Kitora Tumulus Research Articles

UNIFIED ANALYSIS OF OBSERVATION DATES FOR ANCIENT STAR MAPS AND CATALOGUES IN ASIA

It is an intriguing subject in the history of astronomy to determine scientifically observation dates of stars recorded in ancient star maps and catalogues of East Asia. Most of previous studies used leastsquares fitting as a function of time between observation and calculation for a group of selected stars, and they simply took the resulting mean residuals as the dating errors. In 2014, the author of this paper was given an opportunity to perform a dating analysis of a star map drawn on the ceiling of the stone room of the Kitora tumulus discovered in 1998, located at Nara in Japan. The construction time of the tumulus had been inferred by archaeologists to be around the end of the 7th century. The main difficulty in treating stellar coordinates of Chinese origin was that their longitudes had not been measured from a common cardinal point, thus preventing us from directly using the precession theory. After trial-and-error experiments, we developed a method for analyzing the observation epoch of stars exclusively using the 28-Xiu constellations based on an interval estimation in modern statistics. This method is applicable to many historical star maps and catalogues in a unified way. Furthermore, to reduce the error range of dating, the bootstrap method for small sample sizes was introduced. As a result, we obtained 81 BC ± 42 years (for a 90% confidence level) as the observation date of the Kitora star map. The same procedure was applied, with successful outcomes, to data in the Shi shi xingjing, the Almagest catalogue, the famous stone-inscribed Suzhou tianwentu, the 28-Xiu observations by Guo Shoujing, the Japanese paper star map Koshi Gesshin-zu, and Ulugh Beg' s star catalogue. Thus, it is certain that our approach can be effectively used for other old star maps and catalogues yet unexplored.

Two new Cladophialophora species, C. tumbae sp. nov. and C. tumulicola sp. nov., and chaetothyrialean fungi from biodeteriorated samples in the Takamatsuzuka and Kitora Tumuli

During dismantling and relocation of the Takamatsuzuka Tumulus stone chamber, many Cladophialophora and chaetothyrialean black fungi, such as Exophiala and Phialophora, were isolated from samples taken from the joints between the stone walls. However, inside the stone chamber of the Kitora Tumulus, after intermittent UV irradiation in 2009, these black fungi were also isolated from samples taken from the stone walls. Molecular phylogenetic analyses based on only nrLSU and the concatenated (nrLSU D1/D2 + ITS) sequences revealed that the 35 Takamatsuzuka and Kitora Tumuli isolates of Cladophialophora and the chaetothyrialean black fungi were divergent. Two new species of Cladophialophora are described herein: C. tumulicola from the viscous gels and various substrates on the stone walls of the Takamatsuzuka and Kitora Tumuli and C. tumbae from black substances on the plastic cover over the “thief hole,” soil and plaster pieces between the stone walls, and the exterior of the Takamatsuzuka Tumulus chamber. Also, molecular phylogenetic placements for the remaining eight Takamatsuzuka and Kitora Tumuli isolates of chaetothyrialean black fungi have been determined or suggested.

Microbacterium tumbae sp. nov., an actinobacterium isolated from the stone chamber of ancient tumulus

Eight strains characterised as Gram-stain-positive, non-spore-forming and non-motile rods were isolated from samples collected from stone chambers of the Takamatsuzuka and Kitora tumuli in Asuka village, Nara Prefecture, Japan. Among them, one strain, T7528-3-6bT, was shown to form a novel lineage within the genus Microbacterium. The most closely phylogenetically related species to T7528-3-6bT was Microbacterium panaciterrae, with 97.8 % sequence similarity. The major isoprenoid quinones of T7528-3-6bT were MK-12, MK-13 and MK-11. The predominant cellular fatty acids for this isolate were anteiso-C15 : 0, anteiso-C17 : 0, iso-C16 : 0 and iso-C15 : 0. The diagnostic diamino acid of the peptidoglycan of this isolate was ornithine. Major polar lipids of the isolate were phosphatidylglycerol, diphosphatidylglycerol and an unknown glycolipid. The G+C content of the genomic DNA of this isolate was 70.1 mol%. On the basis of the results of physiological, biochemical and chemotaxonomic tests and molecular phylogenetic analysis, T7528-3-6bT is considered to represent a novel species of the genus Microbacterium, for which the name M. tumbae sp. nov. has been proposed. The type strain is T7528-3-6bT (=JCM 28836T=NCIMB 15039T). The results of comparisons of both phenotypic and genotypic (16S rRNA gene sequence) characteristics indicated that the remaining seven isolates were very closely related to Microbacterium shaanxiense. Although the sequence similarity between the two was 99.2 %, further detailed multifaceted comparisons are needed to determine their accurate taxonomic assignment.

Noteworthy anamorphic fungi, Cephalotrichum verrucisporum, Sagenomella striatispora, and Sagenomella griseoviridis, isolated from biodeteriorated samples in the Takamatsuzuka and Kitora Tumuli, Nara, Japan

Phenotypic and genotypic characteristics of five isolates of Cephalotrichum and Sagenomella from various samples collected while dismantling the Takamatsuzuka Tumulus stone chamber were found to represent Cephalotrichum verrucisporum and Sagenomella striatispora. In addition to these, one isolate from the ceiling stone surface in the adjacent small room of the Kitora Tumulus was assignable to Sagenomella griseoviridis. Colonization on biodeteriorated plaster and stone walls as substrata or habitats of these fungi is noteworthy. Physiologically Sagenomella isolates solubilized CaCO3 and may be involved in the deterioration of the plaster walls inside the stone chamber. Cephalotrichum verrucisporum is a new addition to Japan.

Polyphasic insights into the microbiomes of the Takamatsuzuka Tumulus and Kitora Tumulus.

Microbial outbreaks and related biodeterioration problems have affected the 1300-year-old multicolor (polychrome) mural paintings of the special historic sites Takamatsuzuka Tumulus (TT) and Kitora Tumulus (KT). Those of TT are designated as a national treasure. The microbiomes of these tumuli, both located in Asuka village, Nara, Japan, are critically reviewed as the central subject of this report. Using culture-dependent methods (conventional isolation and cultivation), we conducted polyphasic studies of the these microbial communities and identified the major microbial colonizers (Fusarium spp., Trichoderma spp., Penicillium spp., dark Acremonium spp., novel Candida yeast spp., Bacillus spp., Ochrobactrum spp., Stenotrophomonas tumulicola, and a few actinobacterial genera) and noteworthy microbial members (Kendrickiella phycomyces, Cephalotrichum verrucisporum (≡Doratomyces verrucisporus), Sagenomella striatispora, Sagenomella griseoviridis, two novel Cladophialophora spp., Burgoa anomala, one novel species Prototheca tumulicola, five novel Gluconacetobacter spp., three novel Bordetella spp., and one novel genus and species Krasilnikoviella muralis) involved in the biodeterioration of mural paintings, plaster walls, and stone chamber interiors. In addition, we generated microbial community data from TT and KT samples using culture-independent methods (molecular biological methods, including PCR-DGGE, clone libraries, and pyrosequence analysis). These data are comprehensively presented, in contrast to those derived from culture-dependent methods. Furthermore, the microbial communities detected using both methods are analytically compared, and, as a result, the complementary roles of these methods and approaches are highlighted. In related contexts, knowledge of similar biodeterioration problems affecting other prehistoric cave paintings, mainly at Lascaux in France and Altamira in Spain, are referred to and commented upon. Based on substrate preferences (or ecological grouping) and mapping (plotting detection sites of isolates), we speculate on the possible origins and invasion routes whereby the major microbial colonizers invaded the TT stone chamber interior. Finally, concluding remarks, lessons, and future perspectives based on our microbiological surveys of these ancient tumuli, and similar treasures outside of Japan, are briefly presented. A list of the microbial taxa that have been identified and fully or briefly described by us as known and novel taxa for TT and KT isolates since 2008 is presented in Supplementary Materials.

Yamadazyma kitorensis f.a., sp. nov. and Zygoascus biomembranicola f.a., sp. nov., novel yeasts from the stone chamber interior of the Kitora tumulus, and five novel combinations in Yamadazyma and Zygoascus for species of Candida.

Analysis of D1/D2 large-subunit (LSU) rRNA gene sequences predicted that 17 yeast isolates, mainly from viscous gels (biofilms) taken from the stone chamber interior of the Kitora tumulus in Nara, Japan, were placed in the Yamadazyma and Zygoascus clades. Polyphasic characterization, including morphological, physiological and chemotaxonomic characteristics, multigene sequence divergence and DNA-DNA hybridization, strongly suggested the assignment of one novel species to each of the clades; these are Yamadazyma kitorensis f.a., sp. nov., with the type strain JCM 31005T (ex-type CBS 14158T=isolate K8617-6-8T), and Zygoascus biomembranicola f.a., sp. nov., with the type strain JCM 31007T (ex-type CBS 14157T=isolate K61208-2-11T). Furthermore, the transfer of five known species of the genus Candida as novel combinations to the genera Yamadazyma and Zygoascus is proposed; these are Yamadazyma olivae f.a., comb. nov. (type strain CBS 11171T=ATCC MYA-4568T), Yamadazyma tumulicola f.a., comb. nov. (type strain JCM 15403T=ex-type CBS 10917T=isolate T6517-9-5T), Yamadazyma takamatsuzukensis f.a., comb. nov. (type strain JCM 15410T=CBS 10916T = isolate T4922-1-1T), Zygoascus polysorbophila f.a., comb. nov. (type strain NRRL Y-27161T=CBS 7317T) and Zygoascus bituminiphila f.a., comb. nov. (type strain CBS 8813T=MUCL 41424T).

Stenotrophomonas tumulicola sp. nov., a major contaminant of the stone chamber interior in the Takamatsuzuka Tumulus.

During investigation of the biological contamination of the 1300-year-old mural paintings and plaster walls inside the stone chambers of the Takamatsuzuka and Kitora Tumuli (TT and KT) in Asuka-mura, Nara Prefecture, Japan, the identity of 17 bacterial isolates from blackish mouldy spots and viscous gels (biofilms) collected from both tumuli (16 isolates from TT and one from KT) during our 2005-2007 microbiological survey was systematically elucidated. One cluster of the major bacterial isolates was assigned to the genus Stenotrophomonas (class Gammaproteobacteria) by phylogenetic analysis of the 16S rRNA gene sequences. These isolates were divided into two groups A and B. Group A comprised 15 TT isolates that took a phylogenetic position near Stenotrophomonas chelatiphaga LPM-5T. Based on our analysis of the phenotypic (cultural, morphological, physiological and chemotaxonomic) characteristics and genotypic/molecular characteristics (DNA base composition, DNA-DNA relatedness, and 16S rRNA and gyrB gene sequences), the novel species name Stenotrophomonas tumulicola sp. nov. is proposed for the group A isolates with the type strain T5916-2-1bT ( = JCM 30961T = NCIMB 15009T). Group B, which contained only one TT and one KT isolate, was closely related to [Pseudomonas] geniculata, [P.] hibiscicola, [P.] beteli, Stenotrophomonas maltophilia and Stenotrophomonas pavanii. The two isolates were genotypically and phenotypically assignable to S. maltophilia.

“Black particles”, the major colonizers on the ceiling stone of the stone chamber interior of the Kitora Tumulus, Japan, are the bulbilliferous basidiomycete fungus Burgoa anomala

Integrated analysis of phenotypic and genotypic characteristics of 20 isolates of “black particles” (“Kuro-tsubu” in Japanese), that mainly appeared on the plaster wall of the ceiling tuff stone surface of the Kitora Tumulus stone chamber interior in Asuka village, Nara, Japan, has identified them as the bulbilliferous basidiomycetous anamorphic fungus Burgoa anomala. Our 28S rRNA gene D1/D2 (28S) and internal transcribed spacer (ITS) regions sequence-based phylogenies indicated that the 20 isolates displayed no genetic divergence and firmly clustered with the authentic strain B. anomala CBS 130.38. These isolates and B. anomala CBS 130.38 grouped together with species of the teleomorphic genus Sistotrema and its allies in the Cantharelloid clade. Colonization of biodeteriorated murals, plaster, and stone walls as the substrata or habitats by this fungus colonized, are noteworthy. Additionally, B. anomala is recorded for the first time from Japan.

Gluconacetobacter tumulisoli sp. nov., Gluconacetobacter takamatsuzukensis sp. nov. and Gluconacetobacter aggeris sp. nov., isolated from Takamatsuzuka Tumulus samples before and during the dismantling work in 2007

Ten strains of Gram-stain-negative, rod-shaped, non-spore-forming bacteria were isolated from the burial mound soil collected before the dismantling and samples collected during the dismantling work on the Takamatsuzuka Tumulus in Asuka village, Nara Prefecture, Japan in 2007. On the basis of the 16S rRNA gene sequence analysis of the isolates, they were accommodated in the genus Gluconacetobacter (class Alphaproteobacteria) and can be separated into four groups within the cluster containing the genus Gluconacetobacter. One of the groups demonstrated a phylogenetic position identical to that of Gluconacetobacter asukensis, which was isolated from small holes on plaster walls of the stone chamber interior of Kitora Tumulus in Asuka village, Nara Prefecture, Japan. The remaining three groups consisted of novel lineages within the genus Gluconacetobacter. A total of four isolates were selected from each group and carefully identified using a polyphasic approach. The isolates were characterized on the basis of their possessing Q-10 as the major ubiquinone system and C18 : 1ω7c (58.5-65.2 %) as the predominant fatty acid. A DNA-DNA hybridization test was used to determine that the three lineages represented novel species, for which the names Gluconacetobacter tumulisoli sp. nov., Gluconacetobacter takamatsuzukensis sp. nov. and Gluconacetobacter aggeris sp. nov. are proposed. The type strains are T611xx-1-4a(T) ( = JCM 19097(T) = NCIMB 14861(T)), T61213-20-1a(T) ( = JCM 19094(T) = NCIMB 14859(T)) and T6203-4-1a(T) ( = JCM 19092(T) = NCIMB 14860(T)), respectively.

Investigation of acetic acid bacteria isolated from the Kitora tumulus in Japan and their involvement in the deterioration of the plaster of the mural paintings

The Kitora tumulus, which is thought to have been built around the late seventh to early eighth century, has beautiful mural paintings which were executed directly on a very thin layer of plaster in the stone chamber. When the paintings were found, the plaster was partly detached from the stone wall and the murals were therefore considered to be likely to fall off the wall with a casual touch or stimulation. Therefore, it was decided to detach the mural paintings and to store them flat and under controlled environmental conditions. This was initiated immediately after the excavation of the tumulus in 2004. However, fungal growth and biofilm development were observed within the stone chamber during the relocation work. In 2005, small holes containing black substances were observed on certain areas of the ceiling plaster, and following investigation an acetic acid bacterium, Gluconacetobacter sp., was isolated from the black substances. The bacterium was also isolated from the ceiling, floor, and east wall in the stone chamber in 2008 after the relocation of most of the paintings had been completed. These bacteria were shown to decompose calcium carbonate (CaCO3), one of the primary components of the plaster, and to produce organic acids such as acetic acid. Additionally, they were observed to decrease the pH of the culture media significantly in the presence of ethanol and glucose. This is the first example of the characterization of acetic acid bacteria isolated from decayed plaster paintings, and it is likely that microbes such as these bacteria have been involved in the deterioration of the plaster. Chemicals to treat microbes in the Kitora tumulus during the relocation work were selected on the basis of their antimicrobial efficacy, low potential to cause adverse effects on the paintings, and low level of toxicity to humans, depending on the condition of the plaster or stone in each area. However, some chemicals, especially ethanol, may act as a carbon source, which could encourage the growth of microbes and thereby the production of acids by the microbes when diluted to a low concentration or in a degraded state. Moreover, prior contamination by other microbial species in the form of a biofilm could also encourage the growth of the acetic acid bacteria by providing low-molecular-weight organic materials as a nutrient source.

Gluconacetobacter tumulicola sp. nov. and Gluconacetobacter asukensis sp. nov., isolated from the stone chamber interior of the Kitora Tumulus

Six Gram-negative, rod-shaped, non-spore-forming bacterial strains were isolated from small holes on plaster walls of the stone chamber interior of the Kitora Tumulus in Asuka village, Nara Prefecture, Japan. These were investigated by means of a polyphasic approach. All the isolates were strictly aerobic and motile by peritrichous flagella. Phylogenetic trees generated based on 16S rRNA gene sequences identified two novel lineages (comprising five isolates and one isolate, respectively) within the genus Gluconacetobacter. The isolates were characterized by having Q-10 as the major ubiquinone system and C(18:1)ω7c (58.7-63.1% of the total) as the predominant fatty acid. DNA-DNA hybridization experiments endorsed the species rank for the two lineages, for which the names Gluconacetobacter tumulicola sp. nov. (type strain K5929-2-1b(T) = JCM 17774(T) = NCIMB 14760(T)) and Gluconacetobacter asukensis sp. nov. (type strain K8617-1-1b(T) = JCM 17772(T) = NCIMB 14759(T)) are proposed.

Bristle-like fungal colonizers on the stone walls of the Kitora and Takamatsuzuka Tumuli are identified as Kendrickiella phycomyces

Using an integrated analysis of phenotypic and genotypic characterizations, a total of 18 isolates of “bristle-like” fungal colonizers of the Kitora and Takamatsuzuka Tumuli, which had been provisionally identified as Phialocephala phycomyces, were here determined to be Kendrickiella phycomyces (Auersw.) K. Jacobs & M. J. Wingf. The 18 isolates consisted of 10 from stone surfaces or viscous gels (biofilms) of the stone chamber interior and adjacent small room, and air in the adjacent small room of the Kitora Tumulus, and 8 from viscous gels on the stone surfaces of the stone chamber interior, plant roots, and soil in the adjacent space or stone wall interspaces (interstices) in the stone chamber of the Takamatsuzuka Tumulus. Plaster and stone walls in both tumuli were recorded as novel substrates of this fungus. Our 18S sequence-based phylogeny indicated that K. phycomyces and species of the leotiomycetous anamorph genera Chaetomella, Pilidium, Sphaerographium, and Synchaetomella formed a monophyletic lineage distant from the core taxa of the Leotiomycetes (Pezizomycotina, Ascomycota). The relationship between the physicochemical characteristics of these isolates on GYC agar plates, i.e., soluble brownish pigments and dissolution of calcium carbonate (CaCO3), and the biodeterioration of the plaster and plaster walls of both tumuli, are briefly discussed.

Bristle-like fungal colonizers on the stone walls of the Kitora and Takamatsuzuka Tumuli are identified as <i>Kendrickiella phycomyces</i>

Using an integrated analysis of phenotypic and genotypic characterizations, a total of 18 isolates of “bristle-like” fungal colonizers of the Kitora and Takamatsuzuka Tumuli, which had been provisionally identified as Phialocephala phycomyces, were here determined to be Kendrickiella phycomyces (Auersw.) K. Jacobs & M. J. Wingf. The 18 isolates consisted of 10 from stone surfaces or viscous gels (biofilms) of the stone chamber interior and adjacent small room, and air in the adjacent small room of the Kitora Tumulus, and 8 from viscous gels on the stone surfaces of the stone chamber interior, plant roots, and soil in the adjacent space or stone wall interspaces (interstices) in the stone chamber of the Takamatsuzuka Tumulus. Plaster and stone walls in both tumuli were recorded as novel substrates of this fungus. Our 18S sequence-based phylogeny indicated that K. phycomyces and species of the leotiomycetous anamorph genera Chaetomella, Pilidium, Sphaerographium, and Synchaetomella formed a monophyletic lineage distant from the core taxa of the Leotiomycetes (Pezizomycotina, Ascomycota). The relationship between the physicochemical characteristics of these isolates on GYC agar plates, i.e., soluble brownish pigments and dissolution of calcium carbonate (CaCO3), and the biodeterioration of the plaster and plaster walls of both tumuli, are briefly discussed.

Molecular assessment of fungi in “black spots” that deface murals in the Takamatsuzuka and Kitora Tumuli in Japan: <i>Acremonium</i> sect. <i>Gliomastix</i> including <i>Acremonium tumulicola</i> sp. nov. and <i>Acremonium felinum</i> comb. nov

Unidentified black spots (or stains) appeared on the plaster walls of the Takamatsuzuka and Kitora Tumuli in the village of Asuka, Nara Prefecture, Japan. Public attention was drawn to the biodeterioration of the colorful 1,300-year-old murals. A total of 46 isolates of Acremonium sect. Gliomastix were obtained from various samples (mainly black spots) of the Takamatsuzuka Tumulus (TT) (sampling period, May 2004–December 2006) and the Kitora Tumulus (KT) (June 2004–May 2007). These isolates were assignable to four known taxa and a new species in the ‘series Murorum’ sensu W. Gams as inferred from the integrated analysis of phenotypic and genotypic (i.e., ITS and 28S rDNA-D1/D2 sequences) characters: these were Acremonium masseei, A. murorum, A. felinum comb. nov. with the neotype designation, A. polychromum, and A. tumulicola sp. nov., which have been accommodated in the validated series Murorum in the section Gliomastix. The black spots on the murals of the TT and KT were caused mainly by A. masseei and A. murorum, respectively.

Molecular assessment of fungi in “black spots” that deface murals in the Takamatsuzuka and Kitora Tumuli in Japan: Acremonium sect. Gliomastix including Acremonium tumulicola sp. nov. and Acremonium felinum comb. nov

Unidentified black spots (or stains) appeared on the plaster walls of the Takamatsuzuka and Kitora Tumuli in the village of Asuka, Nara Prefecture, Japan. Public attention was drawn to the biodeterioration of the colorful 1,300-year-old murals. A total of 46 isolates of Acremonium sect. Gliomastix were obtained from various samples (mainly black spots) of the Takamatsuzuka Tumulus (TT) (sampling period, May 2004–December 2006) and the Kitora Tumulus (KT) (June 2004–May 2007). These isolates were assignable to four known taxa and a new species in the ‘series Murorum’ sensu W. Gams as inferred from the integrated analysis of phenotypic and genotypic (i.e., ITS and 28S rDNA-D1/D2 sequences) characters: these were Acremonium masseei, A. murorum, A. felinum comb. nov. with the neotype designation, A. polychromum, and A. tumulicola sp. nov., which have been accommodated in the validated series Murorum in the section Gliomastix. The black spots on the murals of the TT and KT were caused mainly by A. masseei and A. murorum, respectively.

The identity of Penicillium sp. 1, a major contaminant of the stone chambers in the Takamatsuzuka and Kitora Tumuli in Japan, is Penicillium paneum

Penicillium appeared as the major dweller in the Takamatsuzuka Tumulus (TT) and Kitora Tumulus (KT) stone chambers, both located in the village of Asuka, Nara Prefecture, in relation to the biodeterioration of the 1,300-year-old mural paintings, plaster walls and ceilings. Of 662 Penicillium isolates from 373 samples of the TT (sampling period, May 2004-2007) and the KT (sampling period, June 2004-Sep 2007), 181 were phenotypically assigned as Penicillium sp. 1 which shared similar phenotypic characteristics of sect. Roqueforti in Penicillium subg. Penicillium. Fifteen representative isolates of Penicillium sp. 1, 13 from TT and 2 from KT, were selected for molecular phylogenetic analysis. The 28S rDNA D1/D2, ITS, beta-tubulin, and lys2 gene sequence-based phylogenies clearly demonstrated that the three known species P. roqueforti, P. carneum and P. paneum in sect. Roqueforti, and all TT and KT isolates grouped together. In addition to this, TT and KT isolates formed a monophyletic group with the ex-holotype strain CBS 101032 of P. paneum Frisvad with very strong bootstrap supports. So far, P. paneum has been isolated only from mouldy rye breads, other foods, and baled grass silage. Therefore, this is the first report of P. paneum isolation from samples relating to the biodeteriorated cultural properties such as mural paintings on plaster walls.

Mycobiota of the Takamatsuzuka and Kitora Tumuli in Japan, focusing on the molecular phylogenetic diversity of Fusarium and Trichoderma

In an effort to clarify the cause of the deterioration of the colorfully painted murals that adorn the inner walls of the small stone chambers in the Takamatsuzuka and Kitora Tumuli in Japan, we enumerated the fungi that were isolated from moldy spots on the plaster walls collected between May 2004 and April 2005. The 262 fungal isolates from 79 samples of both tumuli were identified as approximately 100 species based on their phenotypic characters. Fusarium, Trichoderma, and Penicillium species were the predominant colonizers in the stone chamber interior and adjacent areas of both tumuli. In addition to the 28S phylogeny, neighbor-joining and Bayesian phylogenies of partial EF-1-alpha gene sequences revealed 24 genetically diverse fusaria in the Takamatsuzuka and Kitora Tumuli. Most of the fusaria were nested in clade 3 of the Fusarium solani species complex (FSSC); however, a few isolates were members of the F. oxysporum species complex (FOSC) clade or the F. avenaceum/F. tricinctum species complex clade. The FSSC isolates were compared with those detected in the Lascaux cave in France. In addition, a partial EF-1α gene phylogeny indicated that 13 Trichoderma isolates clustered in the Harzianum-Virens clade and 5 isolates in the Viride clade or Trichoderma sect. Longibrachiatum. Our analyses suggest that most of the fungi recovered from both tumuli are typically soil dwellers.

高松塚・キトラ両古墳石室内壁画面に発生した"黒いしみ"の正体

高松塚・キトラ両古墳石室内壁画面に発生した"黒いしみ"の正体

Mycobiota of the Takamatsuzuka and Kitora Tumuli in Japan, focusing on the molecular phylogenetic diversity of <i>Fusarium</i> and <i>Trichoderma</i>

In an effort to clarify the cause of the deterioration of the colorfully painted murals that adorn the inner walls of the small stone chambers in the Takamatsuzuka and Kitora Tumuli in Japan, we enumerated the fungi that were isolated from moldy spots on the plaster walls collected between May 2004 and April 2005. The 262 fungal isolates from 79 samples of both tumuli were identified as approximately 100 species based on their phenotypic characters. Fusarium, Trichoderma, and Penicillium species were the predominant colonizers in the stone chamber interior and adjacent areas of both tumuli. In addition to the 28S phylogeny, neighbor-joining and Bayesian phylogenies of partial EF-1-alpha gene sequences revealed 24 genetically diverse fusaria in the Takamatsuzuka and Kitora Tumuli. Most of the fusaria were nested in clade 3 of the Fusarium solani species complex (FSSC); however, a few isolates were members of the F. oxysporum species complex (FOSC) clade or the F. avenaceum/F. tricinctum species complex clade. The FSSC isolates were compared with those detected in the Lascaux cave in France. In addition, a partial EF-1α gene phylogeny indicated that 13 Trichoderma isolates clustered in the Harzianum-Virens clade and 5 isolates in the Viride clade or Trichoderma sect. Longibrachiatum. Our analyses suggest that most of the fungi recovered from both tumuli are typically soil dwellers.