Menu
Brasil
home Sumário navigate_before Anterior Atual Seguinte navigate_next
home Sumário
Original Article • Braz. J. Biol. 84 • 2024 • https://doi.org/10.1590/1519-6984.289015 linkcopy

Open-access Structure of fungal community and culturable fungi on the discolored surfaces of pine storage boxes in the tropical region in Dong Nai, Vietnam

Estrutura da comunidade fúngica e fungos cultiváveis nas superfícies descoloridas de caixas de armazenamento de pinheiro na região tropical em Dong Nai, Vietnã

AuthorshipSCIMAGO INSTITUTIONS RANKINGS

    Abstract

    Wood and wood-based materials are commonly used for storage, but their surfaces are prone to biodegradation by microorganisms, especially fungi. This study focuses on the microbial communities on pine wood storage boxes treated with an anti-termite and mold solution in a tropical region in Dong Nai, Vietnam. We isolated 13 fungal strains from these surfaces and classified them into six genera: Rhizopus, Aspergillus, Fusarium, Curvularia, Penicillium, and Trichoderma. Enzyme activity tests revealed that strains Curvularia eragrostidis TD4.2 and Aspergillus sydowii TD5 were the most effective producers of cellulase, amylase, and laccase. Shotgun metagenomics analysis of the biological sample of the discolored surface of pine storage boxes indicated that Ascomycota was the dominant phylum, with Dothideomycetes and Sordariomycetes as the prevalent class. Aureobasidium (0.33%) and Chaetomium (1.1%) were the most abundant genera in the Dothideomycetes and Sordariomycetes, respectively. This research illustrates the complexity of microbial communities on wood surfaces, providing insights into the fungal dynamics affecting wooden storage materials in tropical climates.

    Keywords:
    wood surface microbes; wood-decaying fungi; fungal diversity; discolored wood; pine wood

    Resumo

    Madeiras e materiais à base de madeira são amplamente utilizados para armazenamento, mas suas superfícies são suscetíveis à biodegradação por microrganismos, especialmente fungos. Este estudo foca nas comunidades microbianas presentes em caixas de armazenamento de madeira de pinho tratadas com uma solução antitermites e antifúngica na região tropical de Dong Nai, Vietnã. Isolamos 13 linhagens de fungos dessas superfícies, classificadas em seis gêneros: Rhizopus, Aspergillus, Fusarium, Curvularia, Penicillium e Trichoderma. O gênero predominante foi Aspergillus. Testes de atividade enzimática revelaram que as linhagens Curvularia eragrostidis TD4.2 e Aspergillus sydowii TD5 foram as mais eficazes na produção de celulase, amilase e lacase. A análise metagenômica shotgun da amostra biológica da superfície descolorida das caixas de armazenamento de pinho indicou que Ascomycota era o filo dominante, com Dothideomycetes e Sordariomycetes como classes prevalentes. Aureobasidium (0,33%) e Chaetomium (1,1%) foram os gêneros mais abundantes dentro das classes Dothideomycetes e Sordariomycetes, respectivamente. Esta pesquisa destaca a complexidade das comunidades microbianas nas superfícies de madeira, fornecendo insights sobre a dinâmica fúngica que afeta materiais de armazenamento de madeira em clima tropical.

    Palavras-chave:
    microrganismos da superfície da madeira; fungos decompositores de madeira; diversidade fúngica; madeira descolorida; madeira de pinho

    1. Introduction

    Wood products are widely used in various applications of life. Despite almost all these products being protected by chemical or biological treatments, incidents of treated wood damage are often highly problematic. Degradation was caused by the mutual action of environmental factors (abiotic) and biotic attacks (Rowell, 2005). Biodegradation of treated wooden constructions caused by fungi, including soft rot, molds, and staining fungi, may result in economic losses, reduced product quality, and even endanger human health indoors.

    Fungal biodegradation of wood has been intensively studied for several decades. Soft rot fungi have been found on naturally weathered wood surfaces indoors and outdoors, even wood treated with preservatives and various climatic regions. Two phyla, Ascomycota and Basidiomycota, colonized on naturally weathered wood surfaces with a higher number of CFU (colony forming units) in less antimicrobial wood and higher humidity or amount of rain (Buchner et al., 2019). The ratio of each composition varied depending on wood species, wood decaying stage, and environmental conditions (Kielak et al., 2016). Fungal isolation studies from wood debris showed that basidiomycetes were dominant in the earlier stages of wood decay and that many ascomycetes, such as soft-rot microfungi, occurred in the later stages (Crawford et al., 1983; Fukasawa et al., 2009; Lumley et al., 2001). Research by Fukasawa et al. (2011) indicated that fungal species belonging to Basidiomycota, Ascomycota and Zygomyco isolated from wood debris decayed wood where five species Bjerkandera adusta, Mycena haematopus, Omphalotus guepiniformis, Trametes hirsuta, Trametes versicol (Basidiomycota) caused the highest decay, followed by four species Scytalidium lignicola, Tnchoderma hamatum, T. harzianum, T. koningi (Ascomycota) and finally Zygomycota (Fukasawa et al., 2011). Various atmospheric conditions had different influences on wood products, in which higher densities of fungi and bacteria were found respectively in the exposed indoor and outdoor products, and also microbial agents varied with the wood type (Dan and Nwachukwu, 2020). Under tropical climates, especially in high humidity, fungi could be feasible to grow and propagate, resulting in the decay of the wood (Darmawan et al., 2019). Even in extreme environments such as the Antarctic, for the first time discovered that wood degradation occurred with the presence of two types of specialized lignocellulolytic microbes, soft rot fungi and tunneling bacteria (Björdal and Dayton, 2020), where fungal decay dominates and their hyphae penetrate the outer 2–4 mm of the wood surface.

    Various wood treatment solutions have been widely used to protect wood from fungal colonization, including chemical, physical, and biological methods. However, many researchers reported fungal attacks on the treated wood products (Bridžiuvienė and Raudonienė, 2013; Ma et al., 2013; Pfeffer et al., 2012; van Nieuwenhuijzen et al., 2017). Their findings showed a fungal diversity on treated wood types such as genere Alternaria, Aureobasidium, Cladosporium, Fusarium, Penicillium, Phoma, Superstratomyces, or species Trichoderma sp., Epicoccum sp., Penicillium commune, Aspergillus niger, Paecilomyces variotii, Mucor plumbeus, Trametes versicdor, Gloeophylum trabeum, Aureobasidium pollulans. Thus, the fungi began to resist various preservatives or adapt to environmental conditions. A self-protection mechanism can be due to the production of oxalic acid, extracellular mucilaginous material, or unspecific ligninolytic extracellular enzymes that help degrade (Clausen and Green, 2003; Kim et al., 2007; Szewczyk and Długoński, 2009).

    So far, most research has been based on the culture method and has identified different fungal and bacterial genera or species in decaying wood in different environments (Johnston et al., 2016). However, the cultural proportion was far lower than the whole microbial community, in which the ratio of uncultured microorganisms accounts for a large part (Amann et al., 1990). Metagenomics is the unculture-based analysis of a mixture of microbial genomes (termed the metagenome) using an approach to expression or sequencing (Riesenfeld et al., 2004; Schloss and Handelsman, 2003).

    Fungal extracellular enzymes play a crucial role in degrading wood and lignocellulose materials structured by polymer components such as cellulose, hemicellulose, and lignin. Fungi possess genes encoding laccase, peroxidases, and glycoside hydrolases that work together to break down wood components (Arnstadt et al., 2016). Besides, other polysaccharide-degrading enzymes, including catalytic activities for decaying hemicellulose, starch and glycogen, mutant, chitin, and β-glucans were predicted in P. chrysosporium (Martinez et al., 2004). Studies have also explored the ecological aspects of wood decomposition by saprotrophic fungi, such as Fomes fomentarius, in coarse wood substrates. Significant differences in enzyme production by F. fomentarius have been observed between different wood types, with varying activities of cellulose and xylan-degrading enzymes depending on the wood species (Větrovský et al., 2011). Moreover, fungal communities in wood substrates can influence enzyme production and decomposition processes, highlighting the complex interactions between fungi, bacteria, and wood substrates (Větrovský et al., 2011). Microfungi such as Penicillium chrysogenum, Fusarium solani, and F. oxysporum also showed a degree of lignin decomposition (Kirk and Farrell, 1987). In shotgun metagenomics research, results of fungal gene-encoding enzyme analysis targeted to degrade the oak wood polymers found cellulose-targeting enzymes at different decaying classes (early, intermediate, and late) (Pioli et al., 2023). Additionally, starch-degrading enzymes in fungi were higher at middle decay stages, and the study found abundance Aspergillus oryzae in the dataset, suggesting a possible role in the increased glucoamylase production, while two fungi of Zymoseptoria tritici, Cercospora beticola identified at late decay with increase in α-amylases and a glucoamylase (Pioli et al., 2023).

    To improve effective strategies for fungal prevention in treated wood boxes used in item storage in tropical region Vietnam, a better understanding of the fungal composition as well as the colonized abilities of the causal agents will be essential. The present study was initiated by a mold issue affecting the surface of treated wood storage boxes placed in indoor conditions. Despite the wooden boxes being treated with wood preservatives, fungal growth was observed over time, evidenced by wood discoloration and the presence of dark mycelium. Thus, this study aimed to identify the structure of the fungal community and culturable fungi on treated wood box surfaces by shotgun metagenomics analysis and traditional isolation, respectively. In addition, a biochemical property of enzyme activity important for fungi in wood colonization was determined.

    2. Materials and Methods

    2.1. Materials

    Pinewood storage boxes in warehouse K750, Dong Nai, Vietnam (10° 57’ 0” N, 106° 52’ 26” E) were the objects of the study (Figure 1). Pine wood is harvested and processed into custom parts for packaging in Vietnam. The wood is planed on both sides and cleaned using an air blower. The wood treatment process is carried out by placing the wood in a tank and gradually releasing the prepared treatment solution until the wood is fully submerged. The wood is soaked continuously for 3 hours, then removed and left to dry on racks before being dried at 105 °C for 15 minutes. The treatment solution for anti-termite and mold protection contained 80% Cu2SO4·H2O, 15% K2Cr2O7, 3% Cr2O3, and 2% additives. Material on the surface of wood storage boxes was collected and used as a source for fungi isolation and evaluation of the microbial structure using shotgun metagenomics.

    thumbnail of Figure 1
    Figure 1
    Pine wood storage boxes from warehouse K750, Dong Nai, Vietnam.

    Chemicals for enzyme testing, such as guaiacol, carboxymethyl cellulose, and soluble starch, were purchased from Sigma (Sigma Aldrich, Germany). DNA extraction from isolated strains and specimens was carried out using DNA Extraction Kit (Norgen, Canada) and DNeasy PowerSoil Pro kit (Qiagen), respectively.

    2.2. Sampling method

    A sterile cotton swab was used to wipe the surface of the mold-infected material. The tip of the swab was collected and placed into a sterile tube, which was kept cold during transport to the laboratory. Each fungal-infected spot on the wood was swabbed using 2 swabs over an area of approximately 3×3 cm. A total of 20 swabs were taken, corresponding to 10 locations on the mold-infected wooden box. Samples were collected from five wooden boxes and homogenized for use in the study.

    2.3. Fungal strain isolation

    The biological samples on the swabbed cotton tips were homogenized in sterilized physiological saline with 0.005% Tween 80 and shaken at 200 rpm for 30 minutes. 100 μL of the homogenized solution was taken and spread evenly on the Czapek-Dox petri dish supplemented with a mixture of antibiotics (100 mg/L ampicillin and 100 mg/L tetracycline). The czapek-Dox medium contains saccharose 30 g.L-1; MgSO4 0.5; K2HPO4 1 g.L-1; KCl 0.5 g.L-1; NaNO3 2 g.L-1; FeNO3 0.01 g.L-1; agar 15 g.L-1; water up to 1000 mL; pH 7,3. The inoculated plates were cultivated at 30 °C for 3 - 5 days in the dark. Fungal colonies appaired on the plates were subcultured and purified to obtain pure fungal strains, Czapek-Dox medium was used during the purification process (Ngo et al., 2021).

    2.4. Fungal morphology characterization

    Isolated fungi were inoculated on a Czapek-Dox medium agar plate by inoculating needle and then incubated at 28-30 °C for 3-5 days; the fungal colony morphology was recorded and described by surface color, form, elevation, and margin shape. The reproductive spores and sporulation after 72 hours of cultivation were observed under an optical microscope (Seifert & Gams, 2011).

    2.5. Fungal classification

    The ITS1-5.8S-ITS2 region (ITS region) sequence was adopted to classify isolated fungi phylogenetically. Fungal DNA was extracted using the Fungi/Yeast DNA Extraction Kit (Norgen, Canada). ITS region was amplified with primer pair ITS1F (5'- CTT GGT CAT TTA GAG GAA GTA A - 3') and ITS4R (5' - TCC TCC GCT TAT TGA TAT GC - 3'). ITS regions were sequenced on an ABI PRISM® 3100-Avant Genetic Analyzer (Applied Biosystems, USA) at 1st BASE Company (Singapore). Raw sequences were processed with BioEdit software (ver. 6.0.7, USA) to achieve fungal-isolated ITS sequences. GenBank (NCBI) database and BLAST tool on NCBI (2024) were applied to identify relative ITS sequences to which fungal isolates in this study. The phylogenetic tree was constructed using MEGA software (ver.10, USA) with a neighbor-joining algorithm.

    2.6. Extracellular enzyme activity detection

    Extracellular enzymes such as cellulase, amylase, and laccase, which are involved in the lignocellulose and polymeric carbohydrate metabolism in the wood, were detected by the ability to utilize the corresponding substates of fungi growing on Czapek-Dox agar plates. Czapek-Dox agar plates were supplemented with the substrate as 1% carboxymethyl cellulose (CMC), 0.5% starch, and 0.05% guaiacol for cellulase, amylase, and laccase, respectively (Abdallah et al., 2019; Adegoke and Odibo, 2019; Anita et al., 2013). A 6 mm diameter fungal agar plug was placed on a Czapek-Dox plate with supplemented substrate, each plate placed seven fungal plugs. The testing plates were transferred to a 30 °C incubator for the next 24 hours. Fungal strains that exhibited enzyme activities showed a halo ring around the plug after staining with 1% Lugol's solution. The diameter of the clear zone around the well is measured using a ruler in millimeters. The measurement was done by taking the average of two perpendicular diameters for more accurate results. The test was carried out in triplicate.

    2.7. Shotgun metagenomics

    2.7.1. DNA extraction

    The tips of 10 swabs are cut and placed in 10 mL of sterile distilled water with 2% Tween 80 in a sterilized tube to release the fungal hyphae from the swabs. The mixture is vortexed at maximum speed for 2 minutes, repeated 3 times. The swabs are removed, and the solution is centrifuged at 13,000 rpm to collect the biomass. The subsequent steps are carried out according to the DNeasy PowerSoil Pro Kit Handbook 2023 (Qiagen). DNA metagenome was accumulated to meet the requirements for quality and quantity for next-generation sequencing (NGS) and stored at -80 °C before sequencing.

    2.7.2. Metagenome sequencing

    The extracted DNA was used to generate a library using the NEBnext Ultra II library preparation kit and NEBnext Multiplex Oligos Dual (NEBnext) with four main steps, including cleaving by fragments, adapter ligation, indexing, and cloning by PCR. The library generation procedure was carried out according to the manufacturer's instructions. The library product DNA was attached to adapters and indexes between 300-400 bp in size. The library was then sequenced using next-generation sequencing (NGS) on the Illumina system (USA).

    2.7.3. Bioinformatics analysis

    The quality control of the sequencing data was done using fastp. Key steps included adapter trimming, quality filtering, trimming low-quality bases, removing short reads (<50 bp), and eliminating duplicate reads. These measures ensured high data accuracy and reliability for downstream analyses. Purified reads were used to analyze the microbial structure and predict population function using SqueezeMeta (ver. 1.4.0), a comprehensive workflow ideal for handling the complexity of metagenomic data. The analysis performed in SqueezeMeta v1.4.0 is summarized as follows: the input reads are assembled into longer sequences called contigs. Next, the location of the coding genes and the rRNA sequence on the contig is determined. These genes are aligned with a database to evaluate the microbial composition and function of the community. In addition, the alignment data were used to calculate the number of sequences aligned with a gene in the database and the depth of gene coverage, therefore estimating the relative proportion of microorganisms.

    2.8. Data analysis

    The experiments were triplicate; data was calculated and evaluated using GraphPad Prism (Ver 6.01). ANOVA analysis was applied where relevant.

    3. Result and Discussion

    3.1. Isolation of culturable fungi from the surface of pine wood storage boxes

    From the fungal-harbouring surface of wood storage boxes, 13 fungi were isolated. Isolated fungi were distinguished from each other by colony morphology first and then the characteristics of reproductive structures. Typical colony morphologies of isolated fungi on Czapek-Dox medium after five days of cultivation are 1) powdery and dark brown to black, entire circular; 2) light brown, filamentous margin and form; 3) pastel pink, undulate margin and cottony aerial hyphae; 4) velvety green shade with white margin (Figure 2). In this study, only the Czapek-Dox medium was used as the base medium to cultivate culturable fungi; the number and diversity of isolates were relatively high compared to the status of the boxes in which just black and green mold were dominant, as seen by bared eyes.

    thumbnail of Figure 2
    Figure 2
    Morphology of isolated fungi from the surface of wood storage boxes. (A) Aspergillus niger TS5; (B) Curvularia eragrostidis TD4.2; (C) Fusarium equiseti TD6.2; (D) Penicillium chrysogenum TD12.3.

    All 13 isolates were classified based on their morphology and sequences of ITS region. They belong to six genera, including Rhizopus, Aspergillus, Fusarium, Curvularia, Penicillium, and Trichoderma. The best hit with the highest identity after aligning 13 ITS sequences with the GenBank database is detailed in Table 1. Seven out of 13 are Aspergillus, a dominant genus among culturable fungi on the surface of wood boxes. The phylogenetic relationship based on ITS sequences of fungal isolates is demonstrated in Figure 3.

    Thumbnail
    Table 1
    Best hit in GenBank database of 13 fungal isolates.
    thumbnail of Figure 3
    Figure 3
    Phylogenetic relationship between isolated fungi from the surface of wood storage boxes. The number in the branch indicates the bootstrap value with 1000 bootstrap replications.

    3.2. Production of extracellular enzymes by fungal isolates

    Extracellular enzymes such as cellulase, amylase, and laccase are involved in wood-decaying, with target substrates being the main components of wood structure, including cellulose, starch, and lignin. The enzyme-producing ability of 13 fungal isolates was determined using an agar-plug diffusion plate with corresponding substrates. The isolated fungi showed the ability to produce these three types of enzymes at different levels; some isolates produced all three enzymes, while others did not produce any activity. However, some isolates produced 1 or 2 of the three tested enzymes. A representative illustration of the activity-tested plates is shown in Figure 4, and the transparent halo-ring diameter index is visualized in Figure 5.

    thumbnail of Figure 4
    Figure 4
    Enzyme activity-tested plates. (A) Cellulase; (B) Amylase; (C) Laccase. Positions on the plates: (1) Aspergillus awamori TS1; (2) Aspergillus niger TS5; (3) Penicillium chrysogenum TD12.3; (4) Curvularia eragrostidis TD4.2; (5) Aspergillus sydowii TD5; (6) Fusarium equiseti TD6.2; (7) Aspergillus austroafricanus TD11.1.
    thumbnail of Figure 5
    Figure 5
    Enzyme activity comparison between isolated fungi. The scatter dot graph presents enzyme diffused activity as the diameter of the halo zone. Two-way ANOVA, p < 0.0001.

    Curvularia eragrostidis TD4.2 and Aspergillus sydowii TD5 were two strains that exhibited the ability to produce high levels of all three types of enzymes, including cellulase, amylase, and laccase; the activities of their enzymes were higher than the rest. Meanwhile, Aspergillus austroafricanus TD11.1 and Rhizopus oryzae TD6.1 produced cellulase significantly higher than amylase and laccase. By contrast, Trichoderma atroviride TS10.1 showed more amylase and laccase activity than cellulase. There were two isolates Curvularia umbiliciformis TD7 and Aspergillus austroafricanus TD12.1 of which none of all three tested enzymes was detected. The difference in the enzyme production ability of fungal isolates belonging to the same genus Curvularia (Curvularia eragrostidis TD4.2 and Curvularia umbiliciformis TD7), shows that the enzyme production ability under the surveyed conditions is characteristic that depends on each specific strain. A similar occurrence was also observed with Aspergillus strains; Aspergillus sydowii TD5 displayed outstanding enzyme activities, while other strains of this genus showed no enzymatic activity (Aspergillus austroafricanus TD12.1) or very low activities. Considering the ratio of fungal isolates that produce each enzyme, it was found that most strains were capable of secreting cellulase (11/13 isolates). However, only 8 out of 13 isolates showed amylase activity (Figure 5).

    3.3. Structure of fungal community on the surface of wood storage boxes

    The DNA sample obtained from the biological sample collected from the surface of the wood storage box was used to evaluate the community structure. The shotgun metagenomics approach was applied, and all genetic material in the DNA sample was sequenced. The sequencing yielded 6,185,888 raw reads, of which 65.74% were unclassified reads. The high percentage of unclassified reads in metagenomic data indicates significant microbial diversity that is currently undocumented. Among classified reads, 33.79% were eukaryotic reads, and the number of prokaryotic reads is very low. Bacteria and archaea only account for 0.74% of the total obtained reads, in which the phylum with the largest number is Acidobacteria (0.3075), followed by Proteobacteria and Firmicutes. In contrast, with the eukaryotic group, there were 17 phyla identified, the dominant phylum was Ascomycota, with the largest proportion of 30.19%, and the number of reads belonging to eukaryotes whose phylum was not identified was 3% (Figure 6).

    thumbnail of Figure 6
    Figure 6
    Community structure at domain and phylum levels of biological community on the surface of pine wood storage boxes in the tropical region.

    Ascomycota was the dominant phylum on the surface of the wood box, and the most prominent class was Dothideomycetes. This class accounts for 20.05% of the total obtained reads and 58.5% of the classified reads. There are significant differences between the top two classes in Ascomycota as the second dominant class Sordariomycetes was 2.06%. In addition, up to 7.59% of total reads were unclassified in the Ascomycota phylum. Reads were unclassified into genera in Dothideomycetes held a large number, they consisted of 10.99% Unclassified Dothideomycetes, 5.36% Unclassified Mycosphaerellales and 2.23% for other families. Identified genera that accounted for more than 0.1% were Aureobasidium, Rachicladosporium, Hortaea, Venturia and Periconia, the highest belonged to Aureobasidium with 0.33%. Chaetomium (1.1%) was the most dominant genus in Sordariomycetes. Genera Fusarium and Trichoderma existed in Sordariomycetes. Although the culturable fungal isolates were classified into these genera, Fusarium and Trichoderma possessed a small percentage of 0.2 and 0.018%, respectively (Figure 7).

    thumbnail of Figure 7
    Figure 7
    Taxonomic profiling of dominant groups at Ascomycota phylum (A); Dothideomycetes class (B); Sordariomycetes class (C).

    4. Discussion

    Pine wood is a popular industrial type widely used to produce storage boxes, containers, and pallets. Before being processed into finished products, pine wood is typically treated using various methods to prevent fungal and insect damage (Khademibami and Bobadilha, 2022). However, these wooden containers often suffer from discoloration or blue stain, which diminishes their aesthetic value, impacts the quality of the stored products, reduces their usability, and even leads to decaying the wood material. This discoloration is typically caused by microorganisms, with the main fungal genera frequently mentioned as molds, including Aspergillus, Penicillium, and Fusarium (Rahman and Fuad, 2002; Schmidt, 2006; Singh and Chauhan, 2013). Several studies have identified these genera within the group of decaying fungi on wood; they belong to soft-rot fungi, which can deteriorate wood by virtue of their cellulolytic enzymes (Zabel and Morrell, 2020). These fungal genera have also been identified in microbial samples taken from the pine wood storage boxed surface in this study. Of the 13 fungal strains isolated, they were classified into six genera of filamentous fungi within the Ascomycota phylum. Aspergillus is the predominant genus, with 7 out of the 13 isolated strains belonging to this genus. The ability to produce enzymes involved in wood degradation varies among the isolated strains. Curvularia eragrostidis TD4.2 and Aspergillus sydowii TD5 produced cellulase, amylase, and laccase with relatively high activity. The strain with the highest activity for all three types of enzymes was Aspergillus sydowii TD5. Additionally, the remaining Aspergillus strains exhibited lower enzyme activity, except for Aspergillus austroafricanus TD11.1, which demonstrated superior cellulase activity compared to the other two enzymes.

    This study's pine wood storage boxes were chemically treated with fungal resistance. However, in the tropical climate of Dong Nai, Vietnam, the effectiveness of this treatment in preventing fungal colonization is limited, and discoloration can still occur, even though the wood is stored in a well-ventilated, enclosed space. Identifying the microbial composition on the surface of these stained boxes can provide information to improve wood treatment methods and storage practices during use for better effectiveness (Cao et al., 2024; Darmawan et al., 2019). Microbial community structure obtained through shotgun metagenomics has revealed significant differences from the initial findings based on isolation and classification methods of the isolated strains. The sample collection process only recorded locations with changes in color and the development of fungal hyphae forming cotton-like structures, however, when analyzing the microbial community, we discovered bacteria, viruses, actinomycetes, etc. Unclassified reads accounted for more than 65% of the total genetic information in the sample, indicating unknown biodiversity on the surface of the sample as well as the limited information in the database related to the specific source in this study. Genera Fusarium, Trichoderma, Aspergillus, and Curvularia were all present in the study samples; however, their identification rates varied significantly. Although Aspergillus is a genus with many isolated strains, it only accounted for 0.0427% and could not be classified at the species level. In contrast, Fusarium represented a relatively high percentage of 0.209%, with several species such as F. clavum, F. coffeatum, and F. flagelliforme. In the wooden church in Romani, similar fungal genera, including Aspergillus, Penicillium, Stachybotrys, Scopulariopsis, Arthrinium, Mucor, and Geotrichum, were identified in both wood surface samples and microaeroflora (Ilies et al., 2018).

    Conversely, although Rhizopus was isolated from biological samples from wood surfaces, it was not detected in the obtained sequence data. In addition to the well-studied representative filamentous fungi mentioned above, the non-cultured sample revealed that Chaetomium globosum accounted for 1.0386% of the fungal community on the discoloration surface. This species is the most predominant among the identified fungi in this sample. Chaetomium globosum is an endophytic fungus that can degrade wood and wood products indoors and outdoors. It also has the potential to produce mycotoxins that can cause allergies and opportunistic diseases (Abdel-Azeem et al., 2020; Fogle et al., 2008). When this species is detected, replacing and treating the wood storage boxes and adjusting the storage conditions are necessary. In a warm and humid climate like Dong Nai, Vietnam, the strong development of Chaetomium globosum is inevitable. Pinewood infected with this species has been treated with antifungal chemicals but has not shown effective results. This study suggests the need to treat wood with other mold-resistant agents, especially for soft-rot fungi, such as using essential oils, coating agents, or a combination of several fungicides (Biles et al., 2012; Broda, 2020) while climate and storage conditions are challenging to change.

    At the kingdom level, the results show that bacteria and archaea accounted for a small proportion, while fungi from the phylum Ascomycota were the predominant group. This was quite different from microbial community composition in pine wood samples collected from dead trees in different stages of decomposition, which were dominated by bacterial abundance of Acidobacteria, Alpha-, Beta-, Gammaproteobacteria, Firmicutes and Actinobacteria, whereas fungal abundance almost entirely consisted of two phyla, Basidiomycota and Ascomycota with 95–100% of sequences, respectively (Kielak et al., 2016). The three predominant classes are Dothideomycetes, Sordariomycetes, and Eurotiomycetes, with all isolated fungal genera belonging to these classes. Based on both results from culturable and non-culturable diversity analysis, it can be observed that soft-rot fungi were the primary cause of discoloration stains on the surface of pine wood storage boxes. This group of soft-rot fungi primarily degrades cellulose and hemicellulose using a diverse cellulolytic enzyme system (Saini and Sharma, 2021). Also, itcan produce laccase to degrade lignin, similar to other lignocellulose decaying fungi. This is consistent with the lignocellulolytic enzyme production of the isolated fungal strains (Figure 5). Diversity of microbial community structure in decaying wood is also in relation to wood traits such as carbon content (Pioli et al., 2023). Pioli’s research also combined the results from the metagenomic analysis with in vitro bioassays based on bacterial and fungal isolates. However, only a few cultivated strains of fungi and bacteria matched the metagenomic database for a reliable comparison of intra- and inter-kingdom co-occurrences (Pioli et al., 2023).

    Identifying and understanding these groups of microorganisms will guide the development of methods to mitigate their harmful effects through wood treatment processes or the use of preservatives tailored to each target microbial group. There are various wood preservation methods, such as adjusting temperature and humidity, sterilization with ozone, irradiation, drying, and high-pressure steaming. However, the method applied depends on the size and value of the wooden items. For small, high-value items, adjusting temperature and humidity may be suitable. In the humid tropical climate of Vietnam, drying, steaming, and irradiation methods only temporarily inhibit microorganisms and do not ensure long-term effectiveness. Therefore, the use of chemicals is a suitable solution for larger and more numerous items, with the requirement that they must be safe for humans and the environment. Based on the results obtained, another research is required, such as testing appropriate preservative solutions against Ascomycota fungi, particularly Chaetomium globosum.

    5. Conclusion

    The biological sample from the discoloration area on the surface of pine wood storage boxes shows diversity and predominance of soft-rot fungi. The phylum Ascomycota is the most prevalent, with the predominant classes being Dothideomycetes, Sordariomycetes, and Eurotiomycetes. Chaetomium globosum was the most dominant species in the non-cultured sample. Several fungi isolated from the pine wood surface, such as Curvularia eragrostidis and Aspergillus sydowii, demonstrated the ability to produce lignocellulolytic enzymes such as amylase, cellulase, and laccase.

    Acknowledgements

    This work was supported by the project “Characteristics of microbial deterioration on materials and equipment and the development of protective measures for them under tropical climate conditions” funded by the UBPH, project code T1.3 2022-2023 Additional support was provided by the Ministry of Defense project “Development of specialized am-qs formulations for mold resistance in cellulose-based materials for weapon storage applications,” project code 2022.11.41.

    References

    • ABDALLAH, Y.K., ESTEVEZ, A.T., DIAA EL DEEN, M.T., IBRAHEEM, A.M. and KHALIL, N.M., 2019. Employing laccase-producing Aspergillus sydowii NYKA 510 as a cathodic biocatalyst in self-sufficient lighting microbial fuel cell. Journal of Microbiology and Biotechnology, vol. 29, no. 12, pp. 1861-1872. http://doi.org/10.4014/jmb.1907.07031.
    • ABDEL-AZEEM, A.M., BLANCHETTE, R.A. and HELD, B.W., 2020. Chaetomium as potential soft rot degrader of woody and papery cultural heritage. In: A. ABDEL-AZEEM, ed. Recent developments on genus chaetomium Cham: Springer, pp. 395-419.
    • ADEGOKE, S.A. and ODIBO, F.J.C., 2019. Production, purification and characterization of α-amylase of Aspergillus sydowii IMI 502692. Plant Cell Biotechnology and Molecular Biology, vol. 20, pp. 1050-1058.
    • AMANN, R.I., BINDER, B.J., OLSON, R.J., CHISHOLM, S.W., DEVEREUX, R. and STAHL, D., 1990. Combination of 16S rRNA-targeted oligonucleotide probes with flow cytometry for analyzing mixed microbial populations. Applied and Environmental Microbiology, vol. 56, no. 6, pp. 1919-1925. http://doi.org/10.1128/aem.56.6.1919-1925.1990 PMid:2200342.
      » http://doi.org/10.1128/aem.56.6.1919-1925.1990
    • ANITA, B.B., THATHEYUS, A.J. and RAMYA, D., 2013. Biodegradation of carboxymethyl cellulose using Aspergillus flavus. Science International (Lahore), vol. 1, no. 4, pp. 85-91. http://doi.org/10.5567/sciintl.2013.85.91
      » http://doi.org/10.5567/sciintl.2013.85.91
    • ARNSTADT, T., HOPPE, B., KAHL, T., KELLNER, H., KRÜGER, D., BAUHUS, J. and HOFRICHTER, M., 2016. Dynamics of fungal community composition, decomposition and resulting deadwood properties in logs of Fagus sylvatica, Picea abies and Pinus sylvestris. Forest Ecology and Management, vol. 382, pp. 129-142. http://doi.org/10.1016/j.foreco.2016.10.004
      » http://doi.org/10.1016/j.foreco.2016.10.004
    • BILES, C.L., WRIGHT, D., FUEGO, M., GUINN, A., CLUCK, T., YOUNG, J., MARTIN, M., BILES, J. and POUDYAL, S., 2012. Differential chlorate inhibition of Chaetomium globosum germination, hyphal growth, and perithecia synthesis. Mycopathologia, vol. 174, no. 5-6, pp. 475-487. http://doi.org/10.1007/s11046-012-9572-5 PMid:22903379.
      » http://doi.org/10.1007/s11046-012-9572-5
    • BJÖRDAL, C.G. and DAYTON, P.K., 2020. First evidence of microbial wood degradation in the coastal waters of the Antarctic. Scientific Reports, vol. 10, no. 1, pp. 12774. http://doi.org/10.1038/s41598-020-68613-y PMid:32728072.
      » http://doi.org/10.1038/s41598-020-68613-y
    • BRIDŽIUVIENĖ, D. and RAUDONIENĖ, V., 2013. Fungi surviving on treated wood and some of their physiological properties. Materials Science, vol. 19, no. 1, pp. 43-50.
    • BRODA, M., 2020. Natural compounds for wood protection against fungi: a review. Molecules, vol. 25, no. 15, pp. 3538. http://doi.org/10.3390/molecules25153538
      » http://doi.org/10.3390/molecules25153538
    • BUCHNER, J., IRLE, M., BELLONCLE, C., MICHAUD, F. and MACCHIONI, N., 2019. Fungal and bacterial colonies growing on weathered wood surfaces. Wood Material Science & Engineering, vol. 14, no. 1, pp. 33-41. http://doi.org/10.1080/17480272.2018.1443975
      » http://doi.org/10.1080/17480272.2018.1443975
    • CAO, J., LIU, X., WANG, J., CHEN, H., LIU, D., LI, J. and MAI, B., 2024. Analysis of microbial diversity and its degradation function in wooden piles at Shahe ancient bridge site in Xi’an and protection measures. Heritage Science, vol. 12, no. 1, pp. 33. http://doi.org/10.1186/s40494-024-01157-w
      » http://doi.org/10.1186/s40494-024-01157-w
    • CLAUSEN, C.A. and GREEN, F., 2003. Oxalic acid overproduction by copper-tolerant brown-rot basidiomycetes on southern yellow pine treated with copper-based preservatives. International Biodeterioration & Biodegradation, vol. 51, no. 2, pp. 139-144. http://doi.org/10.1016/S0964-8305(02)00098-7
      » http://doi.org/10.1016/S0964-8305(02)00098-7
    • CRAWFORD, D.L., POMETTO III, A.L. and CRAWFORD, R.L., 1983. Lignin degradation by Streptomyces-viridosporus: isolation and characterization of a new polymeric lignin degradation intermediate. Applied and Environmental Microbiology, vol. 45, no. 3, pp. 898-904. http://doi.org/10.1128/aem.45.3.898-904.1983 PMid:16346253.
      » http://doi.org/10.1128/aem.45.3.898-904.1983
    • DAN, P.H. and NWACHUKWU, J.Q., 2020. Effect of microbes and atmospheric parameters on wood products utilization in Uyo, Akwa Ibom state, Nigeria. Journal of Research in Forestry. Wildlife and Environment, vol. 12, no. 2, pp. 202-211.
    • DARMAWAN, W., HERLIYANA, E.N., GAYATRI, A., HASANUSI, A. and GERARDIN, P., 2019. Microbial growths and checking on acrylic painted tropical woods and their static bending after three years of natural weathering. Journal of Materials Research and Technology, vol. 8, no. 4, pp. 3495-3503. http://doi.org/10.1016/j.jmrt.2019.06.024
      » http://doi.org/10.1016/j.jmrt.2019.06.024
    • FOGLE, M.R., DOUGLAS, D.R., JUMPER, C.A. and STRAUS, D.C., 2008. Growth and mycotoxin production by Chaetomium globosum is favored in a neutral pH. International Journal of Molecular Sciences, vol. 9, no. 12, pp. 2357-2365. http://doi.org/10.3390/ijms9122357 PMid:19330080.
      » http://doi.org/10.3390/ijms9122357
    • FUKASAWA, Y., OSONO, T. and TAKEDA, H., 2009. Microfungus communities of Japanese beech logs at different stages of decay in a cool temperate deciduous forest. Canadian Journal of Forest Research, vol. 39, no. 8, pp. 1606-1614. http://doi.org/10.1139/X09-080
      » http://doi.org/10.1139/X09-080
    • FUKASAWA, Y., OSONO, T. and TAKEDA, H., 2011. Wood decomposing abilities of diverse lignicolous fungi on nondecayed and decayed beech wood. Mycologia, vol. 103, no. 3, pp. 474-482. http://doi.org/10.3852/10-246 PMid:21262989.
      » http://doi.org/10.3852/10-246
    • ILIES, D.C., ONEȚ, A., WENDT, J.A., ILIEȘ, M., BAIAS, Ș. and HERMAN, G.V., 2018. Study on microbial and fungal contamination of air and wooden surfaces inside of a historical Church from Romania. Journal of Environmental Biology, vol. 39, no. 6, pp. 980-984. http://doi.org/10.22438/jeb/39/6/MRN-658
      » http://doi.org/10.22438/jeb/39/6/MRN-658
    • JOHNSTON, S.R., BODDY, L. and WEIGHTMAN, A.J., 2016. Bacteria in decomposing wood and their interactions with wood-decay fungi. FEMS Microbiology Ecology, vol. 92, no. 11, pp. fiw179. http://doi.org/10.1093/femsec/fiw179 PMid:27559028.
      » http://doi.org/10.1093/femsec/fiw179
    • KHADEMIBAMI, L. and BOBADILHA, G.S., 2022. Recent Developments studies on wood protection research in academia: a review. Frontiers in Forests and Global Change, vol. 5, pp. 793177. http://doi.org/10.3389/ffgc.2022.793177
      » http://doi.org/10.3389/ffgc.2022.793177
    • KIELAK, A.M., SCHEUBLIN, T.R., MENDES, L.W., VAN VEEN, J.A. and KURAMAE, E.E., 2016. Bacterial community succession in pine-wood decomposition. Frontiers in Microbiology, vol. 7, pp. 231. http://doi.org/10.3389/fmicb.2016.00231 PMid:26973611.
      » http://doi.org/10.3389/fmicb.2016.00231
    • KIM, J.-J., KANG, S.-M., CHOI, Y.-S. and KIM, G.-H., 2007. Microfungi potentially disfiguring CCA-treated wood. International Biodeterioration & Biodegradation, vol. 60, no. 3, pp. 197-201. http://doi.org/10.1016/j.ibiod.2007.05.002
      » http://doi.org/10.1016/j.ibiod.2007.05.002
    • KIRK, T.K. and FARRELL, R.L., 1987. Enzymatic “combustion”: the microbial degradation of lignin. Annual Review of Microbiology, vol. 41, no. 1, pp. 465-505. http://doi.org/10.1146/annurev.mi.41.100187.002341 PMid:3318677.
      » http://doi.org/10.1146/annurev.mi.41.100187.002341
    • LUMLEY, T.C., GIGNAC, L.D. and CURRAH, R.S., 2001. Microfungus communities of white spruce and trembling aspen logs at different stages of decay in disturbed and undisturbed sites in the boreal mixedwood region of Alberta. Canadian Journal of Botany, vol. 79, no. 1, pp. 76-92. http://doi.org/10.1139/b00-135
      » http://doi.org/10.1139/b00-135
    • MA, X., JIANG, M., WU, Y. and WANG, P., 2013. Effect of wood surface treatment on fungal decay and termite resistance. BioResources, vol. 8, no. 2, pp. 2366-2375. http://doi.org/10.15376/biores.8.2.2366-2375
      » http://doi.org/10.15376/biores.8.2.2366-2375
    • MARTINEZ, D., LARRONDO, L.F., PUTNAM, N., GELPKE, M.D.S., HUANG, K., CHAPMAN, J., HELFENBEIN, K.G., RAMAIYA, P., DETTER, J.C., LARIMER, F., COUTINHO, P.M., HENRISSAT, B., BERKA, R., CULLEN, D. and ROKHSAR, D., 2004. Genome sequence of the lignocellulose degrading fungus Phanerochaete chrysosporium strain RP78. Nature Biotechnology, vol. 22, no. 6, pp. 695-700. http://doi.org/10.1038/nbt967 PMid:15122302.
      » http://doi.org/10.1038/nbt967
    • NATIONAL CENTER FOR BIOTECHNOLOGY INFORMATION – NCBI [online], 2024 [viewed 1 August 2024]. Available from: www.ncbi.nlm.nih.gov
      » www.ncbi.nlm.nih.gov
    • NGO, C.C., NGUYEN, Q.H., NGUYEN, T.H., QUACH, N.T., DUDHAGARA, P., VU, T.H.N., LE, T.T.X., LE, T.T.H., DO, T.T.H., NGUYEN, V.D., NGUYEN, N.T. and PHI, Q.T., 2021. Identification of fungal community associated with deterioration of optical observation instruments of museums in northern vietnam. Applied Sciences (Switzerland), vol. 11, no. 12, pp. 5351. http://doi.org/10.3390/app11125351
      » http://doi.org/10.3390/app11125351
    • PFEFFER, A., HOEGGER, P.J., KÜES, U. and MILITZ, H., 2012. Fungal colonisation of outside weathered modified wood. Wood Science and Technology, vol. 46, no. 1-3, pp. 63-72. http://doi.org/10.1007/s00226-010-0386-7
      » http://doi.org/10.1007/s00226-010-0386-7
    • PIOLI, S., CLAGNAN, E., CHOWDHURY, A.A., BANI, A., BORRUSO, L., VENTURA, M., TONON, G. and BRUSETTI, L., 2023. Structural and functional microbial diversity in deadwood respond to decomposition dynamics. Environmental Microbiology, vol. 25, no. 11, pp. 2351-2367. http://doi.org/10.1111/1462-2920.16459 PMid:37403552.
      » http://doi.org/10.1111/1462-2920.16459
    • RAHMAN, M.A. and FUAD, A., 2002. Biodeterioration of wood by fungi Khulna, Bangladesh: Forestry and Wood Technology Discipline, Khulna University Khulna, pp. 20-38.
    • RIESENFELD, C.S., SCHLOSS, P.D. and HANDELSMAN, J., 2004. Metagenomics: genomic analysis of microbial communities. Annual Review of Genetics, vol. 38, no. 1, pp. 525-552. http://doi.org/10.1146/annurev.genet.38.072902.091216 PMid:15568985.
      » http://doi.org/10.1146/annurev.genet.38.072902.091216
    • ROWELL, R.M., 2005. Handbook of wood chemistry and wood composites Boca Raton: CRC Press. http://doi.org/10.1201/9780203492437
      » http://doi.org/10.1201/9780203492437
    • SAINI, S. and SHARMA, K.K., 2021. Fungal lignocellulolytic enzymes and lignocellulose: a critical review on their contribution to multiproduct biorefinery and global biofuel research. International Journal of Biological Macromolecules, vol. 193, no. Pt B, pp. 2304-2319. http://doi.org/10.1016/j.ijbiomac.2021.11.063 PMid:34800524.
      » http://doi.org/10.1016/j.ijbiomac.2021.11.063
    • SCHLOSS, P.D. and HANDELSMAN, J., 2003. Biotechnological prospects from metagenomics. Current Opinion in Biotechnology, vol. 14, no. 3, pp. 303-310. http://doi.org/10.1016/S0958-1669(03)00067-3 PMid:12849784.
      » http://doi.org/10.1016/S0958-1669(03)00067-3
    • SCHMIDT, O., 2006. Wood discoloration. In: O. SCHMIDT, ed. Wood and tree fungi: biology, damage, protection, and use New York: Springer, pp. 119-133.
    • SEIFERT, K.A. and GAMS, W., 2011. The genera of Hyphomycetes - 2011 update. Persoonia: Molecular Phylogeny and Evolution of Fungi, vol. 27, no. 1, pp. 119-129. http://doi.org/10.3767/003158511X617435 PMid:22403480.
      » http://doi.org/10.3767/003158511X617435
    • SINGH, P. and CHAUHAN, M., 2013. Influence of environmental factors on the growth of building deteriorating fungi: Aspergillus flavus and Penicillium chrysogenum. International Journal of Pharmaceutical Sciences and Research, vol. 4, no. 1, pp. 425.
    • SZEWCZYK, R. and DŁUGOŃSKI, J., 2009. Pentachlorophenol and spent engine oil degradation by Mucor ramosissimus International Biodeterioration & Biodegradation, vol. 63, no. 2, pp. 123-129. http://doi.org/10.1016/j.ibiod.2008.08.001
      » http://doi.org/10.1016/j.ibiod.2008.08.001
    • VAN NIEUWENHUIJZEN, E.J., HOUBRAKEN, J.A.M.P., PUNT, P.J., ROESELERS, G., ADAN, O.C.G. and SAMSON, R.A., 2017. The fungal composition of natural biofinishes on oil-treated wood. Fungal Biology and Biotechnology, vol. 4, no. 1, pp. 2-13. http://doi.org/10.1186/s40694-017-0030-5 PMid:28955471.
      » http://doi.org/10.1186/s40694-017-0030-5
    • VĚTROVSKÝ, T., VOŘÍŠKOVÁ, J., ŠNAJDR, J., GABRIEL, J. and BALDRIAN, P., 2011. Ecology of coarse wood decomposition by the saprotrophic fungus Fomes fomentarius Biodegradation, vol. 22, no. 4, pp. 709-718. http://doi.org/10.1007/s10532-010-9390-8 PMid:20668917.
      » http://doi.org/10.1007/s10532-010-9390-8
    • ZABEL, R.A. and MORRELL, J.J., 2020. Chemical changes in wood caused by decay fungi. In: R.A. ZABEL and J.J. MORRELL, eds. Wood microbiology 2nd ed. London: Academic Press, chap. 8, pp. 215-244. http://doi.org/10.1016/B978-0-12-819465-2.00008-5
      » http://doi.org/10.1016/B978-0-12-819465-2.00008-5

    Publication Dates

    • Publication in this collection
      31 Jan 2025
    • Date of issue
      2024

    History

    • Received
      01 Aug 2024
    • Accepted
      30 Oct 2024
    Creative Common - by 4.0
    This is an Open Access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
    vertical_align_top show_chart
    location_on
    Instituto Internacional de Ecologia R. Bento Carlos, 750, 13560-660 São Carlos SP - Brasil, Tel. e Fax: (55 16) 3362-5400 - São Carlos - SP - Brazil
    E-mail: bjb@bjb.com.br
    rss_feed Acompanhe os números deste periódico no seu leitor de RSS
    Reportar erro