Effect of nitrogen fertilization on yield, N content, and nitrogen fixation of alfalfa and smooth bromegrass grown alone or in mixture in greenhouse pots

Journal of Integrative Agriculture 2015, 14(9): 1864–1876 Available online at www.sciencedirect.com

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RESEARCH ARTICLE

Effect of nitrogen fertilization on yield, N content, and nitrogen fixation of alfalfa and smooth bromegrass grown alone or in mixture in greenhouse pots XIE Kai-yun1, 2*, LI Xiang-lin1, HE Feng1*, ZHANG Ying-jun2, WAN Li-qiang1, David B Hannaway3, WANG Dong1, QIN Yan1, Gamal M A Fadul1 1

Institute of Animal Sciences, Chinese Academy of Agricultural Sciences, Beijing 100193, P.R.China Institute of Grassland Science, College of Animal Science and Technology, China Agricultural University, Beijing 100193, P.R.China 3 Department of Crop and Soil Science, Oregon State University, Corvallis 97331, USA 2

Abstract Planting grass and legume mixtures on improved grasslands has the potential advantage of realizing both higher yields and lower environmental pollution by optimizing the balance between applied N fertilizer and the natural process of legume biological nitrogen fixation. However, the optimal level of N fertilization for grass-legume mixtures, to obtain the highest yield, quality, and contribution of N2 fixation, varies with species. A greenhouse pot experiment was conducted to study the temporal dynamics of N2 fixation of alfalfa (Medicago sativa L.) grown alone and in mixture with smooth bromegrass (Bromus inermis Leyss.) in response to the addition of fertilizer N. Three levels of N (0, 75, and 150 kg ha–1) were examined using 15 N-labeled urea to evaluate N2 fixation via the 15N isotope dilution method. Treatments were designated N0 (0.001 g per pot), N75 (1.07 g per pot) and N150 (2.14 g per pot). Alfalfa grown alone did not benefit from the addition of fertilizer N; dry matter was not significantly increased. In contrast, dry weight and N content of smooth bromegrass grown alone was increased significantly by N application. When grown as a mixture, smooth bromegrass biomass was increased significantly by N application, resulted in a decrease in alfalfa biomass. In addition, individual alfalfa plant dry weight (shoots+roots) was significantly lower in the mixture than when grown alone at all N levels. Smooth bromegrass shoot and root dry weight were significantly higher when grown with alfalfa than when grown alone, regardless of N application level. When grown alone, alfalfa’s N2 fixation was reduced with N fertilization (R2=0.9376, P=0.0057). When grown in a mixture with smooth bromegrass, with 75 kg ha–1 of N fertilizer, the percentage of atmospheric N2 fixation contribution to total N in alfalfa (%Ndfa) had a maximum of 84.07 and 83.05% in the 2nd and 3rd harvests, respectively. Total 3-harvest %Ndfa was higher when alfalfa was grown in a mixture than when grown alone (shoots: |t|=3.39, P=0.0096; root: |t|=3.57, P=0.0073). We believe this was due to smooth bromegrass being better able to absorb available soil N (due to its fibrous root system), resulting in

Received 9 Sepetember, 2014 Accepted 10 March, 2015 XIE Kai-yun, E-mail: [email protected]; Correspondence LI Xiang-lin, Tel: +86-10-62815997-601, E-mail: [email protected] * These authors contributed equally to this study. © 2015, CAAS. All rights reserved. Published by Elsevier Ltd. doi: 10.1016/S2095-3119(15)61150-9

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lower soil N availability and allowing alfalfa to develop an effective N2 fixing symbiosis prior to the 1st harvest. Once soil N levels were depleted, alfalfa was able to fix N2, resulting in the majority of its tissue N being derived from biological nitrogen fixation (BNF) in the 2nd and 3rd harvests. When grown in a mixture, with added N, alfalfa established an effective symbiosis earlier than when grown alone; in monoculture BNF did not contribute a significant portion of plant N in the N75 and N150 treatments, whereas in the mixture, BNF contributed 17.90 and 16.28% for these treatments respectively. Alfalfa has a higher BNF efficiency when grown in a mixture, initiating BNF earlier, and having higher N2 fixation due to less inhibition by soil-available N. For the greatest N-use-efficiency and sustainable production, grass-legume mixtures are recommended for improving grasslands, using a moderate amount of N fertilizer (75 kg N ha–1) to provide optimum benefits. Keywords: alfalfa (Medicago sativa), smooth bromegrass (Bromus inermis), nitrogen (N2) fixation, nitrogen partitioning, 15 N, mixture, monoculture

1. Introduction Nitrogen (N) is often the most limiting factor in agricultural system productivity. For non-N2 fixing plants, the production of 1 kg of dry matter requires 20–30 g of soil-derived N. Thus, the yield of non-leguminous plants is limited by soil-available N (Ukrainetz and Campbell 1988; Robertson and Vitousek 2009). Alfalfa (Medicago sativa L.) is one of the most important forage crops in the world because of its high nutritive quality and yield. In China, alfalfa is commonly grown on sandy and saline-alkali soils where the soil has poor nutrient and water holding capacity. Applying fertilizer N to increase yield potential has been a routine practice for many alfalfa growers (He et al. 2014). Although application of N fertilizer may increase yields, it also can result in environmental pollution through run-off into surface waters or leaching into ground waters (Berry et al. 2003; Deutsch et al. 2006; Anjana and Iqbal 2007). A large amount of N applied to soils is wasted because plants have a low nitrogen use efficiency (NUE) in using commercial N fertilizer (Robertson and Vitousek 2009). Biological nitrogen fixation (BNF) by legumes is an important pathway for N supply and for improving soil fertility (Buerkert et al. 2000). Using legumes in agricultural production systems, including their use as green manure crops and in crop rotations, can reduce dependence on N fertilizer (Jensen et al. 2012), and provide economic and environment benefits (Olivares et al. 2013). Compared with grass grown alone, grass-legume mixtures improve dry matter and protein yield (Gökkuş et al. 1999; Frankow-Lindberg et al. 2009; Nyfeler et al. 2011), and increase soil fertility by increasing organic matter and increasing soil N content (Nguyen 2003; Wichern et al. 2007). Growing legumes can also decrease the need for N fertilizer, and thereby reduce production costs and environmental pollution (Tekeli and Ateş 2005). Interestingly, when grown as mixtures, grasses and

legumes do not compete directly for N (Haynes 1980). In contrast, planting grass-legume mixtures can lead to a significant increase in production and protein yield (Cardinale et al. 2007; Kirwan et al. 2007; Frankow-Lindberg et al. 2009; Nyfeler et al. 2011) resulting from complementary effects between grasses and legumes (Malézieux et al. 2009). These effects include obtaining resources from different niches (their root structures are different; grasses have a fibrous root systems, whereas many legumes have a significant taproot system) and the ability of legumes to fix atmospheric N2 (this provides N not only for their own growth needs, but also for grasses co-cultivated in mixtures (Beschow et al. 2000; Høgh-Jensen and Schjoerring 2000; Schipanski and Drinkwater 2012). N2 fixation also helps to avoid the loss of fertilizer N resulting from the lack of coordination between the soil-available N supply and the plants’ needs (Robertson and Vitousek 2009). Generally, the growth rate of grasses is high, and the amount of N needed to meet the maximal productivity of grasses is far more than that transferred from legumes. Thus, to reach the maximal biomass and protein yield, commercial N fertilizer is applied to grass-legume mixtures (Bijelić et al. 2011). In addition, N fertilizer plays an important role in maintaining the balance between grasses and legumes (Ledgard and Steele 1992). Previous studies have demonstrated a negative correlation between the amount of N2 fixation of legumes and the available soil N (Tanner and Anderson 1964; Hannaway and Shuler 1993; Schipanski et al. 2010). However, little is known about how alfalfa plants make use of the N from various sources, namely soil available N, fertilizer N, and BNF. In alfalfa/grass mixtures, N nutrition is further complicated and it is unknown how BNF is affected by the associated grass. Since both alfalfa and smooth bromegrass are important forage species grown for hay in temperate regions of the world and often used together as a mixture, these species were chosen for evaluation. Studies using the 15N isotope dilution method and the 15N natural abundance method to measure the amount of N2

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fixation by legumes have been conducted under both field and laboratory conditions (Bilgo et al. 2007; Oberson et al. 2007; Wichern et al. 2008). Drawbacks of field experiments include the inability to completely extract plant roots leading to an underestimation of the amount of N2 fixation. For this study, the research hypothesis was that alfalfa’s nitrogen fixation would be decreased by increased levels of soil-available N and optimal yield and nitrogen fixed would occur with a mixture of alfalfa and smooth bromegrass. Specifically, this experiment examined the following research questions: (1) Will nitrogen fixation of alfalfa be inhibited or delayed by increased levels of fertilizer N? (2) With increased soil-available N, will the amount of alfalfa’s N2 fixation be higher when grown with smooth bromegrass, due to absorption of soil N by the grass roots? (3) Will biomass accumulation be highest for alfalfa or smooth bromegrass

grown alone or in a mixture?

2. Results 2.1. Biomass and plant N Dry weight of alfalfa grown alone did not differ significantly when N was increased from 0 to 150 kg ha–1 (Figs. 1 and 2); there was only a 4% increase in shoots biomass accumulation (50.20–52.21 g per pot) and an 8.44% increase in N yield accumulation (1.54–1.67 g per pot; Figs. 3 and 4). Root biomass decreased by 6.84% (53.04–49.41 g per pot) and N yield accumulation was the highest in the N75 treatment (1.11 g per pot). N application increased N content of alfalfa in the 1st harvest of the N150 treatment (Fig. 5). Root N was significantly higher at the 1st and 3rd harvests

Shoot dry weight (g per plant) 8

6

4

2

0

a Monoculture

0

2

4

8

c

N0

A

6

10

12

B

Mixture a A a A a

b

N75

a

N150

a

C b

N75

B a B a

B a

N150

N0

A

A

c

N0

A

A

A b B

a

N75

A

a AB

a A

N150

a A

Fig. 1 Effect of N fertilizer application on dry weight of individual alfalfa and smooth bromegrass plants in shoots. Different capital letters denote significant differences among different N levels in the mixture (P<0.05); lowercase letters denote significant differences among different N levels in monocultures (P<0.05). N0, N75, N150 mean three levels of N (0, 75, and 150 kg N ha–1). The same as below.

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Root dry weight (g per plant) 8

6

4

2

a Monoculture 1st harvest

10

a B

B

B a

N150

A b

N0

a

B ab

N75

A

AB

a B a

3rd harvest

a B a B Alfalfa

a

N150

A b

N0

A

40

b

a B

30

B

N75

a

2nd harvest

20

c

N0

A

Mixture

0

0

B ab

N75

B

N150

a A Smooth bromegrass

Fig. 2 Effect of N fertilizer application on dry weight of individual alfalfa and smooth bromegrass plants in roots.

in the N75 and N150 treatments. Dry weight and N content of smooth bromegrass in shoot and root samples were increased significantly by N application (Figs. 1, 2 and 5, Table 1). When N application was increased from 0 to 150 kg ha–1, smooth bromegrass shoot biomass accumulation increased 58.04% (31.99–50.56 g per pot; Fig. 3) and N yield accumulation of shoots increased 126.3% (0.57–1.29 g per pot; Fig. 4). Similarly, root biomass accumulation increased by nearly 88% (94.24–177.15 g per pot) and N yield accumulation increased by 145% (0.60–1.47 g per pot). In the mixture, individual smooth bromegrass plant dry weight (shoots and roots) was increased significantly by N application for all 3 harvests (Figs. 1 and 2). In contrast, alfalfa shoot dry matter was decreased significantly at the

2nd harvest and root dry matter was decreased by N application at the 1st and 3rd harvests. In addition, individual alfalfa dry weight (shoots+roots) was significantly lower in the mixture than when grown alone at all N levels (shoots: |t|=7.43, P<0.0001; roots: |t|=5.21, P=0.0008). Smooth bromegrass dry weight (shoots and roots) was significantly higher in the mixture than when grown alone regardless of N application level (shoots: |t|=8.72, P<0.0001; roots: |t|=6.28, P=0.0002). In shoot samples, with increased N application, mixture biomass was increased by 25.33% (42.88 to 53.74 g per pot; Fig. 3) and N yield accumulation increased by 46.08% (1.02 to 1.49 g per pot; Fig. 4). In roots, biomass accumulation increased by 43.4% (98.46 to 141.21 g per pot) and N yield accumulation increased by 75.4% (1.18 to 2.07 g per pot).

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1st harvest SB

AL+SB

20

20

AL

SB

10

AL+SB

30

3rd harvest AL

SB

AL+SB

10

10

A A A

SB

AL+SB

20

B A A 0

N 1 N 2 N 3 N 1 N 2 N 3 N 1 N 2 N 3

N 1 N 2 N 3 N 1 N 2 N 3 N 1 N 2 N 3

0

N 1 N 2 N 3 N 1 N 2 N 3 N 1 N 2 N 3

0

80

80

80

SB

AL+SB

AL

SB

AL+SB

AL

SB

AL+SB

200

60

60

60

150

40

40

40

100

N 1 N 2 N 3 N 1 N 2 N 3 N 1 N 2 N 3

A A A

20

SB

AL+SB

B A A

A A A

20

C B A A B B

0

0

50 C B A

N 1 N 2 N 3 N 1 N 2 N 3 N 1 N 2 N 3

C B A

B B A

AL

0

N 1 N 2 N 3 N 1 N 2 N 3 N 1 N 2 N 3

C B A

0

AL

A A A

C B A

A A A

20

3 harvests total

B B A B A A

AL

60

40

20

A A A

C B A

0

Root dry matter (g per pot)

2nd harvest

N 1 N 2 N 3 N 1 N 2 N 3 N 1 N 2 N 3

AL

30

N 1 N 2 N 3 N 1 N 2 N 3 N 1 N 2 N 3

Shoot dry matter (g per pot)

30

Fig. 3 Effect of N fertilizer application on total dry weight of alfalfa and smooth bromegrass shoots and roots at each pot. N1, N2, N3 represent N0, N75 and N150, respectively. AL and SB represent alfalfa and smooth bromegrass grown alone, and AL+SB represents a mixture of alfalfa and smooth bromegrass. The same as below.

From the 1st to 3rd harvest, dry weight of individual

mixture treatments, and the remainder was derived from

smooth bromegrass shoots decreased and alfalfa shoot dry

fertilizer N (0–46.88%) and N2 fixation (0–29.93%). In roots,

weight increased (Fig. 1). In contrast, in the 1st harvest of

most of the N was derived from the soil N (61.15–99.56%),

smooth bromegrass grown alone, shoot dry weight per pot

followed by fertilizer N (0.36–38.33%), and N transferred

was higher than that of alfalfa (|t|=15.53, P<0.0001). During

from alfalfa (0–20.01%) (Table 2).

the 2nd and 3rd harvests, alfalfa shoot dry weight per pot

When grown alone, the N in smooth bromegrass was

was significantly higher than that of smooth bromegrass

derived from soil and fertilizer (Table 2). The proportion of

shoots (2nd harvest: |t|=14.32, P<0.0001; 3rd harvest:

N from fertilizer (%Ndff) of smooth bromegrass increased

|t|=17.13, P<0.0001). Smooth bromegrass root dry weight

with increasing fertilizer and decreased with harvests.

was consistently higher than that of alfalfa when grown alone

When planted with alfalfa in mixtures, the %Ndff in smooth

(1st harvest: |t|=5.92, P=0.0003; 2nd: |t|=5.94, P=0.0001;

bromegrass was increased. When N fertilization increased,

and 3rd: |t|=4.53, P= 0.0015; Fig. 2). In mixtures, dry weight

the %Ntrans to smooth bromegrass decreased. In the 3

per pot was intermediate between that of smooth brome-

harvests, the %Ntrans from the N0 treatment of smooth

grass and alfalfa grown alone, but higher than the mean of

bromegrass in shoots accounted for 16.63, 29.09, and

these 2 monocultures (Fig. 3).

29.93%, respectively. In roots, the %Ntrans accounted for 16.96, 20.01, and 16.28%, respectively. With 150 kg ha–1

2.2. N source

of N, no %Ntrans to smooth bromegrass in shoots and roots was detected.

N content of smooth bromegrass shoots was derived pri-

For alfalfa, the source of N was more complicated

marily from soil N (53.12–99.66%) in both monoculture and

(Table 2). When grown alone, for the 1st harvest, the

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AL+SB

0.8

0.8

0.6

0.6 0.4

0.2

0.2

0.0

0.0

AL

SB

AL+SB

0.6

AL+SB

0.8

AL

SB

AL+SB

AL

SB

AL+SB

0.4

0.2

0.2

0.0

0.0

2.0

0.6

1.5

0.4

1.0

0.2

0.5

0.0

0.0

0.6

AL

SB

AL+SB

0.6

0.4

3 harvests total

3rd harvest

0.4

AL

SB

AL+SB

2.0

AL

SB

AL+SB

1.5

1.0 0.2 0.5

0.0

N 1 N 2 N 3 N 1 N 2 N 3 N 1 N 2 N 3

N 1 N 2 N 3 N 1 N 2 N 3 N 1 N 2 N 3

N 1 N 2 N 3 N 1 N 2 N 3 N 1 N 2 N 3

Root N (g per pot)

0.8

SB

N 1 N 2 N 3 N 1 N 2 N 3 N 1 N 2 N 3

0.4

AL

0.8

N 1 N 2 N 3 N 1 N 2 N 3 N 1 N 2 N 3

SB

2nd harvest

0.0

N 1 N 2 N 3 N 1 N 2 N 3 N 1 N 2 N 3

AL

1.0

N 1 N 2 N 3 N 1 N 2 N 3 N 1 N 2 N 3

1st harvest

N 1 N 2 N 3 N 1 N 2 N 3 N 1 N 2 N 3

Shoot N (g per pot)

1.0

Fig. 4 Effect of N fertilizer application on total N yield of alfalfa and smooth bromegrass shoots and roots at each pot.

%Ndfa was 18.59% in the N0 treatment, with no detectable %Ndfa in the N75 and N150 treatments. For the 2nd harvest, N was derived primarily from N2 fixation in the N0 and N75 treatments (%Ndfa of 75.67 and 54.41%, respectively). With 150 kg ha–1 of N, however, the majority of N was derived from the soil (56.94%), with 27.98% contribution from fertilizer, and 15.08% contribution from N2 fixation. For the 3rd harvest, N was derived primarily from N2 fixation in the N0 and N75 treatments (68.44 and 58.40%). With 150 kg ha–1 of N, however, N was derived primarily from the soil (63.41%), with the secondary source being N2 fixation (22.59%). When planted with smooth bromegrass in the mixture, the %Ndfa of the 1st harvest was 22.17% in the N0 treatment, 17.90% in N75, and 16.28% in N150 (Table 2). For the 2nd and 3rd harvests, N was derived primarily from N2 fixation in all N levels; the %Ndfa for the 2nd harvest of alfalfa accounted for 75.60, 84.07, and 65.12% for the N0, N75, and N150 treatments. Correspondingly, the %Ndfa from the 3rd harvest of alfalfa accounted for 69.30, 83.05, and 69.80%. Similar results were found for roots. In all 3 harvests, the %Ndfa for alfalfa was significantly higher in mixtures than when alfalfa was grown alone (shoots: |t|=3.39, P=0.0096;

roots: |t|=3.57, P=0.0073).

3. Discussion 3.1. Biomass response to N application It is well-known that grass growth and biomass yield is positively correlated with soil-available N (Schipanski and Drinkwater 2012). In most agricultural systems, the amount of soil-available N is not sufficient for optimal grass yield. Even if soil organic N content is relatively high, available N is limited due to the rate of decomposition by microorganisms and mineralization, resulting in sub-optimal N and reduced yield. Thus, for optimal forage grass yield and quality (protein and digestibility), N fertilization is required. When grown in mixtures with legumes, grasses have greater competitive ability for available N (Høgh-Jensen and Schjoerring 1997), and the addition of small amounts of N fertilizer typically increases biomass and N yield. For legumes in general and alfalfa specifically, however, many studies have found that application of N fertilizer does not increase biomass yield or quality (Fishbeck and Phillips 1981; Fan et al. 2011). This is because biologically fixed N

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0.0

0.0

AL Mix

SB Mono

2.5

SB Mix

A

A B

A A

0.0

0.5

N 1 N 2 N 3 N 1 N 2 N 3 N 1 N 2 N 3 N 1 N 2 N 3

0.0

N 1 N 2 N 3 N 1 N 2 N 3 N 1 N 2 N 3 N 1 N 2 N 3

N 1 N 2 N 3 N 1 N 2 N 3 N 1 N 2 N 3 N 1 N 2 N 3

0.0

A

B AB A B AB A

B

0.5

B

B A

A

A

A A

A A

1.0

B

A A A

1.5

1.0

0.5

SB Mix

A

A

A A

SB Mono

2.0

1.5

1.0

AL Mix

AL Mono

A

AL Mono

A A

2.5

SB Mix

A

SB Mono

A A

N 1 N 2 N 3 N 1 N 2 N 3 N 1 N 2 N 3 N 1 N 2 N 3

1.5

AL Mix

2.0

B

Root N (%)

2.0

AL Mono

A

0.0

2.5

A A

0.5

N 1 N 2 N 3 N 1 N 2 N 3 N 1 N 2 N 3 N 1 N 2 N 3

0.5

A A A A

A A B

0.5

N 1 N 2 N 3 N 1 N 2 N 3 N 1 N 2 N 3 N 1 N 2 N 3

1.0

3.0

A

A

A A B

1.5

1.0

1.0

SB Mix

SB Mono

A

2.5 2.0

AL Mix

AL Mono

A

A

A

C C

3.5

3rd harvest

3.0

1.5

1.5

SB Mix

A

A B

2.5

SB Mono

2.0

B

2.5

AL Mix

A

3.5

A

3.0

2.0

AL Mono

4.0

A

SB Mix

3.0

Shoot N (%)

3.5

SB Mono

A A A

4.0

AL Mix

B AB A

AL 4.5 Mono

2nd harvest

4.0

A

1st harvest 5.0

Fig. 5 Effect of N fertilizer application on N content of alfalfa and smooth bromegrass shoots and roots.

Table 1 Analysis of variance results of alfalfa and smooth bromegrass grown alone and in a mixture Source of variance Shoots Species1) N level Species×N level Roots Species N level Species×N level 1)

df

1st harvest F-value P-value

df

2nd harvest F-value

2 2 4

23.04 4.19 1.08

<0.0001 0.0343 0.3982

2 2 4

62.77 12.55 0.77

<0.0001 0.0006 0.5624

2 2 4

98.55 6.39 1.14

<0.0001 0.0091 0.3712

2 2 4

62.24 8.86 6.75

<0.0001 0.0026 0.0022

2 2 4

45.08 6.65 2.06

<0.0001 0.0079 0.1347

2 2 4

13.82 0.69 2.48

0.0003 0.5177 0.0858

P-value

df

3rd harvest F-value

P-value

Species mean alfalfa monoculture, smooth bromegrass monoculture, and alfalfa and smooth bromegrass mixture.

is sufficient to meet the N needs of legumes if the symbiosis is functioning properly. In most agricultural soils, soil-available N is sufficient for early growth of legumes, prior to the establishment of an effective N2 fixation system. However, some research has shown that alfalfa needs supplemental N when it is grown on extremely low N soils (e.g., saline-alkali

and sandy soils with NO3– levels below 15 mg kg–1 or organic matter below 15 g kg–1; Hannaway and Shuler 1993), or in conditions that limit the symbiosis (e.g., low temperature; Hannaway and Shuler 1993; Li and He 2013). Our results support the concept that there is little likelihood for improvement in alfalfa biomass yield or quality with application of N

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Table 2 N source and N partitioning in alfalfa and smooth bromegrass plants grown as a monoculture and a mixture1) Species Shoots Monoculture smooth bromegrass Mixture smooth bromegrass Mixture alfalfa

Monoculture alfalfa

Roots Monoculture smooth bromegrass Mixture smooth bromegrass Mixture alfalfa

Monoculture alfalfa

1st harvest %Ndfa (%Ntrans)

N fertilizer

%Ndff

N0 N75 N150

0.86 29.97 45.78

– –

N0 N75 N150 N0 N75

1.15 29.61 46.88 0.91 24.31

N150 N0 N75 N150 N0 N75 N150 N0 N75 N150 N0 N75 N150 N0

2nd harvest %Ndfa %Ndfs (%Ntrans)

3rd harvest %Ndfa %Ndff %Ndfs (%Ntrans)

%Ndfs

%Ndff 0.89 20.52 32.96

– –

–

99.14 70.03 54.22

(16.63) (1.21) (0.00) 22.17 17.90

82.22 69.18 53.12 76.92 57.79

0.63 19.31 32.76 0.24 3.08

39.25

16.28

44.47

11.42

65.12

23.46

5.63

69.80

24.57

0.89 32.28 47.01

18.59 0.00 0.00

80.52 67.72 52.99

0.22 9.36 27.98

75.67 54.41 15.08

24.11 36.23 56.94

0.11 5.55 14.00

68.44 58.40 22.59

31.45 36.05 63.41

N75

0.56 23.60 33.17 0.76 24.98 38.33 2.60 17.99 32.54 1.41 27.44

– – – (16.96) (0.00) (0.52) 29.04 23.32 14.18 29.92 0.93

99.44 76.40 66.83 82.28 75.02 61.15 68.36 58.70 53.28 68.67 71.63

0.60 22.73 35.63 0.49 20.77 35.45 0.12 8.74 17.11 0.18 10.72

– – – (20.01) (8.63) (0.00) 75.43 57.93 42.17 70.90 52.83

99.40 77.27 64.37 79.50 70.60 64.55 24.45 33.33 40.72 28.92 36.45

0.44 12.57 26.14 0.36 10.80 26.76 0.07 5.50 15.42 0.12 7.21

– – – (16.28) (14.06) (0.00) 73.06 83.22 68.47 68.76 55.62

99.56 87.43 73.86 83.36 75.14 73.24 26.87 11.28 16.11 31.12 37.17

N150

40.11

1.54

58.35

26.23

26.39

47.38

16.79

22.07

61.14

1)

0.34 13.32 18.08

– –

–

99.11 79.48 67.04

–

99.66 86.68 81.92

(29.09) (5.89) (0.60) 75.60 84.07

70.28 74.80 66.64 24.16 12.85

0.54 10.38 18.66 0.08 1.76

(29.93) (10.77) (0.00) 69.30 83.05

69.53 78.85 81.34 30.62 15.19

1)

%Ndff is the proportion of N from nitrogen fertilizer; %Ndfa (%Ntrans) is the percentage of atmospheric N 2 fixation of total N in alfalfa (% N transferred from alfalfa in total N of smooth bromegrass); %Ndfs is the proportion of N from the soil. –, no data.

fertilizer to typical field-grown alfalfa monocultures. In the alfalfa-smooth bromegrass mixture used in this experiment, the biomass of individual alfalfa plants was decreased significantly. This may have resulted from the significant increase in biomass of individual smooth bromegrass plants, thereby causing a reduction in the space available for the growth of alfalfa, especially in the below-ground space provided by the pots used. Competition is only within species in monocultures; however, intra- and inter-specific competition simultaneously exist in mixtures (Hector et al. 1999, 2002). Within a defined growth space, competitive advantage is focused primarily within the root systems, for soil nutrients and water. In this experiment, competition for water can be excluded, since the plants were regularly watered and the volumetric water content of the substrate soil did not fall below 15% during the day. Application of N fertilizer to the mixture only increased the biomass of smooth bromegrass, and not alfalfa. We postulate that the N fertilizer promoted the competitive ability of smooth bromegrass in the mixture, in which it occupied the growth space of alfalfa, as reported in other studies (Yolcu

et al. 2010). In addition, a portion of the increase in biomass of individual smooth bromegrass plants was derived from the N transfer from N2 fixation, further increasing the competitive advantage of smooth bromegrass. Our results support the conclusion that in monocultures, smooth bromegrass responds in a linear fashion to applied N for biomass and N content of shoots and roots whereas alfalfa shows no benefit to fertilizer N when there is a properly functioning symbiosis. In the mixture, application of fertilizer N gives a competitive advantage to the grass, in which it effectively absorbs the applied N through its fibrous root system and also benefits from transferred N from the legume symbiotic N2 fixation.

3.2. N2 fixation response to N application Fertilizer N application typically reduces alfalfa’s N2 fixation (Lamb et al. 1995). In this study, the %Ndfa of alfalfa grown alone was reduced with N application (R2=–0.9376, P=0.0057). This is consistent with the results of HøghJensen and Schjoerring (1994, 1997). We believe that one

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of the most important findings of our study is the relationship between the timing of the initiation of BNF and soil-available N. For alfalfa grown alone, N2 fixation began earlier in the N0 treatment than in the N added treatments (N75 and N150). By the time of the 1st cutting, N0 alfalfa plants had begun N2 fixation, whereas N75 and N150 treatment alfalfa plants had not (Table 2). Thus, the addition of fertilizer N delayed the initiation of N2 fixation by alfalfa grown alone. As anticipated, the rate of N2 fixation by alfalfa in response to N fertilizer application was different when it was grown in the mixture with smooth bromegrass than when it was grown alone. For the mixture, N application did not influence the initiation timing of N2 fixation (Table 2). We believe this is because smooth bromegrass has a greater competitive ability for soil N than alfalfa (Høgh-Jensen and Schjoerring 1997), reducing the nitrate level surrounding alfalfa nodules. This conclusion is supported by the work of Ryle et al. (1979), Phillips (1980), and Thornley and Cannell (2000), who reported that legumes will preferentially absorb the soil-available N, due to its lower plant energy cost. When soil available N is reduced, legumes will utilize atmospheric N2 to supply their N needs. In the initial growth period, fertilizer N preferentially promoted smooth bromegrass, leading to a competition for available resources (nutrients and space), thus, restricting the N2 fixation and growth of alfalfa (Nesheim and Boller 1991; Woledge et al. 1992; Vitousek et al. 2002; Carlsson et al. 2009; Schipanski and Drinkwater 2012). The %Ndfa of alfalfa in mixtures was greatest in the N75 treatment (Table 2) where it accounted for over 80% of total plant N. We believe this was due to a reduction in soil-available N at the nodule surface resulting from grass root absorption (Schipanski and Drinkwater 2012). Over the course of the three harvests, N uptake reduced soil-available N, reducing the competitive advantage of smooth bromegrass (Ledgard and Steele 1992). Thus, in the later harvests, alfalfa dominated the mixture, and N2 fixation increased. These results support our research hypothesis that in mixtures, due to the preferential absorption of N by smooth bromegrass (Høgh-Jensen and Schjoerring 1997), the reduction in N2 fixation caused by fertilizer N would be less, resulting in greater N2 fixation by alfalfa in mixtures than when grown alone (Carlsson and Huss-Danell 2003; Corre-Hellou et al. 2006). This was confirmed by the higher %Ndfa for the mixture, and the earlier initiation of N2 fixation in the mixture.

3.3. N transfer from alfalfa to smooth bromegrass When N application increased from 0 to 150 kg ha–1, the %Ndff for smooth bromegrass increased and the %trans to smooth bromegrass significantly decreased. We believe

this was due to the reduction in the %Ndfa and the smooth bromegrass preferential absorption of N from fertilizer (Table 2). Thus, the %trans of fixed N to smooth bromegrass depends on the soil-available N content; when soil-available N content is high, smooth bromegrass absorbs soil N and fertilizer N until soil-available N content is sufficiently low to allow for N2 fixation by alfalfa in the mixture, some of which is made available to the soil, and is either reabsorbed by the alfalfa or by the companion plants, in this case the smooth bromegrass (Høgh-Jensen 2006; Rasmussen et al. 2007; Paynel et al. 2008).

4. Conclusion N2 fixation by alfalfa in response to N fertilizer was different between plants grown alone and in mixture. In the monoculture, the proportion of N2 fixation by alfalfa was decreased with N application and N2 fixation initiation was delayed. In the alfalfa-smooth bromegrass mixture, smooth bromegrass was a stronger competitor for soil N. This influenced alfalfa’s N2 fixation, as shown by increased growth and N content of smooth bromegrass and reduced growth of the alfalfa. Compared with alfalfa grown alone, in the mixture the proportion of N2 fixation by alfalfa was higher and the time of initiation of N2 fixation was earlier. Thus, to promote economically and environmentally sustainable forage production systems, application of fertilizer N should be limited to the amount that will result in optimal growth of both grass and legume components of mixtures. For alfalfa-smooth bromegrass mixtures, it appears to be 75 kg ha–1. For legumes grown alone, efforts should be focused on establishing an effective N2 fixing symbiosis, not on N fertilization. Increased use of legume-grass mixtures and moderate levels of N fertilization will increase yield and quality of grassland forage, reduce environmental pollution from excessive N application, and increase profitability of forage-livestock systems.

5. Materials and methods Seeds of smooth bromegrass (Bromus inermis Leyss.) and alfalfa (M. sativa L. cv. Sanditi) were obtained from the germplasm repository of the Institute of Animal Sciences, Chinese Academy of Agricultural Sciences. Seeds of smooth bromegrass had been collected from Xinyuan County, Xinjiang Uygur Autonomous Region of China and seeds of alfalfa had been obtained from Barenbrug (Tianjin) International Co., Ltd., China. On November 10, 2012, seeds were placed in glass dishes (12 cm diameter) with wet filter paper and allowed to germinate in a growth chamber maintained at 23°C for the 16 h of light period and 20°C for 8 h of darkness. Relative

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Following transplanting of germinated seedlings, pots were placed in the greenhouse at the College of Resources and Environmental Sciences, China Agricultural University. A soil moisture meter (WET Sensor HH2) was used to measure water content 8 cm beneath the soil surface in pots every 3 days. When the average value was below 15%, all treatments received 1 500 mL of tap water. Weeds were controlled by hand and insect pests were controlled by yellow sticky cards. No pesticides were used.

humidity was maintained at 60%. After 7 days, seedlings of the same approximate size and appearance were selected and transplanted into plastic pots (29 cm diameter and 35 cm height) which had been filled with 22 kg of airdried and sieved fluvo-aquic soil collected from a depth of 0–35 cm from a field in a Beijing suburb previously planted to corn. The physicochemical properties of the soil are presented in Table 3. Available nitrogen was 57.25 mg kg–1.

5.1. Experimental design

5.2. Harvesting and dry weight determination

Experimental treatments included two fixed factors, species and N level. The 3 species were: (1) an alfalfa monoculture, (2) a smooth bromegrass monoculture, and (3) an alfalfa-smooth bromegrass mixture, designated as AL, SB, and AL+SB. Three N levels were evaluated: 0, 75, and 150 kg N ha–1 (0, 1.07, and 2.14 g of N added per pot), designated as N0, N75, and N150. Thus, there were 9 treatments (3 species×3 N levels) (Fig. 6). Treatments were arranged in a randomized complete block design, with 9 replications. Spacing between blocks was 50 cm to allow for convenient watering. Shoots of all pots were harvested when alfalfa was in the 10% bloom stage. For the 1st harvest, smooth bromegrass was in the early (1/3) boot stage. For the 2nd and 3rd harvests, smooth bromegrass was in the late boot stage. Nutrient supplements were applied based on the soil analysis results shown in Table 1. P and K fertilizers were 75 kg P2O5 ha–1 (3.52 g per pot of phosphate fertilizer supplied as calcium superphosphate, 14% P2O5) and 90 kg K2O ha–1 (1.21 g per pot of potassium fertilizer supplied as potassium sulphate, 49% K2O). These were mixed with the soil before transplanting germinated seedlings. Micronutrient fertilizers included borax (2.5 kg B ha–1), zinc sulphate (1 kg Zn ha–1) and ammonium molybdate (0.2 kg Mo ha–1). These were prepared in solution and sprinkled evenly on the soil and mixed thoroughly before transplanting. N fertilizer was 15 N-labeled urea (CO(15NH2)2, 10% abundance, purchased from the Shanghai Research Institute of Chemical Industry, China). The amount of 15N-labeled urea required for each pot was weighed out on an analytical balance (with an accuracy of 0.0001 g), dissolved in 100 mL of deionized water, and sprinkled evenly on the soil surface of each pot. To reduce N volatilization, immediately following application, the surface of each pot was covered with a black plastic sheet. Transplanting was done 1 week after pots were prepared.

At each sampling date, 3 replicates were destructively sampled for determining root biomass. Thus, for the 1st sampling date, there were 9 replicates for shoots, for the 2nd sampling date, there were 6, and for the 3rd date, there were 3 replicates remaining. Roots were separated from the soil by gently washing in tap water. Live and dead roots were not separated. For the alfalfa-smooth bromegrass mixture treatment pots, roots were separated by species. Shoots and roots were dried at 65°C for 48 h and weighed on a balance with an accuracy of 0.01 g.

5.3. N2 fixation determination The 15N isotope dilution method was chosen due to its greater accuracy of in situ measurement of N2 fixation by legumes (Chalk 1991; Carranca et al. 1999). Prior to determination of 15N abundance and total N, the dried shoots and roots samples were ground to a fine powder using a rotary mill (MM200, Retsch, Haan, Germany). Analyses were performed using a 15N isotope ratio mass spectrometer (Mat 253, Finnigan MAT, Bremen, Germany).

5.4. Equations used in N2 fixation data analysis (1) Atom percent excess (15N atom percent excess, APE) of the sample was calculated by the following equation: APE=A–Ao (1) Where, APE is the 15N atom percent excess of the sample, A is 15N abundance of the sample, and Ao=0.365%, the natural abundance of 15N. (2) The percentage of atmospheric N2 fixation to total N in alfalfa (%Ndfa) was calculated by the following equation (McNeill et al. 1994):

Table 3 The physicochemical properties of the soil used in greenhouse experiments Organic matter (g kg–1) 9.18

pH 7.68

Available nitrogen (mg kg–1) 57.25

Nitrate nitrogen (mg kg–1) 24.89

Ammonium nitrogen (mg kg–1) 5.04

Total P (g kg–1)

Available P (mg kg–1)

Total K (g kg–1)

Available K (mg kg–1)

0.69

11.70

18.96

113.32

Field Bulk density capacity –3 (g cm ) (%) 1.28 26.04

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%NdfF ) × 100 (2) %NdfNF Where, %NdfF is the 15N atom percent excess in N2-fixing plants and %NdfNF is the 15N atom percent excess in non-N2-fixing plants. (3) The percentage of N transferred from N2-fixing plants to non-N2-fixing plants in total N (%Ntrans) was calculated by the following equation (Vallis et al.1977): %Ndfa=(1−

%NdfNFmix (3) ) ×100 %NdfNFmono Where, %NdfNFmix is the 15N atom percent excess in non-N2-fixing plants in mixtures and %NdfNFmono is the 15N atom percent excess in non-N2-fixing plants in monocultures. (4) N proportion from fertilizer was calculated as: %Ntrans=(1−

APE ×100%(Af, 15 N atom percent excess in Af fertilizer). (5) N proportion from soil was calculated as: %Ndfs=1–%Ndff–%Ndfa(%Ntrans)

%Ndff=

5.5. Statistical analysis Shoot and root biomass were compared using a mixed model analysis of variance (PROC MIXED) using the Statistical Analysis System (SAS ver. 9.1, SAS Institute, Cary, NC) (Table 2). Effects of species and N level within each of the 3 harvests were analysed by using species, N level, and species×N level interaction as sources of variance. Blocks were considered a random factor. Means were separated by using the least significant difference (LSD) test. Differences of biomass of alfalfa and smooth bromegrass and N2 fixation ability between the monocultures and the mixture was compared using a t-test. Results from the statistical analyses of all data were graphed using Microsoft Excel and GraphPad Prism 6.0 software.

Acknowledgements We would like to thank Profs. Jennifer Kling and Alix Gitelman (Oregon State University, USA), and Prof. Yu X H (Chinese Academy of Agricultural Sciences, China) for their helpful suggestions on data analysis and the three anonymous reviewers for their helpful comments to improve our manuscript. This work was supported by the China Forage and Grass Research System (CARS-35) and the National Key Technology R&D Program of China (2011BAD17B01).

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