Molecular Characterization of Airborne Fungi in Caves of the Mogao Grottoes, Dunhuang, China

International Biodeterioration & Biodegradation 65 (2011) 726e731

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Molecular characterization of airborne fungi in caves of the Mogao Grottoes, Dunhuang, China

Wanfu Wang a,b,1,XuMaa,1, Yantian Ma a, Lin Mao a, Fasi Wu b, Xiaojun Ma a, Lizhe An a,*, Huyuan Feng a,* a School of Life Sciences, Key Lab of Arid and Grassland Ecology of Ministry of Education, Lanzhou University, No 222 Tianshui South Rd, Lanzhou, Gansu 730000, China b The Conservation Research Institute of Dunhuang Academy, Dunhuang 736200, China article info abstract

Article history: In this study, we analyzed air samples collected from several sites within the Mogao Grottoes, Dun- Received 11 October 2010 huang, China. The samples were collected each month from September 2008 to August 2009 from an Received in revised form open cave (OC), a semi-open cave (SC), a closed cave (CC), and the entrance (EN) of the Mogao Grottoes. 13 April 2011 Sampling was carried out using a six-stage Andersen FA-I sampler; then samples were cultured and Accepted 13 April 2011 fungal isolates were identified by partial sequencing of their internal transcribed spacer (ITS) region. Available online 12 May 2011 Eleven different fungal genera were found, and the most prevalent was Cladosporium, followed by Fusarium, Penicillium, Alternaria, and Aspergillus. The fungal community composition varied among the Keywords: fi ¼ ¼ Aerobiology four sites. Fungal community structure was signi cantly related to site (r 0.293, p 0.039) and to ¼ ¼ Molecular biology time of year (r 0.523, p 0.000). The concentrations and abundance of airborne fungi varied greatly Biodeterioration throughout the year at the four sampling sites. Meteorological parameters (e.g., temperature, relative Culturable fungi humidity) and the number of visitors also influenced both abundance and community structure of Mogao Grottoes airborne fungi in the Mogao Grottoes. Ó 2011 Elsevier Ltd. All rights reserved.

1. Introduction measures are taken to prevent their growth, biological damage resulting from microbial growth and biofilm formation can be Cave ecosystems are usually considered to be less complex than avoided. Recently, airborne fungal spores were investigated with other ecosystems because of their relatively stable low temperatures reference to conservation of art works. The most common airborne and nutrient-poor biotopes (Poulson and White, 1969; Barton and fungi, both indoors and out, were Cladosporium spp., Penicillium spp., Northup, 2007). The opening of a cave to tourists can result in Alternaria spp., Fusarium spp., and Aspergillus spp. (Aira et al., 2007; changes in the microclimatic conditions and the food web, as the Saiz-Jimenez and Gonzalez, 2007; Wang et al., 2010a,b). Airborne mass of visitors increases the cave temperature, the CO2 concentra- fungi often show pronounced seasonal periodicity and fluctuations tion, and the amount of water vapor (Hoyos et al., 1998). In addition, related to meteorological conditions (Jones and Harrison, 2004; visitors can serve as a new source of organic matter. These changes Wang et al., 2010a,b). Factors such as temperature, relative can lead to successful invasions of alien organisms. Among numerous humidity, sunlight (ultraviolet light), wind, and even atmospheric biological agents, fungi play a critically important role in stone pollutants can activate free-floating airborne organisms. The deterioration. They cause various types of damage to stone as a result consequences of natural environmental variations and anthropo- of biofilm formation, physical penetration into the substrate, chem- genic influences (e.g., tourists) may change the composition of ical reactions with the substrate, and contamination of the substrate microbial communities in the atmosphere. by pigments (Mitchell and Gu, 2000; Gu, 2003; Li et al., 2008). The Mogao Grottoes of Dunhuang have been called “the art For cultural heritage sites, aerobiological investigations are useful gallery of the world” and “a museum on a wall.” They were officially for detecting potentially harmful or destructive microorganisms. If designated a UNESCO World Cultural Heritage site in 1987. Since the establishment of The Dunhuang Academy in 1943, studies have been undertaken to protect the Mogao Grottoes from the effects of Gobi sandstorms (Zhang et al., 2004; Wang et al., 2006) and * Corresponding authors. Tel./fax: þ86 931 8912537. E-mail addresses: [email protected] (L. An), [email protected] (H. Feng). excretion of Apopestes spectrum imagoes on the murals (Wang et al., 1 Contribution equally to this work. 2005).

With the development of molecular biological techniques, we buffer, 0.2 mM each primer, and 2.0 ml (ca. 10 ng DNA) template. The can identify fungi more precisely and rapidly. Such techniques are amplification program was as follows: initial denaturation at 94 C particularly useful for researchers lacking knowledge about fungal for 5 min, 30 cycles of 94 C for 40 s, annealing at 55 C for 40 s, and identification, and consequently, the field of microbial ecology has extension at 72 C for 40 s, and then final extension for 10 min at advanced markedly. Previously, we determined the concentrations 72 C. The PCR products were detected by electrophoresis on a 1% and seasonal dynamics of culturable airborne fungi in the Mogao agarose gel. Grottoes, and examined the relationship between visitor numbers The similarities among PCR products were determined by and aerial microorganisms (Wang et al., 2010a,b). Here, we evalu- restriction fragment length polymorphism (RFLP) analysis. The ated the diversity of aerial fungi at four sampling sites within the amplified ITS region was digested with the restriction endonucle- Mogao Grottoes by extracting DNA and analyzing the internal ases BsuRI (GG/CC) and HinfI (G/ANTC), and then the digested transcribed spacer region (ITS). In addition, we explored the rela- fragments were visualized on a 2.5% agarose gel. Isolates were tionships between fungal composition and environmental grouped together on the basis of RFLP patterns, and one isolate was parameters. chosen from each group for cloning and sequencing after purification using a quick Midi purification kit (Tiangen Co., Beijing, 2. Materials and methods China). Cloning was performed with the pGM-T Vector System (Tiangen 2.1. Sampling sites Co., Beijing, China) following the manufacturer’s instructions. The ligation product was subsequently transformed into Escherichia coli Details of the four sampling sites at the Mogao Grottoes are DH-5a, which allows blueewhite screening, and plated on LB 1 1 shown in Table 1. medium containing ampicillin (100 mg ml ), X-Gal (20 mg ml ), and IPTG (200 mg ml 1). Positive clones were identified by PCR 2.2. Sampling method amplification with the pGM-T vector primer pairs T7/Sp6 using the same program as that used for ITS amplification. Sampling was conducted each month from September 2008 to Suspensions of expected clones were used for sequencing. The August 2009. A six-stage culturable FA-I sampler (modeled on the sequences were obtained using primer T7 by the Shanghai Major- Andersen sampler), made by the Applied Technical Institute of bio Bio-technology Company. The 30 sequences obtained, each Liaoyang, China, was used to isolate culturable fungi from the air. At approximately 600 bp, were then analyzed with the BLAST program each sampling site, the sampler was mounted 1.5 m above ground of the National Center for Biotechnology Information (NCBI) level on a supporting platform. The sampler consisted of an air (http://www.ncbi.nlm.nih.gov/Blast.cgi). The sequences showing pump, a flow meter, and a glass plate. Each stage of the six-stage the highest similarity to those of the clones were extracted from sampler contains a plate with 400 uniformly sized holes. The GenBank, and a phylogenetic neighbor-joining tree including the sampler separates airborne particles into six fractions on the basis obtained isolates and their closest relatives was constructed using of size, as follows: >7.0 mm (stage 1), 4.7e7.0 mm (stage 2), MEGA 4.0 (Tamura et al., 2007; Kumar et al., 2008). 3.3e4.7 mm (stage 3), 2.1e3.3 mm (stage 4), 1.1e2.1 mm (stage 5), and 0.65e1.1 mm (stage 6). Air was drawn into the sampler at a rate of 2.4. Environmental parameter data 28.3 L/min to impact on petri dishes containing potato dextrose agar (PDA) medium. The run time for collecting samples was 5 min, Meteorological data were provided by the Dunhuang Academy. and each sampling was conducted in triplicate. For each sampling, The monitoring station is located at the top of the Grottoes (N 0 0 the FA-I sampler was loaded with 90-cm petri dishes containing 40 02.261 , E 094 48.196 ). Environmental parameters subject to PDA. The culture dishes were incubated for 5 days at 25 C. statistical analysis included temperature, relative humidity (RH), rainfall, solar radiation, wind speed, wind direction, and surface 2.3. Fungus identification temperature. In addition, each cave had a monitor to provide temperature and RH data. The number of visitors was recorded at After incubation, microbial colonies were counted and fungi the ticket office. Computerized hourly data was collected from Aug. were identified microscopically according to the morphology of the 1, 2008. For all environmental parameters, the values used in data observed hyphae, conidia, and sporangia. Isolates were identified analyses were 10-day averages (collected from 9:00 a.m. to 5:00 using a molecular method as described below. p.m.) from before and after the sampling days. Each isolate was homogenized in liquid nitrogen and then DNA was extracted using the CTAB method (Möller et al., 1992). The 2.5. Statistical analysis internal transcribed spacer (ITS) region of fungal rRNA genes was amplified using the following universal primer set: ITS1 (50- Experimental data were analyzed by one-way analysis of vari- TCCGTAGGTGAACCTGCGG-30) and ITS4 (50- TCCTCCGCTTATTGA- ance (ANOVA) using SPSS version 17.0. Principal coordinate analysis TATGC-30)(White et al., 1990). The reaction mixture (25 ml) con- (PCoA) was calculated by PAlaeontological STatistics (PAST) version sisted of 1 unit Taq polymerase, 0.2 mM dNTP, 2.5 ml10 PCR 2.03 (http://folk.uio.no/ohammer/past/). The relationships

Table 1 Details of sampling sites in the Mogao Grottoes.

Site Location Volume Human impact Number in Mogao Grottoes complex Closed cave (CC) Approx. 50 m away from left side of entrance. 101 m3 Completely closed, no visitors. 54# Open cave (OC) Northwest part of the Mogao Grottoes. 1744.7 m3 Completely open year-round. 16# Semi-open cave (SC) Located on the second ﬂoor level of the cliff. 243.3 m3 Intermittently open in tourist 244# peak season. The Entrance (EN) In front of entrance gate. All visitors’ tickets checked here.

# means the number of the caves. 728 W. Wang et al. / International Biodeterioration & Biodegradation 65 (2011) 726e731

Fig. 1. Phylogenetic tree of fungi ITS sequences derived from airborne fungi in the Mogao Grottoes. W. Wang et al. / International Biodeterioration & Biodegradation 65 (2011) 726e731 729 between airborne fungi diversity and environmental parameters were then tested using Pearson correlation analysis.

3. Results

3.1. Phylogenetic analysis of the fungal communities

The composition of the fungal communities was determined by rRNA phylogenetic analysis based on the ITS region. The phylogenetic tree is shown in Fig. 1. A total of 35 sequences were selected from 640 isolates and classified into 11 different genera. We identified 17 species based on their similarities to sequences in Gen- Bank. The most prevalent genera were Alternaria (35.92%), Fusarium (24.95%), Penicillium (14.15%), Cladosporium (13.96%), and Asper- gillus (7.59%). All of these genera were present at all four sites throughout the entire year (Fig. 2). Other fungi identified in this study included mitosporic Ascomycota (1.57%), Auxarthron (0.45%), Arthrinium (0.53%), Coprinellus (0.16%), and Trametes (0.11%). Prin- cipal coordinate analysis (PCoA) showed that the structure of culturable fungi communities varied greatly among the different Fig. 3. Principal coordinate analysis (PCoA) of airborne fungal communities in the open cave (OC), semi-open cave (SC), closed cave (CC), and entrance (EN) of the Mogao sampling sites (Fig. 3). At the genus level, community composition Grottoes. was related to site (r ¼0.293, p ¼ 0.039), and time of year (r ¼0.523, p ¼ 0.000). The Shannon index represents diversity of fungal communities. 3.3. Contributions of temperature, relative humidity, and other The changes in the Shannon index of fungal communities at the four environmental parameters to community structure of airborne fungi sites during the year are shown in Fig. 4. The mean value of the Shannon index differed among the four sites (1.42 in CC, 1.27 in OC, The relationships between community diversity of airborne fl 1.31 in SC, 1.10 in EN) and uctuated markedly among different fungi and environmental parameters were determined by Pearson months. The highest diversity index values were as follows: July in CC correlation analysis (Table 2). The Shannon index was positively (1.87), June in OC (1.64), August in SC (1.69), and June at the entrance related to temperature in the closed cave (CC) and the open cave (1.57). The lowest diversity index was in July at the entrance (0.65). (OC) (p < 0.05); relative humidity was positively related to fungal diversity in the closed cave (CC) (p < 0.01), but negatively related to 3.2. Distribution of culturable fungi among different stages and fungal diversity in the open cave (OC), the semi-open cave (SC), and sites the entrance (EN). The number of visitors was positively related to fungal diversity in the open cave (OC) and the semi-open cave (SC), The particle size distribution of culturable fungi is shown in but negatively related to fungal diversity at the entrance (EN), Fig. 5a. Alternaria was the most abundant fungus detected from although the difference was not significant. The fungal diversity in > m e m stage 1 ( 7.0 m, 34.48%), stage 2 (4.7 7.0 m, 36.34%), stage 3 the open cave (OC) and the semi-open cave (SC) was significantly e m e m (3.3 4.7 m, 45.10%), and stage 4 (2.1 3.3 m, 44.40%). The most related to season. abundant culturable fungus detected from stage 5 (1.1e2.1 mm) was Cladosporium (24.88% of stage 5 samples) and from stage 6 (0.65e1.1 mm) was Fusarium (44.55% of stage 6 samples). 4. Discussion The proportions of the various culturable fungi obtained at the four samplings sites are shown in Fig. 5b. The genus Alternaria was The ITS region of the nuclear ribosomal repeat unit is the most the most abundant fungus in the closed cave (CC, 39.54%), the open commonly sequenced region for fungal systematics and taxonomy cave (OC, 41.14%), and the semi-open cave (SC, 48.12%). The most in and below the genus level. This region of approximately 650 bp is abundant fungus at the entrance (EN) was Fusarium (48.74%). usually obtainable in a single round of Sanger DNA sequencing, and

Fig. 4. Monthly changes in Shannon index of airborne fungal communities in the open cave (OC), semi-open cave (SC), closed cave (CC), and entrance (EN) of the Mogao Fig. 2. Composition of fungal communities in the Mogao Grottoes. Grottoes. 730 W. Wang et al. / International Biodeterioration & Biodegradation 65 (2011) 726e731

The metabolic activity of fungi is an order of magnitude larger than that of bacteria, and, therefore, they have considerably higher biodeteriorative potential than do bacteria (Bastian et al., 2010). In the Lascaux cave, an aggressive fungus with long white mycelia and a fluffy appearance was identified as a Fusarium species (Bastian et al., 2009a,b). The magnitude and speed of this outbreak prompted an intensive mechanical cleaning treatment (Bastian et al., 2010). The fungi Cladosporium cladosporioides, Alternaria spp., Penicillium spp., Aspergillus spp., and their spores are common in air, and many of them cause allergic responses in humans (Shirakawa et al., 2003). The abundance and community structure of airborne fungi in the Mogao Grottoes were influenced by both site and the time of year. Among the four sites, the closed cave showed the highest Shannon index value and the entrance showed the lowest. This suggests that diversity was decreased by air exchange at the entrance. In general, the highest diversity values were in summer (July in CC, June in OC, August in SC, and June at the entrance). However, the lowest diversity value was also observed in summer (July at the entrance, Fig. 4). This was probably because of the rainfall event just before the sampling day in July. According to Jones and Harrison (2004), temperature and water availability affect the size of the fungal source and control the active release of fungal spores. The domestic climatic factors at the Grottoes, espe- cially temperature and relative humidity, appear to provide an optimal environment for fungal growth. There are numerous visitors to the Mogao Grottoes in summer, about 20,000 visitors in the peak tourist season from May to October, and 2700 in the off- season from November to April. The presence of visitors affects the microclimatic conditions and the food web, because mass visits increase cave temperature, CO2 concentration, and the amount of water vapor (Hoyos et al., 1998), and they add new sources of organic matter. These factors can allow successful invasions of alien organisms, which increase the diversity of microbial communities. Fig. 5. Distributions of particle size and proportions of culturable fungi in the open The most frequently recovered genera of culturable fungi from cave (OC), semi-open cave (SC), closed cave (CC), and entrance (EN) of the Mogao each stage were as follows: stage 1e4, Alternaria; stage 5, Clado- Grottoes. sporium; and stage 6, Fusarium. Alternaria was the most prevalent fungus inside the caves, and Fusarium was the prevalent fungus outside. These results are similar to those of Fang et al. (2005) and of its three subregions (the spacers ITS1 and ITS2 and the 5.8S Polymenakou et al. (2008). The relationship between meteorolog- gene), two (ITS1 and ITS2) show a high rate of evolution and are ical parameters and biological particles is widely documented fi typically species-speci c(Bruns and Shefferson, 2004; Kõljalg et al., (Sabariego et al., 2000; Corden and Millington, 2001; Troutt and 2005). In the present study, the predominant genera were Clado- Levetin, 2001; Burch and Levetin, 2002; Hollins et al., 2004; sporium, Fusarium, Alternaria, Aspergillus, and Penicillium. These Stennett and Beggs, 2004; Gu, 2007). Previously, we reported that genera have been found in the atmosphere in many locations meteorological parameters influenced both the abundance and around the world (Stennett and Beggs, 2004; Fang et al., 2005), and community structure of airborne fungi in the Mogao Grottoes have also been mentioned in previous studies on microorganisms (Wang et al., 2010a). In the closed cave (CC), relative humidity was associated with stone (mineral) materials such as murals, paintings, positively related to airborne fungus diversity but negatively and caves (Gorbushina et al., 2004; Saarela et al., 2004; Jurado et al., related to fungal concentration. The number of visitors was nega- 2009). tively related to airborne fungus diversity near the entrance (EN), but positively related to the concentration of airborne fungi

Table 2 outside. Our results show that meteorological parameters can have Pearson correlation analysis between Shannon index and environmental factors different effects on fungal concentration and community structure from the open cave (OC), semi-open cave (SC), closed cave (CC), and entrance (EN) of in the Mogao Grottoes. the Mogao Grottoes. In summary, the airborne fungal diversity varied throughout the Factor CC OC SC EN year at four sampling sites in the Mogao Grottoes in Dunhuang, Temperature 0.666b 0.655b 0.622 0.069 China. The changes in fungal diversity were related to meteoro- Relative humidity 0.780a 0.071 0.058 0.352 logical parameters and the number of visitors. Visitor amount 0.566 0.547 0.08 b b Month 0.254 0.645 0.743 0.082 Acknowledgements Wind speed 0.078 Wind direct 0.47 Rainfall 0.222 We are grateful to financial support from The Major Project of Surface temperature 0.142 Cultivating New Varieties of Transgenic Organisms (2009ZX08009- a Correlation significant at 0.01 level (2-tailed). 029B), the National Natural Foundation of China (40930533, b Correlation significant at 0.05 level (2-tailed). 30870438), State Key Laboratory of Frozen Soil Engineering, CAS W. Wang et al. / International Biodeterioration & Biodegradation 65 (2011) 726e731 731

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