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Bangladesh J. Mushroom.

2(2): 33-42, 2008 (December)

Effect of Culture Media and Environmental Factors on the Mycelial Growth of Volvariella volvacea
Mahbuba Moonmoon, Md. Nazim Uddin, Abdus Salam Khan and S.M. Ruhul Amin National Mushroom Development and Extension Centre, Savar, Dhaka, Bangladesh

Abstract
Studies on nutritional requirement and environmental factors were carried out on synthetic culture media to evaluate mycelial growth of Volvariella volvacea. Among the different culture media used, the best mycelial growth (0.87 cm/day) and density of mycelium of this mushroom was observed on semi-synthetic, potato dextrose agar (PDA) medium while the least growth (0.57 cm/ day) was recorded on wheat extract yeast agar (WEYA) medium. The optimum temperature that suported the best growth of this fungus was 350 C while the optimum pH was 6.5. In case of light, the best mycelial growth was found in alternation of light (200-280 lux) and dark (0 lux) condition. Among five different carbon sources, sucrose was the best for mycelial growth of V. volvacea. As nitrogen source, peptone was found best for vegetative growth of V. volvacea. No significant variation was observed between the mycelial growth in different C/N ratio (6:1, 12:1, 18:1, 24:1, 36:1, 48:1 and 96:1).

Key words: Volvariella volvacea, culture media, environmental factors, C/N ratio and mycelial growth.

INTRODUCTION Volvariella volvacea is one of the most popular mushrooms in the world, especially in the tropical regions (Zoberi, 1972). In Bangladesh, this mushroom is common during the summer season and commonly known as straw mushroom. It has been reported to be a very good source of essential vitamins, amino acids, glycogen and phosphorus (Alofe, 1985). Ibekwe et al. (2008), Stamet (1993), Kadiri and Kehinde (1999) reported that mycelial stage of mushroom was significantly influenced by the carbohydrate source, nitrogen source, temperature, pH and light intensity. Usually, potato dextrose agar (PDA), malt extract agar (MEA), yeast extract agar (YEA), cane juice agar (CJA), wheat extract agar (WEA), malt extract yeast agar (MEYA), wheat extract yeast agar (WEYA) and potato dextrose yeast agar (PDYA) media have been used as culture media for mycelial growth of mushroom. Fasola et al. (2007) reported that potato dextrose agar (PDA) is the best culture media for mycelial growth of Volvariella speciosa. Fasola et al. (2007) also reported that the maximum, optimum and minimum mycelial growth of Volvariella speciosa at the pH of 9.0, 6.0 and 3.0, respectively while no growth was observed at pH 2.0 and 10.0. Generally, the growth of this fungus reduced at very strong acidic and alkaline pH. Jonathan and Fasidi (2004) observed very good mycelial growth of Volvariella esculenta at pH 6.0. Moreover, mycelial growth of this fungus is very sensitive to environmental factors. The optimum temperature range for the growth of its mycelium is 30-350 C and mycelium does not grow at all when the temperature is raised to 450 C or falls bellow 150 C (Chadha and Sharma, 2005). Ibekwe et al. (2008) found that the absence of light promotes the yield best, while the alternation of light and

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darkness gave better yield and continuous light gave little growth and yield. Volvariella volvacea utilized starch and glucose better than other carbon sources (Ofosu-Asiedu et al., 1984). But the better mycelial growth of Volvariella diplasia on starch, glucose and fructose (Garcha and Kalra, 1979). In case of nitrogen sources, Volvariella requires less nitrogen than carbon (Chadha and Sharma, 2005). In case of Bangladesh, a lot of information exist on the cultivation of oyster mushroom but, very little is known about Volvariella volvacea. Although this mushroom has been cultivated from the very beginning of mushroom cultivation in Bangladesh, but unfortunately, no works had been done to evaluate the factors that enhance the growth and cultivation of Volvariella volvacea. Threfore, this study is aimed to find out useful information about the culture media, nutritional requirements and environmental factors that enhance the optimum growth and yield of Volvariella volvacea, commonly cultivated in Bangladesh.

MATERIALS AND METHODS This experiment was conducted in the tissue culture laboratory of National Mushroom Development and Extension Center (NAMDEC), Sobhanbag, Savar, Dhaka during July to October, 2008. The inocula of Volvariella volvacea were used from the germplasm center of NAMDEC. The inocula used in different experiments were performed with 4 replications. Study of the different growth media on mycelial growth of V. volvacea : Eight different culture media such as potato dextrose agar (PDA), malt extract agar (MEA), yeast extract agar (YEA), cane juice agar (CJA), wheat extract agar (WEA), malt extract yeast agar (MEYA), wheat extract yeast agar (WEYA) and potato dextrose yeast agar (PDYA) medium were used to investigate the mycelial growth of Volvariella volvacea (Table 1).The basal component of each media mixed with agar and the mixture was boiled until the agar dissolved. Chloramphenicol BP 0.5% (100 5l) was aseptically added to the medium after it had cooled to prevent bacterial contamination. The media was poured into petri plates at 20 ml/plates after adjusted pH 6.5. The media were autoclaved for 20 minutes at 121o C temperature and 1kg/cm2 pressure and cooled at normal temperature. Then same size of inocula was transferred from culture grown on PDA to placed in the centre of petri plates of each media. Then the inoculated petri dishes were transferred into incubation room for mycelial growth at 210 to 25o C temperaure. . Study of the different pH on mycelial growth of V. volvacea : The selected medium, PDA was adjusted at different pH level with the addition of 1N HCl or NaOH before autoclave. The pH ranges were 4.5, 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, 8.0, 8.5, and 9.0. After autoclaving, the petri plates were inoculated and transferred into incubation room for mycelial growth at 210 to 25o C temperaure. Study of the temperature on mycelial growth of V. volvacea : The effect of temperature on growth of this fungus was investigated on PDA medium. The medium was adjusted at pH 6.5. Therefore, the medium dispensed into Petri- plates, allowed to solidify, autoclaved and inoculated with a 0.6 cm (diameter) disc of vigorously growing

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culture of Volvariella volvacea and incubated at 12-15o C, 19-22o C, 24-27o C, 28-31o C, 35 and 40o C separately.
Table 1. Different culture media and their constituents used in this study Composition (g/l) PDA PDYA MEA MEYA YEA WEA WEYA CJA Agar 20 20 20 20 20 20 20 20 Dextrose 20 20 Potato 250 250 Malt powder 50 50 Yeast powder 20 20 20 20 120 120 Wheat powder Cane juice 200 PDA: Potato dextrose agar, PDYA: Potato dextrose yeast agar, MEA: Malt extract agar, MEYA: Malt extract yeast agar, YEA: Yeast extract agar, WEA: Wheat extract agar, WEYA: wheat extract yeast agar, CJA: Cane juice agar

Study of the light on mycelial growth of V. volvacea : The selected medium (PDA) was employed to evaluate the suitable light for mycelial growth of V. volvacea. The medium was adjusted at pH 6.5 before autoclaved. After cooling the Petri- plates were inoculated with the inocula of Volvariella volvacea. Then the inoculated plates were incubated at different light condition viz: 0 lux (continuous dark), 40 to 60 lux, alternation of light (40-60 lux) and dark (0 lux),120 to 150 lux, alternation of light (120-150 lux) and dark (0 lux), 250 to 300 lux continuous, alternation of light (250-300 lux) and dark (0 lux) as well as alternation of light(200- 280 lux) and dark (0 lux) separately at 210 to 25o C. Study of the C/N ratio of culture medium on mycelial growth of V. volvacea : Seven different ratio of carbon (Glucose) and nitrogen (Yeast) source such as 6:1, 12:1, 18:1, 24:1, 36:1, 48:1 and 96:1 were used to investigate the mycelial growth of Volvariella volvacea. Glucose, yeast mixed with 2% agar and the mixture was boiled until the agar dissolved. The agar medium was then dispensed into Petri- plates, allowed to solidify, inoculated with a 0.6 cm (diameter) disc of vigorously growing culture of Volvariella volvacea and incubated at 210 to 25o C. Study of the carbon and nitrogen source on mycelial growth of V. volvacea : Five different carbon source such as glucose (pH 6.63), maltose (pH 6.65), sucrose (pH 6.68), fructose (pH 6.50) and lactose (pH 6.47) were used to investigate the mycelial growth of V. volvacea. Two percent of each carbon source mixed with 2% agar and the mixture was boiled until the agar dissolved. The media was poured into petri plates at 20 ml/plates. Same size of inocula grown on PDA was placed in the centre of each media after autoclaving for 20 minutes at 120o C temperature and 1kg/cm2 pressure and cooling. Then petri- plates were transferred in to incubation room for mycelial growth at 210 to 25o C temperature. In case of nitrogen source, peptone (pH 6.45), ammonium nitrate (NH4NO3) (pH 6.02), sodium nitrate (NaNO3) (pH 6.70), yeast (pH 6.51) and urea (pH 7.57) were used to investigate the mycelial growth of V. volvacea.

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Comparative study of potato dextrose agar (PDA) media, sucrose, peptone and their combination on mycelial growth of V. volvacea : Potato dextrose agar (PDA) media, sucrose sgar, peptone agar and their combination (PDA + sucrose, PDA + peptone, sucrose + peptone + PDA, sucrose + peptone) were used as culture media to investigate the mycelial growth of V. volvacea (Table 2). The basal component of each media were mixed with agar. The mixtures were boiled to dissolved the agar, poured into petri plates, autoclaved, cooled at normal temperature, inoculated by V. volvacea and incubated as mentioned before.
Table 2. Culture media and their constituents used in this study Culture media Potato dextrose agar (PDA) Sucrose agar Peptone agar Sucrose agar + peptone Potato dextrose agar (PDA) + sucrose Potato dextrose agar (PDA) + peptone Potato dextrose agar (PDA) + sucrose + peptone Potato 250 250 250 250 Composition (g/l) Dextrose Sucrose Peptone 20 20 20 20 20 20 20 20 20 20 20 20 Agar 20 20 20 20 20 20 20

Analysis of data: The experiment was laid out following by completely randomized design (CRD) with 4 replications. Data on mycelium growth rate (cm/day), days to complete mycelium running (days) were recorded and analyzed following Gomez and Gomez, 1984 and using MSTAT-C computer program. Means were computed following DMRT using the same computer program. The following formula was used to calculate the mycelial growth. Average mycelial growth on each petri- plate = [{(OA1+OB1+OC1+OD1)/ 4}/ days + {(A1A2+B1B2+C1C2+D1D2)/ 4}/ days] / 2

Fig. 1. Method of measuring mycelial growth on Petri plate

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RESULT AND DISCUSSION Study of the different growth media: The effect of mycelial growth and duration of complete mycelium running on eight different culture media is shown in Table 3. Significantly the highest mycelial growth (0.87 cm/day) was found in PDA medium which was statistically similar to that on MEYA (0.83 cm/day). The lowest mycelial growth (0.57 cm/day) was found in WEYA. The density of mycelium on PDA was higher than other culture media. In case of duration of complete mycelium running, the minimum time (5.0 days) was recorded in PDA medium and the maximum time (7.50 days) was found in WEYA medium. This result is similar with the findings of Fasola et al. (2007) who reported potato dextrose agar (PDA) as the best culture media for mycelial growth of V. speciosa. The augment of growth by PDA is not a surprise, because this medium has been widely reported to support mycelial growth of mushroom (Alofe, 1985; Fasidi, 1996; Huang, 1993; Oso, 1977). Jonathan and Fasidi (2001a, 2001b) also obtained better mycelial growth of P. atroumbonata, L. subnudus and S. commune when PDA was supplemented with 0.5% yeast extract.
Table 3. Study of the different growth media on mycelial growth of V. volvacea Mycelial growth (cm/day) Potato dextrose agar (PDA) 0.87 a Potato dextrose yeast agar (PDYA) 0.81 b Malt extract agar (MEA) 0.68 e Malt extract yeast agar (MEYA) 0.83 ab Yeast extract agar (YEA) 0.73 de Wheat extract agar (WEA) 0.80 bc Wheat extract yeast agar (WEYA) 0.57 f Cane juice agar (CJA) 0.76 cd CV (%) 4.13 Culture Media Days to compelete mycelium run (day) 5.0 d 5.57 cd 6.0 bc 5.50 cd 6.75 ab 5.75 cd 7.50 a 5.75 cd 9.0 Density of mycelium A B B C D B C C

In a column, means followed by a common letter are not significantly different at 5% level by DMRT. Here A, B, C and D are the symbol of the density of mycelium and A > B > C > D.

Study of the different pH: To determine the suitable pH on PDA media with the pH range of 4.5 to 9.0 were used for mycelial growth of V. volvacea. The best mycelial growth (0.87 cm/day) was observed in slightly acidic medium (pH 6.5) which was statistically similar to pH 6.0 and pH 7.0. Likewise, Chandra and Purkayastha (1977) and Jonathan and Fasidi (2004) obtained very good growths of Agaricus campestris and V. esculenta at pH 6.0. This result is not similar with the findings of Chang & Miles (1987) who reported 7.5 as the best pH level for mycelial growth of V. volvacea. The lowest mycelial growth (.50 cm/day) was recorded at pH 4.5. In case of duration of complete mycelium running, the minimum time (5.0 days) was recorded at pH 6.5 which was statically similar to pH 6.0 and pH 7.0 and the maximum time (9.25 days) was found at pH 4.5. Fasola et al. (2007) reported that pH 6.0 is best for mycelial growth of V. speciosa. Ibekwe et al (2008) reported that the optimum mycelia growth of Pleurotus ostreatus was recorded at pH 6.5.

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Table 4. Study of the different pH of culture media on mycelial growth of V. volvacea pH level 4.5 5.0 5.5 6.0 6.5 7.0 7.5 8.0 8.5 9.0 CV (%) Mycelial growth (cm/day) 0.51 g 0.53 fg 0.63 de 0.84 a 0.87 a 0.82 ab 0.60 df 0.58 eg 0.67 cd 0.75 bc 8.28 Days to compelete mycelium run (day) 9.25 a 8.00 b 7.00 c 5.25 de 5.00 e 5.25 de 7.00 c 8.00 b 6.00 d 6.00 d 8.76

In a column, means followed by a common letter are not significantly different at 5% level by DMRT.

Study of the temperature: Table 5 shows the effect of temperature on mycelial growth of V. volvacea. The best mycelial growth (1.438 cm/days) was obtained at 350 C which was significantly higher on compare to all the treatment. The lowest mycelial growth (0.050 cm/day) was recorded in 120-150 C. In case of duration of complete mycelium running, the minimum time (3.0 days) was recorded at 350 C. which was statistically similar to 190-220 C. Incase of 120-150 C temperature, the duration of complete mycelium running was more than 25 days. This result is similar with that of Cang and Chu (1969) who reported 350 C as the optimum temperature for mycelial growth of V. volvacea.
Table 5. Study of the temperature on mycelial growth of V. volvacea Temperature ( 0c) 12-15 19-22 24-27 28-31 35 40 CV (%) Mycelial growth (cm/day) 0.050 e 0.38 d 0.735 c 1.085 b 1.438 a 0.443 d 8.24 Days to compelete mycelium run (day) > 25 a 10.75 b 8.0 c 4.0 d 3.0 d 10.50 b 9.91

In a column, means followed by a common letter are not significantly different at 5% level by DMRT.

Study of the light: The effect of different light conditions on the mycelial growth is shown in Table 6. The highest mycelial growth (1.070 cm/day) was found in alternation of light (200-280 lux) and dark (0 lux) condition and the lowest mycelial growth (0.55 cm/day) was as found in alternation of light (120-180 lux) and dark (0 lux) condition. In case of duration of complete mycelium running, the minimum time (4.0 days) was recorded in alternation of light (200-2850lux) and dark (0 lux) condition and the maximum time (7.75 days) was found in alternation of light (40-60 lux) and dark (0 lux) condition. Liao (1990) reported that 12 hours of alternating light gave better than continuous light and darkness for mycelial growth of Coriolus versicolor.

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Table 6. Study of the light on mycelial growth of V. volvacea Light 0 Lux 40- 60 Lux Alternation of light (40-60 lux) and dark (0 lux) 120-180 Lux Alternation of light (120-180 lux) and dark (0 lux) 250-300 Lux Alternation of light (250-300 lux) and dark (0 lux) Alternation of light (200-280 lux) and dark (0 lux) CV (%) Mycelial growth rate (cm/day) 0.7075 c 0.610 de 0.5825 de 0.7500 c 0.55 e 0.8525 b 0.6375 d 1.070 a 5.72 Days to compelete mycelium run 6.0 c-d 6.75 b 7.75 a 5.75 d 7.0 b 5.0 e 6.50 bc 4.0 f 6.04

In a column, means followed by a common letter are not significantly different at 5% level by DMRT.

Study of the C/N ratio of culture medium: The effect of different C/N ratio on the mycelial growth of V. volvacea is shown in Table 7. Although the highest mycelial growth (1.115 cm/day) was shown at 36:1 C/N ratio and the lowest (0.79 cm/day) was found in 6:1, we did not find any significant variation of mycelial growth of V. volvacea in this experiment. In case of duration of complete mycelium running, the minimum time (4.0 days) was recorded at 36:1 C/N ratio.
Table 7. Study of the C/N ratio of culture medium on mycelial growth of V. volvacea. C/N ratio 6:1 12 : 1 18 : 1 24 : 1 36 : 1 48 : 1 96 : 1 CV (%) Mycelial growth rate (cm/day) 0.79 a 1.095 a 1.027 a 1.055 a 1.115 a 1.045 a 0.99 a 20.85 Days to complete mycelium run 5.0 a 5.0 a 4.75 a 5.0 a 4.0 b 5.0 a 5.0 a 3.92

In a column, means followed by a common letter are not significantly different at 5% level by DMRT.

Study of the carbon source: Among the five different carbon sources used to find out the optimal culture condition, the best mycelial growth (0.705 cm/day) of V. volvacea was found in sucrose and the most unsuitable source is lactose (0.190 cm/day). In case of duration of complete mycelium running, the minimum time (8.0 days) was recorded in sucrose which was statistically similar to glucose, maltose and fructose but the maximum time (18.25 days) was found in lactose. This result is similar to that of Imtiaj et al. (2008) where sucrose was found to be the best for mycelial propagation of A. cylindracea and lactose was the most unfavorable carbon source to mycelial growth. It is also similar to that of Shim et al. (1997, 2005).

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Table 8. Study of the carbon source on mycelial growth of V. volvacea Carbon source Glucose Maltose Sucrose Fructose Lactose CV (%) Carbon (%) 40.0 42.1 42.1 40.0 42.1 Mycelial growth (cm/day) 0.535 b .0582 b 0.705 a 0.417 c 0.190 d 8.01 Days to complete mycelium run (day) 8.75 b 8.50 b 8.00 b 10.25 b 18.25 a 20.84

In a column, means followed by a common letter are not significantly different at 5% level by DMRT.

Study of the nitrogen source: Among the five different nitrogen sources used to find out the optimal culture condition, the best mycelial growth (0.705 cm/day) of V. volvacea was found in peptone and the lowest mycelial growth (0.1475 cm/day) was found in ammonium nitrate (NH4NO3). But the most unsuitable source is urea. In case of duration of complete mycelium running, the minimum time (6.0 days) was recorded in peptone which was statistically similar to yeast and the maximum time (24.75 days) was found in ammonium nitrate (NH4NO3). But there was no result found in urea. This result is similar to that of Kadiri and Fasidi (1994) where peptone was found to be the best nitrogen source for mycelial propagation of L. subnudus. Luo (1993) reported that peptone is the preferred nitrogen source for mycelial growth of A. auricular.
Table 9. Study of the nitrogen source on mycelial growth of V. volvacea. Nitrogen source Peptone Ammonium nitrate (NH4NO3) Sodium nitrate (NaNO3) Yeast Urea CV (%) Nitrogen (%) 14.2-15.5 35.0 33.0 * 47.0 Mycelial growth (cm/day) 0.705 a 0.147 d 0.537 c .0612 b 5.97 Days to complete mycelium run (day) 6.0 c 24.75 a 7.50 b 6.50 c 5.62

In a column, means followed by a common letter are not significantly different at 5% level by DMRT. Here * means unknown.

Comparative study of potato dextrose agar (PDA) media, sucrose, peptone and their combination on mycelial growth of V. volvacea : The comparative effect of potato dextrose agar (PDA) media, sucrose, peptone and their combination on the mycelial growth of V. volvacea is shown in Table 10. Significantly the highest mycelial growth (0.87 cm/day) was found in PDA media. The lowest mycelial growth (0.6125 cm/day) was found in the combination of PDA, sucrose and peptone. The density of mycelium on PDA was more than sucrose, peptone and their combination. Fasola et al. (2007) reported that PDA is the best culture media for mycelial growth of V. speciosa. But, Ofosu-Asiedu et al. (1984) reported that the best mycelial growth of V. volvacea was obtained on media containing peptone or potassium nitrate.

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Table 10. Comparative study of potato dextrose agar (PDA) media, sucrose, peptone and their combination on mycelial growth of V. volvacea Culture Media Potato dextrose agar (PDA) Sucrose agar Peptone agar Sucrose agar + peptone Potato dextrose agar (PDA) + sucrose Potato dextrose agar (PDA) + peptone Potato dextrose agar (PDA) + sucrose + peptone CV(%) Mycelial growth (cm/day) 0.87 a 0.7050 b 0.6925 bc 0.6800 bc 0.6425 cd 0.655 b-d 0.6125 d 3.72 Density of mycelium A D D C C B C

In a column, means followed by a common letter are not significantly different at 5% level by DMRT. Here A, B, C and D are the symbol of the density of mycelium and A > B > C > D.

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