1. Introduction

The National Archives of Korea is an organization that collects and stores records worth preserving and nationally important private and foreign records. There are three national archives in Korea. These are the Seongnam Nara Archives, the Daejeon Administrative Archives and Headquarters, and the Busan History Archives. Among them, the National Archives of Korea is promoting records management by collecting, registering, and preserving important national records. The Nara Archives is a record preservation facility equipped with the best preservation facilities such as central air conditioning, constant temperature and humidity facilities, and insect control facilities to safely preserve documents. It was observed that the temperature and humidity within the National Archives facility did not exceed the temperature (18-22℃) and humidity (40-55%) of the National Archives’ paper records preservation environment standards.

Fungi can exist in any space where conditions are conducive to life. If there are fungi in the air, the library's central air conditioning system can help they spread quickly. Due to their remarkable ability to degrade cellulose, the main component of paper, and cause discoloration, fungi represent a serious risk to archival materials (Drougka et al., 2020;Maggi, et al., 2000). Fungi are difficult to control because they produce spores that spread easily through the air. If fungal spores become airborne, records can become contaminated and damaged by fungi from germinated spores.

Previous research has shown the presence of fungi in the air in National Archives libraries that store unsanitized paper-based records (Noh et al., 2021). More research on species analysis of airborne fungal pollutants is needed to improve air quality management. Therefore, in this study, species identification was attempted on fungal samples obtained from the National Archives. We discovered that there are three previously unknown fungal species. Here we describe the morphological and molecular characteristics.

2. Materials and Methods

2.1 Isolation of fungal strains from the archives air samples

From June to August 2020, archival air samples were collected at the Seoul National Archives Library located in Seongnam. Andersen sampling device (Microbial Air Sampler, Model KAS-110, Kemik Corp., South Korea) with DG18 plates was used to collect airborne fungi. The suction flow rate of the measuring device was 28.8 L/min and operated for 3 minutes, inhaling a total of 144 L of air. The air sampled DG18 plates were transported to the laboratory in cooler bags. The air-sampled plates were incubated at 25°C for 7–10 days. To isolate grown colonies into pure cultures, individual colonies of different types were transferred to PDA plates and incubated at 25°C for 7 days. A purification process yielded pure isolates and the obtained pure isolate were stored in 20% glycerol at -80°C.

2.2 Morphological studies

For detailed morphological analyses, the fungal strains were grown on PDA, MEA, CYA, and OA media at 25°C in the dark for 7 days. Colony features were observed by naked eyes. Mycelia was sampled from each fungal colony using a needle to make a slide sample preparation. Slide samples were observed for microstructures under an optical microscope (BX53, Olympus, Japan). Microstructures were measured 30 times each for length and thickness. Colony structures of fungal strains were observed using a stereo microscope (SZ61, Olympus, Japan). For scanning electron microscopy (SEM), the fungal is olate was grown for 5~7 days at 25°C on 2% MEA plates overlaid with cellophane (Bio-Rad Laboratories, Canada) and ultra-structures were examined with a Hitachi S-4300 scanning electron microscope operating at 15 kV (Yun et al., 2009).

2.3 DNA extraction, PCR, and sequencing

Genomic DNA was extracted directly from the mycelia of fungal isolates using the NaviBiotech Direct DNA Prep Kit (NaviBiotech, Cheonan, Korea). Using the extracted DNA as a template, sequences of the internal transcribed spacer (ITS), the 28S LSU rDNA, and β- tubulin gene (BenA), and TEF1-α gene was amplified through PCR (Polymerase Chain Reaction) using the primers and conditions in Table 1. Amplification of the PCR product was verified by electrophoresis on a 1% (w/v) agarose gel. The PCR product was purified using a silica gel column and an 80% ethanol solution. Sequence analysis was entrusted to Macrogen Corp. (Seoul, Korea). After editing the sequence received from Macrogen Corp. using chromas, it was searched for homology through BLAST (Basic Local Alignment Search Tool) in National Center for Biotechnology Information (NCBI, https://www.ncbi.nlm.nih.gov/).

2.4 Phylogenetic analysis

The nucleotide sequence was compared with the nucleotide sequences of fungi registered in the DNA database using the BLAST program located on the web of the NCBI. The nucleotide sequences of the ITS, the 28S LSU rDNA, β-tubulin gene, and TEF1-α gene sequences of the taxa related to the isolated strains were obtained from NCBI’s GenBank for phylogenetic analysis. The obtained sequences were combined and compared with the those of unrecord species sequences obtained in this study. The nucleotide sequences were aligned using the ClustalW by the MEGA-X program (Thompson et al., 1994). The phylogenetic tree was constructed by the Kimura 2-parameter model of MEGA X (Kumar et al., 2018) and maximum likelihood (ML) method (Thorne et al., 1991), using 1,000 bootstraps. The DUCC 16098 (NIBRFGC000508679) strain, DUCC 17764 (NIBRFGC000508672) strain, and DUCC 17767 (NIBRFGC000508678) strain were deposited at the National Institute of Biological Resources of the Republic of Korea and received NIBRFGC numbers. The determined nucleotide sequences of the ITS, the 28S LSU rDNA, and β-tubulin gene and TEF1-α gene of these three strains were deposited in the NCBI database under accession numbers (Table 2-3).

3. Results and Discussion

3.1 Clonostachys farinosa DUCC 16098 (NIBRFGC000508679) strain

The colonies of fungal strain DUCC16098 grew up to 62, 55, 60, and 63 mm on PDA, MEA, CYA, and OA, respectively, after 7 days of incubation at 25°C. The colony color was white o n PDA, M EA, CYA, a nd OA. DUCC16098 strain has conidia that are flat on one side and narrow towards the tip on the other with a size of 6 to 6.5 μm × 1 to 1.5 μm and conidiogenous cells with a size of 12 to 15 μm

Colonies of fungal strain DUCC16098 grew to 62, 55, 60, and 63mm on PDA, MEA, CYA, and OAT, respectively, after culturing for 7 days at 25°C. Colony color was white in PDA, MEA, OAT, and CYA. When observed under a microscope, the DUCC16098 strain is flat on one side and narrows toward the end on the other, with a size of 6-6.5 μm × 1-1.5 μm and a conidial cell of 12-15 μm (Fig. 1).

The length of the sequence obtained through PCR amplification is 493 bp in the ITS, 446 bp in TEF1-α. DUCC16098 strain was identified as Clonostachys farinosa belonging to Sordariomycetes, representing the ITS region sequences similarity of 99.39% (KC806270), and partial TEF-1α gene sequences similarity of 99.76% (KC806270) with C. farinosa. According to the phylogenetic tree based on the combined ITS and TEF-1α gene sequences in Table 2, DUCC16098 strain formed the same clade with C. farinosa strain CML 2311 (Fig. 2). Based on the morphological and molecular analysis, DUCC16098 strain was identified as C. farinosa, an unrecorded species in Korea.

The genus Clonostachys belongs to the subphylum Pezizomycotina, order Hypocreales, family Bionectriaceae. Clonostachys is a common fungus found in soil (Sun et al., 2020). It is both saprophytic and mycoparasitic organisms. Its effect on human is unknown. It is also being extensively researched as a biocontrol agent (Gomes et al., 2017). And C. farinosa is used as a producer of lignocellulose-degrading enzymes that has been extensively explored. C. farinosa’s high protein diversity in these multi-catalytic assemblies confirms it as a novel source of plant biomass-degrading enzymes (Gomes et al., 2017).

3.2 Penicillium cosmopolitanum DUCC 17764 (NIBRFGC000508672) strain

Colonies of fungal strain DUCC17764 grew to 90, 86, 90, and 90 mm on PDA, MEA, CYA, and OA, respectively, a fter culturing for 7 days at 25°C. Colony color was blue with white outline in PDA, CYA, and OA and white in MEA. DUCC17764 was observed by a microscope to have green spherical conidia with a size of 2- 2.5 μm and phialides with a size of 4.5-6 μm × 1.5-2 μm (Fig. 3).

The length of the sequences obtained through PCR amplification was 523 bp for the ITS and 464 bp for the BenA gene. Strain DUCC17764 was identified as Penicillium cosmopolitanum, belonging to the Eurotiomycetes, and showed 99.24% (MH859884) ITS region sequence similarity, and 99.77% (JN606738.1) partial BenA similarity with P. cosmopolitanum. According to the phylogenetic tree based on the concatenated ITS and BenA sequences in Table 3, strain DUCC17764 formed the same lineage as P. cosmopolitanum s train CBS 637.70 (Fig. 4). As a result of morphological and molecular analysis, strain DUCC17764 was identified as P. cosmopolitanum, an unrecorded species in Korea.

The genus Penicillium belongs to the subphylum Pezizomycotina, order Eurotiales, and family Aspergillus family. Penicillium is a well-known and widespread fungus (Steenwyk et al., 2019). Its main function in nature is the decomposition of organic substances. They occur in a variety of habitats, including soil, vegetation, air, indoor environments, and various foods (Yadav et al., 2018). Some species of the genus Penicillium are known to produce penicillin, a molecule used as an antibiotic to inhibit or kill certain types of bacteria (Houbraken et al., 2012). P. aurantiogriseum is a known contaminant during grain storage, P. digitatum and P. italicum are citrus pathogens, and P. camemberti and P. roqueforti are used in cheese production (Visagie et al., 2014). Likewise, P. cosmopolitanum was isolated from the scalp of young and old people (Jeong et al., 2012). However, the morphological and genetic characteristics of the isolated P. cosmopolitanum have not been sufficiently verified. There is no scientific verification. Moreover, that isolate did not exist. Therefore, it cannot be considered a verified record species, which is shown that P. cosmopolitanum discovered through this study was verified and judged to be an unrecorded species in Korea.

3.3 Cephalotrichum purpureofuscum DUCC 17767 (NIBRFGC000508678) strain

Colonies of fungal strain DUCC17767 grew to 90, 86, 90, and 90 mm on PDA, MEA, CYA, and OA, respectively, a fter culturing for 7 days at 25°C. Colony color was olive green with a white outline in PDA, dark blue with a white outline in MEA and OA, and gray in CYA. Strain DUCC17767 has conidia that are flat on one side and narrow toward the end on the other side, with a size of 5-6 μm × 2-3 μm and a conidial cell of 11-14 μm (Fig. 5).

The length of the sequence obtained through PCR amplification is 365 bp in ITS, 852bp in LSU rDNA and 535 bp in BenA. Strain DUCC17767 was identified as Cephalotrichum purpureofuscum, belonging to Sordariomycetes, showing 98.36% ITS region sequence similarity (KY249281), 100% partial LSU rDNA sequence similarity (MH870812), and 99.22% BenA sequence similarity (KY249319) to C. purpureofuscum. (KY249319) (Fig. 6). According to the phylogenetic tree based on the concatenated ITS, LSU rDNA, and BenA sequences in Table 4, strain DUCC17767 formed the same lineage as C. purpureofuscum strain CBS 174.68 (Fig. 6). As a result of morphological and molecular analysis, strain DUCC17767 was identified as C. purpureofuscum, an unrecorded species in Korea.

The genus Cephalotrichum belongs to the subphylum Pezizomycotina, order Microascales, family Microascaceae. The species phylogenetically attributed to Cephalotrichum are characterized by forming synnematous conidiophores with or without sterile distal setae and annellidic conidiogenesis producing smooth or rough conidia arranged in basipetal chains (Sandoval-Denis et al., 2016).

The genus Cephalotrichum belongs to the subphylum Pezizomycotina, order Microascales, and family Microascaceae. Phylogenetically, species belonging to Cephalotrichum are characterized by forming synnematous conidiophores with or without sterile distal setae and annellidic conidiogenesis producing smooth or rough conidia arranged in basipetal chains (Sandoval-Denis et al., 2016).