Improved Nitrogen Utilization of Faba Bean (Vicia faba L.) Roots and Plant Physiological Characteristics under the Combined Application of Organic and Inorganic Fertilizers

Abstract

As one of the most important edible legumes worldwide, faba bean can be grown for grain, feed, vegetable, fertilizer, medicine and deep processing. In this study, experiments were designed to determine the combined effect of organic and inorganic fertilizers on the growth and development of faba bean. Dabaipi (a cultivar of Vicia faba L.) was used for the experiments. Five treatments with different ratios of organic nitrogen (N) to total N were applied, including 0% organic fertilizer (0% OF), 25% OF, 50% OF, 75% OF, 100% OF and 0% OF. Chemical urea was used as an inorganic fertilizer. The experimental results showed that 50% OF resulted in the highest faba bean yield, up to 10,337.39 and 13,595.7 kg ha⁻¹ in 2018 and 2019, respectively. Compared with 0% OF, 50% OF increased the yield by 84.47% and 183.17%, respectively. The regression analysis showed that the yield could be maximized when ROT accounted for 51.1%. The 50% OF treatment significantly increased N accumulation in seeds, resulting in higher N partial factor productivity and N harvest index (NHI). N accumulation in green seeds and aboveground plant parts had a significantly positive linear correlation with the yield and NHI, respectively. The 50% OF treatment maintained appropriate N accumulation in vegetative organs and higher N accumulation in reproductive organs and whole plants. Compared to 0% OF, the 50% OF treatment increased the total nodule number per plant (52.5%), fresh nodule weight (55.8%), nitrate reductase activity (70.7%), glutamine synthetase (18.2%) and glutamate synthase activity (42.4%). Therefore, the combined application of 50% OF and 50% inorganic fertilizer can be recommended for faba bean cultivation. This study will provide a theoretical basis for high-yield cultivation of faba bean.

1. Introduction

Faba bean (Vicia faba L.) contains rich nutrients (particularly proteins). Its protein content can reach 25.4% and is ranked second only to soybean among edible beans. This enables faba bean to be an important source of plant proteins [1]. It has been planted in more than 60 countries and regions. China leads the world in faba bean production, accounting for 36.1% of the world’s total production. It is also important for the structural adjustment of China’s grain production [2]. However, the traditional cultivation mode of faba bean is mainly field cultivation, with a later time to market and lower yield and economic benefit [3]. The green pod picking season of faba bean under greenhouse cultivation can be 2–3 months earlier than that under field cultivation. Thus, greenhouse cultivation has the advantages of high yield and good economic benefits and has become a cropping mode for vegetable farmers to increase their income [2]. However, the cultivation techniques for this cropping mode need to be further improved.

Inorganic fertilizer can increase the content of available soil nutrients and thus the intensity of soil fertilizer supply [4]. Organic fertilizer can improve nutrient storage and soil fertilizer supply capacities. Therefore, the combined application of organic and inorganic fertilizers can facilitate utilizing their respective advantages and thus increase soil fertility and crop yield. Combined fertilizer application is an important measure to ensure stable crop yield increase and promote sustainable and stable agricultural development [4,5,6]. Chaudhary et al. found that applying nanozeolite and nanochitosan along with Bacillus sp. enhanced the bacterial population in the treated soil and helped maintain soil health [7]. Bioformulation offers an environmentally sustainable approach to increasing crop production using microbial bio-inoculants and agriusable nanozeolite. Thus, bioformulation is conducive to improving the productivity of different crops [8,9]. The combined application of organic and inorganic fertilizers has become a more active fertilizer research direction. Combined fertilizer application has been studied on cotton [10], corn [11], cauliflower [12], Hongyang Kiwifruit (Actinidia chinesis) [13], sweet potato [14], wheat [15] and other crops. However, systematic studies on the greenhouse production of green faba bean seeds and nodule changes under the combined fertilizer application are still lacking.

Therefore, this study investigated the effects of the combined application of organic and inorganic fertilizers on the yield, dry matter accumulation, nitrogen absorption and utilization and physiological mechanism in root nodules of greenhouse-grown faba bean under equal nitrogen conditions. This study will provide a theoretical basis and practical guidance for rational fertilizer application in faba bean under greenhouse cultivation.

2. Materials and Methods

2.1. Material Preparation

The experiment was conducted in the greenhouse of the Agricultural College of Yangzhou University (32°24′42″ N, 119°41′29″ E) in 2018 and 2019. The test site was sandy loam soil, with soil total nitrogen content of 1.23 mg g⁻¹, organic matter content of 1.96%, hydrolyzed nitrogen of 69.06 mg kg⁻¹, available phosphorus of 35.78 mg kg⁻¹ and available potassium of 86.22 mg kg⁻¹. Dabaipi (a cultivar of Vicia faba) was planted by seedling transplantation. On 21 September 2018 and 2019, faba bean seeds were selected and washed with clean water to promote germination. The seeds were then put into a refrigerator for vernalization treatment. Seedlings were transplanted into facility greenhouses on 17 October in both years at a density of 45,000 plants ha⁻¹ and harvested when the pods matured in the next year (200 days after transplantation, DAT).

2.2. Experimental Design

Single factor randomized block design was used in the experiment. Five treatments with different ratios of organic Nitrogen (N) to total N (ROT) (

Table 1. The amount of organic and urea N input and the ratio of organic N to total N in different treatments.

Treatment	Organic N (kg ha−1)	Urea N (kg ha−1)	Organic N Ratio %
0% OF (CK)	0.0	97.5	0%
25% OF	24.4	73.1	25%
50% OF	48.8	48.8	50%
75% OF	73.1	24.4	75%
100% OF	97.5	0.0	100%

) were set up. Each treatment was conducted in triplicate. The organic fertilizer tested was an American fish-protein organic fertilizer, which was provided by Pulideng Agricultural Technology (Nanjing) Ltd. The main components of the fertilizer were: organic matter ≥ 58%, nitrogen, phosphorus, potassium ≥ 14% (N-P-K: 10-0-4), water ≤ 5%, particle diameter 2–4 mm, pH 4–5 and 8% amino acid. Pulideng SW fish-protein organic fertilizer contains rich bacterial protein, amino acids, minerals and a variety of trace elements. It is a protein organic granular fertilizer produced by the spray granulation process. The nitrogen fertilizer was urea (inorganic fertilizer; pure nitrogen content was 46%); the potassium fertilizer was potassium chloride (K₂O content was 60%); and the phosphorus fertilizer was calcium superphosphate (P₂O₅ content was 12%). All fertilizers were used as base fertilizers according to the fertilization scheme (N, 97.5 kg ha⁻¹; P₂O₅, 97.5 kg ha⁻¹; K₂O, 60.0 kg ha⁻¹) and were applied before transplanting. After fertilizer application, the fertilizer is evenly mixed with soil through tillage.

Amount of organic nitrogen = Urea nitrogen × Organic N ratio

2.3. Sample Preparation and Assays

2.3.1. Nitrogen Content and Accumulation

At 15 days after transplanting (DAT), 40 DAT, 80 DAT, 170 DAT and 200 DAT, two sample plants were taken from each plot, and the aboveground parts were divided into stems, leaves, pod shells and seeds. The enzymes were deactivated in these samples at 105 °C for 0.5 h; then, the samples were dried at 80 °C to a constant weight and weighed. The H₂SO₄-H₂O₂ digestion method was used to determine the total nitrogen content using a two-channel automatic flow analyzer º [16].

Nitrogen accumulation in each organ was calculated using the following formula: nitrogen accumulation (kg ha⁻¹) = nitrogen content (%) measured in each organ × dry biomass of each organ (kg plant⁻¹) × planting density (plant ha⁻¹) [17].

2.3.2. Total Nodule Number and Weight

At 170 DAT, all treatments were harvested and root systems rinsed with tap and distilled water. Then, the nodules were picked and detached on ice, dried on filter paper and weighed per plant. They were frozen in liquid nitrogen and stored at −80 °C until use. This entire procedure took no more than 30 min.

2.3.3. Nitrate Reductase Activity

The frozen samples of nodules (1 g) were extracted in a chilled mortar with quartz sand, insoluble polyvinylpolypyrrolidone (PVPP: 50%, w/v) and 10 mL of ice-cold extraction medium (50 mM K-phosphate buffer, pH 7.5, 2 mmol L⁻¹ Na₂-EDTA, 2 mmol L⁻¹ DTT and 1.5 soluble casein, w/v). Aliquots (0.4 mL) of the cytosolic fraction were assayed for nitrate reductase activity by the appearance of nitrite in 2 mL reaction mixtures according to Caba et al. [18].

2.3.4. Activities of Glutamine Synthetase (GS) and Glutamate Synthase (GOGAT)

Fresh samples of nodules (1 g) were homogenized on ice with acid-washed quartz sand and 12 mL of an extraction medium containing 100 mmol L⁻¹ maleic acid-KOH, pH 6.8, 100 mmol L⁻¹ sucrose, 2% (v/v) 2-mercaptoethanol and 15% (v/v) ethylene glycol, plus 0.5 g polyvinylpolypyrrolidone. Cell debris was removed by centrifugation, and the supernatant was placed on ice and immediately used to estimate the enzymes’ activities. GS activity was determined by the hydroxamate synthetase assay [19]. Two blanks without enzymes and without L-glutamate were also analyzed. The GOGAT activity was assayed spectrophotometrically at 30 °C by monitoring the oxidation of NADH at 340 nm, essentially as indicated by Groat and Vance [20].

2.4. Statistical Analysis

The partial factor productivity (PFP) of the nitrogen fertilizer was calculated as follows: (PFP, kg kg⁻¹) = Yield/Fertilization amount.

The nitrogen harvest index (NHI) was calculated as follows: (NHI, %) = (Seed nitrogen accumulation/Plant nitrogen accumulation) × 100.

Microsoft Excel 2010 software and SPSS 21.0 software were used for data collection, statistical mapping and significance analysis. The mean separation was performed using the least significant difference (LSD) at p < 0.05.

3. Results

3.1. Effect of Combined Application of Organic and Inorganic Fertilizer on Yield and Composition of Faba Bean Grown in Greenhouses

The ratio of organic Nitrogen (N) to total N (ROT) had a significant effect on the green seed yield of Dabaipi grown in a greenhouse, and the trend was basically the same in both years (

Table 2. Green seed yield and measurements of yield components for different treatments.

Year	Treatment	No. of Effective Branches per Plant	No. of Pods per Plant	Seeds per Pod	100-Seed Weight	Green Seed Yield
		(No. Plant−1)	(No. Plant−1)	(No. Pod−1)	(g)	(kg ha−1)
2018	0% OF (CK)	11.5 ± 0.3 c	30.2 ± 0.8 b	1.8 ± 0.2 a	291.2 ± 16 c	5603.69 ± 308.63 d
	25% OF	17 ± 0.6 b	38 ± 0.6 a	2 ± 0.2 a	326.9 ± 18 b	8720.38 ± 480.28 b
	50% OF	19.3 ± 0.9 a	39 ± 1.2 a	2 ± 0.2 a	362.7 ± 20 a	10,337.39 ± 569.34 a
	75% OF	15.3 ± 0.3 b	34 ± 1.2 ab	1.9 ± 0.2 a	321.2 ± 17.7 b	7627.09 ± 420.07 bc
	100% OF	12.7 ± 0.3 c	33 ± 0.6 ab	1.9 ± 0.2 a	298.3 ± 16.4 c	6556.04 ± 361.08 cd
2019	0% OF (CK)	10 ± 0.3 d	32.8 ± 1.5 c	1.4 ± 0.2 b	285.7 ± 15.7 c	4801.12 ± 264.43 c
	25% OF	12.5 ± 0.8 c	37.2 ± 0.6 ab	1.6 ± 0.2 b	343.7 ± 18.9 b	7586.65 ± 417.84 b
	50% OF	22 ± 0.6 a	40 ± 0.6 a	2.4 ± 0.3 a	394.2 ± 21.7 a	13,595.37 ± 748.77 a
	75% OF	16 ± 0.6 b	33 ± 0.6 bc	1.7 ± 0.2 b	366 ± 20.2 b	7391.06 ± 407.07 b
	100% OF	16 ± 0.6 b	32.4 ± 0.9 bc	1.6 ± 0.2 b	308.8 ± 17 c	5586.33 ± 307.67 c
ANOVA
Year		NS	NS	NS	NS	NS
Treatment		*	**	NS	**	*

Note: Different letters in a column mean significant difference at the 5% level. NS means no significant difference; * indicates significance at the 0.05 probability level; ** indicates significance at the 0.01 probability level.

). Compared with 0% OF (Control Check, CK), replacing some of the inorganic fertilizer with organic fertilizer increased the green seed yield. The difference between 100% OF and CK was not significant, but the yield of other treatments increased significantly (p < 0.05) compared to CK, among which the 50% OF treatment was the highest, followed by the 25% OF treatment. The regression analysis (Figure 1) showed that the relationship of ROT with green seed yield was parabolic with a downward opening and reached a significant level (r = 0.825 *). According to the regression equation, when the ROT reached 51.1%, it was most conducive to improving the green seed yield of Dabaipi grown in the greenhouse.

Further analysis of the yield components showed that the numbers of effective branches and pods per plant, 100-seed weight, seed number per pod and other indicators of organic–inorganic fertilizer combination treatment were increased. Compared with different treatments, the 50% OF treatment was the highest, wherein the numbers of effective branches and pods per plant and 100-seed weight, respectively, increased by 68%, 29% and 25% compared with 0% OF (CK) in 2018, and by 120%, 22% and 38% in 2019.

3.2. Effect of Combined Application of Organic and Inorganic Fertilizer on Dry Matter Accumulation and Distribution among Different Organs

The aboveground dry matter weight showed a gradual increasing trend, reaching the highest level at the green pod harvest (200 DAT;

Table 3. Shoot dry matter accumulation and distribution at green seed harvest for different treatments.

Year	Treatment	Shoot Dry Matter Accumulation					Shoot Dry Matter Accumulation
							at Different Stages
		15 DAT	40 DAT	80 DAT	170 DAT	200 DAT	15–40	40–80	80–170	170–200
							DAT	DAT	DAT	DAT
2018	0% OF (CK)	18.7 ± 1 c	94.5 ± 5.2 c	463.5 ± 25.5 d	2428.3 ± 133.7 e	9976.2 ± 549.4 d	75.8	369.0	1964.8	7547.9
	25% OF	25 ± 1.4 b	160.2 ± 8.8 b	880.2 ± 48.5 b	9620.5 ± 529.9 c	14,738.5 ± 811.7 c	135.2	720.0	8740.3	5118.1
	50% OF	36 ± 2 a	207.5 ± 11.4 a	1141.2 ± 62.9 a	14,719.9 ± 810.7 a	26,994.3 ± 1486.7 a	171.5	933.8	13,578.7	12,274.4
	75% OF	29.5 ± 1.6 ab	137.7 ± 7.6 b	771.3 ± 42.5 b	11,613.7 ± 639.6 b	18,954.7 ± 1043.9 b	108.2	633.6	10,842.4	7341.0
	100% OF	22.7 ± 1.3 bc	104.9 ± 5.8 c	570.6 ± 31.4 c	4444.5 ± 244.8 d	17,664.8 ± 972.9 b	82.1	465.8	3873.9	13,220.3
2019	0% OF (CK)	17.5 ± 1 d	45.5 ± 2.5 d	419.4 ± 23.1 d	2743.4 ± 151.1 e	10,826.1 ± 596.3 d	28.0	374.0	2324.0	8082.7
	25% OF	18.7 ± 1 cd	64.4 ± 3.5 c	618.3 ± 34.1 bc	9467.3 ± 521.4 c	15,864.3 ± 873.7 c	45.7	554.0	8849.0	6397.0
	50% OF	27.9 ± 1.5 a	272.1 ± 15 a	1476.2 ± 81.3 a	14,760.9 ± 813 a	27,029 ± 1488.6 a	244.3	1204.1	13,284.7	12,268.1
	75% OF	23.7 ± 1.3 ab	102.6 ± 5.7 b	631.8 ± 34.8 b	11,318 ± 623.3 b	20,034.9 ± 1103.4 b	78.9	529.2	10,686.2	8716.9
	100% OF	21 ± 1.2 bc	63.9 ± 3.5 c	560.5 ± 30.9 c	4995.2 ± 275.1 d	19,311.9 ± 1063.6 b	42.9	496.6	4434.8	14,316.6
ANOVA
Year		NS	*	NS	NS	NS
Treatment		*	**	**	*	*

Note: Different letters in a column mean significant difference at the 5% level. NS means no significant difference; * indicates significance at the 0.05 probability level; ** indicates significance at the 0.01 probability level.

). Compared with different treatments, the 50% OF treatment had the highest weight over the whole growth period. Taking 2019 as an example, at 200 DAT, the 50% OF treatment increased by 16,202.9 kg hm⁻² compared with CK. However, the trend of the dry matter weight accumulation increase rate (increased mass) in each growth stage was different, wherein the 25% OF, 50% OF and 75% OF treatments showed a trend of first increasing and then decreasing and reached the maximum at 80–170 DAT, while 0% OF and 100% OF showed a continuous increase and reached the maximum at 170–200 DAT. There was no significant difference in dry matter weights between the years, and both years had the same trend.

By the time of green pod harvesting (200 DAT), the proportion of dry matter distribution was the highest in stems, followed by green seeds and leaves, and the lowest in pod shells, with the average distribution rates of 41.8%, 21.7%, 21.4% and 15.1%, respectively (

Table 4. Shoot dry matter distribution at green seed harvest for different treatments (%).

Year	Treatment	Stem	Leaf	Pod Shells	Green Seed
2018	% OF (CK)	54.1 ± 3 a	19.2 ± 1.1 b	15.9 ± 0.9 a	1.8 ± 0.1 c
	25% OF	44.1 ± 2.4 b	15.5 ± 0.9 b	13.9 ± 0.8 a	26.5 ± 1.5 a
	5% OF	42.3 ± 2.3 bc	16.5 ± 0.9 b	14.4 ± 0.8 a	26.8 ± 1.5 a
	75% OF	35.9 ± 2 c	26.8 ± 1.5 a	15.9 ± 0.9 a	21.4 ± 1.2 b
	1% OF	34.6 ± 1.9 c	3.1 ± 0.2 a	14.4 ± 0.8 a	2.9 ± 0.2 b
2019	% OF (CK)	53.1 ± 2.9 a	18.2 ± 1 b	16.9 ± 0.9 a	11.8 ± 0.6 c
	25% OF	43.9 ± 2.4 ab	15.5 ± 0.9 b	12.9 ± 0.7 a	27.6 ± 1.5 ab
	5% OF	41 ± 2.3 b	17.5 ± 1 b	13.4 ± 0.7 a	28 ± 1.5 a
	75% OF	34.9 ± 1.9 c	24.8 ± 1.4 ab	16.9 ± 0.9 a	23.4 ± 1.3 b
	1% OF	33.6 ± 1.9 c	3.1 ± 0.2 a	16.4 ± 0.9 a	19.9 ± 1.1 b
ANOVA
Year		NS	NS	NS	NS
Treatment		*	*	NS	*

Note: Different letters in a column mean significant difference at the 5% level. NS means no significant difference; * indicates significance at the 0.05 probability level.

). Among them, the dry matter distribution rate of green seeds first increased and then decreased with the increase in ROT. The 50% OF and 25% OF treatments had the highest distribution rate of green seeds, followed by the 75% OF treatment; the 100% OF treatment was third, and the 0% OF treatment (CK) was the lowest. Meanwhile, in the pod shells, the 50% OF and 25% OF treatments were lower than the 75% OF, 100% OF and 0% OF treatments.

The correlation analysis (

Table 5. The correlation between the indices of dry matter accumulation in shoot and green seed yield.

	Dry Matter Accumulation (DAT)					Shoot Dry Matter Accumulation between Different Stages				Dry Matter Distribution at Green Seed Harvest
	15	40	80	170	200	15–40 DAT	40–80 DAT	80–170 DAT	170–200 DAT	Stem	Leaf	Pod Shells	Green Seed
Yield	0.690 *	0.946 **	0.983 **	0.862 **	0.798 **	0.949 **	0.982 **	0.842 **	0.181	−0.183	−0.408	−0.653 **	0.759 **

Note: * indicates significance at the 0.05 probability level; ** indicates significance at the 0.01 probability level.

) further showed that the aboveground dry matter weight in each period was significantly different from the green seed yield, but the increase in dry matter was positively correlated with the yield only in the growth periods of 15–40 DAT, 40–80 DAT, 80–170 DAT, while the accumulation of dry matter at 170–200 DAT was not significantly correlated with the yield. The correlation analysis also showed that the dry matter distribution ratio of pod shells was significantly negatively correlated with the green seed yield, while the ratio of green seeds was significantly positively correlated with the yield.

3.3. Effect of Combined Application of Organic and Inorganic Fertilizer on Nitrogen Accumulation

3.3.1. Nitrogen Content

There were no significant differences in the nitrogen content of organs with different treatments among the years, but the differences between treatments reached a significant or highly significant level (

Table 6. Nitrogen contents in different plant components at harvest of green seeds for different treatments (%).

Treatment	Stem		Leaf		Pod Shells		Green Seed
	2018	2019	2018	2019	2018	2019	2018	2019
0% OF (CK)	1.17 ± 0.06 bc	1.14 ± 0.06 b	3.37 ± 0.19 a	3.08 ± 0.17 a	2.37 ± 0.13 b	2.22 ± 0.12 c	3.03 ± 0.17 c	3.16 ± 0.17 c
25% OF	1.1 ± 0.06 cd	1.03 ± 0.06 c	2.5 ± 0.14 c	2.62 ± 0.14 b	2.6 ± 0.14 ab	2.76 ± 0.15 ab	3.43 ± 0.19 b	3.48 ± 0.19 b
50% OF	1.03 ± 0.06 d	1.08 ± 0.06 c	2.1 ± 0.12 c	2.17 ± 0.12 b	1.6 ± 0.09 c	1.79 ± 0.1 d	4 ± 0.22 a	3.73 ± 0.21 a
75% OF	1.2 ± 0.07 b	1.15 ± 0.06 b	3.03 ± 0.17 b	3.3 ± 0.18 a	2.97 ± 0.16 a	2.94 ± 0.16 a	3.37 ± 0.19 b	3.46 ± 0.19 b
100% OF	1.4 ± 0.08 a	1.49 ± 0.08 a	3.1 ± 0.17 b	3.07 ± 0.17 a	2.57 ± 0.14 ab	2.47 ± 0.14 b	3.4 ± 0.19 b	3.42 ± 0.19 b
ANOVA
Year	NS		NS		NS		NS
Treatment	*		*		**		*

Note: Different letters in a column mean significant difference at the 5% level. NS means no significant difference; * indicates significance at the 0.05 probability level; ** indicates significance at the 0.01 probability level.

). The nitrogen content of different organs showed a trend of green seed > leaf > pod shell > stem at 200 DAT. Compared with other treatments, the 50% OF treatment had the highest nitrogen content in green seeds, followed by the 25% OF, 75% OF, 100% OF and 0% OF treatments, with respective averages of 3.9%, 3.5%, 3.4%, 3.4% and 3.1%. The nitrogen content in the leaves was highest in the 0% OF and 75% OF treatments, followed by the 100% OF, 25% OF and 50% OF treatments. In pod shells, the 75% OF and 25% OF treatments had the highest nitrogen content, followed by the 100% OF and 0% OF treatments, and the lowest content was noted for the 50% OF treatment. In stems, the nitrogen content of the 100% OF treatment was the highest, followed by the 0% OF and 75% OF treatments, and the 25% OF and 50% OF treatments had the lowest content.

3.3.2. Nitrogen Accumulation

The nitrogen accumulation of each treatment was in the order of 50% OF > 75% OF > 100% OF > 25% OF > 0% OF, and the treatments with combined application of organic and inorganic fertilizer were significantly higher than that of 0% OF (CK) (

Table 7. The nitrogen accumulation at harvest of green seeds for different treatments (kg ha⁻¹).

	Stem		Leaf		Pod Shells		Green Seed		Total
Treatment	2018	2019	2018	2019	2018	2019	2018	2019	2018	2019
0% OF (CK)	63 ± 3.5 d	65.4 ± 3.6 c	64.5 ± 3.6 c	60.7 ± 3.3 c	37.6 ± 2.1 d	40.6 ± 2.2 c	32.6 ± 1.8 d	40.3 ± 2.2 d	197.7 ± 10.9 d	207 ± 11.4 d
25% OF	71.4 ± 3.9 c	72.1 ± 4 c	57.3 ± 3.2 c	64.6 ± 3.6 c	53.3 ± 2.9 c	56.6 ± 3.1 c	134 ± 7.4 bc	152.3 ± 8.4 b	316 ± 17.4 c	345.6 ± 19 c
50% OF	117.9 ± 6.5 a	119.6 ± 6.6 a	93.8 ± 5.2 b	103.1 ± 5.7 b	62.1 ± 3.4 b	64.7 ± 3.6 b	289.5 ± 15.9 a	283 ± 15.6 a	563.3 ± 31 a	570.3 ± 31.4 a
75% OF	81.6 ± 4.5 bc	80.2 ± 4.4 b	154.1 ± 8.5 a	164.8 ± 9.1 a	89.3 ± 4.9 a	99.5 ± 5.5 a	136.7 ± 7.5 b	162.5 ± 8.9 b	461.8 ± 25.4 b	507 ± 27.9 ab
100% OF	85.7 ± 4.7 b	96.7 ± 5.3 b	164.8 ± 9.1 a	178.7 ± 9.8 a	65.4 ± 3.6 b	78.4 ± 4.3 ab	125.3 ± 6.9 c	131.3 ± 7.2 c	441.1 ± 24.3 b	485.1 ± 26.7 bc
ANOVA
Year	NS		NS		NS		NS		NS
Treatment	*		*		*		**		*

Note: Different letters in a column mean significant difference at the 5% level. NS means no significant difference; * indicates significance at the 0.05 probability level; ** indicates significance at the 0.01 probability level.

). Taking 2019 as an example, the 50% OF, 75% OF, 100% OF and 25% OF treatments, respectively, increased by 363.3, 300.0, 278.1 and 138.6 kg hm⁻² compared with the 0% OF treatment. Compared with the plant parts, the 75% OF and 100% OF treatments were higher in vegetative organs (stem + leaf), followed by the 50% OF, 25% OF and 0% OF treatments. The 50% OF treatment was higher in reproductive organs (pod shell + green seed); the 75% OF and 100% OF treatments were second; the 25% OF treatment was third; and the 0% OF treatment was the lowest.

3.4. Effect of Combined Application of Organic and Inorganic Fertilizer on Nitrogen Utilization Efficiency

There were significant differences in nitrogen use efficiency among treatments, wherein the partial factor productivity (PFP) of nitrogen and nitrogen harvest index (NHI) were in the order of 50% OF > 25% OF > 75% OF > 100% OF > 0% OF, and the 50% OF treatment was significantly higher than other treatments (

Table 8. Dynamic change of N efficiency for different treatments.

Treatment	PFP		NHI
	N Partial Factor Productivity		N Harvest Index
	(kg kg−1)		(%)
	2018	2019	2018	2019
0% OF (CK)	124.9 ± 6.9 d	107 ± 5.9 d	16.51 ± 0.9 d	19.48 ± 1.1 c
25% OF	194.4 ± 10.7 b	169.2 ± 9.3 b	42.41 ± 2.3 b	44.06 ± 2.4 a
50% OF	230.5 ± 12.7 a	303.1 ± 16.7 a	51.4 ± 2.8 a	49.61 ± 2.7 a
75% OF	170.1 ± 9.4 bc	164.8 ± 9.1 b	29.6 ± 1.6 c	32.05 ± 1.8 b
100% OF	146.2 ± 8.1 cd	124.6 ± 6.9 c	28.4 ± 1.6 c	27.07 ± 1.5 b
ANOVA
Year	NS		NS
Treatment	*		**

Note: Different letters in a column mean significant difference at the 5% level. NS means no significant difference; * indicates significance at the 0.05 probability level; ** indicates significance at the 0.01 probability level.

). When the inorganic fertilizer (0% OF) or the organic fertilizer (100% OF) were applied alone, their nitrogen PHP and NHI were significantly lower than those of combined organic and inorganic fertilizer treatment (25% OF, 50% OF, 75% OF).

3.5. Relationship between Nitrogen Accumulation and Yield and Nitrogen Utilization Efficiency

During the pod harvesting period, the relationship between nitrogen accumulation and yield and nitrogen use efficiency in different plant parts (Figure 2) showed that the final nitrogen accumulation in vegetative organs (stem + leaf) had a negative parabolic relationship with yield (r = 0.761 **) and NHI (r = 0.647 *); the maximum yield and NHI were achieved when nitrogen accumulation reached 191.6–194.7 kg ha⁻¹ (Figure 2A).

However, the reproductive organs’ (pod shells + green seed) nitrogen accumulation and the total aboveground nitrogen accumulation were linearly and positively correlated with yield (r = 0.832 **, r = 0.645 *) and NHI (r = 0.843 **, r = 0.621 *), respectively, for reproductive organs (Figure 2B) and shoots (Figure 2C).

3.6. Physiological Change as Affected by Combined Application of Organic and Inorganic Fertilizer

At 170 DAT (the podding stage), the combined application of organic and inorganic fertilizer caused a significant increase in the total nodule number per plant and nodule fresh weight as compared to the control (0% OF) (p < 0.05) (

Table 9. Effects of organic and inorganic fertilizer on total nodule number, nodule weight, NR activity, GS activity and GOGAT activity at 170 DAT (2019).

Parameter	Treatments
	0% OF	25% OF	50% OF	75% OF	100% OF
Total nodule number (No./plant)	21.7 ± 1.2 d	24.9 ± 1.4 c	33.1 ± 1.8 a	27.4 ± 1.5 b	26.2 ± 1.4 bc
Nodule weight (g FW/plant)	5.2 ± 0.3 c	6.4 ± 0.4 c	8.1 ± 0.4 a	7.5 ± 0.4 b	7.2 ± 0.4 bc
NRA [μmol NO2−/(g FW h)]	6.04 ± 0.3 d	6.37 ± 0.4 c	10.31 ± 0.6 a	8.74 ± 0.5 b	7.55 ± 0.4 c
GS activity [μmol/(g FW h)]	380.82 ± 21 d	384.94 ± 21.2 c	450.31 ± 24.8 a	410.15 ± 22.6 b	390.34 ± 21.5 bc
GOGAT activity [μmol/(g FW h)]	70.38 ± 3.9 c	80.58 ± 4.4 bc	100.22 ± 5.5 a	92.31 ± 5.1 ab	77.62 ± 4.3 c

Note: Different letters in a line mean significant difference at the 5% level.

). The largest increase in the two parameters was found with the 50% OF treatment. Compared to the 0% OF treatment, a 52.5% increase in the total nodule number per plant and a 55.8% increase in nodule fresh weight were detected with the 50% OF treatment, respectively.

The conjunctive use of organic and inorganic fertilizers significantly influenced the NR activity (Table 9). The application of the 50% organic Nitrogen to total N combination was superior in increasing it. A 70.7% increase was detected after treatment with 50% OF.

More than 95% of NH₄+ in higher plants is assimilated through the GS/GOGAT cycle. The main function of the GS/GOGAT cycle in root nodules is to assimilate NH₄+ produced by N fixation of rhizobia [21]. The ammonium assimilation activity was measured in nodule cytosol (Table 9). The present results showed that replacing some of the inorganic fertilizer with an organic fertilizer increased the specific activities of GS and GOGAT. The largest increase was found with the 50% OF treatment, reaching 450.31 μmol/(g FW h) and 100.22 μmol/(g FW h), respectively. Compared to the 0% OF treatment, a 18.2% increase in GS activity and a 42.4% increase in GOGAT activity were detected, respectively.

4. Discussion

4.1. ROT Can Increase the Green Seed Yield and Nitrogen Utilization Efficiency of Greenhouse-Grown Faba Bean

The results of Abid et al. show that the combined application of organic and inorganic fertilizer improved the number of grains per cob and the yield of maize [22]. Tian found that the combined fertilizer application had different effects on pea growth, root nodule number, rhizobia reduction and pea yield [23]. Cucci et al. found that wet olive pomace at 140 Mg ha⁻¹ with half of the conventional N, P and K dose of mineral fertilizers allowed the same faba bean productivity of full mineral fertilization [24]. In a field study, Zhang found that at the ratio of 1:3 (organic nitrogen: inorganic nitrogen) and 1:6 (organic phosphorus: inorganic phosphorus), the faba bean yield was significantly improved [25]. In our experiments, under the total nitrogen application of 97.50 kg ha⁻¹, the combined application of organic and inorganic fertilizers increased the number of effective branches and pods per plant and 100-seed weight of greenhouse-grown faba bean (Table 2). At a ROT of 50% (50% OF), the yield reached the highest value, indicating the most significant improvement effects of the combined fertilizer application. This is different from the results of Zhang [25]. This may be because greenhouse-grown faba bean had different growth, development and fertilizer requirements compared with those under traditional field cultivation. Therefore, copying the field cultivation techniques may be inappropriate. The supporting cultivation techniques of greenhouse-grown faba bean need to be systematically studied in the future.

The regression analysis (Figure 1) shows that the maximum theoretical yield can be obtained at a ROT of 51.1%. This may be because, under the combined application of organic and inorganic fertilizers, soil micro-organisms do not decompose organic nitrogen in the early growth stage [26]. The nitrogen required for faba bean growth is mainly supplied by inorganic nitrogen. However, the nitrogen required increases in the middle and late stages. Thus, the organic nitrogen in the fertilizer can be decomposed by micro-organisms and released in time to supply faba bean for absorption and utilization under the condition of insufficient inorganic nitrogen supply. Compound fertilizers can not only avoid the loss of excessive nitrogen in the early stage but also meet the growth and development needs of faba bean in the early stage.

In terms of nitrogen use efficiency, partial factor productivity (PFP) is a comprehensive index for measuring soil fertility and fertilizer application effects. The suitable PFP of a nitrogen fertilizer generally ranges between 40 and 80 kg/kg. A PFP over 60 kg/kg indicates better nitrogen management or lower fertilization [27]. NHI reflects the nitrogen distribution between vegetative and reproductive organs of plants. This study found that the combined application of organic and inorganic fertilizers can improve PFP and NHI (Table 8). When ROT reached 50%, the PFP and NHI were the highest. This indicates that the ability of greenhouse-grown faba bean to convert nitrogen into dry matter and green seeds was improved. This may be because the combined application of organic and inorganic fertilizers supplemented organic carbon in soil, improving the C/N of soil. More carbon was provided for soil micro-organisms, stimulating the activity of soil micro-organisms and accelerating the release of effective nutrients in the soil and fertilizer. Thus, nitrogen promotes absorption and utilization.

4.2. Combined Application of Organic and Inorganic Fertilizers Increases the Ability to Absorb and Assimilate Nitrogen

A favorable rhizosphere environment is highly important for the interaction between root hairs and Rhizobium, since it can encourage the growth and multiplication of rhizobia and ensure the healthy development of root hairs. Organic and inorganic fertilizers have been reported to affect plant root growth and development and increase the abundance of nitrogen-fixing bacteria [28,29]. In the present study, 50% OF significantly increased the nodule weights and total nodule number per plant. This indicates that the increased nodule formation by faba bean under the combined fertilizer application may be due to the beneficial effects of the nodulation initiation process (an event in Rhizobium-legume symbiosis significantly sensitive to fertilizers).

Nitrate reductase is the first enzyme involved in the assimilation of N-NO³⁻, and its activity is crucial for plant development and plays a key role in nitrogen assimilation. During faba bean nodulation, the GS/GOGAT cycle is considered to be responsible for assimilating most of the NH₄+ derived from N₂ fixation in the nodules. In the present study, the combined application of organic and inorganic fertilizers created favorable conditions for faba bean growth and increased the activity of this enzymatic pathway. Our findings show that the largest increase in NR, GS and GOGAT activities occurred under the 50% OF treatment. The NR activity increased more than the GS and GOGAT activities, suggesting that the former is the major factor promoting nitrogen absorption and assimilation of nodules in faba bean.

In addition, compared with the application of inorganic or organic fertilizer alone, the combined application of organic and inorganic fertilizers improved the nitrogen accumulation and distribution among different organs of greenhouse-grown faba bean. This is consistent with the results of previous studies [30,31,32,33]. At a ROT of 50%, the total nitrogen accumulation was appropriate in the final vegetative organs (stem + leaf) and high in the pod (pod shell + green seed) and the whole aboveground part. The regression analysis (Figure 2) also clarifies that the total nitrogen accumulation in vegetative organs had a negative parabolic relationship with the final green seed yield and nitrogen use efficiency. Excessively low and high nitrogen accumulation in vegetative organs indicates insufficient and excessive development of vegetative organs, respectively. This is not conducive to yield formation. However, the nitrogen accumulation in pods and the whole aboveground part showed a positive linear correlation with yield and nitrogen use efficiency. Therefore, the combined application of organic and inorganic fertilizers can also coordinate nitrogen distribution among different organs in greenhouse-grown faba bean.

4.3. Dry Matter Accumulation and Distribution Characteristics of Greenhouse-Grown High-Yield Faba Bean

Nitrogen absorption and utilization directly affect crop growth and development [34,35]. In this study, the dry matter weight in each growth stage was significantly positively correlated with green seed yield. Only the dry matter increases within 15–40 DAT, 40–80 DAT and 80–170 DAT were positively correlated. This indicates that increasing the total dry matter accumulation and the dry matter accumulation rate in the early and middle stages (0–170 DAT) is the key to achieving a high yield of greenhouse-grown faba beans. This differs from the traditional concept, whereby dry matter accumulation in the later crop growth stage contributes more to the yield [36]. This may be attributed to the harvested green seeds of faba bean in this study. The grains were not fully developed and mature. The harvesting period was about 30 days earlier than that of mature grains [37]. Therefore, in practice, the early growth and development of greenhouse-cultivated faba bean need to be promoted to achieve a high yield of green seeds.

However, researchers should still pay attention to adjusting the proportion of dry matter distribution in different plant parts, such as increasing the proportion of green seeds and reducing the proportion of pod shells, stems and leaves. Thus, more nutrients absorbed by plants and/or stored in the pod shells, stems and leaves can be transported to the green seeds. This may be consistent with the rapid transport of nutrients from stems and leaves to green seeds and increased yield and quality in the final maturity stage of Gramineae crops [38].

In addition, this study shows that ROT significantly affected the dry matter accumulation and distribution during the pod harvesting stage of greenhouse-grown faba bean. At a ROT of 50%, the total dry matter accumulation and the final dry matter distribution ratio of green seeds were high. However, when the ROT was too high, the dry matter accumulation and the dry matter distribution ratio in the green seeds were significantly reduced throughout the growth stage, especially during the green pod harvesting stage. This can negatively affect yield formation. In conclusion, dry matter accumulation and distribution of greenhouse-grown high-yield faba bean exhibited the characteristics of high total dry matter accumulation, rapid dry matter accumulation in the early and middle stages and a high dry matter distribution ratio in green seeds.

5. Conclusions

The combined application of organic and inorganic fertilizers increases the green seed yield of greenhouse-grown faba bean. At a ROT of 50%, the yield showed the most significant increase. The 50% OF treatment induced the highest total nodule number, nodule weight, NRA, GS and GOGAT activities, and nitrogen accumulation in the whole aboveground part (especially in seeds). It exhibited the characteristics of high total dry matter accumulation, rapid dry matter accumulation in the early and middle stages and a high dry matter distribution ratio in green seeds. This indicates higher nitrogen utilization efficiency. Therefore, the 50% OF treatment can be recommended for fertilizer application in faba bean cultivation.