Applications of mineral nitrogen increase the yield and content of crude protein in narrow-leaf lupin seeds

Abstract

In a plant pot experiment with non-inoculated seed stock of narrow-leaf lupin (Lupinus angustifolius, L.) of the variety ‘Prima’ we explored the effects of nitrogen compounds applied in the form of NH₄NO₃ on different dates (before sowing, after emergence and during flowering) and at various levels (0–0.5–2–3 g N per pot) on changes in the number of pods per plant, the 1000-seed weight, seed yields and the content of crude protein in seeds. The experiment included four treatments; the first was a control treatment not fertilized with nitrogen. In the other treatments we applied nitrogen either as a single application before sowing or in three split applications. In all the nitrogen-fertilized variants the number of pods per plant, total seed yields per plant and the concentration of crude protein all increased compared to the unfertilized control variant. The highest levels of nitrogen (3 g N per pot) reduced the 1000-seed weight compared to the unfertilized control variant. The total application of 2 g N per pot split into application before sowing (0.5 g N), after emergence (0.5 g N) and during flowering (1 g N) resulted in a highly significant increase in the number of pods per plant (+78%), the 1000-seed weight (+20.1%) and therefore higher seed yields per plant (+139.8%) compared to the control treatment not fertilized with nitrogen. Increasing levels of nitrogen linearly increased the content of crude protein in seeds during harvest from 24.2% to 40.6%, thus increasing the nutritional quality of the seeds.

Keywords:

• Ammonium nitrate
• application
• date
• Lupinus angustifolius L

Introduction

Narrow-leaf lupin (Lupinus angustifolius, L.) is able to obtain a certain amount of nitrogen via symbiosis with nodule bacteria Bradyrhizobium lupinus living on their roots. The percentages of accumulated N derived from N₂ fixation for lupin are below 40% (Haynes et al., 1993). Symbiotic fixation of nitrogen utilizes the energy obtained by photosynthesis in plants to transform N₂ into NH₃ (Marschner, 1995). N₂ reduction is very rapid and it is assumed that it has three forms: diimid, hydrazine, and ammoniac (Mengel & Kirkby, 2001). According to Ma et al. (1997) lupin experiencing transient N deficiency had slower leaf emergence and more delayed flowering than plants with a continuous supply of nitrogen. Seed N concentrations were also decreased by transient N deficiencies at early (floral initiation), mid (flowering), and late (grain, filling) stages (Ma et al., 1998). According to Reddy et al. (1998), nitrogen in the soil advances initial growth and development in leguminous plants, until N₂ fixation begins. Scientific opinions on mineral nitrogen supplementation of lupin differ. The inhibitory effect of NO₃ ⁻ on nodulation and N₂ fixation are also well documented (Garcia-Plazaola et al., 1999; Gibson et al., 1977; Havelka et al., 1985). Ayisi et al. (1992a) discovered that seed yield of inoculated plants at zero N was always higher than that of well fertilized non-inoculated plants.

On the other hand, according to Pearson and Vitousek (2001) the addition of N need not necessarily reduce N₂ fixation. In addition to nitrogen, also optimal nutrition with other macro-biogenic (Gremigni et al., 2003; Brennan & Longnecker, 2001) and micro-biogenic (Pastor et al., 2002; Bakken et al., 2004; Peiter et al., 2000) elements helps to achieve high yields and good quality of legumes.

The objective of the plant pot trial with non-inoculated seeds of narrow-leaf lupin was to estimate the effect of nitrogen applied on various dates and at various levels on seed yields and content of crude protein.

Material and methods

The plant pot trial with narrow-leaf lupin, variety Prima, was established in the spring of 2005 in the plant house of the university. Mitscherlich plant pots (0.20 m diameter and 0.17 m high) were filled with 6 kg of medium heavy soil characterized as fluvisol with a good to high supply of available nutrients (mg kg⁻¹): P: 138, K: 226, Ca: 2784, Mg: 167, S: 24.7 and alkaline exchangeable soil reaction (pH 7.4). The soil was extracted (except sulphur) using the method according to Mehlich III (CH₃COOH, NH₄NO₃, NH₄F, HNO₃ and EDTA).

The colorimetric method was used to determine the content of available phosphorus in the extract. The contents of available potassium, magnesium and calcium were determined by atomic absorption spectrophotometry (AAS). The quantification of sulphate sulphur was preceded by extraction with demineralized water in a ratio of 1:5 and capillary zone electrophoresis with a quartz capillary was applied for the measurement. The activity of hydrogen ions (pH) was measured in a soil extract of 0.2 M KCl using a potentiometer.

The trial involved four treatments (Table I); each was repeated four times. Nitrogen was applied after dissolving the ammonium nitrate fertilizer (34.5% N) in water.

Table I. Dates of application and N application levels to lupin (Lupinus angustifolius, L.).

		Date and level of nitrogen (kg/ha)
Treat.	Pattern	Before sowing	After emergence	Flowering	N application total (kg/ha) 1 pot = 5.34 dm^3
1	N0	0	0	0	0
2	N1	250	0	0	250
3	N2	250	250	500	1000
4	N3	250	250	1000	1500
N0: 0 kg N/ha.
N1: 250 kg N/ha = 0.5 g N per pot.
N2: 1000 kg N/ha = 2 g N per pot.
N3: 1500 kg N/ha = 3 g N per pot.

On 10 May 2005, before sowing, nitrogen was applied to the fertilized treatments 2–4. During plant growth, nitrogen was applied according to the experimental pattern: watering with de-mineralized water to 60% of the maximal capillary capacity, soil loosening and complete weed control. Three plants per pot were harvested at the stage of full maturity on 2 August 2005. Wet mineralization (H₂SO₄+H₂O₂) was conducted and then N was assessed according to the Kjeldahl method. The content of crude protein in seeds was estimated by multiplying the percentage of N by a factor of 6.25. Yields were statistically evaluated using the analysis of variance (ANOVA) and Tukey test (p<0.01).

Results and discussion

The plants did not show any symptoms of excess or toxicity of nitrogen and the yields of all variants were seen to respond to nitrogen fertilization. Table II shows that the number of pods per plant of all the fertilized treatments (treatment 2–4) was statistically highly significantly (p<0.01) higher than in the unfertilized variant (treatment 1). With nitrogen fertilization the number of pods increased by 42–78% (Table II); the maximal number of pods per plant, i.e., 7.3–8.9, was achieved in variants 2 and 3. According to El-Far et al. (2001) the application of fertilizers split into two doses increased both the number of pods per plant and the number of branches. Soheir (2002) reported that increasing nitrogen from 30 to 45 kg N ha⁻¹ stimulated the number of pods. Lopezbellido et al. (1994) discovered a positive correlation between yield and number of pods.

Table II. Results of pot trial with lupin (Lupinus angustifolius, L.).

	Pattern	Number of pods per plant		1000-seed weight		Seed yields
Treat.		pieces	%	g	%	g/plant	%	Crude protein (%)
1	N0	5.0^a	100	107.9^a	100	1,33^a	100	24.2^a
2	N1	7.3^b	146	111.1^a	102.9	2.68^b	201.5	25.1^a
3	N2	8.9^c	178	129.6^b	120.1	3.19^c	239.8	35.9^b
4	N3	7.1^b	142	94.7^c	87.7	2.41^b	181.2	40.6^c
^a,b,cVariants marked with different letters differ statistically significantly at the level of p<0.01.

The 1000-seed weight (TSW) ranged between 94.7 and 129.6 g (Table II) and increased with increasing levels of nitrogen to 2 g N per pot (treatment 3). Nitrogen increased the TSW by 2.9–20.1%; no statistical differences were reported between variants 1 and 2 (Table II). In treatment 4 the highest doses of N reduced the TSW. In a field trial El-Far et al. (2001) discovered that the highest level of TSW was achieved when fertilization was split into two applications. Wiatrak et al. (2004) reported that the TSW of lupin increased by 0.07 g after the application of 1 kg of N.

The total yields of seeds per plant are dependent on the development of the yield-forming elements. The numbers of pods and seeds per plant have the greatest effect on the total yields, to a lesser extent the 1000-seed weight (Hondelmann, 1984). Seed yields increased with increasing nitrogen levels from 1.33 g (treatment 1) to 3.19 g per plant (treatment 3), with no statistical differences between variants 2 and 4 (Table II). Nitrogen split into three applications (prior to sowing, after emergence and at flowering) gave the highest yields in treatment 3; compared to the unfertilized variant (100%), it increased to 239.8%. Schulze et al. (1999) discovered a marked increase in the yields of lupin seeds after application of nitrogen at the flowering stage. Likewise, El-Far et al. (2001) reported increased yields of lupin seeds when the nutrients were split into two applications during growth. According to Barlog (2002), pre-sowing application of 75 kg N ha⁻¹ in the form of (NH₄)₂SO₄ reduced seed yields compared to applications of NH₄NO₃ and Ca(NO₃)₂. Sole application of nitrogen without seed inoculation, much like inoculated seeds without implementing mineral forms of nitrogen, and the combination of both variants, resulted in a significant increase in seed yields compared to control (HobAllah & Kandil, 2001).

Lupin seeds contain 37–50% of crude protein (Schöneberger, 1982) and the seeds are an alternative source of protein (Lopezbellido & Fuentes, 1986). With a nitrogen application this content increased statistically highly significantly from 24.2% to 40.6% (Table II), considerably increasing the nutritional value of the seeds. In a similar trial Rubenschuh (1997) reported that the protein content increased to 45.1–47.3%. According to Ayisi et al. (1992), in all likelihood the increased concentration of protein in the lupin seeds was due to the increased total plant N supply late in the season. According to Barlog and Grzebisz (2000) the application of fertilizers containing nitrogen in the form of NH₄NO₃ and Ca(NO₃)₂ significantly increased the protein content in lupin seeds compared to variants not fertilized with nitrogen or fertilized with nitrogen in the ammonium form.

Conclusions

An adequate level of nitrogen in mineral fertilizer applied either before sowing or as a split application (before sowing, after emergence and flowering) increased yield and crude protein content in the seeds of narrow-leaf lupin. In the case of non-inoculated lupin seeds and a minimum of mineral nitrogen in the soil an optimal N fertilization policy for lupin is recommended.