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Original Article

Mycelial Culture and Spawn Production of two Oyster Mushrooms, Pleurotus florida and Pleurotus eous on Different Substrates E.P.Thulasi1, P. Danial Thomas1*, B. Ravichandran2 and K. Madhusudhanan3 1 Dept. of Microbiology, 2Dept. of Biotechnology, J. J. College of Arts & Science, Pudukkottai - 622 422, 3St. Albert’s College, Kochi, Ernakulam. *Corresponding Author Received:19.07.2010; Revised:23.11.2010;Accepted:29.11.2010;Published:01.12.2010 Abstract Three lignocellulosic substrates like coffee husk, cocoa pod powder and rubber saw dust were evaluated for the mycelial growth characters of two oyster mushrooms (Pleurotus species) in comparison with the traditional substrates like potato dextrose agar (PDA) and oat meal agar (OMA) for the cultivation of Pleurotus florida and Pleurotus eous. Their mycelial growth got completed on the 6th day at 24◦C in both coffee husk and cocoa pod powder media. The results showed an increase in the growth rate as the incubation temperature was increased from 24 to 28ºC. Rubber saw dust medium did not support either of the organisms. The OMA was found to be superior medium for the purpose. However, cocoa pod medium and coffee husk medium can be used as a substitute for PDA and OMA. For the spawn production four substrates such as coffee husk, chopped areca nut leaf sheath, sorghum and paddy grains were used as media. Among them sorghum grains, paddy grains and chopped areca nut leaf sheath supported P. eous for 100% colonization in 10, 16 and 10 days respectively whereas it took 18 days in coffee husk. On the other hand P. florida showed variations in incubation period in which its 100% growth reached on the16th day in sorghum, paddy grains and areca nut leaf sheath and 19 days in coffee husk medium. Among the four substrates sorghum grains was found to be the best and the coffee husk to be the least for the spawn production. Most probably this is due to the less required nutritional content of the substrate. Key words: Pleurotus florida, Pleurotus eous, Substrates, Mycelia, Spawn.

Introduction Edible and medicinal properties of mushrooms were known to many ancient civilizations. Only the reproductive structure comes out of the substrate and forms a fruiting body which is visible, called ‘mushroom’ which may be edible. Some mushrooms may be unpalatable, and others even poisonous, the mushrooms of many species are not only edible but delicious and nutritious (Chang and Miles, 1986). Most of the edible mushrooms belong to Ascomycotina and Basidiomycotina (Pathak et al.,2003). Some mushrooms, such as truffles and morels are Ascomycetes, but most of others are Basidiomycetes (Chang, 1981). The mushroom cultivation is a profitable agribusiness and particularly oyster mushroom which is edible having excellent flavour and taste (Shah et al., 2004). Growing oyster mushrooms has become more popular throughout the world, because of their abilities to grow at a wide range of temperatures utilizing various ligno – cellulosic substrates (Khan and Garcha 1984). Pleurotus species are

found to be efficient lignocellulose decomposing types of white rot fungi. Therefore, many agricultural and industrial wastes can be utilized as substrates for the production of Pleurotus species (Zadrazil and Brunnert, 1981). This is because of their ability to excrete hydrolyzing and oxidizing enzymes (Dougulis and Bone,1977). For spawn running and fruit body development, lignin and cellulosic materials, vegetable and food industry wastes are sufficient (Tautorus,1985). Various works using different substrates like sawdust (Block et al. 1958), paddy straw (Bano and Srivastava, 1962), banana pseudo stems (Jaindaik, 1974), newspaper (Hashimoto and Takahashi, 1974), wheat straw (Zadrazil,1974), hulled cocoa shells (Phettipher, 1987), maize cobs (Sivaprakasam and Kandaswamy,1981), cotton and sugar cane wastes (Chang,1980), sunflowers, millets, coffeebean husks, peanut shoots, pulse husks and tobacco stalks (Lelley, 1988) have been done on this regard. Nowadays sawdust is intensively utilized as a mushroom substrate at commercial scale. 39

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Mushroom cultivation helps conversion of lignocellulosic waste material into high quality protein food and this will naturally open up new job opportunities especially in rural areas. The objectives of this study were to evaluate three different types of substrates for cultivation and two substrates for spawn production and find out the most suitable one for the profitable production of oyster mushroom and also to determine the effects of temperature viz., 24˚C and 28˚C.

Materials and Methods Stock pure cultures Pure cultures of Pleurotus florida and Pleurotus eous maintained on Potato Dextrose Agar (PDA) were obtained from MSSRF, CAbC, Kalpetta(11°36´ N;76°05´E), Wayanad District,Kerala, S. India. Isolatation of spores Mature fruiting bodies of P. florida and P. eous were collected, cleaned with the help of cotton dipped in alcohol so as to remove the surface adhering particles of the fruiting bodies. The pileus was taken with presterilized blade. The cut pieces of fruiting bodies were then surface sterilized with 0.1% mercuric chloride for 1 minute and then washed in sterile distilled water for 2-3 times. PDA medium was used for isolation of the selected Pleurotus species. The medium was poured at rate of 15 ml per sterilized petriplates of 90 mm size in laminar air flow cabinet. The plates containing media was then allowed to solidify. The surface sterilized pieces of fruiting body were placed aseptically on to the media containing plates and were incubated for 7 days at laboratory temperature of 26±2˚C. The fungal growth was subcultured by hyphal top method from the growing tip of fungus on PDA slants. Spawn Production Paddy grain, sorghum seeds, chopped areca nut leaf sheath and coffee husk were soaked in water for 6-8 hrs separately and then sterilized. The sterilized substrates were then spread over a blotting paper and mixed with 4% calcium carbonate (40 gm/kg of substrates). The mixture was filled in 500 ml glucose bottles or milk bottles and the mouth was plugged with non-absorbent cotton and covered with aluminium foil and sterilized. After sterilization the bottles were allowed to cool under room temperature, inoculated with the mycelial disks of P. florida and P. eous under aseptic condition in a laminar air flow © Gayathri Teknological Publication

cabinet, recaped the bottles with cotton plug and placed the bottles at a temperature of about 25-30ºC in the spawn room, preferably in dark room. Observations were recorded on the percentage of mycelium run on the substrate. Substrate preparation For the standardization of P. florida and P. eous three natural media were employed which include a mixture of coffee husk powder (100gm), dextrose (20gm) and agar agar (18gm) in a litter of distilled water. Cocoa pod powder and rubber saw dust were also used in the place of coffee husk powder with the same quantity and with other ingredients as such. Coffee husk, cocoa pod powder and rubber saw dust were considered as wastes hence cheaply available.

Results and Discussion The comparative mycelial growth rate of the two test fungi on three natural culture media showed varying responses. Cocoa pod powder media supported more growth than coffee husk media, whereas rubber saw dust media supported least or nil growth and that may be due to the absence of required nutrients. At 24˚C P. eous in cocoa pod media and coffee husk media showed a maximum growth of 4cm and 2.8cm radius sized colonies respectively, at the end of the 6th day completing their mycelial growth. The results showed an increase in the growth rate as the incubation temperature was increased from 24 to 28˚C (Table 1). Earlier in 1935, Kaufert had already reported that optimum temperature for mycelial growth of P. corticatus was 28˚C. According to Moorthy (1993), 25 to 28˚C were found to be the optimum for P. sajor-caju in in vitro studies. Cartwright and Findlay (1934) had observed that most of the fungi prefer a temperature range of 25 to 30˚C. The OMA was found to be superior medium for the purpose. However, cocoa pod powder medium and coffee husk medium can be used as a substitute for PDA and OMA. A low cost substitute for malt extract agar (MEA) was given by Diwakar and Kapoor in 1991. According to them Bornvita agar can be used instead of MEA which is costlier than Bornvita agar. In our study OMA had given better result than PDA. Suharban and Nair (1991) carried out the growth of Pleurotus species on different culture media and observed that the agar based media determines the mycelial growth rate. For the spawn production four substrates such as coffee husk, chopped areca 40

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P. florida

Species

MEDIA

28˚C

P. florida

P. eous

24˚C

Temperature

colonization in 10, 16 and 10 days respectively nut leaf sheath, sorghum and paddy grains whereas it took 18 days in coffee husk. were used as media and among them sorghum grains, paddy grains and chopped areca nut leaf sheath supported P. eous for 100% Table-1: Comparative mycelial growth (radius in cm) of selected Pleurotus species on different medium at various time intervals*

Days

PDA

OMA

CPA

CHA

RSDA

2 4

0.5 1.4

0.9 1.1

0.5 1

0.8 1.3

_ _

1.8

2.2

1.8

1.2

1.5

1.2

1.1

1.9

2.4

2.7

2.5

2.8

1.3

1.5

1.3

1.2

2.6

2.7

2.3

1.8

P. eous

6 4.2 3 2.5 3 1.2 2 0.9 1 1 1 _ 4 2.6 2.7 2.9 2.7 _ 6 4.5 4.5 4.5 4.5 _ *Average of 3 replication/culture plate PDA- Potato Dextrose Agar, OMA-Oat Meal Agar, CPA-Cocoa Pod Agar, CHA-Coffee Husk Agar, RSDA-Rubber Saw Dust Agar Table -2: Mycelial colonization (%) on different spawn base in spawn bottles Chopped arecanut Sorghum grain Paddy grain Coffee husk Spawn Base leaf sheath 8

P. florida

100

P. oeus

100

Days

Fungi

Average of 3 replication / bottles

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On the other hand P. florida showed variations in incubation period in which its 100% growth reached on the16th day in sorghum, paddy grains and areca nut leaf sheath and 19 days in coffee husk medium. Among the four substrates sorghum grains was found to be the best and the coffee husk to be the least for the spawn production. Most probably this is due to the less nutritional content of the substrate (Table- 2).

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