Pak. J. Bot., 54(5): DOI: http://dx.doi.org/10.

30848/PJB2022-5(45)

MOLECULAR PHYLOGENETICS AND OPTIMIZATION OF GROWTH CONDITIONS

OF INDIGENOUS EDIBLE AND THERAPEUTICALLY SIGNIFICANT PLEUROTUS
FLORIDANUS FROM PAKISTAN
ABDUL REHMAN NIAZI* AND ANEEQA GHAFOOR

Institute of Botany, University of the Punjab, Lahore, 54590, Pakistan

*
Corresponding author's email: drarniazi.botany@pu.edu.pk

Abstract

Poor agricultural practices and unavailability of nutritious food have developed a need to look for some non-
conventional way of food production. Mushrooms and their cultivation are the best alternative way to achieve zero hunger
and no poverty. In the present study, native Pleurotus floridanus a potentially edible and nutritious mushroom was
characterized and evaluated for its cultivability and cultivation potential. P. floridanus is a new record for Pakistan.
Maximum cultural growth was observed on the Compost Extract Agar (CEA) medium at 28ᵒC followed by Potato Dextrose
Agar medium (PDA) and Malt Extract medium (MEA). Cultured strains on CEA medium were used for the spawn
production, Sorghum grains at 28℃ found as the best combination for spawn production. Cultivation potential was
investigated on the mixed substrate of wheat straw and sawdust and pure wheat straw only. A mixed substrate was found
better than pure substrate. Cultivation was only successful at 16ᵒC that showed that P. floridanus is a lower temperature
strain. These results showed that P. floridanus can grow on various types of substrates and medium and its cultivation on
large scale can solve one of the major concerns of growing population that is the lack of nutritious and healthy food .

Key words: Molecular phylogenetics; Cultivability; Spawn; Cultivation potential; Pleurotus floridanus; Nutrient media; Temperature.

Introduction In Pakistan and India, due to oyster like appearance,

Pleurotus is usually called as Dhingri (Khan et al., 2013).
Pleurotus (Fr.) P. Kumm genus exhibit the large range Mushroom cultivation and marketing are around
of plasticity in the phenotypic appearance that hindered the nonexistent in Pakistan. In Pakistan, mushrooms are
correct identification by old phenotypically dependent cultivated in farmhouses, along with but not confined to the
methods (Buchanan, 1993) but the molecular state-owned national logistic cell. Farm production shares
characterization have introduced some explication regarding around one percent of the total mushroom exports, while
the delimitation of species in genus (Vilgalys & Sun, 1994; the remaining of it comes through the natural production in
Vilgalys, 1996; Alberto et al., 2002; Zervakis et al., 2004). Khyber Pakhtunkhwa. (Razi, 2017). There is requirement
The Pleurotus spp. of the class basidiomycetes belong to evolve enriched agronomic system in Pakistan. The
to a group known as “white rot fungi” (Bellettini et al., 2016) agronomist should cultivate organoleptically accepted
mushrooms like Pleurotus spp. with enthusiasm on large
as they produce a white degraded biomass after
scale to meet the needs of a healthy diet. Financial status of
decomposition are generally cultivated on non-composted
cultivars can strengthen through applying the mushroom
lignocellulosic substrates (Xia et al., 2016). P. ostreatus
cultivation as a welcoming industry of agriculture in
(oyster mushroom), P. eryngii (king oyster or Cardoncello), Pakistan (Shah et al., 2004). The aim of this research is the
P. pulmonarius (phenix mushroom), P. djamor (pink oyster morpho-anatomical and molecular characterization and to
mushroom), P. sajor-caju (indian oyster), P. cystidiosus evaluate the optimum temperature and nutritional
(abalone oyster), P. citrinopileatus (golden oyster requirements for the growth of native P. floridanus which is
mushroom) and P. cornucopiae are commercially cultivated previously undescribed, Ishaq et al., (2018) conducted
and have considerable economic, therapeutical and study on the exotic P. floridanus species, the strain they
nutritional values (Zhang et al., 2016). These species are used was not the part of Pakistani mushroom flora.
abundant source of minerals (P, Ca, K, Fe and Na), proteins Cultivation of P. floridanus is rare unlike other species of
and vitamin C, B complex (riboflavin, folic acid, thiamine Pleurotus such as P. florida P. ostreatus and P. eryngii. P.
and niacin) (Patil et al., 2010). floridanus is a new record for Pakistan and also an addition
Pleurotus spp. stimulate immune system, prevent to the edible agaric flora of the country.
swelling or inflammation and tumor growth, possess
antithrombotic, antimicrobial and hypoglycaemic properties, Materials and Methods
reduce blood lipid amounts, retard atherosclerosis and high
blood pressure and possess various other activities (Gunde- Sampling and characterization: Basidiomata of the
Cimerman, 1999). P. cystidiosus and P. floridanus exhibit specimen was collected from Himalayan moist temperate
the strong antioxidant potential (Li et al., 2007; Blanche et forests (khanaspur-Ayubia, KP) Pakistan. The Collected
al., 2019). basidiomata were photographed using a Samsung camera
Worldwide, Pleurotus represented through twenty and field notes were prepared. Systematic characterization
species (Menolli Junior et al., 2010). Eighty five percent was carried out by macro-microscopically and
phylogenetically. The experiments were conducted in Fungal
of all oyster mushrooms is produced in China (Tesfaw et
Biology and Systematics Research Lab, Department of
al., 2015).
2 ABDUL REHMAN NIAZI ET AL.,

Botany, University of the Punjab, Lahore. Micro room temperature. Grains were inoculated with activated
morphological characteristics were observed using a pure mycelium colony. Three discs of the active mycelium
compound light microscope (MX4300H Techno Co., Ltd., of 1cm size of the cultured strains were inoculated in one
Japan) with an oil-immersion lens at a magnification of jar under the aseptic condition. Jars were incubated at
100X. Molecular characterization was done by following the different temperatures and mycelium extension rate on the
Protocol (Gardes & Bruns, 1993) with little modifications. grains (wheat and sorghum) was observed until grains were
The Phylogenetic tree was built through molecular completely covered with the mycelium and mushroom
evolutionary genetic analysis using MEGA 6 software with seeds prepared.
default settings (Tamura et al., 2013). Specimen was
deposited in the Herbarium, Department of Botany, Substrate production: Wheat straw and sawdust were
University of the Punjab, Lahore, Pakistan (LAH). DNA used as the raw materials. Fresh wheat straw collected
Sequence was submitted in GenBank under the accession from the field area of University of the Punjab, Lahore
number MT012089. and sawdust collected from the furniture shop at barket
market, Lahore. Two types of substrates were prepared,
Estimation of culturability: Culturability of Pleurotus one of pure wheat straw and one is the mixture of wheat
floridanus (LAH36079) was assessed according to the straw and sawdust of equal ratio. Raw materials were
method described by Siddiq et al., (2018) on three soaked in water for 24 hours then made pile of them to
different nutrient agar media i.e., Malt extract agar (2% make the substrate suitable for mycelial running and 65%
MEA: agar 20g, malt extract 20g dissolved into 1000mL moisture maintained during the decomposition process of
dH2O), Potato dextrose agar (2% PDA: thin potato slices eight days. Chicken manure and gypsum (one fourth of
200g, glucose 20g, agar 20g per liter of dH2O) and the substrates) were added as supplements for nitrogen
Compost extract agar (CEA: 20g agar,10g glucose and carbon source. Substrate and supplements were piled
dissolved into 1000 mL wheat straw water based filtrate) up and covered properly with polythene sheet to prevent
at different temperatures i.e.,16oC, 22oC and 28oC. air borne contaminants while turning of the pile was done
Inoculated petri plates were sealed with parafilm and kept
after every two days. When substrates were prepared, they
in incubator for 30 days. Mycelial characteristics of all
were filled in polypropylene bags and autoclaved for 3 to
species were observed on regular basis.
Prepared cultures were deposited in the Herbarium, 4 hours to remove any type of contamination if present
Department of Botany, University of the Punjab, Lahore and allowed to cool. Polypropylene bags of 20 x 15cm
(LAH# 11320CA; On PDA medium, LAH# 11320CB; On were used for the cultivation. 1kg of the substrate was
MEA medium, LAH# 11320CC On CEA medium). filled in each bag.

Spawn production: Pal & Thupa, (1979) described Spawning: Sterilized compost bags were inoculated with
methodology was used to prepare spawn. Spawn was the spawn prepared on sorghum grains at the rate of 25g
prepared on cereal grains (sorghum and wheat grains) in per 1kg bag at the sterilized surface and bag mouths were
jars by inoculating the grains with prepared pure culture on tied with the rubber bands and placed on sterilized shelves
CEA medium. Grains (sorghum and wheat) were taken and in the lab under the dim light.
washed properly to remove dust, chaff or any other
particles and soaked for 24 hours for maximum absorption Spawn running, pin head emergence, fruiting and
of water. Soaked grains were again washed with water and flushes: Cultivation potential in the form of spawn
boiled until they become soft. The washed grains were running period, pinhead emergence, fruiting time, and no.
spread on the clean blotting paper to drain off the extra of flushes appeared on both types of substrates were
water from the grains. Two third of the sterilized jars were observed at different temperatures.
filled with the boiled grain (100g in each jar), then calcium
carbonate (0.5g) and gypsum (1g) were added in each jar Biological efficiency (yield): Biological efficiency of
and mixed thoroughly and covered with the lid. Jars with both types of substrates was observed as per kg of the
grains were autoclaved. Autoclaved jars were placed under substrate bag. It used to determine the growth potential of
the sterilized laminar airflow cabinet and allowed to cool at the oyster mushroom based on the following formula:

Biological efficiency dry weight of substrate = Weight of fresh mushroom x 100 %

Statistical analysis of the data: All the experiments were Basidiomata: Found in pairs, creamy white to light gray
conducted in triplicates and data are expressed as mean (8/1 7.5 YR) to yellow (8/6 5Y). Pileus: Smooth margins,
value ± S.E. Analysis of Variance (ANOVA) with color ranged from creamy white to light gray (8/1 7.5 YR)
Duncan’s multiple range tests (p<0.05) by using co-stat to yellow (8/6 5Y), bivalve shape, undulate. Lamellae:
software (Version 3.03) was employed to analyze the Deeply decurrent, yellow (8/6 5Y), crowded.
effect of different treatments on mycelium extension rate
and cultivation potential. Basidiospores: (7.70-) 8.5–9.56(-13.97) x (3.99) (4.40) –
(4.8) (5.024) µm, avl x avw =10.842 x 4.70µm, epiculated,
Results and Discussion subcylinderical, thin walled, inmayloid, hyaline. BASIDIA:
(19.11_) 19.57–24.57(-26) x (5.19-) 5.55–6.0(-6.1) µm, avl x
Morpho-anatomical analysis: Pleurotus floridanus Singer, avw =21.121 x 5.615µm, clavate, two to four spored, thin
Pap. Mich. Acad. Sci. 32: 134 (1948) [1946] (Fig. 1: A-H). walled, basal clamp connection, Basidiols present.
MOLECULAR PHYLOGENETICS & OPTIMIZATION OF GROWTH CONDITIONS OF THE INDIGENOUS PLEUROTUS FLORIDANUS 3

CYSTIDIA: (19.42-) 20.44–25.868(-26.42) x (4.38-) 4.88– clade I. Clade III contains all fives species that placed in
5.50(-5.60) µm, avl x avw = 23.70 x 5.14 µm, clavate, thin subgenus Coremiopleurotus due to their distinct anatomical
walled. PILEUS HYPHAE: (2.45-) 3.671–7.239(-7.39) µm, features and anamorphic stage during their cultural growth
avw = 5.298µm, irregular, thin walled, clamp connection that are absent in all the species of Clade I. Hohenbuenelia
present. STIPE HYPHAE: (3.00-) 3.75–3.96(-4.32) µm, avw petalodes (AF139956), was used for rooting purpose (Fig.
= 3.9272, irregular, thin walled, clamp connection present. 2). P. floridanus is genetically closely related to the P.
Material Examined: Pakistan, Khyber Pakhtunkhwa, ostreatus as shown in fig 2, but Li et al., (2017) clearly
Ayubia, Khanaspur, KP 2575 m a.s.1, on decayed log, 28 demonstrate that it is a distinct species from P. ostreatus.
December 2018. A.R. Niazi (LAH36079), GenBank
accession number; MT012089. Effect of temperature and nutritious media on
It was the first attempt to describe the P. floridanus cultivability: Like every living organism mushrooms also
on both morpho-anatomical and molecular basis from require food to show their growth potential in the form of
Pakistan. Its micro morphological features clearly mycelium extension rate. Three different nutrient media
distinguished it from its genetically closely related P. were used to check their growth potential. Mycelium
ostreatus species (Menolli Junior et al., 2014). texture and growth pattern on all the media used are shown
in (Table 1). Mycelium extension rate (mm/day) on
different media at different temperatures was significantly
different at (p<0.05) showing in (Table 2). Mycelium took
almost the same no. of days to completely colonize the
culture plate on CEA and PDA media while on MEA media
mycelium took more days to completely colonize the petri
plate (Fig. 3). All the media proved the supportive media
for the growth of mycelium of P. floridanus but the growth
is relatively slow on MEA which might be due to nutritious
requirement of the P. floridanus mycelium fulfilled quickly
by the PDA and CEA media as compared to the MEA
medium. The current results were in agreement with the
Zervakis et al., (2004) and Sardar et al., (2015) studied the
mycelium extension rate of Pleurotus species more at high
temperatures 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 to
the Atri et al., (2013). The current study was also in
concurrent with the Ishaq et al., (2017) from Pakistan, they
studied the cultural characteristics of the exotic P.
floridanus but we studied the mycelial characteristics of the
native P. floridanus.

Effect of temperature and grains on the spawn

production potential: Grains are the rich source of
Fig. 1. A – H: Macroscopic and Microscopic Morphology of carbohydrates and proteins. Small discs of mycelium fully
Pleurotus floridanus (LAH36079). A-B. Basidiomata of colonized the grains by using the stored food in the grains
Pleurotus floridanus; C. Basidioles; D. Basidiospores; E. but the colonization rate vary depending on the grains size
Basidia F. Cystidia; G. Pileus hyphae; H. Stipe hyphae. Scale and stored food. Colonization rate was checked at 16ᵒC,
bars: A - B = 2.5cm, C = 9.5µm, D = 5.2µm, E = 13.4µm, F = 22ᵒCand 28ᵒC temperature for both wheat and sorghum
11.6µm, G = 20.12µm, H = 22.7µm. grains. Data (Table 3) showed that the colonization rate
(mm/day) on sorghum and wheat grains at different
Molecular and phylogenetic analysis: Barh et al., (2019) temperatures was significantly differed at (p<0.05).
and Adedokun et al., (2016) reported previously it from Mycelium colonized more quickly on sorghum grains as
India and Nigeria respectively. The specimen was compared to wheat grains at all three temperatures
sequenced using ITS1F/ITS4 region of nrDNA. Targeted because of their smaller size and have greater surface
ITS regions of P. floridanus yielded 662 base pairs. Initial area. Mycelium found more points for colonization so the
blast of our amplified Pakistani strain revealed 98% colonization rate is higher on sorghum grains as compared
similarity with P. floridanus (MK281340). In the to wheat grains. Our findings were similar with the
phylogenetic analysis three clades were formed. All P. Tinoco et al., (2011) and Tsegaye & Tefera, (2017).
floridanus species including Pakistani strain clustered in

Table 1. Mycelial cultural characteristics of Pleurotus floridanus on different media.

Mycelium characteristics
Types of media
Texture Growth Color
CEA Cottony Irregular Creamy white
MEA Cottony Irregular Creamy white
PDA Cottony Irregular Creamy white
CEA, Compost extract agar; MEA, Malt extract agar; PDA, Potato dextrose agar
4 ABDUL REHMAN NIAZI ET AL.,

Fig. 2. Maximum likelihood (ML) analysis was performed using Jukes-Cantor model in MEGA6 software to test the phylogeny at
1000 bootstraps. The analysis involved 32 nucleotide sequences along with Hohenbuehelia petalodes used as an outgroup. All
positions containing gaps and missing data were eliminated. Evolutionary analyses were conducted in MEGA6. Sequences generated
during this study are represented with

Table 2. Mycelium extension rate of Pleurotus floridanus at different temperatures on different media.
Mycelial extension rate (mm/day)
Temperature (ᵒC)
CEA MEA PDA
16 4 ± 0.288e 2.5 ± 0.251f 3.7 ± 0.145e
22 9 ± 0.288c 7 ± 0.210d 8.6 ± 0.145c
28 14.5 ± 0.287a 9.9 ± 0.208b 15.06 ± 0.233a
LSD 0.701
*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

Table 3. Effect of temperature on the mycelium extension rate on the wheat and sorghum grains.
Mycelial Extension Rate (mm/day)
Types of grains
16ᵒC 22ᵒC 28ᵒC
Wheat grains 7.76 ± 0.145e 9.66 ± 0.176d 14.8 ± 0.166b
Sorghum grains 9.83 ± 0.166d 12.5 ± 0.288c 17.8 ± 0.166a
LSD 0.584
*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
MOLECULAR PHYLOGENETICS & OPTIMIZATION OF GROWTH CONDITIONS OF THE INDIGENOUS PLEUROTUS FLORIDANUS 5

Fig. 3. Mycelium extension rate of Pleurotus floridanus (LAH36079) on different media at 28ᵒC after 25 days of inoculation.

Mixed substrate Pure substrate showed that Spawn run rate was faster on the substrate of
120
wheat straw and sawdust combination as compared to the
pure wheat straw only. Lignocellulosic content high in
100 sawdust as compared to the wheat straw that was used by the
mycelium as the protein and carbohydrate source for its
growth that’s why spawn running period completed earlier
Growth rate (%)

80
on the mixed substrate than the pure wheat straw substrate.
Our results were similar to the Das et al., (2015) and Obodai
60
et al., (2003) found the positive correlation between lignin-
cellulosic contents and yield of P. ostreatus. After spawn
40 running completion, specific yellowish- white outgrowths
appeared on all the replicates of both types of substrates and
20 then degenerate, after it primordia appeared. That yellowish
outgrowths may act as the initiator of the primordia or
pinhead emergence (Fig. 5). Time taken from the pinhead
0
appearance to the harvesting was more on the pure substrate
10 20 30 40 50 60
than the mixed substrate.
Number of days
Biological efficiency: Biological efficiency (yield) obtained
Fig. 4. Graph showing the frequency of spawn running high in amount from the substrate of wheat straw and
completion on both type of substrates.
sawdust combination as compared to the pure wheat straw
Effect of temperature and lignocellulosic substrates on only (Table 4, Fig. 6). 57.80±0.137 g yield per 1kgbag was
the cultivation potential: Temperature is a very crucial obtained from the mixed substrate while only 35.7±0.115g
factor for the successful fruiting of any mushroom. Only per 1kgbag was obtained from pure wheat straw substrate
Optimized temperature and humidity can convert the showed that P. floridanus require sawdust substrate for its
dikaryotic mycelium into the fruiting body. Spawn prepared optimum growth. Our findings were in agreement with the
on the sorghum grains was used to determine the cultivation Adedokun & George-David, (2016). Our results were also in
potential on the two types of substrates at various concurrent to the Ishaq et al., (2018) who optimize the
temperatures. There was no fruiting on both types of biological efficiency of the exotic P. floridanus but we
substrates bags kept at 22ᵒC and 28ᵒC while the substrates optimize the growth requirements of the indigenous P.
bags kept at 16ᵒC successfully completed their spawn floridanus species from Pakistan to get maximum economic
running period and initiated primordia formation that proved viability by its cultivation at large scale.
that P. floridanus is a lower temperature strain Data (Fig. 4)

Table 4. Yield obtained from pure and mixed substrate at 16ᵒC.

Types of Biological efficiency (yield)
st nd
substrates 1 Flush yield (g) 2 flush yield (g) 3rd flush yield (g) Total yield (g)
Mixed substrate 18.6 ± 0.152c 20.85 ± 0.175c 18.577 ± 0.295c 57.80 ± 0.137a
Pure wheat straw 17.93 ± 0.145c 17.7 ± 0.115c Not appeared 35.7 ± 0.115b
LSD 0.891
*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
6 ABDUL REHMAN NIAZI ET AL.,

A B C

D E F

G H I
Fig. 5. All stages of cultivation of Pleurotus floridanus from spawn running to harvesting; (A) Spawn Running (B) Fully spawned
compost (C) Yellowish white outgrowth appeared (D) Outgrowth developed fully (E) Pin head emerged (F) Growing Pin heads (G)
Growing Fruiting bodies (H) Matured fruiting bodies (I) Harvesting.

A B C

D E
Fig. 6. Flushes observed on mixed and pure substrates; A-C, 1st, 2nd and 3rd flush on the mixed substrate; D-E, 1st and 2nd flush on the
pure substrate.
MOLECULAR PHYLOGENETICS & OPTIMIZATION OF GROWTH CONDITIONS OF THE INDIGENOUS PLEUROTUS FLORIDANUS 7

Conclusion Ishaq, M., M. Fiaz, Saifullah, M.B. Khan, S. Ullah and R. Khan.
2018. Effect of different substrates and supplements on the
In conclusion, P. floridanus have great potential growth and yield of Pleurotus floridanus Singer. J. Biol. &
Env. Sci., 12(6): 305-310.
to cultivate on a variety of substrates. It is a superfood and
Ishaq, M., M. Fiaz, Saifullah, S. Ullah and M.B. Khan. 2017.
its cultivation on a large scale can help in strengthening Evaluation of mycelial growth of oyster mushroom
the agro-economic status of low-income countries. (Pleurotus floridanus Singer) on different media and cereal
Furthermore, it is previously an unreported species from grains. J. Bio. & Env. Sci., 11(3): 67-72.
Pakistan and an addition to the edible flora of the country. Khan, M.W., M.A. Ali, N.A. Khan, M.A. Khan, A. Rehman and
N. Javed. 2013. Effect of different levels of lime and pH on
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(Received for publication 12 April 2021)