Indian Phytopath.

67 (4) : 412-417 (2014)

RESEARCH ARTICLE

Molecular characterization of Agaricus bisporus strains and

their evaluation for production efficiency

VIJAY KUMAR* and B.C. SUMAN

Mushroom Research Laboratory, Department of Plant Pathology, Dr. Y.S. Parmar University of Horticulture and Forestry, Nauni,
Solan 173 230, Himachal Pradesh, India

ABSTRACT: Ten strains of Agaricus bisporus viz. DMRA-7, DMRA-9, DMRA-14, DMRA-37, DMRA-102, DMRA-103, DMRA-
104, DMRA-121, DMRA-122 and U3, procured from different sources were evaluated for production efficiency. DMRA-7,
recorded highest average yield of 20 kg per 100 kg compost followed by DMRA-37 (19.17 kg) and DMRA-102 (18.67 kg)
based on the average yield of two crops. Strain DMRA-7, in addition to being high yielding also exhibited better morphological
quality traits followed by strains DMRA-37, DMRA-102 and U3. The studies on production efficiency have been further
supported by the results obtained from RAPD-PCR technology. Studies revealed that eight unique markers viz. OPP-3800,
OPP-6700, OPP-6200, OPP-7900,OPP-7700, OPP-9500, OPP-19100 and OPP-19700 have been identified for strain DMRA-7, while only
one unique marker OPP-10800 has been identified for control (U3). Sixteen primers amplified 57 RAPD markers, of which
67.92 per cent were polymorphic. Similarity coefficient obtained from RAPD analysis revealed that similarity among all the
selected strains ranged between 45 to 76 per cent with an average of 60 per cent.

Key words: Agaricus bisporus, polymorphism, RAPD, strains

The production of white button mushroom, Agaricus
bisporus (Lange) Imbach has immense potential in
Himachl Pradesh due to the availability of abundant raw
material and optimum temperature (15-20°C) for its
cultivation under natural conditions during winter season.
In India, since the introduction of white button mushroom
in early sixties, cultivation has been carried out but not
much attention has been paid to strain development and
improvement work. It is well known fact that yield
potentiality of different strains is governed by the
genotype of strains, growing conditions and technology
of cultivation. In the procedure of the evaluation of strains,
favoring one character over another is often a
compromise, since both favorable and unfavorable
characteristics usually are present in the same strain.
Hence, there is always a possibility of obtaining a few
high yielding strains suitable to our conditions and
selection thereof, because of the obvious reason that
strains have different environment optima (Kneebone,
1968; Tschierpe, 1972). The procurement and selection
of new strains in Agaricus bisporus is a continuous
process and the easiest approach to better production,
since improvement at the genetic level has been
restricted mainly because of the homothallic nature of
fungus (Elliot, 1985). Moreover, it possesses
multinucleate hyphal cells and in its life cycle true haploid
stage is almost absent. Thus, strain improvement
programme in Agarcus bisporus , so far has been
achieved mainly by traditional selection methods followed
by production trials. However, reliable estimate of
variation and genetic relationship are pre-requisites for
effective utilization of genetic resources for crop
improvement programme. DNA markers are quick and
*Corresponding author: vnarwal777@yahoo.com
reliable for genetic differentiation of strains within species.
Many workers have used RAPD markers for assessment
of genetic diversity in wild and cultivated strains of A.
bisporus for strain improvement programme (Suman et
al., 2010). In this context, the present investigation on,
molecular characterization of Agaricus bisporus strains
was undertaken.
MATERIALS AND METHODS
Mushroom strains
Ten strains of A. bisporus used in the present study were
procured from different resources DMRA-7, DMRA-9,
DMRA-14, DMRA-37, DMRA-102, DMRA-103, DMRA-
104, DMRA-121, DMRA-122 from Directorate of
Mushroom Research and U3 from university lab.
Mushroom cultivation
The mushroom cultivation experiment was conduced
under controlled conditions in the mushroom house at
Dr. Y.S. Parmar University of Horticulture and Forestry,
Nauni for two seasons. The spawn of each strain was
prepared on wheat grains as per standard procedure
described by Fritsche (1981) and Suman (1989).
Synthetic compost prepared by shor t method of
composting was used. For each strain twenty five bags
of 10 kg capacity ready compost were used and spawed
at the rate of 0.8 per cent. After completion of spawn
run, the bags were cased with casing mixture of well
rotten farm yard manure and garden soil (2:1 w/w) upto
a depth of 4.0 cm. during cropping all the cultural
practices were followed (Munjal and Seth, 1980).
Mushroom from each bag were picked, cut at soil level
and weighed daily for 60 days. Observations on other
Indian Phytopathology 67 (4) : 412-417 (2014) 413

morphological quality traits such as average pileus Table 1. Yield of various strains of Agaricus bisporus by short
diameter, stipe length and weight of fruit bodies. The data method of composting (Kg/100 kg compost)
recorded are of two crops and analyzed statistically. Strains First Second Mean Yield
Flush Flush (Kg/100kg
Molecular characterization of Agaricus bisporus compost)

DMRA-7 20.15 19.86 20.00

DNA extraction and RAPD analysis
DMRA-9 9.17 9.87 9.51
For molecular characterization, DNA was isolated using DMRA-14 12.53 12.47 12.50
the CTAB method (Murray and Thompson, 1980) with DMRA-37 19.10 19.23 19.17
some modifications. Purified DNA was run on a 0.7% DMRA-102 18.43 18.90 18.67
agarose gel with diluted uncut lambda DNA (25 ng/µl) DMRA-103 14.43 14.50 14.47
as standard to assay its concentration and integrity by
DMRA-104 15.23 15.00 15.11
ethidium bromide fluorescence. DNA was also quantified
DMRA-121 13.00 13.10 13.05
with UV/VIS spectrophotometer (BIORAD Smart specTM
DMRA-122 11.07 11.27 11.17
3000). The quantified DNA samples were diluted in TE
U3(control) 18.23 18.33 18.28
buffer to make a final concentration of 50 ng/µl for PCR
reactions. RAPD reactions were performed according to Mean 15.13 15.25
the protocol of Williams et al. (1990) with the modifications Effect CD (0.05)
to enhance reproducibility and consistency of RAPD Yield (Y) 0.08
profiles. RAPD amplifications were performed in 25 µl of Number of crops (NC) 0.03
reaction volume containing 1X PCR buffer (containing
Y × NC 0.11
15 mM MgCl2), 2.5 mM each dNTP (Sigma, USA), 100
pmol of 20 random decamer primers (Operon
Technologies Inc, USA ), 1 unit of Taq DNA polymerase
recorded in strain DMRA-9, followed by strains DMRA-
(Banglore Genei, India) and 50 ng genomic DNA
122 (11.17 kg), DMRA-14 (12.50 kg), DMRA-121 (13.05
template. PCR amplifications were performed in a
kg), DMRA-103 (14.47 kg) and DMRA-104 (15.11 kg)
thermal cycler (Perkin Elmer Thermal Cycler GeneAmp®
which differed significantly among themselves.
PCR System 9700 Model) with heated lid technology for
45 cycles. The PCR conditions were: initial denaturation Morphological quality characters
of genomic DNA at 95°C for 4 min followed by 45 cycles
of DNA template denaturation at 94°C for 1 min, primer The morphological traits of closed fruit bodies of various
annealing at 32°C for 1 min, DNA amplification at 72°C test strains were assessed up to third flush with two
for 2 min and final primer extension at 72°C for 8 min. observations in each flush. The scores for various
Amplicons were separated on a 1.5% agarose gel properties are based on the average of 20 observations
prestained with ethidium bromide solution using 1 X TAE and range from 1 (soft, discolored, scaly, poorly shaped)
buffer. The gels were run for 2 h at 60 V and the RAPD to 5 (firm, white, smooth and round shaped). From the
amplicon profiles were recorded using Syngene Gel data recorded in table 3, it is clear that maximum average
Documentation System with GeneSnap software. The weight of a fruit body was recorded in strain DMRA-7
size of the amplified fragments was determined (11.56 g). However minimum average weight was
using1000 bp plus ladder (MBI Fermentas, Lithuania) recorded in strain DMRA-9 (7.16 g). Strain DMRA-7,
and Gene- Tools software. All RAPD reactions were recorded fruit bodies with significantly maximum (4.86
performed twice to test the reproducibility of the amplicon cm) pileus diameter. Minimum pileus diameter was
profiles. recorded in DMRA-9 (3.00 cm). Similarly maximum
average stipe length was recorded in strain DMRA-103
RESULTS AND DISCUSSION (2.60 cm). Minimum average stipe length was recorded
in DMRA-122 (1.96 cm). Maximum average stipe
Evaluation of various strains of Agaricus bisporus in diameter (1.82 cm) was recorded in strain DMRA-7,
term of production efficiency followed by strain DMRA-37 (1.71 cm) and minimum
average stipe diameter was observed in strain DMRA-9
The average yield of twenty five bags was recorded in (1.20 cm). Fruit bodies of strains DMRA-7 and DMRA-
kg/100kg compost and the data is presented in table 1. 37 were round in shape with slightly raised caps at the
There was a highly significant difference among the centre which was also exhibited by control (U3). While
strains as far as yield was concerned. Maximum (20.00 the fruit bodies of strain DMRA-9 was somewhat
kg/100kg compost) yield was recorded in strain DMRA- flattened. Overall, strain DMRA-7 in addition to being high
7, followed by strain DMRA-37 (19.17 kg), DMRA-102 yielding also exhibited better morphological qualities
(18.67 kg) and U3 (18.28 kg) which differed significantly traits followed by strains DMRA-37, DMRA-102 and U3
among themselves. The strains viz. DMRA-7, DMRA- 37 (control). Strain DMRA-9 which recorded lowest yield of
and DMRA-102 were superior to all other strains of A. 9.51 Kg/100 Kg compost, also lacked many of the quality
bisporus , including control (U3) as far as yield is traits such as toughness, colour and smoothness with
concerned. Minimum (9.51 kg) average yield was poorly shaped fruit bodies.
414 Indian Phytopathology 67 (4) : 412-417 (2014)

Table 2. Morphological qualities traits/characters of different test strains of Agaricus bisporus

Strain Remark on First Av. yield of Av. pileus Av. stipe Av. stipe Av. wt of Judgement of closed
mycelium harvest mushroom dia. (cm) length diameter fresh fruit bodies (pileus)
growth in in days in kg/100kg (cm) (cm) mushroom
bed compost (g) Tough- Shape Smooth Colour
ness -ness

DMRA-7 Very Fast 33.06 20.00 4.86 2.30 1.82 11.56 4.92 5.00 5.00 5.00
DMRA-9 Very slow 40.06 9.51 3.00 2.00 1.20 7.16 4.30 4.50 3.90 3.50
DMRA-14 medium 35.30 12.50 3.06 2.21 1.30 9.73 4.75 4.93 3.90 4.94
DMRA-37 Fast 34.36 19.17 4.61 2.44 1.71 10.73 4.93 5.00 4.91 5.00
DMRA-102 Fast 33.86 18.67 4.50 2.48 1.61 10.93 4.87 4.80 4.88 4.87
DMRA-103 Medium 35.83 14.47 3.66 2.60 1.40 8.16 4.45 4.85 3.87 4.82
DMRA-104 Slow 36.00 15.11 4.00 2.53 1.39 7.53 4.79 4.90 4.11 4.90
DMRA-121 Fast 34.00 13.05 3.43 2.32 1.43 9.06 4.50 4.73 4.31 4.77
DMRA-122 Slow 37.00 11.17 3.20 1.96 1.48 7.73 3.16 4.60 3.99 4.45
U3(control) Fast 34.83 18.28 4.43 2.52 1.56 10.40 4.92 5.00 4.85 5.00
C.D. (0.05) 0.12 0.08 0.08 0.04 0.04 0.19 0.03 0.01 0.03 0.01
1 (Soft, discoulred, scaly, poorly shaped) to 5 (Firm, white, smooth and round shaped)

Molecular characterization of high yielding strains
Genetic diversity was studied among the four strains
including control (U3) (which gave higher yield than
control) using RAPD-PCR technology. Twenty random
oligonucleotide primers (Operon Technologies) were
used and only sixteen primers were able to amplify the
genomic DNA successfully. Total number of RAPD bands
varied from 1-6 with minimum of 1 band in OPP-1, OPP-
10 and OPP-12, and maximum of 6 bands in OPP-3 and
OPP-7. Total number of polymorphic bands varied from
0 to 6 (Fig. 1 & 2), while, total number of monomorphic
bands varied from 0 to 4 (Table 3).
The similarity coefficient revealed that the similarity
among all the selected strains including control ranged

Fig. 1. RAPD profiles of control (U3) and DMRA-7. Circles indicate informative bands
Indian Phytopathology 67 (4) : 412-417 (2014) 415

Fig. 2. RAPD profiles of DMRA-37 and DMRA-102. Circles indicate informative bands

Table 3. Scorable DNA Bands generated by using 16 decamer primers through PCR

Primers Total number of Total number of Total number of Size range of

bands amplified monomorphic bands polymorphic amplified product
amplified bands amplified (bp)

OPP-1 1 1 0 400
OPP-2 4 4 0 200-1100
OPP-3 6 0 6 100-1500
OPP-5 4 4 0 200-500
OPP-6 4 0 4 200-1000
OPP-7 6 0 6 200-1000
OPP-8 3 2 1 400-1100
OPP-9 3 0 3 200-600
OPP-10 1 0 1 800
OPP-12 1 0 1 600
OPP-13 3 0 3 700-900
OPP-14 4 4 0 200-1200
OPP-15 3 0 3 100-900
OPP-16 4 0 4 200-800
OPP-17 3 0 3 200-600
OPP-19 4 2 2 300-1000
416
0.45 0.52 0.60
Coefficient
Fig. 3. Genetic relatedness among selected strains DMRA-7, DMRA-37, DMRA-102 and control (U3) of Agaricus bisporus based on
combined analysis data generated by fifteen random primers
between 45 to 76 per cent, with an average of 60 per
cent. The dendrogram divided four genotypes of Agaricus
bisporus into two different clusters (Fig. 3). First cluster
consisted of strains DMRA-37, control (U3) and DMRA-
102 of A. bisporus. The second cluster consisted of one
strain DMRA-7. However, among all the combinations,
maximum similarity was found between strains DMRA-
37 and controls (U3) (76 per cent), while strains DMRA-
37, control (U3) and DMRA-102 were 69 percent similar
to each other. However, minimum similarity was found in
strain DMRA-7 (45 per cent) with other selected strains,
including control (U3).
It is clear from the yield data of two crops grown on
synthetic compost based on short method of composting,
that strain DMRA-7, produced highest (20.00 kg/100kg
compost) average yield, followed by strains DMRA-37
(19.17 kg), DMRA-102 (18.67 kg) , control (U3) (18.28
kg), DMRA-104 (15.11 kg), DMRA-103 (14.47 kg),
DMRA-121 (13.05 kg), DMRA-14 (12.50 kg), DMRA-122
(11.17 kg) and DMRA-9 (9.51 kg), under the same
environmental conditions. Strain DMRA-7 gave the
maximum average yield of mushrooms in sixty days of
cropping period.
As far as the morphological characteristics and
quality of fruit bodies produced by the different test strains
are concerned, they exhibited different morphological
qualities in addition to their yield. In general, among all
the strains, including control, strain DMRA-7 exhibited
best morphological qualities, while other strains gave one
or more acceptable qualities, but failed to exhibit all the
best qualities. Strain DMRA-9 which recorded lowest
yield, also lacked many of the quality traits. There were
significant variations in the morphological traits of various
strains, like stipe length, stipe diameter, pileus diameter,
colour, toughtness and average weight of fruit body.
The yield studies have been further supported by
the results obtained from RAPD-PCR technology.
Indian Phytopathology 67 (4) : 412-417 (2014)
DMRA-37
U3
DMRA-102
DMRA-7
0.68 0.76
According to this study eight unique markers OPP-3800,
OPP-6700, OPP-6200, OPP-7900, OPP-7700, OPP-9500, OPP-
19 100 and OPP-19 700 have been identified for strain
DMRA-7. Four unique markers OPP-7300, OPP-13900,
OPP-13800 and OPP-13700 have been identified for strain
DMRA-37 while, four unique markers has been identified
for strain DMRA-102 viz. OPP-6900, OPP-81100, OPP-15900
and OPP-17300 and only one unique marker OPP-10800
has been identified for control (U3). Thus, these unique
markers (RAPD markers) can be used for identification
of these strains. Somewhat, similar results were given
by Chiu et al. (1996) who characterized 19 strains of A.
bisporus by three arbitrarily primed PCR profile, seven
RAPD profiles and five RFLP pattern of PCR amplified
rDNAs and Yadav (2003) who successfully used RAPD
markers for the selection of single spore isolates and
two hybrid strain which were superior to parent strain.
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Received for publication: February 17, 2014
Accepted for publication: June 30, 2014