The Culture Conditions for the Mycelial Growth of Phellinus spp.

Phellinus genus belonged to Hymenochaetaceae of Basidiomycetes and has been well known as one of the most popular medicinal mushrooms due to high antitumor activity. This study was carried out to obtain the basic information for mycelial culture conditions of Phellinus linteus, P. baumii, and P. gilvus. According to colony diameter and mycelial density, the media for suitable mycelial growth of them were shown in MEA, glucose peptone, and MCM. The optimum temperature for mycelial growth was 30℃. Carbon and nitrogen sources were mannose and malt extract, respectively. The optimum C/N ratio was 10 : 1 to 5: 1 with 2% glucose concentration, vitamin was thiamine-HCl, organic acid was succinic acid, and mineral salt was MgSO4·7H2O.

The number of mushrooms on Earth is estimated at 140,000, yet maybe only 10% (approximately 14,000 named species) are known (Kirk et al., 2001). For a long time, mushrooms have been valued as an edible and medicinal resource. Phellinus genus is known about 220 species and is found mainly in tropical America and Africa (Dai et al., 1998). The genus is distributed into 7 species and commonly referred to as Sangwhang in Korea (Lee, 1993; Hong, 2000). Many kinds of Phellinus spp. (e.g. P. linteus, P. igniarius, P. gilvus, P. pini and P. hartigii) are known and they have a variety of medicinal effects (Lee et al., 1996). Among them P. linteus, P. baumii and P. gilvus have been known as ones of the most popular medicinal mushrooms due to their high antitumor activity in east Asia (Ikekawa et al., 1968; Han et al., 1999; Bae et al., 2004), and safety of acute oral toxicity test (Han et al., 2001). P. linteus, P. baumii and P. gilvus have been cultivated by mushroom farmers in Korea. P. gilvus is cheaper than P. linteus and P. baumii because of very short cultivating period. Therefore, it has a possibility which can be developed as a functional food and livestock for industrial application in near future. This study was focused on culture conditions affecting the optimal mycelial growth of P. linteus, P. baumii and P. gilvus.

Materials and Methods

Fungal isolates

The isolates of Phellinus species used in this study were listed in Table 1. P. linteus ASI 26099, P. baumii Nongong and P. gilvus KCTC 6653 were presented by Rural Development Administration, Nongong Agricultural Product Company, and Biological Resources Center of Korea, respectively. All isolates were maintained on Potato Dextrose Agar medium (PDA).

Table 1

List of Phellinus spp. strains used in this study

Culture media and temperature

Twelve different culture media were prepared to screen suitable culture media to mycelial growth of P. linteus, P. baumii and P. gilvus (Table 2). The culture media were sterilized for 20 minutes at 121℃ and aseptically poured into plastic petridish. An inoculum was removed from seven days old cultures of Phellinus spp. grown on PDA at 30℃. Mycelial disk (5 mm in diameter) from the cultures was placed in the center of each 85 mm plastic petridishs containing about 20 ml of 12 different media. The fungi were incubated under the dark condition for 9 days at 30℃. Thereafter the mycelial growth, density and color of the colony were examined. To screen temperature condition for a suitable growth of Phellinus spp. The ranges of temperature were 10℃, 15℃, 20℃, 25℃, 30℃ and 35℃, respectively. The fungi cultured on Malt Extract Agar (MEA) for 10 days by the same method above. Then the measurement of mycelial growth was performed.

Table 2

Composition of media used in this study

Effect of favorable nutrient sources

Carbon sources

The experiment was performed on the mushroom minimal media (MMM: dextrose 20 g, MgSO4 0.5 g, KH2PO4 0.46 g, K2HPO4 1 g, asparagine 2 g, thiamine-HCl 120 µg, agar 20 g, DW 1,000 ml) supplemented with each of 10 carbon sources. Each carbon source was added to mushroom minimal media at the concentration of 2%. The fungi were incubated under the dark condition for 10 days at 30℃. Thereafter we examined the mycelial growth, density and color of the colony.

Nitrogen sources

To screen nitrogen source suitable to the mycelial growth of Phellinus spp., the experiment was performed on the mushroom minimal media supplemented with each of 12 nitrogen sources. Each nitrogen source was added to mushroom minimal media at the concentration of 0.2%. A 5 mm diameter plug an inoculum of Phellinus spp. cultures placed in the centure of petridish and incubated under the dark condition for 10 days at 30℃. Thereafter the mycelial growth, density and color of the colony were examined.

C/N ratio

On the mushroom minimal media which were mixed with 10, 8, 6, 4, 2, 1, 0.4 and 0.2% glucose as a carbon source and then mixed continually with 0.2% NaNO3 as a nitrogen source, the mycelial growth of Phellinus spp. was exammined. The C/N ratio was adjusted to 50 : 1, 40 : 1, 30 : 1, 20 : 1, 10 : 1, 5 : 1, 2 : 1 and 1 : 1 in each medium. Inoculated each media incubated under the dark condition for 9 days at 30℃. Thereafter the mycelial growth, density and color of the colony were examined.

Vitamin

On the sterialized mushroom minimal media which were mixed with thiamine-HCl 0.1 mg/l, riboflavin 0.5 mg/l, biotine 0.005 mg/l, pyridoxine 0.5 mg/l and nicotinamide 2.0 mg/l those were filtrated by metrical membrane filter (0.2 µm). Inoculated each media incubated under the dark condition for 9 days at 30℃. Thereafter the mycelial growth, density and color of the colony were examined.

Organic acid

On the mushroom minimal media which were mixed with acetic acid, citric acid, maleic acid, lactic acid, succinic acid and fumaric acid at the concentration of 0.1%, respectively. Inoculated each media incubated under the dark condition for 9 days at 30℃. Thereafter the mycelial growth, density and color of the colony were examined.

Mineral salt

To screen mineral salts suitable to the mycelial growth of Phellinus spp., the experiment was performed on the YM solid media (peptone 5 g, yeast extract 3 g, malt extract 3 g, dextrose 10 g, agar 20 g and DW 1,000 ml) eleminated mineral salt which was supplemented with each of 9 mineral salts. Each mineral salt was added to YM solid media at the concentration of 0.1%. Inoculated each media incubated under the dark condition for 9 days at 30℃. Thereafter the mycelial growth, density and color of the colony were examined.

Results and Discussion

Screening of the suitable culture media

The mycelial growth of Phellinus spp. was favorable in MEA, glucose peptone, and MCM whereas was poor in Czapek dox, Leonian, Hennerberg and Hoppkins medium (Table 3). Lee et al. (2004) reported that the mycelial growth of P. linteus was favorable in MYA and SMS medium. Chi et al. (1996) reported that the mycelial growth of P. linteus was favorable in YM, malt yeast extract, and MCM medium whereas was poor in Czapek dox, Leonian, Lilly, Modified Lutz and Hoppkins medium. We concluded that the above results were similar with this study. The mycelial growth of P. linteus isolate ASI 26099 was less than P. baumii isolate Nongong and P. gilvus isolate KCTC 6653. Colony's color was that P. linteus isolate ASI 26099 was light yellow, P. baumii isolate Nongong was yellow and P. gilvus isolate KCTC 6653 was brown.

Table 3

Effect of culture medium on mycelial growth of Phellinus spp. at 30℃

a): C; compact, SC; somewhat compact, ST; somewhat thin, T; thin

b): Br; brownish, SY; somewhat Yellowish, Y; Yellowish, W; Whitish

c): P. l; P. linteus ASI 26099, P. b; P. baumii Nongong, P. g; P. gilvus KCTC 6653

z): Values in the same line with different literal differ at Duncan's multiple range test (P<0.05) and results are mean ± standard error of three replicates.

Effect of the temperature

The suitable temperature for the mycelial growth of Phellinus spp. was obtained at 30℃ (Fig. 1). Their mycelial growth was suppressed rapidly at the temperature higher than 30℃ and lower than 20℃. Heo et al. (2004) reported that the optimum culture temperature of P. baumii and P. igniarius was 25~30℃, Chi et al. (1996) reported the optimum culture temperature of P. linteus was 25~30℃, Rew et al. (2000) reported that the optimum culture temperature of P. gilvus was 25~30℃. It was concluded that the above results were similar with this study.

Fig. 1

Mycelial growth of P. linteus ASI 26099 (P. l), P. baumii Nongong (P. b), and P. gilvus KCTC 6653 (P. g) on MEA for 10 days at different temperatures. Vertical bars show standard errors (n = 3).

The carbon source promoting a mycelial growth and mycelial density of Phellinus spp. was glucose and mannose (Table 4). Among 10 carbon sources, mannose showed colony diameter of P. gilvus isolate KCTC 6653 was 84 mm. The mycelial density of P. gilvus isolate KCTC 6653 was compact in glucose. Chi et al. (1996) reported that the optimum culture carbon sources of P. linteus were glucose, mannose and dextrose.

Table 4

Effect of carbon source on the mycelial growth of Phellinus spp.

a): C; compact, SC; somewhat compact, ST; somewhat thin, T; thin

b): Br; brownish, SY; somewhat Yellowish, Y; Yellowish, W; Whitish

c): P. l; P. linteus ASI 26099, P. b; P. baumii Nongong, P. g; P. gilvus KCTC 6653

z): Values in the same line with different literal differ at Duncan's multiple range test (P < 0.05) and results are mean ± standard error of three replicates.

The nitrogen source promoting a mycelial growth of Phellinus spp. was malt extract, peptone and potassium nitrate (Table 5). The mycelial density of P. baumii isolate Nongong was compact in malt extract. Among 13 nitrogen sources, malt extract showed colony diameter of P. baumii isolate Nongong was 78 mm. Chi et al. (1996) reported that the optimum culture nitrogen sources of P. linteus were cassamino acid, alanine and glutamic acid.

Table 5

Effect of nitrogen source on the mycelial growth of Phellinus spp.

a): C; compact, SC; somewhat compact, ST; somewhat thin, T; thin

b): Br; brownish, SY; somewhat Yellowish, Y; Yellowish, W; Whitish

c): P. l; P. linteus ASI 26099, P. b; P. baumii Nongong, P. g; P. gilvus KCTC 6653

z): Values in the same line with different literal differ at Duncan's multiple range test (P < 0.05) and results are mean ± standard error of three replicates.

On the culture media which were mixed with 2% glucose as carbon source and then adjusted to the C/N ratio of 10 : 1 and 5 : 1, Phellinus spp. showed the most favorable mycelial growth (Table 6). Lee et al. (2004) reported that the optimum culture C/N ratio of P. linteus was 10 : 1. Also our results were similar to those of Chi et al. (1996).

Table 6

Effect of C/N ratio on the mycelial growth of Phellinus spp.

a): C; compact, SC; somewhat compact, ST; somewhat thin, T; thin

b): Br; brownish, SY; somewhat Yellowish, Y; Yellowish, W; Whitish

c): P. l; P. linteus ASI 26099, P. b; P. baumii Nongong, P. g; P. gilvus KCTC 6653

z): Values in the same line with different literal differ at Duncan's multiple range test (P < 0.05) and results are mean ± standard error of three replicates.

When various vitamins were added to the MMM medium, thiamine-HCl and biotine were very excellent for a mycelial growth of Phellinus spp. (Table 7). After 9 days cultivation, colony diameter of P. gilvus isolate KCTC 6653 for thiamine-HCl and biotine were 77 mm and 82 mm, respectively. Chi et al. (1996) reported that the optimum culture vitamins of P. linteus were biotin and Ca-pantothenic.

Table 7

Effect of vitamins on the mycelial growth of Phellinus spp.

a): C; compact, SC; somewhat compact, ST; somewhat thin, T; thin

b): Br; brownish, SY; somewhat Yellowish, Y; Yellowish, W; Whitish

c): P. l; P. linteus ASI 26099, P. b; P. baumii Nongong, P. g; P. gilvus KCTC 6653

z): Values in the same line with different literal differ at Duncan's multiple range test (P < 0.05) and results are mean ± standard error of three replicates.

When various organic acids were added to the MMM medium, succinic acid and lactic acid were very excellent for a mycelial growth of Phellinus spp., whereas acetic acid was no growth of Phellinus spp. (Table 8, Fig. 3). After 9 days cultivation, colony diameter of P. baumii isolate Nongong for succinic acid and lactic acid were 64 mm and 79 mm, respectively.

Table 8

Effect of organic acid on the mycelial growth of Phellinus spp.

a): C; compact, SC; somewhat compact, ST; somewhat thin, T; thin

b): Br; brownish, SY; somewhat Yellowish, Y; Yellowish, W; Whitish

c): P. l; P. linteus ASI 26099, P. b; P. baumii Nongong, P. g; P. gilvus KCTC 6653

z): Values in the same line with different literal differ at Duncan's multiple range test (P < 0.05) and results are mean ± standard error of three replicates.

Fig. 3

Mycelial growth of P. gilvus KCTC 6653 on the mushroom minimal medium with different organic acid source A-1: Acetic acid, A-2: Citric acid, A-3: Maleic acid, A-4: Lactic acid, A-5: Succinic acid, A-6: Fumaric acid.

When various mineral salts were added to the YM medium, MgSO4·7H2O and KH2PO4 were very excellent for a mycelial growth of Phellinus spp., whereas ZnSO4·7H2O was almost no growth of Phellinus spp. (Table 9). Chi et al. (1996) reported that the optimum culture mineral salt of P. linteus was MgSO4·7H2O.

Table 9

Effect of mineral salt on the mycelial growth of Phellinus spp.

a): C; compact, SC; somewhat compact, ST; somewhat thin, T; thin

b): Br; brownish, SY; somewhat Yellowish, Y; Yellowish, W; Whitish

c): P. l; P. linteus ASI 26099, P. b; P. baumii Nongong, P. g; P. gilvus KCTC 6653

z): Values in the same line with different literal differ at Duncan's multiple range test (P < 0.05) and results are mean ± standard error of three replicates.