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ISSN : 2288-9167(Print)
ISSN : 2288-923X(Online)
Journal of Odor and Indoor Environment Vol.22 No.4 pp.247-257
DOI : https://doi.org/10.15250/joie.2023.22.4.247

First report of two unrecorded fungal species isolated from air conditioner filters in Korea

Hyun Uk Cho, Na Young Yoon, Seong Hwan Kim*
Department of Microbiology, College of Science and Technology, Dankook University, Cheonan 31116, Korea
* Corresponding Author: Tel: +82-41-550-3454 E-mail: piceae@dankook.ac.kr
15/11/2023 17/11/2023

Abstract


Air conditioner filters purify the air of indoor environments by removing air pollutants and supporting the efficiency of the unit’s cooling function. However, an air conditioner filter can become a microenvironment in which some fungi can grow as dust continues to accumulate and favorable humidity conditions are formed. Fungal growth in air conditioner filters could lead to fungal allergies or fungal diseases, in addition to emitting a foul odor. In an effort to understand what species causes this malodorous problem, we investigated the diversity of fungi found in air conditioners. Fungi were sampled from the collected air conditioner filters and grown on DG18 agar media. After purification for pure isolates, species identification was undertaken. Colony morphology was observed on PDA, MEA, CYA, and OA media. Microstructures of fruiting body, mycelia, and spores were examined using a light microscope. Molecular identification was performed by PCR and sequencing of PCR amplicons, and molecular phylogenetic analysis of sequenced DNA markers, including the Internal Transcribed Spacer (ITS), the 28S large subunit of the nuclear ribosomal RNA (LSU rDNA), the β-tubulin (BenA) gene, the Calmodulin (CaM) gene, and the DNA-directed RNA polymerase II subunit 2 (RPB2) gene. Through this identification process, we found two fungal species, Aspergillus miraensis and Dichotomopilus ramosissimus, which are unrecorded species in Korea. We will now report their morphological and molecular features.


Air conditioner filter , Aspergillus miraensis , Dichotomopilus ramosissimum

초록

    © Korean Society of Odor Research and Engineering & Korean Society for Indoor Environment. All rights reserved.

    1. Introduction

    Air conditioners are home appliances that are useful in maintaining the indoor environment of a house or building. Although the filter in your air conditioner is not the main functional unit, it can help filter out environmental contaminants such as dust and dirt. Filter maintenance work ensures the efficiency of your air conditioner. However, air conditioner filters can accumulate dust, form moisture, and become environmental niches for some fungi that can colonize when the air conditioner is briefly out of operation (Chang et al., 1996;Miller and Nazaroff, 1997;Maus et al., 2001).

    Fungi on air conditioner filters can cause indoor air pollution as spores spread through the indoor air when the air conditioner is running (Hamada and Fujita 2002). Indoor air from contaminated filters has been reported to contain human pathogenic airborne fungi that can cause allergies and respiratory diseases (Aquino et al., 2018). Since fungi is an important air pollutant, Korea’s Ministry of Environment regulates it as an indoor air quality pollutant in multi-use facilities (https://www.me.go.kr/ home/web/main.do), and the WHO also limits fungal concentrations to less than 500 CFU/m3 (WHO, 1990;Al-abdalall et al., 2019).

    Fungi in indoor air could cause not only indoor air pollution but also affect human health as allogenic agents and pathogens (Al-Doory and Domson, 1984;Samson et al., 1994). However, fungal diversities present in air conditioning filters has not been much studied. We have been investigating fungi in air conditioners to generate basic scientific data that could help explore what fungi are problematic species and lead to get an insight on solution of fungal problems. In this study, we report two fungal species which are new in Korea and have never been reported from air conditioner filters.

    2. Material and Methods

    2.1 Collecting air conditioner filter and Isolation of fungal strains

    Air conditioner filters were collected in sterilization bags from several homes in Cheonan. The collected filter samples were analyzed in a laboratory at a biosecurity level 2 licensed facility at Dankook University. After cutting into 1 × 1 cm2 pieces, the shredded filters were transferred to a 50 ml conical tube containing 20 ml sterile distilled water and vortexed for 10 min to prepare a dust suspension. The suspension was diluted up to 1000-fold through serial dilution by mixing 1 mL of dust suspension with 9 mL of sterile distilled water. 100 μL of the diluted dust suspension was dispensed onto Potato Dextrose Agar (PDA) medium containing 100 μg/mL antibiotics (Ampicillin, Streptomycin, Kanamycin) and then spread using a sterile glass spreader. PDA agar plates spread with the diluted dust suspension were cultured in an incubator at 25°C for 1 week. Mycelium with different morphology was collected from the growing fungal colonies and purely isolated on PDA. Isolates were assigned Dankook University Culture Collection (DUCC) numbers.

    2.2 Fungal morphological examination and molecular marker gene analysis

    Colony morphology of the purely isolated fungi was observed after growing for one week at 25°C under dark condition on PDA, Malt Extract Aga (MEA), Czapek Yeast Extract Agar (CYA), and Oatmeal Agar (OA). The microstructure was observed using an optical microscope (BX53; Olympus, Shinjuku-ku, Tokyo, Japan). The size of each microstructure was measured (n=50), and the range was indicated. 50 mg of mycelium was scraped off with a surgical blade and collected in a 2 mL tube. Genomic DNA was extracted from the collected mycelium using the Direct DNA Prep kit (NaviBiotech, Cheonan, Korea) according to the protocol provided by the manufacturer. PCR was performed using the extracted DNA as a template using a Bio-Rad T100 Thermal Cycler to amplify the ITS (Internal Transcribed Spacer) region (ITS1-5.8S-ITS2), 28S large subunit of nuclear ribosomal RNA (LSU rDNA), partial β-tubulin (BenA) gene sequences, partial calmodulin (CaM) gene sequences, and partial DNA-directed RNA polymerase II subunit 2 (RPB2) gene sequences. The PCR conditions for each primer set for the maker sequence are listed in Table 1. The PCR-amplified DNA band of each maker gene was confirmed through electrophoresis on a 1% agarose gel. PCR products were purified using the High Pure PCR Product Purification Kit (Roche, Indianapolis, IN, USA) and then sequenced by Macrogen Corp. (Seoul, Korea).

    2.3 Molecular phylogeny analysis

    The determined nucleotide sequences of maker genes were analyzed for homology with the fungal sequence deposited in the U.S. National Center for Biotechnology Information (NCBI, 2023) database using the GenBank BLASTN search engine. Fungal species closely related to the taxa isolated in this study were selected from BLAST search results, and fungal species belonging to the same and different genera were included as reference sequences in the analysis. Reference sequences were obtained from GenBank and are listed in Tables 2-3. Individual sequence datasets from the ITS region, LSU rDNA, β-tubulin gene, Calmodulin gene, and RPB2 gene were aligned using the Clustal W alignment tool in MEGA 11 (Tamura et al., 2021). A maximum likelihood (ML) phylogenetic tree was constructed based on the aligned sequences (Felsenstein, 1998), and 1000 bootstrap analyzes were performed to ensure the reliability of each node in the phylogenetic tree.

    3. Results and Discussion

    Two fungal species, the DUCC 23191 and 23192 strains that were isolated and identified as unrecorded fungi in this study, are Aspergillus miraensis and Dichotomopilus ramosissimus. The analyzed nucleotide sequences of these two DUCC strains were registered in GenBank of the NCBI database, and the accession numbers are shown in Tables 2 and 3, respectively. Morphological comparisons of the two species identified in this study and closely related species are presented in Tables 4 and 5, respectively. Strains DUCC 23191 and 23192 were deposited at the National Institute of Biological Resources, Republic of Korea. The characteristics of the two species observed in DUCC 23191 and 23192 strains are as follows.

    3.1 Aspergillus miraensis Hubka et al., Plant Systematics and Evolution 302 (9): 1288 (2016) [MB#816283]

    Morphological characteristics were observed after culturing in the dark at 25°C f or 8 d ays. Aspergillus miraensis DUCC 23191 (NIBRFGC000510201) showed robust growth on all media, with the fastest growth observed on MEA media (55-58 mm). On PDA, CYA and MEA media, colonies showed a consistently spread, generally flat morphology with a central wrinkle, whereas on OAT media they appeared fluffy. The color of the colonies ranged from yellow to light yellow. After 7 days of culture, green conidia formed in the center of colonies on MEA and PDA media, whereas conidia production was slower on CYA and OAT media. Ascomata irregularly developed around the center of the colonies.

    Both asexual and sexual phases were observed in all media types (PDA, MEA, CYA, OAT), with the observation period for the sexual phase being 1 to 2 weeks later than that for the silent phase. Ascomata have a spherical to quasi-spherical shape, are (200.4-) 292.6 - 395.0 (-413.8) μm long, (173.5-) 273.6 - 365.4 (-418.5) μm wide, and are reddish-brown in color. They were surrounded by Hülle cells. Hülle cells were transparent, light tan in color, measured spherical to ovoid, and were (12.97-) 16.3 – 21.6 (-26.3) μm in size. Ascus was spherical in shape and consist of eight ascospores. Its size is (9.6-) 11.2 - 12.9 (-14.1) × (7.8-) 9.0 - 10.6 (-11.7) μm. Ascospores were purple in color and spherical in shape. The size of the sporophyte was (3.0-) 3.2 – 3.6 (-4.0) × (2.9-) 3.2 – 3.4 (-3.6) μm, and a star-shaped structure was observed on the surface.

    The conidiophores were sturdy and dark yellow in color. The length dimension was highly variable, ranging from 135.6-732.9 μm, and the width ranged from (4.4-) 5.5 to 7.2(-8.5) μm. The vesicles were transparent and light green in color. Their shape varied from subclavate to subglobose, and their width ranged from (6.6-) 9.7 to 14.0 (-17.9) μm. Metulae were transparent and light green. Their shape was cylindrical and their size was (3.6-) 5.8 – 7.8 (-8.4) μm. Fyallids had a pale green, glassy appearance and were flask-shaped. They measured approximately (5.2-) 6.1 – 7.7 (-9.7) × (1.7-) 2.3 – 3.2 (-3.5) μm, with 2-3 phialides attached to each base. Conidia range in shape from spherical to oval and are yellow in color. The size is (2.15-) 2.3 – 2.9 (-3.1) × (1.7-) 2.2 – 2.7 (-3.0) μm. (Fig. 1)

    The ITS region, CaM gene, and RPB2 gene sequences were 487 bp, 650 bp, and 986 bp, respectively. Strain DUCC 23191 showed 100% similarity to the ITS region sequence for A. miraensis strain CBS 140625 (OL711841), 99.79% similarity to the CaM gene sequence for A. miraensis strain DTO 323-B2 (KU866780) and 99.89% similarity to the RPB2 gene sequence for A. miraensis strain CBS 140625 (KU867045). According to the phylogenetic tree based on concatenated sequences of the ITS region, CaM gene and RPB2 gene in Table 2, strain DUCC 23191 formed the same lineage as A. miraensis strain CBS 140625 (Fig. 2). As a result of morphological and molecular analysis, strain DUCC 23191 was identified as Aspergillus miraensis, an unrecorded species in Korea. Morphological differences between DUCC 23191 and phylogenetically related species are shown in Table 4.

    A. miraensis was first described as Emericella miraensis (Zhang et al., 2013). However, E. miraensis was redescribed as Aspergillus miraensis by Chen et al. in 2016. A. miraensis was found in the root of Polygonum macrophyllum var. stenophyllum in Tibet Ningchi and could produce the carcinogens aflatoxin B1, asperthecin, sterigmatocystin (Chen et al., 2016). Aflatoxin B1 is carcinogenic to humans (NCBI, 2023). Asperthecin is an anthraquinone pigment obtained from the same genus, Aspergillus nidulans, a metabolite and biological pigment (Howard and Raistrick, 1955). Sterigmatocystin is the precursor of aflatoxin biosynthesis and small amounts of sterigmatocystin is associated with tumorigenesis in mice (Fujii et al., 1976).

    3.2 Dichotomopilus ramosissimus Wang & Samson, Studies in Mycology 84: 217 (2016) [MB#818869]

    Morphological characteristics were observed after culturing in the dark at 25°C for 8 days. Dichotomopilus ramosissimus DUCC 23192 strain (NIBRFGC000510202) showed the fastest growth on CYA medium (58-59 mm) and the slowest growth on OAT medium (29-30 mm). The colony forms were fluffy and yellow. It grew in the f orm of a f ilament. A f ter 7 days o f g rowth on OAT medium, dark brown asci developed. The ascus is spherical or egg-shaped and has ascus hairs on its surface. The size of the ascus is 123.8 – 237.6 × 94.0 – 186.2 μm. Peridia is reddish-brown to brown in color and exhibits a complex or epidermal texture. The terminal setae exhibit bipartite branches with four or more branches on each side, forming an obtuse angle. It measured (29.7-) 137.7 – 234.2 (-283.9) × (2.1-) 3.0 – 3.4 (-4.4) μm and had a dot-shaped or protruding surface. The side fur is unbranched, resembles bristles, and becomes thinner towards the end. Asci are composed of eight ascospores measuring (10.6-) 10.8 – 14.0 (-16.0) × (7.5-) 7.7 – 9.5 (-9.8) μm and are rapidly destroyed. Ascospores are grey-green, ovate to quasi-spherical in shape, and have a pointed end. They were hyaline, measuring 5.6 – 6.2 × 3.9 – 4.5 μm, with a germination hole at the tapered end. No asexual reproduction stage was observed (Fig. 3).

    The ITS region, LSU rDNA, and BenA gene sequences were 488 bp, 855 bp, and 452 bp, respectively. Strain DUCC23192 showed 100% similarity to the ITS region sequence for D. ramosissimus isolate PUFNI18725ITS (MK990280), 100% similarity to the LSU rDNA sequence for D. ramosissimus strain LC3787 (KP336802), and 99.78% similarity to the BenA gene for D. ramosissimus strain LC3787 (KP336851). According to the phylogenetic tree based on concatenated sequences of the ITS region, LSU rDNA, and BenA gene in Table 3, strain DUCC23192 formed the same lineage as D. ramosissimus strain LC3787 (Fig. 4). As a result of morphological and molecular analysis, strain DUCC 23192 was identified as Dichotomopilus ramosissimus, an unrecorded species in Korea. Morphological differences between strain DUCC23192 and phylogenetically related species are presented in Table 5.

    D. ramosissimus is a mesophilic fungus with optimal growth at 30°C. (Wang et al., 2014). This fungus was isolated from the rhizosphere of Panax notoginseng in China by Wang et al. (2016). D. ramosissimus culture filtrate extract extracted with ethyl acetate showed antibacterial activity against B. cereus, E. coli, and S. aureus, and its components included flavonoids, steroids, terpenoids, aldehydes, ketones, unsaturated compounds, and alcohol (Kodsueb and Lumyong, 2018). Additionally, D. ramosissimus showed a biological control effect against seed-borne Fusarium subglutinans (Koch et al., 2020). Therefore, D. ramosissimus is expected to act as a biological control agent against seed-borne fusarium diseases and the food-borne microorganisms B. cereus and S. aureus (Lowy, 1998).

    4. Conclusion

    Two types of fungi were found in air conditioner filters that have not yet been reported in Korea. One of these species is proven to produce toxins associated with human cancer and other species can cause opportunistic infections in immunocompromised patients. Our results indicate that old or poorly maintained filters provide an environment for fungal growth (Hamada and Fujita, 2002). Therefore, it is important to replace your air conditioner filter regularly to prevent fungal contamination.

    Acknowledgements

    This work was supported by a grant from the National Institute of Biological Resources (NIBR) funded by the Ministry of Environment (MOE), Republic of Korea.

    Conflict of Interests

    The authors declared no conflicts of interest.

    <Author Information>

    Hyun Uk Cho (undergraduate student), Na Young Yoon (graduate student), Seong Hwan Kim (professor)

    Figure

    JOIE-22-4-247_F1.gif

    Colony morphology on different media (A-D) and light microscopic images (E-L) of Aspergillus miraensis DUCC 23191 strain grown at 25°C for 14 days. A: PDA. B: MEA. C: CYA. D: OA. E: conidiophore and Hülle cell. F: conidia. G: ascomata. H: ascomata and Hülle cell. I: Hülle cell. J, K: Asci. L: ascospore. Scale bar: E, F, H, I, J, K, L = 10 μm; G = 100 μm.

    JOIE-22-4-247_F2.gif

    Maximum likelihood phylogenetic tree based on the concatenate ITS, partial CaM gene and partial RPB2 gene sequences of Aspergillus miraensis DUCC 23191 strain. The number of nodes represents the reliability value (>60%) through 1,000 bootstrap replicates. Fungal strain isolated in this study is indicated in bold.

    JOIE-22-4-247_F3.gif

    Colony morphology on different media (A-D) and light microscopic images (E-H) of Dichotomopilus ramosissimus DUCC 23192 strain grown at 25°C for 14 days. A: PDA. B: MEA. C: CYA. D: OA. E, F: ascomata. G: ascomatal hair and ascomata. H: ascospore. Scale bar: E = 100 μm; F, G, H = 10 μm.

    JOIE-22-4-247_F4.gif

    Maximum likelihood phylogenetic tree based on the concatenated ITS, LSU rDNA region, and partial BenA gene sequences of Dichotomopilus ramosissimus DUCC 23192 strain. The number of nodes represents the reliability value (>70%) through 1,000 bootstrap replicates. Fungal strain isolated in this study is indicated in bold.

    Table

    Primer set and PCR conditions used for this study

    Source and GenBank accession numbers of the species closely related to Aspergillus miraensis used for constructing a phylogenetic tree based on the concatenated sequences of ITS, CaM gene and RPB2 gene

    Source and GenBank accession numbers of the species closely related to Dichotomopilus ramosissimus used for constructing a phylogenetic tree based on the concatenated sequences of ITS, LSU rDNA and BenA gene

    The morphological comparison between DUCC 23191 strain and phylogenetically related species

    The morphological comparison between DUCC 23192 strain and phylogenetically related species

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