Journal of Pharmacognosy and Phytochemistry 2019; 8(3): 3167-3171

E-ISSN: 2278-4136
P-ISSN: 2349-8234
JPP 2019; 8(3): 3167-3171
Received: 25-03-2019
Effect of different substrate alone and in
Accepted: 27-04-2019 combination on the sporophore production of elm
Sutha Raja Kumar R oyster mushroom Hypsizygus ulmarius
Department of Plant Pathology,
Faculty of Agriculture
Annamalai University, Sutha Raja Kumar R, Eswaran A, Darwin Christdhas Henry, L Kannan C
Annamalai Nagar, Tamil Nadu,
India
and Jaiganesh V
Eswaran A Abstract
Department of Plant Pathology, Studies were conducted to assess the efficacy of different substrates, viz., banana leaves, casuarina needle,
Faculty of Agriculture coir pith, ground nut shell, paddy straw, sugarcane trash, sugarcane bagasse, saw dust and water hyacinth
Annamalai University, and supplements on the sporophore production. Among the different substrate paddy straw (489.6g bed-1)
Annamalai Nagar, Tamil Nadu was most efficient in enhancing the yield of H. ulmarius. Followed by water hyacinth (474.4g bed-1) and
India,
sugarcane trash (472.7g bed-1). Among the six different methods of sterilization tested, the chemical
Darwin Christdhas Henry
sterilization by soaking in 500 ppm formalin plus 75ppm carbendazim solution and autoclaving for 30 min.
Department of Plant Pathology, was observed to be the most efficient method of substrate sterilization which not only prevented
Faculty of Agriculture contamination by competitive fungi but also enhanced the yield of the mushrooms (489.5g bed-1). Among
Annamalai University, the different supplements, horse gram powder recorded the maximum sporophore yield and biological
Annamalai Nagar, Tamil Nadu, efficiency (498.6g bed-1 and 99.7%, respectively). Among the various agro wastes paddy straw plus water
India hyacinth recorded the maximum sporophore yield (498.9g bed-1) and biological efficiency (99.8%).

L Kannan C Keywords: Substrate pasteurization, organic additives, sporophore yield

Department of Plant Pathology,
Faculty of Agriculture
Annamalai University,
Introduction
Annamalai Nagar, Tamil Nadu, Oyster mushroom is an efficient lignin-degrading mushroom and can grow well on different
India types of lingo cellulosic materials. Oyster mushroom can be grown on various substrates
including paddy straw, wheat straw, maize stalks/cobs, vegetable plant residues, bagasse etc. It
Jaiganesh V is estimated that about 355 million tons of crop residue is generated annually and about 170
Department of Plant Pathology,
Faculty of Agriculture
million is left out for burning and incorporating into soil in manure form (Tewari and Pandey,
Annamalai University, 2002). Even if one per cent of this lignocellulosic agro wastes are diverted to production of
Annamalai Nagar, Tamil Nadu, mushrooms, India will become a major mushroom producing country in the world. An ideal
India substrate should contain nitrogen (supplement) and carbohydrates for rapid mushroom growth.
The nutrient composition of the substrate is one of the factors limiting the sapro biotic
colonization of cultivated mushrooms and particularly the fruiting of Pleurotus spp.
(Tshinyangu and Hennebert, 1995) [26]. The growth of mushrooms as well as quantitative and
qualitative yield of the desired product depends on utilization of nutrients in the medium
(Mukhopadhyay et al., 2002) [15]. Lignocellulosic materials such as cereal straws, corn cobs,
paper, cotton seed hull, bagasse, wood shavings and saw dust as well as food industry wastes
are used for mushroom cultivation (Ragunathan et al., 1996; Baysal et al., 2003; Xing et al.,
2006) [19, 2, 29] . The supplements or additives supply extra nitrogen and/or easily degradable
carbohydrates to increase mushroom yields and hasten the production process (Royse, 2002) [20].
The nutritional content of the substrate can be improved by nitrogen supplementation (Lelley
and Jan Ben, 1993) [12]. Supplementing the substrate with controlled liberation of nitrogen and
MN, shortens the crop period for Pleurotus spp. and also increases mushroom productivity
(Curvetto et al., 2002) [4]. With this background, this research has been carried out to identify
the most suitable substrate, sterilization method, supplements and substrate combination for the
cultivation of Hypsizygus ulmarius mushrooms

Materials and methods

Correspondence Source and maintenance of culture
Sutha Raja Kumar R The pure culture of H. ulmarius Co (OM) 2 was obtained from National Research Centre for
Department of Plant Pathology, Mushroom (NRCM), Sloan and Himachal Pradesh. Subcultures were made periodically and
Faculty of Agriculture maintained on potato dextrose agar (PDA) slants and stored at 252C temp. for further
Annamalai University,
Annamalai Nagar, Tamil Nadu,
investigations.
India
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Journal of Pharmacognosy and Phytochemistry

Bed preparation Coir pith

Cultivation of H. ulmarius was carried out in transparent Partially decomposed coir pith was chemically pasteurized
polythene bags of 60  30cm size with a thickness of 100 gauge using 75 ppm carbendazim and 500 ppm Formalin and used for
and cylindrical beds were prepared using 0.5 kg of paddy straw preparation of beds.
on dry weight basis following layer spawning method as
described by Sivaprakasm (1980) with below mentioned Groundnut shells
modification. The unchopped whole straw was made into coils Well dried groundnut shells crushed into small bits was
and used. A layer of coiled paddy straw was placed at the chemically pasteurized using 75 ppm carbendazim and 500
bottom of polythene bag, over this a twenty g of spawn was ppm formalin and used for preparation of beds.
sprinkled. In this manner five layers of coiled paddy straw and
four layers of spawn were kept in the polythene bag and then Sugarcane trash
bag was tied at the top (modified cylindrical bed method). Properly dried sugarcane trash was chemically pasteurized as
Eight holes of one cm diameter were made at random in the stated earlier and used for preparation of beds.
polythene bags. The mushroom beds were hung from the
ceiling by means of ropes (‘uri‘method). After spawn running Water hyacinth
stage, The beds were kept in cropping room, where the temp. Water hyacinth plants were collected, chopped to convenient
was maintained at 23 to 28 C and relative humidity at 80 to 90 size and sun dried. The dried substrate was chemically
per cent. Water was sprinkled regularly as in the standard pasteurized as stated earlier and used for bed preparation.
cylindrical bed preparation method. The following yield
parameters were observed in all the experiments. Paddy straw
Paddy straw was collected, dried well, pasteurized as stated
Evaluation of different methods of substrate pasteurization earlier and used for preparation of beds.
for the cultivation of H. ulmarius
The different methods of pasteurization of substrates viz., Effect of different bed supplements on the yield of H.
boiling, steaming and autoclaving presoaked paddy straw for ulmarius
30 minutes. at 15 psi; chemical pasteurization by soaking the In the present study, nine different supplements viz., calcium
substrate in water mixed with 0.1 per cent carbendazim carbonate, corn flour, black gram flour, gypsum, horse gram
solution for 16 h.; soaking the substrate in 0.1 per cent flour, rice flour, red gram flour and sorghum flour @ two per
carbendazim plus 500ppm formalin solution for 18 h. and cent were tested for their efficacy in enhancing the yield
autoclaving, pre-soaked in 0.01 per cent carbendazim and 500 attributes of H. ulmarius. The additives were powdered well
ppm formalin solution for 16h were evaluated for their and sterilized in an autoclave at 15 psi for one h. Beds without
suitability and efficacy in reducing the per cent contamination any supplement served as control and each treatment was
by the competitors. The substrate soaked in cold water for 18 replicated thrice. Parameters namely spawn run days, pinhead
h. (without any pasteurization) served as control. A temp of formation, total yield and biological efficiency were assessed
28±2°C and relative humidity of 80-85 per cent was maintained and recorded.
in the cropping room. Three replications were maintained for
each treatment and the observations namely per cent Effect of various combinations of bed substrates on the
contamination by competitors, number of days taken for spawn yield of H. ulmarius
run, button formation, total yield and biological efficiency were In the present study along with paddy straw, the substrates like,
recorded. dried banana leaves, coconut leaves, cotton waste, groundnut
shell, sugarcane trash and water hyacinth were combined at the
Evaluation of different substrates for the cultivation of H. ratio of 1:1 for bed (polybag method) preparation. Beds
ulmarius prepared using paddy straw alone served as control. Each
Nine different locally available substrates viz., banana leaves, treatment was replicated thrice and the beds were incubated at
casuarina needle, coir pith, groundnut shell, paddy straw, 28  2°C temp. With a relative humidity of 85 per cent. The
sugarcane trash, sugarcane bagasse, saw dust and water parameters namely spawn run days, days for pin head
hyacinth were evaluated for their ability in promoting the fruit formation, total duration, total yield and biological efficiency
body formation of H. ulmarius. Polybag method was followed were recorded.
for the preparation of beds and the substrate was pasteurized by
soaking in chemicals viz., carbendazim 75 ppm plus formalin Results and discussion
500 ppm. Horse gram powder @ two per cent was used as Evaluation of different substrates for the cultivation of H.
supplement in all the treatments. A temp. of 28  2°C and a ulmarius
relative humidity of 85 per cent were maintained in the Among the nine different locally available substrates tested for
cropping room. The parameters namely number of days taken their potentiality in supporting the sporophore formation of H.
for spawn run, pinhead formation, number of sporophore, total ulmarius, it was found that the paddy straw (489.6g bed-1) was
yield and biological efficiency were assessed and recorded. the most efficient in enhancing the yield of H. ulmarius,
followed by sugarcane trash (474.4 g bed-1), water hyacinth
Substrate preparation (472.7gbed-1), groundnut shell (458.3gbed-1), banana leaves
Fresh banana, Casuarina needle, leaves were collected, shade (437.1g bed-1), sugarcane bagasse (401.5g bed-1) and casuarina
dried and subjected to chemical pasteurization using 75ppm needle (375.9g bed-1) in the decreasing order of merit.
carbendazim and 500 ppm formalin for 16 h. Then the substrate Minimum fruiting bodies were observed in the beds prepared
was shade dried and used for preparation of beds. by using coir pith as substrate (Table 1).

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Journal of Pharmacognosy and Phytochemistry

Table 1: Effect of bed substrate on the growth and sporophore yield of H. ulmarius
Spawn run Total yield Biological efficiency
Tr. No. Substrates Pinhead formation (days) Total duration No of sporophore bed-1
(days) (g bed-1) (%)
1 Banana leaves 18.9d 23.2c 49.5c 109.6c 437.1d 87.4
2 Coir pith 23.4h 28.9f 57.8f 54.3g 247.2g 49.4
f d d e
3 Casuarina needle 20.2 24.8 52.3 94.9 375.9e 75.2
4 Groundnut shell 18. 4c 22.8c 48.4c 112.5c 458.3c 91.7
5 Saw dust 20.7g 25.1e 54.7e 82.2f 326.8f 65.4
6 Sugarcane bagasse 19.4e 23.7c 51.2d 101.8d 401.5d 80.3
7 Sugarcane trash 17.9b 22.3b 47.8b 115.4b 472.7b 94.5
8 Water hyacinth 17.8b 21.9b 47.6b 115.6b 474.4b 94.9
9 Paddy straw 17.2a 21.1a 47.1a 119.6a 489.6a 97.9
Values not sharing a common superscript differ significantly at P<0.05 (DMRT)

The variations in the growth and yield parameters of the followed by soaking straw substrate in 0.1 per cent
different mushrooms may be due to the biological structure of carbendazim solution for 16 h. and boiling of presoaked straw
the substrate (Mahbuba, 2010) [13]. Mandeel et al. (2005) [14] for 30 min., recorded a contamination percentage of (6.4%,
reported that mushroom grows in a wide variety of 9.2%, 12.4%) and an yield of 472.7g, 454.9g and 449.7g bed-1,
lignocellulosic residues comprising 40-60 per cent cellulose, respectively whereas, the substrate without any sterilization
20-30 per cent hemicellulose and 15-30 per cent lignin. Cereal treatment recorded complete contamination by weed moulds
straws are rich in cellulose, hemicelluloses and lignin from (Table 2).
which the mushroom derives the nutrition (Biswas and Biswas, Sterilization of bed substrate is a must for reducing the
2015) [3]. The enhanced yield observed in paddy straw contamination from competitive fungi and increasing the yield
substrates could be due to the presence of favourable nutrients of mushrooms (Zadrazil and Grabbe, 1983) [31]. Singh and
that are better utilized by the fungus. Pleurotus spp. has the Singh (1991) [22] reported that soybean straw was best sterilized
capacity to degrade cellulose, hemicelluloses and lignin and to by steeping in a solution of formaldehyde at 500 ppm and
produce fruiting bodies. The poor growth and yield observed carbendazim at 75 ppm for 18 h. Pleurotus sp. was able to
with coir pith and saw dust substrate could be attributed to the tolerate higher levels of carbendazim (75-200ppm) during
rich lignin content in them and poor ability of H. ulmarius to chemical sterilization of paddy straw substrate (Nallathambi
degrade lignin like substances. and Marimuthu, 1992) [17]. Earanna and Shetty (1994) found
that when compared to steam sterilization method, the formalin
Evaluation of different methods of substrate pasteurization drench method was more effective in sterilizing paddy straw
for the cultivation of H. ulmarius substrate. Likewise, the yields of Pleurotus spp. were the
Among the six different methods of sterilization tested, the highest on paddy straw pre-treated with carbendazim (75 ppm)
chemical sterilization by soaking in 500 ppm formalin plus 75 plus formaldehyde (500ppm) (Nallathambi and Marimuthu,
ppm carbendazim solution and autoclaving for 30 min. was 1994) [18] and soaking of paddy straw for 20 min. in hot water
observed to be the most efficient method of substrate followed by 10 min. dip in 500 ppm solution of carbendazim
sterilization which not only prevented contamination by resulted in the maximum yield and was free from any
competitive fungi but also enhanced the yield of the contamination (Singh et al., 1997) [23] whereas, Eswaran and
mushrooms (489.5g bed-1) which was on par with chemical Ramabadran (1997) obtained maximum yield of Pleurotus sp.
sterilization alone by soaking in 500 ppm formalin plus 75 ppm from beds treated with carbendazim @ 25ppm and formalin @
carbendazim solution with nil contamination and an yield of 200ppm.
480.2g bed-1. Autoclaving pre-soaked straw for 30 min.

Table 2: Evaluation of different methods of substrate pasteurization for the cultivation of H. ulmarius
Tr. Spawn run Pinhead Total yield Biological
Methods of pasteurization Contamination (%)
No. (days) formation (days) (g bed-1) efficiency (%)
1 Boiling pre- soaked straw for 30 min. 12.4 18.5c 5.3b 449.7d 89.9
d c
2 Steaming pre- soaked straw for 30 min. 18.4 18.9 6.1 368.2e 73.6
3 Autoclaving pre- soaked straw for 30 min. 6.4 17.6b 4.2a 472.7c 94.5
4 Soaking straw in carbendazim 0.1% for 16 h. 2.1 24.1e 8.2d 354.9e 70.9
Soaking straw in carbendazim 0.01% 75 ppm +
5 0.0 17.2a 4.4a 480.2a 96.0
formalin 500 ppm for 16 h.
Soaking straw in carbendazim 0.01% 75 ppm +
6 0.0 17.3a 4.3a 489.5a 96.3
formalin 500 ppm for 16 h. + autoclaving for 30 min.
7 Control 81.3 21.4e 5.4b 116.3f 23.3
Values not sharing a common superscript differ significantly at P<0.05 (DMRT)

Effect of various organic additives to bed substrate on sporophore yield and biological efficiency (498.6g bed-1 and
sporophore production of H. ulmarius 99.7%, respectively) followed by red gram powder (492.2 g
From the data presented in table 3, all other supplements tested bed-1 and 98.4%, respectively) and calcium carbonate (486.1 g
except gypsum and black gram flour recorded increased yield bed-1 and 97.2% respectively). Paddy straw devoid of any
when compared to control. The supplements viz., horse gram, supplements served as control and recorded a sporophore yield
red gram powder and calcium carbonate Significantly of 481.3g bed-1 and 96.3 per cent biological efficiency.
enhanced the yield than all other treatments. Among the Estrada et al. (2009) [6] opined that supplementation is an
supplements, horse gram powder recorded the maximum important step in enhancing oyster mushroom production.
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Journal of Pharmacognosy and Phytochemistry

However, the supplement ratio should not be high due to the There is a direct correlation between the enhanced production
possibility of yield reduction (Fanadzo et al., 2010) [8], of different enzymes viz., cellulose(s), hemicellulose(s), and
possibility of contamination (Yildiz et al., 2002) [30], increase laccase, which degrades cellulose, hemicelluloses and lignin,
in the bed temp. and possibility of mycelium inhibition respectively which is induced by supplementing gram powder.
(Upadhyay et al., 2002) [28]. In the present study, The various organic and inorganic additives mostly influence
supplementation of horse gram and red gram powder recorded the production of enzymes by the mushroom fungi
a significant increase in the yield. Efficient utilization of (Arunprasad, 2004) [1]. Enzyme production by the fungal
lignocellulolytic substrates by mushroom fungi largely mycelium is of paramount importance in the colonization
depends on the activity of extracellular enzymes. In the beds process and also an important determinant of mushroom yield
supplemented with gram powder, an increased rate of (Ferdinandi et al., 2014) [9]. It is appropriate that the enhanced
decomposition was observed in the present study. Jaganathan yield observed in the present study might be attributed to the
(1972) [10] also observed increased rate of decomposition of increased enzyme activity due to the supplementation of gram
straw beds that were treated with amendments. This revealed powder (Munoz et al., 1997) [16] which would have triggered
that the enzymes are possible weapons for substrate quicker degradation of substrates lead to the release of nutrients
decomposition and yield enhancement. which were utilized by the fungus resulting in yield
enhancement.

Table 3; Effect of various organic additives to bed substrate on sporophore production of H. ulmarius
Additives Pinhead formation
Tr. No. Spawn run (days) Total duration Sporophore yield (g bed-1) Biological efficiency (%)
(@ 2 % bed-1) (days)
c c
1 Calcium carbonate 17.2 5.4 48.0d 486.1c 97.2
c c b
2 Corn flour 17.0 4.9 47.8 482.4c 96.5
3 Black gram flour 18.1d 6.2e 48.7c 447.5d 89.5
4 Gypsum 18.9e 7.4f 49.2e 426.8e 85.4
5 Horse gram flour 15.8a 3.9a 46.2a 498.6a 99.7
6 Rice flour 17.2c 5.7d 48.2c 482.9c 96.6
7 Red gram flour 16.3b 4.0b 46.5a 492.2b 98.4
8 Sorghum flour 16.6b 4.3b 47.3b 484.8c 96.9
9 Control 17.4c 5.6d 47.2b 481.3c 96.3
Values not sharing a common superscript differ significantly at P<0.05 (DMRT)

Effect of different combination of bed substrates on the paddy straw plus Sugarcane trash combination recording
yield of H. ulmarius (489.3g bed-1 and 97.9% respectively) while paddy straw alone
Among the various agro wastes viz., banana leaves, coconut recorded 482.6g bed-1 of sporophore yield and 96.5 per cent
leaves, cotton wastes, water hyacinth, groundnut shell and biological efficiency. The minimum biological efficiency
sugarcane trash, in combination with paddy straw, paddy straw (77.3%) and sporophore yield (386.4g bed-1) was recorded with
plus water hyacinth recorded the maximum sporophore yield the combination of paddy straw plus coconut leaves (Table 4).
(498.9g bed-1) and biological efficiency (99.8%) followed by

Table 4: Effect of different combination of bed substrates on the sporophore yield of H. ulmarius
Yield
Tr. No. Substrate combination (1:1) Spawn run (days) Pinhead formation (days) Total duration Biological efficiency (%)
(g bed-1)
1 Paddy straw + Banana leaves 17.3b 6.1d 48.9e 432.7 d 86.5
2 Paddy straw + Coconut leaves 19.2e 7.7e 49.9f 386.4e 77.3
3 Paddy straw + Cotton waste 18.3d 5.3c 48.1d 480.9c 96.2
c
4 Paddy straw + Groundnut shell 17.9 4.8b 47.6c 483.9c 96.8
5 Paddy straw + Sugarcane trash 17.0b 4.2a 46.0b 489.3b 97.9
6 Paddy straw + Water hyacinth 16.7a 4.0a 45.7a 498.9a 99.8
b
7 Paddy straw 17.4 4.7b 46.5b 482.6c 96.5
Values not sharing a common superscript differ significantly at P<0.05 (DMRT)

Paddy straw substrate was reported to be superior for (Shah et al., 2011) [21]. Besides, addition of water hyacinth
cultivation of oyster mushroom (Karthika and Murugesan, along with paddy straw (1:1) will therefore, reduce the
2015) [11], whereas in the present study maximum yield could mushroom production cost and would also help in recycle the
be obtained by using water hyacinth (WH) in combination with nuisance weed (water hyacinth) in an eco-friendly way.
the paddy straw. The significant enhancement in the yield
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