Ectomycorrhizal Colonization Slows Root Decomposition: the Post-Mortem Fungal Legacy

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Ecology Letters, (2006) 9: 955–959 doi: 10.1111/j.1461-0248.2006.00948.x LETTER Ectomycorrhizal colonization slows root decomposition: the post-mortem fungal legacy

Abstract J. Adam Langley,1*Samantha K. The amount of carbon plants allocate to mycorrhizal symbionts exceeds that emitted by Chapman1 and Bruce A. Hungate2 human activity annually. Senescent ectomycorrhizal roots represent a large input of 1Smithsonian Environmental carbon into soils, but their fate remains unknown. Here, we present the surprising result Research Center, Edgewater, that, despite much higher nitrogen concentrations, roots colonized by ectomycorrhizal MD 21037, USA (EM) fungi lost only one-third as much carbon as non-mycorrhizal roots after 2 years of 2 Department of Biological decomposition in a pin˜on pine (Pinus edulis) woodland. Experimentally excluding live Sciences, Merriam-Powell mycorrhizal hyphae from litter, we found that live mycorrhizal hyphae may alter nitrogen Center for Environmental dynamics, but the afterlife (litter-mediated) effects of EM fungi outweigh the influences Research, Northern Arizona University, Flagstaff, AZ 86011, of live fungi on root decomposition. Our findings indicate that a shift in plant allocation USA to mycorrhizal fungi could promote carbon accumulation in soil by this pathway. *Correspondece: E-mail: Furthermore, EM litters could directly contribute to the process of stable soil organic [email protected] matter formation, a mechanism that has eluded soil scientists.

Keywords Decomposition, ectomycorrhizal roots, fine roots, immobilization, 15N, Pinus edulis, soil organic matter.

Ecology Letters (2006) 9: 955–959

laboratory incubations (Ba˚a˚th et al. 2004; Wallander et al. INTRODUCTION 2004), the decomposition rate of EM root litter has never If mycorrhizal symbionts consume an average of 15% of been measured under natural conditions. photosynthate globally, then plants allocate roughly 9 peta- Ectomycorrhizal fungi have long been known to influ- grams (9 · 1015 g) of carbon (C) to mycorrhizal fungi ence decomposition either by competing with saprotrophic annually, which exceeds the sum of all human-caused C decomposers for soil resources (Gadgil & Gadgil 1971) or emissions. This vast amount of C, along with the by producing enzymes that degrade complex organic matter recognition that mycorrhizal abundance responds to global (Read 1991). Evidence from pure-culture incubations change (Treseder 2004), has bolstered interest in the suggests that live mycorrhizal fungi may play an important influence of mycorrhizal colonization on C cycling. Yet, role in the decomposition of their own dead tissues (Kerley the technical difficulty of measuring fungal turnover in soil & Read 1998). In some ecosystems, particularly those with has impeded reaching a consensus. Most dominant trees in poor soils, mycorrhizal fungi can dominate microbial temperate forests, including pinõn pine (Pinus edulis), form biomass and activity. If mycorrhizal fungi do indeed have ectomycorrhizal (EM) associations (Read et al. 2004). When primary access to their own litter, this closed nutrient cycle EM fungi colonize plant roots, they form a sheath around could represent a mechanism by which mycorrhizae sway the outside and penetrate among cortical cells so that EM the balance of competition between plants and free-living roots are composed of roughly 20–40% fungal tissue microbes (Langley & Hungate 2003). (Hobbie 2006). Thus, temperate trees deposit great amounts In the present study, we compared decomposition rates of EM root litter when roots die. This EM litter input holds of EM and non-mycorrhizal (NM) root litter in a pinõn pine great importance in nutrient cycles, in addition to the C woodland. To isolate the influence of live EM hyphae on cycle, because EM roots tend to have higher nutrient root litter decomposition, we buried litterbags in paired concentrations than other roots (Langley & Hungate 2003). cores that were covered with fine-poresize mesh that Although external hyphae appear stable in short-term allowed fungal ingrowth but excluded roots. One core of

2006 Blackwell Publishing Ltd/CNRS 956 J. Adam Langley, S. K. Chapman and B. A. Hungate Letter each pair was rotated frequently to experimentally exclude (a) (b) in-growing hyphae. We compared the inﬂuence of mycorrhizal status of root litter (EM and NM) and the presence of live EM hyphae on C and N dynamics over 2 years of decomposition under ﬁeld conditions.

METHODS In August 2002, we harvested root litter from pinõn pines at Sunset Crater National Monument 10 km NE of Flagstaff, AZ, where EM fungi commonly colonize 30–50% of root tips (Gehring & Whitham 1991) and EM root mass composes roughly one-fourth of total fine root mass. Roots from other species that occur in the soil below pinõn canopies, predominantly Fallugia paradoxa or Juniperus monosperma, were removed based on visible differences in Figure 1 (a) Schematic of a hyphal ingrowth core, and (b) a colour, thickness and architecture. We sorted fine pinõn photograph of core implementation in the field. roots (< 1 mm diameter) according to EM status macro- scopically and verified the distinction under a dissecting microscope. Litter was dried at 60 C. Previous laboratory cohesion; it conformed rapidly to the edges of the cores incubations of pinyon roots that had been surface sterilized following rotation and minimized artificial air gaps. and autoclaved (described in Langley & Hungate 2003) indicate that EM fungi on roots removed from the field and Sampling and analysis subjected to drying do not remain metabolically active. We placed 100 mg of EM root litter and 100 mg of NM root At 6, 10 and 24 months after implantation, 6–10 pairs of litter into separate nylon mesh litter bags. All litter bags were cores were removed. We weighed 10 g subsamples of field- placed, along with displaced soil, in hyphal ingrowth cores wet soil from each core, dried them at 105 C for 72 h, and similar to those described by Johnson et al. (2002). weighed them again to measure gravimetric soil moisture. Litter was removed from bags, dried at 60 C, weighed, ground with a mortar and pestle, and analysed on a Carlo Hyphal ingrowth cores Erba elemental analyzer (Milan, Italy) coupled to a Finnegan PVC pipe (5 cm diameter) was cut into cylinders 15 cm tall. Delta Plus mass spectrometer (Bremen, Germany) for C and Eight square windows totalling 70% of the surface area of N mass and isotopic composition. Samples of initial litter each cylinder were cut from of each core. All cores were were analysed for C, N, lignin and phosphorus as well as covered with 45-lm poresize mesh to allow the passage of scanned for specific root length (n ¼ 3–5). Phosphorus hyphae but not roots (Fig. 1a). In November 2002, cores was measured using a modified micro-Kjeldahl digestion were implanted in the soil under the east and west sides of (Parkinson & Allen 1975) followed by colorimetric analysis 16 pinõn pine canopies at the dripline in pairs (Fig. 1b) on a Lachat flow-injection analyzer (Lachat Instruments, within 1 km of the trees from which root litter was Inc., Milwaukee, WI, USA). Lignin was analysed using a collected. One core from each pair remained static to allow modified acetyl-bromide method and values were compared hyphal ingrowth (+H), and the other was rotated period- with an NIST certified pine standard (Iiyama & Wallis 1990). ically to sever ingrowing EM hyphae ()H). The )H cores, To validate that core rotation did exclude hyphae, hyphal being exactly the same as the +H cores in construction, lengths were measured on the surface of the litterbags in a experienced the same bulk water movement. The mesh manner similar to the inserted membrane technique effectively restricted root growth in both types of cores. The developed by Balaz & Vosatka (2001). Hyphal length was )H cores were rotated at least once weekly during the warm measured in 20 haphazardly located fields on each litterbag at and wet times in the monsoonal window (June to October) 50 · magnification on a dissecting microscope using the and at least once monthly during cold and dry months gridline intersect method (Giovannetti & Mosse 1980). (November to May). Researchers using hyphal exclusion Remaining C, remaining N and d15N from each harvest were cores in clayey soils have expressed concerns that core analysed with a two-factor ANOVA with litter type (EM vs. rotation leaves air gaps in the soil surrounding the )H cores NM) and hyphal ingrowth (+H vs. )H) and the interaction causing treatment artefacts (Johnson et al. 2002). The cinder term as factors. We performed t-tests on soil moisture and soil at Sunset Crater has a large particle size and very low hyphal length measurements (a ¼ 0.05 for all tests).

2006 Blackwell Publishing Ltd/CNRS Letter Afterlife effects of mycorrhizal colonization 957

RESULTS Table 1 Initial chemistry of pin˜on ectomycorrhizal (EM) and non- mycorrhizal (NM) root litter Ectomycorrhizal roots from pin˜on pine decomposed much more slowly than NM roots of the same size class (ANOVA, EM NM tissue type, P < 0.05 at 10 and 24 months; Fig. 2a). After % N 1.57 ± 0.08 0.71 ± 0.03 C : N 28.6 ± 1.0 65.5 ± 2.4 % Lignin 12.3 ± 1.3 13.8 ± 3.4 % P 0.12 ± 0.01 0.08 ± 0.01 (a) 105

100 2 years of ﬁeld decomposition, NM roots lost 19.3% of 95 their initial C mass, whereas EM roots lost only 7.0%. EM roots, which began with much higher N concentration 90 (Table 1), immobilized an additional 15% while NM roots

% C remaining 85 EM – H released 5% of their initial N content (ANOVA, hyphal EM + H ingrowth, P < 0.01 for all dates; Fig. 2b). 80 NM – H The presence of live hyphae had no inﬂuence on the NM + H masses of C and N remaining in litter (P > 0.05 for all dates; (b) 120 Fig. 2a, b). Hyphal ingrowth did generate slight but consistent trends of greater C loss and greater N immobilization in EM litter in unturned +H cores. Hyphal exclusion 110 did affect isotopic composition of litter (ANOVA, hyphal ingrowth, P < 0.05 at 24 months; Fig. 2c). Hyphal ingrowth 100 caused a c. 1& enrichment of d15N in EM litter but not in NM litter resulting in a signiﬁcant interaction between % N remaining 90 hyphal ingrowth and litter type (ANOVA, litter type · hyphal ingrowth interaction, P < 0.05 at 24 months; Fig. 2c). The 80 hyphal lengths measured in +H cores were four times (c) ) 4 greater than in H cores, verifying that core rotation successfully excluded most fungal ingrowth (t-test, 3 P < 0.05, data not shown). Core rotation did not alter 2 gravimetric soil moisture inside the cores (t-test, P > 0.05).

N (‰) DISCUSSION

15 0 δ We found that root litter type (EM vs. NM) drove C and N –1 dynamics throughout decomposition. The 65% depression –2 0 5 10 15 20 25 30 of decomposition rates resulting from EM colonization is comparable with the difference in decomposition rates Months between roots of trees and roots of herbaceous plants Figure 2 Carbon and nitrogen dynamics in ectomycorrhizal (EM) (Silver & Miya 2001). Moreover, though it has much higher and non-mycorrhizal (NM) litter throughout 2 years of decompo- initial [N], EM litter immobilized an additional 15% of its sition in unturned cores which allowed hyphal ingrowth (EM s; initial N in the ﬁrst 10 months of the study period while NM ,) and turned cores which excluded ingrowing hyphae (EM NM litter exhibited a net release over the same time period d;NM ) summarized as means ± SE (n ¼ 7–8). (a) The % of (Fig. 2b). Though EM litters represent a large ﬂux of N-rich initial C mass remaining in each litter type. EM roots lost only one- matter into soil, that litter N appears to remain unavailable third as much C as NM roots and lost no C mass during the final to plants and soil organisms. 14 months. (b) The % of initial N mass remaining in each litter Hyphal ingrowth had no effect on C or N mass remaining type. EM roots, which began with higher initial [N], immobilized N in either litter type (Fig. 2a, b) but sharply increased the for the first 10 months of decomposition while NM litter released 15 N. (c) Natural abundance of 15N in decomposing litter. EM roots d N of EM litter (Fig. 2c), suggesting altered turnover of 15 begin with higher 15N because of natural fungal enrichment. that N pool. Mycorrhizal fungi are strongly enriched in d N A consistent 1& enrichment indicates altered turnover of N in EM (Hobbie et al. 2001), even in comparison with saprotrophic litters exposed to ingrowing hyphae. fungi from the same ecosystem (Henn & Chapela 2001).

2006 Blackwell Publishing Ltd/CNRS 958 J. Adam Langley, S. K. Chapman and B. A. Hungate Letter

Simultaneous enhancements of gross N mineralization and in NM litter, EM root litter immobilized much more N in N immobilization by 15N-enriched, ingrowing mycorrhizal the early stages of decomposition. EM litter represents a hyphae perhaps best explain this effect. The potential for large pool of organic N that mineralizes slowly and so, could mycorrhizal fungi to short-circuit the N cycle by recycling hold importance for N retention in N-limited ecosystems. their own N in this manner has been demonstrated in the Field estimates of mycorrhizal hyphal decomposition rates laboratory (Kerley & Read 1998; Lindahl et al. 2002). are required to constrain the total contribution of mycorrh- Differences in N processing rates may indicate that live izal litters to biogeochemical cycles. EM fungi gain primary access to the N in their own Historically, the live mycorrhizal function as a decom- decomposing litter. Still, after 2 years, hyphal ingrowth did poser was recognized as the primary mechanism by which not strongly influence net C or N dynamics, and accord- mycorrhizal fungi may impart an influence on ecosystem ingly, we conclude that the afterlife effects of EM fungi on processes such as decomposition (Gadgil & Gadgil 1971). root litter quality supercede the effects of live EM fungi on Recent acknowledgement of the large amount of C allocated root litter decomposition in this ecosystem. to, and ultimately deposited by, mycorrhizal fungi has spurred debate regarding the residence time of senesced mycorrhizal structures in soils, most of which focuses on From litter to soil organic matter arbuscular mycorrhizas (Langley & Hungate 2003; Staddon Studies of aboveground plant litter have consistently found et al. 2003; Steinberg & Rillig 2003; Zhu & Miller 2003; that tissues with high [N], or low C : N ratios, tend to Olsson & Johnson 2005). We have shown here that EM decompose more rapidly (Melillo et al. 1982). These indices root litter decomposes much more slowly than NM roots in of litter quality do not appear to predict root decomposition pinõn pine. The influence of mycorrhizal colonization on in the same way. Despite having initial C : N ratios of 29 ecosystem properties of the pinõn woodland appeared to compared with 66 for NM roots (Table 1), EM roots outweigh the influence of live fungal activity. Therefore, we decomposed much more slowly than NM roots, likely owing believe that mycorrhizal influence on resource cycles could to fundamental differences in carbon chemistry between be mediated directly by the turnover of dead fungal fungal and plant tissues. The primary structural polymer in products rather than solely through the impact of live fungi is chitin, which is N-rich (7% N by mass) but resists mycorrhizae on nutrient absorption and decomposition. decomposition (Swift et al. 1979). The surprising recalcit- Understanding these effects will expose the black box of rance of EM roots could explain anomalous trends among belowground C and N cycling and perhaps inform global root decomposition studies such as a strong negative change mitigation strategies. relationship between [N] and decomposition rate in members of Pinaceae (summarized as conifers in Silver & ACKNOWLEDGEMENTS Miya 2001) which commonly host EM fungi. Another controversial precept of soil ecology is that We thank Kitty Gehring, Kris Haskins, Jason Keller and stable soil organic matter largely arises from recalcitrant Arisia Lee for advice and assistance. Support was provided plant compounds such as lignin. However, recent molecular by the Andrew Mellon Foundation’s Program in Conserva- analyses employing isotopic tracers to age carbon found no tion and the Environment. evidence that lignin contributes directly to old soil organic matter (Mikutta et al. 2006). 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