Fungal Community Analyses of a Pirogue from the Tang Dynasty in the National Maritime Museum of China

: The goal of this research was to analyze the fungal community responsible for the biodeterioration of a pirogue in the National Maritime Museum of China and to make recommendations for the protection of this artifact. Molecular identiﬁcation of fungal strains isolated from the surface of the pirogue and the air of the storage room that were most closely related to Cladosporium , Penicillium , Talaromyces and Trichoderma spp. DNA extracted from the samples was sequenced on the Illumina MiSeq platform. The results showed that the predominant fungal genera present were Penicillium sp., Cladosporium sp. and Exophiala sp. Thereafter, cellulose degradation experiments were carried out on the predominant fungi screened by pure culturing. Finally, we tested the sensitivity of the predominant fungal isolates to four biocides. This work suggests that we should pay more attention to Penicillium sp. and Cladosporium sp. in the protection of wooden artifacts, and environmental control is recommended as the main means of protecting the pirogue.


Introduction
The canoe, as the earliest water transportation vehicle in ancient China, has irreplaceable research value. By studying the canoes of different ages unearthed in different places, we can infer information about ancient people's life scenes, manufacturing processes and use of tools.
The National Maritime Museum's collection includes a canoe produced in the Tang Dynasty of China (618-907 A.D.) with a length of 13.8 m and a ventral diameter of approximately 95 cm. The canoe was unearthed in Guangdong province. This is China's largest surviving canoe made by hollowing out a single piece of a wood trunk, and its overall preservation is basically complete. The wood has been identified as Rhodamnia dumetorum.
Biodeterioration can be defined as "any undesirable change in a material brought about by the vital activities of organisms" [1]. Bacteria, archaea, fungi, and lichens as well as insect pests are constantly causing problems in the conservation of cultural heritage because of their biodeteriorative potential [2]. As a natural material, wood can often be found to be biodeterioration by microorganisms. Even if wooden artifacts are preserved in museums, they will still be attacked by microorganisms.

Sampling
Surface samples from the canoe showing visible mycelia were inoculated onto PDA medium with sterile cotton swabs. The sampling positions are marked with red circles as shown in Figure 1B. Then, these petri dishes were transported to the laboratory and placed in incubators for fungal isolation and identification. Samples for high-throughput sequencing were collected from the same points and dipped in sterile water prepared in advance. Microorganism aerosols were collected at four sites around the canoe to analyze the fungal community in the canoe's preservation environment. Air samples were taken at a flow rate of 100 L min −1 with a ZR-2050 air sampler (Junray, China). Three petri dishes with PDA medium were used for cultivating fungi and incubated at 28 °C for 4-7 d.

Isolation and Identification of Fungi
After the petri dishes containing PDA were cultured for 4-7 d at 28 °C; colonies showing different appearances were transferred to new plates to obtain pure isolates, and then the mycelia of fungal isolates were used for DNA extraction via the CTAB method [13]. Molecular identification of fungal strains was performed by amplification of the ITS, 28S rRNA and RNA polymerase II largest

Sampling
Surface samples from the canoe showing visible mycelia were inoculated onto PDA medium with sterile cotton swabs. The sampling positions are marked with red circles as shown in Figure 1B. Then, these petri dishes were transported to the laboratory and placed in incubators for fungal isolation and identification. Samples for high-throughput sequencing were collected from the same points and dipped in sterile water prepared in advance. Microorganism aerosols were collected at four sites around the canoe to analyze the fungal community in the canoe's preservation environment. Air samples were taken at a flow rate of 100 L min −1 with a ZR-2050 air sampler (Junray, China). Three petri dishes with PDA medium were used for cultivating fungi and incubated at 28 • C for 4-7 d.

Isolation and Identification of Fungi
After the petri dishes containing PDA were cultured for 4-7 d at 28 • C; colonies showing different appearances were transferred to new plates to obtain pure isolates, and then the mycelia of fungal isolates were used for DNA extraction via the CTAB method [13]. Molecular identification of fungal strains was performed by amplification of the ITS, 28S rRNA and RNA polymerase II largest subunit (RPB1) genes [14,15]. The primer sequences summarized in Table S1 were used for fungi identification [16]. The PCR reaction programs are summarized in Table S1. The PCR product quality and sequence analyses were performed by GENEWIZ (Beijing, China).

High-throughput Sequencing Analysis
Microorganism DNA was extracted by using a Mo Bio Power Water ® DNA Isolation Kit (Mo Bio Laboratories, Carlsbad, California, USA), and DNA purity and concentration (A260/A280 ratio) were assessed using the BioDrop µLite PC Spectrophotometer (Cambridge, UK). Then, the DNA extractions were used as a template for PCR amplification.
For the study of fungal community, internal transcribed spacer 1 (ITS1) was amplified with the universal fungal primers ITS5-1737F/ITS2-2043R [17,18] (Table S2). The amplification reaction mixture and PCR program were prepared as described Liu et al. [4]. Samples with amplicon bands in the range of 500-700 bp were selected for further analysis. Finally, the PCR products were used for library construction, and sequencing was performed on the Illumina HiSeq2500 PE250 platform. Specific methods of operation can be found in the literature described by Liu et al. [6].

Phylogenetic Tree Analysis
To authenticate genetic associations among identified fungal strains inferred by the ITS gene, the determined sequences were analyzed with the Molecular Evolutionary Genetics Analysis software package (MEGA, version 5.05) [19] and aligned using the Clustal W program included in MEGA v.7.0. We selected 20 species per genus, and for the molecular data, we obtained sequences of species exemplars from GenBank. Based on the adjacency method [20,21], MEGA 5.05 software was used to build the phylogenetic tree for the system. Confidence in the tree topology was estimated using the bootstrap method (1000 bootstrap replicates). The distance scale was estimated to be 0.2 and 0.5, respectively. Figtree v.1.4.3 software was used to visualize and edit the tree.
The raw sequencing data can be downloaded at the NCBI Sequence Read Archive (SRA) under study accession number SRP145540.

Cellulase Production and Enzyme Assays
One milliliter solution containing 1 × 10 8 spores was placed in 500-mL flasks filled with 140 mL of cellulase-producing medium and incubated at 28 • C for 7 days, and enzymatic analysis was performed every 24 h. The medium contained: peptone 10 g L −1 , beef extract 5 g L −1 , KH 2 PO 4 2 g L −1 , MgSO 4 ·7H 2 O 0.5 g L −1 , (NH 4 ) 2 SO 4 2g L −1 , and carboxymethylcellulose sodium (CMC-Na) 10 g L −1 as the carbon source. All cultivations were performed in triplicate. A 1-mL aliquot of the supernatant was obtained by centrifugation (12,000× g) for 5 min and then added to 1 mL of CMC-Na (1% w/v) dissolved in citrate buffer (pH 4.8, 0.05 mol L −1 ). Cellulase activity was estimated by the DNS method, and the procedure was introduced by He et al. [24].

The Susceptibility of Isolated Fungi to Biocides
The selected biocides were P91, BIT 20N, Euxyl ® K100 (Schülke, Norderstedt, Germany), and Preventol ® D7 (Lanxess, Köln, Germany). All of these chemicals are isothiazolinone derivatives, which are considered broad-range biocides. Pure fungal isolates were cultivated on PDA plates and incubated at 28 • C for 5 days. Then a certain amount of 0.1% Tween-80 solution was added to each plate, and the spores were scraped off. The spore suspension was centrifuged at 4500 rpm for 10 min and redissolved in sterile water. The spore suspension of the test fungus was inoculated onto PDA plates, and the sensitivity of the pure fungal isolates to biocides was tested by the disc diffusion method. The four biocides were applied at a 0.5% concentration to inhibit fungal growth on the culture plates. After incubation at 28 • C for 2 d, the diameters of the inhibition zones were measured in centimeters. Each antimicrobial test included three replicates, and sterile water was used as a control.

Identification of Predominant Fungi by High-Throughput Sequencing
The fungal community composition was assessed by amplicon sequencing of three samples taken from different locations using the Illumina HiSeq2500 PE250 platform. Figure 1 shows the distribution of the predominant fungi in the three samples. The dominant phylum in the three samples was Ascomycota. Ascomycota was present in all three samples and accounted for 96.73%, 82.4% and 65.45% of the communities ( Figure 2A). Basidiomycota also existed in three samples and accounted for 2.2%, 15.14%, and 30.85% of the communities. Zygomycota accounted for 0.82%, 1.28% and 1.55% of the communities in the three samples. Neocallimastigomycota (0.03%) was only detected in sample DMZ-11.
Appl. Sci. 2019, 9, x FOR PEER REVIEW 5 of 13 which are considered broad-range biocides. Pure fungal isolates were cultivated on PDA plates and incubated at 28 °C for 5 days. Then a certain amount of 0.1% Tween-80 solution was added to each plate, and the spores were scraped off. The spore suspension was centrifuged at 4500 rpm for 10 min and redissolved in sterile water. The spore suspension of the test fungus was inoculated onto PDA plates, and the sensitivity of the pure fungal isolates to biocides was tested by the disc diffusion method. The four biocides were applied at a 0.5% concentration to inhibit fungal growth on the culture plates. After incubation at 28 °C for 2 d, the diameters of the inhibition zones were measured in centimeters. Each antimicrobial test included three replicates, and sterile water was used as a control.

Identification of Predominant Fungi by High-Throughput Sequencing
The fungal community composition was assessed by amplicon sequencing of three samples taken from different locations using the Illumina HiSeq2500 PE250 platform. Figure 1 shows the distribution of the predominant fungi in the three samples. The dominant phylum in the three samples was Ascomycota. Ascomycota was present in all three samples and accounted for 96.73%, 82.4% and 65.45% of the communities (Figure 2A). Basidiomycota also existed in three samples and accounted for 2.2%, 15.14%, and 30.85% of the communities. Zygomycota accounted for 0.82%, 1.28% and 1.55% of the communities in the three samples. Neocallimastigomycota (0.03%) was only detected in sample DMZ-11. The three samples presented large differences in the diversity and distribution of fungal genera ( Figure 2B). Penicillium (36.63%) and Chaetomium (23.99%) accounted for a large proportion of DMZ- The three samples presented large differences in the diversity and distribution of fungal genera ( Figure 2B). Penicillium (36.63%) and Chaetomium (23.99%) accounted for a large proportion of DMZ-9, while DMZ-10 consisted largely of the genus Exophiala (29.15%). The DMZ-11 sample presented a relatively uniform distribution of the genera Cladosporium (26.4%), Cryptococcus (22.16%) and Aspergillus (20.84%). In addition, Talaromyces, Alternaria, Humicola, Pestalotiopsis, and Trichosporon were detected in the three samples, with prevalence values ranging from 0.04% to 10.9%.

Identification of Main Fungi by Culture-Dependent Approach
A total of 14 species of fungi were isolated from the hull and air using culture-dependent approach and molecular methods; six species were isolated from the hull, and eight species were isolated from the air ( Table 1). Three of the six fungal species from the hull belonged to Penicillium sp., which accounted for 74% of all cultured fungal isolates from the hull samples. Two other species belonging to Talaromyces sp. accounted for 0.36%. One species belonging to Cladosporium accounted for 0.45% of all isolated genera. The strains isolated from air samples were most closely related to Cladosporium sp. (46.69%), Penicillium sp. (47.17%) and Trichoderma sp. (0.47%). To further elucidate the characteristics of these isolated fungi, the morphology of their hyphae and conidia was observed under a microscope ( Figure S1).

Phylogenetic Tree Analysis
From the above results, it was determined that Penicillium sp. and Cladosporium sp. accounted for the largest proportions of the fungal communities, so phylogenetic tree analysis of Penicillium and Cladosporium fungi was carried out. We selected some strains that are often found by researchers on museum artifacts, as well as two strains of Penicillium sp. preserved in our laboratory. Strain NK-NH3 (MH392741.1) was isolated from the surface of the lacquer from the Nanhai No. 1 shipwreck, and strain TJM-F5 (MH171487.1) was isolated from a leather suitcase from the Tianjin Museum.
The results showed that the ITS sequences of the four species of Penicillium sp. were not highly similar, and the sequence homology reached approximately 89%. Phylogenetic tree results showed that the four fungi belonging to Penicillium sp. presented relatively independent evolutionary branches, which was consistent with the results of sequence alignment. The ITS sequences of strain NKDMZ-3 and Penicillium chrysogenum (TJM-F5) showed a closer genetic relationship, while the ITS sequences of strain NKDMZ-15 and NK-NH3 were highly homologous ( Figure 3A). In addition to strain NKDMZ-9, the ITS sequence similarity of other Cladosporium sp. was high, showing differences of only a few base pairs ( Figure 3B). Appl. Sci. 2019, 9, x FOR PEER REVIEW 7 of 13

Degradation of Lignin and Cellulose by Fungal Isolates
The 14 fungi isolated from the pirogue and two strains of Penicillium sp. preserved in our laboratory were inoculated onto two different media to detect cellulase and lignocellulose-degrading enzyme activity. Penicillium pimiteouiense NKDMZ-1 exhibited lignocellulose degradation ability after all fungal isolates were cultured on PDA plates containing 0.04% (v/v) guaiacol at 28 • C for 11 d ( Figure S2). After 3 d, 10 isolates (Penicillium sp., Cladosporium sp., Trichoderma sp.,) produced a clear zone around their colonies, indicating they show good cellulase activity ( Figure S3).
The 10 fungi that showed the cellulose degradation capability were inoculated into a cellulase-producing medium, and their enzyme activity was determined every 24 h. As shown in Figure 4, cellulase activity varied with the fermentation time, and the times at which the cellulase activities of different fungi reached their peaks' were different. Penicillium pimiteouiense NKDMZ-1 and Penicillium sumatrense NKDMZ-15 showed higher enzymatic activity compared to the other eight fungal isolates, reaching 0.0599 IU mg −1 and 0.0443 IU mg −1 , respectively. It was noted that these two species of Penicillium sp. accounted for a large proportion of the results obtained through cultivation. The enzymatic activity was considerably lower in the other eight fungi.

Degradation of Lignin and Cellulose by Fungal Isolates
The 14 fungi isolated from the pirogue and two strains of Penicillium sp. preserved in our laboratory were inoculated onto two different media to detect cellulase and lignocellulose-degrading enzyme activity. Penicillium pimiteouiense NKDMZ-1 exhibited lignocellulose degradation ability after all fungal isolates were cultured on PDA plates containing 0.04% (v/v) guaiacol at 28 °C for 11 d ( Figure S2). After 3 d, 10 isolates (Penicillium sp., Cladosporium sp., Trichoderma sp.,) produced a clear zone around their colonies, indicating they show good cellulase activity ( Figure S3).
The 10 fungi that showed the cellulose degradation capability were inoculated into a cellulaseproducing medium, and their enzyme activity was determined every 24 h. As shown in Figure 4, cellulase activity varied with the fermentation time, and the times at which the cellulase activities of different fungi reached their peaks' were different. Penicillium pimiteouiense NKDMZ-1 and Penicillium sumatrense NKDMZ-15 showed higher enzymatic activity compared to the other eight fungal isolates, reaching 0.0599 IU mg −1 and 0.0443 IU mg −1 , respectively. It was noted that these two species of Penicillium sp. accounted for a large proportion of the results obtained through cultivation. The enzymatic activity was considerably lower in the other eight fungi.

Sensitivity of Fungal Isolates to Four Biocides
In this study, a total of 10 fungi exhibited cellulose degradation capabilities, so these fungi may potentially present risk to the pirogue. Chemical methods such as the application of biocides are considered to be effective methods for controlling deterioration caused by microorganisms. Therefore, Appl. Sci. 2019, 9, 4129 9 of 13 we tested the sensitivity of cellulose-degrading fungi to four biocides. The information of biocides was provided in supplementary materials ( Figure S5).
The experiment was performed using a paper diffusion method. The diameter of the inhibition zones was measured in centimeters ( Figure S4). The four biocides were applied at a 0.5% concentration to inhibit fungal growth on the culture plates. This concentration was significantly lower than the concentration proposed by the manufacturer for common use, which is usually a concentration of 2%. It was observed that Penicillium pimiteouiense NKDMZ-1 and Cladosporium cladosporioides NKDMZ-13 were very sensitive to all four fungicide products, and almost no obvious inhibition zone was observed in Trichoderma afroharzianum NKDMZ-16. Among the four fungicide products, P91 and D7 exhibited better inhibitory effects and almost inhibited all fungi ( Figure 5).
Appl. Sci. 2019, 9, x FOR PEER REVIEW 9 of 13 In this study, a total of 10 fungi exhibited cellulose degradation capabilities, so these fungi may potentially present risk to the pirogue. Chemical methods such as the application of biocides are considered to be effective methods for controlling deterioration caused by microorganisms. Therefore, we tested the sensitivity of cellulose-degrading fungi to four biocides. The information of biocides was provided in supplementary materials ( Figure S5).
The experiment was performed using a paper diffusion method. The diameter of the inhibition zones was measured in centimeters ( Figure S4). The four biocides were applied at a 0.5% concentration to inhibit fungal growth on the culture plates. This concentration was significantly lower than the concentration proposed by the manufacturer for common use, which is usually a concentration of 2%. It was observed that Penicillium pimiteouiense NKDMZ-1 and Cladosporium cladosporioides NKDMZ-13 were very sensitive to all four fungicide products, and almost no obvious inhibition zone was observed in Trichoderma afroharzianum NKDMZ-16. Among the four fungicide products, P91 and D7 exhibited better inhibitory effects and almost inhibited all fungi ( Figure 5).

Discussion
The study involved a 1300-year-old canoe that researchers are trying to protect in a glass structure in a museum. However, fungal mycelia have begun to colonize the surface of the canoe due to excavation work, transportation, storage, surveys, and exhibition activities. For this reason, we analyzed the fungal flora that propagated on the wood to examine possible biodegradative fungi that might become active under new storage conditions [3].
High-throughput sequencing revealed 105 fungal genera; Basidiomycota and Ascomycota accounted for almost all the reads for each sample, and the sum of the two proportions reached 95%. Many articles reported that Ascomycota, Zygomycota, Chytridiomycota, and Rozellomycota can be found on the wooden relics deteriorated by microorganisms [25][26][27].
High-throughput sequencing results showed that the most abundant fungi were Penicillium, Exophiala, Cladosporium, Chaetomium, Cryptococcus, and Aspergillus. Although the three samples

Discussion
The study involved a 1300-year-old canoe that researchers are trying to protect in a glass structure in a museum. However, fungal mycelia have begun to colonize the surface of the canoe due to excavation work, transportation, storage, surveys, and exhibition activities. For this reason, we analyzed the fungal flora that propagated on the wood to examine possible biodegradative fungi that might become active under new storage conditions [3].
High-throughput sequencing revealed 105 fungal genera; Basidiomycota and Ascomycota accounted for almost all the reads for each sample, and the sum of the two proportions reached 95%. Many articles reported that Ascomycota, Zygomycota, Chytridiomycota, and Rozellomycota can be found on the wooden relics deteriorated by microorganisms [25][26][27].
High-throughput sequencing results showed that the most abundant fungi were Penicillium, Exophiala, Cladosporium, Chaetomium, Cryptococcus, and Aspergillus. Although the three samples exhibited different dominant fungal genera, the above fungal genera were present in all samples. Fungal genera Penicillium, Cladosporium and Trichoderma have been isolated from the hull and air samples using a culture-dependent approach. The high-throughput sequencing results showed that the species composition was somewhat different from the results obtained from pure culture, probably because some fungi cannot grow on PDA or MEA medium.
Many researchers have detected the genera Penicillium and Cladosporium in the museum environment. Indoor fungi not only exist in the form of spores in the air but also colonize various materials as mycelia. Museums are colonized by various microbes, and the detailed lists of these species vary according to the type and organization of the museum, the type of exhibit, the indoor microclimate, and the local environment [2,[28][29][30]. The most common colonizing fungal taxa are species of Penicillium, Cladosporium, Alternaria, Aspergillus, and Chaetomium. Abe discovered fungal contamination on a painting stored in an art museum of Tokyo. Afterwards, Cladosporium and Aspergillus were detected in the room where the contaminated painting was stored [31]. Klavker et al. investigated the fungal growth on historical textiles preserved in Slovene museums and religious institutions, using culture-dependent techniques and molecular genus-specific barcodes. The dominant contaminant fungi belonged to the genus Penicillium, followed by Aspergillus and Cladosporium [32]. The genera Penicillium, Aspergillus and Chaetomium have been detected on organic objects such as wooden square stools and suitcases in a museum [6]. Piotrowska et al. observed that the most important degrading component taxa were fungi of the genera Penicillium and Cladosporium [33].
The culture-dependent identification results for the fungal aerosol samples showed that the predominant fungi in the air were Penicillium sp. (47.17%) and Cladosporium sp. (46.69%). In temperate climates, researchers have found that the predominant fungal spores in the air are Cladosporium, Penicillium and Aspergillus. The concentration of these fungal spores varies with the season [34]. The most common fungi in the air are Penicillium, Cladosporium and Aspergillus [35]. Skóra et al. determined microbial contamination in four museums, two libraries and two archives located in Poland. Fungal genera Aspergillus, Cladosporium, Penicillium, Chaetomium, and Trichoderma have been isolated from air aerosols sampled from museum storerooms [30]. The prevailing fungal genera Aspergillus, Penicillium and Cladosporium were isolated from air samples by Borrego et al. [36]. The predominant fungi in the warehouses of museums and other buildings are Aspergillus, Penicillium and Cladosporium [37][38][39]. Thirty-four fungal taxa were isolated by Zielińska-Jankiewicz et al. [40], mainly from the Aspergillus and Penicillium genera.
In our study, Penicillium sp. and Cladosporium sp. were present in all samples and constituted the predominant fungal community. The data in Figure 4 clearly indicate that some members of these two fungal taxa were capable of synthesizing cellulase. Since they are not specific cellulase-producing strains, the cellulase activity of these fungi was not positive. However, these cellulose-degrading fungi may become active if the requirements for growth and environmental conditions become appropriate, so Penicillium sp. and Cladosporium sp. must be regarded as a threat to the pirogue.
In addition, Trichoderma was detected in air samples, accounting for 0.47% of the total number of fungi in the air. Piotrowska et al. detected Trichoderma from fungal aerosols taken from the library [33]. Alternaria, Aspergillus, Cladosporium and Penicillium are airborne fungal spores and are considered to be important causes of respiratory allergies [41]. Within the genus Cladosporium, C. herbarum and C. cladosporioides are the most commonly encountered species, in both outdoor and indoor environments [42]. Therefore, the presence of these fungi may affect the health of museum staff.
Given that microbial contamination of canoes is not serious, environmental control is recommended as the main means of inhibiting microbes because environmental conditions greatly affect the growth of microorganisms and the decomposition of wooden cultural artifacts. First, the temperature and humidity in the glass room where the canoe is stored should be constantly monitored. The temperature in the glass room where the canoe is stored is 18-24 • C, and the humidity is 67%. The excessive humidity is conducive to the colonization of the fungus on the wood. However, due to the serious cracking of the canoe, all environmental adjustments need to be considered from many perspectives. Second, the application of efficient and low-toxicity biocides may be considered to restrain fungal growth and mitigate biodeterioration. However, the chemical-physical nature of the artwork's material and its aesthetic appearance should be taken into account [12].

Conclusions
High-throughput sequencing and culture-dependent approaches showed that Penicillium sp. and Chaetomium sp. accounted for a large proportion of the fungal community composition. Phylogenetic tree results showed that the four fungi belonging to Penicillium sp. presented relatively independent evolutionary branches, but the ITS sequence similarity of Cladosporium sp. was high. Penicillium sumatrense NKDMZ-1 and Penicillium sumatrense NKDMZ-15 showed high cellulose degradation abilities, so they may pose a potential threat to wooden materials. Although the four biocides tested in this study showed efficacy, environmental control is recommended as the main means of protecting this pirogue.
Supplementary Materials: The following are available online at http://www.mdpi.com/2076-3417/9/19/4129/s1, Figure S1: Single colony morphology and micrographs of some fungal isolates, Figure S2: Degradation of lignin by fungal isolates, Figure S3: The cellulose-degrading fungi were screened by measuring the zone of clearance, Figure S4: The diameter of the inhibition zones was measured in centimeter, Figure S5: The information of biocides and active compounds. Table S1: Primer sequences used in this study, Table S2: PCR reaction programs.