A Modified Approach in Substrate Preparation Technique for Small Scale Oyster Mushroom Farming
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The study presents a modified substrate preparation technique for oyster mushroom cultivation aimed at reducing contamination from indigenous microflora. The modified method, which involved a specific immersion process, resulted in complete absence of contaminants, while the control showed a 6.67-20% incidence of various contaminants. The findings suggest that this approach enhances productivity by ensuring contamination-free mushroom production.
![Siddhant et al.; AJAHR, 2(2): 1-5, 2018; Article no.AJAHR.44907
2
control, fresh wheat straw was put in to the steam sterilised gunny bag and steeped directly in the
chemical solution as suggested above. After removal of excess water, the substrate was used for
spawning with inoculums of Pleurotus sajor-caju, Strain- Malaysia.
Results: Beds with modified approach showed a complete absence of contaminants during entire
crop cycle which showed the effectiveness of modified method while beds treated as control
showed little incidence of various contaminants viz., Alternaria alternata, Aspergillus fumigatus,
Curvularia sp., Penicillium sp., and Rhizopus stolonifer with 6.67-20.00 percent incidence.
Conclusion: The results revealed that modified method should be considered to contamination
free mushroom production.
Keywords: Contaminants; mushroom; substrate.
1. INTRODUCTION
Oyster mushroom (Pleurotus sajor-caju) can be
grown on a wide range of agricultural waste such
as straw, saw dust, rice hull etc. due to its strong
enzymatic features [1]. These substrates are
accompanying with indigenous microflora. The
antagonistic interaction between these microflora
and desired fungus contributed to the low
productivity of mushroom [2,3]. Therefore, proper
substrate disinfection is pre requisite to eliminate
weed and obtain good yield. Substrate
disinfection can be achieved by various means
such as using material boiled with hot water [4],
steaming [5], autoclaving [6], radiation and
solarisation [7], composting [8] and chemical
pasteurisation [9] etc. Among these methods, the
chemical pasteurisation is the most popular
method for small scale growers due to its
easiness, effectiveness and moreover minimising
time, labour and expenditure on appliances and
fire wood. In this method, substrates are steeped
in chemical solutions like 1% formaldehyde [10],
0.2% Bavistin [11], 0.5% commercial Lime [12]
and 0.01% Derosal [4] or their combinations viz.,
500 ppm Formaldehyde +75 ppm Bovistin [9,13]
for different time intervals. After removal of
excess water, the substrate becomes ready for
inoculation. Several researchers have been
reported in their work regarding the appearance
of contamination from chemically pasteurised
substrates during the production span [9,13]. The
present study was under-taken with slight
modification in substrate preparation technique to
eliminate dust particles from the substrate and to
assure contamination free mushroom production.
2. EXPERIMENTAL DETAILS
2.1 Mushroom Culture
The pure culture of Pleurotus sajor-caju Strain-
Malaysia was obtained from the Mushroom
Section of Plant Pathology Department, Chandra
Shekhar Azad University of Agriculture and
Technology, Kanpur (U.P.) India. It was
maintained on Potato Dextrose Agar medium
(peeled, sliced and boiled potato, 200g; dextrose,
20g; agar, 20gl-1
) by using serial subculture
method [14].
2.2 Spawn Strategy
2.2.1 Spawn substrate
Wheat grain (Triticum aestivum) was used as a
spawn substrate which was purchased from the
seed market of Faizabad. The spawn was
prepared by the conventional method [14].
2.2.2 Spawn dose
Inoculation of substrate was made with spawn of
P. sajor-caju Strain-Malaysia at 15% w/w on dry
weight basis under aseptic conditions.
2.3 Substrate for Mushroom Cultivation
2.3.1 Substrate
Wheat straw was used as a substrate due to its
easy availability in this area. It was collected at
the time of threshing of harvested crop from
village Madarahia.
2.3.2 Substrate preparation (Pasteurisation)
The wheat straw was immersed in the drum
containing tap water and mixing properly and
allowed to stand for 10-15 min to settle down the
dust particle in the bottom of drum. After that, the
floating substrate was transferred to the slant
surface so that the extra water was decanted off.
This substrate was put in to the steam sterilised
gunny bag and steeped in the chemical solution
consisting of Formaldehyde (500 ppm) and
Bovistin (75 ppm) for 18 h as suggested by Vijay
and Sohi [9]. For the control, fresh wheat straw
was put in to the steam sterilised gunny bag and](https://image.slidesharecdn.com/siddhant222018ajahr44907-181103110701/85/A-Modified-Approach-in-Substrate-Preparation-Technique-for-Small-Scale-Oyster-Mushroom-Farming-2-320.jpg)
![Siddhant et al.; AJAHR, 2(2): 1-5, 2018; Article no.AJAHR.44907
3
steeped directly in the chemical solution as
suggested above. After removal of excess water,
the substrate was used for spawning. A total of
30 bags (15 each for treatment and control) were
prepared with 4.5 kg wet weight (=1.5 kg dry
weight) of the substrate in each replica.
2.4 Method of Cultivation
Plastic bag technology was employed in this
experiment. The beds were prepared from
pasteurised substrate by multilayered spawning
following the procedure adopted by Bano [15].
These were incubated in cultivation room at 22-
30°C temperature for spawn run. When mycelia
had completely covered the beds, the polythene
covering were turned off and relative humidity
was maintained 85-95% with the help of
humidifier.
2.5 Concerning Data
The growth and development of mushrooms
were monitored daily. Data on days require for
mycelia colonisation, initiation and maturation of
fruit bodies, yield and biological efficiency were
recorded. The biological efficiency (%) was
calculated as follows:
%BE=FWm/DWs∗100%;
Where, BE is Biological Efficiency (%); FWm is
total fresh weight (g) of mushroom yield across
all flushes, and DWs is substrate dry weight (g).
Although the yield data of mushroom are given in
present communication, the observation
regarding encountered microorganism was prime
concern to evaluate the efficiency of the modified
method to minimise the appearance of weed.
Daily inspection was made to trace any growth of
unwanted microflora on the beds till harvesting of
the mushrooms. The incidence of competitor
microorganism were recorded as the number of
infected beds with particular microorganism out
of the total beds per treatment and expressed in
percentage. The competitors encountered on the
mushroom beds were examined under a
compound microscope and mycological features
like cultural character, hyphal characteristics,
septations, branching pattern of hyphae,
differentiation and development of sporophore,
measurements etc. were studied and compared
with the relevant literature for identification of the
microflora isolated from contaminated mushroom
beds.
Incidence of contamination =
X 100
2.6 Statistical Analysis
Completely randomized design (CRD) was
followed for the experiment. The yield data was
statistically analysed by using the analysis of
variance (Anova) at P=5%. The critical difference
(CD) was worked out at five per cent probability
level.
3. RESULTS AND DISCUSSION
Beds with modified approach showed a complete
absence of contaminants during the entire crop
cycle which shows the effectiveness of the
modified method (Fig. 1) while control showed
little incidence of contaminants during spawn run
period. Aspergillus fumigatus was predominant
mycoflora appearing on the control beds with
incidence of 20%. The incidence of other
contaminants viz., Penicillium sp., Rhizopus sp.,
Alternaria alternata and Curvularia sp., were in
the range of 6.67 to 13.34% (Fig. 2). Later, the
growth of these mycoflora was suppressed by
mushroom mycelia. It might be due to combined
effect of both higher proportion of inoculums of
Pleurotus against contaminants and
effectiveness of pasteurisation practice. It is well
established when contaminants are scarce in the
substrate they do not offer a competence for the
mycelium of Pleurotus sp which quickly colonised
straw substrate [16]. During the course of study,
five fungal species viz., Aspergillus fumigatus,
Alternaria alternata, Curvularia sp., Penicillium
sp., and Rhizopus stolonifer were encountered
on control. The yield data shows that there was
no significant difference among treatments which
reflects no adverse effect of these contaminants
on mushroom yield (Table 1). It might be due to
the suppression of growth of competitors by
mushroom mycelia. The susceptibility of control
against the contaminants was possibly due to the
presence of dust particles in the wheat straw
substrate that provides inoculum for the
development of competitors [17]. According to
the Chhetri et al. [18] contaminants like
Aspergillus sp. is the major contaminant of
mushroom beds which could cover up the beds
very rapidly and restricted the mycelia run in the
pasteurised straw and caused highest yield loss.
Although, the appearance of lesser incidence of
competitors in our investigation revealed the
efficiency of chemical treatment.](https://image.slidesharecdn.com/siddhant222018ajahr44907-181103110701/85/A-Modified-Approach-in-Substrate-Preparation-Technique-for-Small-Scale-Oyster-Mushroom-Farming-3-320.jpg)
A Modified Approach in Substrate Preparation Technique for Small Scale Oyster Mushroom Farming
- 1. _____________________________________________________________________________________________________ *Corresponding author: Email: siddhant.ani@gmail.com; Asian Journal of Agricultural and Horticultural Research 2(2): 1-5, 2018; Article no.AJAHR.44907 ISSN: 2581-4478 A Modified Approach in Substrate Preparation Technique for Small Scale Oyster Mushroom Farming Siddhant1* , O. P. Ukaogo2 and S. S. Walakulu Gamage3 1 Department of Botany, Durgesh Nandini Degree College, Faizabad (U.P.), India. 2 Environmental/Analytical Units, Department of Industrial Chemistry, Abia State University, Nigeria. 3 Department of Botany, Faculty of Science, University of Ruhana, Matara, Sri Lanka. Authors’ contributions This work was carried out in collaboration between all authors. All authors read and approved the final manuscript. Article Information DOI: 10.9734/AJAHR/2018/44907 Editor(s): (1) Dr. Ahmed Medhat Mohamed Al-Naggar, Professor of Plant Breeding, Department of Agronomy, Faculty of Agriculture, Cairo University, Egypt. Reviewers: (1) Tajudeen Yahaya, Federal University Birnin Kebbi, Nigeria. (2) Teodoro Bernabé-González, Autonomous University of Guerrero, Mexico. Complete Peer review History: http://www.sciencedomain.org/review-history/26942 Received 13 August 2018 Accepted 26 October 2018 Published 31 October 2018 ABSTRACT Aims: Cultivation of oyster mushrooms has increased vastly in a global scale during last few decades. Contaminants and indigenous microflora present in the substrate may led to the low productivity of mushrooms. Keeping this in mind, the present study was under-taken with slight modification in substrate preparation technique to eliminate dust particles from the substrate and to assure contamination free mushroom production. Study Design: Comparative evaluation between modified approach and control beds. Place and Duration: The study was carried out in Shri Laxman Prasad Pyare Lal Agro Products, Ayodhya during 2017. Methodology: The wheat straw substrate was immersed in the drum containing tap water, mixing properly and allowed to stand for 10-15 min to settle down the dust particle in the bottom of the drum. After that, the floating substrate was transferred to the slant surface so that the extra water was decanted off. This substrate was put in to the steam sterilised gunny bag and steeped in the chemical solution consisting of Formaldehyde (500 ppm) and Bovistin (75 ppm) for 18 h. For the Short Communication
- 2. Siddhant et al.; AJAHR, 2(2): 1-5, 2018; Article no.AJAHR.44907 2 control, fresh wheat straw was put in to the steam sterilised gunny bag and steeped directly in the chemical solution as suggested above. After removal of excess water, the substrate was used for spawning with inoculums of Pleurotus sajor-caju, Strain- Malaysia. Results: Beds with modified approach showed a complete absence of contaminants during entire crop cycle which showed the effectiveness of modified method while beds treated as control showed little incidence of various contaminants viz., Alternaria alternata, Aspergillus fumigatus, Curvularia sp., Penicillium sp., and Rhizopus stolonifer with 6.67-20.00 percent incidence. Conclusion: The results revealed that modified method should be considered to contamination free mushroom production. Keywords: Contaminants; mushroom; substrate. 1. INTRODUCTION Oyster mushroom (Pleurotus sajor-caju) can be grown on a wide range of agricultural waste such as straw, saw dust, rice hull etc. due to its strong enzymatic features [1]. These substrates are accompanying with indigenous microflora. The antagonistic interaction between these microflora and desired fungus contributed to the low productivity of mushroom [2,3]. Therefore, proper substrate disinfection is pre requisite to eliminate weed and obtain good yield. Substrate disinfection can be achieved by various means such as using material boiled with hot water [4], steaming [5], autoclaving [6], radiation and solarisation [7], composting [8] and chemical pasteurisation [9] etc. Among these methods, the chemical pasteurisation is the most popular method for small scale growers due to its easiness, effectiveness and moreover minimising time, labour and expenditure on appliances and fire wood. In this method, substrates are steeped in chemical solutions like 1% formaldehyde [10], 0.2% Bavistin [11], 0.5% commercial Lime [12] and 0.01% Derosal [4] or their combinations viz., 500 ppm Formaldehyde +75 ppm Bovistin [9,13] for different time intervals. After removal of excess water, the substrate becomes ready for inoculation. Several researchers have been reported in their work regarding the appearance of contamination from chemically pasteurised substrates during the production span [9,13]. The present study was under-taken with slight modification in substrate preparation technique to eliminate dust particles from the substrate and to assure contamination free mushroom production. 2. EXPERIMENTAL DETAILS 2.1 Mushroom Culture The pure culture of Pleurotus sajor-caju Strain- Malaysia was obtained from the Mushroom Section of Plant Pathology Department, Chandra Shekhar Azad University of Agriculture and Technology, Kanpur (U.P.) India. It was maintained on Potato Dextrose Agar medium (peeled, sliced and boiled potato, 200g; dextrose, 20g; agar, 20gl-1 ) by using serial subculture method [14]. 2.2 Spawn Strategy 2.2.1 Spawn substrate Wheat grain (Triticum aestivum) was used as a spawn substrate which was purchased from the seed market of Faizabad. The spawn was prepared by the conventional method [14]. 2.2.2 Spawn dose Inoculation of substrate was made with spawn of P. sajor-caju Strain-Malaysia at 15% w/w on dry weight basis under aseptic conditions. 2.3 Substrate for Mushroom Cultivation 2.3.1 Substrate Wheat straw was used as a substrate due to its easy availability in this area. It was collected at the time of threshing of harvested crop from village Madarahia. 2.3.2 Substrate preparation (Pasteurisation) The wheat straw was immersed in the drum containing tap water and mixing properly and allowed to stand for 10-15 min to settle down the dust particle in the bottom of drum. After that, the floating substrate was transferred to the slant surface so that the extra water was decanted off. This substrate was put in to the steam sterilised gunny bag and steeped in the chemical solution consisting of Formaldehyde (500 ppm) and Bovistin (75 ppm) for 18 h as suggested by Vijay and Sohi [9]. For the control, fresh wheat straw was put in to the steam sterilised gunny bag and
- 3. Siddhant et al.; AJAHR, 2(2): 1-5, 2018; Article no.AJAHR.44907 3 steeped directly in the chemical solution as suggested above. After removal of excess water, the substrate was used for spawning. A total of 30 bags (15 each for treatment and control) were prepared with 4.5 kg wet weight (=1.5 kg dry weight) of the substrate in each replica. 2.4 Method of Cultivation Plastic bag technology was employed in this experiment. The beds were prepared from pasteurised substrate by multilayered spawning following the procedure adopted by Bano [15]. These were incubated in cultivation room at 22- 30°C temperature for spawn run. When mycelia had completely covered the beds, the polythene covering were turned off and relative humidity was maintained 85-95% with the help of humidifier. 2.5 Concerning Data The growth and development of mushrooms were monitored daily. Data on days require for mycelia colonisation, initiation and maturation of fruit bodies, yield and biological efficiency were recorded. The biological efficiency (%) was calculated as follows: %BE=FWm/DWs∗100%; Where, BE is Biological Efficiency (%); FWm is total fresh weight (g) of mushroom yield across all flushes, and DWs is substrate dry weight (g). Although the yield data of mushroom are given in present communication, the observation regarding encountered microorganism was prime concern to evaluate the efficiency of the modified method to minimise the appearance of weed. Daily inspection was made to trace any growth of unwanted microflora on the beds till harvesting of the mushrooms. The incidence of competitor microorganism were recorded as the number of infected beds with particular microorganism out of the total beds per treatment and expressed in percentage. The competitors encountered on the mushroom beds were examined under a compound microscope and mycological features like cultural character, hyphal characteristics, septations, branching pattern of hyphae, differentiation and development of sporophore, measurements etc. were studied and compared with the relevant literature for identification of the microflora isolated from contaminated mushroom beds. Incidence of contamination = X 100 2.6 Statistical Analysis Completely randomized design (CRD) was followed for the experiment. The yield data was statistically analysed by using the analysis of variance (Anova) at P=5%. The critical difference (CD) was worked out at five per cent probability level. 3. RESULTS AND DISCUSSION Beds with modified approach showed a complete absence of contaminants during the entire crop cycle which shows the effectiveness of the modified method (Fig. 1) while control showed little incidence of contaminants during spawn run period. Aspergillus fumigatus was predominant mycoflora appearing on the control beds with incidence of 20%. The incidence of other contaminants viz., Penicillium sp., Rhizopus sp., Alternaria alternata and Curvularia sp., were in the range of 6.67 to 13.34% (Fig. 2). Later, the growth of these mycoflora was suppressed by mushroom mycelia. It might be due to combined effect of both higher proportion of inoculums of Pleurotus against contaminants and effectiveness of pasteurisation practice. It is well established when contaminants are scarce in the substrate they do not offer a competence for the mycelium of Pleurotus sp which quickly colonised straw substrate [16]. During the course of study, five fungal species viz., Aspergillus fumigatus, Alternaria alternata, Curvularia sp., Penicillium sp., and Rhizopus stolonifer were encountered on control. The yield data shows that there was no significant difference among treatments which reflects no adverse effect of these contaminants on mushroom yield (Table 1). It might be due to the suppression of growth of competitors by mushroom mycelia. The susceptibility of control against the contaminants was possibly due to the presence of dust particles in the wheat straw substrate that provides inoculum for the development of competitors [17]. According to the Chhetri et al. [18] contaminants like Aspergillus sp. is the major contaminant of mushroom beds which could cover up the beds very rapidly and restricted the mycelia run in the pasteurised straw and caused highest yield loss. Although, the appearance of lesser incidence of competitors in our investigation revealed the efficiency of chemical treatment.
- 4. Siddhant et al.; AJAHR, 2(2): 1-5, 2018; Article no.AJAHR.44907 4 Fig. 1. Beds with modified approach showing complete absence of contaminants Fig. 2. Prevalence of competitor moulds in control beds Table 1. Effect of modified treatment on various parameters of Pleurotus sajor-caju Treatment Spawn run (Days) Fruit body initiation (Days) First harvest (Days) Total yield from three flushes (gm/4.5kg wet substrate) Biological efficiency (%) Modified treatment 14.8 20.9 23.2 1404.8 91.96 Control 15.2 21.3 24.8 1379.4 93.65 SE - - - 34.14 2.27 CD (P=0.05) - - - 69.98 4.67 Average of fifteen replications 4. CONCLUSION The Pleurotus sp. can be cultivated on a wide range of agro-industrial wastes which are attacked by several competitors of fungal and bacterial origin. The antagonistic relationship between these microflora and desired fungus contributes a low productivity of mushrooms. In severe cases, complete failure of the mushroom crop may take place. Despite of no significant difference in yield, modified method to eliminate dust particles from the substrate should be considered to contamination free mushroom production.
- 5. Siddhant et al.; AJAHR, 2(2): 1-5, 2018; Article no.AJAHR.44907 5 COMPETING INTERESTS Authors have declared that no competing interests exist. REFERENCES 1. Elisashvili V, Penninckx M, Kachlishvili E, Tsiklauri N, Metreveli E, Kharziani T, Kvesitadze G. Lentinus edodes and Pleurotus species lignocellulolytic enzymes activity in submerged and solid-state fermentation of lignocellulosic wastes of different composition. Bioresour Technol. 2008;99:457-462. 2. Siddique AB, Gogoi R, Puzari KC. Evaluation of phyto-extracts against contaminants of oyster mushroom. Indian J Mycol Pl Pathol. 2004;34(2):329- 332. 3. Siddhant, Yadav S, Mishra R, Singh R. Effect of substrate disinfection on biological efficiency of Pleurotus sajor-caju (Fr.) Singer. Pl Archives. 2014;14(1):205- 209. 4. Ficior D, Indrea D, Apahidean AS, Apahidean M, Rodica Pop, Moldovan Z, et al. Importance of substrat dizinfection on oyster mushroom (Pleurotus sp.) culture. Not Bot Hort Agrobot Cluj. 2006; 34:48-53. 5. Bahukhandi D. Effects of various treatments on paddy straw on yield of some cultivated species of Pleurotus. Indian Phytopath. 1990;43(3):471-472. 6. Tewari RP, Pandey M. An important method of oyster mushroom (Pleurotus sajor-caju) cultivation. Indian J Mycol Pl Pathol. 1988;18(1):104. 7. Ram RC, Thakur D. Evaluation of new substrate preparation techniques for oyster mushroom cultivation. Mush Res. 2005;14(1):37-39. 8. Vieira FR, de Andrade. Optimization of substrate preparation for oyster mushroom (Pleurotus ostreatus) cultivation by studying different raw materials and substrate preparation conditions (composting: Phases I and II). World J Microbiol Biotechnol. 2016;32(11):190. 9. Vijay B, Sohi HS. Cultivation of oyster mushroom Pleurotus sajor-caju (Fr.) Singer on chemically sterilized wheat straw. Mush J Tropics. 1987;7:67-75. 10. Funda CA. Effect of Different substrate Disinfection Methods on the Production of Pleurotus ostreatus. J Agri Stud. 2016;4(4):1-14. 11. Kalita MK. Impact of various sterilization methods on growth and yield of oyster mushroom (Pleurotus florida). Int J Agri Sci. 2015;11(1):104-107. 12. Contreras EP, Sokolov M, Mejía G, Sánchez JE. Soaking of substrate in alkaline water as a pretreatment for the cultivation of Pleurotus ostreatus. J Hort Sci Biotechnol. 2004;79(2):234-240. 13. Pervez Z, Bhuiyan MKA, Islam MS. In vitro control of associated microflora of oyster mushroom substrates by the application of fungicides. Bangladesh Res Pub J. 2009;2(4):737-741. 14. Naraian R, Sahu RK, Kumar S, Garg SK, Singh CS and Kanaujia RS. Influence of different nitrogen rich supplements during cultivation of Pleurotus florida on maize cobs substrate. Environmentalist. 2009;29:1-7. 15. Bano Z. Cultivation of Pleurotus flabellatus. Second Int Symp Pl Pathol. IARI, New Delhi 1971;135. 16. Mejía SJ, Albertó E. Heat treatment of wheat straw by immersion in hot water decreases mushroom yield in Pleurotus ostreatus. Rev Iberoam Micol. 2013; 30(2):125–129. 17. Siddhant, Yadav S, Ahmad A, Singh CS. Effect of wheat straw components on yield of Pleurotus eous. Int J Curr Microbiol Appl Sci. 2013;2(8):221-225. 18. Chhetri K, Senapoty D, Sharma DK. Management of Contaminant Mycoflora of Oyster Mushroom (var-Pleurotus florida) with Botanicals and GRAS Chemicals. Int J Curr Microbiol App Sci. 2018;7(2):1972- 1978. © 2018 Siddhant et al.; This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Peer-review history: The peer review history for this paper can be accessed here: http://www.sciencedomain.org/review-history/26942