Effect of different agro-wastes, casing materials and supplements on the growth, yield and nutrition of milky mushroom (Calocybe indica )
Article Category: Research Article
Publié en ligne: 23 juin 2020
Pages: 115 - 124
Reçu: 23 nov. 2019
Accepté: 13 mai 2020
DOI: https://doi.org/10.2478/fhort-2020-0011
Mots clés
© 2020 Hasan Sardar et al., published by Sciendo
This work is licensed under the Creative Commons Attribution-NonCommercial-NoDerivatives 3.0 License.
Mushrooms are appreciated in most of the countries due to their delicacy, flavour, taste and texture and are considered as an ideal health food (Stamets, 2011). Mushroom cultivation is gaining popularity among growers in urban and peri-urban areas due to inexpensive raw materials including agriculture and industrial waste (Chang, 2007). Mushrooms are considered as a rich source of protein (20–40%) on a dry weight basis (Kurtzman, 2005), dietary fibres, minerals (P, K, Na, Ca and Fe), vitamins such as B1, B2, B12, niacin, folate and ascorbic acid (Mattila et al., 2001) and amino acids while being low in fats. Mushrooms have been renowned also due to high medicinal properties such as anti-ageing, antiviral, antioxidative, anti-hypertensive, antimicrobial, antibacterial, antifungal, anticancer, antitumour, anti-inflammatory and antihypotensive (Patel et al., 2012).
Milky mushroom (
Temperature, amount of moisture in the air as well as in substrate, growing substrate type, spawn age and percentage, culture media, carbon dioxide and oxygen in growth room, air circulation, substrate pH, carbon-nitrogen ratio in substrate and supplements affect the growth of mushroom (Kadiri, 1999; Sardar et al., 2015). It is necessary to find the appropriate culture media, temperature, pH and substrate for higher yield and nutrition of cultivated mushroom. Mushroom fruiting body development depends on lignocellulosic waste material that provides nutrition such as carbon and nitrogen for growth (Miles and Chang, 2004). A large variety of diverse agricultural waste materials including wheat straw, peanut waste, cotton waste, paper waste, olive mill waste, banana leaves, sawdust, sugarcane bagasse, corncobs, coffee waste, soybean straw, peanut hull, and so on are used for cultivation of mushroom (Krishnamoorthy et al., 2000; Amin et al., 2010; Sardar et al., 2017).
In commercial cultivation of milky mushroom, casing is an important agronomic practice on which fruit bodies appear. The casing layer is used to cover the compost after the germination phase and stimulate the transition from vegetative to reproductive growth (Pardo et al., 2004). Casing materials must have high water holding capacity, a good air space ratio to facilitate gaseous exchange, porosity and bulk density (Yadav, 2006). Several casing materials were used by various researchers for mushroom cultivation such as peat moss, loam soil, spent mushroom substrate, coconut coir, biogas slurry, farmyard manure, and so on (Krishnamoorthy et al., 2000). In addition to the casing layer, substrates are supplemented with various materials to influence the spawn run, yield and biological efficiency. Several supplementing materials, such as cotton cake, soybean flour, maize powder, wheat bran, mustard cake, cottonseed, pigeon pea powder, lentil powder, gram powder, neem cake, rice bran, loam soil, spent mushroom substrate, and so on, were used by many workers to enhance the yield and biological efficiency of mushroom (Alam et al., 2010; Amin et al., 2010; Kumar et al., 2012).
Oyster and button mushrooms, which require low temperature (18–20°C) for its growth and production, can only be cultivated during winter months or under controlled conditions (Chang, 2008). The major challenge for mushroom cultivation is maintaining low temperature during the months of summer which ranges between 27 and 45°C (Miles and Chang, 2004). If facilities of low-temperature control are not available during summer months, it necessitates the selection for cultivable mushroom species that are tolerant to high temperature. Milky mushroom is one of the best edible mushrooms which is grown at high temperature during summer months (Vijaykumar et al., 2014). However, there is a need to specify substrates for the cultivation of milky mushroom. Therefore, this study was carried out to determine the best temperature, pH requirement, substrate, casing material and supplementing material for the cultivation of milky mushroom.
Four experiments were conducted in this study. In the first experiment, the mycelial growth of mushroom was recorded at different temperatures, agar media and pH levels. The second experiment was conducted to assess the best growing substrate for nutritional and agronomic characteristics of milky mushroom. The third experiment tested the influence of various casing constituents on the yield and biological efficiency of milky mushroom. In the fourth experiment, supplements were used in substrates for enhancing the growth and yield of milky mushroom.
Milky mushroom
Two types of agar media, that is potato dextrose agar (PDA) and malt extract agar (MEA), were used for the mycelial development at various temperature levels, that is, 25°C, 30°C and 35°C.
For the preparation of PDA was prepared by boiling 200 g peeled potatoes in 1 L water, and extract of potato mixed with 20 g agar and 20 g dextrose in 1 L distilled water. Similarly, the MEA medium was prepared by mixing 20 g agar, 20 g dextrose and 20 g malt extract in 1 L distilled water. Both mediums were poured in volumetric flasks separately and autoclaved to kill microorganisms. The prepared and sterilised media were poured (20 mL) in 90 mm petri dish separately. After solidification of media in petri dish, mushroom culture was inoculated at the centre of the petri dish with 5 mm disc of actively growing mycelium under aseptic conditions with three replications. Mycelium growth in the petri dish was measured after 9 days of inoculated.
After standardisation of agar media and temperature, pH was optimised by using HCL and NaOH. The pH of the media was set at different pH levels (6, 7 and 8) after sterilisation of media in autoclave (Automatic Drying Autoclave ATV1100) at 121°C. Mycelial growth (cm) was also recorded after 9 days of inoculation (Hoa et al., 2015).
Spawn was prepared for the cultivation of milky mushroom. Spawn of milky mushroom was prepared according to the method described by Sardar et al. (2017) with little modifications. Instead of using millet grains, wheat grains were used for the covering of mycelium on them. The wheat grains were half-boiled at 100°C for 30 min (Ruiz-Rodríguez et al., 2011). The pH of the spawn was maintained by adding CaCO3 and CaSO4 in wheat grain. Before inoculating the grains with mycelium grains were autoclaved at 121°C and pressure 15 psi for 30 min to kill the microorganisms and to sterilise grains. The mycelium-coated grains were filled in polythene bags of 1 kg and placed for complete covering of mycelium at temperature 30 ± 2°C. The mycelium took 18–20 days to completely cover the grains.
Substrates for the cultivation of milky mushroom were prepared according to the method described by Amin et al. (2010) with little alterations. The substrates were soaked in water for 24 h and then spread on the floor to constitute the level of moisture at 65% (determined by using hot air oven). The prepared media (1.5 kg) was filled in transparent polypropylene bags of 25.4 cm × 38.1 cm size and pasteurised. The mouths of bags were closed with rubber loose band and opened after the completion of mycelium running.
Substrates filled bags were inoculation with 5% spawn and then incubated at 30°C for mycelium growth in the mushroom growth room. After the completion of mycelium growth in the bags with fungus, 2.5 cm thick layer of casing material was applied uniformly on the substrate. The average relative humidity was maintained at above 85% and the temperature range was set between 30 and 35°C. Humidity was maintained with the help of humidifier (SINBO Hislon SAH-6107). Furthermore, fresh air in the growing room was circulated with an exhaust fan (size 12 inch) to lower the CO2 below 1,000 ppm after every 3–4 h.
The mushroom fruiting bodies were harvested after 25 days (on fruiting body maturity), and observations on growth parameters were recorded. The following data were recorded during the experiment.
Mushroom growth attributes such as time taken to complete mycelium running of bags in days (colonisation of substrate with white mycelium which can be seen through transparent polythene bags), stalk diameter (cm), stalk length (cm), number of fruiting bodies harvested, fruiting body fresh weight (g), pileus diameter (cm), total yield (g/bag) and biological efficiency = (total yield obtained from individual bag/dry weight of substrate used × 100) expressed as percentage (Royse et al., 2004).
Moisture percentage, protein contents, nitrogen and potassium were analysed according to the method of AOAC (1990). Moisture content was determined by drying fresh samples until constant weight at 105°C in a hot air oven. The nitrogen content was determined by micro Kjeldahl method and protein content (N × 6.24) expressed as percentage (Hoa et al., 2015). The ash content was estimated from the sample incinerated at 600 ± 15°C (Raghuramulu et al. 2003). Phosphorus was measured according to the procedure as previously described by Chapman and Parker (1961).
Substrates used for the cultivation of milky mushroom were chemically analysed for nitrogen, phosphorous and potassium before inoculation and after harvesting the mushrooms. The analysis was performed as described for the mushroom fruiting bodies analysis (AOAC, 1990).
Effect of casing materials, that is peat moss, spent mushroom substrate and loam soil on the growth of mushroom, was evaluated. Wheat straw was used as a substrate in this experiment on the basis of the evaluation of the substrates during Experiment No. 2. Experimental conditions and substrate preparation methods were the same as in Experiment No. 2. All the casing materials were subjected to therm treatment at 65°C for 4 h. After therm treatment of casing materials, they were spread on the bags upon completion of mycelium running. A 2.5 cm thick layer of casing material was applied uniformly on the substrate. The following parameters were recorded for this experiment: number of fruiting bodies harvested from per bag, total yield obtained from per bag and biological efficiency in percentage.
Effects of various easily available supplementation materials (i.e. wheat bran and rice bran) were tested with substrate (wheat straw) on the growth of milky mushroom. The supplements were sun-dried and sterilised in an autoclave at 121°C for 20 min. Each of the supplements (4% dry weight) was mixed separately with substrate (wheat straw) before spawning. The substrates were filled in the bags, and spawning was done as described in Experiment No. 2 with the same experimental conditions. The following observations were recorded: days to complete mycelium running of substrate filled bags, number of mature fruiting bodies harvested from per bag, total yield obtained per bag in grams and biological efficiency expressed as percentage.
Data collected were analysed using the Statistix 8.1 software. One way ANOVA was performed using the LSD test at
The effects of two different culture medium (PDA and MEA) on mycelial growth of
Figure 1
The mycelial growth (cm) of
The influence of pH variations on the mycelial growth of milky mushroom is shown in Figure 2. PDA media was used in this experiment with pH levels of 6, 7 and 8. There was a significant difference in the growth of mycelia of milky mushroom as shown in Figure 2. The maximum mycelial growth of
Figure 2
The mycelial growth (cm) of
As summarised in Tables 1 and 2, three different substrates were tested for the highest yield and nutritional components of milky mushroom. The time required to complete mycelium running in days differs significantly (
Effect of different substrates on growth attributes such as mycelial running, number of fruiting bodies per bag, fresh weight of fruiting body, pileus diameter, stem diameter, stem length of
| Treatment | Mycelial running (days) | Pileus diameter (cm) | Stem diameter (cm) | Stem length (cm) | Number of fruiting bodies/bag | Fresh weigh of fruiting body (g) |
|---|---|---|---|---|---|---|
| Wheat straw | 23.00 ± 0.81 c | 5.05 ± 0.07 a | 1.93 ± 0.24 a | 8.17 ± 0.18 a | 13.00 ± 0.40 a | 37.99 ± 0.60 a |
| Rice straw | 28.33 ± 1.03 b | 4.51 ± 0.15 b | 1.57 ± 0.08 a | 7.03 ± 0.20 b | 11.00 ± 0.40 a | 34.40 ± 1.00 ab |
| Cotton waste | 34.00 ± 1.63 a | 4.06 ± 0.08 b | 1.60 ± 0.04 a | 6.53 ± 0.13 c | 7.33 ± 0.23 b | 28.08 ± 1.20 b |
Values are expressed as means ± standard error.
Means with different letters within columns are significantly (
Effect of substrates on growth attributes such as total yield (g), biological efficiency (%) of
| Treatment | Total yield (g/bag) | Biological efficiency (%) |
|---|---|---|
| Wheat straw | 493.67 ± 18.66 a | 54.627 ± 1.91 a |
| Rice straw | 332.00 ± 12.09 b | 36.883 ± 1.34 b |
| Cotton waste | 206.67 ± 12.04 c | 22.957 ± 1.33 c |
Values are expressed as means ± standard error.
Means with different letters within columns are significantly (
The pileus diameter and stem length significantly varied from each other on different substrates, while the stem diameter of mushroom was found non-significant (Table 1). The maximum stem diameter (1.93 cm) was observed in wheat straw, followed by cotton waste (1.60 cm) while the minimum was measured on rice straw (1.57 cm). Although stem diameter was non-significant among substrates, the diameter observed in wheat straw was ~18% greater than in other substrates. The stem length of
Different substrates had a significant influence on number of fruiting bodies (Table 1). The highest number of fruiting bodies per bag was observed in wheat straw (13) followed by rice straw (11), while the lowest (7) in cotton waste substrate. Similarly, the fresh weight of mushroom fruiting bodies differed significantly among substrates. The maximum fresh weight of fruiting body (37 g) was recorded in wheat straw, while the minimum weight of fruiting body (28 g) was noted from cotton waste substrate. Regarding total yield obtained after harvesting of all mushrooms from bags, there was a significant difference (
Nutritional composition (expressed on a dry weight basis) of
Effect of substrates on biochemical attributes such as N, P, K, Ash, moisture, protein of
| Treatments | N (%) | P (mg · 100 g−1) | K (mg · 100 g−1) | Ash (%) | Moisture (%) | Protein content (%) |
|---|---|---|---|---|---|---|
| Wheat straw | 3.78 ± 0.10 a | 389.33 ± 8.08 b | 1,966 ± 62.60 a | 10.00 ± 0.40 a | 85.00 ± 0.81 a | 23.67 ± 0.62 a |
| Rice straw | 3.09 ± 0.10 b | 441.33 ± 6.88 a | 1,700 ± 40.98 b | 7.33 ± 0.85 b | 85.00 ± 1.22 a | 19.33 ± 0.62 b |
| Cotton waste | 3.46 ± 0.10 ab | 356.33 ± 4.51 c | 1,400 ± 40.98 c | 8.33 ± 0.62 b | 83.00 ± 0.40 a | 21.67 ± 0.62 ab |
Values are expressed as means ± standard error.
Means with different letters within columns are significantly (
The N, P, K content significantly varied in fruiting bodies of mushrooms grown on different substrates (Table 3). The maximum N and K contents were present in mushroom grown on wheat straw substrate, among three different types of substrates, rice straw substrate gave the maximum amount of phosphorous in
The substrates were analysed for their nutrient contents before and after cropping (Table 4). After 60 days of incubation with
Biochemical analysis of substrates (waste materials) before and after cultivating mushroom
| Treatment | N (%) | P (%) | K (%) | |||
|---|---|---|---|---|---|---|
| Before cropping | After cropping | Before cropping | After cropping | Before cropping | After cropping | |
| Wheat straw | 1.123 ± 0.039 b | 1.233 ± 0.15 b | 0.760 ± 0.008 b | 0.866 ± 0.025 ab | 1.170 ± 0.014 b | 1.426 ± 0.11 a |
| Rice straw | 0.993 ± 0.008 b | 1.250 ± 0.05 b | 0.683 ± 0.01 c | 0.816 ± 0.040 b | 1.153 ± 0.06 b | 1.396 ± 0.12 a |
| Cotton waste | 1.500 ± 0.04 a | 1.586 ± 0.12 a | 0.853 ± 0.01 a | 1.013 ± 0.113 a | 1.353 ± 0.008 a | 1.566 ± 0.08 a |
Values are expressed as means ± standard error.
Means with different letters within columns are significantly (
The different casing materials (peat moss, loam soil and spent mushroom substrate) were tested for their effect on agronomic characteristics of
Effect of casing materials on growth attributes of
| Treatment | Biological efficiency (%) | Fresh weight of fruiting body (g) | Number of fruits/bags | Total yield (g/bag) |
|---|---|---|---|---|
| Peat moss | 60.79 ± 2.73 a | 41.07 ± 0.65 a | 15.33 ± 0.40 a | 547.18 ± 24.65 a |
| Loam soil | 50.55 ± 1.02 b | 37.99 ± 0.60 ab | 12.00 ± 0.40 b | 455.01 ± 9.23 b |
| Spent mushroom substrate | 41.47 ± 0.72 c | 34.03 ± 0.68 b | 11.00 ± 0.40 b | 373.26 ± 6.51 c |
Values are expressed as means ± standard error.
Means with different letters within columns are significantly (
A significant variation was observed in wheat bran and rice bran supplementation to wheat straw substrate on growth and yield of milky mushroom (Tables 6 and 7). Days required to complete mycelial running in substrates filled bags were minimum on wheat bran supplement while, in control highest days required to colonise substrate (Table 6). Stalk diameter was recorded highest in both wheat and rice bran supplemented substrates, while non-supplemented substrate (control) produced lowest stalk diameter mushrooms. The pileus (cap) diameter of fruiting body was found to be significantly higher on wheat bran supplemented substrate as compared to non-supplemented substrate. The maximum number of fruiting bodies was obtained with wheat bran supplement, followed by rice bran, while the minimum was found with the non-supplemented substrate (Table 7). The equally maximum fresh weight of mushroom fruiting body was obtained with wheat bran and rice bran supplement. However, the minimum numbers were obtained from non-supplemented substrate (control).
Effect of supplements with wheat straw substrates on growth attributes of
| Treatment | Biological efficiency (%) | Fresh weight of fruiting body (g) | Number fruit/bag | Total yield (g/bag) |
|---|---|---|---|---|
| Wheat straw (control) | 43.62 ± 2.42 c | 35.99 ± 1.09 b | 11.00 ± 0.40 c | 393.67 ± 21.84 c |
| Wheat bran | 69.28 ± 1.85 a | 44.590 ± 0.86 a | 16.00 ± 0.40 a | 623.60 ± 16.67 a |
| Rice bran | 58.75 ± 0.84 b | 40.763 ± 0.70 a | 14.00 ± 0.40 b | 528.78 ± 7.62 b |
Values are expressed as means ± standard error.
Means with different letters within columns are significantly (
Effect of supplements with wheat straw substrates on growth attributes of
| Treatment | Mycelial running (days) | Stem length (cm) | Pileus diameter (cm) | Stem diameter (cm) |
|---|---|---|---|---|
| Wheat straw (control) | 26.33 ± 0.62 a | 7.10 ± 0.08 c | 4.88 ± 0.09 c | 1.70 ± 0.08 b |
| Wheat bran | 20.21 ± 0.62 b | 8.33 ± 0.10 a | 6.14 ± 0.14 a | 2.60 ± 0.08 a |
| Rice bran | 24.28 ± 0.62 a | 7.70 ± 0.80 b | 5.44 ± 0.10 b | 2.23 ± 0.06 a |
Values are expressed as means ± standard error.
Means with different letters within columns are significantly (
The total yield obtained of milky mushroom per bag significantly varied with supplementing materials (Table 7). The maximum total yield was obtained with wheat bran supplement as compared to non-supplemented substrate. The highest biological efficiency was obtained with wheat bran supplement followed by rice bran supplement. However, the lowest efficiency was noted in non-supplemented substrate (Table 7). These findings are in agreement with the outcome of Alam et al. (2010) who found that wheat bran supplement added with substrate increased the growth, yield and biological efficiency of milky mushroom. In addition, the effect of wheat bran supplement has also been reported in tropical and sub-tropical oyster mushrooms by Gurjar and Doshi (1995). The most common supplements are the sources of organic nitrogen such as cereal bran, which are necessary for the growth of the mycelial mass but may interfere with the productivity and biological efficiency of mushrooms. Similarly, Alam et al. (2010) reported that the addition of different supplements with the substrates influenced the spawn run, days for pinning, number of pinhead initiation, flushing pattern and overall mushroom yield.
Milky mushroom grows best at 30°C which is higher than required by other cultivated mushrooms. Regarding media used to culture the mycelium that is used for further spawn making, PDA is the best media. Among the locally available lignocellulosic substrates, wheat straw supplemented with wheat bran improved total yield and other agronomic and nutritional characteristics for milky mushroom production. Casing materials, such as peat moss, improved the quality as well as total yield of mushroom. In this study, peat moss proved to be the best casing material.