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# Defoliation-induced changes in carbon allocation and root soluble carbon concentration in field-grown *Lolium perenne* plants: do they affect carbon availability, microbes and animal trophic groups in soil?

S. BAZOT,*† J. MIKOLA,‡ C. NGUYEN* and C. ROBIN*

*Rhizosphere Group, UMR INPL-INRA 'Agronomie et Environnement', Ecole Nationale Supérieure d'Agronomie et des Industries Alimentaires (ENSAIA), BP 172, F-54505 Vandoeuvre-les-Nancy, France, and ‡Department of Biological and Environmental Science, PO Box 35 (YAC), FIN-40014 University of Jyväskylä, Finland

## Summary

1. It is hypothesized that defoliation-induced changes in plant carbon allocation and root soluble C concentration modify rhizosphere C availability and, further, the abundance and activity of soil microbes and their grazers. To test this hypothesis, field-grown *Lolium perenne* swards were defoliated twice during their second growing season at two nitrogen availabilities (added N or no added N). Plant, soil and microbial attributes were measured 2 and 4 days after the last defoliation, and nematode abundance was measured 6 days after the last defoliation.

2. Defoliation decreased shoot production in plots where N was added, but had no significant effect in plots where N was not added. Root biomass and the ratio of root mass to shoot production were not affected.

3. Defoliation increased root soluble C concentration by 26% at the first harvest (2 days after defoliation) and by 18% at the second harvest (4 days after defoliation). Leaf N concentration was 27% lower in defoliated than in non-defoliated swards at the first harvest, while that of stems was 14% higher in defoliated swards at both harvests, and that of roots was not affected. Defoliation increased root C : N ratio, decreased stem C : N ratio, and did not have a statistically significant effect on leaf C : N ratio.

4. Soil attributes (soil soluble C concentration and soil C and N concentrations) were not affected by defoliation. Similarly, microbial attributes such as microbial C and N content, bacterial abundance in rhizosphere soil, and diversity of C sources utilized by the rhizosphere microbial community, did not differ between defoliated and non-defoliated swards.

5. Among nematode trophic groups, defoliation reduced the abundance of fungivorous and herbivorous nematodes by 70 and 47%, respectively, but did not affect the abundance of bacterivorous, omnivorous and predatory nematodes.

6. Although defoliation altered plant C allocation and root soluble C concentration, these changes did not influence C availability, soil microbial growth or the abundance of bacteria-feeding nematodes in the plant rhizosphere. Instead, the effects on root- and fungus-feeding nematodes suggest that the effects of defoliation on soil communities were propagated not through the effects of root-released C on bacteria and bacterial grazers, but through effects of root quality on root-feeders and possibly through effects of mycorrhizal fungi on fungus-feeders.

Key-words: grassland, nematodes, root-released carbon, soil community, soil microbes

Functional Ecology (2005) 19, 886–896
doi: 10.1111/j.1365-2435.2005.01037.x
## Introduction

Removal of photosynthetic shoot tissues (defoliation) by grazing animals may have major effects on the structure and functioning of grassland ecosystems. In addition to affecting the composition, shoot production and root turnover of plant communities (McNaughton 1983; McNaughton et al. 1998), defoliation alters photosynthetic rates and carbon allocation within plants: the flow of energy in the plant-soil system (Painter & Detling 1981; Richards 1993; Morvan-Bertrand et al. 1999). It is well established that, immediately after defoliation, C is remobilized from reserves to sustain the needs of regrowing shoot biomass, whereas thereafter regrowth mainly relies on current assimilates (Welker et al. 1985; Briske & Richards 1995; De Visser et al. 1997). Although remobilized C reserves in grasses originate mainly from remaining leaf and stem rather than root tissues (Richards & Caldwell 1985; Briske & Richards 1995), root growth (Davidson & Milthorpe 1966; Richards 1993) and, with some delay, nitrogen uptake (Jarvis & Macduff 1989; Macduff et al. 1989) may also be depressed after defoliation due to the decreased availability of photoassimilates in roots.

While defoliation-induced changes in plant physiology and C allocation are well documented, there is no consistent picture of how these changes may further affect C availability and the abundance of soil organisms in the plant rhizosphere. For instance, defoliation has been reported to have positive (Holland et al. 1996; Paterson & Sim 1999; Murray et al. 2004); neutral (Todorovic et al. 1999); and negative (Mikola & Kytöviita 2002; Dilkes et al. 2004) effects on the amount of C released from roots to the rhizosphere. These differences may have several reasons: for example, different plant species have different strategies of regrowth and C allocation (Guitian & Bardgett 2000); or different fractions of assimilated C are used to represent root-released C (Mikola & Kytöviita 2002). Defoliation has also been reported to affect organisms living in the plant rhizosphere, such as microbes (Guitian & Bardgett 2000; Hamilton & Frank 2001) and their grazers (Hokka et al. 2004), but it is not clear how widely these effects can be explained by changes in root C release. In addition to affecting root C exudation, defoliation can also change root quality (Seastedt 1985; Merril et al. 1994. Hokka et al. 2004) and root mortality (Jarvis & Macduff 1989; Mikola et al. 2001), but the relative importance of the different mechanisms is not well known. Nevertheless, it is becoming clear that defoliation-induced changes in grassland soils may have wide-ranging consequences in grassland ecosystems as they can feed back to plant growth, plant N uptake and plant N allocation (Hamilton & Frank 2001; Mikola et al. 2005a).

To obtain a comprehensive picture of the effects of defoliation on C transfer from plants to soil, we established a field experiment in which *Lolium perenne* swards were either not defoliated, or defoliated twice during their second growing season. We determined

*L. perenne* growth and C allocation; rhizosphere C availability; and the abundance, activity and C content of the soil microbial community in the *L. perenne* rhizosphere 2 and 4 days after the last defoliation. To examine whether the effects of defoliation would propagate further through upper trophic levels of the soil food web, we also measured the abundance of different nematode trophic groups in the *L. perenne* rhizosphere 6 days after the last defoliation. Moreover, as the availability of nutrients, especially that of N, may affect the release of organic material from roots (Hale et al. 1971; Grayston et al. 1996), we investigated the effects of defoliation in swards of two different N availabilities. We tested the hypothesis that defoliation-induced changes in plant C allocation (Holland et al. 1996) and root soluble C concentration (Paterson & Sim 1999) lead to changes in rhizosphere C availability and, further, to changes in the abundance and activity of soil microbes and their grazers.

## Materials and methods

### EXPERIMENTAL DESIGN

We established three experimental grass swards (each 4 × 4 m) by sowing *L. perenne* cv. Canasta seeds (25 kg ha⁻¹) into clay-loamy soil at the experimental station of INRA-SAD at Mirecourt, France (6°81′ E, 53°6′ N) in September 2002. In February 2003 the grass swards were trimmed to 5 cm above the soil surface to even out plant shoot mass in different swards before the beginning of the second growing season. Each sward was then divided into two plots (2 × 4 m), and each plot further split into two subplots (1 × 4 m). To enhance later sampling of plant roots and rhizosphere soil, four *L. perenne* individuals were randomly selected at each subplot and the soil beneath the plants was isolated from surrounding soil using PVC cylinders (area 50 cm², depth 20 cm). The experimental design consisted of two treatments: N addition and defoliation. Nitrogen addition was applied at plot level: one randomly chosen plot at each sward received additional N, while the other received no additional nutrients. Defoliation, in turn, was applied at subplot level: one randomly chosen subplot at each plot was subjected to defoliation, while the other was left intact. The defoliation treatment included two trimmings, on 30 April and 19 May. For measuring total shoot production, trimmed material was collected, lyophilized and weighted. Nitrogen was added immediately after the February and April trimmings: at each sward, one of the main plots received 50 kg N ha⁻¹ as NH₄NO₃ solution after both trimmings, while the other plot received water only.

### SAMPLING PROCEDURE

The grass swards were sampled three times, 2, 4 and 6 days after the last trimming in May. At first and second samplings, two PVC cylinders, including growing *L.*
perenne individuals, were removed from each subplot for measuring plant, soil and soil microbial attributes. Timing of these samplings was determined based on earlier findings, suggesting that defoliation-induced changes in root C release last for 4–5 days (Paterson & Sim 1999, 2000). At the third sampling, two soil cores (area 9·6 cm², depth 10 cm) were taken from each subplot beneath two randomly selected *L. perenne* individuals for examination of the nematode community. This sampling combines the responses of nematodes to both April (25 days before sampling) and May (6 days before sampling) trimming, as while some nematode genera can respond to defoliation within a week, others require 3–4 weeks (Mikola *et al.* 2005b). At each sampling, soil cores and the plants attached to them were kept intact until further analysis in the laboratory.

At first and second samplings, plant shoots were first removed and sorted into leaf and stem mass. Roots were then picked from the soil by hand and the rhizosphere soil (soil adhering to roots) was detached using a brush. After washing roots with 0·5 M CaCl₂ isotonic solution, all plant samples were frozen in liquid N and stored at –30 °C. Rhizosphere soil was sieved (2 mm) and one half was frozen in liquid N, stored at –30 °C and later used for analysing soil C and N concentrations, while the other half was used fresh for analysing soluble C and microbial attributes.

## PLANT AND SOIL ANALYSES

For measuring plant and soil C and N concentrations, frozen samples were lyophilized, ground to fine powder, and analysed using a C and N auto-analyser (Flash EA 1112 Serie, Thermo Finnigan, Les Ulis, France). Before grinding, lyophilized plant samples were weighted to obtain the dry mass of different plant parts. The concentration of water-soluble carbohydrates in roots (including free sugars, oligosaccharides, polysaccharides and their derivatives) was then measured using the phenol method (Dubois *et al.* 1956). Briefly, 40 mg subsamples of dry roots were extracted with 2 ml 80% ethanol at 80 °C for 15 min. The extract (2 ml), 1 ml 5% phenol and 5 ml of concentrated H₂SO₄ were then mixed and incubated for 20 min at 25 °C. The light absorption of the mixture was measured at 490 nm and the carbohydrate concentration expressed as hexose equivalent (mol kg⁻¹ roots).

The concentration of hot-water-extractable C (HWC) in soil, a measure of soluble organic C composed of microbial and other sources of C (Sparling *et al.* 1998), was determined using the method of Haynes & Francis (1993). Subsamples of fresh soil were first dried at 37 °C for 12 h. Of the dried soil, 5 g was mixed with 25 ml distilled water in a vial and left in an 80 °C water bath for 18 h. At the end of the extraction period, the vial was shaken to ensure the HWC released from the soil was fully suspended in water. Suspension was then filtered through Whatman paper no. 42, and total organic C (TOC) was quantified using a TOC analyser.

## MICROBIAL ANALYSES

Microbial C and N contents were estimated from fresh soil samples using the chloroform fumigation-extraction method (Vance *et al.* 1987), and the C and N contents of the K₂SO₄ solution were measured using a TOC analyser (TOC-VCSH CSH/CNS, Shimadzu, Champs-sur-Marne, France) connected online to an N analyser (TNM-1, Shimadzu).

To evaluate the rhizosphere availability of simple C sources, which are typically released by roots into the soil, we measured the response of soil microbes to the addition of microquantities of labelled ¹⁴C-glucose (Nguyen & Guckert 2001). It has been shown that the proportion of assimilated ¹⁴C stored by microbes increases, and the proportion of assimilated ¹⁴C respired by microbes decreases, with decreasing soil C availability (Nguyen & Henry 2002). The ratio of respired to stored ¹⁴C therefore indicates the availability of C in the soil: the higher the ratio, the higher the C availability. Moreover, respired ¹⁴C gives a relative measure of soil microbial activity (Nguyen & Henry 2002). In the measurement, two glass vials were first filled with 4 g fresh soil (one to be fumigated later with chloroform, the other not) and set in a plastic jar containing 25 ml 1 M NaOH to trap evolving ¹⁴CO₂. In each vial, 100 µl ¹⁴C-glucose solution (37 kBq ml⁻¹; 0·038 µg C ml⁻¹, a negligible amount of C compared with the total pool of soluble C in the soil) was spread over the soil surface and the plastic jar was closed immediately. After 6 days' incubation in the dark at 22 °C, the respired ¹⁴C trapped in NaOH was counted by liquid scintillation (Tri-Carb 2100 Tr, Packard, Perkin-Elmer, Courtaboeuf, France). Soil from one vial was then fumigated with chloroform vapour and the amount of stored ¹⁴C calculated as a difference of ¹⁴C extracted by 0·5 M K₂SO₄ from the fumigated and the non-fumigated soil.

To measure bacterial abundance and to evaluate the catabolic profile of the microbial community in the rhizosphere soil, a dilution series of microbial suspensions was produced by mixing 5 g fresh soil with 50 ml phosphate buffer (7·2 g NaCl, 2·8 g N₂HPO₄, 0·4 g KH₂PO₄ l⁻¹ water, pH 7·2). The mixture was placed on an orbital shaker (40 r.p.m.) for 10 min and centrifuged (750 r.p.m.) for another 10 min, and the supernatant was used to produce a dilution series of 10⁻²–10⁻⁵. Dilutions from 10⁻³ to 10⁻⁵ were spread onto agar plates (Tryptone Soy Agar, Difco, Becton-Dickinson, Le Pont-de-Claix, France) to estimate the number of culturable bacterial cells, measured as colony-forming units (CFU). Two replicates were prepared for each dilution, the agar plates were incubated in the dark at 27 °C, and the number of CFU was recorded after 2 days' incubation.

The catabolic profile of the microbial community was characterized using Biolog GN microplates with 95 wells of different C sources (Biolog Inc., Hayward, CA, USA). Each microplate well was inoculated with 150 µl 10⁻³ dilution, plates were incubated in the dark at 27 °C, and colour development in wells
was recorded automatically after 1, 2, 6, 24, 48 and 72 h incubation at 550 nm using a microplate reader (MR 7000 Dynatech, Issy Les Moulineaux, France). Absorbance values for the wells with C sources were blanked against the control well, and all negative absorbance values were set to zero. In order to measure the diversity of C sources utilized, the area under the colour development curve was calculated for each C source. The diversity of utilized C sources was then expressed as Shannon's diversity index:

$$H = -\sum_{i=1}^{95} P_i \log_{10} P_i,$$

where $P_i$ is the proportion of the area under the colour development curve in well $i$ to the sum of areas in all 95 wells (Preston-Mafham *et al.* 2002).

## NEMATODE COMMUNITY ANALYSIS

To evaluate the response of soil microfauna to the treatments, nematodes were extracted from 30 g (fresh mass) rhizosphere soil using a wet funnel device (Sohlenius 1979). The total number of live individuals was counted; later, using preserved samples, 150 nematodes per sample were identified to genus and allocated into trophic groups according to Yeates *et al.*, 1993).

## STATISTICAL ANALYSES

The mean of the two samples from each subplot was calculated for each variable. The effects of defoliation and N addition on the variables measured were then

analysed with the SPSS statistical package (SPSS 2001) using a nested ANOVA modified to engage the two harvests made for plant, soil, and soil microbial attributes. In this mixed-model ANOVA, (1) N addition, defoliation, harvest time and their second- and third-order interactions were treated as fixed effects; (2) the plot effect, nested within N addition, and its interactions with the fixed effects, were treated as random effects; and (3) random effects were used as error terms for the tests of fixed effects. The nematode data with only one harvest were analysed using nested ANOVA. Homogeneity of variances was tested using Levene's test, and to satisfy the assumption of homogeneity of variances a logarithmic transformation was applied to dependent variables whenever necessary. When the mixed-model ANOVA resulted in a significant interaction between harvest time and defoliation, or between harvest time and N addition, the effects of defoliation and N addition were tested separately within each harvest using a nested ANOVA. When a significant interaction was found between N addition and defoliation, the factors were fixed one by one, and effects of the other factor were analysed within the levels of the fixed factor using a mixed-model ANOVA.

## Results

### PLANT GROWTH

The effects of N addition and defoliation on shoot production were dependent on each other (Table 1; Fig. 1a). Nitrogen addition promoted 3·3-fold plant shoot production in non-defoliated plots ($F = 479, P < 0.001$).

<table><caption>**Table 1.** F statistics from mixed-model ANOVA for the effects of defoliation (no trimming, or trimming twice during second growing season); nitrogen addition (no added N, or N added after trimmings); and harvest time (2 or 4 days after last trimming) on plant attributes in field-grown *Lolium perenne* swards</caption><thead><tr><th rowspan="2">Dependent variable</th><th colspan="7">Source of variation</th></tr><tr><th>Defoliation</th><th>N addition</th><th>Harvest time</th><th>D × N</th><th>D × H</th><th>N × H</th><th>D × N × H</th></tr></thead><tbody><tr><td><strong>Plant growth</strong></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>Shoot production</td><td>0.05</td><td>35.9**</td><td>0.09</td><td>16.5*</td><td>0.14</td><td>0.36</td><td>&lt;0.01</td></tr><tr><td>Root mass</td><td>5.24</td><td>0.83</td><td>3.52</td><td>5.06</td><td>0.11</td><td>1.84</td><td>0.16</td></tr><tr><td>Ratio root mass : shoot production</td><td>2.88</td><td>24.4**</td><td>0.83</td><td>2.64</td><td>0.26</td><td>1.31</td><td>0.64</td></tr><tr><td><strong>Plant C concentration</strong></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>Leaf C concentration</td><td>58.0**</td><td>1.32</td><td>0.48</td><td>0.05</td><td>2.77</td><td>0.34</td><td>0.02</td></tr><tr><td>Stem C concentration</td><td>9.97*</td><td>8.05*</td><td>0.16</td><td>0.09</td><td>7.07</td><td>0.02</td><td>0.01</td></tr><tr><td>Root C concentration</td><td>2.50</td><td>9.75*</td><td>8.50*</td><td>&lt;0.01</td><td>0.68</td><td>0.80</td><td>3.66</td></tr><tr><td>Root soluble C concentration</td><td>31.5**</td><td>0.07</td><td>1.83</td><td>2.66</td><td>10.3*</td><td>0.55</td><td>0.59</td></tr><tr><td><strong>Plant N concentration</strong></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>Leaf N concentration</td><td>9.27*</td><td>336***</td><td>11.1*</td><td>0.05</td><td>9.92*</td><td>1.19</td><td>0.12</td></tr><tr><td>Stem N concentration</td><td>16.3*</td><td>204***</td><td>0.78</td><td>2.65</td><td>0.40</td><td>0.31</td><td>0.69</td></tr><tr><td>Root N concentration</td><td>3.25</td><td>13.5*</td><td>7.81*</td><td>0.12</td><td>0.06</td><td>1.92</td><td>0.48</td></tr><tr><td><strong>Plant C : N ratio</strong></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>Leaf C : N ratio</td><td>0.37</td><td>152***</td><td>17.8*</td><td>0.01</td><td>10.3*</td><td>0.02</td><td>2.22</td></tr><tr><td>Stem C : N ratio</td><td>105***</td><td>204***</td><td>0.95</td><td>4.09</td><td>0.62</td><td>0.02</td><td>0.10</td></tr><tr><td>Root C : N ratio</td><td>21.2**</td><td>10.7*</td><td>3.55</td><td>&lt;0.01</td><td>0.03</td><td>1.25</td><td>1.14</td></tr></tbody></table>

Degrees of freedom: 1,4 for each source of variation.

* , ** , *** , Treatment effect significant at $P < 0.05, 0.01$ and $0.001$, respectively.
<figure><img src="image_48.png" /><figcaption>**Fig. 1.** Plant growth (mean + 1 SE, *n* = 3) in field-grown *Lolium perenne* swards in response to defoliation (def) and nitrogen addition 2 and 4 days after the last defoliation: (a) shoot production (defoliated and harvested shoot mass); (b) root biomass; (c) ratio of root biomass to shoot production. Black and white bars represent swards with and without added N, respectively; D, defoliated swards.</figcaption></figure>

but had no effect in defoliated plots (*F* = 2.07, *P* = 0.223). Defoliation, in turn, decreased shoot production by 27% in plots where N was added (*F* = 41.8, *P* = 0.023), but had no statistically significant effect in plots where N was not added (*F* = 4.17, *P* = 0.178) (Fig. 1a). In contrast to shoot production, N addition and defoliation did not affect harvested root biomass (Table 1, Fig. 1b). The ratio of root mass to shoot production was reduced by N addition on average by 61%, but was not affected by defoliation (Table 1; Fig. 1c).

## PLANT CARBON AND NITROGEN CONCENTRATIONS

Defoliation reduced leaf and stem C concentrations on average by 12 and 7%, respectively, but did not affect root C concentration, whereas N addition increased root and stem C concentrations on average by 7 and 9%,

respectively, but did not affect leaf C concentration (Table 1; Fig. 2a-c). Nitrogen addition had a clear positive effect on plant N concentrations, with 111, 101 and 38% higher leaf, stem and root N concentrations, respectively, found in plots with N addition (Table 1; Fig. 2d-f). Defoliation reduced leaf N concentration by 27% at the first harvest (2 days after defoliation) (*F* = 13.2, *P* = 0.022), but had no effect at the second harvest (4 days after defoliation) (*F* = 0.35, *P* = 0.585). increased stem N concentration at both harvests on average by 14%, and did not affect root N concentration (Table 1; Fig. 2d-f). The C : N ratios of different plant parts were significantly decreased by N addition, whereas defoliation increased the ratio in roots, decreased the ratio in stems, and had no statistically significant effect on the ratio in leaves (Table 1; Fig. 2g-i).

Defoliation increased root carbohydrate concentra-
tion by 26% at the first harvest (*F* = 61.5, *P* = 0.001) and
by 18% at the second harvest (*F* = 13.8, *P* = 0.021), while
N addition had no effect (Fig. 3).

SOIL CARBON AND NITROGEN
CONCENTRATIONS

Soil soluble C concentration (Fig. 4a) and total soil
C and N concentrations (data not shown) were not
affected by defoliation or N addition (Table 2).

SOIL MICROBES

When we analysed in detail the three-way interaction
between the effects of N addition, defoliation and
harvest time on microbial C content (Fig. 4b; Table 2),
a significant interaction between defoliation and N
addition was found at the first harvest (F = 11.2, P =
0.044), but none of the treatments had statistically
significant main effects. Microbial N content was not
affected by defoliation or N addition (Table 2).

In the case of short-term utilization of ¹⁴C-labelled
glucose by soil microbes, the proportion of added ¹⁴C
respired by microbes was decreased by N addition at
first harvest (F = 15.06, P = 0.018), but no other effects
of defoliation or N addition on the amount of respired
¹⁴C, stored ¹⁴C or their ratio were observed (Fig. 4c-e;
Table 2). The number of culturable cells of bacteria in
rhizosphere soil (Fig. 4f) and the diversity of C sources
utilized by the rhizosphere microbial community
(data not shown) were not affected by defoliation or N
addition (Table 2).

NEMATODES

Defoliation decreased the abundance of fungivorous
and herbivorous nematodes on average by 70 and 47%,
respectively, but did not have a statistically significant
effect on the abundance of bacterivorous, omnivorous
and predatory nematodes (Table 3; Fig. 5). None of the
nematode trophic groups was affected by N addition
(Table 3; Fig. 5).
<figure><img src="image_49.png" /><figcaption>Fig. 2. Plant carbon and nitrogen concentrations (mean ± 1 SE, n = 3) in field-grown *Lolium perenne* swards in response to defoliation (def) and N addition 2 and 4 days after the last defoliation: (a–c) leaf, stem and root C concentration; (d–f) leaf, stem and root N concentration: (g–i) leaf, stem and root C : N ratio. Treatment symbols as for Fig. 1.</figcaption></figure>

<figure><img src="image_50.png" /><figcaption>Fig. 3. Root soluble carbon concentration (mean ± 1 SE, n = 3) in field-grown *Lolium perenne* swards in response to defoliation (def) and nitrogen addition 2 and 4 days after the last defoliation. Treatment symbols as for Fig. 1.</figcaption></figure>

Discussion

Our results show that defoliation altered plant C alloca-
tion and root soluble C concentrations in L. perenne
swards. However, against our hypothesis, these changes
did not influence C availability, soil microbial growth
or the abundance of bacteria-feeding nematodes in the
plant rhizosphere. Instead, we found negative effects
of defoliation on root-feeding and fungus-feeding
nematodes. This suggests that the effects of defoliation
were propagated not through the effects of root C release

on bacteria and bacterial grazers, but through effects
of root quality on root-feeders and possibly through
effects of mycorrhizal fungi on fungus-feeders.

Although defoliation had no effect on biomass
allocation in the *L. perenne* swards (the ratio of root
biomass to shoot production did not differ between
defoliated and non-defoliated plants), C allocation
was altered. Defoliation reduced leaf and stem C con-
centration as well as stem C : N ratio, but increased
root C : N ratio and root carbohydrate concentration,
which suggests that defoliated plants allocated relatively
more C to roots and had more water-soluble C in their
roots than non-defoliated plants. This does not sup-
port the common view that plants tolerate defoliation
by mobilizing C into leaf meristems at the expense of
roots after defoliation (Caldwell *et al.* 1981; Miller &
Rose 1992; Strauss & Agrawal 1999), but is consistent
with the hypothesis that plants increase C allocation to
roots after defoliation to create C stores less accessible
for above-ground grazers (Dyer *et al.* 1991; Holland
*et al.* 1996). It has been suggested that increased
allocation of photosynthate to roots after defoliation
(Holland *et al.* 1996) and amplified root concentrations
of soluble C in defoliated plants (Paterson & Sim 1999)
can lead to increased C exudation from roots. Our results
do not support these hypotheses: although concentra-
tions of water-soluble C in *L. perenne* roots were increased
by defoliation, and relatively more C appeared to be
<figure><img src="image_51.png" /><figcaption>Fig. 4. Soluble carbon concentration and microbial attributes of rhizosphere soil (mean + 1 SE, n = 3) in field-grown *Lolium perenne* swards in response to defoliation (def) and nitrogen addition 2 and 4 days after the last defoliation: (a) soil soluble C concentration; (b) microbial C content; (c,d) respired and stored proportion of added glucose-¹⁴C; (e) ratio of respired to stored glucose-¹⁴C; (f) bacterial abundance, measured as colony-forming units on agar plates. Treatment symbols as for Fig. 1.</figcaption></figure>

allocated below ground in defoliated than in non-defoliated plants, none of the soil, microbial or animal attributes indicated increased C availability in the rhizosphere soil. For instance, defoliation did not affect soluble C concentration or microbial utilization of ¹⁴C-labelled glucose in the rhizosphere soil, which suggests that the availability of labile C compounds in the rhizosphere was not affected by defoliation. Similarly, Murray *et al.* (2004) recently reported no effect of defoliation on overall root C exudation in field-grown *Agrostis capillaris* plants. In other studies with graminoids, root C release has been found to increase (Holland *et al.* 1996; Paterson & Sim 1999, 2000; Paterson *et al.* 2003) or decrease (Mikola & Kytöviita 2002; Nguyen & Henry 2002; Dilkes *et al.* 2004; Macdonald *et al.* 2004) after defoliation, suggesting that the influence of defoliation on root C release may be difficult to predict. However, studies differ in terms of the plant species used, type of defoliation and fraction of assimilated C measured in the rhizosphere (Mikola & Kytöviita 2002), and more trials may be needed before predictable patterns emerge. What is noticeable in our study is that we did not find a link between the amount of C available in roots and the amount of C released from roots, which has been recorded in some earlier studies (Mikola & Kytöviita 2002; Dilkes *et al.* 2004; Macdonald *et al.* 2004). A similar discrepancy was reported by Paterson *et al.* (2003), who found that defoliation decreased soluble C concentrations in *Festuca rubra* roots but increased root C exudation.

In accordance with the measurements of rhizosphere C availability, we found no indication of effects of defoliation on soil microbes. Microbial activity –

<table><caption>**Table 2.** F statistics from mixed-model ANOVA for the effects of defoliation (no trimming, or trimming twice during second growing season); nitrogen addition (no added N, or N added after trimmings); and harvest time (2 or 4 days after last trimming) on soil and soil microbial attributes in the rhizosphere of field-grown *Lolium perenne* swards</caption><thead><tr><th rowspan="2">Dependent variable</th><th colspan="7">Source of variation</th></tr><tr><th>Defoliation</th><th>N addition</th><th>Harvest time</th><th>D × N</th><th>D × H</th><th>N × H</th><th>D × N × H</th></tr></thead><tbody><tr><td>Soil C and N content</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>Soil C concentration</td><td>0.65</td><td>0.10</td><td>12.5*</td><td>7.01</td><td>0.58</td><td>1.91</td><td>0.02</td></tr><tr><td>Soil soluble C concentration</td><td>0.50</td><td>0.92</td><td>0.01</td><td>0.02</td><td>1.08</td><td>2.42</td><td>3.98</td></tr><tr><td>Soil N concentration</td><td>&lt;0.01</td><td>2.38</td><td>6.02</td><td>1.92</td><td>1.48</td><td>0.01</td><td>0.86</td></tr><tr><td>Microbial C and N content</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>Microbial C content</td><td>0.14</td><td>0.01</td><td>7.80*</td><td>0.14</td><td>1.76</td><td>0.62</td><td>14.3*</td></tr><tr><td>Microbial N content</td><td>2.64</td><td>0.02</td><td>18.9**</td><td>4.51</td><td>1.02</td><td>11.9*</td><td>1.87</td></tr><tr><td>Microbial utilization of added ¹⁴C glucose</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>Proportion of respired ¹⁴C</td><td>0.26</td><td>0.76</td><td>23.9**</td><td>0.35</td><td>2.51</td><td>1.06*</td><td>0.13</td></tr><tr><td>Proportion of stored ¹⁴C</td><td>3.33</td><td>1.16</td><td>0.02</td><td>6.00</td><td>&lt;0.01</td><td>&lt;0.01</td><td>1.65</td></tr><tr><td>Ratio of respired to stored ¹⁴C</td><td>2.01</td><td>1.36</td><td>4.93</td><td>2.87</td><td>0.46</td><td>1.45</td><td>0.76</td></tr><tr><td>Bacterial abundance</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>Number of colony-forming units</td><td>2.14</td><td>&lt;0.01</td><td>7.65</td><td>2.31</td><td>0.06</td><td>&lt;0.01</td><td>0.64</td></tr><tr><td>Microbial community catabolic profile</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>Diversity of utilized C compounds</td><td>0.01</td><td>0.22</td><td>11.4*</td><td>0.62</td><td>0.04</td><td>0.27</td><td>1.62</td></tr></tbody></table>
<table><caption>**Table 3.** F statistics from nested ANOVA for the effects of defoliation (no trimming, or trimming twice during second growing season) and nitrogen addition (no added N, or N added after trimmings) on abundance of nematode trophic groups in rhizosphere soil of field-grown *Lolium perenne* swards 6 days after last trimming</caption><thead><tr><th rowspan="2">Dependent variable</th><th colspan="3">Source of variation</th></tr><tr><th>Defoliation</th><th>N addition</th><th>D × N</th></tr></thead><tbody><tr><td>Bacterivore abundance</td><td>0.02</td><td>2.74</td><td>1.45</td></tr><tr><td>Fungivore abundance</td><td>11.1*</td><td>4.01</td><td>0.65</td></tr><tr><td>Herbivore abundance</td><td>10.5*</td><td>0.08</td><td>3.22</td></tr><tr><td>Omnivore abundance</td><td>6.28</td><td>0.09</td><td>2.12</td></tr><tr><td>Predator abundance</td><td>1.00</td><td>1.18</td><td>5.67</td></tr></tbody></table>

Degrees of freedom: 1,4 for each source of variation.

\*, \*\* \*\*\*, Treatment effect significant at *P* < 0.05, 0.01 and 0.001, respectively.

<figure><img src="image_52.png" /><figcaption>**Fig. 5.** Nematode abundance in rhizosphere soil (mean + 1 SE, *n* = 3) in field-grown *Lolium perenne* swards in response to defoliation and nitrogen addition 6 days after the last defoliation: (a) bacterivores; (b) fungivores; (c) herbivores; (d) omnivores; (e) predators. Treatment symbols as for Fig. 1.</figcaption></figure>

measured as the proportion of added ¹⁴C-labelled glucose respired by microbes; microbial C content; abundance of bacteria in the rhizosphere soil; and diversity of C sources utilized by the microbial community

was not affected by defoliation. Moreover, the unaffected abundance of bacteria-feeding nematodes in defoliated *L. perenne* swards further suggests that bacterial growth was not affected by defoliation. Microbe-feeding nematodes are good indicators of microbial growth in soil (Sohlenius 1990; Christensen *et al.* 1992; Mikola & Setälä 1998), and their response to defoliation has been shown to be most clear a few weeks after defoliation (Mikola *et al.* 2005b). In the present study, we sampled nematodes 6 days after the May trimming and 25 days after the April trimming, suggesting that we should have observed the response of bacteria-feeding nematodes had it occurred. Like the effects of defoliation on root C release, effects on soil microbes (Garcia & Rice 1994; Guitian & Bardgett 2000; Hamilton & Frank 2001) and microbe-feeding animals (Stanton 1983; Todd *et al.* 1992; Todd 1996; Mikola *et al.* 2001) differ from positive to negative in earlier studies. Our finding that bacterial growth and abundance of bacterial grazers do not respond to defoliation when such a response is not observed in root C release suggests that part of this discrepancy may be accounted for by differences in root C release after defoliation.

In grasses, defoliation typically increases N concentration in shoot and root biomass (Wilsey *et al.* 1997; Fahnestock & Detling 1999; Green & Detling 2000; Hokka *et al.* 2004), which has been suggested to result from improved N availability in soil after defoliation (Holland & Detling 1990). Supporting this idea, Hamilton & Frank (2001) recently showed how increased C release from *Poa pratensis* roots after defoliation stimulated soil microbial growth, increased N availability in the plant rhizosphere, and finally led to elevated shoot N concentration in defoliated plants. In our study, defoliation reduced leaf N concentration 2 days after defoliation by 27%, did not affect root N concentration, and increased stem N concentration by 14%. In comparison with 129 and 56% increases in shoot and root N concentrations, respectively, recorded by Hokka *et al.* (2004) in defoliated *Phleum pratense* plants, defoliation appeared to have little impact on plant N uptake in our study. This is in good agreement with our finding that defoliation did not stimulate root C release and the growth of rhizosphere organisms in our grass swards, and suggests that rhizosphere N availability was not enhanced by defoliation in our study.

In contrast to the response of bacteria-feeding nematodes to defoliation, we found that defoliation reduced the abundance of root- and fungus-feeding nematodes. However root biomass was not significantly reduced by defoliation, suggesting that root mass did not determine the abundance of root-feeding nematodes. Instead, the lower quality of roots (higher C : N ratio) appears to account for the decreased abundance of root-feeding nematodes found in the rhizosphere of defoliated plants, suggesting that changes in root quality after defoliation can substantially affect the
abundance of root-feeding animals. Defoliation
typically enhances root quality in terms of increasing
root N concentration and decreasing root C : N ratio
(Seastedt 1985; Seastedt *et al.* 1988; Hokka *et al.* 2004),
but the effects may vary from positive to negative depend-
ing on the growth phase of defoliated plants (Ilmarinen
*et al.* 2005), and our results show, in contrast to earlier
observations, that defoliation can also increase the
C : N ratio of grass roots. That fungus-feeding nematodes
had a similar negative response to defoliation appears
unexpected considering we did not find any indication
of reduced microbial activity after defoliation. However,
we did not separately measure the response of fungi
to defoliation, and in bacteria-dominated agricultural
soil their response may have gone unnoticed. Also, we
did not measure whether defoliation affected the
abundance of arbuscular-mycorrhizal (AM) fungi in
soil. In earlier studies, negative effects of defoliation
on the root colonization rate of AM fungi are most
common (reviewed by Gehring & Whitham 1994),
although neutral (Ilmarinen *et al.* 2005; Mikola *et al.*
2005a) and positive (Hokka *et al.* 2004) effects in grass
roots have also been found. Finally, the classification
of root-feeding and fungus-feeding nematodes is pro-
blematic, and it is possible that some genera classified
as fungus-feeders in this study also feed on roots,
and vice versa (Yeates *et al.* 1993; Okada *et al.* 2005).
On the other hand, earlier studies in which the same
classification of nematode genera (Yeates *et al.* 1993)
is applied have reported different responses of root-
feeding and fungus-feeding nematodes to plant ageing
and defoliation (Mikola *et al.* 2001; Ilmarinen *et al.*
2005; Mikola *et al.* 2005b). Nevertheless, our results
show clearly that, even though no effects of defoliation
on bacteria and their grazers are found, effects on
root- and fungus-feeders may still appear. Whether
these effects also have significant consequences for
further plant growth and nutrient uptake remains to be
clarified.

We anticipated that soil N availability affects the
response of plants and soil organisms to defoliation, but
found evidence of this only in the case of plant shoot
production. Defoliation reduced shoot production
in plots where N was added, but did not affect plant
growth in non-fertilized plots. Defoliation is known to
reduce root nutrient uptake (Jarvis & Macduff 1989;
Donaghy & Fulkerson 1998) and, as N was added
immediately after trimming, the reduced growth of
defoliated plants in fertilized plots may result from their
reduced capability to utilize the additional N. This is
supported by our finding that N addition did not
significantly increase shoot production when plants
were defoliated, but had a substantial positive effect
on the shoot production of non-defoliated plants. That
N addition had little direct influence on soil microbes
was expected, as microbial growth in soils is usually
constrained by C rather than N availability (Schnürer
et al. 1985; Zak et al. 1994). However, nor did N addi-
tion appear to have much indirect influence on soil

microbes through altered C availability, despite having
positive effects on plant C assimilation (manifested
by increased shoot production in non-defoliated plots)
and root C concentration. The response of microbes to
N addition neither supports results from studies with
woody plants which have shown that, due to increased
fine root turnover, C input to soil increases when plants
are fertilized (King et al. 2002).

Acknowledgements

This study is a part of the projects 'Conservation of soil
organism diversity under global change' funded by the
EU (EU contract EVK2-CT-2002-20112) and 'Linking
below-ground food webs, plants and above-ground
herbivores in grassland ecosystems' funded by the
Academy of Finland. We thank Claude Bazard for
assisting with field work, Patrice Marchal for perform-
ing C and N analyses. Kari Nissinen for helping with
statistical issues and Iuliana Popovici for identifying
the nematodes.

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Received 23 February 2005; revised 14 June 2005; accepted 15 June 2005
<figure><img src="image_53.png" /></figure>
AUTORISATION DE SOUTENANCE DE THESE
DU DOCTORAT DE L'INSTITUT NATIONAL
POLYTECHNIQUE DE LORRAINE

000

VU LES RAPPORTS ETABLIS PAR :

Monsieur Jean-Bernard CLIQUET, Maître de Conférences, Université de Caen, Caen

Monsieur Jean-François SOUSSANA, Directeur de Recherche, INRA, Clermont Ferrand

Le Président de l'Institut National Polytechnique de Lorraine, autorise :

Monsieur BAZOT Stéphane

à soutenir devant un jury de l'INSTITUT NATIONAL POLYTECHNIQUE DE LORRAINE,
une thèse intitulée :

« Contribution à l'étude de l'allocation des photo-assimilats récents dans la plante et la rhizosphère chez une graminée pérenne (Lolium perenne L.) »

en vue de l'obtention du titre de :

DOCTEUR DE L'INSTITUT NATIONAL POLYTECHNIQUE DE LORRAINE

Spécialité : « Sciences agronomiques »

NANCY BRABOIS
2, AVENUE DE LA
FORET-DE-HAYE
BOITE POSTALE 3
F - 5 4 5 0 1
VANDŒUVRE CEDEX

Fait à Vandoeuvre, le 20 mai 2005

Le Président de l'I.N.P.L.,

L. SCHUFFENECKER

<figure><img src="image_54.png" /></figure>
## RÉSUMÉ

L'objectif de notre travail a été étudié le transfert de C dans la plante et vers la rhizosphère de *Lolium perenne* afin d'expliciter le couplage entre le fonctionnement de la plante et la rhizodéposition.

Nous avons d'abord déterminé la contribution du carbone récemment assimilé par la plante à la rhizodéposition. Nous avons ainsi réalisé un double marquage long des parties aériennes des plantes (¹⁴CO₂ et ¹³CO₂) afin d'estimer la répartition dans la plante et les compartiments rhizosphériques du C récent et le C plus anciennement assimilé. Contrairement à ce qui est admis généralement, le C ancien, probablement issu de la remobilisation de composés de réserve, contribue significativement à la rhizodéposition.

Nous avons ensuite évalué sur des dispositifs expérimentaux de terrain l'influence de contraintes subies par les parties aériennes de la plante sur les flux de C récent vers la rhizosphère (augmentation de la concentration en CO₂ atmosphérique et défoliation). La disponibilité en azote dans le sol est un facteur déterminant de la réponse des plantes à l'élévation des concentrations en CO₂ ou à la défoliation. A l'automne, une diminution de la rhizodéposition de C est observée pour les plantes soumises au doublement de CO₂. Les composés organiques libérés par les racines sont minéralisés plus rapidement sous CO₂ élevé que sous CO₂ ambiant. La défoliation n'induit pas de variations notables de la disponibilité en C dans la rhizosphère, suggérant aucune influence significative de ce facteur sur la rhizodéposition.

Les interactions entre les facteurs de l'environnement et de gestion de la parcelle cultivée déterminent le fonctionnement de la plante et modulent les efflux de C à partir des racines, ce qui rend la rhizodéposition difficile à prédire et à modéliser. Il s'avère que les effets nets du doublement de CO₂ ou de la défoliation sur la rhizodéposition sont beaucoup moins marqués au champ qu'en conditions contrôlées. Au delà de la contribution quantitative de la rhizodéposition au bilan de C dans le sol, les interactions entre la nature biochimique des rhizodépôts et l'activité biologique des sols nécessitent d'être approfondies.

## MOTS CLES :

Rhizosphère, rhizodéposition, *Lolium perenne*, carbone, doublement de CO₂, défoliation, marquages longs, marquages courts, ¹⁴C, ¹³C, ¹⁵N, indicateurs de rhizodéposition.

## DISCIPLINE :

Sciences Agronomiques

## INTITULE ET ADRESSE DU LABORATOIRE :

Laboratoire Agronomie-Environnement, UMR INPL-INRA, ENSAIA, 54505 Vandoeuvre les Nancy, France.

## SUMMARY

The aim of our work was to study C fluxes in plant and to rhizosphere of *Lolium perenne* in order to explain the relation between plant functioning and rhizodeposition.

In a first part, we have determined the contribution of recently assimilated carbon to rhizodeposition. We have developed a double long-term labelling of plants shoots (¹⁴CO₂ and ¹³CO₂) in order to estimate partitioning of recent C and older assimilated C in plant and rhizosphere compartment. In contrast to previous results, older C, probably coming from remobilisation of reserve compounds, contribute significantly to rhizodeposition.

In a second part, we have evaluated, with field trials, the influence of constraint affecting plants shoots on recently assimilated C fluxes to rhizosphere (elevated CO₂, defoliation). Soil nitrogen availability is the driving force of plants responses to elevated CO₂ or defoliation. In autumn, a decrease of C rhizodeposition is observed for plants submitted to elevated CO₂. Organics compounds released by roots are mineralised faster under elevated CO₂ than under ambient CO₂. Defoliation involved any change in C availability in the rhizosphere, suggesting any significant influence of this factor on rhizodeposition.

Interactions between environmental and management factors determined plant functioning and modulated C fluxes from roots which give rhizodeposition difficult to predict and to model. Net effect of elevated CO₂ or defoliation on rhizodeposition are less significant under field conditions than under controlled conditions. More than quantitative contribution of rhizodeposition to soil C pool, interactions between biochemical composition of rhizodeposits and soil biological activity need to be complete.

## KEYS WORDS :

Rhizosphere, rhizodeposition, *Lolium perenne*, carbon, elevated CO₂, defoliation, long term labelling and pulse labelling, ¹⁴C, ¹³C, ¹⁵N, rhizodeposition indicators.