INTRODUCTION:

National Agriculture Research Centre (NARC) Islamabad:

National Agricultural Research Centre (NARC), Islamabad established in 1984, is the largest

research centre of the Pakistan Agricultural Research Council (PARC). NARC, with a total land

area of approximately 1600 acres, is located near Rawal Lake, six kilometers south-east of

Islamabad. Physical facilities in term of experimental fields, laboratories, green houses, gene

bank, library/ documentation, auditorium, machinery & lab equipment repair workshops, stores,

hostels, cafeteria, audio visual studios, are also available at NARC.

NARC coordinated programs serve as a common platform for the scientists working in

different federal, provincial agricultural research, and academic institutions to jointly plan their

research activities, avoiding unnecessary duplication of research efforts. Research which can best

be addressed at a National Centre rather than by provincial institutions is undertaken at NARC.

The adaptation of technologies available from the international research system is also managed

by NARC, in collaboration with the provincial research and extension institutions. In particular,

research requiring sophisticated instruments like electron microscopes, ultracentrifuges, and

elaborate analytical and quality testing facilities is undertaken at NARC, supported by highly

qualified and trained manpower.

1 | P a g e

Crop Diseases Research Institute:

Crop Diseases Research Institute (CDRI) is the premier Institute in Pakistan working on Crop

Health Management through applied and basic research in phytopathology under the aegis of

Pakistan Agricultural Research Council. It existed in its nuclear form since 1955 and through

several reorganizations, attained the structure and mandate of Crop Diseases Research Institute

in 1983. Later in 2001 in an institutional re-organization of PARC it merged into a newly

developed Institute of Plant and Environmental Protection with the change of status from

Institute to Program. The Institute is again elevated from program to Institute in 2013. It is the

only one of its kind in the country. It is working with the infrastructure of main laboratories at

National Agricultural Research Centre (NARC), Islamabad and a substation at Murree.

Introduction to Oyster Mushroom:

Mushroom in general and Pleurotus in particular are an important source of nutrition particularly

for the people on cereal-based diet. Edible species of mushroom are low in calories, fats, sodium,

carbohydrates and cholesterol, whereas, rich in proteins, minerals, vitamins and fibers (Nasim et

al. 2001). Besides nutritional importance genus Pleurotus is also known for its medicinal value

(Kothe 2001), paper pulp bleaching, cosmetics and industrial use (Kyung-Ho Ma et al. 2009;

Sigoillot et al. 2005).

The mushrooms are naturally grown in fields, forests; on manure heaps, water channels and hilly

areas, mostly during and just after rains. The most popular varieties are Agaricus bisporus

(European or white button mushroom), Pleurotus spp., (Oyster mushrooms or Dhingri),

Volvariella volvacea (Chinese or paddy straw mushroom) Lentinu sedodes (Shiitake mushrooms)

and Auricu slaria (Black ear mushroom).

Mushrooms are valuable food, which are low in calories, high in vegetable proteins, zinc, chitin,

fiber, vitamins and minerals (Alam and Saboohi, 2001). The mineral salt content in mushrooms

is superior to that of meat and fish and nearly twice that of the most commonly used vegetables

2 | P a g e

(Chang, 1972). Anticancer medicine (Leutinan) is produced recently by some chemical

companies from the extract (Polysaccharides) of Shitake mushroom (Mori, 1986).

The Oyster mushroom is grown under natural conditions on living trees as parasite or dead

woody branches of trees as saprophyte and primary decomposer. The chemical composition of

the fresh fruiting bodies of oyster mushroom, Pleurotus ostreatus indicates a large quantity of

moisture (90.8%), whereas fresh as well as dry oyster mushrooms are rich in proteins (30.4%),

fat (2.2%), carbohydrates (57.6%), fiber (8.7%) and ash (9.8%) with 345 K (cal) energy value on

100 g dry weight basis; while vitamins such as thiamin (4.8 mg), riboflavin (4.7 mg) and niacin

(108.7 mg), minerals like calcium (98 mg), phosphorus (476 mg), ferrous (8.5 mg) and sodium

(61 mg) on 100 g dry weight basis, are also found present (Pandey&Ghosh, 1996). Rambelli &

Menini (1985) reported that this mushroom is reputed to be antitumoral because of its chemical

composition.

The oyster mushrooms can be cultivated successfully under semi controlled conditions in a small

space by using agricultural as well as industrial waste and other refuse as substrate. Badshahet

al., (1992) have grown Pleurotus ostreatus on wheat straw, sugarcane bagasse, corn cobs or

sawdust by mixing 120-130 g of spawn with 2 kg of substrate and placing the mixture in

sterilized polyethylene bags which were kept in the dark at 25 ͦ C for 2-3 weeks.

3 | P a g e

Varietals difference was observed for mycelial colony proliferation of mushroom (Khandakar,

2004). Some growth regulators at different concentrations affect the yield and size of mushroom

(Charles, 1986). Plant growth regulators and media can also play an important role for in vitro

mycelial colony proliferation of mushroom (Muniruzzaman, 2004). Quite a large number of

experiments have so far been carried out throughout the world to find out the appropriate variety

and media for mushroom cultivation. However, in the country sufficient research work have not

yet been done on different variety and media for mushroom cultivation. The present piece of

research work was, therefore, undertaken to investigate the mycelial colony proliferation of

Oyster mushrooms on PDA (Potato Dextrose Agar), CMA (Corn Meal Agar), TWA (Tap Water

Agar) and MEA (Malt Extract Agar) media.

4 | P a g e

MATERIAL AND METHODS:

Sources of materials:

The species of oyster mushroom (strain AG 572) was obtained from Crop Disease Research

Institute (CDRI), Department of Plant and Environmental Protection (DPEP), NARC, Islamabad

and maintained in potato dextrose agar (PDA), corn meal agar (CMA), tap water agar (TWA),

and Malt Extract Agar (MEA) media.

Affect of different Media and Temperature on the growth of mycelium:

The effects of culture media and temperature on the mycelium growth of oyster mushroom

Pleurotus ostreatus for spawn production were studied.

Several trials were designed to evaluate factors affecting mycelium growth of oyster mushroom.

For each test, mycelium discs with similar diameter (1 cm) were used. The discs were obtained

by using the sterilized punch-hole tool to cut the mycelium and place into a new sterilized

medium plate by using a transplant needle. The diameter of the mycelium extension was

measured every 2 days with the help of a transparent ruler at the same time (between 9:00 AM

and 12:00 PM) and surface mycelium density was observed.

Effect of culture media:

Four agar media were used including (PDA) potato dextrose agar medium, (CMA) corn meal

agar medium, (TWA) tap water agar medium (MEA) malt extract agar medium. The ingredients

for those culture media were as follows:

PDA:

200 g potato, 20 g dextrose, 15 g agar powder, and 1000 ml distilled water;

CMA:

30 g corn meal, 20 g dextrose, 15 g agar powder, and 1000 ml distilled water;

5 | P a g e

TWA:

15 g agar powder and 1000 ml distilled water;

MEA:

20 g malt extract, 20 g dextrose, 15 g agar powder and 1000 ml distilled water.

Media and Petri dishes (10 cm diameter) were autoclaved at 121℃ (at a pressure of 1.3 kg/cm2)

for 20 min. The mycelium discs (1 cm diameter) of oyster mushroom was placed in Petri dishes

containing each culture medium (20 ml) under aseptic condition and incubated at ±25ºC &

±28℃ temperature in the darkness. The diameter of the mycelium expansion was measured

every 2 days for 8 days.

Effect of temperature:

The Petri dishes containing sterilized (PDA) potato dextrose agar medium, (CMA) corn meal

agar medium, (TWA) tap water agar medium (MEA) malt extract agar medium (20 ml) each

were inoculated with the mycelium discs (1 cm diameter) of oyster mushroom under aseptic

condition and incubated in the darkness at two levels of temperature (25℃ and 28℃). The

diameter of the mycelium expansion was measured every 2 days for 8 days.

Experimental design:

The experiment was conducted in Crop Disease Research Institute (CDRI), Department of Plant

and Environmental Protection (DPEP), NARC, Islamabad during summer 2015 (June to July).

The experiment was arranged in a completely randomized design (CRD) with three replications

per treatment.

6 | P a g e

RESULT AND DISCUSSION:

Effect of different culture media on the mycelium growth of oyster mushroom:

Every living being requires food for its growth and reproduction and oyster mushrooms are not

an exception to it. In order to culture the oyster mushroom in the laboratory, it is necessary to

furnish those compounds in the media which are requires for its growth and other life process. In

the present study, oyster mushrooms PO was cultured successfully in the laboratory

environment. During the present investigation, different media, i.e., PDA, CMA, TWA, and

MEA were used to identify their effect on mycelium growth.

The observed mycelium colony diameter of mushroom PO on four different media significantly

differed with each other. The Mycelium density of oyster mushroom PO was compact in all

media. The average mycelium diameter of oyster mushroom ranged from 3.92 ± 0.05 to 9.00 ±

0.00 cm at 8 days after inoculation. The mycelium growth of mushroom PO on PDA and CMA

media was better than those on TWA and MEA media at 2, 4, and 6 days after inoculation

(DAI); however, they were not significantly different at 8 DAI.

The result indicated that these four culture media are suitable for mycelium growth of mushroom

while PDA and CMA media are more suitable for the mycelium growth of mushroom PO. This

may be due to availability of required nutrients for mushroom PO in PDA and CMA media.

OYSTER MUSHROOM MYCELIUM ON PDA OYSTER MUSHROOM MYCELIUM ON CMA

7 | P a g e

OYSTER MUSHROOM MYCELIUM ON MEA OYSTER MUSHROOM MYCELIUM ON TWA

The results of the present research showed that the mycelium of PO took shorter time to

complete growth in all media. These findings were similar to the results reported by Mshandete

and Mgonja; Mansur et al. They indicated that PDA and CMA were suitable media for culture

of Pleurotus species to achieve high level mycelium biomass, exo-polysacharides and mycelium

protein. In Pakistan, potato is so much cheaper than corn malt extract, so PDA medium was more

suitable and efficient to use as alternatives for culture of mushroom PO. From this result, we

chose local materials as cheaper materials to prepare media for culture of mushroom PO.

Effect of temperature on mycelium growth of oyster mushroom:

Temperature is a very important environment factor for mycelium growth of fungi. To determine

optimal temperature for mycelium growth, of oyster mushroom was cultivated in PDA, CMA,

TWA, and MEA medium at two temperatures (25℃and 28℃). The trend of mycelium growth

in response to temperature was not very similar, where the optimum temperature for oyster

mushroom was found to be 25℃ followed by 28℃.The mycelium growth of mushroom PO was

significantly faster at 25℃. The temperature tested to grow after 8 days inoculation at 25℃ and 28℃. The mycelium density of mushroom PO was very thin at 28℃ indicated that the

optimum temperature for mycelium growth of oyster mushroom was 25~30℃. This optimum

temperature result indicated that the oyster mushroom PO was able to grow better in summer and

autumn season in subtropical and tropical regions as potential opportunity to develop oyster

8 | P a g e

mushroom production in poor and developing countries in Asia. The result is quite similar to the

results reported by Kashangura, (2008) and Choi et al.(2003). When demonstrating that the

mycelium growth and fruiting formation of oyster mushroom species were affected by

temperature and they could grow at high temperature as summer season in tropical regions.

GROWTH OF MYCELIUM INCUBATED AT 25℃ ON PDA. GROWTH OF MYCELIUM INCUBATED AT 28℃ON PDA.

GROWTH OF MYCELIUM INCUBATED AT 25℃ON CMA. GROWTH OF MYCELIUM INCUBATED AT 28℃ON CMA

9 | P a g e

GROWTH OF MYCELIUM INCUBATED AT 25℃ON MEA. GROWTH OF MYCELIUM INCUBATED AT 28℃ON MEA

GROWTH OF MYCELIUM INCUBATED AT 25℃ON TWA. GROWTH OF MYCELIUM INCUBATED AT 28℃ON TWA.

10 | P a g e

CONCLUSION:

In conclusion, the mycelium growth of oyster mushroom PO was affected by different culture

media and temperature.

It was concluded that PDA, CMA, TWA, and MEA were the most favorable media for the

mycelium growth of mushroom while PDA and CMA were the most suitable media for

mycelium growth of oyster mushroom PO.

Maximum mycelium growth of oyster mushroom PO was achieved by cultivating them at the

optimum temperature of 25℃.

In all studied cases, mycelium growth of oyster mushroom PO was significantly better on PDA

and CMA at 25℃.

11 | P a g e

REFERENCES:

Alam, S. M. and R. Saboohi. 2001. Importance of mushroom. www.mush-world.com.html.

Badu, M., S. K. Twumasi and N. O.Boadi. 2011. Effects of lignocellulosic in wood used as

substrate on the quality and yield of mushrooms. Food Nutr Sci. 2:780–784.

Bai Y. H., Feng Y. Q, Mao D. B., Xu C. P. 2014. Optimization for betulin production from

mycelial cultureof Inonotusobliquus by orthogonal design and evaluation of its

antioxidant activity. J Taiwan Inst Chem Eng. 43:663–669.

Cheng Z., Wu Q., Huang J. B., Hu C.G., Wang Z. L. 2013. Effects of carbon sources, nitrogen

sources and minerals on mycelial growth of Cryphonectriaparasitica. Afr J. Agric.

Res. 8:4390–4395.

Chodchoi, C. 1986. Effects of growth regulators compounds on yield and size of the commercial

mushroom (Auriculariapolytricha). Proceeding of the 24thNational Congress, Bangkok,

pp127-134.

Choi I. Y, Joung G. T., Ryu J., Choi J. S., Choi Y. G. 2003. Physiological characteristics of

green mold (Trichoderma. spp.) isolated from oyster

mushroomPleurotus spp.) Mycobiology. 31:139–144.

Choi K. W. 2004. Mushroom grower's handbook 1: Oyster mushroom cultivation. Seoul:

Mushworld;.Shelf cultivation of oyster mushroom; pp. 153–165.

 Chorváthová V, Bobek P, Ginter E, Klavanová J. Effect of the oyster fungus on glycemia and

cholesterolemia in rats with insulin-dependent diabetes. Physiol Res. 1993;42:175–

179. [PubMed]

Dehariya P, Vyas D, Kashaw SK. Mushroom nutraceuticals on different substrates. Int J Pharm

Pharm Sci.2013;5:88–90.

12 | P a g e

Dung LB. Mushrooms in Tay Nguyen. Ha Noi: Science and Technique; 2003.

 Fu R, Yin C, Liu Y, Ding L, Zhu J, Zheng A, Li P. The influence of nutrient and environmental

factors on mycelium growth and conidium of false smut Villosiclavavirens. Afr J

Microbiol Res. 2013;7:825–833.

Gomez, K.A. and Gomez, A.A. 1984. Statistical procedure for Agricultural Research (2nded).

John Willy and Sons. New York. 680p.

Islam, M.S., Islam, M.M., Al Mansur, M.A.Z. and Ali, M. S. 2007. Study on mycelial growth in

different mushroom species and spawn production of Oyster mushroom in different

substrate. Bangladesh J. Agril. Sci. 34(1):19-22.

Jandaik CL, Goyal SP. Farm and farming of oyster mushroom (Pleurotus sp.) In: Singh RP,

Chaube HS, editors. Mushroom production technology. TehriGarhwal: GovindBallabh

Pant University of Agriculture and Technology; 1995. pp. 72–78.

 Kashangura C. Optimization of the growth conditions and genetic characterisation

of Pleurotus species [dissertation] Harare: Department of Biological Sciences, Faculty of

Science, University of Zimbabwe; 2008.

Khandakar, J. 2004. Effect of media composition and growth regulators on mycelial growth and

spawn production of three mushroom species. MS Thesis, Department of Biotechnology,

BAU, Mymensingh.

 Kim DH, Yang BK, Jeong SC, Park JB, Cho SP, Das S, Yun JW, Song CH. Production of a

hypoglycemic, extracellular polysaccharide from the submerged culture of the

mushroom, Phellinuslinteus. Biotechnol Lett.2001;23:513–517.

Kües U, Liu Y. Fruiting body production in Basidiomycetes.  Appl MicrobiolBiotechnol. 2000;

54:141–152.[PubMed]

Kuforiji OO, Fasidi IO. Factor affecting the yield of fruitbody and sclerotia in Pleurotus tuber-

regium. Adv Food Sci. 2007;29:211–215.

13 | P a g e

Kurtzman RH, Zadražil F. Physiological and taxonomical considerations for cultivation

of Pleurotusmushrooms. In: Chang ST, Quimio TH, editors. Tropical mushrooms

biological nature and cultivation methods.Hong Kong: Chinese University Press; 1989.

pp. 299–348.

 Lee JY. Coloured Korean mushrooms. Seoul: Academy Press; 1993.

Li L, Ng TB, Song M, Yuan F, Liu ZK, Wang CL, Jiang Y, Fu M, Liu F. A polysaccharide-

peptide complex from abalone mushroom (Pleurotusabalonus) fruiting bodies increases

activities and gene expression of antioxidant enzymes and reduces lipid peroxidation in

senescence-accelerated mice. ApplMicrobiol Biotechnol.2007;75:863–869. [PubMed]

Mane VP, Patil SS, Syed AA, Baig MM. Bioconversion of low quality lignocellulosic

agricultural waste into edible protein by Pleurotussajor-caju (Fr.) Singer. J Zhejiang

UnivSci B. 2007;8:745–751. [PMC free article][PubMed]

Maniruzzaman, M. 2004. Influence of media composition and growth regulators on

mycelialgrowth and spawn production of three mushroom species. MS Thesis,

Department of Biotechnology, BAU, Mymensingh.

Mao X.B., Eksriwong T, Chauvatcharin S, Zhong J. J. Optimization of carbon source and

carbon/nitrogen ratio for cordycepin production by submerged cultivation of medicinal

mushroom Cordycepsmilitaris. Proc Biochem.2005;40:1667–1672.

Miles PG, Chang ST. Mushroom biology: concise basics and current developments. Singapore:

World Scientific; 1997. pp. 40-44.

Mori, K. 1986. Cultivated mushrooms in Japan. Prodc. Int’l Sym. Scientific and Technical

aspects of cultivating edible fungi.Penna State Univ.,USA.

Mshandete AM, Mgonja JR. Submerged liquid fermentation of some Tanzanian Basidiomycetes

for the production of mycelial biomass, exopolysaccharides and mycelium protein using

wastes peels media. J Agric Biol Sci. 2009;4:1–13.

14 | P a g e

Munsur MA, Miah A, Rahman MH, Rahman MM, Yahia AS. Effect of varieties and media on

mycelial growth and substrate on spawn production of oyster mushoom. Bangladesh Res

Pub J. 2012;7:361–366.

Naraian R, Sahu RK, Kumar S, Garg SK, Singh CS, Kanaujia RS. Influence of different nitrogen

rich supplements during cultivation of Pleurotusflorida on corn cob

substrate. Environmentalist. 2009;29:1–7.

Nasim, G., Malik, S. H., Bajwa, R., Afzal, M. and Mianm, S.W. 2001. Effect of three different

cultural media onmycelial growth of Oyster and Chinese mushroom.Online Journal

Biological. Sciences. 1(12), 1130-1131.

 Neelam S, Chennupati S, Singh S. Comparative studies on growth parameters and physio-

chemical analysis ofPleurotusostreatus and Pleurotusflorida. Asian J Plant Sci

Res. 2013;3:163–169.

Oei P. Manual on mushroom cultivation: techniques, species and opportunities for commercial

application in developing countries. Amsterdam: TOOL Publications; 2003.

 Purkayastha RP, Nayak D. Analysis of protein patterns of an edible mushroom by gel-

electrophoresis and its amino acid composition. J Food Sci Technol. 1981;18:89–91.

Sánchez C. Cultivation of Pleurotusostreatus and other edible mushrooms. ApplMicrobiol

Biotechnol.2010;85:1321–1337. [PubMed]

Shah ZA, Ashraf M, Ishtiaq CM. Comparative study on cultivation and yield on performance of

oyster mushrooms (Pleurotusostreatus) on different substrates (wheat straw leaves, saw

dust) Pak J Nutr.2004;3:158–160.

Siddiqui, A.B. 2002. Mushroom Production Technology.IHAND Project, GOB/UNDP/FAO,

Rangdhanu Printers, pp1-14.

Sofi B, Ahmad M, Khan M. Effect of different grains and alternate substrates on oyster

mushroom (Pleurotusostreatus) production. Afr J Microbiol Res. 2014;8:1474–1479.

15 | P a g e

Szabová E, Rohaľová L, Hedvigy M. Semi-solid fermentation of Pleurotusostreatus. J

MicrobiolBiotechnol Food Sci. 2013;2:1950–1958.

 Thomas KC, Hynes SH, Ingledew WM. Effect of nitrogen limitation on synthesis of enzymes

inSaccharomycescerevisiae during fermentation of high concentration of

carbohydrates. Biotechnol Lett.1996;18:1165–1168.

 Tinoco R, Pickard MA, Vazquet-Duhalt R. Kinetic differences of purified laccases from

six Pleurotusostreatus strains. LettApplMicrobiol. 2011;32:331–335. [PubMed]

World Bank, 1992.World Development Reports.Oxford University Press, Inc., New York.

World Bank. 2004. World Development Reports.Oxford University Press, Inc., New York.

16 | P a g e