Soil Sci. Plant Nutr., 61 (7), 973-979.2005 973

Characterization of Nitrogen Utilization by Brachiaria Grasses in Brazilian Savannas (Cerrados)

Takuji Nakamura', Cesar H.B. Miranda", Yoshinari Ohwaki**, JosC R. ValCio*, Yeonghoo Kim, and Manuel C.M. Macedo"

National Institute of Crop Science (NICS), Tsukuba, Ibaraki, 305-8518 Japan; *National Beef Cattle Research Center; Brazilian Agricultural Research Corporation (EMBRAPA-CNPGC), BR 262 krn 4 C.P.154 CEP:79002-970 Carnpo

Grande, M S , Brazil; and **National Agricultural Research Center (NARC), Tsukuba, Ibaraki, 305-8666 Japan

Received February 2 1, 2005; accepted in revised form September 30, 2005

Brachiaria species are widely cultivated and the low nitrogen (N) fertility of soils is one of the limiting factors for grass production in the Brazilian savannas (Cerrados). In the present study, we compared the characteristics of N utilization in Brachiaria brizantha (BB), B. decumbens (BD), and B. humidicola (BH), which are common species in these areas. In a soil culture experiment, BB, BD, and BH were grown under 3 levels of N, equiva- lent to 0 application (ON), 0.1 (lN), and 0.3 (2N) g N pot-', with N as ammonium sulfate. The plant dry weight of BB and BD increased with the increase of N levels, whereas BH showed a weaker growth response to N application and the plant dry weight of BH was lower than that of BB and BD. The amount of N accumulated in plant increased with the increase of N levels in BB and BD. The amount of N accumulated in plant in BH was slightly lower than or almost comparable to that in BB and BD. The plant N concentration was higher in BH than in BB and BD. The stronger growth response of BB and BD resulted in a higher fertilizer use efliciency (FUE) and a N use efficiency in fertilizer application (NUEF). The N absorption efficiency (NAE) of BH was higher than that of the other two grasses for 1N and 2N. In the absence of N treatment (hereafter refferred to as "ON treat- ment"), the specific N absorption rate in the roots (SAR) was higher in BH than in BB and BD throughout the growth period. These data suggested that the N absorption ability in roots would be higher in BH than in the other 2 tested grasses, as confirmed by the Michae- lis-Menten kinetic analysis. In a nitrate absorption experiment, the K,,, value of BH was lower than that of BB and BD. As BH showed a higher affinity for nitrate absorption, it could maintain a higher N absorption rate. Therefore, it was considered that the perfop mance of N accumulation of BH would be slightly lower than or almost comparable to that of BB and BD because of the high ability of BH to absorb N, although dry matter production of BH was lower than that of BB and BD.

Key Wonls: Brachiaria grasses, kinetic analysis, N absorption, nitrogen utilization.

Brachiaria species were introduced from Africa in the 1950s and 1960s as pasture grasses and are presently widely distributed throughout the South American savannas, covering approximately 70 million ha (Pizzaro et al. 1996). In the Brazilian savannas (Cerrados), over 40 million ha are now used for pasture and 12 million for annual crops, resulting in a rapid expansion of cattle production (Spain and Ayarza 1992). The Brachiaria species account for 85% of the total planted pasture area in the Cerrados (Macedo 1995), with around 10,OOO

I Corresponding author. Tel & Fax: +81-29-838-8392. E-mail: takuwan@naro.affrc.go.jp

t year-' of Brachiaria seeds being currently produced commercially in Brazil. Thus, Brachiaria grasses are one of the major commercial crops (Fisher and Kerridge 1 996).

The three most common species cultivated in the region are Brachiaria decumbens (BD), 6. brizantha (BB), and B. humidicola (BH). BD is the most widely cultivated species because it is able to adapt to soils with low and medium fertility. BB, introduced recently into the Cerrado area, displays a higher productivity, but its nutrient requirement is high. Although BH can with- stand dry and wet stress and survive under low nutrient conditions, its productivity is lower than that of the oth-

974 T. NAKAMURA et al.

er two species (Pizzaro et al. 1996). Low nutrient status, especially nitrogen (N), is the

main limiting factor for grass productivity of the Brazil- ian Cerrados (Myers and Robbins 1991; Spain and Gualdron 1991; Rao et al. 1996). Decrease of grass pro- ductivity also occurs because of pasture degradation associated with decreased soil nutrient availability when grasses are cultivating over a long period of time (years) (Myers and Robbins 1991; Boddey et al. 1996; Rao et al. 1996). As farms are large and the cost of fertilizers is high, pasture improvement by fertilization is limited. On the other hand, agropastoral systems (crop-pasture rota- tion systems) using small amounts of fertilizers for crops with rapid money returning such as soybean, have recently been considered for the development of sustain- able farming systems with high productivity in environ- mentally degraded areas of the Cerrados (Macedo et al. 2001). The residual effect of fertilizer applied to preced- ing crops has contributed significantly to the promotion of agropastoral systems, in relation to forage production. The residual fertilizer used for the preceding crops can decrease the application levels necessary for the devel- opment and maintenace of subsequently grown pastures (Kanno et al. 2002). In these systems, the investigations on interspecific differences in the adaptive ability of a given pasture species are important for pasture manage- ment and genetic improvement. Therefore, studies on the nutrient utilization of Brachiaria grasses, especially N utilization, are essential to introduce high-quality for- age and to maintain sustainable production. Thus, it is necessary to identify the adaptability of Brachiaria grasses to a broad range of soil-N and applied-N levels.

It has been reported that BH inhibits nitrification (Sylverster-Bradley et al. 1988; Ishikawa et al. 2003), suggesting that it is possible to achieve a higher efficien- cy of soil N utilization of this grass, which may induce low nitrate leaching and denitrification into its surround- ings. These results indicate that Brachiaria grasses dis- play a wide diversity in their strategy for adaptation to soils with low fertility. In the present study, we com- pared the characteristics of N utilization among BB, BD and BH grown under different N conditions.

MATERIALS AND METHODS

Experiment 1: Soil culture experiment. 1) Plant materials and soil culture method:

The experiment was conducted during 2001 in a green- house located at the National Beef Cattle Research Cen- ter, Brazilian Agricultural Research Corporation (EMBRAPA-CNPGC) in Camp0 Grande-MS, Brazil. The mean temperature of the greenhouse during the experiment was 26.0"C. Pots were filled with 2.5 kg of

air-dry Oxisol (dark red Latosol: 3842% Clay) col- lected from a 0-20 cm depth in a native savanna area. This soil contained 3.55 mg kg-' NH,-N, 7.53 mg kg-' NO,-N, 1.00 g kg-' total N and 15.3 g kg-' total C. Limestone (3 t ha-') was applied to adjust the soil to pH 6.0, together with the addition of phosphorus and potas- sium (0.7 g P,O, pot-' and 0.7 g K,O pot-' as Na,HPO,, K,SO,, respectively). Fritted trace elements ( R E ) (0.16 g pot-') were also applied to the soil. Nitro- gen was added, as (NH,),SO,, at 3 levels, 0 addition (ON), 0.1 (1N) and 0.3 g N pot-' (2N).

The seeds were sown in plastic plates filled with soil on September 27, 2001 in the case of B. humidicolu cv. CIAT679 (BH), and September 30, 2001 in the case of Brachiaria brizantha cv. Marandu (BB) and B . decum- hens cv. Basilisk (BD), respectively. Seedlings were ger- minated on October 6, 2001. One seedling each of BB, BD and BH was transplanted to each pot on October 13, 2001. The experimental layout considered of a com- pletely randomized block design with 3 replications. Plants were collected at 70, 91 and 112 d after germina- tion. Plant samples were separated into leaves, stems and roots. The dry weight of each part was determined after oven drying at 80°C for 48 h. After the measure- ment of the dry weight, the samples were ground and a portion was used for N analysis. The N content in the samples was determined by the Kjeldahl method.

Fertilizer use efficiency (FUE), N fertilizer-absorption efficiency (NFAE), N use efficiency for the plants under fertilizer application (NUEF) and N absorption effi- ciency (NAE) were calculated from the data collected at harvest, based on the equations described by Kanno et al. (1999):

2) Analysis and calculation:

FUE = ( WN, - WN,)/FN, NFAE=(TN,-TN,)/FN, NUEF=(WN,- WN,)/(TN,-TN,) NAE = (TN, - TN,)/(RW,, - RW,)

where WN,, is the total dry weight of plants for N levels 1N or 2N levels, WN, is the total dry weight in the absence of N treatment (hereafter referred to as "ON treatment"). TN, is the total N amount of plants for 1N or 2N levels, TN, is the total N amount in the ON treat- ment, FN,, is the amount of N applied for 1N or 2N lev- els, RW,, is the dry weight of roots of plants for 1N or 2N levels, and RW, is the dry weight of roots in the ON treatment (n= 1 or 2).

The model of Hirose (1986) was modified, and the specific absorption rate of N per unit root mass (SAR) was calculated. The rate of N absorption (AR) was given by the equation:

AR = dhJ/dt

N Utilization by Brachiaria Grasses in Cerrados 975

where N is the amount of plant total nitrogen and t is the number of days after germination. SAR was calculated as AR divided by the dry weight of roots (Nakamura et al. 2002).

Experiment 2: Root kinetic absorption expep iment.

1) BB, BD, and BH were sown in plastic trays filled with the same soil as that used in Experiment 1, under the same greenhouse conditions. The mean temperature in the greenhouse was 28°C during the experimental period. At 2 d after germination, the seedlings were transferred to a N-free nutrient solution in 12 L plastic containers, as a modification of the method proposed by Nakamura et al. (1997b) and grown for a period of 2 weeks. The composition of the nutrient solution was as follows: P (NaH,PO,), 5 mg L-I; K (K2S04 : KC1= 1 : l), 30 mg L-I; Ca (CaCl,), 50 mg L-I; Mg (MgSO,), 20 mg L-I; and trace elements including Fe, Mn, B, Zn, Cu, and Mo, 2, 0.5, 0.5, 0.2, 0.01, and 0.005mg L-I, respectively (Nakamura et al. 1997b). The pH was adjusted to 5.3 by the addition of 0.1 mol L-l sodium hydroxide twice a day and the solution was aerated con- tinuously. In order to activate the nitrate or ammonium transporters of roots, seedlings were transferred to the nutrient solution containing 80 pmol L-' NH,-N as (NH,),SO, or NO,-N as NaNO,, respectively.

Ammonium and nitrate absorption studies: Two weeks after germination, absorption studies were conducted in a growth chamber at 28°C and 70% RH. The photosynthetic photon flux density in the chamber was below 200 pmol rn-' s-I. Seedlings were kept in a 0.2 mmol L-I CaCl, solution for 1 h with aeration to remove any excess N in the roots. Ten seedlings (twenty seedlings in the case of BH) were transferred to 200 mL Erlenmeyer flasks containing 150 mL of absorption solution, with three replications. The values of plant dry weight of BB, BD and BH were 58.1, 47.1 and 29.1 mg plant-' respectively. This solution which contained 80 pmol N L-' as NaNO, or as (NH,),SO, with 0.2 mmol L-' CaCl,, was continuously aerated. At 30 min inter- vals, 4 mL of the solution was collected and deep-fro- zen for subsequent nitrate and ammonium analyses. Net absorption of nitrate and ammonium was determined by measuring the nutrient depletion in the solution cor- rected for the loss of volume due to sampling, transpira- tion and evaporation. Nitrate and ammonium concentrations in the solutions collected were deter- mined by ion chromatography (Ion Analyzer PIA-1100, Shimadzu Co., Japan). At the end of the absorption experiment, the seedlings were washed to remove the solution, and separated into roots and shoots. Fresh and dry (80°C) weight was determined. Root length was measured by the line intercept method (Kanno et al.

Plant materials and preculture method:

2)

1999; Nakamura et al. 2002). Root surface area (RSA) was calculated from the root volume, root fresh weight and root length, according to the method of Barber (1984).

Nutrient influx was assumed to follow the Michaelis- Menten kinetics, as described by Claasen and Bar- ber( 1974) and Barber (1984):

3) Calculations of kinetic parameters:

where v is the absorption rate, C is the concentration of either NO,-N or NH,-N in the solution, V,,, is the maxi- mum absorption rate, K, is the Michaelis constant, and Cmin is the minimum concentration of the solution below which net absorption is null. Ammonium and nitrate data were fitted to Eq. (1) and K,,,, V,,,,,, and Cmin were calculated using Sigma Plot software (SYSTAT Soft- ware, Inc., USA).

RESULTS

Experiment 1 The dry weight of all the species increased with the

increase of the N application level (Fig. 1). The dry weight of the plants for ON was 50% of the dry weight for 2N in BH, while that for ON was 33% and 34% in BB and BD, respectively. In particular, in BB and BD, the relative dry weight increased linearly with the increase of the N level, while in BH, it did not increase significantly from 1N to 2N and the dry weight of BH was lower than that of BB and BD.

N concentration of the plants was higher in BH than in BB and BD in all the N treatments (Fig. 1). The amount of N accumulated in plant increased with the increase of the N application level in BB and BD (Fig. 1). In BH, the amount of accumulated N increased from ON to 1N and did not increase from 1N to 2N. In the ON treatment, the amount of accumulated N was not signifi- cantly different among the 3 tested grasses, although the amount of N accumulated in the BH plants tended to be higher than that in BB and BD. The amount of N accu- mulated in BB and BH plants was higher than that in BD for 1N. For 2N, there were no significant differences between BD and BH. Thus, the amount of N accumulat- ed in the BH plants was slightly lower than or almost the same as that in BB and BD. FUE in the 1N treatment was higher in BB than in BD

and BH (Fig. 2). At 2N, FUE in BH was the lowest among all the grasses examined. NFAE of BH was high- er than that of BB and BD in the 1N treatment, and the species difference in NFAE was not significant in the 2N treatment. NUEF of BH was lower than that of BB and BD, while the NAE of BH was higher in both N treat-

976 T. NAKAMURA et al.

'lhble 1. Kinetic constants of NH,+ and NO,- uptake in B . brizunthu (BB), B. decurnbens (BD), and B . humidicolu (BH).

5 % h'

c

z

Fig. 1. Dry weight, N concentration and amount of N accu- mulated in plants at different levels of N application at the end of the experiment. The vertical bars indicate +SE. Bars with the same letter indicate the absence of significance (5% level). Val- ues in parentheses denote the relative dry weight compared to the 2N treatment.

ments. All the grasses tested showed a decrease in SAR with the growth period, while BH exhibited a constantly higher SAR than BB and BD in the ON treatment (Fig. 3).

Experiment 2 Figure 4 depicts the depletion of ammonium and

nitrate in the solution during the absorption experiment. A lag phase of 60 min was observed for all the plants in

NH4+ NO,- BB BD BH BB BD BH

V,,, 117.8a 106.6a 146.2b 152.4a 146.4a 160.6a Kln 25.9a 33.2a 36.7a 11.2b 7.5b 4.4a L" 7.0a 13.4b 14.0b 22.4a 28.h 36.5a

K,,, and C,,, are expressed in pmol L-'; V,,, in kmol h-' m-' RSA. Values with the same letter in the line and N-form do not differ (5% level).

the ammonium solution, and the concentration of ammonium subsequently decreased rapidly until it became constant. In the nitrate solution, a lag phase of 240 min was observed for BB and BD, and only 180 min for BH, after which the nitrate concentration decreased until it reached a constant value.

Based on these results, the kinetic parameters were estimated (Table 1). In the case of ammonium absorp- tion, BH showed a significantly higher V,, value than BB and BD. Its C,, value was also higher (14 pmol L-'), but was not significantly different from that of BD (13 pmol L-I). All the species showed similar K,,, val- ues. There were no significant differences in the V,,, and Cmi, values for nitrate absorption among the species tested, although BH showed a significantly lower K, value (4 pmol L-I) than BB and BD.

DISCUSSION

Growth and N utilization. Plant growth in BB and BD responded more strongly to N application than that in BH (Fig. l) , with an increase of their dry weight at both N levels, while plant growth in BH responded only from ON to 1N. These data suggested that BB and BD displayed a higher productivity under high N condi- tions, while BH productivity was lower one than that of BB and BD.

FUE and NUEF of BB and BD were higher than those of BH in both N treatments (Fig. 2), which reflect- ed the stronger response to N application. Information about the response of Brachiaria grasses to N applica- tion is limited. Alvim et al. (1990) reported that, in field experiments, FUE of BB was higher than that of BD and BH, and the growth response to N application was in the order of BB>BD>BH. The results we obtained for FUE were slightly different, which could be ascribed to the differences in the growth period and conditions. However, it was confirmed that BB and BD strongly responded to N application and that BH displayed only a limited response.

A close relationship was reported between the amount of N absorbed and plant dry weight, with plant growth

N Utilization by Brachiaria Grasses in Cerrados

W p 0.1

977

50 d

5 40 b, - 2 30

20

LL 10

0

W 3

00 BD BH

2oo] b b b b

4 - 150 b,

100 0)

LL Y

$ 5 0 z 0

00 0D BH

0.4 c T

B0 0D

0.10 ~7

BH

- 0.08 I 0.06

b, z 0.04 0) - 2 0.02 z

0.00

500 1 I

BB

60 70 80 90 100 110 120

Days after germination

Fig. 3. (SAR) in the ON treatment. The vertical bars indicate 2SE.

Specific absorption rate of N per root dry weight

being substantially regulated by the amount of N absorbed in plants (Osaki et al. 1992; Nakamura et al. 1997a, 2002). BH showed higher NFAE and NAE, sug- gesting a higher nitrogen absorption ability. In the ON treatment, SAR of BH was higher than that of BD and BB during the growth (Fig. 3). Thus, the amount of N accumulated in plant in BH was slightly lower than or almost comparable to that of BB and BD, although the dry weight value was lower in BH than in BB and BD (Fig. 1). The results obtained in BH could lead to a higher N concentration in plant as well as lower FUE and NUEF (Figs. 1 and 2).

Kinetic analysis. Results from the soil culture experiment suggested that these grasses displayed dif- ferences in the N-absorbing ability from soil. In the Cer- rado soils, the availability of ammonium and nitrate is

BD 0H

Fig. 2. Fertilizer use efficiency (WE), N fertilizer absorption efficiency (NFAE), N use efficiency in fertilizer application (NUEF), and N absorption efficiency (NAE) at the end of the experiment. The vertical bars indicate +SE. Values with the same letter do not dif- - I N

0 2N fer (5% level).

the main limiting factor for the growth of grasses. Thus, further investigations will be conducted using the Michaelis-Menten kinetic analysis, because the charac- terization of N uptake in Bruchiuriu species should pro- vide useful information about the low N availability in the Cerrados.

Ammonium and nitrate uptake rates with the solute concentration were calculated based on Fig. 4. Kinetic parameters were computed with these data, to solve the Michaelis-Menten kinetic equation (Eq. ( 1)). There were no significant differences among the three species in the V,,, and C,, values for nitrate, while the V,,, value for ammonium in BH was higher than that in BB and BD (Table 1). Rao et al. (1996 and 2001) showed that, when ammonium was supplied, the growth of BD and B. dic- fyoneuru was inhibited at higher levels, whereas BH showed an increased production, particularly at higher ammonium levels. It was reported that V,,, is related to plant growth and nutrient absorption (Laine et al. 1993; Abbes et al. 1995; Teo et al. 1995; Swiader and Freiji 1996). In rice, since the differences in the total N absorption among cultivars appeared to be related to the V,,, value of the cultivars (Teo et al. 1992, 1995), rice varieties with a high N absorption ability showed a high V,, value for N. Thus, the superior performance of BH under ammonium-N conditions, which was revealed shown in the results of Rao et al. (1996, 2001), may be due to the high V,,, value.

The range of the K, values was 25-37 pmol L-' for ammonium and 4-11 pmol L-' for nitrate, and the K, value for nitrate in BH was lower than that in BB and

978

+ BB u BB

T. NAKAMURA et al.

0 1 Y

o l , 1 , 1 1 I 0 100 200 300 400 500 600 0 100

Time (min)

BD (Table 1). The range of the K, values for ammonium obtained in the present study was similar to that reported for other crops (15-28 pmol L-' for barley-Bloom 1985; 14 pmol L-' for onions-Abbes et al. 1995; and 50pmol L-' for wheat-Goyal and Huffaker 1986), whereas the range of the K, values for nitrate was slightly lower (34-36 pmol L-' for barley-Aslam et al. 1992; 5.1 pmol L-' for Trifolium incarnatum; 36.4 pmol L-' for Lolium perenne-Laine et al. 1993; 12 pmol L-' for onions-Abbes et al. 1995; 27 pmol L-' for wheat-Goyal and Huffaker 1986; and 9.3 pmol L-' for lettuce-Swiader and Freiji 1996). As the nitrate concentration in upland soil is higher than the ammoni- um concentration, nitrate absorption is more important under N-limiting conditions. Since the K,,, value of BH was significantly lower than that of the other two tested species, and also of other crops, indicating a high affini- ty for nitrate, the ability of N absorption in BH was higher under low N conditions. However, the lower effective utilization of BH could not result in higher dry matter production than that of BD and BB. Assuming that fertilizer might be applied in agropastoral systems, the introduction of BD and BB to the field might be suit- able because of the strong response of production to N application. And the inhibition ability of nitrification in BH, as reported by Sylverster-Bradley et al. (1988) and Ishikawa et al. (2003), may become important because the levels of fertilizer applied could lead to nitrate leach- ing in agropastoral systems. Thus, it is necessary to determine the practical effectiveness of BH cultivation for the prevention of nitrate leaching in agropastoral systems in the Cerrados. In the present report, we exam- ined the early growth stage of the Brachiaria grasses and further investigations should be conducted to deter- mine the dry matter production and N utilization of these grasses over a long period of time, including sec- ond, third and fourth harvests.

Acknowledgments. This study was conducted as a part of international collaboration between the Japan International

I 200 300 400 500

Fig. 4. Depletion of NH,+-N and NO,--N in the nutrient solution by seedlings of Brachiaria species in relation to the absorption time. Plant dry weight values in BB, BD and BH were 58.1, 47.1 and 29.1 mg plant-', respectively.

Research Center for Agricultural Sciences (JIRCAS) and the National Beef Cattle Research Center, Brazilian Agricultural Research Corporation (EMBRAPA-CNPGC), titled "Compre- hensive studies on the development of sustainable agropastoral systems in the sub-tropical zone of Brazil." We would like to thank Dr. T. Kanno, Agriculture, Forestry and Fisheries Research Council, Japan (AFFRC), and Prof. T. Tadano, Tokyo University of Agriculture, for their critical and invaluable comments on the manuscript. We thank Mr. M. M. Borges, Mr. G. M. Pitaluga, and Ms. I. M. Naka (EMBRAPA-CNPGC) for their assistance in the greenhouse and laboratory work.

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