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a Agriculture and Agri-Food Canada, 2560 Hochelaga Bd., Sainte-Foy, QC, G1V 2J3, Canada. Contribution no. 639 Agriculture and Agri-Food Canada
belangergf{at}em.agr.ca
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMaterials and methodsResults and discussionConclusionsREFERENCES |
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Abbreviations: DM, dry matter • LERleaf, leaf elongation rate of an entire leaf • LERlin, leaf elongation rate during the linear portion of leaf growth • LNAR, leaf N accumulation ratio • LWR, leaf weight ratio • N-, population selected for low forage N concentration • N+, population selected for high forage N concentration • NAE, N accumulation efficiency • NCE, N conversion efficiency • NUE, N use efficiency • SLA, specific leaf area
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMaterials and methodsResults and discussionConclusionsREFERENCES |
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Genetic variability for NUE has been reported for many annual species, such as rice (Oryza sativa L.) (Broadbent et al., 1987; DeDatta and Broadbent, 1988), wheat (Triticum aestivum L.) (Ortiz-Monasterio et al., 1997), and pumpkin [Cucurbita moschata (Duchesne) Duchesne ex Poir.] (Swiader et al., 1994), but few studies have been conducted on perennial species. Recent studies indicated the presence of genetic variability for NUE in ryegrass (Lolium perenne L.) (Van Loo et al., 1992; Wilkins et al., 1997) and timothy (Michaud et al., 1998). Studies on Kentucky bluegrass (Poa pratense L.) (Bertauski et al., 1997), wheat (Ortiz-Monasterio et al., 1997), and ryegrass (Van Arendonk et al., 1997) also indicated intraspecific differences of NUE under N fertilization ranging from limiting to optimal conditions.
Because nutritive value is also an important consideration in forage grasses, selection for improved NUE should also aim at maintaining or improving forage N concentration to meet ruminant nutrition requirements. The N concentration in plant tissues decreases with increasing shoot biomass during regrowth, and this decrease can be described by a nonlinear allometric function (Lemaire and Salette, 1984). Under nonlimiting N conditions, Lemaire and Salette (1984) reported that there were no cultivar differences in tall fescue (Festuca arundinacea Schreb.) for the relationship between forage N concentration or accumulation and DM yield, hence suggesting that cultivars with greater DM yield and NUE would necessarily have a lower N concentration. Hence, we hypothesized that under nonlimiting N conditions, timothy populations selected for high forage N concentration will have low DM yield and NUE, whereas populations selected for low forage N concentration will have high DM yield and NUE. Under limiting N conditions, however, we hypothesized that there is genetic variability for the relationship between N concentration or accumulation and DM yield, and that consequently, populations with high N concentration could have a high DM yield and NUE.
Our objectives were (i) to illustrate differences in NUE and N concentration in two timothy populations obtained from divergent selection for high (N+) and low (N-) N concentrations and a reference population, Champ, grown under limiting and nonlimiting N fertilization and (ii) to relate those differences to morphological characteristics.
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Materials and methods |
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TOPABSTRACTINTRODUCTIONMaterials and methodsResults and discussionConclusionsREFERENCES |
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Two populations of timothy obtained from two cycles of divergent selection for high (N+) and low (N-) forage N concentrations and, as a reference, the cultivar Champ, were compared. The base population of the divergent selection consisted of 4485 genotypes, which came from 15 cultivars. During the divergent selection process, only plants with a vigorous growth habit were chosen.
The plants were grown in a Turface (AIMCOR, Deerfield, IL) medium, which is similar to vermiculite but with a higher cationic retention. Plants were started in a greenhouse by placing one seed per pot (100 cm2 of soil surface area; 0.4 L of soil volume). Four weeks after seeding, plants were cut at a 5-cm height to promote tillering and then transferred to a growth chamber. Day and night temperatures were kept at 25 ± 5 and 15 ± 5°C, respectively. Light was provided for a 16-h day by cool white fluorescent and incandescent bulbs with a quantum flux of 350 µmol m-2 s-1 (between 400 and 700 nm) at plant height. Pots were watered every 2 d.
Phase 1
Nutrient Solution
Fifty milliliters per pot of a nutrient solution containing 8.8 mM MgSO4, 4.4 mM K2HPO4, 4.4 mM KH2PO4, 4.4 mM CaCl2, 0.9 mM ferric citrate, 14 mM NH4NO3, and micronutrients (204 µM H3BO3, 40 µM MnCl2 · 4H2O, 3.366 µM ZnSO4 · 7H2O, 1.410 µM CuSO4 · 5H2O, 2.404 µM H2MoO4 · H2O, 0.074 µM CoCl2 · 6H2O) were applied once a week to each pot up to 60 d after seeding, which was the date of sampling at the end of Phase 1. This N rate delivered 20 mg N plant-1 wk-1.
Sampling
Plants were sampled 60 d after seeding. Roots were washed free of soil with water, and plants were separated into leaves, stems (including sheaths), stubble (0–5 cm above the soil surface), and roots. Samples were dried at 55°C for 2 d for determination of DM yield. Dried samples were ground to 1 mm. Forage DM yield was calculated by adding leaf and stem DM yield. Total biomass DM yield was calculated by adding leaf, stem, stubble, and root DM yields.
Total Nitrogen and Insoluble Nitrogen
Total N concentrations of leaves, stems, stubble, and roots were determined by the H2SO4–H2O2 digestion method of Kjeldahl (adapted from Richards, 1993). Total N concentration determined in root tissues was probably underestimated because of leaching of soluble components during root washing. Insoluble N concentration was measured in leaves and stems. Five hundred milligrams of tissue samples were weighed and poured into 125-mL Erlenmeyer flasks with 40 mL of borate–phosphate buffer at pH 6.7 (88.4 mM NaH2PO4 · H2O, 23.4 mM Na2B4O7 · 10 H2O) (Roe and Sniffen, 1990). The samples were shaken in a water bath at 39°C for 1 h and then filtered (vacuum assisted) through glass filtering crucibles (40–60 µm). The residue was washed with 250 mL of borate–phosphate buffer, and the insoluble N concentration of the residue was estimated by the Kjeldahl method. For each type of N compound measured, the forage concentration was estimated from stem and leaf concentrations weighted on the basis of their respective DM yield.
Ash Concentration in Roots
Ash concentration in roots was determined to eliminate the dry weight variation that may occur when root samples are contaminated with soil. Root tissues were ashed at 600°C for 2 h and ashes were weighed. Root DM yield was expressed on an ash-free basis.
Root and Leaf Proportions
Root proportion for DM yield and N accumulation were calculated as root DM yield divided by total biomass DM yield and root N accumulation divided by total biomass N accumulation, respectively. Leaf weight ratio (LWR) was calculated as leaf DM yield divided by forage DM yield. Leaf proportion for N accumulation was calculated as leaf N accumulation divided by forage N accumulation and is referred to below as leaf N accumulation ratio (LNAR).
Tillering
The number of tillers per plant was counted every 3 to 5 d for 25 d preceding sampling. The tillering rate of each population was estimated as the slope of the linear regression between the number of tillers and time expressed in number of days. We also counted the number of tillers, including tillering buds, on the day of sampling.
Leaf Elongation Rate
The length of the most recently emerged leaf of the first secondary tiller having at least one mature leaf (ligule visible) of each plant was measured every day for 5 d. Daily leaf elongation rates (LER) were calculated as the increase in leaf length in 2 d, and the daily LER values were then averaged. The elongation pattern of a newly emerged leaf during a 5-d period corresponds with linear growth (Skinner and Nelson, 1995) and will be referred to below as LERlin.
Specific Leaf Area
Specific leaf area was measured on the leaves previously used for LER determinations. The leaf area was determined using a leaf area meter (LI-3100, LI-COR, Lincoln, NE). The leaves were then dried at 55°C for 2 d and weighed for DM determination.
Phase 2
Nutrient Solution
During the first regrowth cycle, plants used in Phase 2 received a similar nutrient solution as in Phase 1. During the second regrowth cycle, plants of Phase 2 were fertilized twice a week with 50 mL of a half-strength nutrient solution as used in Phase 1 except that four N rates were applied, corresponding with 1 (N1), 5 (N2), 10 (N3), and 20 (N4) mg N plant-1 wk-1.
Sampling
The plants used for Phase 2 were cut at a 5-cm height at the end of the first regrowth cycle (60 d after seeding). At the end of the second regrowth cycle (Phase 2, 30 d later), the sampling was done using the same procedure as described for Phase 1.
Variables
Total N and insoluble N concentrations, root and leaf proportions, ash concentration in roots, tiller number, and SLA were determined as described for Phase 1, except that SLA was measured on a leaf subsample of about 15 leaf blades. Nitrates were extracted from leaves and stems and measured with a specific NO3 electrode (Mod. 93-07, Orion Research, Beverly, MA). In a 100-mL centrifuge tube, 500 mg of plant tissue were weighed out and 50 mL of extraction solution added (26 mM Al2(SO4)3 · 18H2O). Tubes were shaken 30 min and then centrifuged at 1375 g for 20 min. Nitrates were immediately measured in supernatant. A standard solution of KNO3 was used for calibration. The LER was estimated as the difference between the leaf length at emergence and the final leaf length (ligule appearance) divided by the number of days. This measurement of LER over the complete leaf development will be referred to as LERleaf.
Statistical Analysis
All variables were subjected to analyses of variance using SAS Institute (SAS System, Cary, NC). The effect of N rates were partitioned into linear and quadratic contrasts. The effects of the populations were tested using orthogonal contrasts. Statistical significance was postulated at P < 0.10.
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Results and discussion |
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TOPABSTRACTINTRODUCTIONMaterials and methodsResults and discussionConclusionsREFERENCES |
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In Phase 2, the significant quadratic component indicated that the total biomass DM yield tended to a maximum with the N rates used in our study (Fig. 1 and Table 1). However, the total biomass N accumulation increased linearly with increasing N rates, indicating that N accumulation did not reach a maximum value. There was no significant interaction between populations and N rates for DM yield and N accumulation in total biomass, indicating that the three populations responded similarly to N rates.
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The forage N accumulation of N- was significantly greater than that of N+ in Phase 2. The interaction between populations and N rates for forage N accumulation was significant. The forage N accumulation of N+ was equivalent to 73% of that for N- at N1, 86% at N2, and 94% at N3, while N+ had 2.4% more N accumulation at N4. In Phase 1, there were no significant differences of forage N accumulation among the three populations.
The forage DM yield of Champ averaged across N rates was not significantly greater than that of N-, with a 1.0% difference in Phase 1 and a 1.7% difference in Phase 2 (Tables 1 and 2), while it was significantly greater than that of N+ by 5.2% in Phase 1 (P = 0.12) and 18.3% in Phase 2. The forage NUE of N- was similar to that of the reference cultivar Champ, while the forage NUE of N+ was lower. The forage NAE of Champ was also greater than N+ in Phase 2 (Table 1), while it was similar to that of N-.
In Phase 2, the quadratic component was significant, indicating that forage DM yield tended to a maximum under our experimental conditions (Fig. 1 and Table 1). Concurrently, the forage N accumulation increased linearly with increasing N rates, indicating that N accumulation did not reach a maximum value.
Relationship between Nitrogen Accumulation and Dry Matter Yield
In Phase 2, N accumulation in the total biomass and forage increased with DM yield (Fig. 1). However, between the two highest N rates the total biomass and forage N accumulation increased significantly, while the DM yield hardly changed. This resulted in the accumulation of NO3 under the nonlimiting N growing conditions (Fig. 2)
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In a study of tall fescue, Lemaire and Salette (1984) concluded that there was no genetic variability for the relationship between forage N accumulation and DM yield when plants were grown under nonlimiting N conditions. Our results indicate that there is genetic variability in timothy for the relationship between forage N accumulation and DM yield under both limiting and nonlimiting N conditions, that is, for a given DM yield, there is a range in N concentration among populations. However, we observed no genetic variability for the relationship between N accumulation and DM yield of the total biomass.
Biomass and Nitrogen Partitioning
The population N+ had a significantly greater LWR and LNAR than N- and Champ in Phases 1 and 2 (Fig. 3)
. The N effect was not significant, but the interaction of population with N rate was significant. The difference between N+ and N- and Champ decreased as N rates increased. Because stem elongation constitutes a strong sink for assimilates, the greater proportion of stems of N- resulted in a greater proportion of assimilates being used for aboveground growth at the expense of root growth. Stem elongation is beneficial for forage DM yield, but the resulting low LWR may impair forage quality (Bélanger and McQueen, 1996).
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The proportion of roots in the total biomass was significantly less for N- than N+ in Phases 1 and 2 (Fig. 3). In Phase 2, the root proportion of the population Champ was intermediate between N+ and N-, although it was close to that of N- at N3 and N4. The difference in the root proportion between N+ and N- and Champ decreased with increasing N rates, as indicated by the significant interaction between populations and N rates. The root proportion of N- was equivalent to 62% of that of N+ at N1 and 77% at N4. Hence, the population N+ produced a larger root system than N- and Champ, particularly under N-limiting conditions. This may be the cause or the consequence of the lowest proportion of stems in N+ because assimilates not used in shoot growth may be used for root growth.
Similarly to the root biomass, the proportion of roots for N accumulation was significantly less for N- and Champ than N+ in Phases 1 and 2 (Fig. 3). This suggests a greater N immobilization in the underground parts of N+ plants and, therefore, different pools of N for further regrowth. However, the greater investment in root biomass by N+ did not translate into a greater total biomass N accumulation than that obtained by N-. In fact, the N accumulation per unit of root biomass was lower for N+ than for N-. These results were obtained under relatively short periods and with small soil volumes. The benefits of a greater investment in root biomass might not be apparent under such growing conditions. Observing the population characteristics for a longer period of time, beyond two regrowth cycles, may show contrasted regrowth efficiency for N+ and N- (Wilkins et al., 1997).
Although the total biomass DM yield of N+ and N- were similar, forage DM yield of N- was greater due to its greater proportion of aboveground biomass in the total biomass. Hence, the greater forage NUE of N- compared with N+ can be attributed to a change in biomass partitioning between shoots and roots. Even though the divergent selection resulted in contrasted populations for forage NUE, it did not produce a population with a NUE greater than the cultivar Champ. However, using 40 half-sib families of timothy, Michaud et al. (1998) reported that some genotypes had a greater forage NUE than the cultivar Champ.
Along with having a greater proportion of aerial biomass, N- had a greater proportion of stems than N+. Consequently, the population N- had a lower leaf DM yield and a greater stem DM yield than N+ (Table 3) . Further, the forage N concentration of N+ was greater than that of N-. The leaf N concentration of N+ was lower than that of N-, while the stem N concentration of N+ was similar to that of N- (Table 3). Therefore, the greater forage N concentration of N+ was not due to a greater leaf N concentration but to a greater LWR. Colnenne et al. (1998), in a comparison of wheat and rapeseed (Brassica campestris L.), also concluded that the LWR explained species differences in N concentration. This suggests that the improvement of forage N concentration could be obtained through the modification of biomass partitioning between leaves and stems.
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Concentration of soluble N in forage DM was similar in N+ and N- in both Phases 1 and 2, while concentration of insoluble N was significantly greater in N+ than N- (Fig. 2). These results differed from those of Hansen et al. (1992), who reported that selection for higher N concentration in alfalfa (Medicago sativa L.) coincided with higher concentration of soluble N.
Soluble and insoluble N concentrations in forage DM increased linearly with N rates (Fig. 2) concurrently with total N concentration (Table 3). Increasing N rates had no effect on the proportion of soluble or insoluble N in total N (data not shown). We found no interaction between populations and N rates. Assuming a positive relationship between N solubility and rumen protein degradation (Broderick, 1994), we speculate that N+ may have greater forage nutritive value from the standpoint of favoring more rumen bypass of protein.
Morphogenetic Characteristics
In Phase 1, the tillering rates were 0.37, 0.32, and 0.38 tiller d-1 for N+, N-, and Champ, respectively. As a result, at the end of Phase 1, N+ and Champ had a greater tiller density than N- (Table 2). Initial tiller counts in Phase 1 were significantly different among populations (data not shown). In Phase 2, the number of tillers increased with N rates and the quadratic component was significant (Fig. 4)
. The tiller density of Champ was greater than that of N+ and N-, and the tiller density of N+ was greater than that of N-. No interaction between populations and N rates was found. The greater assimilate investment of N- in stem growth compared with N+ may explain its lower tiller development.
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In Phase 1, the SLA was not significantly different for N+, N-, and Champ (Table 2). In Phase 2, the SLA of Champ was significantly greater than that of N+ which, in turn, was significantly greater than that of N- (Fig. 4). This indicates that the leaves of Champ were thinner than those of N-, which were in turn thinner than the leaves of N+. No interaction was found between populations and N rates. The SLA increased linearly with increasing N rates, indicating that leaf thickness tended to decrease as N rates increased. Similar results were reported for tall fescue by Bélanger (1990).
These results indicate the two timothy populations obtained by divergent selection had different morphogenetic characteristics. Further investigations are required to determined the stability of those differences across a wider range of growing conditions.
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Conclusions |
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TOPABSTRACTINTRODUCTIONMaterials and methodsResults and discussionConclusionsREFERENCES |
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ACKNOWLEDGMENTS |
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Received for publication February 5, 1999.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMaterials and methodsResults and discussionConclusionsREFERENCES |
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