1 Introduction

Quinoa (Chenopodium quinoa Willd.) is a stress-resistant plant that has been cultivated in the Andean region for the past 7000 years. Its seeds have a higher nutritional value than traditional cereals, and it is a promising plant for human consumption and nutrition around the world (Vega-Gálvez et al. 2010). Quinoa, known as an edible seed, is classified as a high quality food because of the high content of protein and lysine, which is the most essential limiting amino acid in cereals (Arendt and Zannini 2013; Livingston 2013).

In developing countries, the low soil fertility is the most important factor limiting crop yields (Aleminew et al. 2020). Therefore, one of the most important research areas for the development of crop cultivation is the use and management of chemical fertilizers, particularly nitrogen fertilizers, which play an important role in the healthy and optimal growth of plants. Nitrogen is a key and limiting element for plant growth and yield that participates in all plant metabolic processes (Kakabouki et al. 2018).

Despite improved crop yields in response to nitrogen fertilization, the optimal amount of nitrogen application for many crops has not yet been determined (Almadini et al. 2019). The positive effect of nitrogen on quinoa has been proven in several studies on the growth and seed yield of this plant (Kakabouki et al. 2018). However, the optimal amount of nitrogen application reported for quinoa varies widely. Berti et al. (2000) showed that the highest quinoa seed yield was obtained with the highest nitrogen application rate in Chile (225 kg N ha−1). Erley et al. (2005) reported that quinoa responded strongly to nitrogen fertilizer application in southern Germany. The application of 120 kg N ha−1 doubled the yield of quinoa compared to non-nitrogen conditions. According to the findings of Alvar-Beltrán et al. (2019) in Burkina Faso, quinoa can perform very well in low nitrogen input systems. In this study, the highest yield of quinoa was obtained in the November planting date with application of 25 kg N ha−1. Evaluation of the effect of planting time and nitrogen fertilizer rate on the quinoa growth and yield in Ahvaz, Iran, showed that the highest 1000-seed weight, seed yield, and harvest index were produced on the 20th of October sowing date with application of 320 kg N ha−1 (Saeidi et al. 2020). In China, the application of 240 kg N ha−1 significantly increased the 1000-seed weight and seed protein content of quinoa compared to 80 and 160 kg N ha−1 treatments (Wang et al. 2020). Due to the variability of ecological conditions, extensive studies are already conducted around the world to determine the nitrogen requirement of the quinoa crop.

Although nitrogen has been a key element in increasing the yield and agricultural food production worldwide for more than four decades, only a small fraction of this fertilizer is absorbed by plants. Poor nitrogen recovery is the result of nitrogen volatilization, leaching, biological immobilization, and inefficiency of crops in N uptake and utilization. However, it has been reported that adopting appropriate nitrogen fertilizer management practices can increase nitrogen recovery up to 80%. The split application of nitrogen is one of the ways to improve nitrogen use efficiency in crop plants, which can reduce nitrogen losses through leaching, denitrification, runoff, and evaporation. It usually improves the crops yield and increased the economic benefit from increased yield. Determining the appropriate rate and timing of nitrogen application is a determining factor in achieving higher yields (Belete et al. 2018; Ishfaq et al. 2020).

Some recent studies have found that in comparison to single N application, split applications of nitrogen result in higher recovery efficiency and higher yields. Du et al. (2019) showed that the split application of nitrogen in sweet potatoes resulted in better growth, balanced sink-source ratio, and higher storage root yield, especially under reduced nitrogen conditions. The corresponding net economic benefit increased when sweet potato was planted with split N application rather than conventional one-time basal fertilization. It is reported that the split application of nitrogen before planting and at the beginning of Tillering and panicle formation results in better nitrogen recovery and increased grain yield in rice (Wang et al. 2018). The split application of nitrogen at planting, tillering, and booting stages is reported to be effective for optimal yield production in wheat (Belete et al. 2018).

The split application of nitrogen may enhance the yield and seed quality of quinoa (Jacobsen and Christiansen 2016). In many parts of the world like Iran, no research has been done on the effect of split application of N on quinoa and its relationship with seed yield and quality. This study aimed to investigate the effect of nitrogen application rate and the split application method on the growth characteristics, yield, and yield components of quinoa.

2 Materials and Methods

The experiment was conducted in two cropping years (2017–2018 and 2018–2019) in the research farm of Islamic Azad University, Sabzevar branch, the west of Khorasan Razavi Province, Iran. Sabzevar is located at 36° 21′ N latitude, 57° 67′ E longitude, and 980 m above sea level. The average long-term rainfall and mean annual temperature in this climate are 187.4 mm and 21.6 °C, respectively. Figure 1 shows the statistics on monthly rainfall and average monthly temperatures during the experimental years. The soil of the study area had a sandy loam texture with low organic carbon content and moderate salinity and alkalinity as indicated in Table 1.

Fig. 1
figure 1

Monthly rainfall and average monthly temperature during the experimental years (2018–2019)

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Table 1 Field soil physico-chemical properties at the 0–30 cm depth
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The experiment was performed as a split plot in a randomized complete block design with three replications. The factors studied were nitrogen application rate at four levels (0, 50, 100, and 150 kg N ha−1) and split application method of nitrogen at three levels ((1) four equal splits at planting, 6–8 leaves, anthesis, and the seed filling stages; (2) three equal splits at 6–8 leaves, anthesis, and the seed filling stages; (3) two equal splits at 6–8 leaves and the anthesis stages). Nitrogen application rates and the split application methods of nitrogen were assigned to main plots and subplots, respectively. The source of nitrogen used was urea fertilizer with 46% nitrogen.

Before the experiment, chemical composition of irrigation water and soil physical and chemical properties of the study area were determined (Tables 1 and 2). Land preparation operations included plowing to a depth of 30 cm, discs, and leveling, which was carried out in the spring. To provide the required nutrients for quinoa, 80 kg ha−1 K2O in the form of potassium sulfate and 100 kg ha−1 P2O5 in the form of triple superphosphate were added to the soil and mixed with it before planting. In both years of the experiment, seeds of quinoa were sown manually with high density (12 kg of seeds per hectare) in row spacing of 25 cm in the fourth week of July. Disinfected seeds of the Sajama genotype with carboxin-thiram fungicide were used for planting. The Sajama is a semi-early variety which was released in Bolivia by the IBTAFootnote 1 and provided by PROINPAFootnote 2 to the Seed and Plant Improvement Institute in Iran (Bonifacio et al. 2015; Bazile et al. 2016).

Table 2 Field irrigation water characteristics
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Each subplot size was 8 m2 (2 m × 4 m) including eight planting rows with an inter row spacing of 0.25 m and a length of 4 m. The field was irrigated by flood irrigation system immediately after planting. In the four-split applications method of nitrogen, the first stage of nitrogen application (planting stage) was done simultaneously with the second irrigation, three days after planting. In the other split application methods of nitrogen, the first stage of nitrogen fertilizer was used in the 6–8 leaves stage. Subsequent irrigations were performed at average intervals of 10 days until the end of the growing season in all experimental treatments. Thinning and weeding operations were performed after the emergence and complete establishment of the plant (4–6 true leaves). The final density was about 40 plants per square meter. The time of the split application of nitrogen fertilizer in experimental treatments was when 50% of the plants entered the desired phenological stage.

To evaluate the physiological characteristics, in the seed dough stage, the fully developed upper leaves of the plants were sampled. Leaf samples were immediately frozen in liquid nitrogen and transferred to a freezer at − 80 °C.

2.1 Photosynthetic Pigment Content

The content of photosynthetic pigments was measured by the Lichtenthaler (1987) method. A 0.1 g of fresh leaf tissue was ground in 15 ml of 80% acetone, and the resulting solution was filtered through filter paper. The adsorption of the solutions was read by spectrophotometer (Fullerton, Beckman, CA) at wavelengths of 663.2 nm for chlorophyll a, 646.8 nm for chlorophyll b, and 470 nm for carotenoids. Finally, using the following formulas, the content of chlorophyll a, b, and carotenoids was calculated in mg g−1 fresh weight of the sample.

Chlorophyll a = (12.25 A663.2) − (2.79 A646.8).

Chlorophyll b = (21.51 A646.8) − (5.1 A663.2).

Carotenoids = (100 A470 − 1.8 chl.a − 85.02 chl.b)/19.

2.2 Morphological Characteristics, Seed Yield, and Yield Components

At the physiological maturity stage, 10 plants were randomly selected from the middle rows of each plot, and the plant height was measured. Then, the seeds were separated to determine the number of seeds per plant.

To measure seed yield and aboveground biomass, in the crop ripening stage, two square meters of each plot were completely harvested and oven dried at 70 °C for 48 h. Then, the seeds were separated from the straw and weighed. The seed yield was calculated taking into account 12% moisture content. To determine the 1000-seed weight, three replicates of 200 seeds from each treatment were counted and weighed.

2.3 Statistical Analysis

Due to the heterogeneity of error variances in the years of experiment, the analysis of variance of data was performed separately for each of the experimental years. Analysis of variance and comparison of data means were performed using SAS 9.3 software and in accordance with Duncan’s multiple range test. Tables and graphs were drawn using Word and Excel software.

3 Results

3.1 Number of Days to Flowering

Increasing the rate of nitrogen application increased the time required for plants to enter the flowering stage in the experimental years. The lowest number of days to flowering was recorded in the control treatment (no nitrogen application) and the highest in the treatment of 150 kg N ha−1. In 2019, there was no significant difference between the 100 and 150 kg N ha−1 treatments in relation to the number of days to flowering (Table 3).

Table 3 The effects of nitrogen application rate and split application method on the days to flowering, seed filling period, and content of chlorophyll
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The split application method of nitrogen affected the number of days to flowering. The highest number of days to flowering in both years of experiment was recorded for the two equal split applications of nitrogen at the 6–8 leaves and anthesis stages, and the lowest was obtained for the four equal split applications of nitrogen at the planting, 6–8 leaves, anthesis, and seed filling stages. There was no significant difference between the four-split applications of nitrogen and the three equal split applications of nitrogen at the 6–8 leaves, anthesis, and seed filling stages in both experimental years (Table 3).

Number of days to flowering had positive and significant correlation with seed filling period (r = 0.81**), chlorophyll a content (r = 0.68**), chlorophyll b content (r = 0.64**), total chlorophyll content (r = 0.69**), plant height (r = 0.79**), number of seeds per plant (r = 0.79**), seed yield (r = 0.85**), aboveground biomass (r = 0.81**), harvest index (r = 0.31**) and 1000-seed weight (r = 0.46**). There was no significant correlation between number of days to flowering and carotenoid content (Table 4).

Table 4 Correlation analysis of agro-physiological characteristics of quinoa under different nitrogen application rate and split application method
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3.2 Seed Filling Period

In both years of the experiment, as the rate of nitrogen application increased, the length of the seed filling period increased. The lowest and highest seed filling period were observed in the control and 150 kg N ha−1 treatments, respectively. In 2019, the difference between the application rates of 100 and 150 kg N ha−1 was not significant (Table 3).

The difference between the split application methods of nitrogen was significant in relation to the length of seed filling period. The highest and the lowest seed filling period in both years of the experiment were recorded for the two and four-split applications of nitrogen, respectively. There was no significant difference between the two and three-split applications of nitrogen in relation to the length of seed filling period (Table 3).

Similar to the number of days to flowering, there was a strong and positive correlation between seed filling period and seed yield in quinoa (r = 0.85**). Thus, a reduction in number of days to flowering and seed filling period could reduce the seed yield, as clearly shown in this study (Table 4).

3.3 Photosynthetic Pigments

Nitrogen application increased the content of chlorophyll a, chlorophyll b, total chlorophyll (a + b), and carotenoids during the experimental years. In 2018, the highest chlorophyll a content was obtained for the application rate of 150 kg N ha−1, which was not significantly different from the application rate of 100 kg N ha−1 (Tables 3 and 5). The two-split applications of nitrogen had the highest chlorophyll a content, which was not significantly different from the three-split applications of nitrogen (Table 3). Meanwhile, in 2019, the highest chlorophyll a content was obtained in the two-split applications of 100 kg N ha−1, showing no significant difference with the three and two-split applications of 150 kg N ha−1 and also with the three-split applications of 100 and 50 kg N ha−1 (Table 6).

Table 5 The effects of nitrogen application rate and split application method on the content of chlorophyll b, total chlorophyll (a + b), and carotenoids
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Table 6 Interaction effect of the nitrogen application rate and split application method on the content of chlorophyll a, total chlorophyll (a + b), and carotenoids in 2019
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The highest chlorophyll b content in both years of the experiment was obtained with the application rate of 150 kg N ha−1, which was significantly higher compared with other rates of nitrogen application in 2018. In 2019, the difference between the application rates of 50, 100, and 150 kg N ha−1 in relation to chlorophyll b content was not significant (Table 5). In 2018, there was no significant difference between various split application methods of nitrogen in terms of chlorophyll b content. However, in 2019, the three-split applications of nitrogen produced the highest chlorophyll b content and significantly increased its content compared to the four-split applications of nitrogen (Table 5).

In 2018, the highest total chlorophyll content was observed in the two-split applications of 150 kg N ha−1, which was significantly higher than other experimental treatments (Table 7). However, in 2019, the two-split application of 100 kg N ha−1 had the highest total chlorophyll content, showing no significant difference with the two-split application of 150 kg N ha−1 and the three-split application of 150, 100, and 50 kg N ha−1. The lowest total chlorophyll content was obtained in the absence of nitrogen in both years of the experiment (Table 6).

Table 7 Interaction effect of the nitrogen application rate and split application method on the content of total chlorophyll (a + b), plant height, and 1000-seed weight in 2018
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As the rate of nitrogen application increased, the carotenoid content of the leaves increased similarly to other photosynthetic pigments. In 2018, the application of 150 kg N ha−1 produced the highest carotenoid content, which was significantly higher than other rates of nitrogen application. This year, there was no significant difference between different methods of split application of nitrogen in terms of carotenoid content (Table 5). In 2019, the highest carotenoid content was assigned to the two-split applications of 100 kg N ha−1, which was not significantly different from the three and two-split applications of 150 kg N ha−1. The control treatment had the lowest carotenoid content in both years of the experiment (Table 6).

3.4 Plant Height

Compared to the control treatment, the application of different rates of nitrogen increased the height of quinoa. In 2018, the highest plant height (76.1 cm) was obtained in the two-split applications of 150 kg N ha−1, which was not significantly different from the four-split application of the same rate of nitrogen (74.4 cm) but was superior to other experimental treatments. However, at the application rates of 100 and 50 kg N ha−1, the highest plant height was obtained in the four-split applications of nitrogen (Table 7). In 2019, the highest plant height (61.0 cm) was observed at the application rate of 150 kg N ha−1, which was not significantly different from the application rates of 100 and 50 kg N ha−1. This year, the highest plant height (57.3 cm) was recorded for the four-split application of nitrogen. However, no significant difference was observed between the four-split application and the two equal split applications of nitrogen at the 6–8 leaves and anthesis stages (Table 8).

3.5 Number of Seeds per Plant

The rate of nitrogen application affected the number of seeds per plant. In 2018, the number of seeds per plant increased with increasing nitrogen application rate. The lowest number of seeds per plant was observed in the control treatment and the highest in the application rate of 150 kg N ha−1. There was a significant difference between nitrogen split application methods in terms of the number of seeds per plant. The two equal split applications of nitrogen at the 6–8 leaves and anthesis stages had the highest number of seeds per plant, which was significantly higher than the three-split applications of nitrogen; however, it did not differ significantly from the four-split applications of nitrogen (Table 8). In 2019, the two-split applications of 100 kg N ha−1 produced the highest number of seeds per plant, which was significantly higher than other experimental treatments. However, there was no significant difference between different nitrogen split application methods at the application rate of 150 kg N ha−1. The lowest number of seeds per plant, similar to the first year of the experiment, was produced in the absence of nitrogen application (Table 9).

Table 8 The effects of nitrogen application rate and split application method on the plant height, seed number per plant, and 1000-seed weight
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3.6 The 1000-Seed Weight

In 2018, the three-split applications of 100 kg N ha−1 produced the highest 1000-seed weight, which was not significantly different from the three-split applications of 150 kg N ha−1 (Table 7). In 2019, the highest 1000-seed weight was obtained in the three-split applications of 50 kg N ha−1, which was significantly higher than other experimental treatments. At the application rate of 150 kg N ha−1, the highest 1000-seed weight was observed in the three-split applications of nitrogen, which was not significantly different from the four-split application. However, at the application rate of 100 kg N ha−1, there was no significant difference between different levels of nitrogen split application in relation to the 1000-seed weight (Table 9).

Table 9 Interaction effect of the nitrogen application rate and split application method on the seed number per plant, 1000-seed weight, and aboveground biomass in 2019
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3.7 Seed Yield

The application of different rates of nitrogen significantly increased the seed yield of quinoa compared to the control treatment in both years of the experiment. In 2018, the highest seed yield (3944.4 kg ha−1) was produced by two equal split applications of 150 kg N ha−1, which was significantly higher than other experimental treatments. At the application rate of 100 kg N ha−1, the two-split applications of nitrogen had the highest seed yield (3433.3 kg ha−1), showing no significant difference with the three and four-split applications of 150 kg N ha−1, as well as the four-split applications of 100 and 50 kg N ha−1 (Fig. 2). In 2019, the two equal split applications of 100 kg N ha−1 produced the highest seed yield (2748.9 kg ha−1), which was significantly higher than other treatments. The second rank of seed yield (2266.9 kg ha−1) in this year was allocated to the four-split applications of 100 kg N ha−1, showing no significant difference with the two-split applications of 150 kg N ha−1 and four-split applications of 50 kg N ha−1 (Fig. 3).

Fig. 2
figure 2

Interaction effect of the nitrogen application rate and split application method on the seed yield of quinoa in 2018. Plants were grown under different nitrogen application rates (0, 50, 100, 150 kg N ha−1) and split application methods (4 N splits four equal splits at planting, 6–8 leaves, anthesis, and the seed filling stages; 3 N splits three equal splits at 6–8 leaves, anthesis, and the seed filling stages; 2 N splits two equal splits at 6–8 leaves and the anthesis stages). Bars headed by the same letter are not significantly different at P < 0.05, according to Duncan’s multiple range test. Error bars indicate the standard error of the mean

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Fig. 3
figure 3

Interaction effect of the nitrogen application rate and split application method on the seed yield of quinoa in 2019. Plants were grown under different nitrogen application rates (0, 50, 100, 150 kg N ha−1) and split application methods (4 N splits four equal splits at planting, 6–8 leaves, anthesis, and the seed filling stages; 3 N splits three equal splits at 6–8 leaves, anthesis, and the seed filling stages; 2 N splits two equal splits at 6–8 leaves and the anthesis stages). Bars headed by the same letter are not significantly different at P < 0.05, according to Duncan’s multiple range test. Error bars indicate the standard error of the mean

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Most of the determined characteristics, including number of days to flowering, seed filling period, chlorophyll a content, chlorophyll b content, total chlorophyll content, plant height, number of seeds per plant, aboveground biomass, harvest index, and 1000-seed weight, were positively (P < 0.01) correlated with seed yield in quinoa, while the carotenoid content was negatively (P < 0.01), correlated with seed yield (Table 4).

3.8 Aboveground Biomass

The application of nitrogen fertilizer significantly increased the aboveground biomass of quinoa compared to the control treatment during the experimental years. In 2018, the aboveground biomass increased as the nitrogen application rate increased. The lowest and the highest aboveground biomass (4575.2 kg ha−1 and 7921.2 kg ha−1) were obtained in the conditions of non-application and application of 150 kg N ha−1, respectively (Table 10). There was a significant difference between various split application methods of nitrogen in relation to aboveground biomass. The highest aboveground biomass was produced in the two-split applications of nitrogen, which was not significantly different from the four-split applications of nitrogen; however, it was significantly higher than the three-split applications of nitrogen at the 6–8 leaves, anthesis, and seed filling stages (Table 10). In 2019, the two equal split applications of 100 kg N ha−1 produced the highest aboveground biomass (7628.9 kg ha−1), which was significantly higher than other treatments. At the application rate of 150 kg N ha−1, the highest aboveground biomass (6464.9 kg ha−1) was produced in the two-split applications of nitrogen, which was not significantly different from the four-split applications of 100 kg N ha−1. However, in the application of 50 kg N ha−1, the highest aboveground biomass (5896 kg ha−1) was obtained by the four-split application of nitrogen (Table 9).

Table 10 The effects of nitrogen application rate and split application method on the seed yield, aboveground biomass and harvest index (mean ± SE)
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3.9 Harvest Index

The application of different rates of nitrogen fertilizer reduced the harvest index in both years of the experiment compared to the conditions of absence of nitrogen application. In 2018, there was no significant difference between 50, 100, and 150 kg N ha−1 in relation to the harvest index, whereas in 2019, the harvest index under 100 kg N ha−1 treatment was significantly lower compared to the 50 and 150 kg N ha−1 (Table 10).

There was a significant difference between nitrogen split application methods in terms of the harvest index in both years. The highest harvest index in 2018 and 2019 were obtained under four and three-split applications of nitrogen, respectively. The difference between the four and three-split application methods of nitrogen was not significant in 2019 (Table 10).

Harvest index had positive and significant correlation with number of days to flowering (r = 0.31**), seed filling period (r = 0.27*), number of seeds per plant (r = 0.24*), seed yield (r = 0.42**), and 1000-seed weight (r = 0.62**). However, there were no significant correlations between the harvest index and chlorophyll content, plant height, and aboveground biomass (Table 4).

4 Discussion

Nitrogen management in quinoa production systems is one of the main concerns since it is the most important and primary nutrient for growth and development of the crop. The appropriate rate and timing of nitrogen application can enhance yield productivity and nutrient use efficiencies while reducing the environmental pollution (Abebe and Feyisa 2017).

The results showed that the nitrogen fertilization delayed the flowering and increased the length of seed-filling period compared to unfertilized control. In both experimental years, two-split applications at 6–8 leaves and the anthesis stages delayed the flowering and increased the seed-filling period compared to other methods of nitrogen splitting.

The change from vegetative to reproductive stage is an important event in the life cycle of plants. Plant flowering begins with a very complex network of regulatory processes controlled by the interrelationships between endogenous and environmental factors. Essential nutrients play a special role in regulating the flowering of plants. Nutrients affect the growth and development of plants and, consequently, the flowering through various physiological and metabolic processes (Ye et al. 2019). It has been reported that the application of nitrogen fertilizer has a clear role in stimulating flower development in rice (Wang et al. 2008). In the study of the effect of nitrogen, phosphorus, and potassium on the flowering time of rice, the results showed that the effect of nitrogen fertilizer was stronger than phosphorus and potassium fertilizers (Ye et al. 2019). Non-application of nitrogen accelerated flowering, and increasing the rate of nitrogen application delayed the flowering of rice, which is consistent with the results of the present study. Low soil nitrogen availability and other stress conditions can cause accelerated senescence, reducing the time period of nutrient translocation and thus penalizing yield and quality (Bascuñán-Godoy et al. 2018).

Nitrogen is one of the constituents of many important molecules such as proteins, nucleic acids, some hormones, chlorophyll, and other types of primary and secondary plant materials. Nitrogen is involved in chlorophyll formation, vegetative growth, and plant protein production and is a major component of chlorophyll structure (Leghari et al. 2016). In this study, the application of nitrogen and increasing its rate increased the content of chlorophyll a, chlorophyll b, total chlorophyll, and carotenoids in the leaves of quinoa. This is consistent with the results reported by Adelusi and Oseni (2015) on increasing the chlorophyll a and b content in tomato leaves with increasing nitrogen rate.

It has been reported that the content of carotenoids is directly related to nitrogen content. Mohammadi et al. (2016) reported that nitrogen fertilizer treatments increased the content of chlorophyll a, chlorophyll b, and carotenoids in safflower leaves.

The application of nitrogen fertilizer stimulated vegetative growth and canopy development and, as a result, increased the height of the quinoa during the experimental years so that the plant height increased with increasing the rate of nitrogen consumption. The highest plant height in 2018 and 2019 was obtained in the two-split applications and the four-split application of nitrogen, respectively. In both split application methods, half of the nitrogen fertilizer was used in the vegetative growth stage. This result indicates the importance of using nitrogen at the beginning of the season in increasing the growth characteristics of the plant, especially plant height. It has been reported that the height of quinoa increases with increasing nitrogen rates, mainly due to the role of nitrogen in stimulating metabolic activity, which contributes to increasing the amount of metabolites, which in turn leads to elongation of internodes and increased plant height. Shams (2012) found that the height of quinoa gradually increased with increasing nitrogen rates to 360 kg ha−1, which was consistent with the results reported by Geren (2015) on quinoa.

A number of seeds per plant and 1000-seed weight are two important parameters in determining the yield of crops. Although these two parameters are dependent on genetics and cultivar, environmental factors also play an important role in determining the value of these parameters. In the present study, with increasing nitrogen application up to 150 kg ha−1 in the first year and 100 kg ha−1 in the second year, the number of seeds per plant increased. Improving vegetative growth and availability of more photosynthetic assimilates can be mentioned as the reason for increasing the number of seeds per plant by increasing nitrogen application. The different response of seed number per plant to the rate of nitrogen application in the experimental years can be attributed to the difference in climatic conditions so that more favorable climatic conditions of the first year, especially in the reproductive growth stage, caused a better response of quinoa to nitrogen application. Similar to the results of this study, Shahmansouri (2015) and Saeidi et al. (2020) also showed that increasing the rate of nitrogen application increased the number of seeds per plant in quinoa.

According to the results, the reduction in the number of seeds per plant in the three-split applications of nitrogen (Table 8) increased the seed weight and produced the highest 1000-seed weight due to the increased distribution of photosynthetic assimilates to each seed. In other words, photosynthetic assimilates were divided between fewer seeds, and therefore the 1000 seed weight increased. On the other hand, in the three-split applications of nitrogen, one-third of nitrogen fertilizer was used at the beginning of seed filling stage, which has been effective in increasing the 1000-seed weight by improving photosynthesis and increasing the production of photosynthetic assimilates in this stage.

In the first year, the quinoa seed yield increased with increasing nitrogen application rate from zero to 150 kg ha−1. In the second year, however, the application rate of 100 kg N ha−1 produced the highest seed yield compared to other levels of nitrogen. In the present study, changes in climatic conditions during crop growth periods (Fig. 1) caused large changes in growth characteristics, yield, and yield components of quinoa during the experimental years. The lower average monthly temperature, especially during September and October (reproductive growth period of quinoa) in 2018 compared to the same period of 2019 and also the occurrence of significant rainfall during the seed filling period (October) in 2018, delayed the onset of reproductive growth, increased the length of seed filling period, and as a result increased the yield and yield components of quinoa in 2018. High temperature susceptibility has been reported as a serious obstacle to global quinoa production. Hinojosa et al. (2018) showed that heat stress reduced the pollen viability of quinoa between 30 and 70%. It has been reported that the number of seeds per plant of quinoa cultivars decreases with increasing the average air temperature in the flowering stage and reducing the length of the seed formation period. The coincidence of flowering, pollination, and seed filling stages with end-of-season heat stress reduces seed yield due to the reduction in the number of seeds and the 1000-seed weight (Tavoosi and Sepahvand 2014). Similar results were observed by Belete et al. (2018) regarding seed yield and nitrogen use efficiency in wheat. Lopez-Bellido et al. (2005) also pointed out the correlation between nitrogen fertilizer, wheat yield, and seasonal changes so that wheat yield decreased during rainy years, while in dry years, nitrogen fertilizer had no significant effect or had little effect on wheat yield.

According to the results, the two equal split applications of nitrogen increased the number of seeds per plant, seed yield, and aboveground biomass as compared with the three equal split applications and four equal split applications of nitrogen. The increase in the quinoa yield in the two equal split applications of nitrogen at the 6–8 leaves and anthesis stages may be due to the better adaptation of nitrogen availability to crop requirements during the growing season. Previous studies have shown that the application of nitrogen in the heading phase increases barley seed yield and seed weight. Similar results were obtained for wheat in southwestern Japan (Hattori 1994). However, other studies have shown that its application does not increase barley seed yield or grain weight (Nakano and Morita 2009). Therefore, the response of seed yield and seed weight to the time and rate of nitrogen application may depend on environmental factors such as the year of the climate and location characteristics (Tanaka and Nakano 2019).

The most important role of nitrogen in plants is its presence in the structure of proteins and nucleic acids, which are the most important structural and information material of each cell. Moreover, nitrogen is found in the structure of plant pigments, especially chlorophyll, which enables the plant to absorb solar energy during the process of photosynthesis. Therefore, the availability of nitrogen to the plant affects the amount of protein, amino acids, protoplasm, and chlorophyll formed. In addition, nitrogen affects cell size, leaf area, and photosynthetic activity. Therefore, adequate nitrogen availability is essential to achieve high yield potential in crops (Seyed Sharifi and Namvar 2016).

In the present study, the highest harvest index was obtained in the absence of nitrogen fertilizer application and nitrogen application reduced the harvest index in both years of the experiment. Harvest index, which is the ratio of seed yield to aboveground biomass, indicates the efficiency of distribution of photosynthetic assimilates among different organs of the plant. Harvest index indicates the amount of plant biomass allocated to the seed and is therefore an indicator of the plant’s ability to allocate resources between vegetative and reproductive structures (Carrotero et al. 2010). The distribution of photosynthetic assimilates between vegetative and reproductive parts can be changed by agricultural operations such as planting date, plant density, fertilization, and irrigation (Geren 2015). Considering the increased seed yield and aboveground biomass of quinoa in response to nitrogen application, the greater effect of nitrogen on increasing aboveground biomass can be mentioned as the reason for the decrease in harvest index under nitrogen application conditions. Contrary to the results of the present study, Alandia et al. (2016) reported that harvest index of quinoa was positively affected by N both under irrigation and drought conditions. Geren (2015) showed that the harvest index of quinoa increased with increasing application of nitrogen up to 150 kg ha−1 but later on decreased.

5 Conclusion

Our work clearly demonstrated that quinoa responded strongly to nitrogen application rate and split application method. The effect of nitrogen fertilizer on increasing seed yield was due to increasing the number of seeds per plant and 1000-seed weight, which can be related to increasing the number of days to flowering, seed filling period, increasing the content of photosynthetic pigments and improving vegetative growth. Two equal split applications of nitrogen at the 6–8 leaves and anthesis stages had the better adaptation of nitrogen availability to crop requirements during the growing season so that increased the number of seeds per plant, seed yield, and aboveground biomass as compared with the three equal split applications and four equal split applications of nitrogen. Changes in climatic conditions during crop growth periods affected quinoa response to nitrogen fertilization. The lower average monthly temperature, especially during September and October (reproductive growth period of quinoa) and also the occurrence of significant rainfall during the seed filling period (October) in 2018, caused a better response of quinoa to nitrogen application. Finally, our results indicated that two equal splits adding to 100 kg N ha−1 at the 6–8 leaves and the anthesis stages is recommended to produce the optimal yield of quinoa in similar climatic conditions.