Abstract
This work was designed to evaluate the bioactivity of four monoterpenes, namely carvacrol, p-cymene, eugenol, and iso-eugenol, applied to wood blocks from Pinus sylvestris sapwood using the vapor method against Aspergillus flavus, Aspergillus niger, and Fusarium culmorum. These monoterpenes were prepared at 20, 40, 60, 80, and 100 µL/mL. The highest fungal inhibition percentage (FIP, 24.4%) against the growth of A. flavus was observed for p-cymene when applied to a wood sample at 100 µL/mL. The highest FIPs observed against the growth of A. niger were 21.5% and 16.3%, by p-cymene and iso-eugenol, respectively, at 100 µL/mL. The highest FIPs observed against the growth of F. culmorum were 41.5 and 27.0% by the application of carvacrol at 100 µL/mL and 80 µL/mL, respectively. This study showed the importance of monoterpenes for antifungal activity and may contribute to the most rational use of these compounds as antimicrobial agents for wood protection.
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Antifungal Activity of the Monoterpenes Carvacrol, p-Cymene, Eugenol, and Iso-Eugenol When Applied to Wood against Aspergillus flavus, Aspergillus niger, and Fusarium culmorum
Mohamed Z. M. Salem 
,a,* Wael A. A. Abo-Elgat ,b Maisa M. A. Mansour ,c,* and Shady Selim ,d
This work was designed to evaluate the bioactivity of four monoterpenes, namely carvacrol, p-cymene, eugenol, and iso-eugenol, applied to wood blocks from Pinus sylvestris sapwood using the vapor method against Aspergillus flavus, Aspergillus niger, and Fusarium culmorum. These monoterpenes were prepared at 20, 40, 60, 80, and 100 µL/mL. The highest fungal inhibition percentage (FIP, 24.4%) against the growth of A. flavus was observed for p-cymene when applied to a wood sample at 100 µL/mL. The highest FIPs observed against the growth of A. niger were 21.5% and 16.3%, by p-cymene and iso-eugenol, respectively, at 100 µL/mL. The highest FIPs observed against the growth of F. culmorum were 41.5 and 27.0% by the application of carvacrol at 100 µL/mL and 80 µL/mL, respectively. This study showed the importance of monoterpenes for antifungal activity and may contribute to the most rational use of these compounds as antimicrobial agents for wood protection.
DOI: 10.15376/biores.20.1.393-412
Keywords: Antimicrobial activity; Carvacrol; p-Cymene; Eugenol; Iso-eugenol; Pinus sylvestris sapwood
Contact information: a: Forestry and Wood Technology Department, Faculty of Agriculture (El‑Shatby), Alexandria University, Alexandria 21545, Egypt; b: Restoration Department, High Institute of Tourism, Hotel Management and Restoration, Abu Qir, Alexandria, Egypt; c: Department of Conservation and Restoration, Faculty of Archaeology, Cairo University, Giza 12613,Egypt; d: Department of Pesticide Chemistry and Technology, Faculty of Desert and Environmental Agriculture, Matrouh University;
* Corresponding authors: zidan_forest@yahoo.com; maisamansour@cu.edu.eg
INTRODUCTION
Wood is a naturally occurring, renewable, extremely adaptable, and high-performing material that has been widely utilized by humans from their beginning. Additionally, it holds the greatest amount of carbon trapped in terrestrial ecosystems. However, because of its structure and chemical components, wood is vulnerable to biodeterioration, with fungi being the primary degraders (Goodell et al. 2008; Brischke and Alfredsen 2020; Afifi et al. 2023). Mold stains can cause damage to wood and other organic materials; their activity results in the discoloring of wood, which detracts from its aesthetic value even if they are not seriously damaging structural integrity (Allsopp et al. 2004; Kim et al. 2020; Eldeeb et al. 2022; Taha et al. 2021; Mansour et al. 2023). Certain environmental factors, such as moisture content above 20%, oxygen availability, and temperature between 15 and 45 °C, make wood vulnerable to fungal infestation. The primary target of fungal decay is outdoor wooden structures. The infestation can greatly reduce the mechanical and aesthetic qualities of the wood and shorten its service life (Zabel and Morrell 2012; Meyer and Brischke 2015).
Secondary metabolites of plants called monoterpenes are commonly utilized in industrial processes as starting points for significant fragrance compounds including (−)-menthol and vanillin. Nevertheless, monoterpenes’ physicochemical characteristics make it challenging to convert them conventionally into scents with additional value (Soares-Castro et al. 2020). Because of their low boiling temperatures, monoterpenes are the primary components of most plant essential oils and are responsible for the distinctive odorous qualities of plants. For example, geranyl pyrophosphate, the common acyclic C10 intermediate of the isoprenoid route, is the starting point for their biosynthesis (Rehman et al. 2016; Gershenzon and Croteau 2018). Monoterpene hydrocarbons and oxygenated monoterpenes are the two main categories of monoterpenes. Alcohols, aldehydes, ketones, ethers, and acids are included in the latter category (Zuzarte and Salgueiro 2015; Soares-Castro et al. 2020; Yingngam 2022). Certain monoterpenes have inherent pesticidal qualities that make them suitable starting compounds for the development of safe, efficient, and completely biodegradable insecticides as well as possible substitutes for pesticides (Khursheed et al. 2022; Gupta et al. 2023). Numerous fungicidal actions of monoterpenes (Wuryatmo et al. 2003; Cárdenas-Ortega et al. 2005), and other characteristics, are possessed.
In common practice, the durability issue with wood has been addressed by preservative treatments such as creosote, pentachlorophenol, and inorganic arsenic (Cheng et al. 2008). However, when these preservatives are utilized over time, they cause serious issues with pollution to the environment and human health. Thus, it becomes increasingly important to look for bioactive chemical compounds from plants that are natural, safe, and do not pollute as a substitute for synthetic preservatives (Loh et al. 2011; Hu et al. 2015).
During the authors’ continuous search for potential antifungal substances, four monoterpenes, namely carvacrol, p-cymene, eugenol, and iso-eugenol were applied to wood samples and evaluated for their antifungal activity against three plant pathogenic fungi Aspergillus flavus, Aspergillus niger, and Fusarium culmorum. Monoterpenes, including geraniol, myrcene and thymol were observed to have promising antifungal activity against four plant pathogenic fungi Asperigallus niger, Rhizoctonia solani, Fusarium oxysporum, and Penecillium digitatum (Marei et al. 2012). Among 20 compounds, the antifungal tests revealed that cuminaldehyde, β-citronellol, nerol, geraniol, citral, and α-terpineol exhibited strong antifungal effects against Botryosphaeria dothidea (Zhang et al. 2018). Carvacrol was found to be the most potent of the 41 pure monoterpenes against the wood white-rot fungi Trametes hirsuta, Schizphylhls commune, and Pycnoporus sanguineus. This means that carvacrol may be used as a natural fungicidal agent in the treatment of wood preservation (Zhang et al. 2016). Strong antifungal activity was demonstrated by cinnamaldehyde, α-methyl cinnamaldehyde, (E)-2-methylcinnamic acid, eugenol, and isoeugenol against the brown-rot fungus Laetiporus sulphureus and the white-rot fungus Lenzites betulina (Cheng et al. 2008). Additionally, it was discovered that natural compounds have fungicidal properties against the fungi that cause wood decay. Of these natural compounds, the most potent antifungal ones were eugenol, α-cadinol, τ-muurolol, τ-cadinol, γ-cadinene, cryptomeridiol, chamaecynone, cinnamaldehyde, and ferruginol (Wang et al. 2005 a,b; Cheng et al. 2006; Yen and Chang 2008).
Thus, this study aimed to find a botanical-based compound with potential antifungal activity against wood-decay pathogenic fungus. To reach this goal, the study investigated the fungicidal activity of four monoterpenes, carvacrol, p-cymene, eugenol, and iso-eugenol when applied to wood samples against three pathogenic fungi and compared the antifungal activity potential of these four monoterpenes against each pathogenic type of fungi.
EXPERIMENTAL
Materials
Monoterpenes
Four monoterpenes (Fig. 1), carvacrol, p-cymene, eugenol, and iso-eugenol, were obtained from Sigma-Aldrich (Merck). The monoterpenes were prepared at concentrations of 20, 40, 60, 80, and 100 µL/mL. The respective amount of monoterpene was diluted in 10% dimethyl sulfoxide (10% DMSO and 90% sterile distilled water), and 0.5 mL of Tween 80 (polysorbate-80) as emulsifier was added (Salem et al. 2016; Mohareb et al. 2023).
Fig. 1. Chemical structures of the monoterpenes
Fungi
The antifungal bioassays of the four monoterpenes, carvacrol, p-cymene, eugenol and iso-eugenol were conducted using three molds (Aspergillus flavus AFl375, Aspergillus niger Ani245, and Fusarium culmorum Fcu761) and accession numbers in Gen Bank, MH355958, MH355955, and MH355957, respectively (Abo Elgat et al. 2020; Elshaer et al. 2024).
Methods
Vapor treatment of wood with the monoterpenes for fungi inhibition
Pinus sylvestris wood, which is widely used in Egyptian woodworking, was selected for the work as an expensive imported wood. Therefore, the staining mold fungi can grow over the wood when the appropriate conditions—namely, relative humidity, moisture content, and temperature—are met. Wood blocks from P. sylvestris sapwood (Mohareb et al. 2023) in the dimension of 0.5 × 2 × 2 cm were vapor-treated with each of the prepared monoterpenes at the previous concentrations using the evaporation method (López et al. 2005; Nedorostova et al. 2009). Wood samples were put in Petri dishes that contained 8 layers of Wattman No. 1 filter paper overlaid by a mesh (polyethylene spacer). The dishes were autoclaved at 121 °C for 20 min and left to cool, then each monoterpene compound with the respective concentration was impregnated over the filter papers (three Petri dishes for every monoterpene and concentration) and kept for 48 h to allow the monoterpene evaporation, which was subsequently absorbed by the wood samples.
In vitro antifungal activity of treated wood with monoterpenes
The antifungal activity of wood treated with four monoterpenes samples against the growth of A. flavus, A. niger, and F. culmorum was achieved (Taha et al. 2019; Elshaer et al. 2024). A 15-day-old PDA culture of each fungus was prepared. Three wood samples were used for each concentration. Following the application of each monoterpene compound to wood samples, a Petri dish containing PDA culture was inoculated with a disc (5-mm diameter) of each fungus, and the samples were then incubated for a week at 25 ± 1 °C. As an alternative, 10% DMSO and SDW (1:1 v/v) were combined in the control sample, while fluconazole (0.31%) was used as a positive control. The inhibition zones (IZs, mm) of the monoterpenes around the treated woods against each fungus were measured and recorded (Ali et al. 2021).
The mycelial growth inhibition percentage was measured with the following formula (Correa-Pacheco et al. 2017; Shakam et al. 2022): MGI = [(Ac − At) / Ac] × 100; where MGI is mycelial growth inhibition and Ac and At are average diameters of the fungal colony of the control and treatment, respectively.
After two weeks of inoculation, the visual observation of the fungal growth extent was visually evaluated by the naked eye in accordance with the GOST 9.048–75 (1975) standard, which ranged from 0 (mycelium growth more intense than control) to 5 (no growth).
Statistical analysis
The analysis of variance (ANOVA) tool in SAS version 8.2 was used to statistically examine data from the application of monoterpenes on wood samples against the growth of each fungus. Duncan’s Multiple Range Test at Alpha 0.05 was used to measure the differences among the means. The IC50 (half-maximal inhibitory concentration) value is a measure of the concentration of a compound required to inhibit each fungal growth by 50%. These IC50 values were determined using Probit analysis (Finney 1952).
RESULTS AND DISCUSSION
Visual Observation, Inhibition Zones, and the Fungal Growth on Wood-Treated Monoterpenes
Fungicidal activity was estimated by fungal growth retardation using the visual observation-determined marks (Table 1) and the antifungal bioassay (Figs. 2, 3, and 4). The highest number (4) in Table 1 shows a very marked retardation (colony < 25% of controls) of fungi, especially carvacrol at 100 µL/mL with F. culmorum, p-cymene at 100 µL/mL with A. flavus and F. culmorum, and eugenol and iso-eugenol at 100 µL/mL with F. culmorum.
Table 1. Marks of Fungal Growth Retardation
* Values are measured according to: (GOST-9.048-89 1975; Humar and Pohleven 2005; Krivushina et al. 2022). 0: growth more intense than control, 1: normal growth, insignificant retardation (area of colony ≥ 90% of area of controls); 2: visible signs of retardation (colony < 90% and ≥ 60% of controls); 3: pronounced retardation (colony < 60% and ≥ 25% of controls); 4: very marked retardation (colony < 25% of controls); 5: no growth
As shown in Fig. 2 and Table 2, there was enormous or massive growth of Aspergillus flavus on an untreated wood sample, but this growth decreased when the wood samples were treated with the standard fungicide (fluconazole, 0.31%). The highest inhibition zone (IZ) values were 22.00, 19.00, and 17.00 cm by p-cymene, eugenol, and iso-eugenol at 100 µL/mL, respectively, as well as p-cymene at 80 µL/mL with an IZ value of 17.3 mm, compared to fluconazole (17.7 mm).
Figure 3 and Table 2 present the antifungal activity of monoterpene-treated wood against the growth of A. niger. The highest IZ values were 19.3 and 14.7 mm as wood samples treated with p-cymene and iso-eugenol, respectively, at 100 µL/mL. Additionally, at 80 µL/mL, the treated wood samples showed IZ values of 10.3 and 10.7 mm by p-cymene and iso-eugenol, respectively, compared to fluconazole (7.3 mm).
Figure 4 and Table 2 present the antifungal activity of monoterpene-treated wood against the growth of Fusarium culmorum. The highest IZs were recorded by the application of carvacrol at 100 and 80 µL/mL with values of 37.3 and 24.3 mm, respectively. These were followed by eugenol, p-cymene, and iso-eugenol with values of 21.3, 20.7, and 20.3 mm, respectively, and iso-eugenol at 80 µL/mL with IZ 19.3 mm, compared to fluconazole (17.3 mm).
A: Carvacrol; B: p-cymene; C: Eugenol; D: Iso-eugenol
Fig. 2. Antifungal activity of monoterpenes-treated wood against the growth of Aspergillus flavus
