Introduction

Basidiomycota spores are either ballistosporic or statismosporic when mature and are symbolized as the most fundamental component of their life cycle. These basidiospores after repeated mitotic divisions, give rise to genetically identical monokaryotic or primary mycelium which upon plasmogamy with two distinct hyphae, initiates the development of dikaryotic or secondary mycelium later on responsible for basidiocarp primordium formation (Webster and Weber 2007). Raudaskoski and Viitanen (1982) investigated the early stages of basidiocarp formation. Although the majority of the diagnostic traits used to identify basidiomycetes were provided by basidiocarps, establishing pure cultures derived from those structures makes it very simple to identify unknown fungi (Lodge et al. 2004). Since then, most cultural studies have relied heavily on inner tissue carefully extracted from basidiocarps (Phosri 2004; Fangfuk et al. 2010; Suwannasai et al. 2020). Scrase (1995) observed that the inner tissue of the gilled fungi Hygrocybe (Fr.) P. Kumm. can sometimes be contaminated with bacteria and other fungi, making it challenging to establish a pure culture.

The basidiospore suspension technique is more effective, shorter, and less prone to contamination. Basidiospore isolation is direct, simple, rapid technique where the chances of recovery of a true product of meiosis are quite high (Pandey et al. 2016). The pure cultures of Agaricus brunnescens Peck (Kumar and Munjal 1981); A. bisporus (J.E. Lange) Imbach (Feofilova et al. 2004); oyster mushroom (Pandey et al. 2016) were all obtained from spore suspension culture. Similarly, the effects of liquid culture media, pH, temperature, on the basidiospore germination of Lentinus swartzii and L. strigosus using basidiospore suspension culture was studied (Dulay et al. 2020). However, it has been observed that in many cases in vitro basidiospore germination is a daunting task (Madelin 1962) as there are problem of poor germination and contamination by other microorganisms. To address these issues, several innovative approaches and refinements to existing methods have been suggested. Watling (1981) outlines many strategies for establishing pure cultures from basidiospores using spore suspension in sterile water. Scrase (1995) and Ainsworth (1995) later proposed similar methodologies for isolating the spores and even suggested using colonies of yeast (Rhodotorula mucilaginosa var. sanguinea (Schimon) Lodder, Verh. K. Akad) as a germination inducer, particularly for litter-decomposing gasteromycetes. To minimize the contamination by other fungi and bacteria in the culture medium, Lakkireddy and Kues (2017), used electrostatic attraction to isolate bulk basidiospores from wild mushrooms (Coprinellus domesticus (Bolton) Vilgalys, Hopple & Jacq. Johnson, Schizophyllum Fr. and Psathyrella conopilea (Fr.). However, the prerequisite nutritional and physiological conditions of most fungi remain unknown and may not be fulfilled in culture, which explains why several fungi cannot be cultured or are challenging to cultivate (Rama and Quandt 2021; Rahimlou et al. 2024).

Astraeus Morgan is a common gasteromycetes genus belonging to the Diplocystidiaceae family of the order Boletales. They are found in Dipterocarp forests in Southeast Asia and India, and even Pinaceae and Fagaceae forests in Europe and North America (Phosri et al. 2013). These star-shaped fungi grew as the monsoon season began, and they were always ripe, usually dehisced like a star, and with a whitish or brownish rhizomorph at the bottom of fruitification. The members such as A. asiaticus Phosri, M.P. Martin & Watling and A. odoratus Phosri, Watling, M.P. Martin & Whalley are edible fungi that have substantial markets in Northern Thailand, Laos, Vietnam, Cambodia, and India. However, there is no reliable evidence on the annual yield, preservation, or storage of these mushrooms (Phosri et al. 2014; Vishal et al. 2022).

Till date, eleven species have been identified, out of which Astraeus pteridis (Shear) Zeller, A. hygrometricus Pers., A. odoratus and A. sirindhorniae Watling, Phosri, Sihan., A.W. Wilson & M.P. Martín have had pure culture studies attempted to optimized mycorrhization capabilities of the fungus (Malajczuk et al. 1982; Fangfuk et al. 2010; Kaewgrajang et al. 2013; Suwannasai et al. 2020). In all these studies, inner tissue was extracted from the basidiocarp to establish a pure culture (Phosri 2004; Fangfuk et al. 2010; Suwannasai et al. 2020). Gaie and Heinemann (1980), on the other hand, reported successful pure culture establishment utilising an Astraeus glebal fragment. Their report appears to be the first trustworthy one in this field of research.

However, no such studies for A. asiaticus have been reported to date. The current gasteroid fungus project was part of an endeavor to document the distribution of various Indian Astraeus specimens associated with Indian Dipterocarp forests, particularly Shorea robusta Gaertn. The practices of artificially inoculating agaricomycetes under axenic circumstances have opened up new opportunities in fungi–based restoration, and other fungal biotechnological processes (Kumar et al. 2023).

However, here, we focused on enhancing Astraeus biomass production by cultivating pure culture strains of A. asiaticus and A. odoratus. Our major goal was to develop a reliable method of establishing pure fungal strains. In addition, we used macro– and micromorphology, as well as a combined phylogeny based on ITS and LSU genes, to assess the identity of pure mycelial strains.

Materials and methods

Study site and description

Young fruiting bodies of A. asiaticus (n = 10) and A. odoratus (n = 9) were collected from the dry deciduous tropical Shorea forest of Chainpur (23° 16’ 58.0” N, 84° 14’ 23.1” E) village in the Gumla district of Jharkhand, India (Fig. 1). The district’s landscape is characterized by gradual slopes with hills, valleys, and plateaus, and lush forests dominated by Shorea trees. The elevation ranges from 400 to 1,000 m above mean sea level. The average annual rainfall is 1200 to 1300 mm, and the temperature ranges from 10 to 30 °C. The soil in the forests was red, lateritic, sandy, and less humid. The forests are home to several IUCN-listed endangered wild animals and birds.

Fig. 1
figure 1

Satellite image and protocol for axenic culture establishment of wild edible mushroom. a satellite view of the area of collections (India: Jharkhand: Gumla: Chainpur); b habitat and habit of Astraeus; c immature basidiomes of A. asiaticus; d immature basidiomes of A. odoratus. Bar, B–D = 10 mm

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Isolation and culture preparations

Basidiospores from basidiomata were selected for A. asiaticus and A. odoratus spore cultivation and pure culture. Individual fruiting bodies were sterilized for 20 min with a 10% sodium hypochlorite solution before culture, according to the Watling (1981) approach. PDA, MNC, MMN, and YM have been used (Yamada and Katsuya 1995; Phosri 2004). Streptomycin (100 µg/ml) was added to the melting mediums after autoclaving to prevent contamination; however, it did not affect on the growth of A. asiaticus and A. odoratus. Cultures were first grown on PDA using the spore dropping/dusting method. Basidiospore dropping/dusting is a method in which portions of glebal fragments, containing spores, are sliced transversely and then dropped or dusted directly onto agar petri plates. Approximately 0.1 mg of spores were dropped/dusted in the agar plates. Incubation was done in a dark chamber at 30 °C for 5–6 days (Phosri 2004). The freshly developed primary mycelium was then transferred to MNC, MMN, and YM for pure culture establishment and stored in the incubators (BJPX–H30II, BioBase Pvt Ltd., China) for a month. The pH of the MNC and MMN mediums was merely adjusted to 5.4–5.8 using 1 N HCl or 1 N NaOH. (Phosri 2004; Fangfuk et al. 2010). All of the experiments were carried out in triplicate and with a completely arbitrary design. Uninoculated plates with each medium were utilized as a control. Mycelial growth was measured and recorded every two days during the incubation until mycelial growth reached the edge of 90 mm Petri plates. For subsequent experimentation, established pure mycelium cultures were subcultured monthly. Pure mycelia cultured on PDA, MNC, MMN, and YM agar media were kept at 30 °C for a few months and examined for macro– and micromorphological study. A triplicate of pure fungal stains was deposited at the Department of Botany, DSPMU, Ranchi, Jharkhand, India, under the voucher numbers Rug–10a–JH, Rug–10ab–JH for A. asiaticus and Rug–9a–JH for A. odoratus. A few fresh basidiomata from both specimens were preserved at 4 °C for future basidiospore inoculation experimentation.

Morphological identification of fungi

The main macro– and micromorphological traits of pure culture were studied using an Olympus CX 41 light compound microscope, including line drawing at 1000x magnification. Color and tint on colony, smell or fragrance of culture, surface structure, pattern, margin and growth pigment exuded; hyphal analysis includes hyphal system, wall thickness, septations, branching patterns, and inflations were all noted. The mycelium was mounted in water, 5% KOH, and stained with Melzer reagent. Olympus BX 53 camera was used to capture microscopic images. Colour codes were based on the Methuen Handbook of colour (Kornerup and Wanscher 1967). The images were edited and prepared using Adobe Photoshop 7.0.

DNA extraction, amplification, sequencing and sequence alignment

A plant genomic DNA Extraction kit was used to extract genomic DNA (Farvogen, Taiwan) from the mycelium of the outer part of the potato dextrose agar plates. The sequencing of internal transcribed spacer regions with the intervening 5.8 S nrDNA gene (nrITS) and the partial large subunit nrDNA gene (nrLSU) validated the authenticity of fungal specimens cultured on agar plates.

Table 1 Molecular markers, as well as their PCR primers and cycles, were applied in this study
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The nrITS (ITS1 + 5.8 S + ITS2) and nrLSU (28 S) regions (Table 1) were amplified using primers ITS1/ITS4 and LROR/LR5 (White et al. 1990; Vilgalys and Hester 1990). The PCR reaction and cycles were carried out by Phosri et al. (2014). The resulting amplicons were purified with a PCR/gel purification kit (Farvogen, Taiwan) and sequenced at Macrogen (South Korea). Chromas 2.4 (Technelysium Pty Ltd., South Brisbane, Australia) was used to analyze the chromatograms to determine the quality of each nucleotide base. Five of the consensus sequences were submitted to GenBank to procure accession numbers.

Table 2 List of specimens used in this study
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Phylogenetic analysis

Six newly generated sequences of nrITS and nrLSU from pure culture mycelium of A asiaticus and A. odoratus including their homologues were retrieved (Table 2) from nBLAST runs against the NCBI GenBank nucleotide database (https://blast.ncbi.nlm.nih.gov/Blast.cgi) and other relevant phylogenetic publications in order to infer a functional and phylogenetic linkage between the sequences (Phosri et al. 2007, 2014; Wilson et al. 2011; Fangfuk et al. 2010; Crous et al. 2019). Two distinct datasets were generated, one including ITS sequences and the other comprising LSU nrDNA sequences. The MUSCLE tool was used to align both datasets individually. To represent missing data, missing genes for individual samples were recorded as “–” in the dataset. MEGA software version 10.1.1812 (Kumar et al. 2018) was used to check, trim, and concatenate these datasets. Two locus datasets were employed for maximum likelihood (ML) and Bayesian analysis in the phylogenetic study. To estimate the bootstrap support value, ML analysis was performed with raxmlGUI 2.0 (Edler et al. 2021) using the best substitution model and one thousand replicates. For the Bayesian analysis, the best–fit model, TIM3ef + G4, generated from jMoldelTest, was utilized. MrBayes 3.2.7a (Ronquist et al. 2012) was executed using the CIPRES XSEDE web-server (https://www.phylo.org/portal2/mrbayes xsede! input.action) with the default configuration (Nst = 6, 2 runs and 4 chains per run, each run searching for 106 generations and sampling every 1000th generations). The tree depicts maximum likelihood bootstrap values (MLBS) of ≥ 70% and Bayesian posterior probabilities (PP) of ≥ 0.7.

Results

Culture and growth characteristics of Astraeus

A total of seven strains of Astraeus were established using the basidiospore dropping or dusting method. These strains grow well in natural (PDA) and synthetic media (MMN, MNC and YM). All these strains were placed in a dark room at 30 °C for incubation period of 42 days. Initially after 2–3 days of incubation all the strains produced a pale yellowish (2A3) pigment that diffused through the media, resulting in a change of color. The colonies show radial outgrowth with regular edges. The surface was matted, smooth, fluffy, and velvety. The aerial brown hyphae displayed different growth rate on the medium (Fig. 2G, H). Both A. asiaticus and A. odoratus both have monomitic and generative type of hyphal system with numerous, septate clamp, present at both simple and multiple or in intercalary junctions (Figs. 3B-D and 4B-D). Mycelial cords show further branching and moderately encrusting in KOH.

Growth characteristics of Astraeus asiaticus and A. odoratus

Three strains of Astraeus asiaticus and four strains of A. odoratus were successfully established by this method. Astraeus asiaticus strains was grown primarily on PDA, however A. odoratus strains were cultured on PDA, MMN, MNC, and YM media. Strains of A. asiaticus and A. odoratus showed different pigmentation in PDA, with A. asiaticus (Fig. 2A) showing pale yellow (2A3) on the top, reverse center-light brown(6D8), and dark brownish pigments on the periphery (6FB) whereas A. odoratus (Fig. 2B) shows brownish–green (6E8–27A8) on the top. The Astraeus asiaticus and A. odoratus PDA strains have light brown aerial hypha, which grow slowly in case of A. asiaticus (up to 29 mm in diameter after 42 days) and rapidly in A. odoratus (up to 88 mm in diameter). The radial colony of A. asiaticus grew at 1.2 mm/day during the incubation phase, but decreased to 0.6 mm/day as the duration increased. Astraeus odoratus radial colony grew at 3.5 mm/day but decreased to 2.1 mm/day as the incubation period increased. The study found that A. asiaticus hyphae exude a light brown to slightly dark greenish-brown (27F8–6E8) colored pigment that diffused through the PDA, resulting in a change of color (Fig. 2A) but no watery fluid was observed. However, A. odoratus plates (Fig. 2B) exude watery brown pigments (6D8). Despite several attempts to obtain cultures of A. asiaticus on MMN, MNC, and YM were unsuccessful.

In contrast, A. odoratus grew well on MMN, MNC, and YM agar mediums. MMN plates showed pale brownish (6D8) to light greenish-brown (27A8–6E8) pigments on the top and reverse center light brownish (6D8) (Fig. 2D); MNC plates displayed pale yellow (2A8) to dark greenish-brown (6E8–27AB) pigments on the top and reverse center light brown (6D8) (Fig. 2C). The YM agar plate had a light-brown (6D8) pigment on the top and a dark brown (6D8) pigment on the reverse (Fig. 2E). The MMN, MNC, and YM agar plates had radial aerial hyphae with diameters of 62 mm (Fig. 2D), 33 mm (Fig. 2C), and 20 mm (Fig. 2E) during a 42-day incubation period. The colonies grew at 1.8 mm/day, 1.1 mm/day, and 0.5 mm/day, but decreased as the incubation period grew longer (Fig. 2G). PDA and MNC agar plates exudates watery brown (6D8) pigments (Fig. 2B, C).

Fig. 2
figure 2

Photoplate of pure culture plates of Astraeus asiaticus and A. odoratus. aA. asiaticus obtained from PDA; bA. odoratus obtained from PDA; cA. odoratus obtained from MNC; dA. odoratus obtained from MMN; eA. odoratus obtained from Yeast Media (YM); f scatter with smooth line graph showing colony growth rate of A. asiaticus and A. odoratus after 42 days of incubation; g scatter with smooth line graph showing colony growth rate of A. odoratus obtained from four different media throughout the same incubation period

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Micro–morphological culture characterization of Astraeus asiaticus and A. odoratus

All strains developed monomitic and generative hyphal systems. Astraeus asiaticus and A. odoratus strains produced generative hyphae which were 2.1–5.3 μm and 2.5–6.0 μm wide, clamped, thin to thick-walled (wall up to 1.3 μm thick), long, straight, frequently branched (few branches either at an acute angle from the main branches or nearly at the right angle to them), with frequent paarige branching (is a branching in which a pair of branches is given off immediately below a transverse wall), pigmented, hyaline to greenish in KOH. Some olieferous hyphae (with dense cytoplasmic contents) and a few producing dense contents with irregular bulges turned into chlamydospores were found in A. asiaticus, whereas few dense-content hyphae without bulges had been observed in A. odoratus. Those asexual chlamydospore observed in A. asiaticus were 6.9–38.5 × 5.1–40.5 μm in diam., circular, oval to elliptical, terminal to intercalary of hyphae, moderate to thick-walled, wall smooth (in KOH and Melzer’s) (Fig. 3C-D), bright yellowish (2A8) (Fig. 3A). However, no such asexual chlamydospore was observed in A. odoratus. Furthermore, mycelial cords of A. asiaticus were much wider (22.3–25.5 μm in diam.) than those of A. odoratus (13.5–18.6 μm in diam.). In Astraeus asiaticus, these mycelial cords are moderately encrusted in KOH, but incrustation dissolves in Melzer, orange-yellow (4A8). No such traits were observed in A. odoratus, but some hyphae of the strains have inflated inflations and incrusted hyphal walls.

Fig. 3
figure 3

Line diagram of pure culture hyphal system of Astraeus asiaticus (Rug-10a-JH) at 1000x magnification. a 42days old culture plate; b corded mycelium with multiple clamp-connection and thick-walled hyphae; c olieferous hyphae (with dense cytoplasmic), encrusted hyphae, and chlamydospores in KOH; d paarige branching with multiple clamp-connection and bulges. Bars = b-d 10 μm

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Fig. 4
figure 4

Line diagram of pure culture hyphal system of Astraeus odoratus (Rug-9a-JH) at 1000x magnification. a 42 days old PDA-plate; b-c thick-walled hyphae with infrequent clamp-connection; d dense cytoplasmic with encrusted hyphae and paarige branching. Bars, b-d = 10 μm

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Phylogenetic analysis

We constructed a two-locus (ITS + LSU nrDNA) combined phylogenetic tree to elucidate the placement of the newly generated six sequences from the pure mycelial culture of Astraeus. The sixteen sequences were retrieved from published literature and the GenBank nucleotide database, and six newly generated (Rug–10a–JH, ITS = OQ253540 and LSU = OQ253547; Rug–10ab–JH, ITS = OQ253541; LSU = OQ253558 and; Rug–9a–JH, ITS = OR617082; LSU = OR625446) sequences were used to construct the phyllogram (TABAL 2). For rooting purposes, Pisolithus albus was employed as an outgroup (Fig. 4). The final alignment dataset contains 787 distinctive alignment patterns, with 39.44% of characters undetermined, including gaps. Both RAxML and Bayesian analysis produced trees with comparable topologies, Only the RAxML tree with the final ML optimization likelihood of − 8768.710351 (ln) is taken into account and shown in Fig. 5.

Fig. 5
figure 5

Two gene (nrITS and nrLSU) RAxML cladogram of Astraeus and allied species. a cladogram showing the placements of Indian voucher specimens (Rug–10a–JH, Rug–10ab–JH and Rug-9a-JH); b radial tree showing delimitation of Indian specimens from North American and European specimens. Note- Terminal branches showing vouchers numbers. Number on the nodes represents the percentage of MLBS and PP values. Pisolithus alba was used as an outgroup for rooting purpose

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The length of the tree is 1.103530. The two–locus datasets demonstrated six major clades in the tree. The first three clades include all Southeast Asian specimens, such as A. odoratus, A. asiaticus, and A. sirindhorniae. The remaining three monophyletic clades are represented by European and North American specimens: A. macedonicus, A. hygrometricus, and A. pteridis. The newly discovered sequences from the pure culture of Indian A. asiaticus (Rug–10a–JH and Rug–10ab–JH) form a sister clade with Thai A. asiaticus (ASTRAE–54 and ASTRAE–64) and have a high bootstrap support value (MLBS = 100% and PP = 1.00). Similarly, Indian A. odoratus (Rug–9a–JH) forms a sister clade with Thai A. odoratus (ASTRAE–58, ASTRAE–59, and ASTRAE–62) with a high bootstrap support value (MLBS = 100% and PP = 1.00). However, posterior probability support was often higher than bootstrap support, indicating a more acceptable phylogenetic structure, as demonstrated in the RAxML tree (Fig. 5).

Discussion

The inner cortex tissue of ectomycorrhizal fungi has received the most attention in pure culture studies, rather than basidiospores, rhizomorphs, sclerotia, or other vegetative organs. According to Watling (1963), most of the mushroom’s parts may be dormant, except basidiospore-producing tissue. The basidiospores were the sexual spores of mushrooms, and they were notoriously difficult to germinate (Scrase 1995). Due to the limited survivability of hybrid crosses it was predicted that around 5–20% of basidiospores develop into mature colonies under axenic conditions (Forsythe et al. 2016). The most useful technique for culturing ectomycorrhizal fungi, however, is to germinate their basidiospores following a standard protocol. To obtain pure cultures, one of the simplest methods is to allow a fungus to drop or dust spores directly onto a nutrient-rich agar surface. Fuchs (1911), Kneebone (1950), Gaie and Heinemann (1980), and Nakano et al. (2017) have successfully germinated spores of the ectomycorrhizal fungi Lactarius deliciosus (L.) Gray, L. luteolus Peck, and Astraeus hygrometricus and Rhizopogon roseolus, respectively.

Culturing a particolous ectomycorrhizal fungus from basidiospore has undoubtedly proven challenging for several decades. Astraeus asiaticus and A. odoratus are common earthstar edible ectomycorrhizal fungi that were described and separated from A. hygrometricus by Phosri et al. (2004, 2007). They are well known for their nutritional, nutraceutical, and therapeutic characteristics; nevertheless, little is known about their mycorrhizal symbiosis, host preference, and fruiting ecology. There is a dearth of study on the traits and cultivations of pure cultures.

Therefore, we commence A. asiaticus and A. odoratus cultural studies in a axenic condition by modifying Choi et al. (1999) spore shooting approach to germinating spores. The spore-drop or dusting technique was chosen because the basidiospores, which are enclosed within the hymenium (gleba), are themselves contamination free zones and did not require any special chemical treatment. Obtaining a pure culture from inner tissue, on the other hand, can be contaminated with endobacteria or other microbes, making isolation difficult, as demonstrated in the gilled fungus Hygrocybe sp. (Scrase 1995). Another drawback of tissue-cultured stains is that they are not monokaryons and do not reflect the actual outcome of meiotic division, making them unsuitable for mass genetic breeding programs. The preliminary selection of isolates of interest, accompanied by the breeding of the selected isolates to boost productivity in terms of metabolite synthesis or production of biomass necessitates genetically pure cultures. According to Pandey et al. (2016) they are the genuine outcome of meiosis. Our approach of germinating involves dusting 0.1 mg of spores, which is easier, faster, and more efficient compared to isolating single spores using vaseline or the spore suspension serial dilution method (Watling 1981; Scrase 1995; Ainsworth 1995).

Gaie and Heinemann (1980) are the only mycologists to successfully germinate basidiospores of A. hygrometricus using glebal fragments with 50% success rate; Archer (1962) and Phosri (2004) failed to do so with other Astraeus specimens. Although few mycologists use germination inducers Rhodotorula mucilaginosa var. sanguinea or Rhodotorula glutinis (Fresen.) F.C. Harrison for germination to obtain monosporous mycelia in Amanita Dill. ex Boehm., Tricholoma (Fr.) Staude, Clitopilus (Fr. ex Rabenh.) P. Kumm., Suillus P. Micheli (Fries 1943), and Rhizopogon roseolus (Corda) Th. Fr. (Nakano et al. 2017) for other litter–decomposing gasteromycetes (Scrase 1995).

In this study, no germination inducers were used. On the nutrient medium, a pale yellowish (2A3) to light brownish (6D8) pigment was formed on PDA, MMN, MNC and YM after 2–3 days of incubation, indicating both developmental competency and survivability (Fangfuk et al. 2010; Forsythe et al. 2016). The pigmentation in PDA ranges from yellowish–light brown (2A8–6D8) to slightly dark greenish–brown (27F8–6E8) in A. asiaticus and brownish–green (6E8–27A8) in A. odoratus (Fig. 2A-B). In MMN, MNC, and YM, the pigments were pale brownish (6D8) to light greenish–brown (27A8–6E8), pale yellow (2A8) to dark greenish–brown (6E8–27AB), and light–brown (6D8), respectively, in a few days old agar plates. (Fig. 2C-E). A similar phenomenon was also reported by Phosri (2004); Fangfuk et al. (2010); and Suwannasai et al. (2020). However, Burgess et al. (1995) and Phosri (2004) mentioned that pH and temperature are very crucial parameters for Boletales pure culture, therefore, we maintained a pH of 5.4 to 5.8 for MNC and MMN and a temperature of 30 °C, which were found to be effective conditions for stimulating spore germination.

Table 3 Cultural comparison of the Astraeus and allied species
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After 42 days of incubation, the vegetative hyphae of A. asiaticus develop more slowly (29 mm) than those of A. odoratus (88 mm). Cultures of A. asiaticus on MMN, MNC, and YM were unsuccessful. A. odoratus, on the other hand, grows well on all four mediums (Fig. 2B-E). According to our findings, PDA was the optimal media for cultivating A. asiaticus and; PDA and MMN for A. odoratus. All the PDA triplicates of A. asiaticus and A. odoratus have a fluffy, velvety, smooth, or matted texture, with distinct zones around the edges. The cultural strains were examined on 5% KOH, Melzer reagent, and water successively. On the water and 5% KOH, moderate encrustation was observed with olieferous hyphae (dense cytoplasmic) content (Fig. 3C) observed in A. asiaticus, the same phenomenon was also reported by Gaie and Heinemann (1980) in A. hygrometricus. The vegetative hyphae established many clamp connections at the septa, with occasional paarige branching. These hyphae are monomitic, generative, thick to thin-walled, pigmented and moderately encrusted.

However, A. asiaticus and A. odoratus vegetative hyphae can be distinguished by hyphal cord and generative hypha thickness, the presence or absence of olieferous hyphae, encrustation and asexual chlamydospores. The hyphal cord thickness of A. asiaticus is larger (22.3–25.3 m in diam) than that of A. odoratus (13.5–18.6 m in diam), but the generative hyphal thickness of the latter is larger (2.5–6.0 m in diam) than that of former (2.1–5.3 m in diam) (Fig. 2F-G). A. odoratus agar plates also show no olieferous hyphae or asexual chlamydospore elements (Fig. 3C). Some hyphal branches in A. asiaticus were heavily encrusted (Fig. 3B), but few hyphae were encrusted in A. odoratus (Fig. 4D). Some hyphal branches of A. asiaticus agar plates are either at an acute angle or nearly at a right angle to the major branches, but few hyphal branches of A. odoratus agar plates contain inflated inflations and an incrusted hyphal wall. All of these micromorphological cultural traits were comparable to those of European A. hygrometricus s.l. (Gaie and Heinemann 1980), A. pteridis (Malajczuk et al. 1982), Thai A. odoratus. (Phosri 2004) and Astraeus sp. (Saensuk and Suntararak 2018) and A. sirindhorniae (Suwannasai et al. 2020). Except for Gaie and Heinemann (1980), these mycologists used inner tissue from Astraeus basidiocarps to inoculate and germinate mycelium on artificial media: MMN, MNC, YM, and Malt Extract Agar (MEA), Table 3. Our PDA (Fig. 4A) and MNC (Fig. 2C) culture plates of A. odoratus exuded dark brownish watery droplets. These dark, watery droplets could be pulvinic acid derivatives, according to Watling (2008).

Our phylogeny (ITS and LSU nrDNA) demonstrates the phylogenetic placements of pure culture strains. The vouchers no: Rug–10a–JH and Rug–10ab–JH were placed in the Southeast Asian clade of A. asiaticus. Similarly, voucher no: Rug–9a–JH placed was in the Southeast Asian clade of A. odoratus based on genetic sequence and morphology. Thus, validates the pure culture strain’s identity (Fig. 5A). The radial phylogenetic tree further provides evidence for the Southeast Asian origin of the species (Fig. 5B).

To the best of our knowledge, this is the first report of Astraeus pure culture and cultivation from basidiospores in axenic conditions using the dropping/dusting method. For in vitro culture of A. asiaticus and A. odoratus, potato dextrose agar (PDA) seems to be the most suitable medium. This is because PDA offers several advantages: it has the right nutrients, it’s easy to prepare and maintain, and it doesn’t require any special solutions to keep the fungus growing well. Cultivating ectomycorrhizal fungi provides a low-cost, nature-based strategy that aids in forest restoration, conserves biodiversity, and enhances the yield of forest mycoprotein, ultimately stimulating the local economy. This method not only benefits the environment in these ways, but it also plays a crucial role in green research by capturing greenhouse gases and promoting carbon sequestration (Thomas and Vazquez 2022).

Conclusion

Astraeus is a therapeutic and commercially valuable wild mushroom. Obtaining pure cultures would greatly aid in Mycoforestry, and Myco–economy of the country, although wild mushrooms are extremely difficult to culture. Establishing artificial nutritional media, temperature, and pH may be critical conditions for culture. This study not only describes the cultural traits of Astraeus, but it also suggests that PDA and MMN would be the best natural and synthetic media for the growth of the taxon. However, more study is required to investigate various unexplored areas, such as their mycorrhizal symbiosis, host choice, fruiting ecology, and factors influencing mushroom production longevity.