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BIODEGRADATION AND BIOSYNTHETIC CAPACITY OF MILKY WHITE MUSHROOM (Calocybe indica)

Thesis submitted to the University of Agricultural Sciences, Dharwad in partial fulfillment of the requirements for the Degree of

Master of Science (Agriculture)


in AGRICULTURAL MICROBIOLOGY

By

ADINATH A. KARNAWADI

DEPARTMENT OF AGRICULTURAL MICROBIOLOGY


COLLEGE OF AGRICULTURE, DHARWAD UNIVERSITY OF AGRICULTURAL SCIENCES, DHARWAD - 580 005
OCTOBER, 2006

ADVISORY COMMITTEE

DHARWAD OCTOBER, 2006

(VEENA SAVALGI)
MAJOR ADVISOR Approved by : Chairman : ____________________________ (VEENA SAVALGI)

Members : 1. __________________________ (V. S. SAVALGI)

2. __________________________ (YASHODA HEGDE)

3. __________________________ (N. BASAVARAJ)

4. __________________________ (S. V. HOSMANI)

CONTENTS

Chapter No. I II III IV V VI VII INTRODUCTION

Title

Page No.
1

REVIEW OF LITERATURE MATERIAL AND METHODS EXPERIMENTAL RESULTS DISCUSSION SUMMARY REFERENCES

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LIST OF TABLES

Table No. 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13.

Title Organic carbon content (%) in different substrates at different growth stages of Calocybe indica Nitrogen content (%) in different substrates at different growth stages of Calocybe indica C:N ratio in different substrates at different growth stages of Calocybe indica Lignin content (%) in different substrates at different growth stages of Calocybe indica Cellulose content (%) in different substrates at different growth stages of Calocybe indica Hemicellulose content (%) in different substrates at different growth stages of Calocybe indica Dry matter content (%) in different substrates at different growth stages of Calocybe indica Yield performance of Calocybe indica on different substrates Effect of different substrates on size and shelf life of fruiting bodies of Calocybe indica Proximate analysis of Calocybe indica grown on different substrates Effect of different casing materials on yield of Calocybe indica Effect of different casing materials on the size and shelf life of fruiting bodies of Calocybe indica Proximate analysis of Calocybe indica grown on different casing materials

Page No. 40 42 44 45 47 49 50 52 54 55 57 59 61

LIST OF FIGURES

Figure No. 1.

Title

Between pages 35-36

Flow chart for cultivation of milky white mushroom (Calocybe indica) Effect of Calocybe indica growth on organic carbon content of substrates Effect of Calocybe indica growth on nitrogen content of substrates Effect of Calocybe indica growth on C:N ratio of substrates Effect of Calocybe indica growth on lignin content of substrates Effect of Calocybe indica growth on cellulose content of substrates Effect of Calocybe indica growth on hemicellulose content of substrates Yield performance of Calocybe indica on different substrates Proximate analysis of Calocybe indica grown on different substrates

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40-41

3.

42-43

4.

44-45

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45-46

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47-48

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49-50

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52-53

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56-57

LIST OF PLATES

Plate No.

Title

Between pages

1.

Fruiting bodies of Calocybe indica grown on different substrates

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I. INTRODUCTION
Mushrooms have been favoured as food by mankind since time immemorial being collected from the forests. However, mushrooms could not be domesticated due to their complex nature. Though Chinese were the first to do the artificial cultivation of the tropical and subtropical mushrooms about one thousand years ago, before the real commercial ventures were started in Europe. The mushrooms comprises of large heterogeneous group having various shapes, sizes and colours are quite different in character, appearance and edibility. Out of these large groups with more than 2000 edible species, about 300 species belonging to 70 genera are reported from India (Chadha and Sharma, 1995). However, only a few have been brought under cultivation on commercial scale. Presently mushrooms are being cultivated in about 100 countries with an annual production of 3,206,738 million tones (Anonymous, 2004.) Production of mushrooms is concentrated mainly in three regions viz., Europe (55%), North America (27%) and East Asia (14%). In East Asia, Taiwan, china, Korea and Indonesia are the major mushrooms growers and they export about 89 per cent mushrooms to USA. The mushroom consumption in western countries is 74 per cent of total world production viz., USA (30%), Germany (17%), UK (11%), Italy (10%) and Canada (6%) and remaining 15 per cent by rest of the world (Tewari, 2003). Hence, these countries offers good marketing for mushrooms. During 2002, per capita consumption of mushroom in India was hardly 20-25 gm as compared to 2.5 to 3 kg in developed countries (Verma, 2002). Though mushrooms production in India started in 1960s, it was during 90s, there was a sudden increase in mushroom production due to Hitech projects set up in collaboration with foreign companies. This has resulted in significant increase in mushroom production from 4000 tonnes (1985) to 40,000 million tones at present (Anonymous, 2004). India is blessed with varied agro-climate, abundance of agricultural wastes and manpower making it most suitable for cultivation of temperate, subtropical and tropical mushrooms. The present production of mushrooms in India is low as compared to 13,59,335 million tones in China. In the recent years, the central and state governments have made concentrated efforts in establishing All India Co-ordinated Mushrooms Improvement Project in different parts of the country and established mushroom spawn production units at various agricultural Universities in order to increase mushroom production in India. Mushrooms belong to a separate group of organisms called fungi. They lack chlorophyll present in plants. Mushrooms are classified under sub division Basidiomycotina. They grow on dead and decaying organic material. From these decaying substrates, they absorb their nutrition with the help of very fine thread like structures (mycelium) which penetrates into substratum and generally not visible on the surface. After the mycelium has grown profusely and absorbs sufficient food materials, it forms the reproductive structures which generally comes out of the substrate and forms fruiting body, commonly known as mushrooms. The mushrooms fruiting body may be umbrella like or of various shapes, size and colour. All mushrooms are not edible and some are highly poisonous species which are commonly referred to as toad stools. Mushrooms provide a rich addition to the diet in the form of protein, carbohydrates, valuable salts, minerals and vitamins. As food, the nutritional value of mushrooms lies between meat and vegetables. Like other vegetables it contains about 90 per cent moisture (Crisian and Sands, 1978) and are basically low calorie food (25-30 calorie / 100 gm fresh weight). Carbohydrates are present in the form of chitin and glycogen while starch is absent. Mushrooms are low fat food with 2 to 8% fat on dry weight basis (Crisian and Sands, 1978). It contains high proportion of unsaturated fatty acids especially linoleic acid with no cholesterol. Among sterols, ergosterol is abundant and cholesterol is absent. On dry weight basis mushrooms contains 19-35 per cent protein having 70-90 per cent digestibility. Mushrooms due to high quality and quantity of protein have been recognised by FAO as the food contributing to protein nutrition of the country depending largely on cereals. They are also good source of vitamins and minerals, especially those of B complex group but are relatively poor in fat soluble vitamins, A, D, E and K. Among B complex vitamins, mushrooms are specially rich in thiamine (B1), riboflavin (B2), niacin and biotin (Chang and Miles, 1989). Folic acid and vitamin B12 which are generally absent in plant food present in mushrooms.

Mushrooms also contains vitamin C and minerals like potassium, phosphorus, magnesium, sodium, calcium, zinc and iron in significant quantities. Speciality mushroom is a term given to a group of mushrooms, which are common in a particular area or country. Along with some mushroom genera (Pleurotus sp. Volvariella, Lentinus, Agaricularia) Calocybe indica also comes under speciality mushrooms. Milky mushroom (Calocybe indica) has become the third commercially grown in India after button and oyster mushrooms. This mushroom was first collected in wild form from West Bengal (India) by Purkayastha and Chandra in 1974. Production technology of Calocybe indica has been introduced by Purkayastha and Nayak in 1979 which was improved by Purkayastha and Nayak in 1981. This mushroom is gaining popularity due to its attractive robust, white sporocarps long shelf life and taste (Chadha and Sharma, 1995). Presently button and oyster mushrooms are commercially cultivated in tropical and subtropical regions of India. The oyster mushrooms can be easily grown under natural condition whereas button mushrooms require controlled conditions. Huge input are required to provide ideal condition for button mushrooms. Therefore, button mushroom cultivation is beyond the reach of ordinary farmers. The milky mushrooms require 25-35C temperature. In subtropical region of India, ample quantity of agricultural wastes are available and temperature of 28-35C is prevalent for about 4-5 months. Mostly summer season is suitable for cultivation of Calocybe indica. Keeping the information in view, an attempt was to investigate Biodegradation and Biosynthetic capacity of milky white mushroom (Calocybe indica) to know the yield potential of mushroom, with the following objectives: 1. Screening of suitable substrates for Calocybe indica cultivation 2. Influence of different casing materials on the growth and yield of milky white mushroom

II. REVIEW OF LITERATURE


Scientific thoughts involved in mushroom production technology are reviewed under this chapter. The literature pertaining to milky white mushroom are meager, since the same was recently put a scientific pooling in the field of mushroom biology. All relevant literatures concerning to milky white mushroom cultivation are reviewed here-under.

2.1 IMPORTANT CULTIVATED VARIETIES OF MUSHROOM


So far about 80 species of mushroom have been identified for their commercial cultivation under protected environmental conditions (Chang, 1990). Of these mushrooms, button mushroom (Agaricus bisporus) was first to be enrolled in modern mushroom production technology. Cultivation of this mushroom under protected environment was initiated by melon growers near Paris during 19th century (Nichols, 1992). The cultivated species of oyster mushrooms are Pleurotus ostreatus, P. florida, P. cornucopiae, P. flabellatus, P. citrinopileatus and P. sajor-caju (Zadrazil, 1978). In India both button mushroom and oyster mushrooms have become popular in several states. Further two species of oyster mushroom P. sajor-caju and P. flabulatus have become popular with mushroom farmers (Singh and Rajarathnam, 1977). Milky white mushroom (Calocybe indica) has become the 3 commercially grown mushroom in India after button and oyster mushrooms. This mushroom was first reported from India by Purkayastha and Chandra in 1974 (Purkayastha and Nayak, 1978).
rd

2.2 NUTRITIONAL STATUS OF MUSHROOM


The mushroom is known for its delicacy, flavour and aroma. Nutritionally it is considered as a valuable vegetable, consisting of protein (10-40%), carbohydrate (13-70%), fat (less than 1-8%), minerals and significant amounts of essential aminoacids (Chang et al., 1981). Further, medicinal value of mushroom is very well known, it is composed of linoleic acid which reduces the risk of coronary heart disease. Because of high dietary fibre content (7.4 to 27.66% on dry weight basis), mushroom also recommended for diabetic patients and it reduces risk of colon cancer (Zakia Bano and Rajarathnum, 1988). Chang and Miles (1989a) suggested the possible use of mushroom as food for arthritis patients, because of low amount of nucleic acids. Mineral content of mushroom indicated that 10 per cent ash (dry weight basis) contains potassium, phosphorus, sodium, calcium and magnesium. Mushrooms are also rich source of vitamins like thiamine, niacin, riboflavin, folic acid, ascorbic acid and pro-vitamin D (Rajarathnum et al., 1993). Mushrooms also contain an assay of anti-nutritional factors. These toxic components generally include tannins, oxalates, phytic acid, hydrocyanic acid and alkaloids (Ekanem and Ubengama, 2002).

2.3 EFFECT OF pH ON THE MYCELIAL GROWTH OF Calocybe indica


Aptly, hydrogen ion concentration is the cornerstone factor which adjudges the form and supply of various nutrients from medium to the growing mushroom mycelium under submerged and solid state conditions. A narrow pH range of 5 to 6.5 supported congenial growth of mycelium in milky white mushroom, Calocybe indica under submerged condition (Chandra and Purkayastha, 1977). Kurtzman and Zadrazil (1982) emphasized the use of carbonate and bicarbonate buffers to sustain the optimum hydrogen ion concentration in the medium at the same time without inhibiting the growth of pleurotus mycelium. Bhattacharjee and Samajpati (1989) studied the effect of different H ion concentration on mycelial yield of Pleurotus sajor-caju and suggested that pH of 5.5 was optimum for the same to increase the mycelial yield.

2.4 EFFECT OF CARBON ON MUSHROOM GROWTH


Being an achlorophyllus living being, mushroom fungus is entirely relies on organic carbon for energy source. The correlation between source of carbon and mycelial ramifications was theorized by various authors. Jandaik and Kapoor (1976) reported the

starch was an elite source of carbon for optimum mycelial growth in Pleurotus sajor-caju, although glucose, sucrose, maltose and dextrin supported optimum growth of the mushroom at 3% sugar concentration. However, in case of Calocybe indica, glucose, a monosaccharide supported maximum mycelial growth among 19 carbon compounds tested including starch (Chandra and Purkayastha, 1977).

2.5 EFFECT OF NITROGEN ON MYCELIAL GROWTH


All amino acids are building blocks of protein, which adorned with keystone component of nitrogen. Eventhough, mushroom is the cream source of protein, how it fabricates the entire nitrogen from the lignocellulosics, a poor mate of nitrogen, is an unsolved puzzle in mushroom research. It is obvious that organic and inorganic source of nitrogen can manipulate the mycelial yield and protein content based on sources of evidence provided by various workers. Jandaik and Kapoor (1976) proved that potassium nitrate and Asperagine found to be the best among various inorganic and organic nitrogen sources respectively to increase the mycelial growth of Pleurotus sajor-caju under liquid state condition. However, mycelial growth of Calocybe indica was repressed by all inorganic nitrogen compounds tested, but yeast extract and histidine nurtured the mycelium among all organic nitrogen source used (Chandra and Purkayastha, 1977). The results of Khanna and Garcha (1985), Bhattacharjee and Samajpati (1989) were in harmony towards effect of nitrogen on mycelial yield of Pleurotus sajor-caju. Both workers suggested that nitrate salts of potassium and sodium among inorganic source of nitrogen and aspargine among organic nitrogen sources, positively influenced the mycelial yield. The long run controvert over the possibility of atmospheric nitrogen fixation by Pleurotus spp. was partially slowed down by studies of Kurtzman and Zadrazil (1982), who suggested that presence of free living nitrogen fixing bacteria adhered to the substrate were responsible for enhancement of nitrogen to the growth of mushroom.

2.6 MUSHROOM PRODUCTION SCENARIO


Mushroom production is an eco-friendly activity where agricultural or industrial wastes are utilized and recycled. During last four decades, mushroom has attained the status of commercial crop. At present, it is being produced in about 100 countries. In the Europe and America, it is under hi-tech industry. Total world production is estimated to be 5 million tones, with increasing @ 7% /annum (Tewari, 2003). So far, in modern mushroom production panorama, button mushroom (Agaricus bisporus) reigns supreme with 1,424,000 tonnes of production during 1989-90 (Chang and Miles, 1991), nevertheless recent figures found against button mushroom, compare to speciality mushroom. This is mainly due to the success which attained a fastered rate of oyster mushroom production (nearly 450%) and incredible acceptability of Speciality mushrooms (mushrooms other than Agaricus bisporus) among US and European consumers. According to speculation of Bullman (1993), the consumption rate for button mushroom will be only at 3.5% per annum is lieu of 4.5 per cent by 2000 AD. Special mushroom will overtakes button mushroom in future. It is suggested the ever-increasing production in speciality mushrooms like Calocybe indica pose serious challenge to the current supremacy of button mushroom in the world market (Miller, 1994).

2.7 PHASES IN MUSHROOM CROP HUSBANDRY


Like any other crop cultivation, mushroom crop has commences with choice of seeds with fruiting vigour and ceases with harvesting of fully bloomed fruits (sporophores) (Chang and Miles, 1989). The fruiting ability of Calocybe indica were carefully observed with various lignocellulosic substrates by many workers (Jandaik and Kapoor, 1974, Purkayastha and Nayak, 1978, Trivedi et al., 1991, Krishnamoorthy and Muthusamy 1997, Krishnamoorthy, 2003). Milky white mushroom cultivation in India has witnessed a tremendous revolution in recent years with respect to the types and strains. Series of attempts were made to exploit this mushroom commercially by identifying suitable strain and modified production techniques. Krishnamoorthy (2003) has identified one commercially viable strain and released as a new variety APK 2, which had a yield potential of about 142% bio-efficiency.

2.7.1 Spawn production for mushroom crop cultivation


Sterilized cereal seed material which is on inoculation impregnated with mushroom mycelium is referred as spawn (Zadrazil, 1978). Earlier fermented manure or tobacco stems were used as substrates for spawn production in case of Agaricus bisporus. Sinden during 1931 introduced cereal grains as substrates for the first time for spawn production (Kligman, 1950). Mycelium grows around individual grains and provides better spawning in grain type spawn. Perlite spawn a new type of spawn was developed by Lemke in 1971. Toren (1967) and Dijektra et al. (1972) successfully used mycelia from the submerged cultures as mushroom spawn. An industrial method of spawn production based on fermented substrates was described by Hunke et al. (1973). In anaerobically fermented wheat grains, Pleurotus spp. spawn was successfully prepared (Huhnke et al. 1973). Superiority of grain spawn was established by Szudyga and Mallinowske (1974); Zadrazil (1975) proposed the use of active mycelium as a seed material, which is obtained by allowing the mycelium to spread over the mushroom substrate for a few days, which reduced spawn rate considerably. Based on the information from Stoller (1963), Lemke (1971) proposed a formula for spawn preparation, where he suggested the grain should contain 50 per cent moisture and the medium need to be maintained pH of 6.5 to 6.7 for a better mycelial growth in the spawn bottle. Further, Kumar et al. (1975) suggested the use of calcium carbonate and gypsum in the proportion 1:3 respectively for better growth of grain spawn. Apart from this, recently Sharma (2003) evaluated five cereal grains viz., Jawar, Kutki, Kodo, Maize and Wheat as spawn substrate for Pleurotus djamor. Within this evaluated spawn substrates, Kutki grain showed shortest period of 8 days for spawn development indicating its suitability for efficient spawn production and this duration was significantly less than the wheat grain (12 days).

2.7.2 Substrate preparation


Altogether, the cereal straw production in this planet was about 2946 million tones during 1988-89 (FAO, 1989). Nevertheless, it is misfortune that because of complex chemical nature of the so called lignocellulosics, these abundant resources are considered as recalcitrant molecules which cannot be degraded easily by any means (Ghosh and Singh, 1993). However, quite few microorganisms including white rot fungi degrade the lignocellulosics in an environmentally friendly way and play a key role in organic matter recycling. Mushroom fungi utilize these resources as food substrates for energy. 2.7.2.1 Lignocellulosic substrates for mushroom production All lignocellulosic substrates comprise of lignin and hemicellulose complex, cellulose, extractives and non-extractives (Silica, carbonates and oxalates). However, crop mainly derive the food from sugar complex. Though in nature mushroom fungi utilize these abundant resources, first report on utilization of sterilized straw substrate for cultivation of th Pleurotus spp. came to light during early decades of 20 century (Falck, 1917). In India Zakia Bano and Srivastava (1962) first exploited paddy straw as the elite substrate for production of Pleurotus spp. Chang et al., (1981) reported that in terms of bio-efficiency, paddy straw serves better substrate for Pleusotus sajor-caju than cotton waste. Diwakar and Munjal (1989) showed that groundnut husk, gram seed husk and cotton wastes were marginally better than the commonly used substrate (paddy straw) for cultivation of Pleurotus sajor-caju, Pleurotus sapidus, P. ostreatus and P. florida. Rajarathnum et al. (1993) reviewed the potential of various lignocellulosic substrates to support the growth of Pleurotus spp. Veena Savalgi et al. (1998) suggested to commonly occurring weed Cassia hirsuta and Sugarcane bagasse in 1:1 proportion for cultivation of Pleurotus florida to achieve maximum yield and percent bioefficiency. Tonial et al. (2000) were tried industrial residues of cassava and potato for cultivation of Volvariella volvaceae. In submerged fermentation, growth performance of cultures were evaluated by measuring their rate of radial growth and biomass production. They were reported that medium containing 2.4% (w/v) declassified potato flour and 1.6% (w/v) Cassava bagasse (PFCB) in petri dishes showed, higher growth and biomass production. Rathore and Thakore (2004) reported to use wheat straw as substrate for Pleurotus florida cultivation, since it gives maximum number of sporophores and yield. Purkayastha and Nayak (1978) first reported the propensity of Calocybe indica towards compost prepared from sand, soil and five percent maize meal. Chakrabarty et al.

(1981) reported that Calocybe indica can be cultivated on un-sterilized substrate after or without composting. However, in the same year he suggested to raise Calocybe indica on paddy straw substrate ameliorated with 4 grams of nitrogen, phosphorus, potassium fertilizers favoured the optimal yield than use of compost. Trivedi et al. (1991) scrutinized the accessibility of growing Calocybe indica on various lignocellulosic substrates and concluded that wheat straw with maize meal plus dehydrated lucerne was the suitable to provide maximum bio-efficiency. Pandey and Tiwari (1993) acknowledged that paddy straw was suitable substrate for Calocybe indica which provided better bioconversion efficiency. Abdul Wajeed and Shivappa Shetty (1995) evaluated the potentialities of various locally available mushroom substrates and reported that paddy straw was the prime supporter for production of Calocybe indica. Later Ramalakshmi (1996) tried Calocybe indica on compost prepared by using different combination of substrates. He showed that compost prepared out of paddy straw and poultry manure in the ratio of 1:1 was the best, in terms of bio-efficiency and yield. Krishnamoorthy and Muthusamy (1997) used different substrates namely paddy straw, maize stalks, sugarcane bagasse, palmrosa grass, vetiver grass, groundnut haulms, soybean hay and paddy straw compost for cultivation of Calocybe indica . Among the substrates tried paddy straw and maize stalks gave significantly higher yields with 94 to 99 per cent bioefficiencies. They were also reported that paddy straw compost was not suitable for cultivation. 2.7.2.2 Pre-treatment of substrate Across the board, the substrate milieu not only nourishes the mushroom crop, but also serves as light breeding climate for unwanted fungi. Deliberately, the substrate has to be treated prior to introduction of mushroom seed, just to create favourable environment for mycelial run. Pre-treatment of substrate arrests the growth of competitor molds at the same time makes the substrate more favourable for mushroom mycelial growth. According to postulation of Kurtzman and Zadrazil (1982), an optimum of 70 per cent moisture content in the substrate provides congenial environment for degradation of lignin by lignolytic enzymes of Pleurotus spp. They also stressed that higher moisture content in the substrate stimulated aerial mycelial growth rather substrate mycelium. Substrate pre-treatment can be accomplished with steam sterilization, pasteurization, fermentation and chemical sterilization methods. Zadrazil and Schneidereit (1972) specified that steam pasteurization of substrate at 60C to 100C for 2 to 6 hours created conducive environment for mycelial running of Pleurotus spp. Krishnamoorthy (1981) emphasized the pre-treatment of paddy straw by autoclaving or pasteurization at 15 lb pressure to maximize the bio-efficiency of Pleurotus sajor-caju. Though steam sterilization of substrate at 121C for 1 hour could effectively control the competitor molds and pathogens, it is undue method at industrial scale (Rajarathnum and Bano, 1987). Substrates steeped in hot water at 65 5C for 10 minutes to 1 hour averted the mushroom mycelium from antagonistic fungi and at the same time, imbibitions of water provided benign environment for growing mycelium of Pleurotus spp. (Kurtzman and Zadrazil, 1982). Chemical sterilization of substrate with mixture of 75 ppm carbendazim plus 500 ppm formalin has been considered as trust worthy method for substrate pre-treatment in commercial production of Pleurotus spp. (Vijay and Sohi, 1987). Abdul Wajeed and Shivappa Shetty (1995) showed that treatment of substrate with carbondazim (50 ppm) + formalin (500 ppm) followed by steam pasteulization has enhanced the sporophore yield and there by bio efficiency of Calocybe indica to maximum extent.

2.7.3 Spawning
Tewari (1991) provided an evidence for relationship between spawn rate and sporophore yield in Pleurotus sajor-caju which implied that 4% and 6% of spawn on wet weight basis have enhanced maximum sprophore yield during summer and winter season respectively. Similarly, Calocybe indica yielded more at 4% spawn rate (Doshi et al., 1993). Further, they proclaimed that 3 layer spawning method gave increased sporophore yield in Calocybe indica (Doshi et al., 1993).

2.7.4 Substrate supplementation

All lignocellulosic substrates are known to be deficient in nitrogenous nutrients. It is apparent that addition of nitrogen-rich supplements in substrate could provide an ideal food for mushroom crop, there by sustain the productivity of mushroom. Nutrient enrichment can be done at spawning, during spawn-running, after spawn running and at casing (Rajarathnum and Bano, 1987). The effect of supplements on the sporophore yield of Calocybe indica was evident with the studies of Chakravarthy et al. (1981 a) which showed that amelioration of wheat straw substrate with nitrogen, phosphorus and potassium fertilizers of each four grams confirmed satisfactory yield of sporophore in Calocybe indica. Trivedi et al. (1991) provided a source of evidence for correlation between addition of supplements at spawning and sporophore yield of Calocybe indica. They reported the maize meal (Zea mays) plus dehydrated lucerne proved to be better among various organic nitrogen supplements to increase the sporophore yield and protein content. Though supplementation at spawning enhances the sporophore yield some times it creates unfavourable environment for mushroom fungus which has to compete with weed molds. So in practice, supplements added either after spawn running or at casing to the mushroom beds could enhance the quality of sporophore rather than quantity (Randle, 1985). Ramlakshmi (1996) worked on the supplementation of mushroom compost used in Calocybe indica production. She reported the maximum bioefficiency and yield with treatment of paddy straw, poultry manure, gypsum + rice bran. The sugarcane bagasse supplemented with soybean meal or wheat bran served as valuable substrate for production of Pleurotus sajor-caju, P. eryngii and Agrocybe aegerita (Permana et al., 2000).

2.7.5 Casing
Top dressing of mycelial impregnated substrate with so called casing medium in ad hoc requirement for mild fruiting of Calocybe indica, there by, casing soil provides chief support for standing mushroom canopy. Hayes and Shandilya (1977) investigated the feasible use of various substrates as casing media in production of Agaricus bisporus and they stated that FYM and loamy soil in 1:1 ratio has supported better fruitification in the Agaricus bisporus compared to other substrates. Purkayastha and Chandra (1985) exploited the potentials of the mixture of soil and sand at 1:1 ratio as casing medium for betterment of sporophore yield in cultivation of Calocybe indica. Later Pandey and Tewari (1994) scrutinized various substrates as casing medium for crop production of Calocybe indica and concluded that coir pith or FYM are found to support better fruiting bodies of Calocybe indica. Abdul Wajeed and Shivappa Shetty (1995) evaluated the casing materials in different combinations. They reported that biogas slurry plus soil mixture (1:1 w/w) to be the best for maximum sporophore yield and bio efficiency of Calocybe indica. In 80s letter speaking word CACing (Compost Added at Casing) got into the dictionary of mushroom biology. Gupta et al. (1989) kept a tab on effect of addition of grain spawn on the yield of Agaricus bisporus and the results showed that addition of 0.1 per cent spawn (on wet weight basis of casing soil) assertively influenced sporophore yield in the same. In lieu of compost or grain spawn, hydrated Alginate pellets encompassed with mushroom mycelium added to the casing soil has fastered the initiation of basidiome and synchronized the pinning, resulting in good quality mushroom in Agaricus bisporus (Romaine and Schlagonhaufer, 1992). Gupta and Dhar (1993) advocated the addition of 0.1 per cent 2 grain spawn (on wet weight basis of casing soil) and 1.25 to 1.75 kg/m compost spawn at casing to improve the sporophore yield in Agaricus bisporus. Sharma et al. (1997) evaluated the suitability of different casing materials for Calocybe indica. They showed that two year old cowdung and biogas slurry were best suited casing materials for Calocybe indica. Krishnamoorthy et al. (1997) suggested to use steamed garden soil (clay loam: pH around 8.0) as a casing material for Calocybe indica. Later Szudyga et al. (1999) evaluated the suitability of different peat types for formulation of casing soils, and their influence on yield and average weight of the sporophores in cultivated mushroom (Agaricus bisporus). They reported that the average yield did not differ significantly between peat types but average mushroom weight was greater with a strongly decomposed

peat casing than with a weakly decomposed one. Raina et al. (2002) evaluated the fourteen combination of two year old FYM, three year old spent compost, garden soil and sand as casing material for Agaricus bisporus cultivation. They reported higher yields with the combination of FYM + garden soil + soil (4:2:1) followed by FYM + sand (3:1) and lower yields in garden soil alone.

2.8 MORPHOLOGICAL CHARACTERS OF MUSHROOMS


Purkayastha and Chandra (1985) given the general information on morphological characters of milky white mushrooms and reported that pileus of 10.0 to 14.0 cm diameter, white in colour, at first convex, later expanded and flattened shape; Margin of pileus in regular, incurved, smooth, non-straite, stripe central, sometimes eccentric, cylindrical with bulbous base, upto 10.0 cm long, white, cartilaginous, surface dry and fibrillose, base not hollow, without annulus and volva.

2.9 MUSHROOM SHELF-LIFE


Shelf-life of milky white mushrooms were studied by Krishnamoorthy (2003) and reported that mushrooms have 5 to 7 days of shelf-life when grown on paddy straw.

2.10 MUSHROOMS : ENVIRONMENTALLY PROTECTED CROP


Apparently, the benign ambient environmental conditions are the prime factors which adjudge the kinetics of production in mushroom crop husbandry. Environmentally protected crops or so called green house crops have canopied about 1.5 million hectares in this planet. Indeed, mushroom is also one such environmentally protected crop grown under various forms of cover (Fraser, 1992). An array of thoughts pertinent to influence of eco-physiological factors on sporophore yield of Calocybe indica have been elaborated.

2.10.1 Physical factors


Calocybe indica, a new mushroom known for its temperature tolerance and provides optimum yield in the temperature, range of 25-28C with relative humidity of 75-84 per cent (Trivedi et al., 1991). Jandaik and Kapoor (1976) emphasized the ecological requirements for Pleurotus sajor-caju. The ambient temperature of 25-32C during spawn run and 25C during fructification and 80-85 per cent RH throughout the cropping period have relatively created amicable environment for enhanced sporophore yield. It is evident that most of the basidiomycetes mushroom fungi showed photo-tropism and the enhanced growth of Pleurotus spp. was observed at 40-200 Lux (Kurtzman and Zadrazil, 1982) and Calocybe indica preferred the light intensity in the range of 150-200 Lux for better growth (Trivedi et al., 1991). The results of Rajarathnum et al. (1993) are evident for the substrate moisture content of 70% and particle size of 2-3 cm to acclimatize the friendly environment for mycelial growth of Pleurotus spp. Apart from these factors, hydrogen ion concentration of substrate (6.5- 7.0) and type of container (polythene bag size of 60 x 30 cm) are all the key factors to determine the sporophore yield in Pleurotus sajor-caju (Rajarathnum and Bano 1987).

2.10.2 Chemical factors


The lignocellulosic substrates serves as sole source of energy for growing mushroom crop. Celluose, hemicellulose, lignin and soluble sugar content in substrates could play important role to determine the bio-efficiency of mushroom to digest and absorb the nutrients from substrate through enzymatic attack is considered to be the main factor for selection of mushroom strain (Rajarathnum et al., 1993).

2.10.3 Biological factors


Mallesha and Shetty (1989) first characterized the brown spot disease causing organism as Pseudomonas stutzeri, which is menace in mushroom industries of Karnataka State. Weed molds like Trichoderma sp., Coprinus sp. compete for nutrients in the cultivation

of Calocybe indica (Pandey and Tewari, 1993). Lakshmipathy and Shetty (1993) first isolated and characterized a synergistic bacterial associate from paddy straw substrate as Bacillus polymyxa, which has relatively improved the bio-efficiency of Pleurotus sajor-caju compared to the control. Nair and Fahy (1976) reported the addition of peat containing bacterial antagonistic Pseudomonas fluorescens on blotch disease causal agent (Pseudomonas tolaasii). To control chaetomium (olive green mould) attempts were made by Tautarus and Townsley (1983). They found that thermophyllic Bacillus sp. showed dramatic activity to control mould when inoculated into beds.

2.11

DECOMPOSITION OF MUSHROOM FUNGUS

THE

SUBSTRATE

BY

THE

Styler (1930) found that the lignin could be degraded by mushrooms. Waksman and Mc Grath (1931) and Wakman and Nissen (1932) are the pioneer workers in the field of decomposition of manure by mushroom fungus. They studied the chemical changes that occur during horse manure composting as well as the changes occurring during the actual growth of mycelium. Loss of lignin was seen during last stage of growth where as cellulose and hemicellulose were used during early stage of growth (Cayley, 1938). Gerrits (1969) observed loss in lignin during spawn run and utilization of cellulose and pentosans during fruiting body development. Block et al. (1959) reported that corn steep liquor, molasses, glucose, cellulose and peptone aided the formation of sporophores in Pleurotus ostreatus, where as starch did not. Pleurotus spp. were used for the first time for decomposition studies by Zadrazil (1975a, 1976). He noticed the changes in the hemicellulose, cellulose and lignin status during growth of Pleurotus florida. After cultivation of mushrooms, 20 per cent of original weight remained as spent substrate, changes in water soluble sugars and glucose were also observed. Kaneshiro (1977) studied the lignocellulose status in different agricultural wastes and changes brought about by Pleurotus ostreatus, when grown on these. He recorded 10-40 per cent decrease in lignin and 15-40 decrease in cellulose during growth. Kandaswamy and Sivaprakasam (1980) reported that Pleurotus sajor-caju grows better on substrates with more cellulose and less lignins. They also reported that cellulose was more utilized during mycelial development rather than during sporophore formation. Wood (1979) worked out the biochemical changes during growth and development of mushroom and observed that during growth it behaves like white rot where as during sporophore formation it behaves like brown rot. Moorthy (1981) worked on the biochemical changes in the paddy substrate during the cultivation of Pleurotus sajor-caju. He observed loss of pentosans, lignins and cellulose to a large extent. Some studies have indicated that Pleurotus spp. and few other fungi can fix nitrogen from atmosphere (Ginterova, and Maxinova, 1975; Rangaswamy et al., 1975; Ginterova, 1971, 1973). Kurtzman (1979) reviewed the previous works and concluded through experiments that an increase is caused in nitrogen levels of media when Pleurotus is grown in pure culture. He attributed it to some kind of scavenging activity. Other species of fungi like Aspergillus, Botrytis and Pencillium are known to grow in nitrogen free medium. This is attributed to some kind of scavenging ability there by fixing nitrogenous compounds from atmosphere (Alexander, 1978). Moorthy (1981) also observed increase in nitrogen content of paddy straw after cultivation of Pleurotus sajor-caju. The role of enzymes which are produced by mushroom fungus in biodegradation of lignocellulosic wastes were studied by several workers. Doshi et al. (1987) reported the pectinolytic enzymes viz., polygalacturonase (PG), polymethyl-galacturonase (PMG), polygalacturonase transeliminase (PGTE) and Pectin transeliminase (PTE) as well as cellulolytic enzyme cellulase (Cx) production at various stages of spawn growth of Calocybe indica. Singh et al. (1989) reported production of laccase enzyme by Pleurotus sps. and observed that Pleurotus florida is the best producer of laccase and species of Pleurotus (Pleurotus florida, P. flabellatus and P. ostreatus) for their ability to produce polyphenoloxidase and to degrade lignin in solid state fermentation process. Buswell et al. (1996) worked on the lignocellulolytic enzyme profiles of edible mushroom fungi and reported the varying abilities of edible mushrooms (Lentinula edodus, Volvariella volvacea and Pleurotus sajor-caju) to utilize different lignocellulosies as growth

substrate. Abou Hadid et al. (2003) evaluated the five oyster mushroom species grown on corn stalks and reported that the strains Pleurotus florida was the effective strains for cellulose and hemicellulose degradation. Recently Dhanda et al. (2005) evaluated the potentialities of Pleurotus (oyster mushroom) to breakdown the lingo-cellulose complexes of cereal straws, for making the cellulose constituents available for ruminant feeding. Ball and Jackson (1995) were given the report on the recovery of lignocellulose degrading enzymes from spent mushroom compost. The maximum recoveries of active xylanase activity were detected in extracts from blended spent compost.

III. MATERIAL AND METHODS


Experiment was conducted to improve bio-efficiency of milky white mushroom (Calocybe indica) using both different substrates and casing materials. Studies were conducted in Department of Agricultural Microbiology, Agricultural College, University of Agricultural Sciences, Dharwad, during the year 2004-05. The details of materials used and the methods followed in this investigation are given below.

3.1 SELECTION OF CULTURES AND GROWTH MEDIA


The pure culture of Calocybe indica was obtained from Mushroom culture laboratory, Indian Institute of Horticultural Research, Bangalore and Department of Agricultural Microbiology, University of Agricultural Sciences, Dharwad-580 005. Each culture was separately checked for its fruiting ability and maintained on potato dextrose agar slant at 4C and was sub-cultured at monthly interval. Further to sustain their fruiting vigour they were preserved under refrigerated conditions. The above stock culture was used in all further studies.

3.2 DEVELOPMENT OF SPAWN


Spawn, a seed material for mushroom cultivation was prepared by following standard procedure (Krishnamoorthy, 2003). For this purpose, uninfested, clean sorghum grains were boiled with equal amount of water till grains become soft but were not allowed to split open. The moisture content of boiled grains was adjusted by air drying to obtain 50 to 55 per cent followed by blending with two per cent calcium carbonate and two per cent calcium sulphate. This admixture was filled in to saline bottles or 15 x 20 cm size polypropylene bag. The mouthpart of container was plugged with clean, non-absorbent cotton. The whole set up was sterilized for one hour at 15-pound pressure (121C) for two consecutive days. Upon cooling, the content was inoculated with 10 mm mycelial disc of Calocybe indica fruiting cultures of Calocybe indica under aseptic condition. The containers were incubated for 23-28 days for Calocybe indica to attain complete ramification over the substrate. These seed materials were used freshly without storage for cultivation of aforementioned mushroom.

3.3 SUBSTRATE SELECTION


The potentials of various locally available lignocellulosic substrates were explored to select the best supporter for the production of Calocybe indica. The substrates used for the experiment were as follows: Paddy straw Sugarcane bagasse Maize stalks Potato haulms

3.3.1 Collection of paddy straw


Paddy straw used in the experiment as a substrate was obtained from Agricultural Research Station, Mugad.

3.3.2 Collection of sugarcane bagasse


Sugarcane bagasse used in the experiment as a substrate was obtained from sugarcane juice wanders, Dharwad.

3.3.3 Collection of maize stalks


Maize stalks used in the experiment as a substrate was obtained from Main Agricultural Research Station, Dharwad.

3.3.4 Collection of potato haulms

Potato haulms used in the experiment as a substrate was obtained form Yadavad village of Dharwad district.

3.4 PRE-TREATMENT OF SUBSTRATE


Chopped substrates were soaked in clean water for 8-10 hours and allowed to imbibe water. After soaking period was over, the excess water in the substrate was drained and then, substrates were pretreated by using various methods viz., steam sterilization (80-85C) for one hour. Steam sterilization (121C) for 30 minutes, hot water dipping (80C) for 2 hour, chemical sterilization technique (carbendazim 50 ppm + formalin 500 ppm) and combined treatment (chemical sterilization technique followed by steam pasteurization). The pretreatment methods were followed as recommend by Krishnamoorthy (2003). Then the excess water present in the pasteurized straw substrate was drained out to attain the approximately moisture content of 70 per cent.

3.5 SAMPLING OF SUBSTRATES


A composite sample of 100 g was collected from different bags at different intervals i.e., before spawning, 10th day after spawning, at the time of casing (after complete mycelial colonization), at the first harvest and final harvest. The sample was kept in hot air oven at 60C to a constant weight. The dried material was powdered and samples were stored in butter paper bags. These samples were used for various estimations in order to analyse nutrient status of the substrates.

3.6 CHEMICAL ANALYSIS OF SAMPLES


3.6.1 Dry matter estimation
Ten gram of sample was dried at 105C to reach a constant weight for 8-12 hours and the weight loss was expressed as moisture % from which dry matter % on as such basis or in air dry sample derived after deducting moisture from 100.

3.6.2 Total nitrogen (mg/g)


Total nitrogen was determined by kjeldhal method as outlined by Bremmner (1979). 0.5 g of sample was digested with concentrated sulpharic acid approximately 250 mg of catalyst mixture containing potassium sulphate : copper sulphate : selenium in the ratio of 100:20:1 until clearing of the sample took place. The digest was diluted with distilled water and distilled after addition of sufficient quantity of 40 per cent NaOH to make the digest alkaline. The evolved ammonia was absorbed in 25 ml of 4 per cent boric acid and titrated with 0.05 N sulphuric acid using mixed indicator. The quantity of nitrogen in the sample was calculated as follows. Titre value x N of H2SO4 x 14 x 100 Percent nitrogen = Volume of the sample

3.6.3 Organic carbon (%)


Organic carbon content of the sample was determined by Walkley and Black Wet oxidation method as described by Jackson (1973). To the residue obtained by drying 25g of sample, ten ml of potassium dichromate solution was added along with 20ml of concentrated sulphuric acid. The excess of chromic acid was titrated against 0.5 N ferrous ammonium sulphate until the last traces of blue colour disappears. A blank was also run without the sample percentage of organic carbon was obtained using the following formula. Blank titre x Value N of ferrous ammonium sulphate x 0.003 x 100

Organic carbon = (percentage) Volume of the sample

3.6.4 Cellulose, hemicellulose and lignin content


These were estimated following the method of Goering and Vansoest (1975). Two solutions were prepared. Firstly, Neutral Detergent Fibre solution consisting of 30 g sodium lauryl sulphate, 18.61 g of ethylene diamine tetra acetic acid, 6.81 g of sodium borate decahydrate, 4.56 g of Di sodium hydrogen phosphate and 10 ml of 2 ethoxy ethanol in one litre of distilled water. The second solution being Acid Detergent Fiber solution consisting of 28.4 ml concentrated sulphuric acid and 20 g of C-TAB (Cetyl trimethyl ammonium bromide) with volume made up to 1 liter using distilled water. 3.6.4.1 Neutral Detergent Fiber (NDF) 100 ml of NDF solution was added to a known weight of dried sample in one liter beaker and refluxed for 1 hour. Then the contents of the beaker were filtered through preweighed Gooch crucible. Hot distilled water washing was done to remove all leachates in the gooch followed by two to three washings with acetone. Then, the gooch crucible with NDF was dried over night and weighed. Weight of crucible + NDF Weight of empty crucible % NDF = x 100 Weight of the sample (g) 3.6.4.2 Acid detergent fiber (ADF) To known weight of dried sample in one litre beaker 100 ml of ADF solution was added and refluxed for one hour. The contents were then transferred to gooch crucible and washed with hot distilled water followed by 2 to 3 washings with acetone. This crucible with ADF was transferred to a hot air oven and dried overnight followed by weighing. Weight of the crucible + ADF Weight of empty crucible % ADF = x 100 Weight of the sample (g) The dried crucible containing ADF was treated with 72 per cent H2SO4 for a period of three hours with frequent wetting with 72 per cent sulphuric acid. After three hours the crucible with resulting lignin was washed with hot distilled water with two to three washings with acetone. The gooch crucible with lignin was dried over night and weighed. The dry gooch crucible with lignin was subjected to dry oxidation in a muffle furnace at 600C for a period of two hours. Then the resulting crucible with silica was weighed. % Hemicellulose = % NDF - % ADF

Weight of the crucible + ADFWeight of crucible + lignin % Cellulose = x 100 Weight of the sample (g)

Weight of the crucible + lignin Weight of crucible+ Silica % Lignin = x 100 Weight of the sample (g)

3.7 SPAWNING AND SPAWN RUNNING


In this study, polyprophylene bag with 60 x 30 cm size of 80 gauge thickness were used for substrate filling five per cent of spawn was used on wet basis of substrate. Then mouth part of the container was tightly closed by using rubber bands. Bags were incubated under dark chamber condition for spawn running.

During spawn running period, temperature of 28 2C was maintained and humidity of 80 to 85 per cent was maintained. These partially controlled conditions were maintained for 20 days for complete spawn running period. The spawn running chamber was maintained hygienically by atomizing formalin (40 ppm) + Bavistin (10 ppm) mixture by using aerosol disinfector.

3.8 CROPPING
After complete mycelial formation casing was done. The method of cultivation of C. indica was followed as recommended by Pandey and Tewari, 1993) (Fig. 1).

3.8.1 Casing
The common casing mixture (2 year old FYM + garden soil 1:1 w/w) were partially pasteurized at 80C for 1 hour. Upon cooling, the pH of the medium was adjusted to 7 to 7.2 by addition of calcium carbonate. Then it was added over the mycelial impregnated substrate up to 3 to 3.5 cm thickness. The mouth part of the bag was covered with either formalin treated or autoclaved newspaper to prevent the insect and weed molds. Light watering was done twice a day and ruffling the casing soil surface was done intermittently for good aeration. The case run period was about 18-20 days. After selection of suitable substrate in first experiment, second set of experiment is carried out. Here besides common casing material or mixture (2 year old farmyard manure + Garden soil 1:1 w/w having pH of 7.8) other casing materials were used are as follows. Casing materials Biogas slurry + garden soil (1:1 w/w), Lignite + garden soil (1:1w/w) Vermicompost + garden soil (1:1 w/w) Garden soil + sheep and goat manure (1:1 w/w) pH 8.4 7.4 7.6 7.5

3.9 HARVESTING
Fully matured sporophores of milky white mushroom were harvested when the crop size was about 8-10 cm diameter. Fruiting bodies were harvested prior to watering and fresh weight was determined immediately. Like wise, each bag was allowed stand for 2-3 croppings. Bio-efficiency of mushroom was calculated by using formulae as recommended by Chang and Miles (1989). Fresh weight of mushroom (g) % Bio-efficiency = x 100 Dry weight of substrate (g)

3.9.1 Size and shelf-life of mushrooms


Mushrooms were harvested at fully maturity stage by just twisting the base of stipe or mushroom. Mushroom samples were collected at random from each bag and the following characters were studied. 3.9.1.1 Diameter of fruiting body With the help of thread, diameter of fruiting body was measured and expressed in cm. 3.9.1.2 Stipe length The length of each stipe was measured from the base of stipe to tip with the help of thread and expressed in cm.

Fig 1. Flow chart for cultivation of milky white mushroom (Calocybe indica)

3.9.1.3 Shelf-life The shelf life was calculated and expressed in days based on number of days by which mushrooms can be stored up to marketable or acceptable quality.

3.9.2 Chemical analysis of mushrooms


The mushroom samples were analysed for proximate principles namely moisture, crude protein, total carbohydrates, fats and crude fibre. 3.9.2.1 Moisture The moisture content was determined by difference between the accurately weighed samples before and after drying in an oven at 60C (Anon., 1970). 3.9.2.2 Crude protein The total nitrogen was estimated by micro Kjeldhal method using defatted and dried mushroom powder. Protein was obtained by multiplying the total nitrogen by the factor 6.25 (Anon., 1970). 3.9.2.3 Total Carbohydrates Carbohydrates in the samples were estimated by Anthrone method, 100 mg of sample was hydrolysed in a boiling water bath for 3 hrs with 5 ml of 2.5 N HCl and after boiling, neutralized with sodium carbonate until the effervescence ceases, 0.5 ml of aliquot was taken and cooled rapidly. Absorbance was measured at 630 nm. Standard graph was plotted by using glucose (Sadasivam, 1992). 3.9.3.4 Crude fat Fat content of mushroom sample was estimated by solvent extraction method by circulating petroleum ether in soxhlet apparatus for 14-16 hours (Anon., 1970). 3.9.2.5 Crude fibre Crude fibre was estimated by the acid-alkali digestion method. The residue obtained after digestion in a crucible and its weight was recorded. The dried residue was then ashed in a muffle furnace at 600C and weight was recorded. The difference in these two weights was taken as the weight of crude fibre (Jacobs. 1959). The values of crude protein, fat, crude fibre and total carbohydrates expressed in percentages.

3.10 STATISTICAL ANALYSIS


All experimental data were statistically tested by completely randomized block design. The significance of each data was analysed by calculating critical difference at 1 per cent level.

IV. EXPERIMENTAL RESULTS


An experiment was conducted at Department of Agricultural Microbiology, University of Agricultural Sciences, Dharwad during 2004-05 to screen different substrates for the production of milky white mushroom, Calocybe indica, to find out suitable casing material and nutrient content of mushrooms are presented in this chapter.

4.1 PROXIMATE ANALYSIS OF SUBSTRATES USED FOR THE CULTIVATION OF Calocybe indica
In present study different substrates used for cultivation of milky white mushroom were analysed for various chemical changes brought out by the fungus during its growth. The results are presented here under.

4.1.1 Changes in the organic carbon content of substrates


The data recorded on organic carbon content of different substrates at different intervals due to inoculation of Calocybe indica is presented in Table 1 and Fig. 2. The substrates inoculated with Calocybe indica had positive influences on the organic carbon content. In general, the organic carbon content of substrates was found to be decreased significantly with increase in the intervals up to final harvest stage, but it did not differ significantly between initial stage (87.88%) and 10 days after spawning stage (86.69%). Among the substrates, potato haulms had significantly lower organic carbon content (78.95%) followed by paddy straw (82.78%), maize stalks (86.17%) and sugarcane bagasse (88.99%). The interaction between substrates and intervals on organic carbon content was found to be significant, and it did not differ significantly between paddy straw (72.42%) and potato haulms at final harvest stage (75.28%).

4.1.2 Changes in the nitrogen content of substrates


The data recorded on nitrogen content of different substrates at different intervals due to inoculation of Calocybe indica is presented in Table 2 and Fig. 3. The substrates inoculated with Calocybe indica had positive influence on the nitrogen content. In general, the nitrogen content of substrates was found to be increased significantly with increase in the intervals up to casing stage (1.35%), beyond which it remained practically constant at first harvest stage (1.41%) and final harvest stage (1.41%). Among the substrates, paddy straw recorded significantly highest nitrogen content (1.49%) as compared to maize stalks (1.13%), sugarcane bagasse (1.12%) and potato haulms (1.06%). The interaction between substrates and intervals on nitrogen content was found to be significant at first harvest in paddy straw (1.75%), but on par with paddy straw at casing stage (1.70%) and final harvest (1.70%).

4.1.3 Changes in C : N ratio of substrates


The data pertaining the C:N ratio as influenced growth of Calocybe indica presented in Table 3 and Fig. 4. is

The substrates inoculated with Calocybe indica had positive influence on the C:N ratio. The C:N ratio was found to be decreased significantly with increase in intervals up to final harvest stage (56.80%). Among the substrates, paddy straw had significantly lower C:N ratio (58.74%) followed by other substrates, which are on par with each other (maize stalks with 80.54%, sugarcane bagasse with 81.74%, potato haulms with 81.77%). The interaction between substrates and intervals on C:N ratio was found to be significant at final harvest stage in paddy straw (42.56%), followed by paddy straw at first harvest (48.54%).

Table 1. Organic carbon content (%) in different substrates at different growth stages of Calocybe indica

Intervals Substrates At initial stage 86.62 92.53 88.97 81.43 87.88 10 days after spawning 86.14 91.78 88.26 80.60 86.69 S.Em Intervals (A) Substrates (B) AxB 0.47 0.42 0.95 Mean At casing 85.39 89.85 87.52 80.27 85.76 At first harvest 83.32 86.37 84.76 77.18 82.91 At final harvest 72.42 84.40 81.34 75.28 78.36 CD (P=0.01) 1.77 1.59 3.54 82.78 88.99 86.17 78.95

Paddy straw Sugarcane bagasse Maize stalks Potato haulms Mean

Note: Values represent the average of four replications Cropping period 50 to 55 days

Paddy straw
100

Sugarcane bagasse

Maize stalks

Potato haulms

90

80

70

Organic carbon (%)

60

50

40

30

20

10

At initial stage

10 days after spawning

At casing

At first harvest

At final harvest

Intervals Fig. 2. Effect of Calocybe indica growth on organic carbon content of substrates
Fig. 2. Effect of Calocybe indica growth on organic carbon content of substrates

Table 2. Nitrogen content (%) in different substrates at different growth stages of Calocybe indica

Intervals Substrates At initial stage 1.05 0.86 0.77 0.64 0.83 10 days after spawning 1.24 0.91 0.94 0.89 1.00 S.Em Intervals (A) Substrates (B) AxB 0.01 0.01 0.02 Mean At casing 1.70 1.31 1.24 1.18 1.35 At first harvest 1.75 1.37 1.29 1.22 1.41 At final harvest 1.70 1.20 1.36 1.35 1.41 CD (P=0.01) 0.04 0.04 0.07 1.49 1.13 1.12 1.06

Paddy straw Sugarcane bagasse Maize stalks Potato haulms Mean

Note: Values represent the average of four replications Cropping period 50 to 55 days

Paddy straw
1.8

Sugarcane bagasse

Maize stalks

Potato haulms

1.6

1.4

Nitrogen content (%)

1.2

0.8

0.6

0.4

0.2

At initial stage

10 days after spawning

At casing

At first harvest

At final harvest

Intervals Fig. 3. Effect of Calocybe indica growth on nitrogen content of substrates


Fig. 3. Effect of Calocybe indica growth on nitrogen content of substrates

4.1.4 Changes in the lignin content of substrates


The results with respect to lignin content of substrates are presented in Table 4, which showed significant differences for the substrates, intervals and their interactions as well (Fig. 5). In general, the lignin content of the substrates was found to be decreased significantly with increase in the intervals up to final harvest stages, but it did not differ between initial stages (9.89%) and 10 days after spawning (9.01%). Among substrates, potato haulms had significantly lower lignin content (4.82%) followed by maize stalks (6.65%), paddy straw (9.15%), sugarcane bagasse (10.78%). The interaction between substrates and intervals on lignin content was found to be significant at final harvest stage in potato haulms (3.30%), followed by potato haulms at first harvest (3.64%).

4.1.5 Changes in cellulose content of substrates


The data recorded on cellulose content of different substrates at different intervals, due to inoculation of Calocybe indica is presented in Table 5 and Fig. 6. The substrates inoculated with Calocybe indica at different intervals on cellulose content was found to be decreased significantly with increase in the intervals up to final harvest stage and recorded least with 24.96% of cellulose. Among the substrates, potato haulms had significantly lower cellulose content (21.38%) followed by paddy straw (27.83%) and maize stalks (28.0%) with on par results. While higher cellulose content in sugarcane bagasse (35.32%). The interaction between substrates and intervals on cellulose content was found to be significant at final harvest in potato haulms (18.69%).

4.1.6 Changes in hemicellulose content of substrates


The data recorded on hemi-cellulose content of different substrates at different intervals due to inoculation of Calocybe indica is presented in Table 6 and Fig. 7. In general, the hemicellulose content of substrates was found to be decreased significantly with increase in intervals up to final harvest stage. But decrease was non significant between initial stage (26.01%) to 10 days after spawning(25.46%). Among the substrates, potato haulms inoculated with Calocybe indica had significantly lower hemi-cellulose content (9.08%) followed by paddy straw (27.30%) sugarcane bagasse (28.49%) with on par results, and maize stalks (32.05%). The interaction between substrates and intervals on hemi-cellulose content was found to be significant at final harvest stage in potato haulms (7.10%), but it did not differ with potato haulms at first harvest.

4.1.7 Changes in dry matter content of substrates


The data recorded on dry matter content of different substrates at different intervals due to inoculation of Calocybe indica is presented in Table 7. The substrates inoculated with Calocybe indica had positive influence on the dry matter content. In general, dry matter content of substrates was found to be decreased significantly with increase in the intervals up to final harvest stage. But decrease was nonsignificant between initial stage (24.41%) to 10 days after spawning (24.38%). Among the substrates, sugarcane bagasse had significantly lower dry matter content (19.94%), followed by maize stalks (20.82%), paddy straw (22.46%) and potato haulms (23.40). The interaction between substrates and intervals on dry matter content was found to be significant at final harvest stage in sugarcane bagasse (14.38%), followed by maize stalks at final harvest (15.46%).

Table 3. C:N ratio in different substrates at different growth stages of Calocybe indica

Intervals Substrates At initial stage 82.53 107.17 114.82 131.69 109.05 10 days after spawning 69.30 100.60 93.91 91.38 88.80 S.Em Intervals (A) Substrates (B) AxB 0.66 0.60 1.33 Mean At casing 50.77 67.41 70.49 68.03 64.18 At first harvest 48.54 63.06 65.47 63.02 60.02 At final harvest 42.56 70.43 59.51 54.70 56.80 CD (P=0.01) 2.50 2.23 5.00 58.74 81.74 80.84 81.77

Paddy straw Sugarcane bagasse Maize stalks Potato haulms Mean

Note: Values represent the average of four replications Cropping period 50 to 55 days

Paddy straw
140

Sugarcane bagasse

Maize stalks

Potato haulms

120

100

C:N ratio

80

60

s
40

20

At initial stage

10 days after spawning

At casing

At first harvest

At final harvest

Intervals Fig. 4. Effect of Calocybe indica growth on C:N ratio of substrates


Fig. 4. Effect of Calocybe indica growth on C:N ratio of substrates

Table 4. Lignin content (%) in different substrates at different growth stages of Calocybe indica

Intervals Substrates At initial stage 11.50 13.56 8.29 6.19 9.89 10 days after spawning 10.46 12.21 7.85 5.50 9.01 S.Em Intervals (A) Substrates (B) AxB 0.08 0.07 0.16 Mean At casing 9.23 10.75 6.76 5.46 8.05 At first harvest 8.40 9.77 5.75 3.64 6.90 At final harvest 6.15 7.62 4.60 3.30 5.42 CD (P=0.01) 0.30 0.27 0.60 9.15 10.78 6.65 4.82

Paddy straw Sugarcane bagasse Maize stalks Potato haulms Mean

Note: Values represent the average of four replications Cropping period 50 to 55 days

Paddy straw
14

Sugarcane bagasse

Maize stalks

Potato haulms

12

10

Lignin content (%)

At initial stage

10 days after spawning

At casing

At first harvest

At final harvest

Intervals Fig. 5. Effect of Calocybe indica growth on lignin content of substrates

Fig. 5. Effect of Calocybe indica growth on lignin content of substrates

Table 5. Cellulose content (%) in different substrates at different growth stages of Calocybe indica

Intervals Substrates At initial stage 30.70 38.72 30.86 23.72 31.00 10 days after spawning 29.23 38.74 29.48 22.89 30.08 S.Em Intervals (A) Substrates (B) AxB 0.22 0.19 0.44 Mean At casing 28.62 35.13 27.88 21.74 28.35 At first harvest 26.35 32.84 25.96 19.85 26.25 At final harvest 24.23 31.16 25.77 18.69 24.97 CD (P=0.01) 0.83 0.74 1.66 27.83 35.32 28.00 21.38

Paddy straw Sugarcane bagasse Maize stalks Potato haulms Mean

Note: Values represent the average of four replications Cropping period 50 to 55 days

Paddy straw
40

Sugarcane bagasse

Maize stalks

Potato haulms

35

30

Cellulose content (%)

25

20

15

10

At initial stage

10 days after spawning

At casing

At first harvest

At final harvest

Intervals Fig. 6. Effect of Calocybe indica growth on cellulose content of substrates


Fig. 6. Effect of Calocybe indica growth on cellulose content of substrates

Table 6. Hemicellulose content (%) in different substrates at different growth stages of Calocybe indica

Intervals Substrates At initial stage 28.85 30.44 34.43 10.32 26.01 10 days after spawning 28.41 29.65 33.75 10.01 25.46 S.Em Intervals (A) Substrates (B) AxB 0.25 0.22 0.50 Mean At casing 27.88 29.16 32.30 9.47 24.71 At first harvest 26.24 27.79 30.47 8.47 23.25 At final harvest 25.12 25.36 29.26 7.10 21.71 CD (P=0.01) 0.93 0.84 1.87 27.30 28.49 32.05 9.08

Paddy straw Sugarcane bagasse Maize stalks Potato haulms Mean

Note: Values represent the average of four replications Cropping period 50 to 55 days

Paddy straw
35

Sugarcane bagasse

Maize stalks

Potato haulms

30

25

Hemicellulose content (%)

20

15

10

At initial stage

10 days after spawning

At casing

At first harvest

At final harvest

Intervals Fig. 7. Effect of Calocybe indica growth on hemicellulose content of substrates

Fig. 7. Effect of Calocybe indica growth on hemicellulose content of substrates

Table 7. Dry matter content (%) in different substrates at different growth stages of Calocybe indica

Intervals Substrates At initial stage 26.61 22.95 23.54 24.51 24.41 10 days after spawning 24.97 22.42 24.67 25.44 24.38 S.Em Intervals (A) Substrates (B) AxB 0.21 0.20 0.428 Mean At casing 22.95 21.59 21.82 25.77 23.03 At first harvest 19.68 18.33 18.59 21.58 19.55 At final harvest 18.09 14.38 15.46 19.69 16.91 CD (P=0.01) 0.80 0.72 1.60 22.46 19.94 20.82 23.40

Paddy straw Sugarcane bagasse Maize stalks Potato haulms Mean

Note: Values represent the average of four replications Cropping period 50 to 55 days

4.2 YIELD PERFORMANCE OF MUSHROOM AS INFLUENCED BY DIFFERENT SUBSTRATES


The fruiting bodies of mushrooms are produced in all the substrates were harvested and weighed, to know the yield performance of the substrates. Also the other yield parameters like days for spawn run (DFSR), Days for pin head formation (DFPF), Days for first harvest (DFFH), numbers of buttons and average weight of buttons were harvested bioefficiency was calculated and presented in Table 8. The substrates inoculated with Calocybe indica was found to be enhanced the growth and yield parameters as well. However the substrates differed in their ability to promote growth and yield performance (Fig. 8). The days for spawn run (14.20 days), days for pin head formation (7.40 days), days for first harvest (8.0 days) has taken significantly less time and also yield (347.20 g), average weight (54.24 g) and Bio-efficiency (138.88%) were found to be significantly highest in paddy straw (Plate 1). However Days for spawn run and Days for pin head formation were on par with maize stalks. But yield parameter, number of buttons harvested were significantly maximum in maize stalks (6.8) but did not differ significantly with paddy straw (6.4).

4.3 SIZE AND SHELF LIFE OF MUSHROOMS


The data pertaining to the size and shelf life of mushrooms as influenced by different substrates are presented in Table 9. Between the different substrates the difference in diameter of fruiting bodies, stipe length and shelf life were found to be practically significant. The paddy straw was recorded significantly higher diameter of fruiting bodies (7.62 cm) and stipe length (7.94 cm) followed by maize stalks with 6.9 cm diameter of fruiting bodies and 7.42 cm stipe length. However, shelf life of fruiting bodies were found to be significantly higher in paddy straw (5.8 days) over all other substrates.

4.4 PROXIMATE ANALYSIS OF MUSHROOMS PRODUCED ON DIFFERENT SUBSTRATES


The mushrooms grown on the different substrates were analyzed for their nutrient contents and moisture and results are presented in Table 10. The substrates used were influenced the nutrient contents of mushroom, which were varied significantly with each other except moisture and crude fibre content (Fig. 9). The protein content (32.43%), total carbohydrates (46.96%) were found to be significantly higher in maize stalks. However, it was on par with paddy straw with protein content of 31.58% and 46.34% of total carbohydrates. While fat content (3.63%) was found to be significantly higher in paddy straw followed by maize stalks (3.31%). But moisture and crude fibre content of mushrooms were non significant. The maximum moisture content in paddy straw (91.3%) and crude fibre in sugarcane bagasse (9.51%) and potato haulms (9.51%) were recorded.

4.5 EVALUATION OF CASING MATERIALS FOR GROWTH AND YIELD OF Calocybe indica
The second set of experiment was conducted to screen casing materials for mushroom production. The yield and yield parameters were analysed and data are presented in Table 11. All the yield and yield parameters were varied with respect to the casing materials used. However, this influence was not much affected in early stages of mushroom growth. The days for pin head formation and days for first harvest recorded non significant results with well decomposed FYM + garden soil (1.1 w/w) had minimum days for pin head formation (7.25 days) and minimum days for first harvest (8.0 days). The yield (342.87 g) and yield parameters viz., bio efficiency (137.14%) and average weight (54.8 g) were found to be significantly higher in well decomposed FYM + garden (1:1 w/w). However it was on par with

Table 8. Yield performance of Calocybe indica on different substrates

Substrates

DFSR

DFPF

DFFH

No. of buttons harvested 6.40 5.80 6.80 6.0 0.187 0.77

Average weight (g/button) 54.24 44.83 48.89 45.41 0.464 1.91

Yield (g/250 g bed) 347.20 260.20 332.92 272.34 2.481 10.22

Bio-efficiency (%) 138.88 104.07 133.16 108.93 0.969 3.99

Paddy straw Sugarcane bagasse Maize stalks Potato haulms S.Em CD (P = 0.01)

14.20 16.0 14.60 15.8 0.292 1.20

7.40 9.20 8.0 9.0 0.158 0.65

8.0 11.60 9.0 11.0 0.122 0.50

DFSR- Days for Spawn Run DFFH- Days for First Harvest

DFPF- Days for Pinhead Formation

Note: Values represent the average of five replications Weight of substrates (on dry weight basis) 250 g Polythene bag size : 60 x 30 cm with 80 gauge

140

120

Bio-efficiency (%)

100

80

60

40

20

Paddy straw

Sugarcane bagasse

Maize stalks

Potato haulms

Substrates Fig. 8. Yield performance of Calocybe indica on different substrates


Fig. 8. Yield performance of Calocybe indica on different substrates

Plate 1. Fruiting bodies of Calocybe indica grown or different substrates

Table 9. Effect of different substrates on size and shelf life of fruiting bodies of Calocybe indica

Size of mushrooms Substrates Diameter of fruiting body (cm) Paddy straw Sugarcane bagasse Maize stalks Potato haulms S.Em CD (P = 0.01) 7.62 5.02 6.90 5.62 0.121 0.50 Stipe length (cm) 7.94 6.51 7.42 6.83 0.113 0.46 5.8 5.0 5.0 5.0 0.100 0.41 Shelf life (Days)

Note: Values represent the average of five replications

Table 10. Proximate analysis of Calocybe indica grown on different substrates

Substrates Paddy straw Sugarcane bagasse Maize stalks Potato haulms S.Em CD (P = 0.01)

Moisture content (%) 91.30 91.00 91.10 91.10 0.424 NS

Protein (%) 31.58 28.38 32.43 27.73 0.465 1.91

Carbohydrates (%) 46.34 41.72 46.96 42.02 0.496 2.04

Fat (%) 3.63 3.08 3.31 3.12 0.069 0.28

Crude fibre (%) 9.34 9.51 9.40 9.51 0.042 NS

Note: Values represent the average of five replications

Moisture content (%)


100

Protein (%)

Carbohydrates (%)

Fat (%)

Crude fibre (%)

90

80

70

Per cent

60

50

40

30

20

10

Paddy straw

Sugarcane bagasse

Maize stalks

Potato haulms

Substrates Fig. 9. Proximate analysis of Calocybe indica grown on different substrates


Fig. 9. Proximate analysis of Calocybe indica grown on different substrates

Table 11. Effect of different casing materials on yield of Calocybe indica

Casing materials

DFPF

DFFH

No. of buttons harvested 6.25 6.75 6.25 6.50 6.00 0.100 0.41

Average weight (g/button) 54.80 47.61 50.55 52.24 53.07 0.894 3.72

Yield (g/250 g bed)

Bioefficiency (%) 137.14 128.61 126.48 135.83 127.56 0.599 2.49

Well decomposed FYM + garden soil (1:1 w/w) Biogas slurry + garden soil (1:1 w/w) Lignite + garden soil (1:1 w/w) Vermicompost + garden soil (1:1 w/w) Sheep and Goat manure + garden soil (1:1 w/w) S.Em CD (P = 0.01) DFPF- Days for Pinhead Formation DFFH- Days for First Harvest

7.25 7.75 7.50 7.25 7.75 0.258 NS

8.0 8.0 8.50 8.0 8.25 0.171 NS

342.87 321.72 316.22 339.60 318.92 1.510 6.28

Note: Values represent the average of four replications Depth of casing soil 3 to 3.5 cm thickness Cropping period 50-55 days, size of the polythene bag 60 x 30 cm with 80 gauge

vermicompost garden soil with 339.6 g of yield and 135.83% bio efficiency. But average weight recorded was on par with sheep and goat manure (53.07g). With respect to number of button harvested, significantly higher number of buttons were recorded in biogas slurry + garden soil (6.75) over other substrates.

4.6 SIZE AND SHELF LIFE OF MUSHROOMS INFLUENCED BY DIFFERENT CASING MATERIAL
The data on size and shelf life presented in Table 12 did not show significant differences in shelf life of mushrooms among the casing materials used. The shelf life recorded highest in well decomposed FYM + garden soil (1:1 w/w) with 5.75 days. Between different casing materials used, the differences in diameter of fruiting bodies and stipe length were found to be significant with maximum diameter of fruiting bodies (7.81 cm) and higher stipe length (8.11 cm) were recorded in the well decomposed FYM + garden soil (1:1 w/w) was on par with sheep and goat manure + garden soil (1:1 w/w) (7.72 cm) and stipe length was on par with vermicompost + garden soil (1:1 w/w) (8.06 cm).

4.7 PROXIMATE ANALYSIS OF MUSHROOMS


Mushrooms constituents were analysed from mushrooms produced using different casing materials after moisture percentage was calculated. The results are presented in Table 13. Except moisture and crude fibre content, all other nutritional contents showed significant differences with each other. Protein content (30.98%) and fat content (3.81%) were found to be significantly higher in well decomposed FYM + garden soil (1:1 w/w). However it was on par with vermicompost + garden soil (1:1w/w) with 30.12% protein and biogas slurry + garden soil (1:1 w/w) with 3.74% fat. But, total carbohydrates were found to be significantly highest in sheep and goat manure (1:1 w/w) (47.09%) and was on par with vermicompost + garden soil (1: w/w) (46.87%). While moisture content were highest in well decomposed FYM + garden soil (1:1 w/w) (91.37%) and crude fibre content in vermicompost + garden soil (1:1w/w) (10.01%).

Table 12. Effect of different casing materials on the size and shelf life of fruiting bodies of Calocybe indica

Size of mushrooms Casing materials Diameter of fruiting body (cm) 7.81 6.94 7.33 7.68 7.72 0.105 0.44 Stipe length (cm) 8.11 7.28 7.63 8.06 8.04 0.077 0.32 Shelf life (Days) 5.75 5.25 5.00 5.50 5.00 0.214 NS

Well decomposed FYM + garden soil (1:1 w/w) Biogas slurry + garden soil (1:1 w/w) Lignite + garden soil (1:1 w/w) Vermicompost + garden soil (1:1 w/w) Sheep and Goat manure + garden soil (1:1 w/w) S.Em CD (P = 0.01) Note: Values represent the average of four replications

Table 13. Proximate analysis of Calocybe indica grown on different casing materials

Casing materials

Moisture content (%) 91.37 91.35 91.17 91.30 91.02 0.484 NS

Protein (%) 30.98 29.21 27.46 30.12 30.03 0.532 2.21

Carbohydrates (%) 46.10 44.94 44.80 46.87 47.09 0.351 1.46

Fat (%)

Crude fibre (%) 9.19 9.14 9.33 10.01 9.79 0.192 NS

Well decomposed FYM + garden soil (1:1 w/w) Biogas slurry + garden soil (1:1 w/w) Lignite + garden soil (1:1 w/w) Vermicompost + garden soil (1:1 w/w) Sheep and Goat manure + garden soil (1:1 w/w) S.Em CD (P=0.01) Note: Values represent the average of four replications

3.81 3.74 3.49 3.62 3.52 0.043 0.18

V. DISCUSSION
Cultivation of mushroom is popular in India in recent days. Among the cultivated mushrooms, Calocybe indica has several advantages that can be easily and cheaply taken up by farmers and urbanates for cultivation. At present, it is growing profitably on paddy straw. However, several agricultural and plant wastes have been found to support the growth by Calocybe indica (Krishnamoorthy and Muthuswamy, 1997). These substrates showed a varied performance with respect to the yield and nutritional quality of mushrooms. This variation has been attributed to chemical composition of the substrates used. The present study was conducted to determine the changes in the chemical composition of substrates during the cultivation of mushrooms and to find out the nutritional value of mushrooms produced with the substrates. Different crop wastes namely paddy straw, sugarcane bagasse, maize stalks and potato haulms were used for cultivation of Calocybe indica. The changes in the chemical composition of these substrates were studied by estimating different chemical constituents such as organic carbon, nitrogen, C:N ratio, cellulose, hemicellulose, lignin and also dry matter. Yield parameters and proximate analysis of mushrooms were also carried out. In the second set of experiment, suitable substrate (paddy straw) is selected and evaluated for yield, shelf life and nutritional quality of mushrooms by using different casing materials viz., well decomposed farmyard manure, biogas slurry, lignite, vermicompost and sheep and goat manure with combination of garden soil. The results obtained are discussed in this chapter. From the chemical analysis of the substrates at different intervals, the following conclusions could be drawn. The organic carbon content of substrates were reduced over a period of mushroom growth from initial stage to final harvest stage. Out of the total organic carbon content, maximum reduction occurred during fruitification stage. The similar trend was observed in each substrates with maximum reduction in organic carbon at fruitification stage. In the present investigation, there was a drastic reduction in organic carbon in paddy straw indicated that the carbon has been utilized by the mushroom fungus more effectively in paddy straw than in the other three substrates. These results can be compared to the report of Zadrazil and Brunnert (1980) who investigated the degradation of total organic matter during the solid state fermentation of straw by white rot fungi including some mushroom sps. In their investigation, also found that elevated temperature and prolonged incubation, increase the degradation of total organic matter of the substate. The nitrogen content showed an interesting trend in all the substrates used. Its content was increased during the growth and development of mushroom fungus. Out of the total percent increase, the maximum increase in nitrogen content was noticed during active spawn run stage (10 days after spawning to casing stage) in all the four substrates used. Previous studies, using similar organic wastes by several workers attributed for increase in the nitrogen content of substrates through the atmospheric nitrogen fixation by the mushroom itself. However, some reports showed that, the associative bacteria in the substrate might have fixed atmospheric nitrogen. Lignin degrading bacteria reported by Deschamps et al. (1980) include Klebsiella which is known to fix atmosphic nitrogen. Moorthy (1981) suspected that the increase in nitrogen in the spent substrate for the presence of associative microflora, one of the dominant bacterial isolate was tentatively identified as Beijerinkia sp. The role of such associative bacteria in the nitrogen economy of the mushroom substrates could be interesting study. However, Abou-Habid et al. (2003) conducted a detailed screening study of all species of oyster mushroom reported to fix atmospheric nitrogen. They suggested that Pleurotus sp. has capacity to increase protein content of 90 per cent over the untreated maize stalks. Supporting to this results earlier, Mallesha and Shetty (1989); Lakshmipathy and Shetty (1993); Ramalakshmi and Shivappa Shetty (1996) were also showed a similar trend of increase in the total nitrogen. Although total nitrogen of substrates was increased during the mushroom growth, the substrates at fruitification stage were recorded less per cent of

nitrogen increase. This might be due to conversion of substrate nitrogen in to protein form in mushrooms. Variation in the carbon content or nitrogen content reflects in the value of C:N ratio. It stabilizes when both these constituents are utilized in same proportion which was present in the substrates. The wide variation in substrate C:N ratio itself reflected variation in fungus growth. At optimum C:N ratio, fungus grows best as shown in case of paddy straw (82.5 :1). Quimio (1981) suggested that good growth was obtained with C:N ratio of 90:1. It looks, wide range of C:N ratio in case of sugarcane bagasse (107.1:1), maize stalks (114.82:1) and potato haulms (131.8:1) when compared to paddy straw. Combining these substrates in proper proportion to optimize the C:N ratio. In the present study, all the substrates showed decrease in C:N ratio. However, the reduction in C:N ratio generally from initial stage to final harvest stage with more than 65 per cent reduction of C:N ratio in between initial to casing stage and rest in final harvest stage. Similar trend has been reported by Ramalakshmi (1996). Cellulose, hemicellulose and lignin which constitute the major part of plant waste are known to have direct impact on the growth and development of mushroom fungi (Zadrazil, 1975a). The results from the present investigation are in consonance with Moorthy (1981) and Singh et al. (1989) observed that cellulose, hemicellulose and lignin are degraded up to an extent of 75 per cent during the growth period. In general, during the growth of mushroom the lignin content of substrates were reduced with maximum rate in fruiting stage compared to spawn run stage. The studies on Pleurotus sp. (Singh et al., 1989) indicated that a gradual decrease in lignin up to harvest of crop, and loss was up to 70 per cent during fruitification compare to spawn run stage, which was true with Calocybe indica as observed in the present study. Earlier, Sivaprakasam and Kandaswamy (1980) reported one controversy result to our study that the growth of Pleurotus sajar-caju will reduce the lignin content of different substrates significantly up to 20 days after inoculation and later the reduction was non significant. During the growth of mushroom, the cellulose content of substrates were reduced with maximum rate in fruitification stage compared to spawn run stage. The results of present study are in line with work of Doshi et al. (1987) who reported a reduction of 60 per cent of cellulose in the substrate is due to increase in the activity of cellulase enzyme by the end of spawn run period or casing of Calocybe indica. Utilization of hemicellulose was not as much as lignin and cellulose even though a little variation in the trend of decrease from the beginning until the end. During the growth of mushroom, the hemicellulose of substrates was reduced with maximum rate in fruitification stage and minimum in spawn run stage. The similar results were obtained in the study by Adamovic et al. (1998). They reported the degradation rate of cell wall components of wheat straw by Pleurotus ostreatus with highest in case of lignin (37%) followed by cellulose (17.4%) and lowest in hemicellulose (15%). Summing up all the above observations, it can be concluded that lignin and cellulose were utilized at a moderate rate in sugarcane bagasse, at lowest rate in maize stalks and highest in paddy straw and potato haulms. In case of hemicellulose utilization was maximum in potato haulms and minimum in paddy straw. However, this variation in the substrate utilization is largely depends on the enzymes, which are produced by mushrooms and constituents of the substrates. Buswell et al. (1996) reported the lignolytic enzyme profiles of edible mushroom fungi and observed that Lentinus edodus was cultivated on highly lignified substrate such as wood or saw dust, produces two extra cellular enzymes which have been associated with lignin depolymerization in other fungi, (Manganese peroxide and laccase) conversely, Volveriella volvaceae, which has high cellulose, low lignin containing substrates produces a family of cellulolytic enzymes including at least five endoglucanases, five cellobiohydrolases and 2 beta-glucosidases, but none of the recognized lignin-degrading enzymes. So this reports indicate the varying ability of mushrooms to utilize different lignocellulosics as growth substrates. The dry matter of the substrates not changed significantly during initial stages of mushroom growth. After casing stage, maximum dry matter reduction takes place in all the substrates. This reduction in later stages due to high degradation or utilization of substrates for growth by mushroom.

5.1 SCREENING OF SUBSTRATES FOR GROWTH AND YIELD OF Calocybe indica


A preliminary experiment was conducted to compare various substrates for their ability to support growth of Calocybe indica. The data indicated that paddy straw supported the best growth and given significantly higher yield over maize stalks, sugarcane bagasse and potato haulms. This was similar to earlier report by Krishnamoorthy and Muthuswamy (1997) who reported that paddy straw and maize stalks gave significantly higher yields. Mushroom yields from various substrates were compared by using the parameters viz., days for spawn run, day for pin head formation, days for first harvest, number of buttons harvested, average weight of buttons and percent biological efficiency. Paddy straw realized maximum per cent bio-efficiency (138.88) when compared to other substrates. Mushroom yield was also followed a similar trend with paddy straw (347.20g), maize stalk (332.92g), potato (272.34g) and sugarcane bagasse (260.20g). In the present studies per cent biological efficiency of 138.88 realised in case of paddy straw was comparable to that of 142.0 per cent as reported by Krishnamoorthy (2003). Significant increase in yield of mushroom can be traced back to the significant increase in the yield components, number of buttons harvested and average weight of buttons were indirectly exerted greater influence on the yield of mushroom. Paddy straw recorded 6.40 number of buttons was on par with highest number of buttons (6.8) recorded in maize stalks and 54.24 g of average weight which was significantly higher over the maize stalks (48.89g). It was given significantly higher yields over maize stalks, sugarcane bagasse and potato haulms. In mushroom cultivation, crop growth duration is also play a crucial role along with mushroom species, substrate selection and yield. In the present study, crop growth duration is verified using parameters like days for spawn run, days for pin head formation and days for first harvest. The different substrates used in the experiment showed variation in crop growth duration. Paddy straw was taken minimum time (14.2 days) to complete spawn run was on par with maize stalks (14.6 days). The maximum time was taken by sugarcane bagasse (16 days) was also on par with potato haulms (15.8 days). These results are similar to that of Krishnamoorthy (2003) who reported the days for spawn run lowest in case of paddy straw with 14.0 days and followed by maize stalks (14.8 days). The days taken for the pin head formation was shortest in case of paddy straw with 7.40 days was on par with maize stalks (8.0 days) and highest in sugarcane bagasse (9.2 days). In later stage of first harvest also, paddy straw was recorded significantly shortest duration of 8.0 day over maize stalks (9.0 days). By looking into these results we can conclude that days for pin head formation and days for first harvest were found to be less in substrates that gave better yields. A layer of nutritionally deficient medium (casing material) induces the formation of fruiting bodies by allowing free exchange of gases and supply of necessary water to the developing mushrooms. Along with this, casing material also plays following important role. 1. The regulation of moisture content in casing soil results in lowering of temperature in the vicinity of developing mushrooms through evaporative cooling. 2. Protect the substrate or compost surface from invasion of other micro-organisms. 3. It provides physical support to developing fruit bodies.

5.2 MUSHROOM SIZE AND SHELF LIFE INFLUENCED BY DIFFERENT SUBSTRATES


Morphology is the one of the important characteristic feature of mushroom includes size and surface features of mushrooms. Most of these characters affected by several factors like cultural practices, climatic factors and substrate selection during mushroom cultivation (Theradimani et al., 2001). In the present study, the size of the buttons were varied significantly with the substrates used. The maximum stipe length and diameter of fruiting bodies were recorded in paddy straw and followed by maize stalks which were yielded high. These maximum sized buttons in beds which were grown on paddy straw, as the substrate

may have balanced nutrients to supply and to support good growth of mushroom. It implies good yield of mushroom depends upon quality of the substrates, the cultural practices, environmental condition during cropping and most critical is nutrients available from the substrate. Shelf life is one of the important character of mushroom which is mainly influenced by environmental condition during storage and also cultural practices, substrate selection during mushroom cultivation. In our study paddy straw recorded significantly superior shelf life of mushroom (5.8 days) over other substrates and these results were comparable to earlier reports of Krishnamoorthy (2003), who reported that Calocybe indica grown on paddy straw given significantly superior shelf life (6.0 days) over maize stalks.

5.3 INFLUENCE OF SUBSTRATES ON NUTRITIONAL VALUE OF MUSHROOMS


The nutrients absorbed by the mycelium out of the decaying organic matter accumulate and get transformed in to various constituents of the mushroom fruiting body. Mushrooms are thus a rich source of nutrients, particularly proteins, carbohydrates, vitamins, minerals, fat and fibre contents. These nutritional components varied widely with different species of mushrooms. In the present study, proximate analysis of fruiting bodies of milky white mushroom grown on four substrates was studied. The moisture content in all the mushroom samples grown on four substrates were shown non significant results. But, the carbohydrates and crude protein varied depending upon the substrate on which mushrooms are cultivated. Total carbohydrate content was maximum in mushroom with maize stalks (46.98%) was on par with paddy straw (46.34%). Till now, except Krishnamoorthy (2003), no one worker has done on nutritional evaluation of Calocybe indica. As compare to our investigations, he reported little higher % of carbohydrates (52.6%) in fruiting bodies of milky white mushroom grown on paddy straw. Perhaps this variation in carbohydrates content might be due to variable composition of substrates on which these were produced. Protein content in the mushrooms produced on four substrates varied widely. The maize stalks gave mushroom with maximum protein content of 32.43 per cent and was on par with paddy straw (31.58%) followed by sugar cane bagasse (28.38%). These results are similar to that of Krishnamoorthy (2003), who reported the protein content of 32.3 per cent in mushroom produced on the paddy straw. But, compare to our studies, Purkayastha and Nayak (1978) observed the varied results of protein content with 16 per cent in case of compost + rice husk. For boosting these results, Hayes and Haddad (1976) attributed the conclusion that the variation in the protein content might be due to cultural practices and the combination of substrates used. Analysis of fat and fibre content followed different trend in mushrooms produced on substrates. Fat recorded significantly highest in paddy straw (3.63%) over other substrates. Fibre content was observed as non-significant and recorded highest in sugarcane bagasse (9.51%) and lowest in paddy straw (9.34%). These results are in agreement with the previous work of Rathore and Thakore (2004); and Krishnamoorthy (2003). They reported that mushrooms which are produced on paddy straw contains 4.5 per cent fat and 12.7 per cent crude fibre. The present study showed that plant wastes viz., potato haulms, sugarcane bagasse and maize stalks have inferior biological efficiency when compared to that of paddy straw. This may be due to improper balance of critical chemical components required for milky white mushroom cultivation. This requirement may be satisfied using substrate combination experiments. The nutritional variation in the mushroom produced on four substrates suggested that food components namely carbohydrates, protein, fat and crude fibre were generally high in mushroom from maize stalks and paddy straw. Inspite of being a nutritionally deficient medium, casing layer plays an important role in the productivity of milky white mushroom. It includes the fruiting bodies by allowing free exchange of gasses of supply of necessary water to the developing mushrooms. So, in second set of experiment, various casing materials have been used as casing soil namely well decomposed FYM + garden soil (1:1 w/w), Biogas slurry + garden soil (1:1 w/w), lignite + garden soil (1:1 w/w), vermicompost + garden soil (1:1 w/w) and evaluated yield and nutritional quality of mushrooms. Well growth supported paddy straw in first set of experiment

was used as substrate for milky white mushroom cultivation. The results are discussed in this chapter.

5.4 SCREENING OF CASING MATERIALS FOR GROWTH AND YIELD OF Calocybe indica
An experiment was carried out to evaluate the influence of different casing materials on growth and yield of mushroom. The data indicated that well decomposed FYM + garden soil (1:1 w/w) recorded maximum per cent bio efficiency (137.14%) and is on par with vermicompost + garden soil (1:1 w/w) (135.83%) compared to other casing materials used. Mushroom yield was also followed a similar trend with highest in well decomposed FYM + garden soil (1:1 w/w) (342.87g) and with lowest in lignite + garden soil (1:1 w/w) (316.22g). This higher yield may be due to presence of higher beneficial nutrients in FYM and vermicompost with garden soil compared to other casing materials. Supporting to our results, Raina et al. (2002) recorded the highest yield in Agaricus bisporus with FYM + garden soil + sand (4:2:1 v/v) as casing material and similar higher yield was obtained by using 2 year old cow dung and biogas slurry singly as casing medium in Calocybe indica by Sharma et al. (1997). Significant increase in yield of mushroom can be trace backed to the significant increase in the yield components, number of buttons harvested and average weight of buttons were indirectly exerted greater influence on the yield of mushroom. Higher yield was obtained with well decomposed FYM + garden soil, which recorded 6.50 number of buttons was on par with highest number of buttons (6.75) recorded in biogas slurry + garden soil and 54.80 g average weight of mushroom grown from well decomposed FYM + garden soil (1:1 w/w) was significantly higher over the biogas slurry + garden soil (1:1 w/w). To boosting to these results, Szudyga et al. (1999) reported that the average yields did not differ significantly between peat casing soils but average weight was greater with a strongly decomposed peat casing than with a weakly decomposed one. In mushroom cultivation, crop growth duration is also play a crucial role along with mushroom species, substrate selection, casing and yield. In the present study, crop growth duration is verified using parameters like days for pin head formation and days for first harvest. But different casing materials used in the experiment did not showed variation in crop growth duration.

5.5 MUSHROOMS SIZE AND SHELF LIFE WERE INFLUENCED BY DIFFERENT CASING MATERIALS
The size of mushrooms were significantly varied with the treatments used. The maximum stipe length and diameter of pileus were recorded in well decomposed FYM + garden soil (1:1 w/w) which were yielded high. This results showed that the maximum yield where recorded in beds with maximum sized buttons. However, shelf life of mushrooms were not influenced by different casing materials.

5.6 INFLUENCES OF CASING MATERIALS ON NUTRITIONAL VALUE OF MUSHROOMS


Mushrooms are considered as highly flavoured low calory protenaceous vegetables containing sufficient amount of nutritional components namely carbohydrates, proteins, vitamins, minerals fat and crude fibre. These nutritional components of mushrooms varied widely with several factors like quality of substrates, species of mushrooms and cultural practices followed during cropping. In the present study, proximate analysis of fruiting bodies of milky white mushroom using different casing material were conducted. The moisture content in all the mushrooms grown using different casing material were shown non significant results. But, all nutritional components are varied widely with different casing materials except crude fibre which recorded non significant results. Protein content was maximum in treatment receiving well decomposed FYM + garden soil (1:1 w/w) (30.98%). Similarly, carbohydrate in sheep and goat manure + garden soil (1:1 w/w) (47.09%) and, fat in well decomposed FYM + garden soil (1:1 w/w) (3.81%). Lignite + garden soil (1:1 w/w) recorded lowest protein (27.46%), carbohydrate (44.8%) and fats with 3.49 per cent. This variation in the proximate composition of mushrooms grown by using different casing

materials may be due to variation in the chemical composition and supporting ability of casing materials during cultivation.

VI. SUMMARY
Experiments were conducted to evaluate the four different lignocellulosic substrates, namely paddy straw, sugarcane bagasse, maize stalks and potato haulms for milky mushroom (Calocybe indica) production. Biochemical changes occurred in the substrates during the growth of mushroom fungus were studied. Further, the influence of these substrates on yield, shelf life and nutritional value of mushrooms were examined. Study of chemical composition of the substrates indicated the significant variation in all chemical constituents of four substrates used. The growth of mushroom fungus transformed the substrates into mushrooms and there was reduction in the quantity of chemical constituents of the substrates. A drastic reduction in the organic carbon content of the substrates throughout the growth of Calocybe indica was noticed. Paddy straw recorded maximum loss of carbon (16.39%) especially at fruitification stage. When these substrates were analysed for nitrogen, overall increase in total nitrogen was observed up to casing stage of fungal growth. Later, it showed a reduced nitrogen content due to fruitification. The substrates showed a marked reduction in C:N ratio after the growth of milky mushroom. The range of C:N ratio between 42 to 82 was found to be suitable for better mushroom growth. The substrates when analysed for lignin, cellulose, hemicellulose showed a significant reduction in these components during growth of a fungus. The lignin and cellulose were utilized at a moderate rate in sugarcane bagasse, at lowest rate in maize stalks and high in paddy straw and potato haulms. Incase of hemicellulose its utilization was maximum in potato haulms and minimum in paddy straw. The dry matter was decreased significantly in later stage of mushroom growth due to drastic utilization of substrates. The study on biological efficiency indicated that, out of four substrates, paddy straw alone realized the highest bio-efficiency (138.88%) and followed by maize stalks (133.16%). Higher yielded paddy straw also recorded significantly more number of buttons (6.4), higher average weight of buttons (54.24 g) and minimum time for completion of the spawn run (14.2 days), pin head formation (7.40 days) and days for first harvest (8 days) compare to other substrates. Similarly, size of buttons and shelf life varied significantly and recorded maximum stipe length (7.94 cm), diameter of fruiting body (7.62 cm) and shelf life (5-8 days) in paddy straw. Nutritional studies of mushrooms indicated significant variation in protein, carbohydrate and fat content of mushrooms produced from different substrates. Mushroom from maize stalks recorded high protein (32.43%) and carbohydrates (46.98%), which were on par with paddy straw. The mushroom from paddy straw recorded highest fat (3.63%). Meanwhile both moisture and fibre content of mushrooms were shown non-significant results. Second set of experiments were conducted to evaluate different casing materials on paddy straw for production of milky white mushroom. The casing materials used are well decomposed FYM + garden soil (1:1 w/w), biogas slurry + garden soil (1:1 w/w), lignite + garden soil (1:1 w/w), vermicompost + garden soil (1:1 w/w) and sheep and goat manure + garden soil (1:1 w/w). Further examined the influence of casing materials on yield and nutritional value of mushrooms. Well decomposed FYM + garden soil (1:1 w/w) has shown significantly higher bioefficiency (137.14%) as compared to other casing materials used. The least was observed in lignite + garden soil (1:1 w/w) (126.48%). The yield attributing other characters were also reported significantly higher in well decomposed FYM + garden soil (1:1 w/w) with maximum number of buttons (6.50) and average weight (54.80 g), but influence of casing materials on days for pin head formation and days for first harvest were shown non-significant. With irrespective of different casing materials, all the mushrooms showed shelf life of 5 to 5.5 days. However, size of mushroom varied significantly with maximum stipe length and diameter of pileus was in case of well decomposed FYM + garden soil (1:1 w/w). Nutritionally, mushrooms varied significantly with highest protein (30.98%) and fat (3.81%) in well decomposed FYM + garden soil (1:1 w/w) and carbohydrate in sheep and goat manure + garden soil (1:1 w/w) (47.09%). Meanwhile, moisture and crude fibre content were found to be non-significant. So, both casing materials and different substrates were influenced the bio-efficiency and nutritional value of milky white mushroom. The production of mushroom can be further

improved by combining nutritionally rich substrates or altering the physical and environmental condition for mushroom growth chamber.

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BIODEGRADATION AND BIOSYNTHETIC CAPACITY OF MILKY WHITE MUSHROOM (Calocybe indica)


ADINATH A. KARNAWADI 2006
ABSTRACT
The experiments were conducted at Department of Agricultural Microbiology, University of Agricultural Sciences, Dharwad during 2004-05 for screening and selection of different substrates viz., paddy straw, sugarcane bagasse, maize stalks, potato haulms for the production of milky white mushroom, Calocybe indica. Also different casing materials viz., well decomposed FYM, biogas slurry, lignite, vermicompost and sheep and goat manure in combination with garden soil (1:1 w/w) were tried for mushroom cultivation using paddy straw as a single substrate. The yield and nutrient content of mushrooms were examined. Meanwhile, biochemical changes that occurred in the substrates during the growth of mushrooms were studied. The study of chemical composition of substrates indicated the significant variation in all chemical constituents of four substrates used. The lignin, cellulose, hemicellulose, organic carbon, C:N and dry matter of substrates showed a significant reduction during growth of a fungus. But, nitrogen content was increased over a period of mushroom growth. Out of four substrates studied, paddy straw alone realized the highest bioefficiency (138.88%) and followed by maize stalks (133.16%). Paddy straw also recorded significantly more number of buttons (6.4), minimum time for completion of the spawn run, pin head formation and days for first harvest (14.2, 7.4 and 8 days, respectively) and maximum shelf life (5.8 days). Nutritionally, mushrooms from maize stalks recorded high protein (32.43%) and carbohydrates (46.98%), which were on par with paddy straw. The mushrooms from paddy straw recorded highest fat (3.63%). Meanwhile, both moisture and crude fibre of mushrooms were shown non-significant results. As compared to other casing materials used, well decomposed FYM+garden soil (1:1 w/w) has shown significantly higher bioefficiency (137.14%) and maximum number of buttons (6.50). But, shelflife, days taken for pin head formation and first harvest were shown nonsignificant results. Nutritionally, mushrooms varied significantly with highest protein (30.98%) and fat (3.81%) in well decomposed FYM+garden soil (1:1 w/w) and carbohydrates in sheep and goat manure+garden soil (1:1 w/w) (47.09%). Meanwhile, moisture and crude fibre content were found to be non-significant.

Mrs. VEENA SAVALGI MAJOR ADVISOR