Cucumber plants and tomato seedlings shared the same ERM, because roots of the tomato seedlings were colonized by R. irregularis when inoculum was added solely to the cucumber plants. In agreement with our hypothesis, seedlings were indeed suppressed by linking into a CMN with a large plant. The suppression was caused by P deficiency, and the CMN obviously amplifies competition for P between the large plant and the seedling. We also accepted the second hypothesis that the P in the connective ERM is only poorly accessible to seedlings and is preferentially transported to large plants, which represent the major C source. These whole-plant results accord with observations in mycorrhizal root cultures (Lekberg et al., 2010; Kiers et al., 2011) and offer a likely explanation of the causal relationships underlying similar CMN-induced seedling suppression in previous studies (e.g. Nakano-Hylander & Olsson, 2007; Janouskova et al., 2011). To our knowledge, the present work represents the first evidence in whole-plant CMNs that net transfer of P from an AMF mycelium to its connected hosts favors the larger and older plants over smaller and younger seedlings. This finding represents one answer to the list of key questions raised by van der Heijden & Horton (2009) to provide explanations for the observed variation in CMN effects on seedlings.
Mechanisms underlying CMN effects on seedling growth
Phosphorus was the major growth-limiting nutrient in both experiments, as P amendment increased seedling growth (Figs 3, 4). In Experiment 2, however, the growth of CMN plants given high P was limited by N deficiency, in accordance with the fact that N : P ratios < 14 (Fig. 4) are indicative of N limitation (Koerselman & Meuleman, 1996). In this case, the possibility cannot be excluded that the growth suppression of CMN seedlings was associated with ERM-mediated transfer of N from the side unit compartment to the cucumber compartment. However, it is more likely that transfer via mass flow played the major role, as concentrations were low not only in the soil of CMN seedlings, but also in the soil of nonmycorrhizal seedlings (Fig. 5).
When P is growth-limiting, mycorrhizas usually increase P uptake and growth. In this work, however, 32P in the mycelium was less available, not only to a nonhost plant such as the rmc mutant, but also to the CMN seedlings (Experiment 1; Fig. 3); in Experiment 2, P uptake was much higher in solitary than in CMN seedlings (Fig. 4). Assessment of growth of CMN seedlings against solitary mycorrhizal seedlings excludes the possibility that CMN-induced suppression was caused by a general negative growth response to mycorrhiza. The neutral growth response to mycorrhiza in the solitary seedlings in Experiment 2 (Fig. 4; disconnected, mycorrhizal versus nonmycorrhizal) confirmed that the suppression in the CMN plants was solely attributable to the CMN effect. In previous studies, solitary mycorrhizal control plants also grew much better than corresponding CMN plants (Ocampo, 1986; Moora & Zobel, 1998; Kytoviita et al., 2003; Pietikainen & Kytoviita, 2007). Using uncolonized plants as reference treatments, the CMN responses in the same four studies were calculated as being positive, negative, neutral and neutral, respectively (van der Heijden & Horton, 2009).
Our study demonstrates that the seedling-to-‘donor’ ratio for age or biomass has a great impact on CMN effects on seedling performance (Fig. 2). The importance of seedling age accords with the fact that CMN-induced growth suppression has mostly been reported for relatively young seedlings grown with larger plants (this study and e.g. Nakano-Hylander & Olsson, 2007; Pietikainen & Kytoviita, 2007). In contrast, in reports of CMN-induced benefits, seedlings have mainly been older and associated with a more complex mixture of more mature plant neighbors (Grime et al., 1987; Read & Birch, 1988; van der Heijden, 2004). The latter studies represent plant communities where some plants are grazed or becoming senescent. The importance of this age or biomass ratio for seedlings and ‘donors’ was demonstrated by Carey et al. (2004), who reported that Centauria maculosa seedlings grew better with a Festuca idahoensis companion plant, but not with Bouteloua gracilis, which had a plant dry weight more than threefold that of F. idahoensis. The inherent characteristics of a plant species, for example, growth rate, size and root:shoot ratio, are likely to influence its capacity to supply C to associated AMF. Such differences may also have influenced the relative growth of cucumber and tomato plants in this study. Still, suppression of seedlings has previously been demonstrated in other heterospecific combinations (see Moora & Zobel, 2010; Janos et al., 2013) and was similar in magnitude in conspecific and heterospecific combinations of two species (Nakano-Hylander & Olsson, 2007). Information on exchange of C and P at the level of individual arbuscules will be important for further untangling the mechanisms underlying the observed seedling suppression. Interestingly, a root culture study showed that the abundance of arbuscules was indeed lower in C-starved roots than in roots with an ample C supply (Lekberg et al., 2010).
As the growth suppression of tomato seedlings was mainly caused by P deficiency, the P pools in the ERM must have been less available to the tomato seedlings than to the large cucumber plant. The rapid relief of P deficiency of 76R tomato seedlings upon cutting of ‘donor’ shoots suggests that 32P in the ERM was initially directed toward the large ‘donor’ plant of the CMN, but that the direction of transport shifted toward the small seedlings when the donor no longer represented an active C source. The dominant P transport toward the largest C source prohibited the seedling from receiving significant amounts of P via its mycorrhizal uptake pathway, which in previous studies with tomato accounted for 78–100% of total P uptake (Smith et al., 2003; Nagy et al., 2009). In Experiment 1 of the present study, seedling P uptake would have been more or less confined to Pi uptake at the root epidermis, the direct pathway. The maintained P deficiency of CMN plants revealed that such direct root uptake was small, and two explanations of this are possible: (1) the Pi transporter genes of the direct pathway may have been down-regulated by the presence of mycorrhiza in the tomato roots (see Javot et al., 2007; Grønlund et al., 2013); or (2) available P may have been pre-empted by the ERM colonizing the soil of seedling compartments. The increased P uptake in seedlings in P-amended soil suggests that pre-emption was the causal factor.
The cutting of cucumber shoots had no significant effect on 32P uptake by rmc seedlings, and this indicates that P release from decomposing roots was not responsible for the marked effect of ‘donor’ cutting on the growth of 76R seedlings. This conclusion is supported by the enhanced nutrient uptake and growth of solitary seedlings in Experiment 2 as compared with CMN seedlings.
The role of pre-emption of soil nutrients by the ERM of common mycorrhizal networks
Pre-emption of soil nutrients by ERM was suggested to be responsible for CMN-induced growth depression of seedlings in several studies (Ocampo, 1986; Nakano-Hylander & Olsson, 2007; Janouskova et al., 2011), but actual changes in soil nutrients were not reported. Pool sizes of soil P and N did indeed change with treatment and time in the present study (Fig. 5). Plant available P concentrations decreased over time, even in P0 soil with CMN seedlings, which absorbed only small amounts of P over the 31-d growth period. This confirms that the ERM of the CMN had rather efficient uptake and transfer of P from the seedling soil compartments to the large cucumber plants. The rather low and constant pools over the 17–31-d period may be explained by high nitrification rates and/or high uptake of N by the ERM. The ERM has a high uptake capacity (Govindarajulu et al., 2005; Cruz et al., 2007) and is capable of depleting root-free soil compartments of mineral N (Johansen et al., 1992). Furthermore, the ERM has a stronger affinity for than for (Tanaka & Yano, 2005). The significantly higher concentrations in soil from nonmycorrhizal than from CMN seedlings (and at both soil P concentrations) (Fig. 5) actually suggest that the ERM was partly responsible for the low concentrations.
The decrease in concentrations over time would in the case of CMN seedlings have been caused primarily by mass flow to the large cucumber plants, as seedling shoot N content remained low at 31 d (0.6 and 0.9 mg N at P0 and P30, respectively). The much stronger decrease in concentrations of in soil of solitary seedlings was closely related to the seedling biomass production. Pre-emption by the ERM could partly explain the low concentrations in the CMN treatment as compared with the much higher concentrations in the solitary treatment. However, the maintenance of 11–13 μg g−1 in soil from 17-d-old CMN seedlings suggests that mass flow transport of to the ‘donor’ root compartment was in part replaced by high nitrification activity. This explanation is supported by the observation that soil of solitary seedlings accumulated high concentrations, which may have been derived from a combination of high mineralization/nitrification rates and disruption of mass flow to the ‘donor’ plant sink. Interestingly, this disruption also led to accumulation in mycorrhiza-free soil, but only at 20–30% of the concentration measured in soil from the corresponding solitary mycorrhizal seedlings (24-d harvest). This suggests that the AMF mycelium provided a substrate to stimulate N mineralization, an intriguing finding that suggests a possible key process underlying the reported capacity of AMF to increase N uptake from organic matter patches in soil (Hodge et al., 2001; Leigh et al., 2009). This needs further investigation. A safe conclusion regarding the direct role of ERM in soil N transformation is confounded by the possible difference in composition of the general soil microflora between AMF-inoculated and noninoculated soil. Autotrophic nitrifiers are sensitive to partial soil sterilization (Jakobsen & Andersen, 1982) and introduction of nitrifiers with the inoculum could have been responsible for the increased nitrification rates in soil with mycorrhizal as compared with nonmycorrhizal seedlings. This could also have contributed to the lower concentrations of in soil from mycorrhizal than from nonmycorrhizal plants. It appears that suppression of seedling growth was primarily caused by initial low soil P concentrations and subsequent pre-emption of P (and to some degree N) by the ERM of the CMN. Mycorrhizal colonization did not help to mitigate the suppression of the seedlings, as these had poor access to nutrients in the ERM.