Functional Ecology 2008, 22, 990 999

doi: 10.1111/j.1365-2435.2008.01495.x

BELOWGROUND RESPONSES TO CLIMATE CHANGE

Blackwell Publishing Ltd

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 difcult 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 signicance. 2. Here we describe the advantages and limitations of several commonly employed methods for measuring exudation with respect to their potential adaptability for eld 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 m2 year1 via this ux, which represents 12% 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, ne roots, rhizodeposition, rhizosphere

Functional Ecology (2008) xx, 000000

Introduction
Because they occur in a narrow zone of soil around roots and are rapidly assimilated by soil microbes, root exudates are one of the most poorly quantied components of the belowground C cycle (Wardle 2002; Paterson 2003). Root exudates are soluble, low molecular weight organic compounds that can be passively released to soil due to the concentration gradient between root cells and soil solution or which are actively secreted in response to metal toxicity, nutrient stress and the presence/absence of plant and microbial taxa (Marschner 1995; Jones et al. 2004; Bais et al. 2006). Most exudates
*Correspondence author. Department of Biology, Indiana University, Bloomington, IN 47405, USA. E-mail: rpp6@indiana.edu

consist of sugars, amino acids, and organic acids (Neumann & Romheld 2001), and this ux is believed to represent between 1% and 10% of net assimilated C (Jones et al. 2004). Despite the relatively small magnitude of this ux, root exudates are believed to play an important role in mediating soil nutrient availability in ecosystems due to their chelating properties and their role in stimulating microbial activity (Lynch 1990; Marschner 1995). Furthermore, exudates are primarily derived from recently-assimilated photosynthate (Neumann & Romheld 2001) and thus, may represent a semi-continuous input of labile C to soil in contrast to transient inputs of C resulting from leaf litter inputs (Kuzyakov & Cheng 2001). Signicant methodological challenges limit our ability to accurately measure exudation or to determine how environmental variables affect exudation composition or rates. In

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Root exudation in trees general, all exudation methods attempt to overcome a set of common challenges: (i) capturing exuded C before microbial assimilation, (ii) selecting a medium that does not affect root physiology and exudate recovery, and (iii) distinguishing exuded compounds from other soluble C compounds in the medium. Such challenges become even more formidable when adapting a method for eld use. In most cases, roots need to be temporarily removed from the soil to be studied, which may stress or injure the root and disrupt mycorrhizal networks (Neumann & Romheld 2001). Moreover, root and rhizosphere processes are highly variable in space and time (Hinsinger et al. 2005), which pose challenges to developing experimental protocols that capture this variability, and to scaling measured values to the ecosystem-scale. Not surprisingly, there have been few measurements of exudation from trees in situ (but see Smith 1976). This is particularly true for forest ecosystems where deep roots are difcult to access and whole-system isotopic tracers are difcult to employ. Exudation measurements of tree seedlings grown in controlled experimental systems suggest that differences in biotic factors (e.g. plant species, phenology, mycorrhizal status), abiotic factors (e.g. soil fertility, moisture, temperature), and the experimental system employed may all inuence the rates and composition of exudates (Grayston et al. 1996). This has led many researchers to conclude that exudation in forest ecosystems should be placed into a black box of soil processes and estimated through modelling approaches (Luo et al. 2001) or as residual terms in mass-balance calculations of below-ground C ux (Fahey et al. 2005; van Hees et al. 2005). However, such approaches contribute little to our understanding of the mechanisms that control this process, and by extension, the potential role of exudates in mediating microbial activity, nutrient transformations, and feedbacks to primary productivity and ecosystem C storage (Cheng 1999; Phillips 2007). Below-ground processes such as exudation mediate the ux of energy and materials in terrestrial ecosystems, but our understanding of how such processes inuence feedbacks to ecosystem C and N cycling remains limited owing to signicant methodological obstacles. The goals of this paper are to: (i) review existing methods for collection of exudates, (ii) describe a newly-developed method for collecting exudates from tree roots in situ, and (iii) present data collected over a 15 month period from trees at the Duke Forest Free Air CO2 Enrichment Experiment.

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EXISTING EXUDATION METHODS

Several different approaches have been used to measure exudation, with the specic techniques employed depending upon the investigators primary research interests. In studies where the goal is to examine the total amount of rhizodeposition (sloughed cells, mucilage, etc.) entering soil and the movement of this C into soil pools, isotopic labelling approaches have generally been employed because non-sterile soils can be used (reviewed in Paterson et al. 1997). In studies of the quantity and chemical composition of exudates, culture-based systems

have generally been used because the exudates can be more readily trapped and separated from the medium (Jones et al. 2004). Two primary types of culture-based systems are static and percolating (i.e. non-static) trap solutions. In general, both systems are similar in that root systems are submerged in a medium from which exudates are trapped and collected over a set period of time. The primary difference between the two systems is the replenishment of the trap solution in the percolating system which maintains the diffusion gradient between root cells and trap solution, and minimizes the re-uptake of exuded sugars and amino acids by roots. Thus, an important rst step in employing a static trap solution is deciding when to sample the trap solution. The challenge is to sample the solution after a sufcient amount of C has been exuded (i.e. to reduce signal to noise artefacts), but before exudation rates are affected by C accumulation in the trap solution and re-uptake of exudates by roots (Jones et al. 2004; Personeni et al. 2007). An advantage of percolating solutions is that there is no bias associated with exudate accumulation affecting efux or inux of compounds, and thus percolating systems may better reect a rhizosphere environment where microbes are present. However, percolating solutions have a disadvantage in that they generally require a much greater volume of solution, and several post-collection steps may be necessary to for subsequent chemical analyses. In addition, percolating solutions may not uniformly collect exudates released by roots if the solution follows preferential owpaths in the medium. Because percolating solutions require the continuous pumping of solution through the medium, they may also be less amenable for adaptation to eld studies. In both static and percolating systems, the type of growth medium selected is known to have important consequences for root growth, architecture and exudation. Pure solution cultures (generally a dilute salt or nutrient solution) offer simplicity in maintenance and sample collection. However, the lack of mechanical impedance for roots in non-solid media may affect root morphology and exudation rates (Neumann & Romheld 2001). Small glass beads and acid-washed sand have commonly been employed but such media have limitations as well, as sorption of exuded compounds to these media may result in incomplete recovery of exudates or mischaracterization of the exudate composition (Neumann & Romheld 2001). In our own experiments, we have found that acid-washed sand (3 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 as small amounts of sorbed C or contamination of C from the medium can introduce substantial artefacts to estimated exudation rates and compositional analyses. An important consideration in adapting either trapping system for eld use is the question of sterility of the culture medium. Sterile/axenic culture systems have an advantage in that exudates released from roots will not be metabolized by microbes in the medium, thereby allowing exudation rates to be measured without needing to account for microbial assimilation of released of compounds (Uselman et al. 2000).

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R. P. Phillips et al. and root systems, and the varying degrees of colonization of tree roots by a diverse assemblage of mycorrhizal fungi all of which would presumably affect exudation rates and the chemical composition of the C released (Grayston et al. 1996; Scott-Denton et al. 2006; Van Scholl et al. 2006). Moreover, exudation rates can be scaled to the ecosystem-level using this method so that the consequences of this ux for microbial activity and nutrient cycling can be better quantied.

However, the presence of microbes may stimulate exudation by maintaining a concentration gradient around roots or by inducing exudation via the release of signalling compounds (reviewed in Grayston et al. 1996; Paterson et al. 1997; Neumann & Romheld 2001). Using a sterile medium may thus reduce exudation rates and affect the composition of exudates. The issue of sterility is even more vexing for studies in forest soils as most roots are likely to be extensively colonized 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 efcacy of any antibiotic treatment will likely depend on the type and concentration of antibiotics used as well as the plant species of interest (Neumann & Romheld 2001). In addition to trapping solutions, localized sampling techniques have been used to collect exudates. Agar, specialized resins or lter paper can be placed along desired sections of the root axis to collect compounds released from distinct areas of the root (Neumann & Romheld 2001). These techniques have the benet of providing information on ne-scale spatial patterns of exudation, and can be coupled to experiments with bacteria cloned with reporter gene systems (Killham & Yeomans 2001; Cardon & Gage 2006). Non-sterile conditions are less likely to bias results, as exudates trapped by resins will be unavailable to soil microbes. However, use of resins, lters, etc. may not be ideal when looking for unknown compounds, as the a priori choice of resin will inuence which compounds are trapped. An additional caveat is that exudation rates cannot be readily quantied. Thus, application of this method for eld studies might be appropriate in cases where exudate composition, rather than rates, is of interest. Localized sampling of exudates can also be performed using microsuction cups as tension lysimeters (Gottlein & Blasek 1996; Sandnes, Eldhuset & Wollebaek 2005). This method has advantages in that the exudates are removed from soil before they can be metabolized by microbes, and the cups can be repeatedly sampled to examine temporal trends. However, a major limitation of this method is that only small volumes of solution can be collected under most eld conditions (e.g. irrigation is often necessary), and ux rates cannot be quantied. Moreover, this method must be employed through rhizoboxes or root windows (DessureaultRompre et al. 2006; Shen & Hofand 2007), and thus only a small volume of soil is generally sampled. In this manuscript, we describe our use of a culture-based system for measuring exudation from tree roots which is relatively inexpensive, easy to deploy, and requires minimal maintenance. In addition, this method can account for the spatial heterogeneity and temporal dynamics of forest soils

Materials and methods

SITE DESCRIPTION

The Duke Forest-Atmosphere Carbon Transfer and Storage (FACTS-1) was established in a loblolly pine (Pinus taeda L.) plantation in Orange County, North Carolina (3558N, 7905). The site is dominated by the pine (> 90% of the basal area), although hardwoods have developed in the understorey since the initial planting in 1983. In 1996, replicated 30 m diameter plots containing c.100 trees per plot were established at the site. Three experimental plots at the site are fumigated with exogenous CO2 to maintain an atmospheric concentration c. 200 ppmv above ambient levels (i.e. c. 585 ppmv) while three plots are fumigated with air only (i.e. c. 385 ppmv). The mean annual temperature of the site is 155 C and mean annual precipitation at the site is 11 140 mm year1. Soils at the site (Enon Series) are highly-weathered clay loams (mixed thermic Ultic Hapludalfs) developed from igneous parent materials, and are moderately acidic (pH = 56). In general, the vast majority of the roots (> 90%) reside in the upper 30 cm of soil. Detailed descriptions of the experimental set-up and the site characteristics can be found in Hendrey (1999) and Lichter et al. (2005), respectively. Average daily temperature and photosynthetically active radiation (PAR) were collected during the days of exudate collection from April 2007 to July 2008 (Table 1). These meteorological variables are strongly linked to C assimilation by loblolly pines at the site (Hendrey et al. 1999). Air temperature was collected at 1 min intervals at a mid-canopy location in the middle of each plot using shielded thermocouple with an accuracy of c. 1 C. PAR data were collected at 1 min intervals using a LiCor Quantum sensor (Licor Co., Lincoln, NE) mounted above the forest canopy. Average daily temperature and PAR were calculated from hourly means for each day of exudate collection, and then averaged for each 3 4-day collection period (Table 1).

EXUDATE COLLECTION

Soluble root exudates were collected from loblolly pine trees at 4 8-week intervals from April 2007 to July 2008 using a modication of a static culture-based system which was adapted for eld use (Fig. 1). In this system, intact ne root systems were washed free of adhering soil and incubated in glass cuvettes lled with sterile glass beads 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; mol m2 s1) at the top of the canopy
17/04/2007 Tavg 164 PARavg 1088 27/06/2007 281 950 16/07/2007 285 999 27/08/2007 295 911 25/09/2007 263 937 29/10/2007 153 760 4/12/2007 39 429 25/02/2008 44 637 25/03/2008 150 1024 5/05/2008 211 896 15/07/2008 263 1047

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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 ushing each cuvette with a vacuum pump. Similar designs using bead-lled cuvettes have been used to collect root and mycorrhizal exudates from seedlings of Pinus sylvestris (Ahonen-Jonnarth et al. 2000; Van Scholl et al. 2006), Picea abies [L]. Karst. and Betula pendula Roth (Sandnes et al. 2005) and loblolly pine (Phillips et al. 2008). Similar static bathing solutions have been used to measure nutrient uptake of pine trees in the eld (Lucash et al. 2005). However, this study is the rst study to our knowledge to use this type of system to measure root exudation for eld-grown trees.

EXCAVATION AND CUVETTE ASSEMBLY

Terminal ne root systems (< 2 mm diameter with laterals) were carefully excavated from soil (sensu Lucash et al. 2005) at the interface between the O and A horizons within 05 m from the bole of each

tree. In order to ensure that roots were loblolly pine, all root systems were traced back to a parent tree or coarse root with characteristics unique to loblolly pine (e.g. reddish aky bark). Once unearthed, root segments (1520 cm length) were rinsed with a squirt bottle containing nutrient solution (to minimize osmotic stress to the root) to remove adhering soil. Fine forceps were also used to dislodge adhering soil. Given the intimacy between the roots, mycorrhizal hyphae and organic matter, we took extreme caution to remove soil 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 the root cuvettes (generally 24 48 h later). The re-burial was intended to allow additional time for roots to recover from any potential injury or stress sustained during the excavation and rinsing process. Sieved soil mixed with sand was used to facilitate removal of the soil (by squirt bottle) before placement into the root cuvettes.

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R. P. Phillips et al.

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

Soil-free root systems were placed into a 30-mL glass syringe (Popper & Sons, New Hyde Park, NY) from which the plunger was removed, and each syringe was back-lled with sterile acid-washed glass beads (c. 750 m diameter; La De Da Designs, Baton Rouge, LA). At the narrow end of each syringe, a 30 m mesh cloth was folded into a cone to support the beads and to prevent them from clogging the syringe outlet during removal of the solution via a vacuum pump (Fig. 1). At the top of each syringe, a rubber septum with a small slit cut to accommodate the protruding root was used to seal off each cuvette. To protect the exposed portion of the root from drying out, a moist Kimwipe (Kimberly-Clark Corp., Roswell, GA) was placed around the upper root segment and secured with Paralm (3 Company, Minneapolis, MN). A small volume of dilute nutrient solution (see below) was added to the cuvette to maintain humid conditions during the incubation. The cuvettes were then covered in aluminum foil, returned to the excavated area, and covered with several layers of needle litter to allow the root system to equilibrate with the cuvette environment. Three control cuvettes lled with glass beads only were similarly covered and buried in soil (one in each plot).

procedure described above. We found that three ushes were sufcient to remove over 90% of the soluble C and N in each cuvette in a separate recovery experiment using spiked solutions of low and high C and N concentration (Fig. 2). Following each incubation period, all cuvettes were reopened and glass beads rinsed from roots. We did not analyse the beads following their removal after preliminary tests suggested little residual C accumulation on the beads (data not shown). Washed roots were removed from the tree, photographed, imaged, and analysed for root morphological characteristics using WinRhizo (Regents Instruments Inc., Qubec, Canada). All solutions were ltered immediately through a sterile 022 m syringe lter (Millex PVDF, Millipore Co., Billerica, MA), and refrigerated at 4 C until analyses (< 48 h). All samples were analysed for non-particulate organic C and total N on a TOC analyser (Shimadzu Scientic Instruments, Columbia, MD). Only the C analyses are presented in this article.

EXPERIMENTAL TESTS

EXUDATE COLLECTION

After a 23 day equilibration period, a dilute nutrient solution was added to each glass cuvette to facilitate the removal of accumulated exudates. In order to ensure the complete removal of C from the cuvette before the experimental incubation period, each cuvette was lled to saturation with C-free nutrient solution (05 m NH4NO3, 01 m KH2PO4, 02 m K2SO4, 02 m MgSO4, 03 m CaCl2) and ushed with a vacuum pump. This process was repeated ve consecutive times. After the fth ush, a small volume of nutrient solution was added, and the root-lled cuvette was re-buried as before. At the end of each incubation period, cuvettes were ushed three times with a C-free nutrient solution to remove accumulated exudates using the

Experiment 1: Exudate accumulation and incubation duration

An important consideration for static bathing solution culturesystems is the degree to which exudation rates are affected by the changing C concentration in the trap solution and the potential reuptake of exudates by roots (Jones et al. 2004; Personeni et al. 2007). We initiated a pilot experiment to examine this potential source of bias using loblolly pine roots from two plots (one exposed to ambient CO2, the other elevated CO2) at the Duke Forest FACTS-1 site in July, 2007. We collected exudates at 0, 4, 8, 16, 21, 24 h from a total of eight root systems (two per tree) over four consecutive days (July 1720). Because of the time needed to ush and extract the solutions,

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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 inuence of diurnal variation on exudation (Neumann & Romheld 2001). Root-free controls were ushed at the same time intervals to serve as controls.

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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 sufcient magnitude to be analytically detectable. In both samplings, cuvettes were ushed ve times between dates to ensure that no exudates remained from the incubation of the previous day. Exudates were collected with three ushes and analysed for non-particulate organic C on a TOC analyser (as described above).

Fig. 3. Carbon efux from loblolly pine roots at the Duke FACTs-1 site, NC into a trap solution over the course of a week-long experiment in July, 2007. The data are best t by a logarithmic curve (y = 29205 Ln(x) + 43017; r2 = 094).

Experiment 3: Seasonal variation in exudation rates

We sought to quantify pine root exudation rates at the FACTS-1 site across a range of environmental conditions. Thus, we initiated an experiment to collect exudates every 4 8 weeks from April 2007 to July 2008 in three experimental plots (all at ambient CO2) which vary in annual net N mineralization rates by approximately a factor of three (Finzi & Schlesinger 2003). All exudates were collected from two or more root systems in each of the three plots with the exception of the April, June and July 2007 when exudates were collected from root systems in a single plot. All of the incubations for this experiment lasted for c. 24 h, beginning and ending in the early hours of the photoperiod to account for potential biases associated with diurnal variation in exudation (Neumann & Romheld 2001). All exudates were collected and processed as described above.

Results
EXPERIMENTS 1 AND 2

CALCULATIONS AND STATISTICS

In all three experiments, C accumulation in each cuvette was calculated as the sum of the three ushes minus the total C ushed from the root-free control cuvettes (units of g C). For experiment 2, Pearson correlation coefcients were used to examine day to day variation in C efux from 11 root systems. In addition, a paired t-test was used to evaluate whether the paired means were signicantly different from one another ( = 005). For experiment 3, we used analysis of variance () to examine differences in exudation rates across the N availability gradient of the three plots ( = 005). We also used to examine monthly and seasonal variation in exudation (n = 6 on each sampling date), and linear regression to examine the relationship between C efux and root morphological variables across seasons. Seasons were dened relative to the dates samples were collected: spring (April 2007 and May 2008), summer (June, July and August 2007, July 2008), fall (September and October 2007), and winter (December 2007, February and March 2008). Non-normally distributed data were transformed before statistical analyses with statistical software (v. 6, SAS Institute Inc., Cary, NC).

We evaluated whether C accumulation in the cuvette inuenced exudation by ushing a subset of cuvettes at 05, 4, 8, 16, 21 and 24 h. Carbon released from roots was still increasing over the rst 24 h, and the increase was best described by a logarithmic function ( y = 29205 Ln(x) + 43017; r2 = 094; Fig. 3). Thus, it seems unlikely that exudation rates were inuenced to a large degree by C accumulation in the cuvette. Given the shape of the curve, shorter incubation times (e.g. 8 h) may be desirable under some conditions. However, detecting individual compounds is probably difcult with shorter incubation times particularly under conditions where exudation rates are low. Moreover, a 24 h incubation period has the added advantage in that it spans the diurnal cycle, and thus provides a timeintegrated estimate that accounts for differences in exudation rates which occur between night-time and the peak hours of the day (Kuzyakov & Domanski 2000; Uselman et al. 2000). We examined short-term variation of exudation by collecting exudates from the same root systems on consecutive days in July and September 2007 (Fig. 4). Such variation is important to consider given that only a limited number of root systems can be sampled on a particular day. Overall, exudation rates were positively correlated across the 2-day period (r = 073), and there were no signicant differences between the paired data (P = 0142). Although we cannot directly compare the responses in July and September due to the small number of samples collected, our September data suggest a trend toward reduced rates on the second day. This may have resulted from the slight reduction in average daily air temperature and PAR from day 1 to day 2 (29 C and 926 mol m2 s1 on day 1; 28 C, 866 mol m2 s1on day 2).

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R. P. Phillips et al. unable to detect signicant 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 efux was well-predicted by root morphology but that such relationships depended on season. In the spring months, C efux was predicted by the ne root surface area (r2 = 061; P = 0008, Fig. 6a) and the number of root and mycorrhizal tips (r2 = 060; P = 0008, Fig. 6b). Similarly, C efux was predicted by the root surface area (r2 = 062; P = 0004, Fig. 6c) and the number of tips (r2 = 036; P = 005, Fig. 6d) in the fall months. In the summer months, C efux was somewhat correlated with surface area (r2 = 020) and tip number (r2 = 023) but these relationships were not signicant (data not shown). We found no signicant relationship between C efux and root morphological variables in the winter months (data not shown).

Fig. 4. Relationship between C efux from eleven intact root systems over a 2 day period at the FACTS-1 site. Cuvettes were ushed ve times between the days 1 and 2 collections. Open triangles were collected on 1718 July 2007 while lled triangle were collected on 26 27 September 2007.

Discussion
We have demonstrated a viable new method for collecting exudates from intact root systems of mature trees in the eld 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 (iv) can be deployed anywhere at relatively low costs. While we believe our method is a substantial step forward, linking exudation from a few individual root systems to ecosystem carbon and nutrient cycling presents some formidable challenges.

EXPERIMENT 3

In general, exudation rates varied considerably, with the greatest variation occurring in the plot with the lowest N availability (Fig. 5). In this low N plot, exudation varied by nearly an order of magnitude from 027 g C cm1 root day1 in August 2007 to 242 g C cm1 root day1 in July 2008. In contrast, exudation rates in the plot with the highest N availability varied only by a factor of c. 2 across the same time period. Overall, there were no signicant differences in exudation rates among the plots (P = 097). Averaging across all three plots, we were also

ACCOUNTING FOR SPATIOTEMPORAL VARIABILITY

Accounting for the spatial and temporal variability is a persistent challenge in all root and rhizosphere studies

Fig. 5. Seasonal variability of mass-specic 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 ux from the three plots (n = 3) over the same time interval. 2008 The Authors. Journal compilation 2008 British Ecological Society, Functional Ecology, 22, 990999

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Fig. 6. Relationship between C efux 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 efux and ne root surface area while panels on the right (b, d) represent the relationship between C efux and the number of ne root and mycorrhizal tips. The relationship between C efux and pine root morphology was not statistically signicant for data collected in December 2007 to March 2008.

(Hinsinger et al. 2005). Such heterogeneity presents logistical challenges in deciding how many roots need to be sampled to capture the variability of the site, and how frequently exudates need to be collected and at what time of day in order to account for temporal variation. In our analyses, we sought to reduce some of the variation in exudation by normalizing rates by ne root surface area as exudation is predicted relatively well by this variable (Fig. 6). Still, we were unable to detect signicant effects of month or season on exudation rates. Temporal variation in exudation was low at the daily/weekly scale (Fig. 4) but high over the 15-month period (Fig. 5). The high degree of variability over the course of the study was particularly evident in the plot with the lowest N availability where exudation rates varied by an order of magnitude. Because soils with low N availability often exhibit greater ne root production and mortality (Nadelhoffer et al. 1985), some of the variation in exudation may have resulted from the variable ages of roots in these plots. Fine roots at this site live on average from 1 and 4 years (Matamala et al. 2003; Pritchard et al. 2008a; Strand 2008), and ne root production and mortality at this site occur simultaneously rather than in discrete pulses (King et al. 2002). Such differences in root life span would presumably inuence exudation rates due to the physical and chemical changes that occur as ne roots age (Jones et al. 2004). An additional source of variation may be the colonization of roots by mycorrhizal fungi. Parrent et al. (2006) estimated that loblolly pine roots in these plots are colonized by 64 different ectomycorrhizal taxa, with the highest degree of phylotype richness occurring in the plots with the lowest N availability. Because mycorrhizal taxa vary in their effects on root C efux (Ahonen-Jonnarth et al. 2000; Van Scholl et al. 2006), variability in exudation

rates at the site may reect the spatio-temporal differences in fungal taxa colonizing each root system. Although we did not detect a signicant seasonal effect, exudation rates followed the general pattern of C allocation to ne roots (Pritchard et al. 2008a) and mycorrhizal fungi (Pritchard et al. 2008b) at the FACTS-1 site, with the greatest uxes occurring in late-spring/early summer and mid-fall. Moreover, exudation was well-predicted by the number of root and mycorrhizal tips in each cuvette. These results are consistent with reports of tips being active sites of exudation (Neumann & Romheld 2001), and suggest that mycorrhizal tips may not only be a sink for exudates but a source as well (Grayston et al. 1996). Perhaps more importantly, the results suggest that root images collected from other sources (e.g. minirhizotrons) might provide a way to derive estimates of this ux at the ecosystem-scale. Arguably, the biggest challenge to understanding the role of exudation in forest ecosystems is not merely developing a collection method, but developing an appropriate scaling factor so that annual ecosystem uxes (e.g. in units of g C m2 year1) can be calculated. A simple calculation of the annual contribution of exudates can be made by multiplying the average mass-specic exudation rate from the 15-month sampling period (103 105 g C ne root biomass1 day1) by the average ne root biomass in the O horizon +015 cm of soil in these plots (c. 250 g ne root biomass m2; R. Jackson, unpublished data). Multiplying this ux by 365 days yields an estimated annual ux of 94 g C m2 year1. This value represents 15% of the net primary production (NPP) in the ambient plots at FACTS-1 (625 g C m2 year1; from McCarthy 2006), and closely approximates estimates derived from modelling approaches (c. 1%) at this site (Luo et al. 2001). This estimate

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R. P. Phillips et al. lines of evidence suggest that these uxes are probably appreciable in magnitude and highly responsive to the biotic and abiotic changes anticipated under global environmental change. Thus, a concerted effort is needed to develop novel methods for measuring this process in situ, possibly through adapting commonly used lab methods for eld use. Our method provides an important rst step for measuring exudation from the intact roots of mature trees in forest soils. Although previous methods have been employed for measuring exudation rates from tree roots in situ (Smith 1976), our method offers the advantage that it is inexpensive, relatively simple to set-up, can be employed throughout the growing season, and requires only a minimal amount of soil disturbance. Moreover, our system allows for the chemical characterization of exudates, and thus should be useful in assessing the role of exudates in stimulating microbial activity, nutrient release and the decomposition of soil organic matter in forest soils. The primary caveat of the method is that the disruption of root and mycorrhizal networks present an unknown source of error to the estimates. Scaling up to ecosystem rates poses a further challenge owing to the temporal and spatial variability of exudation. Nevertheless, these uncertainties are an 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 sustained effort to develop in situ methods for exudation will greatly improve our understanding of the role of below-ground processes in mediating ecosystem response to global change. However, it is important to emphasize that no one method is likely to be appropriate in all cases, and we suggest that a suite of complementary approaches (trap solutions, isotopes, reporter gene systems, microlysimetry, etc.) is likely to yield the most information on the role of rhizosphere processes in forest ecosystems.

is probably conservative given that ne roots at lower soil depths, which may account for 3050% of the total standing crop of ne root biomass at the site (Pritchard et al. 2008a), were not sampled. Thus, annual exudation rates may be closer to 3% of net primary production in this forest. The issue of spatial and temporal variability poses a further challenge for understanding the consequences of changing exudation patterns under global change. At the Duke Forest FACTS-1 site, we have also collected exudates from trees exposed to elevated CO2 and N fertilization. Preliminary results from this experiment suggest a consistent CO2 by N interaction over several months of the growing season where CO2-induced increases in mass-specic exudation are greatest at low soil N availability (data not shown). This result is consistent with the exudation response of loblolly pine seedlings grown in a controlled environmental growth chamber (R.P. Phillips, E.S. Bernhardt & W.H. Schlesinger, unpublished) and gives us increased condence in the potential for both independent methods to provide reliable estimates of the exudate response to each treatment. Linking the patterns and rates of exudation in seedlings in growth chambers to those of mature trees in the eld may allow us to overcome the shortcomings and artefacts introduced by each method individually.

FUTURE DIRECTIONS

In this study, we have described a new method for collecting exudates from intact roots of loblolly pine trees, and presented data collected from over 60 root systems over a 15 month period. Given the high degree of spatial and temporal variability of this ux, we suggest that exudates will likely need to be collected over multiple years and analysed using repeated measures similar to the approach used to understand ne root dynamics at this site (Pritchard et al. 2008a) to understand seasonal patterns in this ux. Because the method allows for the characterization of exudate composition, realistic exudate addition experiments should also be used to examine the consequences of changing exudation patterns on soil processes (Landi et al. 2006; Kuzyakov et al. 2007; Paterson et al. 2007). An important limitation of the method is that although root systems are left intact, the rhizosphere is not left intact due to the separation of roots and soil; this will almost certainly result in some severing of mycorrhizal fungal hyphae and may inuence the ux of C from roots. Whether the latter issue can be overcome by keeping roots in the cuvettes long enough to encourage growth of extramatrical hyphae (sensu Van Scholl et al. 2006) warrants further investigation. This issue may also be resolved by coupling trap solution approaches with methods such as isotopic labelling (Hogberg 2008) and reporter gene systems (Cardon & Gage 2006) which permit the sampling of intact rhizosphere systems.

Acknowledgements
Authors thank Elise Pendall, Lindsay Rustad and Josh Schimel for inviting us to participate in the symposium Towards a Predictive Understanding of Below-ground Ecosystem Responses to Global Change at the 2006 SSSA meeting in Indianapolis, IN. Authors also thank Robert Nettles and David Cooley for their technical assistance at the Duke Forest FACTS1 site, Robert Jackson, Seth Pritchard, Adrien Finzi and Jeri Parrent for their insight and willingness to share unpublished data, and the members of the Bernhardt Lab for providing suggestions for improving this article. The bulk of this research was supported by the DOE FACTS-1 grant. Additional funds were provided by the Ofce of Science (BER), US Department of Energy, Grant No. DE-FG0295ER62083. All meteorological data was provided courtesy of the U.S. Department of Energy Contract No. DE-AC02-98CH10886 with Brookhaven National Laboratory.

References
Ahonen-Jonnarth, U., Van Hees, P.A.W., Lundstrom, U.S. & Finlay, R.D. (2000) Organic acids produced by mycorrhizal Pinus sylvestris exposed to elevated aluminium and heavy metal concentrations. New Phytologist, 146, 557567. Bais, H.P., Weir, T.L., Perry, L.G., Gilroy, S. & Vivanco, J.M. (2006) The role of root exudates in rhizosphere interations with plants and other organisms. Annual Review of Plant Biology, 57, 233266. Cardon, Z.G. & Gage, D.J. (2006) Resource exchange in the rhizosphere: molecular tools and the microbial perspective. Annual Review of Ecology Evolution and Systematics, 37, 459488.

Conclusions
The causes and consequences of root exudation in forest ecosystems remain poorly resolved owing to difculties in measuring rhizosphere processes. Nevertheless, several indirect

2008 The Authors. Journal compilation 2008 British Ecological Society, Functional Ecology, 22, 990999

Root exudation in trees

Cheng, W.X. (1999) Rhizosphere feedbacks in elevated CO2. Tree Physiology, 19, 313 320. Dessureault-Rompre, J., Nowack, B., Schulin, R. & Luster, J. (2006) Modied micro suction cup/rhizobox approach for the in-situ detection of organic acids in rhizosphere soil solution. Plant and Soil, 286, 99107. Fahey, T.J., Tierney, G.L., Fitzhugh, R.D., Wilson, G.F. & Siccama, T.G. (2005) Soil respiration and soil carbon balance in a northern hardwood forest ecosystem. Canadian Journal of Forest Research, 35, 244253. Finzi, A.C. & Schlesinger, W.H. (2003) Soil nitrogen cycling in a pine forest exposed to 5 years of elevated carbon dioxide. Ecosystems, 6, 444456. Gottlein, A. & Blasek, R. (1996) Analysis of small volumes of soil solution by capillary electrophoresis. Soil Science, 161, 705715. Grayston, S.J., Vaughan, D. & Jones, D. (1996) Rhizosphere carbon ow in trees, in comparison with annual plants: the importance of root exudation and its impact on microbial activity and nutrient availability. Applied Soil Ecology, 5, 2956. Hendrey, G.R., Ellsworth, D.S., Lewin, K.F. & Nagy, J. (1999) A free-air enrichment system for exposing tall forest vegetation to elevated atmospheric CO2. Global Change Biology, 5, 293309. Hinsinger, P., Gobran, G.R., Gregory, P.J. & Wenzel, W.W. (2005) Rhizosphere geometry and heterogeneity arising from root-mediated physical and chemical processes. New Phytologist, 168, 293303. Hogberg, P. (2008) High temporal resolution tracing of photosynthate carbon from the tree canopy to forest soil microorganisms. New Phytologist, 177, 220228. Jones, D.L., Hodge, A. & Kuzyakov, Y. (2004) Plant and mycorrhizal regulation of rhizodeposition. New Phytologist, 163, 459480. Killham, K. & Yeomans, C. (2001) Rhizosphere carbon ow measurement and implications: from isotopes to reporter genes. Plant and Soil, 232, 9196. King, J.S., Albaugh, T.J., Allen, H.L., Buford, M., Strain, B.R. & Dougherty, P. (2002) Belowground carbon input to soil is control led by nutrient availability and ne root dynamics in loblolly pine. New Phytologist, 154, 389398. Kuzyakov, Y. & Cheng, W. (2001) Photosynthesis controls of rhizosphere respiration and organic matter decomposition. Soil Biology and Biochemistry, 33, 1915 1925. Kuzyakov, Y. & Domanski, G. (2000) Carbon input by plants into the soil. Review. Journal of Plant Nutrition and Soil Science, 163, 421431. Kuzyakov, Y., Hill, P.W. & Jones, D.L. (2007) Root exudate components change litter decomposition in a simulated rhizosphere depending on temperature. Plant and Soil, 290, 293305. Landi, L., Valori, F., Ascher, J., Renella, G., Falchini, L. & Nannipieri, P. (2006) Root exudate effects on the bacterial communities, CO2 evolution, nitrogen transformations and ATP content of rhizosphere and bulk soils. Soil Biology and Biochemistry, 38, 509516. Lichter, J., Barron, S.H., Bevacqua, C.E., Finzli, A.C., Irving, K.E., Stemmler, E.A. & Schlesinger, W.H. (2005) Soil carbon sequestration and turnover in a pine forest after 6 years of atmospheric CO2 enrichment. Ecology, 86, 18351847. Lucash, M.S., Joslin, J.D. & Yanai, R.D. (2005) Temporal variation in nutrient uptake capacity by intact roots of mature loblolly pine. Plant and Soil, 272, 253262. Luo, Y.Q., Wu, L.H., Andrews, J.A., White, L., Matamala, R., Schafer, K.V.R. & Schlesinger, W.H. (2001) Elevated CO2 differentiates ecosystem carbon processes: deconvolution analysis of Duke Forest FACE data. Ecological Monographs, 71, 357376. Lynch, J.M. (1990) The Rhizosphere. John Wiley & Sons, West Sussex, UK. Marschner, H. (1995) Mineral Nutrition of Higher Plants. Academic Press, London. Matamala, R., Gonzalez-Meler, M.A., Jastrow, J.D., Norby, R.J. & Schlesinger, W .H. (2003) Impacts of ne root turnover on forest NPP and soil C sequestration potential. Science, 302, 1385. McCarthy, H.R. (2006) Canopy leaf area constrains [CO2]-induced enhancement of productivity and partitioning among aboveground carbon pools. Proceedings of the National Academy of Sciences of USA, 103, 1935619361.

999

Nadelhoffer, K.J., Aber, J.D. & Melillo, J.M. (1985) Fine roots, net primary production, and soil-nitrogen availability A new hypothesis. Ecology, 66, 1377. Neumann, G. & Romheld, V. (2001) The Release of Root Exudates as Affected by the Plants Physiological Status. The Rhizosphere: Biochemistry and Organic Substances at the Soil-Plant Interface (eds R. Pinto, Z. Varanini & P. Nannipieri), pp. 4193. Dekker, New York. Parrent, J.L., Morris, W.F. & Vilgalys, R. (2006) CO2-enrichment and nutrient availability alter ectomycorrhizal fungal communities. Ecology, 87, 2278 2287. Paterson, E. (2003) Importance of rhizodeposition in the coupling of plant and microbial productivity. European Journal of Soil Science, 54, 741750. Paterson, E., Gebbing, T., Abel, C., Sim, A. & Telfer, G. (2007) Rhizodeposition shapes rhizosphere microbial community structure in organic soil. New Phytologist, 173, 600610. Paterson, E., Hall, J.M., Rattray, E.A.S., Grifths, B.S., Ritz, K. & Killham, K. (1997) Effect of elevated CO2 on rhizosphere carbon ow and soil microbial processes. Global Change Biology, 3, 363377. Personeni, E., Nguyen, C., Marchal, P. & Pages, L. (2007) Experimental evaluation of an efux-inux model of C exudation by a individual apical root segments. Journal of Experimental Botany, 58, 20912100. Phillips, R.P. (2007) Towards a rhizo-centric view of plant-microbial feedbacks under elevated atmospheric CO2. New Phytologist, 173, 664667. Pritchard, S.G., Strand, A.E., Mccormack, M.L., Davis, M.A. & Oren, R. (2008b) Mycorrhizal and rhizomorph dynamics in a loblolly pine forest during 5 years of free-air-CO2-enrichment. Global Change Biology, 14, 1252 1264. Pritchard, S.G., Strand, A.E., Mccormack, M.L., Davis, M.A., Finzi, A., Jackson, R.B., Matamala, R., Rogers, H.H. & Oren, R. (2008a) Fine root dynamics in a loblolly pine forest are inuenced by free-air-CO2-enrichment: a 6-year-minirhizotron study. Global Change Biology, 14, 115. Read, D.J. (2002) Towards Ecological Relevance Progress and Pitfalls in the Path Towards an Understanding of Mycorrhizal Functions in Nature. Mycorrhizal Ecology (eds M.G.A. Van Der Heijden & I. Sanders), pp. 3 32. Springer-Verlag, Berlin, Heidelberg. Sandnes, A., Eldhuset, T.D. & Wollebaek, G. (2005) Organic acids in root exudates and soil solution of Norway spruce and silver birch. Soil Biology and Biochemistry, 37, 259269. Scott-Denton, L.E., Rosenstiel, T.N. & Monson, R.K. (2006) Differential controls by climate and substrate over the heterotrophic and rhizospheric components of soil respiration. Global Change Biology, 12, 205216. Shen, J.B. & Hofand, E. (2007) In situ sampling of small volumes of soil solution using modied micro-suction cups. Plant and Soil, 292, 161169. Smith, W.H. (1976) Character and signicance of forest tree root exudates. Ecology, 57, 324331. Strand, A.E. (2008) Irreconcilable differences: ne-root life spans and soil carbon persistence. Science, 319, 456458. Uselman, S.M., Qualls, R.G. & Thomas, R.B. (2000) Effects of increased atmospheric CO2, temperature, and soil N availability on root exudation of dissolved organic carbon by a N-xing tree (Robinia pseudoacacia L.). Plant and Soil, 222, 191202. Van Hees, P.A.W., Jones, D.L., Finlay, R., Godbold, D.L. & Lundstomd, U.S. (2005) The carbon we do not see the impact of low molecular weight compounds on carbon dynamics and respiration in forest soils: a review. Soil Biology and Biochemistry, 37, 113. Van Scholl, L., Hofand, E. & Van Breemen, N. (2006) Organic anion exudation by ectomycorrhizal fungi and Pinus sylvestris in response to nutrient deciencies. New Phytologist, 170, 153163. Wardle, D.A. (2002) Communities and Ecosystems: Linking the Aboveground and Belowground Components. Princeton University Press, Princeton, Oxford. Received 27 November 2007; accepted 15 September 2008 Handling Editor: Lindsey Rustad

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