Annals of Botany 108: 221 230, 2011 doi:10.1093/aob/mcr102, available online at www.aob.oxfordjournals.

org

TECHNICAL ARTICLE

Is the simple auger coring method reliable for below-ground standing biomass estimation in Eucalyptus forest plantations?
2,3 and Joseph Levillain1,2, Armel Thongo MBou 1, Philippe Deleporte2, Laurent Saint-Andre 2, Christophe Jourdan *
et la Productivite des Plantations Industrielles, BP 1291, Pointe Noire, Republic CRDPI, Centre de Recherche sur la Durabilite ochimie des of Congo, 2CIRAD, UMR Eco&Sols, 2 Place Viala, F-34060 Montpellier, France and 3INRA, UR 1138 - BEF, Bioge ` mes Forestiers, F-54280 Champenoux, France Ecosyste * For correspondence. E-mail christophe.jourdan@cirad.fr
Received: 14 October 2010 Returned for revision: 22 December 2010 Accepted: 18 March 2011 Published electronically: 13 May 2011
1

Background and Aims Despite their importance for plant production, estimations of below-ground biomass and its distribution in the soil are still difcult and time consuming, and no single reliable methodology is available for different root types. To identify the best method for root biomass estimations, four different methods, with labour requirements, were tested at the same location. Methods The four methods, applied in a 6-year-old Eucalyptus plantation in Congo, were based on different soil sampling volumes: auger (8 cm in diameter), monolith (25 25 cm quadrate), half Voronoi trench (1.5 m3) and a full Voronoi trench (3 m3), chosen as the reference method. Key Results With the reference method (01m deep), ne-root biomass (FRB, diameter , 2 mm) was estimated at 1.8 t ha21, medium-root biomass (MRB diameter 210 mm) at 2.0 t ha21, coarse-root biomass (CRB, diameter . 10 mm) at 5.6 t ha21 and stump biomass at 6.8 t ha21. Total below-ground biomass was estimated at 16.2 t ha21 (root : shoot ratio equal to 0.23) for this 800 tree ha21 eucalypt plantation density. The density of FRB was very high (0.56 t ha21) in the top soil horizon (03 cm layer) and decreased greatly (0.3 t ha21) with depth (50100 cm). Without labour requirement considerations, no signicant differences were found between the four methods for FRB and MRB; however, CRB was better estimated by the half and full Voronoi trenches. When labour requirements were considered, the most effective method was auger coring for FRB, whereas the half and full Voronoi trenches were the most appropriate methods for MRB and CRB, respectively. Conclusions As CRB combined with stumps amounted to 78 % of total below-ground biomass, a full Voronoi trench is strongly recommended when estimating total standing root biomass. Conversely, for FRB estimation, auger coring is recommended with a design pattern accounting for the spatial variability of ne-root distribution. Key words: Eucalypt plantation, Eucalyptus urophylla, E. grandis, root biomass, auger, monolith, Voronoi trench, accuracy and precision, labour requirements, Congo.

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IN T RO DU C T IO N Below-ground biomass is an important part of the biosphere and may amount to approx. 30 % of above-ground biomass (Grier et al., 1981; Van Noordwijk et al., 1996; Canellas Rey de Vinas and San Miguel Ayanz, 2000). In addition, ne-root production has been found to be equivalent to, or greater than, above-ground litterfall in a large number of forests, and may account for more than half of plant net primary production (NPP) (Keyes and Grier, 1981; Burke and Raynal, 1994; Fahey and Hughes, 1994). For example, in northern America, . 66 % of the NPP of conifer stands has been attributed to root production (Grier et al., 1981). Furthermore, while net ecosystem exchange can be estimated from eddy ux data (Papale et al., 2006), it can also be assessed from NPP and heterotrophic CO2 respiration (from both above- and below-ground litter decomposition). In the second approach, root production and root turn-over are the key quantities to be assessed to get proper values of NPP and net ecosystem exchange (Matamala et al., 2003; Norby et al., 2004).

One of the most common methods for root biomass estimation is the root : shoot ratio, where root biomass is estimated from easily measured shoot biomass (Mokany et al., 2006). This method is now widely used to estimate below-ground biomass and carbon stocks (Cairns et al., 1997; Snowdon et al., 2000; Eamus et al., 2002), but it is not really precise enough for this purpose due to the considerable variability encountered in the data (Brown, 2002). This variability is probably due not only to natural variability in forests or plantations or the use of different sampling methods, but also to the lack of a systematically, statistically rigorous experimental design executed with the same sampling methods (Brown, 2002). Moreover, reliable estimation of root biomass requires that the root : shoot ratio applied is representative in all systems being studied (Snowdon et al., 2000), which is not the case in all studies (Mokany et al., 2006). Root biomass can also be evaluated by another indirect method, without digging, using allometric equations or et al., 2005), but such equations/models models (Saint-Andre need to be calibrated on large amounts of data collected by reliable excavation methods.

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Levillain et al. Below-ground biomass estimation in forest plantations cropping system. For ne and medium roots, the choice is somewhat determined from the researchers personal preference, experience, equipment, the time taken and/or available nances, rather than accuracy and precision. In this study, the objective was to carry out a comparison in the same area, within the same plot, over the same time span and on the same trees. The experiment took place in large plantations of eucalypt trees near Pointe Noire in Congo (6 years old which is nearly the age of harvesting in that area). Four direct root excavation methods were used to assess ne ( , 2 mm), medium (2 10 mm) and coarse ( . 10 mm) root biomass: two methods using small-volume soil samples (8-cm diameter auger and 25 25 cm2 monolith coring method) were compared with two methods using highvolume soil samples (half and full 3-m3 trench excavation technique). Lastly, the accuracy and precision of the four methods were assessed with regard to labour requirements and nancial costs. M AT E R I A L S A N D M E T H O D S
Study site area and plant material
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Despite the importance of below-ground parts in plant production, estimations of root mass and its distribution in the prole by direct methods are still very difcult and time consuming, and no single reliable study methodology is available (Vogt et al., 1998). Difculties in harvesting roots in their totality, particularly for deep root systems (Stone and Kalisz, 1991; Nepstad et al., 1994, Canadell et al., 1996) may lead to global underestimates of root mass in forest ecosystems. Consequently, depths are not standardized, but the depth selected in a given study is assumed to capture practically all roots. Moreover, no distinctions are usually made between living and dead roots, so root biomass is generally reported as total living and dead roots. Furthermore, it is very difcult to compare, generalize and model root systems due to the scarcity of data and lack of accuracy and precision in the methodology used. Part of the problem lies in substantial below-ground spatial heterogeneity (Haynes and Gower, 1995; Vogt et al., 1998), and the highly variable allocation of photosynthates to roots (Vogt et al., 1996). In addition, ne-root dynamics are subject to many biotic and abiotic factors that vary in time and space. These factors include tree age and species, soil type, soil temperature and moisture, nutrient availability, as well as the impacts of insects, fungi and other soil organisms (Nadelhoffer et al., 1985; Hendrick and Pregitzer, 1993; Haynes and Gower, 1995; Gill and Jackson, 2000). Two types of direct methods have been used to estimate root biomass and involve sampling individuals or multi-tree plots (Snowdon et al., 2002). Single-tree excavation methods consist in removing the tree root system from the soil and tracing each root individually from the stump to root tip. Volumetric soil-root sample methods consist in excavating a given volume of soil and sorting the roots contained in that volume (Pierret et al., 2005). These volumetric samples range from traditional auger cores and monoliths to Voronoi polygons (Honda, 1978; Snowdon et al., 2002; Saint-Andre et al., 2005). With in situ imaging methods, roots are observed through a transparent tube (Lopez et al., 1998; Smit et al., 2000; Tierney and Fahey, 2002; Hendricks et al., 2006; Mainiero and Kazda, 2006) or a transparent pane of glass (Thongo Mbou et al., 2008) inserted into the soil. Biomass can be estimated by these imaging methods (Metcalfe et al., 2008) but it remains inaccurate, because they require the application of a correction factor to convert length to root mass (Steele et al., 1997). Of the direct methods, single-tree excavation is now considered as a standard for coarse-root biomass (Snowdon et al., 2002). Conversely, for ne and medium roots, it is well documented that all the above-mentioned techniques result in highly variable biomass estimates. Furthermore, comparing two different techniques often leads to contradictory results, because most of the studies were not carried out at the same sites (one method per site). For example, Millikin and Bledsoe (1999) found that the root mass density of blue oak using the monolith method was at least 50 % higher than that obtained by the core method for the youngest trees, while the opposite trend was obtained for larger trees. Conversely, Nissen et al. (2008) recorded a good agreement between the soil core and monolith methods for soybean root biomass, and a similar result was obtained by Jose et al. (2001) in a temperate alley

The experimental site was located at Kondi, in the Kouilou region, 30 km north-east of the town of Pointe-Noire, southwestern Congo (4 8S 12 8E). The ecological context was previously described in detail by Laclau et al. (2000). In brief, the site was located on a plateau, 100 m a.s.l., 1020 km from the sea. The soils were classied as Ferralic Arenosols (FAO); they were deep ( . 10 m), homogeneous in colour (grey on the surface and ochre at depth), structure (particulate) and texture ( . 85 % sand content) and chemically poor, except for phosphorus. The climate was characterized by a marked dry season from June to September and 1200 mm of annual rainfall (average of 50 years). The relative humidity was approx. 85 % on average ( + 2 %) and the average temperature was 25 8C with 5 8C of seasonal variations. The site was planted in April 2001 with the highly productive clone 18-52 resulting from a cross between Eucalyptus urophylla and E. grandis, from a tree breeding programme. This clone was chosen for its wide distribution in the local region and its numerous biomass and biogeochemical studies. The stand density was 800 stems ha21. The present study was carried out when the trees were 6 years old.
Sampling design

Four trees were selected after a forest inventory to cover the full range of basal area variations within the stand. Root excavations were performed in the Voronoi polygon (Honda, 1978, et al., 2005), which is the Snowdon et al., 2002, Saint-Andre elementary space dened by the half distances between the sampled tree and its neighbours (Fig. 1D). The hypothesis is that all the roots of the sampled tree that grow outside the Voronoi polygon are balanced by those of neighbouring trees growing inside the polygon. Variability between trees was taken into account by the number of replicates (four trees) and variability along the sampled tree was taken into account by subdividing the Voronoi space into four equal parts and by sampling each of them. A sampled tree was thus delineated by four Voronoi quarters hereafter called

Levillain et al. Below-ground biomass estimation in forest plantations

A
D C D I E G E M A Sampled tree T1 Sampled tree A P13 P12 P1 B A Sampled tree T2 Sampled tree A P1 P22 P23 P24 C P5 P1 D CP 5 x P2 D C P 5 y P24 B A Sampled tree T3 Sampled tree B P14 P13 P12 P1 P22 P23 A K C D H G E F M L B I K J C

223

B B
P14 y

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x P

Sampled tree

Sampled tree

V1 Half trench V2

Monolith

P5 P24 P23 P22 P14 P1 P13 P12 TA TF TG TE TM TL TB

V1

Half and full trench

V2
TD T
H

Full trench V =V1+V2

TI TJ TC

Superimposition of methods

TK

Auger

Neighbour trees

F I G . 1. Design patterns of the different root sampling methods: (A) auger, (B) monolith and (C) half and full Voronoi-trench excavation methods with three option designs in one single Voronoi quarter. All sampling methods were set up around the same sampled tree with the following organization (D): three quarters were selected for replication of methods (auger, monolith and half and full Voronoi trench) and one-quarter was selected to superimpose all the methods. All the quarters were chosen randomly. For the auger method (A), option T1 consisted of ve soil sampling cores located at the four corners and in the centre of the Voronoi quarter (ABCDE). Option T2 was the same as T1 plus four soil sampling cores (G, I, K and M) set in the centre of gravity of triangles AED, DEC, BEC and AEB, respectively. Option T3 was the same as T2 plus four soil sampling cores (H, J, L and F) set in the middle of each diagonal [DE], [CE], [BE] and [AE], respectively. For the monolith method (B) two main lines [Ax] and [Ay] were chosen (x: middle of [CD], y: middle of [BC]). Options P1 and P2 were set up with ve monolith sampling locations (P1, P12, P13, P14 and P5) and (P1, P22, P23, P24 and P5), respectively, and the P option design with eight monolith sampling locations (P1, P12, P13, P14, P22, P23, P24 and P5). For the Voronoi method (C), half Voronoi trenches (V1 or V2) were set up on half of the Voronoi quarter separated by the sampling tree diagonal. The full Voronoi trench (V V1 + V2) was a Voronoi quarter.

Voronoi trench. Four root biomass excavation methods were studied: auger sampling method (auger), monolith sampling method (monolith), half Voronoi-trench excavation (half trench), and full Voronoi-trench excavation (full trench). Each method was randomly allocated to each quarter.

Sampling methods Auger-core method. Coring was done with a root auger (8-cm

inner diameter) in three soil horizons (0 10, 10 50 and 50 100 cm called H1, H2 and H3, respectively). Root mat biomass ( + 3 0 cm, called H0; see Laclau et al., 2004) was

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Levillain et al. Below-ground biomass estimation in forest plantations from the decomposed litter and was dried at 65 8C to constant weight. Weeds and understorey shrubs were controlled by herbicide and no confusion between eucalypt roots and those of other species was possible.
Method comparison Labour requirements and cost benets. The time required for every task in the eld and in the laboratory was recorded: setting up the sampling design on each tree, excavation (coring or digging), sorting roots from soil (sieved or manually), washing and root-size classication. Oven drying and weighing were not taken into account since these operations were performed on sub-samples that were strictly equivalent between the four methods. Labour requirements were transformed into person-days (on a basis of 6 h of eld work per day). The cost/benets analysis between the different methods was applied to the raw dataset without taking account of tree size distribution within a stand. Accuracy and precision. Taking full Voronoi-trench excavation as the reference value, accuracies for auger-core, monoliths and the half Voronoi-trench methods were calculated as the relative differences between those methods and the reference method. Condence intervals at 95 % (IC95), which give the precision of the method, were calculated for each method assuming a Gaussian distribution: IC95 = 1.96 s/ N Npredicted = (1.96 s/IC95 )2

measured above H1, in the soil litter layer above ground. Three different sampling intensities were tested: T1 (ve cores: A, B, C, D and E), T2 (nine cores: T1 + G, I, K and M) and T3 (13 cores: T2 + F, H, J and L) (Fig. 1A).
Monolith method. For the H1 and H2 soil horizons 25 25 cm monoliths were sampled, and for the H3 soil horizon two auger cores (8-cm inner diameter) were collected in each monolith. The Voronoi trench was separated into two equal parts. Two samples (P1 and P5) were set up on the diagonal and were common to the two half trenches. Two lines were then drawn on each side of the diagonal (Fig. 1B) and three additional monoliths were equally distributed on each of these two lines starting from the edge of the trench. Half Voronoi trench. The half Voronoi trench was set up by dividing the entire Voronoi trench into two equal parts (Fig. 1C). The section to be excavated was randomly chosen. The three soil horizons were fully removed. Fine-root biomass values were then corrected by applying a conversion factor to account for losses due to manual sorting compared with the sieving techniques. The conversion factor was calculated by estimating the percentage of roots lost during sieving procedures. The average conversion factor was applied to the H1 soil horizon where the root density was assumed to be higher than the deeper horizons (Bouillet et al., 2002). Full Voronoi trench. This was the reference trench. All soil volume was excavated on the entire Voronoi trench (3 m3). Roots were manually separated from the soil for all horizons without sieving. The same average conversion factor used for the half Voronoi trench was also used here for ne-root biomass estimation. Superimposing of methods. The other three excavation methods were superimposed over the quarter allocated to the full Voronoi trench, for a strict comparison between them: same tree, same place and same period of observations (Fig. 1D). The auger-coring method was applied rst, followed by the monolith method and, nally, the half Voronoi trenches were performed. The root biomass measured in the core and monolith samples was added to the other excavation methods when necessary. Stump and coarse-root system excavation. After completing the volume-sampling methods, the stump and the remaining root system were excavated to assess the total below-ground biomass (up to 1 m in depth). Above-ground biomass was also measured according to the protocol described in et al. (2005). The root : shoot ratio was calculated Saint-Andre from the measured above- and below-ground biomass. Sample treatments. Once collected, root samples were brought to the laboratory where they were gently washed in a standardized sieve (2 mm) to separate roots from soil particles. Three diameter classes were considered (ne roots, 0 2 mm; medium roots, 2 10 mm; coarse roots, . 10 mm). For the stump, a subsample was taken by cutting a longitudinal slice. Roots were oven-dried to a constant weight at 65 8C (approx. 2 d for ne roots, 3 d for medium roots and 1 week for coarse roots and stump sub-samples). For the root mat system (H0), after air drying, the samples were sieved with a 2-mm sieve to remove soil debris. The root mat was then gently and manually separated

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where s is the measured standard deviation and N the number of samples collected for a given method. The formula was reversed to calculate Npredicted, the number of samples to be collected to achieve 10 % precision (i.e. IC95 10 % of the average biomass; Chave et al., 2003). The differences between methods were statistically tested using the GLM procedure of SAS software (SASw Institute Inc., Cary, NC, USA, 2004) combined with Tukeys test. In the Results section, mean root biomass are given with their standard deviation (s.d.) and expressed in term of dry matter (t ha21). R E S U LT S
Below-ground biomass

With the reference trench, 1.8, 2.0 and 5.6 t ha21 were measured for ne-root (FRB), medium-root (MRB) and coarse-root biomass (CRB), respectively, from the root mat to a depth of 1 m (Table 1). Total root biomass varied greatly with tree size. A root density index was calculated by dividing ne and medium-root biomass by the thickness of the soil layer (Fig. 2). The density of ne-root biomass was very high in the top soil horizons (0.19 + 0.04 t ha21cm21 for H0 and 0.04 + 0.02 t ha21cm21 for H1) and decreased with depth (5.7 1023 + 1.3 1023 t ha21cm21 for H3). The same pattern was observed for medium-root biomass. Conversely, CRB was more abundant in the H2 and H3 soil horizons than in H1 (Table 1), without taking into account the below-ground stump. The root : shoot ratio ranged between 14 % and 36 % with an average of 23 % (Table 1).

Levillain et al. Below-ground biomass estimation in forest plantations

TA B L E 1. Tree characteristics [circumference (cm) at 130 cm (C130) and height (m)] and below-ground standing biomass (t ha21) of a 6-year-old eucalypt plantation in Congo
Root : shoot ratio

225

025
Fine- (FRB), medium- (MRB), coarse-root (CRB) and total root biomass (RB) were calculated on all datasets (t ha21) for the full Voronoi trenches (reference method) as a function of soil depth horizons (H0, + 3 0; H1, 0 10; H2, 10 50; H3, 50 100 cm). The total below-ground standing biomass of the trees was calculated by adding total RB and stump biomass (t ha21). Means of tree root biomass are compared by tree; values with the same superscript letters are not signicantly different (Tukey test, P , 0.05).

Root density (t ha1 cm1)

26 % 14 % 36 % 17 % 23 %

020 015 010 005 000 H0 (root mat)

Fine Medium

Total

Below-ground biomass

13.03 16.53 4.71 30.32 16.15

H1 H2 H3 (010 cm) (1050 cm) (50100 cm) Horizon

Stump RB

8.14 7.43 3.24 18.46 9.32

4.89 9.10 1.47 11.86 6.83

All prole

4.11b 4.00b 0.28b 13.85a 5.56

CRB

F I G . 2. Fine- and medium-root densities (in t ha21 cm21 of excavated soil) within four soil horizons. H0, pure organic top soil horizon ( + 30 cm); H1, 0 10 cm; H2, 10 50 cm; H3, 50 100 cm. Vertical bars represent s.d.

MRB

2.28a 1.51b 1.80b 1.39b 1.75

FRB

1.75b 1.92b 1.16b 3.22a 2.01

Coarse-roots and stumps amounted to 78 % of total belowground biomass.

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Comparison between methods

There were no signicant differences between the four methods in estimating mean ne-root biomass (Table 2A), meaning that the auger-core and monolith methods were as accurate as the full Voronoi-trench excavation method. This suggests that ne-root distribution is horizontally uniform around the tree in a 6-year-old stand. The variability was mainly driven by a tree effect in the surface horizon for the monolith method (H1, P , 0.0001; H2, P , 0.0002), and for the auger-core method (H1, P , 0.0001; H2, P , 0.0001), with smaller trees producing fewer ne roots than larger trees. In the deeper horizon (H3, 50 100 cm), root density decreased with the distance from the tree for the augercore (P , 0.0028) and monolith methods (P , 0.0001). As for FRB, there were no statistical differences between methods for estimating MRB (Table 2B). However, standard deviations were higher than for FRB. For H1, the monolith and auger methods showed a distance from tree effect (P 0.0375 and 0.0378, respectively). For H2 and H3, a distance from tree effect was only found for the monolith method (P 0.0010 and 0.0176, respectively). For H2, there was also a tree effect (monolith, P 0.0005; auger, P 0.0198). The auger-core method was not suitable for assessing coarse-root biomass and only the other three methods are given in Table 2C. As for FRB and MRB, there were no statistical differences between the monolith, half and full Voronoi-trench methods. However, variability was very high and the monolith method was found to be less accurate than the half Voronoi-trench method (27 % of underestimation). A distance from tree effect was found for H2 (P , 0.0001), with more CRB near the tree than on the edge of the Voronoi.
Comparison of the methods based on precision and cost

MRB FRB MRB FRB CRB MRB Root mat Height (m) C130 (cm) FRB CRB

44.0 59.4 25.6 71.0 50.0

22.35 27.20 15.53 29.10 23.55

0.62 0.44 0.70 0.48 0.56

H0

0.67a 0.30c 0.41bc 0.22c 0.40

0.33b 0.36ab 0.42ab 0.52a 0.41

H1

0.00a 0.28a 0.00a 1.50a 0.45

0.70a 0.45b 0.42b 0.40b 0.49

1.06a 0.96a 0.48b 1.31a 0.95

H2

3.99b 2.84bc 0.26c 7.75a 3.71

0.30a 0.33a 0.27a 0.30a 0.30

0.37b 0.61b 0.26b 1.39a 0.66

H3

0.12b 0.88b 0.02b 4.61a 1.41

CRB

For FRB, it was necessary to sample 312 auger cores (or 24 trees 13 cores) to achieve 10 % precision for the soil surface horizon (0 10 cm). This gure fell to about 130 auger cores

A1 A2 A3 A4 Mean

Tree

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Levillain et al. Below-ground biomass estimation in forest plantations

TA B L E 2. Comparison of different methods for ne- (FRB, A), medium- (MRB, B) and coarse-root (CRB, C) biomass estimation superimposed on the same Voronoi trench
(A) Fine root H1 Methods Auger Monolith Half trench Full trench (B) Medium root Method Option MRB Auger Monolith Half Trench Full trench (C) Coarse root Method Options CRB Monolith Half trench Full trench P1 P2 P V1 V2 V 0.00a + 0.00 0.00a + 0.00 0.00a + 0.00 0.64a + 1.07 0.48a + 0.81 0.56a + 0.88 H1 RD 100.0 100.0 100.0 14.3 14.3 0.0 CRB 4.05a + 3.91 3.43a + 3.50 3.74a + 3.45 4.12a + 4.02 4.37a + 3.16 4.25a + 3.35 H2 RD 4.7 19.2 11.9 2.9 2.9 0.0 CRB 1.22a + 2.45 1.65a + 3.29 1.43a + 2.70 2.09a + 3.52 2.55a + 5.03 2.32a + 2.70 H3 RD 47.3 29.1 38.2 10.0 10.0 0.0 CRB 5.27a + 6.05 5.08a + 6.28 5.18a + 6.16 6.85a + 8.53 7.41a + 7.95 7.13a + 8.21 Total RD 26.0 28.7 27.4 3.9 3.9 0.0 T1 T2 T3 P1 P2 P V1 V2 V 0.12a + 0.14 0.30ab + 0.13 0.25ab + 0.10 0.45ab + 0.14 0.57b + 0.38 0.51ab + 0.16 0.43ab + 0.10 0.46ab + 0.18 0.45ab + 0.14 H1 RD 73.0 32.2 44.6 1.1 27.7 14.4 3.1 3.1 0.0 MRB 0.54a + 0.41 0.68a + 0.74 0.89a + 0.67 0.77a + 0.61 0.75a + 0.45 0.76a + 0.42 0.98a + 0.42 1.03a + 0.50 1.01a + 0.43 H2 RD 46.0 31.9 11.1 23.8 25.3 24.6 2.1 2.1 0.0 MRB 0.68a + 0.93 0.48a + 0.55 0.43a + 0.39 0.30a + 0.25 0.34a + 0.32 0.32a + 0.28 0.49a + 0.30 0.46a + 0.24 0.48a + 0.25 H3 RD 41.3 0.1 9.4 36.3 30.0 33.1 3.3 3.3 0.0 MRB 1.34a + 0.86 1.47a + 0.94 1.58a + 0.89 1.52a + 0.71 1.66a + 0.76 1.59a + 0.73 1.91a + 0.63 1.95a + 0.63 1.93a + 0.45 Total RD 30.6 23.8 18.1 21.2 14.0 17.6 1.0 1.0 0.0 Options T1 T2 T3 P1 P2 P V1 V2 V FRB 0.37a + 0.27 0.49a + 0.36 0.49a + 0.38 0.46a + 0.21 0.43a + 0.24 0.45a + 0.23 0.49a + 0.30 0.43a + 0.23 0.46a + 0.26 RD 20.4 5.9 7.3 1.1 5.5 2.2 7.2 7.2 0.0 FRB 0.55a + 0.18 0.58a + 0.13 0.57a + 0.14 0.47a + 0.12 0.44a + 0.14 0.46a + 0.12 0.58a + 0.30 0.47a + 0.16 0.52a + 0.23 H2 RD 5.7 11.1 8.1 9.8 15.0 12.4 10.1 10.1 0.0 FRB 0.33a + 0.08 0.36a + 0.05 0.35a + 0.07 0.31a + 0.11 0.25a + 0.11 0.28a + 0.11 0.29a + 0.07 0.31a + 0.08 0.30a + 0.07 H3 RD 9.7 18.9 17.8 2.4 16.8 7.2 4.8 4.8 0.0 FRB 1.25a + 0.41 1.42a 0.50 1.41a + 0.53 1.24a + 0.21 1.13a + 0.28 1.18a + 0.24 1.35a + 0.55 1.21a + 0.36 1.28a + 0.23 Total RD 2.4 11.1 10.1 3.1 12.0 7.5 5.6 5.6 0.0

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Mean root biomass + s.d. are expressed in t ha21 and relative differences (RD) from the Full Voronoi trench as a percentage. Means are compared by method; the values with the same superscripted letters are not signicantly different (Tukeys test, P , 0.05). H1, H2 and H3 represent soil horizons that were 0 10, 10 50 and 50 100 cm thick, respectively.

for H2 and H3. It took 36 person-days to complete this task (Table 3A) on a whole 0 1 m prole. Assuming a Gaussian distribution (Chave et al., 2003), reducing the desired precision from 10 % to 30 % reduced labour costs by 75 %. Compared with auger cores, use of the monolith method reduced the total number of samples by . 2 for H1 but the time required for sieving operations increased by 30 % and total time for a whole 0 1 m prole was approximately double, at 62 persondays. Lastly, the full Voronoi-trench method was the least efcient (in terms of cost/precision) because it required 100, 50 and 20 soil samples to reach 10 % precision for H1, H2, and H3, respectively, and a total of 207 person-days for sampling, sieving and sorting operations. The monolith and auger-core methods were not accurate for estimating medium and coarse-root biomass. Only the half or

full Voronoi-trench methods provided a good estimate. However, given the high variability in the spatial distribution of these roots, a desired precision of 30 % would have been more realistic in terms of cost/precision than 10 %.

Sieving versus manual sorting

Most of time spent in the eld was devoted to separating roots from soil (manually or with a sieve): 40 %, 55 % and 71 % for the auger-core, monolith and full Voronoi-trench methods, respectively. For example, for FRB estimation in the top soil horizon (H1), the time for sieving soil reached 62 % of the total sorting time for a 12 % gain in precision.

Levillain et al. Below-ground biomass estimation in forest plantations

227

TA B L E 3. Precision and labour requirements for auger coring, monolith, half and full Voronoi-trench excavation methods for ne(FRB), medium- (MRB) and coarse-root biomass (CRB) estimation (t ha21)
Sampled Root type Mat FRB Horizon H0 H1 Method Auger Auger Monolith Half trench Full trench Auger Monolith Half trench Full trench Auger Monolith Half trench Full trench Auger Monolith Half trench Full trench Auger Monolith Half trench Full trench Auger Monolith Half trench Full trench Monolith Half trench Full trench Monolith Half trench Full trench Monolith Half trench Full trench Mean + s.d. 0.557 + 0.288 0.420 + 0.374 0.435 + 0.247 0.396 + 0.206 0.396 + 0.203 0.546 + 0.319 0.445 + 0.252 0.503 + 0.187 0.503 + 0.181 0.345 + 0.214 0.321 + 0.181 0.287 + 0.065 0.287 + 0.065 0.340 + 0.752 0.460 + 0.365 0.398 + 0.132 0.398 + 0.092 1.013 + 1.723 1.085 + 1.123 1.043 + 0.353 1.043 + 0.270 0.684 + 2.622 0.541 + 1.124 0.595 + 0.323 0.595 + 0.301 0.019 + 0.128 0.880 + 1.979 0.880 + 1.437 4.820 + 13.976 3.960 + 3.366 3.960 + 3.217 1.302 + 4.775 1.875 + 3.145 1.875 + 2.995 Nsampled 51 103 64 16 8 100 64 16 8 99 61 16 8 104 64 16 8 104 64 16 8 103 61 16 8 64 16 8 64 16 8 61 16 8 Precision 14 % 17 % 14 % 26 % 36 % 12 % 14 % 18 % 25 % 12 % 14 % 11 % 16 % 43 % 20 % 16 % 16 % 33 % 26 % 17 % 18 % 75 % 53 % 27 % 35 % 170 % 111 % 114 % 72 % 42 % 57 % 91 % 83 % 112 % Time NC 2 3 8 8 5 13 31 31 11 15 39 39 2 3 8 8 5 13 31 31 12 15 39 39 3 8 8 13 31 31 15 39 39 Precision 10 % N10% 105 312 127 106 103 134 125 54 50 150 125 20 20 1912 246 43 21 1133 420 45 26 5762 1688 116 100 18578 1982 1046 3297 283 259 5011 1104 1001 Time NC 7 6 52 101 12 25 106 247 17 31 49 98 43 12 21 21 103 83 88 103 652 416 283 491 915 1615 1025 650 555 1014 1234 2703 4902 Precision 30 % N30% 12 35 14 12 11 15 14 6 6 17 14 2 2 212 27 5 2 126 47 5 3 640 188 13 11 2064 220 116 366 31 29 586 123 111 Time NC 1 1 6 11 0 3 12 22 2 3 5 11 5 1 2 2 3 9 10 11 72 46 31 55 102 108 114 72 62 113 144 300 545

H2

H3

MRB

H1

H2

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H3

CRB

H1 H2 H3

Nsampled is the number of samples collected. Precision is calculated with a condence index of 95 % with the hypothesis of normal distribution of the dataset; N10% and N30% are the numbers of samples needed to achieve, respectively, 10 % or 30 % precision on average; labour time requirement (Time) is expressed in person-days. H0, H1, H2 and H3 represent soil horizons that were + 3 0, 0 10, 1050 and 50100 cm thick, respectively.

D IS C US S IO N
Root biomass and distribution

Estimating root biomass under eld conditions is challenging because of unknown root distribution (Costa et al., 2000). Within plant species, root biomass and architecture depend on genetic and environmental factors. The main environmental factors affecting root biomass may be soil fertility, water content and temperature (Delitti et al., 2001; Green et al., 2005). Several studies on eucalypt root biomass and distribution have been conducted in tropical areas (Fabiao et al., 1995; Laclau et al., 2001; Bouillet et al., 2002; Eamus et al., 2002; Teixeira et al., 2002; Harmand et al., 2003), but it is not easy to compare the results because of a diversity of root sizes (stump and coarse roots were not systematically integrated in most below-ground biomass studies), soil depth, plant ages and sampling methods. Another difculty in comparing root biomass data from diverse studies is to account for differences in methodologies and sites. Despite these

difculties, ne-root biomass from the reference trench of the present study was close to that obtained for ne roots ( , 1 mm in diameter) of a 7-year-old Eucalyptus urophylla stand in Brazil sampling to a depth of 60 cm (0.7 t ha21; Teixeira et al., 2002), and a 7-year-old eucalyptus PF1 stand (clone 1-41) in Congo sampling to a depth of 1 m (1.4 t et al., 2005). However, these results were ha21; Saint-Andre in a lower range than those obtained at 0 30 cm in depth on a 2-year-old Eucalyptus globulus stand in Portugal and a 5-year-old Eucalyptus camaldulensis stand in Cameroon (2 t ha21, Jones et al., 1999; Harmand et al., 2003). In addition, the ne-root biomass data of the present study were also lower than those obtained in other temperate and tropical species (Ruess et al., 1996; Jackson et al., 1997; Hertel and Leuschner, 2002; Hendricks et al., 2006; Hwang et al., 2007; Valverde-Barrantes et al., 2007). For example, Jackson et al. (1997) recorded a mean ne-root biomass (0 30 cm depth) of 3 t ha21 in tropical species. The ne-root biomass density was affected by the soil depth effect in the present study. It was higher in H0 and H1 than in

228

Levillain et al. Below-ground biomass estimation in forest plantations was made between root diameter classes (i.e. ne, medium, coarse roots and stumps). The study showed that using an auger core is not a suitable method for estimating coarse-root biomass. This was consistent with previous studies on eucalypt and others species (Millikin and Bledsoe, 1999; Resh et al., 2003; Saint-Andre et al., 2005; Macinnis-Ng et al., 2010). In young and old eucalypt stands in Congo, the half Voronoi-trench and the auger-core methods were unsuitable for correct estimation of et al., 2005). In addition, coarse-root biomass (Saint-Andre Millikin and Bledsoe (1999) obtained at best 25 % of coarseroot biomass with soil coring compared with excavation methods in a Quercus douglasii stand. That result may have been due to heterogeneous distribution of coarse roots around the tree (Macinnis-Ng et al., 2010). Resh et al. (2003) showed that with an increase in coarse-root diameter, their lateral distribution became highly spatially variable. Thus, by enhancing the sampling soil volume, the full Voronoi trench enabled better sampling of the heterogeneity of coarse roots growing in all directions from the stump. This method more effectively took into account the variability of coarse-root distribution and therefore reduced the subsequent biases generated by standard auger-sampling methods (Macinnis-Ng et al., 2010). When the root system is uniformly established in the horizontal soil prole, biomass can be estimated with less risk of error by the auger-core or monolith methods as observed for ne roots in the present study. This result tallied with those found for maize, black walnut and red oak alley cropping systems, which showed a signicant linear relationship between ne-root biomass from auger cores and minirhizotrons (Jose et al., 2001). The study of spatial distribution of root systems in Congo showed that at 1 year old, ne roots extended beyond a depth of 3 m and up to the middle of the inter-row (Bouillet et al., 2002). From this age, we assumed homogeneous colonization of the horizontal horizons by ne roots in undisturbed soil. Fine-root biomass required 312 auger cores to achieve 10 % precision in the 0 10 cm horizon and around one third of that for the 10 50 cm and 50 100 cm horizons. In the surface horizon, ne-root biomass variability may explain this large number of samples. This variability may be introduced locally by factors such as micro-site spatial soil topography or mechanical soil preparation before planting. Consequently, the amounts of ne-root biomass may vary with environmental conditions, crop genotypes, and with soil physical, chemical and biological properties (Bingham and Bengough, 2003). The root mat biomass in the above-ground forest oor amounted to 32 % of the total ne-root biomass (assessed in the 0 1 m soil layer) of a 6-year-old eucalypt stand. This result was consistent with previous studies at the same site (Laclau et al., 2004). In addition, roots may preferentially explore the planting row, where soil compaction is lower, resulting from the ripping treatment carried out during land preparation (Laclau et al., 2000). The smaller number of samples required in the deepest soil layers to achieve a given level of precision may be related to the reduction in biomass variation, as observed in three European forest chronosequences (Claus and George, 2005).

H2 and H3. This result has been observed in many other studies (Fabiao et al., 1990; Castellanos et al., 2001; Bouillet et al., 2002; Resh et al., 2003; Claus and George, 2005) and may be related to a reduction in ne-root formation and mortality (Mainiero and Kazda, 2006) or differences in resource availability (Goransson et al., 2008) along the soil prole. The distance from tree effect on FRB from the monolith and auger methods was not signicant for the H1 and H2 horizons. This result was consistent with other studies on tree plantations that have shown no decrease in ne-root density from the position of the row to the middle of the inter-row (Fabiao et al., 1995; Bouillet et al., 2002). However, the opposite trend was found in a radiata pine stand (Nambiar, 1983). The average root : shoot ratio was estimated at 0.23, which tallied with previous studies carried out at the same site but et al., 2005). This ratio was on another clone (Saint-Andre similar to those (0.26) calculated by Cairns et al. (1997) in more than 160 studies covering tropical, temperate and boreal forests. These authors highlighted the great variability in root biomass estimates, due to the different methods used at a great number of diverse sites and they assumed that the integration of parameters such as latitude, soil texture, tree age and type could reduce that variability. In the present study, the great variability found (range 0.14 0.36) could not be assigned to these parameters as the study was performed in the same 6-year-old plantation, but rather on the great variability in tree height and diameter at breast height of the sampled trees as they were representative of the clonal eucalypt stand. Given the labour requirements of this study, variability between trees associated with a given diameter or height size class (only one tree per diameter at breast height class was sampled) was not investigated, despite its relevance for tting relationships between tree size and the corresponding root biomass. For an accurate root biomass estimation of the stand, this aspect should be taken into account, but for the purpose of the paper (method comparison), and considering the work was on clonal plantations (same genotype) established on sandy soils ( . 90 % sand) with low variability on a stand scale, tree size was the rst and main factor to be considered.
Comparison between methods

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Few studies have compared the different sampling methods for root biomass estimation, probably because of the timeconsuming experiments (Jose et al., 2001) and laborious eld excavation work required. When different root sizes are included, studies are rather scarce (Millikin and Bledsoe, cke et al. (1995) compared four commonly used 1999). Ku direct eld methods for estimating root biomass (i.e. core method, core-break method, trench-prole wall method and root-extraction method). They concluded that the core-break and trench-prole methods delivered no reliable data for comparing rooting intensities between soils and between different crops. On the other hand, the core and root extraction methods were more appropriate for estimating root biomass. Of the four direct eld methods tested in the present study, all of them involved root excavations (i.e. by auger cores, monoliths or large trenches) in a known soil volume, and a distinction

Levillain et al. Below-ground biomass estimation in forest plantations Lastly, this study showed that the full Voronoi-trench method is highly recommended for estimating total standing root biomass, due to the relative share (78 %) of coarse roots and stumps in total below-ground biomass. Moreover, the Voronoi-trench method required a smaller number of samples to achieve good precision for ne-root biomass, but it needed a greater number of person-days for sampling, sieving and sorting operations. This was consistent with the large soil volume of this sampling method. Based on the absence of signicant differences in FRB between the different sampling methods, the auger coring and monolith methods were the best compared with the Voronoi trench for ne-root estimation in a 6-year-old eucalypt plantation and older. However, previous studies have shown uniform ne-root colonization in the root prole (Bouillet et al., 2002) from 1 year old onwards, but radial uniformity was not tested and additional studies need to be carried out in young eucalypt plantations.

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ACK N OW L E DG E M E N T S The study was funded by the EU STREP CARBOAFRICA project. Technical assistance was provided by CRDPI and we thank the nkumbi root team members. We also thank Peter Biggins for revising the English.

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N OTE AD DE D IN P ROO F Photograph of the superimposition of methods illustrated in Fig. 1D.

Full vorono trench Simplified vorono trench Monolith method 010 cm 1050 cm Sampled tree

Auger coring method