Cultivation, Composting, Biochemical and Molecular Characterization of Pleurotus platypus (Cooke and Massee) Sacc

A. Prathiba, P. Madhanraj* and A. Panneerselvam

Department of Botany and Microbiology, A. V. V. M Sri Pushpam College, Poondi – 613 503, Thanjavur District, Tamil Nadu.

ABSTRACT:

In the present investigation, the mushroom yield, chemical composition, nutritional value of Pleurotus platypus cultivated in paddy straw, black gram stem and black gram pods were determined. Fresh mushroom yield amounts obtained from paddy straw, black gram stem and black gram pods substrate were 940, 560 and 595 g / kg respectively. Mushroom cultivated on different substrates were analyzed for protein, carbohydrate, amino acids and fatty acid content. The cellulolytic activity and coir pith compost was also investigated in Pleurotus platypus. Hence P. platypus was also selected for molecular studies. Therefore, the present results suggest it could be used to recycle the agro – waste materials.

KEYWORDS: Pleurotus platypus, Chemical composition, Nutritive value, Cellulolytic activity, coir pith compost.

INTRODUCTION:

Pleurotus spp., commonly known as oyster fungus, is a common primary decomposer of wood and vegetal residues (Zadrazil and Kurtzman, 1982). It can be naturally found in tropical and subtropical rainforests, and can be artificially cultivated (Maziero and Zadrazil, 1994). Appreciated because of its delicious taste, this fungus has high quantities of proteins, carbohydrates, minerals (calcium, phosphorus, iron) and vitamins (thiamin, riboflavin and niacin) as well as low fat (Sturion and Oettere, 1995; Justo et al., 1998; Manzi et al., 1999).

The cultivation of this mushroom constitutes important practice in the modern society due to the fact that this biotechnological process produces food of high nutritious value from agro – industrial substrate, facilitating the reduction of their volume or accelerating its decomposition process. Besides this, it allows the transformed substrate to be used as a fertilizer improving the physical characteristics of the soil or as feed for animal, closing cycle of use of the raw material (Maziero and Zadrazil, 1994).

MATERIALS AND METHODS:

Cultivation and Biochemical analysis of Pleurotus platypus:

Paddy straw, black gram stem and black gram pods used in this study for cultivation of Pleurotus platypus were agricultural lignocellulosic wastes obtained from Thirukanurpatti, Thanjavur district. Fresh mushroom yield amounts were obtained from three different substrates media were calculated. The fruit bodies of Pleurotus platypus were collected from after the first productive flow and dried in oven at 40° C to constant weight and kept under refrigerator at 4° C. Samples of mushrooms were analyzed for chemical composition (Protein, Carbohydrate, amino acids and lipid).

Protein was determined following the method of Lowry et al., 1951, carbohydrate by Dubois et al., 1956, Amino acids by Jayaraman, 1981 and Lipid by Sato, 1988.

Cellulolytic activity:

Sterilized and unsterilized Whatmann No: 1 filter paper was used for this procedure. The mass weight of the filter paper was analyzed before sterilization. These filter papers were placed in petriplates. The Pleurotus platypus was inoculated in to both sterilized and unsterilized filter papers with control. Then it was incubated at 27° C for 21 days. After incubation period, the inoculated filter papers were dried and to remove the fungal mycelium and weighed to determine the cellulolytic activity.

Composting of coir pith:

Coir pith was collected from an organic farm at Thirukanurpatti, Thanjavur district. Composting coir pith was inoculated with P. platypus and incubated for 60 days. Different stages of decomposition were studied for mycoflora for a period of 60 days by taking samples at a regular interval of 15 days using the conventional soil dilution technique on PDA medium (Warcup, 1950). Semi permanent slides were prepared using lactophenol cotton blue and Microphotographs were taken by using Nikon Binocular Microscope (Japan). Identification of the fungi was done by using the standard manuals (Gillman, 1957; Ellis, 1971 and Subramanian, 1971).

Molecular characterization of Pleurotus platypus:

Pleurotus platypus was also selected for molecular studies such as PCR amplification, 18S r DNA sequencing, nucleotide sequence accession, phylogenetic analysis, secondary structure prediction and restriction site analysis.

RESULTS AND DISCUSSION:

To study the mushroom yield, chemical composition and nutritional value of Pleurotus ostreatus (Jacq.) cultivated in wheat stalk (WS), millet stalk (MS), soybean stalk (SS) and cotton stalk (CS) was determined. Fresh mushroom yield amounts (100 g of substrate, 70 % moistre) obtained from WS, CS, MS and SS substrate media were 17.9, 14.3, 22.7 and 31.5 g, respectively. Samples of mushroom cultivated on different culture mediums were analyzed for protein, energy, ash, fat, dietary fibre, carbohydrate, moisture, vitamins (thiamins, riboflavin, pyridoxine and niacin) and amino acid contents (Dundar et al., 2009).

In the present study, fresh mushroom yield of Pleurotus platypus obtained from paddy straw, Black gram stem and black gram pods were 940, 560 and 595 g respectively. Mushroom cultivated on different substrates were analyzed for protein, carbohydrates, amino acids and lipid content (Table 1and 2, Fig 1and 2).

Fig.1 Total harvest of Pleurotus platypus using different substrate

Fig.2 Biochemical analysis of Pleurotus platypus

The colonization of sterilized and unsterilized filter papers by the cereal root rot fungi and weight loss caused by them in 22 days. The various aspects of the cellulosis rate of the cereal root rot fungi in relation to their survival (Garett, 1980 and 1983). In the present study, the colonization of sterilized and unsterilized filter papers by the Pleurotus platypus and weight loss caused by them in 22 days (Table 3).

Nallathambi and Marimuthu (1993) compared the ability of different species of Pleurotus to degrade different agricultural wastes including coir pith. In the present investigation coir pith was quickly composted with the help of P. platypus. The spawn inoculated coir pith took only 60 days for decomposition, the uninoculated coir pith took more than a year (Table 4 and 5).

Table 1: Yield and productivity of Pleurotus platypus using Paddy straw, Black gram stem and Black gram p

Name of the organism

Name of the substrates

Harvest-I g / kg

Harvest - II g / kg

Harvest- III g / kg

Total Harvest

g / kg

P. platypus

Paddy straw

Black gram stem

Black gram pods

560

350

375

220

120

140

160

940

560

595

Table 2: Biochemical analysis of Pleurotus platypus

Name of the Organism

Name of the Substrates

Protein mg / g

Carbohydrate mg / g

Lipid mg / g

Amino acid mg / g

P. platypus

Paddy straw

Black gram stem

Black gram pods

8.2

7.6

7.4

5.9

5.6

5.8

1.6

1.10

1.07

1.15

1.35

1.4

Table 3: Influence of nitrogen contents of nutrient solution (2.0 ml) on the dry weight and loss of filter paper disc by colonization of P. platypus

Mean radial growth (mm)

Mean loss in dry weight of the filter paper (mg)

Radial growth rate over PD agar (mm)

% loss in dry weight of the filter paper (mg)

7 days

15 days

22 days

Unsterilized filter paper

Sterilized filter paper

6.5

10.3

4.5

14.13

22.39

Table 4: Isolation of mycoflora from the coir pith compost Pleurotus platypus inoculated

S. No.	Name of the mycoflora	P1	P2	P3	Average colonies
1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16.	Acrophialophora fusispora Aspergillus awamori A.clavatus A. conicus A. flavus A. luchensis A. nidulans A. niger A. ochraceous A. sydowi A. terreus Penicillium citrinum P. chrysogenum Rhizopus nigricans R. oryzae Trichoderma harzianum	2.0 - 5.0 11.0 - 3.0 - 5.0 7.0 4.0 2.0 3.0 1.0 - 4.0 -	- 1.0 3.0 8.0 2.0 1.0 - 6.0 - 6.0 - 4.0 2.0 2.0 - -	- 3.0 2.0 6.0 1.0 4.0 1.0 6.0 - - 4.0 1.0 3.0 5.0 2.0 3.0	0.66 1.33 3.33 8.33 1.0 2.66 0.33 3.66 4.33 1.33 3.33 2.66 6.0 2.33 2.0 1.0
	Total number of colonies	47	35	41
	Total number of organisms	11	10	13

Table 5: Chemical composition of coir pith compost treated by P. platypus

S. No.

Name of the nutrients

Before compost

After compost

10.

11.

Bulk density (g / cc)

Water holding capacity (%)

Pore space (%)

Power of hydrogen (pH)

Electrical conductivity (Ec)

Organic carbon (%)

Organic matter (%)

Total nitrogen (mg / g)

Available nitrogen (mg / g)

C: N ratio (%)

Lignin content (µg / g)

1.72

103.0

51.5

6.3

0.61

21.2

20.3

0.45

0.037

119

555.0

1.89

121.5

52.2

7.3

0.89

23.3

42.5

0.69

0.71

38.6

417.3

Molecular methods, such as DNA sequencing and RFLP and PCR-RFLP techniques, have been used to identify Pleurotus species. The application of analyses of sequences of ITS regions and large subunits (LSU) of nuclear ribosomal DNA (r DNA) to the determination of Pleurotus species (Vilgalys and sun, 1994; Zervakis et al., 2004) is very powerful at the species level, but these analyses are often used for particular research and have not been used for routine or rapid identification. RFLP and PCR-RFLP methods have also been applied to the study of molecular systematic and the genotyping of Pleurotus (Bao et al., 2004; Iracabal et al., 1995). In the present study, PCR techniques have been used to confirm the identification of Pleurotus platypus.

REFERENCES:

1. Bao D Ishihara H Mori N and Kitamoto Y (2004). Phylogenetic analysis of oyster mushrooms (Pleurotus sp.) based on restriction fragment length polymorphisms of the 5_ portion of 26S rDNA. Journal of Wood Science., 50: 169–176.

2. Dubois M Gilles K A Hamilton J K Robers P A and Smith F (1956). Colorimetric method for determination of sugars and related substances. Analytical Chemistry., 28 : 350 – 356.

3. Dundar A Acay H and Yildiz A (2009). Effect of using different lignocellulosic wastes for cultivation of Pleurotus ostreatus (Jacq.) on mushroom yield, chemical composition and nutritional value. African Journal of Biotechnology., 8 (4) : 662 – 666.

4. Ellis M B (1971). Dematiaceous Hypomycetes, Common Wealth Mycological Institute Pub. Kew Surrey., England. 608p.

5. Garett S D (1983). Factors limiting colonization of unsterilized filter paper by cereal root infecting fungi. Soil Biochemistry., 15 : 101 – 103.

6. Garett S D (1980). Colonization of unsterilized filter paper by cereal root rot fungi, Soil Biochemistry., 74 : 259 – 263.

7. Gillman J C (1957). A manual of soil fungi, Revised 2^nd Edn., Oxford and I. B. H. Publishing Company (Indian reprint), Calcutta, Bombay, New Delhi, 436.

8. Iracabal B Zervakis G and Labarère J (1995). Molecular systematic of the genus Pleurotus: Analysis of the restriction polymorphism in ribosomal DNA. Microbiology., 141: 1479-1490.

9. Jayaraman J (1981). Laboratory manual in Biochemistry, Wiley Eastern Limited, India. 65.

10. Justo M B Guzmán G A Mejía E G Díaz C L G Martínez G and Corona E B (1998). Composition química de tres cepas mexicanas de setas (Pleurotus ostreatus). Archivos Latinoamericanos de Nutricion ., 48 (4): 359-363.

11. Lowry O H Rosebrough N J and Randall R J (1951). Protein measurement with the folin phenol reagent. Journal of Biological Chemistry., 193 : 265 – 275.

12. Manzi P Gambelli L Marconi S Vivanti V and Pizzoferrato L (1999). Nutrients in edible mushrooms: An inter – species comparative study. Food Chemistry., 65 (4): 477 – 482.

13. Maziero R and Zadrazil (1994). Effects of different heat pre – treatments of wheat straw on its microbial activity and colonization by different tropical and sub – tropical edible mushrooms. World Journal of Microbiology and Biotechnology., 10 : 74 – 380.

14. Maziero R and Zadrazil (1994). Effects of different heat pre – treatments of wheat straw on its microbial activity and colonization by different tropical and sub – tropical edible mushrooms. World Journal of Microbiology and Biotechnology., 10 : 74 – 380.

15. Nallathambi P and Marimuthu T (1993). Pleurotus platypus a potent oyster mushroom for organic recycling of agricultural wastes. Mushroom Research., 2: 75 – 78.

16. Sato N (1988). Membrane lipids: In methods in Enzymology (Eds), Packer, L. and Glazer, A. N., 167: 251 – 259.

17. Stutrion G L and Oettere M (1995). Composicao quimica de cogumelos comestiveis (Pleurotus spp.) originados de cultivos em differentes substrates. Ciencia e Technologia de Alimentos., 15 (2): 189 – 193.

18. Subramanian C V (1971). Hyphomycetes, I. C. A. R., Publications, New Delhi.

19. Vilgalys R and Sun B (1994). Ancient and recent patterns of geographic speciation in the oyster mushroom Pleurotus revealed by phylogenetic analysis of the ribosomal DNA sequence. Proceedings of the National Academy Sciences., USA. 91: 4599-4603.

20. Warcup J H (1950). The soil plate method for isolation from soil. Nature., 166 : 117 – 118.

21. Zadrazil F and Kurtzman R H (1982). The biology of Pleurotus cultivation in the tropics In Tropical mushrooms (Chang ST, Quimio TH (Eds.), Hong Kong: The Chinese Press, 493: 277-298.

22. Zervakis G I Moncalvo J M and Vilgalys R (2004). Molecular phylogeny, biogeography and speciation of the mushroom species Pleurotus cystidiosus and allied taxa. Microbiology., 150:715–726.

Received on 25.04.2011

Modified on 08.05.2011

Accepted on 21.05.2011

Research J. Science and Tech. 3(5): Sept.-Oct. 2011: 284-287