Biological nitrogen fixation
We used two strains of Frankia, UGL020604 and UGL020605, which are known to be highly effective on C. cunninghamiana (Wheeler et al., 2000). In our study, N2-fixation by C. cunninghamiana (27–29%) was generally comparable with data from other studies with Casuarina species including C. cunninghamiana (Mariotti et al., 1992; Parrota et al., 1994, 1996; Subbarao & Rodriguez-Barrueco, 1995). We used 15N natural abundance to calculate both percent of total plant N supplied by N2-fixation and the amount of N in the nodules expressed as g N mg−1 dry weight nodule. It is difficult to compare our values with Reddell & Bowen (1985) and Reddell et al. (1988, 1997). Reddell and coworkers calculated the amount of N in the nodule that was derived from fixation as mg N fixed mg−1 nodule dry weight; they did not calculate total nodule N, which includes N from other sources such as soils. In our study, plants received only 5 ml per pot of 4 mmol N as either (14NH4)2SO4 or K14NO3 once a week as a fertilizer. This low N load did not reduce fixation. Other studies reported that low N fertilization even stimulated N2-fixation (Huss-Danell, 1997; Wheeler et al., 2000). There was no effect of N source on N2-fixation in our study, but Zhang and Torrey (1985) found that NH4+ inhibited nodulation of C. cunninghamiana more than NO3−. Other studies with Casuarina species did report reduction of fixation by either NH4+ or NO3− (Kohls & Baker, 1989; Arnone et al., 1994; Baker et al., 1994).
Mycorrhizal colonization in Casuarina (c. 35%) and in Eucalyptus (c. 65%) was generally higher than previously reported by other researchers. With a variety of EM fungi, Casuarina had 10–40% EM (Gardner, 1986; Theodorou & Reddell, 1991; Subbarao & Rodriguez-Barrueco, 1995); and Eucalyptus formed 10–60% EM colonization (Chilvers et al., 1987; Bellei et al., 1992; Burgess et al., 1993, 1994; May & Simpson, 1997; Reddell et al., 1999; Chen et al., 2000). Our seedlings were both EM and nodulated. Gardner (1986) and Subbarao & Rodriguez-Barrueco (1995) also worked with EM and nodulated Casuarinas species. We did not observe any increase in mycorrhization with nodulation, nor did Gardner (1986). Well-controlled aseptic and suitable glasshouse environments may have favored higher mycorrhizal colonization rates.
Common mycorrhizal networks between plants
In our experiment, fungal mycelia grew through nylon mesh and linked the roots of Casuarina and Eucalyptus, establishing a common EM network (He, 2002). We demonstrated that the CMN functioned in two-way N transfer. Mycorrhizal links between plants have been observed in transparent microcosms (Francis & Read, 1984; Newman, 1988; Newman et al., 1994) and by carbon and phosphorus isotope autoradiography (Chiariello et al., 1982; Francis & Read, 1984; McKendrick et al., 2000). Nitrogen movement through mycorrhizal linkage or CMNs from plant to plant has been reported (Haystead et al., 1988; Eissenstat, 1990; Bethlenfalvay et al., 1991; Frey & Schuepp, 1992, 1993; Arnebrant et al., 1993; Johansen et al., 1993a,b; Ikram et al., 1994; Tobar et al., 1994a,b; Ekblad & Huss-Danell, 1995; Ek et al., 1996; Johansen & Jensen, 1996). Read (1997) discussed the ecological importance of nutrient transfer through a CMN and speculated that nutrient transfer affects plant growth and biogeochemical cycling. Two-way nutrient transfer could reduce competition for soil resources between plants.
Responses of plant growth to mycorrhizal colonization and nodulation
Both biomass and N content in Casuarina and Eucalyptus were highest when Casuarina was Nod+Myc+, lower in Nod−Myc+ plants, and lowest in Nod−Myc− plants. Although mycorrhizal infection was double in Eucalyptus compared with Casuarina, this had a slight effect on N accumulation by Eucalyptus. Mycorrhization did, however, enhance N accumulation by Nod−Myc+Casuarina. Compared with Nod−Myc+Casuarina, N content doubled in Nod+Myc+Casuarina, especially when Casuarina was the receiver. As the receiver, Casuarina experienced a 1-month-period with no N fertilizer, perhaps creating nutrient stress. The magnitude of increase in N content in mycorrhizal Eucalyptus was much less when they were partnered with Nod+Myc+Casuarina, rather than with Nod−Myc+Casuarina. Thus, the combination of Frankia and a mycorrhizal fungus had the greatest effect on growth of Casuarina. Both Casuarina and Eucalyptus receivers obtained substantial benefit via CMNs from their donors, when Casuarina was nodulated and mycorrhizal. Biomass production by Casuarina and Eucalyptus was similar whether either species acted as donor or receiver. This suggested a physiological benefit to receivers from N transfer. In our study, Nod−Myc+ seedlings were generally taller, had larger root systems, greater shoot and/or root biomass and higher N content than Nod−Myc− seedlings. Similar results were observed for Nod−Myc+ and Nod+Myc+ seedlings. This has been reported by others (Smith & Read, 1997; Jonsson et al., 2001; Rogers et al., 2001).
Below-ground N-transfer between plants
Our results demonstrated that ectomycorrhizas mediated bidirectional N transfer between Casuarina and Eucalyptus. In the Nod−Myc− treatment, there was almost no N transfer (< 1% in both N transfer and NDFT) to Casuarina or to Eucalyptus, and very low N transfer (c. 2% in both N transfer and NDFT) to Casuarina or to Eucalyptus in the Nod−Myc+ treatment. By contrast, N transfers were greatest in the Nod+Myc+ treatment. More N moved to Nod+Myc+Casuarina than to Myc+Eucalyptus, for both 15NH4+ and 15NO3−. About twice as much N was transferred from Eucalyptus to Nod+Myc+Casuarinas than the reverse, resulting in increased growth by Casuarina due to interspecific N transfer. Since there was virtually no N transfer in the nonmycorrhizal treatment, but significant N transfer in the mycorrhizal treatment, N transfer was mediated by mycorrhizal fungi. The much higher N transfer between nodulated mycorrhizal plants indicated that mycorrhizas and Frankia together enhanced N fluxes between N2-fixing Casuarina and nonN2-fixing mycorrhizal Eucalyptus. The amount of N transfer was significantly greater from Eucalyptus to Nod+Myc+Casuarina than the reverse, contradicting the general hypothesis that N flows from N2-fixing to nonN2-fixing plants (Bethlenfalvay et al., 1991; Frey & Schuepp, 1993). However,% NDFT was similar for Casuarina and Eucalyptus even in Nod+Myc+ treatment, regardless of the direction of N transfer.
Nitrogen transfer from legume to non-legume can take place by indirect or direct routes (McNeill & Wood, 1990). Indirect N transfer via soil solution comes from dead and decayed nodules and roots (Dubach & Russelle, 1994), exudates such as ammonium, amino acids and amides from legume roots (Ta et al., 1989; Paynel et al., 2001), and sloughed-off root-cortical cells (Brophy & Heichel, 1989). These materials are deposited in soil and could be absorbed by adjacent plants. This is the soil pathway (Stern, 1993; Chalk, 1996). Direct N transfer may be mediated by mycorrhizal hyphae where CMNs connect plants. One-way N transfer via CMNs has been demonstrated using fine nylon mesh barriers that allow passage of hyphae but not roots (Bethlenfalvay et al., 1991; Frey & Schuepp, 1992; Arnebrant et al., 1993; Johansen & Jensen, 1996). The one-way N transfer hypothesis states that N flows from an N-rich legume source (N donor) to an N-poor nonlegume receiver (sink) (Bethlenfalvay et al., 1991; Frey & Schuepp, 1993). AM-mediated (15NH4+ or 15NO3−) or EM-mediated (15NH4+ only) one-way N transfer from N2-fixing to nonN2-fixing plants has been reported (Bethlenfalvay et al., 1991; Frey & Schuepp, 1992; Arnebrant et al., 1993; Ekblad & Huss-Danell, 1995; Johansen & Jensen, 1996; Martensson et al., 1998). None of these studies measured bidirectional transfer.
We calculated net N transfer as the difference between N transferred from Casuarina to Eucalyptus and from Eucalyptus to Casuarina. Net N transfer was low in both the Nod−Myc− and the Nod−Myc+ treatments (Table 5). Surprisingly, in the Nod+Myc+ treatment, there was greater net N transfer from nonN2-fixing Eucalyptus to N2-fixing Casuarina (Table 5). A similar phenomenon was reported for grass to legume pairs, resulting in a two-way N-flow between N2-fixing and nonN2-fixing plants (Brophy et al., 1987; Tomm et al., 1994; Hogh-Jensen & Schjoerring, 2000). Transfer was not significant under N-limited conditions and effects of mycorrhization were not examined in those studies. N transfer from barley to pea seedlings connected by Glomus intraradices was very low (Johansen & Jensen, 1996). No N transfer was found between clover and ryegrass through AM hyphal links (Rogers et al., 2001).
Inorganic N in soil is available to plants as NH4+ or NO3− (Forde & Clarkson, 1999). Most EM fungi can assimilate NH4+ (Jongbloed et al., 1991; Martin & Botton, 1993; Martin & Plassard, 2001), but few can efficiently reduce NO3− (Scheromm et al., 1990; Martin & Botton, 1993; Martin & Plassard, 2001). Apparently most AM fungi can assimilate both NH4+ (Smith et al., 1985; Cliquet & Stewart, 1993; Johansen et al., 1996) and NO3− (Ho & Trappe, 1975; Johansen et al., 1996; Faure et al., 1998). The less mobile NH4+ is adsorbed to soil colloids and moves toward plant roots by diffusion. The highly mobile NO3− is transported toward plant roots by mass flow (Brady & Weil, 2002). Mycorrhizal hyphae may play a greater role in transport of NH4+ than of NO3− to plant roots, except in water-deficient soil (Tobar et al., 1994b; Wu et al., 1999).
Our results showed that mycorrhization increased biomass production and N accumulation and that plant biomass increased with N content for both species. Mycorrhizas enhanced N accumulation, biomass production and N transfer between plants, and may thus affect plant fitness. Mycorrhizas had less impact on N accumulation in nonN2-fixing Eucalyptus than in N2-fixing Casuarina, despite the fact that Eucalyptus had nearly double the mycorrhizal infection compared with Casuarina. When Casuarina was nodulated and mycorrhizal and when Eucalyptus was mycorrhizal, biomass yield and N content were maximized in both plants. Our data corroborate previous reports that the main effect of mycorrhization on plant N status was in N2-fixing plants (Hayman, 1986). All these data suggest that N transfer has biological significance. Mycorrhization, together with N availability, plays a vital role in re-distribution of N between plants. Considering that N supply is crucial for plant growth in most terrestrial ecosystems (Vitousek et al., 1997; Moffat, 1998), and that the potential benefits of N transfer are great (CSIRO Publishing, 1998), two-way N transfer may be very significant.