New Approach for Capturing Soluble Root Exudates in Forest Soils

Functional Ecology 2008, 22, 990–999 doi: 10.1111/j.1365-2435.2008.01495.x

BELOWGROUNDBlackwell Publishing Ltd RESPONSES TO CLIMATE CHANGE New approach for capturing soluble root exudates in forest soils

Richard P. Phillips*, Yael Erlitz, Raven Bier and Emily S. Bernhardt

Department of Biology, Duke University, Durham, NC 27708, USA

Summary 1. Soluble root exudates are notoriously difficult to collect in non-hydroponic systems because they are released in a narrow zone around roots and are rapidly assimilated by rhizosphere microbes. This has substantially limited our understanding of their rates of release and chemical composition in situ, and by extension, their ecological significance. 2. Here we describe the advantages and limitations of several commonly employed methods for measuring exudation with respect to their potential adaptability for field use in forest ecosystems. Then, we introduce a novel in situ method for measuring exudation in forest soils, and present preliminary results of the spatial and temporal dynamics of loblolly pine (Pinus taeda L.) exudation at the Duke Forest FACTS-1 site, North Carolina, USA from April 2007 to July 2008. 3. Exudation rates varied by an order of magnitude, with the highest rates occurring in late-June 2007 and mid-July 2008, and the lowest rates occurring during late-August 2007. On an annual basis, we estimate pine roots in the upper 15 cm of soil release c. 9 g C m–2 year–1 via this flux, which represents 1–2% of net primary productivity at the site. 4. The magnitude of exudation rates did not differ across an N availability gradient but did track general patterns of below-ground C allocation at the site. Exudation was well-predicted by root morphological characteristics such as surface area and the number of root and mycorrhizal tips, further supporting a possible link between root C allocation and exudation. 5. Because all methods for estimating exudates introduce experimental artefacts, we suggest that only a limited amount of ecologically relevant information is probably gleaned from a single method. Thus, a complementary suite of experimental approaches will best enable researchers to understand consequences of changing patterns of exudation in the wake of global environmental change. Key-words: below-ground C allocation, fine roots, rhizodeposition, rhizosphere

Functional Ecology (2008) xx, 000–000 consist of sugars, amino acids, and organic acids (Neumann Introduction & Romheld 2001), and this flux is believed to represent Because they occur in a narrow zone of soil around roots and between 1% and 10% of net assimilated C (Jones et al. 2004). are rapidly assimilated by soil microbes, root exudates are Despite the relatively small magnitude of this flux, root exudates one of the most poorly quantified components of the below- are believed to play an important role in mediating soil nutri- ground C cycle (Wardle 2002; Paterson 2003). Root exudates ent availability in ecosystems due to their chelating properties are soluble, low molecular weight organic compounds that can and their role in stimulating microbial activity (Lynch 1990; be passively released to soil due to the concentration gradient Marschner 1995). Furthermore, exudates are primarily derived between root cells and soil solution or which are actively from recently-assimilated photosynthate (Neumann & Romheld secreted in response to metal toxicity, nutrient stress and the 2001) and thus, may represent a semi-continuous input of labile presence/absence of plant and microbial taxa (Marschner C to soil in contrast to transient inputs of C resulting from leaf 1995; Jones et al. 2004; Bais et al. 2006). Most exudates litter inputs (Kuzyakov & Cheng 2001). Significant methodological challenges limit our ability to *Correspondence author. Department of Biology, Indiana University, accurately measure exudation or to determine how environ- Bloomington, IN 47405, USA. E-mail: [email protected] mental variables affect exudation composition or rates. In

Root exudation in trees 991 general, all exudation methods attempt to overcome a set of have generally been used because the exudates can be more common challenges: (i) capturing exuded C before microbial readily trapped and separated from the medium (Jones et al. assimilation, (ii) selecting a medium that does not affect root 2004). physiology and exudate recovery, and (iii) distinguishing Two primary types of culture-based systems are static and exuded compounds from other soluble C compounds in the percolating (i.e. non-static) trap solutions. In general, both medium. Such challenges become even more formidable when systems are similar in that root systems are submerged in a adapting a method for field use. In most cases, roots need to medium from which exudates are trapped and collected over be temporarily removed from the soil to be studied, which a set period of time. The primary difference between the two may stress or injure the root and disrupt mycorrhizal networks systems is the replenishment of the trap solution in the (Neumann & Romheld 2001). Moreover, root and rhizosphere percolating system which maintains the diffusion gradient processes are highly variable in space and time (Hinsinger between root cells and trap solution, and minimizes the et al. 2005), which pose challenges to developing experimental re-uptake of exuded sugars and amino acids by roots. Thus, protocols that capture this variability, and to scaling measured an important first step in employing a static trap solution is values to the ecosystem-scale. deciding when to sample the trap solution. The challenge is to Not surprisingly, there have been few measurements of sample the solution after a sufficient amount of C has been exudation from trees in situ (but see Smith 1976). This is parti- exuded (i.e. to reduce signal to noise artefacts), but before cularly true for forest ecosystems where deep roots are difficult exudation rates are affected by C accumulation in the trap to access and whole-system isotopic tracers are difficult to solution and re-uptake of exudates by roots (Jones et al. 2004; employ. Exudation measurements of tree seedlings grown in Personeni et al. 2007). An advantage of percolating solutions controlled experimental systems suggest that differences in is that there is no bias associated with exudate accumulation biotic factors (e.g. plant species, phenology, mycorrhizal status), affecting efflux or influx of compounds, and thus percolating abiotic factors (e.g. soil fertility, moisture, temperature), and systems may better reflect a rhizosphere environment where the experimental system employed may all influence the rates microbes are present. However, percolating solutions have a and composition of exudates (Grayston et al. 1996). This has disadvantage in that they generally require a much greater led many researchers to conclude that exudation in forest eco- volume of solution, and several post-collection steps may be systems should be placed into a ‘black box’ of soil processes necessary to for subsequent chemical analyses. In addition, and estimated through modelling approaches (Luo et al. percolating solutions may not uniformly collect exudates 2001) or as residual terms in mass-balance calculations of released by roots if the solution follows preferential flow- below-ground C flux (Fahey et al. 2005; van Hees et al. 2005). paths in the medium. Because percolating solutions require However, such approaches contribute little to our under- the continuous pumping of solution through the medium, standing of the mechanisms that control this process, and they may also be less amenable for adaptation to field studies. by extension, the potential role of exudates in mediating In both static and percolating systems, the type of growth microbial activity, nutrient transformations, and feedbacks to medium selected is known to have important consequences primary productivity and ecosystem C storage (Cheng 1999; for root growth, architecture and exudation. Pure solution Phillips 2007). cultures (generally a dilute salt or nutrient solution) offer Below-ground processes such as exudation mediate the flux simplicity in maintenance and sample collection. However, of energy and materials in terrestrial ecosystems, but our under- the lack of mechanical impedance for roots in non-solid media standing of how such processes influence feedbacks to eco- may affect root morphology and exudation rates (Neumann system C and N cycling remains limited owing to significant & Romheld 2001). Small glass beads and acid-washed sand methodological obstacles. The goals of this paper are to: (i) have commonly been employed but such media have limitations review existing methods for collection of exudates, (ii) describe as well, as sorption of exuded compounds to these media may a newly-developed method for collecting exudates from tree result in incomplete recovery of exudates or mischaracterization roots in situ, and (iii) present data collected over a 15 month of the exudate composition (Neumann & Romheld 2001). In period from trees at the Duke Forest Free Air CO2 Enrichment our own experiments, we have found that acid-washed sand Experiment. (3 m HCl) can be both a source and sink for C (R.P. Phillips, unpublished data). Thus, careful consideration should be given to selecting an appropriate medium for the trapping solution EXISTING EXUDATION METHODS as small amounts of sorbed C or contamination of C from the Several different approaches have been used to measure medium can introduce substantial artefacts to estimated exudation, with the specific techniques employed depending exudation rates and compositional analyses. upon the investigators’ primary research interests. In studies An important consideration in adapting either trapping where the goal is to examine the total amount of rhizodeposi- system for field use is the question of sterility of the culture tion (sloughed cells, mucilage, etc.) entering soil and the move- medium. Sterile/axenic culture systems have an advantage in ment of this C into soil pools, isotopic labelling approaches that exudates released from roots will not be metabolized by have generally been employed because non-sterile soils can be microbes in the medium, thereby allowing exudation rates to used (reviewed in Paterson et al. 1997). In studies of the quantity be measured without needing to account for microbial and chemical composition of exudates, culture-based systems assimilation of released of compounds (Uselman et al. 2000).

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However, the presence of microbes may stimulate exudation and root systems, and the varying degrees of colonization of by maintaining a concentration gradient around roots or by tree roots by a diverse assemblage of mycorrhizal fungi – all of inducing exudation via the release of signalling compounds which would presumably affect exudation rates and the (reviewed in Grayston et al. 1996; Paterson et al. 1997; chemical composition of the C released (Grayston et al. 1996; Neumann & Romheld 2001). Using a sterile medium may Scott-Denton et al. 2006; Van Scholl et al. 2006). Moreover, thus reduce exudation rates and affect the composition of exudation rates can be scaled to the ecosystem-level using this exudates. The issue of sterility is even more vexing for studies method so that the consequences of this flux for microbial in forest soils as most roots are likely to be extensively colonized activity and nutrient cycling can be better quantified. by mycorrhizal fungi and rhizoplane bacteria. One possible method for minimizing artefacts associated with non-sterile conditions is to treat roots with antibiotics. However, the Materials and methods efficacy of any antibiotic treatment will likely depend on the type and concentration of antibiotics used as well as the plant SITE DESCRIPTION species of interest (Neumann & Romheld 2001). In addition to trapping solutions, localized sampling tech- The Duke Forest-Atmosphere Carbon Transfer and Storage (FACTS-1) was established in a loblolly pine (Pinus taeda L.) plantation in Orange niques have been used to collect exudates. Agar, specialized County, North Carolina (35°58′N, 79°05′). The site is dominated by resins or filter paper can be placed along desired sections the pine (> 90% of the basal area), although hardwoods have developed of the root axis to collect compounds released from distinct in the understorey since the initial planting in 1983. In 1996, replicated areas of the root (Neumann & Romheld 2001). These tech- 30 m diameter plots containing c.100 trees per plot were established at niques have the benefit of providing information on fine-scale the site. Three experimental plots at the site are fumigated with exogenous spatial patterns of exudation, and can be coupled to experiments CO2 to maintain an atmospheric concentration c. 200 ppmv above with bacteria cloned with reporter gene systems (Killham & ambient levels (i.e. c. 585 ppmv) while three plots are fumigated with Yeomans 2001; Cardon & Gage 2006). Non-sterile conditions air only (i.e. c. 385 ppmv). The mean annual temperature of the site is –1 are less likely to bias results, as exudates trapped by resins will 15·5 °C and mean annual precipitation at the site is 11 140 mm year . be unavailable to soil microbes. However, use of resins, filters, Soils at the site (Enon Series) are highly-weathered clay loams (mixed etc. may not be ideal when looking for unknown compounds, thermic Ultic Hapludalfs) developed from igneous parent materials, and are moderately acidic (pH = 5·6). In general, the vast majority of as the a priori choice of resin will influence which compounds the roots (> 90%) reside in the upper 30 cm of soil. Detailed descriptions are trapped. An additional caveat is that exudation rates cannot of the experimental set-up and the site characteristics can be found be readily quantified. Thus, application of this method for field in Hendrey (1999) and Lichter et al. (2005), respectively. studies might be appropriate in cases where exudate com- Average daily temperature and photosynthetically active radiation position, rather than rates, is of interest. Localized sampling (PAR) were collected during the days of exudate collection from April of exudates can also be performed using microsuction cups as 2007 to July 2008 (Table 1). These meteorological variables are strongly tension lysimeters (Gottlein & Blasek 1996; Sandnes, Eldhuset linked to C assimilation by loblolly pines at the site (Hendrey et al. & Wollebaek 2005). This method has advantages in that the 1999). Air temperature was collected at 1 min intervals at a mid-canopy exudates are removed from soil before they can be metabo- location in the middle of each plot using shielded thermocouple with ° lized by microbes, and the cups can be repeatedly sampled to an accuracy of c. 1 C. PAR data were collected at 1 min intervals using examine temporal trends. However, a major limitation of this a LiCor Quantum sensor (Licor Co., Lincoln, NE) mounted above the forest canopy. Average daily temperature and PAR were calculated method is that only small volumes of solution can be collected from hourly means for each day of exudate collection, and then aver- under most field conditions (e.g. irrigation is often necessary), aged for each 3–4-day collection period (Table 1). and flux rates cannot be quantified. Moreover, this method must be employed through rhizoboxes or root windows (Dessureault- Rompre et al. 2006; Shen & Hoffland 2007), and thus only a EXUDATE COLLECTION small volume of soil is generally sampled. Soluble root exudates were collected from loblolly pine trees at 4– In this manuscript, we describe our use of a culture-based 8-week intervals from April 2007 to July 2008 using a modification system for measuring exudation from tree roots which is of a static culture-based system which was adapted for field use (Fig. 1). relatively inexpensive, easy to deploy, and requires minimal In this system, intact fine root systems were washed free of adhering maintenance. In addition, this method can account for the soil and ‘incubated’ in glass cuvettes filled with sterile glass beads spatial heterogeneity and temporal dynamics of forest soils and C-free nutrient solution. Exudates released by roots were collected

Table 1. Meteorological data from Duke FACTs-1 site, NC on the days of exudate collection. Values are weekly means of daytime averages in air temperature (°C) in the middle of the forest canopy and photosynthetically active radiation (PAR; μmol m–2 s–1) at the top of the canopy

17/04/2007 27/06/2007 16/07/2007 27/08/2007 25/09/2007 29/10/2007 4/12/2007 25/02/2008 25/03/2008 5/05/2008 15/07/2008

Tavg 16·4 28·1 28·5 29·5 26·3 15·3 3·9 4·4 15·0 21·1 26·3

PAR avg 1088 950 999 911 937 760 429 637 1024 896 1047

Root exudation in trees 993

Fig. 1. Sketch of an experimental system employed to trap root exudates from intact loblolly pine roots growing at the Duke Forest FACTS1 site, North Carolina, USA. Note that all root systems are still attached to the tree during exudate collection and only appear unattached for illustrative purposes.

over short time-intervals by flushing each cuvette with a vacuum tree. In order to ensure that roots were loblolly pine, all root systems pump. Similar designs using bead-filled cuvettes have been used to were traced back to a parent tree or coarse root with characteristics collect root and mycorrhizal exudates from seedlings of Pinus sylvestris unique to loblolly pine (e.g. reddish flaky bark). Once unearthed, (Ahonen-Jonnarth et al. 2000; Van Scholl et al. 2006), Picea abies root segments (15–20 cm length) were rinsed with a squirt bottle [L]. Karst. and Betula pendula Roth (Sandnes et al. 2005) and loblolly containing nutrient solution (to minimize osmotic stress to the root) pine (Phillips et al. 2008). Similar static bathing solutions have been to remove adhering soil. Fine forceps were also used to dislodge used to measure nutrient uptake of pine trees in the field (Lucash et al. adhering soil. Given the intimacy between the roots, mycorrhizal 2005). However, this study is the first study to our knowledge to use hyphae and organic matter, we took extreme caution to remove soil this type of system to measure root exudation for field-grown trees. during this process, resulting in average processing time of c. 1 h per root system per person. The intact root systems were placed into a soil-sand mixture (1:1) and re-buried until they could be placed into EXCAVATION AND CUVETTE ASSEMBLY the root cuvettes (generally 24–48 h later). The re-burial was intended to allow additional time for roots to recover from any potential Terminal fine root systems (< 2 mm diameter with laterals) were care- injury or stress sustained during the excavation and rinsing process. fully excavated from soil (sensu Lucash et al. 2005) at the interface Sieved soil mixed with sand was used to facilitate removal of the soil between the O and A horizons within 0·5 m from the bole of each (by squirt bottle) before placement into the root cuvettes.

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Fig. 2. Recovery of soluble C and N from bead-filled cuvettes containing low and high concentration solutions. Panels on the left indicate the total amount of C (a) and N (c) flushed from cuvettes containing a low concentration solution, while panels on the right indicate the total amount of C (b) and N (d) flushed from a high concentration solution.

Soil-free root systems were placed into a 30-mL glass syringe (Pop- procedure described above. We found that three flushes were sufficient per & Sons, New Hyde Park, NY) from which the plunger was to remove over 90% of the soluble C and N in each cuvette in a sep- removed, and each syringe was back-filled with sterile acid-washed arate recovery experiment using spiked solutions of low and high C glass beads (c. 750 μm diameter; La De Da Designs, Baton Rouge, and N concentration (Fig. 2). LA). At the narrow end of each syringe, a 30 μm mesh cloth was Following each incubation period, all cuvettes were reopened and folded into a cone to support the beads and to prevent them from glass beads rinsed from roots. We did not analyse the beads follow- clogging the syringe outlet during removal of the solution via a ing their removal after preliminary tests suggested little residual C vacuum pump (Fig. 1). At the top of each syringe, a rubber septum accumulation on the beads (data not shown). Washed roots were with a small slit cut to accommodate the protruding root was used removed from the tree, photographed, imaged, and analysed for root to seal off each cuvette. To protect the exposed portion of the root morphological characteristics using WinRhizo (Regents Instruments from drying out, a moist Kimwipe (Kimberly-Clark Corp., Roswell, Inc., Québec, Canada). All solutions were filtered immediately through GA) was placed around the upper root segment and secured with a sterile 0·22 μm syringe filter (Millex PVDF, Millipore Co., Billerica, Parafilm (3 m Company, Minneapolis, MN). A small volume of dilute MA), and refrigerated at 4 °C until analyses (< 48 h). All samples nutrient solution (see below) was added to the cuvette to maintain were analysed for non-particulate organic C and total N on a TOC humid conditions during the incubation. The cuvettes were then covered analyser (Shimadzu Scientific Instruments, Columbia, MD). Only in aluminum foil, returned to the excavated area, and covered with the C analyses are presented in this article. several layers of needle litter to allow the root system to equilibrate with the cuvette environment. Three control cuvettes filled with glass beads only were similarly covered and buried in soil (one in each plot). EXPERIMENTAL TESTS

EXUDATE COLLECTION Experiment 1: Exudate accumulation and incubation duration After a 2–3 day equilibration period, a dilute nutrient solution was added to each glass cuvette to facilitate the removal of accumulated An important consideration for static bathing solution culture- exudates. In order to ensure the complete removal of C from the cuvette systems is the degree to which exudation rates are affected by the before the experimental incubation period, each cuvette was filled to changing C concentration in the trap solution and the potential re- m m saturation with C-free nutrient solution (0·5 m NH4NO3, 0·1 m uptake of exudates by roots (Jones et al. 2004; Personeni et al. 2007). m m m KH2PO4, 0·2 m K2SO4, 0·2 m MgSO4, 0·3 m CaCl2) and flushed We initiated a pilot experiment to examine this potential source of with a vacuum pump. This process was repeated five consecutive bias using loblolly pine roots from two plots (one exposed to ambi- times. After the fifth flush, a small volume of nutrient solution was ent CO2, the other elevated CO2) at the Duke Forest FACTS-1 site in added, and the root-filled cuvette was re-buried as before. At the end July, 2007. We collected exudates at 0, 4, 8, 16, 21, 24 h from a total of each incubation period, cuvettes were flushed three times with a of eight root systems (two per tree) over four consecutive days (July C-free nutrient solution to remove accumulated exudates using the 17–20). Because of the time needed to flush and extract the solutions,

Root exudation in trees 995 not all eight root systems could be incubated for the same time intervals on a given sampling day. Thus, we staggered our sampling so that subsets were incubated on different days. This also enabled us to minimize the potential inﬂuence of diurnal variation on exudation (Neumann & Romheld 2001). Root-free controls were ﬂushed at the same time intervals to serve as controls.

Experiment 2: Day to day variation in exudation rates

Given the potential bias of sampling root systems on different days due to variation in environmental conditions (e.g. temperature, PAR, wind speed etc.), we initiated a second experiment at FACTS-1 to examine the variation in exudation rates over consecutive days. We tested this potential source of variation by incubating 11 root systems in two plots (one at ambient CO2, the other at elevated CO2) for 24 h intervals during consecutive days in July and September 2007. The 24 h incubation time was chosen after preliminary tests using loblolly pine roots revealed that exudation rates were still increasing after 24 h (see Experiment 1 results below) and were of sufficient magnitude to be analytically detectable. In both samplings, cuvettes were Fig. 3. Carbon efflux from loblolly pine roots at the Duke FACTs-1 flushed five times between dates to ensure that no exudates remained site, NC into a trap solution over the course of a week-long experi- from the incubation of the previous day. Exudates were collected ment in July, 2007. The data are best fit by a logarithmic curve with three flushes and analysed for non-particulate organic C on a (y = 2·9205 Ln(x) + 4·3017; r2 = 0·94). TOC analyser (as described above).

Experiment 3: Seasonal variation in exudation rates Results

We sought to quantify pine root exudation rates at the FACTS-1 site EXPERIMENTS 1 AND 2 across a range of environmental conditions. Thus, we initiated an experiment to collect exudates every 4–8 weeks from April 2007 to We evaluated whether C accumulation in the cuvette inﬂuenced

July 2008 in three experimental plots (all at ambient CO2) which vary exudation by flushing a subset of cuvettes at 0·5, 4, 8, 16, 21 in annual net N mineralization rates by approximately a factor of and 24 h. Carbon released from roots was still increasing over three (Finzi & Schlesinger 2003). All exudates were collected from the first 24 h, and the increase was best described by a loga- two or more root systems in each of the three plots – with the exception rithmic function ( y = 2·9205 Ln(x) + 4·3017; r2 = 0·94; Fig. 3). of the April, June and July 2007 when exudates were collected from Thus, it seems unlikely that exudation rates were influenced root systems in a single plot. All of the incubations for this experiment to a large degree by C accumulation in the cuvette. Given the lasted for c. 24 h, beginning and ending in the early hours of the shape of the curve, shorter incubation times (e.g. 8 h) may be photoperiod to account for potential biases associated with diurnal desirable under some conditions. However, detecting individual variation in exudation (Neumann & Romheld 2001). All exudates were collected and processed as described above. compounds is probably difficult with shorter incubation times particularly under conditions where exudation rates are low. Moreover, a 24 h incubation period has the added advantage CALCULATIONS AND STATISTICS in that it spans the diurnal cycle, and thus provides a time- In all three experiments, C accumulation in each cuvette was calculated integrated estimate that accounts for differences in exudation as the sum of the three flushes minus the total C flushed from the rates which occur between night-time and the peak hours of root-free control cuvettes (units of μg C). For experiment 2, Pearson the day (Kuzyakov & Domanski 2000; Uselman et al. 2000). correlation coefficients were used to examine day to day variation in We examined short-term variation of exudation by collect- C efflux from 11 root systems. In addition, a paired t-test was used to ing exudates from the same root systems on consecutive days evaluate whether the paired means were significantly different from in July and September 2007 (Fig. 4). Such variation is import- one another (α = 0·05). For experiment 3, we used analysis of variance ant to consider given that only a limited number of root systems anova ( ) to examine differences in exudation rates across the N avail- can be sampled on a particular day. Overall, exudation rates α = anova ability gradient of the three plots ( 0·05). We also used to were positively correlated across the 2-day period (r = 0·73), examine monthly and seasonal variation in exudation (n = 6 on each and there were no significant differences between the paired sampling date), and linear regression to examine the relationship data (P = 0·142). Although we cannot directly compare the between C efflux and root morphological variables across seasons. Seasons were defined relative to the dates samples were collected: responses in July and September due to the small number of spring (April 2007 and May 2008), summer (June, July and August samples collected, our September data suggest a trend toward 2007, July 2008), fall (September and October 2007), and winter reduced rates on the second day. This may have resulted from (December 2007, February and March 2008). Non-normally distri- the slight reduction in average daily air temperature and PAR buted data were transformed before statistical analyses with jmp from day 1 to day 2 (29 °C and 926 μmol m–2 s–1 on day 1; statistical software (v. 6, SAS Institute Inc., Cary, NC). 28 °C, 866 μmol m–2 s–1on day 2).

unable to detect significant differences in exudation between any two sample dates (e.g. July 2007 vs. July 2008) and among the seasons (Fig. 5 inset). We also examined the degree to which root morphological variables predicted exudation (Fig. 6). We found that C efflux was well-predicted by root morphology but that such relationships depended on season. In the spring months, C efflux was predicted by the fine root surface area (r2 = 0·61; P = 0·008, Fig. 6a) and the number of root and mycorrhizal tips (r2 = 0·60; P = 0·008, Fig. 6b). Similarly, C efflux was predicted by the root surface area (r2 = 0·62; P = 0·004, Fig. 6c) and the number of tips (r2 = 0·36; P = 0·05, Fig. 6d) in the fall months. In the summer months, C efflux was somewhat correlated with surface area (r2 = 0·20) and tip number (r2 = 0·23) but these relationships were not significant (data not shown). We found no significant relationship between C efflux and root morphological variables in the winter months (data not shown).

Fig. 4. Relationship between C efflux from eleven intact root systems over a 2 day period at the FACTS-1 site. Cuvettes were flushed five Discussion times between the days 1 and 2 collections. Open triangles were We have demonstrated a viable new method for collecting collected on 17–18 July 2007 while filled triangle were collected on exudates from intact root systems of mature trees in the field 26 –27 September 2007. that overcomes many of the challenges of previous methods. Our method: (i) uses intact root systems, (ii) allows time for recovery from transplant shock, (iii) collects exudates in realistic conditions (moist glass beads rather than static solution), and EXPERIMENT 3 (iv) can be deployed anywhere at relatively low costs. While we In general, exudation rates varied considerably, with the greatest believe our method is a substantial step forward, linking variation occurring in the plot with the lowest N availability exudation from a few individual root systems to ecosystem (Fig. 5). In this low N plot, exudation varied by nearly an order carbon and nutrient cycling presents some formidable of magnitude – from 0·27 μg C cm–1 root day–1 in August 2007 challenges. to 2·42 μg C cm–1 root day–1 in July 2008. In contrast, exudation rates in the plot with the highest N availability varied only by ACCOUNTING FOR SPATIOTEMPORAL VARIABILITY a factor of c. 2 across the same time period. Overall, there were no significant differences in exudation rates among the plots Accounting for the spatial and temporal variability is a (P = 0·97). Averaging across all three plots, we were also persistent challenge in all root and rhizosphere studies

Fig. 5. Seasonal variability of mass-speciﬁc exudation from loblolly pine trees across an N availability gradient at the Duke FACTs-1 site, NC from June 2007 to July 2008. Each data point represents the mean value of two or more root systems in each plot. The inset shows the mean seasonal exudation ﬂux from the three plots (n = 3) over the same time interval.

Fig. 6. Relationship between C efflux and morphological characteristics of loblolly pine roots at the Duke FACTs-1 site, NC across three seasons. Panels on the left represent (a, c) the relationship between C efflux and fine root surface area while panels on the right (b, d) represent the relationship between C efflux and the number of fine root and mycorrhizal tips. The relationship between C efflux and pine root morphology was not statistically significant for data collected in December 2007 to March 2008.

(Hinsinger et al. 2005). Such heterogeneity presents logistical rates at the site may reflect the spatio-temporal differences in challenges in deciding how many roots need to be sampled to fungal taxa colonizing each root system. capture the variability of the site, and how frequently exudates Although we did not detect a significant seasonal effect, need to be collected and at what time of day in order to account exudation rates followed the general pattern of C allocation to for temporal variation. In our analyses, we sought to reduce fine roots (Pritchard et al. 2008a) and mycorrhizal fungi some of the variation in exudation by normalizing rates by (Pritchard et al. 2008b) at the FACTS-1 site, with the greatest fine root surface area – as exudation is predicted relatively fluxes occurring in late-spring/early summer and mid-fall. well by this variable (Fig. 6). Still, we were unable to detect Moreover, exudation was well-predicted by the number of significant effects of month or season on exudation rates. root and mycorrhizal tips in each cuvette. These results are Temporal variation in exudation was low at the daily/weekly consistent with reports of tips being active sites of exudation scale (Fig. 4) but high over the 15-month period (Fig. 5). The (Neumann & Romheld 2001), and suggest that mycorrhizal high degree of variability over the course of the study was tips may not only be a sink for exudates but a source as well particularly evident in the plot with the lowest N availability (Grayston et al. 1996). Perhaps more importantly, the results where exudation rates varied by an order of magnitude. suggest that root images collected from other sources (e.g. Because soils with low N availability often exhibit greater fine minirhizotrons) might provide a way to derive estimates of root production and mortality (Nadelhoffer et al. 1985), some this flux at the ecosystem-scale. of the variation in exudation may have resulted from the Arguably, the biggest challenge to understanding the role variable ages of roots in these plots. of exudation in forest ecosystems is not merely developing a Fine roots at this site live on average from 1 and 4 years collection method, but developing an appropriate scaling factor (Matamala et al. 2003; Pritchard et al. 2008a; Strand 2008), so that annual ecosystem fluxes (e.g. in units of g C m–2 year–1) and fine root production and mortality at this site occur can be calculated. A simple calculation of the annual contri- simultaneously rather than in discrete pulses (King et al. 2002). bution of exudates can be made by multiplying the average Such differences in root life span would presumably influence mass-specific exudation rate from the 15-month sampling exudation rates due to the physical and chemical changes that period (10·3 × 10–5 μg C fine root biomass–1 day–1) by the occur as fine roots age (Jones et al. 2004). An additional source average fine root biomass in the O horizon +0–15 cm of soil in of variation may be the colonization of roots by mycorrhizal these plots (c. 250 g fine root biomass m–2; R. Jackson, unpub- fungi. Parrent et al. (2006) estimated that loblolly pine roots lished data). Multiplying this flux by 365 days yields an esti- in these plots are colonized by 64 different ectomycorrhizal mated annual flux of 9·4 g C m–2 year–1. This value represents taxa, with the highest degree of phylotype richness occurring 1·5% of the net primary production (NPP) in the ambient in the plots with the lowest N availability. Because mycorrhizal plots at FACTS-1 (625 g C m–2 year–1; from McCarthy 2006), taxa vary in their effects on root C efflux (Ahonen-Jonnarth and closely approximates estimates derived from modelling et al. 2000; Van Scholl et al. 2006), variability in exudation approaches (c. 1%) at this site (Luo et al. 2001). This estimate

© 2008 The Authors. Journal compilation © 2008 British Ecological Society, Functional Ecology, 22, 990–999 998 R. P. Phillips et al. is probably conservative given that fine roots at lower soil lines of evidence suggest that these fluxes are probably appre- depths, which may account for 30–50% of the total standing ciable in magnitude and highly responsive to the biotic and crop of fine root biomass at the site (Pritchard et al. 2008a), abiotic changes anticipated under global environmental change. were not sampled. Thus, annual exudation rates may be closer Thus, a concerted effort is needed to develop novel methods to 3% of net primary production in this forest. for measuring this process in situ, possibly through adapting The issue of spatial and temporal variability poses a further commonly used lab methods for field use. challenge for understanding the consequences of changing Our method provides an important first step for measuring exudation patterns under global change. At the Duke Forest exudation from the intact roots of mature trees in forest soils. FACTS-1 site, we have also collected exudates from trees exposed Although previous methods have been employed for measur- to elevated CO2 and N fertilization. Preliminary results from ing exudation rates from tree roots in situ (Smith 1976), our this experiment suggest a consistent CO2 by N interaction over method offers the advantage that it is inexpensive, relatively several months of the growing season where CO2-induced simple to set-up, can be employed throughout the growing increases in mass-specific exudation are greatest at low soil N season, and requires only a minimal amount of soil distur- availability (data not shown). This result is consistent with the bance. Moreover, our system allows for the chemical charac- exudation response of loblolly pine seedlings grown in a terization of exudates, and thus should be useful in assessing controlled environmental growth chamber (R.P. Phillips, E.S. the role of exudates in stimulating microbial activity, nutrient Bernhardt & W.H. Schlesinger, unpublished) and gives us release and the decomposition of soil organic matter in forest increased confidence in the potential for both independent soils. The primary caveat of the method is that the disruption methods to provide reliable estimates of the exudate response of root and mycorrhizal networks present an unknown source to each treatment. Linking the patterns and rates of exudation of error to the estimates. Scaling up to ecosystem rates poses in seedlings in growth chambers to those of mature trees in a further challenge owing to the temporal and spatial vari- the field may allow us to overcome the shortcomings and ability of exudation. Nevertheless, these uncertainties are an artefacts introduced by each method individually. inevitable aspect of all in situ studies of root, rhizosphere and mycorrhizal dynamics (Read 2002). Despite the enormous challenge, it is our opinion that a FUTURE DIRECTIONS sustained effort to develop in situ methods for exudation will In this study, we have described a new method for collecting greatly improve our understanding of the role of below-ground exudates from intact roots of loblolly pine trees, and presented processes in mediating ecosystem response to global change. data collected from over 60 root systems over a 15 month period. However, it is important to emphasize that no one method is Given the high degree of spatial and temporal variability of likely to be appropriate in all cases, and we suggest that a suite this flux, we suggest that exudates will likely need to be collected of complementary approaches (trap solutions, isotopes, reporter over multiple years and analysed using repeated measures – gene systems, microlysimetry, etc.) is likely to yield the most similar to the approach used to understand fine root dynamics information on the role of rhizosphere processes in forest at this site (Pritchard et al. 2008a) – to understand seasonal ecosystems. patterns in this flux. Because the method allows for the characterization of exudate composition, realistic exudate Acknowledgements addition experiments should also be used to examine the consequences of changing exudation patterns on soil processes Authors thank Elise Pendall, Lindsay Rustad and Josh Schimel for inviting us (Landi et al. 2006; Kuzyakov et al. 2007; Paterson et al. 2007). to participate in the symposium ‘Towards a Predictive Understanding of Below-ground Ecosystem Responses to Global Change’ at the 2006 SSSA An important limitation of the method is that although meeting in Indianapolis, IN. Authors also thank Robert Nettles and David root systems are left intact, the rhizosphere is not left intact Cooley for their technical assistance at the Duke Forest FACTS1 site, Robert due to the separation of roots and soil; this will almost certainly Jackson, Seth Pritchard, Adrien Finzi and Jeri Parrent for their insight and willingness to share unpublished data, and the members of the Bernhardt Lab result in some severing of mycorrhizal fungal hyphae and may for providing suggestions for improving this article. The bulk of this research influence the flux of C from roots. Whether the latter issue can was supported by the DOE FACTS-1 grant. Additional funds were provided by be overcome by keeping roots in the cuvettes long enough to the Office of Science (BER), US Department of Energy, Grant No. DE-FG02- 95ER62083. 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