Do Ganoderma Species Represent Novel Sources of Phenolic Based Antimicrobial Agents?

Ganoderma species have been recognized as potential antimicrobial (AM) agents and have been used in traditional Chinese medicine (TCM) for a long time. The aim of this study is to examine the AM potential of autochthonous Ganoderma species (G. applanatum, G. lucidum, G. pfeifferi and G. resinaceum) from Serbia. The extraction of fungal material was prepared in different solvents (ethanol—EtOH, water—H2O, chloroform—CHCl3). Antibacterial activity (ABA) was determined using disk-diffusion, agar-well diffusion, and micro-dilution method, while for antifungal properties disk-diffusion and pour plate method were applied. Antiviral activity was tested on model DNA virus LK3 and determined by plaque assay. Statistical PCA analysis was applied for detection of correlation effects of phenolics and AM activities, while LC-MS/MS was performed for phenolics quantification. G. resinaceum CHCl3 extract expressed the most potent ABA against P. aeruginosa (MIC = 6.25 mg/mL), probably due to presence of flavonoids and 2,5-dihydroxybenzoic acid. Among H2O extracts, the highest ABA was determined for G. pfeifferi against both E. coli and S. aureus (21 and 19 mm, respectively). EtOH extracts of G. pfeifferi and G. resinaceum were the most effective against A. niger (23.8 and 20.15 mm, respectively), with special impact of phenolic acids and flavonoid isorhamnetin, while C. albicans showed the lowest susceptibility. The most potent antiviral inhibitor was G. lucidum (70.73% growth inhibition) due to the high amount of phenolic acids. To the best of our knowledge, this is the first report of a methodical AM profile of G. pfeifferi and G. resinaceum from the Balkan region including PCA analysis.


Introduction
One of the global public health threats to humans is the emergence of multi-drugresistant organisms, which compromise the capacity of medical professionals to treat an expanding range of infectious diseases [1,2]. This follows increased drug-dose consumption, longer hospital stays, higher toxicity of drugs, and increased mortality [3]. One of the most common micro-organisms that have developed multi-resistance are Staphylococcus aureus, Enterococcus faecium, Enterococcus faecalis [4], Streptococcus pneumonia, Acinetobacter baumannii, Mycobacterium tuberculosis, Pseudomonas aeruginosa, Salmonella enterica and Vibrio cholerae [5]. Consequently, the creation of novel, more effective pharmaceuticals to combat current antibiotic-resistant bacteria is urgently needed.
Nonetheless, systemic fungal infections are on the rise, especially in immunocompromised and post-operative patients [6]. Moreover, mucormycosis is becoming a serious

Mycochemical Profile by LC-MS/MS Detection
The results of both chemical analyses (LC-MS/MS) and biological activities (antiinflammatory, antioxidant, antidiabetic, antiproliferative) of selected Ganoderma species (EtOH and H 2 O extracts) have been published in our previous studies [23,25,33] (Table S1). Chemical identifications of G. applanatum and G. lucidum EtOH and H 2 O extracts were published in 2016 [23], while LC-MS/MS results of the same extracts for G. pfeifferi and G. resinaceum were published in 2020 [25]. A new mycochemical profile of all mentioned species was reported in 2020 [33]. On the other hand, in the present study, quantifications of phenolic compounds of CHCl 3 extracts of G. applanatum and G. resinaceum were conducted using LC-MS/MS analysis, due to examination of AB activity and identification of potentially active secondary metabolites (Table 1). <Number: detected compound-peak observed, concentration is lower than the LoQ (limit of quantification), but higher than LoD (limit of detection); d.w.-dry weight.
The scientific data indicate that chemical screening of CHCl 3 extracts of G. applanatum confirmed the presence of flavonoids and phenols, together with saponins and glycosides [34], while alkaloids, flavonoids, terpenoids and saponins were found in EtOH and H 2 O extracts of G. lucidum [1,16]. In contrast to G. applanatum and G. lucidum, from which more terpenoid compounds and polysaccharides have been isolated, G. pfeifferi has received much less attention from mycochemical studies [35].

Antimicrobial Activity
Ganoderma species may exhibit various AM activities based on the polarity of the chemicals produced by the extraction solvent. It was previously summarized by Cowan and Plant [29] that different chemical substances can affect a diverse mechanism of AM activity; caffeic acid is one of the substances within a large group of phenylpropanederived compounds with the greatest oxidation state. Moreover, there is evidence that increasing hydroxylation causes increased toxicity, and that the site(s) and amount of hydroxyl groups on the phenol group are thought to be related to their relative toxicity to microorganisms [29]. Hence, in this study three different solvents were used for detection of AM activity.

Antibacterial Activity
Based on antibiogram results (Table S2), it can be concluded that the largest number of tested strains are sensitive to amikacin, except for E. faecalis ATCC 19433 , which is resistant. Resistance to tetracycline was shown by: P. aeruginosa ATCC 35554 , E. coli ATCC 11229 , whereas the strains K. aerogenes ATCC 13048 , B. cereus ATCC 11778 and E. coli ATCC 11775 were of intermediate sensitivity. All bacterial strains showed resistance to methicillin and kanamycin, except for two strains S. aureus ATCC 6538 and S. aureus ATCC 25923 . Additionally, both B. cereus ATCC 11778 and P. aeruginosa ATCC 35554 were resistant to ceftriaxone, while the other strains proved to be sensitive to this antibiotic. Multi-resistance is observed in E. coli ATCC 11229 , B. cereus ATCC 11778 and P. aeruginosa ATCC 35554 .
The highest AB activity of H 2 O extracts, using diffusion assays, was observed for G. pfeifferi against E. coli ATCC 11775 and B. cereus ATCC 11778 , with inhibition zones of 21 and 19 mm, respectively (Table 2). High susceptibility against these two bacterial strains was also observed for G. lucidum and G. resinaceum (16 and 13 mm, respectively), while G. applanatum showed the highest AB against S. aureus ATCC 255923 . Diffusion-based approaches against P. mirabilis ATCC 12453 yielded no results, while against P. aeruginosa only G. resinaceum extract was active.
Analyses using three different methods showed that H 2 O extracts had the highest activity against Gram-positive bacterial strains, with some minor activity against Gramnegative bacteria. In addition, MIC and MBC against bacterial strains were determined in an in vitro microdilution test (Table 2), with lower values indicating greater efficacy of the tested extract, i.e., higher AB activity. The following order of activity was obtained: G. resinaceum (100 mg/mL) > G. applanatum (100 mg/mL) > G. pfeifferi (200 mg/mL) > G. lucidum (200 mg/mL). The highest MBC activity was demonstrated by G. resinaceum on multi-resistant B. cereus strain, which confirmed the previous conclusion that H 2 O extracts are more effective against Gram-positive bacteria.   All tested CHCl 3 extracts of G. resinaceum and G. applanatum exhibited AB activity ( Table 3). The analyzed strains showed significant variation in their MIC and MBC values, which varied in the range: MIC = 6.125-25 mg/mL and MBC = 25-50 mg/mL, indicating the strain specificity of the analyzed micro-organisms. G. resinaceum exhibited the highest AB activity (MIC = 6.25 mg/mL) against multi-resistant P. aeruginosa ATCC 3554 , which was more than twice the strength of the activity of streptomycin (MIC = 16 mg/mL). The CHCl 3 extracts of this fungal species were the least effective against both K. pneumoniae HP and B. cereus HP , whereas high activities against E. coli ATCC 11229 , S. aureus ATCC 6538 and S. enteritidis ATCC 13076 (MIC = 12.5 mg/mL) were recorded. G. applanatum showed the highest AB activity against multi-resistant E. coli ATCC 11229 and B. cereus HP with MIC of 12.5 mg/mL. The MBC values of both fungal species (G. applanatum and G. resinaceum) against S. aureus ATCC 6538 , S. enteritidis ATCC 13076 and B. cereus HP were recorded at the same concentration of 25 mg/mL, while the values against other bacterial strains differed (Table 2). Table 3. Antibacterial activity of CHCl 3 extracts of G. applanatum and G. resinaceum using microdilution assay. Comparing all Ganoderma species tested in this research, it is evident that G. resinaceum expressed the most potent antibacterial activity. Al-Fatimi et al. [11] compared AB activity of selected basidiomycetes from Yemen, where G. resinaceum also stood out with the highest activity. Moreover, Al-Fatimi et al. [11] compared the level of AB activity of three different types of extracts (CH 2 Cl 2 , MeOH and H 2 O). Different AB activity related to different solvents used was also observed in their study, with H 2 O being the most active, especially against P. aeruginosa and S. aureus (20 mm inhibition zone) [11]. The effectiveness of H 2 O extracts of G. resinaceum against these two bacteria strains was also confirmed in another study [16]. H 2 O extracts of G. lucidum fruiting body from India exhibited weaker AB activity against E. coli, with inhibition zone diameters ranging from 7 to 13 mm [15]. On the other hand, contrary to results from our study, G. lucidum extracts from India showed activity against S. aureus and P. aeruginosa [15], while Mousavi et al. [17] reported good AB activity of MeOH extract against Gram-negative bacteria (E. coli and P. aeruginosa). Furthermore, H 2 O extracts of G. lucidum collected in Turkey and Nepal showed activity against E. faecalis and P. aeruginosa [1,36], which was not consistent with our results. H 2 O extracts of G. applanatum from India exhibited similar AB susceptibility against S. aureus (inhibition zone 12 mm) and no activity against P. aeruginosa, while higher activity was observed against E. coli [34]. Different activities obtained by Karaman et al. [13] on different strains of S. aureus, demonstrated that the fungal extracts were target-specific at the intraspecies level, which is also in accordance with the results of this study. G. pfeifferi exhibited the highest AB susceptibility against E. coli (21 mm), which is in accordance with the report [19]; among Gram-negative bacteria, E. coli was the most affected by volatile oil isolated from this fungus (15 mm). Moreover, G. pfeifferi volatile oil exhibited the largest inhibition zones against Gram-positive bacteria, including Bacillus subtilis (20 mm) and S. aureus (30 mm) [19], which also supports the results obtained in this study ( Table 2).

Bacterial Strain
The inhibitory action of the extracts against particular bacteria strains is directly correlated with the extraction process [37]. The results obtained for the tested CHCl 3 extracts of both G. applanatum and G. resinaceum show stronger AB effects at lower concentrations (25 mg/mL) compared to MeOH (50 mg/mL) [12,38]. Zengin et al. [24] reported weak AB activity of H 2 O and MeOH extracts of these Ganoderma species against E. coli and P. aeruginosa. In contrast, Nagaraj et al. [34] outlined good activity of MeOH extracts against S. aureus, E. coli and B. subtilis, followed by H 2 O, petroleum ether and CHCl 3 extraction.
In this study, H 2 O extract of G. resinaceum was effective against P. aeruginosa, but the highest AB activity was observed for CHCl 3 extract of this fungal species. The fact that AB activity was noticed in extracts with different polarities implies that these Ganoderma species contain substances with AB action. Two new hydroquinones isolated from G. pfeifferi (ganomycin A and ganomycin B) were confirmed to have AB activity by TLC chromatography [27,39]. Two isolated lanostanoid compounds from G. applanatum, 5-ergost-7-en-3-ol, ergost-7,22-dien-3-ol and 5,8-epidioxy-5,8-ergost-6,22-dien-3-ol primarily showed activity against Gram-positive bacteria [40]. Of the non-polar compounds, antimicrobial activity is mainly attributed to terpenoid compounds [40]. The exceptions are ganomycins isolated from G. pfeifferi, which are phenolic in nature. Moreover, Nagaraj et al. [34] associated the good AB activity of MeOH extracts of G. applanatum with higher solubility of detected active compounds (saponins, phenols, steroids, glycosides, terpenoids and flavonoids) in alcohol. High content of phenols in the form of coumarins, flavonoids and tannins were also detected in G. applanatum [23,33,38]. Results are consistent with previous reports which demonstrated that the most active components are typically water insoluble and that low polarity organic solvents would provide more active extracts [12,39,41]. Furthermore, the activity profile of a more lipophilic extract solvent (CHCl 3 ) exhibited similarities between G. resinaceum and G. applanatum, suggesting that the solvent might be extracting comparable chemicals from these Ganoderma species. This is also in accordance with the results obtained in the research of different Ganoderma species from Nigeria, where CHCl 3 :CH 3 COCH 3 extracts were the most active [39]. Significant AB activity was observed in similar tests of CHCl 3 extracts of G. lucidum, where predominantly non-polar molecules were documented as distinct antibacterial agents [12,29,42]. For example, in several studies rutin was documented as a potent AB agent against E. coli and P. aeruginosa [43,44], while Arima et al. [45] reported its synergistic effect with other flavonoids against B. cereus [46]. Thus, we can assume that non-polar compounds, such as rutin, detected in this study in CHCl 3 extract may also be responsible for high AB susceptibility of G. resinaceum.
By comparing methods applied in examination of AB activity, we can conclude that microdilution method was the most accurate since the obtained data is clearer and objective. In addition to solvents and different methods of testing AB activity, growing conditions also represent very important factors for assessing AB susceptibility. Jorcin et al. [47] investigated the effects of culture conditions on AB activity of G. resinaceum and determined that larger-scale cultures in malt extract broth supplemented with 20 g/L glucose and a 15-day incubation should be carried to isolate antibiotic compounds that may be effective against S. aureus. Alves et al. [48] also proved the AM activity of phenolic acids. Based on above results, it is believed that fungal extracts could be a source of substances used as a substitute for commercial antibiotics, to which certain micro-organisms are proven to be resistant.

Antifungal Activity
AF activity of EtOH extracts (range 10-100 mg/mL) of G. pfeifferi and G. resinaceum was examined against S. cerevisiae, C. albicans-yeast and filamentous Aspergillus niger, by measuring the inhibition zone (mm) ( Figure 1). Moreover, AF of these species was determined by applying the pour plate method for lower extract concentrations (0.5, 2.5 and 5 mg/mL) in order to measure the diameter of mycelia growth in a more precise manner for AF activity determination ( Figure 2). tive. In addition to solvents and different methods of testing AB activity, growing conditions also represent very important factors for assessing AB susceptibility. Jorcin et al. [47] investigated the effects of culture conditions on AB activity of G. resinaceum and determined that larger-scale cultures in malt extract broth supplemented with 20 g/L glucose and a 15-day incubation should be carried to isolate antibiotic compounds that may be effective against S. aureus. Alves et al. [48] also proved the AM activity of phenolic acids. Based on above results, it is believed that fungal extracts could be a source of substances used as a substitute for commercial antibiotics, to which certain micro-organisms are proven to be resistant.

Antifungal Activity
AF activity of EtOH extracts (range 10-100 mg/mL) of G. pfeifferi and G. resinaceum was examined against S. cerevisiae, C. albicans-yeast and filamentous Aspergillus niger, by measuring the inhibition zone (mm) ( Figure 1). Moreover, AF of these species was determined by applying the pour plate method for lower extract concentrations (0.5, 2.5 and 5 mg/mL) in order to measure the diameter of mycelia growth in a more precise manner for AF activity determination ( Figure 2).  In most cases regarding the disk diffusion method, a concentration dependence was observed, i.e., the level of AF activity increases with the Ganoderma extract concentration ( Figure 1). However, the highest inhibition was demonstrated by extracts of G. resinaceum tive. In addition to solvents and different methods of testing AB activity, growing conditions also represent very important factors for assessing AB susceptibility. Jorcin et al. [47] investigated the effects of culture conditions on AB activity of G. resinaceum and determined that larger-scale cultures in malt extract broth supplemented with 20 g/L glucose and a 15-day incubation should be carried to isolate antibiotic compounds that may be effective against S. aureus. Alves et al. [48] also proved the AM activity of phenolic acids. Based on above results, it is believed that fungal extracts could be a source of substances used as a substitute for commercial antibiotics, to which certain micro-organisms are proven to be resistant.

Antifungal Activity
AF activity of EtOH extracts (range 10-100 mg/mL) of G. pfeifferi and G. resinaceum was examined against S. cerevisiae, C. albicans-yeast and filamentous Aspergillus niger, by measuring the inhibition zone (mm) (Figure 1). Moreover, AF of these species was determined by applying the pour plate method for lower extract concentrations (0.5, 2.5 and 5 mg/mL) in order to measure the diameter of mycelia growth in a more precise manner for AF activity determination ( Figure 2).  In most cases regarding the disk diffusion method, a concentration dependence was observed, i.e., the level of AF activity increases with the Ganoderma extract concentration ( Figure 1). However, the highest inhibition was demonstrated by extracts of G. resinaceum In most cases regarding the disk diffusion method, a concentration dependence was observed, i.e., the level of AF activity increases with the Ganoderma extract concentration ( Figure 1). However, the highest inhibition was demonstrated by extracts of G. resinaceum with the lowest tested conc. (10 mg/mL) against A. niger, with an inhibition zone of 14 mm, following G. pfeifferi and G. resinaceum extracts against S. cerevisiae (12 mm) at the same concentration. G. pfeifferi was the least effective against C. albicans strains, while AF activity of G. resinaceum was obtained in the following order: A. niger > S. cerevisiae > C. albicans (Figure 1).
Considering the variations in activity, a more detailed examination of the Ganoderma extracts at lower concentrations was conducted by using the pour plate method (Figure 2). AF activity was inversely proportional to the examined concentration of both Ganoderma extracts, demonstrating very strong AF capacity, especially against A. niger. Furthermore, results are in accordance with growth diameters obtained by the disk-diffusion method since the extracts were most effective against A. niger. The lowest activity was shown against C. albicans (Figures 1 and 2). G. pfeifferi revealed better AF potential against A. niger (19.67-25.25 mm) and C. albicans (5.42-8.75 mm) compared to G. resinaceum, where inhibition zones were in ranges of 17.08 to 21.75 mm and 4.33 to 6.92 mm, respectively.
Obtained results are in accordance with the research of Suansia and John [18], where mycelial growth of both A. niger and A. flavus was completely inhibited by Ganoderma sp. EtOH and MeOH extracts. On the other hand, volatile oil isolated from G. pfeifferi was significantly efficient against C. albicans (MIC = 0.6 mg/mL) [19], while C. maltosa showed resistance to MeOH and H 2 O extracts of G. resinaceum [11]. Zengin et al. [24] compared AF activity of G. applanatum and G. resinaceum, where only G. applanatum MeOH extracts showed AF potential against C. albicans at a concentration 2.5 mg/mL. Furthermore, C. albicans and C. maltosa were resistant to sesquiterpenoid hydroquinones and ganomycins A and B from G. pfeifferi [27], suggesting that some other metabolites are responsible for AF activity. p-Coumaric and vanillic acid identified in EtOH extracts of G. pfeifferi and G. resinaceum [25,49] were reported as moderate AF agents against Fusarium oxysporum, F. verticillioides, Penicillium brevicompactum, P. expansum, A. flavus and A. fumigatus with percentage inhibition ranging from 18.70 to 100.00% [26]. Nevertheless, results of a study conducted by Zabka and Pavela [26] showed that phenols had a substantially better efficiency than phenolic acids. Moreover, we can assume that rutin found in this study in the CHCl 3 extract of G. pfeifferi may be responsible for AF activity. Other research also reported this flavonoid as a more potent AF agent against C. krusei compared to fluconazole, while moderate action was observed against C. albicans [50]. On the other hand, numerous literature data indicate the importance of proteins and polysaccharides, such as glucan [51][52][53][54], for exhibiting AF activity by activating and enhancing the immune response; thus, its use is recommended in combination with other antibiotics and immunostimulators in the prevention and treatment of infectious diseases [55]. Wang and Ng [53] isolated ganodermin, a low molecular weight protein (15 kDa) from G. lucidum, that acts by inhibiting the mycelial growth of three phytopathogenic fungi: B. cinerea, F. oxysporum and P. piricola. Moreover, much weaker but not negligible activity was also demonstrated for applanoxidic acid isolated from Ganoderma annulare on Trichophyton mentagrophytes [56]. It can be concluded that the extracts of both examined species have active substances for suppressing fungal growth; however, considering the variations in activity and potential activity of decreased concentrations in G. pfeifferi, a more detailed examination of the extracts at lower concentrations, as well as the purification of individual active components with potential AF action, is necessary.

Antiviral Activity
Animal and plant viruses, as well as bacterial viruses that serve as model organisms for other viruses, are frequently used to assess the AV efficacy of fungal extracts. In this paper, the vB_BbrS_56.1 bacteriophage belonging to the Siphoviridae family was used as a model virus for examination of AV activity of four Ganoderma species. Both EtOH and H 2 O extracts as well as EtOH extract solution in 5% DMSO were analyzed in the range of concentrations from 0.2 to 200 mg/mL, while the AV activity was presented as the percentage of viable phages for each concentration (Table 4). All examined Ganoderma species (G. applanatum, G. lucidum, G. pfeifferi and G. resinaceum) exhibited AV activity during incubation for 24 h, at 37 • C in the tested concentration range. Based on the obtained results, the most potent antiviral agents were EtOH extracts dissolved in 5% DMSO, with the emphasis on G. lucidum where 73.39 ± 1.35 % inhibition was reached at the lowest tested concentration (0.2 mg/mL). Among EtOH and H 2 O extracts, the highest inhibition potential showed G. lucidum and G. resinaceum, followed by G. pfeifferi which demonstrated moderate activity. On the contrary, G. applanatum showed the lowest AV activity in all tested extracts ( Table 4).
The high AV activity of G. lucidum is in line with a previous study, where triterpenoids (lanosta-7,9(11),24-trien-3-one,15; 26-dihydroxy and ganoderic acid Y) isolated from this species exhibited significant inhibition of enterovirus 71 (EV71) cytotoxicity in human rhabdomyosarcoma cells [28]. Furthermore, AV potency of several ganoderic acids, ganoderol A, ganoderol B, ganodermanondiol, and ganodermanontriol was confirmed in another study [20]. According to the findings of Zhang et al. [28], these triterpenoids prevent AV infection by interacting with the viral particle and preventing the virus from adhering to cells; computer molecular docking revealed that they may bind to the viral capsid protein at a hydrophobic pocket (F site) and prevent EV71 from uncoating. Moreover, in a dosedependent way, mycelial extracts of G. resinaceum decreased the levels of Lake Sinai and honey bee deformed wing viruses [21].
Results obtained in this study showed that if EtOH extracts are not dissolved in a highly non-polar solvent, such as DMSO, its inhibitory potential is decreased. In this case, more non-polar molecules with AV potential are likely to be involved in the activity, which is in accordance with previous studies [35,57]. Terpenoid compounds attributed to AV properties were primarily characterized as active components of G. lucidum; however, their presence was also later proven in other species, including G. colossum, G. applanatum, G. tsugae, G. concinna, G. tropicum and G. pfeifferi [58,59]. Furthemore, the AV activity of MeOH extracts of G. pfeifferi against the HSV nie virus (herpes simplex virus I) was demonstrated for isolated compounds of the terpenic and steroid type: aplanoxidic acid, ganoderone, ganoderol, ergosta 7,22-dien-3-ol and lucialdehyde [35]. On the other hand, studies have shown that products rich in phenolic compounds have AV activities against the herpes simplex virus, poliovirus, Coxsackie virus B5 and echovirus 7 [60,61]. For example, caffeic acid, detected in EtOH extracts of all four examined Ganoderma species [23], represents one of the polyphenols with AV activity [62]. In summary, the AV potential of mushrooms has been proven not only for extracts, but also for isolated compounds; AV potential is manifested by inhibition of viral enzymes, viral nucleic acid synthesis or adsorption, i.e., inhibition of virus penetration into mammalian cells [63]. Since AV activity of H 2 O extracts is also expressed in G. pfeifferi, we can assume that the tested activity is also exhibited by more polar compounds, primarily polysaccharides [64]. Nevertheless, AV activity against HSV virus type 1 and type 2 has been demonstrated for both triterpenes and polysaccharide-protein complexes, while water-soluble and MeOH-soluble components displayed in vitro activity against pathogenic viruses, such as influenza A virus and vesicular stomatitis virus [65][66][67]. Moreover, triterpenoids isolated from G. lucidum seem to have anti-HIV activity [67,68], though to create the framework for the use of G. lucidum isolates as anti-HIV drugs, extensive studies are still required. There is strong evidence in the literature that triterpenoids, polysaccharides, nucleotides, sterols, steroids, fatty acids and proteins/peptides contained in G. lucidum may be helpful for the treatment of COVID-19 despite the lack of clinical data [67]. For example, findings by Al-jumaili et al. [69] show that patients who received G. lucidum supplementation also had an increase in lymphocytes; glycosides, which are found in this fungus, also greatly decreased viral reproduction, absorption and penetration to suppress human coronavirus 229E infection in its early stages [67,70].
It can be concluded that the examined extracts of Ganoderma species showed the effect of bacteriophage inactivation due to secondary metabolites, such as phenolics and terpenoids. Furthermore, it should be noted that this is the first report of AV activity of Ganoderma extracts against vB_BbrS_56.1 bacteriophage (Siphoviridae family), which was isolated in 2017 by Petrovic et al. [71]. Further research should be focused on the mechanisms of AV activity, as well as the effect of extracts and/or components on animal viruses.

PCA Analysis
To identify phenolic compounds that are specific to AM activities of each Ganoderma extract (EtOH, H 2 O and CHCl 3 ), principal component analysis (PCA) was carried out for demonstrated AB, AF and AV activities. To the best of our knowledge, this is the first report of PCA analysis conducted on phenolic compounds and AM activity of selected Ganoderma extracts.
Data for microdilution method from Tables 1-3 were subjected to PCA for AB activity, with PC1 variance of 79.19% and PC2 accounting for 11.82%. (Figure 3). Flavonoids, including amentoflavone, rutin and apigenin, had the greatest loadin the positive part of the second principal component, together with 2,5-dihydroxyben acid, gallic acid and quinic acid (Figure 3). Flavonoids and 2,5-dihydroxybenzoic grouped in the positive part of both principal components were detected in high amo in CHCl3 extract of G. resinaceum. Furthermore, AB activity of G. resinaceum CHCl3 ext against E. coli and P. aeruginosa was presented in the positive part of PC1, suggesting these compounds are probably responsible for obtained activity. In addition, gallic quinic acid of H2O extracts of G. resinaceum positively correlated with AB activity aga B. cereus, P. aeruginosa and K. pneumoniae. p-Hydroxybenzoic acid contributed to the gr est loading in the negative part of both axes and positively correlated with activity o applanatum CHCl3 extract against S. aureus. Other phenolic acids and coumarin aescu were separated in IV quadrant, together with G. applanatum H2O extract and MIC aga E. coli.
The multivariate analysis of the AF activity of EtOH extracts of G. pfeifferi and resinaceum permitted the reduction of the variables to two principal components, wi total variance of 17.74% (Figure 4). Flavonoids, including amentoflavone, rutin and apigenin, had the greatest loading in the positive part of the second principal component, together with 2,5-dihydroxybenzoic acid, gallic acid and quinic acid (Figure 3). Flavonoids and 2,5-dihydroxybenzoic acid grouped in the positive part of both principal components were detected in high amounts in CHCl 3 extract of G. resinaceum. Furthermore, AB activity of G. resinaceum CHCl 3 extract against E. coli and P. aeruginosa was presented in the positive part of PC1, suggesting that these compounds are probably responsible for obtained activity. In addition, gallic and quinic acid of H 2 O extracts of G. resinaceum positively correlated with AB activity against B. cereus, P. aeruginosa and K. pneumoniae. p-Hydroxybenzoic acid contributed to the greatest loading in the negative part of both axes and positively correlated with activity of G. applanatum CHCl 3 extract against S. aureus. Other phenolic acids and coumarin aesculetin were separated in IV quadrant, together with G. applanatum H 2 O extract and MIC against E. coli.
The multivariate analysis of the AF activity of EtOH extracts of G. pfeifferi and G. resinaceum permitted the reduction of the variables to two principal components, with a total variance of 17.74% (Figure 4).  The first axes (PC1) accounted for 11.96% and the second (PC2) for 5.78% of the total variability. Stronger separation was conducted in the horizontal plane of the PC2, with all phenolics and isorhamnetin loading in the II quadrant. This indicates that these compounds positively correlated with high AF activity of tested extracts against A. niger The first axes (PC1) accounted for 11.96% and the second (PC2) for 5.78% of the total variability. Stronger separation was conducted in the horizontal plane of the PC2, with all phenolics and isorhamnetin loading in the II quadrant. This indicates that these compounds positively correlated with high AF activity of tested extracts against A. niger and S. cerevisiae, using the pour plate method. Low AF effect of these extracts obtained against C. albicans (Figures 1 and 2) could be a consequence of low amounts of chrysoeriol, detected only in G. pfeifferi [33], since this compound positively correlated with disk-diffusion activity of both Ganoderma species against this pathogen.
A total variance of 62.59% was observed for AV activity of all four Ganoderma species, with PC1 of 36.77% and PC2 of 25.82%. G. lucidum, G. pfeifferi and G. applanatum (EtOH extract and EtOH extract solution in 5% DMSO) were generally separated from G. resinaceum extracts and H 2 O extracts ( Figure 5). Hence, we can assume that high AV activity of G. resinaceum may be related to some other compounds. On the other hand, the highest inhibition of virions by both G. lucidum DMSO solution and EtOH extract is positively correlated with phenolic acids, presented in the II quadrant. Furthermore, the highest loading of gallic, p-coumaric, phydroxybenzoic, quinic and chlorogenic acids is observed in the first quadrant, and thus is related to significant AV activity of G. pfeifferi. Moderate AV activity of H2O extract G. applanatum and G. lucidum may be explained by low content of chrysoeriol detected in these species [23] since this flavone positively correlates with AV activity ( Figure 5).

Fungal Material
Fruiting bodies of selected Ganoderma fungal species were collected in Serbia in September 2010: G. applanatum and G. lucidum from Morović woods (near Fruška gora Mountain); G. pfeifferi from Nature Park Begečka jama in Danube branch; and G. resinaceum from Liman (urban location of Novi Sad Town). Determination and identification were carried out under the authority of Dr. Maja Karaman at the Department of Biology and Ecology, University of Novi Sad. The identifications of the four voucher specimens (G. applanatum, No. 12-00714; G. lucidum, No. 12-00715; G. pfeifferi, No. 12-00723; and G. resinaceum, No. 12-00722) were made using both macroscopic and microscopic morphological characteristics and particular identification keys [72,73]. The specimens were then deposited at the mycological collection of the BUNS Herbarium (the herbarium of the Department of Biology and Ecology at the University of Novi Sad, Serbia).

Preparation of Extracts
The extracts were prepared according to the procedure of Rašeta et al. [23], whereas the chloroform (CHCl3) extracts were prepared in the same way as ethanolic (EtOH) extracts: evaporated EtOH, CHCl3 and lyophilized H2O extracts were dissolved in distilled H2O to a stock concentration of 50 or 100 mg/mL prior to testing for AM activity. To assess antiviral activity, samples were prepared by dissolving evaporated EtOH extracts in 5% DMSO. All samples were stored at +4 °C prior to the analysis. Hence, we can assume that high AV activity of G. resinaceum may be related to some other compounds. On the other hand, the highest inhibition of virions by both G. lucidum DMSO solution and EtOH extract is positively correlated with phenolic acids, presented in the II quadrant. Furthermore, the highest loading of gallic, p-coumaric, p-hydroxybenzoic, quinic and chlorogenic acids is observed in the first quadrant, and thus is related to significant AV activity of G. pfeifferi. Moderate AV activity of H 2 O extract G. applanatum and G. lucidum may be explained by low content of chrysoeriol detected in these species [23] since this flavone positively correlates with AV activity ( Figure 5).

Fungal Material
Fruiting bodies of selected Ganoderma fungal species were collected in Serbia in September 2010: G. applanatum and G. lucidum from Morović woods (near Fruška gora Mountain); G. pfeifferi from Nature Park Begečka jama in Danube branch; and G. resinaceum from Liman (urban location of Novi Sad Town). Determination and identification were carried out under the authority of Dr. Maja Karaman at the Department of Biology and Ecology, University of Novi Sad. The identifications of the four voucher specimens (G. applanatum, No. 12-00714; G. lucidum, No. 12-00715; G. pfeifferi, No. 12-00723; and G. resinaceum, No. 12-00722) were made using both macroscopic and microscopic morphological characteristics and particular identification keys [72,73]. The specimens were then deposited at the mycological collection of the BUNS Herbarium (the herbarium of the Department of Biology and Ecology at the University of Novi Sad, Serbia).

Preparation of Extracts
The extracts were prepared according to the procedure of Rašeta et al. [23], whereas the chloroform (CHCl 3 ) extracts were prepared in the same way as ethanolic (EtOH) extracts: evaporated EtOH, CHCl 3 and lyophilized H 2 O extracts were dissolved in distilled H 2 O to a stock concentration of 50 or 100 mg/mL prior to testing for AM activity. To assess antiviral activity, samples were prepared by dissolving evaporated EtOH extracts in 5% DMSO. All samples were stored at +4 • C prior to the analysis.

LC-MS/MS Analysis of Selected Phenolic Compounds
The quantitative analysis of the phenolic composition of CHCl 3 extracts of G. applanatum and G. resinaceum was performed using LC-MS/MS technique according to the previously described method [74]. A detailed description is available in Supplementary Data S1.

Nutrient Media
The following nutrient media (Torlak, Belgrade, Serbia) were used: nutrient agar (NA) as slanted agar for the cultivation of bacteria that were stored at +4 • C and Müeller Hinton Agar (MHA) medium for determining AB activity. Malt agar-(MA) (Torlak, Belgrade, Serbia) was applied for AF determination.

Antibacterial and Antifungal Activity Bacterial and Fungal Strains
In vitro AB susceptibility of Ganoderma H 2 O extracts (100 mg/mL stock conc.) against four strains of Gram-positive bacteria (Staphylococcus aureus ATCC 255923 , S. aureus ATCC 6538 , Enterococcus faecalis ATCC 19433 , Bacillus cereus ATCC 11778 ) and five strains of Gram-negative bacteria (Escherichia coli ATCC 11775 , E. coli ATCC 11229 , Pseudomonas aeruginosa ATCC 3554 , and Klebsiella aerogenes ATCC 13048 from standard American Type Culture Collection (ATCC), was assessed using the disk-diffusion, agar-well diffusion and microdilution assay (Table 5). AB susceptibility of the CHCl 3 extracts of G. applanatum and G. resinaceum against six bacterial strains (Gram-positive B. cereus HP and S. aureus ATCC 6538 , and Gram-negative P. aeruginosa ATCC 3554 , E. coli ATCC 11229 , Salmonella enteritidis ATCC 13076 , Klebsiella pneumoniae HP ) was evaluated using in vitro microdilution assay, while streptomycin was used as the reference compound (Table 5).
EtOH extracts of G. pfeifferi and G. resinaceum were evaluated for AF activity against laboratory strains Candida albicans, Saccharomyces cerevisiae and Aspergillus niger. Zones of fungal growth inhibition were measured according to the standard microbiological procedures [75,76]. The AF activity was evaluated by applying two methods: disk-diffusion and pour plate.

Antibiogram
Antibiograms (Table S2) provided data on bacterial sensitivity to specific antibiotics according to the Kirby-Bauer procedure and the standard CLSI procedure [77]. For the sensitivity test, five antibiotics disks (d = 9 mm) were used, including amikacin, tetracycline, methicillin, kanamycin and ceftriaxone (all at 10 µg/mL). The sensitivity of the following bacterial strains was tested: B. cereus ATCC 11778 , E. coli ATCC 11229 , E. coli ATCC 11775 , E. faecalis ATCC 19433 , K. aerogenes ATCC 13048 , P. aeruginosa ATCC 35554 , S. aureus ATCC 25923 , and S. aureus ATCC 6538 (Table S2). After incubation (37 • C, 24 h), the diameter of the inhibition zone was measured and the sensitivity of each bacterium was evaluated as sensitive (S), intermediate (I) or resistant (R) based on reference tables developed by the manufacturer (Torlak, Belgrade, Serbia) (Table S2).

Diffusion Assays
The agar-well diffusion assay was used to determine the AM activity of fungal extracts at a concentration of 100 mg/mL. All solid substrates were prepared in 90 mm Petri plates with approximately 22 mL of MHA, which resulted in a final thickness of 4 mm according to the Kirby-Bauer procedure [78]. After drying the substrate, the agar was drilled with 6 wells with a diameter of 6 mm. After 2 h incubation at room temperature, the plates were treated with 50 µL of each individual fungal extract in three replicates and then incubated at 35 • C for 18-24 h. The substrate was inoculated with 0.1 mL suspension of the tested micro-organism, and dilutions of the extracts were made using dH 2 O as a negative control. The diameters of the inhibitory zones were measured after a 24 h incubation and expressed as a distance (mm), including the initial size of the well (6 mm). When applying the disk-diffusion assay, the procedure was repeated, but instead of "drilling" the medium, "empty" white sterile disks (HiMedia Laboratories, Maharashtra, India; 6 mm) were placed on MHA, on which the extract (25 µL) was applied. The same concentrations (100, 50, 25 and 10 mg/mL) were used for each analyzed extract, in three replicates.
The disk-diffusion method for detection of AF activity was performed in 100 × 15 mm Petri plates. The patches of mycelia of tested fungal strain were applied on Petri plates containing MA. At a distance of 0.5 cm from the edge of the mycelium, white sterile disks (HiMedia Laboratories, Maharashtra, India; 0.6 cm) were placed. A 25 µL measurement of each concentration of Ganoderma extracts (10 mg/mL, 50 mg/mL and 100 mg/mL) was applied to the disks. Plates were incubated at 23 • C for 72 h until mycelial growth appeared on control disks. The zone of inhibition around the disks, which manifests AF activity, appears in the shape of a crescent [79].

Pour Plate Method
Additionally, another method was used to determine the AF activity of G. pfeifferi and G. resinaceum species. Three doses of extracts (200 µL, 100 µL, 50 µL) in different concentrations (0.5 mg/mL, 2.5 mg/mL and 5 mg/mL) were used. After sterilization, a medium was cooled to 45 • C and different volumes of extracts were added and mixed rapidly. The mycelia were inoculated centrally, with approximately the same amount placed into each Petri plate, while dH 2 O was used as control. After incubation for 48 h, at 30 • C, the diameter of the mycelial colony was measured, and the inhibition of fungal growth was determined compared to the control.

Microdilution Assay
Antibacterial activity was determined by the standard microdilution CLSI procedure [80]. For the assay in a 96-well micro-plate (Spektar,Čačak, Serbia), Müller Hinton broth for bacteria (MHB, Torlak, Beograd, Serbia) was used. Inoculum was prepared from overnight cultures and bacterial suspension was justified with a turbidity of 0.5 McFarland (equivalent to 1.5 × 10 8 colony-forming units (CFU)/mL). Double dilutions of Ganoderma H 2 O extracts and CHCl 3 extracts of G. applanatum and G. resinaceum were prepared in 1% polysorbate (Tween 80) to a final concentration ranging from 6.25 to 200 mg/mL. After 24 h incubation, at 37 • C, 1 mL of 1% TTC (2,3,5-Triphenyl-2H-tetrazolium chloride) solution (HiMedia Laboratories, Maharashtra, India) was dosed into each microwell and the microtiter plates were incubated again for 2 h, at 37 • C. The TTC solution stains the cells pink-red and allows better visualization of the detection of bacterial culture growth in the wells. The minimal inhibitory concentration (MIC) value in the microtiter plates was evaluated according to CLSI procedure [81]. To determine the minimal bactericidal concentration (MBC) value, 100 µL of the mixture from the wells in which there was no visible growth was seeded on Petri plates with MHA and placed in a thermostat for the next 24 h. After incubation, bacterial colonies were visually counted on each Petri plate (CFU)/mL). The optimal number of colonies is from 30 to 300 on standard Petri plates (90 mm diameter) according to the standard procedure.

Antiviral Activity
DNK virus Bordetella bronchiseptica specific bacteriophage-vB_BbrS_LK3 (Siphoviridae family) was used for AV analysis. The reference strain of Bordetella bronchiseptica ATCC 10580 was used as the original host of phage vB_BbrS_LK3. The phages were multiplied on the appropriate host, precipitated with NaCl (58.4 g/L) and PEG6000 (1:10; w/v), centrifuged (11,000× g, 10 min., +4 • C) and purified by isopycnic ultracentrifugation in a discontinuous CsCl gradient [43]. After dialysis, the phage suspension in SM buffer (50 mM Tris HCl (pH 7.5); 0.1 M NaCl; 8 mM MgSO 4 ; 1 mg/mL gelatin) was stored at 4 • C, and the bacterial strain at −70 • C, in a medium with 10% glycerol. For the purposes of the experiment, the bacteria were grown on a liquid Luria-Bertani medium for 24 h, at 37 • C. Bacteriophages, whose abundance in the final volume was 1 × 10 5 plaque forming unit (PFU)/mL, were mixed with selected Ganoderma extracts with work concentration from 0.20 to 200 mg/mL. The control involved the incubation of the bacteriophage with the highest concentration of solvent, without the addition of the extract or the tested substance. The prepared mixtures of phages and extracts were incubated for 30 min, at 37 • C, following the neutralization by 10-step dilution of the treated bacteriophages in the SM buffer. The control was treated identically. After the treatment, the phage titer was determined by the plaque method [82] and the number of viable phages compared to the control was expressed as a percentage. % inhibition = (1 − N treatment /N control ) × 100 N treatment -number of viruses in the treatment (assay); N control -number of viruses in the control (solvent/buffer).

Statistical Analysis
All AM assays were performed in triplicates, and the results are expressed as mean values ± standard deviation (SD). The data that have a normal distribution were subjected to two-way analysis of variance (ANOVA) and multivariate analysis of variance (MANOVA). Tukey's test was used to determine significant differences (p < 0.05) between the extracts. In examination of AV activity, values represented the percentage inhibition (%) of plaque growth by analyzed extracts and, for statistical analysis, Pillai's Trace statistic test was used together with the Bonferroni correction method; MANOVA was applied as post hoc at p < 0.05 and p < 0.01. The statistical analysis was performed using the IBM SPSS Statistics software version 22.0 for Windows. Principal component analysis (PCA) provided an overview of the relationships between phenolic compounds and AB, AF and AV activities of the analyzed extracts of selected Ganoderma species. PCA analysis was performed using PAST software version 4.03 for Windows, which provided the data's internal structure and allowed good data dispersion.

Conclusions
To the best of our knowledge, this is the first report that documented coherence between AM activity and quantified phenolic compounds of selected autochthonous Ganoderma species from Serbia and the Balkan region in general. The most effective AB and AF agent among all investigated species was G. resinaceum, while G. lucidum dominated in AV activity. It is important to mention that G. resinaceum H 2 O and CHCl 3 extracts showed high activity against multi-resistant strains, including B. cereus, E. coli and P. aeruginosa. Regarding other examined species, G. pfeifferi showed moderate AM activity, while G. applanatum was recorded as a weak AM agent. High AM activity of G. resinaceum was aligned with the detected flavonoids (amentoflavone, apigenin and rutin) and 2,5-dihydroxybenzoic acid, suggesting that phenolic compounds play a key role in tested activities. Possible mechanisms of action could be their ability to complex with extracellular and soluble proteins and with bacterial cell walls, whereas more lipophilic flavonoids might contribute to microbial membrane disruption.
This conclusion was in accordance with PCA analysis, conducted for all three AM activities. However, future research should focus on the AM activity of the identified secondary metabolites, both acting individually and in synergism. The processes behind the AM activity of Ganoderma are still largely unclear.

Conflicts of Interest:
The authors declare no conflict of interest.