Differential effects of soil disturbance and plant residue retention on function of arbuscular mycorrhizal (AM) symbiosis are not reflected in colonization of roots or hyphal development in soil

Soil Biology & Biochemistry 43 (2011) 571e578

Contents lists available at ScienceDirect

Soil Biology & Biochemistry journal homepage: www.elsevier.com/locate/soilbio

Differential effects of soil disturbance and plant residue retention on function of arbuscular mycorrhizal (AM) symbiosis are not reflected in colonization of roots or hyphal development in soil Tingyu Duan a, b, c, Evelina Facelli c, Sally E. Smith c, F. Andrew Smith c, Zhibiao Nan a, b, * a b c

College of Pastoral Agriculture Science and Technology, Lanzhou University, Lanzhou 730020, China Key Laboratory of Grassland Agro-Ecosystems, Lanzhou 730020, China School of Earth and Environmental Sciences, The University of Adelaide, Adelaide, South Australia 5005, Australia

a r t i c l e i n f o

a b s t r a c t

Article history: Received 14 August 2010 Received in revised form 11 November 2010 Accepted 21 November 2010 Available online 10 December 2010

The effects of soil disturbance and residue retention on the functionality of the symbiosis between medic (Medicago truncatula L.) and arbuscular mycorrhizal fungi (AMF) were assessed in a two-stage experiment simulating a crop rotation of wheat (Triticum aestivum L.) followed by medic. Plants were inoculated or not with the AMF, Glomus intraradices and Gigaspora margarita, separately or together. The contribution of the arbuscular mycorrhizal (AM) pathway for P uptake was determined using 32P-labeled soil in a small hyphal compartment accessible only to hyphae of AMF. In general AM colonization was not affected by soil disturbance or residue application and disturbance did not affect hyphal length densities (HLDs) in soil. At 4 weeks disturbance had a negative effect on growth and phosphorus (P) uptake of plants inoculated with G. margarita, but not G. intraradices. By 7 weeks disturbance reduced growth of plants inoculated with G. margarita or AMF mix and total P uptake in all inoculated plants. With the exception of plants inoculated with G. margarita in disturbed soil at 4 weeks, the AM pathway made a significant contribution to P uptake in all AM plants at both harvests. Inoculation with both AMF together eliminated the negative effects of disturbance on AM P uptake and growth, showing that a fungus insensitive to disturbance can compensate for loss of contribution of a sensitive one. Application of residue increased growth and total P uptake of plants but decreased 32P in plants inoculated with the AMF mix in disturbed soil, compared with plants receiving no residue. The AMF responded differently to disturbance and G. intraradices, which was insensitive to disturbance, compensated for lack of contribution by the sensitive G. margarita when they were inoculated together. Colonization of roots and HLDs in soil were not good predictors of the outcomes of AM symbioses on plant growth, P uptake or P delivery via the AM pathway. Ó 2010 Elsevier Ltd. All rights reserved.

Keywords: Arbuscular mycorrhiza Triticum aestivum Medicago truncatula Soil disturbance Crop residue Plant P uptake

1. Introduction Arbuscular mycorrhizas occur widely in terrestrial plants, including important crops such as wheat (Triticum aestivum L.) and pasture species such as medic (Medicago spp.). The symbiosis is normally mutualistic, based primarily on enhanced plant mineral nutrient uptake, particularly phosphorus (P) (Smith and Read, 2008), but also on improved water stress tolerance (Augé, 2001) and resistance to disease (Newsham et al., 1995). The well-

* Corresponding author. College of Pastoral Agriculture Science and Technology, Lanzhou University, Lanzhou 730020, China. Tel.: þ86 931 8661047; fax: þ86 931 8910979. E-mail address: [email protected] (Z. Nan). 0038-0717/$ e see front matter Ó 2010 Elsevier Ltd. All rights reserved. doi:10.1016/j.soilbio.2010.11.024

established effects of arbuscular mycorrhizal (AM) colonization on plant P nutrition are largely a consequence of the development of an extensive fungal mycelium in soil, which absorbs and translocates P to the plant. Thus, AM plants have two potential pathways of nutrient uptake, directly from the soil and via the AM fungal symbiont (Smith and Read, 2008). Improved soil structure and structural stability may also be associated with high AM colonization and are most likely related to the development of the AM hyphal network in soil (Miller and Jastrow, 2000; Tisdall, 1994). Given the ubiquitous occurrence of arbuscular mycorrhizas it is important to evaluate effects of agricultural practices on their development and function. Agricultural practices including reduced or no tillage and residue application have been introduced in many parts of the world, with the aim of improving soil structural and chemical

572

T. Duan et al. / Soil Biology & Biochemistry 43 (2011) 571e578

properties, increasing soil water storage capacity and reducing soil erosion (Triplett and Dick, 2008). There has been considerable interest in the ways in which AM symbiosis is affected by such practices and by crop rotation (e.g. Jansa et al., 2006; McGonigle et al., 1999; McGonigle and Miller, 2000; Miller, 2000). Soil disturbance (such as that caused by tillage) can decrease AM colonization of plant roots via destruction of the AM hyphal network in soil (Evans and Miller, 1988; Jasper et al., 1989), and/or change the AM fungal assemblages and spore numbers in soils (Jansa et al., 2002; Kabir et al., 1998). Although effects of disturbance on AM colonization are not always observed (see Duan et al., 2010; McGonigle and Miller, 2000; Miller, 2000), negative effects on P uptake have been consistently documented and are often accompanied by decreases in plant biomass (Fairchild and Miller, 1988; McGonigle and Miller, 1996; Miller, 2000). The destruction of the external hyphal network by disturbance may decrease the effectiveness of the AM pathway of P uptake and hence the ability of AM fungal hyphae to acquire P beyond the limits of the P depletion zone in the rhizosphere. Soil disturbance may also select AM fungi (AMF) with particular characteristics in relation to plant nutrition, and eliminate others. Jansa et al. (2002, 2003) found that Glomus spp. were dominant in highly tilled fields, whereas Scutellospora spp. were more prevalent in low-tillage fields. McGonigle et al. (2003) investigated the influence of disturbance on the effects of three different AMF, singly and together, on the P uptake and growth of maize (a plant that responds positively to AM symbiosis). They concluded that whereas all three fungi increased plant P uptake, only Glomus mosseae (alone or mixed with Gigaspora margarita) also stimulated plant growth in undisturbed soil. This aspect of ‘functional diversity’ among different plant fungus combinations (Burleigh et al., 2002) has not received a great deal of attention since the direct and quantitative demonstration, using radioactive P (33P or 32P), that even when plants do not respond positively to AM in terms of growth they may take up a considerable proportion of total P uptake via the hyphal network and the hyphal uptake pathway into plants (Smith et al., 2003, 2004). Much of the previous work has concentrated on the effects of tillage/disturbance or residue retention on total P uptake of plants and few investigations have actually measured the contributions of the AM and direct uptake pathways separately. Use of soil labeled with 32P or 33P, available only to the external AM network, has revealed that P uptake via the AM pathway makes large contributions to P uptake, and also that the direct pathway may be decreased in AM plants, compared with non-mycorrhizal (NM) counterparts (Smith et al., 2009). Knowledge of the effects of tillage and rotation on functioning of AM symbioses is limited. McGonigle et al. (1999) predicted that the effects of tillage and rotations on functioning of AM symbioses might extend well beyond the maize and maizeesoybean systems that they had studied extensively, but there have been few direct investigations since then. Nevertheless, rotations are important determinants of yield. Our investigation was carried out in the context of altered management practices on the Loess Plateau of China, where Shen et al. (2004) found that the rotation of wheat (T. aestivum L.) and alfalfa (Medicago sativa L.) increased total N in soils and crops and farmers’ income, compared with continuous wheat monoculture. In the same region of China, Duan et al. (2010) also found that rotation of maize (Zea mays L.), wheat and soybean (Glycine max L.) under reduced tillage had the potential to improve soil structure, again compared with continuous wheat, and did not carry a yield penalty. The newly-introduced practices on the Loess Plateau did not have a large influence on AM colonization of roots (Duan et al., 2010), so that the relationship between AM symbiosis, plant nutrition and agricultural production is not clear.

Residue retention has received much less attention than tillage in the context of effects of conservation practices on AM symbiosis (see references cited in Borie et al., 2002). Duan et al. (2010) observed that whereas no tillage slightly increased AM colonization, this effect was not apparent if residues were also applied to the soil surface. It has long been recognized that returning residues from the previous crop to the field after threshing increases organic matter and returns nutrients to the soil (e.g. Rasmussen et al., 1980). Although AMF are unable to utilize organic C in the residues, it has been suggested that retention of residues may affect AMF indirectly by altering the activity of microorganisms in the rhizosphere or changing soil chemical properties (e.g. pH and nutrient availability). However, two investigations in acid soils (Borie et al., 2002; Hafner et al., 1993) found no effect of application of residues on AM colonization of pearl millet or wheat. The experiment described here was designed to simulate a single rotation of wheat followed by medic, to test the effects of no tillage (undisturbed) or tillage (disturbed), with addition of residue to the soil surface, as is normally done in the Loess Plateau, on the functionality of AM symbiosis. We included one cycle of wheat, grown to maturity, as would occur in the field. This differs from some previous experiments in which several short cycles of growth and disturbance preceded determination of effects of disturbance on crop growth (e.g. Fairchild and Miller, 1988; McGonigle et al., 2003). In addition to measurements of plant growth, AM colonization and total P uptake, we used 32 P-labeled soil supplied in small hyphal compartments (HCs) available only to AM fungal hyphae, to measure the uptake of P by the plants via the AM pathway. This allowed us to directly estimate the contribution of the AM pathway under different simulated soil management conditions. We followed up the findings of McGonigle et al. (2003) by using two AMF (Glomus intraradices and G. margarita, separately and together) which had been shown to have different contributions to P uptake in a previous experiment with tomato (Facelli et al., 2010) and may have different sensitivity to disturbance (McGonigle et al., 2003), although the latter is not certain because the isolates we used were not the same as those used by McGonigle et al. (2003). We hypothesized that: 1) disturbance will decrease AM colonization, P uptake (especially via the AM pathway) and plant growth of medic; 2) G. intraradices and G. margarita will respond differently to disturbance and hence colonization of roots and contributions to P uptake via the AM pathway will differ; 3) when inoculated together, the less sensitive fungus will compensate for reduction in activity of the more sensitive one; and 4) residue will increase plant P uptake and plant growth, but will not affect AM colonization or P uptake via the AM pathway.

2. Materials and methods 2.1. Plants and fungi The plants used were wheat (T. aestivum L. cv Yitpi) and medic (Medicago truncatula L. cv. Jemalong A17), in Stages 1 and 2 respectively (see 2.2). The AMF were G. intraradices Schenck and Smith (DAOM 181602) and G. margarita Becker and Hall (WFVAM 21). It has recently been proposed that the G. intraradices isolate we used falls within a newly described species, Glomus irregulare (Stockinger et al., 2009), but we here continue to use the former name and culture number, to facilitate comparison with previous papers using the same fungus. Inoculum was prepared from pot cultures of the AMF grown on Trifolium subterraneum L. in the same soil mix as used for the experiments, and consisted of dry soil containing spores and colonized root fragments.

T. Duan et al. / Soil Biology & Biochemistry 43 (2011) 571e578

2.2. Experimental design The experiment was set up in two stages, to simulate a rotation involving wheat followed by medic. Stage 1 was designed to establish the undisturbed and disturbed soil treatments and produce plant residues for application to the pots in Stage 2. Wheat was inoculated with the AMF G. intraradices, G. margarita, a mix of G. intraradices and G. margarita, or non-inoculated, with 30 pots for each inoculation treatment. Small hyphal compartments (HCs), covered with 25 mm mesh to prevent root access, were buried in the soil in the main compartment (root hyphal compartment e RHC) of each pot (for details see 2.3). In Stage 1 these remained empty, with the openings at the top covered by lids. Plants were grown for 10 weeks before the shoots were harvested, dried, weighed and straw analyzed for P concentration. The straw was ground and applied to pots in the residue treatments in Stage 2 (see 2.4). To prepare for Stage 2, soil in half the pots of each inoculation treatment was left undisturbed and in the others was thoroughly disturbed. AM colonization was determined on a small sample of roots taken from disturbed pots. Medic was grown in Stage 2. Four disturbance/residue treatments were imposed for each of the inoculation treatments: disturbed plus residue (5 pots), disturbed-no residue (10 pots), undisturbed plus residue (5 pots) and undisturbed-no residue (10 pots) e total 120 pots. Five pots of each disturbance and inoculation treatment without residue (40 pots) were harvested after 4 weeks. The remainder of the pots (5 from each inoculation and disturbance/residue treatment) were harvested after 7 weeks (80 pots). At the start of Stage 2, the HCs were filled from the top with 32Plabeled soil, which could only be accessed by hyphae of AMF growing into the HCs from the medic plants. Thus 32P in the plants could only have been taken up through the AM P uptake pathway. At each harvest, measurements were made of AM colonization, plant growth and P concentration and content, and amounts of 32P in the plants. Hyphal length densities (HLD) in the soil in the RHCs and HCs and 32P remaining in the HCs were determined at 7 weeks. 2.3. Growth medium and compartmented pots Soil collected from Waite Arboretum, South Australia, Australia (Litchfield, 1951) was used to prepare the soil mix used in the experiments. The mix consisted of a mixture of 10% soil and 90% sand (75% coarse sand, 25% fine sand). All components were sieved through a 2 mm sieve, and the soil and sands were sterilized by autoclaving at 121  C for 1 h twice over a period of 3 days and then dried in an oven at 110  C for 36 h. The mix (hereafter referred to as soil) had 3 mg P kg1 plant-available P (resin extraction method, McLaughlin et al., 1994) and pH (CaCl2) of 6.0. P was added as 10 mg P kg1 (KH2PO4) to both RHCs and HCs after harvest of wheat and before planting medic. The RHC was a non-draining plastic pot filled with 1.4 kg soil. Inoculum (10% of total soil by weight) was thoroughly mixed into the soil in this compartment. When both AMF were combined (AMF mix) 5% by weight of inoculum of each fungus was mixed into the soil. Non-inoculated RHCs received an additional 10% sterilized soil. The HC was as described by Johnson et al. (2001). Four ‘windows’ were cut in the wall of polyvinyl chloride (PVC) water pipe (18 mm internal diameter, 14 cm long) and covered with 25 mm mesh. The windows were equivalent to 50% of the total pipe surface area and allowed hyphae of AMF, but not roots, to penetrate from the RHC and absorb labeled P from HCs. The HCs were placed vertically in the middle of the pots, so that the windows faced sideways. During Stage 1 of the experiment (wheat) the HCs contained no soil; during stage 2 (medic) they were filled from the top with 30 g of soil labeled with 32P. Soil for the HCs was well mixed 1 with carrier-free H32 3 PO4 to provide 72 kBq g soil. Thus, the soil in

573

all the HCs was ‘disturbed’, regardless of the treatment in the RHC. No inoculum was added in the HCs. Sufficient reversed osmosis (RO) water was added to the pots to bring the soil as a whole to a water content of 0.1 g g1 oven-dry soil.

2.4. Plant growth and harvesting 2.4.1. Stage 1 Wheat seeds were surface-sterilized with 4% sodium hypochlorite (NaOCl) for 90 s and rinsed five times with RO water. The sterilized seeds were placed on wet filter paper and incubated in the dark at 25  C for 24 h. Four germinated seeds were planted in each pot, and thinned to 2 seedlings after 1 week. White plastic beads were added to the surface of the soil in the pots to reduce evaporation. After 10 weeks growth in a glasshouse, the aboveground parts of wheat were removed, separated into stems (straw) and heads and dried at 75  C for 48 h in an oven. Dry weights (DW) were determined and samples of straw taken for analysis of P concentration. The straw from each inoculation treatment was separately ground, sieved through a 2 mm sieve and applied to the same treatments in Stage 2. The residues comprised approximately 70% fine powder and 30% straw fragments 1e2 mm in length. Heads were not included because they would normally be removed in harvested material. 2.4.2. Stage 2 During preparation each pot was treated separately. Soil in half the pots was thoroughly disturbed by hand mixing. Roots were cut into 1 cm fragments and mixed with the soil, which was then returned to the pots. Wheat residue (4 g per pot) was added to the surface of the soil of the RHC in 5 pots of each inoculation and disturbance treatment. The remaining pots received no residues. No plastic beads were applied during Stage 2. Medic seeds were treated with concentrated anhydrous sulphuric acid (H2SO4) for 5 min, rinsed 5 times with RO water and sterilized as for wheat. Treated seeds were germinated on moist filter paper in the dark at 25  C for 48 h. Three germinated seeds were transplanted to each pot and thinned to one plant per pot after 1 week. Plants were harvested after 4 weeks (no residue treatments only) and 7 weeks (all treatments). Both stages of the experiment were conducted in a glasshouse with irradiance in the range of 220e1000 mmol m2 s1 during the growth period (OctobereMarch, spring and summer in South Australia), depending on weather conditions. The average temperatures were 22e30  C (day) and 14e25  C (night). A modified Long Ashton nutrient solution (-P) was applied to the pots in both stages. The solution contained: 2 mM K2SO4, 1.5 mM MgSO4.7H2O, 3 mM CaCl2$2H2O, 0.1 mM FeEDTA, 4 mM (NH4)2SO4, 8 mM NaNO3. Both wheat and medic were watered with this nutrient solution every other day. Additional N was added as NaNO3 to provide 5, 10, 15, 20, 25, 30 and 35 mg N from the first week to the seventh week during Stage 2 (total 140 mg). Growth was monitored regularly throughout Stage 2 by counting the number of leaves on each plant, and appearance of 32P in the shoots of medic was followed using a hand-held radiation monitor. These data were used to decide the times for harvesting. At each harvest shoots were cut from the plants and used for determination of dry weight (75  C, 48 h), P concentration and content and 32P (kBq plant1). The HCs were carefully removed from the pots, and a small soil sample taken immediately for determination of P concentration and specific activity of 32P. The HCs were then placed in plastic bags and stored at 80  C for later determination of hyphal length density (HLD). A small weighed sample of soil (w20 g) was also taken from the RHCs and stored at 80  C for later

574

T. Duan et al. / Soil Biology & Biochemistry 43 (2011) 571e578

determination of HLDs. P concentrations in the soil samples were determined by the resin extraction method. Roots were carefully washed, blotted and fresh weight (FW) determined. Two weighed subsamples were taken for determination of total root dry weight (from the FW:DW ratios of the subsamples), P concentration and 32P content, and for AM colonization. The ovendried plant material was finely ground and weighed. Phosphate concentrations of plant tissue in both Stage 1 and Stage 2 were determined colorimetrically with a UVeVisible spectrophotometer (Shimadzu, Australia) at 390 nm, using the phosphovanado-molybdate method (Hanson, 1950) after digestion of the dried, ground plant material (w0.5 g) in 5 ml of concentrated nitric acid. 32P in soil and plant extracts was measured in a Wallac (1215 RackBeta II) liquid scintillation counter by measuring the Cerenkov radiation produced by beta particles without any scintillation fluor cocktail, and corrected for decay. AM colonization of roots was determined by the method of Giovannetti and Mosse (1980), following clearing in 10% KOH and staining in 5% ink/vinegar (Vierheilig et al., 1998). Non-quantitative observations of root segments at high magnification indicated the presence of both AMF in dual inoculated roots, based on vesicles formed by G. intraradices and auxiliary cells by G. margarita. HLDs in soil were measured by a modification of the method of Jakobsen et al. (1992). Duplicate 2 g samples of dry soil were placed in a beaker with approximately 100 ml of RO water and stirred for 2 min to break up aggregates. The suspension was gently poured into a 38 mm sieve and washed to remove clay particles. The materials on the sieve were then transferred to a Waring blender with approximately 200 ml of RO water and blended at a high speed for 30 s. The suspension was immediately transferred to a 250 ml flask, which was shaken vigorously for 10 s and allowed to stand for 60 s. Aliquots of 2 ml were taken from the flask, using a pipette tip immersed to a depth of half the suspension height. Each aliquot was transferred to a 25 mm diameter Millipore filter (8 mm pore size) in a multiple filtration device (Carbon 14 Centralen, Denmark). After removing the supernatant using a vacuum, the filters were soaked in 0.05% Trypan blue (v/v) in lactoglycerol solution (1:1:1, lactic acid, glycerol and water) for 15 min. The filters were washed gently with RO water. HLDs were determined at 160 magnification by the grid intersect method, using a bright field microscope. The number of intersects containing AM mycelium were recorded in 25 fields of observation and hyphal length was calculated based on the formula of Tennant (1975).

Inc., Chicago, USA). Comparisons between means were based on the least significant differences at the 0.05 probability level. Data for percent AM colonization were ARCSIN-transformed to achieve normality. 3. Results 3.1. Stage 1 Data for AM colonization, growth and P nutrition of wheat are shown in Supplementary Table 1. By 10 weeks plants inoculated with G. intraradices or the AMF mix were highly colonized, whereas colonization by G. margarita was lower. Non-inoculated (NM) plants did not become colonized. Because the root material provided the inoculum (disturbed or not) for Stage 2, it was not used for other analyses. Dry weights of both heads and straw were lower in all inoculated treatments than in NM plants. Among the AM treatments, growth was least with G. intraradices, greatest with G. margarita and intermediate with the AMF mix. Plants colonized by G. intraradices had higher straw P concentrations than the NM treatment. P concentrations in the straw were very low, probably as a consequence of redistribution to the grain. Due to differences in P concentration in straw the residues (4 g per pot) added in Stage 2 would have contained the following amounts of P: NM, 0.28 mg, G. intraradices 0.40 mg, G. margarita 0.36 mg, and AMF mix 0.28 mg. These additions were very small in relation to the P supplied at the start of Stage 2 (14 mg pot1). 3.2. Stage 2 3.2.1. Plant growth Medic responded positively to AM colonization. Effects were first clearly apparent at 15 d when all inoculated plants had a larger number of leaves than NM plants, except for plants inoculated with G. margarita in the disturbed treatment (results not shown). Root/ shoot (R/S) ratios were not different between treatments at 4 weeks and only small differences were apparent at 7 weeks (Supplementary Tables 2 and 3) so we present total plant DW (Fig. 1a). At 4 weeks total plant DW of all AM plants in undisturbed pots was higher than for NM plants, with no differences between AM treatments. Disturbance had a negative effect on growth of plants inoculated with G. margarita compared with the undisturbed treatment, but growth of plants inoculated with G. intraradices or AMF mix was the same regardless of disturbance. The same differences between inoculation treatments were apparent at 7 weeks (Fig. 1b), but at this time all AM plants were larger than NM plants. Disturbance was associated with reduced growth of plants inoculated with G. margarita or AMF mix compared with the undisturbed treatments, but had no effect on plants inoculated with G. intraradices. Application of residue increased growth of plants regardless of soil treatment or mycorrhizal inoculation (see Supplementary Tables 2 and 3 for ANOVA results).

2.5. Statistical analysis For Stage 1 data are presented as means and standard errors of means of 30 replicates. For Stage 2 data are presented as means and standard errors of means of five replicates, except HLDs which were means of three replicates. Data were analyzed by analysis of variance (ANOVA) using SPSS 11.5.0 statistical analysis software (SPSS

5

undisturbed

b

disturbed

4 3 2 1 0

ab c

ab

a c

b

ab c

Total DW (g)

Total DW (g)

a

5

undisturbed a

4

a

disturbed a

a b

3 c

2 1

d

d

0 NR R NR R NR R NR R

NR R NR R NR R NR R

Fig. 1. Total dry weights (roots plus shoots) of plants of Medicago truncatula grown in undisturbed or disturbed soil and colonized by Glomus intraradices (stippled bars), Gigaspora margarita (horizontally hatched bars), the mix of the two fungi (chequered bars) or non-mycorrhizal (open bars) at 4 weeks (a) and 7 weeks (b). Mean  SEM of five replicates. In b, R ¼ wheat residue applied, NR ¼ no residue. Different letters above bars (a) or pairs of bars (b) show means are significantly different (P < 0.05). In b, residue application increased total DW regardless of soil disturbance or inoculation (P < 0.05). See Supplementary Tables 2 and 3 for ANOVA results.

T. Duan et al. / Soil Biology & Biochemistry 43 (2011) 571e578

undisturbed pots and to plants inoculated with G. intraradices or AMF mix but not to plants inoculated with G. margarita in disturbed pots. At 7 weeks the AM pathway had made a significant contribution to P uptake in all AM plants and the differences in 32P content between plants inoculated with G. margarita in disturbed soil and the other treatments were smaller than at 4 weeks. Disturbance increased 32P in plants inoculated with the AMF mix in the absence of residue, but had no effect in other treatments. Overall residue application had no major effects. Values for 32P in the soil (kBq g1) in the HCs at 7 weeks (Fig. 6) were the converse of 32P uptake into plants (Fig. 5), such that the highest values in the HCs were in pots with NM plants. Thus, considerable 32P depletion occurred from all HCs in inoculated pots, emphasizing the effectiveness of external hyphae in accessing the resin-extractable P fraction in soil. Residue application did not have an effect on 32P depletion from HCs. The percent contribution of the AM pathway to P uptake was not calculated for two reasons: addition of P to the soil at the start of stage 2 and depletion in the HCs, both of which precluded accurate determination of available P in HCs and RHCs (c.f. Smith et al., 2004).

3.3. P nutrition P concentrations in roots and shoots did not vary greatly across treatments or harvests. There were significant effects of inoculation on shoot and root P concentrations (Supplementary Tables 2 and 3), with values for AM plants slightly higher than NM in most cases (results not shown). As the differences were small we present total P per plant as a measure of plant uptake. Total P per plant was significantly affected by soil disturbance, inoculation treatment and residue application (Fig. 2, Supplementary Tables 2 and 3). At 4 weeks, inoculation with AMF increased plant P content, but only marginally in plants inoculated with G. margarita. In contrast, disturbance reduced plant P content regardless of inoculation treatment. At 7 weeks, inoculation with AMF increased plant P content in both disturbed and undisturbed soil. Disturbance had no effect on P uptake in NM plants, but reduced P uptake of AM plants. Residue application increased P uptake regardless of inoculation or soil treatments. 3.4. AM colonization and hyphal length density In medic AM colonization by the two fungi and the AMF mix was well established by 4 weeks (Fig. 3a). At this time colonization reflected the colonization in wheat at the end of Stage 1, with G. intraradices and AMF mix higher than G. margarita. At 7 weeks values for G. margarita were similar to those for G. intraradices and both were higher than AMF mix (Fig. 3b). AM colonization was not affected by disturbance at 4 weeks, but at 7 weeks was higher in disturbed treatments (Fig. 3, Supplementary Table 3). Hyphal length densities (HLDs) in the RHCs and HCs (determined at 7 weeks only) are shown in Fig. 4 (see also Supplementary Table 3 for ANOVA results). Background levels of HLDs in soils in NM pots were low and the same in all treatments and in HCs and RHCs. Soils in AM treatments had much higher HLDs compared with NM treatments. Soil disturbance did not affect HLDs in either RHCs or HCs (Fig. 4, Supplementary Table 3). Values in general reflected the percent colonization of roots at 4 weeks, with G. intraradices similar to or higher than AMF mix and both higher than G. margarita. Hyphae of both AMF had extended into the HCs by 7 weeks, with apparently little or no difference in development between RHCs and HCs. Application of residue did not have an effect on HLDs in RHCs, but increased the HLDs of G. intraradices in the HCs. 3.5. Uptake of

4. Discussion The two-stage experiment simulated a wheat/medic rotation, with disturbance treatments inducing differential effects on AM fungal interactions with medic in Stage 2. AM colonization in wheat (Stage1) was relatively high with G. intraradices and AMF mix, and consistent with levels of colonization sometimes observed in the field and in pot experiments (Li et al., 2008a). Colonization by G. margarita was somewhat lower, as also found previously (Li et al., 2008b). The low colonization may in part reflect lower infectivity of the starting inoculum. During Stage 1, all AM wheat plants grew less well than NM plants, again in line with previous work in soil with low P availability (see Li et al., 2008a, b). It may be that the less negative effect of G. margarita than G. intraradices or AMF mix was due to lower colonization and hence lower carbon drain to the fungus. However, no clear relationship between growth responses (whether positive or negative) and colonization has been previously observed in wheat (Graham and Abbott, 2000; Li et al., 2008a, b) and mechanisms underlying growth depressions require re-evaluation (see Smith et al., 2009, 2010). In any event, well established AM and NM root systems developed in the pots, providing inoculum for Stage 2. Residues prepared from the straw of different treatments of wheat in Stage 1 had generally low P concentrations, and therefore made only small contributions to total P per pot. In Stage 2, and in contrast to wheat, medic responded positively to AM colonization in terms of growth and P uptake, as again shown many times previously (e.g. Burleigh et al., 2002; Smith et al., 2004). In undisturbed treatments this effect was observed with

32

P by plants and depletion in the HCs

The 32P uptake into the plants from the HCs is shown in Fig. 5. There was negligible 32P uptake by the NM plants, as expected. The results clearly show that by 4 weeks the AM pathway was making a significant contribution to P uptake in all AM plants in

10

undisturbed

disturbed

8 6 a

4 2 0

b

b 10 P (mg plant -1)

P (mg plant -1)

a

575

undisturbed

disturbed

a b

b

8 c

c

6 d

4 e

2

e

0 NR R NR R NR R NR R

NR R NR R NR R NR R

Fig. 2. Total P uptake by plants of Medicago truncatula grown in undisturbed or disturbed soil and colonized by Glomus intraradices (stippled bars), Gigaspora margarita (horizontally hatched bars), the mix of the two fungi (chequered bars) or non-mycorrhizal (open bars) at 4 weeks (a) and 7 weeks (b). Mean  SEM of five replicates. In b, R ¼ wheat residue applied, NR ¼ no residue. Different letters above groups of bars (a) or pairs of bars (b) show means are significantly different (P < 0.05). In b, residue application increased total DW regardless of soil disturbance or inoculation (P < 0.05). See Supplementary Tables 2 and 3 for ANOVA results.

T. Duan et al. / Soil Biology & Biochemistry 43 (2011) 571e578

a

undisturbed

AM colonization (%)

80 a

40

a

b b

b

undisturbed

disturbed A

80

a

60

20

disturbed a

AM colonization (%)

576

0

a

B 60

a

a

a

40

b b

20 0

NR R

NR R

NR R

NR R

NR R

NR R

Fig. 3. AM colonization of roots of Medicago truncatula grown in disturbed or undisturbed soil and inoculated with Glomus intraradices (stippled bars), Gigaspora margarita (horizontally hatched bars) or the mix of the two fungi (chequered bars) at 4 weeks (a) and 7 weeks (b). Mean  SEM of five replicates. In b, R ¼ wheat residue applied, NR ¼ no residue. In a, different letters show significant differences between mycorrhizal treatments (P < 0.05) (soil disturbance did not have any effects). In b, different lower case letters above pairs of bars show significant differences between mycorrhizal treatments (P < 0.05). Different upper case letters show significant differences associated with soil disturbance (P < 0.05). Residue application did not have any effects. See Supplementary Tables 2 and 3 for ANOVA results.

both AMF, separately and together, and was accompanied by increased P uptake and transfer of 32P via the AM pathway, as shown by appearance of 32P in the plants. This transfer resulted in depletion of P from the HCs. The pattern of total P uptake per plant across treatments was very similar to uptake of 32P, emphasizing the large contribution of the AMF to P uptake. There was no evidence for reduction in uptake via the direct pathway, as observed in some negatively responsive plants (see Smith et al., 2009). The uniformly high colonization and high P uptake and transfer from HCs demonstrates the importance of the AM fungal network, which must have become rapidly established in the soil of the HCs in all AM treatments. In the undisturbed treatments, lower colonization of medic by G. margarita was not associated with smaller positive growth responses or lower transfer of 32P, even though HLDs in both RHC and HC were lower than for G. intraradices or AMF mix. These results suggest that neither percent colonization during early growth nor HLDs (which presumably represented intact hyphal networks in these treatments) limit the amount of P absorbed via the AM pathway. Nevertheless, depletion of P in HCs of pots inoculated with G. margarita was lower than for the other AM treatments, as was total P uptake and 32P per plant. This indicates that there is functional diversity in arbuscular mycorrhizas in exploitation of soil P depending on fungal species. Furthermore, G. margarita developed slowly compared with G. intraradices when they were inoculated separately and it seems likely that the latter fungus dominated the symbiosis with medic when both fungi were inoculated together. Our hypothesis that disturbance would decrease AM fungal development in medic was not upheld. Inclusion of two harvests allowed us to follow temporal differences between inoculation treatments and hence gain improved insights into effects of disturbance on symbiotic development and function. Considering the no residue treatments at 4 and 7 weeks, there were no reductions in percent colonization or HLDs in either RHC or HC in the disturbed, compared with undisturbed treatments. Inconsistent

a

effects of disturbance on colonization have been observed before with both decrease and no effects observed (see Introduction and Kabir et al., 1997; Miller, 2000; McGonigle and Miller, 2000). We must assume that the infectivity of the soil based on the AM development with wheat roots in Stage 1, whether disturbed or undisturbed, was sufficient to achieve maximum possible colonization for both fungi; in other words inoculum potential in soil was not reduced by disturbance. The colonization process for G. margarita was slower than for G. intraradices, but resulted in equally high colonization percentages at 7 weeks. In any event, our results help explain the lack of effect of disturbance (tillage) on percent colonization in the field (Duan et al., 2010) and emphasize that measurements of percent colonization do not provide information that can predict the outcomes of symbiosis in terms of plant growth and P uptake. Disturbance had no effect on growth or P uptake by NM plants indicating that this treatment mainly affected medic growth via effects on AMF (Miller, 2000). Our findings differ from those of McGonigle et al. (1999) and the lack of an effect on NM plants simplifies the interpretation of our results for AM plants. With G. intraradices there were (with one exception) no reductions in medic growth or P uptake as a result of disturbance. The exception was slightly lower total plant P in disturbed compared with undisturbed treatments at 7 weeks. However, this reduction was not associated with reduced 32P transfer from the HC, which was higher in disturbed treatments at 4 weeks and similar at 7 weeks. These results confirm the ‘aggressive’ (Graham and Abbott, 2000) nature of G. intraradices and show that it is insensitive to disturbance, as also suggested by Jansa et al. (2003). G. intraradices behaved similarly to G. mosseae in the investigation of McGonigle et al. (2003). The lack of sensitivity could be explained by very rapid establishment of colonization from propagules in the soil (as observed at 4 weeks) and development of a new hyphal network growing from medic roots, so that AM P uptake from both RHC and HC is established quickly. Alternatively, this fungus may have

b

Fig. 4. Hyphal length density (HLD) in soils of RHCs (a) and HCs (b) at 7 weeks in association with Medicago truncatula grown in undisturbed or disturbed soil and colonized by Glomus intraradices (stippled bars), Gigaspora margarita (horizontally hatched bars) and the mix of the two fungi (chequered bars), or non-mycorrhizal (open bars). Mean  SEM of three replicates. In b, R ¼ wheat residue applied, NR ¼ no residue. In a, different letters show significant differences between mycorrhizal treatments (P < 0.05). Soil disturbance did not have any effects. In b, different letters show significant differences between means, regardless of soil disturbance treatments (P < 0.05), which did not have any effects. See Supplementary Table 3 for ANOVA results.

T. Duan et al. / Soil Biology & Biochemistry 43 (2011) 571e578

undisturbed

b

disturbed

600

P (kBq plant -1 )

800

a bc

400

ab

abc c

200 d

32

32

P (kBq plant -1)

a

d

d

0

577

undisturbed

800

disturbed a

a

600

ab

ab ab cd

400

bc

bc

200 f

f

bc

cd

cd

de f

f

0 NR R NR R NR R NR R

NR R NR R NR R NR R

Fig. 5. Uptake of 32P (kBq plant1) into shoots of Medicago truncatula grown in undisturbed or disturbed soil and colonized by Glomus intraradices (stippled bars), Gigaspora margarita (horizontally hatched bars), the mix of the two fungi (chequered bars) or non-mycorrhizal (open bars) at 4 weeks (a) and 7 weeks (b). Mean  SEM of five replicates. In b, R ¼ wheat residue applied, NR ¼ no residue. In either a or b, different letters above bars show means are significantly different (P < 0.05). See Supplementary Tables 2 and 3 for ANOVA results.

a marked ability to repair the hyphal network after disturbance by formation of anastomoses (Mikkelsen et al., 2008). In contrast, G. margarita proved to be sensitive to disturbance, as shown for AM symbioses with maize (Fairchild and Miller, 1988; McGonigle et al., 1990). Although fungal development was not affected, growth, P uptake and delivery of 32P to the plants was delayed in disturbed pots, there being no differences between NM plants and those colonized by G. margarita at 4 weeks. At 7 weeks positive growth and P responses were apparent (although these were lower than with G. intraradices) and 32P delivery was as high as in undisturbed treatments, although still lower than with G. intraradices or AMF mix. Inoculation with the two fungi together gave outcomes at 4 weeks that were almost identical to those for G. intraradices in terms of fungal development, growth responses and P delivery, even though structures of both fungi were observed in medic roots at this time. By 7 weeks, colonization was lower than for either fungus inoculated separately and HLDs were lower than for G. intraradices. Nevertheless, growth, P uptake and 32P delivery to plants and depletion from HCs were generally similar to plants inoculated with G. intraradices. Although both AMF were present in the roots it appears that G. intraradices may have ‘dominated’ the activity of the symbiosis, both in terms of rapidity of early colonization and functionality, including tolerance to disturbance. McGonigle et al. (2003) also showed that G. margarita was apparently both a poor competitor (in that case with G. mosseae) and sensitive to disturbance. Our hypothesis that a fungus less sensitive to disturbance would compensate for the reduction of activity of the sensitive fungus was upheld in plants inoculated with AMF mix. This is an example of functional complementarity among AMF as proposed by Koide (2000). The positive effect of G. intraradices was probably enhanced by its ability to colonize quickly and it may well have contributed a much larger fraction of fungal biomass than

10

undisturbed a

a

b

5

disturbed

b

b

c c

32

P (kBq g-1 soil)

15

0

NR R NR R NR R NR R 32

1

c

NR R NR R NR R NR R

Fig. 6. Concentration of P in soil (kBq g soil) in hyphal compartments (HCs) of pots in which Medicago truncatula was grown in undisturbed or disturbed soil and colonized by Glomus intraradices (stippled bars), Gigaspora margarita (horizontally hatched bars) and the mix of the two fungi (chequered bars) or was non-mycorrhizal (open bars). R ¼ wheat residue applied, NR ¼ no residue. Different letters above pair of bars show means are significantly different (P < 0.05). Residue application did not have any effects. See Supplementary Tables 2 and 3 for ANOVA results.

G. margarita, when both were inoculated together. However, the increase in colonization by G. margarita throughout the experiment suggests that this fungus may have contributed more biomass at 7 weeks than at 4 weeks, in competition as well as when inoculated alone. Whereas in undisturbed soil we saw no evidence that concurrent colonization of the plants by the two fungi increased growth or P uptake compared with plants colonized by a single fungus, there was a positive effect of dual inoculation in disturbed treatments on growth, P uptake and 32P delivery compared with G. margarita alone. This finding is similar to that of Jansa et al. (2008) who observed complementarity between G. intraradices (a different isolate) and Glomus claroideum in disturbed soil when both contributed similarly to fungal biomass. Our hypothesis that residue application would increase plant P uptake and plant growth was upheld. It has been suggested that residue improves the available nutrient supply in the soil and/or changes soil chemical properties (pH, nutrient availability) when mixed with soil (Borie et al., 2002) or applied to the soil surface (Hafner et al., 1993). However, in our experiment the available P and pH of soil in RHC were not affected by residue application, regardless of inoculum (results not shown). Our results showed that NM plants had the same P uptake and growth responses to residue application as AM plants suggesting that residue application improved direct plant P uptake. Furthermore, residue application did not affect AM colonization, and had only a marginal effect on the HLDs in HCs which did not translate in any changes on 32P uptake via AMF. 5. Conclusions We simulated a wheat/medic rotation, with disturbance treatments inducing differential effects on AM fungal interactions with medic in Stage 2 of the experiment. Soil disturbance did not affect AM development, indicating that whether disturbed or undisturbed, the propagules in soil were sufficient to achieve maximum possible colonization for both G. intraradices and G. margarita. The results are in accord with some field observations showing no effect of disturbance on colonization. The two fungi had different responses to disturbance in terms of their effects on symbiotic growth and P uptake of the plants, so that the hypothesis that disturbance would decrease plant growth and activity of the AM P uptake pathway was upheld for G. margarita, but not G. intraradices. When inoculated together G. intraradices may have ‘dominated’ the activity of the symbiosis, both in terms of rapidity of early colonization and functionality, including tolerance to disturbance. Overall, the results upheld our hypothesis that a fungus less sensitive to disturbance would compensate for the reduction of activity of a sensitive fungus. Taken together, our results indicate that crop management practices involving reduced tillage (reduced disturbance) and retention of plant residues are likely to have positive effects on AM

578

T. Duan et al. / Soil Biology & Biochemistry 43 (2011) 571e578

symbioses and hence on their effects on plant growth. Combined activities of the assemblage of AMF found in the field are likely to buffer negative effects on individual AMF. Furthermore, effects on plant growth and nutrition cannot be predicted from measurements of AM colonization which, in our experiment, were unaffected by the treatments probably because of the high inoculum potentials generated in Stage 1 of the experiment. Acknowledgements This research was financially supported by the University of Adelaide and the National Basic Research Program of China (973) (2007CB 108902). The authors would like to thank Ms Rebecca Stonor for technical help. Appendix. Supplementary information Supplementary data related to this article can be found online at doi:10.1016/j.soilbio.2010.11.024. References Augé, R.M., 2001. Water relations, drought and vesicular-arbuscular mycorrhizal symbiosis. Mycorrhiza 11, 3e42. Borie, F., Redel, Y., Rubio, R., Rouanet, J.L., Barea, J.M., 2002. Interactions between crop residues application and mycorrhizal developments and some soil-root interface properties and mineral acquisition by plants in an acidic soil. Biology and Fertility of Soils 36, 151e160. Burleigh, S.H., Cavagnaro, T.R., Jakobsen, I., 2002. Functional diversity of arbuscular mycorrhizas extends to the expression of plant genes involved in P nutrition. Journal of Experimental Botany 53, 1e9. Duan, T.Y., Shen, Y.Y., Facelli, E., Smith, S.E., Nan, Z.B., 2010. New agricultural practices in the Loess Plateau of China do not reduce colonisation by arbuscular mycorrhizal or root invading fungi and do not carry a yield penalty. Plant and Soil 331, 265e275. Evans, D.G., Miller, M.H., 1988. Vesicular-arbuscular mycorrhizas and the soiledisturbance-induced reduction of nutrient absorption in maize. I. Causal relations. New Phytologist 110, 67e74. Facelli, E., Smith, S.E., Facelli, J.M., Christophersen, H.M., Smith, F.A., 2010. Underground friends or enemies: model plants help to unravel direct and indirect effects of arbuscular mycorrhizal fungi on plant competition. New Phytologist 185, 1050e1061. Fairchild, G.L., Miller, M.H., 1988. Vesicular-arbuscular mycorrhizas and the soildisturbance induced reduction of nutrient absorption in maize. II. Development of the effect. New Phytologist 110, 75e84. Giovannetti, M., Mosse, B., 1980. An evaluation of techniques for measuring vesicular-arbuscular mycorrhizal infection in roots. New Phytologist 84, 489e500. Graham, J.H., Abbott, L.K., 2000. Wheat responses to aggressive and non-aggressive arbuscular mycorrhizal fungi. Plant and Soil 220, 207e218. Hafner, H., George, E., Bationo, A., Marschner, H., 1993. Effect of crop residue on root growth and phosphorus acquisition of pearl millet in an acid sandy soil in Niger. Plant and Soil 150, 117e127. Hanson, W.C., 1950. The photometric determination of phosphorus in fertilizers using the phosphovanado-molybdate complex. Journal of the Science of Food and Agriculture 1, 172e173. Jansa, J., Mozafar, A., Anken, T., Ruh, R., Sanders, I.R., Frossard, E., 2002. Diversity and structure of AMF communities as affected by tillage in a temperate soil. Mycorrhiza 12, 225e234. Jansa, J., Mozafar, A., Kuhn, G., Anken, T., Ruh, R., Sanders, I.R., Frossard, E., 2003. Soil tillage affects the community structure of mycorrhizal fungi in maize roots. Ecological Applications 13, 1164e1176. Jansa, J., Smith, F.A., Smith, S.E., 2008. Are there benefits of simultaneous root colonization by different arbuscular mycorrhizal fungi. New Phytologist 177, 779e789. Jansa, J., Weimken, A., Frossard, E., 2006. The effects of agricultural practices on arbuscular mycorrhizal fungi. In: Frossard, E., Blum, W., Warkentin, B. (Eds.), Function of Soils for Human Societies and the Environment, vol. 266. Special Publication, Geological Society, London, UK, pp. 89e115. Jasper, D.A., Abbott, L.K., Robson, A.D., 1989. Soil disturbance reduces the infectivity of external hyphae of vesicular-arbuscular mycorrhizal fungi. New Phytologist 112, 93e99. Jakobsen, I., Abbott, L.K., Robson, A.D., 1992. External hyphae of vesicular-arbuscular mycorrhizal fungi associated with Trifolium subterraneum L. 1. Spread of hyphae and phosphorus inflow into roots. New Phytologist 120, 371e380.

Johnson, D., Leake, J.R., Read, D.J., 2001. Novel in-growth core system enables functional studies of grassland mycorrhizal mycelial networks. New Phytologist 152, 555e562. Kabir, Z., O’Halloran, I.P., Fyles, J.W., Hamel, C., 1997. Seasonal changes of arbuscular mycorrhizal fungi as affected by tillage practices and fertilization: hyphal density and mycorrhizal root colonization. Plant and Soil 192, 285e293. Kabir, Z., O’Halloran, I.P., Fylesa, J.W., Hamela, C., 1998. Dynamics of the mycorrhizal symbiosis of corn (Zea mays L.): effects of host physiology, tillage practice and fertilization on spatial distribution of extra-radical mycorrhizal hyphae in the field. Plant and Soil 68, 151e163. Koide, R.T., 2000. Functional complementarity in the arbuscular mycorrhizal symbiosis. New Phytologist 147, 233e235. Li, H.-Y., Smith, F.A., Dickson, S., Holloway, R.E., Smith, S.E., 2008b. Plant growth depressions in arbuscular mycorrhizal symbiosis: not just caused by carbon drain? New Phytologist 178, 852e862. Li, H.-Y., Smith, S.E., Ophel-Keller, K., Holloway, R.E., Smith, F.A., 2008a. Naturally occurring arbuscular mycorrhizal fungi can replace direct P uptake by wheat when roots cannot access added P fertilizer. Functional Plant Biology 35, 125e134. Litchfield, W., 1951. Soil Survey of the Waite Agricultural Research Institute. Commonwealth Scientific and Industrial Research Organization, Division of Soils, Adelaide, p. 38. McGonigle, T.P., Evans, D.G., Miller, M.H., 1990. Effect of degree of soil disturbance on mycorrhizal colonization and phosphorus absorption by maize in growth chamber and field experiments. New Phytologist 116, 629e636. McGonigle, T.P., Miller, M.H., 1996. Mycorrhizae, phosphorus absorption, and yield of maize in response to tillage. Soil Science Society of America Journal 60, 1856e1861. McGonigle, T.P., Miller, M.H., 2000. The inconsistent effect of soil disturbance on colonization of roots by arbuscular mycorrhizal fungi: a test of the inoculum density hypothesis. Applied Soil Ecology 14, 147e155. McGonigle, T.P., Miller, M.H., Young, D., 1999. Mycorrhizae, crop growth, and crop phosphorus nutrition in maize-soybean rotations given various tillage treatments. Plant and Soil 210, 33e42. McGonigle, T.P., Yano, K., Shinhama, T., 2003. Mycorrhizal phosphorus enhancement of plants in undisturbed soil differs from phosphorus uptake stimulation by arbuscular mycorrhizae over non-mycorrhizal controls. Biology and Fertility of Soils 37, 268e273. McLaughlin, M.J., Lancaster, P.A., Sale, P.G., Uren, N.C., Peverill, K.I., 1994. Comparison of cation/anion exchange resin methods for muti-element tesing of acidic soils. Australian Journal of Soil Research 32, 229e240. Mikkelsen, B.L., Rosendahl, S., Jakobsen, I., 2008. Underground resource allocation between individual networks of mycorrhizal fungi. New Phytologist 180, 890e898. Miller, M.H., 2000. Arbuscular mycorrhizae and the phosphorus nutrition of maize: a review of Guelph studies. Canadian Journal of Plant Science 80, 47e52. Miller, R.M., Jastrow, J.D., 2000. Mycorrhizal fungi influence soil structure. In: Kapulnik, Y., Douds, D.D. (Eds.), Arbuscular Mycorrhizas: Physiology and Function. Kluwer Academic Publishers, Netherlands, pp. 1e19. Newsham, K.K., Fitter, A.H., Watkinson, A.R., 1995. Multi-functionality and biodiversity in arbuscular mycorrhizas. Trends in Ecology and Evolution 10, 407e411. Rasmussen, P.E., Allmaras, R.R., Rohde, C.R., Roager Jr., N.G., 1980. Crop residue influences on soil carbon and nitrogen in a wheat-fallow system. Soil Science and Society of American Journal 44, 596e600. Shen, Y.Y., Nan, Z.B., Gao, C.Y., Bellotti, W.D., Chen, W., 2004. Spatial and temporal characteristics of soil water dynamics and crop yield response from a 4-year of lucerne and winter wheat rotation system in the Loess Platau. Acta Ecologica Sinica 24, 640e647. Smith, F.A., Grace, E.J., Smith, S.E., 2009. More than a carbon economy: nutrient trade and ecological sustainability in facultative arbuscular mycorrhizal symbioses. New Phytologist 182, 347e358. Smith, S.E., Li, H.-Y., Grace, E.J., Smith, F.A., 2010. New insights into roles of abuscular mycorrhizas in P nutrition of crops reveal the need for conceptual changes. In: Gupta, V.V.S.R., Ryder, M., Radcliffe, J. (Eds.), Proceedings of the Rovira Rhizosphere Symposium. Crawford Fund, ACT, Australia, pp. 31e40. Smith, S.E., Read, D.J., 2008. Mycorrhizal Symbiosis, third ed. Academic Press, London, UK. Smith, S.E., Smith, F.A., Jakobsen, I., 2003. Mycorrhizal fungi can dominate phosphate supply to plants irrespective of growth response. Plant Physiology 133, 16e20. Smith, S.E., Smith, F.A., Jakobsen, I., 2004. Functional diversity in arbuscular mycorrhizal (AM) symbiosis: the contribution of the mycorrhizal P uptake pathway is not correlated with mycorhizal responses in growth or total P uptake. New Phytologist 162, 511e524. Stockinger, H., Walker, C., Schüßler, A., 2009. ‘Glomus intraradices DAOM197198’, a model fungus in arbuscular mycorrhiza research, is not Glomus intraradices. New Phytologist 183, 1176e1187. Tisdall, J.M., 1994. Possible role of soil microorganisms in aggregation in soils. Plant and Soil 159, 115e121. Triplett Jr., G.B., Dick, W.A., 2008. No-tillage crop production: a revolution in agriculture! Celebrate the Centennial, A Supplement to Agronomy Journal 100, S153eS165. Vierheilig, H., Coughlan, A.P., Wyss, U., Piche, Y., 1998. Ink and vinegar, a simple staining technique for arbuscular-mycorrhizal fungi. Applied and Environmental Microbiology 64, 5004e5007.