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Mobilization of Inorganic Phosphorus from Soils by Ectomycorrhizal Fungi
Pedosphere 24(5): 683–689, 2014 ISSN 1002-0160/CN 32-1315/P c 2014 Soil Science Society of China Published by Elsevier B.V. and Science Press
Mobilization of Inorganic Phosphorus from Soils by Ectomycorrhizal Fungi∗1 ZHANG Liang1,2 , WANG Ming-Xia1 , LI Hua1 , YUAN Ling1 , HUANG Jian-Guo1,∗2 and C. PENFOLD3 1 College
of Resources and Environment, Southwest University, Chongqing 400716 (China) of Biotechnology and Chemical Engineering, Taizhou College, Nanjing Normal University, Taizhou 225300 (China) 3 School of Agriculture, Food and Wine, The University of Adelaide, Adelaide SA 5005 (Australia) 2 School
(Received August 21, 2013; revised April 30, 2014)
ABSTRACT Ectomycorrhizal (EM) fungi could form symbiosis with plant roots and participate in nutrient absorption; however, many EM species commonly found in forest soils, where phosphorus (P) concentration and availability are usually very low, particularly in tropical and subtropical areas, have not yet been investigated for their efficiencies to mobilize soil P. In this study, fungal growth, P absorption, efflux of protons and organic acids, and soil P depletion by four isolates of EM fungi isolated either from acidic or calcareous soils were compared in pure liquid culture using soil as a sole P source. Boletus sp. 7 (Bo 7), Lactarius deliciosus 3 (Ld 3), and Pisolithus tinctorius 715 (Pt 715) from acidic and P-deficient soils of southwestern China showed higher biomass and P concentration and accumulation than Cenococcum geophilum 4 (Cg 4) from a calcareous soil of Inner Mongolia, northern China, after 4 weeks of liquid culture. Oxalate, malate, succinate, acetate, and citrate concentrations in the culture solutions varied significantly with fungal species, and oxalate accounted for 51.5%–91.4% of the total organic acids. Organic acids, particularly oxalate, in the culture solutions may lead to the solubilization of iron-bound P (Fe-P), aluminum-bound P (Al-P), and occluded P (O-P) from soil phosphates. Fungal species also varied greatly in proton efflux, which decreased the culture solution pH and may dissolve calcium-bound P (Ca-P) in soil. This could be the reason for the increment of both inorganic P in the culture solutions and Olsen P in the soil when EM fungi were present. Total inorganic P, the sum of Al-P, Fe-P, O-P, and Ca-P, in the culture solutions was positively correlated with the total concentration of organic acids in the culture solutions (r = 0.918*, n = 5), but negatively with both the total inorganic P in soil (r = −0.970**, n = 5) and the culture solution pH (r = −0.830*, n = 5). These suggested variable efficiencies of EM fungal species to mobilize inorganic P fractions from soil, which could make EM trees to utilize inorganic P in the same way like EM fungi and adapt to the soils with various P concentrations and availabilities. Key Words:
EM fungal species, inorganic P, organic acid, P availability, proton, soil phosphate
Citation: Zhang, L., Wang, M. X., Li, H., Yuan, L., Huang, J. G. and Penfold, C. 2014. Mobilization of inorganic phosphorus from soils by ectomycorrhizal fungi. Pedosphere. 24(5): 683–689.
INTRODUCTION Phosphorus (P) is one of the most important essential elements for plant growth. However, both P concentration and availability are very low in most forest soils, particularly in tropical and subtropical areas because of intensive weathering and leaching. Usually no fertilizers are added into forest soils. Trees therefore must mobilize and extract P from insoluble phosphates in soils to satisfy their P requirement (Barroso and Nahas, 2005). Trees may interact with thousands of ectomycorrhizal (EM) fungal species in natural forests and forest plantations (Smith and Read, 2008). EM fungal species cover the roots and rootlets of trees with a thick man∗1 Supported
tle of hyphae, effectively enlarging the surface areas of the roots in contact with phosphates through soil pores that are not accessible to the root hairs (Hayek et al., 2012; B¨ ucking, 2004). Therefore, EM inoculation might stimulate the growth of host trees in P-deficient soils by improving P nutrition in artificial plantations. The identification of growth and nutrient uptake and mobilization is a key issue for understanding the nutrition of EM plants and selecting the fungal species as inoculants for producing EM tree seedlings in nursery beds (Chalot et al., 2002). Huang and Lapeyrie (1996) reported the improvement of P nutrition in Douglas fir seedlings inoculated by Laccaria bicolor S238N and the concomitant plant growth acceleration. Similarly, the growth of Pinus sylvestris seedlings was stimulated by
by the National Basic Research Program (973 Program) of China (No. 2013CB127405), the National Natural Science Foundation of China (Nos. 40771112 and 41171215), and the Technology Innovation Program of Southwest University of China (No. Ky2009022). ∗2 Corresponding author. E-mail: [email protected].
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inoculation of Paxillus involutus grown in media with strengite as a P source (Gibson and Mitchell, 2004). Courty et al. (2006) studied numerous EM fungi in vitro and found that Xerocomus chrysenteron sp. dissolved mineral phosphates efficiently and also promoted the growth of host trees. This fungal isolate could thus be potentially used in forestation in Pdeficient soils. Moreover, simultaneous release of P from apatite leads to the corruption of mineral structures by EM fungi. This biological decomposition, resulting in the release of otherwise unavailable P from minerals, seems related to the efflux of organic acids, particularly those with low molecular weights, and protons from fungal hyphae (Wang et al., 2011). Wallander (2000) found high concentrations of citric and oxalic acids in the culture media with mineral phosphates as a sole P source when pine roots were colonized by Suillus variegatus. P absorption by EM fungi was increased in liquid culture media with concomitant oxalate efflux (Ahonen-Jonnarth et al., 2000). Thus, the extramatrical mycelia extending from the mantle may play an important role in P mobilization (Tibbett et al., 1998). The release of organic acids may be a mechanism for EM fungi to mobilize and then utilize P from fluorapatite, chlorapatite, and hydroxyapatite (Adeleke et al., 2010; Gui˜ naz´ u et al., 2010). However, EM fungi varied greatly in the abilities to promote the growth and P uptake of trees, such as Pinus pinaster, P. menziesii, P. asperata, P. massoniana, Eucalyptus diversicolor, and E. globulus, after forming ectomycorrhizas (Pampolina et al.; 2002; Thomas et al., 2006; Sousa et al., 2011; Oliveira et al., 2012; Reis et al., 2012). Many EM species commonly found in forests have not yet been investigated on their efficiencies to mobilize soil P. More direct evidence for soil P exploitation by EM fungi is necessary in order to understand the functions of EM fungi in tree nutrition and their applications in artificial forestation. The aims of the present study were i) to evaluate the capacity of EM fungal isolates from different forest ecosystems to mobilize soil P pools in vitro, ii) to examine inorganic P fractions removed from soil using a sequential extraction procedure, and iii) to extend the explanation of P mobilization mechanisms by EM fungi through comparing the efflux of protons and organic acids. MATERIALS AND METHODS Fungal strains Four EM fungi, namely Boletus sp. 7 (Bo 7), Lac-
tarius deliciosus 3 (Ld 3), Cenococcum geophilum 4 (Cg 4) and Pisolithus tinctorius 715 (Pt 715), were used for the experiment. Bo 7, Ld 3 and Pt 715 were obtained from the Microbiology Laboratory, Southwest University, Chongqing, China and Cg 4 was provided by Dr. Shulan Bai of Inner Mongolia Agricultural University, China. The isolates of Bo 7 and Ld 3 were originally isolated from a yellow soil (pH 4.0) in the Jinfu Mountain, Chongqing, southwestern China and Cg 4 from a calcareous soil (pH 7.3) in the Daqing Mountain, Inner Mongolia, northern China. Bo 7, Ld 3 and Cg 4 were all from mycorrhizal Pinus sp. Pt 715 was isolated from eucalyptus mycorrhizas in a red soil (pH 4.5) in Xichang, Sichuan Province, southwestern China. Mycelia for inoculation were grown on Pachlewski agar medium for two weeks (25 ± 1 ◦ C). The medium contained 0.5 g L−1 tartrate, 1.0 g L−1 KH2 PO4 , 0.5 g L−1 MgSO4 , 20 g L−1 glucose, 5.0 g L−1 maltose, 0.1 g L−1 vitamin B1 , 20 g L−1 agar, and 1 mL L−1 microelement solution consisting of 8.45 mg L−1 H3 BO3 , 5 mg L−1 MnSO4 , 6 mg L−1 FeSO4 , 0.625 mg L−1 CuSO4, 2.27 mg L−1 ZnCl2 , and 0.27 mg L−1 (NH4 )2 MoO4 . Soil preparation A purple soil (typical Udorthent according to USDA Soil Taxonomy) derived from muddy sedimentary rocks was collected from the experimental farm (29◦ 49 18 N, 106◦ 25 45 E) of Southwestern University, Chongqing, China, air-dried, ground to pass a 0.5mm sieve, and then steam-sterilized at 121 ◦ C for 30 min. The soil was sandy loam in texture with a pH of 6.8 and contained 0.4 g kg−1 total N, 0.6 g kg−1 total P, 30.0 g kg−1 total K, 10.7 mg kg−1 Olsen P, 21.2 mg kg−1 , aluminum-bound P (Al-P), 28.1 mg kg−1 iron-bound P (Fe-P), 69.9 mg kg−1 occluded P (O-P), and 290.9 mg kg−1 calcium-bound P (Ca-P). 1.00 g soil was placed into a plastic tube (2 cm length × 1 cm diameter) closed at the two ends by microporous membrane (0.22 μm aperture). This membrane allowed water, inorganic ions, and organic acids to pass freely but prevented soil particles from penetration. Experimental procedure Pachlewski liquid medium was prepared with KH2 PO4 replaced by KCl at an equivalent amount of potassium. Into each 150 mL Erlenmeyer flask 20 mL of the Pachlewski liquid medium was transferred and 1.00 g soil prepared in the plastic tube closed at the two ends by microporous membrane was added as a sole P source. After steam sterilization at 121 ◦ C for 30 min, each flask was inoculated with a plug of mycelium (5
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mm in diameter). In order to realize the extraction of soil P pools by fungal isolates, blank control flasks were set up in the same way except that no EM fungi were inoculated. All flasks were incubated statically for 4 weeks at 25 ± 1 ◦ C in the dark. There were 12 replicate flasks of each treatment in a fully randomized design. Sampling and sample analysis After 4 weeks of incubation, EM fungi were harvested by filtration and washed with deionized water to remove culture medium from the mycelium surface. They were then oven-dried, weighed, and digested with H2 SO4 -H2 O2 . The soil in the plastic tubes were air-dried and sequentially extracted with 0.5 mol L−1 NH4 F for Al-P, 0.1 mol L−1 NaOH for Fe-P, 0.5 mol L−1 H2 SO4 for Ca-P, and 0.3 mol L−1 C6 H5 Na3 O7 -1.0 g Na2 S2 O4 -0.5 mol L−1 NaOH for O-P by shaking at 150 r min−1 and 30 ◦ C for 30 min (soil:solution ratio = 1:50) (Chang and Jackson, 1957). Inorganic P in the digests, culture solutions, and extracts were analyzed by the molybdenum blue methods (Ames, 1966). The culture solutions were also analyzed for pH with a pH meter and for oxalate by high performance liquid chromatography (HPLC) with detection at 210 nm. For analysis of organic acids, the culture solutions (20 μL) were injected into an Ion-300 organic acid analysis column (Phenomenex, Torrance, USA) with 2.5 mmol L−1 H2 SO4 mobile phase at 0.5 mL min−1 and 3.1 × 106 Pa. Standard organic acids were prepared and run before and after the culture solutions (Fig. 1).
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(version 9, SAS Institute, Cary, USA). Significant differences between means were tested using Fisher’s protected least significant difference (LSD) at P < 0.05. RESULTS Fungal growth and P absorption As shown in Table I, the four EM fungal isolates varied significantly in growth rate, with Bo 7 showing the highest mean biomass of about 46 mg flask−1 , followed by Pt 715 (39 mg flask−1 ), Ld 3 (34 mg flask−1 ), and Cg 4 (27 mg flask−1 ). EM fungi also varied remarkably in P concentrations in their mycelia, which ranged from 4.01 mg g−1 dry weight (DW) (Ld 3) to 2.41 mg g−1 DW (Cg 4). P accumulation in Pt 715, Ld 3, and Bo 7 (135.6–142.0 μg flask−1 ) did not differ significantly, but was more than 2-fold that of Cg 4. TABLE I Biomass and P concentration and accumulation in ectomycorrhizal (EM) fungal isolates after 4 weeks of incubation in Pachlewski liquid medium with KH2 PO4 replaced by KCl and 1.00 g soil added as a sole P source EM fungal isolatea) Bo 7 Cg 4 Ld 3 Pt 715
Biomass
P concentration
P accumulation
mg flask−1 45.66±2.04c) ad) 27.16±2.59d 33.82±1.71c 39.00±2.71b
mg g−1 DWb) 3.11±0.29b 2.41±0.28c 4.01±0.31a 3.52±0.23ab
μg flask−1 142.00±7.49a 65.46±5.70b 135.62±7.48a 137.28±9.82a
a) Bo 7 = Boletus sp. 7; Ld 3 = Lactarius deliciosus 3; Cg 4 = Cenococcum geophilum 4; Pt 715 = Pisolithus tinctorius 715. b) Dry weight. c) Mean±standard deviation (n = 12). d) Mean values followed by the same letter(s) in a column are not significantly different at P < 0.05 as determined by Fisher’s least significant difference test.
pH, organic acids and inorganic P in culture solutions
Fig. 1 Chromatogram of standard organic acids. AU = arbitrary unit.
Statistical analysis Treatment effects were evaluated by analysis of variance using the SAS statistical software package
The EM fungal isolates decreased the culture solution pH remarkably (P < 0.05). pH values in the culture solutions with Ld 3, Pt 715, and Bo 7 were much lower than that with Cg 4 (Fig. 2). All EM fungi exuded oxalate and acetate, but varied significantly in the exudation rates (Table II). Oxalate accounted for a large proportion (51.5%–91.4%) of the total organic acids. Malate was found in the culture solution with Ld 3, succinate with Ld 3 and Pt 715, and citrate with Bo 7 and Ld 3. The EM fungi species varied significantly in the sum of organic acids, which ranged from 35.4 mg L−1 (Bo 7) to 23.0 mg L−1 (Pt 715) in the culture solutions (Table II). After incubation with fungal inoculation, total inorganic P in culture solutions was remarkably higher than the control without fungal ino-
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TABLE II Organic acids in culture solutions after 4 weeks of incubation with ectomycorrhizal (EM) fungi in Pachlewski liquid medium with KH2 PO4 replaced by KCl and 1.00 g soil added as a sole P source EM fungal straina) Bo 7 Cg 4 Ld 3 Pt 715
Oxalate 30.50±1.86b) ac) 21.75±1.21b 16.07±1.13bc 11.83±1.12c
Malate NDd) ND 1.36±0.12 ND
Succinate mg ND ND 0.39±0.07a 0.51±0.09a
Acetate
Citrate
Total
2.54±0.41a ND 2.33±0.31a ND
35.36±2.68a 23.80±1.51c 28.04±2.49b 22.95±2.21c
L−1 2.32±0.36c 2.05±0.28c 7.89±0.79b 10.61±0.91a
a) Bo
7 = Boletus sp. 7; Ld 3 = Lactarius deliciosus 3; Cg 4 = Cenococcum geophilum 4; Pt 715 = Pisolithus tinctorius 715. deviation (n = 12). c) Mean values followed by the same letter(s) in each column are not significantly different at P < 0.05 as determined by Fisher’s least significant difference test. d) Not detected. b) Mean±standard
Fig. 2 Culture solution pH after 4 weeks of incubation with or without ectomycorrhizal (EM) fungi in Pachlewski liquid medium with KH2 PO4 replaced by KCl and 1.00 g soil added as a sole P source. Values are means with standard deviations shown by vertical bars (n = 12). Bars with the same letter indicate no significant difference (P < 0.05). CK = control without EM fungal inoculation; Bo 7 = Boletus sp. 7; Ld 3 = Lactarius deliciosus 3; Cg 4 = Cenococcum geophilum 4; Pt 715 = Pisolithus tinctorius 715.
Fig. 3 Total inorganic P in culture solutions after 4 weeks of incubation with or without ectomycorrhizal (EM) fungi in Pachlewski liquid medium with KH2 PO4 replaced by KCl and 1.00 g soil added as a sole P source. Values are means with standard deviations shown by vertical bars (n = 12). Bars with the same letter indicate no significant difference (P < 0.05). CK = control without EM fungal inoculation; Bo 7 = Boletus sp. 7; Ld 3 = Lactarius deliciosus 3; Cg 4 = Cenococcum geophilum 4; Pt 715 = Pisolithus tinctorius 715.
culation, and the concentrations of total inorganic P were similar in the solutions (1.56–1.86 mg L−1 ) with fungal inoculation (Fig. 3).
O-P. All four EM fungi significantly lowered soil Ca-P as compared to the control and Ca-P in the soils varied slightly among EM fungi (223.9–236.3 mg kg−1 ). The total inorganic P, the sum of Al-P, Fe-P, O-P, and CaP, in the soils after incubation with EM fungi (Table III) also decreased as compared to the initial soil value, but no significant differences were found among fungal species. Olsen P in soil after incubation with EM fungi ranged from 11.6 to 12.83 mg kg−1 , significantly higher than the initial soil value (10.82 mg kg−1 ). No significant variation in Olsen P in the residual soil was observed among EM fungi inoculated.
Residual soil P fractions Bo 7 and Cg 4 significantly decreased Al-P in the soil from 19.45 mg kg−1 (control without EM fungi) to 14.20 and 13.88 mg g−1 , respectively, whereas there was no significant change in soil Al-P after incubation with Pt 715 and Ld 3 (Table III). Fungal inoculation generally led to a significant depression of Fe-P in the soil, except for Pt 715 which removed little of this P fraction from the soil. Fe-P was similar in the soil with fungal compared to the control without EM fungi. Bo 7, Ld 3, and Cg 4 depleted significantly O-P from the soil in contrast to Pt 715 which extracted scarcely any
Correlation analysis between culture solution pH, inorganic P and organic acids As shown in Table IV, total inorganic P in culture
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TABLE III Variations in inorganic P fractionsa) and Olsen P in soil after 4 weeks of incubation with or without ectomycorrhizal (EM) fungi in Pachlewski liquid medium with KH2 PO4 replaced by KCl and 1.00 g soil added as a sole P source EM fungal strainb) CK Bo 7 Cg 4 Ld 3 Pt 715
Al-P 19.45±1.76d) ae) 14.20±1.23b 13.88±1.45b 15.92±1.61ab 17.14±1.81ab
Fe-P 25.55±2.07a 15.11±1.45b 18.79±1.98b 18.54±1.65b 21.25±1.99ab
O-P
Ca-P
mg 66.51±5.45a 45.19±3.78bc 50.06±4.01b 43.36±3.98c 58.59±4.97ab
kg−1 286.96±13.12a 236.34±12.18b 233.42±13.76b 227.60±10.98b 223.90±11.07b
Total inorganic Pc)
Olsen P
398.47±19.87a 310.84±18.17b 316.15±20.16b 305.42±16.67b 320.88±19.32b
10.82±0.91b 11.82±0.88a 11.65±0.69a 12.83±0.76a 11.60±0.82a
a) Al-P
= aluminum-bound P; Fe-P = iron-bound P; O-P = occluded P; Ca-P = calcium-bound P. = control without EM fungal inoculation; Bo 7 = Boletus sp. 7; Ld 3 = Lactarius deliciosus 3; Cg 4 = Cenococcum geophilum 4; Pt 715 = Pisolithus tinctorius 715. c) Sum of Al-P, Fe-P, O-P, and Ca-P. d) Mean±standard deviation (n = 12). e) Mean values followed by the same letter(s) in each column are not significantly different at P < 0.05 as determined by Fisher’s least significant difference test. b) CK
TABLE IV Correlation coefficients between total inorganic P in culture solutions, total inorganic P in soil, total concentration of organic acids in culture solutions, and culture solution pH after 4 weeks of incubation with or without ectomycorrhizal (EM) fungi in Pachlewski liquid medium with KH2 PO4 replaced by KCl and 1.00 g soil added as a sole P source Factor
Total inorganic P in culture solutions
Total inorganic P in soil
Total concentration of organic acids in culture solutions
Total inorganic P in soil Total concentration of organic acids in culture solutions Culture solution pH
−0.970** 0.918* −0.830*
−0.935* 0.824
−0.796
*, **Significance at P < 0.05 and P < 0.01, respectively.
solutions was positively correlated with the total concentrations of organic acids in culture solutions (r = 0.918*, n = 5), but negatively with both the total inorganic P in soil (r = −0.970**, n = 5) and the culture solution pH (r = −0.830*, n = 5). There was also a negative correlation between the total inorganic P in soil and the total concentration of organic acids in culture solutions (r = −0.935*, n = 5). DISCUSSION Hyphal P uptake from soil was higher in the isolates Bo 7, Ld 3, and Pt 715 than Cg 4. This was due mainly to the larger biomass of Bo 7, Ld 3, and Pt 715 and higher P concentrations in their mycelia than Cg 4. In a previous study, however, we found that Cg 4, Ld 3, and Pt 715 had similar biomass when grown in a Pachlewski liquid medium with KH2 PO4 as a sole P source (Wang et al., 2012). The large reduction in both biomass and P uptake of Cg 4 suggested the poor efficiency of Cg 4 to extract P from soils. Cg 4 was originally isolated from a calcareous soil rich in available P in Inner Mongolia, northern China, while the other three isolates came from subtropical soils of southern China, which
are acidic and deficient in mineral nutrients, in particular P (Yuan et al., 2004). EM fungal species could thus vary in their abilities to adapt to the soils with different P concentrations and availabilities (Miller et al., 1995). EM fungi isolated from P-deficient soils should be able to adapt to poor P conditions and evolve a high efficiency to extract P from soils. Our findings that the EM fungi studied had variable capacities to extract soil P are consistent with the reports of Pampolina et al. (2002), Thomas et al. (2006), Sousa et al. (2011), and Reis et al. (2012), who studied the effectiveness of fungal isolates in forming ectomycorrhizas with the host trees, including E. globulus, P. menziesii, P. asperata, P. massoniana, and E. diversicolor, and found that the fungal isolates varied significantly in their abilities to promote host tree growth and P uptake. EM fungi play a key role in P uptake of host trees. Various mechanisms have been suggested for P uptake by EM trees through the external hyphae from mantles which allow root systems to exploit a greater volume of soil P, including i) extending away from roots and transporting P from some distance to the roots, ii) exploiting smaller soil pores not reached by rootlets, and
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iii) enlarging surface areas for absorption (O’keefe and Sylvia, 1992). However, in recent years greater attention has been paid to P mobilization by microbial processes (Ramani, 2011). We detected malate, succinate, acetate, citrate, and particularly oxalate in culture solution inoculated with EM fungi. Many researchers also detected high concentrations of organic acids (about 1 000 μmol L−1 ) produced by EM fungi in soil in European forests (Van Hees et al., 2005; Plassard and Fransson, 2009; Courty et al., 2010). In this study, all EM fungi could exude oxalate and protons but varied greatly in the efflux rates. Pt 715 effused a larger amount of protons into culture solution in contrast to Bo 7 which showed the highest oxalate efflux rate. Significant negative correlations were found between the total inorganic P and pH in culture solutions (r = −0.830*, n = 5) and between the total inorganic P (the sum of Al-P, Ca-P, Fe-P, and O-P) in soil and the total concentration of organic acids in the culture solutions (r = −0.935*, n = 5). Both (Al (C2 O4 )3 )3− and (Fe (C2 O4 )3 )3− have very high chelation constants of 16.3 and 20.2, respectively. Oxalate may therefore chelate Al3+ and Fe3+ in the insoluble phosphates such as Al phosphate, Fe phosphate, and occluded phosphate, resulting in the solubilization of these phosphates and P release (Cromack et al., 1979; Feng, et al., 2011). Solubility of Ca phosphate increased with decreasing solution pH and protons may thus have a strong capacity to dissolve Ca phosphate in soils (Shen et al., 2011). EM fungi varied significantly in both organic acid and proton efflux, suggesting their different efficiencies to mobilize and utilize inorganic P fractions in soils. The host trees which form symbiosis with EM fungi could therefore utilize inorganic P from soils with various P concentrations and availabilities. The decreased inorganic P fractions in soil as a sole P source in culture solutions could explain why EM fungi increased Olsen P, usually available for plants, in the soil and increased total inorganic P in culture solutions. The biodiversity of these EM fungi which varied significantly in the efflux of organic acids and protons and showed diverse abilities to mobilize and utilize inorganic P fractions in soils could enable EM trees to adapt to different soil P conditions. Trees in natural forests and forest plantations may interact with hundreds of EM fungal species; therefore, further studies are required to investigate the possible biomobilization of P from phosphates by EM fungi in the fields. Biological solubilization of phosphates could be more important and thus merits investigation under field conditions.
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REFERENCES Adeleke, R. A., Cloete, T. E., Bertrand, A. and Khasa, D. P. 2010. Mobilisation of potassium and phosphorus from iron ore by ectomycorrhizal fungi. World J. Microb. Biot. 26: 1901–1913. Ahonen-Jonnarth, U., Van Hees, P. A. W., Lundstr¨ om, U. S. and Finlay, R. D. 2000. Organic acids produced by mycorrhizal Pinus sylvestris exposed to elevated aluminum and heavy metal concentrations. New Phytol. 146: 557–567. Ames, B. N. 1966. Assay of inorganic phosphate, total phosphate and phosphatases. In Colowick, S. P. and Kaplan, N. O. (eds.) Methods in Enzymology. Vol. 8. Academic Press, New York. pp. 115–118. Barroso, C. B. and Nahas, E. 2005. The status of soil phosphate fractions and the ability of fungi to dissolve hardly soluble phosphates. Appl. Soil Ecol. 29: 73–83. B¨ ucking, H. 2004. Phosphate absorption and efflux of three ectomycorrhizal fungi as affected by external phosphate, cation and carbohydrate concentrations. Mycol. Res. 108: 599–609. Chalot, M., Javelle, A., Blaudez, D., Lambilliote, R., Cooke, R., Sentenac, H., Wipf, D. and Botton, B. 2002. An update on nutrient transport processes in ectomycorrhizas. Plant Soil. 244: 165–175. Chang, S. C. and Jackson, M. L. 1957. Fractionation of soil phosphorus. Soil Sci. 84: 133–144. Courty, P. E., Bu´ ee, M., Diedhiou, A. G., Frey-Klett, P., Tacon, F. L., Rineau, F., Turpault, M.-P., Uroz, S. and Garbaye, J. 2010. The role of ectomycorrhizal communities in forest ecosystem processes: New perspectives and emerging concepts. Soil Biol. Biochem. 42: 679–698. Courty, P. E., Pouysegur, R., Bu´ee, M. and Garbaye, J. 2006. Laccase and phosphatase activities of the dominant ectomycorrhizal types in a lowland oak forest. Soil Biol. Biochem. 38: 1219–1222. Cromack, K. Jr., Sollins, P., Graustein, W. C., Speidel, K., Todd, A. W., Spycher, G., Li, C. Y. and Todd, R. L. 1979. Calcium oxalate accumulation and soil weathering in mats of the hypogeous fungus Hysterangium crassum. Soil Biol. Biochem. 11: 463–468. Feng, M. H., Ngwenya, B. T., Wang, L., Li, W., Olive, V. and Ellamd, R. M. 2011. Bacterial dissolution of fluorapatite as a possible source of elevated dissolved phosphate in the environment. Geochim. Cosmochim. Acta. 75: 5785–5796. Gibson, B. R. and Mitchell, D. T. 2004. Nutritional influences on the solubilization of metal phosphate by ericoid mycorrhizal fungi. Mycol. Res. 108: 947–954. Gui˜ naz´ u, L. B., Andr´ es, J. A., Del Papa, M. F., Pistorio, M. and Rosas, S. B. 2010. Response of alfalfa (Medicago sativa L.) to single and mixed inoculation with phosphate-solubilizing bacteria and Sinorhizobium meliloti. Biol. Fert. Soils. 46: 185–190. Hayek, S., Grosch, R., Gianinazzi-Pearson, V. and Franken, P. 2012. Bioprotection and alternative fertilisation of petunia using mycorrhiza in a soilless production system. Agron. Sustain. Dev. 32: 765–771. Huang, J. G. and Lapeyrie, F. 1996. Ability of ectomycorrhizal fungus Laccaria bicolor S238N to increase the growth of Douglas fir seedlings and their phosphorus and potassium uptake. Pedosphere. 6: 217–224. Miller, R. M., Reinhardt, D. R. and Jastrow, J. D. 1995. External hyphal production of vesicular-arbuscular mycorrhizal fungi in pasture and tallgrass prairie communities. Oecologia. 103: 17–23. O’keefe, D. M. and Sylvia, D. M. 1992. Chronology and mecha-
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nism of P uptake by mycorrhizal sweet potato plants. New Phytol. 122: 651–659. Oliveira, R. S., Franco, A. R. and Castro, P. M. L. 2012. Combined use of Pinus pinaster plus and inoculation with selected ectomycorrhizal fungi as an ecotechnology to improve plant performance. Ecol. Eng. 43: 95–103. Pampolina, N. M., Dell, B. and Malajczuk, N. 2002. Dynamics of ectomycorrhizal fungi in an Eucalyptus globulus plantation: effect of phosphorus fertilization. Forest Ecol. Manag. 158: 291–304. Plassard, C. and Fransson, P. 2009. Regulation of low-molecular weight organic acid production in fungi. Fungal Biol. Rev. 23: 30–39. Ramani, V. 2011. Effect of pesticides on phosphate solubilization by Bacillus sphaericus and Pseudomonas cepacia. Pestic. Biochem. Physiol. 99: 232–236. Reis, F. S., Ferreira, I. C. F. R. and Martins, A. 2012. Effect of the mycorrhizal symbiosis time in the antioxidant activity of fungi and Pinus pinaster roots, stems and leaves. Ind. Crop. Prod. 35: 211–216. Shen, J. B., Yuan, L. X., Zhang, J. L., Li, H. G., Bai, Z. H., Chen, X. P., Zhang, W. F. and Zhang, F. S. 2011. Phosphorus dynamics: From soil to plant. Plant Physiol. 156: 997–1005. Smith, S. E. and Read, D. J. 2008. Mycorrhizal Symbiosis. 3rd Ed. Academic Press, London. Sousa, N. R., Franco, A. R., Ramos, M. A., Oliveira, R. S. and Castro, P. M. L. 2011. Reforestation of burned stands: The effect of ectomycorrhizal fungi on Pinus pinaster establishment. Soil Biol. Biochem. 43: 2115–2120.
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Thomas, D. S., Montagu, K. D. and Conroy, J. P. 2006. Leaf inorganic phosphorus as a potential indicator of phosphorus status, photosynthesis and growth of Eucalyptus grandis seedlings. Forest Ecol. Manag. 223: 267–274. Tibbett, M., Sanders, F. E. and Cairney, J. W. G. 1998. The effect of temperature and inorganic phosphorus supply on growth and acid phosphatase production in arctic and temperate strains of ectomycorrhizal Hebeloma spp. in axenic culture. Mycol. Res. 102: 129–135. Van Hees, P. A. W., Jones, D. L., Jentschke, G. and Godbold, D. L. 2005. Organic acid concentrations in soil solution: effects of young coniferous trees and ectomycorrhizal fungi. Soil Biol. Biochem. 37: 771–776. Wallander, H. 2000. Uptake of P from apatite by Pinus sylvestris seedlings colonised by different ectomycorrhizal fungi. Plant Soil. 218: 249–256. Wang, J., Huang, Y. and Jiang, X. Y. 2011. Influence of ectomycorrhizal fungi on absorption and balance of essential elements of Pinus tabulaeformis seedlings in saline soil. Pedosphere. 21: 400–406. Wang, M. X., Yuan, L., Zhou, Z. F., Yang, H. J. and Huang, J. G. 2012. Efflux of oxalate and uptake of nitrogen, phosphorus and potassium by ectomycorrhizal fungal isolates in vitro in response to aluminum stress. Sci. Silvae Sin. (in Chinese). 48: 82–88. Yuan, L., Huang, J. G., Li, X. L. and Christie, P. 2004. Biological mobilization of potassium from clay minerals by ectomycorrhizal fungi and eucalypt seedling roots. Plant Soil. 262: 351–361.
Mobilization of Inorganic Phosphorus from Soils by Ectomycorrhizal Fungi∗1 ZHANG Liang1,2 , WANG Ming-Xia1 , LI Hua1 , YUAN Ling1 , HUANG Jian-Guo1,∗2 and C. PENFOLD3 1 College
of Resources and Environment, Southwest University, Chongqing 400716 (China) of Biotechnology and Chemical Engineering, Taizhou College, Nanjing Normal University, Taizhou 225300 (China) 3 School of Agriculture, Food and Wine, The University of Adelaide, Adelaide SA 5005 (Australia) 2 School
(Received August 21, 2013; revised April 30, 2014)
ABSTRACT Ectomycorrhizal (EM) fungi could form symbiosis with plant roots and participate in nutrient absorption; however, many EM species commonly found in forest soils, where phosphorus (P) concentration and availability are usually very low, particularly in tropical and subtropical areas, have not yet been investigated for their efficiencies to mobilize soil P. In this study, fungal growth, P absorption, efflux of protons and organic acids, and soil P depletion by four isolates of EM fungi isolated either from acidic or calcareous soils were compared in pure liquid culture using soil as a sole P source. Boletus sp. 7 (Bo 7), Lactarius deliciosus 3 (Ld 3), and Pisolithus tinctorius 715 (Pt 715) from acidic and P-deficient soils of southwestern China showed higher biomass and P concentration and accumulation than Cenococcum geophilum 4 (Cg 4) from a calcareous soil of Inner Mongolia, northern China, after 4 weeks of liquid culture. Oxalate, malate, succinate, acetate, and citrate concentrations in the culture solutions varied significantly with fungal species, and oxalate accounted for 51.5%–91.4% of the total organic acids. Organic acids, particularly oxalate, in the culture solutions may lead to the solubilization of iron-bound P (Fe-P), aluminum-bound P (Al-P), and occluded P (O-P) from soil phosphates. Fungal species also varied greatly in proton efflux, which decreased the culture solution pH and may dissolve calcium-bound P (Ca-P) in soil. This could be the reason for the increment of both inorganic P in the culture solutions and Olsen P in the soil when EM fungi were present. Total inorganic P, the sum of Al-P, Fe-P, O-P, and Ca-P, in the culture solutions was positively correlated with the total concentration of organic acids in the culture solutions (r = 0.918*, n = 5), but negatively with both the total inorganic P in soil (r = −0.970**, n = 5) and the culture solution pH (r = −0.830*, n = 5). These suggested variable efficiencies of EM fungal species to mobilize inorganic P fractions from soil, which could make EM trees to utilize inorganic P in the same way like EM fungi and adapt to the soils with various P concentrations and availabilities. Key Words:
EM fungal species, inorganic P, organic acid, P availability, proton, soil phosphate
Citation: Zhang, L., Wang, M. X., Li, H., Yuan, L., Huang, J. G. and Penfold, C. 2014. Mobilization of inorganic phosphorus from soils by ectomycorrhizal fungi. Pedosphere. 24(5): 683–689.
INTRODUCTION Phosphorus (P) is one of the most important essential elements for plant growth. However, both P concentration and availability are very low in most forest soils, particularly in tropical and subtropical areas because of intensive weathering and leaching. Usually no fertilizers are added into forest soils. Trees therefore must mobilize and extract P from insoluble phosphates in soils to satisfy their P requirement (Barroso and Nahas, 2005). Trees may interact with thousands of ectomycorrhizal (EM) fungal species in natural forests and forest plantations (Smith and Read, 2008). EM fungal species cover the roots and rootlets of trees with a thick man∗1 Supported
tle of hyphae, effectively enlarging the surface areas of the roots in contact with phosphates through soil pores that are not accessible to the root hairs (Hayek et al., 2012; B¨ ucking, 2004). Therefore, EM inoculation might stimulate the growth of host trees in P-deficient soils by improving P nutrition in artificial plantations. The identification of growth and nutrient uptake and mobilization is a key issue for understanding the nutrition of EM plants and selecting the fungal species as inoculants for producing EM tree seedlings in nursery beds (Chalot et al., 2002). Huang and Lapeyrie (1996) reported the improvement of P nutrition in Douglas fir seedlings inoculated by Laccaria bicolor S238N and the concomitant plant growth acceleration. Similarly, the growth of Pinus sylvestris seedlings was stimulated by
by the National Basic Research Program (973 Program) of China (No. 2013CB127405), the National Natural Science Foundation of China (Nos. 40771112 and 41171215), and the Technology Innovation Program of Southwest University of China (No. Ky2009022). ∗2 Corresponding author. E-mail: [email protected].
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inoculation of Paxillus involutus grown in media with strengite as a P source (Gibson and Mitchell, 2004). Courty et al. (2006) studied numerous EM fungi in vitro and found that Xerocomus chrysenteron sp. dissolved mineral phosphates efficiently and also promoted the growth of host trees. This fungal isolate could thus be potentially used in forestation in Pdeficient soils. Moreover, simultaneous release of P from apatite leads to the corruption of mineral structures by EM fungi. This biological decomposition, resulting in the release of otherwise unavailable P from minerals, seems related to the efflux of organic acids, particularly those with low molecular weights, and protons from fungal hyphae (Wang et al., 2011). Wallander (2000) found high concentrations of citric and oxalic acids in the culture media with mineral phosphates as a sole P source when pine roots were colonized by Suillus variegatus. P absorption by EM fungi was increased in liquid culture media with concomitant oxalate efflux (Ahonen-Jonnarth et al., 2000). Thus, the extramatrical mycelia extending from the mantle may play an important role in P mobilization (Tibbett et al., 1998). The release of organic acids may be a mechanism for EM fungi to mobilize and then utilize P from fluorapatite, chlorapatite, and hydroxyapatite (Adeleke et al., 2010; Gui˜ naz´ u et al., 2010). However, EM fungi varied greatly in the abilities to promote the growth and P uptake of trees, such as Pinus pinaster, P. menziesii, P. asperata, P. massoniana, Eucalyptus diversicolor, and E. globulus, after forming ectomycorrhizas (Pampolina et al.; 2002; Thomas et al., 2006; Sousa et al., 2011; Oliveira et al., 2012; Reis et al., 2012). Many EM species commonly found in forests have not yet been investigated on their efficiencies to mobilize soil P. More direct evidence for soil P exploitation by EM fungi is necessary in order to understand the functions of EM fungi in tree nutrition and their applications in artificial forestation. The aims of the present study were i) to evaluate the capacity of EM fungal isolates from different forest ecosystems to mobilize soil P pools in vitro, ii) to examine inorganic P fractions removed from soil using a sequential extraction procedure, and iii) to extend the explanation of P mobilization mechanisms by EM fungi through comparing the efflux of protons and organic acids. MATERIALS AND METHODS Fungal strains Four EM fungi, namely Boletus sp. 7 (Bo 7), Lac-
tarius deliciosus 3 (Ld 3), Cenococcum geophilum 4 (Cg 4) and Pisolithus tinctorius 715 (Pt 715), were used for the experiment. Bo 7, Ld 3 and Pt 715 were obtained from the Microbiology Laboratory, Southwest University, Chongqing, China and Cg 4 was provided by Dr. Shulan Bai of Inner Mongolia Agricultural University, China. The isolates of Bo 7 and Ld 3 were originally isolated from a yellow soil (pH 4.0) in the Jinfu Mountain, Chongqing, southwestern China and Cg 4 from a calcareous soil (pH 7.3) in the Daqing Mountain, Inner Mongolia, northern China. Bo 7, Ld 3 and Cg 4 were all from mycorrhizal Pinus sp. Pt 715 was isolated from eucalyptus mycorrhizas in a red soil (pH 4.5) in Xichang, Sichuan Province, southwestern China. Mycelia for inoculation were grown on Pachlewski agar medium for two weeks (25 ± 1 ◦ C). The medium contained 0.5 g L−1 tartrate, 1.0 g L−1 KH2 PO4 , 0.5 g L−1 MgSO4 , 20 g L−1 glucose, 5.0 g L−1 maltose, 0.1 g L−1 vitamin B1 , 20 g L−1 agar, and 1 mL L−1 microelement solution consisting of 8.45 mg L−1 H3 BO3 , 5 mg L−1 MnSO4 , 6 mg L−1 FeSO4 , 0.625 mg L−1 CuSO4, 2.27 mg L−1 ZnCl2 , and 0.27 mg L−1 (NH4 )2 MoO4 . Soil preparation A purple soil (typical Udorthent according to USDA Soil Taxonomy) derived from muddy sedimentary rocks was collected from the experimental farm (29◦ 49 18 N, 106◦ 25 45 E) of Southwestern University, Chongqing, China, air-dried, ground to pass a 0.5mm sieve, and then steam-sterilized at 121 ◦ C for 30 min. The soil was sandy loam in texture with a pH of 6.8 and contained 0.4 g kg−1 total N, 0.6 g kg−1 total P, 30.0 g kg−1 total K, 10.7 mg kg−1 Olsen P, 21.2 mg kg−1 , aluminum-bound P (Al-P), 28.1 mg kg−1 iron-bound P (Fe-P), 69.9 mg kg−1 occluded P (O-P), and 290.9 mg kg−1 calcium-bound P (Ca-P). 1.00 g soil was placed into a plastic tube (2 cm length × 1 cm diameter) closed at the two ends by microporous membrane (0.22 μm aperture). This membrane allowed water, inorganic ions, and organic acids to pass freely but prevented soil particles from penetration. Experimental procedure Pachlewski liquid medium was prepared with KH2 PO4 replaced by KCl at an equivalent amount of potassium. Into each 150 mL Erlenmeyer flask 20 mL of the Pachlewski liquid medium was transferred and 1.00 g soil prepared in the plastic tube closed at the two ends by microporous membrane was added as a sole P source. After steam sterilization at 121 ◦ C for 30 min, each flask was inoculated with a plug of mycelium (5
MOBILIZATION OF P FROM SOILS BY FUNGI
mm in diameter). In order to realize the extraction of soil P pools by fungal isolates, blank control flasks were set up in the same way except that no EM fungi were inoculated. All flasks were incubated statically for 4 weeks at 25 ± 1 ◦ C in the dark. There were 12 replicate flasks of each treatment in a fully randomized design. Sampling and sample analysis After 4 weeks of incubation, EM fungi were harvested by filtration and washed with deionized water to remove culture medium from the mycelium surface. They were then oven-dried, weighed, and digested with H2 SO4 -H2 O2 . The soil in the plastic tubes were air-dried and sequentially extracted with 0.5 mol L−1 NH4 F for Al-P, 0.1 mol L−1 NaOH for Fe-P, 0.5 mol L−1 H2 SO4 for Ca-P, and 0.3 mol L−1 C6 H5 Na3 O7 -1.0 g Na2 S2 O4 -0.5 mol L−1 NaOH for O-P by shaking at 150 r min−1 and 30 ◦ C for 30 min (soil:solution ratio = 1:50) (Chang and Jackson, 1957). Inorganic P in the digests, culture solutions, and extracts were analyzed by the molybdenum blue methods (Ames, 1966). The culture solutions were also analyzed for pH with a pH meter and for oxalate by high performance liquid chromatography (HPLC) with detection at 210 nm. For analysis of organic acids, the culture solutions (20 μL) were injected into an Ion-300 organic acid analysis column (Phenomenex, Torrance, USA) with 2.5 mmol L−1 H2 SO4 mobile phase at 0.5 mL min−1 and 3.1 × 106 Pa. Standard organic acids were prepared and run before and after the culture solutions (Fig. 1).
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(version 9, SAS Institute, Cary, USA). Significant differences between means were tested using Fisher’s protected least significant difference (LSD) at P < 0.05. RESULTS Fungal growth and P absorption As shown in Table I, the four EM fungal isolates varied significantly in growth rate, with Bo 7 showing the highest mean biomass of about 46 mg flask−1 , followed by Pt 715 (39 mg flask−1 ), Ld 3 (34 mg flask−1 ), and Cg 4 (27 mg flask−1 ). EM fungi also varied remarkably in P concentrations in their mycelia, which ranged from 4.01 mg g−1 dry weight (DW) (Ld 3) to 2.41 mg g−1 DW (Cg 4). P accumulation in Pt 715, Ld 3, and Bo 7 (135.6–142.0 μg flask−1 ) did not differ significantly, but was more than 2-fold that of Cg 4. TABLE I Biomass and P concentration and accumulation in ectomycorrhizal (EM) fungal isolates after 4 weeks of incubation in Pachlewski liquid medium with KH2 PO4 replaced by KCl and 1.00 g soil added as a sole P source EM fungal isolatea) Bo 7 Cg 4 Ld 3 Pt 715
Biomass
P concentration
P accumulation
mg flask−1 45.66±2.04c) ad) 27.16±2.59d 33.82±1.71c 39.00±2.71b
mg g−1 DWb) 3.11±0.29b 2.41±0.28c 4.01±0.31a 3.52±0.23ab
μg flask−1 142.00±7.49a 65.46±5.70b 135.62±7.48a 137.28±9.82a
a) Bo 7 = Boletus sp. 7; Ld 3 = Lactarius deliciosus 3; Cg 4 = Cenococcum geophilum 4; Pt 715 = Pisolithus tinctorius 715. b) Dry weight. c) Mean±standard deviation (n = 12). d) Mean values followed by the same letter(s) in a column are not significantly different at P < 0.05 as determined by Fisher’s least significant difference test.
pH, organic acids and inorganic P in culture solutions
Fig. 1 Chromatogram of standard organic acids. AU = arbitrary unit.
Statistical analysis Treatment effects were evaluated by analysis of variance using the SAS statistical software package
The EM fungal isolates decreased the culture solution pH remarkably (P < 0.05). pH values in the culture solutions with Ld 3, Pt 715, and Bo 7 were much lower than that with Cg 4 (Fig. 2). All EM fungi exuded oxalate and acetate, but varied significantly in the exudation rates (Table II). Oxalate accounted for a large proportion (51.5%–91.4%) of the total organic acids. Malate was found in the culture solution with Ld 3, succinate with Ld 3 and Pt 715, and citrate with Bo 7 and Ld 3. The EM fungi species varied significantly in the sum of organic acids, which ranged from 35.4 mg L−1 (Bo 7) to 23.0 mg L−1 (Pt 715) in the culture solutions (Table II). After incubation with fungal inoculation, total inorganic P in culture solutions was remarkably higher than the control without fungal ino-
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TABLE II Organic acids in culture solutions after 4 weeks of incubation with ectomycorrhizal (EM) fungi in Pachlewski liquid medium with KH2 PO4 replaced by KCl and 1.00 g soil added as a sole P source EM fungal straina) Bo 7 Cg 4 Ld 3 Pt 715
Oxalate 30.50±1.86b) ac) 21.75±1.21b 16.07±1.13bc 11.83±1.12c
Malate NDd) ND 1.36±0.12 ND
Succinate mg ND ND 0.39±0.07a 0.51±0.09a
Acetate
Citrate
Total
2.54±0.41a ND 2.33±0.31a ND
35.36±2.68a 23.80±1.51c 28.04±2.49b 22.95±2.21c
L−1 2.32±0.36c 2.05±0.28c 7.89±0.79b 10.61±0.91a
a) Bo
7 = Boletus sp. 7; Ld 3 = Lactarius deliciosus 3; Cg 4 = Cenococcum geophilum 4; Pt 715 = Pisolithus tinctorius 715. deviation (n = 12). c) Mean values followed by the same letter(s) in each column are not significantly different at P < 0.05 as determined by Fisher’s least significant difference test. d) Not detected. b) Mean±standard
Fig. 2 Culture solution pH after 4 weeks of incubation with or without ectomycorrhizal (EM) fungi in Pachlewski liquid medium with KH2 PO4 replaced by KCl and 1.00 g soil added as a sole P source. Values are means with standard deviations shown by vertical bars (n = 12). Bars with the same letter indicate no significant difference (P < 0.05). CK = control without EM fungal inoculation; Bo 7 = Boletus sp. 7; Ld 3 = Lactarius deliciosus 3; Cg 4 = Cenococcum geophilum 4; Pt 715 = Pisolithus tinctorius 715.
Fig. 3 Total inorganic P in culture solutions after 4 weeks of incubation with or without ectomycorrhizal (EM) fungi in Pachlewski liquid medium with KH2 PO4 replaced by KCl and 1.00 g soil added as a sole P source. Values are means with standard deviations shown by vertical bars (n = 12). Bars with the same letter indicate no significant difference (P < 0.05). CK = control without EM fungal inoculation; Bo 7 = Boletus sp. 7; Ld 3 = Lactarius deliciosus 3; Cg 4 = Cenococcum geophilum 4; Pt 715 = Pisolithus tinctorius 715.
culation, and the concentrations of total inorganic P were similar in the solutions (1.56–1.86 mg L−1 ) with fungal inoculation (Fig. 3).
O-P. All four EM fungi significantly lowered soil Ca-P as compared to the control and Ca-P in the soils varied slightly among EM fungi (223.9–236.3 mg kg−1 ). The total inorganic P, the sum of Al-P, Fe-P, O-P, and CaP, in the soils after incubation with EM fungi (Table III) also decreased as compared to the initial soil value, but no significant differences were found among fungal species. Olsen P in soil after incubation with EM fungi ranged from 11.6 to 12.83 mg kg−1 , significantly higher than the initial soil value (10.82 mg kg−1 ). No significant variation in Olsen P in the residual soil was observed among EM fungi inoculated.
Residual soil P fractions Bo 7 and Cg 4 significantly decreased Al-P in the soil from 19.45 mg kg−1 (control without EM fungi) to 14.20 and 13.88 mg g−1 , respectively, whereas there was no significant change in soil Al-P after incubation with Pt 715 and Ld 3 (Table III). Fungal inoculation generally led to a significant depression of Fe-P in the soil, except for Pt 715 which removed little of this P fraction from the soil. Fe-P was similar in the soil with fungal compared to the control without EM fungi. Bo 7, Ld 3, and Cg 4 depleted significantly O-P from the soil in contrast to Pt 715 which extracted scarcely any
Correlation analysis between culture solution pH, inorganic P and organic acids As shown in Table IV, total inorganic P in culture
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TABLE III Variations in inorganic P fractionsa) and Olsen P in soil after 4 weeks of incubation with or without ectomycorrhizal (EM) fungi in Pachlewski liquid medium with KH2 PO4 replaced by KCl and 1.00 g soil added as a sole P source EM fungal strainb) CK Bo 7 Cg 4 Ld 3 Pt 715
Al-P 19.45±1.76d) ae) 14.20±1.23b 13.88±1.45b 15.92±1.61ab 17.14±1.81ab
Fe-P 25.55±2.07a 15.11±1.45b 18.79±1.98b 18.54±1.65b 21.25±1.99ab
O-P
Ca-P
mg 66.51±5.45a 45.19±3.78bc 50.06±4.01b 43.36±3.98c 58.59±4.97ab
kg−1 286.96±13.12a 236.34±12.18b 233.42±13.76b 227.60±10.98b 223.90±11.07b
Total inorganic Pc)
Olsen P
398.47±19.87a 310.84±18.17b 316.15±20.16b 305.42±16.67b 320.88±19.32b
10.82±0.91b 11.82±0.88a 11.65±0.69a 12.83±0.76a 11.60±0.82a
a) Al-P
= aluminum-bound P; Fe-P = iron-bound P; O-P = occluded P; Ca-P = calcium-bound P. = control without EM fungal inoculation; Bo 7 = Boletus sp. 7; Ld 3 = Lactarius deliciosus 3; Cg 4 = Cenococcum geophilum 4; Pt 715 = Pisolithus tinctorius 715. c) Sum of Al-P, Fe-P, O-P, and Ca-P. d) Mean±standard deviation (n = 12). e) Mean values followed by the same letter(s) in each column are not significantly different at P < 0.05 as determined by Fisher’s least significant difference test. b) CK
TABLE IV Correlation coefficients between total inorganic P in culture solutions, total inorganic P in soil, total concentration of organic acids in culture solutions, and culture solution pH after 4 weeks of incubation with or without ectomycorrhizal (EM) fungi in Pachlewski liquid medium with KH2 PO4 replaced by KCl and 1.00 g soil added as a sole P source Factor
Total inorganic P in culture solutions
Total inorganic P in soil
Total concentration of organic acids in culture solutions
Total inorganic P in soil Total concentration of organic acids in culture solutions Culture solution pH
−0.970** 0.918* −0.830*
−0.935* 0.824
−0.796
*, **Significance at P < 0.05 and P < 0.01, respectively.
solutions was positively correlated with the total concentrations of organic acids in culture solutions (r = 0.918*, n = 5), but negatively with both the total inorganic P in soil (r = −0.970**, n = 5) and the culture solution pH (r = −0.830*, n = 5). There was also a negative correlation between the total inorganic P in soil and the total concentration of organic acids in culture solutions (r = −0.935*, n = 5). DISCUSSION Hyphal P uptake from soil was higher in the isolates Bo 7, Ld 3, and Pt 715 than Cg 4. This was due mainly to the larger biomass of Bo 7, Ld 3, and Pt 715 and higher P concentrations in their mycelia than Cg 4. In a previous study, however, we found that Cg 4, Ld 3, and Pt 715 had similar biomass when grown in a Pachlewski liquid medium with KH2 PO4 as a sole P source (Wang et al., 2012). The large reduction in both biomass and P uptake of Cg 4 suggested the poor efficiency of Cg 4 to extract P from soils. Cg 4 was originally isolated from a calcareous soil rich in available P in Inner Mongolia, northern China, while the other three isolates came from subtropical soils of southern China, which
are acidic and deficient in mineral nutrients, in particular P (Yuan et al., 2004). EM fungal species could thus vary in their abilities to adapt to the soils with different P concentrations and availabilities (Miller et al., 1995). EM fungi isolated from P-deficient soils should be able to adapt to poor P conditions and evolve a high efficiency to extract P from soils. Our findings that the EM fungi studied had variable capacities to extract soil P are consistent with the reports of Pampolina et al. (2002), Thomas et al. (2006), Sousa et al. (2011), and Reis et al. (2012), who studied the effectiveness of fungal isolates in forming ectomycorrhizas with the host trees, including E. globulus, P. menziesii, P. asperata, P. massoniana, and E. diversicolor, and found that the fungal isolates varied significantly in their abilities to promote host tree growth and P uptake. EM fungi play a key role in P uptake of host trees. Various mechanisms have been suggested for P uptake by EM trees through the external hyphae from mantles which allow root systems to exploit a greater volume of soil P, including i) extending away from roots and transporting P from some distance to the roots, ii) exploiting smaller soil pores not reached by rootlets, and
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iii) enlarging surface areas for absorption (O’keefe and Sylvia, 1992). However, in recent years greater attention has been paid to P mobilization by microbial processes (Ramani, 2011). We detected malate, succinate, acetate, citrate, and particularly oxalate in culture solution inoculated with EM fungi. Many researchers also detected high concentrations of organic acids (about 1 000 μmol L−1 ) produced by EM fungi in soil in European forests (Van Hees et al., 2005; Plassard and Fransson, 2009; Courty et al., 2010). In this study, all EM fungi could exude oxalate and protons but varied greatly in the efflux rates. Pt 715 effused a larger amount of protons into culture solution in contrast to Bo 7 which showed the highest oxalate efflux rate. Significant negative correlations were found between the total inorganic P and pH in culture solutions (r = −0.830*, n = 5) and between the total inorganic P (the sum of Al-P, Ca-P, Fe-P, and O-P) in soil and the total concentration of organic acids in the culture solutions (r = −0.935*, n = 5). Both (Al (C2 O4 )3 )3− and (Fe (C2 O4 )3 )3− have very high chelation constants of 16.3 and 20.2, respectively. Oxalate may therefore chelate Al3+ and Fe3+ in the insoluble phosphates such as Al phosphate, Fe phosphate, and occluded phosphate, resulting in the solubilization of these phosphates and P release (Cromack et al., 1979; Feng, et al., 2011). Solubility of Ca phosphate increased with decreasing solution pH and protons may thus have a strong capacity to dissolve Ca phosphate in soils (Shen et al., 2011). EM fungi varied significantly in both organic acid and proton efflux, suggesting their different efficiencies to mobilize and utilize inorganic P fractions in soils. The host trees which form symbiosis with EM fungi could therefore utilize inorganic P from soils with various P concentrations and availabilities. The decreased inorganic P fractions in soil as a sole P source in culture solutions could explain why EM fungi increased Olsen P, usually available for plants, in the soil and increased total inorganic P in culture solutions. The biodiversity of these EM fungi which varied significantly in the efflux of organic acids and protons and showed diverse abilities to mobilize and utilize inorganic P fractions in soils could enable EM trees to adapt to different soil P conditions. Trees in natural forests and forest plantations may interact with hundreds of EM fungal species; therefore, further studies are required to investigate the possible biomobilization of P from phosphates by EM fungi in the fields. Biological solubilization of phosphates could be more important and thus merits investigation under field conditions.
L. ZHANG et al.
REFERENCES Adeleke, R. A., Cloete, T. E., Bertrand, A. and Khasa, D. P. 2010. Mobilisation of potassium and phosphorus from iron ore by ectomycorrhizal fungi. World J. Microb. Biot. 26: 1901–1913. Ahonen-Jonnarth, U., Van Hees, P. A. W., Lundstr¨ om, U. S. and Finlay, R. D. 2000. Organic acids produced by mycorrhizal Pinus sylvestris exposed to elevated aluminum and heavy metal concentrations. New Phytol. 146: 557–567. Ames, B. N. 1966. Assay of inorganic phosphate, total phosphate and phosphatases. In Colowick, S. P. and Kaplan, N. O. (eds.) Methods in Enzymology. Vol. 8. Academic Press, New York. pp. 115–118. Barroso, C. B. and Nahas, E. 2005. The status of soil phosphate fractions and the ability of fungi to dissolve hardly soluble phosphates. Appl. Soil Ecol. 29: 73–83. B¨ ucking, H. 2004. Phosphate absorption and efflux of three ectomycorrhizal fungi as affected by external phosphate, cation and carbohydrate concentrations. Mycol. Res. 108: 599–609. Chalot, M., Javelle, A., Blaudez, D., Lambilliote, R., Cooke, R., Sentenac, H., Wipf, D. and Botton, B. 2002. An update on nutrient transport processes in ectomycorrhizas. Plant Soil. 244: 165–175. Chang, S. C. and Jackson, M. L. 1957. Fractionation of soil phosphorus. Soil Sci. 84: 133–144. Courty, P. E., Bu´ ee, M., Diedhiou, A. G., Frey-Klett, P., Tacon, F. L., Rineau, F., Turpault, M.-P., Uroz, S. and Garbaye, J. 2010. The role of ectomycorrhizal communities in forest ecosystem processes: New perspectives and emerging concepts. Soil Biol. Biochem. 42: 679–698. Courty, P. E., Pouysegur, R., Bu´ee, M. and Garbaye, J. 2006. Laccase and phosphatase activities of the dominant ectomycorrhizal types in a lowland oak forest. Soil Biol. Biochem. 38: 1219–1222. Cromack, K. Jr., Sollins, P., Graustein, W. C., Speidel, K., Todd, A. W., Spycher, G., Li, C. Y. and Todd, R. L. 1979. Calcium oxalate accumulation and soil weathering in mats of the hypogeous fungus Hysterangium crassum. Soil Biol. Biochem. 11: 463–468. Feng, M. H., Ngwenya, B. T., Wang, L., Li, W., Olive, V. and Ellamd, R. M. 2011. Bacterial dissolution of fluorapatite as a possible source of elevated dissolved phosphate in the environment. Geochim. Cosmochim. Acta. 75: 5785–5796. Gibson, B. R. and Mitchell, D. T. 2004. Nutritional influences on the solubilization of metal phosphate by ericoid mycorrhizal fungi. Mycol. Res. 108: 947–954. Gui˜ naz´ u, L. B., Andr´ es, J. A., Del Papa, M. F., Pistorio, M. and Rosas, S. B. 2010. Response of alfalfa (Medicago sativa L.) to single and mixed inoculation with phosphate-solubilizing bacteria and Sinorhizobium meliloti. Biol. Fert. Soils. 46: 185–190. Hayek, S., Grosch, R., Gianinazzi-Pearson, V. and Franken, P. 2012. Bioprotection and alternative fertilisation of petunia using mycorrhiza in a soilless production system. Agron. Sustain. Dev. 32: 765–771. Huang, J. G. and Lapeyrie, F. 1996. Ability of ectomycorrhizal fungus Laccaria bicolor S238N to increase the growth of Douglas fir seedlings and their phosphorus and potassium uptake. Pedosphere. 6: 217–224. Miller, R. M., Reinhardt, D. R. and Jastrow, J. D. 1995. External hyphal production of vesicular-arbuscular mycorrhizal fungi in pasture and tallgrass prairie communities. Oecologia. 103: 17–23. O’keefe, D. M. and Sylvia, D. M. 1992. Chronology and mecha-
MOBILIZATION OF P FROM SOILS BY FUNGI
nism of P uptake by mycorrhizal sweet potato plants. New Phytol. 122: 651–659. Oliveira, R. S., Franco, A. R. and Castro, P. M. L. 2012. Combined use of Pinus pinaster plus and inoculation with selected ectomycorrhizal fungi as an ecotechnology to improve plant performance. Ecol. Eng. 43: 95–103. Pampolina, N. M., Dell, B. and Malajczuk, N. 2002. Dynamics of ectomycorrhizal fungi in an Eucalyptus globulus plantation: effect of phosphorus fertilization. Forest Ecol. Manag. 158: 291–304. Plassard, C. and Fransson, P. 2009. Regulation of low-molecular weight organic acid production in fungi. Fungal Biol. Rev. 23: 30–39. Ramani, V. 2011. Effect of pesticides on phosphate solubilization by Bacillus sphaericus and Pseudomonas cepacia. Pestic. Biochem. Physiol. 99: 232–236. Reis, F. S., Ferreira, I. C. F. R. and Martins, A. 2012. Effect of the mycorrhizal symbiosis time in the antioxidant activity of fungi and Pinus pinaster roots, stems and leaves. Ind. Crop. Prod. 35: 211–216. Shen, J. B., Yuan, L. X., Zhang, J. L., Li, H. G., Bai, Z. H., Chen, X. P., Zhang, W. F. and Zhang, F. S. 2011. Phosphorus dynamics: From soil to plant. Plant Physiol. 156: 997–1005. Smith, S. E. and Read, D. J. 2008. Mycorrhizal Symbiosis. 3rd Ed. Academic Press, London. Sousa, N. R., Franco, A. R., Ramos, M. A., Oliveira, R. S. and Castro, P. M. L. 2011. Reforestation of burned stands: The effect of ectomycorrhizal fungi on Pinus pinaster establishment. Soil Biol. Biochem. 43: 2115–2120.
689
Thomas, D. S., Montagu, K. D. and Conroy, J. P. 2006. Leaf inorganic phosphorus as a potential indicator of phosphorus status, photosynthesis and growth of Eucalyptus grandis seedlings. Forest Ecol. Manag. 223: 267–274. Tibbett, M., Sanders, F. E. and Cairney, J. W. G. 1998. The effect of temperature and inorganic phosphorus supply on growth and acid phosphatase production in arctic and temperate strains of ectomycorrhizal Hebeloma spp. in axenic culture. Mycol. Res. 102: 129–135. Van Hees, P. A. W., Jones, D. L., Jentschke, G. and Godbold, D. L. 2005. Organic acid concentrations in soil solution: effects of young coniferous trees and ectomycorrhizal fungi. Soil Biol. Biochem. 37: 771–776. Wallander, H. 2000. Uptake of P from apatite by Pinus sylvestris seedlings colonised by different ectomycorrhizal fungi. Plant Soil. 218: 249–256. Wang, J., Huang, Y. and Jiang, X. Y. 2011. Influence of ectomycorrhizal fungi on absorption and balance of essential elements of Pinus tabulaeformis seedlings in saline soil. Pedosphere. 21: 400–406. Wang, M. X., Yuan, L., Zhou, Z. F., Yang, H. J. and Huang, J. G. 2012. Efflux of oxalate and uptake of nitrogen, phosphorus and potassium by ectomycorrhizal fungal isolates in vitro in response to aluminum stress. Sci. Silvae Sin. (in Chinese). 48: 82–88. Yuan, L., Huang, J. G., Li, X. L. and Christie, P. 2004. Biological mobilization of potassium from clay minerals by ectomycorrhizal fungi and eucalypt seedling roots. Plant Soil. 262: 351–361.