Influence of Fertilization on Growth and Lead Content of Pepper under Lead Stress

To investigate the effect of fertilization on Pb content in vegetables, pepper was planted in L1645 (the 5 influencing factors are fertilizers (N, P, K), organic fertilizers (sheep manure) and Pb2+; the 4 levels are blank, low, medium and high; a total of 16 treatments) pot orthogonal experiment. The effects of fertilizers on the growth and Pb content in various parts of pepper under Pb stress were analyzed. The results showed that: (1) The Pb content in pepper fruit ranged from 0.011 mg·kg−1 to 0.085 mg·kg−1, which did not exceed the limit value (0.1 mg·kg−1) in the National Standard for Food Safety-Limit of Contaminants in Food (GB2762-2017); (2) The effect order of fertilization on pepper fruit weight was P2O5 > sheep manure > N > K2O; The horizontal combination of factors that promoted the maximum fruit weight of pepper was N (0.15 g·kg−1), P2O5 (0.225 g·kg−1), K2O (0.15 g·kg−1) and sheep manure (9 g·kg−1); (3) The order of fertilizer effects on Pb content in pepper fruit was Pb2+ > K2O > N = sheep manure > P2O5; the factor level combination that resulted in the maximum Pb content in pepper fruits was N (0.15 g·kg−1), P2O5 (0 g·kg−1), K2O (0.45 g·kg−1), sheep manure (6 g·kg−1) and Pb2+ (350 mg·kg−1); (4) Based on the soil fertility characteristics of Urumqi, the recommended optimal fertilizer application rate was: high phosphorus fertilizer P2O5 (495 kg·hm−2), low-level potassium fertilizer K2O (330 kg·hm−2), medium-level nitrogen fertilizer N (660 kg·hm−2) (or low-level nitrogen fertilizer N (330 kg·hm−2) + high-level organic manure sheep manure (19,800 kg·hm−2), which can achieve high yield while ensuring that the Pb content in the fruits does not exceed the standard. Strengthening control of effective and reasonable fertilization methods in Urumqi agricultural land is helpful to reduce the Pb content in vegetables.


Introduction
Soil is the basis for plant growth, and the soil-plant system is the basic structural unit of the biosphere, which provides strong productivity for human beings. Soil is often limited by a low level of available nutrients, so fertilization is often necessary to replenish them. But, fertilization can introduce heavy metals into the soil [1]. Excessive use of fertilizers can lead to a decrease in soil pH, increase the availability of natural heavy metal lead (Pb) in soil, cause harm to soil physical properties, and then affect plant growth and development [2]. Lead from anthropogenic sources typically builds up mostly in the top layer of soil, and as depth increases, its concentration declines and only limited amounts of the lead in soil are thought to be soluble and hence available for plant uptake because of its high binding [3]. Due to the detrimental effects on soil microbiology, food safety and crop growth, lead accumulation in soils is a severe concern in agricultural production [4]. Lead's speciation in soil has a significant impact on its bioavailability and, consequently, its toxicity to plants and microorganisms [5]. Studies have found that even short-term exposure of plants to the toxic effects of the heavy metal lead can lead to a significant reduction in their microbial activity; to counteract these effects, many plants and microorganisms have evolved detoxification mechanisms using organic colloidal components [6][7][8].

Test Materials
The pepper variety tested was the improved pig large intestine (PLI) produced by Xi an Qinshu Agriculture Company(Xi'an, Shaanxi Province, China). The tested soil was chernozem soil purchased from Mingzhu Flower Market, Urumqi City (The soil comes from the soil of agricultural land around Urumqi City and is mainly used for planting fruits and vegetables), and the organic fertilizer was pure sheep dung fermented at high temperature from Bafang Zehui Company(Xi'an, Shaanxi Province, China). The basic physical and chemical properties of soil and organic manure (sheep manure) were shown in Table 1. The nitrogen fertilizer used in the experiment was urea (N 46%), the phosphate fertilizer was calcium triple superphosphate (P 2 O 5 52%), the potassium fertilizer was potassium sulphate (K 2 O 54%), and Pb was added as an external compound Pb(NO 3 ) 2 (containing Pb 2+ 62%).

Experimental Design
An orthogonal experiment L 16 4 5 (5 factors, 4 levels, a total of 16 treatments) was used to design and analyze the effects of the combined action of four fertilizers and Pb 2+ on the Pb absorption of PLI. The five factors were N, P 2 0 5 , K 2 O, sheep manure and Pb 2+ . The four levels are blank, low, medium and high. The experiment is done by adding fertilizers and compounds to the soil. The five influencing factors in the experiment were added to the soil in the form of fertilizers and compounds. The orthogonal experimental design is shown in Table 2. In this experiment, potted plants were used. In order to avoid the interference of rainwater, the experiment was carried out in the greenhouse behind the Agricultural Building of Xinjiang Agricultural University. The seedlings were raised on 7 April 2022, and a total of 48 flower pots were treated in 16 groups (3 repetitions in each group) on 14 May. 7 kg of soil were respectively loaded into the pots, and the corresponding fertilizer and Pb(NO 3 ) 2 of each treatment were evenly mixed into the soil according to Table 2 (the fertilizer was applied in the form of base fertilizer (60%) + top fertilizer (40%), and Pb(NO 3 ) 2 was applied at one time during the application of base fertilizer), and 1 L of distilled water was poured and left to stand. On 25 May, the PLI seedlings were transplanted into each flower pot, and one PLI was transplanted in each flower pot. Irrigation was carried out with distilled water. The irrigation amount was 1 L/pot every 5 days, and a total of 22 irrigations were carried out until harvest. The PLI received uniform natural light during the growing period and was harvested on 9 September 2022. Soil pH was measured after the PLI was harvested (Soil pH was extracted using a soil-water ratio of 2.5:1 and determined by a pH meter (Mettler Toledo FE28-Standard)).

Measurements and Methods
After harvesting the PLI, the plant height of the PLI was measured with a tape measure and the number of fruits was recorded. Then the PLI was divided into four parts: root, stem, leaf and fruit. The samples were rinsed and washed with deionized water 2-3 times, the surface moisture was dried, and the samples were weighed fresh. The plant samples were then dried at 105 • C for 30 min, dried in an oven at around 65 • C, ground and sieved, and stored for later use.
The total Pb content of plant and soil samples was determined by System 5000 graphite furnace atomic absorption spectrometer. The content of total nitrogen was determined by Kjeldahl method, the content of total phosphorus was determined by molybdenumantimony colorimetric method, and the content of total potassium was determined by flame photometry. The organic matter content was heated by potassium bichromate. The available phosphorus content was determined by sodium bicarbonate extraction and molybdenumantimony sulfate resistance colorimetric method. The content of available potassium was determined by ammonium acetate extraction and flame photometer method.

Data Analysis
Microsoft Excel 2010, IBM SPSS Statistics 25.0 and Orthogonal Design Assistant II v3.1 were used for statistical analysis of the data. Pb content was calculated as fresh weight, with Mean ± SD value. Plot with Origin 2018 and Sigmaplot 10.0 software.

Effects of Fertilization on Plant Height and Fruit Weight of PLI under Pb Stress
As can be seen from Figure 1, The highest PLI plant height in the 8th group was 49.87 cm, while the lowest PLI plant height in the 11th group was 27.83 cm; the highest number of PLI fruits in the 2nd group was 20, and the lowest number of PLI fruits in the 13th group was 7; the highest PLI fruit weight in the 12th group was 165.47 g, and the lowest PLI fruit weight in the 13th group was 67.50 g. Most of the soil pH was weakly acidic except for groups 1, 3 and 4.
According to Table 3, the factors affecting the plant height of PLI in order of importance were: A (N) > B (P 2 O 5 ) > C (K 2 O) > E (Pb 2+ ) > D (sheep manure), and N fertilizer has the greatest influence on PLI plant height. It can be seen from the K value that the level combination of factors that promote PLI plant height to reach the maximum is: A2 B4 C2 D1 E3, namely N (0.15 g·kg   As can be seen from Figure 2, nitrogen fertilizer was positively correlated with PLI plant height, phosphorus fertilizer was significantly positively correlated with PLI fruit weight (p < 0.05), and nitrogen fertilizer was significantly negatively correlated with soil pH value (p < 0.05).

Analysis of Pb Content in PLI
The results of Pb content determination in PLI were shown in Table 4. As can be seen from Table 4, the Pb content range of different parts of PLI in each treatment was generally in the order of root (0.104~1.086 mg·kg −1 ) > stem (0.032~0.298 mg·kg −1 ) > leaf (0.022~0.072 mg·kg −1 ), indicating that Pb was mainly concentrated in the root. Pb content in fruits ranged from 0.011 mg·kg −1 to 0.085 mg·kg −1 , among which the highest Pb content in fruits treated 16 was 0.085 mg·kg −1 , but it did not exceed the limit (0.1 mg·kg −1 ) in the "National Standard for Food Safety-Limit of Pollutants in Food" (GB 2762-2017).
Note: K1, K2, K3 and K4 are the sum of the indicators at each level of each factor, K1 represents the sum of the values of the test indicators corresponding to the "1" level.; R is called the range, and the largest K is subtracted from the smallest K.
Note: K1, K2, K3 and K4 are the sum of the indicators at each level of each factor, K1 represents the sum of the values of the test indicators corresponding to the "1" level.; R is called the range, and the largest K is subtracted from the smallest K.
As can be seen from Figure 2, nitrogen fertilizer was positively correlated with PLI plant height, phosphorus fertilizer was significantly positively correlated with PLI fruit weight (p < 0.05), and nitrogen fertilizer was significantly negatively correlated with soil pH value (p < 0.05).   The variance analysis of Pb content in different parts of PLI applied with different kinds of fertilizer is shown in Figure 3. The difference in Pb content in different parts of PLI under different treatments shows that there is no significant difference in Pb content in different parts of PLI in groups 1, 7, 15 and 16. Pb content in the roots and stems of PLI in groups 3, 5 and 11 was significantly different from that in the leaves and fruits. The Pb content in the roots of PLI in groups 2 and 6 was significantly different from that in the leaves and fruits. Pb content in the roots of PLI seedlings in groups 4 and 13 was significantly different from that in stems, leaves and fruits. The Pb content in the roots of group 14 was significantly higher than that in stems, leaves and fruits, and the Pb content in stems, leaves and fruits also showed significant differences, The content of Pb in roots was significantly different from that in stems, leaves and fruits.
In addition, the difference between groups in the same part of PLI under different treatments was as follows: the Pb content of group 14 in the root was significantly different from that of group 7 and group 15; Pb content in the stems of group 11 was significantly different from that in the stems of group 4. Pb content in leaf group 7 was significantly different from that in groups 1, 2, 3, 5, 6, 8, 9, 10, 11, 12, 13 and 15. Pb content in fruit group 7 was significantly different from that in groups 2, 3, 8, 9, 12, 13, 14, 15 and 16.

Effects of Fertilization on Pb Content in PLI under Pb Stress
According to the R-value in Table 5

Effects of Fertilization on Pb Content in PLI under Pb Stress
According to the R-value in Table 5

Effect of Fertilization Level on Pb Content in PLI under Pb Stress
The correlation analysis of various indicators of soil and PLI is shown in Figure 4. The Pb content of PLI roots is significantly positively correlated with the Pb content of leaves, and the Pb content of PLI stems and fruits is significantly positively correlated. The ratio of PLI stem to root and shoot showed a significant negative correlation; soil Pb content was significantly positively correlated with plant height, and plant height was significantly positively correlated with fruit weight. The linear relationship between Pb content in different parts of PLI and different fertilizer applications is shown in Table 6. The variation trend of lead content in each part of PLI is shown in Figure 5. According to Table 6 and Figure 5, we can find that there was a significant negative correlation between sheep manure and Pb content in PLI stems (r =−0.990, p = 0.010). Pb 2+ was positively correlated with Pb content in root (r = 0.993, p = 0.007), and Pb content in leaf (r = 0.979, p = 0.021). There was no significant correlation between Pb content in different parts of PLI and other fertilizers. The linear relationship between Pb content in different parts of PLI and d tilizer applications is shown in Table 6. The variation trend of lead content in e PLI is shown in Figure 5. According to Table 6 and Figure 5, we can find that significant negative correlation between sheep manure and Pb content in P =−0.990, p = 0.010). Pb 2+ was positively correlated with Pb content in root (r 0.007), and Pb content in leaf (r = 0.979, p = 0.021). There was no significant between Pb content in different parts of PLI and other fertilizers. Note: "*", p < 0.05, "**", p < 0.01.   The linear relationship between Pb content in different parts of PLI and different fertilizer applications is shown in Table 6. The variation trend of lead content in each part of PLI is shown in Figure 5. According to Table 6 and Figure 5, we can find that there was a significant negative correlation between sheep manure and Pb content in PLI stems (r =−0.990, p = 0.010). Pb 2+ was positively correlated with Pb content in root (r = 0.993, p = 0.007), and Pb content in leaf (r = 0.979, p = 0.021). There was no significant correlation between Pb content in different parts of PLI and other fertilizers. Note: "*", p < 0.05, "**", p < 0.01.

Discussion
Fertilization is one of the important agricultural measures to ensure the increase of agricultural production and income, and it also affects the adsorption and resolution of heavy metals in soil, the physicochemical properties of rhizosphere soil and the absorption of heavy metals by crops [23]. Pb is a non-essential element for plants, thus it is hazardous even in low quantities. It easily passes from the soil and atmosphere to plants [2].
Nitrogen is an essential element for plant growth and is contained in the composition of vitamins and energy systems in plants [24]. In this experiment, there was a positive correlation between nitrogen fertilizer and plant height of PLI, that is, the increase of nitrogen fertilizer would promote the growth of plant height. The study showed that the average growth rate and plant height of plants increased when the nitrogen supply of plants increased, which was consistent with the results of this experiment. However, nitrogen application is considered to be the main driving factor of soil acidification, because the input of nitrogen fertilizer will make the nitrogen in the soil easy to nitrate, and the generated NO3 − will produce H + , which is easy to leach out of the soil with base ions [25]. The study showed that the nitrification of nitrogen in the soil would produce a large number of protons and lead to a decrease in soil pH, while the addition of fertilizer nitrogen in the soil would lead to more significant soil acidification [25]. In this experiment, there was a significant negative correlation between nitrogen fertilizer and soil pH value, that is, the increase of nitrogen fertilizer led to the decrease of soil pH value. The decrease in soil pH can increase the content of extractable heavy metal elements in soil, improve the bioavailability of heavy metals, and enable plants to enrich more heavy metals [26]. Therefore, attention should be paid to the decrease in soil pH value caused by nitrogen fertilizer application.
Rational application of phosphorus fertilizer can increase crop yield, improve crop quality, promote flowering and fruit of jacket vegetables, and improve results. The research showed that after applying phosphorus fertilizer, the yield of PLI increased with the increase of phosphorus application, and the yield was the highest at the highest phosphorus application [18,27]. In this experiment, there was a significant positive correlation between phosphorus fertilizer and PLI fruit weight, that is, the increase of phosphorus fertilizer would increase PLI fruit weight, possibly because phosphorus would participate in the metabolism and transportation of PLI carbohydrates, which is conducive to the growth of PLI fruit.
Organic fertilizer can not only improve soil fertility but also affect the form of heavy metals in soil and their absorption by plants [15,28]. In this experiment, the content of organic fertilizer was negatively correlated with the Pb content in the roots of PLI and significantly negatively correlated with the Pb content in the stems. Pb intake studies in plants revealed that roots have the ability to absorb large amounts of Pb while limiting their translocation to higher parts of the plant [9,29]. This may be because organic fertilizer has a fixed effect, including the adsorption of heavy metals by macromolecules of solid

Discussion
Fertilization is one of the important agricultural measures to ensure the increase of agricultural production and income, and it also affects the adsorption and resolution of heavy metals in soil, the physicochemical properties of rhizosphere soil and the absorption of heavy metals by crops [23]. Pb is a non-essential element for plants, thus it is hazardous even in low quantities. It easily passes from the soil and atmosphere to plants [2].
Nitrogen is an essential element for plant growth and is contained in the composition of vitamins and energy systems in plants [24]. In this experiment, there was a positive correlation between nitrogen fertilizer and plant height of PLI, that is, the increase of nitrogen fertilizer would promote the growth of plant height. The study showed that the average growth rate and plant height of plants increased when the nitrogen supply of plants increased, which was consistent with the results of this experiment. However, nitrogen application is considered to be the main driving factor of soil acidification, because the input of nitrogen fertilizer will make the nitrogen in the soil easy to nitrate, and the generated NO 3 − will produce H + , which is easy to leach out of the soil with base ions [25]. The study showed that the nitrification of nitrogen in the soil would produce a large number of protons and lead to a decrease in soil pH, while the addition of fertilizer nitrogen in the soil would lead to more significant soil acidification [25]. In this experiment, there was a significant negative correlation between nitrogen fertilizer and soil pH value, that is, the increase of nitrogen fertilizer led to the decrease of soil pH value. The decrease in soil pH can increase the content of extractable heavy metal elements in soil, improve the bioavailability of heavy metals, and enable plants to enrich more heavy metals [26]. Therefore, attention should be paid to the decrease in soil pH value caused by nitrogen fertilizer application.
Rational application of phosphorus fertilizer can increase crop yield, improve crop quality, promote flowering and fruit of jacket vegetables, and improve results. The research showed that after applying phosphorus fertilizer, the yield of PLI increased with the increase of phosphorus application, and the yield was the highest at the highest phosphorus application [18,27]. In this experiment, there was a significant positive correlation between phosphorus fertilizer and PLI fruit weight, that is, the increase of phosphorus fertilizer would increase PLI fruit weight, possibly because phosphorus would participate in the metabolism and transportation of PLI carbohydrates, which is conducive to the growth of PLI fruit.
Organic fertilizer can not only improve soil fertility but also affect the form of heavy metals in soil and their absorption by plants [15,28]. In this experiment, the content of organic fertilizer was negatively correlated with the Pb content in the roots of PLI and significantly negatively correlated with the Pb content in the stems. Pb intake studies in plants revealed that roots have the ability to absorb large amounts of Pb while limiting their translocation to higher parts of the plant [9,29]. This may be because organic fertilizer has a fixed effect, including the adsorption of heavy metals by macromolecules of solid organic matter and clay minerals in the soil, limiting its mobility and reducing the availability of heavy metals [30]. Moreover, after entering the root, ions are transported to the xylem through both ectoplasmic and symplast pathways, and then to the aboveground part. However, the low permeability of the ectoplasmic barrier to heavy metal ions makes it difficult for Pb to transfer to the stem of PLI [2].
In this experiment, the Pb content in the roots of group 14 was significantly higher than that in stems, leaves and fruits, and the Pb content in stems, leaves and fruits also showed significant differences, which may be because high levels of Pb 2+ were enriched in PLI roots through cation adsorption, oxidation and reduction reactions of exchange complexes and because heavy metals in roots were not easily transported upward from stems. Pb content in the stems of group 11 was significantly different from that in the stems of group 4, which may be due to the application of high-concentration organic fertilizer, which resulted in the chelation of Pb 2+ with organic matter and other metal oxides, which restricted its mobility and reduced the absorption and transport capacity of Pb in the stems of PLI. Pb content in fruit group 7 was significantly different from that in groups 2, 3, 8, 9, 12, 13, 14, 15 and 16, which may be because potassium plays an important role in plant energy metabolism, acting as a cofactor or activator of many enzymes in carbohydrate and protein metabolism. In addition to helping the roots to absorb Pb from the soil and transfer it from the stem to the leaves, it can also accelerate the process of Pb absorption by the leaf surface itself.
According to the statistics of soil census data, the total nitrogen content of cultivated soil in Urumqi city was 1.13 g·kg −1 , the total phosphorus content was above 0.8 g·kg −1 and the total potassium content was about 19.28g·kg −1 , showing the phenomenon of nitrogen deficiency, phosphorus deficiency and potassium enrichment [31]. Pb content in soil is relatively low, generally < 100 mg·kg −1 .In view of the fact that the Pb content of all PLI fruits treated in this experiment is not beyond the standard, the optimal fertilizer application can be recommended according to the factor level combination (A2 B4 C2 D4) that promotes the maximum fruit weight of PLI: N (330 kg·hm −2 ), P 2 O 5 (495 kg·hm −2 ), K 2 O (330 kg·hm −2 ), sheep manure (19,800 kg·hm −2 ); Or according to the combination of treatment 12 with the maximum fruit weight (A3 B4 C2 D1), namely N (660 kg·hm −2 ), P 2 O 5 (495 kg·hm −2 ), K 2 O (330 kg·hm −2 ), sheep manure (0 kg·hm −2 ), both of which were treated with high-level phosphorus fertilizer and low-level potassium fertilizer. medium level of nitrogen fertilizer (or low level of nitrogen fertilizer + high level of organic fertilizer) can achieve a higher yield and not exceed the standard of Pb in fruit.

Conclusions
(1) The Pb content in PLI fruit ranged from 0.011 mg·kg −1 to 0.085 mg·kg −1 , which did not exceed the limit (0.1 mg·kg −1 ) in the National Standard for Food Safety-Limit of Pollutants in Food (GB 2762-2017).
(3) The effects of fertilization on Pb content in PLI fruit were as follows: Pb 2+ > K 2 O > N = sheep manure > P 2 O 5 ; The level combination of factors that promoted the maximum Pb content in PLI fruit was N (0.15 g·kg −1 ), P 2 O 5 (0 g·kg −1 ), K 2 O (0.45 g·kg −1 ), sheep manure (6 g·kg −1 ), Pb 2+ (350 mg·kg −1 ). There was a significant negative correlation between sheep manure and Pb content in PLI stem (r = −0.990, p = 0.010). There was a significant positive correlation between Pb 2+ and Pb content in the root (r = 0.993, p = 0.007) and in the leaf (r = 0.979, p = 0.021). There was no significant correlation between Pb content in different parts of PLI and other fertilizers.