Molecular Identification of Arbuscular Mycorrhizal Fungi Colonizing Athyrium Yokoscense of the Ikuno Mine Site, Japan

J. Jpn. Bot. 86: 73–81 (2011)

Molecular Identification of Arbuscular Mycorrhizal Fungi Colonizing Athyrium yokoscense of the Ikuno Mine Site, Japan

a a b Nobuhito Nonomura , Yuki Kawada , Yukio Minamiya , Hiroshi

b a, a a Hayakawa , Tatsuya Fukuda *, Yumei Kang and Katsutoshi Sakurai

aFaculty of Agriculture, Kochi University, Monobe, Nankoku, 783-8502 JAPAN; bThe United Graduate School of Agricultural Sciences, Ehime University, Monobe, Nankoku. 783-8502 JAPAN *Correspondence author: [email protected]

(Accepted on February 12, 2011)

The arbuscular mycorrhizal (AM) fungi of Athyrium yokoscense was investigated in the Ikuno mine site of Japan, where a silver mine was active until 1973. We sampled four localities in the Ikuno mine site to identify the AM fungi on At. yokoscense using molecular phylogenetic analysis of the 18S ribosomal DNA. These analyses indicated that at least four types of AM fungi colonized the root of At. yokoscense in the Ikuno mine site. Among AM fungi, we found Acaulospora species from the high arsenic (As) areas of the Ikuno mine site and non-Acaulospora species from other localities. It is possible to identify the AM fungi of At. yokoscense by using this method.

Key Words: 18S ribosomal DNA (rDNA), arbuscular mycorrhizal fungi, Athyrium yokoscense, phylogeny, rbcL.

Phytoremediation is one of most innovative fern Athyrium yokoscense, which is native to technologies that utilize the natural properties the Far East including Japan, often flourishes of plants to remediate hazardous waste sites in mine sites and areas that are highly polluted (Salt et al. 1998, Krämer and Chardonnens with heavy metals. Moreover, it is well known 2001, McGrath and Zhao 2003). For more cost- that the fern accumulates a large amount of effective phytoremediation, hyperaccumulators metals in the tissues, particularly in the roots play an important role of phytoremediation (Nishizono et al. 1987). Nishizono et al. (1988, of heavy metals and it is an attractive option 1989) studied the mechanisms involved in metal to utilize a hyperaccumulating plant after detoxification and accumulation of this fern, and phytoremediation such as the recovery of suggested that a 9.5-kDa cysteine-rich peptide, valuable metals and the production of useful which was induced by the exposure of the fern materials (Baker et al. 2000). to copper, may contribute to the copper-tolerance In Europe and North America, many studies of the fern growing in copper-contaminated have been conducted to find more effective soil. In addition, Xian (1990) reported that the plants for phytoremediation of various pollutants compartmentalization of excess metals in the (e.g., Tarnau et al. 2001). In Japan, on the root cell wall has been believed to be important other hand, studies have been published only for the metal tolerance and accumulation of At. relatively recently (e.g., Saito et al. 2004). The yokoscense.

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The bacteria and fungi living in the the internal transcribed spacer (ITS) region, rhizosphere of these plants also may play suggesting that molecular data are an effective an important role in phytoremediation, but tool to infer AM fungi and the combining with relatively few studies have focused on the morphological data and molecular ones is the effects of these microorganisms on the metal best to identify them. remediation efforts (Pawlowska and Charvat The Ikuno mine site (Fig. 1) was one of the 2004, Vidas et al. 2005, Regvar et al. 2006, most famous mines of Japan and active more Wu et al. 2006). Among them, arbuscular than four hundred years, the operation is stopped mycorrhizal (AM) fungi can form symbiotic now. Mizuno and Fujimura (2003) reported that relationships with the vast majority of land this area had a deep environmental impact on plants including Athyrium ferns (Lee et al. 2001, soil quality of farms downstream, indicating that Zhang et al. 2004, Matsuda et al. 2005), and are the removal or reduction of the existing pollution known to benefit the phosphorus nutrition of hazards is still urgently required including host plants by increasing phosphorus acquisition remediation programs. Therefore, we identified (Smith et al. 2003). Although there are some the AM fungi on At. yokoscense using molecular studies concerned with symbiotic relationships phylogenetic approaches. between AM fungi and ferns, only one study has been conducted on At. yokoscense (Matsuda et Materials and Methods al. 2005). Sampling As for the identification of AM fungi, We collected Athyrium yokoscense from Gerdemann (1965) indicated that it is difficult the Ikuno mine sites of Hyogo Prefecture of to identify AM fungi to species using various Japan in July 2008. We designated the following morphological approaches. Moreover, four localities; Material, Kanamori, Keiju and identification of AM fungi spores holds Daimaru. These localities are shown in Fig. 1. difficulties due to alterations of morphological features during spore ontogeny (Morton Measurement of heavy metal concentration 1993). Additionally, diversity assessments in soils of AM fungi are debatable due to the lack of To compare heavy metal concentration correlation between root colonization and spores in soils of the Ikuno mine site, we collected (Clapp et al. 1995, Renker et al. 2005, Hempel soil samples from all localities. Soils were air- et al. 2007). Recent advances in molecular dried for 7 days before being powdered. For biology and their application have enabled the analysis of concentrations of Cadmium (Cd), exploration of the extent and distribution of Lead (Pb), and Zinc (Zn), the soil samples were genetic variation among or within particular microwave-digested in a concentrated HNO3 taxa. Some studies have managed to identify and HF. Determinations of Cd, Pb, and Zn were AM fungi using molecular phylogenetic conducted by atomic adsorption spectrometry approaches. For example, Whitfield et al. (2004) (AA-6800, Shimadzu, Kyoto, Japan). For indicated that Glomus is the predominant analysis of concentrations of As and P, the soil AM genus colonizing in Thymus polytrichus samples were digested in a concentrated HNO3, (Lamiaceae) by using restriction fragment length HClO4, HF and 2% KMnO4. Determinations polymorphism (RFLP) and DNA sequencing of of As and P were conducted by ICP-Atomic 18S ribosomal DNA (rDNA), moreover, Zarei et Emission Spectrometry (ICPS-1000IV, al. (2008) also reported that the root of Veronica Shimadzu, Kyoto, Japan). rechingeri (Scrophulariaceae) was colonized by some Glomus species by sequencing of April 2011 Journal of Japanese Botany Vol. 86 No.2 75

Fig. 1. Position of the Ikuno mine site including four localities in the Ikuno mine site and localities of Athyrium yokoscense used in phylogenetic analysis. The black box indicates the Ikuno mine site and black circles indicate localities of Athyrium yokoscense.

DNA extraction and sequencing analyses characterized and sequenced using a BigDye Total DNAs were isolated from approximately Terminator ver. 3.1 (Applied BioSystems) and 300 mg of root from At. yokoscense for ABI Prism 3100 Genetic Analyzer (Applied the phylogenetic analyses of the AM fungi BioSystems) according to the manufacturer’s analyses with a Plant Genomic DNA Mini instructions. At least two independent samples Kit (VIOGENE) used according to the of each PCR products were cloned, and both manufacturers’ protocols. The isolated DNA was strands were sequenced. This process was resuspended in TE and stored at −20 °C until repeated twice to confirm the same sequences. use. For the phylogenetic analysis of the AM Data analyses fungi, the 18S rDNA region with primers For phylogenetic analyses of AM fungi, designed by Saito et al. (2004) and Sato et al. a representative of each OTU was compared (2005) were used. Double-stranded DNA was phylogenetically to sequences of known species amplified by incubation at 94°C for 2 min in the GenBank database of the DDBJ/EMBL/ followed by 40 cycles of incubation at 94°C GenBank by using the Blast program. Database for 1.5 min, 48°C for 2 min, and 72°C for sequences yielding the greatest percent similarity 3 min, with a final extension at 72°C for 15 to the clone sequences were chosen as the best min. After the amplification, reaction mixtures match for each OTU. Moreover, some sequences were subjected to electrophoresis in 1% low- of closely related species of the best match melting-temperature agarose gels to purify of the sequences were retrieved from GenBank (Table amplified products. The cloning of purified PCR 1). Phylogenetic trees were then constructed products was performed using the TA cloning with Catenomyces sp. (AY635830) as use for kit (Invitrogen). Approximately 100 clones were outgroups. 76 植物研究雑誌第 86 巻第 2 号 2011 年 4 月

Table 1. Samples used in the phylogenetic study and corresponding GenBank accession numbers Taxa Accession No. Reference Asago01 AB490491 present study Asago02 AB0490492 present study Asago03 AB490494 present study Asago04 AB490495 present study Acaulospora laevis Y17633 Schuessler et al. (2001a) Acaulospora longula AJ306439 Schuessler et al. (2001b) Acaulospora mellea FJ009670 Wu et al. (2006b) Acaulospora rugosa Z14005 Simon et al. (1993) Acaulospora scrobiculata AM746156 Liang et al. (2008) Acaulospora spinos Z14004 Simon et al. (1993) Acaulospora sp. 1 AJ306440 Schuessler et al. (2001b) Acaulospora sp. 2 EF136887 Loke et al. (unpublished) Acaulospora sp. 3 EU332731 Lee et al. (2008) Acaulospora sp. 4 AY129590 Husband et al. (2002) Ambispora fennica AM268192 Walker et al. (2007) Archaeospora leptoticha AB220172 Maki et al. (2008) Archaeospora trappei Y17634 Schwarzott et al. (2001) Catenomyces sp. AY635830 James et al. (2006) Entrophospora colombiana AB220170 Maki et al. (2008) Gigaspora gigantea Z14010 Simon et al. (1993) Gigaspora sp. AB076267 Saito et al. (2004) Glomus claroideum AJ276079 Schwarzott et al. (2001) Glomus consrictum AM946956 Moser and Haselwandter (unpublished) Glomus etunicatum Z14008 Simon et al. (1993) Glomus etunicatum Y17639 Schuessler et al. (2001b) Glomus luteum GSU36591 Simon (1996) Glomus mosseae GMU96144 Vandenkoonhuyse and Leyval (1998) Glomus viscosum Y17652 Schuessler et al. (2001) Glomus sp. 1 EF136911 Loke et al. (unpublished) Glomus sp. 2 AJ301856 Schwarzott et al. (2001) Glomus sp. 3 AB076278 Saito et al. (2004) Glomus sp. 4 DQ090872 de la Pena et al. (2006) Glomus sp. 5 DQ090871 de la Pena et al. (2006) Glomus sp. 6 AJ496109 Opik et al. (2003)

To construct phylogenetic trees, the 18S the 18S rDNA sequences, the GTR model with rDNA sequences were aligned independently gamma shape parameter of 0.2958 (GTR+G) using Clustal W (Thompson et al. 1994), and was chosen (Substitution rates: A, 0.3729; C, were edited manually using MEGA 4 (Tamura et 0.1552; G, 0.1827; T, 0.2891; A-C, 0.0000; A-G, al. 2007). The positions of deletions or insertions 1.6392; A-T, 0.1821; C-G, 1.0085; C-T, 3.8909; were determined by eyes and eliminated in all of and G-T, 1.000). The neighbor-joining (NJ) the phylogenetic analyses. analysis was performed using PAUP 4.0b10 on In the phylogenetic analyses, the most appropriate model of DNA substitution was the most chosen using the Akaike information criteria with PAUP 4.0b10 (Swofford 2002) and Results Modeltest 3.0 (Posada and Crandall 1998). For The results of the comparison of heavy metal April 2011 Journal of Japanese Botany Vol. 86 No.2 77

Fig. 2. Heavy metal concentration in soils of four localities of the Ikuno mine site. (A): Cd, (B): Zn, (C): P, (D): Pb, (E): As. Error bars indicate standard. Columns marked by different letters are significantly different according to Kruskal-wallis test (P < 0.05). concentration in the Ikuno mine site are shown (Fig. 3). In NJ tree, Type1 was identical to in Fig. 2. The concentration of As, Cd and Zn previous published sequences of Acaulospora in the locality ‘Material’ is highest in any of the longula and Ac. rugosa, and consisted of localities (Figs. 2A, B, E), and P and Pb of the monophyletic group containing the following other locality ‘Keiju’ were highest values of all Acaulospora species; Ac. sp. 2, Ac. sp. 16, Ac. sp. (Figs. 2C, D). 18, Ac. laevis, Ac. spinos, and Ac. scrobiculata. From the molecular analysis of the AM Type 2 was consisted of monophyletic group fungi, four sequences were isolated from each with following Glomus species, G. sp. 36, G. of the four localities of At. yokoscense examined sp. 37, G. sp. 38, G. sp. 39, and positioned at in this study. These sequences isolated in this the most basal node in this monophyletic group. study were named from Type 1 to Type 4. Type Type 3 and Type 4 consisted of a monophyletic 1 is the AM fungi isolated from the root of group with Archaeospora leototicha and At. yokoscense collected from the one locality Ambispora fennica. ‘Material’ and ‘Keiju’, Type 2 is from ‘Daimaru’, Type 3 and Type 4 are from ‘Kanamori’. These Discussion four sequences have been also submitted to In this study, some AM fungi colonized DDBJ/EMBL/GenBank nucleotide sequence Athyrium yokoscense distributed in the Ikuno databases under Accession Nos. AB490491, mine site. Our phylogenetic results of the AB490492, AB490494, and AB490495 (Table AM fungi indicated that Type1 isolated from 1). ‘Material’ belonged to a species of Acaulopora We reconstructed phylogenetic trees because Type1 was included in the monophyletic including our isolates and previous published group of species of Acaulospora. Our sequences of AM fungi based on NJ analysis phylogenetic analyses could not identify species 78 植物研究雑誌第 86 巻第 2 号 2011 年 4 月

Fig. 3. Phylogenetic relarionships of Arbuscular mycorrhizal fungal colonization in Athyrium yokoscense roots based on Neighbour-Joining (NJ) method. Numbers above or below branches indicate bootstrap values. or genus for other haplotypes. However, Type of this area still polluted. In this study, why are 2 was positioned at the most basal node of a the AM fungi different between near localities of monophyletic group which consisted of Glomus the Ikuno mine site? It is not surprising because species. This result suggested that Type 2 was these areas had different values of metallic closely related to the genus Glomus, probably concentrations. Our soil data indicated that the belonged to the species of Glomus (Fig. 3). soil of both localities ‘Material’ and ‘Keiju’ had The results of the comparison of heavy metal higher concentrations of As than other localities concentration in the Ikuno mine site indicated (Fig. 2). Therefore, the amount of As might that all localities of the Ikuno mine site involved influence the survival of the AM fungi. In fact, higher concentration of all heavy metals than Wang et al. (2008) indicated that Acaulospora those of previously published standard data species could survive in high concentrations of (Asami and Chino 1983), indicating that the soils As, moreover, some studies have reported that April 2011 Journal of Japanese Botany Vol. 86 No.2 79

Glomus and Sctellospora species were the most the Curator of Tohoku University Herbarium for widespread (Vestberg 1995, Zhang et al. 1998, permitting us to examine herbarium specimens Oehl et al. 2003), although Acaulospora species of Athryium species. This study was partly dominated in some restricted areas (Zhang supported by a Grant-in-Aid for Scientific et al. 2003, Tao et al. 2004). These studies Research from the Ministry of Education, were supported as Acaulospora species could Science and Culture of Japan (to T.F. and Y.K.) be isolated only from the localities ‘Material’ and by the Grant of Sumitomo Environmental and ‘Keiju’ where is a high concentration of Study (to Y.K.). As. Thus, the dominance of Acaulospora in the localities ‘Material’ and ‘Keiju’ and non- References Acaulospora in other localities most likely Asami T. and Chino M. 1983. Environmental and Inorganic Chemistry. Hakuyusha, Tokyo. reflects the influence of specific soil conditions Baker A. J. M., McGrath S. P., Reeves R. D. and Smith and may represent differences in tolerance to J. A. C. 2000. Metal hyperaccumulator plants: A high As concentration, rather than a spatially review of the ecology and physiology of a biochemical dominant individual. Considering these results, resource for phytoremediation of metal-polluted soils. we could hypothesize the best way to remove As In: Terry N. and Banuelos G. (eds.), Phytoremediation of Contaminated Soil and Water. pp. 85–107. Lewis polluted soils of the Ikuno mine site is to use At. Publishers, Boca Raton. yokoscense colonizing Acaulospora species. Clapp J. P., Young J. P. W., Merryweather J. W. and Fitter The interest in trying to link At. yokoscense A. H. 1995. Diversity of fungal symbionts in arbuscular living in various soil types and AM fungi rests mycorrhizas from a natural community. New Phytol. 130: 259–265. on the wide use of such markers for phylogeny de la Pena E., Echeverria S. R., van der Putten W. H., as well as on an increasing number of studies. Freitas H. and Moens M. 2006. Mechanism of control The 18S rDNA analyzed in this work provides of root-feeding nematodes by mycorrhizal fungi in the a robust hypothesis between At. yokoscense dune grass Ammophila arenaria. New Phytol. 169: and the AM fungi. This hypothesis, based on 829–840. Gerdemann J. W. 1965. Vasicular-arbuscular mycorrhizae molecular information, is a timely contribution formed on maize and tuliptree by Endogone fasciculate. that allows unbiased interpretation between Mycologia 57: 562–575. hosts and mycorrhizae. Additional molecular Hempel S., Renker C. and Buscot F. 2007. Differences markers may lend support to this scenario, and in the spe cies composition of arbuscular mycorrhizal fungi in spores, root and soil communities in grassland multiple markers should be surveyed within ecosystem. Environ. Microbiol. 9: 1930–1938. this species to find any variation. This work will Husband R., Herre E. A., Turner S. L., Gallery R. and help to reinterpret existing 18S rDNA data sets Young J. P. 2002. Molecular diversity of arbuscular and facilitate the interpretation of new ones. mycorrhizal fungi and patterns of host association over Moreover, the AM fungi predominated in the time and space in a tropical forest. Mol. Ecol. 11: 2669– 2678. colonized roots of At. yokoscense at all sites, James T. Y., Letcher P. M., Longcore J. E., Mozley- supporting findings from earlier studies that Standridge S. E., Porter D., Powell M. J., Griffith G. suggest that they are particularly tolerant of large W. and Vilgalys R. 2006. A molecular phylogeny of the concentrations of heavy metals. Identification flagellated fungi Chytridiomycota( ) and description of a new phylum (Blastocladiomycota). Mycologia 98: and isolation of the most tolerant strains may 860–871. have important implications for the future Khan A. G., Kuek C., Chaudhry T. M., Khoo C. S. and phytoremediation of heavy-metal-contaminated Hayes W. J. 2000. Role of plants, mycorrhizae and soils (Khan et al. 2000). phytochelators in heavy metal contaminated land remediation. Chemosphere 41: 197–207. Krämer U. and Chardonnens A. N. 2001. The use of We thank Mitsubishi Material Co. Ltd. for transgenic plants in the bioremediation of soils providing soils and fern samples. We also thank contaminated with trace elements. Appl. Microbiol. 80 植物研究雑誌第 86 巻第 2 号 2011 年 4 月

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野々村暢仁 a，川田由紀 a，南谷幸雄 b，早川宗志 b，福田達哉 a，康峪梅 a，櫻井克年 a：生野鉱山のヘビノネゴザに着生するアーバスキュラー菌根菌の分子同定山のヘビノネゴザの根には少なくとも 4 タイプの AM ヘビノネゴザ Athyrium yokoscense のアーバスキュ菌が着生していることが明らかになった．生野鉱山内のラー菌根菌（AM 菌）を，1973 年まで銀山として採鉱ヒ素の高濃度蓄積地点から Acaulospora 属の AM 菌が，されていた生野鉱山で研究した．生野鉱山の 4 地点から，その他の地点から非 Acaulospora 属の AM 菌が検出さ土壌中の重金属蓄積量の解析と，形態学的・分子系統学れた．従って，これらの AM 菌相の違いは，空間的な的手法を用いた AM 菌の同定を行った．分子系統学的優占種の違いではなく，土壌の特殊性に影響しており，解析は，18S ribosomal DNA を用いた．これらの解析これはヒ素蓄積量の差に影響されている可能性が高い．から，形態観察により全ての地点のヘビノネゴザの根に（a高知大学農学部， AM 菌が着生しており，分子系統学的手法により生野鉱 b愛媛大学大学院連合農学研究科）