The Pharma Innovation Journal 2023; 12(10): 2110-2113

ISSN (E): 2277-7695

ISSN (P): 2349-8242
NAAS Rating: 5.23 Studies on the culture media requirements,
TPI 2023; 12(10): 2110-2113
© 2023 TPI temperature and pH on the vegetative growth of
www.thepharmajournal.com
Received: 15-08-2023 Shiitake mushroom (Lentinula edodes (Berk.) Pegler)
Accepted: 22-09-2023

Dr. Deepa Rani CV Dr. Deepa Rani CV, Dr. Safia NE, Ashitha MR and Lulu Das
Assistant Professor, Department
of Plant Pathology, Krishi
Vigyan Kendra, Wayanad, Abstract
Ambalavayal, Kerala, India Shiitake mushroom (Lentinula edodes) is one of the most important culinary and medicinal mushroom
having anticancerous properties in the world. Traditionally, Shiitake has been cultivated on oak logs but
Dr. Safia NE recently there is a trend to cultivate it on sterilized or pasteurized substrates in order to increase yield and
Senior Scientist and Head, Krishi to reduce the time of its growth cycle. Mushroom cultivation has received little attention in most
Vigyan Kendra, Wayanad, developing countries where millions of tons of lignocellulose rich wastes are unused. The present work
Ambalavayal, Kerala, India aimed to determine the ideal culture medium, optimum temperature and pH for the invitro vegetative
growth of Shiitake mushroom. The mushroom was found to grow best in Malt extract peptone dextrose
Ashitha MR
agar medium followed by oat meal agar at an optimum temperature of 20 ºC and pH of 6.00.
Assistant Professor, Department
of Horticulture, Krishi Vigyan
Kendra, Wayanad, Keywords: Lentinula edodes, media, pH, temperature
Ambalavayal, Kerala, India
1. Introduction
Lulu Das Mushrooms are one of the most promising sources of functional food, drug, dietary
Professor, College of Agriculture,
supplements, healthy beverages etc. Lentinula edodes (Berk.) Pegler commonly known as
Vellayani, Kerala, India
Shiitake mushroom, is one of the most widely grown species of mushrooms and a very
efficient biodegrader. The fungus was first described scientifically as Agaricus edodes by
Miles Joseph Berkeley (1877) [1]. It was placed in the genus Lentinula by David Pegler (1975)
[5]
. The mushroom is called ‘elixir of life’ capable of generating stamina, curing colds,
improving circulation and preventing premature ageing. Shiitake is known to contain proteins,
lipids, carbohydrates, fibres, minerals, Vitamin B1, B2, C, D, provitamin, Vitamin E and
Selenium. It is a rich source of antioxidants. The mushroom is used as anticarcinogenic, anti-
inflammatory, antifungal, antibacterial, antiviral and anti-cardiovascular disorders. Mushroom
is well known for unique taste and aroma. Moreover it is a highly priced mushroom. In India,
Shiitake cultivation is mainly reported from North Eastern regions of Meghalaya, Solan,
Himachal Pradesh, Rajasthan etc. where the climatic conditions suited to the mushroom
prevails. There is a vast scope of mushroom cultivation in Kerala where agricultural
byproducts like sawdust, paddy straw and wood shavings of hardwood trees are available in
plenty. The present work was conducted with an objective to study the different culture media
and physiological attributes that enhance the vegetative growth of Shiitake mushroom.

2. Materials and Methods

2.1 Effect of different solid media on the mycelial growth of Lentinula edodes
The experiments were carried out with all the six strains of L. edodes in completely
randomised design (CRD) on seven different media. The growth of L. edodes were evaluated
in terms of radial mycelial growth (cm) to find out the best medium for the growth of the
fungus.
1. Potato dextrose agar (PDA)
2. Oat meal agar (OMA)
3. Malt extract agar (MEA)
4. Malt extract peptone dextrose agar (MEPDA)
Corresponding Author: 5. Carrot agar (CA)
Dr. Deepa Rani CV 6. Czapek’s Dox agar (CDA)
Assistant Professor, Department 7. Yeast extract agar (YEA) Kaur and Lakhanpal (1995) [4].
of Plant Pathology, Krishi
Vigyan Kendra, Wayanad,
Ambalavayal, Kerala, India
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Each of the medium was prepared and transferred to 250 ml Sterilization was done by autoclaving at 15 lbs pressure and
conical flasks at the rate of 150 ml per flask and plugged 121 °C for twenty minutes. After cooling, the media was
tightly with cotton plugs. The flasks were sterilized in an poured into sterile petriplates and allowed to solidify. All the
autoclave at 15 psi pressure and 121 °C for 20 minute. The six strains of L. edodes were used for the experiment. Seven
molten media were poured into sterile petriplates of nine cm day old culture disc of nine mm dia. of all the six strains of L.
diameter and allowed to solidify. The mycelial disc of nine edodes were inoculated at the centre of the dishes that were
mm diameter from seven day old culture of each strain was then sealed with parafilm and incubated at room temperature.
inoculated at the centre of the dish. The dishes were properly (28±2 ºC). Three replications were maintained for each
labelled, sealed with parafilm and incubated at room treatment in CRD. Measurements of radial growth of each
temperature (28±2 oC). Three replications were maintained strain of L. edodes was taken when the mycelial growth of
for each treatment. Nature of mycelial growth and colony any of the tested strains completely covered the entire
diameter, were recorded for each strain. Observations were petriplate.
taken till the mycelial growth covered the entire petriplate. Malt extract peptone dextrose broth was prepared with
different pH concentration as given above. Fifty ml of the
2.2 Effect of different liquid media on the mycelial growth media was taken in 100 ml conical flask and autoclaved at 15
of Lentinula edodes lbs pressure and 121 °C for twenty minutes. The medium
The different liquid media viz., potato dextrose broth, oat were then inoculated with nine mm disc of seven day old
meal broth, malt extract broth, malt extract peptone dextrose culture of Lentinula edodes strains and incubated at room
broth, carrot broth, Czapek’s Dox broth and yeast extract temperature. (28±2 ºC) for 25 days. The mycelial mat was
broth were evaluated to find out the best medium that filtered, dried at 60 ºC until constant weights were obtained.
supported maximum biomass production of all the six strains
of L. edodes (Jung et al. 2001) [2]. The composition was same 3. Results and Discussion
as that of solid media used in the previous experiment except 3.1 Effect of different solid media on the mycelial growth
for the omission of agar maintaining three replications for of Lentinula edodes
each treatment. The growth of L. edodes on petriplate was completed on 12th
The liquid media were prepared and fifty ml of each medium day after inoculation (Fig 1). Among the various media tested,
were dispensed in 100 ml conical flask and autoclaved at 15 malt extract peptone dextrose agar was found to be the best,
psi pressure and 121 °C for twenty minute. The media were inducing maximum radial growth (9.00 cm) for all the strains.
then inoculated aseptically with nine mm culture disc’s of Oat meal agar and malt extract agar were found to be on par
each strain obtained from actively growing culture. The flasks with MEPDA in supporting their mycelial growth. The
were incubated at room temperature. Observations were findings concur with studies by Lata and Sharma (2012) [6],
recorded 25 days after inoculation by filtering the mycelia where malt extract peptone dextrose agar medium supported
through Whatman No: 1 filter paper and drying at 60 ºC. The maximum radial growth followed by potatao dextrose agar.
dry weights were taken until a constant weight was obtained.

2.3 Effect of different temperature on the mycelial growth

and biomass production of Lentinula edodes
Disc of nine mm taken from the actively growing mycelium
of six strains of Lentinula edodes were inoculated into the
sterilised malt extract peptone dextrose agar medium poured
on sterile petriplates. The discs were placed at the centre of
the medium and incubated at 5, 10, 15, 20, 25 °C and room
temperature (28±2 °C) (Khan et al, 1995) [5]. These
experiment was conducted in three replications.
Measurements of radial growth of each strain of L. edodes
was taken when the mycelial growth of any of the tested
strains completely covered the entire petriplate.
Fifty millilitres of malt extract peptone dextrose broth were
taken in 100 ml conical flask and autoclaved at 121 °C and 15 Fig 1: Growth of Lentinula edodes strains in different solid media
lbs pressure for twenty minutes. The medium were then
inoculated with nine mm disc of seven day old culture of six From the above studies, it was observed that mycelial growth
strains of Lentinula edodes and incubated at 5, 10, 15, 20, 25 of 9.00 cm was attained in strains LE-1, 2, 3 and 6 on malt
and room temperature (28±2 °C). Observations were taken 25 extract peptone dextrose agar media twelve days after
days after inoculation. The mycelial mat was then filtered, inoculation. Mycelial growth of LE-4 and LE-5 recorded 8.63
dried at 60 ºC until constant weights were obtained. and 8.80 cm which were on par with other strains. LE-1 also
showed highest growth of 9.00 cm in oat meal agar. LE-2 and
2.4 Effect of different pH on the mycelial growth and LE-6 showed 9.00 cm growth in malt extract and potato
biomass production of Lentinula edodes dextrose agar media. On oat meal agar, LE- 2,3,4,5 and 6
Malt extract peptone dextrose agar medium was used for produced 8.80, 8.63, 8.96, 8.62 and 8.90 cm of radial growth
studying the effect of pH on mycelial growth of different respectively which was on par with LE-1 (9.00 cm). On malt
strains of L. edodes. The medium was prepared and pH was extract, LE-3 and LE-5 showed mycelial growth of 8.33 and
adjusted to 4.0, 5.0, 6.0, 7.0, 8.0 and 9.0 by adding 0.1 N 8.53 cm respectively which were on par with LE-1, 2 and 3.
Hydrochloric acid (HCl) or 0.1 N sodium hydroxide (NaOH). Mycelial growth of all strains except LE-1 and LE-2 were
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comparatively low in potato dextrose agar, Czapek’s Dox and 3.2 Effect of different liquid media on the mycelial growth
carrot agar. Radial growth of all the strains were lowest on of Lentinula edodes: Biomass production of all the strains of
yeast extract agar (Table 1). L. edodes in liquid media were taken 25 days after inoculation
(Fig 2). There was significant difference between each liquid
Table 1: Growth of Lentinula edodes strains in different solid media media in influencing biomass production of L. edodes.
Maximum biomass production of all the six strains were
Radial growth 12 DAI (cm)*
obtained in oat meal broth ranging from 145 mg/ 50 ml to 405
Media Strains
mg/ 50 ml. LE-2 and LE-4 strains also produced highest
LE-1 LE-2 LE-3 LE-4 LE-5 LE-6
MEPDA 9.00a 9.00 a 9.00 a 8.63 a 8.80 a 9.00 a biomass in PDB with 188.46 mg/ 50 ml and 118.66 mg/ 50 ml
OMA 9.00 a 8.80 a 8.63 a 8.96 a 8.62 a 8.90 a respectively which were on par with oat meal broth. Strain
MEA 9.00 a 9.00 a 8.33 a 8.13 b 8.53 a 9.00 a LE-3 produced biomass of 132.66 mg/ 50 ml on MEPDB,
PDA 8.83 a 9.00 a 8.23 a 6.36 c 8.23 b 8.36 b followed by LE-4 (74.40 mg/50 ml), LE-2 (63.80 mg/ 50 ml),
CDA 6.50 c 8.36 b 5.93 c 5.20 d 6.20 d 6.90 c LE-1 (48.13 mg/ 50 ml), LE-6 (40.30 mg/ 50 ml) and LE-5
CA 8.36 b 6.90 c 7.66 b 6.20 c 8.13 b 7.93 b (34.16 mg/ 50 ml). On malt extract broth, LE-2 produced
YEA 1.00 d 0.96 d 1.00 d 1.50e 1.56e 1.43d maximum biomass of 71.40 mg/50ml which was followed by
CD (0.05) 0.23 0.25 0.43 0.28 0.36 0.31 49.93 mg/ 50ml (LE-4), 39.30 mg/50ml (LE-6), 39.06 mg/ 50
* Average of three replications ml (LE-3), 31.00 mg/50 ml (LE-1) and 18.46 mg/ 50 ml (LE-
5). In carrot broth, maximum biomass of 48.16 mg/ 50 ml was
MEPDA: Malt extract peptone dextrose agar, OMA- Oat produced by LE-2 strain followed by 38.96 mg/ 50 ml (LE-4),
meal agar 34.66 mg/ 50 ml (LE-3), 30.70 mg/ 50 ml (LE-6) and 30.40
MEA: Malt extract agar, PDA- Potato dextrose agar mg/ 50 ml (LE-5). Lowest biomass production of L. edodes
CDA: Czapek’s Dox agar, CA- Carrot agar, YEA- Yeast strains were recorded (3.59 mg/ 50 ml to 14.96 mg/ 50 ml) on
extract agar yeast extract broth.

Fig 2: Growth of Lentinula edodes strains in different liquid media

3.3 Effect of different temperature on the mycelial growth Table 2: Growth of L. edodes strains in different temperature in
and biomass production of Lentinula edodes solid media
In solid medium, all strains of L. edodes attained maximum Radial growth 12 DAI (cm)*
Temperature
growth at 20 ºC (8.63 cm to 9.00 cm). This was closely (°C) LE-1 LE-2 LE-3 LE-4 LE-5 LE-6
followed by growth at 25 ºC temperature where LE-6 showed
5 0.90 f 0.90 d 0.90 e 0.90 e 0.90 f 0.90 f
maximum radial growth (8.30 cm) followed by LE-1(8.03
cm), LE-2 (8.17 cm), LE-3 (8.00 cm), LE-4 (7.60 cm) and 10 1.50 e 1.30 d 1.50 e 1.26 e 1.70 e 1.77 e
LE-5 (7.43 cm). Sharma et al. (2013) [8] also reported 15 4.20 d 3.73 c 4.03 d 3.96 d 3.70 d 3.97 d
maximum mycelial growth of L. edodes strains at 20 oC. 20 8.76 a 8.73 a 8.63 a 8.67 a 8.70 a 9.00 a
Radial growth was comparatively high at room temperature 25 8.03 b 8.17 b 8.00 b 7.60 b 7.43 b 8.30 b
(28 ±2 °C) (5.23 cm to 6.03 cm) when compared to lower Room temperature 5.27 c 5.26 c 5.43 c 5.60 c 5.23 c 6.03 c
temperatures of 15 °C and 10 °C. Lowest mycelial growth CD (0.05) 0.37 0.40 0.60 0.58 0.42 0.27
was observed at 5 °C (5.23 cm to 6.03 cm) (Table2). *Average of three replications

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[9]
In liquid medium (Fig 3) highest mycelial biomass of all the and Cristina et al (2001) [2] reported that when various
strains was obtained in broth at 20 ºC (58.73 mg/ 50 ml to Lentinula edodes strains cultivated in PDA medium at
81.90 mg/ 50 ml) (Fig 2). LE-6 produced maximum biomass different pH’s were tested the most suitable pH was reported
of 56.90 mg/ 50 ml at 25 °C, which was followed by LE-5 to be 6.00.
(55.60 mg/ 50 ml), LE-1 (52.40 mg/ 50 ml), LE-3 (50.23 mg/
50 ml), LE-2 (46.87 mg/ 50 ml) and LE-4 (43.67 mg/ 50 ml).
The biomass production at room temperature (28 =-2 °C)
(22.66 mg/ 50 ml to 34.67 mg/ 50 ml) was high compared to
15 °C (11.93 mg/50 ml to 17.10 mg/ 50 ml) and 10 °C (4.20
mg/ 50 ml to 9.63 mg/ 50 ml). The biomass production was
lowest at 5 °C of all the strains of Lentinula edodes.

Fig 4: Growth of L. edodes strains in different pH in liquid media

4. Conclusion
In the present study it can be concluded that Malt extract
peptone dextrose agar was found to be the best culture
medium for the mycelial growth of Shiitake. The optimum
temperature and pH for the mycelial growth of Shiitake in
both solid and liquid medium proved to be 20 oC and 6
Fig 3: Growth of L. edodes strains in different temperature in liquid respectively.
media
5. Acknowledgement
3.4 Effect of different pH on the mycelial growth and The facilities and support provided by Kerala Agricultural
biomass production of Lentinula edodes University has been duly acknowledged.
All the six strains of L. edodes showed highest radial growth
(9.00 cm) at pH 6 which was followed by pH 7. Radial 6. References
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(50.26 mg/50 ml broth). Comparatively less biomass Cultural requirements, enzyme profile, molecular identity
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