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Action Mechanisms of Arbuscular Mycorrhizal Fungi in Phosphorus Uptake by Capsicum annuum L.
Pedosphere 21(4): 502–511, 2011 ISSN 1002-0160/CN 32-1315/P c 2011 Soil Science Society of China Published by Elsevier B.V. and Science Press
Action Mechanisms of Arbuscular Mycorrhizal Fungi in Phosphorus Uptake by Capsicum annuum L.∗1 M. SHARIF1,∗2 and N. CLAASSEN2 1
Department of Soil and Environmental Sciences, NWFP Agricultural University, Peshawar (Pakistan) Department of Crop Sciences (Plant Nutrition), Georg-August-University G¨ ottingen, Carl-Sprengel-Weg 1, D-37075 G¨ ottingen (Germany) 2
(Received October 17, 2010; revised May 4, 2011)
ABSTRACT A pot experiment was conducted to investigate the action mechanisms of arbuscular mycorrhizal (AM) fungi in phosphorus (P) uptake of Capsicum annuum L. in a sterilized fossil Oxisol. Three P levels of 0, 10 and 200 mg kg−1 soil (P0, P10 and P200, respectively) without and with AM fungal inoculation were applied as Ca(H2 PO4 )2 ·H2 O. Shoot dry matter yields and shoot P uptake increased significantly (P > 0.05) by the inoculation of AM fungi at P0 and P10. Root length and P concentration in soil solution increased with the inoculation of AM fungi but the root:shoot ratio decreased or remained constant. Around 50% roots of inoculated plants were infected by AM and the external hyphae amounted to 20 m g−1 soil at P10 and P200. The hyphae surface area of the infected root cylinder amounted to 11 and 2 cm2 cm−2 root at P0 and P10, respectively. The increased P uptake of inoculated plants was mainly because of an up to 5 times higher P influx of the infected root. Model calculations showed that the root alone could not have achieved the measured P influx in both infected and non-infected roots. But the P influx for hyphae calculated by the model was even much higher than the measured one. The P uptake capacity of hyphae introduced in the model was too high. Model calculations further showed that the depletion zone around roots or hyphae was very narrow. In the case of the root only 7% of the soil volume would contribute P to the plant, while in the case of hyphae it would be 100%. The results together with the model calculations showed that the increased P uptake of AM inoculated plants could be explained partly by the increased P concentration in the soil solution and by the increased P absorbing surface area coming from the external hyphae. Key Words:
hyphae, mechanistic model, phosphorus influx, root infection, root morphology
Citation: Sharif, M. and Claassen, N. 2011. Action mechanisms of arbuscular mycorrhizal fungi in phosphorus uptake by Capsicum annuum L. Pedosphere. 21(4): 502–511.
Arbuscular mycorrhizal (AM) fungi have a broad ecological range and are found in tropical to polar regions, wetlands to arid regions and are associated with the roots of approximately 80% of all terrestrial plant species (Sch¨ ußler et al., 2001). Benefits derived by plants from AM fungi include a high uptake of nutrients, especially of phosphorus (P), an increased drought-stress tolerance and an improved tolerance to some pathogens (Koide and Mosse, 2004). They also play an important role in the formation and stability of soil aggregates and contribute to soil fertility and quality (Wright and Upadhyaya, 1998). Mycorrhizas form a symbiotic association with the roots of host plants, where carbon flows to the fun∗1 ∗2
Supported by the Higher Education Commission of Pakistan. Corresponding author. E-mail: [email protected].
gus and inorganic nutrients move to the plant. These fungi develop external hyphae approximately 3 m in diameter (Simard et al., 2002). Hyphae produce arbuscules and in most fungal genera vesicles they act as a bridge connecting the root with the surrounding soil (Allen, 1992). These hyphae increase the P absorbing surface and can absorb P from the place several centimeters away around the root, which is outside the depletion zone of roots (Mosse, 1973), and transfer it to the host roots, thereby shortening the distance of P diffusion to the absorbing surface effectively (Asimi et al., 1980). This is especially important in plants with coarse root systems. The contribution of AM to P uptake by plants has
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been investigated extensively by using different methods. They include simple comparison of mycorrhizal and non-mycorrhizal control plants in terms of P content (Li et al., 1991a), dual labeling with 32 P and 33 P (Pearson and Jakobsen, 1993) and the use of compartmented pot systems (Li et al., 1991b) or together with isotopically labeled P (Smith et al., 2004). But besides evaluating the amount of P contributed by AM, the methods reported little of the possible mechanisms involved in the P uptake by AM hyphae. Besides the hyphae to grow beyond the P depletion zone of roots, their surface is especially suited for uptake P from soils of low P concentration because of their small radius. F¨ohse et al. (1991) demonstrated through model calculations that P depletion on the root hair surface was less than that on the root surface. Similar results can be expected for AM hyphae. The less P depletion will lead to a higher P influx per unit absorbing surface area. Due to the less depletion on the absorbing surface, a more effective P uptake kinetics, given by a low Km (Michaelis constant) or a high Imax (maximum influx) will be found on root hairs or AM hyphae than on the much thicker roots, where P depletion is much stronger (Cress et al., 1979; F¨ohse et al., 1991; Deressa and Schenk, 2008). The multiple interacting processes and factors are difficult to measure directly but can be described by mechanistic models (Claassen and Steingrobe, 1999), which will be used in this investigation to asses root and hyphae properties in relation to P uptake from soil. Arbuscular mycorrhiza may also promote P uptake by increasing its solubility in soil through pH changes or by exudation of P mobilizing compounds like organic acids and phosphatases (Li et al., 1991b; Giri et al., 2005; Mohammad et al., 2005; Tawaraya et al., 2005, 2006). The objective of this investigation was to evaluate the mechanisms by which AM fungi contributed to P uptake of Capsicum annum L. The solubilization or chemical mobilization will be assessed by changes in the P concentration of the soil solution. More emphasis, though, will be given to the increased P absorbing surface from AM hyphae and their morphological, physiological and spatial properties. Their significance for P uptake will be assessed by model calculations.
annuum L. in a growth chamber maintained at 25 ◦ C during a 16 h day and 18 ◦ C during an 8 h night with 70% (day/night) relative humidity and a light intensity of 250 μmol m−2 s−1 . The soil used for the experiment was a fossil Oxisol, containing clay of 500 g kg−1 and calcium-acetatelactate (CAL)-extractable P of 4.7 mg kg−1 with a pH (CaCl2 ) of 5.4. The soil P was mainly bound to Fe and Al. Plastic pots of 3 L capacity were filled with 3 kg soil sterilized at 120 ◦ C for 72 h at a bulk density of 1.43 g cm−3 . Soil surface in each pot was covered with a 1–2 cm layer of quartz sand so that the soil was not disturbed while watering the pots. Water was added to the pots to get moisture content of 260 g kg−1 one week before planting. This moisture content was maintained throughout the growing period by adding water daily to the pots through a fine water sprayer. Three P levels of 0, 10 and 200 mg kg−1 soil (P0, P10 and P200, respectively) were applied as Ca(H2 PO4 )2 ·H2 O. At the P0 and P10 low P availability would limit plant growth and AM fungi were expected to improve plant P nutrition while P supply would not limit plant growth at P200. Basic fertilizers N, K, Mg, B and Mo were applied at the rates of 300, 50, 40, 0.2 and 0.1 mg kg−1 soil in the forms of Ca(NO3 )2 , K2 SO4 , MgSO4 ·7H2 O, H3 BO3 and (NH4 )6 MO7 O24 , respectively. The Ca(NO3 )2 was applied as three split applications. One gram roots of tropical weed “Chromolaena odorata” infected with AM fungal species Glomus mossea (85-1) were inoculated in soil before planting. Seeds of Capsicum annuum L. were surface sterilized by placing them in 2% chloramine solution for 3 min and allowed to germinate in sterilized peat substrate. Four uniform seedlings were transplanted to each pot 18 days after emergence. Soil solution, filtered through a sieve of 30 μm size to remove mycorrhizal spores but all other microorganisms, was applied at the rate of 30 mL to each pot. The experiment was laid out as completely randomized design. There were five treatments including P0 and P10 without and with AM fungal inoculation and P200 without AM fungal inoculation. Each treatment was replicated 8 times, 4 replicates for the first harvest and 4 replicates for the second harvest.
MATERIALS AND METHODS
Soil and plant analyses
Experimental process
Two plant harvests were conducted to calculate shoot and root growth rates and P influx. The first harvest (harvest 1) and second harvest (harvest 2)
A pot experiment was conducted to grow Capsicum
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were 34 and 76 days after transplanting, respectively. Shoots were separated from the roots and oven-dried to constant weight at 60, 80 and 105 ◦ C for 72 h. Shoot dry matter yield was recorded and shoots were milled. Plant-available P content of the soil was determined according to Sch¨ uler (1969) by extracting P using CAL extraction solution. About 20 mL soil solution was collected from each replicate using a modified displacement procedure (Adams and Moore, 1983), watering the soil to field moisture capacity in 250 mL cylinder with drop by drop addition of distilled water at the top. The solution was filtered through 0.45 μm membrane filter paper under the pressure of 3 bars. Concentration of P in the soil solution was determined by molybdate blue method (Murphy and Riley, 1962). Phosphorus in the shoot dry matter was determined by vanadomolybdate yellow method (Gericke and Kumries, 1952) after plant wet digestion with a mixture of HNO3 and H2 O2 in a microwave oven. Soil pH was determined in 0.01 mol L−1 CaCl2 at soil:solution ratio of 1:2.5 (McClean, 1982). Roots were carefully separated from the soil by washing over a sieve with a 200 μm mesh, cleaned off any foreign material and blotted by softly pressing between layers of tissue paper (Schenk and Barber, 1979). Root fresh weight (RFW) was recorded and root length (RL) was determined according to the gridline intersect method of Tennant (1975). The root radius (r0 , cm) was calculated from RL and RFW assuming the root to be a smooth cylinder and having a density of 1 g cm−3 . RFW (1) r0 = π · RL The root surface area (Ar ) is then calculated by Ar = 2π · r0 · RL
(2)
The processes of AM hyphae extraction and staining and the determination of AM hyphae length were conducted according to Brundrett et al. (1994). Infection rate by AM fungi in root was determined by staining roots with trypan blue in lactic acid according to the procedure as described by Phillips and Hayman (1970) and Koske and Gemma (1989). The presence of vesicles, arbuscules or hyphae was measured by the techniques as described by Giovannetti and Mosse (1980). Phosphorus net influx The P influx, as already stated by Sanders et al.
(1977), is the only satisfactory measure of the P uptake efficiency of intact root systems. The net P influx per unit root cylinder surface area (In , mol cm−2 s−1 ) of AM inoculated or un-inoculated roots was calculated as follows: In =
U2 − U1 ln(Ar2 /Ar1 ) × Ar2 − Ar1 t2 − t1
(3)
where U is the P content of the plant (mol), Ar is the root surface area (cm2 ) and t is the growth time of the plant (s). Subscripts 1 and 2 refer to first and second harvest, respectively. To calculate the contribution of AM hyphae (IM ) to the P influx of the inoculated roots, the formula of Sanders et al. (1977) was used: IM =
Ia − Ic Percent of AM infected roots
(4)
where Ia represents the average P influx of the whole root system of the AM inoculated plants and Ic is the average P influx of the whole root system of the uninoculated plants. This calculation assumes that P influx of the root cylinder of AM infected roots is the same as that of un-infected roots. To estimate the influx per unit hyphae surface area (IH ), the contribution of AM to the P influx (IM ) was divided by the surface area of hyphae (Ah ) per unit infected root cylinder surface (Air , cm2 ): IH =
IM Ah × Air
(5)
Simulation model The mechanistic simulation model used in this investigation was the NST 3 of Claassen and Steingrobe (1999). It is based on P release from the soil solid phase into the soil solution, described by the buffer power, transport of P in the soil solution to the absorbing surface (root or hyphae) by mass flow, and diffusion and uptake into the root or hyphae described by a modified Michaelis-Menten kinetic. Model calculations were performed for roots without AM infection and for hyphae alone, assuming that there was only a minor effect of P uptake by roots. The model parameters included P diffusion coefficient in water (DL ), being 8.9 × 10−6 cm2 s−1 according to Edwards and Huffman (1959), soil parameters, morphological root and hyphae parameters, and physiological root and hyphae parameters. Soil parameters comprised initial P concentration in soil solution (CLi , mol cm−3 ), buffer power (b), volumetric water content (θ, cm3 cm−3 ) and impedance or
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tortuosity factor (f ). Assuming a linear buffer curve, the buffer power is calculated by: b = ΔC/ΔCL
(6)
where ΔC (mol cm−3 ) is the change of diffusible P concentration in soil, equal to the CAL-extractable P, and ΔCL (mol cm−3 ) is the change of P concentration in soil solution, equal to CLi . The f value was calculated from θ according to Barraclough and Tinker (1981): f = 0.99θ − 0.17
(7)
The morphological root parameters consisted of root radius (r0 , cm), average half distance among neighboring roots (r1 ) and relative root growth rate (k, s−1 ), calculated as follows: r1 = √
1 RLv · π
(8)
ln(RL2 /RL1 ) (t2 − t1 )
RESULTS Soil solution and CAL-extractable P concentrations Table I shows the effects of P fertilization and AM inoculation on soil solution P and CAL-extractable P. Phosphorus concentration in soil solution was increased by P fertilization. AM inoculation (P0 + AM) increased soil solution P up to 3 times at the time of first harvest. At the time of second harvest the soil solution P did not increase significantly after the inoculation of AM. In contrast to soil solution P, CALextractable P was not affected by AM inoculation. TABLE I Soil solution phosphorus (P) and the calcium-acetatelactate (CAL)-extractable P in the soils planted with Capsicum annuum L.
where RLv (cm cm−3 ) is the root length density. k=
way analysis of variance. Significant differences between the treatment means were calculated with Tukey test at P = 0.05 using SigmaStat 2.03 (Steel and Torrie, 1980).
(9)
where RL (cm) is the root length, t is time and subscripts 1 and 2 refer to the first and second harvest, respectively. Hyphae radius (r0H ) was considered as 1.5 × 10−4 cm as reported by Simard et al. (2002). Surface area of hyphae (AH ), the mean half distance between neighboring hyphae (r1H ) and hyphae growth rate (kH ) were calculated according to the equations for roots. The physiological root parameters included water influx (v0 ), set to 2.7 × 10−7 cm3 cm−2 s−1 from Claassen and Steingrobe (1999), maximum P influx (Imax , 0.32 × 10−12 mol cm−2 s−1 ), Michaelis constant (Km , 1.4 × 10−9 mol cm−3 ) and minimum P concentration (CLmin , 0.04 × 10−9 mol cm−3 ). The Km and CLmin values can usually not be determined directly for plants growing in soil and they were taken from Jungk et al. (1990) for plants grown in solution culture. The physiological parameters Imax , Km and CLmin for hyphae were 0.25 × 10−12 mol cm−2 s−1 , 0.25 × 10−9 mol cm−3 and 0.04 × 10−9 mol cm−3 , respectively, which were taken from Deressa and Schenk (2008). Statistics Logarithmic transformation of the data were performed and treatment effects were determined by one
Treatmenta)
Soil solution P
CAL-extractable P
Harvest 1 Harvest 2 Harvest 1 Harvest 2 μmol P0 0.097ca) P0 + AM 0.293b P10 0.185b P10 + AM 0.301b P200 0.396ab P0 (no plant) 0.106c P10 (no plant) 0.194b P200 (no plant) 0.733a
L−1 0.133b 0.145b 0.139b 0.166b 0.091b 0.185b 0.143b 1.039a
mg 5.23ab 4.65b 5.48ab 5.23ab 9.68a 5.33ab 5.64ab 18.84a
kg−1 4.57b 4.83ab 4.75ab 5.18ab 5.65ab 4.71b 5.50ab 15.87a
a)
Three P levels of 0, 10 and 200 mg kg−1 soil (P0, P10 and P200, respectively) were applied as Ca(H2 PO4 )2 · H2 O and arbuscular mycorrhizal (AM) fungi were inoculated at P0 and P10. b) Means followed by the same letter(s) within each column are not significantly different at P < 0.05.
Shoot and root growth, shoot P content and P uptake The application of P increased shoot dry matter yield of Capsicum annuum L. (Table II). The highest yield was obtained at P200 indicating that P0 and P10 treatments were P deficient. The treatment of AM inoculation increased the shoot yield by a factor of 3 to 4. There was no effect of AM inoculation on shoot P concentration and therefore the effect on shoot P uptake was similar to
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TABLE II Growth status of Capsicum annuum L. as affected by P application of 0, 10 and 200 mg kg−1 soil (P0, P10 and P200, respectively) and arbuscular mycorrhizal (AM) fungal inoculation after 76 days of growth Treatment
P0 P0 + AM P10 P10 + AM P200 a)
Shoot dry matter
Shoot P concentration
Shoot P content
Root length
Half distance between roots
P influx
g plant−1 0.25da) 0.64c 0.56c 2.15b 6.74a
g kg−1 0.72d 0.91cd 0.98bc 1.17b 1.69a
mg plant−1 0.18d 0.59c 0.56c 2.49b 11.35a
cm plant−1 1 650c 2 794c 2 643c 10 521b 24 772a
cm 0.32 0.24 0.25 0.13 0.08
× 10−14 mol cm−2 s−1 0.79 2.41 2.49 4.23 3.21
Means followed by the same letter(s) within each column are not significantly different at P < 0.05.
that on shoot yield. Root growth was also increased by P fertilization and AM inoculation, but the increase of root growth was smaller than that of shoot yield. At P0 AM showed no significant effect on the root growth. Table II also shows that AM inoculation increased the rate of P uptake per unit of root (i.e., P influx). At P0 AM inoculation increased the P influx by a factor of 3. At P10, the increase was by a factor of less than 2. The results showed that the increased shoot P content was contributed by an increased root growth as well as by an increased P influx.
surface area.
Root infection, hyphae growth and P uptake Table III shows that about half of the roots were infected with AM fungi and the external hyphae length was about 20 m g−1 soil at P0 and P10. The surface area of hyphae is about 10 and 2 times the root surface area at P0 and P10, respectively. No hyphae were detected in the pots receiving no AM inoculation and the results for first and second harvests were similar. Fig. 1 shows the proportion of the P influx coming from AM hyphae and from the root infected by AM. It can be seen that at low P supply (P0) about 80% of P uptake came from the AM hyphae and at P10 it was 50%. However the P influx per unit hyphae surface area (Table IV) was about half that per unit root
Fig. 1 Phosphorus (P) influx of the root infected by arbuscular mycorrhiza (AM) and contribution of AM to the total P influx per unit infected root surface area with P application of 0 (P0) and 10 mg kg−1 soil (P10).
Modeling P uptake and P dynamics around roots and hyphae Table V shows the plant and soil parameters used in the model calculation. The model calculated P uptake and the P depletion around roots or hyphae. It was necessary to asses the significance of plant prope-
TABLE III Root infection and external hyphae length of Capsicum annuum L and its relation to root length Treatmenta)
Hypae length
Half distance between hyphae
Infected root
Per unit infected root Hyphae length
−1
P0 + AM P10 + AM a)
mg 20.9 21.4
soil
cm 0.010 0.010
% 48 65
cm cm 1 237 236
−1
Hyphae surface area cm2 cm−2 10.9 2.4
Arbuscular mycorrhizal (AM) fungi were inoculated at the P application of 0 (P0) and 10 mg kg−1 soil (P10).
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TABLE IV Measured and model calculated P influx of arbuscular mycorrhiza (AM) infected roots Treatmenta)
P0 + AM P10 + AM a) b)
Measured P influx
Model calculated P influx
Root cylinder surfaceb)
Hyphae surface
Root cylinder surface
0.79 2.49
× 10−14 mol cm−2 s−1 0.31 0.20 1.12 0.43
Hyphae surface 2.30 2.22
Arbuscular mycorrhizal (AM) fungi were inoculated at the P application of 0 (P0) and 10 mg kg−1 soil (P10). The measured P influx of the root cylinder is the P influx as measured in the AM uninfected root.
TABLE V Plant and soil parameters used for model calculations of P uptake by root and hyphae as affected by P application of 0 and 10 mg kg−1 soil (P0 and P10) and arbuscular mycorrhizal (AM) fungal inoculation Parametera)
P0 (root)
P0 + AM
P10 (root)
P10 + AM
Root
Hypha
Root
Hypha
0.32 1.40 0.04 0.013 2.70 0.13 0.030 3.0
0.25 0.25 0.04 1.500 0.00 0.010 0.010 1 080
3.01 0.37 0.20 802 8.90
3.01 0.37 0.20 802 8.90
Imax (× 10−12 mol cm−2 s−1 ) Km (× 10−9 mol cm−3 ) CLmin (× 10−9 mol cm−3 ) ro (cm) vo (× 10−7 cm3 cm−2 s−1 ) r1 (cm) k (d−1 ) RL (× 103 cm)
0.32 1.40 0.04 0.011 2.70 0.32 0.007 1.2
0.32 1.40 0.04 0.013 2.70 0.24 0.016 1.4
Plant parameter 0.25 0.32 0.25 1.40 0.04 0.04 1.500 0.013 0.00 2.70 0.010 0.25 0.012 0.016 952.5 1.3
CLi (× 10−10 mol cm−3 ) θ (cm3 cm−3 ) f b DL (× 10−6 cm2 s−1 )
0.97 0.37 0.20 2 487 8.90
2.93 0.37 0.20 732 8.90
2.93 0.37 0.20 732 8.90
Soil parameter 1.85 0.37 0.20 1 366 8.90
a)
Imax = maximum P influx, Km = Michaelis constant, CLmin = minimum P concentration, ro = root radius, vo = water influx, r1 = average half distance among neighboring roots, k = relative root growth rate, RL = root length, CLi = initial P concentration in soil solution, θ = volumetric water content, f = tortuosity factor, b = buffer power, DL = P diffusion coefficient in water.
rties, like uptake kinetic parameter or the radius of the absorbing surface (root or hyphae). The results of model calculations in relation to measured values are shown in Table IV. It can be seen that for the root cylinder the model calculates only around 20% of the actual influx but for hyphae it calculates 700% for the P0 + AM and 200% for the P10 + AM treatments. The P influx calculated by the model for hyphae is 5 to 10 times higher than that for the root cylinder. The different calculated influx for the root cylinder and hyphae is mainly related to their different radiuses and the resulting geometry for diffusion
in their vicinity. Fig. 2 showed that after 1 day of P uptake the extension of the depletion zone around the hyphae was less than that around the root. This resulted in a much steeper concentration gradient toward the hyphae. Therefore the flux to the hyphae and so the influx into the hyphae (Table IV) were higher than those into the root. It indicated that hypha surfaces were more effective to absorb P from soils with no P supply. Only by reducing the Imax of the hyphae the calculated influx became close to the actual P influx (Table IV).
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Fig. 2 Comparison of the steepness and extension of the P concentration profiles as calculated by the simulation models around roots and arbuscular mycorrhizal (AM) hyphae after one day of uptake. Data were obtained from the unfertilized but arbuscular mycorrhiza inoculated soil (P0 + AM).
Fig. 3 showed that the width of depletion progressed with time. Since roots were relatively far apart (r1 = 0.24 cm), there was no overlap of the depletion zones of neighboring roots, i.e., there was no inter-root competition. But for hyphae because of their closeness (r1H = 0.01 cm), inter-hyphae competition occurred after 1 day of uptake and after 10 to 20 days 100% of the soil volume would be depleted. In contrast, in the case of roots, only 7% of the soil volume would be partly depleted.
Fig. 3 Progress of the P depletion around roots and arbuscular mycorrhizal hyphae with time of P uptake. Data are for the unfertilized but arbuscular mycorrhiza inoculated soil (P0 + AM).
DISCUSSION The results show that at low P supply an infection with AM fungi significantly increased the yield of Capsicum annuum L. and it was related to an increased P uptake (Table II). The increased P uptake was partly due to a larger root system of AM infected plants but the main reason was the 2 to 3 times higher P uptake rate per unit of root. Similar results were found by others (Sanders et al., 1977; Deressa and Schenk, 2008), where AM inoculation increased the P uptake rate per unit of root by a factor of 2 to 20. The larger root system was probably not due to a specific effect of AM favoring the root growth but because of a higher P uptake. The shoot growth was enhanced and this caused also more root growth. That AM did not enhance root growth specifically is indicated by the lower root:shoot ratio of AM infected plants. The lower root:shoot ratio is probably a consequence of the higher P supply of AM infected plants,
which was also found by Viebrock (1988). In our experiment 50% to 60% of the roots were infected by AM. But the amount of external hyphae per unit of root was higher at P0 than P10 (10.9 versus 2.4 cm2 cm−2 , Table III). Abbott et al. (1984) reported that severe P deficiency increased both the percent of root infection and the length of external hyphae per centimeter of infected root. Reduction of hyphal contribution with increased P addition was also reported by Allison and Goldberg (2002). The total amount of external hyphae of 20 m g−1 soil was in the higher range found by Deressa and Schenk (2008). The contribution of AM to the P uptake per unit of root was about 80% at no P supply (P0) and 50% at P10 (Fig. 1). It is in the same range as found by others (Sanders et al., 1977; Deressa and Schenk, 2008). The main aim of this research, though, was to analyze possible mechanisms by which AM increased the P uptake of plants. The P uptake of a plant depends
MECHANISMS OF AM FUNGI IN P UPTAKE
on the size of its root system and on the rate of P uptake by each root segment, i.e., the P influx. The size of the root system was already discussed above. Table II showed that AM increased the P influx for the whole root system by up to 3 times. But taking the P influx of the infected portion separately (Fig. 1), the increase was by a factor of up to 5. This calculation assumes that the mycorrhizal roots have the same P uptake as the non-mycorrhizal roots do. But it may not have been the case because P concentration in soil solution was higher for AM infected plants at early growth (Table I, Harvest 1). On the other hand the P uptake capacity of the root cylinder may have been reduced due to the infection of roots with AM as was found and extensively discussed by Smith et al. (2004). They found that in AM infected roots the epidermal P uptake path was reduced or even inhibited. To asses the significance of external hyphae for the uptake of P, hyphae length and surface area were determined and compared with the values of the root. There were 230 to 1 200 cm external hyphae per cm of root, but because of the small radius their surface area was only 2 to 10 times larger than that of the root cylinder (Table III). Graham et al. (1982) reported that hyphal surface area was not correlated with that of the root cylinder because of different methods of measurement of external hyphae. This increase of absorbing surface area was more significant than the increase of P influx due to AM (Fig. 1, Table IV), which as a first approach could therefore explain the increased P influx. The comparison of P uptake capacity between hyphae and roots, though, would be insufficient since hyphae have a much smaller radius as roots, which will affect the geometry of diffusion of P towards its surface. Hyphae spread away from the root making more soil volume accessible and furthermore uptake kinetics of hyphae may be different from that of roots. To evaluate these properties of hyphae and roots model calculations were performed. Table IV showed that the P influx of hyphae surface calculated by the model was up to 10 times higher than that of the root cylinder surface. Accordingly hyphae surfaces would be much more effective to absorb P from low P soils than root cylinder surfaces. The higher effectivity of P uptake of hyphae was based on its smaller radius (1.5 versus 110 μm of the root) and lower Km value (0.25 × 10−9 versus 1.4 × 10−9 mol cm−3 ). The smaller radius implicates a larger soil volume per unit surface area of the hyphae, which results in less extension of the P depletion zone (Fig. 2). On the other
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hand the concentration profile becomes steeper, causing a higher P flux to the hyphae by diffusion and consequently a higher P influx. The small Km value still enhanced the P influx at the low P concentration on the hyphae surface. Fig. 3 showed that for roots alone only a fraction of the soil P was accessible to the root (about 7%), while for hyphae the whole soil volume was accessible. With the hyphae length density of 20 m g−1 soil, hyphae were only 0.01 cm apart so that even with a low mobility of P in soil the depletion zones of neighboring roots overlapped. It can be concluded that AM hyphae not only increase the P absorbing surface area but also increase strongly the proportion of soil P spatially available to the plant. The hyphae length density (HLv ) in this study of 20 m g−1 soil is on the high side of data in the literature of 1 to 20 m g−1 (Deressa and Schenk, 2008). But even with 1 m g−1 soil the half distance among neighboring hyphae (r1H ) would be 0.044 cm. The depletion zones would still overlap and the whole soil volume would be spatially available to the plant. This aspect would also be important for the case that hyphae were not uniformly distributed in the soil, i.e., hyphae may concentrate more close to the root surfaces but fade out farther away from them. For hyphae the model calculated P influx was 8 times (P0 + M) or 2 times (P10 + M) higher than the measured value (Table IV). It meant that, assuming the soil parameters were estimated properly, much more P has been supplied to the hyphae by the soil compared to the P taken up by the AM. The results indicated the plants could regulate the amount of P taken up by AM hyphae. Besides regulating the AM hyphae growth, the plants may also regulate the P uptake physiology, e.g., changing Imax or Km . Model calculations (Table IV) showed that the calculated P influx reduced to the values similar to the measured ones due to the strong reduction of the Imax of the hyphae. However, it cannot be excluded that the plant down regulated the P uptake at the hyphae-root interface. It is well known that plants regulate P uptake kinetics according to the P supply. For example at high P supply Imax is down-regulated (Buhse, 1992; Bhadoria et al., 2004). But Smith et al. (2009) reported that there was a fungus-plant signaling, which induced a regulation of the P transporter expression of the AM P uptake path as well as of the epidermal P uptake pathway. Whether the P uptake regulation actually occurred depended on the reliability of the model calculations.
510
The most important soil parameters like the soil solution concentration and soil water content were probably reliably estimated, but the uptake kinetics of hyphae were taken from another experiment of Deressa and Schenk (2008). The corresponding parameters (Imax and Km ) can not be adopted without caution. The authors discussed the insecurities of their estimation and made a conclusion that those parameters may vary according to the conditions like AM strain used and the levels of P in the system. For a better understanding of AM-plant symbiosis concerning P uptake, further research is needed to characterize the uptake kinetics of AM hyphae and to study the fungus-plant signaling concerning the regulation of P transporters of the epidermal and AM P uptake pathways. CONCLUSIONS Arbuscular mycorrhiza (AM) increased the P uptake of Capsicum annum L. in a low P fossil Oxisol with a P concentration of 0.15 μmol L−1 . This was primarily because of a higher P uptake rate (P influx) per unit of AM infected root. This higher P influx was related to a 2 to 10 times larger P absorbing surface area coming from the external hyphae. Model calculations showed that hyphal surfaces were more effective than root cylinder surfaces to absorb P from low P soils and would have been able to absorb more P than that needed by the plant. Model calculations furthermore showed that in the low P soils, P depletion around roots or hyphae only extended to about 0.06 cm and so only 7% of soil P was positionally available to roots. But for hyphae it was about 100%, because the half distance between neighboring hyphae was only 0.01 cm. It can be concluded that the high effectivity of hyphal surfaces to absorb P from soil may be enough in most cases to explain the positive effect of AM on P uptake from soil. ACKNOWLEDGEMENT Thanks is given to Prof. M. Schenk from the Institute of Plant Nutrition, University of Hannover, Germany for offering his laboratory and help in the external hyphae determination. REFERENCES Abbott, L. K., Robson, A. D. and De Boer, G. 1984. The effect of phosphours on the formation of hyphae in soil
M. SHARIF AND N. CLAASSEN by the vesicular-arbuscular mycorrhizal fungus, Glomus fasiculatum. New Phytol. 97: 437–446. Adams, F. and Moore, B. L. 1983. Chemical factors affecting root growth in subsoil horizons of coastal plain soils. Soil Sci. Soc. Am. J. 47: 99–102. Allen, M. F. 1992. Mycorrhizal Functioning: An Integrative Plant-Fungal Process. Chapman and Hall, New York. Allison, V. J. and Goldberg, D. E. 2002. Species-level versus community-level patterns of mycorrhizal dependence on phosphorus: an example of Simpson’s paradox. Funct. Ecol. 16: 346–352. Asimi, S., Gianinazzi-Pearson, V. and Gianinazzi, S. 1980. Influence of increasing soil phosphorus levels on interactions between vesicular-arbuscular mycorrhiza and rhizobium in soybeans. Can. J. Bot. 58: 220–225. Barraclough, B. P. and Tinker, B. P. 1981. The determination of ionic diffusion coefficient in field soils. I. Diffusion coefficients in sieved soils in relation to water content and bulk density. J. Soil Sci. 32: 225–236. Bhadoria, P. S., El Dessougi, H., Liebersbach, H. and Claassen, N. 2004. Phosphorus uptake kinetics, size of root system and growth of maize and groundnut in solution culture. Plant Soil. 262: 327–336. Brundrett, M., Melville, L. and Peterson, L. 1994. Practical Methods in Mycorrhiza Research. The 9th North American Conference on Mycorrhizae. Mycologue Publications, University of Gulph, Canada. Buhse, J. 1992. Wirkung der Wurzelraumtemperature auf das Phosphateaneignungs-vermoegen von Pflanzen und die Phosphateverfeugbarkeit im Boden (in German). Ph.D. Thesis. University of Goettingen, Germany. Claassen, N. and Steingrobe, B. 1999. Mechanistic simulation models for a better understanding of nutrient uptake from soil. In Rengel, Z. (ed.) Mineral Nutrition of Crops: Fundamentals Mechanisms and Implications. The Haworth Press, Inc., New York. pp. 327–367. Cress, W. A., Throneberry, G. O. and Lindsey, D. L. 1979. Kinetics of phosphorus absorption by mycorrhizal and non-mycorrhizal tomato roots. Plant Physiol. 64: 484– 487 Deressa, T. G. and Schenk, M. K. 2008. Contribution of roots and hyphae to phosphorus uptake of mycorrhizal onion (Allium cepa L.): A mechanistic modeling approach. J. Plant Nutr. Soil Sci. 171: 810–820. Edwards, O. W. and Huffman, E. O. 1959. Diffusion of aqueous solution of phosphoric acid at 25 ◦ C. J. Phys. Chem. 63: 1830–1833. F¨ ohse, D., Claassen, N. and Jungk, A. 1991. Phosphorus efficiency of plants. II. Significance of root radius, root hairs and cation-anion balance for phosphorus influx in seven plant species. Plant Soil. 132: 261–272 Gericke, S. and Kumries, B. 1952. Kolorimetrische Phosphrs¨ aurebestimmung mit Ammonium-VanadateMolybdate und ihre Anwendung in der Pflanzenanalyse. J. Plant Nutr. Soil Sci. (in German). 59: 235–
MECHANISMS OF AM FUNGI IN P UPTAKE 247. Giovannetti, M. and Mosse, B. 1980. An evaluation of techniques for measuring vesicular-arbuscular mycorrhizal infection in roots. New Phytol. 84: 489–500. Giri, B., Kapoor, R. and Mukerji, K. G. 2005. Effect of arbuscular mycorrhiza Glomus fasciculatum and G. macrocarpum on the growth and nutrient content of Cassia siamea in a semi-arid Indian wasteland soil. New Forest. 29: 63–73. Graham, J. H., Linderman, R. G. and Menge, J. A. 1982. Development of external hyphae by different isolates of mycorrhiza Glomus spp. in relation to root colonization. New Phytol. 91: 183–189. Jungk, A., Asher, C. J., Edwards, D. G. and Meyer, D. 1990. Influence of phosphate status on phosphate uptake kinetics of maize (Zea mays) and soybean (Glycine max). Plant Soil. 124: 175–182. Koide, R. T. and Mosse, B. 2004. A history of research on arbuscular mycorrhiza. Mycorrhiza. 14: 145–163. Koske, R. E. and Gemma, J. N. 1989. A modified procedure for staining roots to detect VA mycorrhiza. Mycol. Res. 4: 486–488. Li, X., George, E. and Marschner, H. 1991a. Extension of the phosphorus depletion zone in VA-mycorrhizal white clover in a calcareous soil. Plant Soil. 136: 41–48. Li, X., George, E. and Marschner, H. 1991b. Phosphorus depletion and pH decrease at the root-soil and hyphaesoil interfaces of VA mycorrhizal white clover fertilized with ammonium. New Phytol. 119: 397–404. McClean, E. O. 1982. Soil pH and lime requirement. In Page, A. L., Miller, R. H. and Keeney, D. R. (eds). Methods of Soil Analysis. Part 2. Chemical and Microbiological Properties. 2nd Edition. ASA, Madison, WI. pp. 199–223. Mohammad, M. J., Pan, W. L. and Kennedy, A. C. 2005. Chemical alteration of the rhizosphere of mycorrhizalcolonized wheat root. Mycorrhiza. 15: 259–266. Mosse, B. 1973. Advances in the study of vesiculararbuscular mycorrhiza. Annu. Rev. Phytopathol. 11: 171–196. Murphy, J. and Riley, J. P. 1962. Colorimetric method for determination of P in soil solution. Anal. Chim. Acta. 27: 31–36. Pearson, J. N. and Jakobsen, I. 1993. The relative contribution of hyphae and roots to phosphorus uptake by arbuscular mycorrhizal plants measured by dual labeling with 32 P and 33 P. New. Phytol. 124: 489–494. Phillips, J. M. and Hayman, D. S. 1970. Improved procedures for clearing roots and staining parasitic and vesicular-arbuscular mycorrhizal fungi for rapid assessment of infection. T. Brit. Mycol. Soc. 55: 158–160.
511 Sanders, F. E., Tinker, P. B., Black, R. L. B. and Palmerley, S. M. 1977. The development of endomycorrhizal root systems. I. Spread of infection and growth promoting effects with four species of vesicular-arbuscular endophyte. New Phytol. 78: 257–268. Schenk, M. K. and Barber, S. A. 1979. Phosphate uptake of corn as affected by soil characteristics and root morphology. Soil Sci. Soc. Am. J. 43: 880–883. Sch¨ ußler, A., Schwarzott, D. and Walker, C. 2001. A new fungal phylum, the Glomeromycota: phylogeny and evolution. Mycol. Res. 105: 1413–1421. Sch¨ uler, H. 1969. Die CAL-method. Eine neue Methode zur Bestimmung des pflanzenverf¨ ugbare P in B¨ oden. J. Plant Nutr. Soil Sci. (in German). 123: 48–60. Simard, S. W., Jones, M. D. and Durall, D. M. 2002. Carbon and nutrient fluxes within and between mycorrhizal plants. In van der Heijden, M. G. A. and Sanders, I. R. (eds). Mycorrhizal Ecology. SpringerVerlag, Berlin. pp. 33–74. Smith, F. A., Grace, E. J. and Smith, S. E. 2009. More than a carbon economy: nutrient trade and ecological sustainability in facultative arbuscular mycorrhizal symbioses. New Phytol. 182: 347–358. Smith, S. E., Smith, F. A. and Jakobsen, I. 2004. Functional diversity in arbuscular mycorrhizal (AM) symbioses: the contribution of mycorrhizal P uptake pathway is not correlated with mycorrhizal responses in growth or total P uptake. New Phytol. 162: 511–524. Steel, R. G. D. and Torrie, J. H. 1980. Principles and Procedures of Statistics: A Biometrical Approach. McGrawHill, New York. Tawaraya, K., Naito, M. and Wagatsuma, T. 2006. Solubilization of insoluble inorganic phosphate by hyphal exudates of arbuscular mycorrhizal fungi. J. Plant Nutr. 29: 657–665. Tawaraya, K., Ohtaki, M., Tanimura, Y. and Wagatsuma, T. 2005. Mineralization of organic P by hyphal exudates. In Li, C. J. et al. (eds.) Plant Nutrition for Food Security, Human Health and Environmental Protection. Tsinghua University Press, Beijing, China. pp. 790–791. Tennant, D. 1975. A test of a modified line intersect method of estimating root length. J. Ecol. 63: 995–1001. Viebrock, H. 1988. Ursachen der Erh¨ ohung des Phosphat- aneignungsverm¨ ogens von Pflanzen durch VAMykorrhiza (in German). Ph. D. Thesis, Universit¨ at G¨ ottingen, Germany. Wright, S. F. and Upadhyaya, A. 1998. A survey of soils for aggregate stability and glomalin, a glycoprotein produced by hyphae of arbuscular mycorrhizal fungi. Plant Soil. 198: 97–107.
Action Mechanisms of Arbuscular Mycorrhizal Fungi in Phosphorus Uptake by Capsicum annuum L.∗1 M. SHARIF1,∗2 and N. CLAASSEN2 1
Department of Soil and Environmental Sciences, NWFP Agricultural University, Peshawar (Pakistan) Department of Crop Sciences (Plant Nutrition), Georg-August-University G¨ ottingen, Carl-Sprengel-Weg 1, D-37075 G¨ ottingen (Germany) 2
(Received October 17, 2010; revised May 4, 2011)
ABSTRACT A pot experiment was conducted to investigate the action mechanisms of arbuscular mycorrhizal (AM) fungi in phosphorus (P) uptake of Capsicum annuum L. in a sterilized fossil Oxisol. Three P levels of 0, 10 and 200 mg kg−1 soil (P0, P10 and P200, respectively) without and with AM fungal inoculation were applied as Ca(H2 PO4 )2 ·H2 O. Shoot dry matter yields and shoot P uptake increased significantly (P > 0.05) by the inoculation of AM fungi at P0 and P10. Root length and P concentration in soil solution increased with the inoculation of AM fungi but the root:shoot ratio decreased or remained constant. Around 50% roots of inoculated plants were infected by AM and the external hyphae amounted to 20 m g−1 soil at P10 and P200. The hyphae surface area of the infected root cylinder amounted to 11 and 2 cm2 cm−2 root at P0 and P10, respectively. The increased P uptake of inoculated plants was mainly because of an up to 5 times higher P influx of the infected root. Model calculations showed that the root alone could not have achieved the measured P influx in both infected and non-infected roots. But the P influx for hyphae calculated by the model was even much higher than the measured one. The P uptake capacity of hyphae introduced in the model was too high. Model calculations further showed that the depletion zone around roots or hyphae was very narrow. In the case of the root only 7% of the soil volume would contribute P to the plant, while in the case of hyphae it would be 100%. The results together with the model calculations showed that the increased P uptake of AM inoculated plants could be explained partly by the increased P concentration in the soil solution and by the increased P absorbing surface area coming from the external hyphae. Key Words:
hyphae, mechanistic model, phosphorus influx, root infection, root morphology
Citation: Sharif, M. and Claassen, N. 2011. Action mechanisms of arbuscular mycorrhizal fungi in phosphorus uptake by Capsicum annuum L. Pedosphere. 21(4): 502–511.
Arbuscular mycorrhizal (AM) fungi have a broad ecological range and are found in tropical to polar regions, wetlands to arid regions and are associated with the roots of approximately 80% of all terrestrial plant species (Sch¨ ußler et al., 2001). Benefits derived by plants from AM fungi include a high uptake of nutrients, especially of phosphorus (P), an increased drought-stress tolerance and an improved tolerance to some pathogens (Koide and Mosse, 2004). They also play an important role in the formation and stability of soil aggregates and contribute to soil fertility and quality (Wright and Upadhyaya, 1998). Mycorrhizas form a symbiotic association with the roots of host plants, where carbon flows to the fun∗1 ∗2
Supported by the Higher Education Commission of Pakistan. Corresponding author. E-mail: [email protected].
gus and inorganic nutrients move to the plant. These fungi develop external hyphae approximately 3 m in diameter (Simard et al., 2002). Hyphae produce arbuscules and in most fungal genera vesicles they act as a bridge connecting the root with the surrounding soil (Allen, 1992). These hyphae increase the P absorbing surface and can absorb P from the place several centimeters away around the root, which is outside the depletion zone of roots (Mosse, 1973), and transfer it to the host roots, thereby shortening the distance of P diffusion to the absorbing surface effectively (Asimi et al., 1980). This is especially important in plants with coarse root systems. The contribution of AM to P uptake by plants has
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been investigated extensively by using different methods. They include simple comparison of mycorrhizal and non-mycorrhizal control plants in terms of P content (Li et al., 1991a), dual labeling with 32 P and 33 P (Pearson and Jakobsen, 1993) and the use of compartmented pot systems (Li et al., 1991b) or together with isotopically labeled P (Smith et al., 2004). But besides evaluating the amount of P contributed by AM, the methods reported little of the possible mechanisms involved in the P uptake by AM hyphae. Besides the hyphae to grow beyond the P depletion zone of roots, their surface is especially suited for uptake P from soils of low P concentration because of their small radius. F¨ohse et al. (1991) demonstrated through model calculations that P depletion on the root hair surface was less than that on the root surface. Similar results can be expected for AM hyphae. The less P depletion will lead to a higher P influx per unit absorbing surface area. Due to the less depletion on the absorbing surface, a more effective P uptake kinetics, given by a low Km (Michaelis constant) or a high Imax (maximum influx) will be found on root hairs or AM hyphae than on the much thicker roots, where P depletion is much stronger (Cress et al., 1979; F¨ohse et al., 1991; Deressa and Schenk, 2008). The multiple interacting processes and factors are difficult to measure directly but can be described by mechanistic models (Claassen and Steingrobe, 1999), which will be used in this investigation to asses root and hyphae properties in relation to P uptake from soil. Arbuscular mycorrhiza may also promote P uptake by increasing its solubility in soil through pH changes or by exudation of P mobilizing compounds like organic acids and phosphatases (Li et al., 1991b; Giri et al., 2005; Mohammad et al., 2005; Tawaraya et al., 2005, 2006). The objective of this investigation was to evaluate the mechanisms by which AM fungi contributed to P uptake of Capsicum annum L. The solubilization or chemical mobilization will be assessed by changes in the P concentration of the soil solution. More emphasis, though, will be given to the increased P absorbing surface from AM hyphae and their morphological, physiological and spatial properties. Their significance for P uptake will be assessed by model calculations.
annuum L. in a growth chamber maintained at 25 ◦ C during a 16 h day and 18 ◦ C during an 8 h night with 70% (day/night) relative humidity and a light intensity of 250 μmol m−2 s−1 . The soil used for the experiment was a fossil Oxisol, containing clay of 500 g kg−1 and calcium-acetatelactate (CAL)-extractable P of 4.7 mg kg−1 with a pH (CaCl2 ) of 5.4. The soil P was mainly bound to Fe and Al. Plastic pots of 3 L capacity were filled with 3 kg soil sterilized at 120 ◦ C for 72 h at a bulk density of 1.43 g cm−3 . Soil surface in each pot was covered with a 1–2 cm layer of quartz sand so that the soil was not disturbed while watering the pots. Water was added to the pots to get moisture content of 260 g kg−1 one week before planting. This moisture content was maintained throughout the growing period by adding water daily to the pots through a fine water sprayer. Three P levels of 0, 10 and 200 mg kg−1 soil (P0, P10 and P200, respectively) were applied as Ca(H2 PO4 )2 ·H2 O. At the P0 and P10 low P availability would limit plant growth and AM fungi were expected to improve plant P nutrition while P supply would not limit plant growth at P200. Basic fertilizers N, K, Mg, B and Mo were applied at the rates of 300, 50, 40, 0.2 and 0.1 mg kg−1 soil in the forms of Ca(NO3 )2 , K2 SO4 , MgSO4 ·7H2 O, H3 BO3 and (NH4 )6 MO7 O24 , respectively. The Ca(NO3 )2 was applied as three split applications. One gram roots of tropical weed “Chromolaena odorata” infected with AM fungal species Glomus mossea (85-1) were inoculated in soil before planting. Seeds of Capsicum annuum L. were surface sterilized by placing them in 2% chloramine solution for 3 min and allowed to germinate in sterilized peat substrate. Four uniform seedlings were transplanted to each pot 18 days after emergence. Soil solution, filtered through a sieve of 30 μm size to remove mycorrhizal spores but all other microorganisms, was applied at the rate of 30 mL to each pot. The experiment was laid out as completely randomized design. There were five treatments including P0 and P10 without and with AM fungal inoculation and P200 without AM fungal inoculation. Each treatment was replicated 8 times, 4 replicates for the first harvest and 4 replicates for the second harvest.
MATERIALS AND METHODS
Soil and plant analyses
Experimental process
Two plant harvests were conducted to calculate shoot and root growth rates and P influx. The first harvest (harvest 1) and second harvest (harvest 2)
A pot experiment was conducted to grow Capsicum
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were 34 and 76 days after transplanting, respectively. Shoots were separated from the roots and oven-dried to constant weight at 60, 80 and 105 ◦ C for 72 h. Shoot dry matter yield was recorded and shoots were milled. Plant-available P content of the soil was determined according to Sch¨ uler (1969) by extracting P using CAL extraction solution. About 20 mL soil solution was collected from each replicate using a modified displacement procedure (Adams and Moore, 1983), watering the soil to field moisture capacity in 250 mL cylinder with drop by drop addition of distilled water at the top. The solution was filtered through 0.45 μm membrane filter paper under the pressure of 3 bars. Concentration of P in the soil solution was determined by molybdate blue method (Murphy and Riley, 1962). Phosphorus in the shoot dry matter was determined by vanadomolybdate yellow method (Gericke and Kumries, 1952) after plant wet digestion with a mixture of HNO3 and H2 O2 in a microwave oven. Soil pH was determined in 0.01 mol L−1 CaCl2 at soil:solution ratio of 1:2.5 (McClean, 1982). Roots were carefully separated from the soil by washing over a sieve with a 200 μm mesh, cleaned off any foreign material and blotted by softly pressing between layers of tissue paper (Schenk and Barber, 1979). Root fresh weight (RFW) was recorded and root length (RL) was determined according to the gridline intersect method of Tennant (1975). The root radius (r0 , cm) was calculated from RL and RFW assuming the root to be a smooth cylinder and having a density of 1 g cm−3 . RFW (1) r0 = π · RL The root surface area (Ar ) is then calculated by Ar = 2π · r0 · RL
(2)
The processes of AM hyphae extraction and staining and the determination of AM hyphae length were conducted according to Brundrett et al. (1994). Infection rate by AM fungi in root was determined by staining roots with trypan blue in lactic acid according to the procedure as described by Phillips and Hayman (1970) and Koske and Gemma (1989). The presence of vesicles, arbuscules or hyphae was measured by the techniques as described by Giovannetti and Mosse (1980). Phosphorus net influx The P influx, as already stated by Sanders et al.
(1977), is the only satisfactory measure of the P uptake efficiency of intact root systems. The net P influx per unit root cylinder surface area (In , mol cm−2 s−1 ) of AM inoculated or un-inoculated roots was calculated as follows: In =
U2 − U1 ln(Ar2 /Ar1 ) × Ar2 − Ar1 t2 − t1
(3)
where U is the P content of the plant (mol), Ar is the root surface area (cm2 ) and t is the growth time of the plant (s). Subscripts 1 and 2 refer to first and second harvest, respectively. To calculate the contribution of AM hyphae (IM ) to the P influx of the inoculated roots, the formula of Sanders et al. (1977) was used: IM =
Ia − Ic Percent of AM infected roots
(4)
where Ia represents the average P influx of the whole root system of the AM inoculated plants and Ic is the average P influx of the whole root system of the uninoculated plants. This calculation assumes that P influx of the root cylinder of AM infected roots is the same as that of un-infected roots. To estimate the influx per unit hyphae surface area (IH ), the contribution of AM to the P influx (IM ) was divided by the surface area of hyphae (Ah ) per unit infected root cylinder surface (Air , cm2 ): IH =
IM Ah × Air
(5)
Simulation model The mechanistic simulation model used in this investigation was the NST 3 of Claassen and Steingrobe (1999). It is based on P release from the soil solid phase into the soil solution, described by the buffer power, transport of P in the soil solution to the absorbing surface (root or hyphae) by mass flow, and diffusion and uptake into the root or hyphae described by a modified Michaelis-Menten kinetic. Model calculations were performed for roots without AM infection and for hyphae alone, assuming that there was only a minor effect of P uptake by roots. The model parameters included P diffusion coefficient in water (DL ), being 8.9 × 10−6 cm2 s−1 according to Edwards and Huffman (1959), soil parameters, morphological root and hyphae parameters, and physiological root and hyphae parameters. Soil parameters comprised initial P concentration in soil solution (CLi , mol cm−3 ), buffer power (b), volumetric water content (θ, cm3 cm−3 ) and impedance or
MECHANISMS OF AM FUNGI IN P UPTAKE
505
tortuosity factor (f ). Assuming a linear buffer curve, the buffer power is calculated by: b = ΔC/ΔCL
(6)
where ΔC (mol cm−3 ) is the change of diffusible P concentration in soil, equal to the CAL-extractable P, and ΔCL (mol cm−3 ) is the change of P concentration in soil solution, equal to CLi . The f value was calculated from θ according to Barraclough and Tinker (1981): f = 0.99θ − 0.17
(7)
The morphological root parameters consisted of root radius (r0 , cm), average half distance among neighboring roots (r1 ) and relative root growth rate (k, s−1 ), calculated as follows: r1 = √
1 RLv · π
(8)
ln(RL2 /RL1 ) (t2 − t1 )
RESULTS Soil solution and CAL-extractable P concentrations Table I shows the effects of P fertilization and AM inoculation on soil solution P and CAL-extractable P. Phosphorus concentration in soil solution was increased by P fertilization. AM inoculation (P0 + AM) increased soil solution P up to 3 times at the time of first harvest. At the time of second harvest the soil solution P did not increase significantly after the inoculation of AM. In contrast to soil solution P, CALextractable P was not affected by AM inoculation. TABLE I Soil solution phosphorus (P) and the calcium-acetatelactate (CAL)-extractable P in the soils planted with Capsicum annuum L.
where RLv (cm cm−3 ) is the root length density. k=
way analysis of variance. Significant differences between the treatment means were calculated with Tukey test at P = 0.05 using SigmaStat 2.03 (Steel and Torrie, 1980).
(9)
where RL (cm) is the root length, t is time and subscripts 1 and 2 refer to the first and second harvest, respectively. Hyphae radius (r0H ) was considered as 1.5 × 10−4 cm as reported by Simard et al. (2002). Surface area of hyphae (AH ), the mean half distance between neighboring hyphae (r1H ) and hyphae growth rate (kH ) were calculated according to the equations for roots. The physiological root parameters included water influx (v0 ), set to 2.7 × 10−7 cm3 cm−2 s−1 from Claassen and Steingrobe (1999), maximum P influx (Imax , 0.32 × 10−12 mol cm−2 s−1 ), Michaelis constant (Km , 1.4 × 10−9 mol cm−3 ) and minimum P concentration (CLmin , 0.04 × 10−9 mol cm−3 ). The Km and CLmin values can usually not be determined directly for plants growing in soil and they were taken from Jungk et al. (1990) for plants grown in solution culture. The physiological parameters Imax , Km and CLmin for hyphae were 0.25 × 10−12 mol cm−2 s−1 , 0.25 × 10−9 mol cm−3 and 0.04 × 10−9 mol cm−3 , respectively, which were taken from Deressa and Schenk (2008). Statistics Logarithmic transformation of the data were performed and treatment effects were determined by one
Treatmenta)
Soil solution P
CAL-extractable P
Harvest 1 Harvest 2 Harvest 1 Harvest 2 μmol P0 0.097ca) P0 + AM 0.293b P10 0.185b P10 + AM 0.301b P200 0.396ab P0 (no plant) 0.106c P10 (no plant) 0.194b P200 (no plant) 0.733a
L−1 0.133b 0.145b 0.139b 0.166b 0.091b 0.185b 0.143b 1.039a
mg 5.23ab 4.65b 5.48ab 5.23ab 9.68a 5.33ab 5.64ab 18.84a
kg−1 4.57b 4.83ab 4.75ab 5.18ab 5.65ab 4.71b 5.50ab 15.87a
a)
Three P levels of 0, 10 and 200 mg kg−1 soil (P0, P10 and P200, respectively) were applied as Ca(H2 PO4 )2 · H2 O and arbuscular mycorrhizal (AM) fungi were inoculated at P0 and P10. b) Means followed by the same letter(s) within each column are not significantly different at P < 0.05.
Shoot and root growth, shoot P content and P uptake The application of P increased shoot dry matter yield of Capsicum annuum L. (Table II). The highest yield was obtained at P200 indicating that P0 and P10 treatments were P deficient. The treatment of AM inoculation increased the shoot yield by a factor of 3 to 4. There was no effect of AM inoculation on shoot P concentration and therefore the effect on shoot P uptake was similar to
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TABLE II Growth status of Capsicum annuum L. as affected by P application of 0, 10 and 200 mg kg−1 soil (P0, P10 and P200, respectively) and arbuscular mycorrhizal (AM) fungal inoculation after 76 days of growth Treatment
P0 P0 + AM P10 P10 + AM P200 a)
Shoot dry matter
Shoot P concentration
Shoot P content
Root length
Half distance between roots
P influx
g plant−1 0.25da) 0.64c 0.56c 2.15b 6.74a
g kg−1 0.72d 0.91cd 0.98bc 1.17b 1.69a
mg plant−1 0.18d 0.59c 0.56c 2.49b 11.35a
cm plant−1 1 650c 2 794c 2 643c 10 521b 24 772a
cm 0.32 0.24 0.25 0.13 0.08
× 10−14 mol cm−2 s−1 0.79 2.41 2.49 4.23 3.21
Means followed by the same letter(s) within each column are not significantly different at P < 0.05.
that on shoot yield. Root growth was also increased by P fertilization and AM inoculation, but the increase of root growth was smaller than that of shoot yield. At P0 AM showed no significant effect on the root growth. Table II also shows that AM inoculation increased the rate of P uptake per unit of root (i.e., P influx). At P0 AM inoculation increased the P influx by a factor of 3. At P10, the increase was by a factor of less than 2. The results showed that the increased shoot P content was contributed by an increased root growth as well as by an increased P influx.
surface area.
Root infection, hyphae growth and P uptake Table III shows that about half of the roots were infected with AM fungi and the external hyphae length was about 20 m g−1 soil at P0 and P10. The surface area of hyphae is about 10 and 2 times the root surface area at P0 and P10, respectively. No hyphae were detected in the pots receiving no AM inoculation and the results for first and second harvests were similar. Fig. 1 shows the proportion of the P influx coming from AM hyphae and from the root infected by AM. It can be seen that at low P supply (P0) about 80% of P uptake came from the AM hyphae and at P10 it was 50%. However the P influx per unit hyphae surface area (Table IV) was about half that per unit root
Fig. 1 Phosphorus (P) influx of the root infected by arbuscular mycorrhiza (AM) and contribution of AM to the total P influx per unit infected root surface area with P application of 0 (P0) and 10 mg kg−1 soil (P10).
Modeling P uptake and P dynamics around roots and hyphae Table V shows the plant and soil parameters used in the model calculation. The model calculated P uptake and the P depletion around roots or hyphae. It was necessary to asses the significance of plant prope-
TABLE III Root infection and external hyphae length of Capsicum annuum L and its relation to root length Treatmenta)
Hypae length
Half distance between hyphae
Infected root
Per unit infected root Hyphae length
−1
P0 + AM P10 + AM a)
mg 20.9 21.4
soil
cm 0.010 0.010
% 48 65
cm cm 1 237 236
−1
Hyphae surface area cm2 cm−2 10.9 2.4
Arbuscular mycorrhizal (AM) fungi were inoculated at the P application of 0 (P0) and 10 mg kg−1 soil (P10).
MECHANISMS OF AM FUNGI IN P UPTAKE
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TABLE IV Measured and model calculated P influx of arbuscular mycorrhiza (AM) infected roots Treatmenta)
P0 + AM P10 + AM a) b)
Measured P influx
Model calculated P influx
Root cylinder surfaceb)
Hyphae surface
Root cylinder surface
0.79 2.49
× 10−14 mol cm−2 s−1 0.31 0.20 1.12 0.43
Hyphae surface 2.30 2.22
Arbuscular mycorrhizal (AM) fungi were inoculated at the P application of 0 (P0) and 10 mg kg−1 soil (P10). The measured P influx of the root cylinder is the P influx as measured in the AM uninfected root.
TABLE V Plant and soil parameters used for model calculations of P uptake by root and hyphae as affected by P application of 0 and 10 mg kg−1 soil (P0 and P10) and arbuscular mycorrhizal (AM) fungal inoculation Parametera)
P0 (root)
P0 + AM
P10 (root)
P10 + AM
Root
Hypha
Root
Hypha
0.32 1.40 0.04 0.013 2.70 0.13 0.030 3.0
0.25 0.25 0.04 1.500 0.00 0.010 0.010 1 080
3.01 0.37 0.20 802 8.90
3.01 0.37 0.20 802 8.90
Imax (× 10−12 mol cm−2 s−1 ) Km (× 10−9 mol cm−3 ) CLmin (× 10−9 mol cm−3 ) ro (cm) vo (× 10−7 cm3 cm−2 s−1 ) r1 (cm) k (d−1 ) RL (× 103 cm)
0.32 1.40 0.04 0.011 2.70 0.32 0.007 1.2
0.32 1.40 0.04 0.013 2.70 0.24 0.016 1.4
Plant parameter 0.25 0.32 0.25 1.40 0.04 0.04 1.500 0.013 0.00 2.70 0.010 0.25 0.012 0.016 952.5 1.3
CLi (× 10−10 mol cm−3 ) θ (cm3 cm−3 ) f b DL (× 10−6 cm2 s−1 )
0.97 0.37 0.20 2 487 8.90
2.93 0.37 0.20 732 8.90
2.93 0.37 0.20 732 8.90
Soil parameter 1.85 0.37 0.20 1 366 8.90
a)
Imax = maximum P influx, Km = Michaelis constant, CLmin = minimum P concentration, ro = root radius, vo = water influx, r1 = average half distance among neighboring roots, k = relative root growth rate, RL = root length, CLi = initial P concentration in soil solution, θ = volumetric water content, f = tortuosity factor, b = buffer power, DL = P diffusion coefficient in water.
rties, like uptake kinetic parameter or the radius of the absorbing surface (root or hyphae). The results of model calculations in relation to measured values are shown in Table IV. It can be seen that for the root cylinder the model calculates only around 20% of the actual influx but for hyphae it calculates 700% for the P0 + AM and 200% for the P10 + AM treatments. The P influx calculated by the model for hyphae is 5 to 10 times higher than that for the root cylinder. The different calculated influx for the root cylinder and hyphae is mainly related to their different radiuses and the resulting geometry for diffusion
in their vicinity. Fig. 2 showed that after 1 day of P uptake the extension of the depletion zone around the hyphae was less than that around the root. This resulted in a much steeper concentration gradient toward the hyphae. Therefore the flux to the hyphae and so the influx into the hyphae (Table IV) were higher than those into the root. It indicated that hypha surfaces were more effective to absorb P from soils with no P supply. Only by reducing the Imax of the hyphae the calculated influx became close to the actual P influx (Table IV).
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Fig. 2 Comparison of the steepness and extension of the P concentration profiles as calculated by the simulation models around roots and arbuscular mycorrhizal (AM) hyphae after one day of uptake. Data were obtained from the unfertilized but arbuscular mycorrhiza inoculated soil (P0 + AM).
Fig. 3 showed that the width of depletion progressed with time. Since roots were relatively far apart (r1 = 0.24 cm), there was no overlap of the depletion zones of neighboring roots, i.e., there was no inter-root competition. But for hyphae because of their closeness (r1H = 0.01 cm), inter-hyphae competition occurred after 1 day of uptake and after 10 to 20 days 100% of the soil volume would be depleted. In contrast, in the case of roots, only 7% of the soil volume would be partly depleted.
Fig. 3 Progress of the P depletion around roots and arbuscular mycorrhizal hyphae with time of P uptake. Data are for the unfertilized but arbuscular mycorrhiza inoculated soil (P0 + AM).
DISCUSSION The results show that at low P supply an infection with AM fungi significantly increased the yield of Capsicum annuum L. and it was related to an increased P uptake (Table II). The increased P uptake was partly due to a larger root system of AM infected plants but the main reason was the 2 to 3 times higher P uptake rate per unit of root. Similar results were found by others (Sanders et al., 1977; Deressa and Schenk, 2008), where AM inoculation increased the P uptake rate per unit of root by a factor of 2 to 20. The larger root system was probably not due to a specific effect of AM favoring the root growth but because of a higher P uptake. The shoot growth was enhanced and this caused also more root growth. That AM did not enhance root growth specifically is indicated by the lower root:shoot ratio of AM infected plants. The lower root:shoot ratio is probably a consequence of the higher P supply of AM infected plants,
which was also found by Viebrock (1988). In our experiment 50% to 60% of the roots were infected by AM. But the amount of external hyphae per unit of root was higher at P0 than P10 (10.9 versus 2.4 cm2 cm−2 , Table III). Abbott et al. (1984) reported that severe P deficiency increased both the percent of root infection and the length of external hyphae per centimeter of infected root. Reduction of hyphal contribution with increased P addition was also reported by Allison and Goldberg (2002). The total amount of external hyphae of 20 m g−1 soil was in the higher range found by Deressa and Schenk (2008). The contribution of AM to the P uptake per unit of root was about 80% at no P supply (P0) and 50% at P10 (Fig. 1). It is in the same range as found by others (Sanders et al., 1977; Deressa and Schenk, 2008). The main aim of this research, though, was to analyze possible mechanisms by which AM increased the P uptake of plants. The P uptake of a plant depends
MECHANISMS OF AM FUNGI IN P UPTAKE
on the size of its root system and on the rate of P uptake by each root segment, i.e., the P influx. The size of the root system was already discussed above. Table II showed that AM increased the P influx for the whole root system by up to 3 times. But taking the P influx of the infected portion separately (Fig. 1), the increase was by a factor of up to 5. This calculation assumes that the mycorrhizal roots have the same P uptake as the non-mycorrhizal roots do. But it may not have been the case because P concentration in soil solution was higher for AM infected plants at early growth (Table I, Harvest 1). On the other hand the P uptake capacity of the root cylinder may have been reduced due to the infection of roots with AM as was found and extensively discussed by Smith et al. (2004). They found that in AM infected roots the epidermal P uptake path was reduced or even inhibited. To asses the significance of external hyphae for the uptake of P, hyphae length and surface area were determined and compared with the values of the root. There were 230 to 1 200 cm external hyphae per cm of root, but because of the small radius their surface area was only 2 to 10 times larger than that of the root cylinder (Table III). Graham et al. (1982) reported that hyphal surface area was not correlated with that of the root cylinder because of different methods of measurement of external hyphae. This increase of absorbing surface area was more significant than the increase of P influx due to AM (Fig. 1, Table IV), which as a first approach could therefore explain the increased P influx. The comparison of P uptake capacity between hyphae and roots, though, would be insufficient since hyphae have a much smaller radius as roots, which will affect the geometry of diffusion of P towards its surface. Hyphae spread away from the root making more soil volume accessible and furthermore uptake kinetics of hyphae may be different from that of roots. To evaluate these properties of hyphae and roots model calculations were performed. Table IV showed that the P influx of hyphae surface calculated by the model was up to 10 times higher than that of the root cylinder surface. Accordingly hyphae surfaces would be much more effective to absorb P from low P soils than root cylinder surfaces. The higher effectivity of P uptake of hyphae was based on its smaller radius (1.5 versus 110 μm of the root) and lower Km value (0.25 × 10−9 versus 1.4 × 10−9 mol cm−3 ). The smaller radius implicates a larger soil volume per unit surface area of the hyphae, which results in less extension of the P depletion zone (Fig. 2). On the other
509
hand the concentration profile becomes steeper, causing a higher P flux to the hyphae by diffusion and consequently a higher P influx. The small Km value still enhanced the P influx at the low P concentration on the hyphae surface. Fig. 3 showed that for roots alone only a fraction of the soil P was accessible to the root (about 7%), while for hyphae the whole soil volume was accessible. With the hyphae length density of 20 m g−1 soil, hyphae were only 0.01 cm apart so that even with a low mobility of P in soil the depletion zones of neighboring roots overlapped. It can be concluded that AM hyphae not only increase the P absorbing surface area but also increase strongly the proportion of soil P spatially available to the plant. The hyphae length density (HLv ) in this study of 20 m g−1 soil is on the high side of data in the literature of 1 to 20 m g−1 (Deressa and Schenk, 2008). But even with 1 m g−1 soil the half distance among neighboring hyphae (r1H ) would be 0.044 cm. The depletion zones would still overlap and the whole soil volume would be spatially available to the plant. This aspect would also be important for the case that hyphae were not uniformly distributed in the soil, i.e., hyphae may concentrate more close to the root surfaces but fade out farther away from them. For hyphae the model calculated P influx was 8 times (P0 + M) or 2 times (P10 + M) higher than the measured value (Table IV). It meant that, assuming the soil parameters were estimated properly, much more P has been supplied to the hyphae by the soil compared to the P taken up by the AM. The results indicated the plants could regulate the amount of P taken up by AM hyphae. Besides regulating the AM hyphae growth, the plants may also regulate the P uptake physiology, e.g., changing Imax or Km . Model calculations (Table IV) showed that the calculated P influx reduced to the values similar to the measured ones due to the strong reduction of the Imax of the hyphae. However, it cannot be excluded that the plant down regulated the P uptake at the hyphae-root interface. It is well known that plants regulate P uptake kinetics according to the P supply. For example at high P supply Imax is down-regulated (Buhse, 1992; Bhadoria et al., 2004). But Smith et al. (2009) reported that there was a fungus-plant signaling, which induced a regulation of the P transporter expression of the AM P uptake path as well as of the epidermal P uptake pathway. Whether the P uptake regulation actually occurred depended on the reliability of the model calculations.
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The most important soil parameters like the soil solution concentration and soil water content were probably reliably estimated, but the uptake kinetics of hyphae were taken from another experiment of Deressa and Schenk (2008). The corresponding parameters (Imax and Km ) can not be adopted without caution. The authors discussed the insecurities of their estimation and made a conclusion that those parameters may vary according to the conditions like AM strain used and the levels of P in the system. For a better understanding of AM-plant symbiosis concerning P uptake, further research is needed to characterize the uptake kinetics of AM hyphae and to study the fungus-plant signaling concerning the regulation of P transporters of the epidermal and AM P uptake pathways. CONCLUSIONS Arbuscular mycorrhiza (AM) increased the P uptake of Capsicum annum L. in a low P fossil Oxisol with a P concentration of 0.15 μmol L−1 . This was primarily because of a higher P uptake rate (P influx) per unit of AM infected root. This higher P influx was related to a 2 to 10 times larger P absorbing surface area coming from the external hyphae. Model calculations showed that hyphal surfaces were more effective than root cylinder surfaces to absorb P from low P soils and would have been able to absorb more P than that needed by the plant. Model calculations furthermore showed that in the low P soils, P depletion around roots or hyphae only extended to about 0.06 cm and so only 7% of soil P was positionally available to roots. But for hyphae it was about 100%, because the half distance between neighboring hyphae was only 0.01 cm. It can be concluded that the high effectivity of hyphal surfaces to absorb P from soil may be enough in most cases to explain the positive effect of AM on P uptake from soil. ACKNOWLEDGEMENT Thanks is given to Prof. M. Schenk from the Institute of Plant Nutrition, University of Hannover, Germany for offering his laboratory and help in the external hyphae determination. REFERENCES Abbott, L. K., Robson, A. D. and De Boer, G. 1984. The effect of phosphours on the formation of hyphae in soil
M. SHARIF AND N. CLAASSEN by the vesicular-arbuscular mycorrhizal fungus, Glomus fasiculatum. New Phytol. 97: 437–446. Adams, F. and Moore, B. L. 1983. Chemical factors affecting root growth in subsoil horizons of coastal plain soils. Soil Sci. Soc. Am. J. 47: 99–102. Allen, M. F. 1992. Mycorrhizal Functioning: An Integrative Plant-Fungal Process. Chapman and Hall, New York. Allison, V. J. and Goldberg, D. E. 2002. Species-level versus community-level patterns of mycorrhizal dependence on phosphorus: an example of Simpson’s paradox. Funct. Ecol. 16: 346–352. Asimi, S., Gianinazzi-Pearson, V. and Gianinazzi, S. 1980. Influence of increasing soil phosphorus levels on interactions between vesicular-arbuscular mycorrhiza and rhizobium in soybeans. Can. J. Bot. 58: 220–225. Barraclough, B. P. and Tinker, B. P. 1981. The determination of ionic diffusion coefficient in field soils. I. Diffusion coefficients in sieved soils in relation to water content and bulk density. J. Soil Sci. 32: 225–236. Bhadoria, P. S., El Dessougi, H., Liebersbach, H. and Claassen, N. 2004. Phosphorus uptake kinetics, size of root system and growth of maize and groundnut in solution culture. Plant Soil. 262: 327–336. Brundrett, M., Melville, L. and Peterson, L. 1994. Practical Methods in Mycorrhiza Research. The 9th North American Conference on Mycorrhizae. Mycologue Publications, University of Gulph, Canada. Buhse, J. 1992. Wirkung der Wurzelraumtemperature auf das Phosphateaneignungs-vermoegen von Pflanzen und die Phosphateverfeugbarkeit im Boden (in German). Ph.D. Thesis. University of Goettingen, Germany. Claassen, N. and Steingrobe, B. 1999. Mechanistic simulation models for a better understanding of nutrient uptake from soil. In Rengel, Z. (ed.) Mineral Nutrition of Crops: Fundamentals Mechanisms and Implications. The Haworth Press, Inc., New York. pp. 327–367. Cress, W. A., Throneberry, G. O. and Lindsey, D. L. 1979. Kinetics of phosphorus absorption by mycorrhizal and non-mycorrhizal tomato roots. Plant Physiol. 64: 484– 487 Deressa, T. G. and Schenk, M. K. 2008. Contribution of roots and hyphae to phosphorus uptake of mycorrhizal onion (Allium cepa L.): A mechanistic modeling approach. J. Plant Nutr. Soil Sci. 171: 810–820. Edwards, O. W. and Huffman, E. O. 1959. Diffusion of aqueous solution of phosphoric acid at 25 ◦ C. J. Phys. Chem. 63: 1830–1833. F¨ ohse, D., Claassen, N. and Jungk, A. 1991. Phosphorus efficiency of plants. II. Significance of root radius, root hairs and cation-anion balance for phosphorus influx in seven plant species. Plant Soil. 132: 261–272 Gericke, S. and Kumries, B. 1952. Kolorimetrische Phosphrs¨ aurebestimmung mit Ammonium-VanadateMolybdate und ihre Anwendung in der Pflanzenanalyse. J. Plant Nutr. Soil Sci. (in German). 59: 235–
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