In the present study, field investigation, fungi isolation, optimum medium, morphological description, molecular analyses, and preliminary exploration on mycorrhizal synthesis were carried out. The morphological and molecular analyses showed that the same species between Gomphus matijun and Gomphus sp. (zituoluo) might be the related species of Gomphus purpuraceus. Moreover, the root dry weight and first-lateral root number of inoculated seedlings were significantly enhanced by evaluating Pinus massoniana seedlings inoculated with G. matijun. Meanwhile, the levels of nine phytohormones, including five new phytohormones, in the roots of inoculated seedlings were upregulated. This study explored the mycorrhizal synthesis of the wild edible Gomphus species from Southwest China with P. massoniana Lamb. We concluded that G. matijun might be an ECM fungus. The mycorrhizal synthesis of G. matijun under pure culture conditions provided the basis for the next inoculation under controlled soil conditions, making the conservation and cultivation of G. matijun feasible in the future.
The genus Gomphus (Gomphaceae, Gomphales, Basidiomycota) is an ancient group of wild macrofungi. To date, the genus contains 66 records in the Index Fungorum (http://www.indexfungorum.org/Names/Names.asp). However, identifying the taxa of Gomphus is very difficult due to the few reliable morphological characteristics and molecular data available for classification (Giachini & Castellano, 2011; Giachini, Camelini, Rossi, Soares, & Trappe, 2012). The genus Gomphus, which usually grows in old-growth forests, is considered an endangered group by many taxonomists. Gomphus bonarii (Morse) Singer, G. clavatus (Pers.) Gray, G. floccosus (Schwein.) Earle, and G. kauffmanii (A.H. Sm.) Corner, have been listed as threatened in the 1994 Northwest Forest Plan (Giachini & Castellano, 2011). In recent decades, some species of this genus, such as G. floccosus, have been found to have edible or medicinal properties (Arora, 1986; Montoya, Estrada, Kong, & Juárez, 2001).
Many reports suggested that a few species of Gomphus were the ectomycorrhizal fungi, which need to establish ectomycorrhizal (ECM) associations with various plant species (Agerer, Beenken, & Christian, 1998; Trappe, 1960). However, the information on associated tree species and ECM morphological characteristics of this genus is still lacking in the literature. Lamus et al. (2015) successfully synthesized a mycorrhizal system between G. floccosus and Abies religiosa (Kunth) Schltdl & Cham under controlled conditions; the mycorrhizal characteristics were also described in detail. For other ectomycorrhizal fungi, the mycorrhiza of Laccaria, Lactarius, Boletus, and Tuber genera were successfully synthesized under controlled conditions (Flores, Díaz, & Honrubia, 2008; Wang et al., 2019a; Águeda et al., 2008; Wang, Guerin-Laguette, Butler, Huang, & Yu, 2019b). However, developing cheaper and more practical mycorrhizal synthesis techniques has long been the target for researchers. In the United States and Europe, the Tuber and Lactarius genus orchards have been established and corresponding fruiting bodies have been produced successfully (Meadows, Gaskill, Stefanile, Sharpe, & Davis, 2020; Bach et al., 2021; Guerin-Laguette et al., 2014). Ectomycorrhiza usually has a mantle, Hartig net (HN), and emanating hyphae (Peterson & Massicotte, 2004). Besides, host shifts are also an important characteristic of ectomycorrhizal fungi (Looney, Ryberg, Hampe, Sanchez-garcía, & Matheny, 2016; Sato, Tanabe, & Toju, 2017).
Gomphus matijun J.W. Liu & F.Q. Yu is a popular edible fungus at the local wild mushroom markets in Guizhou province, China. Depending on when it is on the market, the price of G. matijun can range from US $14-36 kg-1. Unlike other species in the Gomphus genus (Arora, 1986; Cantrell et al., 2008), G. matijun is edible and cannot cause diarrhea as reported by the local eaters. The fruiting season of G. matijun usually comes under climatic conditions with frequent rain and higher air humidity from May to November. Based on the preliminary morphological identification, G. matijun and Gomphus sp. (zituoluo) may be the same species. Despite the lack of reliable molecular evidence, Gomphus sp. (zituoluo) was first discovered and identified as Gomphus purpuraceus (Iwade) K. Yokoy in the Three Gorges area of China, which was recorded in the colored illustrations of mushrooms of Japan (Tan & Liu, 2002; Imazeki & Hongo, 1987). According to Tan’s preliminary ecological survey of Gomphus sp. (zituoluo) (Tan, Liu, Yu, Lu, & Tan, 2002), Gomphus sp. (zituoluo) usually grows in stony yellow-brown soil under Quercus, Cotinus, and Pinus trees, either fruiting alone, scattered, or gregariously (Aguilar & Villegas, 2010). Besides, Ando, Nagasawa, and Maekawa (2013) described at the meeting of the Mycological Society of Japan that G. purpuraceus was a purple fruiting body with attached spores as verrucose ornamentation on the ground in a Pinus forest. Although studies on putative host trees are available, studies on the mycorrhizal structures of G. matijun in field investigations and in vitro mycorrhizal synthesis trials are lacking. Meanwhile, the molecular evidence of G. matijun is still needed to verify the hypothesis about the evidence of the same species between G. matijun and Gomphus sp. (zituoluo).
This study aimed to provide the molecular evidence of G. matijun describe the morphological characteristics, and investigate the ECM status of G. matijun. The medium formulations for strain isolation and starter propagation were also optimized. The future plan is to use the mycorrhizal synthetic method under controlled conditions. If successful, the morpho-anatomical characteristics of mycorrhiza will be described and the synthetic technologies of large-scale mycorrhizal seedlings will also be explored for the next commercialization process.
The fruiting body of G. matijun was collected in Jul 2020 from a mingled forest with scattered Pinus massoniana Lamb at the Xiazai village of Longli county and Zhaoti village of Anlong county, Guizhou province. The dried specimen was deposited in the herbarium of the Guizhou Academy of Agriculture Sciences (GZAAS). The accession number of samples collected from Xiazhai village of Longli county and Zhaoti village of Anlong county was GZAAS22-0001 and GZAAS22-0002, respectively. The dried fruiting body and mycelium of Gomphus sp. (zituoluo) were obtained from Tan and Liu (2013) of Hubei Three Gorges Polytechnic with accession numbers GZAAS22-0003 and GZAAS22-0004, respectively.
The ectomycorrhiza of G. matijun was also collected from the mingled forest about 10 cm below the fruiting body. The soil cores were approximately 10 × 10 cm2 according to Wang, Yu, Moreno, and Colinas (2020). Fine roots were separated under the Motic ES-18TZLED stereomicroscope (Motic China Group CO., LTD.) for photographs, and the morphotypes were characterized macroscopically.
2.2. Fungal isolation and optimum mediumThe isolates of G. matijun used in this study were obtained from the internal tissue fragments of the fruiting body in Xiazhai village of Longli county with a slightly modified Melin-Norkrans (MMN) medium (2.5 g/L glucose, 0.2 g/L NH4Cl, 0.5 g/L KH2PO4, 0.15 g/L MgSO4 · 7H2O, 2.5 mL of 1% NaCl, 5.0 mL of 1% CaCl2, 1.5 mL of 1% FeCl3, 1 mg/L thiamine-HCl, 15.0 g/L agar powder; pH 6.5 adjusted with 1% NaOH solution) in the dark at 23.5 °C (Shah et al., 2016). Based on early propagation, the optimization of the medium containing a fresh potato extract was only a preliminary exploration of common carbon and nitrogen sources. The composition of the medium is shown in Table 1. The isolates of G. matijun were deposited in the Guizhou Culture Collection with the accession number GZCC22-0002 (GAF-M- 20071601).
| Compounds | A | B | C | D | E | F |
| Glucose (g) | ― | 0.5 | 5.0 | ― | 0.5 | 5.0 |
| Peptone (g) | 2.0 | 2.0 | 2.0 | ― | ― | ― |
| NH4Cl (g) | 0.2 | 0.2 | 0.2 g | 0.2 | 0.2 | 0.2 |
| KH2PO4 (g) | 0.5 | 0.5 | 0.5 | 0.5 | 0.5 | 0.5 |
| MgSO4·7H2O (g) | 0.15 | 0.15 | 0.15 | 0.15 | 0.15 | 0.15 |
| 1% NaCl (ml) | 2.5 | 2.5 | 2.5 | 2.5 | 2.5 | 2.5 |
| 1% CaCl2 (ml) | 5.0 | 5.0 | 5.0 | 5.0 | 5.0 | 5.0 |
| 1% FeCl3 (ml) | 1.5 | 1.5 | 1.5 | 1.5 | 1.5 | 1.5 |
Note: Compounds and micronutrients are given for 1 L of solution. pH about all nutrient solutions was adjusted to 6.5 ± 0.05 with NaOH (1 M).
Two test methods were performed using an optimum medium. (1) Mycelium on a solid substrate, including 20 g/L agar powder, was tested. A 6-mm hole punch was used to pick out the culture for inoculation. Each medium formula had 10 plates. After 2 mo of culture at 23.5 °C, the colony diameter was measured by the cross method. (2) Mycelial slurries were obtained by liquid shock fermentation. The mycelial suspension inoculum was obtained with a mixture of mycelium in Petri dishes and sterile water using a BPD-10A homogenizer (BKMAN Biotechnology, Changde, China). Different formulations of the liquid medium were inoculated with the mycelial suspension (2% v/v) prepared using the homogenizer. The fermentation conditions were 23.5 °C, pH 6.5, and 165 rpm stirring. The mycelia were washed with sterile distilled water and filtered through a 0.5-μm membrane using a suction filter. After filtration, the mycelia were dried and weighed at 105 °C.
2.3. DNA extraction, polymerase chain reaction amplification, and sequencingThe roots were carefully separated from the soil scores of G. matijun, rinsed with sterile water, and used for DNA extraction subsequently. The ITS1-5.8S-ITS2 cluster was used to identify G. matijun existing in the ECM using ITS1: 5’-TCCGTAGGTGAACCTGCGG-3’ and ITS4: 5’-TCCTCCGCTTATTGATATGC-3’ (White, Bruns, Lee, & Taylor, 1990). The ITS2 primer pair (forward primer: 5’-ATGCGATACTTGGTGTGAAT-3’ and reverse primer: 5’-GACGCTTCTCCAGACTACAAT-3’) was used to identify the trees associated with G. matijun (Chen et al., 2010). The 30-μL polymerase chain reaction (PCR) system included 15 μL of 2× PCR Master Mix (BBI Co., Ltd., Shanghai, China), 1 μL of 1/10 dilution DNA extracts, 1 μL of each primer (10 μM), and 12 μL of ddH2O. The cycling conditions of PCR amplification were 95 °C for 5 min for the initial denaturation, 35 cycles of 94 °C for 30 s, 58 °C for 30 s, and 72 °C for 45 s, and a final elongation step of 5 min at 72 °C. The PCR amplification products were purified and sequenced in forward and reverse directions by Huayu Sequence Service (Wuhan, China).
2.4. Phylogenetic analysesBefore phylogenetic analyses, the sequences were converted into a FASTA format. Then sequences were identified by searching databases using the online Basic Local Alignment Search Tool (Nucleotide BLAST) system at the website of the National Center for Biotechnology Information (https://blast.ncbi.nlm.nih.gov/Blast.cgi). The sequences of matching species, closely related species, and representative species were retrieved in BLAST searches in GenBank (http://www.ncbi.nlm.nih.gov/genbank). A total of 39 ITS sequences from 37 Gomphus species (8 generated in this study and 29 retrieved from GeneBank formed the dataset, while two Cantharellus species as out-group taxa) were included in the phylogenetic analysis (Table 2).
| Species name | Voucher | Origin | GenBank no. of ITS | References |
| Gomphus matijun J.W. Liu & F.Q. Yu | GZCC22-0002 | Guizhou, China | ON000132.1 | This study |
| G. matijun J.W. Liu & F.Q. Yu | GZCC22-0003 | Guizhou, China | ON000125.1 | This study |
| G. matijun J.W. Liu & F.Q. Yu | GZAAS22-0001 | Guizhou, China | ON000126.1 | This study |
| G. matijun J.W. Liu & F.Q. Yu | GZAAS22-0002 | Guizhou, China | ON000127.1 | This study |
| G. matijun J.W. Liu & F.Q. Yu | Natural ECM from LL_PM1 | Guizhou, China | ON000128.1 | This study |
| G. matijun J.W. Liu & F.Q. Yu | Natural ECM from AL_PM1 | Guizhou, China | ON000129.1 | This study |
| Gomphus sp. (zituoluo) | GZAAS22-0004 | Hubei, China | ON000130.1 | This study |
| Gomphus sp. (zituoluo) | GZAAS22-0003 | Hubei, China | ON000131.1 | This study |
| Gomphus sp. J. W. Liu | HKAS122604 | Guizhou, China | OL673002.1 | Unpublished |
| Gomphus sp. J. W. Liu | HKAS122605 | Guizhou, China | OL673001.1 | Unpublished |
| Gomphus sp. R. Garibay-Orijel., et al. | RGO-2016 voucher GO-2009-199 | Mexico | KT874994.1 | Unpublished |
| Gomphus sp. R. Garibay-Orijel., et al. | RGO-2016 voucher HC-PNNT-087 | Mexico | KT874995.1 | Unpublished |
| Gomphus ludovicianus R. H. Petersen., et al. | TFB14476 clone c3 | Louisiana, USA | KJ655565.1 | Petersen et al. (2014) |
| G. ludovicianus R. H. Petersen., et al. | DPL11087 | Louisiana, USA | KJ655572.1 | Petersen et al. (2014) |
| Gomphus clavatus J. Kang., et al. | LL-115 | Gansu, China | KX008988.1 | Kang et al. (2016) |
| G. clavatus S. M. Dunham., et al. | EM root tip | Oregon, USA | DQ365637.1 | Dunham et al. (2007) |
| Ramaria aff. Atkinsonii R. H. Petersen., et al. | TFB14475 clone c1 | Louisiana, USA | KJ655555.1 | Unpublished |
| R. aff. Atkinsonii R. H. Petersen., et al. | TFB14475 clone c2 | Louisiana, USA | KJ655556.1 | Unpublished |
| Ramaria obtusissima R. H. Petersen., et al. | TFB14473 | Louisiana, USA | KJ655554.1 | Unpublished |
| Gomphus bonarii M. Berbee., et al. | UBC F-32080 | BC, Canada | MF955206.1 | Unpublished |
| Turbinellus floccosus P. Khaund & S. R.Joshi | PKSR1 | India | KJ411951.1 | Khaund & Joshi (2014) |
| Gomphus flavipes H. J. Dumont., et al. | voucher 10979 | Germany | MW580291.1 | Unpublished |
| G. flavipes H. J. Dumont., et al. | voucher 10978 | Germany | MW580290.1 | Unpublished |
| Gomphus pulchellus H. J. Dumont., et al. | voucher 11851 | Belgium | MW580289.1 | Unpublished |
| G. pulchellus H. J. Dumont., et al. | voucher 11826 | Germany | MW580288.1 | Unpublished |
| Gomphus graslinii H. J. Dumont., et al. | voucher 11111 | Portugal | MW580287.1 | Unpublished |
| Gomphus davidi H. J. Dumont., et al. | voucher 11048 | Israel | MW580286.1 | Unpublished |
| Gomphus kinzelbachi H. J. Dumont., et al. | voucher 11049 | Iran | MW580285.1 | Unpublished |
| G. kinzelbachi H. J. Dumont., et al. | voucher 10994 | Iran | MW580284.1 | Unpublished |
| Gomphus lucasii H. J. Dumont., et al. | voucher 15327F | Algeria | MW580282.1 | Unpublished |
| G. lucasii H. J. Dumont., et al. | voucher 15327 | Algeria | MW580281.1 | Unpublished |
| Gomphus simillimus H. J. Dumont., et al. | voucher 10970 | France | MW580280.1 | Unpublished |
| G. simillimus H. J. Dumont., et al. | voucher 11990 | France | MW580279.1 | Unpublished |
| Gomphus schneiderii H. J. Dumont., et al. | voucher 11525 | Iran | MW580276.1 | Unpublished |
| G. schneiderii H. J. Dumont., et al. | voucher 11481 | Azerbaijan | MW580275.1 | Unpublished |
| Gomphus vulgatissimus H. J. Dumont., et al. | voucher 11526 | Russia | MW580272.1 | Unpublished |
| G. vulgatissimus H. J. Dumont., et al. | voucher 11730 | Russia | MW580271.1 | Unpublished |
| Cantharellus cibarius X. Sun | voucher LL-01 | China | MT566420.1 | Unpublished |
| Cantharellus sebosus V. Hofstetter | BB 08.234 | Madagascar | KF981370.1 | Unpublished |
The nucleotide sequences were aligned using multiple sequence alignment software ClustalX v.1.83 (Sharma, Chadha, Kaur, Ghatora, & Saini, 2008). The sequences were manually adjusted using Bioedit 7.2.5. A phylogenetic tree was constructed using the construct neighbor-joining (NJ) method with MEGA 7.0 (Kumar, Stecher, & Tamura, 2016). NJ analysis was executed applying the rapid bootstrap algorithm with 1000 replicates, and the default values were kept for other parameters.
2.5. Plant materialsThe well-sequenced ITS and ITS2 sequences of roots from the soil cores are shown in Supplementary Table S1. According to the molecular identification of roots using National Center for Biotechnology Information BLAST pairwise comparison, the most likely host tree is P. massoniana Lamb. Hence, the seeds of P. massoniana Lamb from the Huangping forest farm (Huangping, Guizhou, China) were selected for mycorrhizal synthesis. The seeds were rinsed in running water for 12 h, surface-sterilized in 75% ethanol solution for 45 s, and then immersed in 0.1% mercuric chloride for 5 min. Finally, the sterile seeds were rinsed three to five times with sterile distilled water. All seeds were aseptically transferred to tissue-culture containers containing water agar medium and germinated at 25 °C for 15 d under a photoperiod (light/dark) of 16/8 h.
2.6. Mycorrhizal synthesis proceduresTwo inoculation methods were adopted for aseptic mycorrhizal synthesis: the seedlings of P. massoniana were cultivated in square Petri dishes (13 × 13 cm2) with the aforementioned modified Melin-Norkrans (MMN) medium excluding thiamine-HCl and liquid triangular flasks (50 mL) with MMN medium excluding agar powder and thiamine-HCl, which were inoculated with solid inoculum in this study. For solid inoculum preparation, the mycelia were allowed to multiply in the optimum solid medium at 23.5 °C for 45 d. Each square Petri dish and each triangular flask were inoculated with one and three disks (about 1 cm diam), respectively. Further, the sterile seedlings were placed in square dishes and triangular bottles, sealed with parafilm and high-temperature-resistant tissue-sealing film with vaseline, respectively. Meanwhile, 10 uninoculated seedlings in each group were arranged as the control check. Next, both treatments were transferred to a culture chamber at a temperature of 25 °C and under a photoperiod light/dark of 16/8 h (Gomes et al., 2016).
All seedlings and root systems were examined and observed 3 mo after shoot transfer. We found that the depth of seedling root penetration into the solid medium greatly affected its morphological characteristics, leading to inaccurate assessments of mycorrhizal development. Therefore, the assessment was only for the seedlings and root systems in liquid triangular flasks. The assessment criteria referred to in the study by Díaz, Carrillo, and Honrubia (2009): (1) plant height and taproot length (cm), (2) root-collar diameter (diameter was measured with vernier calipers), (3) plant biomass (10 seedlings were randomly selected from each treatment, and shoots and roots were separated completely, cleanly dried at 60 °C for 48 h, and weighed. In order to avoid the influence of the biomass of mycelium, the roots of colonized seedlings were removed excess attached hyphae as much as possible), and (4) lateral root evaluation (average lengths and number of first lateral roots above 0.5 cm were calculated and measured in each group including 10 seedlings).
2.7. Morphological examination and identificationThe appearance characteristics of the fruiting body and mycorrhizae were examined using a Motic ES-18TZLED stereomicroscope (Motic China Group CO., LTD.) The ultrastructure of hyphae and spores was observed under a desktop scanning electron microscope (SEM, EM30-Plus, Coxem, Korea). For SEM preparation, the surface mycelia in the Petri dishes and the part of gills attached to spores were dried in a desiccator with a discoloration silica gel at room temperature to remove the remaining water for 24 h. The dried samples were fixed on an aluminum SEM stub with a conductive adhesive tape and gold-plated for about 2 min using the ion sputtering apparatus (ETD-900M, VPI, China).
For ectomycorrhizal morphological analysis, the representative mycorrhizal branches were put in a fixed solution (2.5% glutaraldehyde and 1.6% paraformaldehyde in buffered saline of 0.05 M phosphate, pH 6.8, 1:1, v/v) for 24 h at 4 °C. The samples were dehydrated in a series of increasing ethanol concentrations (50%, 70%, 80%, 90%, 95%, and 100 %, v/v), for 30 min in each concentration at 4°C. The dehydrated samples were transferred to the solutions with 95% ethanol and base liquid Technovit 7100 (1:1, v/v) for pre-infiltration for 12 h. Then, the samples were infiltrated in the preparation solution (1 g hardener Ⅰ was dissolved in 100 mL of base liquid) for 12 h. Finally, the infiltrated samples were quickly embedded in the disposable embedding box cure-injected with hardener Ⅱ and preparation solution (1:13, v/v) for polymerization at room temperature (23 °C) for 2 h. The embedded specimens were sectioned on a rotary microtome (YD-335, Yidi, China) in increments of 1 µm and mounted with water for light microscopic observation (DS-Ri2, Nikon, China).
2.8. Phytohormone analysisThe roots collected from uninoculated and inoculated seedlings were divided into three parts as parallel repetitions. Six samples were ground into powder under liquid nitrogen. About 50 mg ground samples with 10 μL of 100 ng/mL internal standard mixed solution and 1 mL of methanol/water/formic acid (15:4:1, v/v/v) extract were mixed for 10 min. Then, the solution was centrifuged at 13,000 g for 10 min using a refrigerated centrifuge at 4 °C, and the supernatant was collected and concentrated. The concentrated solution was redissolved in 100 μL of 80% methanol/water solution and filtered with a 0.22-μm filter membrane. The filtrate was used for liquid chromatograph-mass spectrometer analysis.
The data acquisition instrument system mainly included ultra-performance liquid chromatography (UPLC, ExionLCTM AD) and tandem mass spectrometry (MS/MS, QTRAP 6500+). The chromatographic conditions were as follows. UPLC was performed under isocratic elution conditions on the Acquity HSS T3 C18 column (100 mm × 2.1 mm i.d., 1.8 μm particle size). The mobile phase, consisting of ultrapure water with 0.04% glacial acetic acid (solvent A) and acetonitrile with 0.04% glacial acetic acid (solvent B), was pumped at a flow rate of 0.35 mL/min. The gradient program started at 5% B (0-1 min), increased to 95% B (1-8 min) and 95% B (8-9 min), and finally ramped back to 5% B (9.1-12 min). Further, 2 μL was injected into the column at a column temperature of 40 °C (Cai et al., 2014; Niu, Zong, Qian, Yang, & Teng, 2014; Xiao, Cai, Ye, Ding, & Feng, 2018). The mass spectrometric conditions were as follows: the electrospray ionization (ESI) temperature 550 °C, capillary voltage (－) 4500 V/(＋) 5500 V, and curtain gas 35 psi (Pan, Welti, & Wang, 2010; Šimura et al., 2018; Cui et al., 2015).
2.9. Statistical analysisThe statistical analysis of colony diameter, dry weight, plant growth parameters, and different phytohormones was performed using Excel 2013. The statistical procedure was carried out with the software package SPSS 19.0 (IBM, Chicago) for Windows.
Specimens examined: The specimens of Gomphus matijun were located in Xiazai village, Longli county, Qiannan city, Guizhou province, China, mingled in forest stands with Pinus massoniana, 26.5690°N, 106.8556°E, alt. 1149 m, Jul 16, 2020. GAF-20071601 (GZAAS22-0001).
Macromorphology: The fruiting body had a special aroma of the fruit. Basidiomata (Fig. 1B-D) were alone, scattered, or gregarious in distribution and columnar (early stage) to obconical (maturation phase) in shape. The white primordium gradually turned to violet, vinaceous brown to milky-coffee-colored basidiomata. The upper stipe surface was dull “dark purplish” (6E1) with a mottled appearance. The pileus was 2-23 cm broad, and plane to depressed (like Gomphus ludovicianus R.H. Petersen, Justice & D.P. Lewis, 2014). The hymenium (Fig. 1F) was “pale reddish brown” (5E8), wrinkled, generally longitudinally oriented, dichotomous, often to rarely anastomosing, and reticulate to almost poroid, like Gomphus brunneus (Heinem.) Corner (Giachini et al., 2012). The fresh stipe flesh (Fig. 1C) was delicately mottled (16B2).
Micromorphology: The surface of the stipe flesh presented a twist (Fig. 1E). The basidiospores were (4.7-6.3) × (7.1-10.2) μm2. The shape of the spores was similar to ellipsoid, and one side was dented (Fig. 1G). The protuberance of spores was the hilar appendix, which connected the spore to the sterigma of the basidium. Like Ramaria gracilis, the spore surface was covered with obvious short warts, which could be connected to form waves or irregular “cords” (Villegas, Cifuentes, & Torres, 2005). The mycelium in the Petri dish was dense and thickly white (Fig. 1H). The hyphae were 0.6-2.1 μm diam, radiation epitaxial, septate, thin-walled, and branched (Fig. 1I).
Habitat and phenology: The specimens were from the forest of Quercus, Cotinus, and Pinus trees, with yellow-brown soil attached to lime rocks, an altitude of 750-1200 m, an average temperature of 20-25 °C, and a relative humidity of 80%-90% (Fig. 1A).
Distribution: The specimens were distributed in Guizhou and Hubei provinces, central and western China.
Additional specimens: The additional specimens were from Zhaoti village, Anlong county, Qianxinan city, Guizhou province, China, located in a mingled forest with scattered P. massoniana [25.1622°N, 105.4705°E, alt. 1387 m, Jun, 02, 2021, GAF2160201 (GZAAS22-0002)].
3.2. Optimum mediumThe development of mycelia in six media is shown in Figure 2, and the results of diameter and dry weight of mycelia are shown in Table 3. The colony diameter on the solid Petri dish was in the following order: E > B > C > F > A > D. The colony diameter on medium E was the largest, reaching 31.67 mm, indicating that the purpose of the large-scale growth of mycelia on the medium with low carbon and nitrogen deficiency might be to seek more carbon sources. As shown in Figure 2, the mycelia on medium C grew more thickly than on any other medium. The colony diameter on medium D was the smallest (only 9.92 mm), indicating that the natural carbon and nitrogen sources in potato juice could hardly support the growth of mycelia. The dry weight of mycelia in triangular flasks was in the following order: C > B > A > F > E > D. The results of mycelial dry weight further confirmed that the mycelia on the medium with rich nitrogen and carbon sources showed faster growth and maximum biomass.
| Media | Diameter (mm) | Dry weight (mg) |
| A | 11.43 ± 0.93d | 9.63 ± 0.49c |
| B | 21.82 ± 0.86b | 21.44 ± 0.26b |
| C | 19.98 ± 0.38b | 34.34 ± 1.29a |
| D | 9.92 ± 1.36d | 5.59 ± 0.31e |
| E | 31.67 ± 0.76a | 4.11 ± 0.35e |
| F | 15.15 ± 0.57c | 7.80 ± 0.23d |
Note: Each value is expressed as mean ± SE (n = 3). Different letters in column represent statistically significant differences (*P < 0.05).
The target ITS sequences were from two Gomphus basidiomata and their isolates collected from Longli and Anlong counties. Besides, two ITS sequences of ECM roots were collected from a mingled forest with scattered P. massoniana, and the ECM roots were successfully identified by ITS2 as P. massoniana (Supplementary Table S1). Besides, the dried fruiting body and mycelium sequences of Gomphus sp. (zituoluo) from Tan and Liu (2013) were also used to build phylogenetic trees. The phylogeny circle diagram is shown in Figure 3. About eight sequences from G. matijun, Gomphus sp. (zituoluo), and two Gomphus sp. submitted in NCBI by Liu were grouped to form a monophyletic group, which was clearly separated from the other groups. The phylogenetic analysis showed the evidence of the same species between G. matijun and Gomphus sp. (zituoluo), which would most likely be the species of G. purpuraceus. However, due to the lack of molecular evidence for G. purpuraceus, it is not sure that G. matijun is actually G. purpuraceus.
The mycelia existing in soil cores and the colonized root tips from a mingled forest with scattered P. massoniana are shown in Figure 4. The soil core was densely packed with lateral roots and root tips (Fig. 4A), and whitish hyphae closely enclosed the slightly distended root tips. Similar to G. floccosus, the fungus-colonized roots were short, partly curved, and the tips of the colonized roots is swollen. As per the branching, the natural ECM roots were mainly simple or monopodial, while the coralloid roots were not found in natural ECM roots. Meanwhile, the colour of natural ECM roots were darker than that of the uncolonized roots (Fig. 4B). In the micro-morphological characters, a mantle was obviously observed in the natural ECM roots, where the roots were closely enveloped by a mat of fungal symbiont (Fig. 4C). At the same time, the root hairs were absent in the colonized roots, which is also one of the characteristics of ectomycorrhiza. Besides, the Hartig net (HN) confined to the epidermis were found. From the Fig. 4D, the extraradical mycelia (EM) radiating from mantle surface was simple or branched as hyphal network.
After 3 mo of mycorrhizal synthesis under pure culture conditions, the mycelium in square Petri dishes and triangular flasks successfully colonized in the roots of P. massoniana. On the square Petri dishes (Fig. 5A, B), the development of both tap and lateral roots of P. massoniana was poor, probably because the roots did not have good contact with nutrients in a solid medium (Fig. 5A). The roots of P. massoniana were abundant, white, and cotton-wool like with abundant external mycelium; the lateral roots were dark red (Fig. 5B). In the triangular flasks (Fig. 5C, D), the development of both tap and lateral roots of P. massoniana was better (Fig. 5C). The suspended mycelia were like sunken cotton ball, and part of the mycelia extended and developed around tap roots. A portion of the hyphae surrounding the roots protruded above the liquid medium (Fig. 5D). Moreover, the lateral roots of the liquid surface junction showed similar characteristics of mycorrhizae with monopodial or dichotomously branched and complex roots, such as the mycorrhizae of the Lactarius and Tuber genera (Gomes et al., 2016; Wang et al., 2019b).
The colonized roots of P. massoniana were observed 3 mo after inoculation, while no colonization was observed in the uninoculated control. The examination of the transverse cross-sections of roots enclosed with whitish hyphae showed that the hyphae intruded the roots' outer cortex in P. massoniana (Fig. 6A, C). A mantle and an HN were observed in cross-sections of P. massoniana inoculated with G. matijun, which was similar to Phlebopus portentosus (Berk. & Broome) Boedijn (Kumla, Hobbie, Suwannarach, & Lumyong, 2016). In the uninoculated control, the root cells were regular, and the intercellular space was clear and transparent (Fig. 6B, D).
The ITS sequences were identified by searching databases using the Nucleotide BLAST tool in the NCBI, which confirmed the presence of G. matijun in colonized roots of P. massoniana. Meanwhile, PCR products were all more than 700 bp in colonized roots and mycelium (Fig. 7).
Generally, ectomycorrhizal fungi can promote the development of symbiotic plants under a natural environment or controlled soilless conditions (Guerin-Laguette, Plassard, & Mousain, 2000; Guerin-Laguette et al., 2014). Although the composition of the liquid medium in triangular flasks is relatively simple, this study preliminary attempted to understand the effects of colonization of G. matijun on the development of P. massoniana. The results are shown in Table 4 and Figure 8.
| Parameter | Seedlings | Mycorrhized seedlings |
| Height (cm) | 7.44 ± 0.20 | 7.69 ± 0.22 |
| Tap root length (cm) | 21.35 ± 1.83 | 20.57 ± 1.98 |
| Basal diameter (mm) | 0.78 ± 0.02 | 0.82 ± 0.03 |
| Shoot dry weight (mg) | 81.81 ± 9.42 | 86.21 ± 7.41 |
| Root dry weight (mg) | 16.39 ± 2.30* | 26.12 ± 2.03* |
| First-lateral root number | 2.80 ± 0.47* | 6.40 ± 0.97* |
| First-lateral root average length (cm) | 0.90 ± 0.15 | 1.20 ± 0.18 |
Note: Each value is expressed as mean ± SE (n = 3). The symbol * represents a significant difference in the same row (P < 0.05).
After 3 mo of mycorrhizal synthesis in triangular flasks, the height, basal diameter, shoot dry weight, and first-lateral root average length of P. massoniana seedlings inoculated with G. matijun had a slight increase compared with the uninoculated seedlings, which was not found to be significantly different by statistical analysis (P > 0.05). Conversely, slight reductions in the taproot length of the inoculated seedlings were observed (P > 0.05). However, the root dry weight and first-lateral root number significantly increased. Especially, the first-lateral root number of the inoculated seedlings was more than twice that of the uninoculated seedlings. Previous studies showed that the growth of lateral roots and the defense response of plants were reflected by the changes in phytohormones during ectomycorrhizal formation, especially auxins and jasmonic acid (JA). (Plett et al., 2014; Vayssières et al., 2015; Felten et al., 2009). Based on these findings, the phytohormones in the uninoculated and inoculated seedlings were also analyzed and compared.
3.6. Analysis of different phytohormonesA total of 43 phytohormones were detected using LC-MS/MS, of which 22 different phytohormones in six categories were screened out. The results are shown in Table 5. The levels of nine phytohormones in the roots of inoculated seedlings were upregulated, including five new phytohormones. Meanwhile, the levels of 13 phytohormones in inoculated seedlings were downregulated, including 4 missing phytohormones. The levels of auxins affecting lateral root development, including 2-oxindole-3-acetic acid (OxIAA), Indole-3-acetyl-L-glutamic acid dimethyl ester (IAA-Glu-diMe), Tryptamine (TRA), and 3-Indolepropionic acid (IPA), increased after the seedlings were inoculated. At the same time, the content of five types of JA, including N-[(-)-Jasmonoyl]-(l)-phenalanine (JA-Phe), cis(+)-12-Oxophytodienoic acid (OPDA), N-[(-)-Jasmonoyl]-(L)-valine (JA-Val), Jasmonoyl-L-isoleucine (JA-ILE), and JA, decreased after 3 mo of mycorrhizal synthesis. In addition, the content of 5-deoxystrigol (5DS) related to plant symbiosis decreased from 123.94 ng/g to 22.82 ng/g.
| Class | Compounds | Abbreviation | A (ng/g) | B (ng/g) |
| ABA | Abscisic acid | ABA | 35.86 ± 0.51 | 16.52 ± 0.78 |
| Auxin | Indole-3-butyric acid | IBA | 2.33 ± 0.02 | NA |
| 2-oxindole-3-acetic acid | OxIAA | NA | 48.67 ± 3.44 | |
| Indole-3-acetyl-L-glutamic acid dimethyl ester | IAA-Glu-diMe | NA | 1.34 ± 0.10 | |
| Tryptamine | TRA | 0.18 ± 0.02 | 1.07 ± 0.01 | |
| 3-Indolepropionic acid | IPA | 13.36 ± 1.03 | 1.82 ± 0.09 | |
| Indole-3-carboxylic acid | ICA | 1.09 ± 0.10 | 10.53 ± 0.36 | |
| CK | N6-Benzyladenine-7-glucoside | BAP7G | 0.14 ± 0.01 | NA |
| cis-Zeatin-9-glucoside | cZ9G | 1.00 ± 0.05 | 0.29 ± 0.02 | |
| N6-Isopentenyl-adenine-9-glucoside | iP9G | 0.30 ± 0.03 | 0.66 ± 0.02 | |
| 2-Methylthio-cis-zeatin | 2MeScZ | NA | 0.42 ± 0.04 | |
| 6-Benzyladenine | BAP | NA | 0.04 ± 0.001 | |
| cis-Zeatin riboside | cZR | 0.09 ± 0.01 | 0.98 ± 0.03 | |
| GA | Gibberellin A20 | GA20 | NA | 12.59 ± 0.51 |
| Gibberellin A53 | GA53 | 2.96 ± 0.29 | 0.41 ± 0.05 | |
| Gibberellin A7 | GA7 | 0.10 ± 0.01 | NA | |
| JA | N-[(-)-Jasmonoyl]-(l)-phenalanine | JA-Phe | 0.58 ± 0.04 | NA |
| cis(+)-12-Oxophytodienoic acid | OPDA | 412.88 ± 15.16 | 190.18 ± 6.74 | |
| N-[(-)-Jasmonoyl]-(L)-valine | JA-Val | 5.40 ± 0.30 | 0.23 ± 0.02 | |
| Jasmonoyl-L-isoleucine | JA-ILE | 72.38 ± 2.16 | 4.16 ± 0.07 | |
| Jasmonic acid | JA | 65.67 ± 3.48 | 5.21 ± 0.09 | |
| SL | 5-Deoxystrigol | 5DS | 123.94 ± 8.67 | 22.82 ± 0.38 |
Note: Each value is expressed as mean ± SE (n=3). Group A and B refer to the uninoculated and inoculated seedlings group, respectively.
Gomphus matijun is an ancient wild edible macrofungus in China. The fruiting body of G. matijun usually appears under climatic conditions with frequent rain and higher air humidity from May to Nov each year. The present study mainly aimed to investigate the ECM status of G. matijun and analyze G. matijun-P. massoniana compatibility, which was beneficial to the potential of mycorrhizal seedlings in afforestation.
Morphologically, G. matijun is similar to G. ludovicianus found in the southeastern United States (Petersen et al., 2014). The morphological and molecular analyses showed that the same species between G. matijun and Gomphus sp. (zituoluo) might be the species of Gomphus purpuraceus. Although a few species of Gomphus are thought to be the ectomycorrhizal fungi (Agerer et al., 1998; Trappe, 1960), only mycorrhizal synthesis between G. floccosus in this genus and Abies religiosa under controlled conditions has been reported so far. In this study, the colonized roots of G. matijun in field investigations showed similar characteristics of mycorrhizae between G. floccosus in this genus and A. religiosa based on mycorrhizal morphology, which were short, partly curved, and swollen. In order to confirm the ectomycorrhizal status of G. matijun, the artificial mycorrhizal synthesis were performed. Currently, there are two main methods of artificial mycorrhizal synthesis in virto. One is the use of controlled artificial substrates that restore the mycorrhizal soil environment in nature as much as possible. This method is mainly used to confirmed the related trees and ectomycorrhizal morphology for the next cultivation cycle for edible mycorrhizal fungi (EMF). However, the mycorrhizal synthesis is complex and its outcome subject to many factors, including helper bacteria, other biotic and abiotic conditions of the environment (Guerin-Laguette, 2021). Although this method can provide a basis for commercial cultivation of EMF, it is difficult to study the mechanism of mycorrhizal symbiosis due to the influence of environmental factors. To avoid the interference of alternative factors, the mycorrhizal synthesis beneath pure culture conditions has been wont to study the mechanism of mycorrhizal formation, especially in the Laccaria bicolor (Maire) P.D. Orton beneath sandwich culture system in square Petri dish (Felten et al., 2009). According to Felten’s analysis, the micro-morphology of ectomycorrhiza, as well as a mantle, Hartig net (HN) and emanating hyphae, is consistent with those of mycorrhiza under the controlled artificial substrates (nursery conditions). Therefore, the morphology of mycorrhiza under pure culture conditions might also be used as a reference for whether or not ectomycorrhizal fungi. Originally, the mycorrhizal synthesis of G. matijun below nursery conditions has been attempted for two y for the commercial cultivation. Unfortunately, it has not been successful under controlled soil conditions probably due to an unsuitable soil matrix. So the preliminary exploration on the mycorrhizal status of G. matijun under pure culture conditions was performed. With sandwich culture system in square Petri dish, we found the degree of fitting between the roots of seedlings and the surface of solid medium greatly affects the development and formation of mycorrhizas, which leads to the inaccurate evaluation of mycorrhizal biomass. Therefore, the model of ECM synthesis in triangular flask was adopted, though this model was very different compared with nursery conditions. After 3 mo of mycorrhizal synthesis using above 2 model, the emanating hyphae in colonized roots were clearly observed, while a mantle and an HN were also observed in the cross-sections of P. massoniana inoculated with G. matijun. Anyway, it could be sure that tissue structure of roots cross-sections of P. massoniana between inoculated and uninoculated with G. matijun was obviously different. Except for the micro-morphology of the ECM association, fungal infection changes the growth pattern of the root. The fungal sheath reduces the rate of cell division at the root tip, slowing cell elongation and hence reducing the root growth lengthways. Therefore, the roots of ECM are shorter and wider than uninfected ones. Besides, some researchers also confirmed that ectomycorrhizal fungi can reprogramme root architecture by phytohormones or volatile signalling (Vayssières et al., 2015; Felten et al., 2009; Ditengou et al., 2015). Hence, we further assessed plant development between inoculated and uninoculated plants. The results showed that the root dry weight and first-lateral root number of inoculated plants significantly increased. The results showed that G. matijun had the common characteristics of ectomycorrhizal fungi in roots reprogramming. In general, the featured macro-characters of ECM roots have the morphological diversity. The morphological characteristics of various trees infected by the same EMF were slightly different. For example P. portentosus, The mycorrhiza of P. portentosus showed a complex morphological structure in mycorrhizal synthesis with different plants (Kumla et al., 2016). In order to additional perceive the changes of phytohormones in P. massoniana roots once G. matijun infection, phytohormones existed in roots of P. massoniana were additional analyzed. Five different types of JA were decreased in inoculated seedling roots than in the uninoculated group, which was consistent with the findings of Plett et al. (2014). Moreover, the partially different levels of auxins in the roots of inoculated seedlings also increased significantly in this study; the same conclusion was also obtained by Vayssières et al. (2015).
In this study, the colonized roots of G. matijun exhibit the ECM-like structures under in nature. Meanwhile, the micro-morphology of roots infected by G. matijun also showed the typical ectomycorrhizal structures, including a mantle, Hartig net (HN) and emanating hyphae under pure culture conditions. So we speculated G. matijun to be an ECM fungus based on the macro-morphology of colonized roots in field investigations, the micro-morphology of colonized roots under pure culture conditions, changes in phytohormone levels of colonized roots. Considering the commercial prospects and the inoculation efficiency of G. matijun, the mycorrhizal synthesis trials under controlled soil conditions still need to be carried out. Meanwhile, the CAZymes composition characteristics in the G. matijun genome can be considered to assess the ability of carbohydrate metabolism, which can prove the ECM status of G. matijun at the molecular level. If necessary, further exploration is required to clarify the exchange of materials between G. matijun and associated plants.
In this paper, we described morphoanatomical characteristics of G. matijun, investigated its phylogenetic position, optimized medium, preliminarily explored on mycorrhizal synthesis and assessed plant development and phytohormone levels. Through a series of experiments, we have a preliminary understanding of G. matijun, especially in mycorrhizal status of G. matijun, while some questions remain unanswered. Overall, the findings of study provide fundamental insights into morphoanatomical characteristics, phylogenetic position and putative niche of G. matijun. future work need to focus on mycorrhizal synthesis under controlled soil conditions and whole-genome analysis.
The authors declare no competing interests.
This study was financially supported by the Science and Technology Program of Guizhou Province (Grant No. 2022-General 112) and the Major Scientific and Technological Special Project of Guizhou Province (Grant No. 20193006-2). We thank Professor Aihua Tan (Hubei Three Gorges Polytechnic) for providing the dried fruiting body and mycelium of Gomphus sp. (zituoluo). We are also grateful to Wenjun Xu (Kunming Institute of Botany, Chinese Academy of Sciences) for support in field investigation and Fuqiang Yu (Kunming Institute of Botany, Chinese Academy of Sciences) for constructive suggestions about the design and production of the experiment.
