Molecular identification of fungi colonizing art objects in Thailand and their growth inhibition by local plant extracts

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In this initial attempt to identify fungi predominantly colonizing art objects, mural paintings and a bas-relief, at 12 archaeological sites in the central and western parts of Thailand, 13 fungal isolates were identified using morphological
  Vol.:(0123456789)  1 3 3 Biotech (2019) 9:356   ORIGINAL ARTICLE Molecular identification of fungi colonizing art objects in Thailand and their growth inhibition by local plant extracts Witsanu Senbua 1  · Jesdawan Wichitwechkarn 1   Received: 14 June 2018 / Accepted: 20 August 2019 © King Abdulaziz City for Science and Technology 2019 Abstract In this initial attempt to identify fungi predominantly colonizing art objects, mural paintings and a bas-relief, at 12 archaeo-logical sites in the central and western parts of Thailand, 13 fungal isolates were identified using morphological technique and estimated for their prevalence frequency at each site. Five main genera of fungal community found were  Aspergillus , Fusarium , Curvularia , Penicillium , and  Neurospora . These fungi were further identified to species level by molecular method utilizing nucleotide sequence homology analysis of the conserved internal transcribed spacer (ITS) region. Environmental factors such as temperature, relative humidity, and the opening or closure of the temples did not have any influence upon the fungal type. From the area-based distribution,  Aspergillus  was found at all collection sites, while Fusarium  was found in Bangkok, and Ratchaburi and Petchaburi provinces in the western part of the country. Curvularia  was found mostly in Phra Nakhon Si Ayutthaya and Lopburi provinces, and in one temple in Petchaburi. From the phylogenetic relationship, these prevalent fungi were divided into three closely related groups:  Aspergillus  and Penicillium, Fusarium  and  Neurospora , and Curvularia . In addition, growth inhibition of the fungi by local plant extracts of betel leaves, custard apple leaves, mangosteen peel, and guava leaves at 10,000 ppm were investigated. Mangosteen peel extract gave the highest fungal growth inhibition for all the Curvularia  tested, being 68.3%, 65.6%, and 60% for C. verruculosa , C. geniculata , and C. lunata , respectively. Guava leave extract also yielded highest growth inhibition of 64.4% for C. verruculosa . Both betel leave and custard apple leave extracts showed the highest inhibition towards  A. fumigatus , at 65.1% and 61.8%, respectively. The results obtained here are basic information necessary for future applications in the biological prevention of art objects, and the design of appropriate measures for preventive conservation of Thai cultural heritage. Keywords  Art object · Mural painting · Biodeterioration · Biological prevention · Fungal identification · Fungal growth inhibition Introduction Thai traditional art objects such as mural paintings and bas-reliefs, generally found at various archaeological sites located mainly in Buddhist temples, are invaluable cultural heritage that needs special care and attention. Many of these art works dated back several centuries ago and, with the passing of time, have been destroyed by both physical and biological deteriorating factors. Biological deteriorating factors such as plants, insects, lichens, bacteria, and particu-larly filamentous fungi are of great concern. Microorganisms are known to be responsible for the biodeterioration of art materials through invasion by their growth and biochemi-cal alteration process due to their harmful metabolic activi-ties (Allsopp et al. 2004). Fungi, in particular, have been reported to play an important role in deterioration of art objects (Sterflinger 2010; Sterflinger and Piñar 2013). The growth of filamentous fungi could severely damage these invaluable art works both by deep penetration of growing mycelium resulting in cracking, detachment, and swelling in the structure, and by discoloration and staining result-ing in aesthetic problems (Ciferri 1999; Garg et al. 1995; Sterflinger and Piñar 2013). Fungi-induced deterioration of mural paintings was studied both by in situ analysis and a 1-year monitoring of a laboratory mock painting, and *  Jesdawan Wichitwechkarn 1  Department of Biotechnology, Faculty of Engineering and Industrial Technology, Silpakorn University, Sanam-Chandra Palace Campus, Nakhon Pathom 73000, Thailand   3 Biotech (2019) 9:356  1 3  356 Page 2 of 10 concluded that the mural painting deterioration was due to the metabolically active microbial community found there (Unkovic et al. 2016). In Thailand, where hot and humid tropical climate provides favourable conditions for fungal growth, art objects are even more vulnerable to deteriora-tion by fungi. Additionally, ancient Thai mural paintings were performed by Secco technique in which the paintings were done on a dry plaster. Unlike Fresco technique used in Western countries, Secco was complicated and involved the use of pigments mixed with organic binders or lime and special kinds of pigment media. Biological materials such as tamarind seed glue and animal skin glue were used in the creation and restoration of Thai mural paintings for centu-ries. Such biological materials are good nutrients for fungi. This causes Thai mural paintings to be highly susceptible for biodeterioration by fungal growth.Several studies were done on the diversity of microbial community dwelling on works of art worldwide, for exam-ple, those on medieval wall paintings in Austria (Berner et al. 1997), mural paintings of a church in Lower Saxony, Germany (Gorbushina et al. 2004), mural paintings on the rocky habitat in a famous cave in southern Italy (Nugari et al. 2009), rock paintings in Lascaux cave in France (Bas-tian et al. 2010), pre-historic rock paintings in India (Biswas et al. 2013), the ruins of Angkor temples in Cambodia (Bar-toli et al. 2014), and mural paintings at a tomb in Gong-ju, Korea (Lee et al. 2015). While some works used culture and microscopic method to identify and study microbial com-munity, molecular techniques have gained more and more interest from researchers. Molecular identification of fungi to species level is generally based on a conserved rDNA sequence called internal transcribed spacer (ITS) region. With ITS sequence homology analysis, involving the use of DNA amplification by polymerase chain reaction (PCR) and molecular software programs, molecular fungal iden-tification should provide reliable data (Abrusci et al. 2005; Michaelsen et al. 2006, 2010; Kraková et al. 2012; Zucconi et al. 2012).In Thailand, the study of fungi on mural paintings and art objects of other kinds was rare. Due to their enormous deteriorating effects on art works in this area, it is worth identifying the fungi grown on art works in Thailand. Our work focuses on the isolation and identification of the most prevalent fungi dwelling on Thai traditional art objects at archaeological sites in the central and western parts of Thailand, using both culture-dependent morphological and culture-independent molecular methods, with the hope to gain knowledge of the predominant fungal colonization on ancient art works in particular areas in the country. Moreo-ver, to find a safe and simple way to prevent art objects from being destroyed by these fungi, fungal growth inhibition by various local plant extracts was also studied. Since the main objective of preventive art conservation involves the control of the environment around art works such that it does not confer any more deterioration to the art objects, biologi-cal prevention is a challenge for proper conservation. All these results will provide important preliminary information, which is beneficial for the design of appropriate treatments for preventive art conservation. Materials and methods Sample collection The samples were collected from 12 archaeological sites located in temples in various provinces. These sites were Angkaew and Nang-Chi Temples (Bangkok), Yai-suwan-naram and Ko-Kaeo-Suttharam Temples (Petchaburi prov-ince), Mahathat and Kongkaram Temples (Ratchaburi prov-ince), Phutthaisawan, Chang Yai, Muang, and Yai Thepnimit Temples (Phra Nakhon Si Ayutthaya province), Lai Temple (Lopburi province), and Chantaburi Temple (Saraburi prov-ince). Most of the art objects from which the samples were collected were mural paintings, with the exception for that at Lai Temple, which was a bas-relief. Details of the location, collection date, as well as average atmospheric tempera-ture, relative humidity, and rainfall at each site are shown in Table 1. All the samples were aseptically and gently taken, using the cotton-swab technique (adapted from Portnoy et al. 2004; Niemeier et al. 2006; Rojas et al. 2012), by gently rolling sterile cotton swabs over the surface layer of the art objects showing damaged signs, especially with the sign of fungal growth. A piece of paper with a central opening of 1 × 1 cm in dimension was used in each sample collection to control the size of the swabbing area. The cotton swabs were then put into sterile closed glass vials containing sterile dis-tilled water supplemented with 0.1% Tween 80 and shaken well before transferring to the laboratory. The samples were kept at 4 °C until use. Fungal isolation and contaminating frequency analysis All samples were cultivated in potato dextrose agar (PDA) (Schalau, USA) containing 100 µg/ml ampicillin (Fluka, USA) and incubated at room temperature (25 °C) for about 7 days. After 1-week incubation, fungal colonies of different shapes and colours obtained from each sampling site were observed, counted, isolated, and morphologically identified. Pure fungal colonies were analysed to identify genera and each fungus was calculated for the percentage of contaminat-ing frequency using the following formula: % CF  = i g i t × 100  3 Biotech (2019) 9:356  1 3 Page 3 of 10 356 where i g  is the number of isolates in each genus, i t  is the number of total fungal isolates, and %CF is the percentage of contaminating frequency. Slide culture preparation and fungal morphological identification To morphologically identify the fungi, the slide culture technique was used to monitor the conidiophore and conidia production in situ. A cut PDA block (1 × 1×1 cm) was laid on a slide which was placed on cotton completely damped with sterile distilled water in a Petri dish. Fungal conidia or mycelial fragments were inoculated onto the four edges of the PDA block and a sterile cover slip was placed over the agar block. The plate was incubated at 25 °C until growth and sporulation occurred. The morphological identification was done by observing the pure cultures under light micro-scope, Olympus CX21 (Olympus, USA), and identified to their specific genera according to Barnett and Hunter (1987), Samson et al. (1995), and Klich (2002). Scanning electron microscope (SEM) analysis Fungal samples were pre-fixed in 2.5% glutaraldehyde in 0.1 M phosphate buffer at pH 7.2 for 2 h and rinsed with phosphate buffer. They were then dehydrated in a series of ethanol (30%, 50%, 70%, 80%, 90% and 100%) and dried in a critical point dryer. The samples were coated with 20 nm gold particles and examined under SEM (Hitachi, Japan) operating at 10–15 kV. Fungal DNA extraction For fungal DNA extraction, the most highly contaminating fungi at each collection site were cultivated for approxi-mately 5 days at 25 °C in 50 ml of potato dextrose broth (PDB) containing 100 µg/ml ampicillin with shaking at 150 rpm. Mycelium pellets were harvested, washed, and ground in liquid nitrogen. About 0.3 g of mycelium powders was suspended in 400 µl of lysis buffer (50 mM Tris–HCl, pH 7.2, 50 mM EDTA, 3% SDS, 1% 2-mercaptoethanol), extracted with 350 µl chloroform:TE-saturated phenol, mixed, and centrifuged at 10,000× g  for 15 min. The phenol phase of 350 µl was then precipitated to obtain DNA pel-lets by adding 200 µl isopropanol and 10 µl of 3 M sodium acetate, pH 8.0, and centrifuged at 10,000× g  for 5 min. The pellets were then washed gently with 70% ethanol, resus-pended in 100 µl TE buffer (10 mM Tris–HCl, pH 7.2, 0.1 mM EDTA) and stored at − 4 °C until use (Innis et al. 1990). PCR amplification and nucleotide sequencing of ITS region, and phylogenetic analysis The fungal DNA was amplified at the ITS region with the conserved ITS primers (ITS1 and ITS4) (White et al. 1990). A PCR reaction was performed with initial dena-turation at 94 °C for 5 min, followed by 30 cycles of denaturation at 94 °C for 1 min, annealing at 53–55 °C for 1 min, and extension at 72 °C for 2 min, and then final extension at 72 °C for 10 min. All PCR products were confirmed by agarose gel electrophoresis, purified with GenepHlow ™  Gel/PCR Kit (Geneaid, Taiwan), and Table 1 Selected archaeological sitesArchaeological siteProvinceLocationCollection dateAverage atmos-pheric temperature ( o C)Average rela-tive humidity (%)Average rainfall (mm)Central part Angkaew TempleBangkok13°45 ′ 0 ″ N, 100°28 ′ 0 ″ EDec 201023.8–32.56922.7 Nang-Chi Temple Phutthaisawan TemplePhra Nakhon Si Ayut-thaya14°20 ′ 58 ″ N, 100°33 ′ 34 ″ EAug 201323.4–33.780143.5 Chang Yai Temple Muang Temple Yai Thepnimit Temple Lai TempleLopburi14°48 ′ 0 ″ N, 100°37 ′ 37 ″ EJun 201426.6–35.573106.2 Chantaburi TempleSaraburi14°31 ′ 43 ″ N, 100°54 ′ 41 ″ EJun 2014NANANAWestern part Ko-Kaeo-Suttharam TemplePetchaburi13°06 ′ 43 ″ N, 99°56 ′ 45 ″ EJul 201125.3–32.875138.7 Yai-Suwannaram Temple Kongkaram TempleRatchaburi13°32 ′ 8 ″ N, 99°48 ′ 48 ″ EJan 201222.6–32.47413.2 Mahathat Temple   3 Biotech (2019) 9:356  1 3  356 Page 4 of 10 submitted to First BASE Laboratories Sdn Bdh, Malaysia, for ITS nucleotide sequencing using BigDye ®  Termina-tor v3.1 cycle sequencing kit in ABI 3730xl (Applied Biosystems, USA). The obtained rDNA sequences were confirmed with ITSx program (Bengtsson-Palme et al. 2013). The ITS sequence similarity analysis was per-formed by aligning the fungal sequences with those in GenBank database (NCBI) using the BLAST search pro-gram (Zhang et al. 2000).A phylogenetic tree was constructed from the evolu-tionary distance data using Kimura 2-parameter model (Kimura 1980) and the maximum likelihood method (Nei and Kumar 2000) performed with MEGA 7 (Kumar et al. 2016). All positions containing gaps were eliminated. Confidence level of each clade was estimated using boot-strap analysis (1000 replicates). Inhibition of the fungal growth by local plant extracts Certain local plants such as betel ( Piper betle ), custard apple (  Annona squamosa ), mangosteen ( Garcinia man-gostana ), and guava ( Psidium guajava ) were chosen for inhibition tests on these fungi. The parts of these plants used for the investigation were betel leaves, custard apple leaves, mangosteen peel, and guava leaves. The samples were washed with clean water, cut into small pieces if the samples were too hard, dried at 60 °C for 24 h, ground, and kept in a closed container until use. The dried powder of each sample was extracted by soaking in 95% ethanol (1:10 w/v) for 7 days at room temperature with occasional shaking. The mixture was filtered to get rid of debris on the 8th day and the solvent was evaporated in a rotary evaporator at 40–45 °C. One big batch of extract was done for each plant such that all experiments could be performed within the same conditions. The extracts were subjected to antifungal assessment or kept at − 20 °C until use.To test the inhibition activity against the fungal growth, each plant extract was spread onto PDA plate containing 100 µg/ml ampicillin. A piece of agar contain-ing each type of fungus previously grown on another PDA plate for 7 days was transferred using a cork borer to indi-vidual PDA–ampicillin–plant extract plates. The plates were then incubated at 25 °C for 10 days. The diameters of the fungal colonies were measured and the percentages of fungal growth inhibition by individual plant extracts were determined by comparing the colony diameters with those of the control plates (PDA–ampicillin with no plant extract). The plant extract concentration used was 10,000 ppm. These were performed in triplicate. Results and discussion Fungal identification and phylogenetic tree Since fungi are among the most important causes of dete-rioration of art objects which could seriously ruin these invaluable art works both by mycelial penetration causing cracking, detachment, and swelling, and by discoloration and staining, the identification of fungi predominantly colonizing art works would be interesting and informa-tive. From 240 fungal colonies grown on PDA–ampicillin plates, a number of filamentous fungi were isolated to pure colonies. They were subsequently morphologically identi-fied for their genera and analysed for their percentages of contaminating frequency. All of the most prevalent fungi were morphologically identified using light microscope; however, certain fungi which were difficult to identify and easily mistaken were observed under SEM. They were also subjected to molecular identification based on ITS sequences. The most prevalent fungi identified, together with their percentages of contaminating frequency, the accession numbers, and the percentages of identity are illustrated in Table 2. The morphological method could be used to identify most fungi at only genera level, except for  Aspergillus  which could be identified to species level such as  Aspergillus fumigatus  and  Aspergillus unguis . Here, the results of molecular identification were the same as, and hence confirming those of morphological method, with the ability to assign all the fungi to species level. It was found from the identification that there were five main genera of fungal community colonizing art works in the central and western parts of Thailand:  Aspergillus , Fusarium , Curvu-laria , Penicillium , and  Neurospora .In the central part of Thailand, there were four prov-inces, Bangkok, Phra Nakhon Si Ayutthaya, Lopburi, and Saraburi, from which the samples were collected. All of these five fungal genera were found distributed among these areas of collection sites. In Bangkok, the fungi found with highest frequency on mural paintings in both Angkaew and Nang-Chi Temples were identified as  A. fumigatus . The ITS nucleotide sequences of the two strains of  A. fumigatus  found at these two temples were identical (data not shown) which proved that they were the same fungus. Since Angkaew and Nang-Chi Temples are located within the same district, it is not surpris-ing to find this fungus at both temples. Another fungus of equal frequency at Nang-Chi Temple was Fusarium solani . In Phra Nakhon Si Ayutthaya province, the most highly prevalent fungi at Phutthaisawan Temple was  A. unguis , while that found at Muang Temple was Penicil-lium citrinum . However, these two kinds of fungi were found to be closely related to each other, according to  3 Biotech (2019) 9:356  1 3 Page 5 of 10 356 Table 2 Identification of the isolated fungi with highest contaminating frequencies using morphological and molecular methods a  Contaminating frequency b  Shown only for the most prevalent generaArchaeological site% CF a Morphological identification b Molecular identification b Identified microorganismAccession numberIdentity (%)Central part Bangkok Angkaew Temple63.64  Aspergillus fumigatus Aspergillus fumigatus  WSAK10KT581395100 Nang-Chi Temple25.00  Aspergillus fumigatus Aspergillus fumigatus  WSNC10KT58139610025.00  Fusarium  sp.  Fusarium solani  WS2NC10KT58139799 Phra Nakhon Si Ayutthaya Phutthaisawan Temple83.33  Aspergillus unguis Aspergillus unguis  WJPS01KY40417299 Chang Yai Temple28.57  Curvularia  sp.  Curvularia lunata  WJCY01KY40417799 Muang Temple34.78  Penicillium  sp.  Penicillium citrinum  JMU01KY404179100 Yai Thepnimit Temple33.33  Curvularia  sp.  Curvularia lunata  WJYT01KY404178100 Lopburi Lai Temple30.44  Curvularia  sp.  Curvularia verruculosa  WS1L14KT923465100 Saraburi Chantaburi Temple40.48  Neurospora  sp.  Neurospora intermedia  WS1JB14KT84466299Western part Petchaburi Ko-Kaeo-Suttharam Temple50.00  Fusarium  sp.  Fusarium solani  WSKKS11KT58139999 Yai-Suwannaram Temple57.14  Curvularia  sp.  Curvularia geniculata  WSYS11KT581398100 Ratchaburi Kongkaram Temple25.00  Fusarium  sp.  Fusarium proliferatum  WS1KK12KT581405100 Mahathat Temple20.00  Aspergillus  sp.  Aspergillus sclertoiorum  WSMT12KT581403100 Fig. 1 Phylogenetic tree of the fungi with high contamination frequency
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