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. 2019 Sep 5;9(10):356. doi: 10.1007/s13205-019-1879-1

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

Witsanu Senbua 1, Jesdawan Wichitwechkarn 1,✉
PMCID: PMC6728103  PMID: 31501757

Abstract

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 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 particularly filamentous fungi are of great concern. Microorganisms are known to be responsible for the biodeterioration of art materials through invasion by their growth and biochemical alteration process due to their harmful metabolic activities (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 resulting 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 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 deterioration 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 centuries. 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 example, 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 (Bastian et al. 2010), pre-historic rock paintings in India (Biswas et al. 2013), the ruins of Angkor temples in Cambodia (Bartoli 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 community, 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 identification 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. Moreover, 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, biological 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-suwannaram and Ko-Kaeo-Suttharam Temples (Petchaburi province), Mahathat and Kongkaram Temples (Ratchaburi province), Phutthaisawan, Chang Yai, Muang, and Yai Thepnimit Temples (Phra Nakhon Si Ayutthaya province), Lai Temple (Lopburi province), and Chantaburi Temple (Saraburi province). 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 temperature, 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 distilled water supplemented with 0.1% Tween 80 and shaken well before transferring to the laboratory. The samples were kept at 4 °C until use.

Table 1.

Selected archaeological sites

Archaeological site Province Location Collection date Average atmospheric temperature (oC) Average relative humidity (%) Average rainfall (mm)
Central part
 Angkaew Temple Bangkok 13°45′0″N, 100°28′0″E Dec 2010 23.8–32.5 69 22.7
 Nang-Chi Temple
 Phutthaisawan Temple Phra Nakhon Si Ayutthaya 14°20′58″N, 100°33′34″E Aug 2013 23.4–33.7 80 143.5
 Chang Yai Temple
 Muang Temple
 Yai Thepnimit Temple
 Lai Temple Lopburi 14°48′0″N, 100°37′37″E Jun 2014 26.6–35.5 73 106.2
 Chantaburi Temple Saraburi 14°31′43″N, 100°54′41″E Jun 2014 NA NA NA
Western part
 Ko-Kaeo-Suttharam Temple Petchaburi 13°06′43″N, 99°56′45″E Jul 2011 25.3–32.8 75 138.7
 Yai-Suwannaram Temple
 Kongkaram Temple Ratchaburi 13°32′8″N, 99°48′48″E Jan 2012 22.6–32.4 74 13.2
 Mahathat Temple
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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 contaminating frequency using the following formula:

%CF=igit×100

where ig is the number of isolates in each genus, it 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 microscope, 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 approximately 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 pellets 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, resuspended 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 denaturation 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 submitted to First BASE Laboratories Sdn Bdh, Malaysia, for ITS nucleotide sequencing using BigDye® Terminator 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 performed by aligning the fungal sequences with those in GenBank database (NCBI) using the BLAST search program (Zhang et al. 2000).

A phylogenetic tree was constructed from the evolutionary 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 bootstrap 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 mangostana), 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 containing each type of fungus previously grown on another PDA plate for 7 days was transferred using a cork borer to individual 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 deterioration 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 informative. From 240 fungal colonies grown on PDA–ampicillin plates, a number of filamentous fungi were isolated to pure colonies. They were subsequently morphologically identified 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, Curvularia, Penicillium, and Neurospora.

Table 2.

Identification of the isolated fungi with highest contaminating frequencies using morphological and molecular methods

Archaeological site % CFa Morphological identificationb Molecular identificationb
Identified microorganism Accession number Identity (%)
Central part
 Bangkok
  Angkaew Temple 63.64 Aspergillus fumigatus Aspergillus fumigatus WSAK10 KT581395 100
  Nang-Chi Temple 25.00 Aspergillus fumigatus Aspergillus fumigatus WSNC10 KT581396 100
25.00 Fusarium sp. Fusarium solani WS2NC10 KT581397 99
 Phra Nakhon Si Ayutthaya
  Phutthaisawan Temple 83.33 Aspergillus unguis Aspergillus unguis WJPS01 KY404172 99
  Chang Yai Temple 28.57 Curvularia sp. Curvularia lunata WJCY01 KY404177 99
  Muang Temple 34.78 Penicillium sp. Penicillium citrinum JMU01 KY404179 100
  Yai Thepnimit Temple 33.33 Curvularia sp. Curvularia lunata WJYT01 KY404178 100
 Lopburi
  Lai Temple 30.44 Curvularia sp. Curvularia verruculosa WS1L14 KT923465 100
 Saraburi
  Chantaburi Temple 40.48 Neurospora sp. Neurospora intermedia WS1JB14 KT844662 99
Western part
 Petchaburi
  Ko-Kaeo-Suttharam Temple 50.00 Fusarium sp. Fusarium solani WSKKS11 KT581399 99
  Yai-Suwannaram Temple 57.14 Curvularia sp. Curvularia geniculata WSYS11 KT581398 100
 Ratchaburi
  Kongkaram Temple 25.00 Fusarium sp. Fusarium proliferatum WS1KK12 KT581405 100
  Mahathat Temple 20.00 Aspergillus sp. Aspergillus sclertoiorum WSMT12 KT581403 100
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aContaminating frequency

bShown only for the most prevalent genera

In the central part of Thailand, there were four provinces, 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 surprising 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 Penicillium citrinum. However, these two kinds of fungi were found to be closely related to each other, according to the phylogenetic relationship displayed in Fig. 1. For the other two temples in the same province, the most prevalent fungus found at both Chang Yai and Yai Thepnimit Temples was Curvularia lunata. In Lopburi province, north of Phra Nakhon Si Ayutthaya, the main fungus found on the bas-relief at Lai Temple was also of genus Curvularia: C. verruculosa. Neurospora was found only in Saraburi province, at Chantaburi Temple, and was identified as Neurospora intermedia, which was shown to be phylogenetically closely related to Fusarium (see Fig. 1).

Fig. 1.

Fig. 1

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Phylogenetic tree of the fungi with high contamination frequency

As for the western part of the country, in the two connecting provinces Ratchaburi and Petchaburi, there were mainly three genera of Fusarium, Curvularia, and Aspergillus found on the mural paintings at different sites. Fusarium was found in both provinces, although identified as different species, F. solani and F. proliferatum. C. geniculata and A. sclertoiorum were also found in Yai-suwannaram, Petchaburi province and at Mahathat Temple, Ratchaburi province, respectively. Figure 1 is the phylogenetic tree of these fungi. According to the phylogenetic relationship, the most prevalent fungi colonizing art objects were divided into three groups: Aspergillus and Penicillium, Fusarium and Neurospora, and Curvularia. The accession numbers of the fungi were also shown in the figure.

Distribution of predominant fungi colonizing art objects in the central and western parts of Thailand

The distribution of these fungi in the central and western parts of Thailand is illustrated in Fig. 2, which is the map of Thailand showing geographical display of the 12 archaeological sites and their most prevalent fungi contaminating art objects. From the map, these sites are divided into three distinct location zones: zone a, which is Bangkok area; zone b–c–d, which connect three provinces in the central part of the country together including Phra Nakhon Si Ayutthaya, Saraburi, and Lopburi; zone e–f, which links two provinces in the western part including Ratchaburi and Petchaburi. When considering other factors shown in Table 1, the average atmospheric temperature and the average relative humidity of these areas were not drastically different even though the samples from each site were collected at different times of the year. The average relative humidity and the average rainfall were higher in Phra Nakhon Si Ayutthaya and Lopburi provinces, because it was the rainy season during the time of sample collection.

Fig. 2.

Fig. 2

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Map of Thailand showing geographical display of selected archaeological sites and their most prevalent fungi contaminating art objects

From Fig. 2, the area-based distribution of the most prevalent fungi colonizing art objects can be visualized. Aspergillus was found in all these three zones of collection sites, while Fusarium was found in Bangkok area and both provinces in the western part, Ratchaburi and Petchaburi provinces. Curvularia was found mostly in Phra Nakhon Si Ayutthaya and Lopburi provinces in zone b–c–d, and also found in one temple in Petchaburi province in zone e–f. In zone b–c–d, there were other types of fungi, Penicillium and Neurospora, found contaminating the mural paintings with highest frequencies. The fungus with highest percentage of contaminating frequency among all the archaeological sites studied was Aspergillus, which was most predominant in Phra Nakhon Si Ayutthaya province in the central part of the country. The environmental factors such as average atmospheric temperature, average relative humidity, average rainfall, and the season of sample collection, whether in winter (December or January) or the rainy season (June–August) did not seem to have any effect on the type of predominant fungal community on the art objects. Both Aspergillus and Fusarium were found in both winter and the rainy seasons and at sites with high and low average relative humidity and average rainfall. Aspergillus was found in winter at Nang-Chi and Angkaew Temples in Bangkok and Mahathat Temple in Ratchaburi where both average relative humidity and average rainfall were low, and in the rainy season at Phutthaisawan Temple in Phra Nakhon Si Ayutthaya where average atmospheric temperature, average relative humidity, and average rainfall were higher. This also holds true for Fusarium. The fact that some temples were normally closed (Angkaew, Yai Thepnimit, and Ko-Kaeo-Suttharam) and others were open to the public did not seem to have any influence upon the fungal type.

There are numerous reports on the analysis of microbial community on historic art works. Duan et al. (2018) performed a study on microbial communities on the wall paintings at Tiantishan Grottoes in China using the molecular technique and found that they were diverse among samples from different sites depending on the environment. Kim et al. (2016) morphologically and molecularly examined the airborne fungal diversity on the wooden cultural heritages in open and closed buildings in Changdeokgung Palace Complex in Seoul and a building in Namwon. They were observed to vary according to geographical and environmental factors such as the season of sample collection. Ma et al. (2015) compared the microbial communities dwelling on ancient cave wall paintings of the Mogao Grottoes in China among five different caves built during different time periods, using both culture-dependent and culture-independent methods. The fungal community diversity was found independent of environmental factors such as temperature and relative humidity, but corresponded to the building time of the caves. Biswas et al. (2013) studied the bacterial and fungal diversity colonizing on the pre-historic rock paintings of Kabra Pahad in India. Eighteen fungal species were identified, with Aspergillus as the most dominant one, including A. nigger, A. flavus, and A. fumigatus, which were considered the major factors responsible for the deterioration. Capodicasa et al. (2010) studied the microbial community colonizing on a sixteenth century painting in the medieval church of San Giacomo Maggiore in Bologna, Italy, using both culture-dependent and molecular techniques. The fungal isolates obtained were P. chrysogenum and P. namyslowskii which were demonstrated to be associated with the biodeterioration of the painting. Our work presented here is one of the pioneer studies on the analysis of fungal community dwelling on ancient art works in Thailand. With both morphological and molecular identification, the main fungal organisms colonizing on and correlating with the biodeterioration of art objects were Aspergillus, Fusarium, Curvularia, Penicillium, and Neurospora. No influence from the environmental factors such as temperature, relative humidity, and the opening or closure of the temples was noticed upon the fungal type.

Inhibition of the fungal growth by local plant extracts

In an attempt to find a safe and simple biological prevention to cope with these deteriorating fungi, certain local plant extracts: extracts from betel leaves, custard apple leaves, mangosteen peel, and guava leaves were tested for inhibition activity on the growth of prevalent fungi contaminating art works. The plant extracts were tested by incorporating them into PDA–ampicillin plates on which individual types of fungi were cultured, and the percentages of fungal growth inhibition were determined by comparing the diameters of the fungal colonies with those of the control plates. Figure 3 displays a bar chart demonstrating the percentages of fungal growth inhibition by 10,000 ppm local plant extracts. It can be seen that at this concentration, mangosteen peel extract gave the highest fungal growth inhibition for all the Curvularia tested: C. verruculosa, C. geniculata, and C. lunata, conferring 68.3%, 65.6%, and 60% inhibition, respectively. It also showed inhibition activities of 57.6% for A. fumigatus and 51.5% for F. solani. Guava leaf extract gave the highest growth inhibition at 64.4% for C. verruculosa, and 60% for C. lunata and F. solani. Both betel leaf and custard apple leaf extracts yielded the highest growth inhibition towards A. fumigatus, at 65.1% and 61.8%, respectively. Betel leaf extract also inhibited the growth of C. lunata at 52.1% and C. geniculata at 51.6%, while custard apple leaf extract inhibited the growth of C. lunata at 47.1% and P. citrinum at 45.4%.

Fig. 3.

Fig. 3

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Bar chart representing the percentages of fungal growth inhibition by local plant extracts

A. unguis did not respond well to any of these plant extracts, with guava leaves giving the highest inhibition of only about 13%. As for N. intermedia, it was difficult to assess growth inhibition by measuring the colony diameter, since Neurospora species are fungi that have broadly and rapidly spreading colonies, with abundant aerial mycelia. After only 1 day of growth inhibition by plant extracts with apparently reduced colony diameters, the colonies grew out rapidly to cover almost the whole plate area again. All in all, it can be seen that all plant extracts studied here possessed fungal growth inhibition activity, with varying effectiveness against different types of fungi. Apart from A. unguis and N. intermedia which showed no satisfactory responses to any plant extract, mangosteen peel seemed most interesting, because it conferred high inhibitory responses for almost all fungi tested.

Mangosteen is well known for its various pharmacological activities including antifungal activity as reported earlier. The fact that mangosteen peel extract gave rather high antifungal activity may arise from its important constituent, α-mangostin, which was reported to possess numerous pharmacological properties such as anti-oxidant, anti-cancer and cytotoxic, anti-inflammatory, anti-obesity, anti-parasitic, including anti-bacterial and antifungal activities (Gopalakrishnan et al. 1997; Kaomongkolgit et al. 2009; Ibrahim et al. 2016). Fungi such as A. flavus, Penicillium sp., F. roseum, and C. lunata were reported to be susceptible to α-mangostin (Sundaram et al. 1983). Betel leaves were also reported to possess antifungal activity as well as other pharmacological activities (Ali et al. 2010; Chauhan et al. 2016; Shah et al. 2016). Its major phenolic component, hydroxychavicol, was demonstrated to have antifungal activity towards many fungal strains, including Candida albicans, A. flavus, and A. fumigatus (Ali et al., 2010). There were also reports that guava leaves (Mailoa et al. 2014) and custard apple leaves (Kalidindi et al. 2015) had antifungal activity for certain types of fungi.

Future studies on higher concentrations of plant extracts and their minimum inhibitory concentrations (MICs) towards each fungus should also be determined. Other kinds of plant extracts and other potential biological materials should also be further examined for efficient and safe biological prevention. Studies have been done on fungicides and biocides (Garg et al. 1995; Maxim et al. 2012) and nontoxic natural substances such as biocompounds isolated from bacterial cultures (Rosado et al. 2017), natural essential oils (Lee et al. 2015; Veneranda et al. 2018), antibiotics and gamma irradiation (Abdel-Haliem et al. 2013; Emulai et al. 2014) for historic art work protection. Moreover, the effects of art object constituents such as tamarind seed glue and animal skin glue on fungal growth and fungal growth inhibition by biological agents should also be further studied. Unkovic et al. (2016) reported a laboratory investigation of fungal growth on model mural paintings composing of plant-based and animal-based adhesives. Emulai et al. (2014) proposed the use of antibiotic-coated paints for preventing paint deterioration and improving the paint’s shelf life. The results of antifungal activity of natural plant extracts obtained here can provide a safe, simple, non-invasive, and environmentally friendly way to protect priceless art works. It is a big challenge to apply the antifungal activity of these local plants to prevent the deterioration of art objects by fungi. Our proposed method so far would be to formulate the plant extracts in a form that could be used to coat the art objects without any harmful effect to the art work. The use of active constituents extracted and purified from these plants is another option. It still requires a series of experiments, including those involving mock paintings in the laboratory using traditional pigments and components, to investigate how to use these local plants to inhibit fungal growth on paintings efficiently and safely.

Altogether, the elucidation of the predominant fungal community colonizing works of art, together with their safe and effective biological control agents, provides initial information necessary to pave the way for appropriate preventive conservation measures for invaluable cultural heritage.

Acknowledgements

This research was financially supported by Silpakorn University Fund for Research and Creative Work (Faculty of Engineering and Industrial Technology), Thailand. The authors would like to thank the research team for performing various parts of the work. We are especially grateful to Associate Professor Dr. Krisana Houngutain (Art Theory Department, Faculty of Painting Sculpture and Graphic Arts, Silpakorn University, Thailand), a collaborator in our collaborative academic social service project on Thai art preventive conservation, for her fruitful suggestions on Thai art history and culture. We also thank Assistant Professor Dr. Eakaphun Bangyeekhun and Assistant Professor Dr. Rujikan Nasanit for their helpful scientific advice, Miss Tipaporn Subsomboon and Miss Sumarin Sinma for their technical helps, and Mr. Nutthawut Pheungphasutadol for the manuscript artwork.

Author contributions

Witsanu Senbua carried out, as a Ph.D. student, the sample collection, the isolation and identification of the fungi, PCR, the phylogenetic analysis, and assisted the research team in determining the fungal growth inhibition activity by local plant extracts. Jesdawan Wichitwechkarn was in charge, as a principal investigator, of the administration, guidance, and supervision of the research, as well as the preparation of the manuscript. All authors have read and approved the final manuscript.

Compliance with ethical standards

Conflict of interest

The authors declare that they have no conflict of interest in the publication.

References

  1. Abdel-Haliem MEF, Ali MF, Ghaly MF, Sakr AA. Efficiency of antibiotics and gamma irradiation in eliminating Streptomyces strains isolated from paintings of ancient Egyptian tombs. J Cult Herit. 2013;14:45–50. doi: 10.1016/j.culher.2012.03.009. [DOI] [Google Scholar]
  2. Abrusci C, Martín-Gonzáleza A, Amo AD, Catalina F, Collado J, Platas G. Isolation and identification of bacteria and fungi from cinematographic films. Int Biodeter Biodegr. 2005;56:58–68. doi: 10.1016/j.ibiod.2005.05.004. [DOI] [Google Scholar]
  3. Ali I, Khan FG, Suri KA, Gupta BD, Satti NK, Dutt P, Afrin F, Qazi GN, Khan IA. In vitro antifungal activity of hydroxychavicol isolated from Piper betle L. Ann Clin Microb Anti. 2010;9:7. doi: 10.1186/1476-0711-9-7. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Allsopp D, Seal KJ, Gaylarde CC. Introduction to biodeterioration. 2. Cambridge: Cambridge University Press; 2004. [Google Scholar]
  5. Barnett HL, Hunter BB. Illustrated genera of imperfect fungi. 4. New York: Macmillan; 1987. [Google Scholar]
  6. Bartoli F, Municchia AC, Futagami Y, Kashiwadani H, Moon KH, Caneva G. Biological colonization patterns on the ruins of Angkor temples (Cambodia) in the biodeterioration vs bioprotection debate. Int Biodeter Biodegr. 2014;96:157–165. doi: 10.1016/j.ibiod.2014.09.015. [DOI] [Google Scholar]
  7. Bastian F, Jurado V, Nováková A, Alabouvette C, Saiz-Jimenez C. The microbiology of Lascaux Cave. Microbiology. 2010;156:644–652. doi: 10.1099/mic.0.036160-0. [DOI] [PubMed] [Google Scholar]
  8. Bengtsson-Palme J, Ryberg M, Hartmann M, Branco S, Wang Z, Godhe A, et al. Improved software detection and extraction of ITS1 and ITS2 from ribosomal ITS sequences of fungi and other eukaryotes for analysis of environmental sequencing data. Methods Ecol Evol. 2013;4:914–919. [Google Scholar]
  9. Berner M, Wanner G, Lubitz W. A comparative study of the fungal flora present in medieval wall paintings in the chapel of the castle Herberstein and in the parish church of St Georgen in Styria, Austria. Int Biodeter Biodegr. 1997;40:53–61. doi: 10.1016/S0964-8305(97)00062-0. [DOI] [Google Scholar]
  10. Biswas J, Sharna K, Harris KK, Rajput Y. Biodeterioration agents: bacteria and fungal diversity dwelling in or on the pre-historic rock-paints of Kabra Pahad, India. Iran J Microbiol. 2013;5:309–314. [PMC free article] [PubMed] [Google Scholar]
  11. Capodicasa S, Fedi S, Porcelli AM, Zannoni D. The microbial community dwelling on a biodeteriorated 16th century painting. Int Biodeterior Biodegrad. 2010;64(8):727–733. doi: 10.1016/j.ibiod.2010.08.006. [DOI] [Google Scholar]
  12. Chauhan ES, Aishwarya J, Singh A, Tiwari A. A review: nutraceuticals properties of Piper betel (Paan) Am J Phytomed Clin Ther. 2016;4(2):28–41. [Google Scholar]
  13. Ciferri O. Microbial degradation of painting. Appl Environ Microbiol. 1999;65:879–885. doi: 10.1128/aem.65.3.879-885.1999. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Duan Y, Wu F, Wang W, Gu JD, Li Y, Feng H, Chen T, Liu G, An L. Differences of microbial community on the wall paintings preserved in situ and ex situ of the Tiantishan Grottoes, China. Int Biodeterior Biodegr. 2018;132:102–113. doi: 10.1016/j.ibiod.2018.02.013. [DOI] [Google Scholar]
  15. Emulai P, Elumalai EK, David E. Fungi associated with deteriorations of painted wall surfaces: isolation and identification. Eur J Acad Essays. 2014;1(3):48–50. [Google Scholar]
  16. Garg KL, Kamal KJ, Mishra AK. Role of fungi in the deterioration of wall paintings. Sci Total Environ. 1995;167:255–271. doi: 10.1016/0048-9697(95)04587-Q. [DOI] [Google Scholar]
  17. Gopalakrishnan G, Banumathi B, Suresh G. Evaluation of the antifungal activity of natural xanthones from Garcinia mangostana and their synthetic derivatives. J Nat Prod. 1997;60(5):519–524. doi: 10.1021/np970165u. [DOI] [PubMed] [Google Scholar]
  18. Gorbushina AA, Heyrman J, Dornieden T, Gonzalez-Delvalle M, Krumbein WE, Laiz L, Petersen K, Saiz-Jimenez C, Swings J. Bacterial and fungal diversity and biodeterioration problems in mural painting environments of St. Martins church (Greene–Kreiensen, Germany) Int Biodeter Biodegr. 2004;53:13–24. doi: 10.1016/j.ibiod.2003.07.003. [DOI] [Google Scholar]
  19. Ibrahim MY, Hashim NM, Mariod AA, Mohan S, Abdulla MA, Abdelwahab SI, Arbab IA. α-Mangostin from Garcinia mangostana Linn: an undated review of its pharmacological properties. Arab J Chem. 2016;9(3):317–329. doi: 10.1016/j.arabjc.2014.02.011. [DOI] [Google Scholar]
  20. Innis MA, Gelfand DH, Sninsky JJ, White TJ. PCR protocols: a guide to methods and applications. London: Academic Press; 1990. [Google Scholar]
  21. Kalidindi N, Thimmaiah NV, Jagadeesh NV, Nandeep R, Swetha S, Kalidindi B. Antifungal and antioxidant activities of organic and aqueous extracts of Annona squamosa Linn. Leaves. J Food Drug Anal. 2015;23:795–802. doi: 10.1016/j.jfda.2015.04.012. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Kaomongkolgit R, Jamdee K, Chaisomboon N. Antifungal activity of alpha-mangostin against Candida albicans. J Oral Sci. 2009;51(3):401–406. doi: 10.2334/josnusd.51.401. [DOI] [PubMed] [Google Scholar]
  23. Kim MJ, Shin HK, Choi YS, Kim GC, Kim GH. An aeromycological study of various wooden cultural heritages in Korea. J Cult Herit. 2016;17:123–130. doi: 10.1016/j.culher.2015.05.001. [DOI] [Google Scholar]
  24. Kimura M. A simple method for estimating evolutionary rate of base substitutions through comparative studies of nucleotide sequences. J Mol Evol. 1980;16:111–120. doi: 10.1007/BF01731581. [DOI] [PubMed] [Google Scholar]
  25. Klich MA. Identification of common Aspergillus species. Netherlands: Centraalbureau voor Schimmelcultures; 2002. [Google Scholar]
  26. Kraková L, Chovanová K, Selim SA, Šimonovičová A, Puškarová A, Maková A, Pangallo D. A multiphasic approach for investigation of the microbial diversity and its biodegradative abilities in historical paper and parchment documents. Int Biodeter Biodegr. 2012;70:117–125. doi: 10.1016/j.ibiod.2012.01.011. [DOI] [Google Scholar]
  27. Kumar S, Stecher G, Tamura K. MEGA7: molecular evolutionary genetics analysis version 7.0 for biggest databases. Mol Biol Evol. 2016;33:1870–1874. doi: 10.1093/molbev/msw054. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Lee MY, Park HM, Chung YJ. Biochemical characteristics and growth control for fungi isolated from mural painting of Tomb No. 6 at Songsan-ri, Gongju. J Conserv Sci. 2015;31(3):227–241. doi: 10.12654/JCS.2015.31.3.04. [DOI] [Google Scholar]
  29. Ma YT, Zhang H, Du Y, Tian T, Xiang T, Liu XD, Wu FS, An LZ, Wang WF, Gu JD, Feng HY. The community distribution of bacteria and fungi on ancient wall paintings of the Mogao Grottoes. Sci Rep. 2015;5:7752. doi: 10.1038/srep07752. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Mailoa MN, Mahendradatta M, Laga A, Djide N. Antimicrobial activities of tannins extract from guava leaves (Psidium guajava L) on pathogens microbial. IJSTR. 2014;3(1):236–241. [Google Scholar]
  31. Maxim D, Bucşa L, Moza MI, Chachula O. Preliminary antifungal investigation of ten biocides against moulds from two different church frescoes. Ann RSCB. 2012;XVII:139–146. [Google Scholar]
  32. Michaelsen A, Pinzari F, Ripka K, Lubitz W, Pinar G. Application of molecular techniques for identification of fungi communities colonising paper material. Int Biodeter Biodegr. 2006;58:133–141. doi: 10.1016/j.ibiod.2006.06.019. [DOI] [Google Scholar]
  33. Michaelsen A, Pinar G, Pinzari F. Molecular and microscopical investigation of the microflora inhabiting a deteriorated Italian manuscript dated from the thirteenth century. Microb Ecol. 2010;60:69–80. doi: 10.1007/s00248-010-9667-9. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Nei M, Kumar S. Molecular evolution and phylogenetics. New York: Oxford University Press; 2000. [Google Scholar]
  35. Niemeier RT, Sivasubramani SK, Reponen T, Grinshpun SA. Assessment of fungal contamination in moldy homes: comparison of different methods. J Occup Environ Hyg. 2006;3(5):262–273. doi: 10.1080/15459620600637333. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Nugari MP, Pietrini AM, Caneva G, Imperi F, Visca P. Biodeterioration of mural paintings in a rocky habitat: the crypt of the original Sin (Matera, Italy) Int Biodeter Biodegr. 2009;63:705–711. doi: 10.1016/j.ibiod.2009.03.013. [DOI] [Google Scholar]
  37. Portnoy JM, Barnes CS, Kennedy K. Sampling for indoor fungi. J Allergy Clin Immunol. 2004;113(2):189–198. doi: 10.1016/j.jaci.2003.11.021. [DOI] [PubMed] [Google Scholar]
  38. Rojas TI, Aira MJ, Batista A, Cruz IL, González S. Fungal biodeterioration in historic buildings of Havana (Cuba) Grana. 2012;51(1):44–51. doi: 10.1080/00173134.2011.643920. [DOI] [Google Scholar]
  39. Rosado T, Silva M, Dias L, Candeias A, Gil M, Mirão J, Pestana J, Caldeira AT. Microorganisms and the integrated conservation-intervention process of the renaissance mural paintings from Casas Pintadas in Évora—know to act, act to preserve. JKSUS. 2017;29:478–486. [Google Scholar]
  40. Samson RA, Hoekstra ES, Frisvad JC, Filtenborg O. Introduction to food-borne fungi. 4. Utrecht: Centraalbureau voor Schimmelcultures; 1995. [Google Scholar]
  41. Shah SK, Garg G, Jhade D, Pate N. Piper betle: phytochemical, pharmacological and nutritional value in health management. Int J Pharm Sci Rev Res. 2016;34:181–189. [Google Scholar]
  42. Sterflinger K. Fungi: their role in deterioration of cultural heritage. Fungal Biol Rev. 2010;24:47–55. doi: 10.1016/j.fbr.2010.03.003. [DOI] [Google Scholar]
  43. Sterflinger K, Piñar G. Microbial deterioration of cultural heritage and works of art-tilting at windmills? Appl Microbiol Biotechnol. 2013;97:9637–9646. doi: 10.1007/s00253-013-5283-1. [DOI] [PMC free article] [PubMed] [Google Scholar]
  44. Sundaram B, Gopalakrishnan C, Subramanian S, Shankaranarayanan D, Kameswaran L. Antimicrobial activities of Garcinia mangostana. Planta Med. 1983;48:59–60. doi: 10.1055/s-2007-969882. [DOI] [PubMed] [Google Scholar]
  45. Unković N, Grbić ML, Stupar M, Savković Ž, Jelikić A, Stanojević D, Vukojević J. Fungal-induced deterioration of mural paintings: in situ and mock-model microscopy analyses. Microsc Microanal. 2016;22:410–421. doi: 10.1017/S1431927616000544. [DOI] [PubMed] [Google Scholar]
  46. Veneranda M, Blanco-Zubiaguirre L, Roselli G, Girolami GD, Castro K, Madariaga JM. Evaluating the exploitability of several essential oils constituents as a novel biological treatment against cultural heritage biocolonization. Microchem J. 2018;138:1–6. doi: 10.1016/j.microc.2017.12.019. [DOI] [Google Scholar]
  47. White TJ, Bruns T, Lee S, Taylor J. Amplification and direct sequencing of fungal ribosomal RNA genes for phylogenetics. In: Innis MA, Gelfand DH, Sninsky JJ, White TJ, editors. PCR protocols-A guide to methods and applications. London: Academic Press; 1990. [Google Scholar]
  48. Zhang Z, Schwartz S, Wagner L, Miller W. A greedy algorithm for aligning DNA sequences. J Comput Biol. 2000;7:203–214. doi: 10.1089/10665270050081478. [DOI] [PubMed] [Google Scholar]
  49. Zucconi L, Gagliardi M, Isola D, Onofri S, Crocifissa M, Andaloro MC, Pelosi C, Pogliani P, Selbmann L. Biodeterioration agents dwelling in or on the wall paintings of the Holy Saviour’s cave (Vallerano, Italy) Int Biodeter Biodegr. 2012;70:40–46. doi: 10.1016/j.ibiod.2011.11.018. [DOI] [Google Scholar]

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