Abiodun Olusola Salami, Faith Ayobami Bankole, Opeyemi Martins Odubanjo

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1 Research Article SCIFED Publishers Abiodun Olusola Salami,, 2018, 1:3 SciFed Journal of Mycology Open Access Effect of Calcium Supplementation and Sterilization Methods on the Production of Oyster Mushroom * Abiodun Olusola Salami, Faith Ayobami Bankole, Opeyemi Martins Odubanjo * Department of Crop Production and Protection, Obafemi Awolowo University, Ile Ife, Osun State, Nigeria Abstract This study investigated the response of Oyster mushroom (Pleurotus florida) to calcium from different sources and sterilization methods on the production of Oyster mushroom (Pleurotus florida) as well as determined its growth rate. Spawns were produced using 55g ground corncobs mixed with 5% powdered eggshells and limestone of the equivalent weight respectively, these were thoroughly mixed with sterile water before transferring into mayonnaise bottles and then sterilized at 121 C for 30 minutes, allowed to cool and then inoculated. Two hundred and fifty grams of the substrates were weighed and 5% of powdered eggshells and CaCO 3 were added respectively, these were then mixed, packed into polythene bags and sterilized using an autoclave and pasteurizing with hot water for 2 hours. The various substrates were then kept in a dark chamber till complete ramification. Data were collected on days to complete ramification, days to pinhead formation, number of fruiting bodies, weight of fruiting bodies, length of stipe, diameter of cap, width of the stipe and biological efficiency. The collected data were subjected to analysis of variance and significant means were separated using LSD at 0.05 level of probability. The result showed that there was no significant difference in the number of days to complete ramification of spawns for both eggshell and limestone treatment. Also, the mushroom grown on eggshell treatment did not significantly differ from the ones grown on limestone treatment. Also, sterilization method does not have an effect on the yield as there was no difference between both treatments. Also, for a unit increase in time, there was a corresponding increase of cm in the length of stipe, cm in the diameter of cap and cm in the width of stipe. This study concluded that calcium from eggshell can be effectively used for the production of P. florida, hot water treatment of substrates is sufficient for the substrate preparation for optimum yield and for a unit increase in time, a corresponding increase of cm in the length of stipe, cm in the diameter of cap and cm in the width of the stipe with respect to the growth rate of P. florida used in this study was established. It therefore, recommends the use of eggshells in lieu of limestone and also hot water pasteurization instead of using an autoclave provided there is a clean environment. Keywords Calcium Supplementation; Sterilization Methods; Oyster Mushroom Introduction Mushrooms belong to the kingdom fungi, a kingdom distinct from either plant or animal kingdoms. They are also referred to as macrofungi. They are unique with distinctive fruiting bodies which are either epigenous or hypogenous [1]. Within the fungal kingdom, they belong to the division eumycota literally translated as the true fungi. They are saprophytes because they lack chlorophyll and cannot get their food through photosynthesis. Pleurotus florida is widespread in temperate, sub-tropical *Corresponding author: Abiodun Olusola Salami, Department of Crop Production and Protection, Obafemi Awolowo University, Ile Ife, Osun State, Nigeria. sola1salami@yahoo.com; Tel: Received May 30, 2018; Accepted August 2, 2018; Published August 14, 2018 Citation: Abiodun Olusola Salami (2018) Effect of Calcium Supplementation and Sterilization Methods on the Production of Oyster Mushroom. 1:3. Copyright: 2018 Abiodun Olusola Salami. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. page 1 of 7

2 and tropical climatic regions of the world, it is sometimes considered by some to be a sub specie of P. ostreatus and they are of very similar characteristics and appearance [2]. The growth stages of Pleurotus species include the formation of spores by a matured fruiting body; these spores later multiply and develop within a suitable substrate forming a mycelia network. Many species of mushrooms seemingly appear overnight, growing or expanding rapidly. This phenomenon is the source of several common expressions in the English language including to mushroom or mushrooming (expanding rapidly in size or scope) and to pop up like a mushroom (to appear unexpectedly and quickly). In reality all species of mushrooms take several days to form primordia mushroom fruit bodies, though they do expand rapidly by the absorption of fluids [3]. Oyster mushrooms are commonly cultivated on residues from agricultural wastes such as wheat, paddy rice, cotton, sugar-cane, sorghum, soybean and groundnuts to mention but a few [4]. Zhang et al. [5] reported that ground straw yielded better mushroom production than whole or chopped straw, they also reported that the mushrooms grew faster on the ground substrate with their growth cycles being five days shorter than on chopped straw for a similar size. Salmones et al. [6] reported that during inoculation, sugarcane bagasse supported the fastest mycelia growth when mycelia growth rate was compared on fourteen different substrates. Ergun et al. [7] in their report on cultivation of oyster mushroom on waste paper reported that supplementing waste paper with 20% rice husk positively affected spawn running, pin head formation, fruit body formation and mushroom yield compared to sole waste paper. They also reported that the higher the concentration of rice husks in the substrate, the better the yield. Salami et al. [8] reported that both calcium and carbon were elements responsible for the growth of mushrooms. Calcium is essential for the attainment of a slightly basic to neutral medium which favours the growth of the mushroom. Sterilization of substrates is a very intregal part of mushroom production as it helps prevent contamination of substrates. Akhter et al. [9], reported that hot water treatment at 80 C for 3 hours is suitable for substrate treatment in order to produce a good yield and biological efficiency. Manoj [10] recommended that treatment with bavistin be used as a means of sterilization to reduce costs but that hot water treatment is used if the chemical bavistin is not readily available. The objectives of this study were to; evaluate the effect of calcium from different sources (eggshells and limestone) on the production of Oyster mushroom (Pleurotus florida), evaluate the effect of sterilization methods on Oyster mushroom production and establish the growth rate of Oyster mushroom (Pleurotus florida). Materials and Methods Experimental Location This study was carried out at the Mycology Laboratory, Department of Crop Production and Protection, Faculty of Agriculture, Obafemi Awolowo University, Ile- Ife, Osun State. Production of Spawn A bottle of fully ramified grain spawn of Pleurotus florida was collected from the Mycology section of the OAU, Ile-Ife, Osun State Nigeria. This spawn was multiplied using ground corncobs powder. The ground corncob powder was soaked in hot water at 80 C for 30 minutes and pressed to expel excess water. Fifty-five grams of this was then weighed into sterilized welllabeled mayonnaise jars. Five percent equivalent of the weight each of powdered eggshells and calcium carbonate was weighed and then added to the bottles and properly mixed and done for five replicates each This was corked with aluminum foil and autoclaved at 121 C for about 30 minutes after which the container was allowed to cool for 24 hours at room temperature. After 24 hours of cooling, grain to grain spawning was done to multiply the corncob powder spawn [11]. Grain to Grain Spawning An axenic environmental condition was maintained to reduce contamination and this was achieved with the use of a sterile Laminar flow hood. Hand gloves and nose mask were also worn to avoid contamination. The grain spawn obtained from the Mycology section of Department of Crop Production and Protection was scooped out of the bottle into a clean bowl with the aid of spatula; small quantity of the grain spawn was introduced into each of the bottles containing the pasteurized ground corncobs and covered immediately with foil paper and the foil paper was made firm on the bottles with rubber band. The bottles of the newly spawned agro-wastes were incubated at room temperature in complete darkness for mycelia colonization of the corncob powder spawns to occur [4]. Substrate Preparation The corncobs were pound to reduce their sizes page 2 of 7

3 and the dried weight recorded. They were then poured into clean buckets and powdered eggshells and limestone (CaCO 3 ) 5% each of the weight of the corncobs was added and then steamed with hot water by total submergence. They were then drained and allowed to cool. Two hundred and fifty grams of the soaked substrates were then weighed and packed into transparent polythene bags which were labeled for easy identification. Ten bags of the packed substrates were sterilized using an autoclave at 121 C for 30 minutes while ten bags were not sterilized using the autoclave [12]. Upon cooling, the materials were packed into a transparent polythene bag and inoculated with the ramified spawns. Substrate Inoculation The fully ramified spawn of the Pleurotus florida was then inoculated into the transparent polythene bags containing the prepared substrates. This was done in an axenic condition in the lamina flow hood. The inoculated bags were then transferred into a dark chamber to be incubated and were then monitored until full ramification occurred and the number of days taken for each was recorded. After full ramification of the substrate with the mycelia of the mushroom, the fully ramified bags were removed from the dark chamber and then exposed, labeled and watered twice daily. Data Collection The following parameters were collected as the growth occurred: days taken for complete mycelia ramification of substrates (DTCSR), days to pinhead formation (DPHF), number of fruiting bodies (NOFB), weight (g) of harvested fruiting bodies (WTFB) using an electronic weighing balance, length of stipe (LOS) of harvested fruiting bodies using a meter rule, diameter of the Cap (DOC) and diameter of the stipe (DOS) using a thread and a meter rule respectively. Yield and Biological Efficiency Total weight of all the fruiting bodies harvested from all the three pickings were measured as total yield of mushroom. The biological efficiency (yield of mushroom per kg substrate on dry wt. basis) was calculated by the formula below according to Salami et al. [13]. Yield of mushroom 100 BE. (%) = Weight of substrate Experimental Design A completely randomized design was used with two factors in this investigation. Statistical Analysis The data obtained were subjected to ANOVA using Statistical Analysis Software (SAS) version 9.1 and significant means were separated using Fischer s least significant difference at 0.05 level of probability. Microsoft excel 2013 was also used to plot the graphs with standard error bar. Results and Discussion Effect of Calcium Sources on the Spawn Running of Oyster Mushroom The effect of calcium from different sources on the days to complete ramification of the spawn production is presented in figure 1. The growth parameter measured for the spawn production was days to complete spawn ramification (DTCSR), this was recorded as the number of days taken for the mycelia growth to completely ramify from top to bottom of the bottle i.e. the period it took for complete ramification of the spawn bottle from the day of inoculation. It was observed that spawn of Pleurotus florida produced on ground corncobs with eggshell treatment had the lowest number of days to complete ramification followed by the treatment with CaCO 3 but both treatments were not significantly different from each other. However, both treatments were significantly different from the control which had no calcium. Hence, it shows that eggshell treatments can be used to replace calcium carbonate treatment in the production of spawns without an increase in the number of days taken to complete ramification. This is in line with the report of Bankole and Salami [11] who reported that substrate augmentation with calcium is appropriate for spawn production. For fruiting body production, data on days to complete substrate ramification (DTCSR), days to pinhead formation (DPHF), weight of the fruiting body (WOFB), number of fruiting body (NOFB), length of stipe (LOS), diameter of cap (DOC) and biological efficiency (B.E) were collected. Oyster mushrooms grown on substrates containing eggshell treatments were found not to be significantly different from the mushrooms cultivated on the substrates treated with calcium carbonate as seen from Table 1. For DTCSR, there was no significant difference as indicated by the LSD value when used to compare the mean values page 3 of 7

4 Figure 1: Effect of Calcium Supplements on Days taken to Complete Ramification of Spawns Table 1: Growth Parameters of P. Florida As Influenced by Different Calcium Sources Calcium Sources of the growth parameters measured. This applies for DPHF, WOFB, NOFB, LOS, and DOC. For DTCR, it was observed that for eggshell supplements it took an average of days compared to days for CaCO 3, when we compare both using the LSD value, there is no significance difference between them. The values for DPHF for P. florida with eggshell treatment was 23 days while that of CaCO 3 treatment was days. There was no significant difference in the weight of the fruiting bodies of P. florida which was g for eggshell treatment and g for CaCO 3 treatment when compared using the LSD value. Effect of sterilization methods on Oyster mushroom production The effect of sterilization methods on the production of Oyster mushroom was found not to be DTCR DPHF LOS(cm) DOC(cm) NOFB WTFB(g) B.E (%) CaCO Eggshell LSD DTCR: Days to complete ramification, DPHF: Days to pinhead formation, LPH: Length of pinhead, NOFB: Number fruiting body, DOC: Diameter of cap, WTFB: Weight of the fruiting body Table 2: Effect of Sterilization Methods on Oyster Mushroom Production Calcium sources DTCR DPHF LOS(cm) DOC(cm) NOFB WTFB(g) B.E (%) Autoclave Hot water LSD DTCR: Days to complete ramification, DPHF: Days to pinhead formation, LPH: Length of pinhead, DOC: Diameter of cap, WTFB: Weight of the fruiting body, NOFB: Number of fruiting body significantly different from each other and the growth parameters measured were not different from each other (Table 2). Based on this study, sterilization methods did not affect the yield of the mushroom but rather plays other roles such as prevention of contamination of the substrates for the proper growth of the Oyster mushroom. This further supports the findings of Manoj, [10] that substrates treated with hot water sterilization produced yield which was not significantly different with the yield of substrates that was sterilized using an autoclave. An overall view of the growth rate with respect to time is given in Figure 2. The length of pinhead (LPH) was observed to increase from about 2.8 cm to about 5.2 cm between 24 hours and 48 hours and a growth implement of about 2 cm increase between 48 hours and 72 hours. This implies that highest length of pinhead growth was page 4 of 7

5 recorded between 24 and 48 hours. For diameter of cap, a diameter of cap of 1 cm was recorded for 24 hours, 3.2cm for 48 hours and 6 cm for 72 hours, this implies that maximum cap growth occurs between 48 and 72 hours. Width of stipe shows a gradual increase in diameter across the time taken, at 24 hours we have a diameter of 1.5 cm, at 48 hours 3.0 cm and 4.0 cm for 72 hours, this implies that maximum stipe growth takes place in the first 24 hours after growth parameters were taken. The length of the pinheads when measured every 24 hours across a span of 72 hours was found to vary considerably (Figures 3). There was a marked increase in length as we move from 24 hours to 48 hours and then 72 hours before harvesting. From the figure it was observed that for a unit increase in Figure 2: Effect of Time on the Various Growth Parameters of Pleurotus florida LPH: Length of Pinhead, DOC: Diameter of Cap, WOS: Width of Stipe time, there is a corresponding increase of cm in the length of stipe. The diameter of the caps across the same length of time also revealed that the diameter of the caps increased substantially (Figure 4). It is observed that at 24 hours, the diameter of the cap was not noticed but then it grows phenomenally across the remaining hours to give the impressive cap. The graph revealed that for a unit increase in time, there was a corresponding increase of cm in the diameter of the cap. The width of the stipe increased steadily from 24 hours all through to 72 hours (Figure 5). The graph shows that with a unit increase in time a corresponding growth of cm in the width of the stipe was recorded. Figure 3: Growth Rate of the Length of Stipe of Pleurotus florida page 5 of 7

6 Figure 4: Growth Rate of the Diameter of Caps of Pleurotus florida Figure 5: Growth Rate of the Width of Stipe of Pleurotus florida Conclusion From the results, it was concluded that the use of powdered eggshells in lieu of limestone gives the same results without an increase in the number of days taken to completely ramify the substrate. Also, for the growth of the mushrooms on the substrate, the Pleurotus florida cultivated on substrates containing the treatment with powdered eggshells performed as well as those cultivated on limestone treatment when the yield and growth of the fruiting bodies were compared which implies that powdered eggshells can be used instead of limestone on the substrates with the same results. This study also concluded that sterilization methods does not have an effect on the yield and growth parameters as it was shown that the Pleurotus florida cultivated on the substrates sterilized with the autoclave had no significant difference in yield and growth parameters as the ones pasteurized with hot water. The growth rate of Pleurotus florida as determined by this experiment revealed that with a unit increase in time there was a corresponding increase of cm in the length of stipe, cm in the diameter of cap and 0.05 cm in the width of stipe. This study hereby recommends that the use of eggshells in lieu of limestone should be encouraged, this is because it is cheaper, readily available and performs as well as limestone in the production of Oyster mushroom. It also recommends that soaking in hot water be used as an alternative means of sterilization and can be used successfully in the production but clean environment must be ensured to avoid unnecessary contamination. References 1. Chang ST, Miles PG (1992) Mushroom biology-a new discipline. Myco J 6: Khare BK, Achwanya MJ, Mutuku ME, et al. (2014) Developmentand biotechnology of Pleurotus mushroom cultivation. J Sci Tech 6: Rosie Tanabe (2014) Mushroom. New World Encyclopedia. 4. Salami AO, Bankole FA, Salako YA (2017) Nutrient and Mineral Content of Oyster Mushroom (Pleurotus florida) Grown on Selected Lignocellulosic Substrates. J Advan in Bio page 6 of 7

7 and Biotech 15: Zhang R, Li X, Fadel JG (2002) Oyster mushroom cultivation with rice and wheat straw. Bioresource Technology 82: Salmones D, Mata G, Waliszewski KN (2005) Comparative culturing of Pleurotus spp. on coffee pulp and wheat straw: biomass production and substrate biodegradation. Bioresour Technol 96: Ergun B, Huseyin P, Mustafa KY, et al. (2003) Cultivation of oyster mushroom on waste paper with some added supplementary materials. Biores Techn 89: Salami AO, Bankole FA (2018) Don t waste the wastes they are ways to wealth. EC Microbiology 14: Akhter K, Salahuddin M, Chowdhury M, et al. (2017) Effect of sterilization of substrate by hot water on prevalence of contaminants and yield attribute of Oyster mushroom. SJAVS 4: Manoj KK (2015) Impact of various sterilization methods on growth and yield of Oyster mushroom (Pleurotus florida). Int j Agri sci 11: Bankole FA, Salami AO (2017) Use of Agro-Wastes for Tissue Culture Process and Spawn Production of Oyster Mushroom (Pleurotus florida). J Appl Life Sci Inter 14: Salami AO, Bankole FA, Andrew CO, (2018) Effect of organic nitrogen supplements on the yield and nutrient content of Oyster mushroom (Pleurotus florida) cultivated on corncobs. 1: Salami AO, Bankole FA, Olawole OI (2016) Effect of different substrates on the growth and protein content of oyster mushroom (Pleurotus florida). Int J Biol Chem Sci 10: Citation: Abiodun Olusola Salami (2018) Effect of Calcium Supplementation and Sterilization Methods on the Production of Oyster Mushroom. 1:3. page 7 of 7