Effect of Various Sawdusts and Logs Media on the Fruiting Body Formation of Phellinus gilvus.

Present experiments were conducted to determine the possibility of artificial culture with various sawdust of P. gilvus. The pH value was 6.0 of oak sawdust, 6.5 of mulberry sawdust, 6.6 of elm sawdust, 6.3 of acacia sawdust and 6.1 of apple tree sawdust. Mycelial density on elm sawdust and acacia sawdust were lower than those of oak sawdust, and apple sawdust. Weight of fresh fruiting body showed that 179 g on oak tree, 227 g on oak sawdust, 21 g on elm tree, 76 g on elm sawdust, 106 g on apple tree, and 170 g on apple sawdust. Among them, the yield of oak substrates was the highest whereas acacia sawdust was the lowest, and it is concluded that the yields of sawdust substrates were higher than log substrates. P. gilvus grown on various sawdusts and logs used in this study have shown similar in anti-tumor activity against P388.

Genus Phellinus is taxonomically classified into Aphyllophorales in Hymenochaetaceae of Basidiomycota (Larsen and Cobb-Poulle, 1990) and has been also known as a plant pathogen that causes white pocket rot and severe plant diseases such as canker or heart rot in living trees (Gilbertson, 1980). Phellinus spp. is known about 220 species and is found mainly in tropical America, Africa (Dai and Xu, 1998). In Korea, it is distributed into 6~8 species and commonly referred to as Sangwhang (Lee, 1993; Hong, 2000). Recently, many reports demonstrated that Phellinus species contained medicinally valuable substances. In Asian countries such as China, Korea and Japan, Phellinus species have been considered to cure stomachache and arthritis as an oriental medicine (Ying et al., 1987). It was reported that polysaccharide from P. linteus showed immuno stimulating activity (Lee et al., 1996), an inhibitory effect on tumor growth and metastasis (Han et al., 1999) and water extracts of P. linteus, P. baumii, and P. gilvus have shown anti-tumor activity against both sarcoma 180 and P388 (Bae et al., 2004), P. gilvus TMC-1117 showed biphasic vasodilator activity on rat aorta with endothelium (Hosoe et al., 2006). The physiological characteristics, chemical composition and cultivating methods of P. linteus including other species of Phellinus have been intensively researched (Chi et al., 1996; Jung et al., 1997 and Rew et al., 2000). Phellinus species have been isolated from various geographical regions and it was reported that P. linteus occurs mainly on Morus and various species of Quercus in Korea (Kim et al., 1999) and P. gilvus causes a white-pocket rot in Eucalypus crebra F. Muell, Eucalyptus diversicolor F. Muell (Barry et al., 2005) and occurs mainly broadleaf trees (Lee, 1993). Despite such a great medicinal value, study on artificial cultivation of P. gilvus has been rarely conducted. This present study was especially tried to elucidate the possibility of the artificial production of P. gilvus by cultural method using various sawdusts and logs.

Materials and Methods

Substrates analysis

Chemical compositions of substrates analysis applied correspondingly to RDA Soil Physico-Chemistry Analysis (Han, 1988). CaO, MgO and K2O analyzed with Atomic Absorption Spectrometer (Perkin Elmer 2380). Carbohydrate, nitrogen, P2O5 and pH analyzed with Tyurin assay, Kjeldahl assay, Colorimetric assay and pH-Meter (Fisher model 50), respectively.

Fungal isolates

Phellinus gilvus (KCTC 6653) was obtained from Biological Resource Center, KRIBB., and cultured at 30℃ on potato dextrose agar (PDA) medium. The medium was consisted of 0.4% potato extract, 2% dextrose, 1.5% agar and adjusted volume to 1,000 ml. Subcultures were made routinely every 10 days.

Inoculation

The PDA medium was sterilized for 20 minutes at 121℃ and aseptically poured into a petri-dish. After cooling, a piece of mycelia was inoculated on the PDA medium plate to use as an inoculum for the next step.

Mother spawn

The sawdust of oak tree (Quercus spp.) was mixed with rice bran at a ratio of 9 : 1 (v/v) and moisture content was adjusted to about 65% by adding water. Then the mixed medium was put into a 250 ml flask and sterilized at 121℃ for 90 minutes. After cooling to 20℃, a piece of mycelia from the PDA plate was inoculated on the sawdust medium to use as inoculum for the planting spawn.

Planting spawn

The planting spawn medium was prepared by the same method as used for the mother spawn. The medium containing sawdust and rice bran put into 850 ml polyethylene bottle and sterilized at 121℃ for 90 minutes and cooled at 20℃, mother spawn was inoculated to the sawdust culture medium in 850 ml polyethylene bottle. The inoculated sawdust media were incubated at 25℃ for about 35 days until mycelia spreaded all over the media and then used an inoculum for cultivation.

Sawdust cultivation process

The cultivation method for P. gilvus was modified orderly as follows; substrate preparation, transfer substrates to polyethylene bag, sterilization, inoculation, spawn run, initiation of primordium, and growing of basidiocarps.

Substrates and preparation

A variety of sawdusts collected from a local sawmill in Andong City, Gyeongsangbuk-Do, Province. The sawdust was collected and was stored in an enclosed warehouse until it was used. The media were prepared with sawdust of Quercus acutissima (oak), Morus alba (mulberry), Ulmus davidiana (elm), Malus pumila (apple) and Robinia pseudoacacia (acacia). The moisture of each sawdust medium adjusted to about 65% with water. 2 kg each medium put into a polypropylene bag (33 × 38 × 0.02 mm) and sterilized at 121℃ for 90 minutes. After cooling to 20℃, about 100 g inoculum was inoculated in a sawdust medium. The inoculated media were incubated in dark room 35 days at 22 ± 2℃ and duration of mycelial growth, mycelial density were examined. The five kinds logs were cut 20 cm in length, 12~15 cm in diagram and put into polypropylene bags. Sterilization and inoculation were made by the same method as described previously.

Experimental condition

After the completion of spawn run (35 days), the upper 70% part of polypropylene bags were removed and the synthetic media were placed in cultivation house. Relative humidity was maintained 80 to 90% during initiation of primordium and 60 to 70% during fruiting body growing. The temperature was maintained at 26 to 32℃ throughout the experiments. The fruiting body yields of P. gilvus mushroom on various sawdust media displayed wt. of dried individual fruiting body (g), length of pileus (mm), width of pileus (mm), thickness of pileus (mm), wt. of fresh fruiting body (g) and wt. of dried fruiting body (g).

Antitumor activity

Antitumor activity measured by the sulforhodamine B (SRB) assay (Kim et al., 1996) using murine P388 cells. The 180 µl RPMI 1640 medium containing 1 × 104 murine P388 cells added to each well of 96-well format and incubated at 37℃, 5% CO2, 24 hours. 20 µl extract of P. gilvus growing on various substrates added to each well of 96-well format and incubated at 37℃, 5% CO2, 48 hours. Cultures fixed with trichloroacetic acid were stained for 30 minute with 0.4% sulforhodamine B in 1% acetic acid. Unbound dye was removed by five washes with 1% acetic acid and protein-bound dye was extracted with 10 µM unbuffered Tris base. Absorbance was measured at 540 nm using a microplate reader.

Results and Discussion

Physico-chemical analysis

Physico-chemical analysis of medium resources, natural property investigation on each material: Chemical investigation showed us almost similar value of T-C percentage as like 46.6% of oak sawdust, 47.5% of mulberry sawdust, 46.3% of elm sawdust, 47.8% of acacia sawdust, and 43.2% of apple tree sawdust. Their pH value was 6.0 of oak sawdust, 6.5 of mulberry sawdust, 6.6 of elm sawdust, 6.3 of acacia sawdust and 6.1 of apple tree sawdust. It is weak acidity for oak sawdust, apple tree sawdust, neutrality for mulberry sawdust and elm sawdust (Table 1).

Table 1

Chemical compositions of substrates

aTotal carbon, bTotal nitrogen.

A heavy metal examination showed that value of Fe was 112.8 ppm of acacia sawdust, 61.0 ppm of elm sawdust, and their Mn value was 140.5 ppm of oak sawdust, 8.5 ppm of mulberry sawdust, and Cu value was 2.6 ppm of oak sawdust, 2.0 ppm of elm sawdust, and Zn value was 5.77 ppm of elm sawdust, 2.67 ppm of oak sawdust, and Pb value was 0.92 ppm of oak sawdust, 2.59 ppm of acacia sawdust, none of mulberry sawdust, elm sawdust, apple tree sawdust. As value was 1.53 ppm of oak sawdust, 4.27 ppm of elm sawdust, 0.14 ppm of acacia sawdust. Cd and Cr were not detected in all substrates (Table 2).

Table 2

Trace element compositions of substrates

Growth of mycelium in various sawdust substrate

To study for the possibility of artificial cultivation of P. gilvus, we investigated P. gilvus mycelium growing status with five kinds of sawdust or material lumber. It took about 2-times longer period of 54~63 days on cultivation with material lumber than 27~33 days on cultivation with sawdust until completing mycelial growth (Table 3, Fig. 1). Mycelial density on elm sawdust and acacia sawdust were lower than those of oak sawdust, apple sawdust. These result was similar that mycelial growth of P. linteus on oak sawdust, apple tree sawdust, peach tree sawdust were good, whereas acacia sawdust was poor (Chi et al. 1998). The days for primordium formation showed us almost similar as 11 days on oak sawdust and apple tree sawdust, 13 days on mulberry sawdust and apple tree (Fig. 2).

Table 3

Comparison of mycelial growth, mycelial density and primordium formation of P. gilvus with various medium

aMycelial density: ++ = low, +++ = medium, ++++ = high.

Fig. 1

Effect of timber and sawdust substrates on mycelial growth of P. gilvus KCTC 6653 at 25℃. A, Oak; B, Mulberry; C, Elm; D, Apple; F, Acasia.

Fig. 2

Effect of substrate on primordium formation of P. gilvus KCTC 6653. A, Oak; B, Mulberry; C, Elm; D, Apple; F, Acasia.

Character of fruiting body in various sawdust substrate

Weight of dried fruiting body showed that 76 g on oak tree, 84 g on oak sawdust, 10 g on elm tree, 36 g on elm sawdust, 46 g on apple tree, 65 g on apple sawdust. Weight of fresh fruiting body showed that 179 g on oak tree, 227 g on oak sawdust, 21 g on elm tree, 76 g on elm sawdust, 106 g on apple tree, 170 g on apple sawdust. Among them, the yield of oak substrates was the highest whereas acacia sawdust was the poorest. Rew et al. (2000) reported that the wt. of fresh fruiting body was 180 ± 86 g on oak tree, 150 ± 15 g on apple tree and 29 ± 8 g on pine tree. This is in accord with our experimental results, and it is concluded that the yields of sawdust substrates were higher than log substrates. Thickness of pileus (mm) showed that 3.1 mm on oak tree, 2.7 mm on oak sawdust, 2.9 mm on elm tree, 2.5 mm on elm sawdust and 2.5 mm on apple tree and 2.5 mm on apple sawdust. These results showed that the thickness of pileus on log substrates was thicker than sawdust substrates. The length of pileus (mm) showed that 87 mm on oak tree, 82 mm on oak sawdust, 67 mm mulberry tree, 95 mm on mulberry sawdust, 58 mm on elm tree and 65 mm on elm sawdust (Table 4, Fig. 3).

Table 4

Effect of substrate on the fruitbody yields of P. gilvus KCTC 6653

aWeighed as fresh weight, bWeighed as dried weight, cResults are mean ± standard deviation of ten replicates.

Fig. 3

Effect of substrate on fruitbodies of P. gilvus KCTC 6653. A, Cultivation on log; B, Cultivation on sawdust. a, Oak; b, Mulberry; c, Elm; d, Apple; e, Acasia.

Extract of many Phellinus spp. have an anti-tumor activity but a little is known regarding the anti-tumor activity of P. gilvus (Bae et al., 2004). By SRB assay, P. gilvus grown on sawdusts and logs of oak, mulberry, elm, apple, acacia showed similar in anti-tumor activity (Fig. 4).

Fig. 4

Anti-cancer activity against P388 cell line of P. gilvus KCTC 6653 grown on the various tree and sawdust. Superscript (a) indicate significant difference (P < 0.05).