JXB Advance Access originally published online on January 30, 2004
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Journal of Experimental Botany, Vol. 55, No. 397, pp. 743-750, March 1, 2004
© 2004 Oxford University Press
Plants and the Environment |
Interspecific control of non-symbiotic carbon partitioning in the rhizosphere of a grass–clover association: Bromus madritensis–Trifolium angustifolium
Received 10 July 2003; Accepted 4 November 2003
Centre d’Ecologie Fonctionnelle et Evolutive (CNRS, UPR 9056), 1919 Route de Mende, 34293 Montpellier Cedex 5, France
* To whom correspondence should be addressed. Fax: +33 (0)4 67 41 21 38. E-mail: warembourg{at}cefe.cnrs-mop.fr
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Abstract |
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Grass–legume interaction in the rhizosphere was investigated in a greenhouse experiment with two annual species, bromegrass Bromus madritensis (L.) and clover Trifolium angustifolium (L.) grown in mono and mixed cultures. Partitioning of below-ground carbon between roots, respiration, and soil was measured after separate 2 h-labelling of each species with 14CO2 followed by a 9 d chase period. At the time of labelling, clover nodules were not yet fixing N2. Bromegrass grew much faster than clover. Shoot biomass of bromegrass was greater in the presence of clover than in monoculture. By contrast, both shoot and root biomass of clover was less in the presence of bromegrass than in monoculture. Carbon assimilation during the period of labelling was proportional to shoot biomass and partitioning above and below-ground did not differ among treatments. Absolute amounts of labelled C allocated to rhizosphere respiration was more in bromegrass than in clover (respectively 1.38 mg C against 0.75 mg C in monoculture and 1.79 mg C and 0.63 mg C in mixed culture). However, when expressed as a percentage of below-ground C allocation, rhizosphere respiration was lower in bromegrass than in clover, respectively, 38% and 45% in monoculture. In mixed culture, this percentage increased by 7.3% for clover, and 3.5% for bromegrass, thus indicating that the interspecific effect of grass was higher than that of clover. The percentage of below-ground C in a soil solution of clover in mixed culture was more than 2-fold that measured in monoculture. It was also significantly correlated with the percentage of below-ground C in respiration. These results provided evidence that the grass–legume mixture has the potential to influence the rhizosphere processes of each species in more than an additive way and that the effect of the interaction was stronger on clover than on bromegrass. The possible implications of this in grass–legume competition are discussed.
Key words: Bromus madritensis, 14CO2 plant labelling, grass–legume interaction, rhizosphere respiration, root exudation, Trifolium angustifolium.
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Introduction |
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Grass–legume mixtures are important components of many low input grasslands and in Mediterranean regions, pastures and newly abandoned fields are characterized by annual grasses and annual legumes (Houssard et al., 1980). In these systems the legume contributes nitrogen through symbiotic N fixation. However, the grass–legume balance in such associations is sometimes difficult to manage and/or maintain and it has been hypothesized that grass–legume coexistence is the result of a subtle interplay between contrasting responses of grasses and legumes to available soil mineral N (Schwinning and Parsons, 1996). There are many resources for which grasses and legumes compete, including light, water, and soil nutrients (Thornley, 2001) and at low nutrient availability, competition tends to be below-ground. The rate of cycling of mineral nutrients is driven by soil microbial communities whose growth and activity depend on available C sources. The main source of labile organic C in grassland soil being from plant roots through exudation and turnover, there is a tight coupling between plant and microbial productivity (Paterson and Sim, 2000). In addition, enzymes, organic acids, and other molecules released in root exudates can modify plant growth directly by making cations available (Dakora and Phillips, 2002). Plant species have a selective influence on microbial diversity in the rhizosphere via the quality of root C inputs (Grayston et al., 1998). They also release different amounts of C compounds (Warembourg and Estelrich, 2001). Among plant species, the rate of nutrient acquisition will depend on root architecture (spatial distribution, growth, proliferation, branching) (Fitter, 1987) and interaction with adjacent roots either via competition and resource depletion or inhibitory compounds. Any change in rhizosphere processes due to species interactions will induce changes in C partitioning and costs for the plants. To what extent this can contribute to explain coexistence is not known.
Studying C allocation below-ground and partitioning between root and microbial activities is not easy. This is because of the complex and dynamic processes of root growth, rhizodeposition, and decomposition that occur simultaneously in different parts of the root system during growth. The use of tracers, such as 14C, facilitates the study of short-term C fluxes, such as those occurring in the rhizosphere soon after C assimilation (Warembourg and Kummerov, 1991; Van Veen et al., 1991; Killham and Yeomans, 2001). Nevertheless, accurate quantification of the portion of root exudates that is significant for microbial activity is hampered by the fact that it is readily respired and that the CO2 evolved is difficult to distinguish from that originated from root respiration. It is, however, assumed that when plants are pulse labelled with 14CO2, the 14CO2 efflux originating from roots (Warembourg and Billes, 1979; Kuzyakov et al., 1999; Todorovic et al., 2001) and root symbionts (Warembourg and Roumet, 1989) appears earlier than that derived from rhizo microbial respiration. Comparing the dynamics of 14CO2 after pulse-labelling of different plants or different treatments can therefore be a possible way to assess the relative importance of each source (Warembourg et al., 2003).
The purpose of this study was to use separate labelling with 14CO2 of the annual grass Bromus madritensis (L.) and the annual legume Trifolium angustifolium (L.) in monoculture and in mixed culture in order to compare C partitioning to root, rhizosphere respiration, and soil. The detailed analysis of rhizosphere respiration and 14C budgets were used to determine whether C fluxes in two-species cultures could be explained entirely as an additive effect of C fluxes measured on each of the two species grown in monoculture. Interspecific control of rhizosphere C flow by these two species, found together in abandoned fields of the Mediterranean region of the south of France, is discussed in terms of competition.
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Materials and methods |
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Plant material and culture
Seeds of bromegrass Bromus madritensis (L.) and clover Trifolium angustifolium (L.), were germinated at 20 °C in calcinated clay. One week after leaf emergence, seedlings were transplanted into 0.5 l plastic pots (8 cm wide and 11.5 cm deep) with 2 seedlings per pot either in monoculture or two-species mixture. The pots were filled with a 2 mm sieved siliceous sandy-loam soil (pH 5.8; N content 0.06%; C content 0.7%) with a bulk density of 1.15 Mg m–3. The soil was previously adjusted to 85% of field capacity by the addition of deionized water. Plants were grown in spring in a greenhouse. The temperature followed the external fluctuations which ranged from 15 °C at night to 23 °C during the day.
Labelling with 14CO2
Sixty-seven days after sowing, randomly selected pots of each treatment were transferred to a growth cabinet located in an isotope laboratory. Four pots of each species in monoculture and 8 of mixed culture were used. Plastic covers made to fit tightly on top of the culture pots were used to separate shoot and root atmospheres. The covers were cut in half and the two halves presented holes adapted to fit the base of the plant. Glass wool was inserted in the holes and RTV silicone rubber (Dow Corning) poured around the plant stem and over the cut. Along the side of the pots, inlet and outlet ports were used for aeration and collection of CO2. A third hole made in the cover and plugged with a rubber septum was used for watering. The plants were placed under artificial light (600 µmol m–2 s–1) and air was flushed through the soil containers. The next day, prior to labelling, aluminium foil was carefully used to cover the shoot of one or the other species of the mixed culture pots in order to prevent photosynthesis during labelling. Therefore, in monoculture, both plants of a pot were exposed to 14CO2 and the treatment referred to as Gra*Gra* for B. madritensis and Leg*Leg* for T. angustifolium. In mixed culture, only one species was exposed and the treatment referred to as Gra*Leg when B. madritensis was exposed, Leg*Gra when T. angustifolium was exposed. Labelling with 14CO2 was carried out in a chamber according to a method similar to that described by Warembourg and Kummerov (1991). The plants were exposed for 2 h (11.00 h to 13.00 h) to a specific activity of 370 kBq mg–1 C and a constant CO2 concentration (360 µmol mol–1). During this period the soil atmosphere of each pot, tightly separated from the labelled atmosphere, was flushed with outside air. At the end of the labelling period, aluminium foil was removed from the plants in mixed cultures and all pots were placed in an atmosphere without 14CO2.
Respiration measurements
Immediately after labelling, the soil atmosphere was flushed with CO2-free air and the 14CO2 evolved from each soil container was trapped in 0.2 M NaOH solution according to the classical aeration train method (Warembourg and Kummerov, 1991). The chase period lasted for 9 d, a period commonly adopted to account for the majority of net 14CO2 evolution after short-term exposure of plants to 14CO2 (Milchunas et al., 1985; Swinnen, 1994; Warembourg and Estelrich, 2001). NaOH solution was regularly replaced (twice a day during the first 3 d, every day thereafter) and its radioactivity measured by liquid scintillation. At the same time, soil water content was measured and adjusted gravimetrically to 85% of the water-holding capacity using deionized water.
Harvest and analysis
At the end of the chase period, plants were removed from the pots and the soil was immersed in 1.0 l of tap water. The roots of each pair of plants were freed from the soil and carefully separated under water. Shoots and roots were then separated and the water collected. The root fragments remaining in the soil were removed by hand and the soil was weighed to account for water content after drying. Shoot, root, and soil materials were dried at 70 °C, weighed and ground for analysis of C and 14C content by combustion and scintillation methods, respectively (Bottner and Warembourg, 1976). These methods allow for total C recovery and 100% 14C accounting after automatic correction for chemical quenching by the scintillation counter. Aliquots of the wash water were also analysed for 14C content after 48 h sedimentation in a cold room and adjustment to pH 7 in order to avoid counting dissolved 14CO2. In mixed culture, where only one species was exposed to 14CO2, measurements were done on shoot and root material of the non-exposed species in order to check for radioactivity.
Carbon budget calculations
Because all components of the plant soil system (shoots, roots, solution used to wash the roots, soil, and soil respiration) were measured, it was possible to calculate a balance of recovered 14C for each pot. The total amount of 14C represented net 14C assimilation by the labelled plants. In mixed cultures, plants whose shoots were wrapped with aluminium foil did contain some radioactivity, however, it never exceeded 0.01% of that measured in companion plants. Therefore, in these cultures, all 14C measured in the rhizosphere compartments was considered to have originated from the exposed plant.
Respired 14CO2 was the result of root respiration and microbial degradation of part of the root-derived C during the chase period. The amount of 14C measured in solution after washing the roots with water provided relative values for free soluble root exudates and microbial metabolites left in the root zone after microbial respiration. The amount of root-derived 14C left in the soil, exudates or root tissues, as well as any microbial biomass that already contained labelled material was calculated as the total 14C content of the soil minus the 14C content of the water left in the soil before drying. The proportions of 14C in each below-ground compartment (roots, rhizosphere respiration, soil, and solution) have been expressed as a percentage of net assimilated C or a percentage of below-ground C. Net C assimilation during exposure of a plant to 14CO2 was calculated by dividing the total radioactivity recovered by the specific activity of the CO2 in the labelling chamber.
Statistical analyses
The experiment consisted of two species in mono and mixed cultures, each of them labelled separately with 14CO2 and each combination had four replicates. A total of 16 plant/soil systems were used. Carbon allocation to each compartment was compared between treatments. Means and standard error of the means were calculated for all data. Significant differences between species and type of culture were assessed by one-way analysis of variance. Relationships between 14CO2 respired and 14C in solution was tested by linear regression. Statistical analyses were performed with Statgraphics Plus Package (Manugistics, Rockville, Ma, USA) for ANOVA and with EXEL (Microsoft) for regression.
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Results |
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Plant biomass
B. madritensis grew much faster than T. angustifolium in both mono and two-species cultures. Seventy-five days after sowing, the plant biomass of the grass was 2–3-fold higher than the legume biomass (Fig. 1a). In mixed culture, shoot growth was significantly enhanced for B. madritensis, while both shoot and roots biomass of T. angustifolium decreased slightly. The root–shoot biomass ratio of B. madritensis therefore decreased from 1.32 in monoculture to 0.84 in mixed culture (Fig. 1e). For T. angustifolium the R/S ratio was lower and remained around 0.6 in both types of culture. It is noteworthy that clover roots showed few nodules and these were not active at the time of labelling (Acetylene Reduction Assay; Hardy et al., 1973).
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Net 14C assimilation
Data on 14C of plants exposed at day 68 after sowing and followed by a chase period of 9 d were in accord with the results obtained for shoot biomass (Fig. 1a) with more 14C assimilated by grasses than legumes, and for grasses, more 14C assimilated in mixed culture than in monoculture (Fig. 1b). The rate of assimilation per unit of shoot biomass was, however, not significantly different between species and type of culture (Fig. 1c). The root-to-shoot partitioning ratio for labelled C did not differ significantly between species and treatments. It varied from 0.5 to 0.6 (Fig. 1e). For T. angustifolium this was similar to the biomass ratio thus indicating that root sink strength was rather constant for this species during the 75 d growth period. For B. madritensis the difference between R/S biomass ratio (1.32 and 0.84 in mono and mixed culture, respectively) and R/S assimilate distribution ratio at the time of labelling (around 0.5 for both cultures) indicated that root sink strength decreased with plant development. When considering the absolute 14C allocation below-ground including respiration and exudation, and expressed as a percentage of net assimilation, about half was exported below-ground with no significant difference between species, mono or mixed cultures (Fig. 1f). Partitioning between respiration, roots, and soil was, however, significantly different.
Rhizosphere respiration
Cumulative release of 14CO2 in the rhizosphere of each species in mono or mixed culture followed a logarithmic shaped curve (Fig. 2). It sharply increased during the first 2 d after labelling and then levelled off. The initial rates of respiration were higher for T. angustifolium than for for B. madritensis and no difference in the proportion of 14C evolved by respiration was detected for T. angustifolium between mono and mixed culture for the first 2 d. After that, respiration rates decreased and, for T. angustifolium, the decrease was higher in monoculture than in mixed culture. By day 9 after labelling a significant difference which amounted to 7.3% of below-ground 14C was recorded (Fig. 2). B. madritensis also respired significantly less in monoculture than in mixed culture, but the difference was apparent immediately after labelling. After 2 d, the respiration rates in B. madritensis were identical in both types of culture. This is indicated by the parallelism of the 14CO2 evolution curves.
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Partitioning of 14C among rhizosphere components
Following the chase period during which respired 14CO2 was collected and measured, the distribution of 14C in shoots, roots, root solution and soil was determined. The data are presented as percentages of net assimilated 14C for below-ground C and as percentages of below-ground 14C for below-ground compartments (Table 1). Although plants in mixed cultures exported more C below-ground than in monocultures, this was not significant. When grown in monoculture, both species presented differences in the way below-ground C was used in the rhizosphere. The proportion of 14C respired by B. madritensis, (38.1%) was significantly lower than that respired by T. angustifolium (44.6%). Reciprocally, the proportion retained by roots was higher in the former (57% against 50.5%). Percentages of below-ground 14C in the root solution was significantly different between the two species in monoculture with more C recovered for B. madritensis (1.76%) than for T. angustifolium (1.11%). No difference was recorded in soil 14C.
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In mixed cultures, respiration was affected and the effect of B. madritensis on T. angustifolium was more important than the opposing interaction (Table 1; Fig. 2). At the end of the chase period, the difference in the proportion of below-ground 14C respired by B. madritensis was 3.5% between mono and mixed culture. It reached 7.3% for T. angustifolium. Together with increasing respiration, the proportion of below-ground 14C in soil solution significantly increased in mixed culture. For T. angustifolium this proportion was more than 2-fold that measured in monoculture and there was a significant positive correlation (P <0.05) between the proportion of below-ground 14C respired and the proportion of below-ground 14C in solution (Fig. 3).
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Discussion |
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The characteristics and the interest in grass–legume associations as components of many types of grassland is that the plants present contrasting modes of N nutrition: atmospheric N for legumes and soil N for both grass and legumes according to N availability. During plant establishment, however, and before legume root nodules are effectively fixing N, which was the case in our experiment with young plants, this complementarity does not apply and both grass and legume plants rely on soil N. This and the fact that the soil used in this study had a limited supply of nutrients gave emphasis to the importance of specific rhizosphere processes which drive mineral nutrition. These were investigated through C partitioning between root growth, root exudation, and the related rhizosphere respiration which included a root and a microbial component. Studies have been done on C fluxes below-ground in grassland pastures or meadows that included grass and legume species (Warembourg and Paul, 1977; Saggar et al., 1997) but so far, no study has investigated interspecific effects of plants on rhizosphere C partitioning. The experimental design used in this study allowed the comparison of C allocation and use in the rhizosphere of plants grown in the presence of the same species (monocultures) or of another species (mixed cultures). The interactions therefore introduced intra- and inter-species competition in terms of C economy and were investigated using separate short-term labelling of one or other species with 14CO2.
Shoot biomass of grasses was higher in mixed culture than in monoculture whereas root biomass did not differ. By contrast, both shoot and root biomass of clover was less in mixed culture than in monoculture. Carbon assimilation was in agreement with above-ground biomass and there was no difference between grass and clover for the rate of assimilation per unit of green biomass. The proportion of assimilated C allocated below-ground was also very stable between species and treatments (around 50%).
Partitioning among rhizosphere components was, however, differently dependent on whether the companion plant was the same species or another species. Rhizosphere activities, respiration and exudation, were higher in mixed culture than in monoculture and the difference was greater for T. angustifolium than for B. madritensis. The dynamics of 14CO2 efflux from soil after labelling is often used as a comparative indicator of rhizosphere activity, since it includes root and microbial respiration even if the separation of these sources is not absolute (Warembourg and Billes, 1979; Todorovic et al., 2001; Kuzyakov, 2002). Source-separation is based on the assumption that these two processes predominate over different periods following short-labelling of plants with 14CO2. Immediately after labelling and for the first 24–30 h, the efflux of 14CO2 is attributed mainly to root respiration; later-on, microbial respiration of root exudates becomes increasingly predominant. As indicated by the logarithmic shape of the cumulative curves of 14CO2 evolution (Fig. 2), the switch between the predominance of root versus microbial respiration is gradual and may differ between species. At first, respiration (mainly root respiration) increased sharply to a level indicating the magnitude of root activity. Then, the rate of respiration gradually decreased to reach the rate of microbial respiration. With these assumptions, root contribution to rhizosphere respiration of T. angustifolium was higher than that of B. madritensis in monoculture, but the microbial component was less, as indicated by the slope of the curves. In a survey with annual and perennial grass and legume species including B. madritensis and T. angustifolium grown individually in artificial soil (Warembourg et al., 2003) the same difference was observed. Rhizosphere respiration of legume species was reported to be higher than that of grasses with a shorter half-life (23 h against 37 h). In mixed cultures the microbial component was significantly increased for T. angustifolium when the root component remained the same in mono or mixed culture. For B. madritensis the opposite occurred; the root component was slightly increased but the microbial component remained the same in mixed culture as compared with monoculture. This interspecific effect was corroborated by the amount of 14C measured in solution (Fig. 3). This category of C material, which included root and microbial compounds recovered at the end of the chase period, increased in T. angustifolium in the presence of B. madritensis and this was significantly correlated with the increase of rhizosphere respiration. Since the percentage of assimilated C translocated below-ground was not significantly different between plant species and type of culture, root growth was inversely proportional to losses by respiration and exudation. It was less in T. angustifolium than in B. madritensis with a minimum in mixed culture.
These results suggest that different species have the ability to change each other’s rhizosphere activity and that legumes are more affected than grasses. Several factors may explain these observations. First, root systems may respond architecturally to competition in order to adjust the patterns of nutrient uptake according to the architecture of the adjacent plant. Changes in C allocation to root biomass, root length, diameter, branching, and number of root tips will be reflected in root respiration and exudation (Nielsen et al., 1994). Second, there is a selective stimulation of micro-organisms in the rhizosphere according to the nature of C compounds exuded by the roots and a number of studies have assessed the diversity of microbial populations in the rhizosphere of different plant species, including clover and grasses (Sperber and Rovira, 1959; Lawley et al., 1983). A consequence is that metabolic profiles of microbial communities are distinct (Grayston et al., 1998) suggesting differences in quantity and quality of exudates and in microbial respiration, as found in the present study. This may also infer different abilities of the rhizosphere microflora to mineralize and compete with plants for soil nutrients. Studies of the effects that different plant species grown together have on rhizosphere microbial communities and functions are scarce. In a study with grassland plants, Wardle and Nicholson (1996) reported that increasing plant species richness from one to two has the potential to influence soil processes in a positive or negative way depending on species. The present results indicate that the effect of the grass–clover interaction is more than the additive effects of the two species grown in monoculture. Third, as it has been demonstrated that micro-organisms increase root exudation, either by consumption or by altering root permeability (Barber and Lynch, 1977), any changes in microbial population may have altered these functions.
The main result of this study is the insight it gave to the below-ground interaction of grass and legume related to competition. It has already been noted that most legumes are poor competitors with grass for nutrients probably because of differences in root morphology (Haynes, 1980). Furthermore, it is obvious from the present data that during the first weeks of growth, before the onset of N2 fixation, T. angustifolium lost more C in the rhizosphere in the presence of B. madritensis than in monoculture and this was not compensated for by more assimilation and/or allocation below-ground. In fact, both shoot and root growth was affected, thus leading to differences in plant biomass. By contrast, B. madritensis benefited from the interaction with T. angustifolium by an increase of shoot biomass compared with monoculture. Since there was no N effect owing to the absence of N2 fixation by clover, this could have indicated less competition for light in mixed culture. However, the rate of 14C assimilation was not significantly different for the two culture types. The increase of microbial activity through more root exudation by T. angustifolium in mixed culture could have increased nutrient availability to the benefit of B. madritensis only. The increase of root respiration by the latter in mixed culture as compared with monoculture could have been a confirmation of increased root uptake. This interspecific effect on below-ground partitioning contributes to the explanation that clover is the weaker competitor in mixed swards (Haynes, 1980; Frame and Newbould, 1986). Wasting a high proportion of C in respiration and exudation rather than utilizing it for root growth, seems, nevertheless, an inefficient reaction of T. angustifolium to the presence of B. madritensis. It could be a consequence of stress or disturbance, a syndrome described for soil microbial biomass in destabilized ecosystems (Wardle, 1993). The lack of competition of T. angustifolium in association to B. madritensis in the early stages of growth may be corrected for when the plants start fixing N2. However, in Mediterranean grasslands characterized by annual grasses and clovers, which are short-life-cycle plants, early vegetative growth is determinant for grass–legume dynamics and strong competition may affect seed production and the persistence of clover in the associations. In a study of the relationship between plant diversity and ecosystem properties in a Mediterranean grassland, T. angustifolium produced 74% more above-ground biomass at the end of the growing season in monoculture than in mixture with grasses, forbs, and other legumes (Gastine et al., 2003). In future studies, mechanisms that induce an increase of root exudation by clover in mixed culture and the nature of exuded compounds ought to be explored, together with the ecological implication of an increased rhizomicrobial activity.
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Acknowledgement |
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This work was supported by a PNSE grant from the French National Research Council (CNRS).
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References |
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