Accepted Manuscript Selection of Macrocybe crassa mushroom for commercial production Tanapak Inyod, Suriya Sassanarakit, Achara Payapanon, Suttipun Keawsompong PII: S2452-316X(16)30028-X DOI: 10.1016/j.anres.2016.06.006 Reference: ANRES 30 To appear in: Agriculture and Natural Resources Received Date: 14 September 2015 Accepted Date: 14 March 2016 Please cite this article as: Inyod T, Sassanarakit S, Payapanon A, Keawsompong S, Selection of Macrocybe crassa mushroom for commercial production, Agriculture and Natural Resources (2016), doi: 10.1016/j.anres.2016.06.006. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. 1 ACCEPTED MANUSCRIPT 1 Agriculture and Natural Resources. 2016. 50(3): xx −xx 2 Agr. Nat. Resour. 2016. 50(3): xx −xx 3 4 Selection of Macrocybe crassa mushroom for commercial production 5 6 Tanapak Inyod 1, 2, Suriya Sassanarakit 2, Achara Payapanon 3, Suttipun Keawsompong 1* 7 8 1Department of Biotechnology, Faculty of Agro-Industry, Kasetsart University, Chatuchak, 9 Bangkok 10900, Thailand 10 2Thailand Institute of Scientific and Technological Research (TISTR), Technopolis, Klong 5, 11 Klong Luang, Pathumthani 12120, Thailand 12 3Department of Agriculture, 50 Phaholyothin Road, Chatuchak, Bangkok 10900, Thailand 13 MANUSCRIPT 14 Received 14/09/15 15 Accepted 14/03/16 16 17 Keywords: 18 Macrocybe crassa, 19 Nutritional value, 20 Productivity, 21 Quality, ACCEPTED 22 Substrate 23 24 * Corresponding author. 25 E-mail address: [email protected] 2 ACCEPTED MANUSCRIPT 26 Abstract 27 28 Macrocybe crassa or Tricholoma crassum (Berk.) Sacc. (synonym) is a wild, edible 29 mushroom in Thailand. This mushroom has a large fruiting body with meaty texture and a 30 delicious taste. However, this mushroom is not commercially cultivated at a large scale. In 31 this study, five strains of Macrocybe crassa—DOA, DOA-1, DOA-4, DOA-7 and DOA-10— 32 were cultivated on a ratio of rubber tree sawdust to fine rice bran to magnesium sulfate 33 (MgSO 4.7H 2O) to calcium oxide (CaO) of 100:3:0.2:1 (weight per weight). Their growth, 34 productivity and characteristics of the fruiting body were investigated. The results revealed 35 that DOA-10 gave the highest yield (215.10 g per 0.6 kg of substrate) with 59.26% biological 36 efficiency. Observation showed that its pileus diameter, stalk diameter and stalk length were 37 6.78 cm, 2.75 cm and 13.54 cm, respectively. The dried samples of DOA-10 contained 38 protein, carbohydrates, ash and fat at 13.71%, MANUSCRIPT68.08%, 12.06% and 2.49% respectively. 39 Moreover, they contained high contents of both macronutrients and micronutrients:, 40 potassium (43,100 mg/kg), calcium (492.00mg/kg), magnesium (1,046.66 mg/kg), sodium 41 (936.33 mg/kg), iron (283.21 mg/kg), zinc (53.79 mg/kg) and manganese (17.55 mg/kg). 42 These results confirmed that DOA-10 is a promising strain for commercial cultivation. 43 44 Introduction 45 Mushroom cultivation is a major agro-industry and it has become popular as a source of 46 healthy and functionalACCEPTED food, and its production is also increasing (Aida et al. , 2009). 47 Macrocybe crassa or T. crassum (Berk.) Sacc. (its synonym) is a wild, edible mushroom and 48 non-toxic (Teaumroong et al ., 2002; Pradhan et al ., 2010). It is a fungus belonging to the 49 genus Macrocybe , in the family of Tricholomataceae , and the order of Agaricales (Chang and 50 Hayes, 1978). It has a high production yield with outstanding, enormous, fleshy basidiomata 3 ACCEPTED MANUSCRIPT 51 which may exceed 80 kg fresh weight and it shows high biological efficiency, which is more 52 than for many edible mushrooms (Corner, 1993). The large size and flavorful fruiting bodies 53 of some Macrocybe species make them a valuable source of food ,such as lower cholesterol 54 and immuno-modulating effects in humans (Hoshi et al. , 2005). Species of Macrocybe were 55 formerly placed within Tricholoma , but segregated in the section Leucorigida 56 morphologically on the presence of clamp connections, and ecologically on the absence of 57 ectomycorrhizal associations (Pegler et al ., 1998). 58 They produce a fleshy convex pileus and can be found in every region of Thailand and 59 in neighboring countries (Payapanon and Srijumpa, 2008). Several related species such as T. 60 albobrunneum , T. flavovirens , T. paneolum , T. equestre , T. terreum and T. georgii , are found 61 in Europe and America while only a few edible species of this family, such as T. matsutake 62 and M. crassum, are found in the Asian region—in Japan, Thailand and Sri-Lanka— 63 (Huffman et al ., 1989). In Thailand, M. crassum MANUSCRIPT has various common names among local 64 people such as hed-tin-rad (northeast), hed-jan (north), hed-hua-sum (south) and hed yai or 65 hed-tub-tao-khao (central) (Petcharat, 1996). Natural M. crassa is generally rather expensive 66 and rare, because it is usually found only once a year, particularly in the rainy season 67 (Teaumroong et al ., 2002). Moreover, varying the substrate media for mushroom cultivation 68 resulted in different yields due to biological and chemical differences between the substrate 69 media and their quality which may been due to the genotype of the mushroom strains. 70 (Ragunathan and Swaminathan, 2003). However, it can easily and successfully be cultivated 71 on rubber tree sawdustACCEPTED (Payapanon and Srijumpa, 2008). However, still no work has been 72 done to determine the suitability of this locally available lignocellulosic waste for cultivation 73 or to determine the most cost compatible strains under environmental conditions. The main 74 objectives of this research were to evaluate the yield parameters of different strains of M. 75 crassa through traditional cultivation methods on rubber tree sawdust substrates and to 4 ACCEPTED MANUSCRIPT 76 determine their growth and the productivity and characteristics of the fruiting body to identify 77 the best strain of M. crassa mushroom most suitable for commercial cultivation. 78 79 Materials and Methods 80 Mycelial development of Macrocybe crassa mushroom 81 Mycelial discs (10 mm diameter) of five native strains (with substantially high 82 production yield)—DOA, DOA-1 (collected from Bangkok province), DOA-4 (collected 83 from Chiang Rai province), DOA-7 (collected from Chai Nat province) and DOA-10 84 (collected from Prachuap Khiri Khan province)—were obtained from the strain bank of the 85 Department of Agriculture, Ministry of Agriculture and Cooperative, Bangkok, Thailand, and 86 were carefully transferred to the upper surfaces of prepared sorghum grains. Inoculated grain 87 bottles were tightly secured using moist cotton wool and covered with sterile aluminum foil. 88 They were kept in dark, sterile cabinets at ambientMANUSCRIPT room temperature for 14 d until they were 89 fully colonized. The substrates employed in the production and in-depth mycelial 90 development assessments of Macrocybe were prepared using rubber tree ( Hevea brasiliensis ) 91 sawdust mixed with fine rice bran, magnesium sulfate (MgSO 4.7H 2O) and calcium oxide 92 (CaO) in the ratio of 100:3:0.2:1 (weight per weight). Water was added to adjust the moisture 93 content to 65%. Samples of the substrate mixture (each approximately 600 g) were used to 94 fill 1 kg capacity autoclavable polypropylene bags to only three-quarters of their capacity and 95 then steamed for 3 hr in a large cast-iron steamer. When the temperature in the mushroom 96 spawn cooled downACCEPTED to room temperature, 10 −15 seeds of grains spawn were added to the 97 bags and incubated at approximately 25°C for spawn running until the bags were fully 98 covered with mycelia. Data were collected on a daily basis for the mycelium running rate 99 (MRR), days required to complete spawn running (DCS), primordial initiation (DFPI) and the 100 number of fruiting bodies (NFB). 5 ACCEPTED MANUSCRIPT 101 102 Production on sterile substrate 103 The upper end of the bags was cut off and the spawn mushrooms were neatly 104 arranged in a basket and placed on shelves in the growing room. The surface of the compost 105 was covered with a 2.5–5 cm thick layer of steamed casing soil, which was necessary to 106 initiate fruiting. After casing, the relative humidity of the room was maintained between 85% 107 and 90%. Each treatment was composed of 50 replicates of strains in each treatment. After 108 fructification, one flush of mushrooms in each bag was harvested when the cap had started to 109 fold. The cultivation substrates were maintained for 2 mth. The yields of mushrooms and 110 their different quality parameters were recorded regularly. The biological efficiency 111 percentage (BE) was calculated using the following formula: BE = (Fresh weight of fruit 112 body / Dry weight of substrate) × 100 (Chang and Miles, 1989). 113 MANUSCRIPT 114 Morphological characterization procedures of the basidiomata 115 The macroscopic characteristics of the basidiomata collected from each isolate were 116 recorded according to Largent (1977). Qualitative characters such as the color and shape of 117 the cap, the color of the stipe and the color of the spore print of the mushroom were visually 118 evaluated. For microscopic analysis, the dry material was rehydrated in 70% ethanol 119 according to Largent et al. (1977). Free-hand transverse sections 0.1 mm thick were made 120 from rehydrated basidiocarps using a sharp surgical blade.