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Applied and Environmental Microbiology, July 2008, p. 4144-4148, Vol. 74, No. 13
0099-2240/08/$08.00+0     doi:10.1128/AEM.00376-08
Copyright © 2008, American Society for Microbiology. All Rights Reserved.

Phosphorus Availability Influences Elemental Uptake in the Mycorrhizal Fungus Glomus intraradices, as Revealed by Particle-Induced X-Ray Emission Analysis

Pål Axel Olsson,^1* Edith C. Hammer,² Håkan Wallander,² and Jan Pallon³

Department of Plant Ecology and Systematics,¹ Department of Microbial Ecology,² Department of Nuclear Physics, Lund University, Lund, Sweden³

Received 14 February 2008/ Accepted 4 May 2008

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We investigated element accumulation in the arbuscular mycorrhizal fungus Glomus intraradices. Fungal spores and mycelia growing in monoxenic cultures were analyzed. The elemental composition was quantified using particle-induced X-ray emission (PIXE) in combination with scanning transmission ion microscopy. In the spores, Ca and Fe were associated mainly with the spore wall, while P and K showed patchy distributions and their concentrations were correlated. Excess of P in the hyphal growth medium increased the P and Si concentrations in spores and increased the K/Ca ratio in spores. Increased P availability decreased the concentration of Zn and Mn in spores. We concluded that the availability of P influences the uptake and accumulation of several elements in spores. It is demonstrated that PIXE analysis is a powerful tool for quantitative analysis of elemental accumulation in fungal mycelia.

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Arbuscular mycorrhizal (AM) fungal spores are asexual lipid-rich survival organs (32) formed on the soil mycelium. After germination, they provide the energy carbon (C) for the fungus before carbohydrates can be taken up through symbiotic exchange with plants. In addition to lipids, the spores are potential storage organs for mineral nutrients essential for the formation of a germ tube. However, the nutrients stored in spores may reflect not only the need for formation of new fungal biomass but also that of the plants that are being colonized. The establishment of new symbiotic relations could thus depend on the accumulation of plant-limiting nutrients in spores and their transport into the host roots for recognition of a nonparasitic intruder.

The nutrient accumulation and distribution in AM fungal spores have not been studied in as much detail as carbon accumulation has (32), although there are clear links between the nutrient availability and C metabolism (16, 17, 18). Quantitative analysis of elemental composition in AM fungal mycelia is crucial for our understanding of the distribution of nutritional resources.

Electron-based methods, such as energy-dispersive X-ray spectroscopy or electron probe microanalysis (5, 30), can be used to obtain elementary maps of thin sections of fixed biological tissues. However, quantification of intact structures is not possible with these methods.

Particle-induced X-ray emission (PIXE) analysis is based on the analysis of characteristic X-rays emitted from specific elements bombarded with protons from an accelerator (10). PIXE can be used to analyze elemental distributions in three-dimensional structures, and no chemical fixation is needed. Use of this technique, in combination with scanning transmission ion microscopy (STIM), enables quantification of the amounts and distribution of elements in structures as small as AM fungal spores and hyphae. PIXE is analogous to energy-dispersive X-ray spectroscopy, but its sensitivity is much higher, allowing quantification at levels of µg g^–1. Limitations of the PIXE method are that it requires an extensive amount of time and the number of samples that can be analyzed is therefore limited.

Various types of fungal and plant material have been analyzed using PIXE. Earlier studies have reported the elemental compositions of fungal fruit bodies (11), ectomycorrhizal roots and hyphae (31, 34), the bladders of the fungus Geosiphon pyriforme (13, 28), AM-colonized roots (27), and AM fungal spores (23, 24, 35). The focus of previous studies on AM fungi has been on their potential to accumulate heavy metals in order to investigate whether they can be used to remediate contaminated soil.

We analyzed the elemental concentration and distribution within AM fungal spores, using a monoxenic mycorrhizal culture of Glomus intraradices, in order to investigate the effects of P addition to the medium on nutrient uptake by the fungus.

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Monoxenic AM cultures.
The AM fungus Glomus intraradices Schenck & Smith (DAOM 197198; Biosystematics Research Center, Ottawa, Canada) was grown monoxenically in mycorrhizal association with root organ cultures of carrot (Daucus carota L.). The cultures were clones of carrot roots (line DC1) that had been transformed with the T-DNA of the root-inducing (Ri) plasmid from the bacterium Agrobacterium rhizogenes (2). The cultures were originally established as described by St.-Arnaud et al. (29). AM-colonized cultures were maintained at 24°C in petri dishes on a minimal nutrient medium (Table 1) containing 10 g liter^–1 sucrose as the C source and 35 µM P (4.8 mg liter^–1 KH₂PO₄), gelled with 0.3% Phytagel (Sigma Chemical Co., St. Louis, MO).

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TABLE 1. Elemental composition of minimal growth medium (6) and AM fungal spore-to-medium ratio for elements in liquid minimal medium without sucrose and with a low P concentration of 25 µM (n = 3)

Cylindrical plugs (diameter, 1.5 cm; height, 0.5 cm) of solid medium containing carrot roots and mycelium and spores of G. intraradices were transferred from 4-month-old cultures to the experimental two-compartment petri dishes (14). One plug was transferred to each new dish and inserted into a hole in the solid minimal nutrient medium (about 20 ml medium in total) on the root side. The cultures were sealed with Parafilm M (American National Can, Chicago, IL). At the start of the experimental treatment (see below), the second compartment was filled with liquid minimal medium without sucrose.

Monoxenic P enrichment experiment.
The liquid medium had two different P treatments applied to each of five petri dishes. The amount of P added was 25 µM (low-P treatment) or 2.5 mM as KH₂PO₄ (high-P treatment). The systems were harvested 100 days after addition of the liquid medium. Samples from three replicates (the three petri dishes for each treatment that had the largest amounts of mycelium) of each treatment were used for PIXE analysis. Two spores from each replicate plate were analyzed, and the mean value was used for each.

In order to investigate hyphal samples of mycelium that were still in an early stage, when sporulation had just started, we also sampled young mycelia from two replicate petri dishes of the low-P treatment 50 days after adding the liquid medium. These samples were collected from separate petri dishes that were not used further. We analyzed mycelium from each of the dishes (see Fig. 2) and selected two areas from each mycelium for determination of elemental concentrations (Table 2; see Fig. 2 for selected areas). The areas were selected because they showed divergent concentrations of P.

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FIG. 2. P distribution in hyphae from two selected areas of mycelium (a and b) from monoxenic cultures of the AM fungus Glomus intraradices. Mycelium with a low density was selected in order to differentiate individual hyphae. We selected spots that showed variation in color intensity in order to obtain quantitative data on this variation. The color scale shows the gradient from low to high elemental concentrations. (Data from selected areas are presented in Table 2.) The color scale indicates the variation in elemental concentration within a specific graph and should not be used to compare different graphs. Bars = 10 µm.

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TABLE 2. Elemental content in young hyphae of Glomus intraradicesa

Mounting of mycelia.
Mycelia from monoxenic cultures were washed with deionized water by replacing the nutrient medium with water five times in intact cultures, i.e., the mycelium was still attached to the roots while being washed. After the washing step, the liquid medium compartment was filled with water. The mycelium was detached using a razor and left floating in the water. The mounting ring (covered with double-sided adhesive tape) was inserted into the water below the mycelium by use of two pairs of forceps, and the mycelium was then lifted vertically up. The mycelium stuck to the adhesive tape, which had a diamond-shaped slit in the middle (2 mm x 4 mm) (Fig. 1). The mounted mycelium was freeze-dried and stored dry at –20°C until analysis. Only mycelium in the slit was analyzed.

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FIG. 1. Schematic illustration of sample preparation for PIXE analysis. The mounting ring was covered with double-sided adhesive tape with a diamond-shaped slit in the middle. Only mycelium in the slit was analyzed.

Elemental analyses.
The PIXE analysis was performed at the Lund Nuclear Microprobe Laboratory. A 2.5-MeV proton beam with a current of 300 pA was focused to a spot of about 5 µm in diameter. Each sample was scanned accumulatively over an area of about 250 by 250 µm for 25 to 35 min, or until the data collected presented sufficiently detailed elemental maps of at least P, S, K, Ca, and Fe. STIM, which is based on the detection of energy loss when protons pass through a sample (12, 20), was performed in on-axis/off-axis geometry, which has the advantage that it permits simultaneous PIXE and STIM measurements (21, 22). The STIM data were used to create elemental maps of the sample and to determine the mass per unit area of spores and hyphae (no area outside the biological material was included).

All data were collected in event-by-event mode, i.e., raw data (in coded protocol) were recorded in time sequence as a large computer file, using KMAX-based software (5). This permitted a later resorting of the data and applying, e.g., position restrictions to limit the results to a certain area of the investigated sample.

After analysis, quantitative images were created using the CSIRO dynamic analysis method GeoPIXE II (http://www.nmp.csiro.au/dynamic.html), which enables true elemental images to be extracted from the generally complex PIXE energy spectrum (25). The elemental images were inspected, and regions within spores and hyphae were selected for precise quantitative evaluation. Using GeoPIXE II, detailed PIXE spectra were first constructed by resorting the data and then evaluated to provide quantitative information. From the corresponding STIM spectra, the local mass per unit area of the sample was determined and the elemental content per unit of dry weight (freeze-dried) was calculated.

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Elemental distribution in spores and hyphae.
Phosphorus was the element with the highest level of accumulation in spores compared with the concentration in the medium (Table 1). Zn and Cu also showed high levels of accumulation. S and Cl, on the other hand, showed almost the same concentrations in spores as in the medium (Table 1). Al and Si were found in small amounts in spores (Table 3), although they were not specifically added to the substrate.

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TABLE 3. Effects of phosphorus addition on elemental composition in sporesb

Table 2 shows the variation in mass and elemental concentration between four young hyphae from two separate petri dishes, and Fig. 2 illustrates the P distribution in the same selection. As can be seen, the hyphae showed large variations in elemental composition, and the P concentration also varied between different parts of a mycelium.

The elemental concentrations were analyzed in several spores and hyphae. Figure 3 shows the relative distributions of P, K, Ca, Fe, and S in one sample containing mainly spores (from the high-P treatment) and one sample containing only hyphae (from the low-P treatment). The hyphal mass was too low to provide any detectable pattern in the graph. The spores showed great variation in spore mass as well as elemental composition. P and K had similar distributions, often in patches, in the spores. Ca and Fe, on the other hand, seemed to be associated with the spore wall. A few runner hyphae (the thicker and older hyphae) contained much more P than the other hyphae. Fe, in particular, was more evenly distributed throughout all the hyphae (Fig. 3).

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FIG. 3. Scanned areas of spores and hyphae selected specifically to illustrate the distributions of spore mass, P, K, Ca, Fe, and S in the AM fungus Glomus intraradices. The samples were selected from the P enrichment experiment; the spore sample comes from a plate with high-P treatment, and the hyphae come from a plate with low-P treatment. The particular spore sample was chosen because it covers a large number of spores, and the hyphal sample was chosen because it covers both thick runner hyphae and thinner absorbing hyphae. No pattern can be seen for mass because the intensity is too low. The color scale indicates the variation in elemental content or mass. Bars = 50 µm.

Effects of P availability on nutrient accumulation in spores.
The spore mass per unit area tended to increase as a result of excess P in the medium (from 1.0 ± 0.1 to 2.3 ± 0.6 mg cm^–2 [n = 3]; P = 0.067 [not significant] by two-tailed t test on logarithmic data). Marked differences in nutrient accumulation were seen when mycelium was formed in liquid medium with excess P (Table 3). The P content increased 6-fold with a 100-fold P enrichment in the medium, but other elements also increased, for example, 3-fold higher concentrations of Si, S, and K were found. However, the only statistically significant increase, apart from that of P, was that of Si. There was also a significant increase in the K/Ca ratio. Ca was associated with the cell wall and may be a good indicator of the structural part of the spore, and it tended to decrease in content due to increased P availability. The uptake and accumulation of Mn and Zn were significantly decreased by P enrichment; the Mn concentration decreased 10-fold and the Zn concentration decreased 5-fold. The concentrations of Cl, Fe, and Cu were constant, irrespective of the P availability.

Spatial distribution of elements in spores.
Six pairs of spores, each from a separate plate subjected to high- or low-P treatment (three plates for each treatment), were used to analyze the elemental distribution across the diameter of the spores. The profiles for Ca, P, and K distributions were compared with the spore masses (Fig. 4).

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FIG. 4. Distributions of P, K, Ca, and spore mass as averages for three replicate plates (n = 3; each replicate represents the average for two spores) following low-P treatment and high-P treatment. Error bars represent 1 standard error.

The spores had diameters ranging between 50 and 130 µm, and the diameter of the spores was therefore expressed as a percentage of the distance from one side to the other. Following low-P addition, Ca was the dominant element in the spores. Its distribution was slightly higher in the spore wall. The distributions of P and K were similar to each other. Following the high-P treatment, both P and K showed higher abundances in the spores than that of Ca. P and K were present mainly in the middle of the spores.

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Using the PIXE technique, we estimated the P concentration in AM fungal spores to be the same as the concentration usually found in plants, i.e., about 2,000 µg g^–1 (15, 26). The spores are used for carbon storage and can thus be expected to contain less of the elements normally found in metabolically active tissues. This was found to be the case for elements such as S and K, which in some cases had concentrations in the spores that were 10 times lower than those normally found in plants. The high P content in spores indicates that these may act as storage organs for P as well as for C. The fungi may take up P during the late autumn and winter, when decomposition occurs (7, 8) but there is little plant growth.

The abundance and distribution of K were correlated with the abundance of P in spores. In AM fungi, P is present mainly as polyphosphate, and it has been suggested that K acts as a counterion to P in polyphosphate, thereby stabilizing the molecule (19). The precipitate of calcium polyphosphates previously reported for AM structures is now believed to be an artifact induced by chemical fixation of the samples (19). Polyphosphate granules do occur in fungi, but K and Mg seem to be the main counterions (3). The spatial distributions of P and K in spores were closely related to each other. No other element was related to P in this way, indicating a specific interaction between K and P. Furthermore, the K content tended to increase in spores when the P content increased. The increase in the K/Ca ratio could also be due to the fact that additional K was added with the P (given as KH₂PO₄), but the hyphae already had an excess of K in the medium. Ashford et al. (1) and Orlovich and Ashford (19) found high levels of P and K in AM fungal vacuoles, and just as we noted for the AM fungal spores in this study, their abundances seemed to be linked. This may be due to polyphosphate transport mechanisms in the vacuolar system.

Maetz et al. (13) analyzed the bladders of Geosiphon pyriforme fungi by using PIXE and found the concentration of P to be 13,000 µg g^–1, while that of K in the same structure was as high as 50,000 µg g^–1. In studies of ectomycorrhizal fungal rhizomorphs collected in the field, the P content determined using PIXE was generally about 1,000 µg g^–1, while the K content of Rhizopogon sp. was found to be 5,000 to 10,000 µg g^–1 in one study (34) and those in Paxillus involutus, Telephora terrestris, and Tylospora fibrillosa in another study were 200 to 300 µg g^–1 (34). Indeed, the relationship between P and K in fungal mycelia warrants further study to elucidate the importance of K in P metabolism.

The elemental analysis of nutrient accumulation in AM fungal structures using PIXE allows for quantitative evaluation on the micrometer scale. Finely resolved elemental maps over single spores can be obtained using PIXE. We were thus able to reveal differences in the elemental compositions of hyphae and spores reflecting their different functions. Compounds known to be active in membrane transport, such as Cl and K (15), were particularly common in hyphae, highlighting the role of spores in storage, not in uptake and transport.

Excess P decreased the uptake of Zn and Mn in the AM fungal mycelium. This implies that there is some link between these compounds and the regulation of the high-affinity P transporter in Glomus. Excess P repressed the expression of the high-affinity P transporter gene in extraradical AM fungal mycelia (14). In plants, the high-affinity P transporter is regulated by Zn limitation, leading to increased P uptake (9). Our observations show that the uptakes of these compounds influence each other, although we do not yet know the mechanism behind this.

We concluded that AM fungal spores may act as an important P storage structure for AM fungi as well as their host plants. Accumulation of nutrients in spores may prove much more important than hitherto believed, especially for nutrients that are rapidly precipitated at mineral surfaces or taken up by other microorganisms.

 
This work was funded by grants from The Swedish Research Council for the Environment, Agriculture Sciences and Spatial Planning (FORMAS), The Crafoord Foundation, and The Swedish Research Council.

 
* Corresponding author. Mailing address: Department of Plant Ecology and Systematics, Ecology Building, Lund University, SE-223 62 Lund, Sweden. Phone: 46 46 222 4247. Fax: 46 46 222 4158. E-mail: pal_axel.olsson{at}ekol.lu.se

Published ahead of print on 9 May 2008.

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Applied and Environmental Microbiology, July 2008, p. 4144-4148, Vol. 74, No. 13
0099-2240/08/$08.00+0     doi:10.1128/AEM.00376-08
Copyright © 2008, American Society for Microbiology. All Rights Reserved.

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