Vegetation at the former open-pit Ningyo-toge mine, 36 years after closure treatment: Impact of soil cover on woody plant establishment and dominance of the perennial herb Miscanthus sinensis.

Soil cover is a prevailing method used at mine sites to ensure the safety of hazardous materials and restore ecological functions when the base materials are unfavorable for plant growth. The former open-pit Ningyo-toge Mine was backfilled with overburden and neutralized smelting residues and covered with soil in 1987. After 36 years, the vegetation remained dominated by the perennial herb Miscanthus sinensis, and woody plant establishment did not progress successfully. This study investigated the factors that inhibited woody plant establishment at the site. The soil profile survey revealed that the soil cover formed Bg horizons (pseudogley soil) with cloudy mottling, representative of poorly drained soil. In the Bg horizon, woody plant roots of Pinus densiflora and Weigela hortensis exhibited growth inhibition. Elemental analysis revealed that in the Bg horizon the roots of P. densiflora and W. hortensis accumulated high Fe concentrations exceeding 10,000 mg/kg DW at critical levels. Our results suggested that woody plant roots in the Bg horizon may have suffered from chronic oxygen deficiency accompanied by excessive Fe stress in the soil cover. Topsoil water content (<50 mm) and microtopographic features were not critical factors disrupting woody plant establishment because some individuals were growing in areas with high soil water content, exceeding 60%. Considering that woody plant roots were developed primarily in the shallow A horizon, A horizon formation by M. sinensis is a key step in initiating woody plant establishment by improving the soil structure and physiochemical characteristics of the soil cover, such as carbon content, exchangeable nutrients, and air-filled porosity. For successful mine pollution control and vegetation recovery, implementing an appropriate topsoil system, such as native forest soil, loosely graded and minor infiltration layer above the overburden would be necessary.

Ontogenetic changes in root and shoot respiration, fresh mass and surface area of Fagus crenata.

BACKGROUND AND AIMS: To date, studies on terrestrial plant ecology and evolution have focused primarily on the trade-off patterns in the allocation of metabolic production to roots and shoots in individual plants and the scaling of whole-plant respiration. However, few empirical studies have investigated the root : shoot ratio by considering scaling whole-plant respiration at various sizes throughout ontogeny. METHODS: Here, using a whole-plant chamber system, we measured the respiration rates, fresh mass and surface area of entire roots and shoots from 377 Fagus crenata individuals, from germinating seeds to mature trees, collected from five different Japanese provenances. Non-linear regression analysis was performed for scaling of root and shoot respiration, fresh mass and surface area with body size. KEY RESULTS: Whole-plant respiration increased rapidly in germinating seeds. In the seedling to mature tree size range, the scaling of whole-plant respiration to whole-plant fresh mass was expressed as a linear trend on the log-log coordinates (exponent slightly greater than 0.75). In the same body size range, root and shoot respiration vs. whole-plant fresh mass were modelled by upward-convex (exponent decreased from 2.35 to 0.638) and downward-convex trends (exponent increased from -0.918 to 0.864), respectively. The root fraction in whole-plant respiration, fresh mass and surface area shifted continuously throughout ontogeny, increasing in smaller seedlings during early growth stages and decreasing in larger trees. CONCLUSIONS: Our results suggest a gradual shift in allocation priorities of metabolic energy from roots in seedlings to shoots in mature trees, providing insights into how roots contribute to shoot and whole-plant growth during ontogeny. The models of root : shoot ratio in relation to whole-plant physiology could be applied in tree growth modelling, and in linking the different levels of ecological phenomena, from individuals to ecosystems.

Isoavenaciol and 7-hydroxy-isoavenaciol: Zn-chelating metallophores produced by root-endophytic Pezicula ericae in a Zn-accumulating plant, Aucuba japonica.

Metallophores are low-molecular-weight compounds capable of chelating heavy metals, which have recently been reported to alleviate heavy metal stress in plants. We isolated two undescribed compounds as Zn-chelating metallophores from the culture broth of the root endophytic Pezicula ericae w12-25, which was collected from a Zn-accumulating plant, Aucuba japonica Thunb. These two compounds were determined to be (3aS,4S,6aR)-3a-hydroxy-3-methylene-4-octyldihydrofuro[3,4-b]furan-2,6(3H,4H)-dione and (3S,3aS,4S,6aR)-3a-hydroxy-3-(hydroxymethyl)-4-octyldihydrofuro[3,4-b]furan-2,6(3H,4H)-dione using spectroscopic methods (HRMS, 1H and 13C NMR, and 2D NMR) and X-ray crystallography, respectively. The two compounds, classified as furofurandiones, were named isoavenaciol and 7-hydroxy-isoavenaciol. After the hydrolysis of the lactone moiety, isoavenaciol would release the carboxyl group to show Zn-chelating activity. Their antifungal activities were confirmed using Cladosporium herbarum (AHU9262).

Miscanthus sinensis contributes to the survival of Pinus densiflora seedlings at a mining site via providing a possible functional endophyte and maintaining symbiotic relationship between P. densiflora and endophytes from high soil temperature stress.

At a sedimentary site in an old mine site, Miscanthus sinensis formed patches, where Pinus densiflora seedlings could grow better compared with those outside the patches, indicating that M. sinensis would improve P. densiflora seedling establishment. The purpose of this study was to understand the mechanisms by which M. sinensis facilitates the survival of P. densiflora seedlings by considering the soil properties, heavy metal tolerance, and root endophytes in P. densiflora seedlings at the sedimentary site. The sedimentary site, which is a bare ground, contained high concentrations of Fe, indicating that plants should be exposed to Fe and high soil temperature stresses. Measurement of soil temperature revealed that M. sinensis suppressed sharp increases and alternation of soil temperature, resulting in reducing high soil temperature stress in P. densiflora seedlings. To adapt to the Fe stress environment, P. densiflora outside and inside the patches produced Fe detoxicants, including catechin, condensed tannin, and malic acid. Ceratobasidium bicorne and Aquapteridospora sp. were commonly isolated from P. densiflora seedlings outside and inside the patches as root endophytes, which might enhance Fe tolerance in the seedlings. Aquapteridospora sp., which is considered as a dark-septate endophyte (DSE), was also isolated from the roots of M. sinensis, suggesting that M. sinensis might play a source of a root endophyte to P. densiflora seedlings. Ceratobasidium bicorne could be classified into root endophytes showing symbiosis and weak pathogenicity to host plants. Therefore, high soil temperature stress would weaken P. densiflora seedlings, causing root endophytic C. bicorne to appear pathogenic. We suggested that P. densiflora could adapt to the Fe stress environment via producing Fe detoxicants, and M. sinensis would facilitate the establishment of P. densiflora seedlings in the sedimentary site by providing a DSE, Aquapteridospora sp., and maintaining symbiosis of C. bicorne from high soil temperature stress.

Oosporein Produced by Root Endophytic Chaetomium cupreum Promotes the Growth of Host Plant, Miscanthus sinensis, under Aluminum Stress at the Appropriate Concentration.

Chaetomium cupreum, a root endophyte in Miscanthus sinensis, enhances Al tolerance in M. sinensis by changing aluminum (Al) localization and the production of a siderophore, oosporein, which chelates Al for detoxification. Oosporein has various functions, including insecticidal activity, phytotoxicity, antifungal activity, and a siderophore. In our study, we focused on the detoxification effect of oosporein as a siderophore and on the growth of M. sinensis under Al exposure. In addition, the phytotoxicity of oosporein to M. sinensis was confirmed to compare with those in Lactuca sativa and Oryza sativa as control plants. Under Al stress, oosporein promoted plant growth in M. sinensis seedlings at 10 ppm, which was the same concentration as that detected in M. sinensis roots infected with C. cupreum in our previous study. Oosporein also showed low phytotoxicity to M. sinensis compared with L. sativa at even high concentrations of oosporein. These results suggest that the concentration of oosporein in M. sinensis roots would be maintained at the appropriate concentration to detoxify Al and would promote M. sinensis growth under Al stress, although oosporein would show low phytotoxicity to the natural host plant, M. sinensis, compared with the non-host plant, L. sativa.

Zn tolerance in the evergreen shrub, Aucuba japonica, naturally growing at a mine site: Cell wall immobilization, aucubin production, and Zn adsorption on fungal mycelia.

Aucuba japonica Thunb. is an evergreen understory shrub that grows naturally at a mine site. The mine soil contains high concentrations of heavy metals, and A. japonica appears to maintain detoxification mechanisms against heavy metals in the study site's understory. This study aimed to investigate the heavy metal tolerance mechanisms in A. japonica, considering the possible roles of arbuscular mycorrhizal and root-endophytic fungi. We conducted fieldwork in summer (canopy-foliation season) and winter (canopy-defoliation season) to measure the heavy metal concentrations in leaves, branches, and roots and analyze possible detoxicants in the roots. The infection rates of arbuscular mycorrhizal and root-endophytic fungi were evaluated via microscopic observation, and heavy metal (Zn) localization in A. japonica roots was observed using confocal laser scanning microscopy. Field analysis showed that A. japonica accumulated excessive Zn and produced aucubin and citric acid in the roots in both summer and winter. Zn localization observations clarified that Zn was distributed in thickened epidermal and cortical cell walls, suggesting that the cell walls functioned as Zn deposition sites, reducing Zn toxicity. It was further clarified that Zn was contained within cortical cells, indicating that Zn might be detoxified by aucubin and citric acid. Arbuscular mycorrhizal and root-endophytic fungi within cortical cells adsorbed Zn on fungal cell walls, indicating that these fungi would reduce Zn content within root cells and might alleviate Zn toxicity. Our results indicated that A. japonica would maintain Zn tolerance in both summer and winter via Zn immobilization in the cell walls and production of aucubin and citric acid, and that arbuscular mycorrhizal and root-endophytic fungi might play important roles in the Zn tolerance of A. japonica.

Root-endophytic Chaetomium cupreum chemically enhances aluminium tolerance in Miscanthus sinensis via increasing the aluminium detoxicants, chlorogenic acid and oosporein.

Miscanthus sinensis Andersson is a pioneer plant species that grows naturally at mining sites. Miscanthus sinensis can detoxify aluminium (Al) by producing phytosiderophores, such as chlorogenic acid, citric acid, and malic acid, and localizing Al in cell walls. Root-endophytic Chaetomium cupreum, which produces microbial siderophores, enhances Al tolerance in M. sinensis. However, we could not determine whether the siderophores produced by C. cupreum actually enhance Al tolerance in M. sinensis, because the microbial siderophores have not yet been identified in previous research. The purpose of this study was to clarify how C. cupreum chemically increases Al tolerance in M. sinensis under acidic mining site conditions, especially considering siderophores. Using instrumental analyses, the siderophore produced by C. cupreum was identified as oosporein. Comparison of the stability constant between Al and phytosiderophores and oosporein indicated that oosporein could detoxify Al similarly to chlorogenic acid, which shows higher stability constant than citric acid and malic acid. Inoculation test of C. cupreum onto M. sinensis in acidic mine soil showed that C. cupreum promoted seedling growth, and enhanced Al tolerance via inducing chlorogenic-acid production and producing oosporein. These results suggested that C. cupreum could chemically enhance Al tolerance and might promote growth via reducing excessive Al in cell walls, the main site of Al accumulation. In addition, the chemical enhancement of Al tolerance by C. cupreum might be important for M. sinensis to adapt to acidic mining sites.

Root Fungal Endophytes Enhance Heavy-Metal Stress Tolerance of Clethra barbinervis Growing Naturally at Mining Sites via Growth Enhancement, Promotion of Nutrient Uptake and Decrease of Heavy-Metal Concentration.

Clethra barbinervis Sieb. et Zucc. is a tree species that grows naturally at several mine sites and seems to be tolerant of high concentrations of heavy metals, such as Cu, Zn, and Pb. The purpose of this study is to clarify the mechanism(s) underlying this species' ability to tolerate the sites' severe heavy-metal pollution by considering C. barbinervis interaction with root fungal endophytes. We measured the heavy metal concentrations of root-zone soil, leaves, branches, and fine roots collected from mature C. barbinervis at Hitachi mine. We isolated fungal endophytes from surface-sterilized root segments, and we examined the growth, and heavy metal and nutrient absorption of C. barbinervis seedlings growing in sterilized mine soil with or without root fungal endophytes. Field analyses showed that C. barbinervis contained considerably high amounts of Cu, Zn, and Pb in fine roots and Zn in leaves. The fungi, Phialocephala fortinii, Rhizodermea veluwensis, and Rhizoscyphus sp. were frequently isolated as dominant fungal endophyte species. Inoculation of these root fungal endophytes to C. barbinervis seedlings growing in sterilized mine soil indicated that these fungi significantly enhanced the growth of C. barbinervis seedlings, increased K uptake in shoots and reduced the concentrations of Cu, Ni, Zn, Cd, and Pb in roots. Without root fungal endophytes, C. barbinervis could hardly grow under the heavy-metal contaminated condition, showing chlorosis, a symptom of heavy-metal toxicity. Our results indicate that the tree C. barbinervis can tolerate high heavy-metal concentrations due to the support of root fungal endophytes including P. fortinii, R. veluwensis, and Rhizoscyphus sp. via growth enhancement, K uptake promotion and decrease of heavy metal concentrations.

Root endophytic bacteria of a (137)Cs and Mn accumulator plant, Eleutherococcus sciadophylloides, increase (137)Cs and Mn desorption in the soil.

We found that root endophytes of (137)Cs accumulator plant produce siderophores, resulting in the desorption of (137)Cs from the contaminated soil collected at Fukushima, Japan. We selected an endemic Japanese deciduous tree, Eleutherococcus sciadophylloides (Franch. et Sav), that accumulates high concentrations of (137)Cs and Mn. Root endophytic bacteria were isolated from E. sciadophylloides and microbial siderophore production was evaluated via chrome azurol S (CAS) Fe and CAS Al assays. Of the 463 strains that we isolated, 107 (23.1%) produced the siderophores. Using eight strains that showed high siderophore production in our assays, we examined desorption of (137)Cs, Mn, Fe and Al by the bacterial culture filtrates from (137)Cs-contaminated soil after decomposing the soil organic matter using hydrogen peroxide. We found (137)Cs and Mn desorption concomitant with Al and Fe desorption, as well as a decrease of pH. We also detected succinic acid, a well-known siderophore, in the bacterial culture filtrates of our two root endophytic bacteria. Our results strongly suggest that the root endophytic bacteria of E. sciadophylloides produce the siderophores that enhance (137)Cs and Mn desorption in the rhizosphere, making the resulting (137)Cs and Mn ions easier for E. sciadophylloides to absorb from the rhizosphere.