**Reduction in Nitrogen Rate and Improvement of Nitrogen Use Efficiency without Loss of Peanut Yield by Regional Mean Optimal Rate of Chemical Fertilizer Based on a Multi-Site Field Experiment in the North China Plain**

**Jiayu Hu 1, Yang Yang 2, Hongyan Zhang 1, Yuhao Li 2, Shuhong Zhang 2, Xinhua He 3,4, Yufang Huang 1, Youliang Ye 1, Yanan Zhao 1,\* and Jungying Yan 5,\***


**Abstract:** It is important to quantify nutrient requirements and optimize fertilization to improve peanut yield and fertilizer use efficiency. In this study, a multi-site field trial was conducted from 2020 to 2021 in the North China Plain to estimate nitrogen (N), phosphorus (P), and potassium (K) uptake and requirements of peanuts, and to evaluate the effects of fertilization recommendations from the regional mean optimal rate (RMOR) on dry matter, pod yield, nutrient uptake, and fertilizer use efficiency. Results show that compared with farmer practice fertilization (FP), optimal fertilization (OPT) based on the RMOR increased peanut dry matter by 6.6% and pod yield by 10.9%. The average uptake rates of N, P, and K were 214.3, 23.3, and 78.4 kg/ha, respectively, with 76.0% N harvest index, 59.8% P harvest index, and 41.4% K harvest index. The OPT treatment increased N, P, and K uptake by 19.3%, 7.3%, and 11.0% compared with FP, respectively. However, the average of yield, nutrition uptake, and harvest indexes of N, P, and K were not significantly affected by fertilization. The peanut required 42.0 kg N, 4.6 kg P, and 15.3 kg K to produce 1000 kg of pods. The OPT treatment significantly improved the N partial factor productivity and N uptake efficiency but decreased the K partial factor productivity and K uptake efficiency. The present study demonstrates that fertilizer recommendations from RMOR improve N use efficiency, and reduce N and P fertilizer application without yield loss in regions with smallholder farmers, and the corresponding estimation of nutrient requirements helps to make peanut fertilization recommendations.

**Keywords:** *Arachis hypogaea* L.; fertilizer use efficiency; nutrient uptake; optimal fertilization; pod yield

#### **1. Introduction**

Peanut is an important oilseed crop, an N2-fixing legume plant with drought tolerance, and a very efficient cash crop with relatively low production input, high yield, and higher price and greater income than other oil crops [1]. The extraction rate of peanut oil is high, and the oil quality is good. Therefore, peanut plantations play an important role in meeting edible oil demand and supporting local economies. China is a major peanut producer, ranking second in planting area after India and first in total production [2]. In 2020, the planting area reached five million hectares, and the total production reached 18 million tons, accounting for more than 20% and 40%, respectively, of the global totals [2]. Nitrogen

**Citation:** Hu, J.; Yang, Y.; Zhang, H.; Li, Y.; Zhang, S.; He, X.; Huang, Y.; Ye, Y.; Zhao, Y.; Yan, J. Reduction in Nitrogen Rate and Improvement of Nitrogen Use Efficiency without Loss of Peanut Yield by Regional Mean Optimal Rate of Chemical Fertilizer Based on a Multi-Site Field Experiment in the North China Plain. *Plants* **2023**, *12*, 1326. https:// doi.org/10.3390/plants12061326

Academic Editors: George Lazarovits, Christian Dimkpa, Jörg Gerke and Dimitris L. Bouranis

Received: 5 November 2022 Revised: 7 March 2023 Accepted: 8 March 2023 Published: 15 March 2023

**Copyright:** © 2023 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

(N), phosphorus (P), and potassium (K) are essential nutrients for crops. Application of NPK fertilizer can improve crop yield [3].

Although peanut is a legume with the ability to fix atmosphere N, its growth requires adequate N, P, and K nutrients by fertilization [3]. The N fixation by rhizobia can meet 40% to 50% of the N requirement of peanut plants [4]. However, the N fixation level by peanut rhizobia is significantly and negatively correlated with increasing N application [4]. China is the world's largest consumer of chemical fertilizer [5]. In recent years, unreasonable fertilization in pursuit of higher crop yields has become common, while yield response to fertilization has become lower. Unreasonable fertilizer application not only increases production costs and reduces FUE but also causes environmental pollution [5,6]. Therefore, it is an important challenge for sustainable peanut production to ensure high peanut yield, quality, and FUE through rational fertilization.

In recent decades, studies have attempted to develop various methods to optimize the application rates of chemical fertilizer to solve the associated economic and environmental problems caused by improper application. These techniques of fertilization recommendation were based on soil testing [7], aboveground plant analysis [8], yield–fertilizer rate response modeling [9], digital and information technology [10–12], and integrated soil-crop system management [13,14]. Most of these recommended techniques have focused on sitespecific optimal nutrient rates and have required field trials, soil samples, plant collection, and laboratory analysis, factors that can be rather time-consuming, labor-intensive, and expensive. Although these methods are useful for precision fertilization, they are rather difficult to implement widely for many smallholders in developing countries including China [15]. Firstly, arable land in these countries/regions is limited, with small sizes and scattered field plots. Secondly, in the rural regions of these areas, there is a lack of testing conditions and technical guidance for agrochemical services, with insufficient testing equipment and technical personnel. Finally, these field sites are highly intensive with two or sometimes three crops planted annually in the same field, and testing is difficult to perform in time to avoid delaying planting.

In the practice of agricultural production, it is critical to establish a simple method for the overall regulation and promotion of the rational application of fertilizer on a regional scale rather than a field scale [16]. Numerous factors affect crop yields, and some are difficult to predict accurately. Therefore, even for the same field, the suitable fertilizer application rate and related parameters obtained from an experiment in one year may not be accurately applied to other years [17]. Accordingly, a very high degree of accuracy is unnecessary to calculate the appropriate fertilizer application rate for obtaining a certain yield. For agricultural production, the determination of a suitable fertilizer application range can basically meet the needs of large-scale production.

A regional mean optimal rate (RMOR) of N fertilizer application has been proposed to resolve this technology gap in Tai Lake Region, China [18]. The purpose of the resolution is to provide a baseline fertilization recommendation for a specific region by determining the RMOR of this region to obtain the maximum total production and economic benefits. The RMOR can be the average of the optimal fertilizer rate obtained from the multi-site trial, and is further used as the fertilization recommendation for the whole region [18]. However, the optimal fertilizer rate can be determined by other methods, such as nutrient requirement to produce unit yield. The method has been proven feasible for crops such as rice, wheat, and oilseed rape; the recommendations have produced increasing yields, income, and nutrient use efficiency and have reduced environmental impacts [19,20]. Adjusting the RMOR for different regions, crop types, and soil conditions can provide a more accurate estimation of the optimal rate of fertilizer applied. However, the effectiveness of this resolution in other regions, crop types (N-fixing crops such as peanut), and nutrients (P and K fertilizer) is unknown.

Here, a two-year period (2020 and 2021) of multi-site field experiments were conducted in the largest peanut-growing area of the North China Plain. The hypothesis is that the ROMR method can maintain peanut yield and FUE while reducing the chemical fertilizer at a regional scale. The overall goals of this study were to quantify (1) N, P, and K uptake and requirements; (2) effects of the RMOR on peanut yield, nutrient uptake, and utilization efficiency; and hence (3) effectiveness of this RMOR method in peanut farming at the county scale.

#### **2. Materials and Methods**

#### *2.1. Experimental Site*

The field experiments were conducted in 2020 and 2021 in several townships in Zhengyang County (N32◦16'-32◦47', E114◦12 -114◦53 ), Henan, China (Figure 1). Both the peanut planting area and production of Zhengyang county rank first among all counties in China. The area is located in the transition region from the north subtropics to the warm temperate zone, and it has a continental monsoon humid climate. The average annual temperature is 15.3 ◦C, and the average annual precipitation is 935 mm. The monthly temperature and rainfall are shown in Figure 2 [21].

**Figure 1.** The geographical locations of experimental field sites. (**a**) Henan Province is shown as a red background in the China map; (**b**) the blue border is the Zhengyang County; (**c**) distribution of experimental field sites in the Zhengyang County.

#### *2.2. Field Experiment Design*

There were eight field sites in 2020 and five sites in 2021 for the experiment (Figure 1). The planting pattern was summer peanut and winter wheat rotation. The soil type was Alfisol according to the Soil Taxonomy with clay texture and low pH value. The basic soil physiochemical properties are shown in Table 1.

Each field experiment was set up with two treatments, farmers' practice fertilization (FP) and optimized fertilization (OPT). All management of the FP treatment was determined by farmers based on their practices, and the rate of fertilizer applied was accurately recorded (Table 1). As a management strategy focused technology promotion, the determination of RMOR must be based on other approaches or data. Therefore, for the OPT treatment, the RMOR was determined based on the results of previous studies with similar environment, in which, the optimized amount of fertilizer was determined according to the yield–fertilizer

rate response modeling [22]. In brief, a total of 46 field trials including four NPK fertilizer rates were collected. The average value of yield and fertilizer application under the same treatment was obtained. The yield–fertilizer rate response modeling was simulated by quadratic equation (y = ax<sup>2</sup> + bx + c, y: yield; x: N, P, or K fertilizer rates). Then the RMOR for NPK fertilizer was calculated at the maximum yield according to the modeling (-b/2a). The fertilizer recommendations were further adjusted by nutrient experts based on local conditions. The N, P, and K fertilizer rates for OPT in 2020 and 2021 were 181.5 kg/ha, 39.3 kg/ha, and 93.4 kg/ha, respectively (Figure S1). The other management measures were completely consistent with the FP treatment. Each treatment was repeated three times in plots of greater than 100 m2.



All peanuts were sown in early June and harvested at the end of September. The peanut varieties were decided by the farmers themselves, and they all used the widely grown varieties of momordica fruit type. Before peanut sowing, soil was plowed and then tilled. The peanuts were planted in ridges with a width of 80 cm at the bottom and 60 cm at the surface. Two rows of peanuts were planted on the ridge surface with a spacing of 20 cm. The overall sowing rate was 225 kg/ha. Ridging, sowing, and fertilizing were completed by mechanized methods performed once. Other management such as weed, pest, and disease control was consistent with FP treatment.

#### *2.3. Sampling and Measurement*

At peanut maturity, 2 m<sup>2</sup> peanut samples were taken for yield measurement in each plot of all experiment sites. The air-dried sample was weighed and its moisture content was determined, and further converted to the weight with a moisture content of 8%. Meanwhile, five representative plants were taken at random for dry matter (DM) and nutrient measurement. The haulm and pod were cleaned and sterilized at 105 ◦C for 30 min, then dried at 75 ◦C. After the plant and pod were dried to a constant weight, the DM weight (plant and pod) was recorded. The dried samples were crushed and passed through a 2 mm sieve for the determination of N, P, and K contents. A 0.2 g sample was digested with H2SO4-H2O2 to obtain a solution. The total N and P contents were determined using a flow injection analyzer (AA3, Seal, Germany), and the K content was determined by a flame photometer [23]. The fatty acid composition of peanut seeds was measured by the gas chromatography.

#### *2.4. Data Processing and Analysis*

Taking N as an example, parameters of NHI (N harvest index), RIEN (N reciprocal internal efficiency), PFPN (N fertilizer partial factor productivity), NUpE (N uptake efficiency), and NUtE (N utilization efficiency) were used to evaluate the characteristics of nutrient uptake and utilization [24,25]. The parameters were calculated as follows:


The data were processed by Microsoft Excel 2016 software and graphed by Origin Lab Origin 2018 software. The statistical analysis of data were performed in SPSS 20.0 software. A one-way ANOVA was used for significant tests at *p* < 0.05.

#### **3. Results**

#### *3.1. Dry Matter (DM), Pod Yield (PY), and Harvest Index (HI)*

The PY ranged from 1958 kg/ha to 6915 kg/ha and averaged 4879 kg/ha for 13 field sites in 2 years. Compared with the FP treatment, OPT significantly increased PY in six field sites (Figure 3a). The yield increases by the OPT treatment ranged from -784 kg/ha to 1549 kg/ha and averaged 10.9%. The response of DM to different treatments was consistent with PY at 13 sites over 2 years (Figure 3b). Compared with FP, the DM increase in OPT ranged from −1179 kg/ha to 1544 kg/ha with an average rate of 6.6%. The variation range of peanut HI was 39.9%–64.0% with an average of 54.0% (Figure 3c). The peanut HI under OPT increased significantly in three field sites while decreasing significantly in other three field sites. There was no significant change in HI in total between FP and OPT treatments.

**Figure 3.** Variations in dry matter (DM, (**a**)), pod yield (PY, (**b**)), and harvest index (HI, (**c**)) of different fertilization treatment at different field sites in 2020 and 2021. FP: farmers' practice fertilization; OPT: optimized fertilization. A: experiment in 2021; B: experiment in 2022. Tentacle lines on the bars are standard deviation. Asterisks (\*) on the bars indicate a significant difference at *p* < 0.05 between fertilization treatments for the same field site.

#### *3.2. N, P, and K Uptake*

There was significant variation in peanut nutrient uptake at different sites (Figure 4). For the two years, total N, P, and K uptake rates were 96.3–364.2, 6.2–42.6, and 23.0–110.3 kg/ha, and averaged 214.3, 23.3, and 78.4 kg/ha, respectively (Figure 4a,c,e). The total N, P, and K uptake rates under OPT were generally higher than in the FP treatment, with increasing rates of 19.3%, 7.3%, and 11.0%, respectively. In general, the N and P uptake rates by peanut straw were lower than for pods, while the K uptake was higher by straw than by pods, with a lower KHI (Figure 4f). The N, P, and K uptake rates were 164.3, 14.0, and 31.8 kg/ha in pods, and 50.0, 9.3, and 46.6 kg/ha in straw, respectively. For these 2 years, the NHI, PHI, and KHI were 61.7%-86.1%, 48.6%-74.7%, and 20.9%-60.7%, respectively, and averaged 76.0%, 59.8%, and 41.4%, respectively (Figure 4b,d,f). Although there were differences between FP and OPT in several field sites, in general, the OPT treatment did not significantly affect nutrient harvest indexes of peanuts in 13 field sites.

#### *3.3. Reciprocal Internal Efficiency (RIE)*

For 13 field sites in the 2 years, the RIE of N (RIEN) averaged 42.0 kg with a range of 26.8–54.7 kg (Figure 5a). The averaged RIEN was 6.9% higher under OPT than under FP. The RIE of P (RIEP) in the 2 years varied from 2.7 kg to 7.5 kg with an average of 4.6 kg (Figure 5b). The RIE of K (RIEK) varied from 11.1 kg to 19.9 kg with an average of 15.3 kg (Figure 5c). There was no significant difference in averaged RIEK or RIEP between FP and OPT treatments.

**Figure 4.** Uptake and nutrient harvest indexes of N (**a**,**b**), P (**c**,**d**), and K (**e**,**f**) in peanuts under different fertilization treatments. FP: farmers' practice fertilization; OPT: optimized fertilization. A: experiment in 2021; B: experiment in 2022. Tentacle lines on the bars are standard deviation. Asterisks (\*) on the bars indicate a significant difference at *p* < 0.05 between fertilization treatments for the same field site.

**Figure 5.** Requirements of N (**a**), P (**b**), and K (**c**) to produce 1000 kg pod yield of peanuts (reciprocal internal efficiency (RIE) under different treatments. FP: farmers' practice fertilization; OPT: optimized fertilization. A: experiment in 2021; B: experiment in 2022. Tentacle lines on the bars are standard deviation. Asterisks (\*) on the bars indicate a significant difference at *p* < 0.05 between fertilization treatments for the same field site.

#### *3.4. Partial Factor Productivity of NPK Fertilizer*

Compared with the FP treatment, the average PFPN of OPT increased by 15.1% in 2020 and by 29.8% in 2021 (Figure 6a). The average PFPP of the OPT treatment decreased by 2.4% in 2020 and increased by 3.4% in 2021 compared with FP (Figure 6b). However, the average PFPK of OPT decreased by 23.7% and 31.5% in the 2 years, respectively (Figure 6c).

**Figure 6.** Partial factor productivity of N (**a**), P (**b**), and K fertilizer (**c**) under different fertilization treatments. FP: farmers' practice fertilization; OPT: optimized fertilization. For each boxplot, the top, middle, and bottom solid lines of the box represent the 75% quantile, 50% quantile, and 25% quantile, respectively; the top and bottom horizontal lines outside the box represent the 90% quantile, and 10% quantile, respectively. The square inside the box represents the mean. Blue or red diamonds: data in 2020 or 2021.

#### *3.5. Nutrient Uptake Efficiency*

There was a significant variation in NUpE among different field sites (Figure 7a). From 2020 to 2021, the average NUpE of the OPT treatment was increased by 19.3% and 45.2% compared with FP. The average PUpE of OPT decreased by 6.5% in 2020 and increased by 3.4% in 2021 compared with FP (Figure 7b). However, the average KUpE of OPT decreased by 25.5% and 32.2% compared with FP (Figure 7c).

**Figure 7.** Uptake efficiency of N (**a**), P (**b**), and K (**c**) in peanuts under different fertilization treatments. FP: farmers' practice fertilization; OPT: optimized fertilization. For each box plot, the top, middle, and bottom solid lines of the box represent the 75% quantile, 50% quantile, and 25% quantile, respectively; the top and bottom horizontal lines outside the box represent the 90% quantile, and 10% quantile, respectively. The square inside the box represents the mean. Blue or red diamonds: data in 2020 or 2021.

#### *3.6. Nutrient Utilization Efficiency*

Compared with FP, the average NUtE for OPT was reduced by 3.8% in 2020 and by 11.7% in 2021 (Figure 8a). The average PUtE for OPT had a 4.8% increase in 2020 but an 11.7% decrease in 2021 compared with FP (Figure 8b). No clear difference in averaged KUtE between FP and OPT treatments was found in the 2 years (Figure 8c).

**Figure 8.** N (**a**), P (**b**), and K utilization efficiency (**c**) of peanuts under different fertilization treatments. FP: farmers' practice fertilization; OPT: optimized fertilization. For each box plot, the top, middle, and bottom solid lines of the box represent the 75% quantile, 50% quantile, and 25% quantile, respectively; the top and bottom horizontal lines outside the box represent the 90% quantile, and 10% quantile, respectively. The square inside the box represents the mean. Blue or red diamonds: data in 2020 or 2021.

#### *3.7. Fatty Acid Composition of Peanut Kernels*

In terms of the fatty acid composition of peanut kernels, oleic and linoleic acids accounted for 42.1% and 36.7%, respectively, followed by palmitic acid and stearic acid at 10.7% and 5.0%, respectively (Figure 9a). The ratio of oleic acid to linoleic acid (O/L ratio) was only 1.2 (Figure 9b). In general, the OPT treatment did not significantly affect the fatty acid composition or O/L of peanut kernels compared with FP.

**Figure 9.** Fatty acid composition (**a**) and oleate/linoleate ratio (O/L, (**b**)) of peanut kernels under different fertilization treatments. FP: farmers' practice fertilization; OPT: optimized fertilization. For each box plot, the top, middle, and bottom solid lines of the box represent the 75% quantile, 50% quantile, and 25% quantile, respectively; the top and bottom horizontal lines outside the box represent the 90% quantile, and 10% quantile, respectively. The square inside the box represents the mean. Blue or red diamonds: data in 2020 or 2021.

#### **4. Discussion**

#### *4.1. Feasibility of Fertilizer Recommendation Based on RMOR*

There are some common concerns in developing countries such as small and scatted field sites, lack of test conditions, and lack of technical guidance for agrochemical services [15,26]. The present study demonstrated that the unified fertilization recommendations (i.e., RMOR) can achieve a reduction in chemical fertilizer and N use efficiency without yield loss in peanuts for smallholders in country scale. In these regions, this method has high practical value; the maximum benefit can be obtained, and the fertilizer recommendation is easy to promote. The theoretical basis of the RMOR method is to optimize the fertilizer application in the region scale, thus improving soil condition, the growth characteristics, and nutrient utilization of peanuts [22,27]. In this study, the N and P fertilizer rates according to RMOR were lower than the application rate in peanuts by the farmer practice in this region. Therefore, the simplified method can also save on N and P fertilizer. This is of great practical significance for decreasing the chemical fertilization rate while improving FUE, and thus decreasing the environmental risk, especially in areas with excessive chemical fertilizer consumption [28]. Once the RMOR for a specific crop in a certain region is determined, it can be applied for several years as long as there are no significant changes in climate or cultivation conditions. Therefore, the RMOR can be used as a basis for fertilizer development planning in the region, and as a basis for recommended fertilizer application rates for the crop in different field sites.

#### *4.2. Limitation and Uncertainty*

The RMOR is a management strategy for the optimization of regional fertilization, rather than a calculations method. Therefore, the determination of RMOR for a specific region needs to be based on other methods [19]. Meanwhile, applying the RMOR in the entire region, the benefits for individual farmers vary in this study; for some field sites, yields and nutrient efficiency even decrease, though it would not result in a significant yield loss for the region as a whole. The appropriate fertilizer rate for a given crop is affected by diverse factors such as management, soil, climate, and tillage [26,29]. This study showed that even within the same county there was significant variation in peanut yield and nutrient requirement. Therefore, the RMOR and its control range when applied should be delimited according to different conditions in specific regions and field sites and should be determined again with the changes in production environments. Furthermore, the adverse effects of the simplified practice can be decreased when combined with the specific conditions of each field, and then one can make appropriate adjustments of increase or decrease such as according to the soil fertility level, organic fertilizer application, and previous stubble [19,30]. Especially, for counties and regions with high requirements for precision fertilization, adjustment according to the specific situation of the local field and/or the necessary testing is needed to avoid the loss of yield and economic benefits from individual field sites.

#### *4.3. Nutrient Requirements of Peanut*

Compared with other studies with large sample sizes, the N requirement to produce 1000 pods in this study was similar to the result of 42.2 kg [25], but slightly lower than in other studies [31,32]; P and K demand was lower than what was estimated across China [31] and in the same region of central north China [32]. Estimation variation in nutrient requirement may be related to differences in planting regions, cultivars, soil conditions, yield level, and tillage [30,33]. Crucsiol et al. [3] demonstrated that N absorption for older cultivars remains high, while newer cultivars were less demanding in N. Xie et al. [25] found that the values of nutrient requirement simulated by modeling were lower than the average observed values, and they explained that the N, P, and K predicted by the model were the optimal nutrient requirements under the conditions of the balanced absorption of N, P, and K. However, high soil nutrient supply and excessive fertilization practices may have resulted in excess nutrient uptake [31]. A large number of studies have demonstrated that crop nutrient uptake positively correlates with yield level, but the yield increase decreases when the yield reaches a certain level [32]. As a result, nutrient requirements per unit yield tended to decrease as yields increased, especially when yields were above 70% of the potential yield [25]. The peanut yield has been strongly promoted in the past few decades in China due to cultivar renewal, adequate fertilization, soil improvement, and other management changes [32]. This means that the nutrient uptake requirements to produce unit pods may be decreasing.

#### **5. Conclusions**

The results of multi-site peanut experiments supported the hypothesis that compared with the FP treatment, the OPT treatment based on the RMOR method promoted N use efficiency (PFPN and NUPE), and decreased the nutrient inputs by chemical fertilizer, especially the N and P fertilizers, without the loss of peanut yield and NPK uptake. The NPK nutrient requirements of peanuts are quantified in this study, which is important for the regional recommendation of fertilization based on the RMOR. Therefore, the RMOR method is feasible for NPK fertilizer recommendations for peanut plantations, as it can simultaneously realize the optimization of agronomic, economic, and environmental benefits at a regional scale. The RMOR method can also be generally adopted in countries and regions with widespread smallholder farms. Furthermore, combined with precision fertilization technologies, the RMOR method is promising to realize agronomic and environmental optimization at the field scale.

**Supplementary Materials:** The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/plants12061326/s1, Figure S1: the yield-fertilizer rate response modeling for the RMOR of NPK fertilizer.

**Author Contributions:** Conceptualization, Y.Z. and J.Y.; methodology, Y.Z., Y.L., and S.Z.; validation, J.H., Y.Y. (Yang Yang), H.Z., and Y.L.; formal analysis, J.Y. and Y.Y. (Youliang Ye); investigation, H.Z. and Y.H.; data curation, Y.Z. and J.Y.; writing—original draft preparation, J.H.; writing—review and editing, Y.Z., X.H., and J.Y.; visualization, J.H.; supervision, J.Y.; project administration, Y.Z. All authors have read and agreed to the published version of the manuscript.

**Funding:** This study was financially supported by the Key Scientific Research Project of the Higher Education Institutions of Henan Province (21A210022), the Research and Practice Project of Higher Education Teaching Reform in Henan Province (2021SJGLX086), and the Open Research Project of the Innovation Center for the Efficient Use of Nitrogen Fertilizer from Henan Xinlianxin Chemical Industry Group Co., Ltd. (30801723).

**Institutional Review Board Statement:** Not applicable.

**Informed Consent Statement:** Not applicable.

**Data Availability Statement:** The data presented in this study are available on request from the corresponding author.

**Acknowledgments:** We thank Songhua Yue, Hongbo Bai, and Qun Li for their assistance during the experiments, Hui Yu of Zhengyang Peanut Institute, Henan, China for suggestions to the RMOR of chemical fertilizer, and LetPub (www.letpub.com) for its linguistic assistance during the preparation of this manuscript.

**Conflicts of Interest:** The authors declare no conflict of interest. Improvement of peanut yield and nutrient use efficiency by regional mean optimal rate of chemical fertilizer based on a multi-site field experiment in the North China Plain.

#### **References**


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## *Article* **Response of Normal and Low-Phytate Genotypes of Pea (***Pisum sativum* **L.) on Phosphorus Foliar Fertilization**

**Petr Škarpa 1,\*, Marie Školníková 1, Jiˇrí Antošovský 1, Pavel Horký 2, Iva Smýkalová 3, Jiˇrí Horácek ˇ 3, Radmila Dostálová <sup>3</sup> and Zdenka Kozáková <sup>4</sup>**


**Abstract:** Phosphorus (P) is an important nutrient in plant nutrition. Its absorption by plants from the soil is influenced by many factors. Therefore, a foliar application of this nutrient could be utilized for the optimal nutrition state of plants. The premise of the study is that foliar application of phosphorus will increase the yield of normal-phytate (*npa*) cultivars (CDC Bronco a Cutlass) and low-phytate (*lpa*) lines (1-2347-144, 1-150-81) grown in soils with low phosphorus supply and affect seed quality depending on the ability of the pea to produce phytate. A graded application of phosphorus (H3PO4) in four doses: without P (P0), 27.3 mg P (P1), 54.5 mg P (P2), and 81.8 mg P/pot (P3) realized at the development stages of the 6th true leaf led to a significant increase of chlorophyll contents, and fluorescence parameters of chlorophyll expressing the CO2 assimilation velocity. The P fertilization increased the yield of seeds significantly, except the highest dose of phosphorus (P3) at which the yield of the *npa* cultivars was reduced. The line 1-2347-144 was the most sensible to the P application when the dose P3 increased the seed production by 42.1%. Only the *lpa* line 1-150-81 showed a decreased tendency in the phytate content at the stepped application of the P nutrition. Foliar application of phosphorus significantly increased ash material in seed, but did not tend to affect the protein and mineral content of seeds. Only the zinc content in seeds was significantly reduced by foliar application of P in *npa* and *lpa* pea genotypes. It is concluded from the present study that foliar phosphorus application could be an effective way to enhance the pea growth in P-deficient condition with a direct effect on seed yield and quality.

**Keywords:** chlorophyll content; fluorescence parameters; seed yield; seed quality; seed nutrient content; pea; foliar application

#### **1. Introduction**

Pea (*Pisum sativum* L.) is a plentifully grown leguminous plant in many countries, and it can be utilized both in human nutrition as well as a part of the feed for farm animals. It is considered as one of the most important sources in human nutrition, because its pods contain a great content of proteins, carbohydrates, vitamins, and minerals. By its ability to hold nitrogen from the air and return it back to the soil, the pea contributes to sustainable agriculture [1]. Besides high demands for nitrogen, the pea belongs to those plants with relatively high demands for phosphorus (P), too.

Crop production on more than 30% of the world arable land is limited by the P availability [2]. P is an essential nutrient required by all plants to grow, photosynthesize,

**Citation:** Škarpa, P.; Školníková, M.; Antošovský, J.; Horký, P.; Smýkalová, I.; Horáˇcek, J.; Dostálová, R.; Kozáková, Z. Response of Normal and Low-Phytate Genotypes of Pea (*Pisum sativum* L.) on Phosphorus Foliar Fertilization. *Plants* **2021**, *10*, 1608. https://doi.org/10.3390/ plants10081608

Academic Editors: Przemysław Barłóg, Jim Moir, Lukas Hlisnikovsky and Xinhua He

Received: 26 June 2021 Accepted: 4 August 2021 Published: 5 August 2021

**Publisher's Note:** MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations.

**Copyright:** © 2021 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

and form proteins. It is especially limiting in organic environments for legumes, which need more P than cereals to form root nodules for nitrogen fixation [3,4]. In particular, P is important for the growth of the field pea, and for protein synthesis [4]. Sufficient amounts of phosphorus taken by plants are also necessary for an optimal production yield. In the case of low P supplies in the soil or conditions decreasing its intake by plants, fertilization by phosphorus significant increases not only legume production, but also its quality [5–11]. One of the phosphorus forms naturally presented in plants is phytic acid. Phytic acid is the major storage form of phosphorus in seeds of legumes. In the case of the pea, its highest concentration is in the endosperm [12]. Since phytate can form complexes with proteins and minerals, reducing the digestive availability of phosphorus [13], it is usually regarded as an antinutrient, although recent works indicate that it has important beneficial roles as an antioxidant for plants [14]. Therefore, there is an interest in the assessment and manipulation of phytate contents in important food grains such as peas. Through plant breeding, it is possible to prevent the phosphorus transformation into phytic acid and, thus, to decrease the content of phytic acid by even up to 50–95% [15]. For example, Wilcox et al. [16] reported an 80% decrease in P-phytate content in the low-phytate soybean. Reduction of phytate phosphorus concentration in the low-phytate pea seed by about 60% accompanied by an increase in free inorganic phosphorus was presented by Warkentin [17]. The content of phytic acid in the pea is influenced not only by various properties, but also by the climate, irrigation, and soil conditions as well as by the fertilization. The decrease of phytate concentration caused by phosphorus fertilization was observed in the case of soya [18] oat [19] and corn [20].

One possible way to provide necessary nutrients to the plants during vegetative stages is a foliar application of fertilizers. The foliar fertilization directly applies the nutrient to the plant tissue, by-passing a potential fixation, and losses that may arise from the soil application. However, the efficacy of the foliar fertilizer is relatively uncertain [21]. An uptake potential of foliar P is generally considered to be low, but the increased efficiency of the P usage has been reported with foliar application [22]. The greatest benefits of the foliar P fertilization were observed under low moisture and highly P-deficient soil conditions. In these aforementioned conditions, the foliar P application has a potential to increase the yield and quality of seeds [23,24]. However, there have been relatively few reports on analyses of the productivity and seed quality of legumes [10] or normal [11] and low-phytate pea cultivars grown under varying foliar P fertilization levels.

This work contributes to the extension of knowledge on the foliar phosphorus application and its influence on selected growth parameters, production, and quality of normal-phytate cultivars, and low-phytate pea lines. It offers an alternative approach to optimize phosphorus nutrition of peas, i.e., the use of foliar fertilization with this nutrient, which is a suitable procedure especially in conditions where soil application seems ineffective (e.g., inappropriate soil pH, which immobilizes phosphorus in the soil). A positive effect of the foliar P application on pea seed production and its quality in plants grown under P deficient conditions, especially a reduction of the portion of P bounded to phytate, is expected. An increase of free P (inorganic) for nutritional purposes is expected. At the same time, it is determined whether the low-phytate lines will use the P supplied by fertilization directly for binding to phytate, i.e., will this increase the phytate phosphorus content or will it only increase the total P content of the seeds and the phytate level will remain unchanged?

#### **2. Results and Discussion**

#### *2.1. The Effect of Foliar P Fertilization on Content of Chlorophyll and Chlorophyll Fluorescence Parameters*

The foliar application of phosphorus had a significant effect (*p* ≤ 0.05) on the content of chlorophyll in leaves of all tested pea genotypes. The increase of the N-tester values was significant (*p* ≤ 0.05, 0.01) in both measurements, especially at variant P2 and P3. A significant effect of the foliar phosphorus application on the chlorophyll content of wheat was presented by Waraich et al. [25]. Besides wheat, the increased content of

chlorophyll after the phosphorous fertilization was also achieved in the case of mung bean [26] aubergine [27] maize [28] and cluster bean [29]. The positive effect of the applied phosphorus can be explained by its direct involvement into the structure of cell membranes [30] a whole range of proteins, nucleic acids, and nucleotides [25] with direct effects on photosynthesis [31]. This fact explains the decrease of the chlorophyll content by the phosphorus deficiency in rice [32]. The mean N-tester values of all tested pea genotypes measured in the first term (T1) were enhanced by the stepped P application by 2.9% (P1), 4.2% (P2), and up to 4.9% (P3). The highest increase of the N-tester values was determined in plants of the line 1-150-81 at variant P3. The effect of the phosphorus fertilization lasted up to the second term of the measurement (T2), as presented in Table 1.

**Table 1.** The effect of the foliar phosphorus application on chlorophyll contents (N-tester value) and chlorophyll fluorescence parameters (*ΦPSII* and *RFd*).


The mean values marked with asterisk are significantly different (\* *p* ≤ 0.05; \*\* *p* ≤ 0.01) from the variant without P fertilization (P0) by Fisher's LSD test (each of the genotypes was statistically evaluated separately). The values in the table represent the arithmetic mean (*n* = 60) ± SD (standard deviation). Determinations were carried out 14 (T1) and 28 (T2) days after the P-application.

> The quantum yield of the electron transport of the photosystem II (*ΦPSII*), which expresses the real capacity of the photosystem II (PSII) for photochemical reactions, and represents the availability of reaction centers of the PSII, was significantly (*p* ≤ 0.05, 0.01) influenced by the phosphorus application. Its mean value measured at the variant without the P fertilization (P0) was 0.800. By the stepped P fertilization, this value was enhanced at all doses (P1−3) to 0.822 in the first term (T1). The results corresponded with conclusions in the study of Xu et al. [32], which found out that the phosphorus deficit had induced changes in efficiency of excitation energy absorption of reaction centers of the PSII in rice plants and had reduced the quantum yield *ΦPSII*. For example, the decrease of the quantum yield (*ΦPSII*) caused by the phosphorus deficit was observed in *Lonicera pampaninii* [33]. The photosynthetic rates in the *lpa* and *npa* genotypes of soybean were 1.3 and 1.5 times higher, respectively, in the treatment with the high P dose than with the low P dose [34]. In the first measurement term (T1), the *ΦPSII* value was most significantly enhanced after the phosphorus application in the case of the line 1-150-81. According to Fryer et al. [35], a strong linear correlation between quantum yield of the electron transport of the PSII (*ΦPSII*) and the carbon fixation efficiency was found. Plant species with phosphorus deficit like *Lotus japonicus* showed a decrease of the maximal rate of photosynthesis. In the case of the ratio of the dark respiration to the maximal photosynthesis, it declined significantly [36]. The significantly (*p* ≤ 0.05; 0.01) increased *ΦPSII* values of variants with the P fertilization on the *lpa* lines lasted until the later vegetation stages (T2). Contrary to this, the quantum

yield values of the *npa* cultivars were reduced (Table 1). In the case of the cultivar CDC Bronco, the decrease was significant (*p* ≤ 0.01). The increase of the *ΦPSII* values at the *lpa* pea lines was followed by a significant enhancement of the fluorescence decrease ratio (*RFd*), which is measured at saturation irradiance, and which is directly proportional to the net CO2 assimilation rate. In the second term T2, a significant correlation (*r* = 0.635; *p* ≤ 0.001) between the *ΦPSII* and *RFd* values was determined at these pea lines. Therefore, it can be stated that the foliar phosphorus application has a significant effect on the availability of reaction centers on the photosystem II at the *lpa* pea lines.

#### *2.2. The Effect of Foliar P Fertilization, on Yield Parameters of Pea*

The seed yield of normal phytate varieties was on average 27.3% higher than the seed yield of low-phytate lines. Research comparing yield levels of *lpa* and *npa* pea genotypes indicates that the low-phytate lines were similar in agronomic performance to normal phytate cultivars, except for somewhat slower time to flowering and maturity, slightly lower seed weight, and slightly lower grain yield [17,37]. However, the primary focus of our study was to evaluate the effect of phosphorus foliar fertilization on yield and seed quality of the tested pea genotypes.

The foliar application of phosphorus significantly influenced the yield of the pea seeds (*p* ≤ 0.05; 0.01). Its enhancement was evident at all tested genotypes, as presented in Figure 1. A significantly increased grain yield was also achieved according to the literature dealing with the evaluation of the phosphorus application influence on legumes. The attention of contemporary research is mostly focused on the phosphorus uptake by the root system after the application of P-fertilizers in stepped doses to the soil. This application is used for the legumes growing both in a monoculture: pea [5], faba bean [6], chickpea [7], and mung bean [8], and in a mixed cultures [9]. Only a few literal sources evaluate the efficiency of the foliar P application on the seed production of legumes, and their quality [10,11]. Comparing the different techniques, the foliar P fertilization has a better potential to improve its nutritional deficiencies in plants caused by the low content of P in soil or limited availability of this nutrient by the root [23,38]. The reaction of the tested pea lines on the foliar P fertilization was different. In the case of the *npa* cultivars, the seed production was enhanced by the stepped doses of the P-fertilizer, except of the highest level (P3) which reduced the seed production. The yield was decreased by the mean value of 10% compared to the variant P2 at both tested cultivars. This fact corresponded to the parameters of photosynthesis determined during vegetation and indicated that the dose of 81.8 mg P (P3) is not beneficial to plants of the *npa* cultivars. Froese et al. [11] also observed a reduced yield caused by the highest dose of P (20 kg/ha P2O5) applied on leaves of pea. Contrary to the *npa* cultivars, the *lpa* line 1-2347-144 was positively affected by all fertilization variants, including P3, and provided significantly enhanced seed production compared to the variant P0. The stepped phosphorus application increased the production by 17.8% (P1), up to 22.2% (P3). The most significant effect of the foliar phosphorus application was achieved at the *lpa* line 1-150-81. In this case, the seed yield was also linearly increased by the applied phosphorus doses (*r* = 0.849; *p* < 0.001), and the highest level of the P fertilization (P3) induced the enhanced yield of 42.1%. The result corresponded to the response of the low-phytate lines of soyabean on the P fertilization, where the seed yield was significantly (*p* ≤ 0.05) higher than at the normal-phytate genotypes [39]. However, these presented responses of pea on the foliar phosphorus fertilization were contradictory to the conclusions of Froese et al. [11]. In this study, the foliar P application was unable to substitute the seed-placed monoammonium phosphate and, overall, it had a marginal effect on the grain yield, P uptake as well as the seed nutritional value. According to the yield response on the phosphorus fertilization, the studied pea lines can be divided in two groups. While in the case of the *lpa* lines (1-2347-144; 1-150-81), the yield significantly correlated with the seed weight (*r* = 0.597, *p* = 0.041; *r* = 0.752, *p* = 0.005), in the case of the *npa* cultivars (CDC Bronco, and Cutlass), the pea production was significantly influenced by the number of seeds (*r* = 0.828, *p* = 0.001; *r* = 0.600, *p* = 0.039). According to the available

literature, a positive influence of the foliar phosphorus application on the seed weight of common bean [39], chickpea [40], wheat, and maize [38] etc. was proved. Although the seed weight was relatively enhanced at all tested genotypes (Table 2), the influence of the phosphorus nutrition on the seed weight was significant (*p* ≤ 0.01) only at the application of the highest P dose (P3) on the *lpa* line 1-150-81. The number of seeds produced by the pea plants was also influenced by the P nutrition. After the application of the P doses P2 and P3, the number of seeds was significantly increased (*p* ≤ 0.01) at cultivars CDC Bronco and Cutlass by the mean 17.9% and 18.1%, respectively, compared to the non-fertilized variant (P0). In the case of the low-phytate lines, the number of seeds was also significantly (*p* ≤ 0.01) increased by 8.0% and 13.0%, respectively, compared to P0 (Table 2).

**Figure 1.** The effect of the foliar phosphorus application on the seed yield. The mean values (*n* = 8) marked with an asterisk are significantly different (\* *p* ≤ 0.05; \*\* *p* ≤ 0.01) from the variant without the P fertilization (P0) by the Fisher's LSD test (each of the genotypes was statistically evaluated separately). Error bars represent the standard deviation of arithmetical mean (SD).

The foliar phosphorus application also significantly influence the height of plants, which correlated with the seed yield at the tested genotypes (*r* = 0.786, *p* < 0.05). While in the case of the *lpa* pea lines, the plant height was significantly (*p* ≤ 0.01) increased even at the application of the highest phosphorus dose (P3), in the case of the cultivars CDC Bronco and Cutlass, the P3 dose did not influence the plant height. The cultivar Cutlass had no response on the P fertilization. By the mean values of the tested pea genotypes, the plant height was increased after the P fertilization by 3.4 (P1), 8.8 (P2), and 11.6 cm (P3) in the case of the low-phytate lines, and by 6.8 (P1), 7.0 (P2), and 1.4 cm (P3) in the case of the conventional cultivars (*npa*), compared to the control P0. According to the available literature, the phosphorus application to the soil had a significant influence on the height of cowpea [41]. Maize and wheat plant heights were also significantly increased by the foliar P application [38]. Interactions of the foliar P application with magnesium fertilization significantly increased the plant height of faba bean [10].


**Table 2.** The effect of the foliar phosphorus application on the plant height, seed weight and number of seeds.

The mean values marked with an asterisk are significantly different (\* *p* ≤ 0.05; \*\* *p* ≤ 0.01) from the variant without P fertilization (P0) by the Fisher's LSD test (each of the genotypes was statistically evaluated separately). The values in the table represent the arithmetic mean (*n* = 8) ± SD (standard deviation).

#### *2.3. The Effect of Foliar P Fertilization, on Pea Seed Quality*

Within the seed, P is primarily stored as phytic acid and/or phytate that accumulate in protein vacuoles. Phytate comprises up to 80% of the total seed phosphorus, and can comprise as much as 1.5% of the seed dry weight [42]. Significantly, the lowest (*p* ≤ 0.05) content of phytate in seeds was determined at the *lpa* line 1-150-81. By the P application, its content was reduced by 19.9% (P1), 24.9% (P2), and 9.1% (P3), respectively, but not significantly (*p* ≤ 0.01). Nevertheless, the line 1-150-81 was the only one that tended to the decrease of phytate in seeds by the stepped phosphorus application. The foliar P application had a limited effect on phytate in seeds of canola, wheat, and pea [11]. In that experiment, phytate concentration in the pea seeds was decreased, comparing to the P non-fertilized variant, only by the application of the P-fertilizer (10 kg/ha P2O5) that was safely placed in the seed row with pea in combination with the foliar P application (10 kg/ha P2O5). The other tested genotypes, including the *lpa* line 1-2347-144, shown a relative increase of the phytate content by the stepped P application, significantly in the cultivar Cutlass, only (Table 3). It has been reported that the phytate concentration in seeds gradually increased and was positively correlated with the applied P levels in soybean [18], oat [19], and maize [20]. In context with the phosphorus content in seeds (Table 3), which was also significantly enhanced by fertilization only in the case of the cultivar Cutlass (*p* ≤ 0.01), the portion of phytate-P amount to the total P content in seeds was evaluated for the studied genotypes (Figure 2). While in the case of the cultivar Cutlass, the fertilization by doses P2 and P3 increased the phytate-P portion from 65.6% (P0) to 73.4% and 73.7%, respectively, in the case of the line 1-150-81, a provable (*p* ≤ 0.05) decrease of the phytate-P portion from 47.8% (P0) to 40.9% (P3) was induced by the foliar P nutrition.


**Table 3.** The effect of the foliar phosphorus application on the content of phosphorous, phytate, and crude protein in pea seeds.

The mean values marked with an asterisk are significantly different (\*\* *p* ≤ 0.01) from the variant without P fertilization (P0) by the Fisher's LSD test (each of the genotypes was statistically evaluated separately). The values in the table represent the arithmetic mean (*n* = 8) ± SD (standard deviation). (DM) dry matter.

**Figure 2.** The effect of foliar phosphorus application on portion of phytate-P amount to the total P content in seeds. The mean values (*n* = 8) marked with an asterisk are significantly different (\* *p* ≤ 0.05) from the variant without P fertilization (P0) by the Fisher's LSD test (each of the genotypes was statistically evaluated separately). Error bars represent the standard deviation of arithmetical mean (SD).

> The positive effect of the foliar phosphorus application on the crude protein content in the pea seeds was proved for the cultivar Cutlass, only (Table 3). In the case of the *lpa* line, content of the crude protein was not influenced by the P nutrition. The study by Klimek-Kopyra et al. [43] showed that phosphorus had a limited effect on this parameter. The phosphorus application in doses of 70 and 140 kg/ha P2O5, respectively, to the soil at 6 lines of pea did not influence content of the crude protein. Our study also does not confirm a consistent tendency towards the increase of the crude protein under higher

1-150-81

Cutlass

CDC Bronco phosphorus doses for cultivars of pea. Conversely, various *studies* [44,45] presented that the P fertilization increased the crude protein content of cowpea grain as well as low-phytate and normal-phytate cultivars of soybean [34].

The content of ash material and particular nutrients in the pea seeds of the tested genotypes is presented in Table 4. The relatively highest content of ash material was determined at the variant P3 for all tested genotypes. In this case, the mean increase of ash content was 0.13% compared to the variant P0. Besides the cultivar CDC Bronco, a significantly effect of the phosphorus application in doses P2 and/or P3 on the content of ash material was proved in tested genotypes. An important nutrient contained in the pea grain is potassium. Although its amount significantly correlated with the ash content (*r* = 0.870, *p* < 0.05), the foliar phosphorus application (P3) increased its content significantly only in the case of the line 1-2347-144 (Table 4). Potassium content was increased insignificantly by application of the highest dose of phosphorus (P3), from 0.92% to 0.98% DW on average across all tested pea genotypes. By contrast, the enhancement of potassium content in the tissue of the peanut plant observed after the phosphorus application was presented by Malakondaiach and Rajeswararao [46]. A significant increase in potassium uptake due to the increasing doses of phosphorus application was also found out in cowpea [47], mung bean [48], and urd bean [49]. The content of magnesium did not change by the P fertilization in seeds of the *lpa* lines, but it was increased in the *npa* cultivars (Table 4). A diverse response of the *npa* and *lpa* genotypes on the phosphorus fertilization was observed in the case of calcium utilization, too. While its content was not influence by the P fertilization of the cultivar CDC Bronco, it was significantly enhanced in the cultivar Cutlass. In the case of the low-phytate lines, Ca content was decreased, and in the case of the line 1-150-81 significantly. Since the effect of foliar P fertilization on seed mineral content in some genotypes is only statistically significant for some treatments, it is not possible to say that foliar P application will increase seed nutritive value.


P0 3.26 ± 0.13 1.02 ± 0.04 0.121 ± 0.008 0.040 ± 0.007 28.7 ± 0.6 P1 3.13 ± 0.29 0.99 ± 0.09 0.114 ± 0.010 0.035 ± 0.003 28.5 ± 1.2 P2 2.97 ± 0.06 \* 0.95 ± 0.00 0.112 ± 0.006 0.032 ± 0.007 \* 26.6 ± 5.8 \* P3 3.09 ± 0.04 1.01 ± 0.00 0.121 ± 0.004 0.030 ± 0.004 \*\* 25.6 ± 1.2 \*

P0 2.68 ± 0.06 0.82 ± 0.06 0.109 ± 0.002 0.032 ± 0.002 24.7 ± 2.4 P1 2.68 ± 0.07 0.83 ± 0.01 0.114 ± 0.009 0.033 ± 0.005 25.6 ± 4.0 P2 2.96 ± 0.06 \* 0.90 ± 0.03 0.116 ± 0.002 0.035 ± 0.005 25.6 ± 3.7 P3 3.02 ± 0.18 \*\* 0.87 ± 0.08 0.129 ± 0.007 \*\* 0.045 ± 0.006 \*\* 25.2 ± 1.6

P0 2.96 ± 0.14 0.93 ± 0.07 0.104 ± 0.004 0.030 ± 0.002 34.9 ± 3.7 P1 2.96 ± 0.08 0.96 ± 0.03 0.111 ± 0.004 0.029 ± 0.002 34.4 ± 4.1 P2 3.02 ± 0.11 0.96 ± 0.04 0.116 ± 0.007 \* 0.029 ± 0.002 27.1 ± 2.4 \* P3 3.08 ± 0.01 1.01 ± 0.01 0.108 ± 0.003 0.031 ± 0.005 29.1 ± 4.2

**Table 4.** The effect of the foliar phosphorus application on ash content, and mineral content in pea seeds.

The mean values marked with an asterisk are significantly different (\* *p* ≤ 0.05; \*\* *p* ≤ 0.01) from the variant without P fertilization (P0) by the Fisher's LSD test (each of the genotypes was statistically evaluated separately). The values in the table represent the arithmetic mean (*n* = 8) ± SD (standard deviation).

> Zinc absorption capacity was reduced by the high phosphorus utilization, and zinc in the plant and soil was in a state of antagonism with phosphorus. According to the available studies, a deficiency of zinc in plants was caused by phosphorus fertilization to the soil [50,51]. The foliar phosphorus application significantly decreased the zinc content stored in seeds both in the *lpa* line (1-150-81) as well as in the *npa* cultivar (CDC Bronco).

One of possible explanations can be the fact that the increased P concentration in seeds due to the foliar phosphorous application can aggravate Zn deficiency. Another explanation is that zinc concentration in seeds of pea was decreased by the effect of the induced growth response on the P fertilization. In other words, zinc was diluted in the plant tissues. Thus, the foliar phosphorus application can decrease the nutrition value of the pea seeds.

The results of the pot experiment show the possibilities of using the foliar phosphorus fertilization in the growing of normal and low-phytate pea genotypes. It is shown that the foliar application increases not only the seed yield, but also their nutritional quality, usability in the food industry and human nutrition. It is clear that the phytate content of the seeds can be regulated in this way, which is variety dependent. A decrease in phosphorus binding to phytate was obtained in the low phytate line 1-150-81 and an increase in the Canadian variety Cutlass after foliar application of phosphorus. However, verification of the results obtained from the pot experiment in field trials will be necessary.

#### **3. Materials and Methods**

#### *3.1. Plant Materials, Plant Cultivation and Conditions of Growth*

The effect of the foliar phosphorus application on photosynthetic parameters, seed yields and quality of four pea genotypes (*Pisum Sativum* L.) was investigated in this study. The experiment was conducted in growth box (PlantMaster, CLF Plant Climatics GmbH, Wertingen, Germany) at the Mendel University in Brno located at 49◦21 03 N and 16◦61 38 E. The low-phytate pea lines (*lpa*) 1-150-81 and 1-2347-144 [17] were chosen for this study. In these lines, the content of phytate phosphorus is reduced by approximately 60% compared to the normal phytate genotype, with a compensating increase in inorganic phosphorus [17]. Both lines were derived from the cultivar CDC Bronco [52] through chemical mutagenesis [17]. Further, the normal-phytate cultivars (*npa*) CDC Bronco and Cutlass [53] were also used for this study. Pea plants were grown under the control condition: 12 h of light (light intensity 550 μmol m−<sup>2</sup> s−1) and 12 h of darkness; day temperature 22 ◦C and night temperature 15 ◦C; humidity 60% during the day and 90% at night. Mitscherlich pots with a volume of 6.2 L were used to grow the pea plants. Each container contained 6000 g of arable soil with a constant composition (Table 5).

**Table 5.** Chemical composition of soil used in this study.


\* low available phosphorus content. Soil parameters were determined according to Zbíral [54].

Six pea seeds of each genotype per pot were sown on 27 April 2020. Four plants were grown in each pot for all pea genotypes. The foliar fertilization of phosphorus was carried out on the plant development stages of the 6th true leaf unfolded at the 6th node (3 June 2020). The following four treatments with three doses of the foliar phosphorous application were included in the experiment: P0—without P (0 mg P/pot), P1—62.5 mg P2O5 (27.3 mg P) per pot; P2—125 mg P2O5 (54.5 mg P) per pot and P3—187.5 mg P2O5 (81.8 mg P) per pot. These treatments of the foliar phosphorus application were carried out for all genotypes (for each genotype separately). The phosphorous foliar application (phosphoric acid, H3PO4) in 5 mL of water solution per pot at each treatment (P1−P3) was used. The control variant (P0) was treated with water. Water and phosphorus solutions were regularly applied using a pressurized hand pump sprayer (DPZ 1500, ProGlass, Weilheim an der Teck, Germany). The vegetation pots were arranged randomly in the growth box. A total of 32 pots were established for each genotype: four treatments with the graded dose of P (P0–P3); each treatment was established in eight replications (pots).

#### *3.2. Measurement of Photosynthetic Parameters of Pea Plants*

Selected photosynthetic parameters of pea plant The content of chlorophyll (like Ntester value), and selected parameters of fluorescence (quantum yield of photosystem II, and chlorophyll fluorescence decrease ratio) were evaluated. The measurements were performed 14 (T1) and 28 days (T2) after the foliar application.

#### 3.2.1. Content of Chlorophyll (N-Tester Value)

The content of chlorophyll in pea leaves, expressed as N-tester values, was determined using an N-Tester instrument (Yara International ASA, Oslo, Norway) in the wavelength range 650–940 nm [55]. Chlorophyll content was determined from leaves located in the middle part of the plants.

#### 3.2.2. Chlorophyll Fluorescence Parameters

Photochemical *efficiency* of *photosystem II* (PSII) in pea plants was determined. Chlorophyll fluorescence determination was performed with a PAR-FluorPen FP 110-LM/S (Photon Systems Instruments, Drásov, Czech Republic). The measured data were subsequently evaluated using FluorPen 1.1 software [56]. The leaves of the pea plant were dark adapted for 30 min prior to the measurement. Protocol for the measurement of chlorophyll fluorescence parameters is presented in Table 6.


**Table 6.** Measurement protocol of the chlorophyll fluorescence.

*λ* = 454 nm, L—light, DR—Dark recovery.

The Quantum yield of the PSII (*ΦPSII*) and the Chlorophyll fluorescence decrease ratio (*RFd*) were measured as a photochemical quenching parameter (Table 7).

**Table 7.** The photochemical quenching parameter.


(F0) minimal fluorescence from the dark-adapted leaves, Fm means maximal fluorescence from the dark-adapted leaves; (Fd) fluorescence decrease from (Fm to Fs; Fs) steady state chlorophyll fluorescence.

#### *3.3. Yield Parameters and Seed Quality*

The pea plants were harvested at the full ripeness on 29 July 2020. The height of plants, the yield of seeds per pot, the weight of one thousand seeds, and the number of seeds were determined. Plant height was measured before harvesting. After cutting the plants, the pea seeds were harvested by hand. The pea seeds were then weighed (laboratory scale PCB Kern, KERN and Sohn GmbH, Balingen, Germany), and counted (seed counter Contador, Pfeuffer GmbH, Kitzingen, Germany). The weight of 1000-seeds was subsequently determined.

Subsequently, the pea seeds were analyzed in order to evaluate selected qualitative parameters, namely: the content of phytic acid (phytate), content of phosphorus in the phytate, content of crude protein, ash material, and content of nutrients in seeds (P, K, Mg, Ca, and Zn).

#### 3.3.1. Determination of Phytic Acid (Phytate)

Content of phytic acid was determined using a commercial kit "Phytic acid (phytate)/Total phosphorus" (Megazyme, Bray, Ireland) [59]. Pea seeds were finely ground using the Foss Tecator Cyclotec 1093 (Foss Analytical, Hillerød, Denmark). For analysis, 1 g of flour was weighted. Phytic acid from the sample was extracted with 0.66 M HCl. The neutralized aliquot of the sample was treated with phytase that was specific for phytic acid, and the lower myo-inositol phosphate forms. Then, the sample was treated with alkaline phosphatase that hydrolyzed myo-inositol phosphates and released free phosphate. The total phosphate was measured using a colorimetric method with molybdenum blue (Spekol 1300, Analytik Jena AG, Jena, Germany). The amount of molybdenum blue was proportional to the amount of phosphate in the sample.

#### 3.3.2. Analysis of Crude Protein and Ash

Ash material was determined by weighing the material remaining after the burning of a fixed-weight sample at 550 ◦C and specified conditions. Nitrogenous substances were measured by the Kjeldal method (N × 6.25 coefficient) using the Kjeltec 2300 device (Foss Analytical, Hillerød, Denmark) [60].

#### 3.3.3. Determination of Nutrient Contents

The samples of pea seeds were dried at temperature of 50 ◦C, then crushed in the grinder (Foss Tecator Cyclotec 1093, Foss Analytical, Hillerød, Denmark), and homogenized. After the microwave closed vessel acid digestion (HNO3/H2O2) in ETHOS One (Milestone Srl, Sorisole, Italy), the contents of nutrients were determined (Table 8).


**Table 8.** The methods for the determination of nutrients in pea seed.

(UV/VIS) ultraviolet–visible.

#### *3.4. Statistical Data Analysis*

Measured data were statistically evaluated using STATISTICA 12 program (TIBCO Software Inc., Palo Alto, CA, USA) [62]. The normality and homogeneity of variances were verified, respectively, by Shapiro-Wilk's and Levene's test at *p* ≤ 0.05. The influence of the monitored factors was analyzed via two-way ANOVA (level of significance *p* ≤ 0.05). All evaluated parameters are expressed in tables and graphs as the arithmetic mean ± standard deviation (SD). The differences between the arithmetical means were evaluated by the Fisher's (*LSD*) test at the 95% (*p* < 0.05), and 99% (*p* < 0.01) level of significance.

**Author Contributions:** Conceptualization, P.Š.; methodology, P.Š., M.Š. and J.A.; formal analysis, P.Š., M.Š., J.A. and Z.K.; investigation, P.Š., M.Š., J.A., P.H. and J.H.; writing—original draft preparation, P.Š., M.Š., J.A., P.H., I.S., J.H. and R.D., writing—review and editing P.Š., M.Š. and Z.K. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research was financially supported by the Ministry of Agriculture of the Czech Republic under the project No. QK1810072, Development of biofortified pea breeding lines with low phytic acid content.

**Institutional Review Board Statement:** Not applicable.

**Informed Consent Statement:** Not applicable.

**Data Availability Statement:** The data presented in this study are available on request from the corresponding author. Due to the nature of this research, participants of this study did not agree for their data to be shared publicly, so supporting data is not available.

**Acknowledgments:** The authors thanks Professor Tom Warkentin, Centre/Department of Plant Sciences, University of Saskatchewan, Canada, for providing Canadian varieties and LP pea lines.

**Conflicts of Interest:** The authors declare no conflict of interest.

#### **References**

