Influence of various substrate treatment methods on yield and biological efficiency of Pleurotus florida mushroom

Due to their excellent flavour and taste, Pleurotus species, which flourish in temperate and subtropical environments, are well-liked among edible mushrooms. Oyster mushrooms could be grown on a variety of substrates due to their powerful enzymatic properties. Pleurotus sp. grows more easily, more profitably, and more nutrient-densely on a variety of organic waste raw materials and environmental factors. Sterilization of the substrate is one of the most crucial steps in oyster mushroom cultivation. There have been numerous reports of contaminated mushroom substrate. Suitable sterilization techniques must be used on the substrates. There are several treatments for mushroom substrate that can be used to manage the frequent moulds that infest edible mushrooms. Sterilization of the substrate is necessary to get rid of pathogenic and rival microorganisms and to encourage the mycelial growth of the desired mushroom species. The goal of the current study was to identify the best treatment strategy for the chosen substrates. For substrate treatment for Pleurotus florida cultivation, hot water, boiling water, steam sterilization in an autoclave, and chemical treatment methods were used. It was observed that the steam sterilization method was superior to other methods as it supported relatively short spawn run duration, amazing yield, and high biological efficiency.


Introduction
Mushrooms have probably been used as food since the dawn of civilization.Around the globe, over 200 mushroom species have long been consumed as functional foods.With the passage of time, a greater understanding of the nutritional value of mushrooms and its medicinal value has increased [1,2].Pleurotus species, which thrive in temperate and subtropical environments, are popular among edible mushrooms due to their excellent flavour and taste.Because of their strong enzymatic properties, oyster mushrooms could be cultivated on a variety of substrates.Pleurotus sp. is simpler to cultivate, more nutritious to eat, and more cost-effective to grow on various organic waste raw resources and weather conditions [3,4].Oyster mushrooms have significant economic, medicinal, and nutritional value [5,6].These are widely and regionally grown on non-composted lignocellulosic substrates.Numerous studies on different substrates have reported the successful cultivation of Pleurous sp., by using paddy straw, maize stalks and cobs, vegetable plant residues, bagasse, sawdust, chopped office papers, cardboard, plant fibres, and so on [7,8].
One of the most important steps in oyster mushroom cultivation is substrate sterilization.There have been several reports of mushroom substrate contamination.Substrates must be sterilized using an appropriate method.Several mushroom substrate treatments are available to control the common moulds found in edible mushrooms [9,10].Substrate sterilization is required to eliminate pathogenic and competitive microorganisms and to promote mycelial growth of desired mushroom species.Sterilization of substrates is primarily conducted to avoid the presence of pathogens that appear to compete for nutrient uptake [11].The composition of substrates used to grow various mushrooms varies greatly, as does the preparation of substrates used for each cultivated species [12,13].Most substrates are boiled in water for a set amount of time.Ideally, all seeds, nematodes, insects, and other organisms that thrive at the temperatures used to grow the mushrooms must be killed by the efficient procedure [14].Steam sterilization, immersion in hot water, and chemical treatment methods are described in the oyster mushroom cultivation protocol [15].However, very few studies have been published in the literature to evaluate the efficacy of these sterilization methods that influence mushroom's quality and quantity [16].In this context, the current study was conducted to determine the most appropriate treatment method for the selected substrates.

Obtaining a spawn sample and processing selected agro-industrial residues
Pleurotus florida spawns were obtained for the first time from Peeper Agro Industry in Vimanapura, Bengaluru, India, and it's pure culture was grown on potato dextrose agar plates (pH 7.2).Bread grass (Brachiaria brizantha), soybean straw, saw dust, rice straw, wheat straw, cotton straw, and sugarcane bagasse were collected in dried form.All substrates, with the exception of sawdust, were cut into fine pieces and pulverised with a grinder [2].

Different treatment methods for prepared substrates
Five different treatment methods were used for the substrates prepared in aforementioned combinations.

 Hot water treatment
Each substrate consortium was placed in a net bag, tied with thread, and dipped to pasteurize in hot water at 70 o C in a large container for 60 minutes.The excess water was then drained by hanging the bag for 20 hours.Following that, the contents of each bag were spread on different plastic sheets to evaporate excess moisture, resulting in a moisture content of 55 to 60% [16].

 Boiling water treatment
In this method, each consortium of substrates was immersed in boiling water for 60 minutes before repeating the method described above [15].

 Steam sterilization in an autoclave
The prepared substrate consortiums were placed in net bags, tied with thread, and soaked in fresh water for 8 hours.The extra water was removed.Each formulation was individually wrapped in cloth before being sterilized in an autoclave at 121 °C for 20 minutes.The sterilized contents of the cloths were placed in a laminar air flow chamber for 4-5 hours [2].

 Chemical treatment method
Fungicide solutions A and B were prepared and labeled separately.The solution A contained Bavistin (50 ppm) and Formalin (500 ppm).SAAF TM (UPL Limited, Gujarat) was used in solution B (500 ppm).The substrate bags were allowed to soak in these chemical solutions for 18 hours.The solution was then drained from each net bag, and a moisture content of 55-60% was maintained in the wet substrates before spawning [16].
 Ordinary water/control: By this method the substrates were treated in simple water [13].

Spawning, incubation and harvesting
An equal quantity of spawns was added while filling the sterilized substrates into each polypropylene bag of 100-gauge thickness.In this way, all the bags were packed and tied with pieces of thread.Then, with the tip of a dull pen, uniform holes were made in all the bags.The holes were approximately 2-3 mm in size.Bags were incubated at 28 ℃ and a relative humidity of 80-85% was maintained in the cropping room under dark conditions until a complete spawn run was achieved.The fruiting bodies of Pleurotus florida were harvested by twisting to uproot them from the base [2,21,25].

Observation and calculations
The time required for complete substrate colonization is called spawn run duration.The observation was recorded for time taken for spawn run.The biological efficiency (BE) for each substrate consortium was calculated as follows [2,[10][11][12][13][14][15].Biological efficiency (%) = (Fresh weight of mushroom / Dry weight of substrate) × 100

Data analysis
All experiments were performed in three replications.Data was analyzed in MS-Excel 2013 software [2].During the incubation period, no contamination was observed in the steam sterilized bags.The highest contamination of Aspergillus sp., Penicillium sp., Trichoderma sp., Mucor sp. and Rhizopus sp. was observed in substrates treated in hot water, while substrates treated in boiling water had the lowest contamination of these fungi.However, minor contamination of these fungi was observed in chemically treated substrates.The substrates treated with ordinary water (control) without any heat or chemical treatment completely failed to support the growth of Pleurotus florida mycelium, resulting in the formation of a large number of maggots and infesting molds in the bags.Figure 1 (a, b and c) depicts the bags processed by different treatments.Figure 1

Measurement of observed parameters
Spawn run duration was compared across substrate treatment methods (Figure 2).Pleurotus florida mycelium was completely spread after 10 days in the ninth consortium, and the bag was fully transformed into white.The growth of the fruiting body was initiated after 13 days, and the complete fruiting body appeared after 16 days.Figure 3 depicts the appearance of fruiting bodies with their different growth stages on steam sterilized consortium of substrates.
According to Khan et al., complete spawn running on substrates like sawdust, wheat straw, rice husk, etc. takes 15 to 20 days [11].

Effect of different substrate treatment methods on yield and biological efficiency
According to the findings, steam sterilization in an autoclave was one of the best substrate treatment methods for all designed consortiums and it supported the production of maximum yield in each consortium.Table 1 shows the yield of Pleurotus florida that was obtained using various substrate treatment techniques on all consortiums.The consortium no. 9 composed of dried bread grass (8%), soybean straw (15%), saw dust (25%), rice straw (20%), wheat straw (15%), cotton straw (10%), and sugarcane bagasse (7%), treated by steam sterilization method, had the highest biological efficiency (75.55%) than any other method.Figures 4 and 5 show the biological efficiencies obtained by various treatment methods for consortiums 1-7 and 8-15, respectively.Based on yield, steam sterilization was found to be the most effective method for treating substrates, followed by treatment with fungicide solutions A and B, boiling water, and hot water treatment.There are numerous different kinds of mushrooms available around the world, and only 280 species of the 2,500 known edible varieties are grown in India [1,15].The actual disinfection of the mushroom substrate is the most crucial unit process.It takes a lot of time and effort to prepare the mushroom substrate by disinfecting the agricultural residues because the process must be done in a clean environment [15,24,25].The disinfection procedure is a crucial step in mushroom growth for a number of reasons.Lignin is said to act as a barrier to the availability of carbohydrates because it is encrusted with both cellulose and hemicellulose in the cell wall [26].In order to overcome this barrier and obtain a high level of fermentable sugars for mushroom growth, hydrothermal treatment is crucial.The substrate is pasteurized at 65 °C for 6-8 hours to soften the texture and eradicate mesophilic microorganisms [15,16].While mould spores can be killed at temperatures above 80 °C, they are stable at 65 °C.Most importantly, substrate disinfection is carried out to eradicate rival mould species like Trichoderma, Coprinus, Penicillium, and Aspergillus that emerge during colonization and fruiting.One of the most prevalent and destructive diseases in the mushroom farming industry is green mould, which is brought on by these species.It is possible to create low-cost, energy-efficient substrate treatment techniques by utilizing solar energy to generate hot water and steam, which would otherwise go to waste in the fight against the energy crisis.There aren't many reports on microwave pasteurization of substrate.Microwave energy can be used to quickly and uniformly disinfect a large amount of substrate after soaking because it can quickly and ubiquitously heat any quantity of substance.The creation of low-cost, suitable pasteurization equipment is what mushroom growers urgently need to do in order to address their most pressing issues [15].

Conclusion
For the efficient and smooth cultivation of Pleurotus florida mushrooms, substrates must be treated properly.The steam sterilization method was found to be more effective with fairly short spawn run duration, truly amazing yield, and the greatest biological efficiency than other methods.In our study, the different substrate treatment methods had an impact on the spawn run duration, mushroom yield, and biological efficiency of Pleurotus florida mushrooms.Steam sterilization was therefore determined to be the best method for treating substrates for Pleurotus florida cultivation.

Figure 1a
Figure 1a Substrates treated by steam sterilization method, b: Substrates treated by fungicide solution A, c: Substrates treated by hot water, d: Contaminants isolated from the substrates treated with hot water

Figure 2 Figure 3
Figure 2 The time required for complete substrate colonization compared with all substrate treatment methods

Table 1
Yield and biological efficiency (B.E.) of Pleurotus florida obtained using various substrate treatment techniques on all consortiums