Analysis of fungal diversity of the rotten wooden pillars of a historic building

High-throughput sequencing technology was used to analyze the fungal community structure and its association with the cause of decay on the wooden pillars of an ancient archway in Beijing. The dominant fungi on the rotten pillars belonged to Ascomycetes regardless of the sampling position. Compared with the fungal community composition of discolored wood previously studied, the proportion of Basidiomycetes in rotten wood pillars increased at the highest value of 37.9%. High-throughput sequencing showed that the main fungi in the first pillar were Ascomycetes ( Phoma , Lecythophora , and Scedosporium ) and Basidiomycetes (Sporidiobolales). Ascomycetes Lecythophora and Basidiomycetes Cryptcoccus and Postia were the main fungi in pillar 2. Phoma , Trichoderma, and Entoloma were isolated from pillar 1, whereas Alternaria and Phaeosphaeriaceae were obtained from pillar 2 using culture isolation. Traditional isolation failed to obtain all dominant fungi. The importance of high-throughput sequencing technology in ancient wooden structure building biodeterioration analysis was further explained. At the three sampling sites, the contact-ground fungal community composition was similar to that of in-ground wood, whereas above-ground fungal community composition was significantly different from the other two sites. The high moisture content of the wood caused decay. The bottom of the pillar was immersed in groundwater, whereas that cement coating prevents the evaporation of water,cause the wood moisture content to be high. By comparing the fungal diversities of decaying wood and discolored and dry, decayed wood, the relative content of Basidiomycetes may be used as an indicator of wood decay state. This study detected fungal diversity on rotten wooden pillars of an ancient building archway in Beijing by amplicon high-throughput sequencing and analyzed the cause of decay. Comparison of microbial composition of discolored and dry rotten woods showed the increased proportion of Basidiomycetes with the highest value of up to 37.9%, although Ascomycetes remained the dominant phylum. Fungal composition analysis showed the first pillar mainly contained Ascomycetes (Phoma, Lecythophora, and Scedosporium) and Basidiomycetes Sporidiobolales. Ascomycetes Lecythophora and Basidiomycetes Cryptcoccus and Postia were the main fungi in pillar 2. Phoma, Trichoderma and Entoloma were isolated from pillar 1, and Alternaria and Phaeosphaeriaceae were obtained from pillar 2 using culture isolation method. Traditional isolation failed to determine the dominant fungi. The importance high-throughput sequencing technology in biodeterioration analysis of ancient wooden structural buildings was further


Introduction
Historical buildings, as landmarks of culture and diversity, could be conserved, reused, and be expected to deliver a good shape for future generations [1]. As Chinese ancient buildings are characterized by wooden structures, wood biodeterioration and protection are important concerns.
Proposed effective maintenance projects for ancient wooden structures require information on wood deterioration degree. In the past, visual inspection and tapping methods were used, and they were later replaced several non-destructive or semi-destructive evaluation methods, such as the Pilodyne Wood Tester, Ultrasonics and stress wave technology, [2][3][4][5]. However, none of these tests revealed 3 the cause of biodeterioration.
The most serious biological deterioration of wood is caused by fungi. Fungi not only cause serious decay of timber but can also spread through buildings from one timber location to another across non-nutritional surfaces, thus seriously affecting historical buildings [6]. Fungi have both direct and indirect effects on the health of building materials, structures, and occupants; thus, "building mycology" is defined as the branch of mycology dealing with the study of fungi in and around built environments [7,8].
Traditional methods for identifying wood fungi include isolation of fungi from degrading building timbers and identification of fungal species based on culture characteristics. Several molecular methods based on DNA analyses have been used to provide efficient, sensitive, and rapid diagnostic tools for the detection and identification of wood decay fungi without requiring a prior fungal isolation step [9][10][11][12].
Wood decay is caused by various fungi. Microbial diversity has been traditionally assessed by cultivation techniques. The biodiversity of forest fungi can serve as an indicator of forest health [10].
The relationship among microbial diversity, environmental factors, and wood determines the degradation rate of timbers [13,14]. Kahl et al. observed that the activity of wood-degrading enzymes laccase and endocellulase and fungal diversity are the most important predictors for wood decay when applying a multi-model inference approach [15]. Der Wal et al. and Pastorelli et al. reported that two basic parameters, namely, microbial community structure and diversity, are good indicators to predict the rate of wood mass loss and determine the rate of wood decay [16][17][18].
The two essential parameters of fungal diversity, namely, species richness (the number of species within a community) and species evenness (the sizes of species populations within a community), can be detected using amplicon high-throughput sequencing, which is the best method for in-depth analysis of fungal community composition [19,20]. Amplicon high-throughput sequencing, also known as next-generation sequencing, provides the best understanding of microbial community diversity on any substrate, including building timbers [21][22][23]. Another advantage of amplicon high-throughput sequencing method is that testing can be conducted with as low as 0.1 g wood sample, minimizing the damage to in-service building timbers [22].
Experiencing more than 200 years of wind and rain, a historical memorial archway in Beijing has decayed to different degrees. With a history of almost 40 years, the two large round wood pillars buried in the ground were eventually replaced. Serious decay occurred in the pillars. Thus, the degree of decay and the assessment of decayed organisms should be carried out. In this study, a small amount of sawdust was sampled from the contact-ground wood (HC) pillar, 100 cm in-ground wood (HUP), and 100 cm above-ground wood (HD), and microbial DNA was directly extracted from the sawdust. High-throughput sequencing was used to analyze the fungal structure composition and the cause of decay on the wooden pillars to provide basis for the analysis of wood decay degree and disease development in historical buildings.

On-site inspection and sampling
The decay on sample location was observed, and the moisture content of the rotten pillar was measured using JINGTAI wood moisture meter SD-C50. Routine desk survey was performed. A more than 200-year-old decayed old wood beam of the ancient building was selected as the control (G6) ( Fig. 1). Small pieces of wood (0.1 g) were sampled by knife aseptically.

Fungi isolation
The small wood chips detached from each of the five samples were surface-sterilized and then placed onto 2% agar plates. The plates were incubated at constant temperature and humidity incubator at 28±10 °C. Fungal isolation and purification were conducted as described by Fang [24].
PCR, amplicon quantification, and MiSeq high-throughput sequencing test were performed thrice.

On-site inspection and sampling
The ancient memorial archway in Beijing was established more than 200 years ago. The two large, round wooden pillars buried in the ground were numbered No. 1 and No. 2. They were nearly 40 years old and seriously decayed and replaced in a later stage (Fig. 1). The wood species of No. 1 and No. 2 pillar were Pseudotsuga menziesii and Picea sp., respectively, according to maintenance records. The in-ground part was covered in cement, and the pillar base was submerged in underground water. The in-ground wood was seriously decayed. The sampling was conducted on in-ground, decaying wood (HUP); contact-ground, decaying wood (HC); and above-ground wood without progressing decay (HD).
Samples from No. 1 pillar were coded as HC1, HUP, and HD1, and those from No. 2 pillar were HC2 and HD2. The buried part of No. 2 pillar had been cut off during the second maintenance, and the inground foundation was connected to the cement pillar. Thus, only the contact-ground and aboveground samples were available. The moisture content was measured at the corresponding sampling location. At least three points were measured in each location, and the average was obtained. All data are shown in Table 1. Table 1 shows that the moisture content at the bottom of the wood pillar was extremely high (average moisture content: 56.3%). The contact-ground wood moisture content was also notably high and exceeded 20%, but that above-ground was less than 14%. The average moisture content of the control ancient wood (dry and rotten wood) was notably low at 8.9%. Mold infections start with 14% wood moisture content, whereas wood decaying fungi need more than 20% to initiate decay [27]. The moisture content in contact-ground and in-ground wood is suitable for the growth of decaying fungi and occurrence of wood decay.
6 What caused the high moisture content of the in-ground and above-ground wooden pillars? During onsite observation, the bottom of the wood pillar was soaked in ground water, whereas the in-ground pillar was encased in cement. The water at the bottom of pillar originated partly from underground water or accumulated rain, whereas the cement coating prevented water evaporation.

Fungal isolation
By traditional tissue isolation and purification, one species of mold Phoma sp. was isolated from HC1, Trichoderma atroviride from HUP, and Entoloma from HD1. Meanwhile, two species of Ascomycetes Alternaria and Phaeosphaeriaceae were isolated from HC2 of No. 2 pillar. However, no fungus was isolated from HD2 (Fig.2). Entoloma is a decaying fungus, whereas the rest are molds. Good's coverage of six samples were close to 100%, with the mean obtained from the full sequence.

Microbial diversity estimation on five samples α-Index diversity analysis
The community diversity indicator Shannon index was 1.9-3.9, indicating the relatively simple fungal community composition of the sample. The abundance index Chao1 was 103.02-294.51, which was used to evaluate the community richness, and the value of phylogenetic diversity (PD_whole tree) was 21.89-69.39 (Table 2).

Fungal composition analysis
The community structure and relative abundance of the five samples in different taxonomic levels (phylum, class, order, family, and genus) are shown in a colored column chart (Fig. 3). Fig. 3 and Table 3 show that the dominant fungi were Ascomycota, followed by Basidiomycetes. Basidiomycetes accounted for the largest proportion (37.86%) in HC2. The second highest proportion was observed in 7 HD2 at 27.02%. Similar to the control G, the proportion of Ascomycota in HD1 reached more than 90%, whereas that of Basidiomycetes approximated 5%. Compared with the microbial diversity structure of the discolored wood, the overall proportion of Basidiomycetes in rotten wood pillars increased but was less than 10% in the discolored wood [22].
Basidiomycetes are the main degradation fungi of wood decay. The proportion of Basidiomycetes is a possible indicator to predict whether ancient wood decay progresses. Further research is needed to prove this assumption.

Principal component analysis (PCA) and cluster tree
The relationship among the samples could be analyzed by PCA (Fig. 6) and cluster tree analysis (Fig.   7) based on the composition of each sample OTU. The closer the position in the PCA figure, a more clustering relationship occurs, and the more similar composition of samples. Fig. 7 shows that PC1 factor reached 53.78%, and PC2 totaled 28.82%. Three sequencing tests have shown excellent identity (Fig. 7). The cluster tree showed the close relationship between HC1 and HC2, whereas HC (HC1+HC2) and HUP exhibited a closer relationship. HD (HD1+HD2) was distantly located from HC and HUP, consistent with fungal composition analysis (Fig. 4).

Venn diagram and core microbiome analysis
Venn diagrams showed the common and exclusive fungal OTUs of the different wood samples (Fig. 8).
A total of 23 OTUs were observed in all wood samples. Meanwhile, 75 OTUs were detected in three different locations, and 168 OTUs were identified in different pillars.

Dominant fungal species analyzed by MiSeq sequencing and isolation
One fungus (Phoma) was isolated by traditional method. Thus, Phoma is the dominant genus in HC1.
Thus, the development of molecular identification of fungi is needed.
The fungal composition of HUP comprised 74.07% Ascomycota and 24.23% Basidiomycota. The fungal structure at the genus level was composed of 50.8% Lecythophora, 20.66% Sporidiobolales, 9.16% Phoma, 6.97% Cladophialophora, and 3.32% Cryptococcus. Trichoderma accounted for 0.006%, which had been obtained by isolation method. The dominant fungi (50.8% Lecythophora) was not obtained. Not only is fungal infection of timber an unsightly and potentially hazardous to human health, it can also adversely affect the structural integrity of timbers and disrupt the use of buildings [6]. The tests revealed numerous mold species at certain proportions in rotten pillars. Degradation substances by wood decay fungi provides most of the nutrients for the growth of saprophyte molds. Mold spores can cause a great risk to the human body. Therefore, timely repair of wood decay of ancient buildings is also important for tourist health.
On the other hand, the moisture content at the bottom of the wood pillar is extremely high; thus, the role of bacteria in decay formation should not be ignored based on the progressive infection mechanism of microorganisms on wood [30]. The bacterial diversity of rotten pillars should be further analyzed.

Conclusions
This study detected fungal diversity on rotten wooden pillars of an ancient building archway in Beijing by amplicon high-throughput sequencing and analyzed the cause of decay. Comparison of microbial composition of discolored and dry rotten woods showed the increased proportion of Basidiomycetes with the highest value of up to 37.9%, although Ascomycetes remained the dominant phylum.
Fungal composition analysis showed the first pillar mainly contained Ascomycetes (Phoma, Lecythophora, and Scedosporium) and Basidiomycetes Sporidiobolales. Ascomycetes Lecythophora and Basidiomycetes Cryptcoccus and Postia were the main fungi in pillar 2. Phoma, Trichoderma and Entoloma were isolated from pillar 1, and Alternaria and Phaeosphaeriaceae were obtained from pillar 2 using culture isolation method. Traditional isolation failed to determine the dominant fungi. The importance high-throughput sequencing technology in biodeterioration analysis of ancient wooden structural buildings was further explained.      Cluster tree analysis based on the composition of each sample OTU. Venn diagrams of fungal OTUs of the wood samples.