Introduction

Functional foods like mushrooms have a wide range of biomolecules with nutritional and medicinal substances (Thaper and Lakshmi, 2017; Mirzaei et al., 2018). Mushrooms are macrofungi (Mansoury, 2019; Al-Zahrani and Bukhari, 2019), and there are approximately 27,000 species widely distributed around the world (Chang and Miles, 2004), with especially high diversity in tropical regions. They are found in a great variety of habitats. Some can grow on rotting logs and animal dung (e.g., saprophyte and parasite), and some mushrooms fruit on the ground beneath or nearby a tree (e.g., ectomycorrhiza). Ectomycorrhizal mushrooms normally live symbiotically with the hairy roots of living plants (Smith and Read, 1997). Currently, more than 100 species of saprophyte mushrooms can be cultivated at the industrial level (Stamets, 1993), such as Pleurotus ostreatus, Pleurotus sajor-caju, and Lentinus squarrosulus. Mycorrhizae represent symbiotic mutualistic relationships between soil fungi and fine plant roots. Ectomycorrhiza (ECM) is a type of mycorrhiza associated with the plant root system, enhancing plant uptake of minerals and drought tolerance. In contrast, the host plant provides photosynthetic products and a place to live to ECM. Dipterocarpaceae contains a group of dominant timber species found in Southeast Asia, including Thailand, and is associated with various types of ECM fungi (Cheevadhanarak and Ratchadawong, 2006). Most of them are Basidiomycetes and Ascomycetes, of them some are edible mushrooms, for example, the most famous and expensive food, truffle (Hall et al., 1998). In addition, most wild edible mushrooms are ectomycorrhizal fungi. Edible mycorrhizal mushrooms have been collected and consumed for centuries. 

The classical method for identifying mushrooms is by observing the morphology of the macroscopic (shape and surface of cap, shape and position of stipe, and color), and microscopic structures (structure, shape, size, and color of spores). Though this method takes a long time and is difficult to identify closely related species (Appiah et al., 2017). Therefore, this method needs a well-trained mycologist for identification. The molecular technique is a rapid method, highly specific, and accurate to identify (Aslam et al., 2017). Therefore, this study used morphological structures and molecular techniques to analyze the internal transcript spacer region for identifying and phylogenetic relationships between mushrooms.

Materials and Method

Sample collection

All mushrooms were collected from the local forest of Na Si Nuan sub-district (in a plant genetic conservation project under the Royal Initiative of Her Royal Highness Princess Maha Chakri Sirindhorn at Mahasarakham University), Kantarawichai, Maha Sarakham. The site is located between 16°20’20.5”-16°20’34.6”N and 103°12’44.1”-103°12’45.6”E. The average area covered is approximately 0.065 km². Small pieces of fresh mushrooms were kept in absolute ethanol and stored at -20°C until use. Complete and fresh sporocarps were identified by morphology using relevant literature (Chandrasrikul et al., 2008; Soithong, 1994; Arora, 1986). The samples were dried at 50°C. Some parts of dried mushrooms were grounded for antioxidant determination (Yuwa-amornpitak et al., 2020).

DNA Extraction and Phylogenetic Tree Construction

The mushroom sample was immersed in absolute ethanol and dried by adsorbing with paper to remove ethanol before DNA extraction with an extraction kit (Vivantis, GF1). The ITS rRNA was amplified by PCR. A diluted genomic DNA template (1 µl) was mixed with 1 µl of each primer (ITS5F: 5’-GGAAGTAAAAGTCGTAACAAGG-3’ and ITS4R:  5’-TCCTCCGCTTATTGATATGC-3’) at a concentration of 20 pmol/µl and master mix with Taq DNA polymerase (Vivantis) for a total volume of 25 µl. Amplification conditions were as follows: denaturation at 94°C for 3 min, 30 cycles of denaturation at 94°C for 40 s, annealing at 55°C for 40 s, and extension at 72°C for 1 min. The final extension was performed at 72°C for 7 min.

The PCR products of the ITS region from each mushroom were sequenced by Sanger Coulson’s method (Sanger et al., 1980) using ABI BigDye (R) Terminator v3.1 Cycle Sequencing Kit (Applied Biosystems). The sequences were searched for the most similar sequence from the GenBank database with the blastn tool (https://blast.ncbi.nlm.nih.gov/ Blast), as shown in Table 2. The sequences of mushrooms and from the GenBank database were aligned by Clustal Omega (https://www.ebi.ac.uk/Tools/msa/clustalo/). The alignment of all sequences was examined and adjusted manually by Texpad 8 software. The final alignment was formatted as a MEGA file (MEGA version 6). Neighbor-joining (NJ) analysis was used to generate the distance matrix and produce a tree with 1000 bootstrap replicates. Maximum parsimony (MP) analysis was also conducted by bootstrapping 1000 replicates with MEGA6.0.6. The NJ tree and MP tree are shown in Figures 2 and 3, respectively.

Results and Discussion

Twenty-seven wild mushroom species from the community forest of the Na Si Nuan district were collected and identified (Figure 1). They were studied and identified by morphology. The results are presented in Table 1 with the local name and code. They were placed in 12 families: Russulaceae (8), Boletaceae (7), Tricholomataceae (2), Clavariaceae (2), Plutaceae (2), Lycoperdaceae (1), Schizophyllaceae (1), Auriculaceae (1), Hymeogasteraceae (1), Astracaceae (1), and Sclerodermataceae (1). The code of each species was used to easily remember and represent the local and scientific names. Only in the rainy season (June–October) all mushrooms were found. However, Hed-Poa and Hed-Ra-ngok-luang were found only in the early rainy season (June – July). Hed-Kai-na-kao, Hed-Kai-na-luang, and Hed-Hai-na-keaw were often found in the rainy season, especially when they were abundant at the end of the rainy season. Hed-Pluak-jig and Hed Pluak-tab are termite mushrooms and were also abundant at the end of the rainy season. Termite mushrooms are preferred for eating by local people, including three types of Hed-Kai (Hed-Kai-na-kao, Hed-Kai-na-luang, Hed-Hai-na-keaw). Hed-Kho-na-dang, Hed-Tan-lek, Hed-na-kao, and Boletus species were often found in the rainy season but not in abundance. Hed-Klum-ma, Hed-Sai-duan, Hed-Ta-poo, Hed-Teen-took-kae, Hed-Hoo-noo, and Hed-Pa-ka-lung1 were not abundant. Furthermore, Scleroderma sp. was not abundant and was found only at the end of the rainy season.

From the blastn search tool, the most similar sequence was aligned again with Clustal Omega (https://www.ebi.ac.uk/Tools/msa/clustalo/). The similarity and gap of each sample are shown in Table 2, with the length between 512-810 base pairs. The similarity and gap of each pair were between 59-100% and 0-8.33%, respectively. Most mushroom samples were identified at the genus level. Only 9 samples were fitted to both genus and species levels. These 9 included Russula densifolia, Russula cf. rosaceae, Amanita hemibapha, Termitomyces cylindricus, Termitomyces fuliginosus, Calvatia craniiformis, Clavulina cf. cristata, Phylloporus gajari, and Mycoamaranthus cambodgensis, with percentage similarity (and gap) of 92.98 (0.79), 94.64 (1.17), 96.28(0), 99.70 (0), 82.25(0.49), 99.69(0), 99.69(0), 100(0), and 98.76 (0.15), respectively. Although the similarity of some species was greater than 90%, they were not determined at the species level. These included FLAV, RHET, RALB, SCY, SCHI, AUAR, BOSP, BOL2, BOL3, and ASTA. These results may be because the morphology of each mushroom in the different areas is different and there is no sequence database for those mushrooms in GenBank.

A	B	C
D	E	F
G	H	I
J	K	L
M	N	O
P	Q	R
S	T	U
V	W	X
Y	Z	Ω

Figure 1. Wild mushrooms collected from the local forest of Na Si Nuan district; mushroom codes: A = RAMY, B = RLIC, C = FLAV, D = RHET, E = RDEN, F = RALB, G = REME, H = LAC, I = AHEM, J = AVAT, K = TCLY, L = TALB, M = CMIS,N = SCY, O = CTA, P = SCHI, Q = AUAR, R = BOSP, S = BOL1, T = BOL2, U = BOL3, V = PHY, W = BOL5, X = BOL6, Y = ALPO, Z = ASTA, Ω = MSU1.

Table 1. Identification of wild mushrooms by morphology, with the local name, code, and scientific name.

Table 2:  Similarity of ITS sequences of wild mushrooms with the GenBank database.

Table 2 (continued)

*Scientific name by morphological identification

Molecular phylogenetic trees from neighbor-joining and maximum parsimony methods were analyzed by MEGA 6.0.6 and are shown in Figures 2 and 3, respectively. Both trees showed the same placement of each species with homologous ITS sequence from GenBank database and percentage confident placing with the 1000 replicate bootstraps. The NJ and MP tree consisted of 3 clades (A, B, and C). The Russulales, Agaricales and Aphyllophoreales orders were closely related and grouped in clade A. However, the results also showed that the order Boletales was divergent, and it separated into 2 clades, B and C. However, the two clades were sisters.

Figure 2. Neighbor-joining tree generated from the ITS regions of 27 wild mushrooms (code). Numbers on nodes indicate the bootstrap percentage of 1000 replicates.

Figure 3. The consensus tree of the maximum parsimony trees generated by the ITS regions of 27 wild mushrooms (code). The number on each node indicates the bootstrap percentage of 1000 replicates.