Wang et al. Herit Sci (2021) 9:36 https://doi.org/10.1186/s40494-021-00508-1 RESEARCH ARTICLE Open Access Fungal communities in the bioflms colonizing the basalt sculptures of the Leizhou Stone Dogs and assessment of a conservation measure Yali Wang1, Huan Zhang1* , Xiaobo Liu2, Xiaoqing Liu1 and Wei Song1 Abstract The Leizhou Peninsula in China is very famous for its unique folk art creation, especially for the stonework, like the basalt sculptures of the Leizhou Stone Dogs, which have a history of more than two thousand years. Since these sculptures are usually exposed outdoors, many of them are sufering from severe damage by lichen and fungi. To pro- tect them from microbial attack, we determined fungal communities in the bioflms colonizing the stone dogs and explored an efective conservation measure. Specifcally, the micromorphology analysis of the bioflms on the stone dogs was performed through both on-site and laboratory-based microscopies in order to provide an overview of the fungal community structure. High-throughput sequencing analysis revealed that the predominant fungi in the bio- flms belong to the following genera Caloplaca, Chaetomium, Clitopilus, Acanthostigma, Tolypocladium, Aspergillus, and Toxicocladosporium. The energy dispersive X-ray spectrometry (EDS) showed that the basalt is mainly composed of silica (53% by weight), followed by ­Al2O3 (14.4%), ­Fe2O3 (10.1%), CaO (8.4%), and MgO (7.4%) that are oxides suscepti- ble to corrosion of biogenic acids. The EDS analysis of the hyphae provided the direct evidence on the leaching of the minerals of the sculptures. To control microbial attack, three kinds of antimicrobial agents were tested for 2 years. As a result, the mixture of biocide and water repellent showed the best efectiveness. Our fndings provide an overview of fungi diversity in the bioflms on the stone dogs and help the investigation of fungi-induced biodeterioration and the exploration of specifc conservation measure. Keywords: Biodeterioration, Bioflms, Fungi, Biocide, Stone dog sculptures, Conservation Introduction to their robust reproductive capacity. From then on, the Leizhou Stone Dog sculptures in Guangdong, China are stone sculptures of dogs have been widely popular in typical cultural creative products that blend the ances- the Leizhou Peninsula and, thus, have been well known tors’ tradition of totem worship with diverse ethnic cul- as the Leizhou Stone Dogs, representing the unique cul- tures [1, 2]. In ancient times, the Leizhou Peninsula was tural heritage that could be dated back to around 200 a wild land and having children was very difcult here BC, the Han Dynasty [4, 5]. Nowadays, a total of about because of the local harsh environment [3]. Terefore, 10,000 stone dogs of diverse meanings and shapes are the indigenous inhabitants started to worship dogs due estimated to be distributed over the Leizhou Penin- sula corners, such as the village entrances, the sides of roads or farmlands, and the gate of houses. Te major- *Correspondence: [email protected] ity of them are made of the basalt originated from a local 1 Guangdong Conservation Centre, Guangdong Museum, 2 Zhujiang East Road, Guangzhou 510623, Guangdong, China quarry (E109° 31′–110° 55′ and N20°–21° 35′). However, Full list of author information is available at the end of the article like other stone monuments exposed to the outdoor © The Author(s) 2021. 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The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdo- main/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. Wang et al. Herit Sci (2021) 9:36 Page 2 of 12 environment, such stone dogs have also been sufer- Penicillium frequentans) cause extensive deterioration ing from both natural weathering of rains or winds and and destruction of stone materials, including cement/ biodeterioration of diverse organisms colonizing on the concrete, limestone, granite and sandstone by dissolv- stone surfaces (Fig. 1), resulting in a considerable loss of ing the minerals [8, 20]. In addition to acidolysis, these the appearance [6]. organic acids are capable of complexation or adsorp- Importantly, a typical tropical monsoon climate pre- tion of metal cations (e.g., Fe2+ and Mg2+) to the dete- vails in the Leizhou Peninsula, where microbial activities rioration of the minerals [8, 11, 20–23]. Te formation on stone objects are highly active due to the preferable of calcium oxalate on stone matrices is a consequence temperature and humidity over the year. In such environ- of the precipitation of calcium carbonate by oxalic acid ments, diverse microorganisms (e.g., bacteria, archaea, released by lichens or fungi [20, 24, 25]. Although many fungi, lichens, and cyanobacteria) often colonize and biodeterioration mechanisms have been reported for grow into multispecies bioflms that can lead to the long- fungal biodeteriogens, their diversity and community term biodeterioration of the stone dogs through their characterises in the natural multispecies bioflms on vital activities, such as cell proliferation [7], excretion of stone materials are still under study. Understanding of corrosive acids [8, 9], bioflm matrix formation [10–12], the fungal communities on the stone dogs is of great redox reactions [13, 14], and biogeochemical cycles of importance for the diagnosis of specifc biodeteriora- elements [15–17]. Interestingly, fungi and lichens are tion processes that are proceeding on the surface and widely found in the bioflms on the stone dogs and, thus fnally helps the development of the corresponding mit- their activities might be one of the important factors that igation strategies. have led to the biodegradation there. Our study aims to characterize the fungal diversity Such biodeteriogens have been known to promote and community structure in the bioflms colonizing the the biodeterioration of rocks through the physical stone dogs during their two-year exposure to outdoor disruptive pressure of the growing hyphae, further conditions, examine the biodeterioration activities, and penetration, and bioleaching of mineral constitu- assess the feasibility of an on-site conservation meas- ents [15, 18, 19]. For example, organic acids produced ure. Overall, our fndings indicate that a great diversity by the flamentous fungi (e.g., Aspergillus niger and of fungal communities are present in these bioflms and Fig. 1 Stone dogs exposed to the outdoor environment in the Leizhou Peninsula. a An overview of the subject stone dogs displayed in a yard. b–e Are the four stone dogs where bioflms are sampled for the analysis of fungal communities. The labelled sites from M1 to M7 indicate the seven sampling areas (c.a. 5 cm 5 cm for each) × Wang et al. Herit Sci (2021) 9:36 Page 3 of 12 some of the essential considerations should be taken DNA extraction while a conservation practice is performed. Bioflm samples were evenly suspended into the ster- ile distilled water. After that, the suspension was cen- trifuged at 10,000×g for 15 min; then, the pellets were Materials and methods re-suspended in the lysis bufer of the ZymoBIOMICS Sampling strategy DNA Miniprep Kit D4300 (Zymo Research) and trans- Seven bioflm samples (M1 to M7) were taken from the ferred into the ZR BashingBead™ Lysis Tubes. DNA surfaces of four stone dogs exposed to an outdoor envi- was extracted following the manufacturer’s instructions ronment in the same yard in the Leizhou Peninsula in using the FastPrep-24 5G homogenizer. Finally, the the September of 2019 (Fig. 1). Each sampling area was concentration and purity of the extracted DNA were measured using the NanoDrop One Microvolume UV– c.a. 5 cm × 5 cm and bioflms were sampled with a ster- ile scalpel. Ten, samples were separately collected into Vis Spectrophotometer (Termo Fisher Scientifc, MA, a sterile petri dish layered with a piece of wet sterile flter USA). paper to keep the humidity. All the dishes were packed into a bag cooled with ice bags and immediately sent to Amplicon generation the lab for further analysis. ITS genes of distinct regions (ITS2) were amplifed with a pair of special primers (GCA TCG ATG AAG AAC GCA Analyses of microscopy and energy dispersive X‑ray GC and TCC TCC GCT TAT TGA TAT GC) with 12 bp spectrometry barcode. Primers were synthesized by Invitrogen (Inv- itrogen, Carlsbad, CA, USA). Te PCR reaction system Te on-site analysis of microscopy for bioflms on the (a total volume of 50 μl) contained 25 μl of the 2 Pre- stone dogs was performed using the Cordless Digi- × mix Taq (Takara Biotechnology, Dalian Co. Ltd., China), tal Microscope DG-3x (Scalar Corporation, Japan). 1 μl of each primer (10 mM), 3 μl of the DNA (20 ng/μl) Some bioflm samples were also collected and sent to template, and 20 μl of distilled water. Amplicons were the lab for further microscopy analysis. For a brief opti- amplifed by a thermocycling reaction: 5 min at 94 °C cal microscopy, a drop of the suspension of each sample for initialization, 30 cycles of 30 s denaturation at 94 °C, was transferred onto a microscope slide and examined by annealing at 52 °C for 30 s, extension at 72 °C for 30 s, the Microscope Axio Imager.A2m (ZEISS) under difer- and followed by fnal elongation at 72 °C for 10 min.