Isolation, Culture and Bioactive Substances Elicitation of Sanghuangporus Baumii

Sanghuangporus baumii is a forest pathogenic fungus and also a medicine mushroom. In the wild, S. baumii parasitize on living host plants and there have been no reports of cultivating fruiting bodies under articial conditions. In this study, we identied and isolated a S. baumii strain, and successfully cultivated the fruiting bodies on sawdust medium by optimizing culture conditions. The optimum medium, culture temperature and pH for mycelial growth of S. baumii were WBA and YPA, at pH 5.5–7.5 and 28 ℃ , respectively. The contents of total avonoids, total polysaccharides and total triterpenoids in S. baumii were compared with those in other two medicinal sanghuang, S. vaninii and S. sanghuang. The results showed that both the fruiting bodies and mycelia of S. baumii were rich in bioactive substances. The content of total avonoids was higher in the fruiting bodies (34.91 mg/g), while the contents of total polysaccharides and total triterpenoids were higher in the mycelia (45.41 mg/g, 14.06 mg/g, respectively). In addition, the mycelia of S. baumii could be elicited to produce more bioactive substances. The use of sealing lm in culture increased the polysaccharides content in mycelia by 51.8%, while the light increased the avonoids content by 151.0%. The cultivation of fruiting bodies and the elicitation of bioactive substances from mycelia provide biological materials for the study and utilization of S. baumii.


Introduction
Sanghuang is a lethal or pathogenic fungus that damages a variety of hardwood species, causing living wood to decay and form large fruiting bodies (Kim, Hohenlohe, Kim, Seo, & Klopfenstein, 2016). However, the economic value of this pathogenic fungus outweighs the damage it causes. The fruiting bodies formed by Sanghuangporus spp. are well-known mushrooms that have been used as traditional medicine for thousands of years in China and other East Asian regions (Han et al., 2016). The most ancient record of sanghuang that is currently available is the "Shen Nong Materia Medica", which was written around 102-200 A.D. Although this record was written before the advent of modern science, it was empirically found that Sanghuangporus spp. were indeed effective against diseases. In recent decades, Sanghuangporus has received widespread attention for its anti-in ammatory and anti-cancer effects ( Sanghuangporus vaninii can produce fruiting bodies under arti cial conditions, so it is widely sold in medicinal markets (Zhou et al., 2020). Sanghuangporus baumii parasitic on the trunk of Syringa reticulata and cannot be cultivated under arti cial conditions. It also contains bioactive substances with anti-tumor and immunomodulation activity, but it is not as well-known as S. sanghuang and S. vaninii. The failure of cultivation greatly restricted the research and the industrialization of S. baumii. This failure is most likely due to a lack of knowledge of appropriate cultivation conditions and fruiting management.
To date, it remains unknown how the medium, pH, and temperature affect the mycelial growth of S. baumii, and there have been very few reports on fruiting body cultivation and management.
Compared with fruiting bodies, mycelia also contain a large number of bioactive substances, and mycelia are easier to obtain through fermentation culture (Rathore, Prasad, Kapri, Tiwari, & Sharma, 2019).
However, many bioactive substances are secondary metabolites whose gene clusters are silent under standard laboratory conditions. Optimization of culture conditions (medium, pH, temperature, etc.) can achieve high biomass yield, but may not increase the content of bioactive substances. Elicitation is one the most effective techniques currently used for improving the bioactive substances production (Ramirez-Estrada et al., 2016). The elicitors are mainly carbon sources, inorganic compounds or physical factors such as oxygen and light (Tian, Dai, Song, Xu, & Ng, 2015). To the best of our knowledge, no work has been published on the elicitation bioactive substances of S. baumii.
In this paper, we described, isolated and cultured the medicinal mushroom S. baumii, and successfully cultivated the fruiting bodies. In addition, since S. baumii, like S. vaninii and S. sanghuang, is known for its medicinal properties, we compared the contents of total polysaccharides, avonoids, and triterpenoids in mycelia and fruiting bodies of the three species. We also tested the contents of bioactive substances in the elicited S. baumii mycelia.

Materials And Methods
Strain collection, isolation and morphological study Basidiocarps of S. baumii, S. vaninii, and S. sanghuang were collected in China in September 2019 (Details are shown in Table S1). The internal tissue sections of the fruiting bodies were transferred onto potato dextrose agar (PDA) and incubated at 25°C under dark conditions until the agar surface was covered with mycelium. The strains were preserved at the College of Forestry, Northeast Forestry University.
The fresh samples were photographed, and macroscopic details were described. Notably, the macromorphological characteristics were described following the methods reported in Lodge et al. (Lodge, Ammirati, dell, & Mueller, 2004). Moreover, the Ridgeway color standards (Zimmer, 1948) were used (the corresponding keys are displayed in parentheses after the color descriptions in the Results section). Permanent sections were cut using a freezing microtome (Leica CM1520; Leica, Wetzlar, Germany), and microphotography was performed with a compound microscope (ECLIPSE Ni-U; Nikon, Tokyo, Japan).
The size of basidiospores was followed by length × width, and the length or width is in the form of (a-) b-c(-d), where a and d are the minimum and maximum values respectively, and 90% of the measured values were within the range of b-c. The samples were air-dried and deposited in the Herbarium of the College of Forestry, Northeast Forestry University, Harbin, China.

Phylogenetic analysis
Genomic DNA was extracted from the internal tissues of basidiocarps using the cetyltrimethylammonium bromide method (Allen, Flores-Vergara, Krasnyanski, Kumar, & Thompson, 2006). The ITS sequences were ampli ed by polymerase chain reaction (PCR) using the primers ITS-1 (5'-TCCGTAGGTGAACCTGCGG-3') and ITS-4 (5'-TCCTCCGCTTATTGATATGC-3'). The PCR conditions were as follows: 95°C for 3 min, followed by 35 cycles at 95°C for 30 s, 56°C for 30 s, 72°C for 1 min, and a nal extension at 72°C for 10 min. The PCR products were puri ed and sequenced by Boshi Biotech Co., Harbin, China. Sequence similarity searches were performed using GenBank and the basic local alignment search tool algorithm

Optimization of mycelium culture conditions
Three different culture media were used to determine the optimal medium for mycelial growth. These three media consisted of PDA (20 g/L potato infusion, 20 g/L dextrose, and 15 g/L agar), wheat bran extract agar (WBA; 15 g/L wheat bran extract, 20 g/L dextrose, and 15 g/L agar), and yeast extract peptone agar (YPA; 10 g/L yeast extract, 10 g/L peptone, and 15 g/L agar), respectively. The PDA medium was used to determine the optimal pH for mycelial growth, and pH was adjusted to 5.5, 6.0, 6.5, 7.0, or 7.5, with 1 N HCl and 1 N NaOH. The PDA medium with a natural pH was used to determine the optimal temperature for mycelial growth, and the temperature was set to 18°C, 21°C, 23°C, 25°C, or 28°C. For all treatments, ve replicates were used, and incubation was performed under dark conditions for 10 days. The culture temperature used to determine the optimum medium and optimum pH was 25°C.
Mycelial growth was evaluated based on the mycelial growth rate and mycelial density. The mycelial growth rate was calculated by averaging the vertical and horizontal lengths of the colony diameter.

Fermentation culture and cultivation
The three strains of Sanghuangporus were inoculated in potato dextrose broth media and maintained at 25°C and 160 rpm for 7 days before harvesting their mycelia.
The sawdust medium consisted of a mass fraction of 60% water, 34% sawdust, 5% wheat bran, 0.5% potassium dihydrogen phosphate, and 0.5% magnesium sulfate. The medium was adjusted to the optimum pH to cultivate the basidiocarps of the three strains. The mixed medium was placed in polypropylene bags and sterilized in an autoclave sterilizer at 121°C for 2 h. The strains were inoculated on sawdust medium, and each strain was cultured in 10 bags. The inoculated bags were incubated under dark conditions and at the optimum temperature for each strain until the medium was fully colonized by the mycelia. The bags were then transferred to a mushroom chamber for subsequent fruiting management.

Elicitation of bioactive substances in S. baumii
Eleven different treatments (a ~ k) were evaluated to elicit the bioactive substances of S. baumii. The semi-solid media formulae used are shown in Table 1. All elicitation cultures were performed in glass Petri dishes. After inoculation, the Petri dishes of treatment a and treatment d-k were wrapped around the circumference twice with air-permeable Para lm (PM-996 Para lm® M Laboratory Film). The Petri dishes of treatment b were wrapped with air-impermeable grafting membrane (LINGS company, China). The Petri dishes of treatment c were not wrapped. Except for treatment k, all Petri dishes were incubated at 25°C under dark conditions for 12 days. The petri dishes of treatment k underwent 10 days of dark culture and 2 days of light culture with a light intensity of 200 umol s −1 m −2 . After the elicitation culture, the colony of S. baumii was picked out to harvest mycelium. Assay of bioactive substances The fruiting bodies and mycelia were sampled, dried to a constant weight at 50°C, ground into powder and sifted through a 60-mesh sieve. The total polysaccharides were determined by the phenol-sulfuric acid method (

Morphological and phylogenetic analysis
The morphological characteristics of the basidiocarps of S. baumii are displayed in Figure 1. The S. baumii basidiocarps were perennial and sessile with a cork texture when they were fresh, and a woody hard texture when they were dry. The pileus was semicircular, 7-12 cm in length, 3.5-6.0 cm in width, and up to 3.0 cm thick. The pileus surface was dark brown (7F5), glabrous, and rough, and it displayed radial cracking. There were also dense to sparse concentric annular grooves from the center to the edge of the pileus. The pore surface was light brown (6D8), and the margin was yellow ochre (5C7). There were 9-11 pores per millimeter. The setae were conical, 11-20 µm in length, and 4-8 µm in width. The basidiospores were ellipsoid, mostly sunken, and were ( The morphological characteristics of the basidiocarps of S. vaninii are presented in Figure S1. The basidiocarps of S. vaninii were perennial and sessile, with a cork texture when they were fresh, and a hard woody texture when they were dry. The pileus was semicircular, 6-10 cm in length, 3.5-5.0 cm in width, and up to 3.5 cm thick. The pileus surface was Chinese yellow (4B7) to olive brown (4F7), and glabrous. It was characterized by dense to sparse concentric annular grooves from the center to the edge of the pileus. The pore surface was Chinese yellow (4B8), and there were 9-10 pores per millimeter. The setae were conical, 10-20 µm in length, and 4-7 µm in width. The basidiospores were ellipsoid, mostly sunken, and (2. The morphological characteristics of the basidiocarps of S. sanghuang are shown in Figure S2. The S. sanghuang basidiocarps were perennial and sessile, with a cork texture when they were fresh, and a hard woody texture when they were dry. The pileus was irregularly semicircular, 8-15 cm in length, 5-9 cm in width, and up to 4.0 cm thick. The pileus surface was brown (6E8), glabrous, rough, and displayed irregular longitudinal cracking, as well as dense to sparse concentric annular grooves from the center to the edge of the pileus. The pore surface was yellow ochre (5C7), and the margin was buttercup yellow µm in size. The generative hyphae were 2.0-3.0 µm in diameter, transparent, brown, and branched.
Likewise, the skeletal hyphae were 2.0-3.0 µm in diameter, but golden brown, thick-walled, and arranged closely and in parallel.
The phylogenetic tree included 20 species from the Hymenochaetaceae family and was based on the ITS sequences ( Figure 2). The clustering of these sequences in the phylogenetic tree showed that the species phylogeny was divided into three clades (i.e., Sanghuangporus, Tropicoporus, and Phellinus). The phylogenetic analysis indicated that S. sanghuang, S. vaninii, and S. baumii belonged to the Sanghuangporus clade.
Optimal culture conditions and fruiting management After 10 days of cultivation, the media surfaces were colonized with lamentous colonies. However, the growth status of the three strains varied between the three media ( Figure S3). The mycelial growth rates and mycelial density of the three species in each culture medium are presented in Table 2.
Sanghuangporus baumii formed white lamentous colonies on the three media. The highest mycelial growth rate was observed on WBA and YPA media, while the greatest mycelial density was observed on PDA and YPA media. Sanghuangporus vaninii formed white colonies on YPA media and white to yellowish colonies on PDA and WBA media. The highest mycelial growth rates of S. vaninii were observed on PDA and YPA media, while the greatest mycelial densities were observed on WBA and YPA media. Sanghuangporus sanghuang formed white to yellow colonies on the three media, and the mycelium growth rate and mycelial density did not signi cantly differ between the media. All pH values between 5.5 and 7.5 were suitable for the mycelium growth of the three species (Table 3).
There was no signi cant difference in the mycelium growth rate or mycelial density of S. baumii as the pH changed. As for S. vaninii and S. sanghuang, although they grew well on media of pH 5.5-7.5, the most favorable pH was 7.5, with the highest mycelium growth rates and mycelial densities, followed by pH 7.0. The mycelial growth rates of the three species increased when the temperature increased from 18°C to 28°C (Table 4). Notably, the mycelial density of S. baumii remained abundant across different temperatures. In contrast, the highest mycelial density was observed between 21°C and 25°C for S. vaninii and at 28°C for S. sanghuang.  The bags full of mycelia were transferred to the mushroom chamber for further culture until the color of the mycelia changed from white to dark yellow, which took approximately 10 days. A sterile scalpel was then used to make incisions, which were 5-8 cm long and 0.5 cm deep, on the surface of the bags. In the mushroom chamber, the temperature was maintained at 25°C-28°C, the air humidity was of 85-95%, the light intensity was of 200-300 lx, and the chamber was ventilated twice a day. The basidiocarps of S. baumii and S. vaninii developed from the incisions (Figure 3), whereas S. sanghuang did not form fruiting bodies.
Comparison of bioactive substance contents in mycelia and fruiting bodies of the three species The contents of bioactive substance of three species were signi cantly different. In the fruit bodies, the contents of total avonoids and total triterpenoids of S. vaninii were the highest among the three species, and there was no signi cant difference in polysaccharides contents among the three species (Table 5). In the mycelia, the total polysaccharides content of S. vaninii was the highest, while the total avonoids and triterpenoids contents of S. sanghuang were the highest. In addition, there were signi cant differences in bioactive substances between fruiting body and mycelia of the same strain. The total polysaccharides contents in the mycelia of S. baumii, S. vaninii, and S. sanghuang was 5.1-, 5.4-, and 4.5-fold, respectively, higher than that in their fruiting bodies, and the total triterpenoids contents in their mycelia was 2.6-, 1.1-, and 3.5-fold, respectively, higher than that in their fruiting bodies. The total avonoids contents in the fruiting bodies of S. baumii, S. vaninii, and S. sanghuang was 5.4-, 19.6-, and 4.3-fold, respectively, higher than that in the mycelia.

Elicitation of bioactive substances of S. baumii
Eleven different treatments (a ~ k) had different effects on the growth and bioactive substances contents of S. baumii (Figure 4 and 5). The mycelia of S. baumii grew poorly under the treatment g so that there were not enough mycelia to measure the bioactive substances. The mycelia of S. baumii could not grow under the treatment j. The treatment a used common culture conditions (PDA semi-solid media plates, sealed with Para lm, dark culture) and was therefore used as a control. The mycelial growth rate of treatment b was 3.61 ± 0.05 mm/day, which was the only one higher than that of the control (3.48 ± 0.11 mm/day). The biomass of the treatment c was 201.4 ± 3.26 mg, which was the only one higher than that of control (133.7 ± 8.87 mg). Other treatments inhibited the growth rate and biomass of mycelia to varying degrees. The avonoids contents of the treatment c and k were 14.12 ± 0.38 mg/g and 26.7 ± 0.42 mg/g, respectively, which were signi cantly higher than that of the control (10.64 ± 0.35 mg/g). The polysaccharides contents of the treatment b (38.43 ± 1.79 mg/g), d (30.49 ± 0.52 mg/g), f (33.97 ± 0.79 mg/g), h (32.55 ± 1.30 mg/g), and k (34.90 ± 2.43 mg/g) were signi cantly higher than that of the control (25.31 ± 1.05 mg/g). The triterpenoids contents in all treatments were not signi cantly higher than that of the control.

Discussion
This study describes and culturates a medicinal mushroom, S. baumii, and compares it with S. vaninii and S. sanghuang. The three species shared common characteristics of Sanghuangporus, such as the perennial basidiocarps with a dimitic hyphal system, a hard woody texture when they are dry, and conical setae in the hymenophore. Notably, the morphological analysis revealed that typical individuals of these three species can be distinguished by their macroscopic characteristics, despite high levels of phenotypic plasticity.
The effects of the medium, pH, and temperature on the mycelial growth of the S. baumii were assessed. The best growth rates and mycelium density were obtained on WBA and YPA media, which indicated that organic nitrogen (wheat bran extract and yeast extract) was bene cial to the growth of mycelia. Therefore, 5% wheat bran was added into the sawdust medium for S. baumii cultivation. There was no difference in the mycelial growth of S. baumii at pH of 5.5-7.5, while the optimum pH of S. vaninii and S. sanghuang was 7.5. Therefore, sawdust medium at pH 7.5 was used to cultivate S. baumii. Within the temperature range of this study, 28°C was the most suitable temperature for the mycelial growth of S. baumii. The optimum temperature for the mycelial growth of S. vaninii and S. sanghuang was also 28°C, indicating that the three species had the same sensitivity to temperature. In the cultivation of S. baumii, 28°C is the most suitable temperature for mycelial growth. When the sawdust medium is complete colonization by the mycelium, a low temperature stimulation is required to form the fruiting body. In view of the consistent temperature sensitivity of S. vaninii and S. baumii, we referred to the stimulation temperature of S. vaninii and successfully cultivated the fruiting body of S. baumii at 18°C-23°C.
There was no signi cant difference in the contents of the three bioactive substances in fruit bodies between S. baumii and S. sanghuang, and no signi cant difference in mycelia between S. baumii and S. vaninii. This means that S. baumii contains as much bioactive substances as S. sanghuang and S. vaninii. In addition, it was found in S. baumii that the content of avonoids in fruit bodies was much higher than that in mycelia, while the contents of polysaccharides and triterpenoids in mycelia were much higher than that in fruit bodies. This indicated that the fruit bodies of S. baumii had advantages in the production of avonoids, while the mycelia had advantages in the production of polysaccharides and triterpenoids.
Most of the elicitors used in this study had adverse effects on the mycelial growth and bioactive substances contents of S. baumii. Among them, different sealing methods and light obtained satisfactory effects. In biological laboratories, agar culture plates are often wrapped to avoid dehydration and contamination, but this sealing also severely limits the rate of gas ow in and out of the culture container, Data availability The datasets generated during the current study are available from the corresponding author on reasonable request.
Con ict of interest The authors have no con ict of interest to declare.
Ethical approval This article does not contain any studies with human participants or animal experiments. 3.04 ± 0.11 b 3+ 6.5 7 7.5 2.87 ± 0.08 c 3.16 ± 0.12 ab 3.20 ± 0.09 a 3+ 3+ 4+ Note: For each strain, values followed by the same lowercase letter are not significantly different (p < 0.05).    Phylogenetic tree obtained using the neighbor-joining method from internal transcribed sequence (ITS) datasets.

Figure 4
Page 19/19 The front and back sides of the Sanghuangporus baumii mycelia after elicitation. Treatment a to k were: a control; b grafting membrane; c Para lm; d lactose; e saccharose; f phosphate ; g phosphate ; h ammonium sulfate; i peptone; j urea; k light.

Figure 5
The growth and bioactive substances contents of S. baumii mycelia after elicitation. (A) The growth rates and biomass. (B) The contents of total avonoids, polysaccharides and triterpenoids.

Supplementary Files
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