Introduction
Volvariella volvacea (Bull.) Singer belongs to the Volvariella genus from the Pluteacea family, is an
edible cultivated mushroom and commonly known as paddy straw mushroom or Chinese
mushroom (Chang 1977; Chen et al.
2019). Volvariella volvacea is widely grown in tropical and subtropical climates like China,
Thailand, Philippines, and Malaysia (Bao et al. 2013). Due to V. volvacea qualities
of having a nice texture and aroma, pleasant flavour, fast growth rate, and
easy cultivation technology, this mushroom became a popular choice amongst
consumers and growers (Ahlawat and Tewari 2007; Thiribhuvanamala et al.
2012; He et al. 2018). Besides that, the demand and cultivation practices of
V. volvacea have increased worldwide, given their rich protein content
of 19 to 35% of protein on a dry weight basis with all essential amino acids
required by the human body (Kumud et al.
2014). Furthermore, Volvariella volvacea additionally has significant
pharmaceutical values, such as immunosuppressive proteins, anti-tumor
polysaccharides, and immunomodulatory lectins (Liu et al. 2011).
Therefore, the cultivation of V. volvacea in Malaysia could promote
societal livelihood through economic, nutritional, and medicinal values
(Marshall and Nair 2009; Rosmiza et al. 2016).
The cultivation of V. volvacea is
increasingly popular worldwide because of factors like prevailing external
climatic conditions, short growing times, low input requirements, and since
agricultural waste is highly available, only a small investment is needed
(Ahlawat and Tewari 2007; Rosmiza et al. 2016). There are several
factors that affect the growth and production of V.
volvacea, such as spawn production, a substrate for the mushroom bed, and
conditions during cultivation like temperature, relative humidity, and hydrogen
potential (Miles and Chang 2004). Moreover, Ukoima et al. (2009) stated
that using an empty fruit bunch (EFB) as the substrate could increase the production of V. volvacea and
reduce biomass waste in the environment. The highest possible production of V.
volvacea is obtainable through high-quality mycelium and substrate in the
spawn preparation. Therefore, improving the quality of the mycelium enhances
the quality of the spawn, which in turn might result in high production of the
mushroom.
In recent studies, the application of mutation
techniques using acute gamma radiation on the mycelium was suggested to improve
mushroom production by developing new varieties of mushroom species (Rashid et
al. 2014). Irradiation refers to a process of exposing a substance to
ionizing radiation from a variety of different sources, such as electron beams
and gamma rays (Akram and Kwon 2010; Fernandes et al. 2012). Gamma rays
have been reported as an efficient ionizing radiation technique and one of the
most convenient methods in performing radiation-induced mutation for developing
new varieties as it can cause mutation over a wide spectrum (Nakagawa 2009;
Noordin et al. 2014). Whether spontaneous or induced, the mutations are
crucial for genetic variability, which can improve crop yield (Manjaya 2009).
Furthermore, Ibrahim et al. (2017) showed the exposure of gamma
irradiation on the mycelium of L. edodes, proving that irradiation can affect
the mycelial growth of the mushroom due to the alteration of the genetic
compatibility of these mycelia. In addition, gamma irradiation is usually
applied as an improved post-harvest technology on fresh mushrooms (Akram et
al. 2012). However, there were no reported trials on acute gamma radiation
used to the mycelia of V. volvacea during the spawn preparation.
Therefore, this study aimed to determine the optimal doses of radiation
exposure for improving the growth and production of V. volvacea.
Materials and Methods
Irradiation treatment
The PDA culture of V. volvacea with complete
mycelial inoculation was irradiated using Biobeam GM 8000 Gamma Irradiator with
Cesium-137 (Cs-137) as radioactive sources. Mycelia samples were exposed at six
different low doses of irradiation, i.e.,
0 (control), 300, 600, 900, 1200 and 1500 Gy at a rate of 12.5 Gy min-1.
Each treatment contained three samples of irradiated mycelia. The irradiated
mycelia for each treatment were sub-cultured on the new PDA. All the subcultures
were next incubated at room temperature, RT (25 ± 2°C). The growth of mycelium
on PDA was measured by drawing two perpendiculars of a straight line at the
back of each Petri dish. The measurement unit used was in millimetres (mm). The
mycelial growth on PDA was recorded every day for seven days. The growth rates
were determined by fitting the linear growth function, following the formula of
y = krx + c (where, kr is the growth rate, y is the
distance covered by mycelium, and x is the respective time) and expressed in mm
day-1 (Koutrotsios and Zervakis 2014).
Inoculation of irradiated mycelia on wheat
The selected mycelia grown from each treatment were
inoculated on a wheat substrate prepared in test tubes with dimensions of 60 Χ 25
mm. The samples were incubated at room temperature, RT (25 ± 2°C). The growth
of mycelium along the test tubes was observed at two-day intervals, with the
growth rate of mycelium being determined. Moreover, the growth of mycelium on
the wheat substrate was recorded by measuring the mycelia running down along
the test tube. The growth of mycelia was observed and recorded in two-day
intervals, and the growth rate was determined.
Preparation of spawn and mushroom bed
Spawn's preparation: The method of preparation of
the spawn followed Miles and Chang (2004) with a slight modification. First of
all, the paddy straw was soaked in water for about four hours. The paddy straw
was then washed and left to remove excess water. After that, the paddy straw
was cut into a shorter length (57 cm). Subsequently, it was mixed with 1%
calcium carbonate, CaCO3. Then, the paddy straw was transferred into
a polypropylene plastic bag and covered with a cap. Each treatment consists of
four replicates with 100 g of paddy straw for each bag. The prepared spawn bags
were autoclaved at 121°C for 60 min and left to cool at
room temperature (25 ± 2°C). About 10 to 15 grains covered
with mycelium from each irradiation dose were transferred into the prepared
spawn bag. This process was done under a sterilized condition. The inoculated
spawn bags were left at room temperature (25 ± 2°C) until the spawn run for each treatment was completed.
Mushroom bed preparation: The empty fruit bunch (EFB) was prepared and used as
the substrate for the mushroom bed. The EFB was composted for nine days,
covered by canvas, and watered at three-day intervals. The pH of the EFB was
adjusted using calcium carbonate, CaCO3 to achieve pH 68. After nine days, the EFB was watered until reaching
7080% moisture and was feet-pressed to remove excess
water and reduce the air gap in the bed to ensure more compact mycelial growth.
The EFB was then arranged in a basket with a measurement of 45 Χ 35 cm.
Hundreds of grams of treated spawn from each treatment were used for one
mushroom bed. There were four replicates of mushroom beds for each treatment.
All the mushroom beds were covered with black polythene sheets for seven days.
After seven days, the polythene cover was removed to prepare the polypipe into
a dome shape before the polythene sheet was lifted using polypipe as the
support.
Determination of V. volvacea production and
texture analysis
Production of the fruiting body: After 15 days from the spawning process, the first
flush of V. volvacea was observed and harvested. The mushrooms were
harvested at the button stage. The numbers and weight of the mushrooms for each
treatment were recorded for each harvest time or flush.
Texture analysis: Five replicates of the button stage of V. volvacea
weighing in the range of 25 ± 2 g from each treatment were chosen. The texture
analysis was done using an Instron Texture Analyzer to compare the firmness of
the mushroom.
Statistical analysis
The raw data were processed using Microsoft Excel
2016, and all the statistical tests included Analysis of Variance (ANOVA), and
Duncan Multiple Range Test, which is for multiple mean comparisons, were performed using Statistical Analysis System (SAS)
9.4 Software.
Results
Growth of V.
volvacea mycelia on PDA and wheat grains
Mycelia growth on PDA: All treatments showed thick white cottony mycelia
before light orange formation began to appear as early as day 6. The time taken
for spawn run for mycelia irradiated with 1500 Gy was longer, reaching nine
days compared to other treatments, which were only seven days. Besides that, there
was no noticeable difference in mycelia colour and growth characteristic
between all treatments (Fig. 1). However, the treatments of 600 and 900 Gy
resulted in significantly higher growth rates of V. volvacea mycelia, which are 11.78 ± 0.10 mm day-1 and
11.85 ± 0.13 mm day-1, respectively to control (10.72 ± 0.16 mm/day)
and other treatments (Fig. 2).
Mycelia growth on wheat: There was no significant difference (P > 0.05) in the mean growth rate of
mycelia irradiated with 300, 600 and 900 Gy (9.57 ± 0.20, 9.82 ± 0.16, and 9.83
± 0.17 mm day-1, respectively) compared to 0 Gy which at 9.70 ± 0.16
mm day-1 (Fig. 3). Even though the mycelia irradiated with 1200 and
1500 Gy had slower growth rates (8.62 ± 0.20 and 8.23 ± 0.09 mm day-1,
respectively), there were no noticeable in the colour of mycelia and no
mortality of mycelia observed (Fig. 4).
Evaluation of V. volvacea production for each
treatment
Harvesting periods: The cultivation period is the time between the
spawning and production of the first flush of mushrooms. The spawn with
mycelium irradiated at 1500 Gy took the longest time to produce a fruiting body
compared to other treatments. The spawn with 300, 600 and 900 Gy irradiated
mycelia showed a shorter time for fruiting body production compared to control
and spawn with 1200 Gy irradiated mycelia (Table 1).
Physical appearance: This study resulted in no noticeable differences in
the colour appearance of the fruiting body of V. volvacea produced
between all treatments. However, there was a noticeable presence of hairy
structure on the fruiting body produced by all spawn with irradiated mycelia
(Table 1). In addition, the fruiting body produced from spawn treated with 1200
Gy irradiated mycelia showed a clustered form.
Number and weight of fruiting
body: The mean number of mushrooms produced by 300, 600 and
900 Gy showed no significant difference (P
> 0.05) to control except for treatments 1200 and 1500 Gy (Table 2). The mean weight of the fruiting body
showed no significant difference between control treatment and spawned treated
with 300, 600, 900 and 1200 Gy. However, spawn with 1500 Gy irradiated mycelia
showed very poor production of V. volvacea, with only one out of four
replicates of the mushroom bed producing V.
volvacea fruiting body. The other beds of 1500 Gy spawn showed stunted
growth of fruiting bodies, which only grows until the pinhead stage.
The texture of fruiting body: The mean texture (firmness) of the fruiting body of V.
volvacea showed no
significant difference (P > 0.05)
between all treatments (Table 2).
Discussion
Results obtained from this study suggested that
the mycelial growth rate of V. volvacea
on PDA and wheat was significantly affected by gamma irradiation. The growth
rate of V. volvacea mycelia increased from control (0 Gy) until 900 Gy
before it started showing a decreasing mycelial growth on PDA. On the other
hand, on the wheat substrate, the mycelia irradiated with 1200 and 1500 Gy
showed a significantly slower growth rate compared to others. The
growth rate of a mycelium depends on the formation of clamps, which enables the
exchange of genes and extension to more areas as compatible mated mycelia was
observed, showing a faster growth (Kothe 2001; Rosnina et al. 2016). Ibrahim et al. (2017) study on Lentinula edodes that were exposed to a
higher dose of gamma irradiation showed decreasing numbers of clamp connections
compared to control. The study indicates that there are genetic compatibility
changes between individual hyphal cells within the irradiated L. edodes mycelia. In short, exposure to
higher doses of gamma radiation could decrease the number of clamps, thus
reducing mycelial growth. This also caused the possibility of sterile mycelia
being selected during the subculture process to increase.
Moreover, Beejan and Nowbuth
(2009) studied the gamma irradiation on a different strain of Pleurotus species
resulted in no definitive trend alterations observed in mycelia colonization
rates on media with the increasing doses of gamma radiation. They concluded
that irradiation could improve the Pleurotus spp. strains, and at
certain strains under different doses, the mushroom yield can be enhanced.
Therefore, the mycelial growth on the PDA and substrate can be different
depending on the mushroom species and types of substrate used.
The clustered
fruiting body of V. volvacea at the button stage is perhaps induced by
gamma-ray mutagenesis. According to Nie et al. (2017), all the
abnormalities in the morphology of V. volvacea produced compared to
control can indicate mutation. The gamma radiation could modify some
physiological characteristics and thus create new mutants. This could help Table 1: The
evaluation of V. volvacea characteristics
produced from each treatment
|
Dose (Gy) |
Cultivation period (Four replicates of mushroom's
bed) |
Characteristic of the fruiting body at button stage |
|
0 (control) |
18 days |
i) White colour ii) No hairy structure presence |
|
300 |
15 days |
i) White colour ii) Presence of hairy structure at the base of the
fruiting body |
|
600 |
15 days |
i) White colour ii) Presence of hairy structure at the base and the surface of fruiting body |
|
900 |
15 days |
i) White colour ii) Presence of hairy structure at the base and the
surface of fruiting body |
|
1200 |
18 days |
i) White colour ii) Presence of hairy structure at the base and the
surface of fruiting body iii) Most fruiting bodies produced in clustered form |
|
1500 |
24 days (only one mushroom's bed produce the
fruiting body) |
i) White colour ii) Presence of hairy structure at the base and the
surface of fruiting body |
%2053-59_files/image002.png)
Fig. 1: The mycelial growth on PDA at day 7 for each
irradiation doses; (a) 0, (b) 300, (c) 600, (d) 900, (e) 1200 and (f) 1500 Gy
%2053-59_files/image004.png)
Fig. 2: The mean growth rate of mycelia (mm/day ± S.E)
on PDA for each dose of irradiation throughout seven days
%2053-59_files/image006.png)
Fig. 3: The growth rate of mycelia (mm/day ± S.E) on
wheat for each dose of irradiation for 12 days
produce higher amounts of
essential metabolites, developing agriculturally and economically significant
varieties besides potentially increasing productivity (Noordin et al.
2014). In this study, only spawn colonized with 1500 Gy irradiated mycelia
showed very poor production, while other treatments showed no significant
difference to control treatment. Therefore, any increase in the induction of
gamma radiation on mycelium more than 1500 Gy possibly shows no mushroom
production. Apart from that, Lee et al. (2000) investigated the gamma irradiation exposure on P.
ostreatus mycelia at 2000 Gy, which showed a reduction in mycelial growth
compared to mycelia irradiated at 1000 Gy. However, the study concluded that
the mycelia irradiated at 2000 Gy also able to produce a fruiting body.
Table 2: Mean
number, weight (g), and texture of fruiting body for each treatment
|
Treatment (Gy) |
Number ± SE |
Weight ± SE (g) |
Texture ± SE (N) |
|
0 |
6.00 ± 1.22a |
105.38 ± 26.82ab |
13.75 ± 0.55a |
|
300 |
6.75 ± 1.25a |
104.00 ± 22.58ab |
13.04 ± 0.22a |
|
600 |
7.75 ± 1.31a |
140.25 ± 21.43a |
12.41 ± 0.10a |
|
900 |
8.00 ± 0.58a |
162.88 ± 10.54a |
12.25 ± 0.33a |
|
1200 |
2.75 ± 0.48b |
43.88 ± 16.81bc |
12.48 ± 0.84a |
|
1500 |
0.25 ± 0.25b |
2.50 ± 2.50c |
Nd |
Nd is not determined. The same superscript letter for each
value in the same column showed no significant difference (P
> 0.05) between the treatments
%2053-59_files/image008.png)
Fig. 4: The mycelial growth on wheat at Day 12 for
each irradiation dose; (a) 0, (b) 300, (c) 600, (d) 900, (e) 1200 and (f) 1500 Gy. The mycelia completely
covered the wheat at Day 12 for treatments 0, 300, 600 and 900 Gy. The mycelial growth of treatments 1200 and 1500 Gy at Day 12 showed the arrow in Fig. 4 (e) and (f)
Besides, Ramchander
et al. (2015) stated that the nature of mutation could be decided by
determining the correct radiation doses. Therefore, a mutation can happen to
the mycelial strain at certain radiation doses, causing no production of
fruiting bodies of V. volvacea. The texture analysis of a mushroom
indicates that the mushroom has high firmness. However, this study concluded
that acute gamma irradiation does not affect the firmness of the fruiting body.
Other than that, Shrivastava (2006) also showed a similar result for oyster mushrooms where
there was no significant difference in the texture of mushrooms between all
treatments during storage. However, Hou et al. (2018) indicate that
gamma irradiation (0.8 kGy) on the fruiting body of V. volvacea helps in
maintaining their good quality and firmness for seven days storage period under
16 ± 0.5°C compared to other irradiation doses. Another study was done by
Xiong et al. (2009) on gamma irradiation of Pleurotus nebrodensis
at 1200 and 2000 Gy, which reduced fruiting bodies firmness by 45 and 62%,
respectively. Meanwhile, the reduction percentage of the fruiting body firmness
control group (non-irradiated) was 58%. Although the study on gamma irradiation showed a significant
effect on the mushroom morphology, the IAEA (1992) reported that irradiation
with an average dose of 10000 Gy shows no toxicological hazards and introduces
no specific microbial or nutritional problems. Therefore, the irradiation
method can be implemented in mushroom production for commercial purposes.
Conclusion
Acute gamma radiation on mycelia showed a significant effect on the spawning
rate of V. volvacea. Further study on the effect of acute gamma
radiation on nutrient content and physiological changes in fruiting bodies of V.
volvacea is highly recommended. It is hoped that this study will stimulate improvement
in current mushroom cultivation methods, thereby contributing to the production
and quality of the V. volvacea simultaneously.
Acknowledgments
All the work was carried out by facilities provided at
the Faculty of Agriculture, Universiti Putra Malaysia and Malaysian Nuclear
Agency. This work was supported by Malaysian Higher Education (MOHE) for the
FRGS grants (FRGS/1/2016/WAB01/UPM/02/15. We are thankful to Mohamad Yuzaidi
Azmi from the Department of Agriculture (DOA) Padang Terap, Kedah, for his
willingness to participate in this study.
Authors Contributions
FNF performed
the methodology, collected and analyzed the result. Also, FNF wrote the
manuscript. SA, AM, designed the conceptual ideas and interpreted the results.
SA, SS and BA participated and revised the manuscript. Apart from that, SA,
MTMM, AM participated and supervised the project. All authors have read and
agreed to the published version of the manuscript.
Conflicts of Interest
All authors declare no conflicts of interest.
Data Availability
Data presented in this study will be available on a
fair request to the corresponding author.
Ethics Approval
Not applicable.
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