The experiment was performed in accordance with relevant guidelines and regulations.
Mother Culture. Pure cultures were procured from Directorate of Mushroom Research, Chambaghat, Solan, Mushroom Research and Training Centre, Haryana Agricultural University, Murthal, Haryana and Mushroom Research Laboratory, Indian Institute of Horticultural Research, Hesarghatta, Bengluru. Cultures were multiplied on malt extract agar and maintained in test tubes. We prepared six combinations from these three cultures through mycelial anastomosis. Out of them, one strain was found to be the most suitable under sub-tropical climate, which we later named as “Pratap King Oyster-1” (P.K.O-1) and this strain has been used in our experiment.
Preparation of master culture. The study complies with local and national regulations. For the collection of seeds or plants, all relevant permissions or licenses have been obtained. Healthy wheat grains were soaked in water overnight and were boiled up to semi cooked conditions in the next morning. Excess water was drained off and seeds were left for surface drying. These seeds were then mixed with 1% CaCO3 and were filled in milk glass bottles or polypropylene bags which we later sterilized at 20 lbs psi pressure (126.5oC) for 2 hrs. After cooling, each bag was inoculated aseptically with two mycelial agar bits of P.K.O.-1 culture in Laminar Air Flow and incubated at 20±1oC in incubator for 20-25 days till the grains were fully impregnated with the mycelium of inoculated mushroom culture  (Fig. 5).
Preparation of substrate. The experiment was laid out by using straw of wheat crop, since; it is easily available and cheap. Straw was chopped into 3-5 cm pieces and chemically treated by soaking in mixture of Bavistin (8g), Formalin (300ml) and Nuvan (30ml) with water in drum of 200 litre capacity and kept overnight. On the next day, after decanting excess water, it was used for spawning purpose  (Fig. 6).
Spawning. The experiment was performed for the four winter months viz. November, December, January and February. For layer spawning, substrate filled in bag was pressed to a depth of 2-3 inches and broadcasted with spawn above it. Similarly, 2nd and 3rd layers of entire substrate were put simultaneously and after spawning, some amount of substrate is put over it, and then the bags tied up with rubber bands (Fig. 7). Thus, five bags in each month were spawned and they were maintained under optimum conditions of mushroom growth. The date of spawning was also kept same in every month. Thus, four treatments with five replications of each were placed using Completely Randomized Design to reach the conclusion so that a consolidated recommendation can be made as to how the King Oyster can be grown with maximum BE.
Crop management and harvesting. After 20-22 days of spawning, when spawn run gets completed and bags turns white in colour due to white mycelial run, the polythene covers were teared off. Pin heads came out after 2 to 3 days of removing the polycovers which later developed into mature fruit bodies . At this stage, mushrooms were plucked off by twisting in clockwise direction at the substrate level, counted and weighed separately for each replication at every harvest (Fig. 8). Yield was recorded and the Biological efficiency was also calculated.
Biological efficiency. The Biological efficiency was also calculated by following the given formula .
Statistical analysis. Statistical Analysis Software (SAS software) developed by Indian Statistical Research Institute (ISRI), New Delhi was used for performing statistical analyses of data. The critical differences among treatment groups were determined using the ANOVA test at level of significance (p= 0.05). All the experiments were carried out in five replications in Completely Randomized Design (CRD).
The data analysis for this paper was generated using SAS software. Copyright  SAS Institute Inc. SAS and all other SAS Institute Inc. product or service names are registered trademarks or trademarks of SAS Institute Inc., Cary, NC, USA.