Contenuto totale di flavonoidi, perossidazione lipidica e attività antiossidante di <em>Hericium coralloides</em>, <em>Fomes fomentarius</em> e <em>Schizophyllum commune</em> coltivati col metodo del confronto diretto
10.6092/issn.2531-7342/10668
Utilizzando il metodo del confronto diretto sono stati studiati la crescita miceliare, la fruttificazione  il contenuto di flavonoidi totale, la perossidazione lipidica e le  proprietà antiossidanti di  Hericium coralloides, Fomes fomentarius e Schizophyllum comune su agar patata con glucosio. La co-coltivazione è stata accompagnata da una inibizione della crescita delle colonie. L'effetto inibitorio più elevato è stato riscontrato in H. coralloides e il più basso in S. commune.Nei corpi fruttiferi di H. coralloides, F. fomentarius e S. commune formatisi in co-coltura (tranne che nella combinazione  H. coralloides + F. fomentarius ), è stato osservato un aumento del contenuto di flavonoidi totali e dell'attività antiossidante totale. Le condizioni di confronto diretto hanno dimostrato essere uno stress per le culture perché è stato osservato un aumento del livello di perossidazione lipidica nei corpi fruttiferi.La tendenza all'accumulo del contenuto totale di flavonoidi nei corpi fruttiferi di H. coralloides, F. fomentarius e S. commune si è rilevato è un importante aspetto legato all’attività antifungina nella co-coltivazione di macromiceti, in quanto l'accumulo di flavonoidi è stato direttamente correlato all'entità dell'inibizione di crescita di H. coralloides e F. fomentarius. Ciò significa che i funghi hanno chiari meccanismi di contrapposizione a eventuali altri concorrenti che sviluppano nel medesimo substrato.
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Mariia Pasailiuk
Italian Journal of Mycology
stress
Total flavonoid content, lipid peroxidation and total antioxidant activity of <em>Hericium coralloides</em>, <em>Fomes fomentarius</em> and <em>Schizophyllum commune</em> cultivated by the method of direct confrontation
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https://italianmycology.unibo.it/
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Articles
2020-05-26
2020-03-17
2020-02-10
2020-09-28
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Ital J Mycol
Mariia Pasailiuk
2020/05/26
10.6092/issn.2531-7342/10668
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Hutsulshchyna National Nature Park, 84 Druzhba St., UA-78600, Kosiv, Ukraine
2531-7342
stress
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Zhang ZL, Qu W (2004) Experimental Guidance of Plant Physiology, High Education Press, Beijing, China.
Total flavonoid content, lipid peroxidation and total antioxidant activity of <em>Hericium coralloides</em>, <em>Fomes fomentarius</em> and <em>Schizophyllum commune</em> cultivated by the method of direct confrontation
https://italianmycology.unibo.it/article/download/10668/11049
49
Pasailiuk                                                                  Italian Journal of Mycology 49: 25-37 (2020)

DOI: 10.6092/issn.2531-7342/10668


Original Paper

   Total flavonoid content, lipid peroxidation and total antioxidant
      activity of Hericium coralloides, Fomes fomentarius and
          Schizophyllum commune cultivated by the method
                        of direct confrontation

                                               Mariia V. Pasailiuk

Hutsulshchyna National Nature Park, 84 Druzhba St., UA-78600, Kosiv, Ukraine

Corresponding author e-mail: mariia.pasailiuk@gmail.com

Article info
Received 17/03/2020; accepted 21/05/2020
DOI: 10.6092/issn.2531-7342/10668

Abstract
The patterns of culture growth, fruiting, total flavonoid content, lipid peroxidation and antioxidant properties were
established for Hericium coralloides, Fomes fomentarius and Schizophyllum commune, and cultivated on Potato Glucose
Agar by the method of direct confrontation. Co-cultivation is accompanied by the inhibition of the colonies’ growth. The
highest inhibitory effect was found to H. coralloides, and minimal inhibitory values were established to S. commune.
In the fruiting bodies of H. coralloides, F. fomentarius and S. commune that were formed on a different co-culture
composition (except H. coralloides + F. fomentarius composition), an increase of total flavonoid content and total
antioxidant activity were observed. Direct confrontation conditions are a stress for cultures because an increase of lipid
peroxidation level in the fruiting bodies was observed.
Tendency of accumulation of total flavonoid content in the H. coralloides, F. fomentarius and S. commune fruiting
bodies is an important antifungal aspect in co-cultivation of macromycetes, because the accumulation of flavonoids
directly correlates to the magnitude of inhibition of the H. coralloides and F. fomentarius culture growth. This means
that fungi have clear mechanisms of counteraction to competitors. However, practical application of this phenomenon is
questionable.

Keywords
antagonism; culture; fruiting bodies; Malondyaldehyde; stress

Introduction

Hericium coralloides (Scop.) Pers. is an edible fungus of great significance in medicine (Pallua et al.
2012). It produces whitish basidiomata growing mostly on dead wood of Fagus sylvatica L., causing
white rot (Ko et al., 2005; Boddy et al., 2011; Pallua et al., 2012). Hericium coralloides is a rare
fungus found in most countries of Europe and Asia http://iucn.ekoo.se/iucn/species_view/120231/,
including Ukraine.
       The main conservation measures for H. coralloides on the territory of Ukraine and other countries
include the protection of its gene pool in culture collections (ex-situ method) (Saxena and Gupta,
2019) and in fungal populations of nature reserves and nature parks (in situ conservation method). Re-
situ cultivation technology of rare mushrooms in nature has been developed and successfully tested
in Hutsulshchyna National Nature Park (NNP), Kosiv, Ukraine (Petrichuk et al., 2017, Pasailiuk et
al., 2018). Re-situ is a method that provides introduction and support of vital mushroom functions in
nature by forming basidiomata (Pasailiuk et al., 2018). Some steps of growing H. coralloides by re-situ

ISSN 2531-7342 Department of Agricultural and Food Science in partnership with the Italian Mycological Society (UMI).
The journal is hosted and mantained by ABIS-AlmaDL. © 2020 The Authors. This work is licensed under CC BY 4.0
Pasailiuk                                                       Italian Journal of Mycology 49: 25-37 (2020)

DOI: 10.6092/issn.2531-7342/10668


method include growing the mycelium in the laboratory and inoculating mycelium of H. coralloides
in beech logs found nature. When re-situ technology of cultivation was initiated, it was established
that beech logs were colonised by Fomes fomentarius (L.) Fr. and Schizophyllum commune Fr. and
were not suitable for H. coralloides.
       Fomes fomentarius has a wide distribution. It is a white-rot fungus that has been recorded on a
wide range of hardwood trees (beech, birch, oak, poplar, maple, etc.) (https://mycocosm.jgi.doe.gov/
Fomfom1/Fomfom1.home.html). It is found that F. fomentarius has significant properties, including
its role in antioxidant enzyme activities, enzymatic activities, antimicrobial activities, antifungal
activities (Bal and Akgül, 2018).
       Schizophyllum commune is one of the most widely distributed and common mushrooms on
Earth (Kuo, 2003). It grows anywhere that dead wood is found (http://www.michigannatureguy.com/
blog/2013/11/28/split-gill-fungus/) causing white-rot (Ujor, 2010). Schizophyllum commune is an
edible mushroom having medicinal value and it is also a very good source of protein, vitamins, lipids
and mineral elements (Zahida et al., 2015).
       We deduce that there is an antagonism between H. coralloides and other widely distributed
xylotrophic fungi sharing the same ecological niche, on beech wood. This conclusion for F.
fomentarius and S. commune was confirmed on malt extract agar and wort agar (Pasailiuk et al., 2019).
The mechanisms providing the base of antagonistic effect and biochemical landscape of changes that
occurred during direct confrontation remains unclear.
       Competition between mushrooms is a phenomenon well-described between macromycetes
and micromycetes within one Petri dish (Barinova et al., 2008). Recent works demonstrate the
mechanisms of so-called “fungi wars” (Hiscox et al., 2018), the existence of which is possible in
natural conditions due to the synthesis by certain fungal species of biologically active substances.
In this case, obviously, mushrooms with better “vitality” will exhibit better adaptability and higher
viability in the same conditions. The properties and ability to counteract other fungi are attributed to
compounds of flavonoid nature. The process of such struggle, in our view, is stressful and must be
accompanied by biochemical changes typical of stress.
       Therefore, the aims of this study are: 1) to investigate the fungal mechanisms of counteraction
to competitors; 2) to study the patterns of culture growth and opportunity of fruiting of H. coralloides,
F. fomentarius and S. commune cultivated on Potato Glucose Agar (PGA) by the method of direct
confrontation; 3) to determinate the total flavonoid content (as a possible antifungal factor), the MDA
content (as the level of lipid peroxidation and the marker of stress), and the antioxidant properties of
H. coralloides, F. fomentarius and S. commune fruiting bodies growing on PGA in the competition
conditions.


Materials and Methods

Mycelial culture
The pure cultures of H. coralloides (strain 2332), S. commune (strain 1763), F. fomentarius (strain
(1528) were obtained from the Mushroom Culture Collection of the M.G. Kholodny Institute of
Botany of the National Academy of Sciences of Ukraine (IBK), #– 1152 in the international database
of the World Federation for Culture Collections – WFCC (Bisko et al., 2016, http://www.wfcc.info/
ccinfo/index.php/collection/by_id/1152/).

Nutrient media and culturing fungi
Single fungal isolates and their combinations were cultivated on PGA nutrient medium (Potato 200 g
l-1, Glucose 10 g l-1, Agar 20 g l-1, pH 5.8) at 22 ± 1 °C to complete overgrowth of the Petri dishes by
the mycelium and at 14 ± 1 °C after complete overgrowth to fruiting.

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Determination of antagonist activity
The antagonist activity on PGA was studied according to Camporota (1985) and Bouziane et al.
(2011). The method of direct confrontation was used. It means fungal cultures of H. coralloides (Hc),
F. fomentarius (Ff) and/or S. commune (Sc) were placed in the same Petri dish (culture combinations:
Hc + Ff; Hc + Sc, Hc + Ff + Sc). Each Petri dish contained 15 ml of PGA and two or three pieces
of the inoculum (diameter of 5 mm), which were positioned in a diametrically opposite manner at a
distance of 5 cm apart and 2 cm from the edge of the Petri dish. We assessed mycelial growth on the
66th days by measuring the diameter of the colony.
      Inhibition of H. coralloides was quantified as the percentage of mycelial growth inhibition
using the following formula:
                                         I% = (1 – Cn/Co) × 100

      where Cn is the average diameter of the Hc colonies in the presence of Ff and/or Sc, and Co is
the average diameter of Hc colonies in the control (Camporota, 1985; Bouziane et al., 2011).
      Inhibition of Ff (Sc) was quantified as the percentage of mycelial growth inhibition using the
following formula:
                                          I% = (1 – Cn/Co) × 100

     where Cn is the average diameter of the Ff (Sc) colonies in the presence of Hc and/or Sc (Ff),
and Co is the average diameter of Ff (Sc) colonies in the control.

Sample Collection
Fruiting bodies (teleomorphs) of H. coralloides, F. fomentarius and S. commune were collected in
according fruiting time, i.e. on day 66 in co-cultures and on day 115 (H. coralloides), on day 68 (F.
fomentarius) and on day 69 (S. commune) in single cultures.

Preparation of Mushroom Extracts
Organic extraction of the mushroom was carried out according to the method described in Jinting et
al. (2017). Fruiting bodies (without medium) were minced and dried at room temperature for 2 hours.
Accurately, 20 ml of methanol and chloroform were added at 1:1 volume ratio. The homogenate was
agitated for 2 hours, filtered with normal filter paper, refluxed for 3 hours. The dried extract was re-
dissolved in 100% dimethyl sulfoxide (DMSO) for highest concentration and finally stored at 4 ˚C.

Determination of Total Flavonoid Content
Total flavonoid content was determined following the method by Singh et al. (2012) and Jinting et al.
(2017). Accurately, 1 ml of sample or standard was diluted with 4 ml distilled water and 0.3 ml of 5%
sodium nitrate solution was added. After 6 minutes, 0.3 ml of a 10% aluminium chloride solution was
added to the mixture. The mixture was incubated at room temperature for 5 minutes. Then, 2 ml of 1M
sodium hydroxide was added to the mixture after 5 minutes of incubation. The mixture was vortexed
thoroughly and the absorbance of the pink colour was measured at 510 nm against a blank by using
UV-1100 Spectrophotometer. Quercetin was used for the calibration curve with a concentration range
of 1 – 100 µg ml-1. Results were expressed as micrograms quercetin equivalent (μg QE g-1) of dried
extract. All experiments were carried out in four replicates.

Determination of total antioxidant activity by phosphomolybdenum assay
Different concentration of Butylated Hydroxy Toluence (BHT) (5-125 μg ml-1) in distilled water were
prepared. 0.3 ml of mushroom extracts were taken in test tubes. The reagent solution was prepared
by mixing 10 ml of 0.6 M sulphuric acid, 10 ml of 28 nM sodium phosphate and 10 ml of 4 mM
ammonium molybdate into a beaker and 3 ml reagent solution was added to all the tubes. Three

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DOI: 10.6092/issn.2531-7342/10668


hundreds microliters of methanol served as blank. All the tubes were incubated at 95 ºC for 90 min.
The tubes were cooled to room temperature and the optical density was measured at 695 nm using UV-
1100 spectrophotometer. Results were expressed as a μg equivalent of Butylated Hydroxy Toluene
× gram of fruiting body of H. coralloides, F. fomentarius and S. commune in phosphomolybdenum
assay by the method of Prieto et al. (1999) as described by Imran et al. (2011).

Estimation of lipid peroxidation
The measurement of Malondialdehyde (MDA) has been used as an indicator of lipid peroxidation.
MDA was measured according to the thiobarbituric acid reaction as described by Zhang and Qu (2004)
and Chen et al. (2014). Two hundreds milligrams of fruiting bodies samples was homogenized with
5% trichloroacetic acid (TCA) and centrifuged at 4,000 g for 10 min. Two ml of extract was added
to 2 ml 0.6% TBA placed in a boiling water bath for 10 min, and the absorbance at 532, 600, and
450 nm, namely, A532, A600, and A450, respectively, was determined spectrophotometrically. The
MDA concentration was calculated according to the formula: 6.45 × (A532 − A600) − 0.56 ×A450 and
expressed as nmol (nM) MDA × gram of fruiting body.

Data analysis
All experiments were carried out in four replicates. Data obtained were analyzed by using Statistica
8.0 (StatSoft Inc., USA) and Pearson’s correlation coefficient was performed. The data are presented
as mean ± standard deviation. Significant differences between means are indicated at P ≤ 0.05 level.


Results

Effects of joint cultivation on PGA on mycelia growth
To compare the mycelial growth of H. coralloides with and without competitive fungi, we inoculated
mycelium of F. fomentarius and/or mycelium of S. commune on the same Petri dishes. After 66 days
of joint cultivation, (the time where all competitive fungi had fruited on PGA) the colony diameters
of H. coralloides, F. fomentarius and S. commune were measured. We found all culture growth was
inhibited by joint cultivation. The most greater inhibition effects were observed for H. coralloides in
using combination Hc + Sc – 90% and Hc + Ff – 84% (Table 1).

      Table 1 - Growth of Hericium coralloides cultivated with confronting cultures on PGA,
      day 66
                                          Colony diameter of
          Combination of cultures1
                                          H. coralloides (cm)                 Inhibition (%)
                                        Experiment        Control
          Hc + Ff + Sc                  3.93 ± 0.17*                          51.24 ± 2.12
          Hc + Ff                       1.30 ± 0.14*       8.05 ± 0.21        84.07 ± 1.81
          Hc + Sc                       0.80 ± 0.08*                          90.06 ± 1.01
      1
       Hc = Hericium coralloides, Ff = Fomes fomentarius, Sc = Schizophyllum, * Significant
      differences from the control, Р ≤ 0.05

      Cultivation of H. coralloides in triple composition is accompanied by the less values of inhibition
(51%), than in double (84% and 90%). Cultivation of H. coralloides and F. fomentarius together has
significant inhibition effect on F. fomentarius growth (Table 2) not only on H. coralloides. To S.
commune inhibition effect was the less (Table 3).

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      Table 2 - Growth of Fomes fomentarius cultivated with confronting cultures on PGA, day
      66
                                            Colony diameter of
          Combination of cultures1
                                            F. fomentarius (cm)                 Inhibition (%)
                                        Experiment         Control
          Hc +Ff +Sc                     4.40 ± 0.08*                           51.11 ± 0.91
          Hc+Ff                          3.00 ± 0.36*      9.00 ± 0.08          66.67 ± 3.96
          Ff +Sc                         5.80 ± 0.08*                           35.56 ± 0.91
      1
       Hc = Hericium coralloides, Ff = Fomes fomentarius, Sc = Schizophyllum, * Significant
      differences from the control, Р ≤ 0.05

      Table 3 - Growth of Schizophyllum commune cultivated with confronting cultures on
      PGA, day 66
                                           Colony diameter of
          Combination of cultures1
                                           S. commune (cm)                      Inhibition (%)
                                        Experiment        Control
          Hc +Ff +Sc                    6.95 ± 0.31*                            22.78 ± 3.45
          Hc+Ff                         7.05 ± 0.13*      9.00 ± 0.08           21.67 ± 1.43
          Ff + Sc                       6.90 ± 0.29*                            23.33 ± 3.27
      1
       Hc = Hericium coralloides, Ff = Fomes fomentarius, Sc = Schizophyllum commune
      * Significant differences from the control, Р ≤ 0.05

Effects of joint cultivation on PGA on culture fruiting
Starting from day 61 to day 66 we observed fruiting of confronting cultures (Fig. 1). The combination
of Hc + Ff was a single combination where fruiting bodies were not formed and the content of
flavonoids, total antioxidant activity and MDA were not studied accordingly. We observed fruiting of
single cultures, i.e., and the control, too (Fig. 2).




            Figure 1 - Formation of teleomorphs of Hericium coralloides (Hc), Fomes
            fomentarius (Ff) and Schizophyllum commune (Sc) (except the combination Hc+ Ff)
            for direct confrontation between cultures, day 66




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               Figure 2 - Formation of teleomorphs of Hericium coralloides (Hc,
               day115), Fomes fomentarius (Ff, day 68) and Schizophyllum commune
               (Sc, day 69) on PGA

       We found a difference in the time frame of fruiting for monocultures compared to the cultures
in direct confrontation conditions. For the monoculture of H. coralloides on PGA, almost total over
growth of the Petri dishes happened on days 30 ± 1, with teleomorphs forming on days 110–115.
For the monoculture of F. fomentarius on PGA, total overgrowth of the Petri dishes happened on
days 15 ± 1, with teleomorphs forming on days 63–68. For the monoculture of S. commune on PGA,
total overgrowth of the Petri dishes happened on days 18 ± 1, with teleomorphs forming on days
64–69. Therefore, fruiting bodies of H. coralloides, F. fomentarius and S. commune were harvested
accordingly on days 115, 68 and 69 and total flavonoid content, total antioxidant activity and lipid
peroxidation by content of MDA were studied.
       Total overgrowth of the Petri dishes by confronting cultures happened on days 32 ± 2 (except the
combination of Hc + Ff). Starting from day 61 to day 66, we observed fruiting of confronting cultures
(except the combination of Hc +Ff). Therefore, fruiting bodies of H. coralloides, F. fomentarius and
S. commune growing in competitive conditions were harvested on day 66 and total flavonoid content,
total antioxidant activity and lipid peroxidation by content of MDA were studied.

Comparison of total flavonoid content in fruiting bodies of confronting fungi
Total flavonoid content (μg QE g-1 dried extract) of fruiting bodies of control variants and their
experimental combinations are presented in Figure 3.
       In this study H. coralloides cultivated on PGA, exhibited the total flavonoid content in the
teleomorphs with 1.14 ± 0.03 μg QE × gram dried extract, F. fomentarius cultivated on PGA exhibited
the total flavonoid content in the teleomorphs with 0.44 ± 0.02 QE g-1, Schizophyllum commune
cultivated on PGA exhibited the total flavonoid content in the teleomorphs with 0.64 ± 0.16 QE g-1
(Fig. 3).
       The present study indicates that co-cultivation of xylotroph cultures is accompanied by a
significant increase of total flavonoid content in the fruiting bodies compared to the monoculture
conditions. Moreover, during the confrontation in all fruiting bodies, we observed the increase of total
flavonoid content. Thus, the total flavonoid content for H. coralloides fruiting bodies in the conditions
of the triple combination increased in 4.7 times, and for the combination of Hc+ Sc, it increased
almost 10-fold (Fig. 3).
       In the cases with high inhibition effect to H. coralloides culture, we detected higher total
flavonoid content of H. coralloides fruiting bodies. Between these values, the coefficient of correlation
was + 0.99. In a similar occurrence, the dependence of high total flavonoid content in the teleomorphs
correlated to high levels of inhibition of culture growth for F. fomentarius (coefficient of correlation
was + 0.84) but not for S. commune (coefficient of correlation was + 0.24).
       We observed an increase of total flavonoid content in the teleomorphs of F. fomentarius in 14.8-
fold (triple combination), and in 11.7-fold (combination of Ff + Sc). For S. commune the increase of

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total flavonoid content was maximal and ranged from 4.5 (in 7-fold) to 13.16 μg QE g-1 dried extract
(in 20.6-fold regarding control).




                       Figure 3 - Total flavonoid content in the fruiting bodies of fungi
                       on PGAin single cultures (control) and in different co-culture
                       combinations, day 66. * Significant differences from the control,
                       Р ≤ 0.05


Studying of total antioxidant activity and lipid peroxidation in the fungi competitive mechanism
By studying of lipid peroxidation, we observed that the direct confrontation is accompanied by an
increase MDA level in formed fruiting bodies, with the exception of S. commune fruiting bodies in
the variant Hc+ Sc. MDA significantly increased, ranging from 15% for S. commune in the variant Ff
+ Sc to 35% for H. coralloides in the combination Hc+ Sc (Table 4).

    Table 4 - Total antioxidant activity and lipid peroxidation in the teleomorphs of Hericium
    coralloides, Fomes fomentarius and Schizophyllum commune
        Combination of cultures     Sample    Total antioxidant activity      Lipid peroxidation (nM
                                              (μgBHT/mg DW)                   MDA/g fruiting body)
        Hc + Ff + Sc                Hc        16.33±0.32*                     365.00±15.34*
                                    Ff        25.30±1.57*                     354.00±26.14*
                                    Sc        15,56±1.02*                     531.25±17.97*
        Hc + Sc                     Hc        32.35±5.57*                     428.00±24.10*
                                    Sc        21,85±1.46*                     445.25±30.67
        Ff + Sc                     Ff        19,37±1.18*                     317.00±18.57*
                                    Sc        56,65±1.54*                     487.25±21.84*
        Hc (control)                Hc         4.20±0.08                      316.75±6.18
        Ff (control)                Ff         2.06±0.06                      263.25±12.31
        Sc (control)                Sc         3.12±0.45                      423.00±19.82
    1
     Hc = Hericium coralloides, Ff = Fomes fomentarius, Sc = Schizophyllum commune
    * Significant differences from the control, Р ≤ 0.05

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Discussion

The co-cultivation of H. coralloides, F. fomentarius and S. commune cultures on PGA presented
growth inhibition. Thus, testing cultures counteracting one another was important. Interestingly, each
fungus is listed as a white rot fungi, therefore each of them has similar ecological-trophic requirements
and this fact maybe is a reason of antagonism.
       The inhibition of H. coralloides growth in triple composition was almost 2-fold lower than when
H. coralloides was cultivated in dual combinations. This type of counteraction is similar to additive
antagonism type (Folt et al., 1999). Not only was bilateral inhibition of culture growth established on
the PGA medium for the Hc + Ff combination, but it was too fixed stopping of the colonies growth
before their contact and fruiting bodies did’nt form. Therefore, no further studies (total antioxidant
activity, total flavonoid content, MDA level) have been performed for this variant.
       As it has been shown the chemical composition of the nutrient medium plays an important
role in the manifestation of the antagonistic strengths of the cultures (Pasailiuk et al., 2019). Type
of interaction ranged from inhibition H. coralloides growth on WA to stimulation H. coralloides
growth on Czapek + cellulose medium. Thus, antagonistic interactions are determined by the type
of combination, the chemical composition of the nutrient medium. It addition, these antagonistic
interactions can be mediated at a distance and following contact. Similar conclusions are demonstrated
by Boddy (2000), Boody et al. (2008) and Hiscox et al. (2015). Undoubtedly, the inhibitory effect is a
consequence of the complex and diverse morphological, physiological and biochemical changes that
are affected by the present species and environmental conditions.
       The scholarly work of Griffith et al. (1994), Boddy (2000), Baldrian (2004), Hynes et al.
(2007), Boddy et al. (2008), Evans et al. (2008) under score that the result of a culture’s interaction
could be determinated by pigment production, release of volatile organic compounds (which also act
as info-chemicals), diffusible enzymes, toxins and antifungal metabolites. Additionally, it has been
demonstrated that the interaction of fungi with plant hosts or microorganisms may be accompanied
by oxidative stress (Breitenbach et al., 2015).
       Our results demonstrate that co-cultivation of cultures is accompanied by antagonism, the
inhibition of growth and oxidative stress too, because we register an increase of MDA (Table 4).
Thus, cross-contamination of cultures is a stress factor.
       Oxidative stress happens due to an increase of reactive oxygen byproducts. Free radicals induce
lipid peroxidation, playing an important role in pathological processes. The stress mediated by free
radicals can be measured by conjugated dienes, malondialdehyde, 4-hydroxynonenal, and others.
However, MDA has been pointed out as the main product to evaluate lipid peroxidation (Grotto et al.
2009). In the last 20 years, MDA has been recognized as an important lipid peroxidation indicator,
and could be used as a biomarker to detect damage in the body (Simioni et al., 2018).
       An increase of MDA levels in H. coralloides fruiting bodies is directly related to the inhibitory
effect on the H. coralloides mycelium growth which occurred in the presence of other cultures. When
we observed a high percentage of growth inhibition, we also observed a hight MDA concentration,
with the correlation coefficient of + 0.88. Consequently, co-cultivation can be considered a stress
factor for H. coralloides culture. At the same time, the accumulation of MDA in the F. fomentarius
fruiting bodies and its inhibition in the different combinations of F. fomentarius culture growth are
values that correlate less closely, with the correlation indicator of + 0.64. In the S. commune fruiting
bodies, the significant increase of MDA levels and the inhibition indicators of the growth of this
culture, are not completely correlated. Therefore, co-cultivation is a reason the increase of MDA can
be considered a stress factor for F. fomentarius and S. commune cultures too.
       It is known that there are two principal mechanisms of protection against oxidative stress –
regulation of membrane permeability and antioxidant potential (Lushchak, 2011). When cultures are

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grown together on the same Petri dish, a large number of stress factors can arise from competitive
conditions. There is a limited surface of free space for growth, exhausting nutrient resources of the
medium.
       As a rule, many species of fungi form teleomorphs in the Petri dishes during the last stages of
mycelium growth, in fact before death. Teleomorphs are important for macromycetes resettlement in
nature, and formation of them is genetically programmed (Bukhalo, 1988). Thus, despite the limited
resources of the nutrient medium and competition from each to other, mushrooms not only survive,
but also perform a standard conservation program and form the fruiting bodies (Fig. 1). What are the
mechanisms of the cultures counteraction (in fact antifungal) in this struggle?
       The antifungal properties are attributed to compounds of flavonoids nature. In vitro experimental
systems show that flavonoids possess anti-inflammatory, anti-allergic, antiviral, and anti-carcinogenic
properties (Kavuru, 2008). Flavonoids promote physiological survival of plant by protecting it
from pathogenic microorganisms and UV radiations (Hayat et al., 2017). Flavonoids are secondary
metabolites. Phenolic and flavonoid molecules are important antioxidant components which are
responsible for deactivating free radicals based on their ability to donate hydrogen (Aryal et al.,
2019). Total flavonoid content is one of the most important quality indexes of living organisms and it
is concerned with total antioxidant activity through the scavenging of oxygen – derived free radicals.
Various health benefits arise due to the high proportion of antioxidants (Shi et al., 2019).
       After studying total flavonoid content in the fruiting bodies of the monocultures of H. coralloides,
F. fomentarius and S.commune, we found a relatively low level of flavonoid content. The established
indicators are much less than the ones described in the literature for the content of flavonoids in the
fruiting bodies of wild-growing exemplars of these species (Heleno et al., 2015; Vamanu and Voica,
2017). Co-cultivation was accompanied a significant increasing of total flavonoid content in the
fruiting bodies of all cultures against to control (Fig. 3). Besides, the accumulation of total flavonoid
content in the fruiting bodies of H. coralloides and F. fomentarius is a direct consequence of the
culture growth inhibition.
       An increase in total flavonoid content in our experiment is not the only example where the
content of these compounds increases as a response to stress factors. Stress triggered by salinity and
drought are also accompanied by an increase in total flavonoid content. For example, under salinity
stress, total phenolics and flavonoids, contents of vanillin and protocatechuic acid in tolerant varieties
of rice cultivar (Oryza sativa L.) increased (Minh et al., 2016). The result, influenced by drought on
oxidative stress and flavonoid production in the cell suspension culture of Glycyrrhiza inflata Bat.
indicated that water deficit enhanced biomass accumulation and flavonoid production, which was
about 2-fold and 1.5-fold of the control, respectively (Yang et al., 2007). Literature has shown that
drought stress elevates bioactive compounds, vitamins, phenolics, flavonoids and antioxidant activity
in many leafy vegetables (Sarker and Oba, 2018). Abiotic stresses induce an increase of total phenolic,
total flavonoid and antioxidant properties in endemic, Malaysian microalgae and cyanobacterium
(Azim et al., 2018).
       In addition, oxidative damage to cellular components is often associated with aging in a wide
range of organisms, including fungi (Aung-Htut et al., 2012). Storage of postharvested mushrooms
is accompanied by an increase of MDA and total flavonoid content in fruiting bodies (Meng et al.,
2008). Therefore, we selected well-formed, mature (but not old) fruiting bodies of H. coralloides,
F. fomentarius and S. commune from co-cultivation conditions and from monocultures in our
experiment. Experiments were conducted after drying of fruiting bodies on the same day of collection
of exemplars. Therefore, the changes we observed for total flavonoid content and MDA regarding the
control are a reaction to competitive conditions, not age-related symptoms.
       So, our study showed that co-cultivation positively affected flavonoid accumulation in
mushroom, which may further promote the improvement of antioxidant status in the fruiting bodies.

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Pasailiuk                                                        Italian Journal of Mycology 49: 25-37 (2020)

DOI: 10.6092/issn.2531-7342/10668



We assume that the increase of total flavonoid content is clearly evidence of the antifungal properties
of flavonoids and their potential in scavenging oxygen-derived free radicals. This assumption seems
plausible due to the fact that the composition of Hc + Sc, in the S. commune fruiting bodies, showed
that the MDA level did not exceed the control values. Therefore, the higher level of total flavonoid
content in the S. commune fruiting bodies exhibits a lower level of oxidative stress and ultimately a
higher competitive ability relative to other cultures.
       Total antioxidant capacity is an analysis frequently used to assess the antioxidant status of
biological sample. It can evaluate the antioxidant response against the free radicals produced in a
given situation. In physiological conditions, oxy-radicals are part of the normal regulatory course of
the organism and the cellular redox state is closely controlled by antioxidants. When the levels of free
radicals increases and both the enzymatic systems and low molecular antioxidants are not sufficient
to protect the organism, these radicals attack membranes and cells (Rubio et al., 2016).
       The results of studying total antioxidant activity supports the hypothesis that flavonoid
accumulation in mushrooms positively affects total antioxidant activity and it was demonstrated that
co-cultivation was accompanied by a significant increase of total antioxidant activity in the fruiting
bodies of each studied culture (Table 4).
       Therefore, a high level of oxidative stress in the joint cultivate subjects leads to an imbalance in
the fruiting body antioxidant capacity. The increased antioxidant capacity may signify an increase of
antioxidant protection in co-cultures and demonstrate high amounts of total flavonoid content.


Conclusions

Co-cultivation of fungal cultures with the same ecological and trophic requirements is accompanied
by the inhibition of their colonies’ growth. On PGA, F. fomentarius and S. commune are confirmed
to be antagonists to H. coralloides. Co-cultivation is a stress for cultures, and is accompanied by
an increase of MDA and biologically active substances in the fruiting bodies. Our study suggest
that accumulation of total flavonoid content in the H. coralloides, F. fomentarius and S. commune
is an important antifungal aspect in co-cultivation of these fungi. This means that fungi have clear
mechanisms of counteraction to competitors. However, practical application of this phenomenon
is questionable. A full spectrum of metabolites of secondary synthesis and their ratios needs to be
studied.


Acknowledgements. The author thanks Nina Fontana, PhD candidate for editorial assistance with
the manuscript.


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