The influence of laser irradiation on the development of vegetative myceliumPleurotus ostreatus
Kateryna Reshetnyk,
Yurіy Prysedsky,
Dmytro Yuskov
Department of Plant Physiology and Biochemistry, Vasyl’ Stus Donetsk National University, 600-richya St. 21, Vinnytsia 21021, Ukraine
The article presents the results of the study on the influence of
laser irradiation on the development of vegetative mycelium and
the period of the occurrence of the corcules of the fruit bodies of
Pleurotus ostreatus. It has been established the growth processes
of P. ostreatus, can be best stimulated by applying laser irradiation
of mycelium with a green spectrum of light for 10 s. According
to this mode of exposure, the best increase in the growth rate of
mycelium of 38.3% and the appearance of the largest number of
rudiments of the fruiting bodies were recorded. Laser irradiation
of mycelium for 10 s with red and blue light spectrum increased
the growth rate of mycelium from 7.41 to 20.4%, respectively, and
the number of rudiments of the fruiting bodies increased by 1.5
to 2 times. Laser irradiation of the mycelium with 5 s, 15 s, and
20 s with red, blue, and green light spectra did not have a significant effect on the growth processes of P. ostreatus. These data
open significant prospects for the modification of the existing
cultivation technologies, which would increase the economic efficiency of the biotechnological cultivation process of P. ostreatus.
After Agaricus bisporus, Pleurotus ostreatus is
known as the second most cultivated species of
edible fungi in the world. Due to the ability of
the Pleurotus genus to grow on a variety of lignocellulosic wastes, their cultivation can be an effective technology for the processing of organic
waste products in agriculture, which would enable the production of protein-rich products and
contribute to the reduction of environmental
pollution (Bhattacharjya et al., 2015; Figlas et al.,
2016). On the other hand, the use of fungi as food
will overcome the deficiency of protein in the developing countries. Relying on standard mushroom cultivation techniques, researchers add
new technological components to this process
that allow shortening the cultivation time, increasing the yield and the quality of the fruiting
bodies. However, the solution of these issues
is impossible without the use of knowledge on
the physiology of fungi, combined with information on the environmental factors that affect
the growth and development of macromycetes
(Tisch, Schmoll, 2010).
One of these factors is the light that regulates
morphogenetic processes in many types of fungi, although they are not phototrophic organisms. The nature of the effect of light depends
on its spectral characteristics and the duration
of the light (Kamada et al., 2010). Mushrooms
can absorb almost ultraviolet, blue, green, red,
and distant red light, using up to 11 photoreceptors and signalling cascades to control most
of the genome and adapt to environmental
conditions (Zhenzhong, Reinhard, 2018). Recently, the mechanisms of the photoreceptor of
fungi have attracted intensive research (Kritskiy et al., 2010; Tisch, Schmoll, 2010; Poyedinok, 2015). Several types of photoreceptors in
mushrooms were described (Herrera–Estrella,
Horwitz, 2007). Thus, in the basidymicetes Coprinus cinereus, Pleurotus ostreatus, and Lentinula edodes the genes encoding the receptors
responsible for the perception of blue light
were found. The study into the fungal genome
revealed the photoreceptor genes encoding
proteins that are sensitive to red light (Kamada et al., 2010). The green light is perceived
by opsin systems based on retinal, the biological functions of which still need to be clarified
(Zhenzhong, Reinhard, 2018). The use of artificial light to stimulate biological processes in
fungi is limited by the methods that require
prolonged illumination of crops at different
stages of morphogenesis, which leads to additional energy expenditure.
On the other hand, studies conducted by
Karu at the cellular level for some biological objects showed that short-term irradiation
(a few seconds) with lowintensity light in relatively small doses (102–103 J/m2) contributes
to a long-lasting macro effect (Karu, 1986).
The positive influence of UV and γ irradiation
on the yields of P. ostreatus fungus was also
investigated, and it was found that laser irradiation at doses of 45–230 mJ/cm2 stimulated
sprout growth and mycelium growth in Hericium erinanceus. The known influence of lowintensity light on the linear growth and biomass
accumulation by different types of macromycetes (Agaricus bisporus, Inonotus obliguus,
Ganoderma lucidium, Hericium erinanceus,
and Lentinula ededes) (Poyedinok et al., 2013).
According to Poyedinok, at different stages of
ontogenesis, macromycetes are sensitive to
low-intensity light in the visible wavelength
range with different spectral and energy characteristics (Poyedinok, 2015). According to
modern mechanisms of photoregulation of living organisms, photochemical reactions occur
due to the excitation of electrons in the atoms
of the absorbing light of matter. At the molecular level, this is expressed as photoionization of matter, its reduction, photooxidation,
photodissociation of molecules, and their reorganisation – photoisomerization (Karu et al.,
1994). Analysis of the results of research on
photoreceptor mechanisms in mushrooms obtained by local and foreign scientists proves
the possibility of using laser radiation in order
to increase the biological activity of cultivated
macromycetes, to stimulate their growth and
intensify the technological stages of cultivation.
Nonetheless, the above methods of intensifying
growth processes are complex in terms of practical use, since they require the use of stationary
laser installations, argon and helium-neon lasers that have significant energy intensity, large
dimensions and masses. LED lasers, which
have high efficiency (up to 50%), speed (up to
10–11 s), comfort of excitation, the possibility
of generating radiation with the required wavelength, small dimensions and technological
compatibility with elements of optical integrated circuits are much more effective for the intensification of metabolic processes of macromycetes (Vasiura, 1998). Also, they are low cost
and require little power consumption when
applied. However, the literature data on the influence of LED laser systems on the parameters
of fungal growth are limited, thus this issue requires further study. The purpose of the work
was to investigate the effect of laser irradiation
on the development of vegetative mycelium
and the period of the appearance of the buds of
the fruiting bodies of P. ostreatus.
MATERIALS AND METHODS
The P-192 strain of Pleurotus ostreatus (Jacq.:Fr.)
Kumm mushroom from the collection of
cabbage mushrooms of the Department of
Plant Physiology and Biochemistry of Vasyl’
Stus Donetsk National University was studied.
The strain under research was isolated in a pure
culture of wild-growing fruiting bodies of basidium mushrooms collected in different areas
of the Donetsk region. In order to study the effect of laser irradiation on the development of
the vegetative mycelium of the P. ostreatus fungus, the strain P-192 was cultivated for seven
days in standard wort-agar environment (4° by
Baling) in standard Petri dishes (9 cm in diameter), prior to the inoculation of mycelia in Petri
dishes separated by a sterile steel tube into pieces 5 × 5 mm in size. Then each inoculum was
irradiated with LED lasers: BRP-3010-5 witha 635 nm red wavelength radiation, BBP-3010-5 with the radiation of the blue spectrum at
a wavelength of 405 nm, and BGP-3010-5 with
the green spectrum of radiation at a wavelength
of 532 nm (BOB LASER Co., China). The power
of each laser was 100 mW. The density of the energy of laser irradiation was calculated by Vakarchuk (Vakarchuk, 2012). The energy dose
of irradiation (the energy of light falling into
the unit area) was determined as the product of
the energy density and the time of irradiation.
In all variants of the experiment, the irradiation
energy was within the range of 25–102.5 mJ/
cm2. This value was selected according to the results of the literature (Poyedinok, 2015). Irradiation of mycelium was carried out in keepeing
with the scheme presented in Table 1. Unsaturated mycelium was used for control culturing.
Surface cultivation of mycelium P. ostreatus was
carried out in a thermostat at a temperature of
26°C in a standard wort-agar medium (4° Baling). Each day of the cultivation period, the radius of the colonies on the media in Petri dishes
was measured in four perpendicular directions.
The presence of “buckles” – specific morphological formations on the P. ostreatus hyphae – was
determined by microscopic control. After complete overgrowth of Petri dishes with mycelium,
they were transferred to a growth chamber
at a temperature of 12–16°C to be tested for
the ability to form the buds of the fruiting bodies (primordia).
Table 1. The scheme of irradiation of mycelial fungus P. ostreatus
Sample variant
Duration of irradiation, s
Energy of irradiation, mJ/cm2
Red
Blue
Green
1
0
0
0
0
2
5
0
0
25.05
3
0
5
0
25.05
4
0
5
5
25.05
5
10
0
0
51.1
6
0
10
0
51.1
7
0
0
10
51.1
8
15
0
0
77.3
9
0
15
0
77.3
10
0
0
15
77.3
11
20
0
0
102.5
12
0
20
0
102.5
13
0
0
20
102.5
Based on the obtained data, the average
daily gain of mycelium, the average speed of
the radial growth (mm/day), the time interval
before the formation of primordia (number of
days), and the average number of primordia per
Petri dish were determined. In order to evaluate the growth of the cultures, a method based
on the study and analysis of the dynamics of increasing the radius of colonies from the time of
cultivation was applied (Bysko et al., 1983).
All the necessary experiments were replicated three times. The method of dispersion analysis was applied to determine the probability of
exposure to laser irradiation. The comparison
of the mean values was based on the Dunnet
method. The processing was carried out usinga package of statistical programs designed at
the Department of Plant Physiology and Biochemistry of the Donetsk Vasyl’ Stus National
University (Prysedskyy, 2005).
RESULTS AND DISCUSSION
Since the photoreceptor system of mushrooms
is known to be adapted to light in the range
of wavelengths from 350 to 730 nm (Corrochano, 2007), this spectral row is represented
by the wavelengths chosen by us (red, blue and
green spectra). The results of the research demonstrate the positive effect of laser irradiation
on the development of vegetative mycelium
of the studied strain P. ostreatus. The response
to irradiation varied according to the spectral
characteristics of light and the duration of exposure (Table 2).
From day 3 of cultivation, a significant probable difference was observed in the growth of
micelles irradiated with red (Option 5), blue
(Option 6), and green (Option 7) light for 10 s
compared to non-irradiated mycelium (Option 1). In particular, the average daily gain of
mycelium was 3.27, 2.76, and 3.72 mm respectively, i.e., these figures exceeded the control
figures by 32.7%, 27.6%, and 37.2%, respectively. Laser irradiation of mycelium for 5 s in
the 2nd and 3rd experimental variants accelerated the average daily gain of the mycelium of
the investigated strain by 1.42 mm (red spectrum of irradiation), 1.91 mm (blue irradiation
spectrum), and 1.61 mm (green spectrum of
irradiation) compared with the control sample. Laser irradiation of mycelium for 15 s with
red, blue and green spectra probably increased
mycelium growth by only 14.8% (option 8)
and 12.7% (options 9 and 10), respectively. Because of irradiation of mycelium for 20 s, no
significant change in average daily sprout was
observed. On day 4 of cultivation, a significant
increase in mycelium was observed because of
laser irradiation for 10 s with the green light:
by 38.3% more than the control sample. The reaction in response to the effect of the red and
blue light spectra at the same duration of exposure was quite high: the average daily gain
of mycelium increased by 28.5% and 21.5%,
respectively. Micellar irradiation for 5, 15, and
20 seconds did not cause significant changes in
mycelium growth. For the effects of green (variant 7) irradiation with a duration of 10 s already
for 8 days of cultivation, the radius of the colonies reached 45.05 mm, which is 19.2% more
than the control sample. The Petri dishes were
completely overgrown with mycelium. In other
variants of radiation, the change in the radius
of colonies of mycelium was negligible and only
for the effects of red and blue laser irradiation
this figure increased by 11.2% and 4.5%, respectively.
Table 2. Increase of the radius ofP. ostreatuscolonies due to laser irradiation
Note. * – the difference is statistically significant compared to the control variant (P < 0.05), (variants of the experiment according to Table 1).
We found out that laser irradiation positively
influenced the average rate of the radial growth
of mycelial P. ostreatus. Thus, for the effects
of red laser irradiation of mycelium for 10 s,
the average speed of radial growth was 20.4%
higher than the control ones, while the effects
of exposure to the blue spectrum increased by
7.4% (Fig. 1).
The best growth was recorded as a result of
irradiation with the green light for 10 seconds,
the average speed of the radial growth exceeded the control sample by 27.8%. Laser irradiation of micelles for 5 s, 15 s, and 20 s with
red, blue, and green light spectra did not have
a significant effect on the growth processes of P. ostreatus.
The next stage of our research was the study
of the effect of laser irradiation of mycelium on
the formation of the buds of the fruiting bodies (primordia). The appearance of the buds
was recorded as early as dya 13 of cultivation
in almost all variants of experiment, including
the control one. In the dishes with unpexed mycelium, the first primordia appeared on day 15
of cultivation. On day 13 of cultivation, most of
the primordia (nine pieces) were formed under
the effects of laser irradiation with the green
spectrum of light for 10 s. Irradiation with red
and blue light for 10 s contributed to the formation of primordia in six pieces. Under the
effect of laser irradiation of mycelium for 5 s
and 15 s of red, blue, and green light, there were
5–6 pieces of primordia. For these modes of
micellar irradiation for 20 s, the number of rudiments of the foetal bodies was insignificant
(1–3 pieces). On day 15 of cultivation, the largest number of primordia was formed under
the action of radiation for 10 s with the green
light – 180.2% more than the control ones. Irradiation with the red and blue light for 10 s
increased the number of primordia from 80.3%
to 120.4%, respectively. Laser irradiation of
mycelium over 5 s and 15 s with red, blue and
green light increased the number of formed
primordia from 40.3 to 60.4%, respectively. Under the effects of radiation for 20 s, the number
of primordia was almost the same as in the control sample. On day 18 of cultivation, the maximum number of buds of the fruiting bodies was
recorded under the irradiation of mycelium for
10 s with the green spectrum of light, which is
137.5% higher than the control sample. Micellar irradiation for 5 s, 15 s, and 20 s with red,
blue and green light spectra increased the number of primordia by 1.5–2 times. On day 21 of
cultivation, the largest number of primordia
(26 pieces) was formed due to laser irradiation of mycelium with the green light for 10 s.
The irradiation with blue and red light for 10 s
increased the number of primordia by 46.1%
and 38.4%, respectively. In other studied modes
of exposure, the number of primordia increased
from 23.5 to 30.1% (Fig. 2).
Fig. 1. Influence of laser irradiation on the average rate of the radial growth of P. ostreatus mycelium
When irradiated for 20 s, the number of primordia almost did not exceed the control sample. On day 24 of cultivation, the most buds of
the fruiting bodies formed due to the effects of
green laser irradiation that lasted for 10 s; their
number was 73.7% higher than the control variant. Laser irradiation with the blue light for 10 s
contributed to an increase in the number of primordias by 36.8% compared to the control variant. Irradiation with the red spectrum increased
the number of buds of fruiting bodies by 26.3%.
The irradiation of mycelium for 5 s and 15 s with
red, blue and green spectra increased the number of primordia by 15.8–26.3%. Under irradiation of mycelium for 20 s, a small difference was
found between the number of buds of the fruiting bodies compared with the control variant.
It was established that laser irradiation affects
the growth processes of P. ostreatus. The reaction in response to the laser exposure depends
on the duration of exposure and the spectrum
of light. The effect of green laser irradiation that
lasted for 10 s is the most effective. In this mode
of exposure, the best increase in the growth
rate of mycelium was found to be 38.3%, and
the greatest number of buds of the fruiting bodies appeared. Laser irradiation with red and blue
spectra increased the average radial growth rate
from 7.4% to 20.4%, respectively, and the number of buds of the fruiting bodies increased by
1.5– 2 times, respectively. The irradiation of mycelium that lasted for 5 s, 15 s, and 20 s did not
have a significant effect on the growth processes
of P. ostreatus. The results of our research coincide with the literature data (Doroshkevich, 2007
Poyedinok, 2015). Responses to the action of laser irradiation are associated with the changes
in the parameters of cellular homeostasis, and
they fit into the theory of universal mechanisms
of photostimulation. According to this theory,
the main physical or chemical changes caused
by exposure to photoconceptor molecules are
accompanied by a cascade of biochemical reactions in cells that do not require further activation by light (Karu et al., 1994). The stimulation
of growth processes for the production of uterine
and sown micelles is crucial in the cultivation of
macromycetes, since it reduces the probability of
pathogenic microflora contamination with mycelium and improves its adaptive properties.
Fig. 2. The effect of laser irradiation on the period of appearance and the number of rhizomes of the fruiting bodies of P. ostreatus (variants of the experiment according to Table 1)
CONCLUSIONS
The research validates the fact that the best
way to stimulate P. ostreatus growth processes,
is to apply laser irradiation of mycelium with
the green light spectrum for 10 s. The effect of
this irradiation regime resulted in a maximum
increase in the growth rate of mycelium by
38.3% and the appearance of the largest number of buds of the fruiting bodies. Laser irradiation of mycelium for 10 s with red and blue
light spectra increased the growth rate of mycelium from 7.4% to 20.4%, respectively, and
the number of buds of the fruiting bodies increased by 1.5–2 times. Laser microwave irradiation for 5 s, 15 s, and 20 s with red, blue
and green spectra of light did not significantly
affect the growth processes of P. ostreatus. These
data open significant prospects for the modification of existing cultivation technologies,
which would increase the economic efficiency
of the process of biotechnological cultivation of
P. ostreatus.
Received 19 June 2019
Accepted 8 November 2019
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LAZERIO SPINDULIUOTĖS POVEIKIS GLUOSNINĖS KREIVABUDĖSPLEUROTUS OSTREATUSGRYBIENAI
Santrauka
Straipsnyje pateikiami lazerio spinduliuotės poveikio
vegetatyviniam grybienos vystymuisi ir Pleurotus ostreatus vaisiakūnių formavimuisi tyrimų rezultatai.
Nustatyta, kad norint stimuliuoti P. ostreatus augimo
procesus, geriausia grybieną 10 sek. veikti žalios šviesos spektro spinduliuote. Taikant šį būdą buvo užfiksuotas ryškus grybienos augimo greičio padidėjimas
(38,3 %) ir didžiausias vaisiakūnių užuomazgų skaičius. Paveikus grybieną raudonos ir mėlynos šviesos
lazeriu 10 sek., grybienos augimo greitis padidėjo
atitinkamai 7,41 ir 20,4 %, o vaisiakūnių užuomazgų
skaičius išaugo 1,5–2 kartus. Lazerinė spinduliuotė
raudonos, mėlynos ir žalios šviesos spektru 5 sek.,
15 sek. ir 20 sek. neturėjo reikšmingo poveikio P. ostreatus augimo procesams. Šie duomenys atveria
didelę esamų auginimo technologijų modifikavimo
perspektyvą, o mūsų tiriamuoju atveju patvirtina
P.ostreatus biotechnologinio auginimo proceso didesnį ekonominį efektyvumą.