Growth dynamics and morphological response of Macrocybe gigantea to different casing soil treatments

Introduction

According to Chang and Miles (1992) [5] , mushrooms are macro fungi characterized by distinctive fruiting bodies visible to the naked eye and are manually harvested. Historically, mushrooms have been gathered from natural habitats for both food and medicine, and are regarded as a culinary marvel and a forgotten source of nutrients likened to "white vegetables" (Feeney et al., 2014; Raut and Adhikari, 2021) [1,19] . Owing to their delicacy and flavour, mushrooms are also known as vegetable meat, and their thread-like mycelial structures allow efficient nutrient absorption, making mushroom cultivation an important income-generating venture in many countries. Mushrooms possess a wide range of medicinal benefits, including anti-inflammatory, antidiabetic, antiallergic, anticancer, hepatoprotective, antioxidant and antimicrobial effects (Jeitler et al., 2020) [10] , and have shown effectiveness against diabetes, high cholesterol, stress and cancer. Ancient civilizations valued mushrooms highly -the Greeks regarded them as a royal dish, the Egyptians as food of the Gods, and the Chinese as an elixir of life (Kotowaski, 2019). References to mushrooms are found in Ayurveda, the Vedas, and Islamic and Biblical texts alike (Adhikari, 1981; Wasson, 1971; Marwat et al., 2009) [1,21] . Purkayastha and Chandra (1974) [18] were the first to report Calocybe indica (morphologically resembling Macrocybe gigantea), a popular edible mushroom locally known as 'Dudh chatta' (milk umbrella) in West Bengal, while the giant mushroom itself is commonly referred to as 'Boro Dudh Chattu' or 'Bada Dudh Chattu'. Macrocybe gigantea belongs to Kingdom Fungi, Phylum Basidiomycota, Class Agaricomycetes, Order Agaricales and Family Callistosporiaceae (Kui et al., 2021; Kirk et al., 2008), and was first documented in West Bengal (Pegler et al., 1998) [17] . It is found throughout the tropical regions of Asia and Africa, is characterised by its pure white colour, and is larger and more robust than both Calocybe indica and Agaricus bisporus (Akhtar et al., 2018) [2] . The genus Macrocybe, segregated from Tricholoma on morphological and molecular grounds (Pegler et al., 1998; Kui et al., 2021) [17] , comprises eight species, most of which are edible, with M. gigantea also valued for tolerating a high temperature range of 30-38℃ (Amin et al., 2010) [3] . Macrocybe gigantea is rich in carbohydrates, fats, proteins, amino acids and mineral elements, particularly Ca, Mg and Zn (Wang et al., 2004; Liu et al., 2012) [15] , comprising 24.1% protein, 10.2% carbohydrate and 5.6% glycogen on a dry weight basis (Pamitha and Latha, 2014). It also possesses notable antioxidant, antibacterial and antitumour properties (Gaur and Rao, 2016; Mau et al., 2002; Dai et al., 2009; Giri et al., 2012; Chatterjee et al., 2011). By converting otherwise valueless agro-waste into an effective growing substrate, mushroom cultivation provides additional income and labour-intensive employment to farmers while making productive use of agricultural residues. Macrocybe gigantea, like other agaric mushrooms, requires a 'casing layer' once the mycelium has fully colonised the substrate. The casing layer, applied 14-16 days after spawning, harbours microorganisms that trigger the shift of the fungus from vegetative growth to the reproductive (fruiting) stage (Gülser and Pekşen, 2003; Pardo-Giménez et al., 2020a), and is regarded as one of the key growing parameters influencing the productivity, quality and uniformity of commercial mushroom crops (Kaur and Rampal, 2017). Cocopeat is generally preferred as a casing base owing to its water-holding capacity, neutral pH and granular structure, which favour gaseous exchange and the release of hormonelike microbial substances that stimulate fruit body formation (Eger, 1972). Spent mushroom substrate can also be used as casing to lower production costs and reduce pollution (Pardo et al., 2011). Effective casing materials should possess high water-holding capacity, porosity, bulk density and a good air-space ratio, and the quality of the casing material determines fruiting body initiation and regulates moisture, temperature and other physical, chemical, microbiological and nutritional factors that govern the suitability of a material for casing Macrocybe gigantea. Different casing soil formulations for Macrocybe gigantea cultivation were tested, using varying proportions of coir pith combined with lime, soil, SMS, and FYM. The treatments ranged from 70:30 to 90:10 ratios, with the control using coir pith and FYM in an 80:20 proportion. These formulations aimed to identify the optimal casing material for the mushroom growth.

Materials and Methods

The present investigation was carried out at the All India Coordinated Research Project (AICRP) on Mushroom, Govt. College of Agriculture, Pune (MPKV), Maharashtra. India during 2026. The materials and methodology used for evaluating the growth and fruit body characters of Macrocybe gigantea under different casing soil formulations are described below.

Procurement of culture of Macrocybe gigantea

The pure culture of Macrocybe gigantea was obtained from AICRP on Mushroom, Govt. College of Agriculture, Pune, and maintained on Potato Dextrose Agar (PDA) medium, with Malt Extract Agar (MEA) used for mass multiplication.

Preparation of spawn and spawning process:

The bold seeded whole wheat grains were weighed and rinsed 2-3 times in tap water. The vessel was filled with water until the grains were just immersed. The grains were boiled in hot water for 25-30 minutes to prevent starch from escaping. After cooking, the grains were spread over a large wire gauze mesh to drain extra water. Overnight, the grains were maintained in a ventilated atmosphere. Based on the weight of these boiled grains, 4% calcium carbonate and calcium sulphate were mixed in the next morning to maintain pH to 6.5 to 7 and to prevent the gains from sticking together. For mother spawn preparation, sterilized conical flask having wheat grain mixed with CaCO3 were inoculated with small mycelial fragments of seven days old Macrocybe gigantea culture grown on test tube and quarter size bottle slants. The flasks were gently swirled to evenly distribute the inoculum, ensuring extensive contact between grains and mycelium. The flasks were then incubated at 25±1 0 c for approximately 10-12 days and the resulting spawn used as master spawn to prepare commercial spawn. The mother spawn was inoculated into sterilized polypropylene bags containing autoclaved wheat grains under laminar airflow conditions. Aseptic procedures were meticulously followed throughout the process. Mother spawn was added to each polypropylene bag, which contained 500 grams of wet sterilized wheat grains using 4% spawn rate. After the inoculation, the bags were replugged and the contents were gently shaken to ensure even distribution of the inoculum throughout the substrate. These bags were then incubated for 12-15 days at 25 ± 1°C in an incubator. Once the commercial spawn substrate was fully colonized by the mycelium, the spawn was considered ready for use.

Preparation of substrate

Wheat straw was chopped into 2-3 cm pieces and chemically pasteurised by soaking in a solution of formalin (125 ml) and Bavistin (7.5 g) in 100 litres of water for 18 hours, after which excess water was drained.

Bed Preparation

Macrocybe gigantea was cultivated using the polythene bag method. Polythene bags of size 35x55 cm were disinfected with 5% formalin solution. Beds were prepared by the layer method of spawning at 4% on wet weight basis of straw. The necks of the bags were tightly tied with thread. About 35-40 pinholes were made all over the surface of the bags. Four replications for each casing treatment were kept for incubation (spawn run) in the incubation room.

Casing of substrate bags

Casing is a top dressing applied to the mycelium colonised substrate with soil or other casing material having a high water-holding capacity and a neutral pH (Amin et al., 2010) [3] . Once the substrate was completely run, a layer of 2-4 cm casing material consisted of coir pith combined with lime, soil, SMS, and FYM. The treatments ranged from 70:30 to 90:10 ratios, with the control using coir pith and FYM in an 80:20 proportion. Before use, casing materials were sterilized in an autoclave at 15 lbs psi for 60 minutes to ensure pathogen free environment (Panda and Biswas, 2021). Spraying of water was done1-2 times a day. The temperature was maintained in the range of 23-25 0 C and relative humidity of more than 80 percent with the help of humidifier throughout the cropping period.

Harvesting of mushroom

Sporophores were harvested on maturation when caps became flat with incurved margins (Fig. 1 and Fig. 2). Harvesting of fruit bodies was done by gently twisting the stipes of the fruiting bodies both clockwise and anticlockwise, which ensured that young sporophores growing nearby remain unaffected.

Fresh weight of mushroom Biological efficiency (B. E.) = - -------------------------X [9] , who reported that Macrocybe gigantea mushroom's pileus diameter ranged from 4 to 7 cm. However, Akhtar et al., (2019) [2] observed morphological characteristics of four isolates of Macrocybe gigantea and recorded that the average pileus diameters range between 8.46 and 11.63 cm

Colour

No significant effect of different casing materials on the colour of Macrocybe gigantea basidiocarps was observed. Regardless of the casing type, the basidiocarps remained mostly white to creamy white, with the cap turning pale cream or buff upon maturity, while the gills and stipe stayed white to cream. The findings align with those of G Suresh and R Sutha Raja Kumar (2022) [20] who reported that the pileus of Tricholoma gigantea was creamy white, with milky white gills.

Odour

The odour of Macrocybe gigantea was not influenced by the casing materials. Fresh basidiocarps across all treatments emitted a mild, pleasant, dehydrated milk-like, and characteristic fungoid (mushroom-like) scent, with no noticeable differences between casing types. As reported by Dutta and Acharya (2014) [8] , M. gigantea, M. lobayensis and M. crassa, are locally known in India as 'boro dhoodh chhatu,' or 'dhoodh chhatu' meaning they emit a milk-like smell when dried.

Stipe length

The data ( [2] who reported that the average stipe length of four different isolates of Macrocybe gigantea ranged from 9.73 to 12.53 cm. while, Anurag Kerketta et al., (2019) [12] reported that stalk length varied between 2.63 and 3.45 cm and remained statistically comparable across the casing materials i.e. cocopeat, vermicompost + cocopeat (1:1), vermicompost + soil (1:1), cocopeat + soil (1:1) vermicompost + cocopeat + soil (1:1:1) and soil + FYM + sand (1:1:1).

Stipe girth

The highest average stipe girth (Table 1, Fig. 1 and Plate 3.) of 9.82 cm was observed in T4, closely followed by T5 (9.40 cm), which were statistically at par. Treatments T2 and T3 produced stipe girths of 8.88 and 7.82 cm, respectively, while the lowest girth (6.96 cm) was recorded in T1. The conclusion drawn by Galappaththi et al., (2022) [9] states that the girth of Macrocybe gigantea mushroom ranges between 2.5 to 4. Mownika et al., (2024) [16] described the cylindrical stipe of Macrocybe gigantea as about 6 cm in diameter, while Pegler & Lodge (1998) [17] stated the stipe is centrally positioned, solid, and measures 4 -8 cm in thickness.

Acknowledgement

AcknowledgementThe authors are thankful to the All India Coordinated Research Project on Mushroom and Department of Plant Pathology and Agricultural Microbiology, Govt. College of Agriculture, Pune, and BNF Mahatma Phule Krishi Vidyapeeth, Rahuri, Maharashtra, for providing the necessary laboratory facilities to carry out this study.

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