Efficacy of Fungus Comb Extracts Isolated from Indo-Malayan Termite Mounds in Controlling Wood-Decaying Fungi

Abstract

The authors have recently investigated the chemical components and bioactivity of fungus comb from Macrotermes gilvus Hagen mounds. The ethyl acetate, methanol, and water extracts of the fungus comb contained active compounds which are preventing the growth of Aspergillus foeti-dus, one of the most economically important wood-staining fungi in Indonesia. In this present study, the bioactivity of the fungus comb extracts was examined against the white-rot fungus Schizophyllum commune Fr. For the purpose of generating a realistic in-service type of environment, the extracts were evaluated according to modified EN-113 after impregnated into wood samples by the vacuum-pressure method, following in-vitro antimicrobial susceptibility test. The results showed that ethyl acetate extract at concentrations ranging from 2 to 6% and methanol extract at a concentration of 6% presented high bioactivity against S. commune. This result was established through optical microscopy images, which demonstrated the absence of fungal mycelia in the vessels of wood samples treated with EtOAc extract at concentrations of 2%, 4%, and 6%, as well as MeOH extract with a concentration of 6%. The toxic values of the ethyl acetate and methanol extracts were determined to be 6.17% and 7.72%, respectively. Based on UPLC-HRMS analysis, azelaic acid, and erucamide were discovered as the dominant components in ethyl acetate extracts, which are anticipated to be the most active compounds. It appears that ethyl acetate extract, as well as methanol extract, can be considered as novel preservative sources for controlling wood-decaying fungi.

1. Introduction

Schizophyllum commune Fr. LPH 01 is one of the most economically important wood-decaying fungi for residential buildings in Java Island, the most populated island in Indonesia. In the year of 2010, 87% of houses in the island were severely attacked by the decay fungi, causing an economic loss of almost USD 35 million/year [1]. The fungi attacked more than 25 wood species commonly used as building components in the country and could cause 70% weight loss of the attacked wood [2]. Supporting this finding, according to a report by Djarwanto et al. [3], S. commune is highly aggressive and has the ability to infect nearly all types of wood. It has been reported that S. commune has a wide range of substrates, including at least 150 genera of woody plants, softwood, and grass silage. The ability of S. commune to degrade all components of lignocellulosic biomass is due to the presence of numerous gene candidates in its genome, such as 240 for glycoside hydrolases, 89 of which are involved in plant polysaccharide degradation. Thus, S. commune is an economically significant agent in the biodeterioration of wood structures due to its plant cell wall degrading and modifying enzymes, including 75 for glycosyl transferases, 16 for polysaccharide lyases, 17 for expansin-related protein, 30 for carbohydrate esterase, and 16 for lignin-degrading oxidoreductases [4]. Even Schmidt [5] noted that the fungus was a serious wood destroyer in tropical regions and potentially colonized wood faster than any other wood-rot fungi.

The protection of wooden-based materials in residential buildings against the attack of wood-decaying fungi is challenging due to the environmental conditions in Indonesia, with average annual air temperature at 26.8 °C and humidity levels ranging from 80%–100% [6], which are ideal for the growth of different fungi that inhabit wood [7,8]. Priadi et al. [1] reported that precipitation is the climatic factor that has the greatest impact on wood decay in house construction in Java Island, Indonesia. During the rainy season, wood is exposed to rainwater containing nutrients, which facilitates the activities of wood-degrading fungi, leading to biodeterioration [9]. As such, preventing the attacks of wood-decaying fungi on wooden components of residential buildings in Indonesia is crucial. In addition, since most of the wood components used in the country are highly susceptible to such attacks [1] the prevention effort is even more important to undertake.

Furthermore, 85% of the log production in Indonesia, which amounts to 47.9 million m³/year, is obtained from plantation forests [10], which are highly vulnerable to biodeterioration [11]. Consequently, it is reasonable that most residential buildings in the country are constructed using non-durable wood species that are susceptible to wood-decaying fungi [1]. To address this situation, various approaches are needed, including the development of organic fungicides that are safe for both humans and the environment. These fungicides should be composed of active ingredients that are derived from indigenous natural resources. This effort is relevant to the emerging development of natural wood preservatives to substitute synthetic substances.

The authors recently examined the chemical constituents of the fungus comb from the Indo-Malayan termite Macrotermes gilvus Hagen (Isoptera: Termitidae) and its bioactivity against wood-staining fungi. Phytochemical analysis revealed abundant secondary metabolites, primarily from phenolic hydroquinone, steroid, terpenoid, and saponin groups. Gas chromatography-mass spectrometry (GC-MS) analysis showed that the ethyl-acetate (EtOAc) extract was dominated by a phenolic group. The results also showed that the fungus comb extracts inhibited one of the most economically important wood-staining fungi in Indonesia, Aspergillus foetidus, with inhibition rates ranging from 24.17% to 100% and EtOAc extract as the most active extract compared to water and methanol extract [12]. However, the efficacy of the fungus comb extract against wood-decaying fungi has not been reported. Hence, this present research aims to determine the chemical component and bioactivity of the fungus comb extract against the wood-decaying fungus S. commune. Ultra-performance liquid chromatography-high resolution mass spectrometry (UPLC-HRMS) was used to explore the active compounds in the fungus comb extract. In addition to the in-vitro antimicrobial susceptibility test, the study extended the method with the EN-113 European Standard. The objective of the study is to ascertain the biological activity of the extracts of fungus comb from Indo-Malayan termite M. gilvus mounds against wood-decaying fungus S. commune. In this circumstance, it is expected that we could identify the active compounds of the extract and its toxic value on S. commune fungi. We believe that these inventions would contribute to the effort of developing environmentally friendly organic fungicides in order to control these fungi attacks on various wood species that are frequently used as components in residential buildings.

It is important to note that Indonesia’s tropical climate is home to a diverse range of termite species. Out of the 3106 species of termites recorded globally, 300 of them (11.05%) have been discovered in Indonesia [13,14]. Even, the Indo-Malayan termite M. gilvus has the widest geographical distribution in the country [15]. Hence, any attempts to utilize fungus comb from the termite mound as a source of environmentally friendly organic fungicides may potentially add value to the idle local resource.

2. Materials and Methods

2.1. Fungus Comb Preparation and Extraction Process

We collected fungus combs from 18 mounds of M. gilvus located in Yanlappa, West Java, Indonesia, at coordinates 6°25′3.05″ S–106°29′59.36″ E and 6°25′21.00″ S–106°29′46.20″ E. The preparation and extraction of the fungus comb followed the procedure described by Nandika et al. [12]. Initially, the n-hexane solvent was used for extraction, followed by EtOAc for semipolar extractive substances. Methanol (MeOH) was used for further extraction, while distilled water was used to obtain the water extract. The polar extractive substances of the fungus comb were determined by MeOH and water extracts.

2.2. Extract Compounds Analysis Using UPLC-HRMS

Prior to injection to UPLC-HRMS (UHPLC Vanquish Tandem Q Extractive Plus Orbitrap HRMS, Thermo Scientific, Karlsruhe, Germany), the extract was diluted in MeOH and filtered with Polytetrafluoroethylene (PTFE) 0.2 um membrane. The column used was Accucore C18, 100 × 2.1 mm, 1.5 μm (ThermoScientific, Germany) with a 0.2 mL/min flow rate. Elution was performed using a gradient system with water + 0.1% formic acid as solvent A and acetonitrile + 0.1% formic acid as solvent B. From 0–1 min, the eluent was 5% B; from 1–25 min was 5%–95% B; from 25–28 min was 96% B, and from 28–30 min was 5% B. The column temperature was 30 °C, mass range of 100–1500 m/z with positive ion mode.

2.3. Preparation of Extract Solutions

To obtain different concentrations of the EtOAc, MeOH, and distilled-water extracts from the fungus comb, they were dissolved in 5 mL of a 5% dimethyl sulfoxide (DMSO, Sigma Aldrich, St. Louis, MO, USA) solvent. The concentrations used were 2%, 4%, and 6% (w/v). A negative control solution was also prepared, which consisted of a 5% DMSO solvent without the fungus comb extract. After preparing the extract solutions, each was filtered using a 0.45 µm microsolve filter and passed through a syringe to obtain sterile extracts, which were then transferred to glass vials.

2.4. In-Vitro Antimicrobial Susceptibility Test of the Extract Solutions

To investigate the impact of different extract solutions on the growth of S. commune fungus on potato dextrose agar (PDA) media, an in-vitro anti-fungal activity test of the fungus comb extracts was conducted according to the method in the previous study of Nandika et al. [12].

2.5. Wood Samples and the Vacuum-Pressure Treatment

Wood samples were obtained from air-dried rubberwood (Hevea brasiliensis Muell. Arg.) boards of a twenty-year old tree in Bogor, West Java, Indonesia. The boards measured 3.5 cm in thickness, 20 cm in width, and 40 cm in length, with the length parallel to the grain, and mainly consisted of sapwood. The mean moisture content and density of the air-dried boards were 15.23% and 0.64 g/cm³, respectively. The boards were then cut into wood samples of dimensions 0.5 cm in thickness, 1.5 cm in width, and 2.5 cm in length, oven-dried at 103 ± 2 °C, and sterilized using steam in an autoclave (GEA Medical, YX-24LDJ, Jiangyin Binjiang Medical Equipment Co., Ltd., Jiangyin, China) for 15 min. The extract solutions, including the negative control, were introduced into the wood samples with a procedure following Nandika et al. [16]. In this procedure, the wood samples were placed in a beaker glass (cap. 250 cc), then each extract solution with a particular concentration (150 cc for a single pressure application) was poured into the beaker glass, so that all of the samples were submerged. In addition, negative control solutions were also applied to the wood samples with the same procedures. Furthermore, the container was placed in a closed stainless-steel autoclave, and a vacuum of 50 bar was created and maintained for 15 min, followed by increasing the pressure to 2.5 psi for 2 h. Afterward, the pressure was released, and the wood samples were extracted from the residual solution. Each extract treatment, as well as the untreated samples (negative control), had six replications. After impregnation, wood samples were left at 22 °C and 65% relative humidity in a conditioning room for two weeks prior to the anti-fungal test according to EN-113 Standard in which wood species, size, and climatic room were modified.

2.6. Anti-Fungal Test of Extract Solutions According to EN-113 Standard

An anti-fungal efficacy test was conducted to evaluate the efficacy of fungus comb extract solutions against S. commune (IPBCC 22 1531, Bogor, Indonesia) following the EN-113 standard, with a total of 72 wood samples and six replicates for each solution (including untreated control and various concentrations of extraction solvent). One treated and one untreated wood sample were placed into a 9 cm diameter petri dish containing a 7-day-old fungal culture of S. commune, and the samples were incubated at 28 ± 2 °C and 85% relative humidity for 16 weeks. The weight loss calculation of each wood sample was done according to Simeto et al. [17].

$$\mathrm{Weight\; loss} (\%)=({\mathrm{D}\mathrm{W}}_1-{\mathrm{D}\mathrm{W}}_2)/{\mathrm{D}\mathrm{W}}_1·100$$

(1)

DW₁ represents the oven-dried weight of the wood sample prior to the test, while DW₂ represents the oven-dried weight of the same sample after being exposed to the fungus. The toxic value of the active extract is expressed as the lowest concentration that can prevent weight loss of the wood sample from exceeding 3%, which is considered adequate in controlling the growth of S. commune. If the lowest concentration does not meet the adequate level in controlling the fungi growth, we will predict the toxic value of the active extract using regression analysis between the extract concentration and the weight loss of the wood sample demonstrated by any extract.

2.7. Wood Degradation Monitoring

Wood sample degradation after exposure to S. commune culture was monitored using optical microscopy. 30 µm microtome cuts were taken from the top surface of the wood sample using a microtome apparatus (American Optical Company, New York, NY, USA). These were analyzed with a digital camera Keyence VHX-6000 transmission optical microscope (Keyence Corporation, Osaka, Japan).

2.8. Data Analysis

The study reported the mean extraction yields of different solvents from the fungus comb, the weight percentage gain (WPG) on the treated sample, the inhibition rate of the fungus comb extracts on S. commune growth, and the weight loss of the wood samples. The data were analyzed using a completely randomized design with a 3 × 4 factorial structure. The first factor (A) was the solvent used in the extraction process (EtOAc, MeOH, and water), while the second factor (B) was the concentration of the extract solution (0%, 2%, 4%, and 6%). The data were analyzed using two-way analysis of variance (ANOVA) with a significance level of 5% in IBM SPSS Statistics 24 software, followed by Duncan’s multiple range test. The mathematical model for this data analysis could be expressed as followed: Y_ijk = µ + A_i + B_j + A_iB_j + ε_ijk, where Y_ijk is observation value at i extraction solvent, j extraction concentration, and k replication, $\mathsf{\mu }$ is mean value of observation, A_i is extraction solvent at i level, B_j = extract concentration at j level, A_iB_j is effect of interaction between extraction solvent at i level and extract concentration at j level and ε is error at i extraction solvent, j extraction concentration, and k replication.

3. Results

3.1. Compounds Identified in All Extracts

The chromatogram of 3 extracts (Figure 1) showed that the fungus comb extract consists of different components. The detected compounds based on mass spectrometry data are shown in

Table 1. The detected compounds in three extracts of the fungus comb.

No	Calc. MW	Formula	Predicted Compound	Detected in Extract of (% Area)
				Water	MeOH	EtOAc
1	103.09988	C5H13NO	Choline	11.57	21.35	0.50
2	117.07912	C5H11NO2	Betaine	5.48	-	-
3	131.0945	C6H13NO2	L-Isoleucine	1.61	5.93	-
4	145.05209	C9H7NO	4-Indolecarbaldehyde	-	-	2.17
5	174.08826	C8H14O4	Suberic acid	-	-	1.89
6	182.07829	C6H14O6	D-(-)-Mannitol	5.84	4.31	-
7	188.10395	C9H16O4	Azelaic acid	1.11	1.01	8.98
8	296.23495	C18H34O4	NP-008993	-	-	1.67
9	300.19326	C15H26O3	NP-004917	-	-	2.33
10	322.17485	C16H28O5	(6E)-10-Heptyl-5,8,9-trihydroxy-3,4,5,8,9,10-hexahydro-2H-oxecin-2-one	-	-	0.55
11	326.19126	C18H30O3S	4-Dodecylbenzenesulfonic acid	14.59	2.69	3.02
12	337.33266	C22H43NO	Erucamide	-	-	6.52
13	470.33739	C30H46O4	18-β-Glycyrrhetinic acid	-	-	3.60

. Nine compounds were detected in the EtOAc extract, with erucamide as the major compound. Choline is the major compound in water and MeOH extracts.

3.2. Bioactivity of Fungus Comb Extract against S. commune

3.2.1. Results of In-Vitro Antimicrobial Susceptibility Test

According to the findings, the EtOAc extracts completely inhibited the growth of the fungus at all tested concentrations. Additionally, the growth of S. commune fungus was inhibited by the MeOH and distilled water extracts at rates ranging from 38.22% to 100% and 3.67% to 17.56%, respectively. However, the negative control solution, which contained no extracts (0%), did not have any inhibitory effect on the fungus growth (0% growth inhibition), as shown in Figure 2.

The results of the study showed that the extracts obtained from EtOAc, MeOH, and water inhibited the S. commune growth at 2%, 4%, and 6% concentrations. The two-way ANOVA and further data analysis of Duncan’s test indicated that the inhibition rates of all tested extracts on the growth of S. commune were mostly significantly different, except for the inhibition rate caused by the EtOAc extract at any tested concentration (2%, 4%, and 6%) and the MeOH extract at a concentration of 6%. The EtOAc and MeOH extracts displayed higher inhibition rates than the water extract at the same concentration, with the highest inhibition rates being observed with the EtOAc extract at all tested concentrations and the MeOH extract at a concentration of 6%.

Upon visual observation, it was noted that the diameter of the S. commune colony on the PDA media treated with all tested concentrations of ethyl-acetate extract (2%, 4%, and 6%), and the 6% MeOH extract was smaller after seven days of incubation compared to the water extract (Figure 3).

This investigation also demonstrated that the inhibition of S. commune fungus growth was influenced by the solvent used during the extraction process and the concentration of the extract solution added to the PDA media. As presented in Figure 2, each solvent used in the extraction process exhibited different results concerning the inhibition of fungus growth. Moreover, the extracts’ ability to suppress growth increases with increasing fungus comb extract solution concentration. It was also established that the ethyl-acetate extract at all tested concentrations (2%, 4%, and 6%) and the 6% MeOH extract exhibited the highest bioactivity by inhibiting the growth of S. commune fungus (achieving a growth-inhibiting rate of 100%).

3.2.2. Results of the Anti-Fungal Test According to EN-113 Standard

The most significant efficacy of the fungus comb extract in controlling S. commune attack was demonstrated by the wood samples treated with EtOAc and MeOH extracts at 6% concentration, which showed relatively low average weight loss of only 3.72% and 4.01%, respectively. Meanwhile, untreated wood samples (0% concentration) in EtOAc, MeOH, and water extracts demonstrated average weight loss of 14.08%, 15.28%, and 14.75%, respectively (Figure 4).

Figure 4 presents the average weight loss percentages of wood samples exposed to different concentrations of fungus comb extracts after 16 weeks of exposure to S. commune. The statistical analysis confirmed significant differences (p ≤ 0.05) between the treated and untreated samples for all extraction solvents used (EtOAc, MeOH, and water). The results showed that the weight loss of treated wood samples ranged from 3.72% to 12.43%, while the untreated samples had weight loss percentages between 14.08% and 15.28%. These findings suggest that higher extract concentrations resulted in lower weight loss for wood samples treated with any extraction solvent. The lowest weight loss percentages were observed for the wood samples treated with a 6% concentration of EtOAc and MeOH, respectively (p ≤ 0.05). Additionally, the EtOAc extract at the lowest concentration applied (2%) demonstrated anti-fungal properties. The results also suggest a clear correlation between the extract concentration and the weight loss of the wood samples.

Since the lowest weight loss of the wood sample demonstrated by each extract was higher than 3% (threshold value considered adequate in controlling the growth of S. commune by EN 113), we predict the toxic value of each extract by regression analysis. Based on the analysis, the ethyl acetate extract meets the adequate level in controlling S. commune at a concentration of 6.17%, while the methanol extract is predicted to prevent weight loss to less than 3% after reaching the concentration of 7.72%. The water extract has a much higher predicted toxic value, up to 26.93%, as shown in Figure 5.

The study showed that the EtOAc and MeOH extracts had notable anti-fungal properties against S. commune fungi. This finding was further confirmed by observing the absence of fungal mycelia in the vessel of wood samples treated with EtOAc extract at concentrations of 2%, 4%, and 6%, and MeOH extracts with a concentration of 6%. Conversely, fungal mycelia were present in the vessel of wood samples treated with water extracts and untreated wood samples (Figure 6).

4. Discussion

According to the results, the extraction yield resulting from EtOAc was the lowest, followed by MeOH and distilled water. This finding was consistent with a study conducted by Nandika et al. [12], which also reported lower extraction yields from EtOAc compared to MeOH and distilled water (1.73%, 2.53%, and 4.61%, respectively). According to Syafii [18], the amount of extract yield is influenced by the quality of the solvent used, as well as the particle size of the extracted material, the extraction process, and the water content of the extracted material. Since the extraction method performed in this study followed Nandika et al. [12], it could be confirmed that these mentioned factors involved in both studies were similar, resulting in a quite similar extraction yield.

Hidayah et al. [19] reported that EtOAc is able to extract phenolic, terpenoids, alkaloids, aglycones, and glycosides compounds. Based on our result, the EtOAc extract consists of some compounds that are found in MeOH and water extract (Figure 1 and Table 1). These results can be explained by the semipolar compound inherent in EtOAc. Meanwhile, MeOH, which is more polar than EtOAc, can dissolve more polar compounds [20]. MeOH, as an extracting agent, is able to extract phenolic compounds, steroids, terpenoids, alkaloids, and glycosides [21]. Based on the chromatogram (Figure 1), the component in MeOH extracts is similar to water extract because the nonpolar compounds had been extracted in EtOAc extract. The rest of the component in the fungus comb was extracted with water. Water is the most polar solvent compared to other solvents, such as MeOH [22], and can dissolve bioactive compounds such as phenolic, including flavonoids, tannins, and alkaloids [23]. Based on the chromatogram (Figure 1 and Table 1), components extracted in water content mostly are quaternary ammonium, amino acid, sugar alcohol, and other acids. The different content in each extract will produce different activities, including different anti-fungal activities.

In relation to the differences in anti-fungal bioactivity of each fungus comb extract, we argue that these variations are due to different chemical compounds contained in each fungus-comb extract, which depends on the solvent used in the extraction process. Our study shows that of 13 detected compounds in three extracts of the fungus comb, 8 of them were reported to have antimicrobial and specifically anti-fungal activity (

Table 2. Reported antimicrobial activity of the detected compounds in three extracts of the fungus comb.

Detected Compounds	Targeted Microbe	Reported Activity	References
Betaine	Malassezia restricta	Anti-fungal	[24]
L-Isoleucine	Candida albicans	Antifungal (Icofungipen)	[25]
4-Indolecarbaldehyde	Candida albicans, Candida dubliniensis	Antifungal	[26]
Suberic acid	Altenaria Alternata, Phytophtora infestans, Fusarium Oxysporum, F. equiseti	Antifungal	[27,28]
D-(-)-Mannitol	Fusarium latenicum, Mucor hiemalis, Aspergillus niger, Aspergillus ochraceus, Candida mycoderma, Alternaria alternata, Geotrichum candidum	Antifungal	[29]
Azelaic acid	Staphylococcus epidermidis, Propioni bacterium, S. capitis, S. hominis, P. granulosum, P. aviduni	Antifungal	[30,31,32]
Erucamide	Agrobacterium tumefaciens, Erwinia carotovora, Ralstonia solanacearum	Antibacterial	[33]
18-β-Glycyrrhetinic acid	Staphylococcus aureus	Antibacterial	[29]

). In line with the bioactivity data of the fungus-comb extracts (Figure 2), the compounds whose bioactivity functioned as antimicrobial, including anti-fungal, were found scattered in all extracts.

The inhibition growth of S. commune against fungus comb extract is different depending on the concentration. This difference in inhibition rate could be attributed to the varying concentrations of secondary metabolites present in the media treated with different extract concentrations. As explained by Sitepu et al. [34], a higher concentration of extract in the medium leads to a greater diffusion of the extract into the fungus cells, which can disturb their growth. Furthermore, the weight loss of the wood samples was found to differ depending on the extraction solvent used. As a result, the fungus comb extract exhibits remarkable bioactivity against S. commune. This result also supported the lower weight loss of wood samples impregnated with the fungus comb extract with concentrations 2%, 4%, and 6% (Figure 4). The EtOAc extract is relatively effective in preventing weight loss by the fungi attack (3.72%), and it is not so different with weight loss of wood samples treated with MeOH extract (4.01%). However, the weight loss of wood samples treated with water extract (10.06%) was quite higher than the other extract. Those weight loss differences were the effect of the active components of the solvents, which acted as anti-fungi. According to Witasari et al. [35], phenolic compounds such as guaiacol and syringol are present in the EtOAc extract of fungus combs and are believed to be the main antimicrobial substances. The lowest weight loss among the wood samples was demonstrated by the ethyl acetate extract at 3.72%. However, this value still exceeds the 3% requirement set by EN 113. By using regression analysis, the predicted toxic value of the ethyl acetate extract is 6.17%, with an R-squared value of 0.92. Similarly, the predicted toxic value of the methanol extract is 7.72%, with an R-squared value of 0.95. Additionally, the predicted toxic value of the water extract is 26.93%, with an R-squared value of 0.94.

The MeOH extract could inhibit 100% of the S. commune growth at a high concentration of 6%. The major component in the MeOH extract is choline which is also detected in the water extract. No report related to choline as an anti-fungal, but this compound was reported as a nutrient for humans and animals [36]. Choline has been reported to play a role in the synthesis of neurotransmitters, transport of lipids, signaling in cell membranes, and metabolism of methyl groups. It means the water and MeOH extract of fungus comb is prospective to be utilized for humans’ or animals’ nutrients. In addition, the MeOH extract of fungus comb has high antioxidant activity [37].

The EtOAc extract showed 100% inhibition against S. commune, even in the lowest concentration of 2%. This extract consists of azelaic acid and erucamide as the main component, as much as 8.98 % and 6.54%, respectively. Azelaic acid is reported as antimicrobial against skin bacteria such as Staphylococcus epidermidis, S. capitis, S. hominis, Propionibacterium acnes, P. granulosum, and P. aviduni [26,27]. This carboxylic acid was reported to have anti-fungal activity [28]. Erucamide is reported as antibacterial agent against Agrobacterium tumefaciens, Erwinia carotovora, and R. solanacearum [29]. The EtOAc extract could inhibit the growth of S. commune predicted not only because of the two compounds but also from the unknown compounds, which mark with * in Figure 1. The peaks with * mark mostly are specific peaks of EtOAc extract, which could not be found in other extracts. For the next research, isolating and identifying unknown compounds is important. However, further investigations are needed to comprehensively clarify the bioactivity of the compound and its chemical structure without neglecting the other active compounds detected in this study. In addition, it is important to note that the given figures possibly show some variations within the stages of the termite’s colony maturity.

5. Conclusions

The bioactivity of fungus-comb extracts (ethyl acetate, methanol, and water) against wood-decaying fungi S. commune was observed. The highest bioactivity was displayed by ethyl acetate and methanol extracts of the fungus comb, with a concentration of 6%, in inhibiting the attack of the wood-decaying fungus S. commune. The regression analysis predicts that the toxic value for ethyl acetate, methanol, and water extract were 6.17%, 7.72%, and 26.93%, respectively. The performance of ethyl acetate extract at 6% concentration was particularly notable, which could be attributed to its specific active components, including azelaic acid and erucamide, along with other unidentified compounds that might act as the most active components. More research is necessary to separate and identify the chemical structure of specific active components of the fungus comb extract that are accountably for inhibiting the wood-decaying fungus.