THE BIOLOGICAL CONTROL OF FUNGI FROM WOODEN CULTURAL PROPERTIES

Abstract

In this study, there has been investigated the decontamination of Korean cultural artifacts by gamma irradiation. Tyromyces and Coriolus were isolated from naturally contaminated wooden artifacts stored in the warehouse of a forest museum. The identification was conducted using 18S rRNA sequence analysis. The two isolated fungi were cultivated and treated in a ^sup 60^Co irradiation unit with doses up to 20 kGy. Tyromyces and Coriolus were inactivated at a dose of 5 kGy. These results demonstrate the effectiveness of gamma irradiation for the recovery of damaged wooden artifacts, which will facilitate the preservation of important historical materials. [PUBLICATION ABSTRACT]

Headnote

ABSTRACT

In this study, there has been investigated the decontamination of Korean cultural artifacts by gamma irradiation. Tyromyces and Coriolus were isolated from naturally contaminated wooden artifacts stored in the warehouse of a forest museum. The identification was conducted using 18S rRNA sequence analysis. The two isolated fungi were cultivated and treated in a ^sup 60^Co irradiation unit with doses up to 20 kGy. Tyromyces and Coriolus were inactivated at a dose of 5 kGy. These results demonstrate the effectiveness of gamma irradiation for the recovery of damaged wooden artifacts, which will facilitate the preservation of important historical materials.

Key words: Gamma irradiation, Tyromyces, Coriolus, biological control.

INTRODUCTION

Cultural artifacts made of wood are vulnerable to biodégradation by fungi. Fungi can hydrolyze a wide variety of polymers, including cellulose and hemicellulose. Under environmental conditions favourable to their growth, cellulolytic fungi can destroy wooden materials in a short period of time. Methyl bromide has previously been used as a fumigation agent to control the fungal biodegradation of wooden cultural artifacts. However, methyl bromide was banned in 1992 based on the Montreal Protocol, due to its toxicity to humans and the environment (Da Silva et al., 2006). As a substitute for chemical treatment, gamma irradiation methods have been developed for the preservation of organic materials. Gamma irradiation is a well-known treatment for the preservation of food and sterilization of medical devices, and has been used for theses purpose for the last 50 years. Although several recent studies have described the application of gamma irradiation to control fungal growth on paper and wood products, the optimal dose of radiation for the wood products has not been specifically identified (Hanus, 1985). Thus, in this study, fungi were isolated from Korean cultural artifacts stored in a museum, and identified using 18S rRNA sequencing. Experiments were conducted to determine the irradiation dose required to inactivate these fungi.

MATERIALS AND METHODS

The cultural artifacts used in this study were traditional Korean agricultural artifacts including Hongdukkae (the wooden roller used in smoothing cloth) and Holtae (tools stripping off the grains), which have been stored in the warehouse of Wando Forest Museum (Wando, South Korea).

Strains were isolated from different positions on each artifact by using a sterilized cotton bud, and subsequently incubated on a PDA medium (potato dextrose agar; Difco Laboratories, Sparks, MD, USA) at 25°C. To identify the fungi on the artifacts, method based on DNA sequencing analysis was used in this study. Genomic DNA was isolated from the fungi by using a preparation kit (MP Biomedicals LLC, Irvine, CA, USA). To obtain 18S rDNA, PCR was performed with the 18S rRNA gene primers of ITS (Internal Transcribed Spacers) 1 (TCC GTA GGT GAA CCT GCG G) and ITS4 (TCC TCC GCT TAT TGA TAT GC) (Korabecna et al., 2003). The gene sequence of the PCR products was analyzed by comparison with previously reported 18S rDNA sequences from the National Center for Biotechnology Information (http: //blast. ncbi.nlm.nih.gov/Blast.cgi).

Fungi were irradiated by a 60cobalt irradiator (point source, AECL, IR- 79, Nordion, Canada) with doses of 0, 1, 5, 10, and 20 kGy for inactivation. The temperature of the irradiation room was controlled at 22 ± 2°C with a relative humidity of 50 ± 5%. The source strength was approximately 11.1 PBq with a dose rate of 10 kGy/h at the location of the sample. Dosimetry was carried out using alanine dosimeters (Bruker Instruments, Rheinstetten, Germany) and measured with a Bruker EMS 104 EPR Analyzer. The actual doses were within 2% of the target dose.

To assess the radiosensitMty of isolated fungi, fungi were inoculated on plastic Petri dishes with MA2% (Malt Extract Agar 2%; Difco Laboratories) and irradiated. After irradiation, the plates were incubated at 250C for 7 days. The controls were also cultivated on MA2% and were maintained in the same conditions as the treated fungi. The colony diameter was measured to verify fungal growth comparing to the controls. The fungal inactivation was reconfirmed with the inoculation of the irradiated fungi into Yeast Malt Extract Broth (Difco Laboratories) and the survival was recorded over a 10-day period at 25°C.

RESULTS AND CONCLUSION

In this study, Korean agricultural artifacts, including Hongdukkae (a wooden roller used for smoothing fabrics) and Holtae (a tool for stripping grains), were tested for fungal contamination. These historical implements were used in the late 19th century and are currently stored in a forest museum (Wando Forest Museum, Wando, South Korea). Several fungi were isolated from the artifacts; 2 most abundant strains were cultivated and their genomic DNA was prepared for sequence analysis. From the 18S rDNA sequence analyzed, the isolates were identified as species of the genera Tyromyces and Coriolus. Tyromyces and Coriolus are known to degrade cellulose and hemicellulose in wood (Xu and Goodell, 2001; Tsujiyama et al., 1998). These fungi, capable of breaking down fibers via the activity of several cellulase enzymes and of reducing sugars in wood fibers, were shown to have contaminated the cultural artifacts used in this study, despite storage of the artifacts under controlled museum conditions. Although the storage conditions did not stimulate fungal growth, long-term storage allows for biodegradation in the absence of active treatment. Counter-measures aimed at controlling biodegradation are, therefore, necessary for the preservation of such cultural artifacts.

To assess the radiosensitivity of these isolated fungi, the cultivated fungi were exposed to gamma irradiation. Growth of Coriolas and Tyromyces was measured after gamma irradiation at different doses. As shown in Fig. 1 , fungal growth was inhibited by gamma irradiation. At a dose of 1 kGy, growth of Coriolas was significantly inhibited compared to the non-irradiated control. At a dose of 5 kGy, Coriolas was completely inactivated. The inhibition of Tyromyces growth was considerably more marked compared with Coriolas at 1 kGy. As shown in Fig. 2, non-irradiated Coriolus and Tyromyces grew well, but after irradiation at 1 kGy, fungal growth was retarded and strains irradiated at doses above 5 kGy did not show any growth on the plate.

Gamma rays are used extensively for sterilizing microflora and for killing insects, particularly on organic materials. A dose of at least 500 Gy is required to kill larvae and to prevent the emergence of adult insects. Fungi are less sensitive to ionizing radiation than insects, and different strains show different levels of sensitivity. Generally, most fungi are inactivated by a dose of 10 kGy. Blank and Corrigan (1995) investigated the resistance of Penicilliam and Aspergillus spores to gamma rays, and reported that radiation doses of 0.236-0.416 and 0.209-0.319 kGy, respectively, were necessary to inactivate 90% of the initial cells. In a study investigating the use of gamma rays for controlling the biodegradation of cultural artifacts, a gamma irradiation dose of 6-15 kGy successfully inactivated fungi found in various materials, including paper, wood, and clothes (Da Silva et al., 2006). In the case of waterlogged archaeological wood, a dose of 10 kGy was sufficient to inactivate all marine and terrestrial fungi (Pointing et al., 1998).

These results confirmed that radiation treatment is extremely efficient for the control of fungi. Recently, Choi et al. (2012) reported that gamma irradiation up to an absorbed dose of 50 kGy did not cause any deleterious effects on paper. With its effectiveness at decontamination, its high penetration depth, and lack of mechanical damage to the objects on which it is used, irradiation technology can be utilized for the preservation of cultural material.

ACKNOWLEDGEMENTS

This work was co-supported by Basic Research Support Program, PaAERI, and by Korea Science and Engineering Foundation and Ministry of Education, Science and Technology.

References

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