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Keywords:

  • ammonia;
  • arbuscular mycorrhiza;
  • external mycelium;
  • fungal cell wall;
  • hyphal transport;
  • internal mycelium;
  • nitrate;
  • nitrification;
  • nitrogen nutrition;
  • symbiosis

ABSTRACT

  1. Top of page
  2. ABSTRACT
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. CONCLUSION
  8. ACKNOWLEDGMENTS
  9. REFERENCES

Arbuscular mycorrhizal fungi can enhance nutrient acquisition by a plant via their extraradical hyphae. This is particularly true for phosphorus, but the case for nitrogen (N) has been less clear. In our growth systems there was a small air-gap between root and hyphal compartments, which eliminated diffusion of nutrients between compartments. Moreover, our methods allowed us to distinguish between nitrate and ammonium. We found that N transfer to Zea maize L. depends on the sources fed to the hyphae of Glomus aggregatum Schenck & Smith. In experiment 1, despite the fact that plant demand for N was already met, plants received 10 times as much 15N from ammonium than from nitrate. In experiment 2, 74% of shoot-N was derived from the slow-release urea added to the hyphal compartment while only 2.9% was derived from the nitrate-N. Intraradical hyphae isolated from roots contained a considerable amount of 15N in the cell wall even when 15N-nitrate was the source. We conclude that the mycorrhizal fungus can rapidly deliver ammonium-N to the plants, and that while the fungus can absorb nitrate, it apparently lacks the capacity to transfer it to the plant.


Abbreviations
AM

arbuscular mycorrhiza

HC

hyphal compartment

NHC

nitrogen derived from hyphal compartment

RC

root compartment.

INTRODUCTION

  1. Top of page
  2. ABSTRACT
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. CONCLUSION
  8. ACKNOWLEDGMENTS
  9. REFERENCES

Plants with arbuscular mycorrhizas (AM) are enabled to acquire nutrients from comparatively distant sites through the extraradical hyphae, which elongate into the soil. As a result, the nutritional status of the host plant can be improved. This function is particularly remarkable for phosphorus (P) (Bolan 1991; Clark & Zeto 2000). As for nitrogen (N), however, the function of AM remains uncertain despite a number of reports on this topic because previous experiments suffered from significant methodological problems.

The major problem has been an inability to distinguish between N sources. Both ammonium and nitrate are available to most plants. The former is relatively immobile and the latter is readily mobile in the soil solution (Tinker & Nye 2000). On the basis of the mobility, AM function was previously predicted to be different between them (Smith & Read 1997; Clark & Zeto 2000). In soil, however, ammonium is rapidly nitrified by soil microbes (Barber 1995), hence there is concern that the ammonium supplied might be transformed into nitrate before the hyphae were able to capture it.

Another problem has been the inability to assess whether there was direct transfer of N from hyphae to root. It has been common to adopt a container system that is divided into two compartments by fine nylon mesh to restrict roots in a root compartment (RC) while allowing extraradical hyphae to enter the hyphal compartment (HC) (Ames et al. 1983; Johansen, Jakobsen & Jensen 1992, 1993a, b; Frey & Schüepp 1993; Tobar, Azcon & Barea 1994a, b; Subramanian & Charest 1999). By adding 15N into the HC several centimetres away from the RC, it was assumed that the plant would only receive 15N via the extraradical hyphae. However, the assumption may not be valid because considerable 15N can be detected in non-AM plants, probably due to solute movement across the mesh.

Some attempts have been made to solve these problems. For example, Johansen et al. (1992, 1993a) and Johansen, Jakobsen & Jensen 1994) used ammonium in conjunction with a nitrification inhibitor. Although this allowed them to distinguish between N sources, even if it was provided 5 cm away from the root, absorption by non-AM roots still occurred (Johansen et al. 1993a). Mader et al. (2000) proposed a method of compartmentalization using a polytetrafluoroethylene (PTFE) membrane that could pass water vapour, but inhibited passage of liquid water. However, N movement between the compartments could not be completely prevented even by this method.

A major problem in the evaluation and quantification of AM function in the mineral nutrition of plants arises from changes in growth, and root morphology and physiology, brought about by mycorrhizal colonization (Marschner 1995; Yano, Yamauchi & Kono 1996). This might also affect results of the previous experiments using non-AM controls. It is possible that AM plants had greater capacity for uptake of nutrients arriving by mass flow because of greater rates of transpiration, but also for nutrients arriving by diffusion because AM plants are usually larger due to improved nutrition and thus may possess larger root systems.

In this study therefore the following attempts were made. First, a small air-gap was positioned between the RC and the HC (Fig. 1) to prevent solute movement between them. This system was first used by Faber, Zasoski & Munns (1991) to evaluate the ability of AM fungi to transport water. Next, in order to reduce nitrification of ammonium by soil microbes, agar gel instead of soil was used as a medium for the HC in experiment 1, and a resin-coated urea that releases ammonia slowly in soil was used in experiment 2. As far as we know, this is the first attempt to apply agar gel and also resin-coated urea to the HC. This improves our ability to distinguish between N sources. By so doing, we aimed to identify direct N delivery via AM hyphae, and to compare N sources (ammonium versus nitrate) in their suitability for transfer to the root by mycorrhizal fungi.

image

Figure 1. A compartment system used in the present study. Root development is restricted within root compartment (RC) due to the nylon mesh, but extraradical hyphae elongating from the roots enable to enter hyphal compartment (HC) passing through the mesh. A wire net inserted between RC and HC prevents solute movement across the mesh otherwise via extraradical hyphae.

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MATERIALS AND METHODS

  1. Top of page
  2. ABSTRACT
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. CONCLUSION
  8. ACKNOWLEDGMENTS
  9. REFERENCES

Experiment 1

Glomus aggregatum Schenck & Smith was used as the AM fungus considering its rapid colonization of maize (McGonigle, Yano & Shinhama 2003). An inoculum of the fungus was added to Andosol that had been sterilized by autoclaving (121 °C, 180 min) previously. Crimson clover (Trifolium incarnatum L) was grown for 6 months in this soil to produce inoculum, and the shoots were removed. The soil was passed through a 7-mm-screen. The roots on the screen were cut into pieces 20–30 mm in size and mixed thoroughly with the soil. The sieved soil containing the root pieces was used as the medium for the RC.

The RC (250 mm in length, 400 mm in depth, and 20 mm in width) (Fig. 1) was filled with the medium and one germinated maize seed (Zea mays L.) hybrid Robust 30–71 (Takii Seed Co. Ltd, Kyoto, Japan) was sown into each RC. During the experimental period, they were kept in a natural light growth chamber (14 h day, day/night temperature 30/25 °C, relative humidity 75%) and water was given to each RC when necessary. At 6 and 16 d after sowing, 20 mL of Hoagland solution (Hoagland & Arnon 1950) were added into each RC. At 6 d the solution contained full nutrients and at 16 d the solution excluded N.

On day 8 after planting, the HC (250 mm in length, 400 mm in depth, and 25 mm in width) was installed. Each HC was filled with 2% (w/v) agar gel (1.6 L) that was vertically divided into two; one side contained NaNO3 (50 mg N L−1) and the other side (NH4)2SO4 (50 mg N L−1). This was done to avoid the possibility of the mycelium preferentially taking up one N source over another. Nylon mesh (50 µm in opening) was inserted between the RC and the HC in all the containers to restrict root growth to the RC while allowing penetration of extraradical hyphae into the HC. In addition, a stainless wire net (1.5 mm in thickness, 6 mm-mesh) to create an air-gap was inserted between the compartments according to the method of Shibata & Yano (2003). Furthermore, the space between compartments was sealed with vinyl tape in order to prevent the agar from dehydrating. On day 15 following the HC installation, a new HC, prepared as above with the exception that it contained 15N, was exchanged for the old HC. Using Na15NO3 (5.01 atom percentage 15N, Shoko Co. Ltd, Japan) and (15NH4)2SO4 (5.06 atom percentage 15N, Shoko Co. Ltd), two treatments were established; one contained 15NO3 and 14NH4+ whereas another contained 14NO3 and 15NH4+ in a HC. Shoots were sampled at 22 d after HC was exchanged.

Experiment 2

The mycorrhizal inoculum was prepared in the same manner as for experiment 1 except using loamy sand. Each RC (100 mm in length, 180 mm in depth, and 20 mm in width) was filled with a mixture of inoculum (100 g) and autoclaved (121 °C, 180 min) loamy sand (250 g). Three germinated maize seeds (Zea mays L.) were sown into each RC, and thinned to two plants 1 week later. The plants were grown in a glass house and water was given every day until the HC was installed. At 7 and 14 d after sowing 20 mL of a 200-fold dilution of full nutrient solution (Hyponex 6-6-6; Hyponex Japan Co. Ltd, Osaka, Japan) was given to each RC.

Five containers were sampled at 18 d after sowing and the rate of colonized root length by the fungus was determined to be approximately 30%. Following this, the HC (100 mm in length, 180 mm in depth, and 10 mm in width) was immediately installed. This HC was filled with loamy sand (200 g) that had been autoclave sterilized (121 °C, 180 min) in the same manner as for the RC, and 5 mL of loamy sand leachate was added in advance. Then 1 g of one of two different sources of N was then added to each container; NaNO3(10.3 atom percentage 15N, Shoko Co. Ltd), or a resin-coated urea (LP40, 40%N, 3.22 atom percentage 15N, Shoko Co. Ltd) releasing ammonium-N slowly. In soils the hydrolysis of urea usually takes place before it is absorbed (Marschner 1995).

Nylon mesh (50 µm in opening) was inserted between the compartments in all the containers to restrict root growth to the RC while allowing penetration of extraradical hyphae into HC (Fig. 1). For each N source, in half of the containers a stainless wire net (1.5 mm in thickness, 6 mm-mesh) was placed as an air-gap adjacent to the nylon mesh so that the soils of the HC and the RC were not in contact (Gap+). In the remaining half of the containers, the wire net was not installed hence both compartments of soil could be in contact (Gap–). In this system, 15N movement from the HC into the RC due to ammonium volatilization was negligible.

Water was supplied every day following HC installation in order to maintain 20% (w/w) moisture content in the soil. As water movement between compartments took place freely in Gap, the content of water in soil was maintained by measuring the total weight of both compartments and supplied the amount of water reduction. As water movement between compartments did not take place in Gap+, we measured the amount of water reduction in each compartment separately. For this reason, other containers (n = 6) were prepared separately, the reduction in the amount of water from the HC was estimated, and the water ratio in each soil was made constant by replacing the required amount of water to the RC and the HC.

Beginning on day 3 after HC installation and continuing every 5 d, a sample of leaf-disc (approximately 1 cm2) from the youngest leaf at the time from one of the two individual plants in each container was collected, and used for time course changes in percentage NHC. The same plant was used for all leaf samples in this part of the experiment.

At 30 d after the HC was installed, all plants were sampled. Shoots and roots of the intact plant in each pair (the plant which had not been used for regular leaf sampling) were used for N analyses. The root system of the plant which had been used for the regular leaf samples was preserved in FAA (folmaldehyde 1: acetic acid 1: 70% ethanol 18 by volume) and used for N analyses of intraradical hyphae and colonized root length estimation.

Isolation of cell walls of intraradical hyphae

Roots preserved in FAA were thoroughly rinsed with deionized water and cut into 5–10 mm segments, and then autoclaved with 1 N NaOH for 120 min at 90 °C to remove the cytosol of the fungus and the roots. Each sample was thoroughly washed with distilled water and digested with enzymes (20 m m Mes-KOH buffer, pH 5.5, containing 5% (w/v) Cellulase ‘Onozuka’ RS (Yakult Pharmaceutical Ind., Tokyo, Japan), 0.25% (w/v) Macerozyme R-10 (Yakult Pharmaceutical Ind.), 0.075% (w/v) Pectolyase Y-23 (Kyowa Chemical Products Co. Ltd., Osaka, Japan) for 90 min at 30 °C according to the methods of Karahara & Shibaoka (1992). After washing with distilled water, intraradical hyphae were collected using a needle under a stereoscopic microscope. At the time of collecting, the presence of vesicles was checked as the specific structure of intraradical hyphae (Fig. 4). Intraradical hyphae were dried at 80 °C for 48 h (approximate dry weight of 0.2–0.8 mg) and the 15N concentration was measured by the same method as for the plant materials.

image

Figure 4. (a) Mycorrhizal maize root. (b) and (c) Intraradical hyphae isolated from the root using 1 N NaOH and enzymes digesting root cell wall. The hyphae is stained with chlorazol black E for the contrast. Arbuscules (A) and vesicles (V) as specific structure in intraradical hyphae are observed.

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15N analyses

The samples were dried at 80 °C for 48 h, and the dry weight was determined. Each sample was ground and approximately 0.1 mg of the ground sample was used to measure N concentration by an elemental analyser (NA2500; CE Instruments, Milan, Italy). A portion of the combustion gases was introduced into an isotopic ratio mass spectrometer (Delta Plus; Thermo Electron Corp., San Jose, CA, USA), and the 15N concentration was determined. Based on the following formula, the percentage of N derived from the HC (%NHC) and the 15N recovery of total supply were calculated.

  • %NHC  =  [atom% 15N excess (sample)/atom% 15N excess          (N source)]  ×  100
  • Content of NdfHC  =  (%NHC  ×  N content)/100

Colonized root length

Roots preserved in FAA were rinsed and cut into 5 to 10 mm segments. Root segments were cleared with 10% (w/v) KOH (Phillips & Hayman 1970) for 15 min at 90 °C and stained with 0.05% (w/v) chlorazol black E (Brundrett, Piché & Peterson 1983) for 90 min at 80 °C. Then, the percentage of colonized root length to total root length was estimated according to the method of McGonigle et al. (1990).

RESULTS

  1. Top of page
  2. ABSTRACT
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. CONCLUSION
  8. ACKNOWLEDGMENTS
  9. REFERENCES

Experiment 1

The growth of each plant was vigorous without any nutrient deficiency symptom due to the previous supply of full nutrients into the RC (Fig. 1), and also similar between the treatments as shown in shoot dry weight and total N content (Table 1). The percentage of N derived from the HC (%NHC) in the shoot was very small regardless of which N source was labelled, indicating that the demand for N in each plant was fully met without the labelled N delivered via extraradical hyphae. Despite the similar growth and sufficient N, 15N content in the shoot was significantly different depending on the form of labelled N supplied (P = 0.0029). When nitrate was labelled, only 0.2 mg of the labelled N was detected in the shoot. By contrast, the amount was 2.7 mg when ammonium was labelled. The results therefore show that the ability of the fungus to deliver N to maize was higher for ammonium than for nitrate even when plant demand for N was small.

Table 1.  Dry weight, N content and percentage of N derived from hyphal compartment (%NHC) in maize shoot at 22 d after installation of HC amended with 15N sources (experiment 1)
15N sourceShoot dry weight (g plant−1)Shoot N Content (mg plant−1)Shoot 15N Content (mg plant−1)%NHC (%)
  1. Probability (P-value) was calculated by anova (n = 5)

15NO3, 14NH4+2.6566.70.240.35
14NO3, 15NH4+2.6459.42.664.67
P-value0.9688 0. 18190.00290.0029

Experiment 2

Time course change in N derived from HC in upper-most leaf

The difference in ability to deliver N based on the form of N supplied was further examined using soil as the medium of the HC. In this case, the N source was a resin-coated urea, which releases ammonia slowly. In the Gap– treatment, in which both roots and hyphae were able to capture 15N, the %NHC increased sharply until 18 d after the installation of the HC and then reached a plateau at approximately 75% regardless of the form in which N was supplied (Fig. 2). In contrast, %NHC was quite different between N sources in the presence of the air-gap (Gap+) where only extraradical hyphae could access 15N supplied to the HC. When ammonium was given, %NHC exhibited a transition that was similar time course of Gap– but with a delay of about 5 d, and reached a similar maximum of around 75% (Fig. 2). However, when nitrate was given in the Gap+ treatment, the %NHC was almost negligible throughout the experimental period.

image

Figure 2. Time course in percentage of N derived from hyphal compartment (%NHC) in upper-most leaf of maize plants (experiment 2). Hyphal compartment (HC) was amended with 15N-nitrate or a resin-coated fertilizer releasing 15N-ammonium slowly. Gap+ and Gap– indicates the presence or absence of an air-gap between compartments, respectively. Each plot represents mean ± standard error (n = 5).

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Plant dry matter and mycorrhizal colonization

The plants were harvested 30 d after the installation of the HC. Dry weights of the shoot and the root were significantly lower in the Gap+ treatment given nitrate, probably due to a N deficiency (Table 2). A greater dry matter was observed in the Gap+ treatment given the resin-coated urea, which was similar to the Gap– treatment. The interaction between the two main effects, N source and Gap, was significant for the shoot (P = 0.0690) and root dry weight (P = 0.0392), indicating that the presence of the air-gap had different effects depending on the N source supplied in the HC.

Table 2.  Rate of mycorrhizal root to total root length, dry weight, N content and percentage of N derived from hyphal compartment (%NHC) in maize plant at 30 d after installation of HC amended with 15N sources (experiment 2)
15N SourceGapMycorrhizal root length (%)Dry weight (g plant−1)Total N content (mg plant−1)%NHC (%) 
ShootRootShootRootShootRoot
NO3Gap +64.0 a1.48 a0.81 a13.0 a5.2 a2.9 a8.2 a
Gap–61.2 a2.23 b1.01 b50.2 b14.3 b73.1 b66.9 b
NH4+Gap +73.6 ab2.17 b0.98 b48.2 b12.3 b74.0 b65.8 b
Gap–77.6 b2.17 b0.93 ab63.5 b18.9 c80.2 c75.0 c
anova P-value      
  1. Means (n = 5) followed by the same letter within a column are not significantly different (P < 0.05) from each other by LSD.

N source 0.01010.11950.45260.00040.0002< 0.0001< 0.0001
Gap 0.89470.06680.20330.0002< 0.0001< 0.0001< 0.0001
Interaction 0.45690.06900.03920.06130.3393< 0.0001< 0.0001

The rate of root length colonized by the fungus exceeded 60% in all the treatments (Table 2). The N source supplied in the HC affected the percentage (P = 0.0101), resulting in approximately 10% higher colonization with ammonium-N than with nitrate-N, although the presence of the air-gap did not affect colonization (P = 0.8947).

N derived from HC in plant

The N content of shoots and roots was lower in the Gap+ treatment given nitrate than in the other treatments (Table 2). For shoot N content, the interaction was also significant (P = 0.0613) in addition to N source (P = 0.0004) and Gap (P = 0.0002), implying different effects of the air-gap depending on N source. Specifically, the absence of the air-gap increased shoot N content only with nitrate. For root N content, the interaction was not significant (P = 0.3393) whereas N source (P = 0.0002) and Gap (P < 0.0001) were significant. Thus the absence of an air-gap increased root N content to a similar extent irrespective of the N source, and ammonium resulted in higher N content than nitrate, independent of the air-gap.

The shoot %NHC showed high values of 70–80% in the three treatments other than Gap+ given nitrate, which had a value of only 2.9%. There was a strong interaction (P < 0.0001) between the two main effects of N source (P < 0.0001) and Gap (P < 0.0001), supporting a specific effect of the air-gap on nitrate-N transfer to the plants. Interestingly, %NHC became higher in roots than in shoots only when 15N-nitrate and the air-gap were provided, suggesting retention of N by intraradical hyphae, as will be discussed later.

When the resin-coated urea was given, 36 mg of labelled N was detected from the shoot in Gap+ (Fig. 3). That amount was equivalent to 71% of the value for the Gap–, in which both root and extraradical hyphae could access the labelled N. On the other hand, when nitrate was given, 37 mg labelled N was detected in the shoot of Gap–, whereas only 0.4 mg was detected in the Gap+. The two main effects of N source and Gap were significant (P < 0.0001) in addition to their interaction (P = 0.0300). This pattern was similar to that of the root, however, the interaction was not significant.

image

Figure 3. Content of N derived from hyphal compartment (NHC) in maize plant at 30 d after installation of HC amended with each of 15N sources (experiment 2). Two-way anova detected a significant interaction effect (P < 0.001) between two main effects (N source P < 0.001; Gap P < 0.001). Means (n = 5) followed by the same letter are not significantly different (P < 0.05) from each other by LSD.

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N derived from the HC in cell wall of intraradical hyphae

The fungal cell wall was isolated from the roots (Fig. 4). The %NHC of the fungal cell wall showed different values depending on N sources supplied (P < 0.0001). When the resin-coated urea was given, the %NHC was approximately 40% regardless of the presence or absence of the air-gap (Fig. 5). In contrast, when nitrate was supplied, although the difference was not significant, a contrast in %NHC was observed depending on the air-gap; Gap– 18% versus Gap+ 8%. However, these values either in the presence or absence of the air-gap were apparently closer than those corresponding to the root; namely Gap– 67% versus Gap+ 8% (Table 2).

image

Figure 5. Percentage of N derived from hyphal compartment (%NHC) in fungal cell wall from intraradical hyphae isolated from maize roots colonized. Means (n = 5) followed by the same letter are not significantly different (P < 0.05) from each other by LSD.

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DISCUSSION

  1. Top of page
  2. ABSTRACT
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. CONCLUSION
  8. ACKNOWLEDGMENTS
  9. REFERENCES

Delivery of ammonium-N via the hyphae

On the basis of the results of experiments 1 and 2, there is no doubt that the AM fungus transferred ammonium-N to the host plant. In experiment 1, we found that the fungus delivered ammonium-N much more than nitrate-N (Table 1) even if the plant demand for N was fully met without fungal N delivery. In experiment 2, when a resin-coated urea, which releases ammonium-N slowly, was supplied, the fungus alone delivered 71% of the amount of N that both extraradical hyphae and roots were able to capture (Fig. 3). Considering the contrasts of ammonium-N over nitrate-N in this study, it would appear that using agar gel medium or the resin-coated urea substantially reduced nitrification of ammonium.

Our results suggest that delivery of ammonium-N via extraradical hyphae is active and rapid. For example, in experiment 1, the fungus delivered ammonium-N at a rate more than 10-fold that of nitrate-N (Table 1). The rate of ammonium-N delivery could be equivalent to that of P. Using a container similar to that used in the current study, Johansen et al. (1993a) supplied 32P into the HC at 1 cm away from the RC and then sampled the second youngest leaf on the host plant. As a result, 32P was detected in a similar time course to our results in Gap+ (Fig. 2). This rapid delivery of ammonium-N may be related to the small fungal amino acid pool as suggested by Cliquet & Stewart (1993).

A number of experiments have been conducted to date with respect to the ability of AM fungi to transfer ammonium-N (Johansen et al. 1992, 1993b, 1994; Frey & Schüepp 1993; Tobar et al. 1994b). While these studies have provided generally positive results for N transfer, the extents vary from negligible (Johansen et al. 1992) to considerable (Johansen et al. 1994). As pointed out earlier, these previous experiments might not fully control nitrification of ammonium or prevent solute movement between the compartments.

The present study reveals the importance of controlling nitrification and solute movement driven by roots. In Table 2, the plants had full access to 15N regardless of the form supplied into HC if the air-gaps were removed (Gap–). This was the case in most previous studies. On the basis of this result alone, one might have concluded that the fungal N delivery is high regardless of the form. Moreover, in the previous papers, if ammonia supplied in the HC had been transformed into nitrate by soil microbes before hyphal capture, then the ability to acquire this more mobile form of N in the HC was probably determined by the root volume and the transpiration rate of the plants rather than by the fungus.

Delivery of nitrate-N via the hyphae

The results indicate that the fungus had only a poor capacity to deliver nitrate-N to maize (Table 1, Fig. 3). Some studies have suggested that the mycorrhizal fungi have ability to transfer nitrate-N (Tobar et al. 1994a; Subramanian & Charest 1999). The difference between the previous studies and ours might be attributed to the species difference of fungi or plant used. Again, however, we cannot eliminate a possibility that those results were caused by absorption by roots of N moving in mass flow from the other compartment. Indeed, 15N was detected in non-AM plants (Tobar et al. 1994a; Subramanian & Charest 1999).

There are two possible reasons why N delivery by the fungus was negligible for nitrate-N; (1) extraradical hyphae could not acquire nitrate, or (2) extraradical hyphae acquired nitrate, but did not deliver it to the plant. In order to distinguish between these two possibilities, intraradical hyphae were isolated from roots (Fig. 4), and the amount of labelled N contained in the cell wall was measured. A major component of the fungal cell wall is chitin (a polymer of N-acetyl glucosamine) that contains approximately 6% N (Leake & Read 1990). Considerable labelled N was detected in the fungal cell wall in Gap+ even when nitrate was given (Fig. 5). Therefore, while it was confirmed that the hyphae did not readily deliver nitrate-N to the host, they did nevertheless assimilate nitrate-N in part to build the fungal cell wall inside the root. Consistent with this, %NHC was higher in roots than in shoots only when nitrate and the air-gap were provided (Table 2).

N utilization by the fungus for the cell wall construction was lower when nitrate-N was supplied in comparison with ammonium-N (Fig. 5). The following two possibilities were considered as explanations: (1) the hyphal ability to acquire nitrate was very low due to poor development of the extraradical hyphae in the HC in comparison with the ammonium supply; and (2) the assimilation rate of nitrate was low. Since we measured 15N content only for the cell wall, it is not known which one is the cause.

AM function in plant N nutrition

As far as we know, this is the first report to determine the capability of N delivery to a plant by AM fungal hyphae without any interference by roots, and consequently to demonstrate that the N delivery depends on the form of N that was fed to the fungus. Interestingly, nitrate-N was scarcely delivered to the plant despite the fact that the fungus utilized it in the building of its cell wall. Rapid delivery was apparent for ammonium-N. George et al. (1992) and Hawkins & George (1999, 2001) provided an air-gap in their experimental system and investigated AM function in plant N nutrition. However, their interest seemed to be in the form of N fed to plants rather than to extraradical hyphae; and thus the reports did not suggest discrimination in N delivery to plants according to the form fed to extraradical hyphae as we have found.

Previously, a lower effectiveness by extraradical hyphae of nitrate-N transfer was based on the high mobility of nitrate. From this viewpoint, it is expected that nitrate-N transfer by AM fungi may become more significant as its mobility is reduced, such as when soil moisture content declines (Tobar et al. 1994a; Smith & Read 1997; Subramanian & Charest 1999; Clark & Zeto 2000). However, that may be difficult even in dry soil if hyphal discrimination in N transfer such as we found is common to all AM fungi. To confirm this, it will be worth examining other species of mycorrhizal fungi.

Our results suggest that N acquisition by AM fungi does not necessarily indicate that the N will be transferred to the host plant. Bago et al. (1996) showed indirectly that extraradical hyphae can take up nitrate using an agar culture medium containing a pH indicator. Johansen, Finlay & Olsson (1996) directly demonstrated that AM fungi acquired both nitrate and ammonium, and assimilated them into amino acids, by analysing 15N substances in extraradical hyphae. However, it was unknown from those experiments whether the N acquired by AM fungi was delivered to the host plants or not. To confirm fungal delivery of N to the host, N analysis of roots containing the hyphae would not be suitable because intraradical hyphae may accumulate N without transferring it. Thus, shoot or xylem sap analysis should be performed.

Compared with P, there is little information on the role of AM in plant N acquisition, at least in non-legumes, although both natural and agricultural ecosystems are often limited by N rather than P (Marschner 1995). One of the reasons for this may have been the inability of previous researchers to distinguish between ammonium and nitrate. Considering the high capability of the fungus for ammonium transfer, but the low capability for nitrate transfer, enhanced N acquisition via extraradical hyphae may be especially significant in soils that are rich in organic matter, which can release ammonium gradually into the soil. Hodge, Campbell & Fitter (2001) reported that an AM fungus accelerates decomposition of organic matter through strong N capture. We assume that high capability for delivering ammonia-N by the fungi enables this.

CONCLUSION

  1. Top of page
  2. ABSTRACT
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. CONCLUSION
  8. ACKNOWLEDGMENTS
  9. REFERENCES

By solving two experimental problems, nitrification of ammonium-N and solute movement between the compartments, we could identify direct N delivery to the host plant via extraradical hyphae. As a result, we clearly showed for the first time that the ability of an AM fungus to deliver N differs depending on the N source supplied to the extraradical hyphae. Specifically the fungus can rapidly transfer ammonium-N to maize at an equivalent rate to P. In contrast, the fungus seems not to be able to deliver nitrate-N, even if nitrate-N was absorbed.

ACKNOWLEDGMENTS

  1. Top of page
  2. ABSTRACT
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. CONCLUSION
  8. ACKNOWLEDGMENTS
  9. REFERENCES

The authors would like to express our best thanks to Professor Roger T. Koide, The Pennsylvania State University, for reading our manuscript and providing many valuable suggestions. We are grateful to Yasuko Katoh for her assistance in laboratory works, and to Waka Takasoh for her help in computer drawing. The authors also appreciate very much to anonymous referees for reviewing our manuscript.

REFERENCES

  1. Top of page
  2. ABSTRACT
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. CONCLUSION
  8. ACKNOWLEDGMENTS
  9. REFERENCES
  • Ames R.N., Reid C.P.P., Porter L.K. & Cambardella C. (1983) Hyphal uptake and transport of nitrogen from two 15N-labelled sources by Glomus mosseae, a vesicular-arbuscular mycorrhizal fungus. New Phytologist 95, 381396.
  • Bago B., Vierheilig H., Piché Y. & Azcon-Aguilar C. (1996) Nitrate depletion and pH changes induced by the extraradical mycelium of the arbuscular mycorrhizal fungus Glomus intraradices grown in monoxenic culture. New Phytologist 133, 273280.
  • Barber S.A. (1995) Soil Nutrient Bioavailability, 2nd edn. John Wiley & Sons, New York, USA.
  • Bolan N.S. (1991) A critical review on the role of mycorrhizal fungi in the uptake of phosphorus by plants. Plant and Soil 134, 189207.
  • Brundrett M.C., Piché Y. & Peterson R.L. (1983) A new method for observing the morphology of vesicular-arbuscular mycorrhizae. Canadian Journal of Botany 62, 21282134.
  • Clark R.B. & Zeto S.K. (2000) Mineral acquisition by arbuscular mycorrhizal plants. Journal of Plant Nutrition 23, 867902.
  • Cliquet J.B. & Stewart G.R. (1993) Ammonia assimilation in Zea mays L. infected with a vesicular-arbuscular mycorrhizal fungus Glomus fasciculatum. Plant Physiology 101, 865871.
  • Faber B.A., Zasoski R.J. & Munns D.N. (1991) A method for measuring hyphal nutrient and water uptake in mycorrhizal plants. Canadian Journal of Botany 69, 8794.
  • Frey B. & Schüepp H. (1993) Acquisition of nitrogen by external hyphae of arbuscular mycorrhizal fungi associated with Zea mays L. New Phytologist 124, 221230.
  • George E., Haussler K.U., Vetterlein D., Gorgus E. & Marschner H. (1992) Water and nutrient translocation by hyphae of Glomus mosseae. Canadian Journal of Botany 70, 21302137.
  • Hawkins H.J. & George E. (1999) Effect of plant nitrogen status on the contribution of arbuscular mycorrhizal hyphae to plant nitrogen uptake. Physiologia Plantrum 105, 694700.
  • Hawkins H.J. & George E. (2001) Reduced 15N-nitrogen transport through arbuscular mycorrhizal hyphae to Triticum aestivum L. supplied with ammonium vs. nitrate nutrition. Annals of Botany 87, 303311.
  • Hoagland D.R. & Arnon D.J. (1938) The Water Culture Method for Growing Plants without Soil. California Agricultural Experimen Station Circular no. 347. In College of Agriculture, University of California, Berkeley.
  • Hodge A., Campbell C.D. & Fitter A.H. (2001) An arbucular mycorrhizal fungus accelerates decomposition and acquires nitrogen directly from organic material. Nature 413, 297299.
  • Johansen A., Finlay R.D. & Olsson P.A. (1996) Nitrogen metabolism of external hyphae of arbuscular mycorrhizal fungus Glomus intraradices. New Phytologist 133, 705712.
  • Johansen A., Jakobsen I. & Jensen E.S. (1992) Hyphal transport of 15N-labelled nitrogen by a vesicular-arbuscular mycorrhizal fungus and its effect on depletion of inorganic soil N. New Phytologist 122, 281288.
  • Johansen A., Jakobsen I. & Jensen E.S. (1993a) External hyphae of vesicular-arbuscular mycorrhizal fungi associated with Trifolium subterraneum L. New Phytologist 124, 6168.
  • Johansen A., Jakobsen I. & Jensen E.S. (1993b) Hyphal transport by a vesicular-arbuscular mycorrhizal fungus of N applied to the soil as ammonium or nitrate. Biology and Fertility of Soils 16, 6670.
  • Johansen A., Jakobsen I. & Jensen E.S. (1994) Hyphal N transport by a vesicular-arbuscular mycorrhizal fungus associated with cucumber grown at three nitrogen levels. Plant and Soil 160, 19.
  • Karahara I. & Shibaoka H. (1992) Isolation of casparian strips from pea roots. Plant and Cell Physiology 33, 555561.
  • Leake J.R. & Read D.J. (1990) Chitin as a nitrogen source for mycorrhizal fungi. Mycological Research 94, 993995.
  • Mader P., Vierheilig H., Streitwolf-engel R., Boller T., Frey B., Christie P. & Wiemken A. (2000) Transport of 15N from a soil compartment separated by a polytetrafluoroethylene membrane to plant roots via the hyphae of arbuscular mycorrhizal fungi. New Phytologist 146, 155161.
  • Marschner H. (1995) Mineral Nutrition of Higher Plants, 2nd edn. Academic Press, London, UK.
  • McGonigle T.P., Miller M.H., Evans D.G., Fairchild G.L. & Swan J.A. (1990) A new method which gives an objective measure of colonization of roots by vesicular-arbuscular mycorrhizal fungi. New Phytologist 115, 495501.
  • McGonigle T.P., Yano K. & Shinhama T. (2003) Mycorrhizal phosphorus enhancement of plants in undisturbed soil differs from phosphorus uptake stimulation by arbuscular mycorrhizae over non-mycorrhizal controls. Biology and Fertility of Soils 37, 268273.
  • Phillips J.M. & Hayman D.S. (1970) Improved procedures for clearing roots and staining parasitic and vesicular-arbuscular mycorrhizal fungi for rapid assessment of infection. Transactions in British Mycological Society 55, 158160.
  • Shibata R. & Yano K. (2003) Phosphorus acquisition from non-labile sources in peanut and pigeonpea with mycorrhizal interaction. Applied Soil Ecology 24, 133141.
  • Smith S.E. & Read D.J. (1997) Mycorrhizal Symbiosis, 2nd edn. Academic Press, San Diego, USA.
  • Subramanian K.S. & Charest C. (1999) Acquisition of N by external hyphae of an arbuscular mycorrhizal fungus and its impact on physiological responses in maize under drought-stressed and well-watered conditions. Mycorrhiza 9, 6975.
  • Tinker P.B. & Nye P.H. (2000) Solute Movement in the Rhizosphere. Oxford University, New York, USA.
  • Tobar R., Azcon R. & Barea J.M. (1994a) Improved nitrogen uptake and transport from 15N-labelled nitrate by external hyphae of arbuscular mycorrhiza under water-stressed conditions. New Phytologist 126, 119122.
  • Tobar R., Azcon R. & Barea J.M. (1994b) The improvement of plant N acquisition from an ammonium-treated, drought-stressed soil by the fungal symbiont in arbuscular mycorrhizae. Mycorrhiza 4, 105108.
  • Yano K., Yamauchi A. & Kono Y. (1996) Localized alteration in lateral root development in roots colonized by an arbuscular mycorrhizal fungus. Mycorrhiza 6, 409415.