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Characterizing nitrogen transfer from red clover populations to companion bluegrass under field conditions

Publication: Canadian Journal of Plant Science
1 October 2012

Abstract

Thilakarathna, R. M. M. S., Papadopoulos, Y. A., Rodd, A. V., Gunawardena, A. N., Fillmore, S. A. E. and Prithiviraj, B. 2012. Characterizing nitrogen transfer from red clover populations to companion bluegrass under field conditions. Can. J. Plant Sci. 92: 1163–1173. The ability of two red clover (Trifolium pratense L.) cultivars, AC Christie (diploid) and Tempus (tetraploid), to transfer fixed nitrogen (N) to companion bluegrass (Poa pratensis L.) was evaluated under field conditions. Plant samples were harvested three times during the 2009 growing season and N transfer from the red clover cultivars to bluegrass was determined using the natural abundance method for first harvest and 15N dilution techniques for second and third harvests. Soil and soil water samples were used to evaluate cultivar effects on soil N conditions. Both red clover cultivars derived more than 90% of their N from biological N fixation. The proportion of bluegrass N derived from interplant N transfer was 7, 11, and 26% for the first, second, and third harvests, respectively. Soil KCl extractable nitrate increased along the three cuts for Tempus in the 0 to 15-cm soil zone. Soil-water nitrate content increased periodically for AC Christie and remained constant for Tempus throughout the growing season. This result indicates that the two cultivars have distinctly different N cycling patterns.

Résumé

Thilakarathna, R. M. M. S., Papadopoulos, Y. A., Rodd, A. V., Gunawardena, A. N., Fillmore, S. A. E. et Prithiviraj, B. 2012. Caractérisation au champ du transfert de l'azote entre le trèfle rouge et le pâturin des prés cultivé comme plante-abri. Can. J. Plant Sci. 92: 1163–1173. Les auteurs ont évalué la capacité au champ de deux cultivars de trèfle rouge (Trifolium pratense L.), AC Christie (diploïde) et Tempus (tétraploïde), à transférer l'azote (N) qu'ils ont fixé à la plante-abri qu'est le pâturin des prés (Poa pratensis L.). Les échantillons végétaux ont été prélevés à trois reprises pendant la période végétative de 2009 et on a mesuré la quantité de N transférée des variétés de trèfle rouge au pâturin par la méthode de la teneur isotopique naturelle, à la première coupe, et celle de la dilution du 15N, à la deuxième et à la troisième coupes. Des échantillons de sol et d'eau du sol ont permis d’évaluer l'incidence du cultivar sur les conditions du N du sol. Les deux variétés de trèfle rouge tirent plus de 90% du N par fixation biologique. La proportion de N issue du transfert entre plantes chez le pâturin s’élève respectivement à 7, 11 et 26% à la première, à la deuxième et à la troisième coupe. Chez Tempus, la concentration de nitrate extractible au KCl augmente d'une coupe à l'autre, dans la couche de 0 à 15cm de profondeur du sol. La concentration de nitrate dans l'eau du sol augmente périodiquement avec AC Christie, alors qu'elle demeure la même durant la période végétative avec Tempus. Ce résultat laisse croire que le cycle de l'azote diffère radicalement chez les deux cultivars.
Red clover is one of the most important forage legumes grown in Canada, especially for livestock production and as a rotation crop. Its high biological N fixing ability (BNF) and compatibility with different grasses makes it an ideal companion legume in forage mixtures (Carlsson et al. 2009). Red clover can also transfer fixed N to companion non-legumes (Ta and Faris 1987; Høgh-Jensen and Schjoerring 1994, 2000; Pirhofer-Walzl et al. 2011), which helps farmers to reduce inorganic N fertilizer applications and lessens subsequent nitrate leaching into ground water. The transfer of N is the “movement of N from a legume to another plant, either during growth of an interplant associated with a legume component or as residual N for the benefit to a succeeding plant” (San-nai and Ming-pu 2000). Below-ground N transfer can be categorized as being direct or indirect. Direct N transfer results from mycelia networks formed by arbuscular mycorrhizal fungi and directly supplies N from the donor plant to the companion plant by interconnecting the root systems of both species (Haystead et al. 1988; McNeill and Wood 1990; Dubach and Russelle 1994; He et al. 2003). Indirect N transfer occurs through the soil compartment. This takes place through the rhizodeposition of N into the soil followed by uptake by grass (Paynel et al. 2008). Proposed sources of rhizodeposition include: death and decay of nodules and roots (Ta et al. 1986; Dubach and Russelle 1994; Trannin et al. 2000; Sierra and Desfontaines 2009) and exudates from legume roots (Paynel et al. 2001, 2008; Jalonen et al. 2009a, b; Sierra and Desfontaines 2009). Among the different N compounds exuded by legume roots and nodules are ammonium, amino acids, ureides, peptides, and proteins, which have been identified in leachates of legumes (Ta et al. 1986; Wacquant et al. 1989; Paynel and Cliquet 2003; Paynel et al. 2008; Fustec et al. 2010). The most abundant amino acids found in clover root exudates are serine and glycine (Paynel and Cliquet 2003).
The N requirement of grasses grown in legume–grass mixtures can be met, in part, via transfer of symbiotically fixed N from legumes to non-legumes (Walley et al. 1996). When considering the transfer distance of N from legumes to adjacent non-legumes, Gebhart et al. (1993) report that N transfer occurred over a distance of at least 25 cm, and that N transfer increased as the proportion of legumes increased in the mixture. Also, Paynel et al. (2008) report that N fertilizer increase N transfer between legumes and grasses because the resultant increase in soil exploration by grasses provides greater access to available N sources, including the N compounds exuded by clover.
It is important to investigate the major aspects associated with N transfer during the growth of pasture stands. The objectives of this field study were: (1) to quantify N transfer during the growing season, (2) to evaluate the effect of cultivar and legume/grass mixture on N transfer, and (3) to determine the effect of N transfer on soil mineral N. To our knowledge, this is the first field research study to evaluate the N transfer ability of a pasture legume on an individual plant basis.

MATERIALS AND METHODS

Site Description

The experimental area was located at the Agriculture and Agri-Food Canada Research Farm in Nappan, NS (lat. 45°N, long. 64°W, 20 m above mean sea level). Mean monthly temperatures during the June, July, and August 2009 growing periods were 15.4, 17.5, and 18.6°C, respectively. At the experimental site, total precipitation during the study period (June to August 2009) was 355 mm; 81.4, 141.8, and 131.4 mm during the months of June, July, and August, respectively. The experiment was conducted on a Gleyed Brunisolic Gray Luvisol fine sandy loam of the Queens series (Webb and Langille 1995). From 2003 to 2006, the field was mainly cropped to corn and was not treated with fertilizer (organic or inorganic). Oats and peas were grown for silage in 2007, followed by fall rye.

Plant Establishment

A pure bluegrass stand was established in March 2008 (variety Ginger, seeded at 14 kg ha−1). Two red clover cultivars were selected based on different ploidy levels; diploid (AC Christie, Martin et al. 1999) and tetraploid (Tempus, http://www.inspection.gc.ca/english/plaveg/variet/regvare.shtml). The red clover plants were grown in a greenhouse at the Nova Scotia Agricultural College for 90 d in plastic rootrainer trays (Beaver Plastics Ltd., Acheson, AB). Pro-mix was used as the growing media, and plants were inoculated with Rhizobium leguminosarum biovar trifolii. Three weeks before transplanting, the red clover plants were clipped, leaving 5-cm above-ground growth to induce the growth of vigorous plants. The two red clover cultivars were transplanted individually to the field on 2008 Jul. 15. The experimental layout was a completely randomized design with 12 field replicates per red clover cultivar.

15N Labelling to Determine N2 Fixation and Transfer

Nitrogen fixation and transfer were determined using a 15N isotope dilution technique as well as natural abundance methods during the second year after red clover establishment. During the 2009 growing season, individual red clover plants surrounded by bluegrass were permanently marked with 45-cm-diameter plastic rings in the field. The pure bluegrass control was also permanently marked with 45-cm-diameter plastic rings. Both the red clover and bluegrass inside the plastic rings were uniformly labelled with 10 atom% 15N-ammonium sulphate (Sigma-Aldrich, Oakville, ON) on 2009 Jun. 10 (just after the first harvest in the first production year) according to the 15N dilution technique (Mallarino et al. 1990). The 15N labelled ammonium sulphate (10 atom%15N) was applied at 1 kg 15N ha−1. A water-based solution of 15N labelled fertilizer was sprayed on the marked areas (10 mm m−2) and was carefully watered down with approximately 10 mm m−2 of water. The plot outside the 15N-labelled area received an equivalent top-dressing of ammonium sulphate.

Harvest and Analysis

Plants were harvested when 50% of the red clover plants reached the early blooming stage. The red clover plants, the bluegrass surrounding each red clover plant, and the pure bluegrass (control) were harvested 5 cm above the soil surface with scissors. One harvest was taken during the establishment year (late September 2008) and three harvests were taken during the growing season, on 2009 Jun. 08, Jul. 14 and Aug. 14. Herbage was harvested from the 0.159-m2 area enclosed by the plastic rings. Herbage from the bluegrass/red clover stand was separated by species. All the harvested plant material was dried individually at 65oC for 48 h in a forced-air oven to measure dry matter yield. Dry plant samples were ground using a Wiley mill, standard model 3 (Arthur H Thomas Co., Philadelphia, PA), to pass through a 1-mm sieve followed by a mixer mill (Retsch Germany). The plant materials were analyzed for 15N and total N using a mass spectrometer (Costech ECS4010 Elemental Analyzer coupled to a Delta V mass spectrometer).
Since the plant materials from the first harvest (2009) were not labelled with enriched 15N, the proportion of N derived from the atmosphere (% Ndfa) of the two red clover cultivars was calculated using the following formula, according to the natural abundance technique (Høgh-Jensen and Schjoerring 1994),
Where d15N is the 15N enrichment relative to atmospheric N. The B value is the 15N enrichment relative to atmospheric N, for the clover grown solely on atmospheric N. A B value of –0.76 δ15N was used (Nimmo 2011).
The % Ndfa of the two red clover cultivars for the second and third harvests was calculated using the following formula, according to the isotope dilution technique (Jørgensen et al. 1999),
Where atom% 15N excess = atom% 15N (clover or grass) − 0.3663.The 15N natural abundance of 0.3663 was used to adjust for the 15N abundance of plant samples from the background contribution.
The amount of N fixed (g) by different red clover cultivars was determined by
The apparent transfer of N from clover to grass during the first harvest was calculated using the method cited by Høgh-Jensen and Schjoerring (1994):
The apparent transfer of N from clover to grass during the second and third harvests was calculated using methods cited by Jørgensen et al. (1999); the amount of biologically fixed N transferred from red clover to bluegrass was calculated, and the 15N enrichment for bluegrass in pure stand was compared with that from a mixed stand:
Total N yield of the red clover and bluegrass was calculated by multiplying their dry mass by their tissue N concentration.

Soil Mineral Nitrogen Study

During the 2009 growing season, the same set of plants was used to evaluate the impact of two red clover cultivars (AC Christie and Tempus) under a mixed stand and a pure bluegrass stand on soil N conditions. In early spring, ceramic suction lysimeters, consisting of round-bottom porous ceramic cups (Hoskins Scientific, Burlington, ON) with polyvinyl chloride rubber tubing attached, were permanently inserted into the soil 5 cm from the base of the red clover plant within each ring to a depth of 15 cm. As a reference control, a second set of ceramic suction lysimeters was installed outside the plot area in a pure bluegrass stand. After each rainfall that exceeded 10 mm, soil water samples were collected from the lysimeters by applying a vacuum of 0.8 bar using a mobile vacuum pump (Bouman et al. 2010). Soil water samples were collected six times over the 2009 growing season. The accumulated soil water from the ceramic cups was vacuumed up through the rubber tubing into an outside collecting amber glass bottle. Samples were immediately stored at −20°C until the nitrate analysis. The nitrate concentration of the samples was analyzed using a Waters Ion Chromatography System (Waters Canada Ltd.) which comprised of a Waters Model 1525 Binary HPLC Pump, a Waters Model 717-Plus Autosampler, and a Waters Model 432 Conductivity Detector. Soil solution samples were syringe-filtered with 0.45-µm nitrocellulose membrane filters in preparation for single-column ion chromatography with direct conductivity detection (Eaton et al. 2005). A Waters IC-PAK Anion HC 4.6 × 150 mm was the anion-exchange column used. The detection limit of analysis was 0.08 mg L−1. For the soil water analysis, samples with concentrations below the detection limit were assigned a value of 0.04 mg L−1, as an option outlined in McBean and Rovers (1998).

Soil Sampling

Soil samples were collected in the establishment year (2008) and on three harvest dates of the first production year. Soil cores were obtained randomly within each ring from soil depths of 0–15 and 15–30 cm. These soil samples were extracted using 2.0 M KCl according to Maynard et al. (2008) and analyzed for available N ( and ) using Technicon Auto Analyzer II (Tarrytown, NY). Total N content was also determined from the 0 to 15-cm soil depth samples using the combustion method on a LECO protein/ N determinator FP-528 according to the Dumas method (Williams et al. 1998).
Initial chemical properties of the soil (pH, organic matter, cation exchange capacity, P, K, Ca, Mg, Na, S, Fe, Mn, Cu, Zn, and B) during the establishment year (2008) were analyzed (Table 1). Soil samples were dried at 35°C and ground to pass through a 2-mm sieve. Soil pH was determined on a 1:1 ratio of soil to distilled water (Schofield and Taylor 1955). Soil organic matter was determined by loss on ignition according to Donald and Harnish (1993). Phosphorus, K, Ca, Mg, Na, S, Fe, Mn, Cu, Zn, and B was extracted by a Mehlich 3 extractant solution and were analyzed using Jarell-Ash ICAP-9000 Plasma Spectrometer (Mehlich 1984).
Table 1.
Table 1. Chemical properties of the soil during the establishment year (2008) at two soil depths (0–15 and 15–30 cm)
z
zSoil organic matter was determined by loss on ignition according to Donald and Harnish (1993). Soil pH was determined on a 1:1 ratio of soil to distilled water (Schofield and Taylor 1955). Total N content was determined from the 0 to 15-cm soil-depth samples using the combustion method on a LECO protein/ N determinator FP-528 according to the Dumas method (Williams et al. 1998). P, K, Ca, Mg, Na, S, Fe, Mn, Cu, Zn, and B were extracted by a Mehlich 3 extractant solution and were analyzed using Jarell-Ash ICAP-9000 Plasma Spectrometer (Mehlich 1984).

Statistical Analysis

A completely randomized study of two red clover cultivars (AC Christie and Tempus) in mixture with bluegrass was established with 12 field replicates to evaluate N transfer. Yield, shoot total N, and 15N were collected across three harvests of the red clover. Data were analyzed by ANOVA with repeated measurements across harvests expressed as linear and quadratic trends across the growing season. Pure stands of bluegrass were selected to compare soil mineral N status. Orthogonal contrasts, within an ANOVA for a completely randomized design, were used to compare the pure bluegrass stand with the red clover cultivars in mixed stands with bluegrass, and between the two red clovers at a significance level of P < 0.05. Principal component analysis was used to compare soil N measurements and soil water nitrate response to the treatments (AC Christie, Tempus, and pure bluegrass). Data were analyzed using GenStat® (VSN International 2011).

RESULTS

Forage Yield

During the 2008 establishment year, the yield of the two red clover cultivars and their associated bluegrass yield under a mixed stand were not significantly different (data not shown). On average, the dry matter yield of AC Christie was 7.9 g plant−1, whereas that of Tempus was 8.6 g plant−1 during the establishment year. During the 2009 growing season, cumulative yield of the AC Christie and Tempus cultivars across the three harvests was 36 g and 41g plant−1, respectively (Table 2). The dry weight of the two red clover cultivars at the first, second, and third harvests, as well as total seasonal yield, were not significantly different. However the first red clover harvest in June had greater yields (mean of 15.7 g plant−1) for both red clover cultivars than the July and August harvests. Similarly, mixed stand bluegrass yield was greatest for the first harvest and decreased for the second and third harvests. However, this trend was neither linear nor quadratic. Bluegrass yield in mixed stands for the three harvests, and for the cumulative seasonal yield, was not significantly different between the red clover cultivars.
Table 2.
Table 2. Yield of two red clover cultivars (AC Christie and Tempus) grown in mixed swards with bluegrass across three harvests during the 2009 growing season
z
zAll measurements are on a dry weight basis.
y
ySEM, standard error mean.
x
xF prob, F probability.
w
wNS = P value greater than 0.05.

Plant Nitrogen Content

Tissue N concentration of AC Christie and Tempus was not significantly different for each harvest during the 2009 production year (Table 3) nor in the 2008 establishment year (data not shown). Mean N concentration of the two red clover cultivars ranged from 3.18% to 3.54% in 2009. The greatest mean N yield per plant was from the first harvest at 0.58 g per plant. The N yield at three harvests and the cumulative total seasonal N yield were not significantly different between the two red clover cultivars. Bluegrass N concentration from the mixed stand increased from 1.42, 1.78, and 2.06% for the first, second, and third harvests, respectively. However, this trend was neither linear nor quadratic. Total N yield for the bluegrass in mixed stands decreased from first harvest to third harvest.
Table 3.
Table 3. Nitrogen concentration and nitrogen content of the two red clover cultivars (AC Christie and Tempus) under mixed stands with bluegrass across three harvests during the 2009 growing season
z
zAll measurements are on a dry weight basis.
y
ySEM, standard error mean.
x
xF-prob, F probability.
w
wNS = P value greater than 0.05.

Nitrogen Fixation and Transfer

The N fixing capacity (%) and total N fixation for the two red clover cultivars during the 2009 production year were not significantly different across the first, second, and third harvests (Table 4). However, both cultivars reported high N-fixing capacity across the three harvests with more than 90% of the red clover N being derived from biological N fixation. Nitrogen fixation for both red clover cultivars increased from the first to third harvest, but the increase was not statistically significant. The greatest N fixing capacity was found during the third harvest at 98.7%.
Table 4.
Table 4. Percentage (%) nitrogen derived from atmosphere (%Ndfa) by two red clover cultivars (AC Christie and Tempus) and the amount of N fixed under mixed stand with bluegrass at three harvests during the 2009 growing season
z
zAll measurements are on a dry weight basis.
y
ySEM, standard error mean.
x
xF-prob, F probability.
w
wNS = P value greater than 0.05.
Nitrogen in bluegrass derived from red clover N-transfer increased gradually over the season; mean N was 7, 11, and 26% for the first, second, and third harvests of 2009 respectively (Fig. 1). The quantity of N transferred from red clover to bluegrass on a per-plant basis was 33, 41, and 67 mg per red clover plant for the first, second, and third harvests, respectively (data not shown). There were no significant differences between the N transfer ability of the diploid AC Christie and that of the tetraploid Tempus cultivar. The seasonal total of N transferred to bluegrass was 140 mg per red clover plant with no significant difference between the two red clover cultivars (data not shown).
Fig. 1.
Fig. 1. Nitrogen (%) in bluegrass transferred from AC Christie and Tempus red clover cultivars throughout the 2009 growing season. Vertical bars indicate standard error of the mean.

Soil Mineral Nitrogen Study

Based on soil N results for the 2008 establishment year, the soil nitrate and ammonium content of AC Christie and Tempus under mixed stands of bluegrass and pure bluegrass at a 0 to 15-cm depth were not significantly different (Table 5). However, at a depth of 15 to 30-cm, the soil nitrate content associated with the pure bluegrass sward was significantly greater compared with the mixed stands which contained either Tempus or AC Christie (P = 0.006).
Table 5.
Table 5. Soil extractable nitrate, ammonium, and total nitrogen concentration of the 0 to 15-cm and 15 to 30-cm soil depths during the establishment year (2008) followed by the production year (2009) for AC Christie and Tempus under mixed stand with bluegrass compared to pure bluegrass stand
z
zAll measurements are on a dry weight basis.
y
ySEM, standard error mean.
Based on the orthogonal contrasts, soil extractable nitrate contents of the 0 to 15-cm and 15 to 30-cm soil depths were not significantly different between the two red clover cultivars (under mixed stand with bluegrass) and pure blue grass over three harvests during the 2009 production year (Table 5). At 0–15 cm, soil nitrate content below Tempus-bluegrass mixed stand increased along the three different cuts (4.75, 9.60, and 12.04 mg kg−1 dry soil), but the nitrate content under AC Christie–bluegrass and pure bluegrass did not follow the same trend. Soil extractable ammonium content of the 0 to 15-cm and 15 to 30-cm soil was also not significantly different under the two red clover/bluegrass mixed stands and the pure stand of bluegrass at the three harvests during the 2009 production year. Based on the total soil N data at the top 0 to 15-cm soil depth, total soil N content was not significantly different between AC Christie, Tempus (mixed stand with bluegrass), and pure bluegrass at three harvesting points (Table 5). The mean total N concentration of the top 15 cm soil was 0.070, 0.064, and 0.064% with respect to first, second, and third harvests.
Soil water samples collected from 15-cm deep lysimeters were analyzed for nitrate and plotted as cumulative nitrate values (Fig. 2). Tempus showed the lowest soil water nitrate throughout the growing season. Soil water nitrate concentration under AC Christie mixed stand fluctuated over the season compared with Tempus under mixed stand and pure bluegrass. Soil water nitrate increased at the end of July (Jul. 21 and 31) for AC Christie in mixed stands but remained constant for Tempus in mixed stands. In pure bluegrass stands, soil water nitrate levels were comparatively higher in late June and remained constant throughout the growing period.
Fig. 2.
Fig. 2. Variation of cumulative soil water nitrate-nitrogen in top 15 cm of two red clover cultivars (AC Christie and Tempus) vs. pure bluegrass during the 2009 growing season. The regression line for each species was fitted using linear regression analysis (n = 15).
In the principal component analysis biplot, the first two principal components (PC) explained 76% of total variation for the five quantitative traits studied (Fig. 3). The PC1 and PC2 accounted for 47 and 29% of the total variation, respectively. Score 1 depicts a contrast between soil-water nitrate at 15 cm and for soil nitrate at the two sampling depths (0–15 and 15–30 cm). At the third harvest, Tempus had higher soil nitrate concentration at both sampling depths (Fig. 3). AC Christie released more N into the soil water later in the season than Tempus. Score 2 is a weighted average dominated by soil ammonium at 0 to 15-cm and 15 to 30-cm depths. Tempus had a substantial increase in soil ammonium over the season, while bluegrass and AC Christie were not significantly affected by the score 2 soil ammonium response. Principal component analysis depicts a positive correlation between soil ammonium for both sampling depths. There was also a positive correlation between the two depths for nitrate, but a negative correlation between soil nitrate and soil-water nitrate. Since soil nitrate and ammonium are dominated on two different scores, it is unlikely that they have any relationship to each other.
Fig. 3.
Fig. 3. Principal component analysis of soil extractable nitrate and ammonium at two soil depths (first 15 cm and 15–30 cm) representing two red clover cultivars (AC Christie and Tempus) and pure bluegrass. T, Tempus; C, AC Christie; B, pure bluegrass; 1, first harvest; 2, second harvest; 3, third harvest.

DISCUSSION

Although AC Christie and Tempus have different ploidy levels (diploid vs. tetraploid), yield differences were not significant for the three harvests during the 2009 production year. Similarly, bluegrass yield associated with the two red clover cultivars in mixed stand did not show any significant difference. Greater nutrient availability and more favourable growing conditions in early spring (May–June) may have stimulated plant growth, and may explain the higher yields for the first harvest of red clover and bluegrass. Repeated defoliation reduces root mass (Carrillo et al. 2011), which can reduce yield in later harvests. There were no significant differences between the two red clover cultivars for N concentration and no change over the growing period. However, bluegrass N concentration in the mixed swards did increase from the first harvest to the third, which may indicate N transfer. Total N content (g) of the bluegrass in the red clover mixed sward decreased over the growing season.
Both red clover cultivars showed high biological N fixing capacity under bluegrass mixed stands across the three harvests, which highlights the importance of using red clover in forage mixtures. High biological N fixation is especially valuable because biologically fixed N is less susceptible to volatilization, denitrification, and leaching. Our findings corroborate results from Nyfeler et al. (2011) and Dahlin and Stenberg (2010a); red clover can derive 90–98% of its own N from BNF under mixed stand with ryegrass. The intensity of BNF by pasture legumes can vary widely according to soil properties and environmental conditions (Hardarson 1993). Ta and Faris (1988) report that high light intensity, long days, and cool temperatures (20/16oC day/night) are optimal for N fixation in alfalfa. Low soil mineral N also promotes high N fixation in legumes (Ledgard 2001). Low total N concentration (0.05–0.08 DW basis) in the top 15 cm of soil in our experimental site may have contributed to the high BNF for the two red clover cultivars during the 2009 growing season. Cutting regimens also induce N fixation (Dahlin and Mårtensson 2008; Dahlin and Stenberg 2010a) and our results show an increase in Ndfa (%) over the growing season. Cutting helps keep plants at a vegetative stage which may contribute to a continuous demand for N.
Based on the natural abundance method, 0 to 17% of N in grasses may be derived from clover N transfer (Høgh-Jensen and Schjoerring 1994). According to our results, 7% of N in bluegrass derived from red clover N transfer during the first harvest. Our results also show that bluegrass N derived up to 11 and 26% from clover N transfer during the second and third harvests, respectively. These results are in agreement with those of Høgh-Jensen and Schjoerring (2000).
Although there was no significant difference in N transfer ability between the two red clover cultivars, N transfer from clover to bluegrass increased over the season. Apparent N transfer tends to increase with sward age in legume sward mixtures (Jørgensen et al. 1999; Høgh-Jensen and Schjoerring 1994, 2000; Dahlin and Stenberg 2010b). Grasses prefer to uptake N released by legumes, possibly due to a lower energy cost compared with soil N absorption (Daudin and Sierra 2008). This N transfer could result from the release of N from the breakdown and decomposition of dead tissues and direct excretion from living root systems (Fustec et al. 2010). Shoot harvesting may also increase below-ground N release (Trannin et al. 2000; Ayres et al. 2007; Carrillo et al. 2011). Also, defoliation affects rhizosphere respiration, stimulation of root exudates followed by rhizosphere priming affect the decomposition of soil organic matter (Fu and Cheng 2004; Hamilton et al. 2008). However, positive effects of legume defoliation on grass N nutrition mainly occurs by direct transfer of fixed N rather than from changes in the availability of soil organic matter N (Ayres et al. 2007; Saj et al. 2008).
Chu et al. (2004) report that N transfer from legume to non-legume was high under low soil-N availability using a peanut-rice model. However, Paynel et al. (2008) found more N transferred at high N levels because the increase in soil exploration by grasses provided greater access to other N sources, including N compounds exuded from clover. In contrast, Høgh-Jensen and Schjoerring (1994) report that N application does not impact N transfer from clover to associated grasses. Since we applied 15N ammonium sulphate to label the plants with 15N after the first harvest, N transfer from clover to bluegrass during the second and third harvests may have been affected. Since total N and ammonium content in the top soil at each harvest were low, application of 15N fertilizer should not have impacted the N transfer.
Ta and Faris (1988) report that high light intensity, long days, and cool temperatures (20/16oC day/night) were optimal for high N fixation-transfer in an alfalfa–timothy stand. Nitrogen transfer also depends on the root density of legume plants (Sierra and Nygren 2006). Since we analyzed N transfer based on an individual plant basis, our results might differ because dense clover populations affect root density and their interaction with grass roots. Therefore, it is important to consider legume plant density when evaluating N transfer data from different studies. Interestingly, Nyfeler et al. (2011) report that an increase in the percentage of grasses in a sward also increased the apparent N transfers from legumes to grasses. Therefore, below-ground N transfer depends not only on the N donor plant, but also on the plant receiving the N (Pirhofer-Walzl et al. 2011).
Under mixed swards, soil extractable nitrate was greater than ammonium at both soil depths (0–15 cm and 15–30 cm) during the 2009 growing season. Based on a micro-lysimeter study, using sand as the growing medium, Paynel and Cliquet (2003) report that clover releases a large amount of ammonium compared to nitrate from root exudates. However, based on our results, nitrate was the dominant N source in the soil extracts compared with ammonium. This result is particularly relevant for producers. Even though the majority of N exuded by clover is ammonium, it rapidly converts to a nitrate form in the soil. Soil extractable nitrate levels in deep soil (15–30 cm) were consistently greater than in shallow soil (0–15 cm) for pure bluegrass stands, and were greater than in the mixed stands, which included either the red clover cultivars Tempus and AC Christie in 2008 and for the first cut of 2009. These results may have implications for nitrate leaching since further movement of nitrate in the profile will be out of the active root zone and be lost to the environment.
Tempus showed increasing soil nitrate availability over the growing season. This result strongly supports our finding of increasing N transfer from red clover to bluegrass over the growing season. Once plants mature, they may release more N from the root system into the rhizosphere, which can be used by neighbouring plants. Also, nodule senescence and subsequent decomposition may enhance the N transfer capacity of red clover over the growing season. The soil N behaviour of the two red clover cultivars differed during the growing season, with Tempus contributing more N in the top 15 cm of soil. In our in vitro study using the same two red clover cultivars, we found that Tempus had a greater fraction of active nodules out of total nodules, and a greater root dry weight and root surface area, compared with AC Christie (Thilakarathna et al. 2012). Having more root mass supported by a greater fraction of active nodules enhances biological N fixation and N release from below-ground parts.
The PCA shows that the soil nitrate associated with Tempus increased over the three harvests while soil water nitrate levels remained low. In contrast, AC Christie had low soil nitrate late in the season. However, AC Christie released increasing amounts of nitrate (soil water nitrate) over the season, which was not retained in the soil for use by other species, probably due to leaching. This result may indicate that the nitrate supplied by AC Christie late in the season was not fully utilized by the companion bluegrass in this study.
In summary, this study quantifies and describes the cultivar effect on N transfer from two diverse red clover cultivars to companion bluegrass under field conditions. N transfer increased as the season advanced. The impact of the two red clover cultivars on soil mineral N status showed genetic variability between the two cultivars.

ACKNOWLEDGEMENTS

The technical support provided by Matthew Crouse, the editorial assistance of Christina McRae, EditWorks, and the many helpful comments provided by the internal review process at the Atlantic Food and Horticulture Research Centre, are greatly appreciated. The authors also wish to acknowledge the technical assistance of the Farm Service of the Nappan Research Farm during this study.This work was supported by Agriculture and Agri-Food Canada and a National Science and Engineering Research Council grant to Dr. Y. Papadopoulos.

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Information & Authors

Information

Published In

cover image Canadian Journal of Plant Science
Canadian Journal of Plant Science
Volume 92Number 6November 2012
Pages: 1163 - 1173

History

Received: 10 February 2012
Accepted: 17 June 2012
Version of record online: 1 October 2012

Key Words

  1. N transfer
  2. red clover cultivars
  3. soil N conditions

Mots-clés

  1. Transfert du N
  2. cultivars de trèfle rouge
  3. conditions du N du sol

Authors

Affiliations

R. M. M. S. Thilakarathna
Department of Biology, Dalhousie University, Halifax, NS, Canada B3H 4J1
Y. A. Papadopoulos
Agriculture and Agri-Food Canada, Nova Scotia Agricultural College, Truro, Nova Scotia, Canada B2N 5E3
A. V. Rodd
Agriculture and Agri-Food Canada, Charlottetown, Prince Edward Island, Canada CIA 4N6
A. N. Gunawardena
Department of Biology, Dalhousie University, Halifax, NS, Canada B3H 4J1
S. A. E. Fillmore
Agriculture and Agri-Food Canada, Kentville, Nova Scotia, Canada B4N 1J5
B. Prithiviraj
Department of Environmental Sciences, Nova Scotia Agricultural College, Truro, Nova Scotia, Canada B2N 5E3

Notes

Abbreviations: BNF, biological nitrogen fixation; Ndfa, nitrogen derived from the atmosphere; PC, principal component

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