Journal of Applied Botany and Food Quality 95, 167 - 173 (2022), DOI:10.5073/JABFQ.2022.095.021 1Institute of Botany, University of the Punjab, Lahore, Pakistan Domestication and element analysis of the giant edible Macrocybe gigantea from Pakistan Aneeqa Ghafoor1, Abdul Rehman Niazi1*, Najam-ul-Sehar Afshan1 (Submitted: January 11, 2022; Accepted: June 22, 2022) * Corresponding author Summary During a survey of mushrooms of Pakistan, Macrocybe gigantea was collected from University of the Punjab, Lahore, Pakistan under the Morus species. For the domestication of this wild species, its culturability and cultivation potential was assessed by using different synthetic culture media and substrates. Among these different media used, maximum cultural growth was observed on Potato Dextrose Agar (PDA) medium at 30 ℃ followed by Malt Extract Agar (MEA), Compost Extract Agar (CEA), Glucose Peptone Agar (GPA), and Saboraud Dextrose Agar (SDA). Strains on PDA medium were used for production of spawning material on wheat, sorghum and barley grains. Sorghum grains at 30 ℃ were the best combination for spawn production. A mixed substrate of wheat straw and Tea waste at 30 ℃ produced the highest yield. Mineral analysis of the wild and culti- vated strain revealed that both forms enrich Potassium and Calcium. These findings show that this giant edible mushroom species could be domesticated on the number of media and substrates. Its domestica- tion can provide nutritional, economical, medicinal and tasty food to the growing population that would otherwise be restricted to natural production at a specific time of the year. Keywords: Biological efficiency, Culturability, Cultivation potential, Macrocybe gigantea, Spawn. Introduction The genus Macrocybe Pegler & Lodge emerged out in 1998 as sepa- rate entity from the genus Tricholoma on the basis of morphological and molecular evidences (Pegler et al., 1998). Species of Tricholo- ma are obligatory ectomycorrhizal with clampless hyphae while the species of Macrocybe are large, saprophytic with clamped hyphae (Pegler et al., 1998; razzaq et al., 2017). Recent comparative genomic approaches also support that Macrocybe is a stand-alone genus from Tricholoma (Kui et al., 2021). It has nine well-recognized species, distributed in the tropical regions of the world, which in- clude M. gigantea (Massee) Pegler & Lodge, M. pachymeres (Berk. & Broome) Pegler & Lodge, M. praegrandis (Berk.) M. spectabi- lis (Peerally & Sutra) Pegler & Lodge, M. crassa (Sacc.) Pegler & Lodge, M. praegrandis (Berk.) Pegler & Lodge, M. titans (H.E. Bigelow & Kimbr.) Pegler, Lodge & Nakasone, M. lobayensis (R. Heim) Pegler & Lodge and Macrocybe sardoa Vizzini, Consiglio & M. Marchetti and most of these species are edible (Pegler et al., 1998; Duong et al., 2017; Vizzini et al., 2020). Initially, the Macrocybe species were only studied for pharmaceutical purposes (Chatterjee et al., 2011), while their farming is relatively new as compared to other commonly cultivable mushrooms. M. gigantea (Massee) Pegler & Lodge belongs to family Tricholomataceae under the order Agaricales and was first reported as Tricholoma giganteum (Massee, 1912). This giant mushroom is mainly confined to the pantropical regions (Asia and Africa) and commonly found in the West Bengal region. However, it is also reported from the Japan, USA, and Korea (Khatua and aCharya, 2016; ghosh and aCharya, 2022). It grows gregariously in shady, grassy areas or with angiospermic trees in high temperature and humidity and occurs in hylaea and subtropical rain forests in Africa and Asia (huang, 2001). It is mostly grown in clus- ter form of about 20 to 30kg (Kui et al., 2021). It has a smooth cap, whitish to greyish white in color, the gills are straw yellow in color and are crowded. The stipe is 15-18 cm in length and 6 cm in diam- eter and its color is same as that of the cap. The spore print is white (Pegler et al., 1998; Kui et al., 2021). It contains water-soluble polysaccharides and certain bioactive com- pounds that have a pharmaceutical value (Kui et al., 2021) as anti- tumor, anticancer, antioxidant and antimicrobial agent (Chatterjee et al., 2011; gaur and rao, 2016). Macrocybin, a natural triglyce- ride present in Macrocybe species reduces tumor cells both in vitro and in vivo by interacting with the actin cytoskeleton (Vilarino et al., 2020). Ethanolic extracts of M. gigantea revealed the presence of antioxidants like Vitamin E, colchicines, and 5-methyldioadenosine. (aCharya et al., 2012). It is also rich in mineral elements such as Ca (38-470 mg/kg), Mg (84-550 mg/kg) and Zn (16-160 mg/kg) (liu et al., 2012). M. gigantea is also valuable because it sustains high temperature range 30~38 ℃ in addition to nutrient and taste (aMin et al., 2010). Due to these nutritional and therapeutic attributes, (Chatterjee et al., 2011; niazi and ghafoor, 2021) it could be advantageous to grow this fungus at industrial scale for maximum benefits. M. gigantea can meet the demand of food of a growing population due to both nutri- tional and therapeutic peculiarities. However, in wild form, there is a chance of radioactive contamination, which can be overcome by the cultivation under controlled conditions (falanDysz et al., 2015). Cultivation practices of M. gigantea, M. crassa, and M. lobayense are described in literature but none of the species farming status reached the industrial scale in any country. Initially, M. gigantea was com- monly cultivated as Tricholoma gigentium (yoshiKazu and taKashi 1997; DaDwal, 1984; lu, 1992; KiM et al., 1998; Kinjo and Miyagi, 2006; Chen et al., 2012; inyoD et al., 2016) as one of the largest edibles tricholomatoid agarics of southeast Asia. It is being cultivated on a small scale in India and China but requires more research and refinement. Generally, it requires a temperature between 25-35 °C, 70-80% relative humidity and light of 8-10 h for its growth (razaq et al., 2016; inyoD et al., 2016; VerMa et al., 2017; aKhtar et al., 2019; DeVi and suMbali, 2021). From Pakistan, only one species of M. gigantea is reported (razaq et al., 2016). Pakistan’s climatic conditions are favourable for its natural growth but its domestication was never tried before. The aim of this study was to optimize the standard cultivation require- ments of the M. gigantea and to compare the element composition of the wild and artificially cultivated fruiting bodies to get maximum benefit from this edible mushroom. Its domestication can compete with the nutritional peculiarities of the widely growing edible fungal species like button and oyster strains. 168 A. Ghafoor, A.R. Niazi, N.-u.-S. Afshan A step towards Sustainable Development Goal 2 “Zero Hunger” The findings will help open new possibilities for food production, namely large scale production of Macrocybe gigantea, and contribute to sustainability by optimizing the use of waste materials as substrates for food production. Material and methods Sampling and experimental design Basidiomata of the M. gigantea were collected in gregarious form un- der the Morus species from University of the Punjab, Lahore Pakistan. The collected specimen were photographed using an Android camera and identified by macro-microscopically and phylogenetically ac- cording to the literature available (razzaq et al., 2016). The experi- ments were carried out in Fungal Biology and Systematics Research lab, Institute of Botany, University of the Punjab, Lahore. Specimen M. gigantea (LAH03821), was deposited in the Herbarium, Institute of Botany, University of the Punjab, Lahore, Pakistan (LAH) for ready reference. Evaluation of culturability Culturability of M. gigantea was assessed according to the method described by siDDiq et al., 2018. Small tissues from inner unexposed part of the fruiting body were placed onto five different nutrient agar media i.e, Malt extract agar (2% MEA: agar 20 g, malt extract 20 g dissolved into 1000 mL dH2O), Potato dextrose agar (2% PDA: thin potato slices 200 g, glucose 20 g, agar 20 g per liter of dH2O), Glucose peptone agar medium (2% GPA: 20 g peptone, 20 g dex- trose, 5 g Nacl, 15 g agar dissolved into 1000 ml dH2O), Saboraud dextrose agar ( 2% SDA: 15 g agar, 40 g dextrose, 10 g peptone dis- solved into 1000 ml dH2O) and Compost extract agar (2% CEA: 20 g agar,10 g glucose dissolved into 1000 mL wheat straw water based filtrate). Inoculated petri plates were sealed with paraffin film and then incubated at different temperatures i.e., 15 ℃, 20 ℃, 25 ℃, 30 ℃, and 35 ℃. Mycelial growth characteristics were observed on regular basis. Each effect was determined in triplicates. These mush- room cultures were also deposited in the Herbarium Culture Collection of University of the Punjab, Lahore (as LAH#072021C(ABCDE). Spawn production Spawn was prepared by following Pal and thuPa (1979) described methodology. Three types of cereal grains viz., sorghum, wheat, and barley grains were used as the substrate to determine the spawn pro- duction efficiency. For spawn preparation, grains were washed and soaked for overnight, boiled for half an hour and excess water from grains was removed by spreading them on blotting paper. Three quarters of the each 1 L filter jars was filled with boiled grains sup- plemented with gypsum (2 g) and lime (1 g) and then autoclaved. Spawns were prepared by inoculating mycelial discs from pure PDA culture on sterilized grains in a laminar air flow cabinet. Inoculated grains were incubated at 30 ℃. Effect of grains on production of spawning material was determined in triplicates. Substrate production Wheat straw, sawdust and tea waste were used as the raw material for substrate production. Dried wheat straw collected from the field area, University of the Punjab, Lahore, Sawdust collected from the furniture shop, while tea-waste collected from the Hostel Canteens, University of the Punjab, Lahore. Tea waste is just the left-over resi- dues of tea (water containing tea) after usage. It is enriched with cel- lulose, hemicellulose, proteins, lipids, polyphenols as well as many minerals (zhu et al., 2013). Six types of substrates were prepared. Three of pure types i.e., wheat straw, sawdust and tea-waste while three were of mixed type i.e., sawdust and wheat straw, tea waste and saw dust and tea waste and wheat straw. For substrate produc- tion (pure and mixed types), raw materials were sprinkled with water and piled up, 65% moisture maintained during the substrate produc- tion process of ten days. Piles were turned every second day. Chicken manure and urea (one fourth of the substrates (25 g/1 kg) were added as supplements for nitrogen (N) and carbon (C) source on the second and last turning while gypsum (15 g/kg) was added and thoroughly mixed before the pasteurization process. When substrates were pre- pared, 700 g/bag were filled in polypropylene bags (20 × 15cm) and autoclaved for 3 to 4 hours for sterilization purpose. Experiment was performed in triplicates. Spawning Sterilized substrate bags were kept until their temperature reached 28 ℃ (for spawning), then they were inoculated with the spawn pre- pared on sorghum grains (25 g/700 g). The bag mouths were loosely tied with the rubber bands and incubated at different temperatures. Spawn running Spawn running period was observed on different substrates at differ- ent temperatures. During the spawn running, relative humidity (70%) was maintained by humidifier and ventilation fan at different incuba- tion temperatures. When the spawn running was almost completed, casing with autoclaved tea-waste (tea containing water) was done to maintain the moisture of the substrates. After pinhead emergence, bags were transferred to the cropping room with relative humidity of 85%, maintained by continuous ventilation. Biological efficiency Biological efficiency (dry weight basis) of different types of sub- strates up to three flushes was observed as per 700 g of the substrate bags. weight of fresh mushroom Biological efficiency = × 100 % dry weight of substrate Element analysis Element analysis of the wild and cultivated fruiting bodies on the mixed substrate of wheat straw and Tea waste was pursued by us- ing standard procedures described by horwitz et al. (1970). Powder samples were subjected to digestion by wet digestion method to check the mineral contents present in the samples. Minerals i.e., Potassium (K), Zinc (Zn), Calcium (Ca), Nickle (Ni), Copper (Cu) and Cobalt (Co) were analyzed through wet digestion method. Standard solutions of 5, 10, 15 20 and 25 ppm were prepared from the stock solution of the five required metals except potassium. For potassium, standard solution of 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10 ppm were prepared from the stock solution (1000 mL) of potassium and were analyzed in Atomic Absorption (AA) Spectrophotometer (PerkinElmer AAnalyst100) and in Flame Photometer (JANWAY PFP 7). Statistical analysis of the data Completely randomized design was used to determine the differ- ent parameters i.e., culturability, spawn production and cultivation potential. All the treatments were evaluated in triplicates and two- way Analysis of variance was applied to determine the significant differences between different treatments. SPSS software package was used for the statistical analysis. Data is also expressed as mean value ± S.E. Domestication and element analysis of the Macrocybe gigantea 169 Results and discussion Screening the most effective culture medium and optimization of temperature Mycelium extension rate and density always rely on the suitable culture medium utilized for culturing (reyes et al., 2009). Mycelial features of M. gigantea were evaluated on various nutritive solid media at different temperatures. White colored mycelium appeared in cultures without any exudates, with regular pattern and moderate to abundant density on different solid media. (Fig. 1). Cottony myce- lium with fibrillar growth was observed on all the media used for the evaluation of mycelial growth potential. Findings of this study were similar with Dulay et al., 2021; in which they observed the same mycelial characteristics for L. tigrinus on the majority of carbon sources used for its culturing. Amongst the different media utilized for the culturing of M. gigan- tea, PDA proved the most efficient in terms of mycelium extension rate (13.96±0.033 mm/day) and density (abundant). SDA medium (6.43±0.035 mm/day) at 15 ℃ was found to be the least appropriate for the mycelial growth of this species (Fig. 1). The current results coincide with sarDar et al., 2015 as they found that PDA is the best medium for the growth of mycelium of various Pleurotus species. Our findings were also in concurrent with the roy and KrishnaPPa, 2018; they evaluated the medium growth specificity of the M. gigan- tea on different nutritive solid media such as PDA, MEA, SDA and Czapek Dextrose Agar (CDA). They determined the maximum mycelial growth on the PDA medium followed by MEA. jo et al., 2002 evaluated the cultural potential of Ganoderma lucidum and screened the PDA as the appropriate medium for the mycelial growth. However, our findings were contrasted with the shiM et al., 2005 as they found the minimum mycelial density of Macrolepiota procera on the PDA medium. Vegetative growth of mushrooms needs a specific range of tempera- ture for proliferation due to its effect on metabolic reactions. Different basidiomycetes species grow in a wide range of temperatures, while the best temperature is between 20-30 ℃ (VahiDi et al., 2004; lai et al., 2014). For the vegetative growth of M. gigantea, temperature suitability was evaluated and 30 °C was determined as the optimum temperature on all the solid nutritive media used for culturability test- ing. At 35 ℃, mycelium growth slowed down. Our findings were also in line with the temperature response of various tropical mush- rooms like Lentinus squarrosulus (leon et al., 2017), Collybia re- inakeana (reyes et al., 1997) and Volvariella volvacea (reyes et al., 1998). All media used for the evaluation of culturability of M. gigantea were proven to be supportive for its growth, with PDA being the best option at 30 ℃. The mycelium extension rate (mm/ day) on different media at different temperatures differed significant- ly at p<0.001 as shown in Tab. 1. Spawn production Spawn is the medium for the transformation of the mushroom my- celium to the growing substrate (wozniaK, 2009). It promotes quick colonization of the mushroom substrate and initiates successful fruit- ing (Chang, 2009). The colonization rate of the active mycelium (cultured on the PDA medium) was checked on cereal grains (sor- ghum, wheat and barley) at 30 ℃. Mycelium colonized more quickly on sorghum (Sorghum bicolor) followed by wheat (Triticum aesti- vum) and barley (Hordeum vulgare) (Fig. 2). Full spawning material (whole grains covered with the mycelium) was ready on sorghum grains after 13 days of inoculation. The ample growth of mycelium on sorghum grains was due to its moisture and nutrient composition (leDer, 2004). This is in agree- ment with DeVi and suMbali (2021) for the spawn production of Fig. 1 (A-F): A: Basidiomata of M. gigantea; B-F: Cultures on different nutri- ent agar media at 30 oC after 12 days of inoculation; B: on PDA; C: on MEA; D: on CEA; E: on GPA; F: on SDA. Scale bar: A: 2 cm, B-F: 1 cm Table 1: Mycelial growth rate (mm/day) of M. gigantea on different media at different temperatures. CEA, Compost Extract Agar; PDA, Potato dextrose agar ; MEA, Malt Extract Agar ; SDA, Saboraud Dextrose Agar ; GPA Glucose Peptone Agar Values given are mean ± Standard error. Media type and temperature have significant impact over Mycelium growth rate (p<0.001). Moreover, the joint effect of media and temperature has also a significant impact over Mycelium extension rate (p<0.001). In addition, LSD test was applied and found the significant differences between all possible combinations of media types (p<0.001) and between all possible combinations of temperatures (p<0.001). Ty p e s o f media Mycelium extension rate (mm/day) Temperatures 15 °C 20 °C 25 °C 30 °C 35 °C P-value PDA 7.33±0.033 10.27±0.057 12.33±0.035 13.96±0.033 12.85±0.033 <0.001 MEA 7.25±0.036 9.83±0.035 11.28±0.033 12.87±0.033 11.95±0.033 <0.001 CEA 6.95±0.043 9.3±0.033 10.4±0.057 12.26±0.631 11.36±0.036 <0.001 GPA 6.76±0.033 8.96±0.033 9.9±0.057 11.24±0.033 10.96±0.036 <0.001 SDA 6.43±0.035 6.96±0.033 7.86±0.033 8.56±0.036 7.93±0.035 <0.001 P-value <0.001 <0.001 <0.001 <0.001 <0.001 25 Tab. 1: Mycelial growth rate (mm/day) of M. gigantea on different media at different temperatures. CEA, Compost Extract Agar; PDA, Potato dextrose agar; MEA, Malt Extract Agar; SDA, Saboraud Dextrose Agar; GPA Glucose Peptone Agar Types of media Mycelium extension rate (mm/day) Temperatures 15 °C 20 °C 25 °C 30 °C 35 °C P-value PDA <0.001 MEA <0.001 CEA <0.001 GPA <0.001 SDA <0.001 P-value <0.001 <0.001 <0.001 <0.001 <0.001 Values given are mean ± Standard error. Media type and temperature have significant impact over Mycelium growth rate (p<0.001). Moreover, the joint effect of media and temperature has also a significant impact over Mycelium extension rate (p<0.001). In addition, LSD test was applied and found the significant differences between all possible combinations of media types (p<0.001) and between all possible combinations of temperatures (p<0.001). (Green-Yellow-Red) Scheme was applied on the tables for quick readability of the most and least effective treatment on all the parameters. Green color indicated the best treatment while red color showed the least effective. 170 A. Ghafoor, A.R. Niazi, N.-u.-S. Afshan M. gigantea. leon et al. (2017) observed the shortest incubation period for spawn production of wild strain of L. squarrosulus on sorghum grains from the Philippines. These grains were also found to be the good spawning material for Agaricus blazei and Agrocybe aegerita (galaMgaM, 2009; MarCelo, 2011). bharti (2019) found wheat grains while PaMitha (2014) and Duong et al. (2017) found paddy grains as the suitable substrate for spawn preparation of M. gigantea. Tab. 2 shows that the days required to complete spawn production of M. gigantea on sorghum, wheat and barley grains at 30 ℃ significantly differed at (p<0.001). Determination of efficient lignocellulosic substrate for fruiting and yield of M. gigantea The success of a newly cultivated strain depends on both economical and biological factors (thawthong et al., 2014). Temperature is one of the key biological factors for successful fruiting of any mushroom or the conversion of the dikaryotic mycelium into the fruiting body. Mata et al. (2005) reported that different factors like, temperature, light and humidity of the incubation room influence the spawn runn- ing of mushrooms. Spawn prepared on sorghum grains was used to determine the spawn running time of M. gigantea on six different sub- strates (three were of pure type and three mixed substrate) at various temperatures, 20 ℃, 25 ℃, 30 ℃, 35 ℃, and 40 ℃. At 30 ℃, mini- mum spawn running period was observed on the tea waste + wheat straw (19.86±0.033d) followed by pure wheat straw (21.34±0.035d) with 70% humidity level of the incubation rooms (Tab. 3). This is similar to the work of DeVi and suMbali (2021), who found the low- est spawn running period (16 days) on wheat straw substrate. Furthermore, cultivation potential, in the form of fruiting bodies and yield, was evaluated on six types of substrates, three were of pure substrates, (wheat straw, tea waste and saw dust) and other three were the combination of two substrates (wheat straw+ tea waste, tea Fig. 2 (A, B, C): Spawn production of M. gigantea on A: sorghum grains; B: wheat grains; C: barley grains at 30 oC after 17 days of inoculation. Scale bar: A-C: 1 cm. Tab. 2: Efficiency of wheat, sorghum and barley grains for spawn production of M. gigantea Types of grains Days required to complete spawn production at 30 °C Sorghum 13 d 15 h ±1.15h Wheat 16 d 22.3 h ± 1.20 h Barley 20 d 22 h ± 1.15 h The results reported were run in triplicates and stated as Mean± Standard error. Table 3: Days required to complete Spawn Running Period on different substrates at variable temperatures Values given are mean ± Standard error. Substrate types and temperature have significant impact over spawn running time (p<0.001). Moreover, the joint effect of substrates and temperature has also a significant impact over spawn running time (p<0.001). In addition, LSD test was applied Types of Days required to complete Spawn running period substrates Temperatures 15 °C 20 °C 25 °C 30 °C 35 °C P - value Tea waste & wheat straw 29.96±0.033 25.93±0.035 22.96±0.033 19.86±0.033 21.86±0.006 <0.001 Pure wheat straw 27.93±0.035 26.93±0.035 24.96±0.036 21.34±0.035 24.93±0.035 <0.001 Saw dust & wheat straw 28.92±0.039 27.96±0.033 26.85±0.033 24.96±0.033 26.96±0.033 <0.001 Tea waste & saw dust 31.93±0.035 28.96±0.033 28.33±0.035 27.85±0.036 29.95±0.033 <0.001 P u r e s a w dust Not Initiated 30.93±0.035 29.96±0.033 27.96±0.033 30.93±0.035 <0.001 P u r e t e a waste Not Initiated 32.91±0.044 30.56±0.036 29.93±0.035 31.3±0.033 <0.001 P-value <0.001 <0.001 <0.001 <0.001 <0.001 27 Tab. 3: Days required to complete Spawn Running Period on different substrates at variable temperatures Types of substrates Days required to complete Spawn running period Temperatures 15 °C 20 °C 25 °C 30 °C 35 °C P-value Tea waste & wheat straw Pure wheat straw Saw dust & wheat straw Tea waste & saw dust Pure saw dust Pure tea waste P-value <0.001 <0.001 <0.001 <0.001 <0.001 Values given are mean ± Standard error. Substrate types and temperature have significant impact over spawn running time (p<0.001). Moreover, the joint effect of substrates and temperature has also a significant impact over spawn running time (p<0.001). In addition, LSD test was applied and found the significant differences between all possible combinations of substrate types (p<0.001) except between Tea waste & wheat straw and pure tea waste; and between all possible combinations of temperatures (p<0.005) except 15 oC & 20 oC (p=0.086) and 20 oC and 25 oC (p=0.737). (Green-Yellow-Red) Scheme was applied on the tables for quick readability of the most and least effective treatment on all the parameters. Green color indicated the best treatment while red color showed the least effective. <0.001 <0.001 <0.001 <0.001 <0.001 <0.001 Domestication and element analysis of the Macrocybe gigantea 171 waste+ saw dust and saw dust+ wheat straw) at 30 ℃ with humidity level 85%. The mixture of tea waste and wheat straw was found to be the best substrate for the cultivation of M. gigantea at 30 ℃ in the term of fruiting and yield (86.77±0.035g) followed by the wheat straw (79.85±0.035). Tea-waste was used for the first time as the growing medium for the cultivation of M. gigantea that was proved effective. yang et al. (2016) revealed the tea-waste as the economic and suitable substrate for the fruiting and yield of P. ostreatus. Lignin cellulosic waste supplemented with tea-waste could be an efficient source of growing substrate for the cultivation of various saprophy- tic mushrooms like Ganoderma lucidum (PeKsen and yaKuPoglu, 2009). As far as the efficiency of substrates were concerned, inyoD et al. (2016) obtained the maximum yield of M. crassa on saw-dust. atri and lata (2013) obtained the maximum yield of L. cladopus from the substrate of the mixture of wheat straw and paddy straw. Our research was also related to the findings of Dulay et al., 2021. They observed the combination of rice straw and saw dust at different tempera- tures as the suitable substrate for the cultivation of Lentinus species. Tab. 4 revealing the yield obtained from varieties of substrates and Fig. 3 representing the different stages of fruiting body production of M. gigantea. Element analysis The Element analysis was conducted to determine macro and trace elements in the nutritionally and medicinally significant Macrocybe gigantea. This species was enriched with macronutrients like potas- sium and calcium while trace elements (nickel and cobalt) were not detected in both wild and cultivated basidiomata (Tab. 5). Enrichment of essential elements and absence of toxic metals showed their suit- ability to enrich a diet. These results were in agreement with the find- ings of liu et al., 2012 and MalliKarjuna et al., 2013. Conclusion It can be concluded that M. gigantea could easily be grown on differ- ent media but PDA medium at 30 °C was proved the best combina- tion for the growth of this mushroom. Tea waste medium was used for the first time as the growth medium for M. gigantea and found very effective. However, different combinations of the substrates and media like paddy straw with tea waste, cotton waste with tea waste, tea waste with banana peels etc at 30 ℃ should be investigated in more detail to enhance the yield and biological efficiency of this nutritious mushroom. Conflict of interest No potential conflict of interest was reported by the authors. Authors contribution Aneeqa Ghafoor: Collection, analysis, methodology and writing Abdul Rehman Niazi: Identification, supervision, analysis and methodology Najam-ul-Sehar Afshan: Formal analysis, visualization Tab. 4: Yield (g/700 g) obtained from different types of substrates at 30 ℃ Types of substrates Biological Efficiency (yield g/700 g) 1st flush yield 2nd flush yield 3rd flush yield Total yield Tea waste & wheat straw 39.96±0.033 25.96±0.033 20.85±0.033 86.77±0.035 Pure wheat straw 37.96±0.036 22.96±0.033 18.93±0.033 79.85±0.035 Sawdust & wheat straw 30.96±0.036 21.93±0.035 17.95±0.033 70.84±0.033 Tea waste & saw dust 26.96±0.033 19.94±0.047 Not appeared 46.9±0.035 Pure saw dust 21.40±0.057 18.93±0.065 Not appeared 40.33±0.057 Pure tea waste 20.93±0.033 17.93±0.033 Not appeared 38.86±0.044 P-value <0.001 <0.001 <0.001 <0.001 Values given are mean ± Standard error. Substrate types have significant impact over the total yield (p<0.001). Fig. 3 (A, B, C): A: Spawned compost; B: pinheads and C: Harvested fruiting body of M. gigantea. Scale bar (A-C): 2 cm Tab. 5: Minerals concentration present in the Macrocybe gigantea Mushrooms Essential and Trace minerals (mg/g) Ca Co Cu K Zn Ni M. gigantea wild 1.54