Molecular Characterization of Airborne Fungi in Caves of the Mogao Grottoes, Dunhuang, China
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International Biodeterioration & Biodegradation 65 (2011) 726e731 Contents lists available at ScienceDirect International Biodeterioration & Biodegradation journal homepage: www.elsevier.com/locate/ibiod 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). 0964-8305/$ e see front matter Ó 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.ibiod.2011.04.006 W. Wang et al. / International Biodeterioration & Biodegradation 65 (2011) 726e731 727 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 purifi- cation 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 À1 À1 shown in Table 1. medium containing ampicillin (100 mg ml ), X-Gal (20 mg ml ), À 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).