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Mycorrhizae confer aluminum resistance to tulippoplar seedlings Heidi B. Lux and Jonathan R. Cumming
Abstract: Aluminum (Al) toxicity may limit the growth and nutrient acquisition of sensitive tree species in regions receiving acidic deposition. Symbioses between tree roots and mycorrhizal fungi may offset the negative impacts of Al in the root zone. Liriodendron tulipifera L. (tulip-poplar) is an important tree species in the Appalachian Mountains of the southeastern United States and may be at risk from the high levels of acidic deposition in that area. Mycorrhizal and non-mycorrhizal tulip-poplar seedlings were exposed to Al levels of 0, 50, 100, and 200 µM in sand culture for 6 weeks. Mycorrhizal plants accumulated two to seven times the shoot and root biomass of non-mycorrhizal plants and demonstrated no decreases in biomass with Al exposure. Non-mycorrhizal plants exhibited significant reductions in biomass at and above 100 µM Al. Aluminum toxicity in non-mycorrhizal plants appears to be the result of the disruption of P translocation to leaves and Ca, Mg, P, Cu, and Zn uptake in roots. Mycorrhizal plants accumulated 2 and 1.5 times the concentration of Al in shoots and roots, respectively, indicating that Al resistance was not associated with the exclusion of Al from the plant. Patterns of labile Al in solution, nutrients, and Al accumulation in tissues suggest that arbuscular mycorrhizal fungal ecotypes may alter the form or compartmentation of Al within the rhizosphere and plant, thus protecting seedlings from the effects of exposure to Al in the soil solution. Résumé : La toxicité à Al peut limiter la croissance et le prélèvement des nutriments chez les espèces arborescentes sensibles dans les régions où il y a des dépôts acides. Les symbioses entre les racines des arbres et les champignons mycorhiziens peuvent compenser les effets néfastes de Al dans la zone des racines. Le Liriodendron tulipifera L. (tulipier de Virginie) est une essence importante dans les Appalaches du sud-est des États-Unis qui pourrait être menacée par le niveau élevé de dépôts acides dans cette région. Des semis mycorhizés et non mycorhizés de tulipier ont été exposés pendant 6 semaines à des niveaux de 0, 50, 100 et 200 µM de Al dans un substrat de sable. Les plants mycorhizés ont accumulé deux à sept fois plus de biomasse épigée et hypogée que les plants non mycorhizés et n’ont subi aucune réduction de biomasse quel que soit le niveau de Al. Les plants non mycorhizés ont subi des réductions significatives de biomasse à la concentration de 100 µM de Al et plus. Chez les plants non mycorhizés, la toxicité à Al semble être due à une perturbation de la translocation de P vers les feuilles et de l’adsorption de Ca, Mg, P, Cu et Zn par les racines. Les plants mycorhizés ont accumulé respectivement 2 et 1,5 fois la concentration de Al dans les pousses et les racines, ce qui montre que la résistance à Al n’est pas associée à l’exclusion de Al dans la plante. Les patrons de Al labile en solution, les nutriments ainsi que de l’accumulation de Al laissent croire que les champignons mycorhiziens à arbuscules pourraient modifier la forme et le compartimentage de Al dans la rhizosphère et la plante; ce qui protégerait les semis des effets d’une exposition à Al dans la solution de sol. [Traduit par la Rédaction]
Lux and Cumming
Introduction Elevated levels of nitrogen and sulfate deposition from human activity are acidifying forest ecosystems in the United States and Europe (Aber et al. 1998; Johnson and Siccama 1983). As soils acidify in these ecosystems, available aluminum (Al) increases and may become phytotoxic (Godbold et al. 1988a; Joslin and Wolfe 1992; Aber et al. 1998). While many tree species are resistant to Al toxicity (McCormick and Steiner 1978; Kelly et al. 1990), some are Received August 13, 2000. Accepted December 20, 2000. Published on the NRC Research Press Web site on April 13, 2001. H.B. Lux1,2 and J.R. Cumming. Department of Biology, West Virginia University, Morgantown, WV 26506, U.S.A. 1 2
Corresponding author (e-mail:
[email protected]). Present address: The Ecosystems Center, Marine Biological Laboratory, 7 MBL Street, Woods Hole, MA 02543, U.S.A.
Can. J. For. Res. 31: 694–702 (2001)
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highly sensitive, exhibiting reductions in root and shoot growth as well as perturbations in nutrient uptake at Al concentrations observed in the field (Godbold et al. 1988b; Raynal et al. 1990). The presence of Al-sensitive species in areas of elevated acidic deposition may lead to declines in the health of these trees (Aber et al. 1989) and, ultimately, to changes in the composition of these forests. Liriodendron tulipifera L. (tulip-poplar) is a commercially important species in the southeastern United States, an area which receives elevated levels of acidic deposition (National Atmospheric Deposition Program 1993; Adams et al. 1995). In a previous study, we found that tulip-poplar seedlings exhibited sensitivity to Al in the rhizosphere at concentrations as low as 200 µM (Lux and Cumming 1999). Although these seedlings had been field collected and were colonized by arbuscular mycorrhizal (AM) fungi, the lack of nonmycorrhizal controls prompted us to evaluate the response of tulip-poplar with and without AM fungi. The role of mycorrhizal relationships in altering plant response to Al exposure is poorly understood, particularly in tree species (Jones et al.
DOI: 10.1139/cjfr-31-4-694
© 2001 NRC Canada
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Lux and Cumming
1986; Koslowsky and Boerner 1989). Fungal mechanisms, including the maintenance of ion uptake, the binding of Al to fungal hyphae, or the extracellular detoxification of Al in the rhizosphere, may be important for overcoming the stresses associated with Al exposure (Gadd 1993; Meharg and Cairney 2000). Here, we assess the role that mycorrhizal fungi play in modulating the effects of Al on biomass and nutrient acquisition of tulip-poplar seedlings. This study included a nonmycorrhizal treatment and a combined AM inoculum treatment consisting of acidic ecotypes of Glomus clarum (Nicolson and Schenck) and Glomus diaphanum (Morton and Walker). We hypothesized that acidic ecotypes of AM fungi would confer Al resistance to tulip-poplar seedlings by reducing the availability of reactive Al and the associated effects of Al on nutrient acquisition.
Materials and methods Seeds of L. tulipifera (obtained from the Pennsylvania Department of Natural Resources, Springmills, Pa., U.S.A.) were stratified at 4°C for 90 days in damp sand. Following stratification, seeds were watered with deionized H2O until germination and then with a nutrient solution containing 1.2 mM NO3, 0.4 mM NH4, 0.5 mM K, 0.2 mM Ca, 0.05 mM P, 0.1 mM Mg and SO4, 50.5 µM Cl, 25 µM B, 2 µM Mn and Zn, and 0.5 µM Cu, Na, Co, and Mo adjusted to pH 4.0 for 2 months. Seedlings were transplanted into D16 tree tubes (650 cm3 volume, Stuewe and Sons, Corvallis, Oreg., U.S.A.) containing acidwashed sand. One-half of the tubes contained mycorrhizal inoculum cultured on sorghum and sudan grass roots, and the other half contained a pseudoinoculum consisting of sterile sorghum roots. The lower third of these tubes was filled with acid-washed sand, and the top two-thirds of the tube was filled with acid-washed sand and mycorrhizal inoculum (1:10, concentrated inoculum : sand), composed of the species G. clarum and G. diaphanum. Fungal species were cultured from an abandoned coal mine site in West Virginia with a soil pH of 4.1 and Al concentrations in surface water as high as 600 µM (International Culture Collection of Arbuscular and Vesicular-Arbuscular Mycorrhizal Fungi (INVAM) collection of Dr. Joseph Morton, West Virginia University, Morgantown, W. Va., U.S.A.). The inoculum was sieved to remove as much soil as possible before mixing with sand. Funnels were installed under a subset of all tubes to collect and monitor leachate chemistry. Blank tubes, filled with sand and inoculum or pseudoinoculum, were used to monitor the effects of the tubes and sand on solution chemistry. Transplanted seedlings were placed in a growth chamber with a 14 h light : 10 h dark photoperiod and a day:night regime of 24:19°C. Photosynthetically active radiation was 220 µmol·m–2·s–1, and relative humidity was maintained at 70%. Plants were watered three times daily with 25 ml of nutrient solution with a pH adjusted to 4.0. The sand matrix in each tube retained 125 ml of nutrient solution. Seedlings were acclimated to the growth chamber environment for 3 weeks prior to the commencement of Al treatments. Seedling shoots and roots weighed 0.100 ± 0.013 g and 0.065 ± 0.007 g (mean ± SE), respectively, and mean shoot height, measured from the bottom of the cotyledons, was 2.21 ± 0.07 cm at the start of Al treatments. At the end of the acclimation period, Al treatments of 0, 50, 100, and 200 µM Al (as Al2(SO4)3) were added to nutrient solutions of the same composition listed above. All solutions were titrated to a pH of 4.0 with HCl or NaOH to prevent precipitation of Al. Leachate was collected once every other week throughout the experiment, and pH and reactive Al concentrations (American Public Health Association 1985) were measured.
695 Plants were harvested at the end of a 6-week exposure period. Stems were clipped at the sand surface, washed in a 0.1% Tween 80 solution and rinsed with deionized H2O to remove surface contamination. Leaves were separated from stems, and leaf area was measured with a LI-COR 3100 leaf area meter (LI-COR, Lincoln, Nebr., U.S.A.). Leaves were then dried at 60°C for 24 h and weighed. Roots were rinsed free of sand, weighed, and a subsample was stained to quantify mycorrhizal colonization. These root subsamples were cleared with boiling KOH and then stained using trypan blue dye. Colonization was quantified using the gridline-intersect method (Giovannetti and Mosse 1985). The remaining root sample was dried at 60°C for 24 h and weighed. All dried leaf and root tissue was ground to pass a 1-mm screen. Subsamples of tissue taken from the entire ground, homogenized sample were digested using a H2SO4–H2O2 digest (Parkinson and Allen 1975) and analyzed on a Thermo Jarrell-Ash 965 inductively coupled argon plasma emission spectrophotometer for Al and nutrients by the Analytical Laboratory at the University of Georgia (Athens, Ga., U.S.A.). Treatments were arranged in a 2 × 4 factorial design with 11 replicate seedlings per treatment. Treatment factors were mycorrhizal inoculation (two levels) and solution Al (four levels). Three replicates of each treatment were pooled for nutrient analyses (n = 3 for these tests). Treatment effects on seedling leaf area, biomass, and elemental ion concentrations of leaf and root samples were evaluated using a two-way analysis of variance. Differences between treatments were considered significant at P < 0.05. Analysis of covariance, using initial seedling height measurements, revealed initial differences in height between treatments, which account for 0.41 and 2.16% of the variation in the model for final shoot and root mass, respectively (data not presented). Residuals of root biomass, shoot biomass, and leaf area exhibited heterogeneous variances, and data were log transformed prior to analyses. Least square means and standard errors for nontransformed data are presented in the figures and tables. Means for mycorrhizal and non-mycorrhizal treatments were examined separately for the significance of the Al response, using the Tukey–Kramer honestly significant difference (HSD) test. Regression analyses were used to assess patterns of accumulation and relationships between nutrients and biomass for nutrition variables. All analyses were conducted using the statistical package JMP, version 3 (SAS Institute Inc., Cary, N.C., U.S.A.).
Results Experimental treatments Mycorrhizal colonization of seedlings by an inoculum containing the AM species G. clarum and G. diaphanum was highly successful, with root colonization ranging between 82 and 100% in inoculated seedlings (data not presented). There was no relationship between colonization and Al treatment. Noninoculated seedlings had 0 to 5% root colonization. While Al and H+ concentrations in leachate solutions for mycorrhizal and non-mycorrhizal seedlings were initially similar, solution chemistry deviated as the experiment progressed (Fig. 1). Solutions collected from mycorrhizal plants exhibited significantly lower reactive Al concentrations and higher pH than reference (blank) lysimeters, whereas solutions from non-mycorrhizal seedlings exhibited higher solution Al concentrations and lower pH than reference lysimeters (Fig. 1). Biomass The biomass of mycorrhizal tulip-poplar seedlings was greater than that of non-mycorrhizal seedlings across all Al © 2001 NRC Canada
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696 Fig. 1. Mean change in reactive Al (top panel) and H+ (bottom panel) concentrations in leachate between lysimeters containing mycorrhizal (myc) or non-mycorrhizal (non-myc) tulip-poplar seedlings and lysimeters containing no plants. Error bars are standard errors.
treatments. Shoot and root mass were three- to five-fold greater in mycorrhizal seedlings than in non-mycorrhizal seedlings (P < 0.01) (Fig. 2). Leaf area and mass followed a similar pattern, and were, respectively, 5 and 6.5 times greater in mycorrhizal plants than in non-mycorrhizal plants (Table 1). Mycorrhizal plants allocated proportionally less carbon to roots than non-mycorrhizal plants, as demonstrated by a root/shoot ratios of 0.66 for mycorrhizal plants compared with a value of 0.77 for non-mycorrhizal plants, at the 0 µM Al level (Table 1). The biomass of non-mycorrhizal, but not mycorrhizal, tulip-poplar seedlings was significantly less when exposed to Al in solution (Fig. 2). Leaf area and mass of non-mycorrhizal seedlings exposed to Al were up to 52% less than the values obtained for the non-mycorrhizal controls, whereas there were no differences in leaf area and
Can. J. For. Res. Vol. 31, 2001 Fig. 2. Final biomass of shoots and roots of mycorrhizal (top panel) and non-mycorrhizal (bottom panel) tulip-poplar seedlings exposed for 6 weeks to Al in sand culture. Error bars are standard errors.
mass of mycorrhizal seedlings exposed to Al treatments and their controls (Table 1). Aluminum also influenced the allocation of carbon between roots and shoots in nonmycorrhizal seedlings, having a greater negative effect on shoot biomass (Fig. 2, Table 1). Elemental concentrations The accumulation of Al in seedling leaves was dependent on mycorrhizal colonization (P < 0.01 for the mycorrhizae × Al interaction). Leaves of mycorrhizal plants accumulated Al at concentrations up to 423 µg·g–1, in direct proportion with the solution Al concentration. The concentration of Al in leaves of non-mycorrhizal plants increased to approximately 200 µg·g–1 at and above 50 µM Al (Fig. 3). Roots of mycorrhizal plants accumulated greater concentrations of Al than roots of non-mycorrhizal plants (P < 0.01), although © 2001 NRC Canada
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Table 1. Least square means for leaf area, leaf mass, and root/shoot ratio of tulip-poplar seedlings as influenced by mycorrhizal inoculation and aluminum (Al) in solution. Inoculation
Al (µM)
Leaf area (cm2)
Leaf mass (mg)
+Mycorrhiza
0 50 100 200 0 50 100 200
315.8 289.3 278.8 287.6 66.7a 47.3ab 36.7bc 32.6c
1137.9 1137.1 1059.7 1057.3 241.4a 177.8ab 147.4bc 116.7bc
10.7
