Unveiling Nitrogen Fertilizer in Medicinal Plant Cultivation

Abstract

Nitrogen fertilizer is the most frequently used fertilizer in the cultivation of medicinal plants, and has a significant contribution to their yields and quality. Yet, there is biased and excessive N application in medicinal crops. This study aims to quantitatively analyze the recommended N application concentrations for diverse medicinal species and disentangle the intricate relationships between soil fertility, N application rate (NAR), and the quality/yield of medicinal crops. We first characterized 179 medicinal species and 7 classes of phytometabolites therein, including terpenoids, flavonoids, phenylpropanoids, phenolics, alkaloids, etc., reported during the past three decades from the phylogenetic and spatial perspectives. The relationships between soil fertility, NAR, and medicinal crops were then subjected to statistical analyses. The pharmaco-phylogenetic and geographic distributions of NAR suggest that the impact of ecological/environmental factors on the N demand of medicinal plants was much greater than that of genetic endowments. We found that different medicinal species were distinct in N demand, which is related to soil fertility levels in different production areas. The NAR reported by China, 215.6 ± 18.6 kg/ha, was higher than that of other countries (152.2 ± 20.3 kg/ha; p = 0.023). Moderate N application generally increases the yield and phytometabolite content of medicinal crops, but excessive N application has the opposite effect. It is necessary to plan N concentration and formula fertilization on a case-by-case basis and with reference to empirical research. Our results provide baseline information and references for the rational application of N fertilizer in the precision agriculture of medicinal crops.

1. Introduction

Plants require at least 14 mineral elements for their nutrition, including the macronutrients nitrogen (N), phosphorus (P), potassium (K), calcium (Ca), magnesium (Mg), and sulphur (S), and numerous micronutrients, which are generally obtained from the soil. Crop production is often limited by the low phytoavailability of essential mineral elements; plant nitrogen nutrition is essential for sustainable development and global health, and studies have explained how nitrogen is taken up by roots and distributed within plants [1]. N-containing amino acids are precursors of numerous specialized metabolites. For instance, aromatic amino acids are the biosynthetic precursors of phenylpropanoids, flavonoids, and phenolics [2]. Glucogenic amino acids provide precursor molecules for the MEP (2-C-methyl-d-erythritol-4-phosphate) and MVA (mevalonate) pathways [3], which involve the functions of multiple enzyme proteins and are responsible for the terpenoid biosynthesis; they also provide precursors for polysaccharide biosynthesis. Moreover, many structural classes of alkaloids are derived from amino acids. These interconnected cellular pathways and empirical observations justify the application of N fertilizers in medicinal plant cultivation. However, it is challenging to determine the amount of nitrogen applied, and it is necessary to consider the genetic background of cultivated species, the characteristics of cultivation environments, and the relevant technical and economic factors.

Based on soil nitrogen studies [4,5,6], introducing the concept of the nitro-genome is beneficial; this refers to the sum of various forms of nitrogen, e.g., ammonium N and nitrate N, and the sum of all possible ways in which nitrogen-containing substances can be meaningfully associated with each other. There is no independent single nitrogen form; rather, there are two or more nitrogen forms and their relationships. Therefore, heterogeneity in the internal composition of the nitro-genome is emphasized. The plant nitro-genome refers to the nitrogen compositions of subcellular structures, plant cell, tissue, or organism, which are interconnected with soil nitro-genome via the activities of transport proteins and other mechanisms [7]. We hypothesize that N fertilization could partially determine the species-specific tissue and cellular nitro-genomes, thus impacting the growth and phytometabolite of the respective medicinal species. The current research addresses the N fertilization of bulk medicinal crops, addressing the related problems and presenting solutions for agricultural soils that can guarantee the supply chain of phytomedicines and optimize fertilizer applications for economic and environmental sustainability [8,9].

Agriculture must produce medicinal crops that contribute sufficient medicinal compounds and mineral elements for adequate human efficacy, and there is a widespread belief that the use of N fertilizer can increase the yields of traditional medicine crops [10,11]. However, there is a serious problem of biased and excessive nitrogen application in the cultivation of medicinal plants [12,13], which fails to increase the yield of medicinal plants, and even reduces their yield and quality. So far, research on N application to medicinal plants has mostly focused on the yield of a certain medicinal herb and the contents of one or several medicinal components. However, there has been little comparison and comprehensive analysis between different N fertilization studies, and there is a lack of research analyzing the current status of N fertilizer application from the perspectives of phylogenetic and spatial distribution, which is not conducive to elucidating the regulatory mechanisms of nitrogen application or formulating optimal regimes that further improve the yield and quality of medicinal crops. Based on analyzing the nitrogen application all over the world during the past 30 years, we can perceive and outline the impact and regulatory mechanism of nitrogen application on the yield and quality of diverse medicinal plants, so as to provide baseline references for rational N application to medicinal plants.

Reasonable nitrogen concentration and optimized nitrogen application technologies are highlighted in the medicinal plant agriculture field; these are also a concern for pharmaceutical farmers [1,14]. In the past three decades, research on the formula/N fertilization of medicinal plants has been thriving [15,16], with the aim of improving the efficacy components and yields of medicinal varieties. In field and microcosm experiments [12,17], using methods such as orthogonal design [18], different concentrations, and different ratio fertilization schemes have been set up across various soil types, and the yields, secondary metabolites, volatile oils, and other contents of medicinal plants were evaluated. The optimal nitrogen application concentration for different medicinal varieties in different production areas was fitted through the mathematical model [19,20]. However, such a model is far from being practical, perfect, or universal, given the complicated genetics and ecologies of target species, as well as the diverse technical and economic considerations.

Based on the above understanding, we hypothesize that examining the phylogenetic and spatial distribution of medicinal plants and the N fertilization thereof could help improve their characterization and contribute to knowledge on the novel research activities surrounding nitrogen application in herbs. Therefore, the objectives of this study were to provide the first evaluation that has been conducted on the extent to which N fertilizers are applied to medicinal plants and relate this to species-level phylogenies and their spatial patterns. Based on the reported N fertilization of medicinal crops on English and Chinese journals during the past three decades, we utilized a set of measures to show how research efforts on N fertilization with medicinal importance are conducted. The intricate relationships between N fertilization and NPK levels in the original soil, as well as the effects of N fertilization on medicinal plants, were qualitatively and quantitatively investigated.

2. Materials and Methods

2.1. Nitrogen Fertilization Database of Reported Medicinal Plants

In Table S1, we can see that some studies have recommend a fixed value of nitrogen application concentration, which was selected based on the highest yield or active ingredient content among different treatments [21,22]; meanwhile, others have recommended an interval of nitrogen application concentration [23,24]. In order to better understand the fertilizer requirements of medicinal plants and avoid engaging in blind nitrogen application activities or copying the nitrogen application of local food crops by herbal medicine growers, and in the interest of providing basic data for the formula fertilization of herbs, we searched for relevant research on nitrogen application in medicinal plant cultivation from 1995 to 2024. We used the following keywords: “medicinal plant”, “nitrogen fertilizer”, “nitrogen application in medicinal plant”, “nitrogen fertilizer in medicinal plant”, and others. Relevant articles were retrieved from the following databases: China National Knowledge Infrastructure (CNKI), Taylor & Francis, Science Direct, MDPI, Frontiers, Wiley, and Nature-Springer. The best nitrogen fertilizer application concentrations in different regions and herbs, as well as in field studies and indoor ones, were summarized (Tables S1 and S2), along with region-specific information on soil fertility levels (Table S3). The phytometabolite database was first digitalized on the basis of journal articles reporting various classes of medicinal phytometabolites, involving 132 species of 119 genera from 54 families. In terms of biosynthetic pathways, all metabolites were classified into seven major categories: terpene (e.g., [25]), alkaloid [26], flavonoid [27], phenolic [28], phenylpropanoid [29], polysaccharide [30], and others [22]. N fertilization and phytometabolites were coded with binary characters, respectively: when they are present in a species, it is coded with 1; otherwise, it is coded with 0.

2.2. Phylogenetic Tree of Medicinal Species

The in-house expanded phylogenetic tree sp_tree_chm.tre (sp, species; chm, Chinese herbal medicine) with 30,742 species was utilized; a total of 32 species which were included in our N fertilization database but which were not in the sp_tree_chm.tre were inserted using the phylo.maker function of V.PhyloMaker package in R 3.5.3 https://www.r-project.org/ (accessed on 1 May 2024). An expanded phylogenetic tree sp_tree_nit (nit, nitrogen) of 30,774 species was obtained. The bladj function of V.phylomaker was used to correspond the branch length to the evolutionary distances between the species. A single subtree involving the phytometabolites of six major classes was extracted from sp_tree_nit.tre; the names of the species were proofread and standardized in https://www.iplant.cn/pnc, accessed on 1 May 2024, developed by Institute of Botany, Chinese Academy of Sciences. The R packages Picante and Ape (https://cran.r-project.org/web/packages/ape/, accessed on 1 May 2024) were used to generate the subtree. iTOL v6 (https://itol.embl.de/, accessed on 1 May 2024) was used to draw and visualize the subtrees.

2.3. Statistics and Calculation of Phylogenetic Distribution of N Fertilization

NRI (net relatedness index) measures the extent of deep-level (e.g., family/genus) clustering, while NTI (nearest taxon index) calculates the extent of terminal (i.e., species level) lumping [31]. NRI and NTI values were calculated using the ses.mpd and ses.mntd functions of the Picante package, respectively. The positive values of these two indices suggest the phylogenetic clustering of the reported medicinal species with N fertilization, while negative values indicate that species with N fertilization are over-dispersed on the phylogenetic tree. The observed patterns of N fertilization distribution were compared with the expected ones to assess whether the values of NRI and NTI were statistically significant (p < 0.05). The strength of phylogenetic signal of N fertilization was quantified using the D statistic, which was implemented by the function phylo.d in R package caper (http://CRAN.R-project.org/package=caper, accessed on 1 May 2024). There was a strong phylogenetic signal when p (D < 1) < 0.05 and p (D > 0) > 0.05.

2.4. Geographical Distribution of Reported Medicinal Plants and N Fertilization

The 753,182 county-/district-level occurrence records of 2305 China plant species [32] involve 2798 county-level administrative units, and the occurrence records of 85 out of 179 species included in our N fertilization database were retrieved. In order to understand the county-/district-level distribution of medicinal species with N fertilization and their phytometabolites in 2789 counties of China, we conducted data mining in the occurrence database by writing Python scripts. Two data files, data1 and data2, were introduced. The data1 file contained two fields: species name and county name. The data2 file included the species name and the names of seven structural classes. Through merging in terms of the species name field, 85 species with N fertilization and their phytometabolites, in our phytometabolite database, were matched to the corresponding county. The number of species and compounds from each county was counted. In order to figure out the distribution of medicinal plants and their phytometabolites in 34 provincial-level administrative regions of China, we searched Flora of China https://www.iplant.cn/frps (accessed on 1 May 2024) to record the province(s) in which each of the 179 species is distributed. The global distribution of medicinal plants with N fertilization in 232 countries and regions was explored using the literature data and the Resource and Environmental Science Data Platform of the Institute of Geographic Sciences and Natural Resources Research, Chinese Academy of Sciences (https://www.resdc.cn/, accessed on 1 May 2024).

2.5. Statistical Analyses

One-way analysis of variance (ANOVA), multiple linear regression (MLR), Student’s t test, chi-square test, non-parametric Kruskal–Wallis rank test (H test), and Spearman rank correlation test were conducted in Stata 17.0, with p < 0.05 being statistically significant.

3. Results and Discussion

3.1. Phylogenetic Distribution of Reported Medicinal Species with N Fertilization

Of 236 reports involving 179 medicinal species of 63 families, 45 reports (involving 41 species) proposed an optimal nitrogen application rate (NAR) of 0–99 kg N/ha (Figure 1), followed by 64 reports (involving 59 species) of NAR 100–199, 28 reports (involving 24 species) of NAR 200–299, 17 reports of NAR 300–399, etc. The reported medicinal species with N fertilization and their phylogenetically close taxa constitute a vast biologic/chemical space for the further exploration of N fertilization (Figure 1; Table S1).

When 179 species were taken into account, the NTI and NRI values were 1.53 and 1.40, respectively, indicating the clustered structure on the species level phylogeny; as p values were 0.064 and 0.128, respectively, such a cluster was not statistically significant. A total of 128 medicinal species, whose NARs were shown as kg N/ha in the original report or can be converted into kg N/ha, were used to calculate the indices of the phylogenetic structure. A statistically significant clustered structure was only suggested by an NTI value of >0 for NAR 100–199 kg N/ha (p = 0.011). Asteraceae, Lamiaceae, Apiaceae, Poaceae, and Fabaceae had more species with this NAR interval, and Artemisia, Salvia, Atractylodes, Chrysanthemum, Polygonatum, and Panax were the hotspot genera. When conducting cultivation and fertilization research on other species of these genera, one can first try this level of NAR. On the other hand, NAR levels of 200–299, 300–399, 400–499, and 500–599 were of overdispersion (NTI < 0) on the phylogenetic tree, which were supported by NRI < 0 except for 300–399, implying that phylogenetically closer species do not necessarily have analogous NAR. Overall, values of D statistic suggest the stochastic distribution of all NAR levels (p(D < 1) > 0.05). On the other hand, p(D > 0) < 0.05 in NAR levels of 0–99, 100–199, 200–299, and 300–399 suggests that their distribution on the phylogeny was not clumped as significantly as expected by the standard Brownian model of evolution. The results of D statistic largely support those of NRI/NTI, suggesting that the environmental factors, rather than genetic ones, could have greater effect on the N demand of medicinal plants, especially when they need to synthesize defensive secondary metabolites in the face of abiotic/biotic stress.

3.2. Global-, Provincial-, and County-Level Distribution of Medicinal Plants with N Fertilization

The different NAR levels of 79 key medicinal species were widely distributed in 25 provinces/autonomous regions of China (Figure 2A, Table S1). The median NAR of Guangdong was the highest, i.e., 310 kg N/ha (Desmodium styracifolium, Momordica charantia), followed by 272.25 of Anhui (Andrographis paniculata, Prunella vulgaris, Chrysanthemum morifolium), 261.42 of Henan (Achyranthes bidentata, Allium sativum, Carthamus tinctorius, Rehmannia glutinosa), and 247 of Guangxi (Chrysanthemum indicum, Euonymus fortunei, Pueraria thomsonii). Heilongjiang, Inner Mongolia, Hunan, and Taiwan had fewer NAR of medicinal plants. This may be due to the abundant phytomedicine resources and salient soil fertility in the specific region, where medicinal plants can be produced well without the need for N application. Alternatively, the protection of soil ecological environment is prioritized, and low N application is conducive to protecting the soil, atmosphere, and water from pollution as much as possible. The distribution differences of NAR also reflect the differences in agricultural and economic development among provinces, with developed or agricultural intensive areas potentially investing more resources in medicinal plant growth and production. In the Flora of China-based analysis, the top ten families in terms of reported numbers of N fertilization species were widely distributed in all 34 provinces. Sichuan possessed the highest number of reported medicinal species (49) (Figure 2B, Tables S1 and S2), followed by Shaanxi (47) and Yunnan (42). In most provinces, the Asteraceae family had the highest number of reported species (Figure 2B), followed by Lamiaceae and Apiaceae. There were also many NAR reports in Fabaceae and Poaceae; meanwhile, there is a lack of NAR studies in Ranunculaceae, which is a family with the highest proportion of medicinal species. One or several phytometabolites of each medicinal species were reported (Figure 1 and Figure 2, Table S1). All seven phytometabolite classes were widely distributed in all provinces (Figure 2C, Tables S1 and S2). The most numerous phytometabolites were identified in Sichuan, followed by Yunnan and Jiangsu. Most provincial administrative units had the highest numbers of terpenoids (Figure 2C), followed by flavonoids and phenolics; the reported polysaccharides and alkaloids were also abundant in most provinces.

In county-/district-level analysis, different NAR levels of 79 species were widely distributed in 102 county-level administrative units (Figure 2D). The median NAR of Xiangfang District, Harbin, Heilongjiang Province, was the highest, i.e., 1050 kg N/ha (Acanthopanax senticosus), followed by 983.75 of Libo county, South Guizhou Buyi, and Miao Autonomous Prefecture (Strobilanthes cusia), and 575 of Yuzhong County, Lanzhou, and Gansu Province (Anredera cordifolia). Mao County, Aba Tibetan, and Qiang Autonomous Prefecture and Wenjiang District, Chengdu, Sichuan Province, had lower NARs for medicinal plants. NAR may be influenced by comprehensive factors such as ecological and geographical conditions, plant physiological and biochemical characteristics, local economic development level, environmental protection policies, and so on, and there is great potential for further case-by-case research. Lin’an, Hangzhou, possessed the highest number of reported medicinal species (53) (Figure 2E, Tables S1 and S2), followed by Gulou District, Nanjing (52), and Pukou District, Nanjing (48). There were also many medicinal species found in counties of Henan, Hubei, and Hunan, among others. If fertilization research is carried out in these production areas, published reports of NAR can be referred to, and the NAR design can be better optimized based on local edaphic and climate conditions. Seven phytometabolite classes of 85 species were widely distributed in 2755 counties, spanning all 8 terrestrial TCM (traditional Chinese medicine) production areas (Figure 2F). The distribution pattern of seven classes of reported phytometabolites was not significantly different from that of the species (Figure 2E), and the counties of Jiangsu, Zhejiang, and Sichuan had the highest numbers of reported compounds. These results suggest that, in provinces with large-scale production of medicinal plants and strong phytomedicine industries, there is broad development potential to improve the yield and quality of medicinal crops through rational N fertilization.

Due to the difficulty in collecting native language research from other countries, this study collected distribution data of 80 medicinal species of 34 families in 28 countries outside of China from 97 reports (Figure 2G). There were 139 NAR studies in China, possibly due to China’s emphasis on N fertilizer research and the convenience of collecting Chinese research. Twenty-eight and five species were reported in Iran and the US, respectively, with respect to NAR studies. These countries have a long history of application and cultivation of medicinal plants, with a more developed medicinal plant cultivation industry and a greater emphasis on fertilization research. In Turkey, India, Egypt, Nigeria, and Italy, there are also some reports on the N fertilization of medicinal plants, while in Nepal, Uzbekistan, Lithuania, and Bangladesh, there are few reports on the NAR of medicinal species. Possible limitations include climate conditions, human activities, low biodiversity, and limited knowledge of N application for medicinal plants among the local indigenous people. The extreme imbalance in global N fertilizer application has left enormous development space for future related research.

3.3. Amount of Nitrogen Fertilizer Applied to Medicinal Crops

The recommended application concentrations of N, P, and K fertilizers for medicinal plants in 166 field studies were summarized (Table S1), involving various medicinal parts of 133 medicinal species, e.g., [26]. There are 82 species with root/rhizome as the medicinal part, followed by 56 species with stem/bark, 51 with leaf, 45 with flower, and 34 with fruit/seed. A total of 141 studies reported the recommended concentration of pure N [33], 20 reported the recommended concentration of urea [34], 4 presented the amount of poultry manure/vermicompost [35], and 1 presented the amount of ammonium sulfate (33.5% N) [36]. The recommended concentration of urea was converted into that of pure N based on the 46% N content of urea, and the N content of biofertilizer was converted into pure N content. Moreover, 26 reports of pot experiments with seedlings or individual plants, 20 hydroponic reports, and 21 reports of biofertilizers (containing organic N), for which pure N was difficult to calculate, were included in the present study (Table S2A–C).

Urea was the most commonly used N fertilizer in the field studies of medicinal plants, which was applied in 121 studies. Other forms of N fertilizer, such as NH₄NO₃ (13), (NH₄)₂SO₄ (6), (NH₄)H₂PO₄ (4), Ca(NO₃)₂ (3), and NH₄HCO₃ (3), etc., were also reported. There was no significant difference in NAR between medicinal plants with different medicinal parts (Kruskal–Wallis H test, p = 0.592), and the recommended NAR varied between 0 and 1565 kg/ha in various plants. Labisia pumila (Kacip Fatimah, Primulaceae) had the lowest NAR [13], and Sesamum indicum (Pedaliaceae) received the highest NAR [37]. A total of 62 studies suggested an NAR of 100–199 kg/ha, accounting for approximately 37.3% of all studies (Figure 1, Table S1). For example, the NAR of Calendula officinalis, Erigeron breviscapus, and Stevia rebaudiana fell within this interval. Forty-eight studies suggested NAR of 0–99 kg/ha, involving Bupleurum chinense, Nigella sativa, and Rosa damascena, etc. Twenty-nine studies suggested an NAR of 200–299 kg/ha, such as Andrograp his paniculata, Isatis tinctoria, and Ligusticum sinense ‘Chuanxiong’. The fertilization of Salvia miltiorrhiza, Astragalus membranaceus var. mongholicus, Panax notoginseng, Isatis tinctoria, and Bupleurum angustissimum was the most extensively studied, and there was no significant difference in the NARs among the five species (ANOVA, p = 0.153). The hotspot families of fertilizer research were Asteraceae (25 studies), Lamiaceae (19), Apiaceae (18), and Fabaceae (16), and no significant difference of NAR was identified among the four families (ANOVA, p = 0.464). In China and other countries, NAR was not different between plants with medicinal root, stem, leaf, flower, and fruit (Kruskal–Wallis H test, p = 0.706, 0.999, respectively). However, the NAR reported by China, 215.6 ± 18.6 kg/ha, was higher than that reported by other countries (152.2 ± 20.3 kg/ha; Student’s t test, p = 0.023). The chi-square test showed that the distribution pattern of NAR between China and other countries was significantly different (p = 0.003), e.g., the number of NAR studies within 0–99 kg/ha in China was less than that in other countries.

The differences in NAR among different medicinal plants could be related to multiple factors. A total of 135 studies reported the soil nitrogen, phosphorus, and potassium content before fertilization, and multiple linear regression (MLR) was conducted to clarify the relationship between the background values of soil N, P, and K and NAR. We found that, without distinguishing medicinal parts, there was no significant correlation between NAR and soil N, P, and K, whether all species or the top four families were examined (p = 0.985, 0.638, respectively). Interestingly, in China and in other countries, there was significant correlation between NAR and soil elements (p ≤ 0.0002); NAR was positively correlated with available N (AN; e.g., inorganic N and easily hydrolyzable organic N) in the former, and positively correlated with available K (AK) and AN in the latter. Moreover, there was correlation between the NAR of plants with medicinal root/rhizome and the soil N, P, and K (p < 0.001;

Table 1. Summary of MLR between NAR and background values of soil N, P, and K.

MLR	Root	Stem	Leaf	Flower	Fruit	Five Most Reported Species a
Total
p value	0.00 ***	0.00 ***	0.00 ***	0.00 ***	0.0019 **	0.0105 *
Effect	AP(+), TN(+), AN(+)	AP(+), TN(+), AK(+)	AP(+), AK(-), TN(+)	AP(+)	AP(+), TN(-), AN(+)	TN(+), AN(+), AK(-)
China
p value	0.0253 *	0.0005 ***	0.0109 *	0.0172 *	0.5434	0.0002 ***
Effect	AN(+)	AN(+)		AK(+), AP(-), TN(+)		AP(+)
Other countries
p value	0.6319	0.0001 ***	0.0017 **	0.0007 ***	0.002 **	0.00 ***
Effect		AK(+), TN(+)	--	AK(+)	--	AK(+), AN(+)

Note: +, positively correlated with NAR (N application rate); -, negatively correlated with NAR. * p < 0.05, ** p < 0.01, *** p < 0.001. ^a Salvia miltiorrhiza, Astragalus membranaceus var. mongholicus, Panax notoginseng, Isatis tinctoria, Bupleurum angustissimum. MLR, multiple linear regression.

). The available P (AP) had the greatest positive effect on NAR, followed by total N (TN) and AN. In China, the NAR of plants with medicinal root significantly positively correlated with AN (p = 0.025). There was significant correlation between NAR of plants with medicinal stem/bark and the soil N, P, and K content (p < 0.001). AP had the greatest positive effect on NAR, followed by TN and AK. In China, the NAR of plants with a medicinal stem positively correlated with AN (p = 0.0005); meanwhile, in other countries, it was positively correlated with AK and TN (p = 0.0001). The correlation between NAR with medicinal leaf and soil N, P, and K was also significant (p < 0.001); AP had the greatest positive effect on NAR, followed by TN, while AK had a significant negative effect on NAR. This significant correlation holds true in China and in other countries (p ≤ 0.010); in the latter, NAR with medicinal leaf positively correlated with AK. The correlation between NAR with medicinal flowers and soil N, P, and K was significant (p < 0.001), and only AP had a significant positive effect on NAR. This significant correlation holds true in China and in other countries (p ≤ 0.017). In the former, NAR with medicinal flower was positively correlated with AK and TN and was negatively correlated with AP; in the latter, it was positively correlated with AK. The correlation between NAR with medicinal fruit/seed and soil N, P, and K was significant (p = 0.0019); AP had the greatest positive effect on NAR, followed by AN, whereas TN had a significant negative effect on NAR. This significant correlation holds true in other countries (p = 0.002) rather than in China. In hotspot plants S. miltiorrhiza, A. membranaceus var. mongholicus, P. notoginseng, I. tinctoria, and B. angustissimum, there was significant correlation between NAR of these plants and the soil N, P, and K (p = 0.010); TN had the greatest positive effect on NAR, followed by AN, while AK had a significant negative effect on NAR. These results preliminarily characterize the close relationship and mutual influence between soil nitro-genome (Figure 3) and plant nitro-genome. Research on nitrogen application should focus on both N and P/K supply to meet the needs of plant growth and biosynthesis of secondary metabolites.

Due to uneven variances, non-parametric tests were used for comparing species with different medicinal parts. The average value of soil TN was not significantly different between species with different medicinal parts (Kruskal–Wallis H test, p = 0.11); this was the case for AN, AP, and AK as well. The average value of soil TN in China was significantly higher than that in other countries (Student’s t test, p = 0.008), which was particularly evident in species with the stem, flower, and fruit as the medicinal parts. However, the background AN of China species with stem/bark as the medicinal part (80.0 mg/kg) was significantly lower than that of other countries (297.3 mg/kg; p = 0.008). Concerning the question of whether or not we should distinguish between medicinal parts, the AP and AK of China species were not significantly different from those of other countries’ species, respectively; the exception to this is that the AK of the former with a medicinal stem was significantly lower than that of the latter with a medicinal stem (p = 0.009). These results indicate the importance of the soil test and formula fertilization in medicinal plant cultivation, whatever the medicinal parts. Only by determining the nutrient content in the soil before nitrogen application can a reasonable fertilization scheme for medicinal crops be formulated. Moreover, attention should be paid to the simultaneous use of N and other macro/microelements to achieve synergistic results.

However, it must be noted that the improper management of N fertilization in agriculture has led to a large amount of nitrate leaching and NH₃/N₂O emissions [4,5,6,38], which is especially the case in grain/forage and fruit production systems [39,40]. High levels of nitrate in drinking water can cause harm to the human body, excessive nitrate in rivers leads to eutrophication and damage to the ecological environment of the water, and excessive NH₃/N₂O emissions have negative effects on the global climate. Although there are no specific studies of the potential environmental impacts of N fertilization of medicinal plants, the preventive and alleviating measures and methods for reducing them have been reported, such as using grass cover, applying controlled-release N fertilizer [41], adding nitrification inhibitors [42], etc. More importantly, comprehensive measures are entailed to achieve agricultural sustainability and environmental protection goals.

3.4. Effects of N Fertilizer on Medicinal Phytometabolites

Precise N fertilization calls for a deeper understanding of plant uptake and utilization to optimize sustainable production. However, limited information is available about the effects of N supplements on physiological and chemical properties of medicinal crops, as well as their potential role in standardizing the active compounds in the plant material supplied to patients. MLR suggested that NAR was not significantly correlated with the content of six classes of medicinal phytometabolites (p = 0.432). In Spearman correlation analysis, NAR was not significantly correlated with the content increment of terpenoids, flavonoids, phenolics, alkaloids, polysaccharides, and others (p ≥ 0.16). NAR was also not significantly correlated with the biomass increment, but was marginally and positively correlated with the content increment of phenylpropanoids (p = 0.082). The N fertilizer increased the terpenoid content by 0.58–460.32% [43], with a mean of 74.32%, an SD of 95.73%, and a median of 37.98%. It increased the flavonoid content by 1.1–272.7% [44], and the mean (42.1%), SD (54.1%), and median (27.4%) were lower than those of terpenoids. N fertilizer increased the phenolic content by 1–252% [45], with the highest mean (109.9%) and median (109.4%) among the seven classes of phytometabolites. N fertilizer also increased the content of phenylpropanoid [46], alkaloid [47], polysaccharide [48], and others [49] to different degrees. There are also reports on the reduction in metabolites caused by N fertilizer (Table S1). The relative content of most flavonoids, phenylpropanoids, and phenolic acids was decreased with increasing N supply in some plants [50], but N promoted carotenoid biosynthesis.

Different types of metabolites are produced with varying concentrations of N sources and can be used as metabolic markers to improve the N use efficiency (NUE) [51]. Different levels of N application can modify the chemical composition and increase essential oil production [36]. When Origanum marjorana was cultivated in sandy loam, moderate–high N level, i.e., 150–200 kg ha⁻¹, increased the yield of essential oil monoterpenes terpinene-4-ol and (Z)-β-terpineol [52]. In Arnica montana, 60 kg N ha⁻¹ increased the content of sesquiterpene lactones, flavonoids, and essential oils [11], as well as the anticancer activity of A. montana water extracts; but further increases in the N dose decreased the contents of these metabolites.

In hemp, nitrate transporters, N-metabolism-related genes, transcription factors, and genes involved in secondary metabolism are co-regulated [53] as part of a complex machinery to counteract abiotic stress. The cannabinoid (diterpenoid) metabolism of Cannabis sativa was sensitive to N nutrition [54], and the N supplements affected cannabinoid content differentially, which were location- and organ-specific, and varied between cannabinoids. NPK supplementation increased cannabigerol (CBG) levels in flowers by 71%, and lowered cannabinol (CBN) levels in both flowers and inflorescence leaves by 38 and 36%, respectively. Reducing N fertilizer input lowered the inflorescence yield of C. sativa [55], but increased the cannabidiol (CBD) concentration. Differences in CBD yield between fertilizer types occurred only at the final harvest, indicating limitations in N uptake due to N forms in the organic fertilizer. In five essential oil hemp cultivars, NAR of >50 ppm N significantly reduced plant growth, biomass accumulation, and cannabinoid concentrations [56]. N of >300 ppm led to compliant Δ9-tetrahydrocannabinol (THC) levels (<0.3%), while CBD concentration showed higher sensitivity to increased NAR compared to THC and CBG (>300 vs. >450 ppm N). In regions with a short growing season, N treatment increased overall CBD yield [57], which was driven by increases in inflorescence biomass. High NAR was not recommended due to reduced cannabinoid concentration and biomass yield [9], and the ideal N supply could be between 60 and 210 mg/L. The identified effects of N supplementation are useful in the chemical control and standardization of cannabis.

The NO₃⁻ and NO₂⁻ in the plasma-treated water increased the production of free amino acids and ginsenosides in the Panax ginseng sprout [58]. In Panax notoginseng, N deficiency promoted the accumulation of amino acids L-proline, L-leucine, L-isoleucine, L-norleucine, L-arginine, and L-citrulline [59], as well as sugars arabinose, xylose, glucose, fructose, and mannose, thus providing precursor metabolites for the biosynthesis of flavonoids and triterpenoid saponins. N deficiency might increase the expression of key genes for N uptake [60], transport, assimilation, and signal transduction to enhance NUE in the rhizomatous species. Fertilization with 75 mg N/plant led to the greatest seedling height, ground diameter, crown width, and flavonoid phlorizin content [16], and upregulated the gene expressions of phenylalanine ammonia-lyase (PAL), 4-coumarate-CoA ligase (4CL), and phlorizin synthase (PGT1). At low or intermediate N levels (0–1.25 mM Ca(NO₃)₂, Table S2B), the flavonoid content in the roots and stalks of Coreopsis tinctoria remained stable, while that in leaves peaked [61]. Similarly, as the N levels decreased from 270 to 0 kg N/ha, the production of GSH and GSSG, anthocyanin, total flavonoids, and ascorbic acid increased steadily in Labisia pumila [13]. A N slow-release fertilizer significantly increased the plant growth, phenolics, flavonoids, polysaccarides, essential oil, caffeic acid derivatives, and anti-radical scavenging activities of Echinacea purpurea [41].

In the Southeast Asian tree Mitragyna speciosa (kratom), NPK had little influence on the concentrations of mitragynine, paynantheine, speciociliatine, mitraphylline, and corynoxine per leaf dry mass [15], while low–medium rates of NPK fertilizer maximized concentrations of speciogynine, corynantheidine, and isocorynantheidine per leaf dry mass, suggesting a promotion of N allocation for secondary metabolism of specific alkaloids. A low-N-responsive bHLH gene facilitated the development of Atropa belladonna with high-yield tropane alkaloids under the decreased usage of N fertilizer [62]. In Dendrobium denneanum, the polysaccharide content was the highest at 1500 mg·L⁻¹ N and 3000 mg·L⁻¹ P [18], which was 26.8% higher than the control. The flavonoid content increased by 36.2% at 500 mg·L⁻¹ N, 2000 mg·L⁻¹ P, and 300 mg·L⁻¹ K. N had the most significant impact on the various indicators of D. denneanum, followed by P and K. Treatments with 1500 mg·L⁻¹ N, 3000 mg·L⁻¹ P, and 500 mg·L⁻¹ K significantly altered the expressions of five phenylpropanoid biosynthetic genes and two flavonoid biosynthetic genes. In Hokkaido, 12 kg N/10-year was the optimal amount of N for healthy growth of Angelica acutiloba [63], and the content of (Z)-ligustilide, a lactone in the essential oil of A. acutiloba root, increased with an increasing supply of N.

3.5. Pot Experiments, Hydroponic Studies, and Others

In pot experiments, the minimal and maximal NAR was 0.036 g/pot and 35 g/pot, respectively [64], with a mean of 6.13 g/pot, a median of 2.22 g/pot, and an SD of 10.02 g/pot. In individual plant experiments, the minimal and maximal NAR was 1.333 g/individual and 500 g/individual, respectively [65], with a mean of 84.6 g/individual, a median of 27.6 g/individual, and an SD of 140.73 g/individual. In hydroponic studies, the minimal and maximal NAR was 4 mg/L and 3750 mg/L, respectively [18], with a mean of 683.5 mg/L, a median of 293.2 mg/L, and an SD of 999.9 mg/L, indicating great variation.

When the nitrogen concentration was 20 mg·L⁻¹ (ammonium nitrate 57.14 mg/plant, pure N 20 mg/plant), the net photosynthetic rate and chlorophyll content of ginseng leaves were the highest [66], and the relative expression levels of ginsenoside biosynthetic genes PgHMGR and PgSQE were highest in the roots. The research on fine cultivation of pollution-free ginseng with N fertilizer is beneficial for the production of quality ginseng materials, providing a scientific basis for fertilizer reduction and efficiency improvement, as well as the development of environmentally friendly and sustainable ecological planting.

The effect of additional N fertilization, in the form of ammonium nitrate and urea, on the content and yield of nutrients and polyphenol in fragrant agrimony (Agrimonia procera), depends on the dose and the N form [67], as well as the morphological part of the plant. Among the diverse N forms of the nitro-genome, ammonium N and nitrate N are the main sources for N uptake by plants, where NH₄⁺/NO₃⁻ ratios have a significant effect on the biomass, quality, and metabolites composition of plants grown in soil, substrate, and hydroponic cultivation systems [51]. For example, as the proportion of ammonium increased, the growth of centipedegrass showed a “bell-shaped” response [68], with the maximum biomass and C/N accumulation under the NH₄⁺:NO₃⁻ treatment ratio of 50:50. In purple coneflower (Echinacea purpurea), an increase in NO₃⁻/NH₄⁺ ratio significantly increased plant growth parameters and the content of terpene hydrocarbons, e.g., germacrene D, myrcene, α-pinene, and others [69]. The biosynthesis of diterpene oridonin in Rabdosia rubescens was boosted under the supply of 75% NO₃⁻ + 25% NH₄⁺ [29], and 50–75% NH₄⁺ promoted the accumulation of rosmarinic acid. In a hydroponic culture of Echinacea purpurea, increasing of NO₃⁻/NH₄⁺ ratio enhanced the production of phenolic compounds [70]. In contrast, altering the NO₃⁻/NH₄⁺ ratio did not have any significant effect on the phenolic content of Echinacea angustifolia in the controlled environment systems [71]. The yield of preferred bioactive ingredients could be increased in medicinal plants by adding different ratios of nitrate N and ammonium N.

Exogenous KNO₃ supply to ginseng seedlings significantly increased the root secondary growth [72], which was attributed to the increase in the cambium stem cell activity and the subsequent differentiation of cambium-derived storage parenchymal cells. The formation of a transcriptional network comprising auxin, brassinosteroid (BR)-, ethylene-, and jasmonic acid (JA)-related genes contributed to the secondary growth of ginseng storage roots, and the accumulation of starch granules was inhibited in storage parenchymal cells. Given that excessive nitrate N content in agricultural products is harmful to human health, the nitrate contamination in medicinal plants can be mitigated and N recovery can be increased by combining the nitrification inhibitor dicyandiamide, moringa oil, and zeolite with N fertilizer [42].

Vermi-compost, cow manure, and chicken manure are commonly used organic N fertilizers [73]. The aquaponic water, a natural source of fish-derived nutrients [74], increased leaf formation in jewel orchid Ludisia discolor, enhanced chlorophyll content and photosystems’ productivity, and stimulated and prolonged flowering. Compared to chemical fertilizers alone, the combination of biological fertilizers and chemical fertilizers or biofertilizers alone can better promote the growth of medicinal plants or the accumulation of their bioactive components. The biofertilizers are frequently applied in food and medicine continuum (FMC) plants [75]. Yet, the acquisition and utilization efficiency of the organic fertilizer was relatively lower [55], and it is necessary to improve the timing of bioavailability of organic fertilizers and use soil amendments.

4. Conclusions

In summary, nitrogen application plays an important regulatory role in the yield and phytometabolite content of medicinal crops. NAR varied significantly among medicinal crops and geographic regions; 100–199 kg N/ha was most frequently reported, followed by 0–99 and 200–299. The bearing of N fertilizer on the yield and quality of different medicinal materials varies greatly, and the amount of N application can also differentially impact their yield and quality. Pharmaco-phylogenetic and geographic investigations showed that environmental factors, rather than genetic ones, could have greater effect on the N demand of medicinal plants. However, most studies on the regulatory effect of fertilization on the yield and quality of medicinal crops are still preliminary. So far, research on genes involved in N fertilizer utilization and metabolism in medicinal plants is still largely lacking. Studies on the regulatory mechanism of N fertilizer at the molecular level should be undertaken to elucidate the key roles of nitrogen-responsive genes and nitrogen-utilization-regulating genes in the roots of medicinal crops. To date, the biosynthesis pathways of some secondary metabolites in medicinal plants have been clarified, and studying the regulatory effect of nitrogen application on the biosynthesis genes will provide new avenues for improving the quality of medicinal materials. Moreover, increased research on the regulatory effects of commonly used fertilizers, such as phosphorus fertilizer, potassium fertilizer, and trace elements, on the yield and quality of medicinal materials should be undertaken. These nutrients also play an important role in the growth and accumulation of medicinal components in medicinal plants, which are often applied in combination with N fertilizer in the field. Our knowledge of how these nutrients interact with the soil/plant nitro-genome remains limited. Such studies are crucial for formula fertilization, which will help maximize the planting benefits of medicinal plants and promote their sustainable development through scientific fertilization.