ISSN: 00845841

Volume 51, Issue 01, June, 2021

Comparative study of the mycelial characteristics of

Pleurotus species with the first time description of
anamorphic stage of Pleurotus cystidiosus from
Pakistan
Abdul Rehman Niazi1, Aneeqa Ghafoor1*

Department of Botany, University of the Punjab, Lahore, 54590, Pakistan1

Corresponding Author: 1*

Keywords: ABSTRACT
Mycelium, Pleurotus In the present study, potentially edible and nutritious Pleurotus spp. i.e.,
cystidiosus, Pleurotus
P. cystidiosus, P. floridanus, P. ostreatus were collected to figure out
floridanus, Pleurotus ostreatus
their cultural characteristics and to find out the optimum requirements
for their mycelial growth which are the pre-requisite requirements for
their successful cultivation. Their mycelial features were observed on the
different nutrient media at different temperatures. Mycelium of each
species was assessed morpho-anatomically. Mycelium growth pattern
and texture of three Pleurotus species were irregular and cottony while a
special anamorphic stage (Antromycopsis macrocarpa) in the form of
Coremium and coremioliquid appeared during the cultural growth of P.
cystidiosus. Anatomically mycelium of each species was septate and
clampedwith the addition of toxocyst and arthroconidia production in the
anamorphically grown myceliumof P. cystidiosus, which is unreported
feature for this species from Pakistan. P. floridanus took more time to
completely colonize the petriplates on all media followed by P.
cystidiosus and P. ostreatus. All species have spawn production potential
but spawn production rate was more of P. ostreatus followed by P.
floridanus and P. cystidiosus. The linear growth rate was assessed on the
mixed substrate of sawdust and wheatstraw. P. floridanus, P. ostreatus,
P. cystidiosus mycelium showed their best colonization rate at 16ᵒC,
22ᵒC, and 28ᵒC respectively. From these results it could be assessed that
these nutritionally and medicinally significant Pleurotus spp. possess the
cultivation potential and their cultivation on large scale can meet the
nutritious need of the growingpopulation of the world.

This work is licensed under a Creative Commons Attribution Non-Commercial 4.0

International License.

1. INTRODUCTION
Mushrooms are acknowledged not only because of their flavor and texture but also due to nutritional and
chemical properties [6]. Mushrooms are a superfood, and one of the world's healthiest foods.
Approximately, 50% of edible mushrooms are recognized functional foods, meaning that over and beyond
basic nutrition they have a potentially beneficial health impact [13]. Edible mushrooms are nutriment
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endued fungi that propagate or sprout wildly on the logs, roots of trees and leaves along with the detoriated
ligneous mass [10], [31], [21]. These edible and nutritive mushrooms entail Pleurotus spp, Volvariella
volvacea, Auricularia auricula, as well as Agaricus spp. [36]. Worldwide through human culture, Pleurotus
(Fr.) P. Kumm spp. have been utilized because of their therapeutic values, nutritional worth and other useful
effects. Pleurotus spp. are a healthy source of dietary fibers and other worthful nutrients. Pleurotus spp.
stimulate immune system, prevent swelling or inflammation and tumor growth, possess antithrombotic,
antimicrobial and hypoglycaemic properties, reduce blood lipid amounts, retard atherosclerosis and high
blood pressure and possess various other activities [16]. P. cystidiosus and P. floridanus possess the strong
antioxidant potential [20], [7] while Pleurotus ostreatus exhibit strong antitumor activity [11]. As the
number of wild mushrooms reduces due to the deteriorated environment and costlier activities, cultivated
mushrooms will provide not only food security but also a healthy and balanced diet [35]. Mushroom
cultivation technology is quite a recent revolution. The economic and social position of poor farmers can be
improved by the embodiment of the non-conventional crop in subsisting agricultural system. After yeast,
mushrooms cultivation is referred to as the largest economical microbial science [25]. Study of mycelial
nature is helpful in understanding the life cycle and various aspects of cultivation of therapeutically
important mushrooms [17], [29], [2], [3], [1], [8], [14], [15]. Mushrooms developed from mycelium that
sprout on a base of wet sterilized cereal grain commonly sorghum, rye or millet. This mycelium-infused
grain is named as spawn and is used to inculcate into the mushroom substrate [27]. Usage of poor spawn
may often lead the failure to get a satisfactory harvest [9]. The aim of this research was to assess the
mycelial characteristics and figure out the optimum temperature for mycelium expansion that can help to
understand the crucial aspects of cultivation of these functional Pleurotus species as their cultivation on
large scale can solve two major concerns of developing countries like Pakistan that are of health and food.
Anamorphic stage of P. cystidiosus mycelium was previously undescribed feature for this species from
Pakistan. CEA medium was first time used for culturing of Pleurotus spp. in Pakistan.

2. Material Methods

2.1 Sampling and Experimental design

Basidiomata of the P. floridanus and P. ostreatus were collected from Himalayan moist temperate forests
(Khanaspur-Ayubia, KP) in Pakistan; while Basidiomata of the P. cystidiosus was collected from Girls
Hostel Area, University of the Punjab, Lahore, Punjab, Pakistan. The Collected specimens were
photographed using a Samsung camera and identified by macro-microscopically and phylogenetically
accordingly to the literature already reported [23], [19], [5]. The experiments were carried out in Fungal
Biology and Systematics Research lab, Department of Botany, University of the Punjab, Lahore. Specimens
(Pleurotus floridanus (LAH36079), P. cystidiosus (LAH36080) and P. ostreatus (LAH36081) were
submitted in the Herbarium, Department of Botany, University of the Punjab, Lahore, Pakistan (LAH).

2.2 Evaluation of Culturability of wild mushrooms

Culturability of Pleurotus spp. was assessed according to the method described by [30]. Small tissues from
inner unexposed part of the fruiting bodies of P. floridanus, P. cystidiosus, and P. ostreatus were placed
onto three different nutrient agar media i.e, Malt extract agar (2% MEA: agar 20g, malt extract 20g
dissolved into 1000mL dH2O), Potato dextrose agar (2% PDA: thin potato slices 200g, glucose 20g, agar
20g per liter of dH2O) and Compost extract agar (CEA: 20g agar,10g glucose dissolved into 1000 mL
wheat straw water based filtrate) at different temperatures i.e.,16oC, 22oC and 28oC. Inoculated petri plates
were sealed with parafilm and kept in incubator for 30 days. Mycelial characteristics of all species were
observed on regularbasis.

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2.3 Microscopic observation of mycelium

To study the mycelium anatomical features, a fine thread of mycelium from pure mycelium was taken using
sterilized tweezer and mounted on the glass slide. Mycelium was stained with 2% tryphan blue and covered
with the cover slip and observed under a compound light microscope (MX4300H Techno Co., Ltd., Japan)
with an oil-immersion lens at a magnification of 100X.

2.4 Spawn production

[24] described methodology was used to prepare spawn. For spawn preparation, sorghum grains were
washed and soaked for overnight, boiled for half an hour and excess water from grains was removed by
spreading the grains on blotting paper. Half fill jars with boiled grains supplemented with gypsum (1g) and
lime (0.5g) were autoclaved. Seeds of mushrooms were prepared by inoculating small mycelial discs of
1cm from pure culture onto the sterilized sorghum grains in a laminar air flow cabinet. Inoculated grains
were incubated at different temperatures i. e16 oC, 22 oC and 28 oC for 30 days.

2.5 Evaluation for spawn-running performance on substrate

A mixed substrate of sawdust and wheat straw was prepared for determining the spawn running rate of three
Pleurotus species in test tubes. Saw dust and wheat straw was taken in equal ratio and made pile of them to
make the substrate suitable for mycelial running and 65% moisture maintained during the decomposition
process of eight days. Manure and urea (one fifth of the substrates) were added as the supplements while
gypsum (one fourth of the substrates) was added and thoroughly mixed before the pasteurization process.
To find out the spawning rate in test tubes, seven seeds of mushrooms (spawn) were placed at the bottom of
test-tubes and filled with the prepared substrate, closed with the cotton plugs and aluminium foil and
incubated them at different temperatures i.e 16 oC, 22 oC and 28 oC for 30 days.

2.6 Statistical Analysis of the Data

All the experiments were conducted in triplicates and data is expressed as mean value ± S.E. Analysis of
Variance (ANOVA) with Duncan’s multiple range tests (p<0.05) by using co-stat software (Version 3.03)
was employed to analyze the effect of different treatments on mycelium extension rate.

3. Results and Discussion

Effect of temperature and nutrient media on culturability of different Pleurotus species Mycelial
characteristics of Pleurotus species were assessed on different nutrient agar media atdifferent temperatures.
Mycelium extension pattern was irregular and texture is cottony for allthree Pleurotus species on each
media used for the determination of the culturability potentialwhile a distinct anamorphic anamorphic stage
(Antromycopsis macrocarpa) that possessed theCoremium (white colored aerial hyphae and coremioliquid
(black colored slimy liquid) on its topappeared during the cultural growth of P. cystidiosus. Anatomically
mycelium of all species wasseptate and clamped but in P. cystidiosus, toxocyst was present with hyphae
and arthroconidiadevelop from the division of the upper cells of coremium hyphae present inside the
coremioliquid (Fig 1), this specialized feature of P. cystidiosus (enriched with the medicinal values) make it
to becharacterized in the subgenus Coremiopleurotus. Mycelium extension rate (mm/day) of
Pleurotusspecies on different media such as PDA, MEA and CEA media at different temperatures was
observed and it was found that P. floridanus exhibit relatively slowest growth rate(mm/day) among each
species except on CEA media at 28C it showed maximum growth rate(mm/day) followed by P. ostreatus
and P. cystidiosus which is because every mushroom species requires different requirement to expand their
mycelial network. Mycelium extension rate (mm/day) of Pleurotus species on different media at different
temperatures was significantly differed at (p<0.05) showingin table 1. The current results were in agreement
with the [37], [28] studied that the mycelium extension rate of Pleurotus species more at high temperatures
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and found the Potato dextrose agar medium (PDA) as the best medium for the growth of mycelium of
various Pleurotus species. Our results were also similar with the [4] studied the growth rate of Pleurotus
species on PDA media and found that the P. floridanus possessed slowest growth rateand Pleurotus
cystidiosus showed the special coremium structure during the cultural growth. [34] observed the
arthroconidia formation process from the coremia hyphae.

Table 1. Mycelium extension rate of Pleurotus species at different temperatures on different Media
Temp Mycelial Extension Rate (mm/day) on media
ᵒC
CEA MEA PDA
PC PF PO PC PF PO PC PF PO
16 2.9±0. 2.96±0 3.8±0. 1.86± 1.93±0. 3.83±0. 3.83±0. 2.96±0.0 3.92±0.0
1f .03f 1e 0.13e 06e 08c 08d 3e 8d
22 4.93±0 3.91±0 5.83±0 2.93± 2.96±0. 4.9±0.1 5.86±0. 3.92±0.0
6.9±0.1b
.06d .08e .08c 0.06d 03d b 13c 7d
28 5.86±0 6.93±0 6.73±0 4.86± 3.93±0. 5.9±0.1 7.85±0. 5.93±0.0 7.9±0.05
.08c .03b .26a 0.08b 06c a 14a 6c a
LSD 0.343 0.257 0.276
*The results reported were run in triplicates and stated as Mean± Standard error. *LSD stands for the least
significant difference *Different alphabets indicate significant (p<0.05) difference between the mean
according to Duncan’s new multiple range test while ± indicates standard error.
CEA, Compost Extract Agar; MEA, Malt Extract Agar; PDA, Potato dextrose agar
PC, Pleurotus cystidiosus; PF, Pleurotus floridanus; PO, Pleurotus ostreatus

A B C

D E F

H I J
G

K L
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Figure 1. Basidiomata, Macroscopic and Microscopic features of mycelium of Pleurotus ostreatus (A, B,
C); Pleurotus floridanus (D, E, F); Pleurotus cystidiosus (G, H, I, J, K, L); I (Coremia and Coremioliquid), J
(septate mycelium), K (toxocyst), L (arthroconidia) grown on PDA medium.

3.1 Spawn production potential

Cereal Grains are the rich source of nutrition for mushrooms. Sorghum grains are smaller in size than other
types of cereal grains and mycelium find more point for colonization. Mycelium colonization rate was
regular for P. ostreatus while irregular for P. floridanus and P. ostreatus on sorgum grains (Fig 2). Each
species showed the spawn production potential but the Spawn production rate (mm/day) was more for the P.
ostreatus followed by the P. cystidiosus and P. floridanus. Spawn production potential of Pleurotus species
on sorghum grains (mm/day) at different temperatures was significantly differed at (p<0.05) showing in
table 2. Our results were in concurrent with the [32]. [33] observed that mycelium extension rate more on
the substrates having pores and greater surface area.

Figure 2. Spawn formation rate of P. ostreatus, P. cystidiosus and P. floridanus at 22ᵒC after 20 days of
inoculation on sorghum grains.

Table 2. Spawn production rate (mm/day) of different Pleurotus species on sorghum grains at different
temperatures.

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*The results reported were run in triplicates and stated as Mean± Standard error. *LSD stands for the least
significant difference *Different alphabets indicate significant (p<0.05) difference between the mean
according to Duncan’s new multiple range test while ± indicates standard error.

PC, Pleurotus cystidiosus; PF, Pleurotus floridanus; PO, Pleurotus ostreatus

Spawn running rate on lignocellulosic substrate at different temperatures in test tubes

Lignocellulosic substances such as wheat straw and sawdust have large amount of cellulosic and lignin
content. Mushrooms mycelium degrade them and used as the source of nutrition. Optimum temperature can
enhance the mycelium expansion process on these lingo-cellulosic substrates. Only Optimized temperature
and humidity can convert the dikaryotic mycelium into the fruiting body. P. cystidiosus showed best
mycelial expansion rate at 28ᵒC, while P. floridanus mycelium extend best at 16ᵒC (Fig 3). Each species
showed the different optimum temperature requirementsfor the spawn running rate (mm/day) on
lignocellulosic waste such as P. cystidiosus showed the maximum spawn running rate at 28ᵒC while P.
floridanus showed the minimum mycelium extension at 28ᵒC which is due to the specific temperature
requirement of each species. Mycelial colonization rate (mm/day) on mixed substrate in testtubes showing
in table 3. Our findings were similar with the [22] found that the Pleurotus spp. can grow at varied ranges of
temperatures, from 18 to 30 °C. [26] studied that P. ostreatus can be cultivated at different temperatures.
[18] who studied the mycelium extension rate of P. cystidiosus and P. ostreatus at different temperatures
found the 28ᵒC as the optimum temperature for the mycelium extension. Our results were also in agreement
with the [12].

Table 3. Effect of different temperatures on the Spawn running rate of Pleurotus species in test tubes

*The results reported were run in triplicates and stated as Mean± Standard error. *LSD stands for the least
significant difference *Different alphabets indicate significant (p<0.05) difference between the mean
according to Duncan’s new multiple range test while ± indicates standard error.

PC, Pleurotus cystidiosus; PF, Pleurotus floridanus; PO, Pleurotus ostreatus

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Pleurotus Pleurotus Pleurotus

floridanus cystidiosus ostreatus

A B C

D E F

H I
Figure 3. SpawnG running rate of P. floridanus, P. cystidiosus, P. ostreatus in test tubes at threedifferent
temperatures; 16ᵒC (A, D, G), 22ᵒC (B, E, H) and 28ᵒC (C, F, I).
4. Conclusion
It is, concluded that, all the, three species, of Pleurotus, possess the cultivation potential on the variety of
media and temperatures but with different optimum, requirements. These species act as the superfood and
with the proper understanding of their mycelial growth requirements, they can cultivate on a large scale
which can help in strengthening the agro-economic status of low-income countries. Furthermore, P.
cystidiosus, showed the, unique anamorphic, stage during, the cultural growth which is unreported feature
for this species from Pakistan.

5. Acknowledgements
We are highly thankful to the Prof. Dr. Muhammad Asif Ali, Institute of Horticulture Sciences, University
of Agriculture, Faisalabad for providing valuable guidance which helped a lot to accomplished this study.

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