The influence of below-ground herbivory and defoliation of a legume on nitrogen transfer to neighbouring plants

Authors

  • E. AYRES,

    Corresponding author
    1. Soil and Ecosystem Ecology Laboratory, Institute of Environmental and Natural Sciences, Lancaster University, Lancaster LA1 4YQ, UK,
      †Author to whom correspondence should be addressed (present address: Natural Resource Ecology Laboratory, Colorado State University, Fort Collins, CO 80523-1499, USA). E-mail: edayres@nrel.colostate.edu
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  • K. M. DROMPH,

    1. Soil and Ecosystem Ecology Laboratory, Institute of Environmental and Natural Sciences, Lancaster University, Lancaster LA1 4YQ, UK,
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    • Present address: Department of Ecology, Royal Veterinary and Agricultural University, Thorvaldsensvej 40, DK-1871 Frederiksberg C, Denmark.

  • R. COOK,

    1. Institute of Grassland and Environmental Research, Plas Gogerddan, Aberystwyth, Ceridigion SY23 3EB, UK, and
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  • N. OSTLE,

    1. Centre for Ecology and Hydrology Lancaster, Library Avenue, Bailrigg, Lancaster LA1 4AP, UK
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  • R. D. BARDGETT

    1. Soil and Ecosystem Ecology Laboratory, Institute of Environmental and Natural Sciences, Lancaster University, Lancaster LA1 4YQ, UK,
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†Author to whom correspondence should be addressed (present address: Natural Resource Ecology Laboratory, Colorado State University, Fort Collins, CO 80523-1499, USA). E-mail: edayres@nrel.colostate.edu

Summary

  • 1Both foliar and root herbivory can alter the exudation of carbon from plant roots, which in turn can affect nitrogen availability in the soil. However, few studies have investigated the effects of herbivory on N fluxes from roots, which can directly increase N availability in the soil and uptake by neighbouring plants. Moreover, the combined effects of foliar and root herbivory on N fluxes remains unexplored.
  • 2We subjected the legume white clover (Trifolium repens L.) to defoliation (through clipping) and root herbivory (by an obligate root-feeding nematode, Heterodera trifolii Goggart) to examine how these stresses individually, and simultaneously, affected the transfer of T. repens-derived N to neighbouring perennial ryegrass (Lolium perenne L.) plants using 15N stable-isotope techniques. We also examined the effects of defoliation and root herbivory on the size of the soil microbial community and the growth response of L. perenne.
  • 3Neither defoliation nor root herbivory negatively affected T. repens biomass. On the contrary, defoliation increased root biomass (34%) and total shoot production by T. repens (100%). Furthermore, defoliation resulted in a fivefold increase in T. repens-derived 15N recovered in L. perenne roots, and increased the size of the soil microbial biomass (77%). In contrast, root herbivory by H. trifolii slightly reduced 15N transfer from T. repens to L. perenne when T. repens root 15N concentration was included as a covariate, and root herbivory did not affect microbial biomass. Growth of L. perenne was not affected by any of the treatments.
  • 4Our findings demonstrate that defoliation of a common grassland legume can substantially increase the transfer of its N to neighbouring plants by directly affecting below-ground N fluxes. These finding require further examination under field conditions but, given the prevalence of N-limitation of plant productivity in terrestrial ecosystems, increased transfer of N from legumes to non-N-fixing species could alter competitive interactions, with implications for plant community structure.

Introduction

Ecologists have long recognized that both above- and below-ground herbivory can have major consequences for primary producers, altering plant community structure and influencing rates of vegetation succession (McNaughton et al. 1989; Brown & Gange 1990; Brown & Gange 1992; Van der Putten, Van Dijk & Peters 1993; Bardgett 2005). However, over the past decade there has been a growing appreciation of the importance of indirect effects of above- and below-ground herbivory on plant communities, via their influence on soil biological properties and processes (Bardgett, Wardle & Yeates 1998; Bardgett & Wardle 2003; Wardle et al. 2004; Bardgett et al. 2005). In particular, it has become apparent that plant physiological responses to both foliar and root herbivory that alter rates of root exudation can influence the activity of rhizosphere microbes, which rely on exudates as their primary nutrient source, thereby altering rates of nutrient mineralization (Yeates et al. 1998, 1999; Hamilton & Frank 2001). Such responses of plants to herbivory therefore have the potential to alter nutrient availability and acquisition by plants, thereby influencing local-scale plant–plant interactions.

To date, most studies investigating the relationship between herbivory and plant inputs below ground have focused on carbon rhizodeposition. For instance, at the individual plant level, defoliation of a range of plants has been shown to increase the exudation of isotopically labelled C from roots into the soil (Holland, Chang & Crossley 1996; Hamilton & Frank 2001; Paterson et al. 2003, 2005; Murray et al. 2004; but cf. Todorovic et al. 1999; Mikola & Kytöviita 2002; Dilkes, Jones & Farrar 2004), which can stimulate microbial activity in the root zone (Mawdsley & Bardgett 1997; Guitian & Bardgett 2000) and increase soil nitrogen availability and its uptake by defoliated plants (Hamilton & Frank 2001; Ayres et al. 2004). Similarly, below-ground herbivory of clover has been shown to enhance transfer of C from plant roots to the rhizosphere, resulting in a stimulation of soil microbial biomass (Yeates et al. 1998, 1999; Bardgett, Denton & Cook 1999; Denton et al. 1999). These studies point to an indirect and short-term pathway whereby both above- and below-ground herbivory can enhance soil N availability to plants, via increased C rhizodeposition, leading to a stimulation of microbial populations and enhanced rates of N mineralization. However, few studies have investigated the direct effect of herbivory on the rhizodeposition of N from plants, which may also enhance N availability in the soil. For instance, below-ground herbivory of clover by root-feeding nematodes has been shown to increase the exudation of N into the soil, resulting in increased transfer of clover-derived N to a neighbouring grass species, stimulating its growth (Bardgett et al. 1999; Dromph et al. 2006). The effect of above-ground herbivory, and combined effects of root and foliar herbivory, on short-term N rhizodeposition and below-ground N transfer have not been investigated.

In this study, we investigated the effects of both defoliation through clipping and root herbivory on the transfer of N from a legume (Trifolium repens) to a neighbouring grass (Lolium perenne). We also measured the growth response of each plant species to defoliation and root herbivory of T. repens, and effects on the soil microbial biomass. Trifolium repens is the most abundant legume in European and New Zealand grasslands, and often co-occurs with grass species, including L. perenne (Whitehead 1995). Moreover, T. repens is frequently subject to both defoliation (by livestock and other herbivores, as well as cutting) and root herbivory (Cook et al. 1992; Whitehead 1995). In addition, up to 80% of grass N may be derived from clover (Broadbent, Nakashima & Chang 1982; Boller & Nösberger 1987; Ledgard 1991). In this study, we experimentally controlled root herbivory by inoculating microcosms with Heterodera trifolii, a widespread obligate root-feeding nematode that parasitizes T. repens throughout the UK and New Zealand, and is associated with reduced T. repens biomass (Skipp & Christensen 1983; Cook & York 1985; Cook et al. 1992). We tested the hypothesis that both above-ground clipping and below-ground herbivory would increase the transfer of T. repens-derived N to neighbouring L. perenne plants, thereby benefiting its growth. We predicted that the transfer of T. repens-derived N to neighbouring plants would be greatest when defoliation and root herbivory operate together. These hypotheses were tested in model T. repens–L. perenne systems, using T. repens plants whose foliage was labelled with 15N to enable measurement of N transfer.

Materials and methods

A brown earth soil was collected from an unfertilized hay meadow at Colt Park, Ingleborough National Nature Reserve, north-west England (54°12′ N, 2°21′ W). Colt Park meadow is an L. perenne–Cynosurus cristatus grassland on a soil of moderate–high residual fertility (Smith et al. 2003). The soil was passed through a 6-mm sieve and then defaunated by three freezing (−20 °C) and heating (50 °C) cycles. Several other studies have employed a similar method to defaunate soil (Laakso & Setälä 1999a, 1999b; Liiri et al. 2002). Although this defaunation procedure results in the death of some microbes and may alter their community composition, a soil microbial community remains (Laakso & Setälä 1999a). Defaunated soil was added to 40 0·75-l pots (10 cm diameter) into which two T. repens cuttings and two L. perenne tillers were planted. The plants were placed in a growth chamber maintained at 15 °C with 16/8 h light/dark cycles.

The treatments (± H. trifolii, ± clipping and ± labelling with 15N) were applied in a fully factorial randomized block design with five replicate blocks. Treatments were not replicated within each block. After 90 days, half the pots were inoculated with H. trifolii (an obligate root-feeding nematode of T. repens that does not attack L. perenne) by adding a suspension of ≈1000 infective juveniles to the soil. The H. trifolii inoculum was prepared from full cysts extracted from pots of sandy soil planted with T. repens. Clean cysts were placed on sieves standing in shallow trays filled with water, and infective juveniles emerging from eggs within these cysts were collected daily and stored at 2 °C. Suspensions of juveniles were bulked and concentrated by settling and decanting at 2 °C, and aliquots taken from the suspension were pipetted into soil around the T. repens plants. The clipping treatment of T. repens (removal of ≈50% of leaves) was applied on two occasions (days 174 and 186); L. perenne plants were not clipped. Removal of 50% leaf area was considered appropriate as it is similar to defoliation of clover-dominated pasture (Orr et al. 2004). The short time between clipping treatment and labelling, compared with the H. trifolii treatment, was due to the ephemeral (days) nature of defoliation on nutrient fluxes from plant roots to soil (Paterson & Sim 1999, 2000). In contrast, H. trifolii needed time to colonize the roots of T. repens after inoculation. Half the pots were labelled with 15N for 7 days starting on day 188; the remaining unlabelled pots were used to determine the natural abundance of 15N stable isotopes and were not included in the statistical analysis of any measure. Labelling with 15N consisted of immersing three trifoliate leaves of each T. repens plant into a 10 at %15N-labelled 300-mm KNO3 solution for 7 days immediately before the end of the experiment (Bardgett et al. 1999).

The pots were destructively harvested 195 days after establishment, immediately after labelling with 15N. Roots from each species were carefully separated from the soil. Nodules were clearly visible on clover roots, indicating that they had successfully formed an association with Rhizobium. Shoot and root biomass for each species was determined by drying and weighing plant material (70 °C). A subsample of shoot and root material was ground for stable isotope analysis, which was conducted on a Carlo Erba elemental analyser (CE Elantech, Lakewood, USA) coupled to a isotope ratio mass spectrometer (Denis Leigh Technologies, Manchester, UK). Separate clover and grass-root subsamples, taken from block 1 pots only, were analysed for root infestation by H. trifolii: only a single measurement of clover and grass-root infestation was made for each treatment. Roots were cleared, mixed with distilled water (25 ml) and acid fuchsin stain solution (1 ml), and boiled for 30 s (Byrd, Kirkpatrick & Barker 1983), then stained nematodes within the roots were counted using a stereomicroscope. In addition, H. trifolii cysts were extracted from 100 g (FW) soil from each microcosm by elutriation for 90 s at 4·5 l min−1, collected from the floated debris by hand under a stereomicroscope, and counted. Soil microbial biomass C and N were determined using the fumigation–extraction procedure (Brookes et al. 1985; Vance, Brookes & Jenkinson 1987).

The concentration of 15N in plant tissues was calculated using the following equation:

15Nconc = Nconc × (15Nat%/100)( eqn 1)

where 15Nconc is the concentration of 15N in the plant tissue, Nconc is the concentration of N in the plant tissue, and 15Nat% is the percentage of N atoms in the plant tissue that are 15N rather than 14N. The natural abundance of 15N in unlabelled plants was averaged across the five replicates of each treatment combination, and this value was subtracted from the 15N concentration of each of the labelled plants subjected to that treatment, to determine excess 15N concentrations.

Data were analysed using generalized linear models (Nelder & Wedderburn 1972) with the GENMOD procedure in sas ver. 8·0 (SAS Institute 1990). Defoliation, H. trifolii and block were independent variables in each analysis, and T. repens root excess 15N concentration was used as a covariate for analysis of grass excess 15N data, as 15N transfer to L. perenne was expected to relate to the concentration of 15N in T. repens roots (Bardgett et al. 1999; Dromph et al. 2006). Heterodera trifolii inoculation was not included as an independent variable for the analysis of H. trifolii cyst abundance in soil, as only a single cyst was found in the uninoculated pots. The number of H. trifolii cysts in the soil, T. repens and L. perenne shoot and root biomass, and microbial biomass C and N were log-transformed to meet assumptions of normality and homogeneity of variance. Statistical significance was taken as P < 0·05.

Results

Inoculation of T. repens with the root-feeding nematode was successful, resulting in an average of 917 cysts per kg soil in the H. trifolii treatments; only a single cyst was found in one sample of the uninoculated soil, confirming that the initial defaunation procedure had been successful. Defoliation did not significantly affect cyst abundance in the inoculated soil; mean ± SE number of cysts per kg dry soil were 587 ± 237 in the unclipped treatment and 1248 ± 415 in the clipped treatment (F1,4 = 0·1, P > 0·1). Consistent with the soil data, no H. trifolii were observed in T. repens roots from uninoculated pots, whereas 1318 H. trifolii per g root were observed in roots of undefoliated T. repens, and 411 H. trifolii per g root were observed in roots of defoliated T. repens. This level of infection is equivalent to 66 nematodes per plant in the undefoliated treatment and 21 nematodes per plant in the defoliated treatment; around 25 nematodes per plant is typical of lightly infested grass–clover pastures in the UK (Cook et al. 1992). No H. trifolii were observed in L. perenne roots from any of the treatments.

Neither defoliation nor infection with H. trifolii affected T. repens shoot biomass at the end of the experiment (Fig. 1a). However, defoliation doubled T. repens total shoot biomass production, when calculated as the biomass of accumulated clippings taken from the two defoliation events and shoot mass at the end of the experiment (F1,4 = 43·9, P = 0·003; Fig. 1b), indicating that T. repens compensated for the tissue lost as a result of defoliation. Defoliation also increased T. repens root biomass by 34% at the end of the experiment (F1,4 = 5·1, P = 0·024; Fig. 1c). Labelling of T. repens with 15N was successful: clover-root excess 15N values were 3·9 µg per g root when averaged across all treatments (Fig. 1d). The allocation of 15N to roots of T. repens was unaffected by any of the treatments.

Figure 1.

Effect of the root herbivore Heterodera trifolii and defoliation on Trifolium repens (a) shoot biomass; (b) total shoot biomass production (shoot biomass plus clippings); (c) root biomass; and (d) root excess 15N at end of experiment. Results from a generalized linear model are also shown (main effects and interaction). Values are means (± SE) of five replicates. Defol, defoliation.

Defoliation of T. repens significantly increased the size of the soil microbial biomass (microbial biomass C) by 77% (F1,4 = 15·4, P < 0·001), but had no effect on the amount of N contained within microbes (microbial biomass N) (Fig. 2). Inoculation with H. trifolii, and the interaction between H. trifolii and defoliation, did not affect microbial biomass C or N.

Figure 2.

Effect of the root herbivore Heterodera trifolii and defoliation on microbial biomass (a) carbon; (b) nitrogen. Results from a generalized linear model are also shown (main effects and interaction). Values are means (± SE) of five replicates. Defol, defoliation.

Both inoculation of T. repens with H. trifolii and defoliation significantly affected the recovery of T. repens-derived 15N in grass roots when T. repens root 15N concentration was included as a covariate (H. trifolii, F1,3 = 4·5, P = 0·034; defoliation, F1,3 = 6·8, P = 0·009; Fig. 3a). Defoliation of T. repens resulted in a fivefold increase of 15N recovery in L. perenne roots, while inoculation with H. trifolii reduced 15N transfer from T. repens to L. perenne by 13%. The concentration of T. repens-derived 15N in grass shoots was not affected by the treatments (Fig. 3b), and neither treatment altered L. perenne root or shoot biomass at the end of the experiment (Fig. 4).

Figure 3.

Effect of the root herbivore Heterodera trifolii and defoliation on Trifolium repens-derived excess 15N concentration in L. perenne (a) roots per unit excess 15N in T. repens roots, and (b) shoots. Results from a generalized linear model are also shown (main effects and interaction). Values are means (± SE) of five replicates. Defol, defoliation. Denominator df = 3 in these analyses because T. repens root excess 15N was included as a covariate.

Figure 4.

Effect of the root herbivore Heterodera trifolii and defoliation on Lolium perenne (a) shoot, and (b) root biomass. Results from a generalized linear model are also shown (main effects and interaction). Values are means (± SE) of five replicates. Defol, defoliation.

Discussion

The aim of our study was to test the hypothesis that both defoliation and root herbivory of clover (T. repens) stimulates soil microbial biomass and the flux of T. repens-derived N to neighbouring plants. This was achieved by measuring the effect of defoliation and root herbivory on the soil microbial biomass and the transfer of T. repens-derived 15N to the neighbouring grass L. perenne. Defoliation resulted in a fivefold increase in concentrations of 15N in L. perenne roots, indicating increased transfer of N from T. repens. The concentration of 15N in L. perenne shoots was also greater in the defoliated treatment; however, this increase was not significant, possibly because the shoots are an extra ‘step’ removed from the source of 15N. Although we cannot be certain of the mechanism behind the increase in N transfer from T. repens to L. perenne, the short period between 15N labelling of T. repens and 15N sampling of L. perenne suggests a direct physiological mechanism, such as increased exudation of N to the soil and/or transfer of N by mycorrhizae. Nitrogen transfer from legumes to non-legumes via mycorrhizae has been demonstrated in other studies, for example, from Trifolium alexandrinum to Zea mays (Frey & Schüepp 1992). Regardless of the mechanism, this is the first time, as far as we are aware, that defoliation has been shown to increase N transfer from one plant species to a neighbouring plant species (‘direct pathway’, Fig. 5). Defoliation has been shown to stimulate soil N availability through effects on the physiology of trees (Ayres et al. 2004) and a grazing-tolerant grass (Hamilton & Frank 2001). However, this was thought to be the result of increased C inputs enhancing the mineralization of soil organic N via a stimulation of the soil microbial community (‘indirect pathway’, Fig. 5; Hamilton & Frank 2001), rather than increased N inputs into the soil by the defoliated plants. The positive effect of defoliation on microbial biomass that we observed indicates that the indirect pathway may also have operated in this study, although we did not measure N mineralization in the soil. The results presented here, in combination with evidence from the literature, suggest that defoliation can increase N availability via both a direct and indirect pathway (Fig. 5). First, defoliated plants can increase N inputs below-ground, resulting in enhanced transfer to neighbouring plants (direct pathway). Second, defoliated plants often increase soil C inputs, stimulating the activity of the soil microbial community that mediates N-mineralization rates (indirect pathway) (Hamilton & Frank 2001). Further studies are required to determine if defoliation of other legumes, and non-leguminous species, influences N transfer to neighbouring plants.

Figure 5.

Conceptual diagram showing how defoliation can increase soil nitrogen availability via direct (grey dotted box) and indirect (solid lined box) pathways.

Inoculation of T. repens with H. trifolii also altered N transfer from T. repens to L. perenne, resulting in a 13% reduction in N transfer when T. repens root excess 15N concentration was used as a covariate. This is surprising given that two other studies, using a similar approach, have shown increased 15N transfer from T. repens to L. perenne in response to root feeding by nematodes. Bardgett et al. (1999) observed that H. trifolii increased 15N flux from T. repens to L. perenne; and Dromph et al. (2006) observed a positive relationship between the density of two root-feeding nematodes (H. trifolii and Pratylenchus sp.) in T. repens roots, and 15N transfer to, and growth of, L. perenne. The study by Bardgett et al. (1999) had a long period between labelling and sampling (84 days), whereas we sampled immediately after labelling with 15N. Thus our contrasting findings may indicate that increased transfer of T. repens-derived N as a result of root herbivory may become apparent only after several weeks or months. Dromph et al. (2006) observed increased T. repens-derived N transfer associated with greater densities of plant-parasitic nematodes; however, two nematode species were present in this study (H. trifolii and Pratylenchus sp.), which may have caused the different results.

We found that the growth of T. repens, measured as shoot and root biomass at the end of the experiment, was unaffected by root herbivory by an obligate root-feeding nematode. In contrast, defoliation of T. repens significantly increased both total accumulated shoot biomass (100%), indicating compensatory growth, and root biomass (34%), relative to undefoliated plants. Denton et al. (1999) found that low-level infestation of T. repens with H. trifolii did not affect shoot or root biomass, but high-level infestation reduced shoot biomass (Denton et al. 1999). In contrast, Bardgett et al. (1999) observed greater clover root biomass in the presence of H. trifolii 84 days after inoculation. It has long been known that defoliation can promote compensatory growth in a range of plants (McNaughton 1985; Hamilton & Frank 2001; Ayres et al. 2004) including T. repens (del Val & Crawley 2004). Our findings indicate that moderate rates of defoliation can promote root and shoot production by T. repens, and simultaneously increase N transfer to L. perenne. Given that N-limitation is common in terrestrial ecosystems (Vitousek & Howarth 1991), increased N supply has the potential to influence plant production in grassland ecosystems. Several studies have observed positive effects of herbivory on plant production in natural ecosystems, which have been attributed to a variety of mechanisms, such as selective feeding altering plant community structure, or stimulation of soil nutrient availability due to the addition of labile organic matter in animal waste (McNaughton et al. 1997a, 1997b; Augustine & McNaughton 1998; Frank & Groffman 1998); although negative effects of herbivory on plant productivity are also common (Pastor et al. 1993; Bardgett & Wardle 2003). Our finding may represent a previously unrecognized mechanism for the stimulation of primary productivity, which requires further testing under field conditions.

The growth of L. perenne, measured as shoot and root biomass, was not affected by any of the experimental treatments. This is surprising given the increase in N transfer to L. perenne as a result of defoliation and the N-responsive nature of this species (Daepp, Nösberger & Lüscher 2001; Wagner et al. 2001). However, the lack of response may be due to the relatively short period between the first defoliation event and the end of the experiment (3 weeks), and/or the inherently high availability of N in the soil due to its moderate–high fertility and a pretreatment of sieving and defaunation (several freeze–thaw cycles). Both these disturbances are likely to have resulted in a flush of N to the soil which, in combination with the N fixed by rhizobia on the roots of T. repens, may have meant that L. perenne was not N-limited. Contrary to our findings, Bardgett et al. (1999) and Dromph et al. (2006) observed increased L. perenne growth when neighbouring T. repens was subjected to root herbivory. In these experiments, increased growth of L. perenne coincided with increased transfer of T. repens-derived N to L. perenne (Bardgett et al. 1999; Dromph et al. 2006). Both Bardgett et al. (1999) and Dromph et al. (2006) used soil of low N status, which might have resulted in N-limitation of L. perenne, and potentially explains why L. perenne biomass increased in response to enhanced N transfer from T. repens in their studies, but not in ours.

As far as we are aware, this study is the first to investigate the effects of both defoliation and below-ground herbivory on nutrient fluxes to neighbouring plants. The results show that both types of herbivory may influence N transfer between plant species. However, they do not support our hypothesis that defoliation and below-ground herbivory interact positively to increase N transfer from T. repens to L. perenne. Consistent with our findings, studies of plant responses to above- and below-ground herbivory often report few interactive effects (Müller-Schärer & Brown 1995; Maron 1998; Rudgers & Hoeksema 2003), despite evidence that above- and below-ground herbivores of the same plant can influence each other (Moran & Whitham 1990; Masters & Brown 1992; Masters, Brown & Gange 1993). The results presented here warrant further investigation under field conditions and with real above-ground herbivores. However, given the prevalence of N-fixing plants in grassland and early successional communities (Stevens & Walker 1970; Whitehead 1995), foliar herbivory could have important implications for plant community structure and succession, as increased N transfer from N-fixers to neighbouring non-N-fixing species might alter their competitive interactions.

Acknowledgements

Technical assistance was provided by Helen Quirk, Kate Harrison and Tony Mizen. Isotope analysis was undertaken at the Natural Environment Research Council's Stable Isotope Facility by Helen Grant, Darren Sleep and Andy Stott. Diana Wall, Jim Detling, Breana Simmons and three anonymous reviewers provided valuable comments on an earlier version of this manuscript. We are very grateful for their assistance. This research was funded by a grant awarded to R.D.B. from the UK Biotechnology and Biological Sciences Research Council (BBSRC), under its Biological Interactions in the Root Environment (BIRE) thematic programme.

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