Mycorrhiza DOI 10.1007/s00572-015-0629-4
ORIGINAL PAPER
Growth and photosynthetic responses of ectomycorrhizal pine seedlings exposed to elevated Cu in soils Yahua Chen & Kazuhide Nara & Zhugui Wen & Liang Shi & Yan Xia & Zhenguo Shen & Chunlan Lian
Received: 16 September 2014 / Accepted: 26 January 2015 # Springer-Verlag Berlin Heidelberg 2015
Abstract It is still controversial whether ectomycorrhizal (ECM) mycelia filter out toxic metals in nutrient absorption of host trees. In this study, pine (Pinus densiflora) seedlings colonized by Cu-sensitive and Cu-tolerant ECM species were exposed to a wide spectrum of soil Cu concentrations to investigate functions of ECM fungi under Cu stress. The photosynthetic rates of intact needles were monitored in situ periodically. The biomass and elements of plants were also measured after harvest. The ameliorating effect of ECM infection on host plants exposed to toxic stress was metal concentration specific. Under lower-level Cu stress, ECM fungi increased seedling performance, while ECM seedlings accumulated more Cu than nonmycorrhizal (NM) seedlings. Under higher-level Cu stress, photosynthesis decreased well before visible symptoms of Cu toxicity appeared. The reduced photosynthesis and biomass in ECM seedlings compared to NM seedlings under higher Cu conditions were also accompanied by lower phosphorus in needles. There was no marked difference between the two fungal species. Our results indicate that the two ECM fungi studied in our system may not have an ability to selectively eliminate Cu in nutrient absorption and may not act as effective barriers that decrease toxic metal uptake into host plants. Y. Chen : Z. Wen : L. Shi : Y. Xia : Z. Shen : C. Lian (*) The Collaborated Lab. of Plant Molecular Ecology (between College of Life Sciences of Nanjing Agricultural University and Asian Natural Environmental Science Center of the University of Tokyo), Nanjing Agricultural University, Nanjing 210095, China e-mail: [email protected] K. Nara Department of Natural Environmental Studies, Graduate School of Frontier Science, The University of Tokyo, 5-1-5 Kashiwanoha, Kashiwa, Chiba 277-8563, Japan C. Lian Asian Natural Environmental Science Center, The University of Tokyo, 1-1-8 Midori-cho, Nishitokyo, Tokyo 188-0002, Japan
Keywords Copper uptake . ECM fungi . Heavy metal-contaminated soils . Metal toxicity . Photosynthesis . Pine (Pinus densiflora)
Introduction Mining activities have produced large quantities of tailings that have caused extensive environmental damage (Adriaensen et al. 2004; Wu et al. 2011). At sites of spoil accumulation, establishment of arboreal vegetation cover helps minimize the migration of heavy metals into surrounding areas and contributes to stabilization and pollution control (Khan et al. 2000; Estaun et al. 2007; Roy et al. 2007; Mendez and Maier 2008). Mine tailings often have toxic levels of metal waste and are deficient in plant macronutrients (Huang et al. 2012), making re-vegetation difficult. Ectomycorrhizal (ECM) fungi are ubiquitous in forested soils, including those highly contaminated with metals; these fungi are thought to play an important role in the metal tolerance of some plants (Godbold et al. 1998; Bellion et al. 2006; Smith and Read 2008; Colpaert et al. 2011). Most studies indicate that inoculation of ECM fungi ameliorates metal toxicity in host plants and promotes the re-vegetation of polluted soils (Jentschke and Goldbold 2000; Van Tichelen et al. 2001; Roy et al. 2007). However, a few investigations have demonstrated that amelioration of metal toxicity by ECM fungi is not universal. Indeed, the fungi sometimes enhance metal uptake by plants or increase the toxicity of heavy metals (Godbold et al. 1998). For example, Jones and Hutchinson (1986) examined the effects of four ECM fungal species (Scleroderma flavidum, Lactarius hibbardae, Lactarius rufus, and Laccaria proxima) on Cu and Ni tolerance in Betula papyrifera. Only inoculation with S. flavidum increased seedling Ni tolerance. Colpaert and van Assche (1992) demonstrated that
Mycorrhiza
inoculation with Thelephora terrestris increases Zn accumulation in the needles of Pinus sylvestris seedlings compared with nonmycorrhizal ones. Although there is no doubt that in many cases ECM fungi do indeed ameliorate metal stress for their host plants, unequivocal evidence for the process and clear understanding of the mechanisms involved are limited. The marked variability in the effects of various mycorrhizal fungi on host plant metal tolerance indicates that this topic warrants further investigation. Diagnosing metal toxicity in plants is not easy and diagnostic methods vary among studies. Many parameters, including plant growth (biomass), nutrient (P or N) uptake (van Tichelen et al. 2001; Adriaensen et al. 2004; Krznaric et al. 2009), water consumption (Colpaert and van Assche 1992), metal uptake, and metal distributions in various organs, tissues, cells, and subcellular components (Denny and Wilkins 1987; Marschner et al. 1996; Frey et al. 2000), have been used as indicators or measures of the Bmetal tolerance capacity^ of plants. Diagnosing metal toxicity is much more difficult in woody plants, which generally grow slowly and react slowly to nutrient deficiencies or metal toxicities (Ingestad and Kahr 1985; Van Tichelen et al. 2001). Thus, visible toxicity symptoms in trees usually appear after long periods of stress exposure (Adriaensen et al. 2006). It is for this reason that previous studies using woody plants have been so protracted, e.g., 8 weeks (Hartley-Whitaker et al. 2000a), 12 weeks (Bradley et al. 1981, 1982; Dixon and Buschena 1988; Kozdroj et al. 2007; Krupa and Kozdroj 2007; Jourand et al. 2010), 16 weeks (Dixon 1988; Colpaert and van Assche 1992), 18 weeks (Jones and Hutchinson 1986), 6 months (Colpaert and van Assche 1993; Hartley-Whitaker et al. 2000b; Zimmer et al. 2009), 8 months (Kim et al. 2004), and 9 months (Adriaensen et al. 2006). A sensitive tool for rapid detection and monitoring of metal toxicity symptoms would promote progress in this field. Photosynthesis is among the most important basic processes of plants and is highly sensitive to environmental changes, including exposure to metal stress (Taiz and Zeiger 2010). There are currently only limited data about the relationship between photosynthesis responses and ECM colonization on plants under metal stress (Jones and Hutchinson 1988a; Huang and Tao 2004; Nguyen et al. 2006). Among the few studies on this topic, most have measured photosynthetic rates at the end of experiments. Consequently, we still poorly understand the change of photosynthetic activities in ECM plants after the initial exposure to metal stress. In this study, we investigate (1) whether ECM colonization alleviates Cu toxicity in plants along various Cu concentrations using a soil system that is relevant to natural conditions; (2) how the photosynthesis of ECM seedlings changes after the exposure to metal stress, with special focus on whether the changes are detectable before visible symptoms of Cu toxicity; and (3) whether the colonizing ECM fungal species (i.e.,
Cu-tolerant and Cu-sensitive fungal species in this study) affects plant performance under Cu stress.
Materials and methods ECM synthesis Based on the results of our previous pure culture experiments using modified Melin-Norkrans (MMN) medium for screening Cu-tolerant ECM fungal species, two ECM fungal species, Pisolithus sp. (Pisolithus sp. 4 sensu Martin et al. 2002) (hereafter, Pt, a Cu-tolerant species) and Cenococcum geophilum (Cg, a Cu-sensitive species), were selected, of which the 50 % effective concentrations (EC50) of Cu (Cu concentration which reduced fungal biomass by 50 %) were 200 and 50 μmol L−1, respectively. These fungi were also chosen for their ease of cultivation, rapid growth rates, and vigorous ECM formation. In total, 120 forty-day-old Japanese red pine (Pinus densiflora) seedlings were prepared. The main root of each was cut at 1 cm below the node to enhance lateral root formation. These seedlings (each with 1 cm of main root remaining) were transplanted into plastic pots (200-mL capacity) in April, filled with an autoclaved (121 °C, 1 h) mixture (1:1, v/v) of Tanashi nursery soil (black sand loam) and Shibanome soil (volcanic sand; Setogahara Co., Gunma, Japan). Physicochemical properties of soils in this study are summarized in Table 1. We combined two inocula to induce ECM formation on the seedlings: cultured mycelia on agar media and soil mycelia radiating from pre-colonized mycorrhizal mother trees. In order to get mother trees, we placed pure cultured mycelia on MMN agar media adjacent to the roots of 40day-old pine seedlings planted in autoclaved mixture mentioned above and cultivated them for 120 days in a clean room (AEG-1000AL, AS ONE, Osaka, Japan) settled in a greenhouse with natural lighting, 65±10 % relative air humidity, and 25/20 °C day/night temperatures. Because the clean room facility has HEPA filter systems that prevent airborne spores Table 1 Selected physicochemical properties of soil used
Values are means±SE (n=3)
Parameter
Value
pH (CaCl2) Sand (%) >0.05 mm Silt (%) 0.05–0.001 mm Clay (%)
ORIGINAL PAPER
Growth and photosynthetic responses of ectomycorrhizal pine seedlings exposed to elevated Cu in soils Yahua Chen & Kazuhide Nara & Zhugui Wen & Liang Shi & Yan Xia & Zhenguo Shen & Chunlan Lian
Received: 16 September 2014 / Accepted: 26 January 2015 # Springer-Verlag Berlin Heidelberg 2015
Abstract It is still controversial whether ectomycorrhizal (ECM) mycelia filter out toxic metals in nutrient absorption of host trees. In this study, pine (Pinus densiflora) seedlings colonized by Cu-sensitive and Cu-tolerant ECM species were exposed to a wide spectrum of soil Cu concentrations to investigate functions of ECM fungi under Cu stress. The photosynthetic rates of intact needles were monitored in situ periodically. The biomass and elements of plants were also measured after harvest. The ameliorating effect of ECM infection on host plants exposed to toxic stress was metal concentration specific. Under lower-level Cu stress, ECM fungi increased seedling performance, while ECM seedlings accumulated more Cu than nonmycorrhizal (NM) seedlings. Under higher-level Cu stress, photosynthesis decreased well before visible symptoms of Cu toxicity appeared. The reduced photosynthesis and biomass in ECM seedlings compared to NM seedlings under higher Cu conditions were also accompanied by lower phosphorus in needles. There was no marked difference between the two fungal species. Our results indicate that the two ECM fungi studied in our system may not have an ability to selectively eliminate Cu in nutrient absorption and may not act as effective barriers that decrease toxic metal uptake into host plants. Y. Chen : Z. Wen : L. Shi : Y. Xia : Z. Shen : C. Lian (*) The Collaborated Lab. of Plant Molecular Ecology (between College of Life Sciences of Nanjing Agricultural University and Asian Natural Environmental Science Center of the University of Tokyo), Nanjing Agricultural University, Nanjing 210095, China e-mail: [email protected] K. Nara Department of Natural Environmental Studies, Graduate School of Frontier Science, The University of Tokyo, 5-1-5 Kashiwanoha, Kashiwa, Chiba 277-8563, Japan C. Lian Asian Natural Environmental Science Center, The University of Tokyo, 1-1-8 Midori-cho, Nishitokyo, Tokyo 188-0002, Japan
Keywords Copper uptake . ECM fungi . Heavy metal-contaminated soils . Metal toxicity . Photosynthesis . Pine (Pinus densiflora)
Introduction Mining activities have produced large quantities of tailings that have caused extensive environmental damage (Adriaensen et al. 2004; Wu et al. 2011). At sites of spoil accumulation, establishment of arboreal vegetation cover helps minimize the migration of heavy metals into surrounding areas and contributes to stabilization and pollution control (Khan et al. 2000; Estaun et al. 2007; Roy et al. 2007; Mendez and Maier 2008). Mine tailings often have toxic levels of metal waste and are deficient in plant macronutrients (Huang et al. 2012), making re-vegetation difficult. Ectomycorrhizal (ECM) fungi are ubiquitous in forested soils, including those highly contaminated with metals; these fungi are thought to play an important role in the metal tolerance of some plants (Godbold et al. 1998; Bellion et al. 2006; Smith and Read 2008; Colpaert et al. 2011). Most studies indicate that inoculation of ECM fungi ameliorates metal toxicity in host plants and promotes the re-vegetation of polluted soils (Jentschke and Goldbold 2000; Van Tichelen et al. 2001; Roy et al. 2007). However, a few investigations have demonstrated that amelioration of metal toxicity by ECM fungi is not universal. Indeed, the fungi sometimes enhance metal uptake by plants or increase the toxicity of heavy metals (Godbold et al. 1998). For example, Jones and Hutchinson (1986) examined the effects of four ECM fungal species (Scleroderma flavidum, Lactarius hibbardae, Lactarius rufus, and Laccaria proxima) on Cu and Ni tolerance in Betula papyrifera. Only inoculation with S. flavidum increased seedling Ni tolerance. Colpaert and van Assche (1992) demonstrated that
Mycorrhiza
inoculation with Thelephora terrestris increases Zn accumulation in the needles of Pinus sylvestris seedlings compared with nonmycorrhizal ones. Although there is no doubt that in many cases ECM fungi do indeed ameliorate metal stress for their host plants, unequivocal evidence for the process and clear understanding of the mechanisms involved are limited. The marked variability in the effects of various mycorrhizal fungi on host plant metal tolerance indicates that this topic warrants further investigation. Diagnosing metal toxicity in plants is not easy and diagnostic methods vary among studies. Many parameters, including plant growth (biomass), nutrient (P or N) uptake (van Tichelen et al. 2001; Adriaensen et al. 2004; Krznaric et al. 2009), water consumption (Colpaert and van Assche 1992), metal uptake, and metal distributions in various organs, tissues, cells, and subcellular components (Denny and Wilkins 1987; Marschner et al. 1996; Frey et al. 2000), have been used as indicators or measures of the Bmetal tolerance capacity^ of plants. Diagnosing metal toxicity is much more difficult in woody plants, which generally grow slowly and react slowly to nutrient deficiencies or metal toxicities (Ingestad and Kahr 1985; Van Tichelen et al. 2001). Thus, visible toxicity symptoms in trees usually appear after long periods of stress exposure (Adriaensen et al. 2006). It is for this reason that previous studies using woody plants have been so protracted, e.g., 8 weeks (Hartley-Whitaker et al. 2000a), 12 weeks (Bradley et al. 1981, 1982; Dixon and Buschena 1988; Kozdroj et al. 2007; Krupa and Kozdroj 2007; Jourand et al. 2010), 16 weeks (Dixon 1988; Colpaert and van Assche 1992), 18 weeks (Jones and Hutchinson 1986), 6 months (Colpaert and van Assche 1993; Hartley-Whitaker et al. 2000b; Zimmer et al. 2009), 8 months (Kim et al. 2004), and 9 months (Adriaensen et al. 2006). A sensitive tool for rapid detection and monitoring of metal toxicity symptoms would promote progress in this field. Photosynthesis is among the most important basic processes of plants and is highly sensitive to environmental changes, including exposure to metal stress (Taiz and Zeiger 2010). There are currently only limited data about the relationship between photosynthesis responses and ECM colonization on plants under metal stress (Jones and Hutchinson 1988a; Huang and Tao 2004; Nguyen et al. 2006). Among the few studies on this topic, most have measured photosynthetic rates at the end of experiments. Consequently, we still poorly understand the change of photosynthetic activities in ECM plants after the initial exposure to metal stress. In this study, we investigate (1) whether ECM colonization alleviates Cu toxicity in plants along various Cu concentrations using a soil system that is relevant to natural conditions; (2) how the photosynthesis of ECM seedlings changes after the exposure to metal stress, with special focus on whether the changes are detectable before visible symptoms of Cu toxicity; and (3) whether the colonizing ECM fungal species (i.e.,
Cu-tolerant and Cu-sensitive fungal species in this study) affects plant performance under Cu stress.
Materials and methods ECM synthesis Based on the results of our previous pure culture experiments using modified Melin-Norkrans (MMN) medium for screening Cu-tolerant ECM fungal species, two ECM fungal species, Pisolithus sp. (Pisolithus sp. 4 sensu Martin et al. 2002) (hereafter, Pt, a Cu-tolerant species) and Cenococcum geophilum (Cg, a Cu-sensitive species), were selected, of which the 50 % effective concentrations (EC50) of Cu (Cu concentration which reduced fungal biomass by 50 %) were 200 and 50 μmol L−1, respectively. These fungi were also chosen for their ease of cultivation, rapid growth rates, and vigorous ECM formation. In total, 120 forty-day-old Japanese red pine (Pinus densiflora) seedlings were prepared. The main root of each was cut at 1 cm below the node to enhance lateral root formation. These seedlings (each with 1 cm of main root remaining) were transplanted into plastic pots (200-mL capacity) in April, filled with an autoclaved (121 °C, 1 h) mixture (1:1, v/v) of Tanashi nursery soil (black sand loam) and Shibanome soil (volcanic sand; Setogahara Co., Gunma, Japan). Physicochemical properties of soils in this study are summarized in Table 1. We combined two inocula to induce ECM formation on the seedlings: cultured mycelia on agar media and soil mycelia radiating from pre-colonized mycorrhizal mother trees. In order to get mother trees, we placed pure cultured mycelia on MMN agar media adjacent to the roots of 40day-old pine seedlings planted in autoclaved mixture mentioned above and cultivated them for 120 days in a clean room (AEG-1000AL, AS ONE, Osaka, Japan) settled in a greenhouse with natural lighting, 65±10 % relative air humidity, and 25/20 °C day/night temperatures. Because the clean room facility has HEPA filter systems that prevent airborne spores Table 1 Selected physicochemical properties of soil used
Values are means±SE (n=3)
Parameter
Value
pH (CaCl2) Sand (%) >0.05 mm Silt (%) 0.05–0.001 mm Clay (%)
