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a Département de Phytologie, Faculté des Sciences de l'Agriculture et de l'Alimentation, Université Laval, Québec, QC, Canada, G1K 7P4
b Present address: Dep. of Agron. and Plant Breeding, Aburaihan Campus, P.O. Box 4147, Univ. of Tehran, Tehran, Iran
* Corresponding author (francois-p.chalifour{at}plg.ulaval.ca).
Received for publication July 3, 2001.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONCONCLUSIONREFERENCES |
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Abbreviations: CL, clay loam • DM, dry matter • DPS, de-inking paper sludge • DPSL, linear effects of de-inking paper sludge • DPSQ, quadratic effects of de-inking paper sludge • DPS0, de-inking paper sludge applied at 0 Mg dry matter ha–1 • DPS50, de-inking paper sludge applied at 50 Mg dry matter ha–1 • DPS100, de-inking paper sludge applied at 100 Mg dry matter ha–1 • Ndfa, nitrogen derived from atmosphere • 15N-E, 15N enrichment (method) • 15N-NA, 15N natural abundance (method) • SCL, silty clay loam • 
15N, natural abundance of 15N
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONCONCLUSIONREFERENCES |
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Considerable efforts have been directed to the development of methods for measuring N2 fixation (Rennie and Rennie, 1983; Hauck and Weaver, 1986; Shearer and Kohl, 1986; Danso et al., 1993; Chalk and Ladha, 1999). Selection of a suitable method to estimate N2 fixation is of outmost importance. Available methods have distinct advantages and disadvantages, i.e., experimental conditions, complexity, and cost of analyses. The methods that are currently the most reliable for determining N2 fixation imply the use of stable isotopes of N (Unkovich and Pate, 2001).
Nitrogen-15 isotope dilution techniques, including 15N natural abundance (15N-NA) and 15N enrichment (15N-E) methods, to quantify N2 fixation by legumes, have been used extensively. With these methods, N2 fixation in forage legumes can be estimated over long periods of time, including entire growth cycles and growth seasons (Heichel et al., 1984, 1985; Carranca et al., 1999; Seguin et al., 2000). The 15N-NA method uses the variation in natural 15N abundance of the N2–fixing and non-N2–fixing plants to estimate percentages of N derived from the atmosphere (Ndfa) (Rennie and Rennie, 1983; Shearer and Kohl, 1986; Unkovich and Pate, 2001). The 15N-E method requires the use of a 15N-labeled fertilizer, which must be added to the legume and reference (non N2–fixing) species but at a sufficiently low rate so as not to counter N2 fixation (Chalifour and Nelson, 1987). The application of DPS to soil induces N immobilization (Fierro et al., 2000). Under these conditions, the 15N-NA method may be a more suitable alternative to the 15N-E method in soils newly amended with DPS since the former method does not require the use of a 15N-labeled fertilizer.
Although several studies have examined sludge mixtures on plant growth (Chong and Cline, 1993; Norrie and Gosselin, 1996; Aitken et al., 1998; Chong et al., 1998; Phillips et al., 1998; Fierro et al., 1999), the current literature lacks information on the effect of high rates of DPS on symbiotic N2 fixation of forage legumes. The objective of the present study was to quantify and compare the contribution of N2 fixation in forage legumes by the 15N-NA method under high rates of application of DPS applied once before legume establishment, during a 2-yr period.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONCONCLUSIONREFERENCES |
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19.4 g C kg–1), and in St-Louis-de-Pintendre (hereafter Pintendre), on a poorly drained Kamouraska silty clay loam (SCL) soil (fine, mixed, frigid, Typic Humaquept) with an average of 27.0 g C kg–1. The altitude, longitude, and latitude of experimental sites are 73 m, 71°48'56'' W, and 46°38'09'' N, respectively. On the Kamouraska SCL, hay cultivation was done during 1992 and 1993, whereas on the Tilly CL, alfalfa and millet (in 1992) and cereals (in 1993) were cultivated. Both sites were under fallow during the 1994 growing season.
Experimental Design and Management
Treatments
The experiment was a two-factor factorial, comprising four replicates arranged in a split-plot design:
Establishment, Sowing, and Fertilizer Application
In October 1994, DPS was incorporated in the soils at the specified rates. This DPS was obtained from Papiers Stadacona Inc. (formerly Les Produits Forestiers Daishowa Ltée) located in Quebec City. Before incorporation, representative samples of DPS were oven-dried (at 105°C for 24 h) to determine their moisture percentage. Selected properties of DPS and soil types are presented in Table 1. Chemical characteristics were determined for soils and DPS previously air-dried and sieved. All analyses were made in triplicate. Soil water pH and electrical conductivity (EC) were taken from a 1:2 mix (mass/volume), except for DPS, for which a 1:9 ratio was required (adapted from CPVQ, 1997). Bulk density was measured as described by Culley (1993). Water content was determined after drying 10 g of soil or DPS samples at 105°C until constant weight (CPVQ, 1997). Total C and total N were measured in the gas evolved from combustion of the ground material with a LECO elemental analyzer, Model CNS-1000 (LECO Co., St. Joseph, MI) (Jimenez and Ladha, 1993).
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Legumes were inoculated before sowing with their corresponding rhizobial inoculant: Sinorhizobium meliloti for sweetclover and alfalfa, Rhizobium leguminosarum biovar trifolii for red clover, and Mesorhizobium loti for birdsfoot trefoil. Forage species were sown on 10 May 1995 in St-Augustin and on 15 May 1995 in Pintendre at a rate of 600 viable seeds m–2 (12 kg seeds ha–1). Each plot was 4.3 m wide (i.e., 28 rows spaced 15.3 cm apart) and 7 m in length.
In 1995, the Kamouraska SCL was fertilized once with 38 kg P2O5 ha–1 and 79 kg K2O ha–1 while the Tilly CL received 30 kg P2O5 ha–1 and 99 kg K2O ha–1. Before establishment of forage crops in the spring of 1995, an additional 0.23 kg of P was added to each megagram of DPS incorporated to counter P immobilization by DPS (Fierro et al., 1999). In 1996, total P and K fertilizer applications were 40 kg P2O5 ha–1 and 30 kg K2O ha–1 on the Tilly CL, whereas 60 kg P2O5 ha–1 and 50 kg K2O ha–1 were applied on the Kamouraska SCL. In 1996, and at both sites, total quantities of these fertilizers were applied equally in two fractions, one early in the growing season and the second fraction after the first cut.
Plant Sampling and Analytical Methods
The plant samples for determining DM yield and N uptake by forage crops were collected by cutting two 0.612- by 7-m strips in the central portion of each experimental unit. The forage crops were cut at a 10-cm height, except sweetclover, which was cut at a 20-cm height. Dry matter content was determined using a 500-g subsample from the entire harvested material in each plot. Each subsample was weighed, dried in a forced air oven at 60°C until constant weight, and weighed to determine the DM content.
Plant sampling for estimating N2 fixation by the 15N-NA method was done on two 1-m central rows, within each experimental unit. Plant samples were dried at 60°C until constant weight, subsampled, finely ground (0.5 mm) with a Retsch Centrifugal Mill Model ZM-1 (Brinkmann Instruments Canada Ltd., Rexdale, ON, Canada), and further ground by using a ball mill (Retsch Mixer Mill Model MM-2, Brinkmann Instruments Canada Ltd., Rexdale, ON, Canada) to obtain the powder sample, which was analyzed for 15N/14N ratio (natural abundance samples) and total N concentration. The ratios of 15N/14N were measured by a continuous-flow isotope ratio mass spectrometer (CF-IRMS) (Europa Scientific, Crewe, England) interfaced with a RoboPrep Sample Converter (Europa Scientific); analyses were performed at the Stable Isotope Laboratory, Department of Soil Science, University of Saskatchewan, Saskatoon, SK, Canada. Total N concentration of shoots was determined in the gas evolved from combustion of the ground material with a LECO elemental analyzer, Model CNS-1000 (LECO Co., St. Joseph, MI) (Jimenez and Ladha, 1993).
Only one harvest was effected in the year of establishment (7 Sept. 1995 on the Tilly CL and 10 Sept. 1995 on the Kamouraska SCL). In addition, in 1995, Leptosphaerulina leaf spot (Leptosphaerulina briosiana Poll.) was encountered in alfalfa plots at both sites. The disease symptoms were observed two months after planting (June 1995). To overcome this problem, metalaxyl [methyl N-(2,6-dimethylphenyl)-N-(methoxyacetyl)-DL-alaninate; formulated as Ridomil fungicide (Novartis Crop Protection Canada Inc., Guelph, ON)] was applied at a rate of 4.2 L ha–1 in July 1995. Leptosphaerulina leaf spot was controlled by fungicide application, and disease symptoms were not observed thereafter. During the second year (1996), three cuts were effected: (i) first cut, 12 June on the Tilly CL and 14 June on the Kamouraska SCL; (ii) second cut, 16 August on the Tilly CL and 19 August on the Kamouraska SCL; and (iii) third cut, 10 October on the Tilly CL and 12 October on the Kamouraska SCL.
Estimation of Dinitrogen Fixation by the Nitrogen-15 Natural Abundance Method
Dinitrogen fixation was determined as the percentage Ndfa and the amount of Ndfa on an area basis by the 15N-NA method, according to Rennie and Rennie (1983) and Peoples et al. (1989). The calculations used were as follows:
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15N) value of an effectively nodulated legume grown in media totally lacking combined N and thus totally dependent on symbiotic N2 fixation for its N requirements. Effects of host plant on 15N of the four forage legumes solely dependent on fixed N2 (B value) were assessed in minus-N silica sand culture under growth chamber conditions (Peoples et al., 1989). Plants were supplied with a N-free nutrient solution (Chalifour and Nelson, 1987). The average shoot B values obtained were –0.24
for alfalfa, –1.57 for birdsfoot trefoil, –0.15 for red clover, and 0.33 for sweetclover. The B values did not vary significantly with time of harvest for the different forage legumes.
In our experiment, bromegrass and ineffective alfalfa were used as the non-N2–fixing species. At both sites and during the 2 yr of the experiment, ineffective alfalfa had inconsistent growth in the presence of DPS, and the ineffective alfalfa 15N values showed inconsistent trends as well. Seguin et al. (2000) have also reported problems for the regrowth of ineffective ‘Agate’ alfalfa under Minnesota field conditions and used annual ryegrass (Lolium multiflorum Lam.) as an alternate reference species for birdsfoot trefoil and Kura clover (Trifolium ambiguum M.B.). In contrast, bromegrass showed consistent trends in 15N values in response to DPS and was used as the reference crop in the present study. Bromegrass was established at both sites in the absence of DPS but did not show significant growth for yield determination in the presence of DPS in the year of establishment. Nevertheless, enough plant material could be collected within each plot to estimate 15N values of bromegrass at DPS50 and DPS100. Furthermore, bromegrass established itself adequately during the 1996 growing season in the DPS50 and DPS100 treatments, without the need of reseeding. Therefore, all calculations were done using bromegrass as the reference species in the present study.
Statistical Analyses
The General Linear Models Procedure (GLM) of the SAS statistical package (Release 6.12, SAS Inst., 1996) was used to test the significance of the associations between each dependent variable and the treatments. Interpretation of statistical analyses was done on interactions when these were significant. Scatter plots of the residuals from the respective statistical models as well as Bartlett's test (Steel and Torrie, 1980) were used to test homogeneity of the experimental error variances and to determine if data transformations were required. Since there was homogeneity of pooled error variances between the soils of the two sites for nearly all of dependent variables (Gomez and Gomez, 1984), combined statistical analyses were effected. Differences among treatments were determined by simple, first- and second-order class and trend contrasts (Little and Hills, 1978). Sweetclover had no regrowth at the third cut on the Tilly CL and at the second and third cuts on the Kamouraska SCL; therefore, the statistical analyses were done on the other three forage legumes for corresponding cuts at the two sites. Differences were declared significant at P < 0.06.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONCONCLUSIONREFERENCES |
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Year 1996
At the first cut in 1996, and on both the Kamouraska SCL and the Tilly CL, the DM yield and N uptake of bromegrass decreased with DPS application while those of all legumes were either unaffected or slightly increased by DPS (DPSL x legumes vs. bromegrass, P < 0.0001; Table 2), in contrast with the situation in 1995, and especially for alfalfa and birdsfoot trefoil. Bromegrass showed significant growth at DPS50 and DPS100 at both sites, but in contrast to forage legumes, DM yield and N uptake were reduced by DPS, the greatest reductions occurring at the highest DPS rate (DPSL x legumes vs. bromegrass, P < 0.0001; Table 3).
At the second cut in 1996, the presence of DPS was generally without effect on the DM yield and N uptake of all species, including bromegrass (Table 2). At the second cut, sweetclover did not show regrowth on the Kamouraska SCL.
As for the second cut, DPS did not affect DM yield and N uptake at the third cut (Tables 2 and 3). At the third cut and at both sites, sweetclover showed no regrowth. Dry matter yield and N uptake of all species were generally highly decreased at the third cut compared with the other cuts (Tables 2 and 3). Bromegrass showed similar N uptake for the different rates of DPS for the second and third cuts at each site.
Symbiotic Dinitrogen Fixation
Percentages of Nitrogen Derived from Atmosphere
Year 1995. In the year of establishment, the percentages of Ndfa increased in all forage legumes in response to DPS, the highest percentages of Ndfa being associated with the highest rate of DPS. Also, there were differential increases among forage legumes in percentages of Ndfa in response to DPS. For instance, there was a greater increase in percentage Ndfa in alfalfa than in red clover at both sites (DPSL x alfalfa vs. red clover, P < 0.0006; Table 4). The low percentages of Ndfa for alfalfa, especially at DPS0, may be due in part to stunted growth following infection by Leptosphaerulina leaf spot in 1995. In that year, there was a greater increase in percentage Ndfa in response to DPS for birdsfoot trefoil than for red clover, on the Kamouraska SCL only; indeed, similar responses were observed on the Tilly CL (site x DPSL x birdsfoot trefoil vs. red clover, P < 0.0001 and site x DPSQ x birdsfoot trefoil vs. red clover, P < 0.05; Table 4). This was mostly due to the very low percentage of Ndfa observed for birdsfoot trefoil at DPS0 on the Kamouraska SCL. In that year, the Tilly CL was more conducive to N2 fixation than the Kamouraska SCL [means averaged over DPS and forage legumes on the Kamouraska SCL and Tilly CL: 72.5 and 83.1%, respectively; Table 4].
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At the second cut, the effect of DPS was attenuated compared with the first cut, but differential effects of DPS among forage legumes were still observed between sites; percentages of Ndfa did not vary in alfalfa in response to DPS at both sites while for red clover, there were decreases on the Kamouraska SCL and increases on the Tilly CL (site x DPSL x alfalfa vs. red clover, P < 0.007; Table 4). Different responses to DPS were also observed between birdsfoot trefoil and red clover; similar to red clover, percentages of Ndfa increased for birdsfoot trefoil on the Tilly CL in response to DPS but remained stable on the Kamouraska SCL (DPSL x birdsfoot trefoil vs. red clover, P < 0.05; Table 4). No effect of DPS was observed at the third cut.
As for 1995, the Tilly CL was generally more conducive to N2 fixation than the Kamouraska SCL in 1996, at all three cuts (means averaged over DPS and forage legumes on the Kamouraska SCL were 72.8, 72.3, and 75.0% for the first, second, and third cuts in 1996, respectively, and on the Tilly CL, they were 89.8, 88.7, and 84.7% for the first, second, and third cuts in 1996, respectively; Table 4).
Amounts of Nitrogen Derived from Atmosphere
The total amounts of Ndfa, measured in both years, are a function of the N uptake and percentages of Ndfa reported above. Only in the establishment year (1995) was there an overall significant effect of DPS (P < 0.04) on amounts of Ndfa, but there were differential responses to DPS in 1995 and for the first and second cuts in 1996, as shown by significant DPS x species and site x DPS x species interactions (see below). It is only for the third and last cut in 1996 that the effect of DPS on amounts of Ndfa had subsided and was nonsignificant.
Year 1995. In that year, the lowest amounts of Ndfa were found in alfalfa on the Tilly CL, and low but higher amounts were found for this species on the Kamouraska SCL and showed very slight increase (Kamouraska SCL) or decrease (Tilly CL) in response to DPS; in contrast, the amounts of Ndfa in red clover were higher than for alfalfa and showed little response to DPS on the Kamouraska SCL but increased with the level of DPS on the Tilly CL (site x alfalfa vs. red clover, P < 0.006; Table 5). The amounts of Ndfa were higher for sweetclover than for red clover on the Tilly CL but were more in the same range for both species on the Kamouraska SCL (site x sweetclover vs. red clover, P < 0.0001; Table 5). Amounts of Ndfa for birdsfoot trefoil either increased (Kamouraska SCL) or decreased (Tilly CL) in response to DPS, departing from the response observed in red clover where amounts of Ndfa were unaffected or increased in response to DPS (site x DSL x birdsfoot trefoil vs. red clover, P < 0.002; Table 5). This differential response is attributable to a reduced N uptake in birdsfoot trefoil in response to DPS, with a concomitant increased dependence on N2 fixation (i.e., increased percentages of Ndfa), which was very strong from DPS0 to DPS50 for birdsfoot trefoil on the Kamouraska SCL; in contrast, red clover showed no response or a greater N uptake in response to DPS and also an increased dependency on N2 fixation (i.e., increased percentages of Ndfa) in response to DPS (Tables 3, 4, and 5).
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At the third cut, the impact of DPS had totally subsided, and there were mainly differences among forage species. Alfalfa produced the highest amounts of Ndfa, followed by red clover and then by birdsfoot trefoil (alfalfa vs. red clover and birdsfoot trefoil vs. red clover, P < 0.004 and 0.0001, respectively; Table 5). While alfalfa produced similar amounts of Ndfa at both sites, red clover produced lower amounts on the Kamouraska SCL compared with the Tilly CL (site x alfalfa vs. red clover, P < 0.02; Table 5).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONCONCLUSIONREFERENCES |
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Dry Matter Yield and Nitrogen Uptake
In the year of establishment (1995) and at both sites, DM yield and N uptake of sweetclover and red clover were generally unaffected by DPS addition; in contrast, DM yield and N uptake of alfalfa and birdsfoot trefoil were reduced by DPS application in that year. Differential responses to DPS of similar composition have also been reported by Fierro et al. (1997) in various forage species; indeed, under greenhouse conditions, these authors also found contrasting responses to DPS among different forage legumes. Sweetclover and galega showed little response or increased growth when amended with DPS and sufficient P compared with the legume black medic (Medicago lupulina L.), which showed reduced growth at all DPS rates compared with its unamended control. The reasons for the observed differences among legume species in their response to DPS have not been well documented and may be related to differences in the establishment and functioning of the N2–fixing symbiosis in each species (see below).
Our results also compare well with those of Heckman and Kluchinski (1995) in which pot-grown soybean [Glycine max (L.) Merr.] was amended with tree leaf or crop residues with C/N ratios ranging from 17 to 75, providing a range in N immobilization potential. These authors compared nodulating and nonnodulating isolines of soybean; amendment with leaf and crop residues caused severe N deficiency in nonnodulating soybean, whereas the nodulating isoline showed a gradual alleviation of N deficiency as N2 fixation became effective.
In the present study, DM yield and N uptake of forage legumes were generally unaffected by DPS application in 1996 compared with the first year, presumably due to the greater decomposition of DPS in the second year (Chantigny et al., 2000a). In contrast, bromegrass DM yield and N uptake continued to be affected by DPS during the second year because of low availability of N, which is likely to have a significant impact on a non-N2–fixing crop. Similarly, in a greenhouse study on the effects of bleached, primary papermill sludge on bermudagrass (Cyanodon dactylon L.), Feagley et al. (1994) reported that addition of the sludge reduced N uptake. In the present study, the impact of DPS on bromegrass in the 1996 growing season was the greatest at the first cut. In subsequent cuts, differences among DPS treatments were much less pronounced, suggesting similar soil N availability in the different DPS treatments.
In contrast with the results obtained with bromegrass, in a field experiment on the plant and soil responses to DPS (0 and 105 Mg ha–1) and N and P applications, Fierro et al. (1999) reported an increased aboveground biomass of tall wheatgrass (Agropyron elongatum) in the presence of sludge during the first and second growing seasons. However, they applied 3, 6, and 9 g N kg–1 sludge, once at the beginning of the experiment, to reduce the high C/N ratio of soil due to the sludge application. In another study, Fierro et al. (1997) demonstrated the adverse effects of DPS on the growth of grasses Agropyron elongatum, meadow foxtail (Alopecurus pratensis), and hard fescue [Festuca ovina var. duriuscula (L.) Koch]. In their experiment, these adverse effects were alleviated by the provision of N fertilizer to the soil–sludge mixtures, thereby allowing adequate C/N ratios for plant growth.
The absence of regrowth in sweetclover by the second (on the Kamouraska SCL) and third cuts (both sites) in 1996 is related to the growth habit of this species, i.e., a biannual crop for which most of its growth and mineral nutrient uptake take place during the first year compared with the second year when reserves are used for flowering.
Symbiotic Dinitrogen Fixation
In the year of establishment (i.e., 1995), the presence of DPS led to increases in percentages of Ndfa by forage legumes, especially in birdsfoot trefoil (despite decreased N uptake), and the highest percentages of N2 fixation in all legumes were associated with the highest rate of DPS. This can be attributed to soil N immobilization by DPS (Fierro et al., 2000), resulting in a greater dependency of legumes on symbiotic N2 fixation. Similar DPS effects were observed at the first cut in 1996. The results from the present field study agree with the results obtained by Croteau and Zibilske (1998) on the growth of snap bean (Phaseolus vulgaris), which showed the immobilization of N with paper sludge application. In greenhouse (Heckman and Kluchinski, 1995) and field experiments (H
gh-Jensen and Kristensen, 1995), soil N immobilization was also linked to the increased dependency of legumes on symbiotic N2 fixation. The enhancing effects of DPS on N2 fixation of forage legumes due to its high C/N ratio may be further enhanced by the high pH (alkaline) and Ca concentration of DPS (Beauchamp et al., 2002), similar to the effect achieved by liming, for example, in common bean (Buerkert et al., 1990) or alfalfa (Pijnenberg and Lie, 1990).
Our results obtained for N2 fixation estimates in the seeding year at DPS0 for alfalfa, red clover, and birdsfoot trefoil agree partially with earlier observations of Heichel et al. (1984)( 1985) obtained with the 15N-E method for these three species in Minnesota. These authors found seasonal means (of three cuts) for percentage Ndfa of 39.8 and 65% for birdsfoot trefoil and red clover, respectively (Heichel et al., 1985), and 55.9 and 62.4% for alfalfa cultivars Agate and Saranac, respectively (Heichel et al., 1984). Thus, our data confirm the lower efficiency of N2 fixation of birdsfoot trefoil compared with alfalfa and red clover in the seeding year. The very low value found on the Kamouraska SCL for birdsfoot trefoil at DPS0 may be also linked to a higher initial NO–3–N content in the 1995 spring at this site for the DPS0 treatments 
compared with the Tilly CL 
, which may have inhibited the establishment and functioning of the symbiosis (Chalifour and Nelson, 1987) in birdsfoot trefoil more than in the other forage legumes. This would agree with the findings of Bergersen et al. (1989), who found that for soybean inoculated with Bradyrhizobium sp. at the standard rate and following fallow, there was a very low percentage of Ndfa (9.9%) (estimated by the 15N-NA method) compared with soybean following oat (Avena sativa L.) (58.0%). The corresponding plant-available N was 37.6 mg N kg–1 soil following fallow and 18.5 mg N kg–1 soil following oat. The low value for percentage of Ndfa in alfalfa in the absence of DPS may be partly attributed to Leptosphaerulina leaf spot; it is quite probable that substantially higher percentages of Ndfa would have been obtained otherwise.
During the second year of the present study (1996), the presence of DPS had no obvious effect on the percentages of Ndfa at the second and third cuts. This could be attributed to the greater decomposition of C materials from DPS (Chantigny et al., 2000a; Fierro et al., 2000), which thus had lesser effects on N2 fixation. Therefore, in this year and after the first cut, differences among species were mostly due to the varying capacity of forage legumes to fix atmospheric N2. The percentages of Ndfa estimated for second-year forage legumes are higher than those reported by Heichel et al. (1984)(1985) for alfalfa, birdsfoot trefoil, and red clover [seasonal means of 36.4 (two cultivars), 29.9, and 34.9%, respectively]; however, these authors reported an unusually wet second year, which would have been unfavorable to N2 fixation. Indeed, also under Minnesota conditions, Seguin et al. (2000) have reported percentages of Ndfa (15N-E method) for birdsfoot trefoil that are in the same range (seasonal mean of 62.5% for two sites) of those found in the present study with 15N methodology. Environmental factors may thus have a profound impact on N2 fixation efficiency.
Percentages of Ndfa were generally higher on the Tilly CL than on the Kamouraska SCL. Reasons for the Tilly CL being more conducive to N2 fixation both in the absence and presence of DPS may be numerous and include both environmental and biological constraints (Bohlool et al., 1992; Peoples and Baldock, 2001). Some of these constraints may have been alleviated by amending with DPS. In a parallel study, Chantigny et al. (2000b) found that microbial growth and activity in both soils were increased by DPS addition due to improved C and water availability. It is quite likely that DPS favored the growth and survival of indigenous and inoculated rhizobia in soils, thus favoring nodulation and N2 fixation. This may be important for N2 fixation in a species such as birdsfoot trefoil, which requires the formation of new nodules at each growth cycle (Vance et al., 1982). Improvement in soil physical properties may also contribute to increased N2 fixation (Bohlool et al., 1992). In line with this, Chantigny et al. (1999) have also shown that DPS addition increased the proportion of water-stable aggregates >1 mm, an effect which was observed throughout the duration of the present study.
In 1996, at both sites, percentages of Ndfa in birdsfoot trefoil were generally lower than those observed in the three other legumes. Differences between birdsfoot trefoil vs. alfalfa and red clover were particularly noticeable at the first and third cuts; percentages of Ndfa in birdsfoot trefoil were the lowest at these two cuts. These data are consistent with the morphogenesis of nodules and the mechanisms of recovery of N2 fixation following shoot removal in alfalfa and birdsfoot trefoil (Vance et al., 1979, 1980, 1982). Following shoot removal, nitrogenase (i.e., N2 fixation) activity declines and then resumes upon regrowth. Alfalfa and birdsfoot trefoil have contrasting differences in the maintenance of nodule tissue following harvesting (Vance et al., 1979, 1980, 1982). Alfalfa nodules have apical meristems that function over prolonged periods; upon harvesting, nodules undergo a partial and temporary senescence process, upon which nodules regrow and fix N2 after shoot regrowth has resumed (Vance et al., 1979, 1980). In contrast, in birdsfoot trefoil nodules, the meristematic cells are active only in the first stages of nodule development, and extended senescence of existing nodules occurs following harvest; thus, new nodules must be formed at the beginning of each regrowth cycle for N2 fixation to resume (Vance et al., 1982). These studies by Vance et al. (1979)(1980, 1982) have led to the proposal that the lower efficiency of N2 fixation in birdsfoot trefoil compared with alfalfa is correlated to the above-mentioned differences in nodule development and function in the two species and would explain the lower persistence and amounts of Ndfa of birdsfoot trefoil compared with alfalfa.
Red clover and sweetclover had similar percentages of Ndfa as alfalfa at both sites in the second year. On that basis, it can be proposed that the nodules in these three species respond in a similar manner to shoot removal, leading to similar estimates of percentage Ndfa at the end of the following growth cycle, unlike birdsfoot trefoil. This remains to be ascertained since, to our knowledge, there are no histological and ultrastructural studies of nodules in red clover and sweetclover subjected to shoot harvesting.
The lower efficiency of N2 fixation in birdsfoot trefoil compared with the three other legumes is noteworthy for the first cut in 1996. Although N uptake of birdsfoot trefoil was in the same range or higher than that of the other legumes, percentages of Ndfa were the lowest. The results obtained at the third cut in 1996 emphasize even more the differences between birdsfoot trefoil and the three other legumes as not only were percentages of Ndfa lower, but also N uptake. Thus, it can be speculated that the need for new nodules to be formed on birdsfoot trefoil lowered the benefits of N2 fixation in this species since it had to rely on more soil N to compensate for the lower efficiency of N2 fixation. Opportunities for rapid and efficient renodulation in birdsfoot trefoil after the second cut in mid-August were probably more remote than in the spring, and may explain, at least in part, the very low DM yield measured at the third cut. The presumed differential senescence of nodules that occurs in birdsfoot trefoil nodules compared with alfalfa and/or red clover following harvesting would need to be addressed in more detail by 15N isotopic methods to better assess the impact of shoot removal on N2 fixation recovery in these species.
Although yearly totals of Ndfa and yearly means of Ndfa in the first year of production (1996) were not calculated and statistically analyzed, since DPS effects varied among harvests, a comparison with published values from the literature is interesting. In the absence of DPS and on the Kamouraska SCL, yearly mean percentages of Ndfa were 83, 60, and 88% for alfalfa, birdsfoot trefoil, and red clover, respectively, and were 96, 62, and 90% on the Tilly CL. Based on their relative dependence on N2 fixation for growth (percentage Ndfa) and subsequent contributions of fixed N to cropping systems, Peoples and Baldock (2001) indicate that pasture legumes with <65% Ndfa are poor for N2 fixation, those with 65 to 80% Ndfa are adequate, and those with >80% Ndfa would be considered excellent for N2 fixation. On that basis, in the present study, alfalfa would be classified adequate to excellent, red clover would be excellent, and birdsfoot trefoil poor for N2 fixation. Variation in overall efficiency for N2 fixation (percentage Ndfa) can thus vary among sites, as shown for alfalfa, in agreement with data reported by Peoples and Baldock (2001) for alfalfa and other pasture legumes under Australian conditions. Variation in efficiency for N2 fixation is also variable among growth cycles, as shown in the present study and by others (Heichel et al., 1984, 1985; Seguin et al., 2000). Examination of data for percentage Ndfa indicates that a species such as birdsfoot trefoil, which was poor for N2 fixation in absence of DPS, became adequate with DPS on the Tilly CL, a site generally more conducive to N2 fixation. In the absence of DPS and on the Kamouraska SCL, yearly amounts of Ndfa were 170, 130, and 188 kg N ha–1 yr–1 for alfalfa, birdsfoot trefoil, and red clover, respectively, and were 178, 138, and 204 kg N ha–1 yr–1 on the Tilly CL. The data for alfalfa and red clover agree with those compiled from the literature by Vance (1998), with median N2 fixation levels of 180 and 170 kg N ha–1 yr–1 for alfalfa and red clover, respectively. Our data for birdsfoot trefoil are also in agreement with those of Seguin et al. (2000), who reported yearly amounts of Ndfa of 145 kg N ha–1 yr–1 for 2- and 3-yr-old stands.
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CONCLUSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONCONCLUSIONREFERENCES |
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ACKNOWLEDGMENTS |
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONCONCLUSIONREFERENCES |
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gh-Jensen, H., and E.S. Kristensen. 1995. Estimation of biological N2 fixation in a clover–grass system by the 15N-dilution method and the total-N difference method. p. 203–219. In L. Kristensen, C. Stope, P. Kolster, A. Granstedt, and D. Hodges (ed.) Nitrogen leaching in ecological agriculture. Royal Veterinary and Agric. Univ., Copenhagen, Denmark.
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