Culture Conditions for the Mycelial Growth of Ganoderma applanatum.

Ganoderma applanatum is one of the most popular medicinal mushrooms due to the various biologically active components it produces. This study was conducted to obtain basic information regarding the mycelial culture conditions of Ganoderma applanatum. Based on the colony diameter and mycelial density, PDA, YMA and MCM media were suitable for the mycelial growth of the mushroom. The optimum temperature for mycelial growth was found to be 25~30℃. The optimum carbon and nitrogen sources were mannose and dextrin, respectively, and the optimum C/N ratio was 2 to 10 when 2% glucose was used. Other minor components required for the optimal growth included thiamine-HCl and biotin as vitamins, succinic acid and lactic acid as organic acids, and MgSO4·7H2O, KH2PO4 and NaCl as mineral salts.

It is estimated that there are 140,000 species of mushrooms worldwide, yet only 10% have been identified to date (Kirk et al., 2001). n class="Species">Mushrooms have long been valued as edible and medicinal resources. Ganoderma applanatum (Pers.) Karst, which belongs to Polyporaceae of basidiomycetes, grows spontaneously on the branches of the broad leaf tree. Specifically, G. applanatum forms semicircular carpophores on the branch in parallel. G. applanatum is found worldwide, including in Korea (Park, 1991), where it has long been used as a medicine for the treatment of various human tumorigenic diseases (Kim et al., 1980). The mushroom reportedly contains a variety of biologically active components, including bitter triterpenoids (Nishitoba et al., 1989), alnusenone, friedelin (Protiva et al., 1980), α-D-glucan and β-D-glucan (Mizuno et al., 1981; Usui et al., 1983). Moreover, G. applanatum has been reported to exert biological effects such as modulation of the humoral immune response and antibacterial and antiviral activities against pathogenic microorganisms via FDP (Fibrinogen Degradation Product) and its aqueous extract, respectively, without any acute toxicity (Kim et al., 1994a, b, c; Rym et al., 1999). This study was conducted to evaluate the culture conditions for the optimal mycelial growth of G. applanatum.

Materials and Methods

Fungal isolates

The isolates of G. applanatum used in this study are listed in Table 1. n class="Species">G. applanatum ASI 50167, ASI 52821, ASI 52822, ASI 52823, ASI 53399 and G. lucidum ASI 7125 were obtained from the Rural Development Administration in Korea. G. applanatum GBGA-01 and GBGA-02 were collected in the wild. All isolates were maintained on Potato Dextrose Agar (PDA).

Table 1

List of G. applanatum strains used in this study

Effect of pH

To screen for the optimal pH value for the favorable growth of G. applanatum species, 5 mm diameter plugs were removed from 5 day old cultures of n class="Species">G. applanatum grown on PDA using a cork borer. The plugs were then placed in the center of PDA plates with pHs that had been adjusted to 4~9 using 1 N NaOH or HCl. The samples were then incubated in the dark for 5 days at 25℃, after which the mycelial growth was measured according to the method described by Shim et al. (1997).

Temperature

The growth of the mushrooms was evaluated at temperatures ranging from 10~35℃. The fungi were cultured on n class="Chemical">PDA for 6 days and the mycelial growth was determined as described above.

Culture media

Twelve different culture media were screened to determine if they were suitable for the mycelial growth of G. applanatum and n class="Species">G. lucidum (Table 2). All media were sterilized for 20 minutes at 121℃ and then aseptically poured into plastic Petri dishes. An inoculum was then removed from five day old cultures of G. applanatum grown on PDA at 25℃ and a mycelial disk (5 mm in diameter) was placed in the center of the prepared media. The fungi were then incubated in the dark for 5 days at 25℃, after which the mycelial growth, density and color of the colonies were examined.

Effect of favorable nutrient sources

Carbon sources

Suitable carbon sources were screened by culturing the n class="Species">mushroom on mushroom minimal media (MMM: 20 g dextrose, 0.5 g MgSO4, 0.46 g KH2PO4, 1 g K2HPO4, 2 g asparagine, 120 µg thiamine-HCl 120, 20 g agar, 1,000 ml distilled water) supplemented with one of 10 carbon sources at a concentration of 2%. The fungi were incubated in the dark for 7 days at 25℃, after which the mycelial growth, density and color of the colonies were evaluated.

Nitrogen sources

To screen for nitrogen source suitable for the mycelial growth of n class="Species">G. applanatum mushrooms were cultured on the mushroom minimal media supplemented with each of 12 nitrogen sources at a concentration of 0.2%. A 5mm diameter plug an inoculum of G. applanatum was placed in the center of the Petri dish, which was then incubated in the dark for 6 days at 25℃. The mycelial growth, density and color of the colonies were then examined.

C/N ratio

To screen for the optimal C/N ratio, n class="Species">mushroom minimal media were prepared using 10, 8, 6, 4, 2, 1, 0.4 and 0.2% glucose as the carbon source and 0.2% NaNO3 as the nitrogen source to give C/N ratios of 50 : 1, 40 : 1, 30 : 1, 20 : 1, 10 : 1, 5 : 1, 2 : 1 and 1 : 1, respectively. The Petri dishes were then inoculated with G. applanatum and incubated in the dark for 6 days at 25℃, after which the mycelial growth, density and color of the colonies were examined.

Vitamins

To determine which vitamins were suitable for the mycelial growth of G. applanatum, n class="Species">mushrooms were cultured on sterilized mushroom minimal media that had been amended with thiamine-HCl (0.1 mg/l), riboflavin (0.5 mg/l), biotin (0.005 mg/l), pyridoxine (0.5 mg/l) or nicotinamide 2.0 (mg/l) and then filtered through a metrical membrane filter with a pore size of 0.2 µm. The Petri dishes were incubated in the dark for 5 days at 25℃, after which the mycelial growth, density and color of the colonies were examined.

Organic acid

To screen for mineral salts suitable for the mycelial growth of n class="Species">G. applanatum, mushroom minimal media was prepared using acetic acid, citric acid, maleic acid, lactic acid, succinic acid or fumaric acid at a concentration of 0.1%. The Petri dishes were then inoculated with G. applanatum and cultured in the dark for 5 days at 25℃, after which the mycelial growth, density and color of the colonies were examined.

Mineral salt

To screen for mineral salts suitable for the mycelial growth of n class="Species">G. applanatum, the mushrooms were cultured on YM solid media (5 g peptone, 3 g yeast extract, 3 g malt extract, 10 g dextrose, 20 g agar and 1,000 ml DW (distilled water). Consider specifying, also specify if Millipore water supplemented with one of 9 mineral salts at a concentration of 0.1%. The Petri dishes were inoculated with G. applanatum and then cultured in the dark for 7 days at 25℃, after which the mycelial growth, density and color of the colonies were examined.

Results and Discussion

A pH of 6~9 was found to be suitable for the growth of G. applanatum. However, the mycelial growth and density of n class="Species">G. applanatum was almost identical at all pHs within this range (Table 3). The optimum pH range for the growth of Ganoderma spp. has been reported to be 5~9 and 5.5~6.0 (Jayasinghe et al., 2008; Cho et al., 1993). The results of the present study suggest that the growth of G. applanatum mycelia can occur at a broad pH range.

Table 3

Effect of pH on the mycelial growth of G. applanatum at 25℃

aValues in the same line with different letters differ significantly according to Duncan's multiple range test (P<0.05). Results shown are the mean ± standard deviation of three replicates.

bC, compact; SC, somewhat compact; ST, somewhat thin; T, thin.

Effect of temperature

Temperatures ranging from 25~30℃ were found to be suitable for the mycelial growth of G. applanatum (Fig. 1, 2). However, the mycelial growth of n class="Species">G. applanatum was suppressed rapidly at temperatures above 30℃ and below 20℃. These findings are similar to those of a study conducted by Jeong et al. (2005), who reported that the optimum temperature for the growth of G. applanatum was 25℃.

Fig. 1

Mycelial growth of G. applanatum on PDA for 6 days at different temperatures. Vertical bars show the standard errors (n = 3). 1: 10℃, 2: 15℃, 3: 20℃, 4: 25℃, 5: 30℃, 6: 35℃.

Fig. 2

Colonies of G. applanatum grown on PDA for 6 days at 30℃. A, GBGA-01; B, GBGA-02; C, ASI 50167; D, ASI 52821; E, ASI 52822; F, ASI 52823; G, ASI 53399; H, ASI 7125.

Screening for suitable culture media

The mycelial growth of G. applanatum was favorable in n class="Chemical">PDA, YMA, and MCM, whereas it was poor in Czapek Dox, Leonian, Hennerberg and Hoppkins medium (Table 4). The mycelial growth of G. applanatum isolate ASI 52821 was lower than that of GBGA-01, GBGA-02, ASI 50167, 52821, 52822, 52823, 53399, and 7125. The mycelial densities of G. applanatum isolates GBGA-01, GBGA-02, ASI 50167, 52821, 52822, 52823, and 53399 were greater than that of G. lucidum isolate ASI 7125. The colonies formed by the seven G. applanatum isolates and G. lucidum isolate ASI 7125 were white. Shim et al. (2005) also reported that PDA, YM, Mushroom Complete Medium and Hamada were the suitable for the growth of Macrolepiota procera, whereas Czapex Dox and Glucose Peptone media were not.

Table 4

Effect of culture medium on the mycelial growth of G. applanatum at 25℃

aValues in the same line with different letters differ significantly according to Duncan's multiple range test (P < 0.05). Results shown are the mean ± standard deviation of three replicates.

bC, compact; SC, somewhat compact; ST, somewhat thin; T, thin.

Dextrin and n class="Chemical">mannose were found to promote mycelial growth and increased mycelial density of G. applanatum (Table 5). Of the 10 carbon sources evaluated, mannose led to the formation of G. applanatum isolate GBGA-01 colonies with the greatest diameter (64.3 mm). The mycelial density of G. applanatum isolate GBGA-02 was compact in mannose. Jeong et al. (2005) reported that the optimum carbon source for the growth of G. applanatum was glucose. Jayasinghe et al. (2008) reported that dextrin was the best carbon source for the mycelial growth of G. lucidum. Griffin (1994) suggested that mannose and fructose were the most commonly utilized sugars after glucose.

Table 5

Effect of carbon source on the mycelial growth of G. applanatum at 25℃

aValues in the same line with different letters differ significantly according to Duncan's multiple range test (P < 0.05). Results shown are the mean ± standard deviation of three replicates.

bC, compact; SC, somewhat compact; ST, somewhat thin; T, thin.

The nitrogen sources that promoted the mycelial growth of n class="Species">G. applanatum were malt extract and yeast extract (Table 6). The mycelial density of G. applanatum isolate GBGA-01 was compact when grown in the presence of yeast extract. Among the 13 nitrogen sources evaluated, yeast extract resulted in the formation of G. applanatum isolate GBGA-01 colonies with a diameter of 85 mm. Jeong et al. (2005) reported that the optimum nitrogen source for the culture of G. applanatum was corn steep power (10%).

Table 6

Effect of nitrogen source on the mycelial growth of G. applanatum at 25℃

aValues in the same line with different letters differ significantly according to Duncan's multiple range test (P < 0.05). Results shown are the mean ± standard deviation of three replicates.

bC, compact; SC, somewhat compact; ST, somewhat thin; T, thin.

C/N ratios

The C/N ratios promoting the mycelial growth of n class="Species">G. applanatum were 1 : 1, 2 : 1, 5 : 1 and 10 : 1 (Table 7, Fig. 3). The mycelial density of G. applanatum isolate GBGA-01 was compact when it was cultured in the presence of C/N ratios of 10 : 1, 5 : 1 and 2 : 1. Among the 8 C/N ratios evaluated, a C/N ratio of 5 : 1 resulted in the growth of G. applanatum isolate GBGA-02 colonies with a diameter of 52.3 mm. Jo et al. (2006) reported that the optimum culture C/N ratios for Phellinus spp. were 10 : 1 and 5 : 1.

Table 7

Effect of C/N ratio on the mycelial growth of G. applanatum at 25℃

aValues in the same line with different letters differ significantly according to Duncan's multiple range test (P < 0.05). Results shown are the mean ± standard deviation of three replicates.

bC, compact; SC, somewhat compact; ST, somewhat thin; T, thin.

Fig. 3

Mycelial growth of G. applanatum GBGA-01 on PDA containing different C/N ratios. A: 50, B: 40, C: 30, D: 20, E: 10, F: 5, G: 2, H: 1.

When various vitamins were added to the MMM medium, n class="Chemical">thiamine-HCl and biotin produced excellent growth of the mycelium of G. applanatum (Table 8). After 5 days of cultivation, the diameter of colonies of G. applanatum isolate GBGA-01 grown in thiamine-HCl and biotin were 77.0 mm and 76.7 mm, respectively. Cho et al. (1993) reported that the optimum culture vitamins of G. lucidum were nicotinic acid and pantothenic acid.

Table 8

Effect of vitamins on the mycelial growth of G. applanatum at 25℃

aValues in the same line with different letters differ significantly according to Duncan's multiple range test (P < 0.05). Results shown are the mean ± standard deviation of three replicates.

bC, compact; SC, somewhat compact; ST, somewhat thin; T, thin.

Organic acids

When various organic acids were added to the n class="Chemical">MMM medium, succinic acid and lactic acid were found to be excellent for the mycelial growth of G. applanatum (Table 9). After 5 days of cultivation, the colony diameters of G. applanatum isolate GBGA-01 grown in the presence of succinic acid and lactic acid were 47.3 mm and 45.0 mm, respectively.

Table 9

Effect of organic acid on the mycelial growth of G. applanatum at 25℃

aValues in the same line with different letters differ significantly according to Duncan's multiple range test (P < 0.05). Results shown are the mean ± standard deviation of three replicates.

bC, compact; SC, somewhat compact; ST, somewhat thin; T, thin.

Mineral salts

When various mineral salts were added to the n class="Chemical">YM medium, MgSO4·7H2O, KH2PO4 and NaCl were found to be excellent for the mycelial growth of G. applanatum, whereas ZnSO4·7H2O resulted in almost no growth of G. applanatum isolates GBGA-01, ASI 52821, ASI 52822 and ASI 52823. (Table 10). Chi et al. (1996) reported that the optimum growth of P. linteus occurred when MgSO4·7H2O was provided as the mineral salt.

Table 10

Effect of mineral salts on the mycelial growth of G. applanatum at 25℃

aValues in the same line with different letters differ significantly according to Duncan's multiple range test (P < 0.05). Results shown are the mean ± standard deviation of three replicates.

bC, compact; SC, somewhat compact; ST, somewhat thin; T, thin.

Table 2

Composition of media used in this study