Mining activities destroy vegetation and remove surface soil to reach buried ore deposits. Mineral extraction processes are often accompanied by pulverization of rocks and chemical use. The residue of the extracted substrates is stored in tailings, producing large areas of bare ground and piles of tailings. Ideally, these mine areas should be restored to the original vegetation. However, the toxicity of heavy metals (HMs), lack of macronutrients, and abnormal soil structures in mining areas make restoration difficult without careful planning (e.g., storage of surface soil to cover the bare ground after mining activities). In China, many mining areas have been abandoned, causing severe ecological and environmental problems. Soils in these abandoned mine areas are highly contaminated by HMs, which are dispersed by wind or dissolved in water, causing human health disorders in the surrounding regions. Many of the abandoned mine areas in China were originally covered by forests dominated by Pinaceae and Fagaceae. These tree species depend on ectomycorrhizal fungi (EMF) for nutrient absorption and cannot grow in their absence.

Some reports have demonstrated that EMF helps trees survive in soils contaminated with HMs by alleviating the toxicity of the metals. Thus, application of EMF in abandoned mine areas may help with forest restoration. Unfortunately, our knowledge about the function of EMF in heavy-metal tolerance is largely from in vitro experiments using a few easily culturable strains. To develop an effective reforestation strategy in the mining areas, we need better understanding of EMF on major forest tree species in mining areas.

In this dissertation, ectomycorrhizal fungal communities associated with two dominant forest trees, Masson pine (Pinus massoniana) or oak (Quercus spp.), were investigated in four mine sites (three types of HM, including lead-zinc (Chapter II), manganese (Chapter III) and copper (Chapter IV)) in the central south of China. In addition, seedling bioassay experiment was applied to screen EMF propagule communities in the mine tailing soil and guest soils covering tailings (Chapter V). The goals of this dissertation were to characterize EMF communities on Masson pine or oak growing in these different mining areas and to evaluate the effects of HMs and other soil factors.

Root systems were collected from selected trees in each mine sites and the corresponding rhizospheric soil samples and tree leaves were also collected for chemical analysis. Soil pH and electrical conductivity were determined by mixing the soil in water. For the element determination (K, Pb, Cd, Cu, Zn, and Mn) in soil and leaf samples, soils and leaves were digested by nitric acid and perchloric mixture and then determined using the atomic absorption spectrometer. For the determination of total N and P in soils, soils were digested by sulfuric acid and perchloric acid, and then N and P were determined by indophenol-blue method and molybdenum blue method, respectively.

Ectomycorrhizas were identified by combining morphotyping and molecular analyses. For molecular analysis, fungal internal transcribed sequence region of nuclear rDNA were amplified by polymerase chain reaction and then subjected to sequencing analysis.

As a summary, in total, 107 species-level operational taxonomic units (OTUs) based on 97% ITS sequence similarity were identified from the Masson pine (103 OTUs) and oak (20 OTUs) ectomycorrhizal root tips from all of study sites. Of these OTUs, 84 belong to basidiomycete, 22 belong to Ascomycete. The richest family is Thelephoraceae, including 24 OTUs, followed by Russulaceae (22 OTUs) and Cenococcum (7 OTUs). These OTUs formed a phylogenetically diverse community associated with Masson pine. However, different structures of ectomycorrhizal fungal community were found among the different mine sites investigated.

1.Ectomycorrhizal fungal communities associated with Masson pine in Pb-Zn mine sites

The diversity and composition of EMF associated with Masson pine and soil chemistry were investigated in Taolin Pb-Zn mine tailings (TLT), two fragmented forest patches in Huayuan Pb-Zn mineland (HY1 and HY2), and a non-polluted forest in Taolin (TLC). The two study sites in the Huayuan mineland (HY1 and HY2) were significantly different in soil Pb, Zn, and Cd concentrations, but no significant difference was observed in ectomycorrhizal colonization, EMF richness, diversity, or rank-abundance. In addition, the similarity of communities between HY1 and HY2 was quite high (Sorensen similarity index = 0.47). Thus, the concentration of HMs may not be determining factors in the structure of these communities. In TLT, however, significantly lower ectomycorrhizal colonization and EMF richness were observed. The amounts of Pb and Zn in the tailing sand were higher than those in the non-polluted forest but far lower than in HY1. Thus, these HMs did not account for the reduced colonization and EMF richness in TLT. The community in TLT was dominated by four pioneer species (Rhizopogon buenoi, Tomentella ellisii, Inocybe curvipes, and Suillus granulatus), which collectively accounted for 93.2% of root tip colonization. The immature soil conditions in tailing (low N and P, sand texture and lack of organic matter) may only allow certain pioneer EMF species to colonize the site. When soil samples from four sites combined, I found the occurrences of major EMF taxa were not clearly related to the concentrations of Pb, Zn, and Cd. In conclusion, my results suggest that ectomycorrhizal fungal communities in these Pb-Zn mining areas are not necessarily affected by HMs themselves but could be largely determined by soil maturity.

2.Ectomycorrhizal fungal communities associated with Masson pine and oak growing in a manganese mine site

The ectomycorrhizal fungal communities associated with Masson pine and oak were investigated in Xiangtan Mn mining area. The sampled trees were divided into three groups based on soil color: pine trees growing in yellow soils (normal color in local site), pine trees in black soils (polluted with Mn tailing wastes) and oak trees in gray-black soils. The results showed that average Mn concentrations in black soils (4034.5 ± 4692.5 mg kg(-1)) and gray-black soils (4246.8 ± 3983.0 mg kg(-1)) were significantly higher than those in yellow soils (2019.8 ± 2205.2 mg kg(-1)). On the contrary, ectomycorrhizal colonization rates of Masson pine growing in black soils (39.1 ± 34.4%) and oak trees (38.9 ± 15.2%), being not significantly different from each other, were lower than that of pine in yellow soils (54.6 ± 18.7%). In total, 53 OTUs were identified at 97% similarity threshold, of which 48 were found in pine trees and 20 in oak trees. Compared to pine trees in yellow soils, a lower EMF richness of pine in the black soils and oak was observed. Correlation analysis indicated a significantly negative dose effect of Mn on EMF richness. The three ectomycorrhizal fungal communities associated with the three groups of trees showed different structure. The pine growing in yellow soils were dominated by Atheliaceae with relative abundance of 38.2%, followed by Russulaceae (34.9%) and Thelephoraceae (8.7%). The pine growing in black soils were dominated by Atheliaceae with relative abundance of 37.8%, followed by Thelephoraceae (34.3%) and Cenococcum (17.0%). On the other hand, the most dominant species in oak was members of Thelephoraceae, followed by Cenococcum with relative abundance of 61.9% and 19.4% respectively. Atheliaceae only accounted for 3.6% of ectomycorrhizal tips of oak. Russulaceae being dominant in pine from yellow soils became rare in pine in black soils (2.8%) and absent in oak. Russulaceae preferred to soils with lower Mn concentrations (163.0~3132.8 mg kg(-1)) compared to Atheliaceae, Thelephoraceae, and Cenococcum (one-way ANOVA, P < 0.01). I concluded that the overburden of Mn tailing wastes may inhibit the ectomycorrhizal development and eliminate some Mn-sensitive ectomycorrhizal taxa, such as Russulaceae, resulting in the decrease of the EMF richness.

3.Ectomycorrhizal fungal communities associated with Masson pine growing on guest soils on copper (Cu) mine tailing slope in Dexing Cu mine

Ectomycorrhizal fungal communities associated with Masson pine were investigated in the guest soils covering Dexing No.2 tailing dam slope (DXT) and one adjacent non-polluted forest (DXC) in Jiangxi Province. Soil chemical analysis showed significantly lower N concentration (394 ± 143.6 mg kg(-1)) and relatively high Cu concentration (100.7 ± 71.2 mg kg(-1)) in the DXT as compared to those in the DXC (N: 2492.9 ± 703.5 mg kg(-1); Cu: 39.7 ± 10.8 mg kg(-1)), but the Cu concentrations in guest soils were significantly lower than that in tailings in DXT, suggesting that covering guest soils could significantly obstruct the movement of HMs from tailing substrates into surrounding environments. As compared to DXC, the ectomycorrhizal fungal communities in DXT showed a very uneven structure and low species richness. In addition, the similarity of communities between the two sites was low (Sorensen similarity index = 0.25). In DXT, ectomycorrhizal fungal community were overwhelmed by two OTUs of Atheliaceae (Atheliaceae sp.1 and Tylospora sp.1), which accounted for 78.2% ectomycorrhizal tips and collectively colonized 29 of 30 trees. In DXC, the community was mainly comprised of Cenococcum (28.9%), Thelephoraceae (21.6%), Atheliaceae (16.9%) and Russulaceae (16.4%), collectively accounting for 83.8% relative abundance. The concentrations of Cu in the guest soils did not affect the ectomycorrhizal fungal community. The guest soils originating from the subsoils and plant community might be responsible for the low diversity and poor species richness of EMF in Dexing tailing slope.

4.EMF propagule communities in Taolin Pb-Zn tailings and guest soils on Dexing Cu tailing slope

The ectomycorrhizal propagule communities of in the tailing sites and HM contaminated soils have been given relatively little attention. A seedling bioassay method was used to investigate the resistant propagule communities of EMF in the Taolin Pb-Zn tailing (TLT) and the guest soils on Dexing tailing slope (DXT). Rhizosphere soils were collected for seedling growth from TLT, DXT and one non-disturbed mature forest (DXC). After eight months of sawing, the seedlings of Masson pine were harvested and analyzed. The growth of seedlings in the TLT and DXT soils was significantly inhibited with comparison to those growing in the soils of DXC. Correlation analysis showed that N concentrations in soils were the limiting factor for seedling growth (R = 0.538, P < 0.01). Poor ectomycorrhizal colonization were observed in the seedlings growing in TLT (9.0 ± 14.9%) and DXT (22.4 ± 17.7%) soils, in contrast, DXC seedlings were colonized well (47.5 ± 24.9%). Molecular analysis revealed that Cenococcum, Atheliaceae sp., Rhizopogon, Inocybe and Suillus were frequently encountered in seedling roots, but species assemblage was significantly different among the three sites. Comparing with the forest EMF communities of the same sites investigated, high similarity was observed in the TLT site (Sorensen similarity index = 0.71, 5 OTUs sharing). In DXC, little overlap was found (Sorensen similarity index = 0.12). Some early-stage fungi at TLT (Inocybe curvipes) and DXT (S. granulatus and S. luteus) were abundant in seedling roots, whereas DXC seedlings were dominated by the members of Cenococcum and Rhizopogon. In TLT, EMF species (5 OTUs shared between seedling and forest investigations) may be available candidates for culturing ectomycorrhizal seedlings for reforestation in the bare tailings.

In conclusion, Masson pine in central south China harbored phylogenetically diverse EMF, which is similar to that observed in many subtropical or boreal forest trees. In present study, the elevated concentrations of HMs (Pb, Zn, and Cd) may not be the determinants for ectomycorrhizal colonization and low diversity of EMF except Mn in the mine areas. Poor ectomycorrhizal colonization and low diversity in the Pb-Zn tailing site can be explained from the immaturity (low N and organic matter, poor texture) of tailing substrates and the stage of succession. The overburden of Mn tailing wastes may inhibit the ectomycorrhizal development and eliminate some Mn-sensitive ectomycorrhizal taxa, such as Russulaceae. Moreover, the occurrence of most of the EMF were not related to the concentrations of HMs in soils except Russulaceae. Russulaceae only occurred in the soils with low HMs (Mn, Cu) and rich N and may be sensitive to HMs stresses.

重金属鉱山の開発は植生と耕地を直接破壊するだけでなく、選鉱で生じる廃水が周辺の河川と土壌を汚染する。また、砂礫など大量の尾鉱が広大な土地に山積みされ、雨水によって重金属が溶出し、周辺の広い土地を汚染する。中国には、数多くの重金属鉱山が存在し、重金属汚染による環境問題が深刻になっている。鉱山荒廃地から溶出する有害物質を軽減するには、植生被覆によって雨水による重金属溶出を抑制することが重要である。また、植生が回復することによって植物体や土壌有機物へ有害物質が固定されることも有害物質の溶出抑制に繋がる。しかし、鉱山荒廃地では有害重金属の濃度が高く、植物の生長に必要な養分も乏しいことから、植生の回復は非常に困難である。本論文では、中国中南部の異なる重金属荒廃地において、樹木の重金属耐性や生長を促進する機能を持つとされる外生菌根菌(以下、菌根菌)の群集構造を調べた。

第1章の序論に続く第2章では、湖南省のPb・Zn鉱山荒廃地における馬尾松(Pinus massoniana)に共生する菌根菌の群集構造を調べた。調査地は湖南省の花垣市Pb・Zn鉱山2ヵ所(HY1とHY2)、岳陽市桃林Pb・Zn鉱山の尾鉱荒廃地(TLT)、およびその近辺の植生非破壊地(対照地、TLC)に設定した。HY1とHY2では、土壌PbとZn濃度が著しく高い地点を含んでいたものの、馬尾松根系の菌根菌の感染率や多様性などは、対照地の値と有意な差が認められなかった。一方、TLTでは土壌のPbとZnの濃度はHY1より顕著に低かったものの、菌根菌感染率と菌種の豊かさは他の三つの調査地より著しく低く、4つの先駆菌種(Rhizopogon buenoi、Tomentella ellisii、Inocybe curvipes、Suillus granulatus)が93.2%の菌根を占めていた。以上の結果から、土壌中のPbやZn濃度の直接の影響よりも、土壌中の養分や有機物が少ないことがTLTの菌根菌群集に影響しているものと考えられた。

第3章では、湖南省湘潭市のMn鉱山荒廃地において、馬尾松とカラカシワナラ(Quercus fabri)に共生する菌根菌の群集構造を調べた。検出された菌根菌の種数は、馬尾松から48種、カラカシワナラから20種で、合計53種であった。Atheliaceaeは馬尾松の菌根菌群集では優占していたが、カラカシワナラの菌根では3.6%の根端にしか見られなかった。同様に、Russulaceaeは馬尾松で優占していたが、カラカシワナラの菌根からは検出されなかった。菌根菌の種の豊かさはMn濃度と有意な負の相関を示したほか、高Mn濃度ではRussulaceaeやThelephoraceaeの出現が減少する傾向が見られた。RussulaceaeとThelephoraceaeはMnに感受性が高いため、汚染土壌では菌根の形成が抑制されたため、菌根菌の種の豊かさが減少したものと考えられる。

第4章では、江西省徳興市のCu鉱山尾鉱ダム斜面の客土上に植栽された馬尾松と隣接した馬尾松優占林分(対照地)で菌根菌群集を調べた。その結果、対照地の菌根菌群集は多くの菌種で構成され、各菌種の優占度の均一性も高かったのに対し、尾鉱ダム斜面ではAtheliaceaeに属する2菌種の優占度が著しく高く、その相対菌根数は78.2%に達した。各菌の出現頻度および相対菌根数と客土中のCu濃度には有意な関係が認められなかった。客土中のN濃度は対照地土壌よりも有意に低いなど土壌養分に乏しいこと、客土は山の心土由来で菌根菌感染源が少ないことなどが、尾鉱ダム斜面客土中の菌根菌の群集構造に影響しているものと考えられる。

第5章では、桃林Pb・Zn尾鉱(TLT)、徳興Cu鉱尾鉱の客土(DXT)、植生非破壊森(DXC)における菌根菌の埋土胞子(菌核等の感染源を含む)の群集構造について調べた。それぞれの林分から採取した土壌を充填したポットに馬尾松の苗を植えて、8ヵ月栽培し、埋土胞子から苗の根に形成された菌根を調べることによって埋土胞子群集を調べた。DXCと比べ、TLTとDXTの菌根菌の感染率と苗の成長量は低かった。サイト間で埋土胞子群集の優占種は全く異なり、TLTではI. curvipes、DXTではS. granulatusとSuillus luteus、DXCではCenococcumとRhizopogonがそれぞれ優占していた。各サイトにおける埋土胞子群集と、前章までに明らかにした成木の菌根菌群集と比較すると、TLTでは群集の類似度が高かったものの、DXCでは低かった。

第6章では、すべての調査地の結果を統合・再解析して、総合討論した。本研究で検出された菌根菌の種数は、馬尾松から104種、カラカシワナラから20種の合計107種であり、84種は担子菌に、23種は子嚢菌に属していた。菌根菌の群集構造に及ぼす重金属の影響については、重金属の種類によって異なることが示された。Mn濃度の高い土壌では、Mnに感受性が高い菌種の菌根形成を抑制することにより菌種組成が大きく変わっていたほか、菌根菌の種の豊かさも減少した。一方、土壌中のPbやZn濃度の直接的な影響は認められなかった。さらに、重金属鉱山地域では、重金属以外の要因(例えば土壌中の養分や有機物の含量)の影響も大きいことが示唆された。

以上のように、異なるタイプの重金属鉱山荒廃地において菌根菌の群集構造の特性を明らかにしたことは、国際的にも新規性の高い研究成果であると評価できる。また、樹木の生長に重要な役割を持つ菌根菌が重金属に対してどのような生態的反応を示すのかを示した本研究の成果は、鉱山跡地の植生回復への応用へ繋がる重要なものである。よって審査委員一同は本論文が博士(農学)の学位論文として価値あるものと認めた。