Antifungal Efficacy Of Essential Oils Against Wood-Degrading Fungi: A Strategy For Environmentally Sustainable Wood Preservation

INTRODUCTION

Wood is a renewable, organic material widely used in industries such as construction and furniture. However, the presence of organisms like fungi, bacteria, or termites in homes and buildings poses potential health risks. This problem is a major concern for homeowners, builders, contractors, and insurance companies who are involved in ensuring the safety and integrity of structures.^[1] Carbohydrates (hemicelluloses and cellulose) and phenols (lignin) are the main organic compounds in wood. Wood comprises trace amounts of organic extractives, primarily low-molecular-weight molecules, alongside inorganic minerals such as calcium, potassium, magnesium, manganese, and silica.^[2] Additionally, wood constituents include structural cell wall polysaccharides such as cellulose and hemicelluloses, lignin, and extractives that influence various material properties. Consequently, wood is vulnerable to various deteriorating agents, as they recognize these attacks as a vital source of energy.^[3] Wood-decaying fungi are crucial organisms that decompose woody materials, playing a vital role in nutrient recycling and carbon cycling in forest ecosystems.^[4]

Wood-decomposing fungi secrete a wide range of enzymes that act synergistically to break down the major constituents of wood, including cellulose, hemicellulose, and lignin. Studies indicated that cellulases play a key role in cellulose degradation. Cellulases encompass several enzyme classes, including endoglucanases, exoglucanases, and β-glucosidases, which work together to hydrolyze cellulose into smaller sugar units.^[5] These enzymes cleave the glycosidic bonds within cellulose, releasing glucose as a readily available carbon source for the fungi. In addition to cellulose, hemicellulose is another major component of wood. Wood-decomposing fungi produce hemicellulases, such as xylanases and mannanases, which target the complex structure of hemicellulose. Xylanases specifically break down xylan, a major hemicellulosic polysaccharide, into xylose and other sugar units.^[6] Mannanases, on the other hand, degrade mannans, another type of hemicellulosic polysaccharide, releasing mannose and other sugar derivatives. By secreting these hemicellulases, wood-decomposing fungi efficiently utilize the diverse range of substrates present in wood.

Various methods and techniques are used to prolong the lifespan of wood and wood products, including chemical processing and chemical treatments for wood protection against biocorrosion. However, these chemical substances can have detrimental impacts on the ecosystem.^[7] Consequently, studies have aimed to explore natural alternatives that can act as environmentally friendly preservatives for wood and effectively inhibit fungal growth. Numerous natural substances, mainly of plant origin, including plant extracts, essential oils and their components, phenolic compounds, or alkaloids, have been investigated as potential antifungal agents in wood protection.^[8]

Essential oils are volatile, extremely concentrated substances derived from various parts of specific plant species. Every essential oil possesses distinct energetic and therapeutic attributes. These volatile liquids are very intricate compounds exhibiting exceptionally potent and precise effects.^[9] Essential oils are relatively widespread in the plant kingdom, some families being wealthy in such substances, both in number and quantity.^[10] Pharmaceuticals, healthcare, food, and packaging applications extensively utilize essential oils and their derivatives for their antimicrobial properties.^[11] Essential oils obtained from plants are complex mixtures of natural compounds, consisting of both polar and non-polar substances. These oils are well-known for their antiseptic and medicinal properties, and more and more people around the world are interested in how they could be used in research on how to protect wood in an environmentally friendly way. The number of essential oils with biological activity is constantly growing, making them a significant and diverse group of ingredients with potential use in eco-friendly wood preservation.^[12] This work aims to investigate the potential of essential oils as a source of chemicals for wood preservative formulations. In vitro tests will be conducted to evaluate antifungal activity against various fungi that degrade wood. The findings will be open, affordable, and sustainable options for the extraction of antifungal components for application in preservatives for wood. The wood industry may be able to replace some of the more hazardous preservative ingredients with the antifungal components, which would lessen the preservatives’ detrimental effects on the environment. It would also discover a purpose for many wastes that are currently underutilized.

MATERIAL & METHODS

RESULTS AND DISCUSSION

Isolation and qualitative evaluation of cellulase production of isolated fungal strains

Wood deterioration is a significant concern, necessitating the isolation and identification of the fungus responsible. Results show that five fungal strains were obtained from different rotten wood samples. These isolates were qualitatively examined for their ability to produce cellulase enzymes. The results show variations in the production of extracellular cellulase enzymes by the isolated fungal strains. Isolate No. 1 showed the highest enzyme production among the isolates. While Isolate No. 4 was recorded as the minimum enzyme producer. Isolate No. 2 and Isolate No. 5 show intermediate cellulase enzyme production as illustrated in Figure 1.

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Figure 1:

The cellulase enzyme activities of isolated fungal strains. Vertical bars represent standard error (± SD).

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Identification of fungal isolates

As presented in Table 1 and Figure 2, five fungal isolates, obtained from rotten wood, were morphologically identified. Most of these isolates belong to Ascomycota. Among these isolates, molecular identification of the highly cellulase-producing isolate, as reported in the previous investigation, was carried out. The BLAST and phylogenetic analyses showed that the isolate was related to Neurospora crassa, as shown in the phylogenetic tree [Figure 3]. The ITS sequence obtained was submitted to NCBI GenBank under Accession No. PX620420

Table 1: Fungal strains isolated from different rotten wood samples.

Fungal isolates	Name	Family
Isolate 1	Neurospora sp.	Sordariaceae
Isolate 2	Rhizopus sp.	Mucoraceae
Isolate 3	Alternaria sp.	Pleosporaceae
Isolate 4	Penicillium sp.	Aspergillaceae
Isolate 5	Aspergillus sp.	Aspergillaceae

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Figure 2:

Microscopic images of isolated fungal strains: (a) Isolate 1, (b) Isolate 2, (c) Isolate 3, (d) Isolate 4 and (e) Isolate 5.

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Figure 3:

Phylogenetic tree based on ITS sequences of isolated fungal strains and fungal ITS sequences from the GenBank.

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Evaluation of the effect of essential oils on fungal growth and cellulase activity

level of cellulase

Chemical preservatives used in wood have negative environmental effects. Therefore, attention turned to the availability of safer alternatives, so this experiment was conducted to determine the possibility of using these alternatives as promising materials for preserving wood from decaying by Neurospora sp. due to their virulence toward wood decay. As shown in Figure 4, wood samples treated with the tested essential oils weren’t suitable for the growth of Neurospora sp. compared with the +ve control.

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Figure 4:

The effect of wood treatment with the biocontrol agents on growth of Neurospora sp: (a) Licorice oil, (b) Jojoba oil, (c) Rosemary oil, (d) Garlic oil, (e) Peppermint oil, (f) Cinnamon oil, (g) Lavender oil, (h) Lavender + tea tree oils, (i) negative control, and (j) positive control.

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As shown in Table 3 and Figure 5, among all tested oils, compared with the control condition, the presence of licorice oil and cinnamon oil in the media significantly reduces CMCase production by Neurospora sp. Reaching activity 0.13 and 0.28 IUg^-1, respectively. Also, garlic oil has a relatively similar effect on enzyme production. The remaining tested oils have a moderate impact on enzyme activity.

Table 3: Effect of wood treatment with the biocontrol agents on CMCase production by Neurospora sp.

Tested biocontrol agents	CMCase activity (IU g-1)
control	3.12 ±0.006a
Licorice oil	0.13 ± 0.008h
Jojoba oil	2.16 ± 0.084c
Rosemary oil	1.81 ± 0.016 d
Garlic oil	0.94 ± 0.053f
Peppermint oil	2.34 ± 0.029b
Cinnamon oil	0.28 ± 0.012g
Lavender oil	1.81 ± 0.042d
Lavender oil + tea tree oil	1.57 ± 0.076e

In each column, values followed by the same letter are not significantly different according to Tukey’s HSD test (p ≤ 0.05), each value represents the mean of three replicates ± SD.

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Figure 5:

Effect of wood treatment with the biocontrol agents on CMCase production by Neurospora sp.

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DISCUSSION

The search for novel wood preservatives has been encouraged by the growing resistance of fungi to chemical preservatives, heightened societal environmental consciousness, and more stringent chemical laws governing conventional wood preservatives.^[20] The initial stage of a program to investigate various biological resources involves in vitro analysis, which identifies the most bioactive products and establishes their working concentration range. This work successfully isolated five fungal strains from naturally deteriorated wood, which were subsequently identified based on morphological and cultural characteristics. Most of these isolates belong to Ascomycota. These results are in accordance with those of.^[21] The diversity of isolates obtained in this investigation aligns with previous research highlighting the rich mycobiota associated with the degradation of hardwood substrates.

Wood-rot fungi play a crucial role in nutrient and carbon cycling in forest ecosystems because they are primarily responsible for breaking down lignocellulose, one of the most resistant polymers in wood.^[22] For this purpose, the present study evaluated the efficacy of the isolated fungal strain for cellulase production, and the results showed that Neurospora sp. was superior to its peers. These findings are corroborated by, which asserts that Sordariomycetes are proficient lignocellulose decomposers capable of generating substantial quantities of lignocellulose-degrading enzymes due to possessing genes that degrade lignocellulose.^[23]

Plant derivatives, such as essential oils and extracts, obtained from various plant materials, exhibit biological effects. Therefore, chemical compounds and plant-derived substances may be of interest for the development of new, environmentally friendly wood preservatives. Moreover, a significant benefit of plant-derived products is their abundant diversity and accessibility, as well as the ability to extract bioactive compounds from plants. Research demonstrates that several plant-derived chemicals have been evaluated for wood preservation, including essential oils, tannins, terpenes, plant extracts, phenolic compounds, alkaloids, plant oils, and resins.^[24-26]

Before analyzing target locations or mechanisms of action, the antifungal activity of essential oils must be demonstrated.^[27] This method is accurate, as, to the best of our knowledge, essential oils’ activity ranged widely depending on the target pathogen, from extremely effective to ineffectual and from general to specific efficacy. In this study, several essential oils were used, including cinnamon oil, which has powerful antifungal activity.^[28] Showed similar results by testing cinnamon volatile oils against six fungi that caused post-harvest decay of grapes. This decent efficiency is because E-cinnamaldehyde and cinnamyl acetate are the main constituents of cinnamon oil. In Taiwan, researchers evaluated the in vitro antifungal activity of cinnamon leaf EO against fungi that cause wood decay. According to this study, at 50–100 ppm, this EO and its primary ingredient, cinnamon aldehyde, had potent antifungal action.^[29] Our results are consistent with the study, which reported that cinnamon essential oils possess broad-spectrum antifungal activity.^[16]

Lavender oil exhibited both fungistatic and fungicidal activities. The primary constituents of lavender oil, linalool and linalyl acetate, accounted for approximately 75% of the oil. It was found that linalool was responsible for the fungicidal activity, while linalyl acetate inhibited germ tube formation.^[30] Likewise, rosemary oil exhibited promising antifungal properties. It contains bioactive compounds such as 1,8-cineole, which possess antifungal characteristics. These compounds have been reported to disrupt fungal cell membranes and inhibit fungal enzyme activity, thereby inhibiting fungal growth.^[31]

Peppermint oil also displayed significant antifungal effects. The main active component in peppermint oil, menthol, exhibits broad-spectrum antimicrobial activity, including antifungal properties. Menthol disrupts fungal cell membranes, resulting in fungal death or growth inhibition.^[32] Furthermore, tea tree oil and its constituents were found to alter the permeability and fluidity of fungal membranes, thereby affecting membrane properties and disrupting membrane-related processes.^[12] Garlic oil also showed significant antifungal activity, which may be due to its richness in organic sulfur compounds. Moreover, it has various medicinal properties, including antioxidant, antifungal, antidiabetic, and anti-inflammatory.^[33]

Many plants, especially those in the Lamiaceae family, such as Rosmarinus officinalis L. (rosemary), are known for their antifungal activity, particularly in their essential oils and leaves.^[34] Studies indicate that rosemary essential oil yields vary depending on plant species, the collection area’s ecology, whether the plant is cultivated or spontaneous, and the extraction method used.^[35] Antifungal activity of this oil is due to the chemical structure, which includes many important compounds, such as bornyl acetate, rosmarinic acid, α-pinene, camphor, carnosol, carnosic acid, rosmarinic acid, rosmanol, and rosemaridiphenol.^[36,37] These active compounds facilitate the permeation of the fungal cell wall and penetrate the lipid bilayer, rendering the cell membrane permeable, resulting in cytoplasmic leakage, cell lysis, cell death, or prevention of sporulation and germination.^[38]

Previous studies reported that rosemary oil exhibited relatively weak to moderate antifungal activity against various fungal species, including Penicillium sp., Aspergillus sp., Fusarium sp., and Trichoderma sp.^[39] Antimicrobial essential oils disrupt membrane permeability and osmotic equilibrium and depend on their hydrophobicity and partitioning into microbial membranes, resulting in structural and functional damage to microbes.^[40]

The present study demonstrated that the tested essential oil significantly inhibited cellulase activity of the most potent wood-decomposing isolate, identified as Neurospora crassa, isolated from rotted wood samples. In particular, the presence of licorice and cinnamon oils significantly reduced the extracellular cellulase activity, suggesting that these oils can disrupt the cellulolytic mechanisms of this fungal strain.^[41,42] Reported that cinnamon and licorice oils have an extensive antimicrobial effect, and this adversely influences fungal metabolism and enzyme production. According to a recent study that emphasized the sensitivity of cellulase expression to environmental and metabolic stress, our findings indicate that essential oils may reduce cellulase production in Neurospora crassa, either directly by inhibiting enzyme secretion or indirectly by interfering with signaling pathways.^[43]

CONCLUSION

Recent studies have significantly advanced our knowledge of wood-decaying fungi, unveiling their enzymatic capabilities, ecological significance, and evolutionary relationships. The diverse enzymatic machinery employed by wood-decaying fungi highlights their remarkable adaptability and efficiency in wood-decay processes. Understanding the ecological roles and classification of wood-decaying fungi is crucial for forest management and conservation efforts, as they play a fundamental role in nutrient cycling and carbon dynamics. Ongoing research in this field will continue to deepen our understanding of these fascinating organisms and further unravel the intricate mechanisms of wood decay.