- Top of page
- Materials and methods
The external mycorrhizal mycelium plays a crucial role in nutrient uptake of plants (Marschner & Dell, 1994; Smith & Read, 1997). This has predominantly been shown for arbuscular mycorrhizas (e.g. George et al., 1992; Johansen et al., 1993). For ectomycorrhizas, although a number of tracer studies have demonstrated the potential of ectomycorrhizal hyphae to increase N or P nutrition of their hosts (e.g. Melin & Nilsson, 1950, 1952; Finlay & Read, 1986; Kammerbauer et al., 1989; Ek et al., 1994), direct evidence supporting a significant role of the external mycelium in mineral nutrition of forest tree seedlings is still scarce. Recently, using a compartmental culture technique, in which hyphal nutrient uptake could be isolated from root uptake, we have demonstrated that the external mycelium of Paxillus involutus contributed significantly to N and P nutrition of N and P deficient Norway spruce seedlings (Brandes et al., 1998). However, as the seedlings were deficient in both N and P it was unclear from this experiment whether N or P limitation were the driving forces for mycelial foraging and translocation from patches of increased nutrient supply. Hyphal foraging for P, independently of N, may be important in P limited ecosystems to maximize P uptake from a heterogeneous soil environment.
Phosphate taken up by ectomycorrhizal fungi may readily be incorporated into polyphosphates located in fungal vacuoles (Ashford et al., 1994; Gerlitz & Gerlitz, 1997). As the pH may range between 4.3 and 7.5 in fungal vacuoles (Rost et al., 1995), polyphosphates are strongly negatively charged. To neutralize these charges polyphosphates must be associated with cations. Positively charged nitrogen compounds, such as basic amino acids (e.g. arginine), or metal cations, for example K+, Mg2+ or Ca2+ may play a role in neutralizing these negative charges. Recently, microanalytical studies have provided direct evidence for the association of vacuolar P with K and Mg (Orlovich & Ashford, 1993; Bücking & Heyser, 1999), and have shown that Ca plays no significant role in this association.
Different mechanisms of hyphal translocation have been postulated but the importance of these remains unclear (Finlay, 1992). Ashford (1998) has hypothesized that vacuoles may be involved in longitudinal translocation in fungi. At present, the available evidence supports a role of the pleiomorphic vacuolar system only within hyphal tips and their involvement in long-distance transport is less certain. Nevertheless, if indeed translocation occurs within motile vacuoles or ‘pumping’ of vacuolar contents through a tubular vacuole system, a strong coupling of K, Mg and possibly N compounds with P translocation may be expected. Thus, elements may even be translocated when not in short supply at the sink location (the host plant). Although translocation of mineral nutrients, such as N, P, Rb (for K), Ca (Finlay, 1992) or Mg (Jentschke et al., 2000), has been studied in ectomycorrhizal systems, only a few investigations have looked at simultaneous translocation of more than one nutrient.
The aim of this study was to demonstrate: that hyphae of ectomycorrhizal plants deficient only in P forage for P and translocate it to the host, thereby increasing plant growth; and that translocation of elements not in short supply in the host plant, such as N, K or Mg, may be coupled to P translocation.
- Top of page
- Materials and methods
Addition of P available only to mycorrhizal hyphae strongly stimulated hyphal growth at the location of P application. The hyphae took up and translocated significant amounts of P to their P-deficient host plant and strongly improved host P status. Improved P nutrition, in turn, stimulated seedling growth. This series of events in our experiment provides direct evidence for the active role of the external ectomycorrhizal mycelium in foraging for P and stimulation of host P nutrition and growth. Our data confirm earlier results that showed simultaneous hyphal foraging for N and P ameliorated N and P deficiency in mycorrhizal Norway spruce seedlings (Brandes et al., 1998). As the seedlings in our experiment were solely deficient in P the results demonstrate that P deficiency alone may trigger hyphal foraging and translocation of P from P-rich sites.
Paxillus involutus hyphae, besides P, translocated significant amounts of N, K and Mg to the host plant. Based on both 15N labelling and mass balance data, hyphal NH4+ acquisition contributed 12% to total plant N uptake. Ek et al. (1994) found NO3− translocation in P. involutus hyphae connected to Picea abies and Betula pendula in presence of NH4+ was approx. 50% of NH4+ translocation (measured at pH 4). Our estimates of NO3− translocation based on N mass balance confirm these figures. Thus, total hyphal N translocation including NO3− amounted to 17% of total plant N uptake and, in absolute numbers, exceeded P translocation five-fold (Table 7). Potassium translocation was of a similar order of magnitude to P translocation, though estimates based on mass balance were somewhat less precise, as K uptake was relatively small compared to K fluxes through the system. Yet, hyphal K acquisition was significant and contributed a minimum of 6% to total plant uptake. These data confirm the potential role of the ectomycorrhizal mycelium in K acquisition, which has so far only been demonstrated using radioisotopes of Rb as analogue for K (Finlay, 1992). Potassium fluxes in our experiment were in the same order of magnitude as P fluxes, confirming the earlier results of Finlay (1992). Magnesium translocation by the external mycelium, estimated by stable isotope labelling, was approx. 3–4% of total host Mg uptake as published in detail elsewhere (Jentschke et al., 2000). In absolute terms, Mg fluxes through the hyphal network were lowest of all element fluxes determined, one or two orders of magnitude lower than P or N fluxes, respectively (Table 7).
The nutrient solution added to the root compartment was designed to limit plant growth by P. The molar ratio of N : P, K : P, Mg : P and other element : P ratios by far exceeded critical values for spruce seedlings determined by Ingestad (1979). Phosphorus limitation was directly evident from the strong growth response of nonmycorrhizal plants receiving extra P (mass-flow-control plants). Even in these plants, supply of nutrients other than P was still super-optimal as relevant element : P ratios, measured in both nutrient solutions and plant tissues, exceeded critical values (Ingestad, 1979). Mycorrhizal plants which had access to a similar amount of P as the fertilized nonmycorrhizal seedlings were similar in size, but had slighty lower N : P ratios (but still in the optimal range) than seedlings of the mass flow control. In addition, N supply to the roots exceeded N uptake by a factor of 3, suggesting that N was not limiting growth. Similar estimations for K and Mg indicate that these nutrients were also not in short supply. Thus, here we demonstrate, for the first time to our knowledge, a cotransport of limiting (P) and nonlimiting nutrients (N, K, Mg) in the ectomycorrhizal mycelium.
Different mechanisms of hyphal translocation have been postulated but the relative importance of these under different conditions and for different elements remains unclear (Finlay, 1992). However, there is increasing evidence that P translocation in ectomycorrhizal hyphal systems occurs by active translocation rather than by transpiration-driven mass flow of water or diffusion (Timonen et al., 1996; Ashford, 1998). If translocation is indeed an active process, it may then be expected for reasons of energy efficiency that it is controlled by external factors such as the host nutritional status and demand. This may be achieved either indirectly by varying growth of the external mycorrhizal mycelium (Wallander & Nylund, 1992; Arnebrant, 1994), or directly by selectively down-regulating translocation processes for nutrients not in short supply. The translocation of significant amounts of nutrients not limiting host growth (N and K) suggests that these nutrient fluxes were not strongly down-regulated. In a similar experiment (Brandes et al., 1998), in which the same symbionts but a different nutrient regime were used, we found that the molar ratio of N to P transported through the mycorrhizal mycelium was 12. As the seedlings were deficient in both N and P, the higher relative N transfer (N : P ratio 12 vs 5 as found in this experiment) suggests that translocation may indeed be affected by host demand, albeit moderately. Acquisition of N not needed at the time of uptake, however, may be stored in the host plant and may provide an additional N source in times with insufficient N supply. Thus, in terms of long-term plant success, the simultaneous delivery of N, P and other nutrients by the external mycorrhizal mycelium may be an advantegeous strategy.
When no P was supplied to the hyphal compartment and, thus, no P was translocated to the host plant, the transfer of N, K, and Mg decreased or ceased. As both the growth rate of seedlings, which determines nutrient demand, and the hyphal density in the hyphal compartment differed between +P and −P mycorrhizal seedlings, care must be taken in interpreting these results. However, while seedling N uptake during the experimental period and hyphal density were only reduced to 74% (Table 4, final minus initial seedling N content) or 33% (Fig. 1) of the values in the +P seedlings, respectively, nitrogen transfer was reduced to 10% of the rate with simultaneous P transfer (i.e. much more than expected from the combination of the reductions (0.74 × 0.33 = 24%)). Potassium translocation may additionally have been influenced by differences in K supply to the hyphal compartment between −P and +P seedlings; however, these differences (approx. 30%) were probably too small to explain the strong reduction in K translocation in −P mycorrhizal seedlings. Mg translocation completely ceased when no P was translocated. Although our experiment does not provide conclusive evidence, it is likely that these changes were a result of a strong coupling of N and especially Mg and K fluxes to P translocation. Finlay (1992) suggested that N transported as positively charged amino acid, such as arginine, may play a role in neutralizing negative charges of simultaneously translocated (poly)phosphates. In fact, microanalytical studies have provided evidence for association of N and P compounds in mycorrhizal fungal vacuoles (Turnau et al., 1993; Kottke et al., 1995). As N translocation may exceed P translocation severalfold (Table 7), it is, however, clear that only a small portion of N as arginine would be needed to neutralize negative charges of P compounds. In P. involutus, however, arginine as a transport form of N may play no role at all (Finlay, 1992; Ek et al., 1994). Thus, in this fungus, negative charges from translocated P compounds must be neutralized by other means. Our data support the idea that K and, to a minor extent, Mg may act as neutalizing agents. Ashford (1998) has hypothesized that longitudinal P transport in fungi may be carried out through the tubular vacuole system, which is also present in P. involutus (Rees et al., 1994). As X-ray microanalysis has repeatedly shown that P in fungal vacuoles is mainly associated with K and Mg (Orlovich & Ashford, 1993; Bücking & Heyser, 1999), translocation of vacuolar contents through the tubular system would most likely require K and Mg fluxes to be strongly coupled with P translocation.
This work has confirmed the active role of ectomycorrhizal fungal hyphae in acquisition of P in P deficient Norway spruce seedlings. Although not in short supply, the hyphae also translocated N, K and Mg to the host plant. The translocation of nonlimiting nutrients depended upon the simultaneous translocation of P. This supports the idea that nutrient fluxes within fungal hyphae are interdependent and that for reasons of charge neutralization cation fluxes may be strongly coupled to P translocation.