Nitrogen effects on temperate forest AMF
The decrease in AMF root colonization with N addition is very apparent from the results using both techniques. This is the first study to demonstrate such a response in temperate hardwood forests treated for so long with realistic amounts of simulated N deposition. Hutchinson et al. (1998) found a significant decrease in percentage AMF colonization of sugar maple roots at one site after 3 yr of 1000 kg ha−1 yr−1 N addition, while another site showed no difference after 2 yr of N addition. Lansing (2003) also found a reduction in AMF colonization rates for sugar maple after 4 yr of 100 kg ha−1 yr−1 N addition. Interestingly Lansing's reduction in AMF colonization for sugar maple in Michigan was similar to that found in our study (R-values of 0.88 and 0.80, respectively), where R is the response ratio (R = mean of treatment divided by mean of control) (Treseder, 2004). Our total N addition over 12 yr (360 kg ha−1) was comparable to their total N addition over 4 yr (400 kg ha−1).
Several factors could be causing the reduction of the AM symbiont in an N-amended environment. One hypothesis is that N addition reduces host C allocation to AMF. This is consistent with the significant results found in the analyses of proportional allocation to AMF by the maples (above-ground and litter biomass), which is lower in the N-amended plots. The high N deposition sites (sites C and D) also had a lower proportional allocation to AMF independent of treatment, which might be the result of long-term differences in ambient N deposition. If less C is being allocated to the fungal symbiont this could also explain some of the reduced soil respiration found in the N-amended plots. This decline in soil respiration has not been explained by other factors, that is, root respiration or microbial respiration in mineral soil (Burton et al., 2004; Zak et al., 2006). Furthermore the N-amended plots have shown increased tree growth (K. S. Pregitzer et al., unpublished), which suggests that more C is invested in above-ground biomass.
Another hypothesis for reduced AM fungal biomass with N addition could be that the mycorrhizas are directly affected by the higher amounts of N in the soil. Wallander (1995) suggested that reduced fungal growth was not caused by reduced C flow to ectomycorrhizal fungi (EMF), but that the increased amount of N supply caused the mycorrhizal fungi to use more C in the costly process of N assimilation instead of using the C for growth. This hypothesis is consistent with the increased N content of the foliage and leaf litter of the N-amended plots at our study sites (K. S. Pregitzer, unpublished). Fungal growth response can also differ among species depending on their capacity for N assimilation and the pathway of N assimilation (Wallander, 1995), and this might also explain some of the differences in treatment response among sites. However, N uptake costs may be lower for AMF than EMF, because of the difference in their N-assimilation pathways. In AMF symbioses studied so far, N is transferred to the host plant as ammonium and not, as in EM symbiosis, as an amino acid (Govindarajulu et al., 2005). Therefore AMF retain most of the C from the amino acids, while EM fungi lose the C in the transfer of N as amino acids to the host plant (Govindarajulu et al., 2005). However, the N-uptake by AMF still has energetic and C costs that could affect AMF growth.
Sites varied in the strength of the reduction in AMF colonization with N addition. Site D showed only a marginal decline in AMF abundance with N addition in July and a trend toward an increase in October. Although the ambient N deposition of site D is about the same as that of site C, the lack of strong reduction of AM fungal root abundance with N addition could possibly be caused by site-level differences in C allocation to, or N-assimilation by, AMF caused by variation in mean annual temperature, precipitation, tree growth, N-mineralization rates, C : N ratio in litter or phosphorus availability. Alternatively, site-level differences in AMF abundance could be driven by changes in AM fungal community structure. Functional diversity (e.g. variation in C demand vs nutrient supply) exists among AMF, and compositional and functional community responses have been found in previous studies of AM fungal response to N (Johnson, 1993; Corkidi et al., 2002). For example, Johnson (1993) found a change in AM fungal community with N (and other nutrients) fertilization and suggested that the AM fungal species dominant at the fertilized sites were more parasitic than those dominant at low-N sites. We will address these alternative hypotheses in a future paper.
Staining vs fatty acid methods
The positive linear relationship of the fatty acid 16:1ω5c with percentage AMF colonization in stained roots found in this study is consistent with findings from other studies that have performed both staining and fatty acid analysis (Olsson et al., 1997; Van Aarle & Olsson, 2003; R. M. Miller, unpublished). In a controlled glasshouse study with cucumber plants inoculated with a single AMF species, very strong relationships were found between colonized root length and both PLFA and NLFA 16:1ω5c (R2 = 0.92 and 0.95, respectively) (Olsson et al., 1997). In another glasshouse study, Van Aarle & Olsson (2003) found weaker significant relationships between both PLFA and NLFA 16:1ω5c and percentage AMF colonization (R2 = 0.44 and 0.57, respectively). The higher R2 values within the Olsson et al. (1997) and the Van Aarle & Olsson (2003) study compared with our study could be caused by the much more controlled environment vs a field study; a single AMF species vs greater AMF diversity combined with differences between AMF species in fatty acid composition and amounts (Bentivenga & Morton, 1996; Olsson & Johansen, 2000); and/or a bigger range and better distribution of the values of root colonization.
The relationship of NLFA 16:1ω5c with the amount of storage structures (Fig. 4c) was stronger than that of PLFA 16:1ω5c with the percentage total AMF colonization (Fig. 4a). It is unclear exactly why this is, but possibilities include the inability to distinguish live and dead hyphae using staining methods; poor staining of some AMF species (Morton & Redecker, 2001); vesicles’ larger size and distinctive shape compared with hyphae, which minimizes error in counting; and the larger potential for variability in hyphal density compared with vesicle density at an intersect.
The steeper slope of the relationship between NLFA 16:1ω5c and vesicle colonization in October compared with July is indicative of vesicle filling, that is, the accumulation of storage lipids through the growing season. This suggests that most of the AMF storage structures (vesicles) are already present earlier on in the colonization process of the roots, and more lipids are added to these vesicles during the growing season for storage and use for the next year. A similar observation was made by Van Aarle & Olsson (2003) in their glasshouse study. NLFA 16:1ω5c is therefore perhaps a better indicator of the amount of stored energy than the numbers of vesicles present in the roots.
We saw a similar, but weaker, effect of season on the relationship of PLFA 16:1ω5c and percentage total AMF colonization. The distinction between the two regression lines in this relationship (Fig. 4a) is less obvious than for NLFA 16:1ω5c vs vesicle colonization (Fig. 4c). The threefold steeper slope in October compared with July is a much smaller relative increase compared with the fivefold steeper slope for the neutral lipids vs percentage vesicles. PLFA 16:1ω5c also appeared to be a more sensitive biomass indicator than our frequency-based ocular measurements of AMF colonization, probably because the ocular method does not take colonization intensity into account. As a result, when only ocular measurements are performed, changes in biomass could be overlooked or underestimated.
The improvement of the relationship of lipid and ocular estimates after rescaling to a volumetric (cm3 soil) basis was striking, indicating that the strength of the relationship of the two metrics depends on the form of their expression. Since mycorrhizas and roots exploit space rather than mass, the stand-level values (Fig. 4b,d), which show the actual mycorrhizal biomass in a volume of soil, are perhaps more relevant to use than concentration values (Fig. 4a,c). Both root biomass and specific root length, which were used to calculate AM fungal biomass on a stand-level basis, were not affected by treatment. However, the percentage colonization decreased with an increase in specific root length (R2 = 0.47, P < 0.0001) and root biomass decreased at all sites from July to October (P = 0.001). By expressing the AMF abundance on a volumetric basis, these length and biomass differences were taken into account, and improved the relationships between ocular measurements and fatty acid 16:1ω5c.
In conclusion, after 12 yr of simulated N-addition, the abundance of AMF within the active fine root system of maples and proportional investment in AMF decreased significantly, as estimated by both lipid analysis and staining. Positive linear relationships were found between the fatty acid 16:1ω5c and the percentage total AMF colonization and number of storage structures. The phospholipid fraction seems to be a good indicator of active AMF biomass, and NLFA 16:1ω5c was found to be a better indicator of AMF stored energy than the number of vesicles present. The fatty acid analyses gave a better insight into changes in AMF total biomass and stored energy over time compared with the staining method, and avoided possible under- or overestimation of the total AM fungal abundance. However, the staining method can elucidate changes in specific fungal structures (arbuscules, coils, etc.), which is not possible with fatty acid analyses. The observed decrease in AMF abundance and investment could suggest either reduced C allocation to these fungi or a direct soil N-mediated decline. The observed reduction in the abundance of, and investment in, AMF below ground is consistent with the reduction in soil respiration reported earlier for this study (Burton et al., 2004). Future research will focus on the effects of increased N inputs on AMF extraradical hyphae and community analyses designed to understand if N deposition is altering AMF community composition, structure and function.