INTRODUCTION:

The milky mushroom (Calocybe indica) is a potentially new species to the world mushroom growers. It is a robust, fleshy, milky white, umbrella like mushroom, which resembles button mushroom. The species is suitable for hot humid climate and can be cultivated indoor in high temperature and high humidity areas. It grows well at a temperature range of 25-35°C and relative humidity more than 80%. Milky mushrooms can be cultivated throughout the year in the entire plains of India.

The cultivation technology is very simple, involves less cost and no special compost is needed for the cultivation. The cultivation process resembles that of oyster mushroom but for the additional process of casing. The mushroom can be harvested from 24-28 days after spawning and the total crop cycle is only 45-50 days. Most importantly, the milky mushroom has an extended shelf life of 3-5 days compared to other cultivated species, making it more amenable to handling, transportation and storage. So, there is a growing interest among the farmers towards milky mushroom.

MATERIALS AND METHODS:

For cultivation of milky mushroom, various substrates viz. paddy straw, teak leaves and sugarcane trash were attempted. The fruit bodies of harvested mushrooms were dried in an oven at 40°C for 48 hours. The dried samples were obtained for the analysis of protein, carbohydrate, amino acid and lipids. 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. The cultivated mushrooms were also tested the ability of cellulolytic activity by using Whatman filter paper No: 1 at the interval of 7, 15 and 22 days. After incubation period, the inoculated filter papers were dried and to remove the fungal mycelium and weighed to determine the cellulolytic activity.

Composting coir pith was inoculated with Calocybe indica 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 Calocybe indica:

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

RESULT AND DISCUSSION:

The results of cellulolytic activity and composting of coir pith of the mushroom was shown in Table 3 and 5. The production of cellulase (carboxymethylcellulase, filter paper, cellobiase) and xylanase by different mushroom strains on the Reese mineral medium supplemented with Carboxymethylcellulose as carbon source was assayed after 7 and 15 days of incubation. The cultural filtrate of these mushroom strain exhibited relatively higher activity of all four enzymes after 15 days interval during the course of its growth (Mishra, 2009). In the present study cellulolytic activity of C. indica was studies at an interval of 7, 15 and 22 days respectively (Table 3).

Purkayastha et al., (1981) cultivated C. indica using 49 different substrates including various plant products, crop residues and leaves and recorded the higher yield from paddy straw supplemented with 5 per cent maize meal. In the present study, three different substrates were used for the cultivation of Calocybe indica. From the cultivation, paddy straw showed maximum yield when compared to teak leaves and Sugarcane trash (Table 1 and Fig 1).

Fig.1 Total harvest of Calocybe indica using different substrate

Fig.2 Biochemical analysis of Calocybe indica

According to Sivaprakasam et al (1986) crude protein, fat and total carbohydrate contents of C. indica and P. sajor – caju analysed at various growth stages exhibited strikingly different results. In the present study, protein, carbohydrate, amino acids and lipid contents of C. indica was analyzed on different substrates (Table 2 and Fig 2).

Calocybe indica showed the least enzyme activity. P. djamor degraded the coir pith to the maximum level of decreasing the cellulose, lignin and carbon content from 27.13, 28.25 and 28.97 to 10.25, 23.14 and 18.15 percent respectively. The nitrogen content was increased from 0.28 to 1.148 per cent and narrowing down the C: N ratio was fixed as index for the composting process. P. djamor was found to be an effective degrader of coir pith. Application of coir pith compost made by using P. djamor effectively reduced the black gram dry root rot disease incidence and was comparable to carbendazim 0.1% (Ramamoorthy et al., 1999). In the present study, C. indica degraded the coir pith to the maximum level of decreasing the lignin and carbon from 555.0, 23.2 to 409.3, and 21.2 percent respectively. The nitrogen content was increased from 0.45 to 0.67 per cent. C. indica was found to be an effective degrader of coir pith (Table 5).

Table. 1 Yield and productivity of Calocybe indica using Paddy straw, Sugarcane trash and Teak leaves.

Name of the organism

Name of the substrates

Harvest-I g/ kg

Harvest-II g / kg

Harvest-III g / kg

Total Harvest g / kg

C.indica

Paddy straw

Sugarcane trash

Teak leaves

570

250

325

340

170

240

230

150

180

1140

570

745

Table. 2 Biochemical analysis of Calocybe indica

Name of the Organism

Name of the Substrates

Protein mg / g

Carbohydrate mg / g

Lipid mg / g

Amino acid mg / g

C. indica

Paddy straw

Sugarcane trash

Teak leaves

7.6

7.3

7.4

5.3

5.4

4.9

1.7

1.05

1.1

1.5

1.3

Table. 3 Influence of nitrogen content of nutrient solution (2.0 ml) on the dry weight and loss of filter paper disc by colonization of C. indica.

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

5.2

7.2

3.2

3.9

11.30

15.65

Table 4: Mycoflora isolated from spawn treated coir pith compost by C. indica, their abundance and percentage of frequency occurrence

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.	Aspergillus candidus A.clavatus A.flavipes A. flavus A. luchensis A. nidulans A.niger A. ochraceous A.repens A. sydowi A. terreus A.versicolor Penicillium janthinellum Rhizopus nigricans R. oryzae Trichoderma koeningi	- 2.0 1.0 8.0 - 2.0 8.0 2.0 1.0 5.0 2.0 8.0 1.0 5.0 3.0	3.0 4.0 - 6.0 - 4.0 9.0 5.0 - 6.0 - 7.0 3.0 - 2.0	1.0 - - 5.0 1.0 - 10.0 4.0 1.0 6.0 - 4.0 3.0 5.0 1.0	1.3 2.0 0.3 6.3 0.3 2.0 9.0 3.6 0.6 5.6 0.6 6.3 3.6 2.6 1.3
	Total number of colonies	48	49	41
	Total number of organisms	13	10	11

Table. 5 Chemical composition of coir pith compost treated by C. indica

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 (mg/g)

Total nitrogen (mg/g)

Available nitrogen (mg/g)

C: N ratio (%)

Lignin content (%)

1.72

103.0

51.5

6.3

0.61

23.2

20.3

0.45

0.037

119

555.0

1.85

116.3

53.2

7.3

0.90

21.2

39.3

0.67

0.065

37.3

409.3

Nineteen cultures of Calocybe indica were molecularly identified and characterized using ITS and RAPD profiles. All the 19 accessions exhibited identical ITS lengths of approximately 650 bp on the gel. This confirmed that all the accessions belong to one species. No intra-specific polymorphism could be observed in ITS lengths in all the 19 accessions studied. The RAPD profiles of C. indica group of accessions generated with six primers exhibited significant polymorphism in scorable banding patterns. All the six random defamer RAPD primers amplified the scorable DNA fragments in all the 19 accessions of C. indica and separated them into seven distinct phylogenetic sub-clades. Present study clearly indicates the existence of intra-specific genetic variation in C. indica accessions (Mahesh and Yadav, 2006). In the present study C. indica was molecularly identified and characterized.

REFERENCES:

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

3. 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.

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

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6. Mahesh A and Yadav C (2006). Nineteen strains of milky mushroom Calocybe indica were molecularly identified and characterized using ITS sequencing and RAPD profiles. Mushroom Newsletter., 12 (1) :2.

7. Purkayastha R P Mondal T and Jana K K (1981). An improved method of cultivation of C. indica an edible white mushroom, Indian J. Mush., VII (1 and 2) : 3.

8. Ramamoorthy V Meena B Muthusamy M Seetharaman K and Alice D (1999). Composting of coir pith using lingo cellulolytic fungi for the management of root rot of black gram. Mushroom Research., 8 (2) : 13 – 17.

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

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11. Subramanian CV (1971). Hyphomycetes, I. C. A. R., Publications, New Delhi.

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

Received on 19.07.2011 Accepted on 10.08.2011

Asian J. Pharm. Res. 1(3): July-Sept. 2011; Page 55-57