Effects of mycorrhizal colonization and nodulation on plant growth
Biomass of both plant species in Casuarina/Eucalyptus pairs was greater when plants were mycorrhizal, and then greatest when Casuarina was both mycorrhizal and nodulated. Compared with the Nod−Myc−Casuarina, N content was twice as high in the Nod−Myc+ and four times as high in the Nod+Myc+Casuarina. Compared with the Myc−Eucalyptus, N content of Eucalyptus increased 1.5 times in the Nod−Myc+/Myc+ and twice in the Nod+Myc+/Myc+ treatment. Our data agree with previous reports that the main effect of mycorrhization on plant N status was in N2-fixing plants (Hayman, 1986; Vassilev et al., 2001). Common host genes could potentially enhance the formation of mycorrhizal associations, and are required for both nodulation and mycorrhizal formation in N2-fixing plants (Albrecht et al., 1999; Anéet al., 2004) for their high N-demanding requirements (McKey, 1994; Sprent, 1994; Sprent, 2001). About twice as much N was transferred from Myc+Eucalyptus to Nod+Myc+Casuarinas than the reverse, resulting in increased growth by Casuarina caused by interspecific N transfer. Our data support the hypothesis that N2-fixing plants may have evolved in an N-rich environment (McKey, 1994; Stewart & Schmidt, 1999), and are consistent with the observation that a non-N2-fixing plant (Baccharis pilularis) facilitates the establishment of an N2-fixing plant (Lupinus arboreus) (Rudgers & Maron, 2003). Furthermore, regardless of whether Casuarina or Eucalyptus was the donor or the receiver, and whether 15NH4+ or 15NO3− was the 15N source, seedlings in the mycorrhizal treatments were taller, and had larger root systems than those in the nonmycorrhizal treatment (He, 2002). Similar results were obtained by others (Smith & Read, 1997; Jonsson et al., 2001). For both donors and receivers, both biomass and N content in Casuarina and Eucalyptus were similar, suggesting N transfer through CMNs from donors to receivers. This indicates that CMNs can provide direct conduits for nutrient exchange between plants, and that CMNs are effective in supplying growth-supporting amounts of nutrients to a receiver plant. The increased growth of recipients suggests that they benefited physiologically from mycorrhizal-mediated nutrient transfer between plants.
Two-way nitrogen transfer between plants and its potential ecological significance
Roots had greater 15N at % excess and 15N content than shoots for both plant species, whether Casuarina or Eucalyptus was the donor or the receiver, and whether 15NH4+ or 15NO3− was the 15N source. Both 15N at % excess and 15N content were greater in the 12-month-old seedlings than in the 6-month-old ones (He et al., 2004). Our results demonstrated two-way N transfers, generated from the 15N content of both donor and receiver plants, between 12-month-old paired Casuarina and Eucalyptus seedlings supplied with either 15NH4+14NO3− or 14NH4+15NO3−. In the nonnodulated nonmycorrhizal (Nod−Myc−/Myc−) treatment, amounts of N transfer were less than 2% of the total plant N. This low N transfer may have occurred from diffusion along the interface between mesh and perforated Perspex barriers, or perhaps from leaf absorption of volatilized N-containing compounds (Frank et al., 2004). In the nonnodulated mycorrhizal (Nod−Myc+/Myc+) treatment, amounts of N transfer were 16% of total plant N. This N transfer was enhanced by mycorrhizal interconnections. In the nodulated mycorrhizal (Nod+Myc+/Myc+) treatment, much greater amounts of N were transferred, up to 30% of the total plant N. Thus mycorrhizas and Frankia together had the greatest positive effect on N flux between nodulated mycorrhizal Casuarina and mycorrhizal Eucalyptus. Furthermore, significantly greater amounts of N were transferred from non-N2-fixing mycorrhizal (Nod−Myc+) Eucalyptus to nodulated mycorrhizal (Nod+Myc+) Casuarina than the reverse. These data were consistent with our previous study with 6-month-old Casuarina/Eucalyptus pairs. Thus our two-way N transfer results only partially support the hypothesis of Bethlenfalvay et al. (1991) and Frey & Schuepp (1993) that one-way N flows from legume donors to nonlegume receivers.
Nitrogen transfer between legumes and nonlegumes can take place by indirect or direct routes (McNeill & Wood, 1990). Indirect N transfer occurs through recapture of N-containing materials from rhizodeposition (Stern, 1993; Dubach & Russelle, 1994; Chalk, 1996; Paynel et al., 2001; Walker et al., 2003), although rhizodeposition is regulated by interactions between plants and mycorrhizas (Jones et al., 2004). Direct N transfer occurs when nutrients are obtained through mycorrhizal hyphae where CMNs connect plants (Newman, 1988; Frey & Schuepp, 1993; He et al., 2003). To eliminate transfer through indirect pathways, we used fine mesh screen and air gaps to exclude root contact and water movement, and to allow passage of hyphae. Similar techniques have been used previously in other studies to demonstrate one-way N transfer via CMNs (Bethlenfalvay et al., 1991; Frey & Schuepp, 1992, 1993; Arnebrant et al., 1993; Johansen & Jensen, 1996).
We investigated two-way or net N transfer as the difference from Casuarina to Eucalyptus and from Eucalyptus to Casuarina. Net N transfer was greater in mycorrhizal treatments when supplied with 15NH4+ than with 15NO3−. Independent of N source, net transfer was very low (<1 mg per plant) in the nonnodulated nonmycorrhizal treatment. Mycorrhizas doubled this amount (≈2 mg per plant) in the nonnodulated mycorrhizal treatment, but the greatest increase (127–185 mg per plant) was in the combination of mycorrhizas and nodulation treatment (Fig. 4c,d). Amounts of net N transfer from 12-month-old pairs supplied with 15NH414NO3 or 14NH415NO3 was greater than that from 6-month-old pairs supplied with (15NH4)2SO4 or K15NO3 (He et al., 2004). In the present study, for both the Nod−Myc−/Myc− and the Nod−Myc+/Myc+ treatment, 12-month-old Casuarina was the N sink plant when 15NH4+ was supplied, while 12-month-old Eucalyptus was the N sink plant when 15NO3− was supplied. However, for both Nod−Myc−/Myc− and Nod−Myc+/Myc+ treatments in our previous study, either the 6-month-old Casuarina or Eucalyptus was the N-sink plant, independent of 15NH4+ or 15NO3− (He et al., 2004). Both studies demonstrated a much greater net flow of N from mycorrhizal Eucalyptus to nodulated mycorrhizal Casuarina, suggesting that Casuarina was the stronger N sink in the Nod+Myc+/Myc+ treatment. Our data are consistent with the hypothesis proposed by McKey (1994) that N fixation by legumes may have evolved to help fuel an N-rich lifestyle. We found only one other study that reported <1% N transfer from non-N2-fixing barley to N2-fixing pea connected by an AM fungus, G. intraradicesJohansen & Jensen (1996). Other two-way N-transfer studies did not report effects of mycorrhizas, and N transfer was insignificant from grass to legume (Brophy et al., 1987; Tomm et al., 1994; Hogh-Jensen & Schjoerring, 2000) or from rice to peanut (Shen & Chu, 2004).
We assume that the following scenario may exist in nature for plants that have different N requirements (McKey, 1994; Sprent, 1994; Sprent, 2001). Differences in net N transfer with different nodulation/mycorrhizal combinations could have important ecological implications both for nutrient cycling and for the structure and function of agricultural or natural plant communities, particularly with respect to those plants that can potentially construct a ‘wood-wide web’ or common mycorrhizal network to transfer nutrients underground. Our data support the hypothesis that mycorrhizal colonization can result in resource equalization or sharing (Read, 1997; Perry, 1998), thus reducing dominance of aggressive species and promoting coexistence and biodiversity (Read, 1997; van der Heijden et al., 1998). Benefits of N nutrition to non-N2-fixing plants by neighbouring N2-fixing plants have been reported in both agriculture (Stern, 1993; Chalk, 1996; Anon., 1998) and forests (Binkley, 1992; O’Connell & Grove, 1996; Binkley et al., 2003). However, non-N2-fixing plants may also benefit N2-fixing plants by increasing germination, survival and growth; such facilitation could benefit other members in a plant community (Rudgers & Maron, 2003). From an evolutionary perspective, plants may evolve with increased competitive ability for resource requirements, and may respond differently to a more nutrient-rich condition. For instance, an N2-fixing plant may not exploit its capacity to fix atmospheric N2, but may use soil available N (McKey, 1994; Marschner, 1995), especially early in establishment, and thus become relatively N-poor (receivers). In the meantime, plants adapted to extremely nutrient-low environments, especially those with an evolving capacity to live in nutrient-impoverished forests and woodlands (Attiwill & Leeper, 1987; Stewart & Schmidt, 1999), such as many native Australian plants, may become relatively N-rich (donors). If such provisional N-rich and N-poor plants become neighbours, they may alter their donor and receiver behaviour and N-transfer may switch from the low-N-demand non-N2-fixing plant to the high-N-demand N2-fixing plant in order to satisfy the latter's higher N requirements.
Nitrogen transfer between plants: ammonium vs nitrate
Patterns of 15N distribution and N transfer between plants in this study were similar to patterns from our previous study using single (15NH4)2SO4 or K15NO3 (He et al., 2004). As NH4+ and NO3− have different chemical mobility in soil, and their uptake by plants and fungi may be different, it is important to compare effects of NH4+ and NO3− on mycorrhizal mediation of N transfer between plants. In this study we were able to compare differences in N transfer by fertilizing plants with the same N-containing fertilizer (14NH414NO3) but monitoring 15N movement between plants by labelling distinctive 15N labels (15NH4+14NO3− and 14NH4+15NO3−).
In this study our data showed that more N was transferred when plants were supplied with the cation 15NH4+ than with the anion 15NO3−, although NO3− is more mobile in plants and in soil (Fig. 4a,b). This effect of inorganic form of 15N source on differences in amount of N transfer supplied with 15NH4+ vs 15NO3− was modest (−2.5 less to Eucalyptus vs −1.0 mg per plant less to Casuarina) in the nonnodulated nonmycorrhizal/nonmycorrhizal (Nod−Myc−/Myc−) treatment; greater (+23 more to Eucalyptus vs +27 mg per plant more to Casuarina) in the nonnodulated mycorrhizal/mycorrhizal (Nod−Myc+/Myc+) treatment; and substantially greater (+118 more to Eucalyptus vs +175 mg per plant more to Casuarina) in the nodulated mycorrhizal/mycorrhizal (Nod+Myc+/Myc+) treatment.
Was the applied 15NH4+ transformed into 15NO3−? We were concerned that the concentration of NO3− might increase because of rapid nitrification of NH4+ by microbes (Paul & Clark, 1996; Brady & Weil, 2002). To prevent nitrification in the soil, a nitrification inhibitor, N-serve, was added (5 mg kg−1 media). N-serve retards the first step in nitrification of NH4+ to hydroxylamine by Nitrosomonas sp. (Slangen & Kerkhoff, 1984). We did not observe any toxic effects of N-serve on plant growth or mycorrhizal development in this study, or in our earlier study (He et al., 2004). Our observations agree with other studies. Chambers et al. (1980) showed that there was little or no effect of N-serve on mycorrhizal development under high concentration of 15 mg kg−1 soil. In addition, N-serve had little effect on other soil microorganisms when 10 mg kg−1 soil was used (Laskowski et al., 1975). We were also concerned that the concentration of NH4+ and NO3− might be changed because of leaking out of the experimental system. Our experimental pots were closed, so there was no NH4+ or NO3− leaching. Thus 15NH4+ or 15NO3− concentrations should be maintained at the same level, whether 15NH4+ or 15NO3− was the 15N source.
Why was more N transferred when 15NH4+ was supplied, although NO3− is more mobile? We suggest that the following three factors could be responsible. First, the cation NH4+ is bound to negatively charged sites of clay lattices in soil, reducing mobility and leading to reduced availability, so that diffusion to the root surface becomes the rate-limiting step (Marschner, 1995; Tinker & Nye, 2000; Brady & Weil, 2002). Therefore plants may require more extensive root systems to access NH4+ (Raven et al., 1992; Engels et al., 2000), such as mycorrhizal roots (Marschner & Dell, 1994; Smith & Read, 1997) or cluster roots in both Casuarina (Reddell et, 1986; Diem et al., 2000; Neumann & Martinoia, 2002) and Eucalyptus species (Grierson & Adams, 2000; Neumann & Martinoia, 2002). Both mycorrhizas and cluster roots can enhance nutrient acquisition by increasing root surface area and by physiological adaptations (Marschner, 1995; Diem et al., 2000; Neumann & Martinoia, 2002). However, cluster roots, which are adapted to habitats of extremely low soil fertility as an effective nutrient-uptake mechanism, were not observed in either Casuarina or Eucalyptus in our experiments, as suitable N-nutrition and glasshouse growth conditions might not favour their formation. In contrast, the anion NO3−, which is not bound to negatively charged soil particles, is more mobile in the soil, and mass flow to the root surface becomes the rate-limiting step (Marschner, 1995; Tinker & Nye, 2000; Brady & Weil, 2002). Theoretically, NO3− in soils could be extracted by a nonmycorrhizal root system assuming high fine-root density. Thus mycorrhizas would add little advantage to NO3− uptake (Tinker & Nye, 2000). However, NH4+ is often the dominant inorganic N form in Australian forest soils (Adams & Attiwill, 1986; Attiwill & Leeper, 1987; Connell et al., 1995). Both Eucalyptus (Shedley et al., 1995; Barros & Novais, 1996) and Casuarina (Torrey, 1983; Subbarao & Rodriguez-Barrueco, 1995) have mycorrhizal association and inherently prefer NH4+ rather than NO3−. Therefore it is possible that N uptake and translocation could be promoted by mycorrhizal colonization in soils where NH4+ is the main N form. Our data from both mycorrhizal and the nodulated mycorrhizal treatments support this hypothesis, because a greater amount of N was transferred to Casuarina or to Eucalyptus when 15NH4+, rather than 15NO3−, was the 15N source.
Second, increased ammonium transfer may be related to differences in uptake and assimilation of 15NH4+ and 15NO3− by mycorrhizal fungi. Mycorrhizas play important roles in N nutrition of trees by increased uptake at the root surface and by extensive mycelial growth and soil exploration (Smith & Read, 1997). Most EM fungi can assimilate NH4+ (Jongbloed et al., 1991; Martin & Plassard, 2001), but only some can reduce NO3− (Sarjala, 1990; Scheromm et al., 1990; Martin & Plassard, 2001). EM fungal mycelia are abundant in humus and litter layers in soils where NH4+ concentrations are greater than NO3− (Alexander, 1983, 1989; Read, 1992). Thus hyphae might be effective absorbing structures for NH4+ in ectomycorrhizal roots. EM associations may enhance NH4+ uptake (Bledsoe & Zasoski, 1983; Littke et al., 1984; Rygiewicz et al., 1984; Javelle et al., 1999; Plassard et al., 2000). Disruption of external mycelia can decrease NH4+ uptake (Javelle et al., 1999). However, the influence of EM associations on NO3− uptake is controversial. NO3− uptake can be either increased (Bledsoe & Zasoski, 1983; Gobert & Plassard, 2002; Plassard et al., 2002) or reduced (Kreuzwieser et al., 2000; Plassard et al., 2002). In general, NH4+ uptake was greater than NO3− uptake by ectomycorrhizas (Bowen & Smith, 1981; Littke et al., 1984; Kreuzwieser et al., 2000). Uptake rates for NH4+ were greater than for NO3− for the EM fungus Hebeloma crustuliniforme (Littke et al., 1984). Lower NO3− uptake rates by mycorrhizal seedlings indicate that mycorrhizas may play a less important role in accessing more mobile NO3−. However, nothing is currently known about its N-uptake ability for the Pisolithus species used in our experiment. We only knew that all the 37 isolates from three unidentified Australian Pisolithus species utilized a range of amino acids, and most of them showed a preference for NH4+ rather than NO3− (Anderson et al., 1999).
In our study, high water-holding-capacity crystals were added to the potting mix in order to minimize diffusion and mass flow. In this way, roots could obtain water while the growth media remained relatively dry. Nutrient uptake is inhibited in dryer soils as decreased soil water leads to a decrease in rates of mass flow and diffusion, and consequently to reduced rates of nutrient transport to root surfaces (Marschner, 1995; Tinker & Nye, 2000; Brady & Weil, 2002). External mycelia might be of great importance under drier conditions because they occupy a larger soil volume for greater access to nutrients (Davies et al., 1992; Smith & Read, 1997). Mycorrhizal hyphae may thus play a greater role in transport of less mobile NH4+ than in transport of more mobile NO3− to plant roots, except when the mobility of NO3− from soil to root surface is reduced in water-deficient soil (Tobar et al., 1994; Wu et al., 1999). Nevertheless, information about the water-use capacity of the mycorrhizal fungus Pisolithus sp. is needed.
The third factor may be associated with differences in energy required to metabolize NH4+ and NO3−. Uptake and assimilation of inorganic NH4+ or NO3− by mycorrhizal fungi or by plants is a prerequisite to transfer of assimilated N between plants. For conversion of inorganic to organic N, NH4+ is directly assimilated by plants, whereas NO3− must first be reduced to NO2− and then to NH4+ in roots and/or shoots before it can be assimilated. Reduction of NO3− requires energy, C and protons (Marschner, 1995; Forde & Clarkson, 1999). Similar assimilatory mechanisms occur in mycorrhizal fungal mycelia (Smith & Read, 1997; Hawkins et al., 2000; Martin & Plassard, 2001). As a consequence, NH4+ fluxes within fungal and plant tissues, or between plants through hyphal connections, might require less energy and be cost effective, leading to more rapid fluxes or translocations of NH4+ than NO3−. We also found one report that NO3− could be toxic to Eucalyptus (Shedley et al., 1995), and two other reports that NO3− inhibited root-hair development in Casuarina (Kohls & Baker, 1989; Arnone et al., 1994). The presence of NH4+ often depresses NO3− uptake when both are supplied together, so many plants appear to take up NH4+ preferentially (Marschner et al., 1991; Paul & Clark, 1996; Kreuzwieser et al., 1997; Nordin et al., 2001). AM-mediated one-way N transfer (15NH4+ alone or 15NO3− alone) or EM-mediated one-way N transfer (15NH4+ alone) from N2-fixing to non-N2-fixing plants has been reported (Bethlenfalvay et al., 1991; Frey & Schuepp, 1992, 1993; Arnebrant et al., 1993; Ekblad & Huss-Danell, 1995; Johansen & Jensen, 1996; Martensson et al., 1998). No previous studies compared N transfer when plants were supplied with ammonium nitrate labelled as 15NH414NO3 and as 14NH415NO3.
In summary, biomass and N content were increased by mycorrhization. Mycorrhizal networks have the capacity to mediate significant N transfer among interconnected plants. Net N transfer occurred between mycorrhizal Casuarina/Eucalyptus pairs. Our results showed that, compared with the nonnodulated nonmycorrhizal pairs, amounts of N transfer were greater in the nonnodulated mycorrhizal pairs, and substantially greater in the dual-nodulated mycorrhizal/mycorrhizal pairs, whether 15NH4+ or 15NO3− was applied. Nitrogen transfer was greater between mycorrhizal pairs when 15NH4+, rather than 15NO3−, was supplied. Unexpectedly, we measured much greater net N transfer to Casuarina than to Eucalyptus when Casuarina was both nodulated and mycorrhizal, and Eucalyptus was also mycorrhizal. Mycorrhizas alone had less impact on N accumulation in non-N2-fixing Eucalyptus than in N2-fixing Casuarina, despite the fact that Eucalyptus had about twice the mycorrhizal colonization of Casuarina. When Casuarina was both nodulated and mycorrhizal, and when its partner Eucalyptus also became mycorrhizal through hyphal interconnections, biomass and N content were maximized in both plant species; plant performance was similar between donors and receivers through mycorrhizal-mediated N transfer. Our data agree with previous reports that the main effect of mycorrhization on plant N status was in N2-fixing plants (Hayman, 1986; Vassilev et al., 2001). Our data also support the hypothesis that N2-fixing species are high N-demand plants, and N fixation may have evolved to help fuel an N-rich lifestyle (McKey, 1994; Sprent, 1994, 2001). This suggests that mycorrhization, together with N availability, plays a vital role in N redistribution between plants, resulting in resource sharing or facilitation. Thus N transfer could be important for plants that encounter either N excess or N limitation (Vitousek et al., 1997; Anon., 1998; Moffat, 1998). Nitrogen transfer might also reduce the success of invasive species and promote coexistence and biodiversity (Read, 1997; van der Heijden et al., 1998). This may have practical consequences for environmental restoration or phytoremediation (Franco et al., 1997; Pattinson et al., 2004; Pennisi, 2004). For these reasons, more research is warranted on two-way N transfers mediated by mycorrhizal networks.