Current Research in Environmental & Applied Mycology (Journal of Fungal Biology)

8(3): 323–330 (2018) ISSN 2229-2225

[Link] Article
Doi 10.5943/cream/8/3/4
Copyright © Beijing Academy of Agriculture and Forestry Sciences

Mycelia growth performance of Agaricus bisporus in culture media of

composts supplemented with Sesbania sesban straw and phosphate
rock
Rashid HM1, Abed IA2, Owaid MN3,4*
1
State Company for Foodstuff Trading, Ministry of Trade, Ramadi, Anbar 31001, Iraq
2
Desert Studies Center, University of Anbar, the University Campus, Ramadi, Anbar 31001, Iraq
3
Department of Heet Education, General Directorate of Education in Anbar, Ministry of Education, Hit, Anbar 31007,
Iraq
4
Department of Ecology, College of Applied Sciences, University of Anbar, Hit, Anbar 31007, Iraq

Rashid HM, Abed IA, Owaid MN 2018 – Mycelia growth performance of Agaricus bisporus in
culture media of composts supplemented with Sesbania sesban straw and phosphate rock. Current
Research in Environmental & Applied Mycology (Journal of Fungal Biology) 8(3), 323–330,
Doi 10.5943/cream/8/3/4

Abstract
Alternate composts are used to improve button mushroom growth. Egyptian pea (Sesbania
sesban) straw is used for the first time in growing mushrooms. WHS2 medium (30% wheat straw,
45% horse manure, 15% Egyptian pea (Sesbania sesban) straw, 5% CaSO4 and 5% phosphate rock)
is considered best significant (p<0.05) compost extract for mycelial growth rates of Agaricus
bisporus which reached 86.33 mm compared with the control (WH) 80.83 mm after 14 days.
However, the lower growth rates have been recorded 46.50 mm and 53.83 mm on WCS2 and
WCS1 media, respectively. As in the solid media, WHS2 broth is exhibited higher biomass weight
of A. bisporus 4.24 g/L significantly (p<0.05), whereas, lower biomass weights were 1.60 and 1.73
g/L in WCS2 and WCS1 broths, respectively. Generally, Agaricus bisporus C9 (brown strain) has
been growing best than A. bisporus F599 (white strain) as observed in the current study. In
conclusion, using Egyptian pea (S. sesban) extract plus phosphate rock enhances the importance of
the compost with horse manure compared with chicken manure for growing A. bisporus, in vitro.

Key words – Biomass – button mushroom – C:N ratio – decomposing – Egyptian pea

Introduction
Agaricus bisporus, is a famous edible fleshy mushroom, has the first production in the world.
It belongs to genus Agaricus of Basidiomycota. Its common name is button mushroom (Chang &
Miles 2004). It is rich in macro and microelements (Owaid 2015), proteins, polyphenols (Owaid et
al. 2017b), fatty acids and vitamins, thus its fruiting bodies have an important role in nutrition as
healthy fresh foods or useful herbs (Atila et al. 2017) in treating bacterial, candidal (Waithaka et al.
2017), fungal (Kumar & Yadav 2014) and cancer cases (Salih & Al-Mosawy 2010) and it is a
potent antioxidant agent (Al-Jubori et al. 2016, Cardoso et al. 2017) and enhancing
immunostimulatory, antitumor (Zhang et al. 2014) and anti-diabetic activities (Atila et al. 2017).
Recently, A. bisporus fruiting bodies have been used to biosynthesize silver nanoparticles which
enter into medical and industrial applications (Owaid et al. 2017c).
Submitted 9 February 2018, Accepted 22 June 2018, Published 27 June 2018
Corresponding Author: Mustafa Nadhim Owaid – e-mail – mustafanowaid@[Link] 323
 This mushroom has biodegradability of lignocelluloses by excretion of enzymes like laccase
for utilization of substrates as carbon and an energy source (Kabel et al. 2017). The genome of
Agaricus bisporus encodes a limited repertoire of lignin-modifying enzymes compared to wood-
decaying white rot species (Morin et al. 2012). During composting phases, 66% of polysaccharides
of walls were consumed by microbes, and only 17% of the polysaccharides were used during
mushroom production (Iiyama et al. 1994). Thus, composting process is an essential and critical
point for the cultivation of A. bisporus (Vieira & Pecchia 2018).
In Iraq, A. bisporus was cultivated on various composts consisted from Triticum wheat straw,
Oryza rice straw, Zea mays corn stalk (HaoLin et al. 2017), Phoenix dactylifera date-palm trunk
(Hamoodi & Hameed 2013), Helianthus sunflower residues (Muslat et al. 2014) and straw of
Phragmites australis reed plant (Muslat et al. 2011, Owaid et al. 2018) mixed with some
supplements like chicken manure, urea, and gypsum. Sesbania sesban is a species of plant belongs
to the legume family (Fabaceae). Its common name is seseban in Arabic or sesbania or Egyptian
pea or the Egyptian Riverhemp in English. The exact origin of Sesbania sesban is unclear, but it is
widely distributed and cultivated throughout tropical Africa and Asia (Orwa et al. 2009). It has
crude protein 209 g/kg, and tannin contents 31 g/kg (dry weight) (Kiatho 1997). Different parts of
Sesbania sesban are considered for different purposes like weed control, anti-inflammation,
antioxidant and antimicrobial activities (Nigussie & Alemayehu 2013).
The value of nitrogen in Egyptian pea (seasaban) residues and is reputed an unused cellulosic
source for feed of livestock, so it is introduced in the compost formula instead of others carbon and
nitrogen sources as supplements to enhance mycelial growth of button mushroom and reduce cost
of production when it is applied in farms thus this investigation is considered the first attempt to
use Egyptian pea straw with phosphate rock in compost composition of A. bisporus and applied
toward the mycelia growth in vitro.

Materials & Methods

Mushroom strains
Two strains of mushrooms are Agaricus bisporus F599 (white button mushroom) and
Agaricus bisporus C9 (brown button mushroom), obtained from ITALSPAWN, Italy by
Hameediyah Mushroom Farm (HMF), Ramadi, Kilo 18, Anbar, Iraq. They are subcultured on PDA
(Oxoid, England) and used in this investigation.

Chemicals and Organic matters

There are six matters viz.; wheat straw, Egyptian pea (Sesbania sesban, Fig. 1) straw, horse
manure and chicken manure obtained from agricultural fields in Ramadi, Iraq. The powder of
phosphate rock obtained from State Company for Phosphate in Anbar in raw form. This fertilizer
contains macro and microelements viz.; Co, Cr, Ni, Pb, Fe, Zn, Cu, Mn and Cd (Owaid & Abed 2015).
CaSO4 obtained from the local market. As in Table 1, six formulas were prepared after composting
processes achievement as mentioned by Owaid (2009).

Preparing compost extract media

Six compost extracts were used in this work. About 250 g of each the ready compost after
three weeks of composting was extracted in 400 ml D.W, stand for 24 h, then crushed using a
blender for 10 min and filtered twice by the gauze. The produced aqueous extracts were completed
to the volume 500 ml by D.W individually. For broth culture, they are used directly while in case
the solid media, 15 g agar per liter was added to the aqueous extracts to prepare the solid media.
These media were sterilized within Autoclave at temperature 121 °C and pressure 15 psi for 15
min. The solid media were poured in 9 cm Petri dishes whereas the liquid media (50 ml broth) were
poured into 100 ml-volumic flasks separately.
One 7-days mycelial disk put in the center of Petri dish and incubated in the incubator at 25 C
while the one disk put in 50 ml broth and incubated at the same temperature. PDA and PDB were

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used as a control. The measurements are mycelial growth rate after 14 days and the biomass weight
after 30 days was recorded and statistical analysis applied by two-way analysis of variance using
CRD (Completely Randomized Design) by GenStat software at a probability less than 5%.

Fig. 1 – Straw of Egyptian pea (Sesbania sesban)

Table 1 Compositions of composts and its carbon:nitrogen (C:N) ratio (On the basis of dry weight)

Wheat Horse Chicken Egyptian Phosphat

Treatments CaSO4 C:N ratio
straw manure manure pea straw e rock
WH 45% 45% - - 5% 5% 42.5:1
WHS1 40% 20% - 30% 5% 5% 38.8:1
WHS2 30% 45% - 15% 5% 5% 35.8:1
WC 45% - 45% - 5% 5% 37.5:1
WCS1 40% - 20% 30% 5% 5% 36.6:1
WCS2 30% - 45% 15% 5% 5% 30.8:1
Legend: C:N ratio of Wheat straw: 60:1, horse manure: 25:1, chicken manure: 15:1, Egyptian pea (Sesbania
sesban) straw: 20:1.

Characteristics of extracts
Take 1 g of the dried powder of each formula (compost) individually as in Table 1, mix with
5 ml D.W and put in a shaker for 1 hr. Use these aqueous extracts to determine pH and EC (Owaid
et al. 2017a). Also, C:N ratios were calculated after assessing carbon content as in method of Page
(1982) and nitrogen content by Kjeldahl method using Gallenkamp Kjeldahl Apparatus (Sawhney
& Singh 2000)

Results and Discussion

Properties of compost extracts

Properties of compost extracts are including pH, EC, nitrogen content, and carbon:nitrogen
(C:N) ratio as shown in Table 2. The pH of extracts was alkaline close to neutral which ranged
from 7.24 to 7.75. C:N ratio of extracts ranged from 12.6 to 24.1. WCS1 extract exhibited lower
C:N ratio (12.6) and higher nitrogen content (2.7%) compared with the control, WH medium which
showed higher C:N ratio (24.1) and lower nitrogen content (1.63%) respectively. This phenomenon
appears because of the compositions of WCS1 compost which have a high percentage of Egyptian
pea (Sesbania sesban) straw which is rich in nitrogen content compared with the wheat straw
(Table 1). This straw leads to the best microbial growth that reflects on biomass of compost and
finally on the nitrogen content in WCS1 and WHS1 composts compared with the control during
composting processes (Vos et al. 2017).

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 The neutral values of pH in those composts may be returned to using CaSO4 with phosphate
rock, and this is suitable for button mushroom growth and close to the optimal pH (Royse 2008). At
the same time, this fertilizer, phosphate rock, is considered the main source for phosphor element
which is regarded an important source for a microbial community in the compost through the
composting processes (Abed & Rasheed 2013). Thus, the phosphate rock was used to increase
production of some mushrooms like A. bisporus (Abed & Rasheed 2013) and Pleurotus spp.
(Owaid et al. 2015). The role of phosphate-rock-solubilizing bacteria and fungi in compost is
important for increasing phosphate rock solubility after mixing with cellulosic wastes during
composting processes (Al-Tae'e 2008, Faraj 2014).
Also, the value of C:N (carbon:nitrogen) ratio is a benefit for production this type of
mushroom which needs this feature a ratio of 20:1 as an optimal value which comes from the
degradation of bacterial biomass through the composting processes outdoor (Buscot & Varma
2005). The higher electrical conduction (EC) showed 17.67 ds/m in WCS1 compost followed 15.08
and 14.74 ds/m WCS2 and WC composts respectively compared with 9.85, 11.22 and 11.86 ds/m
for WH, WHS2 and WHS1 composts respectively that may be return to increase EC and TDS in
chicken manure compared as horse manure.

Table 2 Physical and chemical characteristics of aqueous extracts of composts

Features WH WHS1 WHS2 WC WCS1 WCS2 LSD

(p<0.05)
pH 7.67 7.73 7.75 7.64 7.58 7.24 0.02
EC (ds/m) 9.58 11.86 11.22 14.74 17.67 15.08 0.09
Nitrogen content % 1.63 2.5 2 2.3 2.7 2.6 0.15
C:N ratio 24.1 14.8 16.9 15.9 12.6 12.7 1.29
Legend: EC: electrical conductivity, WH compost (control): 45% wheat straw, 45% horse manure, 5% CaSO4 and 5%
phosphate rock. WHS1 compost: 40% wheat straw, 20% horse manure, 30% Egyptian pea (Sesbania sesban) straw, 5%
CaSO4 and 5% phosphate rock. WHS2 compost: 30% wheat straw, 45% horse manure, 15% Egyptian pea (Sesbania
sesban) straw, 5% CaSO4 and 5% phosphate rock, WC compost: 45% wheat straw, 45% chicken manure, 5% CaSO4
and 5% phosphate rock. WCS1 compost: 40% wheat straw, 20% chicken manure, 30% Egyptian pea (Sesbania sesban)
straw, 5% CaSO4 and 5% phosphate rock. WCS2 compost: 30% wheat straw, 45% chicken manure, 15% Egyptian pea
(Sesbania sesban) straw, 5% CaSO4 and 5% phosphate rock.

Mycelial growth of A. bisporus in the solid media of compost extracts

Generally, Agaricus bisporus C9 (brown strain) is considered the best mushroom than A.
bisporus F599 (white strain), Table 3, Fig. 2. This is relating to genetic characteristics of A.
bisporus that have been limited so far (Hildén et al. 2013). Also, those results show that type of
compost extract affected significantly (p<0.05) on mycelial growth of A. bisporus F599 and A.
bisporus C9. The medium WHS2 shows best growth reaches 88.00 mm and 84.66 mm, followed by
82.33 mm and 79.33 mm in WH medium for A. bisporus C9 and A. bisporus F599 respectively
after 14 days. A. bisporus C9 and A. bisporus F599 exhibit mycelial growth 68.33 mm and 64.33
mm in WC medium and 66.33 mm and 63.33 mm in WHS1 medium respectively. The lower
growth rate was recorded 44.00 mm and 49.00 mm in WCS2 medium, followed 52.00 mm and
55.66 mm in WSC1 medium for A. bisporus F599 and A. bisporus C9 respectively. The differences
among composts due to different specifications of pH, EC, and carbon:nitrogen ratio (Table 2).
The medium WHS2 showed best mycelial growth because it has optimal nitrogen content 2%
compared with the higher nitrogen content of WCS1 and WCS2 media reached to 2.7% and 2.6%
respectively (Table 2) which negatively reflected on mycelial growth rate in WCS1 and WCS2
media because that lead to dispersed colonies in the medium (Calvo 2010) as in Fig. 2.
Also, higher values of EC in the media of WC, WCS1, and WCS2 (containing horse manure)
may be slightly affected on the growth in vitro (Table 2) compared with WH, WHS1 and WHS2
media (containing chicken manure). Generally, the finding horse manure was suitable more than
chicken manure in this test that agrees with many studies in button mushroom cultivation. But in
the mushroom farm, usually, electrical conductivity (EC) did not influence on the growth of many

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types of mushroom in the mushroom farms thus values of EC cannot influence on this investigation
(Royse 2008).

Effect of compost type on the mycelial growth rate

According to Table 3, generally, WHS2 medium is considered best significant (p<0.05)
compost extract for growth rates of A. bisporus which recorded 86.33 mm after 14 days, compared
with the control (WH) 80.83 mm. However, the lower growth rate was 46.50 mm and 53.83 mm on
WCS2 and WCS1 media respectively.
WH and WC media are superior due to the difference of carbon sources as through large
percentages of various lignocellulosic residues. The availability or scarcity of carbon and type of its
sources determine the rate of mycelial growth of mushroom, development, and appearance of A.
bisporus (Calvo 2010). The high content of nitrogen in the compost extracts influences on mycelial
growth negatively (Royse 2008) as shown in other media (WHS1, WHS2, WCS1, and WCS2).
WHS2 medium is regarded the best formula/compost in case of mycelial growth rate that
might return to its adequate properties of pH, EC, and C:N ratio. Subsequently, it improves
conditions of the microbial growth in the compost during decomposing stages (Owaid 2009).

Table 3 Mycelial growth rate of A. bisporus on the solid media of different compost extracts after
14 days

Mushroom Strains WH WHS1 WHS2 WC WCS1 WCS2 Mean

A. bisporus F599 79.33 63.33 84.66 64.33 52.00 44.00 64.60
A. bisporus C9 82.33 66.33 88.00 68.33 55.66 49.00 68.27
Mean of media 80.83 64.83 86.33 66.33 53.83 46.5 66.44
LSD(p<0.05) 15.4 0.51
Legend: WH compost (control): 45% wheat straw, 45% horse manure, 5% CaSO4 and 5% phosphate rock. WHS1
compost: 40% wheat straw, 20% horse manure, 30% Egyptian pea (Sesbania sesban) straw, 5% CaSO4 and 5%
phosphate rock. WHS2 compost: 30% wheat straw, 45% horse manure, 15% Egyptian pea (Sesbania sesban) straw, 5%
CaSO4 and 5% phosphate rock, WC compost: 45% wheat straw, 45% chicken manure, 5% CaSO4 and 5% phosphate
rock. WCS1 compost: 40% wheat straw, 20% chicken manure, 30% Egyptian pea (Sesbania sesban) straw, 5% CaSO4
and 5% phosphate rock. WCS2 compost: 30% wheat straw, 45% chicken manure, 15% Egyptian pea (Sesbania sesban)
straw, 5% CaSO4 and 5% phosphate rock.

Biomass weight of Agaricus bisporus in broth of compost extracts

It is difficult to determine the suitability of the solid medium for the biomass weight because
it is not possible to measure the growth density, so the biomass measurement is used in the liquid
medium (broth). In general, A. bisporus C9 recorded best significant (p<0.05) biomass weight (2.75
g/L) compared with A. bisporus F559 (2.63 g/L) in liquid media of compost extracts after 30 days.
Table 3 shows that WHS2 broth significantly (p<0.05) recorded the best result of formation
Agaricus bisporus biomass reached to 4.36 g/L and 4.13 g/L for A. bisporus C9 and A. bisporus
F599 respectively, that return to the adequate C:N ratio in those composts which determine the rate
of mycelial growth of A. bisporus (Calvo 2010). Next, WHS1 exhibits biomass weight of 3.50 g/L
and 3.42 g/L, followed by 2.69 g/L and 2.57 g/L in WC broth compared with the control (WH
broth) 2.50 g/L and 2.45 g/L for A. bisporus C9 and A. bisporus F599, respectively. While WCS1
and WCS2 recorded lower biomass reached to 1.56 g/L and 1.66 g/L for A. bisporus F599 and then
1.65 g/L and 1.81 g/L for A. bisporus C9 respectively. WHS2 compost is suitable for growth this
mushroom in solid and liquid media of compost due to its characteristics which let for performance
best growths (Tables 3 & 4).

Effect of compost type on the biomass of mushroom in broths

In the broths as in the solid media, WHS2 broth is exhibited best biomass weight of A.
bisporus 4.24 g/L significantly (p<0.05), followed rates 3.46 and 2.63 g/L by WHS1 and WC
broths respectively. Furthermore, the lower biomass weight was 1.60 and 1.73 g/L on WCS2 and
WCS1 broths respectively as shown in Table 4. WHS2 broth is regarded the best medium to

327
synthesis the fungal biomass that might return to the suitable properties of this compost (Table 2).
Indeed, using Egyptian pea (Sesbania sesban) straw reflects on improving nitrogen content of
WHS2 compost which consists of 30% wheat straw, 45% horse manure, 15% Egyptian pea straw,
5% CaSO4 and 5% phosphate rock (dry matter basis).
Also, WHS1 and WHS2 broths exhibited best mushroom biomass because of their suitable
nitrogen content or C:N ratio compared to other broths and the control. The suitability these
composts extracts may be returned to nitrogen content which obtained from microbial community
biomass during composting phases at optimal condition because of constituents of these formulas
(Vieira & Pecchia 2018). This investigative work agrees with other studies and agriculture reports
in the button mushroom farms globally.

Fig 2 – Pattern of mycelial growth of A. bisporus after 14 days

Legend: WH compost (control): 45% wheat straw, 45% horse manure, 5% CaSO4 and 5% phosphate rock. WHS1
compost: 40% wheat straw, 20% horse manure, 30% Egyptian pea (Sesbania sesban) straw, 5% CaSO4 and 5%
phosphate rock. WHS2 compost: 30% wheat straw, 45% horse manure, 15% Egyptian pea (Sesbania sesban) straw, 5%
CaSO4 and 5% phosphate rock, WC compost: 45% wheat straw, 45% chicken manure, 5% CaSO4 and 5% Phosphate
rock. WCS1 compost: 40% wheat straw, 20% chicken manure, 30% Egyptian pea (Sesbania sesban) straw, 5% CaSO4
and 5% phosphate rock. WCS2 compost: 30% wheat straw, 45% chicken manure, 15% Egyptian pea (Sesbania sesban)
straw, 5% CaSO4 and 5% phosphate rock.

Table 4 Biomass weights of A. bisporus in the broth of various compost extracts after 30 days

Mushroom Strains WH WHS1 WHS2 WC WCS1 WCS2 Mean

A. bisporus F599 2.45 3.42 4.13 2.57 1.56 1.66 2.63
A. bisporus C9 2.50 3.50 4.36 2.69 1.65 1.81 2.75
Mean of media 2.48 3.46 4.24 2.63 1.60 1.73 2.69
LSD (p<0.05) 0.04 0.01
Legend: WH compost (control): 45% wheat straw, 45% horse manure, 5% CaSO4 and 5% phosphate rock. WHS1
compost: 40% wheat straw, 20% horse manure, 30% Egyptian pea (Sesbania sesban) straw, 5% CaSO4 and 5%
phosphate rock. WHS2 compost: 30% wheat straw, 45% horse manure, 15% Egyptian pea (Sesbania sesban) straw, 5%
CaSO4 and 5% phosphate rock, WC compost: 45% wheat straw, 45% chicken manure, 5% CaSO4 and 5% phosphate
rock. WCS1 compost: 40% wheat straw, 20% chicken manure, 30% Egyptian pea (Sesbania sesban) straw, 5% CaSO4
and 5% phosphate rock. WCS2 compost: 30% wheat straw, 45% chicken manure, 15% Egyptian pea (Sesbania sesban)
straw, 5% CaSO4 and 5% phosphate rock.

Conclusion
WHS2 extract showed significant (p<0.05) growth in the solid and liquid media compared
with the control. In solid media, Agaricus bisporus recorded best growth rate on WHS2 medium

328
(30% wheat straw, 45% horse manure, 15% Egyptian pea (Sesbania sesban) straw, 5% CaSO4 and
5% phosphate rock) is 86.33 mm after 14 days, compared with 80.83 mm by the control (WH, 45%
wheat straw, 45% horse manure, 5% CaSO4 and 5% phosphate rock). Also, WHS2 broth is
exhibited best biomass weight of A. bisporus 4.24 g/L significantly (p<0.05). Finally, Agaricus
bisporus C9 (brown strain) is considered the best mushroom than A. bisporus F599 (white strain),
generally. In conclusion, using Egyptian pea (Sesbania sesban) extract with phosphate rock
enhances the compost property.

Acknowledgement
Authors are thanking Hameediyah Mushroom Farm (HMF), Ramadi, Kilo 18 for obtaining
spawn of mushrooms, and they are thanking College of Agriculture, University of Anbar for
achievement this work.

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