Bioactivity and toxicity of polysaccharides derived from the phytopathogenic mushroom Ganoderma orbiforme cultured in a bioreactor

Abstract

The phytopathogenic status of G. orbiforme as the causative agent of basal stem rot on oil palm has masked its biotechnological potential. This study explored its growth profile in an Air-L-Shaped Bioreactor along with the antibacterial and antioxidant activities of its extracellular (EPS) and intracellular polysaccharides (IPS), and evaluated their toxicity using the zebrafish embryo toxicity (ZFET) assay. The biomass pellet peaked at 2.53 ± 0.44 g/L, with EPS and IPS yields reaching 0.15 ± 0.06 g/L (Day 6) and 0.06 ± 0.01 g/L (Day 10), respectively. Morphological observations revealed that the hairy starburst-shaped pellet correlated with peak EPS release. Then, the antibacterial assays revealed EPS as the better antibacterial agent compared to IPS, highlighted by a broader spectrum activity with the most prominent inhibition against Streptomyces griseus and Staphylococcus epidermidis with a minimum inhibitory concentration of 5 mg/mL. Both EPS and IPS showed strong antioxidative capabilities, demonstrated in DPPH assay—IC₅₀ of 15.59 ± 0.42 mg/mL for EPS and 26.85 ± 1.60 mg/mL for IPS, and FRAP assay—74.30 ± 0.38 mM Fe(II)/g for EPS and 74.18 ± 0.62 mM Fe(II)/g for IPS. Lastly, the LC₅₀ values of 1.88 mg/mL and 1.56 mg/mL for EPS and IPS, respectively, demonstrated their harmless nature in the ZFET assay. After 120 h of post-fertilization, the zebrafish embryos did not show abnormalities, with vital organs and structures remaining intact (fins, guts and melanophores). Collectively, this study underscores the importance of exploring the untapped potential of G. orbiforme while ensuring its safety status, opening a new possibility of bioprospecting a phytopathogen for oil palm. From a sustainable perspective, the production pattern and bioactivities are well aligned with SDG 15: Life on Land and SDG 3: Good Health and Well-Being, respectively.

Introduction

Medicinal mushrooms, particularly those belonging to the Ganoderma genus, have long been valued in traditional Asian medicine. Species such as G. lucidum, G. applanatum, and G. sinense have been extensively utilized in Asian countries like Japan, China and Korea⁶². These ancient practices were validated by modern-day discoveries, reporting useful bioactive compounds such as phenolics, flavonoids, fatty acids, polysaccharides, steroids, triterpenes, vitamins and minerals extracted from various fungal components, i.e. fruiting bodies, mycelia and spores⁶. Among these, polysaccharides and terpenes are regarded as the primary contributors to Ganoderma’s medicinal potential⁴.

As of 2023, Ganoderma spp. market value stands at USD 6.24 billion and is expected to have a healthy economic growth at a compound annual growth rate (CAGR) of 12% from 2024 to 2030, with Alphay International Inc., DXN Holdings Bhd and Hokkaido Reishi Co., Ltd. as some of the major stakeholders²². Driven by the growing awareness of natural health solutions and preventive healthcare, the utilization of Ganoderma-based supplements has gained popularity among mainstream consumers, reflecting their significant economic and therapeutic relevance⁷⁰.

Despite these diverse applications, several Ganoderma spp. have been classified as phytopathogenic towards oil palm. As of 2020, the Malaysia Palm Oil Board (MPOB) has classified G. boninense (synonym: G. orbiforme), G. zonatum and G. miniatocinctum as the main causative agents of a lethal oil palm disease–basal stem rot (BSR). The widespread distribution of the phytopathogens is a great concern for oil palm-producing countries such as Malaysia and Indonesia as it poses detrimental effects towards the sustainability of the industry⁵⁴. A loss of yield up to 80% is expected if the disease is not contained as it severely disrupts the productivity and shortens the life span of the oil palm⁸⁴. Consequently, research on G. boninense or G. orbiforme has mainly focused on detection, prevention and curation strategies, overshadowing its other potential applications⁶⁶.

Interestingly, despite its phytopathogenic nature, emerging evidence suggests that G. orbiforme exhibits promising bioactivities. Extracts such as triterpenes demonstrated antibacterial, antidiabetic, anti-inflammatory and antituberculosis properties^28,38,82,89. These findings urge the redirection of research focus on G. orbiforme, especially in the context of the antimicrobial resistance (AMR) crisis. AMR is a condition where bacteria and fungi are resistant to well-established antimicrobial agents⁵⁶. AMR microbes do not respond to the prescribed medicines, making the infections harder to treat, and in a worst-case scenario, can cause widespread disease, severe illnesses and potentially death¹⁵. Therefore, the need to find novel antimicrobial agents is of the essence to tackle this global issue.

Recently, basidiomycetes especially Ganoderma spp. have been touted as a competent green source of antimicrobial compounds due to their long-standing use in traditional medicines⁴⁶. Both the fruiting bodies and the mycelium are known to harbour compounds with useful pharmaceutical potential⁵³. Several species such as G. lucidum, G. applanatum, G. formosanum and G. capense have demonstrated antimicrobial and antioxidant properties^17,27,42,49. However, similar investigations on G. orbiforme remain limited, particularly regarding its polysaccharide content and functional properties.

Given its phytopathogenic status, evaluating the safety profile of G. orbiforme is crucial before considering therapeutic applications. Zebrafish embryo acute toxicity (ZFET) has emerged as a reliable model for toxicity testing, drug discovery, and developmental biology studies due to their ease of breeding, rearing, maintenance and high-throughput under laboratory conditions². The fully developed morphological structures and internal organs such as the heart, liver, kidney, brain and eyes can be directly examined using a light microscope, five days after conception due to the embryo’s transparency. The permeability of zebrafish embryos is important as the chemicals or extracts added to the fish medium can permeate directly into the embryos, offering a simple drug administration step and assay processing⁶¹. Recent publications on G. lucidum and Lignosus rhinocerus successfully employed the zebrafish embryo model to determine the toxicity level of the mushroom extracts^75,78.

Traditionally, Ganoderma spp. are cultivated via solid substrate cultivation using mushroom spawn bags with sawdust as their main substrate. However, several recurring issues such as contamination, inconsistent quality, time-consuming and labour-intensive are some of the notable drawbacks of utilizing this method in the long run³⁴. Additionally, the method employed is mainly for consumption purposes which restricts its other potential applications. As an alternative, submerged liquid fermentation has gained traction as an efficient strategy to produce mycelium and associated bioactive metabolites. This approach allows for precise control of culture conditions and is more amenable to industrial-scale production using bioreactors^10,14. Cultivation in a bioreactor allows the manipulation of the parameters such as temperature, pH, dissolved oxygen, etc. to be suited for the cultivated species, ensuring a reproducible outcome. Recently, Supramani et al.⁶⁵ fabricated a cost-effective bioreactor, named air-L-shaped bioreactor (ALSB) which was proven to be more effective in cultivating G. lucidum, recording a higher yield compared to the shake flask cultivation. It was later supported by Lim et al.⁴⁰, who utilized the ALSB for L. rhinocerus cultivation.

In this study, we investigated the growth profile of phytopathogenic G. orbiforme in the ALSB system, with a focus on the production of mycelial biomass, extracellular polysaccharides (EPS), and intracellular polysaccharides (IPS). The antibacterial and antioxidant properties of the extracted polysaccharides were evaluated, alongside their toxicity profiles using the ZFET assay. To the best of our knowledge, this is the first report detailing the bioactivity and toxicological assessment of EPS and IPS from G. orbiforme cultivated in an ALSB, offering a novel perspective on the potential therapeutic utility of this understudied phytopathogenic fungus.

Results

Growth profile of G. orbiforme in ALSB

Figure 1A and B illustrate the production profile of G. orbiforme’s mycelial biomass and polysaccharides in the ALSB over a 12-day cultivation period. The production pattern of the biomass manifested three distinct regions: log (Day 2–6), stationary (Day 6–10) and death phase (Day 10–12). The absence of the lag phase could be due to the usage of 2^nd seed mycelium from the shake flasks, whereby the G. orbiforme biomass has comfortably adapted to the liquid environment and thus, shortens or makes the initial lag phase negligible²⁵.

Fig. 1.

(A) Growth profile of G. orbiforme and EPS. (B) Growth profile of G. orbiforme and IPS. (C) Boltzmann model fitting on biomass. (D) Gaussian model fitting on EPS. (E) Linear model fitting on IPS.

The highest production of biomass and EPS, and IPS was recorded on Day 6 and Day 10, respectively. The mycelial biomass started with 1.52 ± 0.91 g/L on Day 2 and proceeded to multiply by close to two-fold, reaching its maximum value of 2.53 ± 0.44 g/L on Day 6. Then, it experienced a stationary period until Day 10 before entering the early stage of the death phase, indicated by a slight decrease in its biomass (2.22 ± 0.34 g/L). The transition between these phases also influenced the G. orbiforme’s polysaccharide productivity. For instance, the peak value of biomass is accompanied by the highest release of EPS at 0.15 ± 0.06 g/L on Day 6. Meanwhile, the highest IPS output was recorded on the onset of death phase entry of G. orbiforme on Day 10 at 0.06 ± 0.01 g/L. This observation suggests that in a liquid environment, G. orbiforme tends to give the highest output of polysaccharides at different time points of its growth with EPS being released the highest during the transition period (between the end of log phase and the stationary phase) and IPS at the end of the growth cycle as a result of internal accumulation⁷⁹. From Day 8 onwards, G. orbiforme cultivation in ALSB showed a steady decline in terms of its product, reaching its minimum output of biomass (2.17 ± 1.19 g/L) and EPS (0.07 ± 0.05 g/L) on Day 12. This can be attributed to nutrient limitation in the ALSB batch culture, whereby G. orbiforme entered sequential processes of deceleration, stationary and death. Over time, essential nutrients such as carbon and nitrogen sources become limited, hampering growth and development. When the nutrient is completely depleted, toxic metabolites build up, leading to cell lysis¹⁶. This observation is also in agreement with the previous Ganoderma spp. grown in a liquid setting, demonstrating the distinct regions of growth curve within the first 15 days of cultivation^11,64,65.

Meanwhile, Fig. 1C–E illustrate the nonlinear fitting of G. orbiforme cultivation outputs: biomass, EPS and IPS which can help in predicting the outcome of the desired product at specific time points. The biomass data were best fitted using a Boltzmann sigmoidal model, reflecting a classical growth trend with distinct lag, log and stationary phases. EPS production followed a Gaussian distribution, indicative of a peak yield occurring mid-cultivation, likely tied to metabolic activity during the transition from exponential to stationary phase. Meanwhile, IPS accumulation was best fitted to a linear regression model, suggesting a steady internal accumulation throughout the cultivation period²⁰. The fitted models demonstrated strong agreement with the experimental data, each yielding a coefficient of determination (R2) above 0.85, underscoring their reliability for predictive modelling³⁵. Table 1 summarizes the kinetic model fitting of biomass, EPS and IPS of G. orbiforme produced in ALSB.

Table 1.

Kinetic model fitting of biomass, EPS and IPS production in ALSB.

	Model	Equation	R2
Biomass	Boltzmann		0.8661
EPS	Gaussian		0.8748
IPS	Linear regression		0.9728

Visual observation of pellet morphology

Additionally, Fig. 2 depicts the morphological changes of the G. orbiforme’s mycelial biomass throughout the cultivation period that served as an indicator for the polysaccharide production trend. On Day 2, the mycelial biomass closely resembles a hairy circular structure (0.99 ± 0.10 µm) that is associated with the self-immobilisation process as a form of protection from the shear stress created in the agitated liquid environment⁴⁰. Then, at the end of the logarithmic period (Day 6), the size of the pellet increased to 1.27 ± 0.24 µm which could suggest the production of new pellets as indicated by the slight protuberance observed on the parent pellet, resembling a starburst appearance⁷⁹. The liberation and release of the new pellets is accompanied by the exponential release of the EPS (0.15 ± 0.06 µm). The release of newly formed pellets is also confirmed by the decrease of the mycelial pellet to 1.10 ± 0.08 µm on Day 12, signalling the entry of the death phase. The morphological behaviour of G. orbiforme in liquid culture is comparable to that of previous Ganoderma spp. studies especially the exponential release of EPS during the transition period^31,77.

Fig. 2.

Morphological behaviour of G. orbiforme mycelium pellet in ALSB for 12 days cultivation. (Bar = 0.5 µm).

Productivity of G. orbiforme in comparison with previous ALSB studies

Meanwhile, Table 2 compares the productivity of G. orbiforme with other previously studied basidiomycetes in the ALSB setting. G. orbiforme’s biomass, EPS and IPS productivity are comparatively lower than G. lucidum and L. rhinocerus. This suggests that G. orbiforme tend to grow at a very slow pace (0.42 ± 0.07 g/L/day) which directly reflects its native growth behaviour that usually takes two months before it can fully colonize a rubber wood block in most BSR studies⁴⁵. This stark difference is also due to the fast-growing nature of G. lucidum among other Ganoderma spp. as demonstrated by Nussbaum et al.⁵⁰. Besides, the ALSB is equipped with a steady aeration which could significantly affect the G. orbiforme’s growth³. Furthermore, the potential heat generated and accumulated in the ALSB cultivation system might create thermal stress, especially if temperatures exceed the fungus’s optimal growth range⁵⁵. These factors collectively suggest that while the ALSB system benefits fast-growing Ganoderma spp., further parameter optimization may be needed to support slower, more sensitive species like G. orbiforme.

Table 2.

Productivity of G. orbiforme in comparison to previous ALSB studies.

Species	Biomass		EPS			IPS			References
	Max (g/L)	Prod. (g/L/day)	Max (g/L)	Prod. (g/L/day)	S.P.R. (gEPS/gBiomass)/day	Max (g/L)	Prod. (g/L/day)	S.P.R. (gIPS/gBiomass)/day
G. orbiforme	2.53 ± 0.44	0.42 ± 0.07	0.15 ± 0.06	0.03 ± 0.01	0.01 ± 0.01	0.06 ± 0.05	0.01 ± 0.00	< 0.01 ± 0.00	This study
G. lucidum	33.96 ± 0.03	2.264 ± 0.03	15.52 ± 0.05	1.035 ± 0.03	0.305 ± 0.03	N.A	N.A	N.A	65
Lignosus rhinocerus	5.32 ± N.A	0.532 ± N.A	N.A	N.A	N.A	N.A	N.A	N.A	40

Prod. productivity; S.P.R specific production rate; NA not available.

Antibacterial activity of EPS and IPS

Then, the EPS and IPS produced were screened for their antibacterial activity against both positive and negative grams strains (Table 3). Inhibition zones exceeding 8 mm were observed in all strains, indicating moderate inhibitory activity except for EPS extract against S. epidermidis (7.93 ± 0.19 mm; weak), IPS extract against E. coli (7.87 ± 0.15; weak), and K. pneumoniae (7.47 ± 0.05; weak) according to the classification category reported by Sundari et al.⁶³. Meanwhile, the largest inhibition zone was observed from the EPS extract against Proteus sp. (8.79 ± 0.33 mm), a species clinically classified as a cause of urinary tract infections²⁶. Overall, the EPS extract moderately inhibited more strains than IPS (EPS = 13 strains; IPS = 12 strains), suggesting a more potent antibacterial activity. This could be attributed to differences in molecular structure and solubility²⁴.

Table 3.

Antibacterial activity of EPS and IPS from G. orbiforme; zone of inhibition, MIC and MBC.

Bacterial species	Zone of inhibition (mm)			MIC (mg/mL)		MBC (mg/mL)
	EPS	IPS	Ampicillin	EPS	IPS	EPS	IPS
Enterococcus faecalis†	8.46 ± 0.54e	8.17 ± 0.14e	24.23 ± 2.44c	20.0	15.0	N.A	N.A
Streptomyces griseus	8.42 ± 0.22e	8.38 ± 0.25e	8.86 ± 0.00e	5.0	N.A	N.A	N.A
Staphylococcus epidermidis	7.93 ± 0.19e	8.01 ± 0.62e	35.74 ± 0.83a	5.0	N.A	N.A	N.A
Staphylococcus aureus†	8.37 ± 0.17e	8.24 ± 0.07e	35.62 ± 2.21a	10.0	N.A	N.A	N.A
Escherichia coli†	8.47 ± 0.14e	7.87 ± 0.15e	32.81 ± 1.53b	10.0	15.0	N.A	N.A
Erwinia sp.	8.09 ± 0.21e	8.57 ± 0.05e	10.00 ± 0.00e	20.0	N.A	N.A	N.A
Klebsiella pneumoniae†	8.73 ± 0.07e	7.47 ± 0.05e	20.41 ± 0.22d	N.A	N.A	N.A	N.A
Proteus sp.	8.79 ± 0.33e	8.59 ± 0.16e	20.97 ± 0.82d	N.A	N.A	N.A	N.A
Pseudomonas sp.†	8.21 ± 0.09e	8.10 ± 0.17e	8.48 ± 0.06e	N.A	N.A	N.A	N.A
Ralstonia sp.	8.26 ± 0.17e	8.77 ± 0.27e	9.54 ± 0.00e	N.A	N.A	N.A	N.A
Serratia marcescens†	8.10 ± 0.40e	8.44 ± 0.44e	8.04 ± 0.11e	20.0	N.A	N.A	N.A
Salmonella sp.†	8.32 ± 0.33e	8.35 ± 0.25e	7.91 ± 0.43e	N.A	N.A	N.A	N.A
Vibrio anguillarum	8.01 ± 0.06e	8.27 ± 0.38e	23.02 ± 0.00cd	15.0	N.A	N.A	N.A
Xanthomonas sp.	8.50 ± 0.12e	8.23 ± 0.02e	22.36 ± 0.00cd	N.A	N.A	N.A	N.A

^†WHO priority pathogens. N.A. not measurable activity at tested concentrations. Data is expressed in mean ± SD (n = 3) where applicable.

Within each row, overall means with different superscript letters are significantly different (p < 0.05).

In the MIC assay, EPS extract inhibited 7 out of 14 tested bacterial strains, with MIC values ranging from 5 to 20 mg/mL. The lowest MIC value (5 mg/mL) was observed in EPS extract against S. griseus and S. epidermidis, both of which are Gram-positive bacteria. While the EPS extract also inhibited Gram-negative bacteria such as E. coli, S. marcescens, Erwinia sp., and V. anguillarum, the observed MIC values were generally higher, ranging from 10 to 20 mg/mL. Conversely, the IPS extract inhibited only two strains: E. faecalis and E. coli. The observed MIC values for both strains were 15 mg/mL, suggesting lower antibacterial efficacy of IPS compared to EPS. However, neither EPS nor IPS showed any bactericidal effects in the MBC assay. Together, the agar well diffusion and MIC assays consistently indicate that EPS is a more effective antibacterial agent compared to IPS. These findings are supported by Mehta and Jandaik⁴⁷ who studied mycelial extracts (rich in EPS) and fruiting body extracts (rich in IPS) of the closely related G. lucidum for their antimicrobial properties.

Antioxidant activity of EPS and IPS

Additionally, EPS and IPS were also tested for their antioxidant capabilities via DPPH and FRAP assays (Table 4). In the DPPH assay, both EPS and IPS showed dose-dependent radical scavenging ability. At the highest tested concentration (20 mg/mL), EPS achieved 65.20% inhibition, while the IPS extract showed lower inhibition of 34.19%. Comparatively, EPS exhibited a lower IC₅₀ (15.58 mg/mL) compared to IPS (26.75 mg/mL), indicating EPS was approximately 1.7 times more effective in scavenging free radicals. The radical scavenging ability of EPS was comparable to previously reported values for crude polysaccharide extracts of G. boninense³⁷. Although IPS showed a lower IC₅₀ value than expected, it still fell within the expected range for fungal polysaccharide extracts³³.

Table 4.

Antioxidant activity of G. orbiforme polysaccharide extracts.

Concentration of EPS and IPS extracts	DPPH	FRAP
	IC50 (mg/mL)	Ferrous sulphate (mM Fe (II)/g)	Ascorbic acid equivalent (AAE) (mM AAE/g)
	0–20 mg/mL	20 mg/mL	20 mg/mL
EPS	15.59 ± 0.42a	74.30 ± 0.38a	7.60 ± 0.04a
IPS	26.85 ± 1.60b	74.18 ± 0.62a	7.57 ± 0.07a
Ascorbic acid (0–0.1 mg/mL)	0.08 ± 0.01c	1.27 × 104 ± 14.02b	N.A

N.A. not applicable. Data is expressed in mean ± SD (n = 3) where applicable.

Within each row, overall means with different superscript letters are significantly different (p < 0.05).

Meanwhile in the FRAP assay, both extracts demonstrated strong reducing capacity at 20 mg/mL. Both EPS and IPS demonstrated high FRAP activity values of 74.30 ± 0.38 mM Fe(II)/g and 74.18 ± 0.62 mM Fe(II)/g, respectively. These values are consistent with those reported for extracts derived from similar Ganoderma species such as G. lucidum³². Additionally, FRAP activity values of both EPS and IPS extracts were also expressed in ascorbic acid equivalents (AAE) to provide a benchmark for antioxidant potency. The EPS extract showed a slightly higher AAE value (7.60 ± 0.04 mM AAE/g) compared to the IPS extract (7.57 ± 0.07 mM AAE/g), supporting the relatively superior antioxidant capacity of EPS observed in the DPPH IC₅₀ and FRAP assays.

Survival rate and hatching rate of zebrafish embryos treated with EPS and IPS

Due to the phytopathogenic nature of G. orbiforme⁷², the proven antibacterial and antioxidant activities would be useful if the EPS and IPS are deemed safe (nontoxic). Therefore, the use of zebrafish embryos in determining the acute toxicity of EPS and IPS extracted from liquid cultures of G. orbiforme will support future product development and implementation. Figure 3A and B show the survival rate (%) of zebrafish embryos at 0 to 120 h of post-fertilization (HPF) with EPS and IPS (0–10 mg/mL) of G. obiforme. At the lowest EPS inclusion (0.16 mg/mL), the zebrafish embryos’ survival rate at 120 HPE (96%) is comparable with the untreated group (98%). The survival rate decreased steadily below 90% at 24–120 HPE when 2.5–10 mg/mL of EPS was used. Notably, EPS at 5–10 mg/mL resulted in a drastic decline in survival rate (< 30%) after 48 HPF, with zero embryo survival at 10 mg/mL. Relatively, IPS treatment had similar outcomes as the concentration above 2.5 mg/mL would severely affect the survival rate (< 30%) of the zebrafish embryos at 72 HPF with notable total death at 10 mg/mL. The IPS concentration between 0.16 and 0.63 mg/mL resulted in a > 90% survival rate at 120 HPE. Overall, EPS and IPS inclusion at 0.16–1.25 mg/mL had a > 90% survival rate of zebrafish embryos between 24 and 120 HPF.

Fig. 3.

(A) Survival rate of zebrafish embryos treated with EPS at 0 to 120 h. (B) Survival rate of zebrafish embryos treated with IPS at 0–120 h. (C) Hatching rate of zebrafish embryo treated with EPS at 0 to 120 h. (D) Hatching rate of zebrafish embryo treated with IPS at 120 h. *p < 0.05 significantly different from the untreated (control) group at 120 HPF.

Meanwhile, Fig. 3C and D show the hatching rate (%) of zebrafish embryos. Generally, a normal period of hatching for zebrafish is between 48 and 72 HPF⁸³. However, increased concentrations of EPS and IPS negatively affected the embryo’s hatchability, with maximum inclusion at 10 mg/mL halting hatching completely. At 0.16–1.25 mg/mL of EPS and IPS, the hatching rate (> 90%) is comparable to the untreated group, considered harmless. However, the usage of > 2.5 mg/mL results in a low hatching rate (< 40%) due to death as observed at 48–72 HPF. This indicated that the inclusion of < 1.25 mg/mL of both EPS and IPS in zebrafish embryos is still conducive to hatching.

Mortality rate and heartrate of zebrafish embryos treated with EPS and IPS

Furthermore, Fig. 4A and B illustrates the mortality rate (%) of zebrafish embryos at 120 HPF, setting the foundation for the determination of LC₅₀ i.e. the lowest concentration that results in 50% death of the tested samples. Generally, EPS and IPS effects on zebrafish embryos are time- and dose-dependent. At 0.160–1.25 mg/mL, minimum mortality (< 10%) was recorded for EPS. However, the tested EPS of > 2.5 mg/mL showed an exponential increase in mortality (> 90%) until a total death at 10 mg/mL EPS inclusion. As a result, the LC₅₀ for EPS was recorded at 1.88 mg/mL. Meanwhile, IPS treatment at 0.16–0.63 mg/mL had a low mortality rate (< 10%). But the mortality increases gradually to 30% at 1.25 mg/mL IPS and > 90% from concentrations of 2.5–5 mg/mL. Likewise, total mortality was recorded at 10 mg/mL for IPS treatment, with an LC₅₀ value recorded at 1.56 mg/mL. Although both EPS and IPS were extracted from G. orbiforme, the difference in terms of the LC₅₀ values could be due to the compounds having different compositions, owing to the extraction procedure⁸⁸. Similar outcomes were also observed for Lignosus rhinocerus and G. lucidum extracts employing the same toxicity study^67,75.

Fig. 4.

(A) Mortality rate of zebrafish embryo treated with EPS at 120 HPF. (B) Mortality rate of zebrafish embryo treated with IPS at 120 HPF. (C) Heartbeat of zebrafish embryo treated with EPS at 96 HPF. (D) Heartbeat of zebrafish embryo treated with IPS at 96 HPF. *p < 0.05 significantly different from the untreated (control) group.

The heart represents the main functioning organ throughout the developmental stage of the zebrafish embryos⁸⁷. Therefore, the heartbeat (beat/min) of the treated zebrafish embryos was measured at 96 HPF (Fig. 4C and D). The heart rate at > 2.5 mg/mL was not determined due to the high mortality rate (> 90%). A normal zebrafish embryo’s heartbeat closely resembles a human’s which is around 120–180 bpm²¹. As such, the treatment with both EPS and IPS at 0.16–1.25 mg/mL did not negatively affect the heartbeat of zebrafish embryos as they fall within the normal heartbeat count at 150–170 bpm. The treated zebrafish embryos’ heartbeats are also comparable with the untreated group (168 bpm) with no significant difference (p > 0.05), signalling little to no effect on the heartbeat when both EPS and IPS (0.16–1.25 mg/mL) were included.

Zebrafish embryos morphology

Moreover, morphological observations were conducted from 0 to 120 HPF to detect any malformations or abnormalities in the developing embryos. Untreated zebrafish embryos (Fig. 5) served as the healthy benchmark, while embryos treated with EPS and IPS at 0.16 and 10 mg/mL were examined for morphological changes⁶⁷.

Fig. 5.

Untreated zebrafish embryo showing normal embryogenesis at different HPF development i.e. blastula stage (0 HPF), segmentation stage (24 HPF), pharyngula stage (48 HPF) and hatching stage (72 HPF)⁶⁷. Label; A-eye enlarge, An-anus, Bc-blood cells, C-chorda, Ch-chorion, F-fin, G-gut, M-melanophores, O-ear bud, P-pericardium, S-somites, Y-yolk sac.

There were no obvious teratogenic symptoms on embryos from 0 to 120 HPF for both EPS and IPS treatments at 0.16 mg/mL (Figs. 6, 7), comparable to the healthy untreated embryos. This indicates that EPS and IPS extracted from G. orbiforme do not exhibit adverse implications upon zebrafish embryo development, both prior to and post-hatching. However, at 10 mg/mL of EPS and IPS, numerous defects and abnormalities could be observed, ultimately resulting in unhatched embryos. The abnormalities became apparent at 24 HPF in EPS treatment, signalled by the coagulated embryos and followed by the loss of the yolk sac (burst) at 48 HPF. Similar symptoms were observed in IPS-treated embryos where the coagulation and yolk sac bursting happened at 48 HPF and 72 HPF, respectively. At 120 HPF, the unhatched embryos in both EPS and IPS treatments suffered from structural deformity where they lost the common shape of a zebrafish embryo i.e. with missing major organs such as fins, guts and melanophores etc., possibly due to shrinkage and death⁷⁸.

Fig. 6.

Illustrations of zebrafish development and embryogenesis after treatment with EPS of G. orbiforme—0.16 mg/mL (Row 1) and 10 mg/mL (Row 2) from 0 to 120 HPF at 40× magnification.

Fig. 7.

Illustrations of zebrafish development and embryogenesis after treatment with IPS of G. orbiforme—0.16 mg/mL (Row 1) and 10 mg/mL (Row 2) from 0 to 120 HPF at 40× magnification.

Discussion

To the best of our knowledge, G. orbiforme is the third basidiomycete that was cultivated in ALSB after G. lucidum and L. rhinocerus^40,65. ALSB was specifically designed to cater for the needs of growing basidiomycetes in the liquid environment as extensively described by Supramani et al.⁶⁵. Having direct access to the liquid media will enhance mycelial growth and bioactive compound production as they are readily available, making liquid cultivation a fast, quick and efficient process as opposed to traditional solid cultivation³⁴. Recent examples of successful attempts in using a bioreactor to grow the species include the cultivation of G. lucidum to produce its beneficial bioactive metabolites such as ganoderic acids and polysaccharides^30,65. Even different types of bioreactors were utilized to cultivate Ganoderma spp. such as stirred tank bioreactors (STRs) and airlift bioreactors (ARs), demonstrating its cultivation versatility. Despite most bioreactor applications on Ganoderma spp. cultivation was conducted on a small scale, this continuous trend shows the potential of bringing this biotechnological tool into the industrialization level in producing both biomass and metabolites from Ganoderma spp.²⁹.

However, several problems arose during cultivation using STRs, particularly the formation of wall growth on the vessel surface and extended probes, which created dead zones and reduced process efficiency⁴⁸. As such, ALSB was chosen as the platform to cultivate G. orbiforme to eliminate such problems. Despite having a lower biomass productivity for G. orbiforme (0.42 ± 0.07 g/L/day) than G. lucidum⁶⁵ and L. rhinocerus⁴⁰, the attempt to shift towards liquid cultivation in a bioreactor is deemed to be more sustainable by Bakratsas et al.¹⁰. According to Liu and Zhang⁴³, the slow growth of Ganoderma sp. can be overcome by increasing its hyphal tips through pellet homogenization and optimizing the culture conditions. As such, crucial aspects such as medium ingredients, inoculum concentrations and airflows must be further optimized to bring about the best production trend as demonstrated by Rosales-López et al.⁵⁹. This ultimately opens a potential scaling-up strategies using a modified industrial-scale bioreactor¹⁴.

Then, both EPS and IPS extracts demonstrated broad-spectrum activity, particularly against E. faecalis and E. coli, as supported by the observed MIC values (5–20 mg/mL). However, stronger inhibition was observed for Gram-positive bacteria (E. faecalis, S. griseus, and S. epidermidis), suggesting a degree of Gram-positive selectivity, which is reflected in previous findings for related Ganoderma species⁵⁸. This may result from the absence of an outer membrane made from lipopolysaccharide or phospholipid layer that is present in Gram-negative bacteria as opposed to Gram-positive bacteria which facilitates polysaccharide penetration⁵². However, no minimum bactericidal concentration (MBC) was determined, indicating that EPS and IPS extracts are bacteriostatic but not bactericidal, suggesting that they inhibit growth but do not kill pathogens. This observation is consistent with previous studies showing fungal polysaccharides disrupt quorum sensing or biofilm formation without directly lysing bacterial cells²³. Meanwhile, in the antioxidant assays, the DPPH IC₅₀ of EPS was also comparable to reported values from other related species. For example, Tseng et al.⁷¹ found that the DPPH radical scavenging rate of polysaccharides extracted from G. tsugae fermentation broth achieved 74.9% at a concentration of 20 mg/mL. However, IPS showed lower radical scavenging ability compared to EPS. The superior performance of EPS could be attributed to several factors, including the difference in molecular structure and the presence of specific functional groups, which enhance hydrogen-donating capacity³⁷. Despite the difference in DPPH radical scavenging, both EPS and IPS showed nearly identical FRAP activity values, suggesting that both extracts contain potent electron-donating compounds, such as the hydroxyl functional groups present in G. lucidum polysaccharides¹⁹. These findings suggest that IPS may be less efficient at neutralising free radicals, but it could still contribute significantly to redox balance through metal ion reduction¹². Both extracts showed significantly lower FRAP values as compared to the positive control, ascorbic acid (p < 0.05), but their performance was still comparable with the values reported for other fungal polysaccharides used in food and nutraceutical applications³³. Collectively, these findings highlight the potential of G. orbiforme EPS and IPS as natural antioxidants, with diverse applications in various industries such as functional foods, cosmeceuticals, and pharmaceuticals.

The antibacterial and antioxidant properties demonstrated by both EPS and IPS can be explained by their structural characteristics. Qian-Zhu et al.⁵⁷ discovered the functional groups that typically exist within a polysaccharide structure in G. orbiforme’s such as hydroxyl (–OH), carbonyl (C=O) and ether linkage (C–O–C), while noting glucose and mannose make up most of the constituents. Generally, heteropolysaccharides having a higher composition of uronic acid, galactose and mannose are better antioxidative agents compared to homopolysaccharides⁷. The presence of the functional groups within EPS and IPS of G. orbiforme may contribute to their antioxidative capacity either through radical scavenging, metal chelation or reductive capacity⁶⁹. Meanwhile, the observed antibacterial activity could be attributed to the EPS and IPS ability in disrupting the bacterial cell membranes, leading to leakage of ions and loss of membrane potential⁹⁰. Other possible mechanisms of action of EPS and IPS in demonstrating their antibacterial properties include chelation of metal ions (Fe, Ca, Mg, etc.) which limits the nutrient availability, and inhibition of enzyme activities such as ATPase (Ca²⁺–Mg²⁺ ATPase) that can destabilize ion balance^39,90.

Additionally, the antibacterial and antioxidant properties demonstrated by EPS and IPS of G. orbiforme in this study paved the way for bioprospecting i.e. obtaining valuable compounds such as medicinal drugs and biochemicals from plants or animals. This can then be translated into a biotechnological application in various fields—skincare, food and health products. For instance, in today’s world of prioritizing green and sustainable beauty, G. lucidum has become more prevalent as its polysaccharides–infused beauty products take care of numerous skin concerns such as nourishment, elasticity, colour, and wrinkles⁴¹. Besides, the widespread practice of a healthy diet has prompted the inclusion of polysaccharides from Ganoderma sp. into coffee, fruit juice, tea, yogurt, cakes and functional foods, adding health and wellness values to the easily accessible products¹⁸. Additionally, its long usage in ancient Chinese practices has fast-tracked its adoption in modern medicine as Ganoderma-based healthcare products are available in various dosage forms such as powder, oral liquid, capsules and tablets to cater to all consumer needs³⁶. Due to its wood-degrading ability, G. orbiforme’s lignin-modifying enzymes such as laccases and peroxidases can also be utilized for bio-pulping, producing digestible animal feed, bioremediation and bioconversion of lignocellulosic residues into valuable items such as ethanol⁸⁵. These proven applications further justify the need for bioprospecting G. orbiforme especially in oil palm-producing countries such as Malaysia and Indonesia to fully reap the unexplored benefits of the phytopathogen.

Moreover, the ever-increasing experimentation utilizing animals has raised concerns regarding its ethics, necessity, feasibility and the potential harm caused. This necessitates the urgency of utilizing alternatives, especially to mammals and higher vertebrates which can result in similar valuable outcomes¹³. One of them is a small fish model, specifically zebrafish (Danio rerio) that is predominantly used in biomedical laboratories such as in this study. Besides, zebrafish has also been extensively used in a variety of research such as in bone studies⁴⁴, cancer studies⁷⁶, immunology⁸ and cardiovascular disease⁶⁸. The high usage of the zebrafish in the research field is highly associated with their numerous advantages, both practically and biologically. Their highlighted features such as cost-efficiency, ease of handling, external fertilization, rapid embryo formation, and optical transparency along with well-established molecular tools make it highly feasible for laboratory use¹³. In this study, the LC₅₀ of EPS (1.88 mg/mL) and IPS (1.56 mg/mL) extracted from G. orbiforme were categorised as harmless as their values exceeded 0.1 mg/mL. Comparably, LC₅₀ of EPS from G. lucidum, G. applanatum and L. rhinocerus were 2.6 mg/mL, 1.41 mg/mL and 0.41 mg/mL, respectively^67,75,78. This indicates that the LC₅₀ values obtained in this study agree with the rest of the medicinal mushroom species, which are historically known to promote health benefits yet are safe for consumption⁶².

Viewing from a sustainability perspective, this study aligns with the current Sustainable Development Goals (SDGs), particularly Goal 3: Good Health and Well-Being, and Goal 15: Life on Land. Since this study explored the potential usage of EPS and IPS from the phytopathogenic G. orbiforme as antibacterial and antioxidant agents and demonstrated their safety via zebrafish embryo toxicity assays, it supports the ideology of Goal 3 which focuses on ensuring healthy lives and promoting well-being for all at all ages. The development of cost-effective and accessible therapeutic agents is a favourable criterion which can be achieved through bioprocessing and biotechnological tools as demonstrated in this study. The cultivation technique through ALSB or any bioreactor systems also minimized land use, prompting more protection, restoration and promotion of sustainable use of terrestrial ecosystem, aligning with Goal 15. Furthermore, the discovery of the bioactive potential of G. orbiforme opens new avenues for bioprospecting, particularly in oil palm-producing countries such as Malaysia and Indonesia, where the phytopathogen is prevalent. Overall, this study not only highlights the biotechnological value of an often-overlooked Ganoderma species due to its phytopathogenic nature but also underscores its relevance in contributing to global sustainability efforts.

This study highlights the untapped biotechnological potential of the phytopathogen Ganoderma orbiforme, best known as the causative agent of basal stem rot (BSR) in oil palm. Its successful cultivation in a liquid system such as the Air-L-Shaped Bioreactor (ALSB) demonstrates its suitability for submerged fermentation. The established growth profile, including mycelial biomass, EPS, and IPS yields, provides a foundation for future scale-up strategies. Both EPS and IPS exhibited antibacterial and antioxidant activities, supporting their potential applications in the medicinal, pharmaceutical, and food industries. These findings were reinforced by the zebrafish embryo toxicity (ZFET) assay, which confirmed the safety of both polysaccharides, with no observed malformations or developmental defects. Overall, this study presents G. orbiforme not merely as a plant pathogen but as a promising candidate for bioprospecting, particularly in oil palm-producing countries such as Malaysia and Indonesia—turning a threat into a valuable resource.

Materials and methods

Stock culture of Ganoderma orbiforme

G. orbiforme (IMI 375,382) mycelium plate retrieved from the Centre of Agriculture and Biosciences International (CABI) was subcultured by transferring a plug (2 cm × 2 cm) on Potato Dextrose Agar (PDA, Oxoid CM0139—4 g/L Potato extract, 20 g/L glucose and 15 g/L agar with pH 5.6 ± 0.2) for 14 days. The fully colonized plate was subcultured again for 7 days before any laboratory procedures to ensure its viability (Fig. 8A). The temperature of G. orbiforme culture was maintained at 28 °C throughout the study unless stated otherwise¹.

Fig. 8.

(A) G. orbiforme on PDA plate. (B) 1^st seed culture of G. orbiforme. (C) 2^nd seed culture of G. orbiforme. (D) G. orbiforme culture in ALSB. (E) Mycelium biomass of G. orbiforme. (F) Polysaccharides of G. orbiforme.

Liquid and air-L shaped bioreactor (ALSB) cultivation of G. orbiforme

Liquid cultivation of G. orbiforme was conducted via a two-stage cultivation method according to Lim et al.⁴⁰ with slight modifications. In the first stage, 5 mycelial plugs of G. orbiforme were transferred into 100 mL of Potato Dextrose Broth (PDB, Oxoid CM0962B—4 g/L potato extract, 20 g/L glucose at pH 5.6 ± 0.2) in 250 mL Erlenmeyer flasks followed by agitation at 120 rpm for 7 days (Fig. 8B). In the second stage, the grown mycelial pellets were homogenized using a sterilised hand-blender (PerySmith® PS-850) for 10 s before being transferred (40 mL) into 160 mL of new PDB in 500 mL Erlenmeyer flasks. The culture was left to agitate for 10 days at 120 rpm (Fig. 8C).

The second-stage G. orbiforme culture was used as the inoculum for the ALSB cultivation, supplemented with new PDB, with a 2L working volume (20% of culture and 80% of PDB)⁶⁵. The G. orbiforme culture in ALSB was maintained at 120 rpm with a sterile airflow of 1 L/min using an air filter (0.22 µm) for 14 days to establish its growth profile (Fig. 8D).

Mycelium biomass, extracellular polysaccharide (EPS) and intracellular polysaccharide (IPS) from G. orbiforme

The formed mycelium biomass (pellets) of G. orbiforme were filtered through 90 mm filter paper (filtraTECH, ST61) and collected via a Buchner filtration system and an air pump (Finetech, VS300-2), followed by drying (40 °C) using a dehydrator (COCCA, ST-06) until the weight was constant (Fig. 8E)⁸⁰.

The resulting filtrate was collected and added to cold 95% ethanol (1:4, v/v) and stored at 4 °C for three days, causing EPS precipitation. The EPS was separated via centrifugation at 10,000 rpm at 25 °C (Frontier™ FC2706), where the isolated EPS was rinsed with sterile dH₂0 and dried at 40 °C until constant weight was achieved (Fig. 8F)⁷⁵.

The dried mycelium biomass was mixed with dH₂0 at a 1:10 ratio (g:mL). The mixture was subjected to hot water extraction via sterilization for 30 min at 121 °C¹¹. The IPS extracts were filtered as previously described, where the filtrate was mixed with cold 95% ethanol (1:4, v/v). The IPS was then isolated and dried as described for EPS.

The stock solutions (mg/mL) of EPS and IPS were prepared via gentle heating at 60 °C for one hour using dH₂0, diluted accordingly and stored at 4 °C until further assays.

Productivity calculations

The productivity for biomass, EPS, and IPS produced by G. orbiforme in the ALSB were calculated following the methodology of Usuldin et al.⁷³. The calculations included productivity and specific production rate as stated in Eqs. (1) and (2).

1

2

where:

• Productivity_X represents the production rate of biomass, EPS, or IPS,

• Specific production rate_X​ denotes the specific production rate of EPS or IPS,

• X_max is the maximum concentration of biomass, EPS, or IPS (g/L) obtained during cultivation,

• B_max is the maximum biomass concentration (g/L), and

• Day refers to the duration (in days) taken to reach X_max.

Antibacterial activity

All bacteria tested for the antibacterial activity of EPS and IPS were retrieved from the Functional Omics & Bioprocess Development Laboratory, Faculty of Science, Universiti Malaya. The stock bacteria were revived via single loop inoculation into fresh nutrient broth (NB, Oxoid CM0001B—1 g/L Yeast extract, 5 g/L peptone, 5 g/L sodium chloride and 1 g/L Lab-Lemco powder at pH 7.4 ± 0.2) at 37 °C, 24 h before the assay. The concentration of the revived bacteria was adjusted to a 0.5 McFarland standard (equivalent to 1.5 × 10⁸ CFU/mL) using 0.85% NaCl, followed by the spreading of 200 µL bacterial culture on Mueller Hinton Agar (MHA, HIMEDIA® M173-500G—2 g/L beef heart infusion, 17.5 g/L casein acid hydrolysate, 1.5 g/L starch and 17 g/L agar with pH 7.3 ± 0.1). Using the agar well diffusion method, 100 µL of EPS and IPS (20 mg/mL) was pipetted into the pre-prepared well (6 mm) on the inoculated MHA and incubated overnight (37 °C) to observe the inhibition zones (mm). Ampicillin (30 µg/mL) and dH₂0 served as the positive and negative control, respectively⁸¹. The antibacterial potential was assessed as follows: < 8 mm → weak, 8–11 mm → moderate, 11–20 mm → strong and > 20 mm → very strong⁶³.

Bacterial cultures with inhibition zones (> 8 mm) in the previous step were selected for minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) screening. In MIC, 25 µL of EPS and IPS (0–20 mg/mL), and 75 µL of microbial culture (0.5 McFarland) were mixed in 96-well microtiter plates, incubated overnight (37 °C) and assessed for microbial inhibition, indicated by a clear suspension and reduced absorbance at 600 nm. MIC was defined as the lowest concentration of EPS or IPS required to inhibit visible microbial growth. Then, MBC was determined by inoculating (100 µL) the mixture that showed microbial inhibition on new MHA and incubated overnight (37 °C). The MBC of EPS and IPS was defined as the lowest polysaccharide concentration at which no microbial growth was observed ⁵.

Antioxidant activity

2, 2-diphenyl-1—picrylhydrazyl (DPPH) and ferric reducing antioxidant power (FRAP) assays were conducted according to Usuldin et al.⁷⁴ with slight modifications. For DPPH, the EPS or IPS (50 µL) was mixed with fresh 0.2 mM DPPH in methanol, incubated in the dark for 30 min followed by absorbance reading at 517 nm. The percentage of DPPH inhibition was calculated using Eq. (3).

3

The IC₅₀ value, determined by the concentration required to reach 50% DPPH inhibition, was calculated from the inhibition curve.

Meanwhile, for the FRAP assay, fresh FRAP reagent was prepared by mixing 300 mM acetate buffer (pH 3.6), 10 mM TPTZ in 40 mM HCl, and 20 mM FeCl₃·6H₂0 in a volume ratio of 10:1:1 (v/v), respectively. Then the EPS or IPS (20 µL) was mixed with 180 µL of the FRAP reagent followed by dark incubation (30 min) and absorbance reading at 593 nm. Results were expressed as mM Fe(II)/mg extract and mM ascorbic acid/mg extract using calibration curves from FeSO₄ (R² = 0.9721) and ascorbic acid (R² = 0.9682) standards, respectively. In both antioxidative assays, ascorbic acid and dH₂O were used as the positive and negative controls, respectively.

Zebrafish embryos toxicity (ZFET) assay

Stock solutions of EPS and IPS were prepared at 50 mg/mL by dissolving samples in embryo media (Danio-SprintM solution). Then, a working solution at 10 mg/mL was prepared by diluting the stock at 5 × in embryo media (Danio-SprintM solution). After that, samples were diluted in embryo media (Danio-SprintM solution) at twofold serial dilution to seven concentrations ranging from 0.16 to 10 mg/mL for both samples in a 96-well microplate. In this toxicity test, embryos in the embryo media (Danio-SprintM solution) only were used as a control (untreated).

Keeping and raising of zebrafish (Danio rerio F. Hamilton, 1822) brood stocks in Danio Assay Laboratories Sdn. Bhd. facilities are performed under permission of Institutional Animal Care and Use Committee (IACUC), Universiti Putra Malaysia (UPM/IACUC/AUP-R044/2022). Briefly, a pair of adult zebrafish was placed into a breeding tank prior to the day of breeding setup. In the next day, embryos were collected from egg collector, washed and incubated in embryo media, Danio-SprintM solution for approximately 2 h. Dead/coagulated embryos were discarded, and healthy fertilized embryos were selected for zebrafish embryo toxicity assay⁹.

The zebrafish embryo toxicity assay was carried out based on Organization for Economic Cooperation and Development (OECD) guideline for fish embryo toxicity (FET) test⁵¹, Test No. 236. Briefly, zebrafish embryos (one embryo/well) at < 10 h of post-fertilization (considered 0 dpf) were exposed to samples (200 µL) in 96-well microplates at seven different concentrations ranging from 0.16 to 10 mg/mL for both samples, EPS and IPS. The samples and untreated were tested with a total of 12 embryos per exposure group. Treated embryos were incubated at room temperature (25–28 °C) for 5 days. The cumulative mortality and developmental malformations of embryos and larvae were observed and determined every 24 h from 0 to 120 h of post fertilization (HPF). Survival rate, hatching rate, heart rate, morphological malformation or teratogenic defects were observed, and images/videos were captured/recorded using an inverted microscope inverted microscope with build-in digital camera (DMIL LED/LEICA, Germany)⁸⁶. The heartbeat was counted from three selected embryos using a stopwatch for 1 min. Lethal endpoints were characterized by coagulation and no heartbeat. Developmental anomalies include pericardial oedema, yolk sac oedema, non-hatched, curved body and bent tail. After five days, live zebrafish larvae will be euthanized by addition of a bleach (5% sodium hypochlorite) to aquarium water at the rate of 1-part bleach to 5 parts of water. They should remain in this solution at least 5 min to ensure death⁹.

Ethics declaration

The approval of the Zebrafish broodstocks (Danio rerio F. Hamilton, 1822) breeding and in vivo toxicity test was granted by a licenced Danio Assay Laboratories Sdn. Bhd. (1,075,617-T), Institutional Animal Care and Use Committee (IACUC), Director of Animal Biochemistry & Biotechnology Laboratory (ABBTech), Department of Biochemistry, Faculty of Biotechnology & Biomolecular Sciences of Universiti Putra Malaysia (UPM), Selangor, Malaysia. The assay was conducted in was conducted in accordance with OECD Guideline No. 236⁵¹, under IACUC approval from Universiti Putra Malaysia (UPM), and complied with ARRIVE (Animal Research: Reporting of In Vivo Experiments) guidelines⁶⁰.

Statistical analysis

All graphs, model fitting and interpolation were conducted using GraphPad Prism 10.5 software (GraphPad Software, Inc) where the data presented are the mean of triplicates with their respective ± SD wherever applicable. The absence of error bar indicates it is shorter than the symbol size. The lethal concentration at 50% (LC₅₀) of treated samples toward zebrafish embryos was also measured using the same software. Heart rate was presented as mean ± standard error of mean (S.E.M) from three different animals. One-way analysis of variance (ANOVA) was used to carry out the significant differences with a post hoc test using Dunnett’s Multiple Comparison. The significant difference was considered at *p˂0.05, **p < 0.01 and ***p < 0.001 between the means of treated group as compared to embryos in embryo media only (untreated).

Author contributions

Conceptualization, W.A.A.Q.I.W.-M., N.A.R. and Z.I.; writing—original draft preparation, D.‘A.N. and J.C.; writing—review and editing, D.‘A. N., J.C., N.A.R., Z.I., N.A.Z.-A. and W.A.A.Q.I.W.-M.; visualisation, D.‘A. N., J.C., N.A.R., Z.I., N.A.Z.-A. and W.A.A.Q.I.W.-M.; supervision, W.A.A.Q.I.W.-M., N.A.R. and Z.I; acquisition, analysis, or interpretation of data, D.‘A. N., J.C. and N.A.Z.-A.. All authors have read, reviewed and agreed to the published version of the manuscript.

Funding

The authors thank the Ministry of Higher Education (MOHE) under the Fundamental Research Grant Scheme (FRGS:FP089-2023 (FRGS/1/2023/WAB04/UM/02/1) for this project ''Antagonistic effects of non-pathogenic mushroom against the Basal Stem Rot disease caused by Ganoderma boninense'' and the Universiti Malaya for the Research University Grant (GPF084B-2020).

Data availability

Data will be available upon request and provided by the corresponding author qadyr@um.edu.my.

Declarations

Conflict of interest

The authors declare that they have no conflict of interest.