- Email: [email protected]
An aeromycological study of various wooden cultural heritages in Korea
G Model CULHER-2959; No. of Pages 8
ARTICLE IN PRESS Journal of Cultural Heritage xxx (2015) xxx–xxx
Available online at
ScienceDirect www.sciencedirect.com
Original article
An aeromycological study of various wooden cultural heritages in Korea Min-Ji Kim a,1 , Hyun-Kyeong Shin b,2 , Yong-Seok Choi b,3 , Gwang-Chul Kim c,4 , Gyu-Hyeok Kim d,∗ a
BK21 Plus Eco-Leader Education Center, Korea University, Seoul 136-713, Republic of Korea Division of Wood Engineering, Department of Forest Products, Korea Forest Research Institute, 57, Hoegiro, Dongdaemun-gu, Seoul 130-712, Republic of Korea c Department of Housing Environmental Design, Chonbuk National University, Jeounju 561-756, Republic of Korea d Division of Environmental Science & Ecological Engineering, College of Life Sciences & Biotechnology, Korea University, 5-1 Anam-dong, Seongbuk-gu, Seoul, 136-713, Republic of Korea b
a r t i c l e
i n f o
Article history: Received 11 February 2015 Accepted 6 May 2015 Available online xxx Keywords: Airborne fungi Fungal diversity Korea Wood deterioration Wooden cultural heritage
a b s t r a c t Korea has many wooden cultural heritages (WCHs), which should be preserved, along with various other cultural properties. WCHs, however, have undergone biodeterioration because of various fungal attacks in the past centuries; this type of biodeterioration is one of the significant problems faced during preservation of WCHs. To prevent this damage, it is important to investigate the fungal diversity of the WCHs. This aim of this study was to analyze the diversity of airborne fungi at 3 WCHs in Korea: Yeonghwadang (YHD; open building) and Juhamnu (JHN; closed building) in Changdeokgung Palace Complex located in Seoul and Unbong hyanggyo (UH; closed building) in Namwon. The airborne fungi were isolated twice in spring (March) and summer (August) using the gravity settling culture plate method and were identified using morphological and molecular techniques. There were differences in fungal diversity depending on the geographical location, climatic conditions, and the open or closed status of a building. During spring, in the open and closed buildings, a total of 671 fungal isolates (20 genera and 25 species) were collected in YHD and 125 isolates (19 genera and 25 species) were isolated in JHN. In summer, 175 isolates (11 genera and 12 species) and 66 isolates (12 genera and 13 species) were collected from YHD and JHN, respectively. The number of fungal isolates was greater in the open building than in the closed WCHs, but these buildings had similar fungal diversity. In UH, 180 isolates (13 genera and 15 species) were recovered in spring season and 58 isolates (14 genera and 17 species) in summer. There was no significant difference in the number of fungal isolates, but the fungal diversity was different depending on the environmental factors. Finally, fungal diversity was richer in spring than in summer because dusty and windy weather in spring was conducive to the release and transmission of fungal spores. In summer, there were a substantial number of basidiomycetes probably because their spores germinate better at higher temperatures and humidity. © 2015 Elsevier Masson SAS. All rights reserved.
1. Research aims
∗ Corresponding author. Tel.: +82 2 3290 3014. E-mail addresses: [email protected] (M.-J. Kim), [email protected] (H.-K. Shin), [email protected] (Y.-S. Choi), [email protected] (G.-C. Kim), [email protected] (G.-H. Kim). 1 Tel.: +82 2 3290 3468. 2 Tel.: +82 2 961 2708. 3 Tel.: +82 2 961 2704. 4 Tel.: +82 63 270 4847.
The present study was focused on preservation of wooden cultural heritages (WCHs) in Korea against fungal biodeterioration. The first aim was to analyze the diversity of airborne fungi at 3 WCHs in Korea, Juhamnu (JHN; closed building) and Yeonghwadang (YHD; open building) in Changdeokgung Palace Complex in Seoul and Unbong hyanggyo (UH; closed building) in Namwon. These buildings are under different environmental conditions and are managed in different ways such as open to the public or closed. The second aim was to determine the relationship of fungal diversity with environmental and geographic factors. Understanding of
http://dx.doi.org/10.1016/j.culher.2015.05.001 1296-2074/© 2015 Elsevier Masson SAS. All rights reserved.
Please cite this article in press as: M.-J. Kim, et al., An aeromycological study of various wooden cultural heritages in Korea, Journal of Cultural Heritage (2015), http://dx.doi.org/10.1016/j.culher.2015.05.001
G Model CULHER-2959; No. of Pages 8
ARTICLE IN PRESS M.-J. Kim et al. / Journal of Cultural Heritage xxx (2015) xxx–xxx
2
fungal diversity between various WCHs could provide useful clues for the selection of target fungi for preventive measures.
Palace Complex and UH in Namwon. In addition, we compared the observed fungal diversity among different climatic conditions.
2. Introduction
3. Materials and methods
Since ancient times, wood has generally been considered as a great source of building materials because of its firmness and endurance. Numerous architects have used wood in Korea. However, it is inevitable that WCHs would be exposed to outdoor environment, and subjected to high humidity and temperature, in the course of several centuries. This situation is expected to aggravate the deterioration problem. Recently, serious biological deterioration of WCHs around the world was continuously reported along with growing concerns about preservation [1–3]. Among the risks, fungal attacks are considered one of the main causes of damage [4]. There are four types of fungal damage. One is decay caused by basidiomycetes and three other types of damage are soft rot, sapstain, and surface mold caused by ascomycetes [5]. This fungal damage can decrease the aesthetic value and cause serious structural problems. Thus, the causes of deterioration, such as climatic effects, moisture, and pollutants, should be eliminated and their effects mitigated. Appropriate measures should be taken to protect WCHs. Generally, fungal diversity in indoor and outdoor environments is closely related to air particulate matter because fungal spores are airborne. Therefore, constant air circulation is a particularly important factor for the fungal diversity and preservation of WCHs. To enhance the value of WCHs, there have been several efforts to protect WCHs from biodeterioration. The European Council has implemented policies to reduce air pollution and other deterioration factors [6]. In 2009, the World Health Organization explained indoor biological pollution caused by the problem of excess moisture and recommended air quality guidelines [7]. In Korea, the first law on the protection of the cultural heritage was passed in 1962. Furthermore, there are many restoration and repair projects at WCHs, but scientifically valid protection techniques have not been developed yet. Nowadays, in Korea, some WCHs are open to the public to help the people to learn about and understand the wisdom of ancestors and to offer enjoyment and appreciation to public users, other WCHs are closed to the public in order to reduce the inherent risks associated with air pollution and inappropriate handling. The diversity and growth of fungal species depend on the outdoor environment including the presence of water that comes from atmospheric precipitation, condensation phenomena, and dampness of the soil as well as indoor relative humidity influenced by the exchange with the outdoor environment and by weather changes. Although an open building is well ventilated and exposed to outdoor air, when the doors of WCHs are closed, the airflows are blocked, resulting in high humidity levels and risk of mold growth. Therefore, identification and assessment of biological risks in open and closed WCHs are needed for systematic protection. Management of these risks is expected to be facilitated by characterization of the differences between the environmental effects and biodeterioration. In order to preserve WCHs from the fungal biodeterioration, it is necessary to determine the kinds of fungal species that are found in WCHs and mechanism of WCHs damage caused by fungi in combination with environmental factors such as temperature and humidity. Although numerous attempts are made to enhance our understanding of inhabiting fungi involved in deterioration of WCHs [8,9], the data on WCHs in Korea have yet not been collected thoroughly. Therefore, the aim of this study was to investigate the fungal diversity at 3 WCHs: JHN and YHD in Changdeokgung
3.1. Sampling sites JHN and YHD in Changdeokgung Palace Complex, which is located in Seoul (37◦ 58 N, 126◦ 99 E) were selected to compare the diversity of indoor airborne fungi between buildings with closed and open doors. All doors of YHD are always open, and those of JHN are closed all year around (Fig. 1). JHN is about 30 m away from YHD, so airborne spore levels of the outdoor environment of these 2 buildings are not different. Fig. 2 shows UH which is located in Namwon (35◦ 25 N, 127◦ 31 E), Jeollabuk-do, was selected to compare the diversity of indoor airborne fungi depending on geographical locations. The fungal diversity of UH, which is a closed building, was compared with that of JHN. The difference in geographical coordinates between JHN and UH, including the altitude above the sea level, may affect fungal diversity because the climatic conditions of the 2 locations are different.
3.2. Isolation of the fungi Indoor airborne fungal spores were collected at the 3 WCHs twice in spring (May) and summer (August) using the gravity settling culture plate method. Fifteen petri dishes were exposed to the air for 30 minutes in each building. The petri dishes were placed on the floor. The culture medium was 2% malt extract agar (MEA), composed of 20 g of Difco malt extract and 15 g of Difco agar per 1 L of distilled water, with 100 ppm streptomycin to inhibit bacterial growth. After the exposure, the plates were sealed with parafilm and incubated at room temperature for 2 weeks, and the mycelial margins were routinely sub-cultured onto new plates to obtain pure cultures. For the preferential isolation of the basidiomycetes, 2% MEA with 4 ppm benomyl and 100 ppm streptomycin was used. After further purification, most of the airborne fungal species were identified to the genus or species level according to morphological and molecular biological characteristics.
3.3. DNA extraction, PCR amplification, and DNA sequencing Genomic DNA was extracted from fungal mycelia using the Accuprep Genomic DNA Extraction Kit (Bioneer, Korea). The nuclear ribosomal DNA (rDNA) gene cluster was amplified by PCR using the Accupower PCR Premix Kit (Bioneer). The nuclear internal transcribed spacer (ITS) region including the 5.8S rDNA gene was amplified by means of the following primer set: ITS5 (5 -GGAAGTAAAAGTCGTAACAAGG-3 )/ITS4 (5 -TCCTCCGCTTATTGATATGC-3 ) [10]. In addition, partial large subunit (LSU) was sequenced to identify basidiomycetes more accurately. According to Vigalys and Hester (1990) [11], partial LSU gene with primer set LROR (5 -ACCGCGTGAACTTAAGC-3 )/LR3 (5 GGTCCGTGTTTCAAGAC-3 ) was amplified. PCR conditions for the ITS and LSU regions were as follows; a 7 min at 95 ◦ C, followed by 30 cycles of 40 s at 95 ◦ C, 40 s at 51 ◦ C, and 1 min 20 s at 72 ◦ C, with the final extension for 7 min at 72 ◦ C. The PCR products were purified using a PCR purification kit (Bioneer) and sequenced by the MACROGEN DNA Synthesis Sequencing Facility (Seoul, Korea). All sequences were compared with reference strains by BLAST search of the GenBank database [12]. The sequences were deposited in GenBank under accession numbers shown in Table 1.
Please cite this article in press as: M.-J. Kim, et al., An aeromycological study of various wooden cultural heritages in Korea, Journal of Cultural Heritage (2015), http://dx.doi.org/10.1016/j.culher.2015.05.001
GenBank accession
Number of isolatesb YHD M
KP288228 KP288229 KP288230 KP288231 KP288232 KP288233 KP288234 KP288235 KP288236 KP288237 KP288238 KP288239 KP288240 KP288241 KP288242 KP288243 KP288244 KP288245 KP288246 KP288247 KP288248 KP288249 KP288250 KP288251 KP288252 KP288253 KP288254 KP288255 KP288256 KP288257 KP288260 KP288258 KP288259 KP288261 KP288262 KP288263 KP288264 KP288265 KP288266 KP288267 KP288268 KP288269 KP288270 KP288271 KP288272 KP288273 KP288274 KP288275 KP288276
A
M
A
M 10
1 2 1
3
36d
92d
5d 1
7 21 20 33d
6d 2
54d 8
1 2 2
8
3
16 48d
2
1 1
3 1
2 7
3 1 3
17 12 31d
7d 10d
1
2
10
17d 1
2 3
4 2 8 12 d
8
5 1
2 25d
1* 6d 7d
1 8 39d
4 4 6 8 7 4
2 6d
Alternaria alternata (AB667801) Arthrinium phaeospermum (GU266274) Aspergillus niger (HQ170509) Aspergillus versicolor (JN997427) Aureobasidium pullulans (EU715320) Botryosphaeria dothidea (JQ936677) Cladosporium cladosporioides (HM148014) Clonostachys rosea (EU326187) Coniochaeta velutina (GQ154542) Coniothyrium fuckelii (AB665313) Diatrypella vulgaris (HQ692592) Discosia artocreas (AB594773) Dothideomycete sp. (EU680553) Epicoccum nigrum (HQ728258) Eutypella sp. (AB693818) Eutypella vitis (FJ537071) Gaeumannomyces graminis (U17212) Graphiopsis chlorocephala (EU009456) Lecanicillium psalliotae (AB083034) Leptosphaerulina chartarum (HQ607815) Leucostoma persoonii (GU062285) Meyerozyma guilliermondii (JF508433) Nectria sp. (GU934544) Neofusicoccum parvum (FJ904817) Neurospora sp. (FJ176470) Paraconiothyrium brasiliense (JF439492) Penicillium brevicompactum (HM469408) Penicillium chermesinum (AY742693) Penicillium glabrum (HM469402) Penicillium herquei (EU833220) Penicillium marneffei (HM595497) Penicillium purpurogenum (HM469414) Penicillium paneum (AB479311) Penicillium simplicissimum (HM469430) Penicillium sp. (HM469421) Penicillium sp. (HQ608086) Phaeosphaeria fuckelii (EF151447) Phaeosphaeria microscopica (AF455494) Phaeosphaeriopsis triseptata (KJ522476) Phoma exigua (EU343168) Phoma glomerata (EU273521) Phoma sp. (JN578632) Phoma sp. (JN207319) Phoma sp. (JQ936186) Phomopsis sp. (JF288552) Phomopsis sp. (GU462143) Seimatosporium discosioides (EF600969) Stagonosporopsis cucurbitacearum (GU045304) Trichoderma harzianum (KC847190)
559/559 (100.0) 567/567 (100.0) 588/588 (100.0) 557/557 (100.0) 570/570 (100.0) 572/572 (100.0) 540/540 (100.0) 557/558 (99.8) 532/532 (100.0) 530/530 (100.0) 563/563 (100.0) 540/541 (99.8) 490/491 (99.8) 532/534 (99.6) 539/540 (99.8) 554/556 (99.6) 528/530 (99.6) 605/615 (98.4) 583/584 (99.8) 603/603 (100.0) 554/554 (100.0) 596/596 (100.0) 510/515 (99.0) 568/568 (100.0) 575/575 (100.0) 594/594 (100.0) 557/557 (100.0) 539/539 (100.0) 554/554 (100.0) 575/576 (99.8) 542/542 (100.0) 558/558 (100.0) 572/572 (100.0) 575/576 (99.8) 568/569 (99.8) 576/579 (99.5) 535/535 (100.0) 569/572 (99.5) 549/558 (98.4) 521/530 (98.3) 526/528 (99.6) 518/519 (100.0) 531/532 (99.8) 531/532 (99.8) 569/571 (99.7) 556/560 (99.3) 556/556 (100.0) 524/531 (98.7) 608/608 (100)
Alternaria alternata Arthrinium phaeospermum Aspergillus niger Aspergillus versicolor Aureobasidium pullulans Botryosphaeria dothidea Cladosporium cladosporioides Clonostachys rosea Coniochaeta velutina Coniothyrium fuckelii Diatrypella vulgaris Discosia artocreas Dothideomycete sp. Epicoccum nigrum Eutypella sp. Eutypella vitis Gaeumannomyces graminis Graphiopsis chlorocephala Lecanicillium psalliotae Leptosphaerulina chartarum Leucostoma persoonii Meyerozyma guilliermondii Nectria sp. Neofusicoccum parvum Neurospora sp. Paraconiothyrium brasiliense Penicillium brevicompactum Penicillium chermesinum Penicillium glabrum Penicillium herquei Penicillium marneffei Penicillium purpurogenum Penicillium paneum Penicillium simplicissimum Penicillium sp.1 Penicillium sp.2 Phaeosphaeria fuckelii Phaeosphaeria microscopica Phaeosphaeriopsis triseptata Phoma exigua Phoma glomerata Phoma sp. 1 Phoma sp. 2 Phoma sp. 3 Phomopsis sp. 1 Phomopsis sp. 2 Seimatosporium discosioides Stagonosporopsis cucurbitacearum Trichoderma harzianum
A
3 5 165d
Fungal identity
UH
4 2 18
Similarityc (%)
3 2
3 1
4 3
ARTICLE IN PRESS
Ascomycetes KUC5301 KUC5302 KUC5303 KUC5304 KUC5305 KUC5306 KUC5307 KUC5309 KUC5310 KUC5311 KUC5312 KUC5313 KUC5314 KUC5315 KUC5318 KUC5319 KUC5320 KUC5321 KUC5322 KUC5323 KUC5324 KUC5325 KUC5328 KUC5329 KUC5330 KUC5331 KUC5333 KUC5334 KUC5335 KUC5336 KUC5337 KUC5338 KUC5339 KUC5340 KUC5341 KUC5342 KUC5344 KUC5345 KUC5346 KUC5347 KUC5348 KUC5350 KUC5351 KUC5352 KUC5353 KUC5354 KUC5356 KUC5357 KUC5358
JHN
Closest fungal match (accession number)
G Model
CULHER-2959; No. of Pages 8
Isolate numbera
M.-J. Kim et al. / Journal of Cultural Heritage xxx (2015) xxx–xxx
Please cite this article in press as: M.-J. Kim, et al., An aeromycological study of various wooden cultural heritages in Korea, Journal of Cultural Heritage (2015), http://dx.doi.org/10.1016/j.culher.2015.05.001
Table 1 Fungi isolated from wooden architectural heritages in Korea.
3
G Model
CULHER-2959; No. of Pages 8
4
Isolate numbera
GenBank accession
Number of isolatesb JHN
YHD
M
KP288277 KP288278 KP288279 KP288280 KP288281
89d 58d
12d
2
4
Zygomycetes KUC6020
KP288282
9
Basidiomycetes KUC9261 KUC9262 KUC9263 KUC9264 KUC9265 KUC9266 KUC9267
KP288283 KP288284 KP288285 KP288286 KP288287 KP288288 KP288289
3
Total isolates a
M
8d 4 1
20d 31d
20d 9d
2 2
5d 5d 4d
16d 6d 2
671
175
125
Ascomycota sp. (HQ607923) Uncultured fungus (GU053979) Uncultured ascomycota (AM901802) Diatrypaceae sp. (JF773650) Fungal sp. (KC507210)
509/524 (97.1) 576/576 (100.0) 524/530 (98.9) 558/585 (95.4) 405/517 (78.3)
Unidentified ascomycota sp. 1 Unidentified ascomycota sp. 2 Unidentified ascomycota sp. 3 Unidentified ascomycota sp. 4 Unidentified ascomycota sp. 5
Cunninghamella elegans (FJ792589)
691/692 (99.9)
Cunninghamella elegans
Irpex lacteus (JX290579) Peniophora sp. (AB808410) Phanerochaete sordida (FJ471547) Phanerochaete velutina (FJ471550) Phanerochaete sp. (GQ470660) Schizophyllum commune (FJ471576) Trametes versicolor (JF416687)
560/560 (100.0) 541/541 (100.0) 575/575 (100.0) 580/580 (100.0) 576/577 (99.8) 579/579 (100.0) 584/584 (100.0)
Irpex lacteus Peniophora sp. Phanerochaete sordida Phanerochaete velutina Phanerochaete sp. Schizophyllum commune Trametes versicolor
A
3
59d 11
Fungal identity
UH A
KUC5359 KUC5361 KUC5362 KUC5363 KUC5364
Similarityc (%)
78
180
58
KUC: Korea University Culture Collection. YHD: Yeonghwadang; JHN: Juhamnu; UH: Unbong hyanggyo; M: May; A: August. c Similarity (%) was calculated as the ratio of matched nucleotides to compare nucleotides in the GenBank database. The internal transcribed spacer region was sequenced for ascomycetes and zygomycetes and the large subunit region was sequenced for basidiomycetes. d Dominant species, a species is considered dominant if Pi > 1/S, where Pi (frequency of species I divided by the total frequency for all species) is the portion of the total sample represented by species i. and S. (species richness) is the number of competing species present in the community [13]. b
ARTICLE IN PRESS
A
M
Closest fungal match (accession number)
M.-J. Kim et al. / Journal of Cultural Heritage xxx (2015) xxx–xxx
Please cite this article in press as: M.-J. Kim, et al., An aeromycological study of various wooden cultural heritages in Korea, Journal of Cultural Heritage (2015), http://dx.doi.org/10.1016/j.culher.2015.05.001
Table 1 (Continued)
G Model
ARTICLE IN PRESS
CULHER-2959; No. of Pages 8
M.-J. Kim et al. / Journal of Cultural Heritage xxx (2015) xxx–xxx
5
Fig. 1. Juhamnu (A-a) and Yeonghwadang (A-b) in Changdeokgung Palace Complex; an initial view of Yeonghwadang (B) and Juhamnu (C).
4. Results
in summer (Table 2). According to Camargo’s index [13], the dominant species in YHD in spring were 7 ascomycetes, Cladosporium cladosporioides, Discosia artocreas, Epicoccum nigrum, Leucostoma persoonii, Penicillium brevicompactum, Phoma glomerata, unidentified ascomycota sp. 1 and unidentified ascomycota sp. 2. On the other hand, in summer, 3 basidiomycetes, Irpex lacteus, Schizophyllum commune and Phanerochaete sp. were dominant species with an ascomycete, Penicillium marneffei. One hundred twenty-five fungal isolates including 19 genera and 25 species and 66 isolates including 12 genera and 13 species were recovered from JHN in spring and
A total of 1259 fungal isolates were obtained from 45 sampling plates at 3 WCHs (Table 1). The majority of fungal species belonged to 56 taxa of ascomycetes, followed by 7 taxa of basidiomycetes and a taxon of zygomycetes. In YHD, 671 fungal isolates including 20 genera and 25 species were collected in spring, whereas 175 isolates including 11 genera and 12 species were recovered in summer. Ascomycetes were dominant (98.2% of isolates) in spring, whereas basidiomycetes were dominant (69.1% of isolates)
Table 2 Frequency of isolates at the 3 wooden cultural heritages. Fungal type
Percentage frequency of isolates (number of isolates) Yeonghwadang
Ascomycetes Basidiomycetes Zygomycetes All isolates
Juhamnu
Unbong hyanggyo
Spring
Summer
Spring
Summer
Spring
Summer
98.2 (659) 60.5 (3) 61.3 (9) 671
30.9 (54) 69.1 (121) – 175
96.0 (120) 60.8 (1) 63.2 (4) 125
34.6 (27) 65.4 (51) – 78
96.7 (174) 63.3 (6) – 180
75.9 (44) 24.1 (14) – 58
Please cite this article in press as: M.-J. Kim, et al., An aeromycological study of various wooden cultural heritages in Korea, Journal of Cultural Heritage (2015), http://dx.doi.org/10.1016/j.culher.2015.05.001
G Model CULHER-2959; No. of Pages 8 6
ARTICLE IN PRESS M.-J. Kim et al. / Journal of Cultural Heritage xxx (2015) xxx–xxx
Fig. 2. Unbong hyanggyo.
summer, respectively. Ascomycetes constituted 96.0% of the total number of isolates in spring, but a high proportion of basidiomycetous fungi representing 65.4% was isolated in summer, which was similar to the pattern of the isolates from YHD. C. cladosporioides, P. brevicompactum and unidentified ascomycota sp. 1 which were the dominant species of YHD in spring were frequently isolated in JHN along with Coniothyrium fuckelii, Paraconiothyrium brasiliense, Penicillium sp. 1 and Phomopsis sp. 1. In addition, in summer, I. lacteus was most frequently detected, followed by Phanerochaete sp., Peniophora sp., and S. commune. Finally, fungal diversity in UH included 180 strains (13 genera and 15 species) and 58 strains (14 genera and 17 species) in spring and summer, respectively. In contrast with YHD and JHN, ascomycetous fungi were dominant not only in the spring (96.7% of isolates), but also in summer (75.9% of isolates). Two ascomycetes, C. cladosporioides and Phaeosphaeria microscopica, were isolated with a high frequency in spring, whereas 4 ascomycetes including the 2 species mentioned above and 3 basidiomycetes — C. cladosporioides, P. microscopic, Phaeosphaeria frankly, and unidentified ascomycota sp. 5 — were dominant in summer. It should be noted that C. cladosporioides was the most frequently obtained species at all sampling sites in spring. This species is ubiquitous on various substrates and is known to form dark mold. Because of its morphological characteristics, this species was previously confused with C. tenuissimum but was later clearly distinguished and identified [14]. Furthermore, the authors of that study found that the discoloration ability of this species is strongest on Japanese red pine and radiata pine blocks within 14 days. As for the genus Penicillium, P. marneffei was found only in summer at 3 WCHs. Four species, P. brevicompactum, P. glabrum, P. purpurogenum, and Penicillium sp. 1 were isolated from both the open building YHD and closed building JHN, while P. chermesinum, P. herquei, P. paneum, P. simplicissimum, and Penicillium sp. 2 were detected only in UH. The discoloration ability of P. brevicompactum and P. simplicissimum was characterized previously [15]. Among the
2 Penicillium species, P. simplicissimum occurred only in UH reached the highest discoloration level on Japanese red pine within 1 week, and it also considerably discolored on radiata pine for 3 weeks. Besides, Alternaria alternata, Aspergillus niger, and Phoma spp. were observed that are common molds found in the diversity from YHD, JHN, and UH. These species could reduce the disfigurement of WCHs, and require more research attention. Only one species of zygomycete, Cunninghamella elegans, was obtained from YHD and JHN in spring. Biodeterioration effects of this zygomycete on Changdeokgung Palace Complex were negligible because this species constituted only 4.5% of isolates. Compared to the research performed at historic sites located in the Antarctic Peninsula [16], zygomycetes were isolated at a low frequency here. Seven basidiomycete species were obtained, of which 4 species were found in YHD and JHN and 5 species were found in UH. All basidiomycete isolates in this study were white rot fungi, which can degrade lignin and carbohydrates at similar rates [17]. Moreover, I. lacteus and Peniophora sp. were collected from all WCHs under study. The decay capability of the isolated basidiomycetes, I. lacteus, Peniophora sp., Phanerochaete spp., S. commune and Trametes versicolor, was reported previously [18–21]. 5. Discussion 5.1. Differences between the open and closed buildings To understand the differences in fungal distribution between environments inside the open and closed buildings, we compared the fungal diversity of YHD (where all doors have always been open to the public and visitors are sometimes allowed) and JHN (a completely closed building) in Changdeokgung Palace Complex. As a result of twice-fungal isolation in spring and summer, the diversity of airborne fungi was not significantly different between YHD and JHN. Many of the same fungal species were isolated from both sites. Among these fungi, C. cladosporioides, P. brevicompactum, and
Please cite this article in press as: M.-J. Kim, et al., An aeromycological study of various wooden cultural heritages in Korea, Journal of Cultural Heritage (2015), http://dx.doi.org/10.1016/j.culher.2015.05.001
G Model CULHER-2959; No. of Pages 8
ARTICLE IN PRESS M.-J. Kim et al. / Journal of Cultural Heritage xxx (2015) xxx–xxx
unidentified ascomycota sp. 1 were found to be dominant in YHD and JHN, whereas D. artocreas and L. persoonii frequently occurred only in YHD. Generally, indoor airborne fungi can appear in a building through dispersal from the outdoor environment, afterwards, they grow and become resuspended in the indoor environment and the air. If the indoor environment is not suitable for the growth of some fungal species, they do not survive there. Accordingly, open and closed buildings have a similar aero fungal community, despite the change of seasons. Nonetheless, the difference in the number of isolates between YHD and JHN is evident. JHN shows a substantially lower frequency of fungal isolates than YHD. It is believed that air circulation that carries airborne spores between indoor and outdoor environments and the fungi introduced by visitors increase the chances of fungal presence in YHD. Adams et al. [22] reported that composition of an indoor fungal community is related to that of outdoor samples and that, quantitatively, fungal biomass is detected more frequently outdoors than indoors.
7
although this phenomenon is not influenced strongly by their strength. For that reason, further research is needed to assess the discoloration ability of ascomycetes in order to preserve WCHs more effectively. 6. Conclusions The results of the present study suggest that WCHs have been exposed to various airborne fungi and the diversity of fungi that might damage WCHs varies depending on the seasonal factors, the open or closed status of a building, and geographical differences. During the spring season, there seems to be rich fungal diversity at all sampling sites, and ascomycetes are mainly isolated. On the other hand, basidiomycetes dominate in summer, at low levels of fungal diversity. In addition, due to the airborne circulation of spores with airflow, a large number of fungal isolates can be detected in an open building. The diversity of indoor airborne fungi also seems to vary depending on the temperature, humidity, and sea level altitude.
5.2. Differences between geographical locations Acknowledgements The patterns of distribution of airborne fungi between UH in Namwon and JHN in Seoul are significantly different in this study, although both of these WCHs are closed buildings. In JHN, the results demonstrated that ascomycetes and basidiomycetes are dominant in spring and summer, respectively. However, ascomycetes are the predominant fungal phylum in all seasons in UH. Namwon and Seoul have different climatic conditions due to their geographical location. Specifically, Namwon has a higher altitude above the sea level, temperature, and humidity than Seoul. Amend et al. [23] identified some of the factors that affect fungal distribution, in particular, local outdoor environmental variables, such as rainfall and temperature, as well as substrate preference, propagule size, and trophic status show statistical correlations with the composition of indoor fungal community. Furthermore, it has been suggested that the geographical location is one of the major factors that affect inhabitation by fungi [22,24]. 5.3. Seasonal differences Among the 3 WCHs, we observed seasonal variation in the number and diversity of isolates. First, at all sites in this study, the total number of isolates in spring us greater than that of summer. Spring weather is windy and dusty. Dust is considered a fungal spore binding agent and wind plays a role in airflow. In addition, ascomycete spores tend to be released with a decrease in relative humidity [25]. These factors ensure that the spores get around more easily and consistently in spring than in summer. During summer, there may be relatively fewer spores in the air owing to inhibition of the spore dispersal by rainfall and greater wind speed compared to that in spring. Second, because the dominant species in summer are basidiomycetes, whereas the dominant species in spring were ascomycetes, it can be inferred that basidiomycetes prefer the summer season (i.e. a hot and humid climate). In other words, ascomycetes are likely to live comfortably under conditions of cooler temperatures and lower humidity. The present results demonstrated that seasonal variation can influence the pattern of fungal diversity, in line with some other studies [26,27]. A lot of ascomycetes, so-called molds, are known for their discoloration ability on wood surfaces. The discoloration characteristics of some genera, Aureobasidium, Coniochaeta, Cladosporium, Discosia and Graphiopsis, which were frequently isolated in this study, have been described in numerous other studies [14,15,28,29]. Nevertheless, there is still a lack of information on fungal discoloration, because many studies have been limited to only well-known fungi. These kind of fungal attack may lower the aesthetic value of WCHs,
This work was supported by the project of National Research Institute of Cultural Heritage, Korea (NRICH-1207-B11F-1) and the BK21 Plus program in 2013 (Project No. 21A20130012270) funded by National Research Foundation of Korea (NRF). References [1] P. Brimblecombe, C.M. Grossi, I. Harris, The effect of long-term trends in dampness on historic buildings, Wearther 61 (2006) 278–281. [2] L.K. Herrera, C. Arroyave, P. Guiamet, S.G. de Saravia, H. Videla, Biodeterioration of peridotite and other constructional materials in a building of the Colombian cultural heritage, Int. Biodeter. Biodegrad. 54 (2004) 135–141. [3] K. Sterflinger, Fungi: their role in deterioration of cultural heritage, Fungal Biol. Rev. 24 (2010) 47–55. [4] V. Kozlov, M. Kisternaya, Biodeterioration of historic timber structures: a comparative analysis, Wood Mater. Sci. Eng. 9 (2014) 156–161. [5] R.A. Blanchette, B.W. Geld, J.A. Jurgens, D.L. McNew, T.C. Harrington, S.M. Duncan, R.L. Farrell, Wood-destroying soft rot fungi in the historic expedition huts of Antarctica, Appl. Environ. Microbiol. 70 (2004) 1328–1335. [6] J.M. Ballester, Sustained Care of the Cultural Heritage Against Pollution. Cultural Heritage. No. 40, Council of Europe Publishing, Sweden, 2000. [7] World Health Organization (WHO), WHO Guidelines for Indoor Air Quality: Dampness and Mould, WHO Regional Office for Europe, Denmark, 2009. [8] R.A. Blanchette, A review of microbial deterioration found in archaeological wood from different environments, Int. Biodeter. Biodegrad. 46 (2000) 189–204. [9] I. Irbe, M. Karadelev, I. Andersone, B. Andersons, Biodeterioration of external wooden structures of the Latvian cultural heritage, J. Cult. Herit. 13 (2012) S79–S84. [10] T.J. White, T.D. Bruns, S. Lee, J.W. Taylor, PCR Protocols: a Guide to Methods and Applications, Academic Press, New York, 1990. [11] R. Vilgalys, M. Hester, Rapid genetic identification and mapping of enzymatically amplified ribosomal DNA from several Cryptococcus species, J. Bacteriol. 172 (1990) 4238–4246. [12] S.F. Altschul, M.S. Boguski, W. Gish, J.C. Wootton, Issues in searching molecular sequence databases, Nat. Genet. 6 (1994) 119–129. [13] J.A. Camargo, Must dominance increase with the number of subordinate species in competitive interactions? J. Theor. Biol. 161 (1993) 537–542. [14] Y.M. Lee, Y. Jang, G.H. Kim, J.J. Kim, Phylogenetic analysis of major molds inhabiting woods and their discoloration characteristics. Part 3. Genus Cladosporium, Holzforschung 66 (2012) 537–547. [15] Y. Jang, N. Huh, J. Lee, J.S. Lee, G.H. Kim, J.J. Kim, Phylogenetic analysis of major molds inhabiting woods and their discoloration characteristics. Part 2. Genus Penicillium, Holzforschung 65 (2011) 265–270. [16] B.E. Arenz, R.A. Blanchette, Investigations of fungal diversity in wooden structures and soils at historic sites on the Antarctic Peninsula, Can. J. Microbiol. 55 (2009) 46–56. [17] E.B. Cowling, Comparative Biochemistry of the Decay of Sweet Gum Sapwood by White Rot and Brown Rot Fungi, No. 1258, US Department of Agriculture Technical Bulletin, United States, 1961. [18] R.A. Blanchette, Delignification by wood decay fungi, Annu. Rev. Phytopathol. 29 (1991) 381–398. [19] M. Capelari, F. Zadrazil, Lignin degradation and in vitro digestibility of wheat straw treated with Brazilian tropical species of white rot fungi, Folia Microbiol. 42 (1997) 481–487.
Please cite this article in press as: M.-J. Kim, et al., An aeromycological study of various wooden cultural heritages in Korea, Journal of Cultural Heritage (2015), http://dx.doi.org/10.1016/j.culher.2015.05.001
G Model CULHER-2959; No. of Pages 8 8
ARTICLE IN PRESS M.-J. Kim et al. / Journal of Cultural Heritage xxx (2015) xxx–xxx
[20] W.E. Eslyn, F.F. Lombard, Fungi associated with decayed wood in stored willow and cottonwood logs, Mycologia 76 (1984) 548–550. [21] J.J. Worrall, S.E. Anagnost, R.A. Zabel, Comparison of wood decay among diverse lignicolous fungi, Mycologia 89 (1997) 199–219. [22] R.I. Adams, M. Marzia, J.W. Taylor, T.D. Bruns, Dispersal in microbes: fungi in indoor air are dominated by outdoor air and show dispersal limitation at short distances, ISME J. 7 (2013) 1262–1273. [23] A.S. Amend, K.A. Seifert, R. Samson, T.D. Bruns, Indoor fungal composition is geographically patterned and more diverse in temperate zones than in the tropics, Proc. Natl. Acad. Sci. U S A 107 (2010) 13748–13753. [24] D.W. Li, B. Kendrick, Functional relationships between airborne fungal spores and environmental factors in Kitchener-Waterloo, Ontario, as detected by Canonical correspondence analysis, Grana 33 (1994) 166–176.
[25] A.M. Jones, R.M. Harisson, The effects of meteorological factors on atmospheric bioaerosol concentrations—a review, Sci. Total Environ. 326 (2004) 151–180. [26] H.J. Chao, J. Schwartz, D.K. Milton, H.A. Burge, Populations and determinants of airborne fungi in large office buildings, Environ. Health Perspect. 110 (2002) 777–782. [27] B.G. Shelton, K.H. Kirkland, W.D. Flanders, G.K. Morris, Profiles of airborne fungi in buildings and outdoor environments in the United States, Appl. Environ. Microbiol. 68 (2002) 1743–1753. [28] M.A. Shirakawa, V.M. John, C.C. Gaylarde, P. Gaylarde, W. Gambale, Mould and phototroph growth on masonry facades after repainting, Mater. Struct. 37 (2004) 472–479. [29] H. Viitanen, Factors affecting the development of biodeterioration in wooden constructions, Mater. Struct. 27 (1994) 483–493.
Please cite this article in press as: M.-J. Kim, et al., An aeromycological study of various wooden cultural heritages in Korea, Journal of Cultural Heritage (2015), http://dx.doi.org/10.1016/j.culher.2015.05.001
ARTICLE IN PRESS Journal of Cultural Heritage xxx (2015) xxx–xxx
Available online at
ScienceDirect www.sciencedirect.com
Original article
An aeromycological study of various wooden cultural heritages in Korea Min-Ji Kim a,1 , Hyun-Kyeong Shin b,2 , Yong-Seok Choi b,3 , Gwang-Chul Kim c,4 , Gyu-Hyeok Kim d,∗ a
BK21 Plus Eco-Leader Education Center, Korea University, Seoul 136-713, Republic of Korea Division of Wood Engineering, Department of Forest Products, Korea Forest Research Institute, 57, Hoegiro, Dongdaemun-gu, Seoul 130-712, Republic of Korea c Department of Housing Environmental Design, Chonbuk National University, Jeounju 561-756, Republic of Korea d Division of Environmental Science & Ecological Engineering, College of Life Sciences & Biotechnology, Korea University, 5-1 Anam-dong, Seongbuk-gu, Seoul, 136-713, Republic of Korea b
a r t i c l e
i n f o
Article history: Received 11 February 2015 Accepted 6 May 2015 Available online xxx Keywords: Airborne fungi Fungal diversity Korea Wood deterioration Wooden cultural heritage
a b s t r a c t Korea has many wooden cultural heritages (WCHs), which should be preserved, along with various other cultural properties. WCHs, however, have undergone biodeterioration because of various fungal attacks in the past centuries; this type of biodeterioration is one of the significant problems faced during preservation of WCHs. To prevent this damage, it is important to investigate the fungal diversity of the WCHs. This aim of this study was to analyze the diversity of airborne fungi at 3 WCHs in Korea: Yeonghwadang (YHD; open building) and Juhamnu (JHN; closed building) in Changdeokgung Palace Complex located in Seoul and Unbong hyanggyo (UH; closed building) in Namwon. The airborne fungi were isolated twice in spring (March) and summer (August) using the gravity settling culture plate method and were identified using morphological and molecular techniques. There were differences in fungal diversity depending on the geographical location, climatic conditions, and the open or closed status of a building. During spring, in the open and closed buildings, a total of 671 fungal isolates (20 genera and 25 species) were collected in YHD and 125 isolates (19 genera and 25 species) were isolated in JHN. In summer, 175 isolates (11 genera and 12 species) and 66 isolates (12 genera and 13 species) were collected from YHD and JHN, respectively. The number of fungal isolates was greater in the open building than in the closed WCHs, but these buildings had similar fungal diversity. In UH, 180 isolates (13 genera and 15 species) were recovered in spring season and 58 isolates (14 genera and 17 species) in summer. There was no significant difference in the number of fungal isolates, but the fungal diversity was different depending on the environmental factors. Finally, fungal diversity was richer in spring than in summer because dusty and windy weather in spring was conducive to the release and transmission of fungal spores. In summer, there were a substantial number of basidiomycetes probably because their spores germinate better at higher temperatures and humidity. © 2015 Elsevier Masson SAS. All rights reserved.
1. Research aims
∗ Corresponding author. Tel.: +82 2 3290 3014. E-mail addresses: [email protected] (M.-J. Kim), [email protected] (H.-K. Shin), [email protected] (Y.-S. Choi), [email protected] (G.-C. Kim), [email protected] (G.-H. Kim). 1 Tel.: +82 2 3290 3468. 2 Tel.: +82 2 961 2708. 3 Tel.: +82 2 961 2704. 4 Tel.: +82 63 270 4847.
The present study was focused on preservation of wooden cultural heritages (WCHs) in Korea against fungal biodeterioration. The first aim was to analyze the diversity of airborne fungi at 3 WCHs in Korea, Juhamnu (JHN; closed building) and Yeonghwadang (YHD; open building) in Changdeokgung Palace Complex in Seoul and Unbong hyanggyo (UH; closed building) in Namwon. These buildings are under different environmental conditions and are managed in different ways such as open to the public or closed. The second aim was to determine the relationship of fungal diversity with environmental and geographic factors. Understanding of
http://dx.doi.org/10.1016/j.culher.2015.05.001 1296-2074/© 2015 Elsevier Masson SAS. All rights reserved.
Please cite this article in press as: M.-J. Kim, et al., An aeromycological study of various wooden cultural heritages in Korea, Journal of Cultural Heritage (2015), http://dx.doi.org/10.1016/j.culher.2015.05.001
G Model CULHER-2959; No. of Pages 8
ARTICLE IN PRESS M.-J. Kim et al. / Journal of Cultural Heritage xxx (2015) xxx–xxx
2
fungal diversity between various WCHs could provide useful clues for the selection of target fungi for preventive measures.
Palace Complex and UH in Namwon. In addition, we compared the observed fungal diversity among different climatic conditions.
2. Introduction
3. Materials and methods
Since ancient times, wood has generally been considered as a great source of building materials because of its firmness and endurance. Numerous architects have used wood in Korea. However, it is inevitable that WCHs would be exposed to outdoor environment, and subjected to high humidity and temperature, in the course of several centuries. This situation is expected to aggravate the deterioration problem. Recently, serious biological deterioration of WCHs around the world was continuously reported along with growing concerns about preservation [1–3]. Among the risks, fungal attacks are considered one of the main causes of damage [4]. There are four types of fungal damage. One is decay caused by basidiomycetes and three other types of damage are soft rot, sapstain, and surface mold caused by ascomycetes [5]. This fungal damage can decrease the aesthetic value and cause serious structural problems. Thus, the causes of deterioration, such as climatic effects, moisture, and pollutants, should be eliminated and their effects mitigated. Appropriate measures should be taken to protect WCHs. Generally, fungal diversity in indoor and outdoor environments is closely related to air particulate matter because fungal spores are airborne. Therefore, constant air circulation is a particularly important factor for the fungal diversity and preservation of WCHs. To enhance the value of WCHs, there have been several efforts to protect WCHs from biodeterioration. The European Council has implemented policies to reduce air pollution and other deterioration factors [6]. In 2009, the World Health Organization explained indoor biological pollution caused by the problem of excess moisture and recommended air quality guidelines [7]. In Korea, the first law on the protection of the cultural heritage was passed in 1962. Furthermore, there are many restoration and repair projects at WCHs, but scientifically valid protection techniques have not been developed yet. Nowadays, in Korea, some WCHs are open to the public to help the people to learn about and understand the wisdom of ancestors and to offer enjoyment and appreciation to public users, other WCHs are closed to the public in order to reduce the inherent risks associated with air pollution and inappropriate handling. The diversity and growth of fungal species depend on the outdoor environment including the presence of water that comes from atmospheric precipitation, condensation phenomena, and dampness of the soil as well as indoor relative humidity influenced by the exchange with the outdoor environment and by weather changes. Although an open building is well ventilated and exposed to outdoor air, when the doors of WCHs are closed, the airflows are blocked, resulting in high humidity levels and risk of mold growth. Therefore, identification and assessment of biological risks in open and closed WCHs are needed for systematic protection. Management of these risks is expected to be facilitated by characterization of the differences between the environmental effects and biodeterioration. In order to preserve WCHs from the fungal biodeterioration, it is necessary to determine the kinds of fungal species that are found in WCHs and mechanism of WCHs damage caused by fungi in combination with environmental factors such as temperature and humidity. Although numerous attempts are made to enhance our understanding of inhabiting fungi involved in deterioration of WCHs [8,9], the data on WCHs in Korea have yet not been collected thoroughly. Therefore, the aim of this study was to investigate the fungal diversity at 3 WCHs: JHN and YHD in Changdeokgung
3.1. Sampling sites JHN and YHD in Changdeokgung Palace Complex, which is located in Seoul (37◦ 58 N, 126◦ 99 E) were selected to compare the diversity of indoor airborne fungi between buildings with closed and open doors. All doors of YHD are always open, and those of JHN are closed all year around (Fig. 1). JHN is about 30 m away from YHD, so airborne spore levels of the outdoor environment of these 2 buildings are not different. Fig. 2 shows UH which is located in Namwon (35◦ 25 N, 127◦ 31 E), Jeollabuk-do, was selected to compare the diversity of indoor airborne fungi depending on geographical locations. The fungal diversity of UH, which is a closed building, was compared with that of JHN. The difference in geographical coordinates between JHN and UH, including the altitude above the sea level, may affect fungal diversity because the climatic conditions of the 2 locations are different.
3.2. Isolation of the fungi Indoor airborne fungal spores were collected at the 3 WCHs twice in spring (May) and summer (August) using the gravity settling culture plate method. Fifteen petri dishes were exposed to the air for 30 minutes in each building. The petri dishes were placed on the floor. The culture medium was 2% malt extract agar (MEA), composed of 20 g of Difco malt extract and 15 g of Difco agar per 1 L of distilled water, with 100 ppm streptomycin to inhibit bacterial growth. After the exposure, the plates were sealed with parafilm and incubated at room temperature for 2 weeks, and the mycelial margins were routinely sub-cultured onto new plates to obtain pure cultures. For the preferential isolation of the basidiomycetes, 2% MEA with 4 ppm benomyl and 100 ppm streptomycin was used. After further purification, most of the airborne fungal species were identified to the genus or species level according to morphological and molecular biological characteristics.
3.3. DNA extraction, PCR amplification, and DNA sequencing Genomic DNA was extracted from fungal mycelia using the Accuprep Genomic DNA Extraction Kit (Bioneer, Korea). The nuclear ribosomal DNA (rDNA) gene cluster was amplified by PCR using the Accupower PCR Premix Kit (Bioneer). The nuclear internal transcribed spacer (ITS) region including the 5.8S rDNA gene was amplified by means of the following primer set: ITS5 (5 -GGAAGTAAAAGTCGTAACAAGG-3 )/ITS4 (5 -TCCTCCGCTTATTGATATGC-3 ) [10]. In addition, partial large subunit (LSU) was sequenced to identify basidiomycetes more accurately. According to Vigalys and Hester (1990) [11], partial LSU gene with primer set LROR (5 -ACCGCGTGAACTTAAGC-3 )/LR3 (5 GGTCCGTGTTTCAAGAC-3 ) was amplified. PCR conditions for the ITS and LSU regions were as follows; a 7 min at 95 ◦ C, followed by 30 cycles of 40 s at 95 ◦ C, 40 s at 51 ◦ C, and 1 min 20 s at 72 ◦ C, with the final extension for 7 min at 72 ◦ C. The PCR products were purified using a PCR purification kit (Bioneer) and sequenced by the MACROGEN DNA Synthesis Sequencing Facility (Seoul, Korea). All sequences were compared with reference strains by BLAST search of the GenBank database [12]. The sequences were deposited in GenBank under accession numbers shown in Table 1.
Please cite this article in press as: M.-J. Kim, et al., An aeromycological study of various wooden cultural heritages in Korea, Journal of Cultural Heritage (2015), http://dx.doi.org/10.1016/j.culher.2015.05.001
GenBank accession
Number of isolatesb YHD M
KP288228 KP288229 KP288230 KP288231 KP288232 KP288233 KP288234 KP288235 KP288236 KP288237 KP288238 KP288239 KP288240 KP288241 KP288242 KP288243 KP288244 KP288245 KP288246 KP288247 KP288248 KP288249 KP288250 KP288251 KP288252 KP288253 KP288254 KP288255 KP288256 KP288257 KP288260 KP288258 KP288259 KP288261 KP288262 KP288263 KP288264 KP288265 KP288266 KP288267 KP288268 KP288269 KP288270 KP288271 KP288272 KP288273 KP288274 KP288275 KP288276
A
M
A
M 10
1 2 1
3
36d
92d
5d 1
7 21 20 33d
6d 2
54d 8
1 2 2
8
3
16 48d
2
1 1
3 1
2 7
3 1 3
17 12 31d
7d 10d
1
2
10
17d 1
2 3
4 2 8 12 d
8
5 1
2 25d
1* 6d 7d
1 8 39d
4 4 6 8 7 4
2 6d
Alternaria alternata (AB667801) Arthrinium phaeospermum (GU266274) Aspergillus niger (HQ170509) Aspergillus versicolor (JN997427) Aureobasidium pullulans (EU715320) Botryosphaeria dothidea (JQ936677) Cladosporium cladosporioides (HM148014) Clonostachys rosea (EU326187) Coniochaeta velutina (GQ154542) Coniothyrium fuckelii (AB665313) Diatrypella vulgaris (HQ692592) Discosia artocreas (AB594773) Dothideomycete sp. (EU680553) Epicoccum nigrum (HQ728258) Eutypella sp. (AB693818) Eutypella vitis (FJ537071) Gaeumannomyces graminis (U17212) Graphiopsis chlorocephala (EU009456) Lecanicillium psalliotae (AB083034) Leptosphaerulina chartarum (HQ607815) Leucostoma persoonii (GU062285) Meyerozyma guilliermondii (JF508433) Nectria sp. (GU934544) Neofusicoccum parvum (FJ904817) Neurospora sp. (FJ176470) Paraconiothyrium brasiliense (JF439492) Penicillium brevicompactum (HM469408) Penicillium chermesinum (AY742693) Penicillium glabrum (HM469402) Penicillium herquei (EU833220) Penicillium marneffei (HM595497) Penicillium purpurogenum (HM469414) Penicillium paneum (AB479311) Penicillium simplicissimum (HM469430) Penicillium sp. (HM469421) Penicillium sp. (HQ608086) Phaeosphaeria fuckelii (EF151447) Phaeosphaeria microscopica (AF455494) Phaeosphaeriopsis triseptata (KJ522476) Phoma exigua (EU343168) Phoma glomerata (EU273521) Phoma sp. (JN578632) Phoma sp. (JN207319) Phoma sp. (JQ936186) Phomopsis sp. (JF288552) Phomopsis sp. (GU462143) Seimatosporium discosioides (EF600969) Stagonosporopsis cucurbitacearum (GU045304) Trichoderma harzianum (KC847190)
559/559 (100.0) 567/567 (100.0) 588/588 (100.0) 557/557 (100.0) 570/570 (100.0) 572/572 (100.0) 540/540 (100.0) 557/558 (99.8) 532/532 (100.0) 530/530 (100.0) 563/563 (100.0) 540/541 (99.8) 490/491 (99.8) 532/534 (99.6) 539/540 (99.8) 554/556 (99.6) 528/530 (99.6) 605/615 (98.4) 583/584 (99.8) 603/603 (100.0) 554/554 (100.0) 596/596 (100.0) 510/515 (99.0) 568/568 (100.0) 575/575 (100.0) 594/594 (100.0) 557/557 (100.0) 539/539 (100.0) 554/554 (100.0) 575/576 (99.8) 542/542 (100.0) 558/558 (100.0) 572/572 (100.0) 575/576 (99.8) 568/569 (99.8) 576/579 (99.5) 535/535 (100.0) 569/572 (99.5) 549/558 (98.4) 521/530 (98.3) 526/528 (99.6) 518/519 (100.0) 531/532 (99.8) 531/532 (99.8) 569/571 (99.7) 556/560 (99.3) 556/556 (100.0) 524/531 (98.7) 608/608 (100)
Alternaria alternata Arthrinium phaeospermum Aspergillus niger Aspergillus versicolor Aureobasidium pullulans Botryosphaeria dothidea Cladosporium cladosporioides Clonostachys rosea Coniochaeta velutina Coniothyrium fuckelii Diatrypella vulgaris Discosia artocreas Dothideomycete sp. Epicoccum nigrum Eutypella sp. Eutypella vitis Gaeumannomyces graminis Graphiopsis chlorocephala Lecanicillium psalliotae Leptosphaerulina chartarum Leucostoma persoonii Meyerozyma guilliermondii Nectria sp. Neofusicoccum parvum Neurospora sp. Paraconiothyrium brasiliense Penicillium brevicompactum Penicillium chermesinum Penicillium glabrum Penicillium herquei Penicillium marneffei Penicillium purpurogenum Penicillium paneum Penicillium simplicissimum Penicillium sp.1 Penicillium sp.2 Phaeosphaeria fuckelii Phaeosphaeria microscopica Phaeosphaeriopsis triseptata Phoma exigua Phoma glomerata Phoma sp. 1 Phoma sp. 2 Phoma sp. 3 Phomopsis sp. 1 Phomopsis sp. 2 Seimatosporium discosioides Stagonosporopsis cucurbitacearum Trichoderma harzianum
A
3 5 165d
Fungal identity
UH
4 2 18
Similarityc (%)
3 2
3 1
4 3
ARTICLE IN PRESS
Ascomycetes KUC5301 KUC5302 KUC5303 KUC5304 KUC5305 KUC5306 KUC5307 KUC5309 KUC5310 KUC5311 KUC5312 KUC5313 KUC5314 KUC5315 KUC5318 KUC5319 KUC5320 KUC5321 KUC5322 KUC5323 KUC5324 KUC5325 KUC5328 KUC5329 KUC5330 KUC5331 KUC5333 KUC5334 KUC5335 KUC5336 KUC5337 KUC5338 KUC5339 KUC5340 KUC5341 KUC5342 KUC5344 KUC5345 KUC5346 KUC5347 KUC5348 KUC5350 KUC5351 KUC5352 KUC5353 KUC5354 KUC5356 KUC5357 KUC5358
JHN
Closest fungal match (accession number)
G Model
CULHER-2959; No. of Pages 8
Isolate numbera
M.-J. Kim et al. / Journal of Cultural Heritage xxx (2015) xxx–xxx
Please cite this article in press as: M.-J. Kim, et al., An aeromycological study of various wooden cultural heritages in Korea, Journal of Cultural Heritage (2015), http://dx.doi.org/10.1016/j.culher.2015.05.001
Table 1 Fungi isolated from wooden architectural heritages in Korea.
3
G Model
CULHER-2959; No. of Pages 8
4
Isolate numbera
GenBank accession
Number of isolatesb JHN
YHD
M
KP288277 KP288278 KP288279 KP288280 KP288281
89d 58d
12d
2
4
Zygomycetes KUC6020
KP288282
9
Basidiomycetes KUC9261 KUC9262 KUC9263 KUC9264 KUC9265 KUC9266 KUC9267
KP288283 KP288284 KP288285 KP288286 KP288287 KP288288 KP288289
3
Total isolates a
M
8d 4 1
20d 31d
20d 9d
2 2
5d 5d 4d
16d 6d 2
671
175
125
Ascomycota sp. (HQ607923) Uncultured fungus (GU053979) Uncultured ascomycota (AM901802) Diatrypaceae sp. (JF773650) Fungal sp. (KC507210)
509/524 (97.1) 576/576 (100.0) 524/530 (98.9) 558/585 (95.4) 405/517 (78.3)
Unidentified ascomycota sp. 1 Unidentified ascomycota sp. 2 Unidentified ascomycota sp. 3 Unidentified ascomycota sp. 4 Unidentified ascomycota sp. 5
Cunninghamella elegans (FJ792589)
691/692 (99.9)
Cunninghamella elegans
Irpex lacteus (JX290579) Peniophora sp. (AB808410) Phanerochaete sordida (FJ471547) Phanerochaete velutina (FJ471550) Phanerochaete sp. (GQ470660) Schizophyllum commune (FJ471576) Trametes versicolor (JF416687)
560/560 (100.0) 541/541 (100.0) 575/575 (100.0) 580/580 (100.0) 576/577 (99.8) 579/579 (100.0) 584/584 (100.0)
Irpex lacteus Peniophora sp. Phanerochaete sordida Phanerochaete velutina Phanerochaete sp. Schizophyllum commune Trametes versicolor
A
3
59d 11
Fungal identity
UH A
KUC5359 KUC5361 KUC5362 KUC5363 KUC5364
Similarityc (%)
78
180
58
KUC: Korea University Culture Collection. YHD: Yeonghwadang; JHN: Juhamnu; UH: Unbong hyanggyo; M: May; A: August. c Similarity (%) was calculated as the ratio of matched nucleotides to compare nucleotides in the GenBank database. The internal transcribed spacer region was sequenced for ascomycetes and zygomycetes and the large subunit region was sequenced for basidiomycetes. d Dominant species, a species is considered dominant if Pi > 1/S, where Pi (frequency of species I divided by the total frequency for all species) is the portion of the total sample represented by species i. and S. (species richness) is the number of competing species present in the community [13]. b
ARTICLE IN PRESS
A
M
Closest fungal match (accession number)
M.-J. Kim et al. / Journal of Cultural Heritage xxx (2015) xxx–xxx
Please cite this article in press as: M.-J. Kim, et al., An aeromycological study of various wooden cultural heritages in Korea, Journal of Cultural Heritage (2015), http://dx.doi.org/10.1016/j.culher.2015.05.001
Table 1 (Continued)
G Model
ARTICLE IN PRESS
CULHER-2959; No. of Pages 8
M.-J. Kim et al. / Journal of Cultural Heritage xxx (2015) xxx–xxx
5
Fig. 1. Juhamnu (A-a) and Yeonghwadang (A-b) in Changdeokgung Palace Complex; an initial view of Yeonghwadang (B) and Juhamnu (C).
4. Results
in summer (Table 2). According to Camargo’s index [13], the dominant species in YHD in spring were 7 ascomycetes, Cladosporium cladosporioides, Discosia artocreas, Epicoccum nigrum, Leucostoma persoonii, Penicillium brevicompactum, Phoma glomerata, unidentified ascomycota sp. 1 and unidentified ascomycota sp. 2. On the other hand, in summer, 3 basidiomycetes, Irpex lacteus, Schizophyllum commune and Phanerochaete sp. were dominant species with an ascomycete, Penicillium marneffei. One hundred twenty-five fungal isolates including 19 genera and 25 species and 66 isolates including 12 genera and 13 species were recovered from JHN in spring and
A total of 1259 fungal isolates were obtained from 45 sampling plates at 3 WCHs (Table 1). The majority of fungal species belonged to 56 taxa of ascomycetes, followed by 7 taxa of basidiomycetes and a taxon of zygomycetes. In YHD, 671 fungal isolates including 20 genera and 25 species were collected in spring, whereas 175 isolates including 11 genera and 12 species were recovered in summer. Ascomycetes were dominant (98.2% of isolates) in spring, whereas basidiomycetes were dominant (69.1% of isolates)
Table 2 Frequency of isolates at the 3 wooden cultural heritages. Fungal type
Percentage frequency of isolates (number of isolates) Yeonghwadang
Ascomycetes Basidiomycetes Zygomycetes All isolates
Juhamnu
Unbong hyanggyo
Spring
Summer
Spring
Summer
Spring
Summer
98.2 (659) 60.5 (3) 61.3 (9) 671
30.9 (54) 69.1 (121) – 175
96.0 (120) 60.8 (1) 63.2 (4) 125
34.6 (27) 65.4 (51) – 78
96.7 (174) 63.3 (6) – 180
75.9 (44) 24.1 (14) – 58
Please cite this article in press as: M.-J. Kim, et al., An aeromycological study of various wooden cultural heritages in Korea, Journal of Cultural Heritage (2015), http://dx.doi.org/10.1016/j.culher.2015.05.001
G Model CULHER-2959; No. of Pages 8 6
ARTICLE IN PRESS M.-J. Kim et al. / Journal of Cultural Heritage xxx (2015) xxx–xxx
Fig. 2. Unbong hyanggyo.
summer, respectively. Ascomycetes constituted 96.0% of the total number of isolates in spring, but a high proportion of basidiomycetous fungi representing 65.4% was isolated in summer, which was similar to the pattern of the isolates from YHD. C. cladosporioides, P. brevicompactum and unidentified ascomycota sp. 1 which were the dominant species of YHD in spring were frequently isolated in JHN along with Coniothyrium fuckelii, Paraconiothyrium brasiliense, Penicillium sp. 1 and Phomopsis sp. 1. In addition, in summer, I. lacteus was most frequently detected, followed by Phanerochaete sp., Peniophora sp., and S. commune. Finally, fungal diversity in UH included 180 strains (13 genera and 15 species) and 58 strains (14 genera and 17 species) in spring and summer, respectively. In contrast with YHD and JHN, ascomycetous fungi were dominant not only in the spring (96.7% of isolates), but also in summer (75.9% of isolates). Two ascomycetes, C. cladosporioides and Phaeosphaeria microscopica, were isolated with a high frequency in spring, whereas 4 ascomycetes including the 2 species mentioned above and 3 basidiomycetes — C. cladosporioides, P. microscopic, Phaeosphaeria frankly, and unidentified ascomycota sp. 5 — were dominant in summer. It should be noted that C. cladosporioides was the most frequently obtained species at all sampling sites in spring. This species is ubiquitous on various substrates and is known to form dark mold. Because of its morphological characteristics, this species was previously confused with C. tenuissimum but was later clearly distinguished and identified [14]. Furthermore, the authors of that study found that the discoloration ability of this species is strongest on Japanese red pine and radiata pine blocks within 14 days. As for the genus Penicillium, P. marneffei was found only in summer at 3 WCHs. Four species, P. brevicompactum, P. glabrum, P. purpurogenum, and Penicillium sp. 1 were isolated from both the open building YHD and closed building JHN, while P. chermesinum, P. herquei, P. paneum, P. simplicissimum, and Penicillium sp. 2 were detected only in UH. The discoloration ability of P. brevicompactum and P. simplicissimum was characterized previously [15]. Among the
2 Penicillium species, P. simplicissimum occurred only in UH reached the highest discoloration level on Japanese red pine within 1 week, and it also considerably discolored on radiata pine for 3 weeks. Besides, Alternaria alternata, Aspergillus niger, and Phoma spp. were observed that are common molds found in the diversity from YHD, JHN, and UH. These species could reduce the disfigurement of WCHs, and require more research attention. Only one species of zygomycete, Cunninghamella elegans, was obtained from YHD and JHN in spring. Biodeterioration effects of this zygomycete on Changdeokgung Palace Complex were negligible because this species constituted only 4.5% of isolates. Compared to the research performed at historic sites located in the Antarctic Peninsula [16], zygomycetes were isolated at a low frequency here. Seven basidiomycete species were obtained, of which 4 species were found in YHD and JHN and 5 species were found in UH. All basidiomycete isolates in this study were white rot fungi, which can degrade lignin and carbohydrates at similar rates [17]. Moreover, I. lacteus and Peniophora sp. were collected from all WCHs under study. The decay capability of the isolated basidiomycetes, I. lacteus, Peniophora sp., Phanerochaete spp., S. commune and Trametes versicolor, was reported previously [18–21]. 5. Discussion 5.1. Differences between the open and closed buildings To understand the differences in fungal distribution between environments inside the open and closed buildings, we compared the fungal diversity of YHD (where all doors have always been open to the public and visitors are sometimes allowed) and JHN (a completely closed building) in Changdeokgung Palace Complex. As a result of twice-fungal isolation in spring and summer, the diversity of airborne fungi was not significantly different between YHD and JHN. Many of the same fungal species were isolated from both sites. Among these fungi, C. cladosporioides, P. brevicompactum, and
Please cite this article in press as: M.-J. Kim, et al., An aeromycological study of various wooden cultural heritages in Korea, Journal of Cultural Heritage (2015), http://dx.doi.org/10.1016/j.culher.2015.05.001
G Model CULHER-2959; No. of Pages 8
ARTICLE IN PRESS M.-J. Kim et al. / Journal of Cultural Heritage xxx (2015) xxx–xxx
unidentified ascomycota sp. 1 were found to be dominant in YHD and JHN, whereas D. artocreas and L. persoonii frequently occurred only in YHD. Generally, indoor airborne fungi can appear in a building through dispersal from the outdoor environment, afterwards, they grow and become resuspended in the indoor environment and the air. If the indoor environment is not suitable for the growth of some fungal species, they do not survive there. Accordingly, open and closed buildings have a similar aero fungal community, despite the change of seasons. Nonetheless, the difference in the number of isolates between YHD and JHN is evident. JHN shows a substantially lower frequency of fungal isolates than YHD. It is believed that air circulation that carries airborne spores between indoor and outdoor environments and the fungi introduced by visitors increase the chances of fungal presence in YHD. Adams et al. [22] reported that composition of an indoor fungal community is related to that of outdoor samples and that, quantitatively, fungal biomass is detected more frequently outdoors than indoors.
7
although this phenomenon is not influenced strongly by their strength. For that reason, further research is needed to assess the discoloration ability of ascomycetes in order to preserve WCHs more effectively. 6. Conclusions The results of the present study suggest that WCHs have been exposed to various airborne fungi and the diversity of fungi that might damage WCHs varies depending on the seasonal factors, the open or closed status of a building, and geographical differences. During the spring season, there seems to be rich fungal diversity at all sampling sites, and ascomycetes are mainly isolated. On the other hand, basidiomycetes dominate in summer, at low levels of fungal diversity. In addition, due to the airborne circulation of spores with airflow, a large number of fungal isolates can be detected in an open building. The diversity of indoor airborne fungi also seems to vary depending on the temperature, humidity, and sea level altitude.
5.2. Differences between geographical locations Acknowledgements The patterns of distribution of airborne fungi between UH in Namwon and JHN in Seoul are significantly different in this study, although both of these WCHs are closed buildings. In JHN, the results demonstrated that ascomycetes and basidiomycetes are dominant in spring and summer, respectively. However, ascomycetes are the predominant fungal phylum in all seasons in UH. Namwon and Seoul have different climatic conditions due to their geographical location. Specifically, Namwon has a higher altitude above the sea level, temperature, and humidity than Seoul. Amend et al. [23] identified some of the factors that affect fungal distribution, in particular, local outdoor environmental variables, such as rainfall and temperature, as well as substrate preference, propagule size, and trophic status show statistical correlations with the composition of indoor fungal community. Furthermore, it has been suggested that the geographical location is one of the major factors that affect inhabitation by fungi [22,24]. 5.3. Seasonal differences Among the 3 WCHs, we observed seasonal variation in the number and diversity of isolates. First, at all sites in this study, the total number of isolates in spring us greater than that of summer. Spring weather is windy and dusty. Dust is considered a fungal spore binding agent and wind plays a role in airflow. In addition, ascomycete spores tend to be released with a decrease in relative humidity [25]. These factors ensure that the spores get around more easily and consistently in spring than in summer. During summer, there may be relatively fewer spores in the air owing to inhibition of the spore dispersal by rainfall and greater wind speed compared to that in spring. Second, because the dominant species in summer are basidiomycetes, whereas the dominant species in spring were ascomycetes, it can be inferred that basidiomycetes prefer the summer season (i.e. a hot and humid climate). In other words, ascomycetes are likely to live comfortably under conditions of cooler temperatures and lower humidity. The present results demonstrated that seasonal variation can influence the pattern of fungal diversity, in line with some other studies [26,27]. A lot of ascomycetes, so-called molds, are known for their discoloration ability on wood surfaces. The discoloration characteristics of some genera, Aureobasidium, Coniochaeta, Cladosporium, Discosia and Graphiopsis, which were frequently isolated in this study, have been described in numerous other studies [14,15,28,29]. Nevertheless, there is still a lack of information on fungal discoloration, because many studies have been limited to only well-known fungi. These kind of fungal attack may lower the aesthetic value of WCHs,
This work was supported by the project of National Research Institute of Cultural Heritage, Korea (NRICH-1207-B11F-1) and the BK21 Plus program in 2013 (Project No. 21A20130012270) funded by National Research Foundation of Korea (NRF). References [1] P. Brimblecombe, C.M. Grossi, I. Harris, The effect of long-term trends in dampness on historic buildings, Wearther 61 (2006) 278–281. [2] L.K. Herrera, C. Arroyave, P. Guiamet, S.G. de Saravia, H. Videla, Biodeterioration of peridotite and other constructional materials in a building of the Colombian cultural heritage, Int. Biodeter. Biodegrad. 54 (2004) 135–141. [3] K. Sterflinger, Fungi: their role in deterioration of cultural heritage, Fungal Biol. Rev. 24 (2010) 47–55. [4] V. Kozlov, M. Kisternaya, Biodeterioration of historic timber structures: a comparative analysis, Wood Mater. Sci. Eng. 9 (2014) 156–161. [5] R.A. Blanchette, B.W. Geld, J.A. Jurgens, D.L. McNew, T.C. Harrington, S.M. Duncan, R.L. Farrell, Wood-destroying soft rot fungi in the historic expedition huts of Antarctica, Appl. Environ. Microbiol. 70 (2004) 1328–1335. [6] J.M. Ballester, Sustained Care of the Cultural Heritage Against Pollution. Cultural Heritage. No. 40, Council of Europe Publishing, Sweden, 2000. [7] World Health Organization (WHO), WHO Guidelines for Indoor Air Quality: Dampness and Mould, WHO Regional Office for Europe, Denmark, 2009. [8] R.A. Blanchette, A review of microbial deterioration found in archaeological wood from different environments, Int. Biodeter. Biodegrad. 46 (2000) 189–204. [9] I. Irbe, M. Karadelev, I. Andersone, B. Andersons, Biodeterioration of external wooden structures of the Latvian cultural heritage, J. Cult. Herit. 13 (2012) S79–S84. [10] T.J. White, T.D. Bruns, S. Lee, J.W. Taylor, PCR Protocols: a Guide to Methods and Applications, Academic Press, New York, 1990. [11] R. Vilgalys, M. Hester, Rapid genetic identification and mapping of enzymatically amplified ribosomal DNA from several Cryptococcus species, J. Bacteriol. 172 (1990) 4238–4246. [12] S.F. Altschul, M.S. Boguski, W. Gish, J.C. Wootton, Issues in searching molecular sequence databases, Nat. Genet. 6 (1994) 119–129. [13] J.A. Camargo, Must dominance increase with the number of subordinate species in competitive interactions? J. Theor. Biol. 161 (1993) 537–542. [14] Y.M. Lee, Y. Jang, G.H. Kim, J.J. Kim, Phylogenetic analysis of major molds inhabiting woods and their discoloration characteristics. Part 3. Genus Cladosporium, Holzforschung 66 (2012) 537–547. [15] Y. Jang, N. Huh, J. Lee, J.S. Lee, G.H. Kim, J.J. Kim, Phylogenetic analysis of major molds inhabiting woods and their discoloration characteristics. Part 2. Genus Penicillium, Holzforschung 65 (2011) 265–270. [16] B.E. Arenz, R.A. Blanchette, Investigations of fungal diversity in wooden structures and soils at historic sites on the Antarctic Peninsula, Can. J. Microbiol. 55 (2009) 46–56. [17] E.B. Cowling, Comparative Biochemistry of the Decay of Sweet Gum Sapwood by White Rot and Brown Rot Fungi, No. 1258, US Department of Agriculture Technical Bulletin, United States, 1961. [18] R.A. Blanchette, Delignification by wood decay fungi, Annu. Rev. Phytopathol. 29 (1991) 381–398. [19] M. Capelari, F. Zadrazil, Lignin degradation and in vitro digestibility of wheat straw treated with Brazilian tropical species of white rot fungi, Folia Microbiol. 42 (1997) 481–487.
Please cite this article in press as: M.-J. Kim, et al., An aeromycological study of various wooden cultural heritages in Korea, Journal of Cultural Heritage (2015), http://dx.doi.org/10.1016/j.culher.2015.05.001
G Model CULHER-2959; No. of Pages 8 8
ARTICLE IN PRESS M.-J. Kim et al. / Journal of Cultural Heritage xxx (2015) xxx–xxx
[20] W.E. Eslyn, F.F. Lombard, Fungi associated with decayed wood in stored willow and cottonwood logs, Mycologia 76 (1984) 548–550. [21] J.J. Worrall, S.E. Anagnost, R.A. Zabel, Comparison of wood decay among diverse lignicolous fungi, Mycologia 89 (1997) 199–219. [22] R.I. Adams, M. Marzia, J.W. Taylor, T.D. Bruns, Dispersal in microbes: fungi in indoor air are dominated by outdoor air and show dispersal limitation at short distances, ISME J. 7 (2013) 1262–1273. [23] A.S. Amend, K.A. Seifert, R. Samson, T.D. Bruns, Indoor fungal composition is geographically patterned and more diverse in temperate zones than in the tropics, Proc. Natl. Acad. Sci. U S A 107 (2010) 13748–13753. [24] D.W. Li, B. Kendrick, Functional relationships between airborne fungal spores and environmental factors in Kitchener-Waterloo, Ontario, as detected by Canonical correspondence analysis, Grana 33 (1994) 166–176.
[25] A.M. Jones, R.M. Harisson, The effects of meteorological factors on atmospheric bioaerosol concentrations—a review, Sci. Total Environ. 326 (2004) 151–180. [26] H.J. Chao, J. Schwartz, D.K. Milton, H.A. Burge, Populations and determinants of airborne fungi in large office buildings, Environ. Health Perspect. 110 (2002) 777–782. [27] B.G. Shelton, K.H. Kirkland, W.D. Flanders, G.K. Morris, Profiles of airborne fungi in buildings and outdoor environments in the United States, Appl. Environ. Microbiol. 68 (2002) 1743–1753. [28] M.A. Shirakawa, V.M. John, C.C. Gaylarde, P. Gaylarde, W. Gambale, Mould and phototroph growth on masonry facades after repainting, Mater. Struct. 37 (2004) 472–479. [29] H. Viitanen, Factors affecting the development of biodeterioration in wooden constructions, Mater. Struct. 27 (1994) 483–493.
Please cite this article in press as: M.-J. Kim, et al., An aeromycological study of various wooden cultural heritages in Korea, Journal of Cultural Heritage (2015), http://dx.doi.org/10.1016/j.culher.2015.05.001