Académique Documents
Professionnel Documents
Culture Documents
Thesis submitted to the University of Agricultural Sciences, Dharwad in partial fulfillment of the requirements for the Degree of
By
ADINATH A. KARNAWADI
ADVISORY COMMITTEE
(VEENA SAVALGI)
MAJOR ADVISOR Approved by : Chairman : ____________________________ (VEENA SAVALGI)
CONTENTS
Title
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REVIEW OF LITERATURE MATERIAL AND METHODS EXPERIMENTAL RESULTS DISCUSSION SUMMARY REFERENCES
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LIST OF TABLES
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
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LIST OF FIGURES
Figure No. 1.
Title
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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LIST OF PLATES
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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
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).
(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).
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.
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
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.
Potato haulms used in the experiment as a substrate was obtained form Yadavad village of Dharwad district.
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)
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)
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.
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.
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
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
60
50
40
30
20
10
At initial stage
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
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
1.2
0.8
0.6
0.4
0.2
At initial stage
At casing
At first harvest
At final harvest
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
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
At casing
At first harvest
At final harvest
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
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
At initial stage
At casing
At first harvest
At final harvest
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
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
25
20
15
10
At initial stage
At casing
At first harvest
At final harvest
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
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
20
15
10
At initial stage
At casing
At first harvest
At final harvest
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
Note: Values represent the average of four replications Cropping period 50 to 55 days
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
Substrates
DFSR
DFPF
DFFH
Paddy straw Sugarcane bagasse Maize stalks Potato haulms S.Em CD (P = 0.01)
DFSR- Days for Spawn Run DFFH- Days for First Harvest
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
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)
Substrates Paddy straw Sugarcane bagasse Maize stalks Potato haulms S.Em CD (P = 0.01)
Protein (%)
Carbohydrates (%)
Fat (%)
90
80
70
Per cent
60
50
40
30
20
10
Paddy straw
Sugarcane bagasse
Maize stalks
Potato haulms
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
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
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).
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
Fat (%)
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
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.
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.
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.
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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