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Article

Effects of Zinc, Manganese, and Molybdenum Fertilizers on Growth and Main Medicinal Metabolites of Stauntonia leucantha Leaves

by
Yang Zhou
1,
Junxin Zhou
2,
Jianyong Chen
2,
Yunni Chang
3,
Xiaoqing Lin
2,
Ziqing Zhong
2 and
Baoyin Li
1,*
1
College of Geographical Sciences, Fujian Normal University, Fuzhou 350007, China
2
College of Forestry, Fujian Forestry Vocational and Technical College, Nanping 353000, China
3
Tea Research Institute, Fujian Academy of Agricultural Sciences, Fuzhou 350013, China
*
Author to whom correspondence should be addressed.
Horticulturae 2025, 11(2), 123; https://doi.org/10.3390/horticulturae11020123
Submission received: 26 December 2024 / Revised: 19 January 2025 / Accepted: 20 January 2025 / Published: 23 January 2025
(This article belongs to the Section Medicinals, Herbs, and Specialty Crops)
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Abstract

:
Zinc, manganese, and molybdenum have a significant impact on plant growth and the accumulation of metabolites. However, the impact of zinc, manganese, and molybdenum on the growth and metabolic substances of Stauntonia leucantha leaves is still unclear. To explore the effects of zinc, manganese, and molybdenum on growth and main medicinal metabolites of Stauntonia leucantha leaves, to provide a reference for the fertilizer application method of Stauntonia leucantha, a three-factor, three-level orthogonal design with five-year-old field Stauntonia leucantha as the test material. The results showed that fertilization with zinc and molybdenum significantly enhanced the medicinal value of Stauntonia leucantha leaves. The leaf growth of Stauntonia leucantha mainly concentrated in 0–120 days after fertilizer application and gradually slowed down in 120–180 days after fertilizer application, significantly affected by zinc and manganese. Zinc significantly increased the content of chlorophyll, oleanolic acid, rutin, and calceolarioside B. Manganese significantly increased the content of chlorophyll and rutin while inhibiting the accumulation of oleanolic acid and calceolarioside B. Molybdenum significantly increased the content of oleanolic acid and rutin but did not affect chlorophyll b content. Based on a comprehensive analysis, the optimum fertilizer combination for leaf quality of Stauntonia leucantha leaves was identified as Zn3Mn1Mo3 (T7), 6 g/plant of zinc sulfate, 3 g/plant of ammonium molybdate, but no manganese sulfate.

1. Introduction

Stauntonia leucantha, also known as inverted-ovate leaf wild papaya, “Na Teng Bao” or “Ma Teng Bao”, is a member of the genus Stauntonia in the Lardizabalaceae family. This distinctive evergreen, woody vine, native to China, is recognized for the medicinal value of its roots, stems, leaves, and fruits. sweet and palatable, are widely consumed [1]. In Fujian Province, the genus Stauntonia comprises three species, among which Stauntonia leucantha stands out due to its abundance in wild resources compared to Stauntonia chinensis and Stauntonia urophylla [2]. Investigations and comparisons have highlighted Stauntonia leucantha’s strong adaptability, rapid growth, large fruit size, and excellent flavor [2]. Therefore, Stauntonia leucantha has considerable potential for development and utilization.
However, to date, research on Stauntonia has mainly focused on Stauntonia chinensis and Stauntonia cavalerieana Gagnep, with relatively few reports on Stauntonia leucantha. Studies have primarily focused on investigating the medicinal value, functional medicinal components, nutritional composition of the fruit, as well as processing and utilization technologies. Currently, oleanolic acid (OA) and rutin, which have been identified as the main medicinal compounds present in Stauntonia leucantha [3,4], are considered key constituents, while calceolarioside B is regarded as an indicator for evaluating the medicinal value of Stauntonia leucantha [1]. OA possesses anti-inflammatory [5], antibacterial [6], and antioxidant properties [7]. Rutin has been confirmed to have anti-inflammatory, antioxidant, and antibacterial effects [8]. Calceolarioside B is thought to have anti-virus effects [9]. At present, research on artificial cultivation techniques for Stauntonia leucantha is limited, and changes in nutritional components, metabolites, and accumulation of functional substances in this plant under the influence of environmental factors have not been investigated.
Plant growth and development require various nutritional elements, including macronutrients such as nitrogen, phosphorus, and potassium, and micronutrients like zinc, copper, manganese, and molybdenum [10]. Although plants and animals have a relatively low demand for micronutrients, they are as important as macronutrients [11]. These trace elements play an indispensable role in the normal physiological activities of plants [12]. Zn is an essential micronutrient that guarantees normal growth and development [13]. It plays a crucial role in core processes such as photosynthesis [14], production and transport of auxins [15], maintenance of biomembrane stability [16], and cell division [17]. Mn is an important element for plant growth. It plays a significant role in plant photosynthesis [18], serves as an activator and component of multiple enzymes, and ensures the integrity of plant cell membranes [19]. Manganese also affects the synthesis of indole-3-acetic acid (IAA). Changes in Mn levels in plants directly impact the activity of IAA oxidase, with Mn deficiency leading to increased IAA oxidase activity and accelerated IAA decomposition [20]. However, Mn deficiency in plants often occurs without visible leaf symptoms, making it challenging to promptly identify and address latent manganese deficiency [18]. As a micronutrient, molybdenum is essential for nitrogen fixation in organisms and a key element of nitrate reductase and several other molybdenum-dependent enzymes in plants [21]. In addition, Mo can significantly enhance phosphatase activity, facilitating the conversion of phosphate into organic phosphorus compounds [22]. This action stabilizes the chlorophyll structure, enabling crops to mature earlier and enhancing their resistance to environmental stress.
No studies have reported the effects of micronutrient fertilization on plants in the Stauntonia genus. For the entire Lardizabalaceae family, only Xiao et al. [23] have reported on Akebia trifoliata, another plant in this family, suggesting that combined fertilization of Zn, B, and Mo could significantly increase the yield of Akebia trifoliata. Despite the growing demand and rising quality requirements for Stauntonia leucantha, there is a lack of research aimed at enhancing its yield and medicinal value. This gap is significant, as the impact of trace elements on the growth and medicinal quality of Stauntonia leucantha remains unclear, hindering efforts to optimize cultivation practices. Therefore, research on the types and application rates of micronutrient fertilizers, exploring their impact on leaf growth and changes in medicinal components, and identifying the optimal micronutrient fertilization scheme for the growth of Stauntonia leucantha are of importance for enhancing the cultivation benefits of this species. Our study investigated the effects of co-fertilization with Zn, Mn, and Mo on leaf area, leaf thickness, chlorophyll a, chlorophyll b, OA, rutin, and calceolarioside B content in the leaves of Stauntonia leucantha. The objective was to establish a fertilization reference to enhance the medicinal value of Stauntonia leucantha leaves.

2. Materials and Methods

2.1. Experimental Site

The experiment was conducted at the Stauntonia leucantha planting base of the Guangze Shengxiang Xiangmi Fruit Agricultural Cooperative in Zhaili Town, Guangze County (27°71′ N, 117°33′ E). It belongs to the mid-subtropical marine monsoon climate, with an annual sunshine duration of 1780 h, annual average temperature of 18.2 °C, average temperature of 7.9 °C in the coldest month of January, average temperature of 28.1 °C in the hottest month of July, and annual average precipitation ranging from 1500 to 1700 mm. The experimental field is mountainous red soil with a pH value of 5.5–6.5, an organic matter content of 3.38%, a total nitrogen content of 0.1750%, a total phosphorus content of 0.8867 g/kg, a total potassium content of 0.8867 g/kg, a total zinc content of 0.0143 g/kg, a total manganese content of 0.4733 g/kg, and a total molybdenum content of 0.001 g/kg.

2.2. Plant Material

Five-year-old clonal plants of Stauntonia leucantha with similar growth conditions were selected as experimental materials. The plants had an average ground diameter of 3–4 cm and grew upright on a vine trellis.

2.3. Experimental Design

The experiment was conducted in a field environment using an orthogonal L9(33) experimental design among a population of 5-year-old artificially cultivated Stauntonia leucantha plants in the same plot. The experiment comprised three factors: type of micronutrient fertilizers, zinc (Zn), manganese (Mn), and molybdenum (Mo) fertilizers. Each factor included three treatments: zinc and manganese sulfate application rates of 0, 3, and 6 g/plant and ammonium molybdate application rates of 0, 1.5, and 3 g/plant (Table 1 and Table 2). We selected the fertilization levels based on the current practices and applications for Akebia trifoliata observed in the local region while also referencing the study by Wu [24] and Li et al. [25]. The zinc sulfate heptahydrate (content ≥99.8%) was produced by Xilong Scientific Chemical Co., Ltd., Shantou, China; the manganese sulfate monohydrate (content ≥99.0%) and ammonium molybdate tetrahydrate (content ≥99.0%) were produced by Sinopharm Chemical Reagent Co., Ltd., Shanghai, China. The experiment consisted of nine treatments, each with three replicates; each replicate contained three test plants spaced 2 m apart. The treatment without fertilizer application served as the control group.
In early April 2023, a base fertilizer was applied to the roots once at a fertilizer application rate of 100 g/plant of 15N:15P:15K compound fertilizer produced by Xindu Chemical Fertilizer Co., Ltd., Chengdu, China. In late April 2023, circular cultivation was conducted centered on the root neck of the participating plants, with a cultivation depth of 5–10 cm, shallower inside and deeper outside. Micronutrient fertilizers were uniformly mixed with fine soil and then applied to the soil. The fertilizer application area was covered with ~5 cm of soil to prevent the loss of micronutrient fertilizers due to watering or rainwater erosion of the soil surface. After that, normal water and fertilizer management was implemented.
This table represents the fertilization methods for all treatments. The levels (1, 2, 3) of Zn, Mn, and Mo in the table represent different fertilization concentrations, as follows: Zn (1, 2, 3) indicates ZnSO4 fertilization amounts of 0 g, 3 g, and 6 g per plant. Mn (1, 2, 3) indicates MnSO4 fertilization amounts of 0 g, 3 g, and 6 g per plant. Mo (1, 2, 3) indicates Na2MoO4·2H2O fertilization amounts of 0 g, 1.5 g, and 3 g per plant. T1 is the control group, with no fertilization.

2.4. Method of Index Determination

2.4.1. Sample Collection

Leaf samples were collected at 0, 60, 120, and 180 days after fertilization, with collections occurring at the same time each day, for a total of four collections. For each treatment, 50 g of leaves and 2 cm segments of the basal stem were randomly collected from each plant, totaling 150 g, thoroughly mixed, and considered as one sample. Some of the leaves were used for leaf area (LA) and leaf thickness (LT) measurement; others were collected immediately and placed in liquid nitrogen.

2.4.2. Determination of Leaf Indexes

Freshly picked leaves were washed and leaf images were scanned using an EPSONv 39 scanner (Epson, Nagano, Japan). The LA was calculated using Image J 1.53 (National Institute of Health, Bethesda, MA, USA). The LT was measured using calipers (accurate to 0.01 mm), avoiding the main leaf vein. Different parts along the main vein on the same side of the leaf measured three times the average value as the LT.
The contents of chlorophyll a and b in leaves were determined by spectrophotometer [26].

2.4.3. Determination of Physiological Characteristics

The OA, rutin, and calceolarioside B of Stauntonia leucantha leaves were determined using UPLC-Waters XEVO-TQS (Waters, Milford, MA, USA). Detailed methods are provided in Appendix A.

2.5. Data Processing

SPSS 25 (IBM, Armonk, NY, USA) was used for one-factor analysis of variance and Duncan’s multiple comparisons. Excel (Microsoft, Redmond, WA, USA) was used for data processing and orthogonal experiment analysis of variance. GraphPad Prism 8 (GraphPad Software, La Jolla, CA, USA) was used for graphing. Origin 2024 (OriginLab, Wellesley/Newton, MA, USA) was used for correlation analysis. The method of comprehensive score proposed by Liu et al. [27] and Chen et al. [28] to select the best fertilization method.

3. Results

3.1. Effects of Different Fertilization Methods on Leaf Growth Characteristics at Different Stages

Leaf area of Stauntonia leucantha continuously increased under all treatments from 0–180 d after fertilization (Table 3). The LA of all fertilized treatments was higher than the non-fertilized treatments (Figure 1A). The LA increased the fastest in 0–60 d after fertilization, slowed down in 60–120 d, and was almost constant from 120–180 d (Figure 1A). The LA reached a maximum value of 28.59 cm2 in T9 treatment, with an increase rate of 78%, and a minimum value of 20.66 cm2 in T1 treatment, with an increase rate of 23%.
Within 0–60 d of fertilization, the LT showed a continuous increase under all treatments (Table 3). The increase in the LT slowed within 60–180 d after fertilizer application, with most treatments showing almost no significant changes (Figure 1B). The LT increased the fastest in 0–60 d after fertilization and slower in 60–180 d (Figure 1B). The LT reached a maximum value of 0.24 mm in T9 treatment, with an increase rate of 140%, and a minimum value of 0.14 mm in T1 treatment, with an increase rate of 17%.
According to Table 4, both Zn and Mn had significant effects (p < 0.01) on LA and LT. Mo had no significant effect on LA at 60 days after fertilization but had a significant effect (p < 0.05) on LA at 120 and 180 days. In addition, Mo only had a significant effect (p < 0.05) on LT at 60 days.
Zn and Mn significantly enhanced the LA, LT, and chlorophyll a and b contents in the leaves of Stauntonia leucantha (Figure 2A,B,D,E). These indicators escalated with increasing fertilization level, peaking at a fertilization level of three. Mo promoted the LA at 120 and 180 days (Figure 2C). Furthermore, Mo promoted LT to some extent but not significantly (Figure 2F).

3.2. Effects of Different Fertilization Methods on the Content of Leaf Metabolic Substances

The contents of chlorophyll a, chlorophyll b, OA, rutin, and calceolarioside B in fertilized treatments were significantly higher than those in unfertilized treatments from 0 to 180 days after fertilization (Figure 3).
Chlorophyll a content showed a continuous increase under most treatments (Figure 3A). In contrast, the chlorophyll a content under treatments T1 and T3 decreased slightly within the 120–180 days. Chlorophyll b content increased most rapidly within 0–60 days (Figure 3B). The chlorophyll content increased slowly within 60–120 days and even slightly decreased in the T3 and T4 treatments. In addition, the chlorophyll b content of all fertilized treatments increased between 120 and 180 days. OA content showed a continuous increase within 180 days (Figure 3C). The content of OA increased at a gradually slowing rate over time, but T7, T8, and T9 showed a higher increment between 120 and 180 days. Similarly, rutin content showed a continuous increase (Figure 3D), with the most rapid increase between 0 and 60 days, almost no increase between 60 and 120 days, and a slight increase between 120 and 180 days. Similarly, the content of calceolarioside B also showed continuous growth (Figure 3E), but the increase was most rapid between 60 and 120 days.
According to Table 5, the chlorophyll a reached a maximum value of 8.75 mg·g−1 in the T9 treatment with an increase rate of 94% and a minimum value of 5.15 mg·g−1 in the T1 treatment, with an increase rate of 14%; the chlorophyll b reached the maximum value of 2.93 mg·g−1 in the T9 treatment, with an increase rate of 78%, and the minimum value of 1.72 mg·g−1 in the T1 treatment, with an increase rate of 5%. OA content reached the maximum value of 2792.87 ng·mL−1 in the T7 treatment, with an increase rate of 127%, and the minimum value of 1539.92 ng·mL−1 in the T1 treatment, with an increase rate of 25%; the rutin content reached the maximum value of 5442.55 ng·mL−1 in the T9 treatment, with an increase rate of 2300%, and the minimum value of 2426.63 ng·mL−1 in the T1 treatment, with an increase rate of 1019%; the content of calceolarioside B reached the maximum value in the T7 treatment (2648.60 ng·mL−1), with an increase rate of 6712%, and the minimum value in the T1 treatment (301.81 ng·mL−1) with an increase rate of 681%.
As shown in Table 6, both Zn and Mn had a significant effect (p < 0.01) on chlorophyll a, chlorophyll b, OA, and rutin contents at 60, 120, and 180 days. Mo significantly affected the content of chlorophyll a (p < 0.05), but not chlorophyll b (p > 0.05). Mo also significantly influenced the contents of OA and rutin (p < 0.01); however, the impact of Mo on rutin content at 180 days only reached p < 0.05. In addition, calceolarioside B content on different days was significantly affected by Zn (p < 0.01). Although Mn and Mo did not significantly affect the content of calceolarioside B at 60 days (p > 0.05), they significantly affected it at 120 and 180 days (p < 0.01).
Based on the F-ratios (Table 6), we found that zinc had a stronger effect on the contents of chlorophyll a, chlorophyll b, OA, and rutin across different time periods compared to Mn and Mo. In addition, the effect of Mn on chlorophyll and rutin was greater than Mo. However, at 120 days, the effect of Mo on calceolarioside B was greater than that of Mn.
Zn and Mn significantly enhanced the chlorophyll a and b contents in the leaves of Stauntonia leucantha over all periods (Figure 4A,B,D,E). These indicators escalated with increasing fertilization level, peaking at a fertilization level of 3. Mo promoted the chlorophyll a content at 60 and 120 days; however, excessive application led to a decrease in chlorophyll a content at 180 days (Figure 4C). In addition, the content of chlorophyll b was not affected by the change of Mo fertilizer application (Figure 4F).
Zn and Mo significantly enhanced the OA content in the leaves of Stauntonia leucantha (Figure 5A,C), whereas Mn did not contribute to the accumulation of OA at any period (Figure 5B). A significant variation in the OA content was observed under different Zn release levels, with Zn-2 and Zn-3 levels exhibiting markedly higher levels than Zn-1, and Zn-3 reaching the peak value (Figure 5A). Conversely, the OA content in the Mn-2 level was significantly lower than that of the Mn-1 level at any period, but no significant difference was found between the Mn-3 and Mn-1 levels; the maximum value was reached at the Mn-1 level (Figure 5B). Similarly, significant differences in the OA content were observed among various Mo fertilization levels at every period (Figure 5C). Both Mo-2 and Mo-3 levels demonstrated substantial increases in the OA content compared to the Mo-1 level. A significant difference was found between the Mo-2 and Mo-3 levels. The highest OA content was recorded at the Mo-3 level.
The rutin content exhibited a pronounced difference among various fertilization levels of Zn. Specifically, the rutin content at Zn-2 and Zn-3 was significantly higher than that of Zn-1, with Zn-3 reaching the maximum value at every period (Figure 5D). This pattern of response to Zn fertilization was mostly mirrored by Mn, where rutin content at Mn-3 levels was significantly higher than Mn-1 and Mn-2 at every period, indicating a similar efficacy to Zn in enhancing rutin accumulation (Figure 5E). No significant difference in the rutin content between Mo-2 and Mo-1 at 180 days; however, Mo-3 exhibited a significant difference compared to both Mo-2 and Mo-1, with the highest rutin content observed in Mo-3 (Figure 2F). Within 120 days, rutin reached the maximum value at Mo-2, indicating that excessive Mo fertilization is detrimental to the early accumulation of rutin.
The calceolarioside B content in the leaves of Stauntonia leucantha differed among different Zn fertilization levels. It increased with increasing Zn fertilization levels, with its content in Zn-2 and Zn-3 being significantly higher than that in Zn-1; however, no significant difference was observed between Zn-2 and Zn-3 at any period (Figure 5G). Mn fertilization did not enhance the accumulation of calceolarioside B in the leaves of Stauntonia leucantha (Figure 5H). Instead, the calceolarioside B decreased with increasing Mn fertilizer concentrations at 120 and 180 days. Specifically, the contents at Mn-2 and Mn-3 fertilization levels were significantly lower compared to Mn-1. The variation in calceolarioside B content under different Mo fertilization levels was mostly consistent with that of Zn (Figure 5I). Specifically, the calceolarioside B was higher in Mo-3 than in Mo-1 and Mo-2, and no significant differences observed between Mo-1 and Mo-2 at 120 and 180 days. At 60 days, although there was no significant difference in calceolarioside B content under different Mo fertilization levels, the calceolarioside B content under Mo-3 was slightly higher than that under Mo-1 and Mo-2. The maximum calceolarioside B content was reached at Mo-3 at every period.

3.3. Correlation Analysis of Leaf Indicators in Leaves of Stauntonia leucantha Under Zinc, Manganese, and Molybdenum Co-Fertilization

At 60 days after fertilization (Figure 6), the correlation between the LA and LT as well as the content of chlorophyll a, chlorophyll b, OA, and rutin in the leaves of Stauntonia leucantha under co-fertilization of Zn, Mn, and Mo was determined to be significant (p < 0.05). The LT was significantly correlated with the content of chlorophyll a, chlorophyll b, OA, and rutin (p < 0.05), indicating that an increase in LA and LT may promote the accumulation of metabolic substances and photosynthetic pigments within the leaves. Both chlorophyll a and b contents significantly correlated with the OA and rutin content (p < 0.05), suggesting that the accumulation of photosynthetic pigments during this period significantly affected the OA and rutin metabolism. Additionally, calceolarioside B content was significantly correlated with the OA and rutin content (p < 0.05).
As shown in Figure 7, the correlation between rutin and each indicator had changed at 120 days, and it only showed a significant correlation with OA (p < 0.05). In addition, the correlations between calceolarioside B and LA, LT, and chlorophyll a became significant (p < 0.05).
At 180 days, there was nearly an identical pattern of correlations among the various indicators as seen at 60 days (Figure 8). The only noteworthy alteration was the emergence of a significant correlation between LA and calceolarioside B (p < 0.05).

3.4. Selection of Optimal Fertilization Methods

According to the results of the comprehensive scoring method (Table 7), the comprehensive score of experiment T7 was the highest (61.2), the treatment method was Zn3Mn1Mo3, and the comprehensive score of experiment T9 was the second highest (61.0); R(Zn = 35.23) > R(Mn = 10.46) > R(Mo = 10.33). for the leaf quality of Stauntonia leucantha, the influencing factors in descending order of importance (Table 8).

4. Discussion and Conclusions

4.1. Trends in Leaf Growth of Stauntonia leucantha

LA and LT are important indicators of a plant’s growth status and are related to nutrient addition and environmental changes. In this study, the increase in the LA and LT of Stauntonia leucantha leaves mainly occurred in the first 0–120 days after fertilizer application and gradually slowed down in the 120–180 days after fertilizer application (Figure 1). This may be attributed to the fact that fertilization improves resource availability for plants. Under nutrient-sufficient conditions, plants tend to reduce the synthesis of secondary metabolites and invest resources in growth and propagation [29]. This also explains the high medicinal value of the old leaves of Stauntonia leucantha, as fertilization reduces the plant’s defense needs; it can devote more resources to primary metabolism and growth [30]. The gradual loss of nutrients from the soil after fertilization, as well as the tendency of plants to allocate nutrients to reproductive organs as they enter the flowering and fruiting stages, may be responsible for slowing leaf growth.
We found that the impact of micronutrients on leaf growth and metabolite content varies over different periods (Figure 2, Figure 4 and Figure 5). This may be due to the fact that the absorption of nutrients by plants is a continuous and slow process. It is only when nutrients accumulate to a sufficient concentration within the plant that they can exert a significant impact. In addition, the experiment was carried out in the field; environmental changes may also have an effect. Moreover, the nutrient utilization strategies of plants may be different at different periods, and this variation could potentially contribute to the phenomenon.

4.2. Effect of Zinc, Manganese, and Molybdenum on Leaf Quality of Stauntonia leucantha

Irrational fertilization widely exists in cultivation, resulting in insufficient or excessive micronutrients or macronutrients and inhibiting the growth and development of plants, such as the growth of leaves, stems, and roots. This inhibition will further affect plant photosynthesis, nutrient absorption, and stress resistance, resulting in plant yield reduction. Inappropriate Zn fertilization negatively affected plants, resulting in a significant decrease in yield and metabolites [31]. In addition, Mn is one of the most abundant trace elements in the lithosphere, but its concentration in soils is highly variable [32]. Moreover, appropriate Mo content in plants is the key factor to ensure the accumulation of molybdenum-containing enzymes, which ensures the accumulation of metabolites [21]. This is a crucial factor in ensuring the medicinal value of plants. Therefore, in the fertilization process, it is necessary to continuously optimize according to the actual situation. Reasonable co-fertilization of Zn, Mn, and Mo effectively improves the medicinal value of plant leaves. The results of this study show that co-fertilization of different levels of Zn, Mn, and Mo significantly affects the leaf quality of Stauntonia leucantha (Table 3 and Table 5).
LA is an important indicator that directly reflects the plant growth conditions. To a certain extent, LT reflects a leaf’s ability to retain water, resist external mechanical damage, and retain resources, all of which are related to nutrient addition, environmental changes, and other factors. In this experiment, Zn fertilization resulted in a significant increase in LA and LT (Figure 2A,D), consistent with the results of Hasani et al. [33] and Ojeda-Barrioset et al. [34]. This may be attributed to the association of Zn with the metabolism and synthesis of IAA and gibberellin (GA3) in plants [35,36]. IAA promotes plant growth and delays senescence [37], while GA3 promotes cell growth and photosynthesis [38]. Fertilization with Zn can increase the content of IAA and GA3 [39], which promotes plant growth and results in increased LA and LT. Mn fertilization also significantly increases the LA and LT (Figure 2B,E), which is consistent with the studies of Hasani et al. [33] and Papadakis et al. [40]. This may be attributed to the involvement of Mn in the metabolic processes. Fluctuations in Mn levels within plants have a direct impact on IAA oxidase [41], which is related to the promotion of plant growth. Therefore, Mn fertilization promotes an increase in the LA and LT. Mo fertilization significantly increased LA between 120 and 180 days (Figure 2C), likely due to the key role of Mo in nitrogen metabolism and assimilation [42]. Knops et al. [43] found a significant positive correlation between nitrogen and LA in a study on rice, suggesting that Mo fertilization increases the metabolism and uptake efficiency of nitrogen in plants, indirectly causing an increase in LA. Mo fertilization promoted LT at 120 and 180 days, but it was not significant. However, LT was promoted within 60 days (Figure 2F). Yu et al. [44] showed that Mo fertilization increased the leaf water retention and LT, which contrasts the experimental results in this paper. This discrepancy may be due to the complex interactions between Mo and other nutrient elements. Excessive or inadequate levels of other nutrient elements in the soil may affect the uptake and utilization of Mo by plants, thus weakening the effect of Mo application on LT. Additionally, the month with higher temperature is expected to arrive after 60 days. Increased temperature may result in lower leaf water content and possibly lower LT [45], which could potentially counteract the promoting effect of Mo.
Chlorophyll content reflects, to some extent, the efficiency of photosynthesis, which determines crop yield and quality [46]. In this study, we observed that Zn fertilization significantly increased the chlorophyll a and b in leaves (Figure 4A,D). This increase in chlorophyll content corresponds to higher levels of Zn fertilization. Zn participates in the synthesis of chlorophyll and prevents its degradation [47,48], thereby increasing the chlorophyll content in leaves. The effect of Mn fertilization on the chlorophyll a and b in leaves is analogous to that of Zn fertilization, both demonstrating an increase in chlorophyll content as the fertilization dosage rose (Figure 4B,E). This may be related to the fact that Mn is one of the essential elements required to maintain the normal structure of chloroplasts, which is integral to the oxygen release process in photosynthesis [18]. In addition, Mn participates in the antioxidant system of plants [49], alleviating the damage caused by oxidative stress. Therefore, the application of Mn fertilizer at a certain concentration can increase the chlorophyll content. Mo significantly promoted the content of chlorophyll a (Figure 4C). In addition, although molybdenum promoted the content of chlorophyll b to some extent, it was not significant (Figure 4F). This trend may be attributed to the fact that Mo is mostly present in nitrite reductase [50], an enzyme mainly found in chloroplasts or preplastids and involved in various reactions, including photosynthesis. Mo fertilization led to an increase in this type of enzyme, which affects photosynthesis and increases the chlorophyll content. However, excessive Mo fertilization can decrease chlorophyll content, likely due to antagonistic effects that interfere with the absorption of other nutrients.
OA, a triterpenoid saponin, was first identified in Stauntonia leucantha by Hu et al. [51]. OA has antitumor, lipid-lowering, antibacterial, and anti-inflammatory effects [52]. In this study, Zn fertilization significantly increased the OA content in leaves of Stauntonia leucantha (Figure 5A), which is consistent with the findings of Tang et al. [53] on Ganoderma lucidum and Ali [54] for triterpenoid compounds of Calendula officinalis. The synthesis of triterpenoid saponins is regulated by a variety of enzymes, such as squalene synthase (SQS), squalene epoxidase (SE), and oxidosqualene cyclase (OSC) [55,56,57]. Zn is a component of various enzymes; it may directly or indirectly affect the activity of enzymes associated with triterpenoid saponins. Furthermore, the synthesis of triterpene saponins occurs in the cytoplasm and plastids [58,59,60]. Chloroplasts, a type of plastid, are crucial for this process. Zn plays a role in ensuring the stability of chloroplast structure. Disruption of the chloroplast envelope and abnormalities in the lamellar structure can be due to Zn deficiency [47]. He et al. [61] demonstrated that the chloroplast ultrastructure in triticale leaves is optimal under sufficient Zn application. This suggests that Zn application may stabilize the synthesis site of triterpene saponins, thereby increasing the content of these compounds. Mn application did not significantly promote the content; in fact, it exhibited an inhibitory effect at low release levels (Figure 5B). This result differs from the promotional effects reported by Zhang et al. [62] on Ganoderma lucidum but is largely consistent with the changes observed by Vidović et al. [63] in olive leaves. However, it cannot be concluded that Mn does not promote the OA content in Stauntonia leucantha leaves. This difference may be due to differences in species, which have different strategies for nutrient uptake and utilization, as well as differences in soil nutrients, which may have affected the uptake and accumulation of nutrients in plants. Differences in fertilizer application may also be responsible for this difference, or it may be due to insufficient release to reach the threshold for promoting content accumulation, so the effect of higher Mn fertilization levels on OA content in the leaves of Stauntonia leucantha needs to be further investigated. Fertilization of Mo can also significantly increase the content of OA in leaves (Figure 5C), which is consistent with the results of the study of triterpenoid compounds in Lily by Zhou et al. [64]. This may be due to Mo being able to maintain the stability of the chloroplasts [22] to ensure the place of the synthesis of triterpenoid saponin compounds.
Rutin is a flavonoid commonly found in Lardizabalaceae and has a variety of pharmacological functions and physiological activities, such as anti-tumor, anti-inflammatory, and anti-aging [65]. In this study, Zn fertilization significantly increased leaf rutin content (Figure 5D), which was consistent with the study of Naguib et al. [66]. The synthesis of rutin is regulated by phenylalanine ammonia-lyase (PAL) [67]. The first step is the conversion of phenylalanine to cinnamic acid by PAL, which is subsequently converted to rutin after a series of reactions [68]. Appropriate Zn concentrations could improve the gene expression and its synthesis of PAL [69], and Zn can increase phenylalanine content in plants [70,71,72], providing sufficient substrate for the synthesis and transformation of rutin. Fertilization of Mn and Mo can also significantly increase the content of rutin in leaves (Figure 5E,F), which is consistent with the results reported for tobacco by Liu [73]. Mn and Mo have the potential to enhance PAL activity [73,74], thereby promoting the synthesis of rutin.
Calceolarioside B is a phenylethanoside that is considered as the identification index of the medicinal quality of Stauntonia leucantha [1]. It has antibacterial, antiaging, and other effects [75]. In this study, Zn and Mo application significantly increased the calceolarioside B in the leaves of Stauntonia leucantha, while Mn application significantly reduced the calceolarioside B in the leaves of Stauntonia leucantha (Figure 5G–I). It has been suggested that the phenylpropanoids in phenylethanol glycosides are partially derived from phenylalanine or tyrosine [76,77,78] and that the synthesis of cinnamic acid derivatives from phenylalanine is catalyzed by PAL, cinnamate 4-hydroxylase (C4H), and 4-coumarate: Coenzyme A ligase (4CL) [76,77,78]. It is possible that Zn and Mo increase the PAL activity [69,73], thereby increasing the calceolarioside B content; however, the reason for the inhibition of calceolarioside B content by Mn remains unclear. The possible reason is that, structurally, phenylethanoid glycosides are derived from the phenylethanol skeleton. They are characterized by aromatic acids (such as caffeic acid, cinnamic acid, etc.) and hydroxyethyl moieties linked to β-pyranose units through ester and glycosidic bonds, respectively [79]. β-pyranose is a type of carbohydrate. The change of Mn may cause the disorder of carbohydrate synthesis and transport in plants [80], which may lead to the decrease of calceolarioside B. At present, research on calceolarioside B in Stauntonia leucantha mainly focuses on the determination method and content detection. Research on the effect of micronutrient fertilization on the change of the content of calceolarioside B has not yet been found; its metabolic pathway, related synthetic enzymes in the blank, synthetic pathway, and key enzymes must be subjected to further investigation.
We found significant correlations existed among LA, LT, and chlorophyll content (Figure 6, Figure 7 and Figure 8). Chlorophyll content reflects the efficiency of photosynthesis, which indicates the plant’s ability to capture energy [46]. Plants require substantial energy and nutrients for growth. A higher chlorophyll content ensures that plants can capture more energy, leading to improved growth conditions. This explains the significant correlation among chlorophyll content, LA, and LT. In addition, we found that the correlation between rutin content and other indicators was stronger at 60 and 180 days, which may be related to plant defense mechanisms. This phenomenon may indicate that rutin acts as an antioxidant and is involved in resisting environmental stress during early and late leaf growth. Significant correlations also existed among calceolarioside B, OA, and rutin (Figure 7). Calceolarioside B, OA, and rutin are secondary metabolites with similar pharmacological activities. The biosynthetic pathways for these compounds in plants may overlap, indicating collaborative functions in plant defense mechanisms and adaptive responses. For instance, their metabolism often involves common enzymes such as PAL [68,69], or their metabolic pathways may involve the same intracellular sites, including the Golgi apparatus and endoplasmic reticulum, which needs further investigation. This overlap likely underlies the significant correlation observed among these compounds. The interaction between these compounds and their collective influence on plant growth, development, and ecological adaptation is of considerable importance.
This study only focused on the effect of trace element fertilization on the leaf growth and metabolic substance content changes of Stauntonia leucantha leaves. In order to complement the research vacancy about this plant, the effects of fertilization on different parts of the plant, such as fruit and flower, need to be studied in the future. In addition, the metabolic pathways of these metabolites in Stauntonia leucantha need to be further verified. Furthermore, the field environment is highly complex and variable. Our current study, which focuses on the period of 0-180 days following fertilization, is insufficient to fully capture the long-term effects and dynamics of the processes under investigation. Given the transient nature of our observations, it is imperative that we extend the duration of our experiments to encompass a more extensive timeframe. Such an extension will enable us to gain a more comprehensive understanding of the phenomena at play and to better assess the long-term implications of our findings. Consequently, conducting experiments with a longer duration will be a key priority in our future research endeavors. This limitation in the temporal scope of our current study represents one of the areas where we recognize the need for improvement and further exploration.

4.3. Best Combined Application of Zinc, Manganese, and Molybdenum

In orthogonal experiments, when multiple indicators are investigated. This makes it difficult to reach a unified conclusion. Through analysis of variance and multiple comparisons, the indexes were further scored, the importance and measured value of each index were compared, and a comprehensive score was obtained for each experimental result. The score was used as a single index for statistical analysis to evaluate the treatment effect more comprehensively and objectively and eliminate one side of a single index. Suitable treatments can be selected scientifically. In the actual cultivation of Stauntonia leucantha, due to the decrease in temperature after October, the plant enters a period of slow growth or even dormancy. Therefore, leaves are typically collected in October for use. Consequently, we selected the samples collected at 180 days for comprehensive analysis. This approach is anticipated to hold enhanced practical significance and utility.
In this study, we found that Zn3Mn1Mo3 (T7) was the best combined application scheme with a comprehensive score of 61.2, followed by Zn3Mn3Mo2 (T9) with a comprehensive score of 61.0, a small difference between the two schemes, but all the indicators evaluated for the T7 treatment, including LA, LT, chlorophyll content, OA content, rutin content, and calceolarioside B content, were above average (Table 7). Moreover, the T9 treatment scored only 2.5 points on calceolarioside B, a metabolite considered as the identification index of medicinal quality of Stauntonia leucantha. This low score on calceolarioside B indicates that the T9 treatment may not be suitable for practical medicinal use. Therefore, we conclude that T7 treatment (zinc sulfate 6 g/plant, manganese sulfate 0 g/plant, ammonium molybdate 3 g/plant) is the best treatment for the medicinal value of Stauntonia leucantha leaves. In addition, it is well known that plants live in a constantly changing environment, which results in poor application effects of trace element fertilizers in actual production. Therefore, we need to investigate the content of micronutrients in the soil before fertilization, adjust the optimal fertilizer concentration according to the actual situation, and monitor it during the cultivation process.

5. Conclusions

This study investigated the effects of Zn, Mn, and Mo on the growth and medicinal value of the leaf of Stauntonia leucantha through a three-factor, three-level orthogonal experiment. We found reasonable fertilizer concentrations of Zn significantly increased LA, LT, chlorophyll, OA, rutin, and calceolarioside B content. In addition, Mn significantly increased LA, LT, chlorophyll, and rutin content but was not beneficial to the accumulation of OA and calceolarioside B in the leaves. Moreover, Mo significantly increased LA and calceolarioside B content between 120 and 180 days and LT within 60 days. The contents of chlorophyll a, OA, and rutin were increased by Mo at each period, while the content of chlorophyll b was not affected by Mo. The influence of Zn, Mn, and Mo on the comprehensive medicinal value of Stauntonia leucantha was in the order of Zn > Mn > Mo, with the optimal fertilizer treatment being zinc sulfate 6 g/plant, manganese sulfate 0 g/plant, and ammonium molybdate 3 g/plant. This study provides reference for the artificial cultivation and fertilization method of Stauntonia leucantha, filling part of the research gap for this plant.

Author Contributions

Conceptualization, Y.Z., J.Z. and B.L.; methodology, Y.Z. and J.Z.; software, Y.Z. and Y.C.; validation, Y.Z., J.Z. and J.C.; formal analysis, Y.Z., Y.C. and Z.Z.; investigation, Y.Z., J.Z. and J.C.; resources, J.C. and X.L.; data curation, Y.Z., X.L. and Z.Z.; writing—original draft preparation, Y.Z.; writing—review and editing, Y.Z., J.Z. and B.L.; visualization, Y.Z. and J.Z.; supervision, B.L.; project administration, B.L. and J.Z.; funding acquisition, J.Z. and B.L. All authors have read and agreed to the published version of the manuscript.

Funding

This research was funded by the Forestry Science and Technology Project of Fujian Province (application of comprehensive development and utilization technology of Stauntonia leucantha, grant number [B23002/108]).

Data Availability Statement

The data presented in this study are available on request from the corresponding author due to laboratory confidentiality regulations.

Conflicts of Interest

The authors declare no conflicts of interest.

Abbreviations

The following abbreviations are used in this manuscript:
ZnZinc
MnManganese
MoMolybdenum
LALeaf area
LTLeaf thickness
OAOleanolic acid
IAAIndole-3-acetic acid
GA3Gibberellin A3

Appendix A

Methods for the Determination of OA, Rutin, and Calceolarioside B by UPLC-Waters XEVO-TQS

The collected leaf samples were removed from liquid nitrogen and immediately put into a mortar and pestle for grinding, while liquid nitrogen was added to prevent the samples from deliquescing. The ground samples were freeze-dried in a vacuum freeze-dryer until constant weight and passed through a 100-mesh sieve. A precision balance scale was used to weigh 0.25 mg of sample, and 1 mL of 70% methanol solution was added to each sample at low temperature, vortexed and mixed well, sonicated for 20 min, centrifuged at 4 °C and 12,000 rpm for 10 min, pipetted 900 uL supernatant into a centrifuge tube, centrifuged at 4 °C and 12,000 rpm for 10 min, and then pipetted 700 uL supernatant for UPLC test.
Instrument type: Waters XEVO-TQS.
Positive Ion Mode Parameters: Capillary: 0.85 kV; Cone: 30 V; Source Temperature: 150 °C; Cone Gas Flow: 150 L/hr; Desolvation Gas Flow: 800 L/hr; Desolvation Gas Temperature: 400 °C.
Negative Ion Mode Parameters: Capillary: 1.50 kV; Cone: 30 V; Source Temperature: 150 °C; Cone Gas Flow: 150 L/hr; Desolvation Gas Flow: 800 L/hr; Desolvation Gas Temperature: 400 °C.
Scanning mode: MRM (Multiple Reaction Monitor).
Solvent A: Water+5mM Ammonium Acetate; Solvent B: MeOH.
The mobile phase gradients are shown in Table A1 below.
Table A1. Gradient method of binary solvents.
Table A1. Gradient method of binary solvents.
Time (min)Flow Rate (mL/min)%A%B
00.37030
10.34060
20.30100
70.30100
7.10.37030
100.37030
Column temperature: 40 °C; sample chamber temperature: 10 °C; sample volume: 2 μL; chromatographic column type and specification: Waters BEH C18 100 mm × 2.1 mm 1.7 μm.
The transition parameters for the scanning channels are shown in Table A2 below.
Table A2. Transition parameters used in MRM scanning.
Table A2. Transition parameters used in MRM scanning.
IndicatorsMRM TransitionCollision Energy (V)ESI Mode
Oleanolic acid455.0 > 406.9 *40ES-
455.0 > 97.055
Calceolarioside B476.8 > 160.8 *21ES-
476.8 > 315.020
400.9 > 134.7 *30
Rutin609.1 > 271.156ES-
609.1 > 300.2 *36
* indicates quantitative ions.
Oleanolic acid standard solutions with concentrations of 0.2, 2, 20, 800, and 1200 ng/mL were injected and tested. Rutin standard solutions with concentrations of 0.1, 1, 10, 40, 100, and 600 ng/mL were injected and tested. Calceolarioside B standard solutions with concentrations of 0.2, 2, 20, 80, 200, and 800 ng/mL were injected and detected. The standard curve was drawn with the concentration of standard solution as the abscissa and the peak area as the ordinate. The limit of detection (LOD) was the concentration at a S/N ratio of 3:1. The limit of quantification (LOQ) was the concentration at a S/N ratio of 10:1.
Table A3. Standard curves of compounds, limits of detection, and limits of quantification.
Table A3. Standard curves of compounds, limits of detection, and limits of quantification.
IndicatorsStandard CurveR2Limit of DetectionLimit of Quantitation
Oleanolic acidy = 34.7451x − 2.815130.99956.33 ng18.14 ng
Calceolarioside By = 742.58x + 2218.930.99525 ng33.3 ng
Rutiny = 1185.31x + 25394.20.99660.685 ng34.8 ng

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Horticulturae 11 00123 g001
Figure 1. Changes of (A) leaf area and (B) leaf thickness during 0–180 days after zinc (Zn), manganese (Mn), and molybdenum (Mo) co-fertilization. Different colored curves represent different treatments. Zero, 60, 120, and 180 represent the measurement time of the sample after fertilization.
Figure 1. Changes of (A) leaf area and (B) leaf thickness during 0–180 days after zinc (Zn), manganese (Mn), and molybdenum (Mo) co-fertilization. Different colored curves represent different treatments. Zero, 60, 120, and 180 represent the measurement time of the sample after fertilization.
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Figure 2. The effect of zinc (Zn), manganese (Mn), and molybdenum (Mo) co-fertilization on leaf area and leaf thickness of Stauntonia leucantha leaves at 60, 120, and 180 days. (A) Effects of different Zn fertilization levels on leaf area at 60, 120, and 180 days. (B) Effects of different Mn fertilization levels on leaf area at 60, 120, and 180 days. (C) Effects of different Mo fertilization levels on leaf area at 60, 120, and 180 days. (D) Effects of different Zn fertilization levels on leaf thickness at 60, 120, and 180 days. (E) Effects of different Mn fertilization levels on leaf thickness at 60, 120, and 180 days. (F) Effects of different Mo fertilization levels on leaf thickness at 60, 120, and 180 days. Different colored columns represent the average values of the metabolite under different levels of micronutrients. Vertical bars above the mean indicate the standard error of replicates. Different lowercase letters represent significant differences by the Duncan’s multiple range test (p < 0.05, n = 3). Zn-1, Zn-2, and Zn-3 mean 0 g, 3 g, and 6 g of Zn fertilizer application; Mn-1, Mn-2, and Mn-3 mean 0 g, 3 g, and 6 g of Mn fertilizer application; Mo-1, Mo-2, and Mo-3 mean 0 g, 1.5 g, and 3 g of Mo fertilizer application.
Figure 2. The effect of zinc (Zn), manganese (Mn), and molybdenum (Mo) co-fertilization on leaf area and leaf thickness of Stauntonia leucantha leaves at 60, 120, and 180 days. (A) Effects of different Zn fertilization levels on leaf area at 60, 120, and 180 days. (B) Effects of different Mn fertilization levels on leaf area at 60, 120, and 180 days. (C) Effects of different Mo fertilization levels on leaf area at 60, 120, and 180 days. (D) Effects of different Zn fertilization levels on leaf thickness at 60, 120, and 180 days. (E) Effects of different Mn fertilization levels on leaf thickness at 60, 120, and 180 days. (F) Effects of different Mo fertilization levels on leaf thickness at 60, 120, and 180 days. Different colored columns represent the average values of the metabolite under different levels of micronutrients. Vertical bars above the mean indicate the standard error of replicates. Different lowercase letters represent significant differences by the Duncan’s multiple range test (p < 0.05, n = 3). Zn-1, Zn-2, and Zn-3 mean 0 g, 3 g, and 6 g of Zn fertilizer application; Mn-1, Mn-2, and Mn-3 mean 0 g, 3 g, and 6 g of Mn fertilizer application; Mo-1, Mo-2, and Mo-3 mean 0 g, 1.5 g, and 3 g of Mo fertilizer application.
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Figure 3. Changes of (A) chlorophyll a, (B) chlorophyll b, (C) oleanolic acid, (D) rutin, and (E) calceolarioside B during 0–180 days after zinc (Zn), manganese (Mn), and molybdenum (Mo) co-fertilization. Different colored curves represent different treatments. Zero, 60, 120, and 180 represent the measurement time of the sample after fertilization.
Figure 3. Changes of (A) chlorophyll a, (B) chlorophyll b, (C) oleanolic acid, (D) rutin, and (E) calceolarioside B during 0–180 days after zinc (Zn), manganese (Mn), and molybdenum (Mo) co-fertilization. Different colored curves represent different treatments. Zero, 60, 120, and 180 represent the measurement time of the sample after fertilization.
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Figure 4. The effect of zinc (Zn), manganese (Mn), and molybdenum (Mo) co-fertilization on chlorophyll a and chlorophyll b content of Stauntonia leucantha leaves at 60, 120, and 180 days. (A) Effects of different Zn fertilization levels on chlorophyll a content at 60, 120, and 180 days. (B) Effects of different Mn fertilization levels on chlorophyll a content at 60, 120, and 180 days. (C) Effects of different Mo fertilization levels on chlorophyll a at 60, 120, and 180 days. (D) Effects of different Zn fertilization levels on chlorophyll b content at 60, 120, and 180 days. (E) Effects of different Mn fertilization levels on chlorophyll b content at 60, 120, and 180 days. (F) Effects of different Mo fertilization levels on chlorophyll b content at 60, 120, and 180 days. Different colored columns represent the average values of the metabolite under different levels of micronutrients. Vertical bars above the mean indicate the standard error of replicates. Different lowercase letters represent significant differences by the Duncan’s multiple range test (p < 0.05, n = 3). Zn-1, Zn-2, and Zn-3 mean 0 g, 3 g, and 6 g of Zn fertilizer application; Mn-1, Mn-2, and Mn-3 mean 0 g, 3 g, and 6 g of Mn fertilizer application; Mo-1, Mo-2, and Mo-3 mean 0 g, 1.5 g, and 3 g of Mo fertilizer application.
Figure 4. The effect of zinc (Zn), manganese (Mn), and molybdenum (Mo) co-fertilization on chlorophyll a and chlorophyll b content of Stauntonia leucantha leaves at 60, 120, and 180 days. (A) Effects of different Zn fertilization levels on chlorophyll a content at 60, 120, and 180 days. (B) Effects of different Mn fertilization levels on chlorophyll a content at 60, 120, and 180 days. (C) Effects of different Mo fertilization levels on chlorophyll a at 60, 120, and 180 days. (D) Effects of different Zn fertilization levels on chlorophyll b content at 60, 120, and 180 days. (E) Effects of different Mn fertilization levels on chlorophyll b content at 60, 120, and 180 days. (F) Effects of different Mo fertilization levels on chlorophyll b content at 60, 120, and 180 days. Different colored columns represent the average values of the metabolite under different levels of micronutrients. Vertical bars above the mean indicate the standard error of replicates. Different lowercase letters represent significant differences by the Duncan’s multiple range test (p < 0.05, n = 3). Zn-1, Zn-2, and Zn-3 mean 0 g, 3 g, and 6 g of Zn fertilizer application; Mn-1, Mn-2, and Mn-3 mean 0 g, 3 g, and 6 g of Mn fertilizer application; Mo-1, Mo-2, and Mo-3 mean 0 g, 1.5 g, and 3 g of Mo fertilizer application.
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Figure 5. The effect of zinc (Zn), manganese (Mn), and molybdenum (Mo) co-fertilization on oleanolic acid, rutin, and calceolarioside B content of Stauntonia leucantha leaves at 60, 120, and 180 days. (A) Effects of different Zn fertilization levels on oleanolic acid a content at 60, 120, and 180 days. (B) Effects of different Mn fertilization levels on oleanolic acid content at 60, 120, and 180 days. (C) Effects of different Mo fertilization levels on oleanolic acid at 60, 120, and 180 days. (D) Effects of different Zn fertilization levels on rutin content at 60, 120, and 180 days. (E) Effects of different Mn fertilization levels on rutin content at 60, 120, and 180 days. (F) Effects of different Mo fertilization levels on rutin content at 60, 120, and 180 days. (G) Effects of different Zn fertilization levels on calceolarioside B content at 60, 120, and 180 days. (H) Effects of different Mn fertilization levels on calceolarioside B content at 60, 120, and 180 days. (I) Effects of different Mo fertilization levels on calceolarioside B content at 60, 120, and 180 days. Different colored columns represent the average values of the metabolite under different levels of micronutrients. Vertical bars above the mean indicate the standard error of replicates. Different lowercase letters represent significant differences by the Duncan’s multiple range test (p < 0.05, n = 3). Zn-1, Zn-2, and Zn-3 mean 0 g, 3 g, and 6 g of Zn fertilizer application; Mn-1, Mn-2, and Mn-3 mean 0 g, 3 g, and 6 g of Mn fertilizer application; Mo-1, Mo-2, and Mo-3 mean 0 g, 1.5 g, and 3 g of Mo fertilizer application.
Figure 5. The effect of zinc (Zn), manganese (Mn), and molybdenum (Mo) co-fertilization on oleanolic acid, rutin, and calceolarioside B content of Stauntonia leucantha leaves at 60, 120, and 180 days. (A) Effects of different Zn fertilization levels on oleanolic acid a content at 60, 120, and 180 days. (B) Effects of different Mn fertilization levels on oleanolic acid content at 60, 120, and 180 days. (C) Effects of different Mo fertilization levels on oleanolic acid at 60, 120, and 180 days. (D) Effects of different Zn fertilization levels on rutin content at 60, 120, and 180 days. (E) Effects of different Mn fertilization levels on rutin content at 60, 120, and 180 days. (F) Effects of different Mo fertilization levels on rutin content at 60, 120, and 180 days. (G) Effects of different Zn fertilization levels on calceolarioside B content at 60, 120, and 180 days. (H) Effects of different Mn fertilization levels on calceolarioside B content at 60, 120, and 180 days. (I) Effects of different Mo fertilization levels on calceolarioside B content at 60, 120, and 180 days. Different colored columns represent the average values of the metabolite under different levels of micronutrients. Vertical bars above the mean indicate the standard error of replicates. Different lowercase letters represent significant differences by the Duncan’s multiple range test (p < 0.05, n = 3). Zn-1, Zn-2, and Zn-3 mean 0 g, 3 g, and 6 g of Zn fertilizer application; Mn-1, Mn-2, and Mn-3 mean 0 g, 3 g, and 6 g of Mn fertilizer application; Mo-1, Mo-2, and Mo-3 mean 0 g, 1.5 g, and 3 g of Mo fertilizer application.
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Figure 6. Pearson correlation analysis of leaf indicators of Stauntonia leucantha under co-fertilization of zinc (Zn), manganese (Mn), and molybdenum (Mo) at 60 days after fertilization. * represent significant differences between indicators at p < 0.05. The number at the intersection of two indicators represents the Pearson correlation coefficient between the two indicators.
Figure 6. Pearson correlation analysis of leaf indicators of Stauntonia leucantha under co-fertilization of zinc (Zn), manganese (Mn), and molybdenum (Mo) at 60 days after fertilization. * represent significant differences between indicators at p < 0.05. The number at the intersection of two indicators represents the Pearson correlation coefficient between the two indicators.
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Figure 7. Pearson correlation analysis of leaf indicators of Stauntonia leucantha under co-fertilization of zinc (Zn), manganese (Mn), and molybdenum (Mo) at 120 days after fertilization. * represent significant differences between indicators at p < 0.05. The number at the intersection of two indicators represents the Pearson correlation coefficient between the two indicators.
Figure 7. Pearson correlation analysis of leaf indicators of Stauntonia leucantha under co-fertilization of zinc (Zn), manganese (Mn), and molybdenum (Mo) at 120 days after fertilization. * represent significant differences between indicators at p < 0.05. The number at the intersection of two indicators represents the Pearson correlation coefficient between the two indicators.
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Figure 8. Pearson correlation analysis of leaf indicators of Stauntonia leucantha under co-fertilization of zinc (Zn), manganese (Mn), and molybdenum (Mo) at 180 days after fertilization. * represent significant differences between indicators at p < 0.05. The number at the intersection of two indicators represents the Pearson correlation coefficient between the two indicators.
Figure 8. Pearson correlation analysis of leaf indicators of Stauntonia leucantha under co-fertilization of zinc (Zn), manganese (Mn), and molybdenum (Mo) at 180 days after fertilization. * represent significant differences between indicators at p < 0.05. The number at the intersection of two indicators represents the Pearson correlation coefficient between the two indicators.
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Table 1. Experimental factor levels.
Table 1. Experimental factor levels.
FactorABC
levelZn (g/plant)Mn (g/plant)Mo (g/plant)
1000
2331.5
3663
Table 2. Orthogonal experimental design table.
Table 2. Orthogonal experimental design table.
TreatmentMethods of FertilizationFactor
Zn (ZnSO4)Mn (MnSO4)Mo (Na2MoO4·2H2O)
T1 (CK)Zn1Mn1Mo1111
T2Zn1Mn2Mo2122
T3Zn1Mn3Mo3133
T4Zn2Mn1Mo2212
T5Zn2Mn2Mo3223
T6Zn2Mn3Mo1231
T7Zn3Mn1Mo3313
T8Zn3Mn2Mo1321
T9Zn3Mn3Mo2332
Table 3. Results of leaf area and leaf thickness at 0, 60, 120, and 180 days after zinc (Zn), manganese (Mn), and molybdenum (Mo) co-fertilization.
Table 3. Results of leaf area and leaf thickness at 0, 60, 120, and 180 days after zinc (Zn), manganese (Mn), and molybdenum (Mo) co-fertilization.
IndicatorTreatmentDays after Fertilization
060120180
Leaf area/cm2T116.79 ± 0.34 a18.89 ± 0.49 e20.52 ± 0.43 f20.66 ± 0.49 e
T217.03 ± 0.22 a20.07 ± 0.20 d21.59 ± 0.70 e22.16 ± 0.55 d
T316.73 ± 0.43 a21.19 ± 0.86 d22.76 ± 0.05 d23.21 ± 0.15 d
T417.51 ± 0.42 a23.17 ± 0.18 c25.25 ± 0.07 c25.71 ± 0.12 c
T516.45 ± 0.91 a23.97 ± 0.46 abc25.98 ± 0.21 bc26.46 ± 0.27 bc
T616.79 ± 0.80 a24.23 ± 0.20 abc26.73 ± 0.05 b26.96 ± 0.23 bc
T717.58 ± 0.39 a23.72 ± 0.10 bc26.91 ± 0.18 b27.55 ± 0.61 ab
T817.08 ± 0.79 a24.60 ± 0.33 ab26.92 ± 0.46 b27.37 ± 1.10 ab
T916.82 ± 1.19 a24.92 ± 0.26 a28.13 ± 0.11 a28.59 ± 0.40 a
Leaf thickness/mmT10.12 ± 0.01 a0.14 ± 0.01 f0.14 ± 0.00 e0.14 ± 0.00 f
T20.13 ± 0.02 a0.18 ± 0.00 e0.18 ± 0.00 d0.19 ± 0.00 e
T30.13 ± 0.01 a0.19 ± 0.00 d0.19 ± 0.00 d0.20 ± 0.00 de
T40.11 ± 0.01 a0.19 ± 0.00 c0.20 ± 0.00 cd0.20 ± 0.00 cd
T50.12 ± 0.01 a0.20 ± 0.00 bc0.21 ± 0.01 bc0.21 ± 0.00 c
T60.12 ± 0.00 a0.21 ± 0.00 a0.21 ± 0.00 b0.23 ± 0.00 a
T70.12 ± 0.01 a0.20 ± 0.00 b0.22 ± 0.00 b0.22 ± 0.00 b
T80.12 ± 0.01 a0.21 ± 0.00 a0.24 ± 0.00 a0.23 ± 0.01 ab
T90.10 ± 0.01 a0.21 ± 0.00 a0.23 ± 0.01 a0.24 ± 0.01 a
Data are presented as mean ± standard error. Different lowercase letters in the same column indicate significant differences between treatments (p < 0.05). Zero, 60, 120, and 180 represent the measurement time of the sample. T1 represents the fertilization method of Zn1Mn1Mo1, T2 represents the fertilization method of Zn1Mn2Mo2, T3 represents the fertilization method of Zn1Mn3Mo3, T4 represents the fertilization method of Zn2Mn1Mo2, T5 represents the fertilization method of Zn2Mn2Mo3, T6 represents the fertilization method of Zn2Mn3Mo1, T7 represents the fertilization method of Zn3Mn1Mo3, T8 represents the fertilization method of Zn3Mn2Mo1, and T9 represents the fertilization method of Zn3Mn3Mo2, the same as below.
Table 4. Results of one-way ANOVA for leaf area and leaf thickness of Stauntonia leucantha leaves at 60, 120, and 180 days after zinc (Zn), manganese (Mn), and molybdenum (Mo) co-fertilization.
Table 4. Results of one-way ANOVA for leaf area and leaf thickness of Stauntonia leucantha leaves at 60, 120, and 180 days after zinc (Zn), manganese (Mn), and molybdenum (Mo) co-fertilization.
IndicatorSource of VariationDays After FertilizationdFMSF
Leaf areaZn60250.112265.906 **
12079.946751.627 **
18082.782308.363 **
Mn6025.32428.253 **
1206.27358.977 **
1805.87421.881 **
Mo6020.3441.824 ns
1200.5595.253 *
1801.2704.732 *
Leaf thicknessZn6020.00462.7 **
1200.007114.856 **
1800.008120.2 **
Mn6020.00224.477 **
1200.00229.805 **
1800.00339.565 **
Mo6020.0003.586 *
1200.0002.624 ns
1800.0002.176 ns
Zn, Mn, and Mo represent different sources of variance. MS represents the mean square. dF represents variance degrees of freedom. F represents the F-ratio. Sixty, 120, and 180 represent the measurement time of the sample. The level of significance (p-value) is shown in the table: * represents significant differences at p < 0.05, and ** represents significant differences at p < 0.01; ns represents no significance.
Table 5. Result of chlorophyll a, chlorophyll b, oleanolic acid, rutin, and calceolarioside B content at 0, 60, 120, and 180 days after zinc (Zn), manganese (Mn), and molybdenum (Mo) co-fertilization of the leaves of Stauntonia leucantha.
Table 5. Result of chlorophyll a, chlorophyll b, oleanolic acid, rutin, and calceolarioside B content at 0, 60, 120, and 180 days after zinc (Zn), manganese (Mn), and molybdenum (Mo) co-fertilization of the leaves of Stauntonia leucantha.
IndicatorsTreatmentDays after Fertilization
060120180
Chlorophyll a/mg.mL−1T14.52 ± 0.08 a5.28 ± 0.03 g6.05 ± 0.07 e5.15 ± 0.06 e
T24.53 ± 0.04 a5.71 ± 0.05 f6.29 ± 0.06 e7.45 ± 0.11 c
T34.48 ± 0.03 a5.94 ± 0.06 e6.66 ± 0.26 d6.55 ± 0.28 d
T44.53 ± 0.03 a6.17 ± 0.06 d7.16 ± 0.08 c7.56 ± 0.03 c
T54.57 ± 0.12 a6.30 ± 0.06 c7.30 ± 0.12 c7.45 ± 0.09 c
T64.47 ± 0.07 a6.42 ± 0.02 c7.59 ± 0.06 b8.65 ± 0.01 a
T74.48 ± 0.01 a6.35 ± 0.02 c7.70 ± 0.02 b8.03 ± 0.12 b
T84.53 ± 0.03 a6.56 ± 0.02 b7.62 ± 0.04 b8.50 ± 0.02 a
T94.51 ± 0.03 a6.67 ± 0.07 a8.05 ± 0.06 a8.75 ± 0.05 a
Chlorophyll b/mg.mL−1T11.64 ± 0.01 a1.82 ± 0.02 f1.71 ± 0.08 e1.72 ± 0.06 e
T21.65 ± 0.03 a1.94 ± 0.02 e1.93 ± 0.01 d2.45 ± 0.10 d
T31.66 ± 0.01 a2.06 ± 0.05 d1.97 ± 0.08 cd2.34 ± 0.18 d
T41.69 ± 0.03 a2.12 ± 0.04 c2.04 ± 0.08 c2.51 ± 0.04 cd
T51.66 ± 0.02 a2.16 ± 0.01 bc2.17 ± 0.01 b2.49 ± 0.02 d
T61.66 ± 0.03 a2.26 ± 0.01 a2.27 ± 0.03 a2.69 ± 0.06 bc
T71.63 ± 0.01 a2.14 ± 0.01 c2.18 ± 0.03 ab2.68 ± 0.04 bc
T81.64 ± 0.01 a2.21 ± 0.02 ab2.21 ± 0.03 ab2.83 ± 0.01 ab
T91.65 ± 0.01 a2.27 ± 0.01 a2.27 ± 0.04 a2.93 ± 0.03 a
Oleanolic acid/ng.mL−1T11228.91 ± 23.59 a1350.76 ± 13.62 g1576.49 ± 9.27 g1539.92 ± 6.75 h
T21233.07 ± 23.06 a1417.25 ± 9.24 f1682.10 ± 14.01 f1670.88 ± 10.98 g
T31229.65 ± 6.34 a1791.56 ± 20.30 c2060.54 ± 15.31 c2133.15 ± 3.13 d
T41238.24 ± 13.82 a1929.05 ± 9.25 b2229.09 ± 15.07 b2280.40 ± 5.61 c
T51231.53 ± 20.50 a1909.07 ± 9.49 b2213.32 ± 13.57 b2263.80 ± 2.48 c
T61242.85 ± 12.00 a1568.31 ± 6.31 e1818.93 ± 8.06 d1852.12 ± 5.93 f
T71232.39 ± 16.27 a1957.75 ± 12.77 a2248.68 ± 22.61 ab2792.87 ± 9.93 a
T81236.28 ± 17.13 a1621.31 ± 5.39 d1741.78 ± 16.18 e2017.54 ± 17.48 e
T91243.29 ± 22.93 a1978.78 ± 8.51 a2268.41 ± 19.87 a2588.24 ± 5.22 b
Rutin/ng.mL−1T1216.88 ± 15.04 a2031.11 ± 17.16 f2118.46 ± 10.26 f2426.63 ± 16.46 f
T2219.54 ± 1.23 a2746.69 ± 189.83 e2961.78 ± 106.92 e3236.60 ± 4.57 e
T3222.03 ± 3.00 a3026.23 ± 184.91 de3360.94 ± 185.02 cd4037.92 ± 65.44 cd
T4220.32 ± 2.56 a3762.83 ± 19.50 cd3380.58 ± 493.81 cd3455.83 ± 50.63 e
T5222.49 ± 2.23 a2857.54 ± 5.29 e3121.41 ± 232.96 de3996.47 ± 51.50 d
T6226.41 ± 5.57 a3583.18 ± 10.44 bc3662.79 ± 14.59 bc4266.67 ± 65.68 c
T7222.19 ± 2.74 a3678.92 ± 212.32 b4037.22 ± 175.29 b4717.24 ± 387.29 b
T8224.67 ± 4.92 a3459.45 ± 179.77 bc3797.23 ± 159.88 b5272.81 ± 114.38 a
T9226.73 ± 5.02 a4571.19 ± 8.50 a4728.62 ± 16.09 a5442.55 ± 12.66 a
Calceolarioside B/ng.mL−1T138.66 ± 0.36 a121.88 ± 4.04 i235.54 ± 10.15 i301.81 ± 21.76 i
T238.78 ± 0.21 a370.31 ± 5.13 e843.08 ± 12.06 e1063.45 ± 51.17 e
T339.13 ± 0.09 a322.33 ± 10.00 f751.01 ± 8.39 f941.97 ± 7.24 f
T439.12 ± 0.09 a550.34 ± 4.35 b1265.55 ± 9.07 b1848.37 ± 15.29 b
T539.03 ± 0.12 a436.69 ± 3.21 d1024.48 ± 12.01 d1236.95 ± 8.48 d
T638.64 ± 0.28 a273.59 ± 5.69 g631.75 ± 14.05 g793.09 ± 7.34 g
T738.88 ± 0.19 a852.52 ± 3.00 a2044.85 ± 25.11 a2648.60 ± 58.32 a
T838.78 ± 0.37 a478.95 ± 9.02 c1104.45 ± 10.07 c1341.60 ± 33.45 c
T938.95 ± 0.28 a232.13 ± 8.50 h514.39 ± 12.22 h617.83 ± 18.79 h
Data are presented as mean ± standard error. Different lowercase letters in the same column indicate significant differences between treatments (p < 0.05). Zero, 60, 120, and 180 represent the sampling time of the sample.
Table 6. Results of one-way ANOVA for chlorophyll a, chlorophyll b, oleanolic acid, rutin, and calceolarioside B content of Stauntonia leucantha leaves at 60, 120, and 180 days after zinc (Zn), manganese (Mn), and molybdenum (Mo) co-fertilization.
Table 6. Results of one-way ANOVA for chlorophyll a, chlorophyll b, oleanolic acid, rutin, and calceolarioside B content of Stauntonia leucantha leaves at 60, 120, and 180 days after zinc (Zn), manganese (Mn), and molybdenum (Mo) co-fertilization.
IndicatorSource of VariationDays after FertilizationdFMSF
Chlorophyll aZn6021.86296.89 **
1205.017440.573 **
18010.09349.064 **
Mn6020.37860.295 **
1200.52846.335 **
1802.9614.389 **
Mo6020.0355.552 *
1200.0393.46 *
1800.8494.125 *
Chlorophyll bZn6020.193173.739 **
1200.3283.313 **
1800.37884.697 **
Mn6020.06558.132 **
1200.08622.351 **
1800.18641.611 **
Mo6020.0010.724 ns
1200.0041.107 ns
1800.0030.773 ns
Oleanolic acidZn602289,440.74665.845 **
120295,145.14294.087 **
1801,055,649.842567.061 **
Mn60241,195.7459.372 **
12073,917.33323.563 **
180137,411.853334.149 **
Mo602329,462.77674.95 **
120520,563.641165.946 **
180811,478.8911973.302 **
RutinZn6023,814,249.64789.052 **
1204,285,419.43279.731 **
1808,452,023.637220.513 **
Mn6021,560,621.32936.436 **
1201,422,262.54926.462 **
1802,513,437.11765.575 **
Mo602591,059.70213.8 **
120569,547.3810.597 **
180170,967.774.461 *
Calceolarioside BZn602180,692.9856.06 **
120882,287.78910.147 **
1801,464,922.5099.193 **
Mn60236,969.4211.24 ns
120739,938.2938.509 **
1801,493,718.9589.374 **
Mo60256,448.3161.893 ns
120899,248.46610.342 **
1801,490,451.859.353 **
Zn, Mn, and Mo represent different sources of variance. MS represents the mean square. dF represents variance degrees of freedom. F represents the F-ratio. Sixty, 120, and 180 represent the measurement time of the sample. The level of significance (p-value) is shown in the table: * represents significant differences at p < 0.05, and ** represents significant differences at p < 0.01; ns represents no significance.
Table 7. Results of the comprehensive score of leaf quality of Stauntonia leucantha.
Table 7. Results of the comprehensive score of leaf quality of Stauntonia leucantha.
TreatmentMethods of FertilizationExperimental Index Score
Leaf AreaLeaf ThicknessChlorophyll aChlorophyll bOleanolic AcidRutinCalceolarioside BComprehensive Score
T1Zn1Mn1Mo11.01.01.01.01.01.01.07.0
T2Zn1Mn2Mo22.74.66.86.41.93.43.929.7
T3Zn1Mn3Mo33.96.44.55.65.25.83.534.9
T4Zn2Mn1Mo26.76.47.06.86.34.16.944.2
T5Zn2Mn2Mo37.67.36.86.76.25.74.644.9
T6Zn2Mn3Mo18.29.19.88.23.26.52.947.9
T7Zn3Mn1Mo38.88.28.28.110.07.910.061.2
T8Zn3Mn2Mo18.69.19.39.24.49.55.055.1
T9Zn3Mn3Mo210.010.010.010.08.5102.561.0
Table 8. Analysis of the range of comprehensive score results of leaf quality of Stauntonia leucantha.
Table 8. Analysis of the range of comprehensive score results of leaf quality of Stauntonia leucantha.
Value NameZn (A)Mn (B)Mo (C)
k123.8737.4736.67
k245.6743.2346.97
k359.1047.9347.00
R35.2310.4610.33
k: average sum of each factor at each level. R: range = Kmax − Kmin.
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Zhou, Y.; Zhou, J.; Chen, J.; Chang, Y.; Lin, X.; Zhong, Z.; Li, B. Effects of Zinc, Manganese, and Molybdenum Fertilizers on Growth and Main Medicinal Metabolites of Stauntonia leucantha Leaves. Horticulturae 2025, 11, 123. https://doi.org/10.3390/horticulturae11020123

AMA Style

Zhou Y, Zhou J, Chen J, Chang Y, Lin X, Zhong Z, Li B. Effects of Zinc, Manganese, and Molybdenum Fertilizers on Growth and Main Medicinal Metabolites of Stauntonia leucantha Leaves. Horticulturae. 2025; 11(2):123. https://doi.org/10.3390/horticulturae11020123

Chicago/Turabian Style

Zhou, Yang, Junxin Zhou, Jianyong Chen, Yunni Chang, Xiaoqing Lin, Ziqing Zhong, and Baoyin Li. 2025. "Effects of Zinc, Manganese, and Molybdenum Fertilizers on Growth and Main Medicinal Metabolites of Stauntonia leucantha Leaves" Horticulturae 11, no. 2: 123. https://doi.org/10.3390/horticulturae11020123

APA Style

Zhou, Y., Zhou, J., Chen, J., Chang, Y., Lin, X., Zhong, Z., & Li, B. (2025). Effects of Zinc, Manganese, and Molybdenum Fertilizers on Growth and Main Medicinal Metabolites of Stauntonia leucantha Leaves. Horticulturae, 11(2), 123. https://doi.org/10.3390/horticulturae11020123

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