Implications of Vegetal Protein Hydrolysates for Improving Nitrogen Use Efficiency in Leafy Vegetables

Abstract

Climate change and the degradation of ecosystems is an urgent issue to which the agricultural sector contributes through the overuse of productive inputs such as chemical fertilizers. A disproportionate use of nitrogenous fertilizers combined with low efficiency inevitably results in worsening environmental problems (greenhouse gas emissions, soil degradation, water eutrophication, and groundwater pollution). Nevertheless, increasing population growth puts additional pressure on the already struggling agricultural world. Awareness of these problems has pushed the world of research towards the development of more sustainable but equally efficient strategies in terms of production. The use of biostimulant substances and/or micro-organisms promoting yield, resilience to abiotic stresses in plants, and increasing the functional quality of products have been indicated as a valid strategy to improve the sustainability of agricultural practices. In modern horticulture, the use of vegetable–protein hydrolysates (V-PHs) is gaining more and more interest. These biostimulants could influence plants directly by stimulating carbon and nitrogen metabolism and interfering with hormonal activity, but also indirectly as V-PHs could improve nutrient availability in plant growth substrates and increase nutrient uptake and utilization efficiency. By exploiting this aspect, it would be possible to reduce the use of chemical fertilizers without affecting potential yields. After a brief introduction to the issues related to the intensive use of nitrogen fertilizers, this review focuses on the use of V-PHs as a strategy to increase nitrogen use efficiency (NUE). Starting with their heterogeneous origins and compositions, their effects on nitrogen metabolism, as well as the physiological and biochemical processes involved in these products, this review concludes with an in-depth discussion of the effects of V-PHs on major leafy vegetables.

1. Introduction

One of the challenges currently facing the agricultural sector is meeting the ever-increasing demand for food production as the world population grows. According to current estimates, to feed the world population, which will reach 9 billion in 2050, it will be necessary to increase agricultural production by about 1.7 times [1,2]. Occupying 40% of the world’s land area and consuming about 70% of global fresh water, the agricultural sector is struggling to meet this demand, despite the fact that climate change and resource scarcity problems (such as arable land and fresh water) negatively affect the yield and quality of agricultural products [3]. As if this were not enough, it must be emphasized that conventional agricultural practices further influence the degradation of natural resources that are not always renewable [4].

Specifically, the agricultural sector will have to integrate circular economy strategies to decrease farm inputs while continuing to obtain an adequate or even higher yield. For this reason, it would be advisable to re-use the agro–industrial residues produced by farms both to add value through the production of new bioproducts and to avoid the high disposal costs of residues, which would otherwise be disposed of as special waste according to the European Waste Code [2]. Among the various bioproducts that can be obtained from the recycling of agro–industrial waste, it is worth mentioning, by way of example, compost, vermicompost, compost tea, oil-free seed meal, digestates, biofumigants, soil biofertilizers, and plant biostimulants [5]. The use of the latter due to the transition from a linear to a circular economy would be an excellent strategy to mitigate climate change thanks to increased resource-use efficiency and the consequent resilience of crops to increasing environmental stresses [6]. The integration of biostimulants in agriculture is attracting considerable interest from growers and the research community [7,8,9]. By definition, a biostimulant is ‘any substance or micro-organism capable, when applied to plants, of improving nutritional efficiency, tolerance to abiotic stresses and/or crop quality traits, regardless of its nutrient content’ [10].

Humic and fulvic acids, protein hydrolysates (PHs), algae extracts, silicon, and growth-promoting fungi and bacteria are the main types of biostimulants used by the agricultural world [9]. Among these, we decided to focus on PHs as they are obtained from agro–industrial by-products and are also interesting from an environmental and economic point of view [11,12]. Indeed, the raw materials from which PHs can be obtained are by-products of the agri-food industry, animal husbandry, and livestock effluents [13,14,15]. From these, through partial hydrolysis, a mixture of amino acids, oligopeptides, and polypeptides are obtained, which turn out to be the main constituents of PHs [9].

2. Protein Hydrolysates: What They Are and How They Work

Depending on the production process (chemical or enzymatic hydrolysis) and the origin of the raw material (animal or plant), PHs have different chemical characteristics (chirality of the amino acids, molecular weight of the elements, electrical conductivity, and the rate of free amino acids in peptides/proteins) [16,17]. A study by Cavani et al. [18] found that, after testing 22 different PHs, the electrical conductivities ranged from 3.9 to 20.0 dS m⁻¹ while the free amino acid contents ranged from 0.8 to 20 percent. Based on the above, this review will discuss protein hydrolysates of vegetable origin (V-PHs) obtained instead through an enzymatic hydrolysis process. This process appears to be more environmentally friendly due to low energy requirements and carbon dioxide emissions. In this case, protein hydrolysis is mediated by proteolytic enzymes of a different nature (animal, plant, or micro-organism), which do not require high activation temperatures (<60 °C) and target specific peptide bonds. The resulting V-PH consists of a mixture of amino acids and peptides of variable length, low salinity, and constant composition over time [16,19]. In addition to the well-known and discussed compounds of a nitrogenous nature (amino acids and peptides), V-PHs contain a plethora of other compounds that synergistically contribute to their biostimulating action (Figure 1). It is important to remember that the efficacy of these products, whose maximum benefits are by definition obtained at very low dosages [20,21], depends on the species and cultivar, environmental conditions, mode and time of application, phenological stage, and their mutual interactions [22,23]. Generally, V-PHs, like most non-microbial biostimulants, are applied by foliar or root application [12,24]. The organic constituents of plant biostimulants, after being absorbed, are translocated and finally transformed into the compounds used by the plants [25]. Due to microbial competition in the rhizosphere, root application may result in plants taking up only part of the amino acids present (about 6–25%) [16]. In contrast, foliar uptake is mediated by moisture levels, wind speed, stomatal reactivity, and leaf cuticle thickness [12]. Several authors have observed that plants treated with V-PHs exhibited increased water and nutrient use efficiency [26,27]. To explain this, V-PHs act by promoting root development, as they are able to trigger an auxin-like mechanism of action [28,29]. Improved nutrient use efficiency is also related to soil microbial activity, as this can influence crop response to V-PH application through the production of enzymes that hydrolyse V-PH peptides into smaller fragments that can act as signalling compounds for the crop [30]. Another interesting feature of protein hydrolysates is the improved assimilation of nitrogen, as amino acids, being a source of organic N, are more easily assimilated in proteins than inorganic N [31]. It has been hypothesized that the increase in nitrogen use efficiency (NUE) may be related to an increase in photosynthesis, resulting in more energy being available for amino acid biosynthesis [16]. As reported by Ertani et al. [32], the V-PH-induced improvement in photosynthetic performance could be attributable to increased chlorophyll production. In addition to positively influencing crop productivity under optimal conditions, V-PHs, by triggering increased production of secondary metabolites, allow the plant to respond more quickly to abiotic stresses such as drought, oxidative salinity conditions, and nutritional deficits [33]. Enzymatic antioxidant responses to biostimulants in maize and soybean subjected to drought showed that the application of V-PHs increased the activity of enzymes such as superoxide dismutase, L-ascorbate peroxidase, and catalase, suggesting an antioxidant action that helped plants to overcome stress conditions. The “defense system” modulation of plants is likely due to more efficient transduction of messages from cell to cell after product application. A study of tomato plants found that the plants’ responses to biostimulant application revolves around the plants’ reactive oxygen species (ROS) signaling network, which would drive plants to increase their contents of antioxidant compounds such as phenols and carotenoids [34]. The increased content of antioxidant molecules and thus antioxidant capacity is particularly favorable from a product quality perspective and has been widely reported in the literature. A study on the effect of commercial lignosulfonate–humate on Zea mays L. metabolism showed that increased production of phenyl-propanoids correlated with increased enzyme activity of phenyl–alanine–ammonia–lyase (PAL) mitigated the negative effects of salt stresses. Under conditions of otherwise low nutrient availability, plant biostimulants could specifically activate the biosynthetic pathway of salicylic acid, which in turn may lead to increased absorbent root surface area and consequently improve plant production performance [35]. Last but not least, the use of V-PHs by enhancing nitrate reductase activity [9,36] would lead to a lower nitrate accumulation in leafy vegetables and thus to a positive influence on the final quality of these crops. Considering the close relationship between N metabolism and V-PH application, in the present review we will analyze, by discussing several studies on the most important leafy vegetables, how this category of biostimulants affects the uptake of this crucial macronutrient.

3. Nitrogen Use Efficiency and Nitrate Uptake

Since their introduction, synthetic nitrogen fertilizers have been the main tool farmers use to increase crop yields [37,38]. Nitrogen (N) is a crucial macroelement that significantly influences the growth and/or development of plants. The significance of this element is directly linked to its importance in key biological functions [39], as nitrogen absorbed by plants is a constituent of compounds such as amino acids, proteins, and nucleic acids directly involved in primary and secondary metabolism [40]. Regardless of the type of nitrogen available, nitrate is the primary nitrogen source used by plants. After being absorbed by specific root transporters, nitrate is first reduced to nitrite and then reduced to ammonia, mediated by the enzymes nitrate reductase and nitrite reductase, respectively [41]. Although, an increase in the supply of synthetic nitrogen fertilizers does not result in a proportional increase in biomass. In fact, the approximately 7.5-fold increase in nitrogen fertilization has only increased crop yield by 2.4 times [42]. Furthermore, the excessive use of nitrogen fertilizers could make crops more susceptible to biotic and abiotic stress, adversely affecting production [39]. The environmental impact of the use of indiscriminate fertilizers is also notable, as it is closely related to the emission of large amounts of greenhouse gases from the production and distribution process, especially the risk of leaching of nitrates [43]. In super intensive agricultural systems, nitrate leaching losses may exceed 50% of applied nitrogen. Nitrate-contaminated water becomes undrinkable and continuous exposure to high concentrations can pose a serious risk to human health [44]. To avoid this, the water industry must incur additional expenses to remove nitrates from groundwater [45]. The harmful impacts of nitrate loss from agricultural soils have toxicological implications for animals and humans and also for the environment, leading to water eutrophication with deleterious impacts on the preservation of marine ecosystems [46]. This phenomenon is mainly linked to the proliferation of green algae, a drastic reduction of oxygen in surface water, reduced light infiltration, and the production of toxins harmful to fish, livestock, and humans [47]. In these scenarios, improving nitrogen use efficiency (NUE) by simultaneously reducing nitrogen losses to the environment and the use of synthetic nitrogen fertilizers is imperative. To achieve this goal, it is necessary to delve into the concept of NUE, defined as ‘the yield obtained per unit of available nitrogen supplied by the fertilizer and/or already present in the soil’ [48]. Despite its apparent simplicity, the major contribution to increasing NUE would come from genetic parameters (species and/or cultivar), environmental parameters (including climate, soil type, organic matter content, soil microflora, and related biological activity determining nitrogen fixation, transformation, and storage), and agronomic management parameters (related to the type of fertilizer applied to the soil, application time, and administration method) [48]. In general, NUE is determined by the interaction of these parameters, which influence both nitrogen uptake efficiency and nitrogen use efficiency within the plant. Among various agronomic management practices, the use of biostimulants could be a promising strategy to improve NUE [49]. Specifically, vegetal-origin protein hydrolysates, through up-regulation of genes involved in nitrogen assimilation, could serve as an interesting tool to integrate into achieving this goal, potentially helping to reduce the nitrate content in plant tissues [16,50,51]. This is noteworthy because excessive accumulation of nitrate is associated with high nitrate assimilation from the soil and low conversion to nitrogenous metabolites useful for crop growth (and therefore low NUE). Leafy vegetables are commonly termed nitrate accumulators since nitrates are transported through the xylem by transpiration flow into the vacuoles of leaf mesophyll cells [52,53]. The direct impact of nitrates on human health remains uncertain. Several clinical studies have reported no correlations between food-derived nitrates and carcinogenic phenomena, probably due to simultaneous intake of antioxidant metabolites such as vitamin C and phenolic acids from the same food sources [54,55]. In addition to this, it should be noted that methods of boiling and cooking vegetables drastically reduce the content of this anti-nutritional element. Plant species belonging to the families Amaranthaceae (Chenopodiaceae), Umbelliferae, Asteraceae, and Brassicaceae are often identified as nitrate accumulators [56]. Within this large group are important horticultural crops: amaranth, spinach, chard, celery, fennel, aromatic herbs (such as cilantro, dill, parsley, basil, oregano, mint, and thyme), arugula, mustard, radish, watercress, and lettuce. However, thresholds defining their marketing have not been set for all of them. Indeed, the European Commission, through Regulation No. 1881/2006 and subsequent Regulation No. 1258/2011, has precautiously regulated the nitrate content in three leafy vegetables: spinach (Amaranthaceae), lettuce (Asteraceae), and arugula (Brassicaceae). Specifically, fresh spinach [3500 mg/kg fresh weight (FW)]; frozen and deep-frozen spinach (2000 mg/kg FW); fresh lettuce (3000–5000 mg/kg FW); iceberg-type lettuce (2000–2500 mg/kg FW); and wild arugula and salad [Eruca sativa Mill., Diplotaxis tenuifolia (L.) DC., Brassica tenuifolia (L.) Baill., Sisymbrium tenuifolium L. (6000–7000 mg/kg FW)].

The urgent attention being paid to the content of nitrates is primarily due to its conversion into harmful by-products. Salivary enzymes and oral bacteria (e.g., Streptococcus intermedius and Staphylococcus sciuri) in humans convert nitrates to nitrites, which can pose a serious threat to human health due to their involvement in methemoglobinemia, better known as ‘blue baby syndrome,’ and in gastric and bladder tumors [57,58]. The close and well-established link between food and health has prompted numerous researchers to analyze in detail the nitrate contents of leafy vegetables [59,60,61]. This research has shown that the most impactful factor is genetic material followed by environmental conditions and agronomic management practices. In recent years, several studies [62,63,64] have summarized the effects of different pre-harvest factors (nitrogen fertilization, temperature, and light conditions) in addition to nitrate accumulation on nitrogen utilization efficiency. However, scientific works concerning the use of biostimulants on these issues are limited. It is crucial to consider that the efficacy of biostimulants is closely dependent on the crop, climate, type of biostimulant used, and dose [65,66]. In the case of V-PHs, the final composition of biostimulants, as well as the results achievable in plants after their application, are strongly influenced by the botanical origin of the biomass used in the production process. This review aims to present the most recent research on the application of V-PHs in leafy vegetables by reporting their impact on nitrogen metabolism as well as on marketable yield. The effects on NUE, some possible mechanisms of nitrate concentration regulation, and perspectives for future research are discussed.

4. Biostimulant Action of Protein Hydrolysates in Increasing NUE: A Case Study on Leafy Vegetables

The capacity to improve growth and enhance the qualitative aspects of horticultural products has positioned biostimulants as one of the most intriguing technological advancements in the agricultural sector over recent decades. While positive feedback underscores the efficacy of these products, our comprehension of the potential mechanisms orchestrating the ‘regulation’ of nitrogen metabolism in plants, especially under limited conditions, remains a substantial enigma. In the context of V-PHs, several authors have emphasized their pivotal role in nitrogen absorption processes (Table 1). This is attributed to the up-regulation of enzymes not only within the Krebs cycle but also directly involved in assimilating this crucial microelement [10,16,67]. Despite such compelling evidence, a comprehensive understanding of the precise mechanisms through which biostimulants, particularly V-PHs, influence nitrogen metabolism—especially in challenging environmental conditions—demands further exploration.

Lettuce plants (Lactuca sativa L.) subjected to weekly V-PH treatments showed a 26% increase in yield, attributed to the inclusion of tryptophan in the commercial formulation used [68]. This essential amino acid, serving as a vital precursor to indole-3-acetic acid (IAA), likely played a role in fostering a more robust development of both roots and shoots in the treated plants. The observed increase in productive performance was complemented by a considerable reduction in nitrogen accumulation within plant tissues, indicating an enhancement in NUE indices. This underscores the active involvement of V-PHs in the intricate pathways of nitrogen metabolism. Moreover, the plants treated with V-PHs demonstrated a heightened level of amino acids in the phloem compared to their untreated counterparts, potentially leading to a diminished absorption and accumulation of nitrates. Beyond these effects on primary metabolism, Sabatino et al. [68] noted a 6.6% increase in vitamin C content in V-PH-treated plants compared with control plants. This observation confirms the positive influence of these stimulants on the biosynthesis of specialized metabolites. Noteworthy is the response observed in basil plants (Ocimum basilicum L.) subjected to V-PH treatment under nitrogen-deficient conditions (0 and 50 kg of N ha^–1). An increase of 14% in NUE was recorded in comparison to the untreated group, emphasizing the beneficial impact of V-PHs under nitrogen-limited and non-limiting conditions [69]. The positive impact on nitrogen metabolism was associated with an increase in the fresh saleable yield (+16%) of basil plants subjected to V-PH treatment, which was characterized by higher chlorophyll content (+5%).

The application of V-PHs at low nitrogen levels is a plausible factor inducing a positive up-regulation of glutamine synthetase and amino acid transporters. This, in turn, promotes improved nitrogen absorption, yielding positive consequences for the growth of treated plants. Regardless of the nitrogen fertilization treatment, the application of V-PHs reduced nitrate content in plant tissues by 10%. This phenomenon is likely attributed to an over-regulation of nitrate reductase, facilitating a more efficient conversion of assimilated nitrate into organic compounds [70]. In the specific case of arugula (Diplotaxis tenuifolia (L.) DC.) [71], compared to control conditions, foliar application of V-PHs resulted in an increase in the yield and number of rosettes by 15 and 29%, respectively. Caruso et al. [71] postulated that soluble peptides and key amino acids present in the tested protein hydrolysate formulation stimulated metabolic processes in perennial arugula. This stimulation contributed to the enhancement of absorption, translocation, and accumulation of key macroelements such as potassium (K) and magnesium (Mg). The biostimulant employed may have induced, alongside a structural modification of the root system, an increased activity of specific transporters for macroelements through the up-regulation of involved genes. Rouphael et al. [72] demonstrate that the application of the same V-PH (Trainer^®®) resulted in an augmentation of the area, number, and fresh weight of leaves in Genovese basil. This enhancement in yield parameters coincided with heightened assimilation of carbon dioxide (+54%), improved water use efficiency (WUE) (+46%), and an elevation in K (+15%), Mg (+33%), and sulfur (S) (+82%) contents. The authors posit that the superior nutritional status of plants treated with V-PH could be linked to a remodulation of the root system’s shape and growth, promoting more efficient absorption and transport of minerals. This phenomenon, commonly recognized as the ‘nutrient acquisition response,’ forms the basis for results obtained by Carillo et al. [73] on baby spinach (Spinacia oleracea L.) cultivated in a protected environment. Specifically, V-PH integration improved the production (by 29% on average) and nutritional status of spinach plants grown under non-optimal nitrogen levels (0 and 15 kg ha^–1 of N). Notably, foliar application of V-PH not only heightened the assimilation and translocation of phosphorus (P) (+13%), calcium (Ca) (+13%), and Mg (+10%) in plant tissues but also triggered a re-mobilization of nitrogen reserves from roots to shoots, thereby enhancing overall nutritional status and yield. Similar results were also observed in lettuce [74] and arugula [75]. Specifically, the tested application of V-PH on lettuce [74] and arugula [75] compensated for the biomass loss resulting from lower nitrogen availability, highlighting the heightened efficacy of V-PH under deficient conditions. Furthermore, the same authors [74,75] noted a significant enhancement in nitrogen use efficiency (NUE) indices following the application of V-PH. This improvement was attributed not only to an increase in fresh yield but also to a more efficient accumulation of total nitrogen in treated lettuce plants. The authors emphasized that, particularly in nitrogen-depleted soils, the use of these biostimulant products could facilitate a judicious reduction in the application of nitrogen fertilizers without compromising crop yields.

As reported by Navarro-León et al. [74] and Di Mola et al. [75], a prominent stimulating effect of V-PH was observed in both non-nitrogenous fertilizer and non-optimally-fertilized treatments. However, irrespective of the nitrogen fertilization regime, the foliar application of V-PH increased leaf nitrate content by about 1.7 times, while still remaining within the limits set by EU Regulation 1258/2011 for arugula [75]. In contrast, the study conducted on hydroponic spinach revealed a more pronounced biostimulation in response to V-PH treatment when plants were provided with an optimal nitrogen regime [76]. The V-PH treatment appeared to prompt spinach plants grown under optimal nitrogen conditions to increase nitrogen uptake, thereby stimulating greater leaf expansion that would have led to improved light interception. The authors hypothesized that the influence of V-PH treatment on the regulation of genes involved in nitrogen metabolism might be contingent upon nitrogen availability. Regarding V-PH, Cristofano et al. [77] observed significant differences in the magnitude of the biostimulating effect in response to two different application methods (foliar and root). In comparison to foliar application, the root application of V-PH resulted in a more potent biostimulating effect (increased yield and leaf area by 12 and 11 percent, respectively) and a reduction in nitrate content for both lettuce cultivars of 15.7 percent. The constant and direct ‘contact’ of V-PH with the root system of lettuce plants grown in the floating raft system (FRS) likely contributed to the enhanced bioavailability of the product. Additionally, considering that foliar absorption is passive, the lower efficiency of the foliar treatment could be a direct consequence of not-always-optimal climatic conditions, influencing the biological responses of the plant. Consistent with the findings of Cristofano et al. [77], Choi et al. [78] observed, also in lettuce, greater efficiency of the root treatment with V-PH compared to foliar treatment. In addition to this, the comparison with untreated plants highlighted how the root application of V-PH was more efficient under nitrogen-deficient conditions. Similarly to the different application method, Di Mola et al. [79] emphasized, in their study, a species-specific response. Despite the overall improvement in production performance for both baby spinach and lamb’s lettuce due to V-PH application, the positive effects were more pronounced in spinach, likely due to different leaf morphology and anatomy. For both evaluated species, V-PH application allowed plants grown with a suboptimal nitrogen dose (N-50%) to achieve productivity values comparable to those obtained from untreated plants but subjected to an optimal nitrogen dose (N-100%). However, regardless of fertilization level and species, compared to control conditions, V-PH treatment resulted in both higher nitrogen absorption efficiency and a higher nitrogen use efficiency (NUE) value.

In addition to significant changes in primary metabolism, V-PH greatly enhanced the antioxidant capacity of both baby spinach and lamb’s lettuce. By directly applying V-PH in the nutrient solution, compared with non-biostimulated plants, a reduction in nitrate content in both peppermint and spearmint grown in the floating raft system (FRS) was observed without, however, observing an improvement in yield parameters [80]. It is likely that, in nutrient solutions containing V-PH, the co-presence of different nitrogen forms may have slowed down nitrate absorption due to an antagonistic effect, especially of the ammonium and amino acids present in the biostimulant (as previously described in the same study). This result was partially observed in two cultivars of basil (Ocimum basilicum L.) grown hydroponically [81]. Regardless of the concentration of the nutrient solution used, the integration of V-PH at the lower dose (0.15 mL L^–1) resulted in a significant reduction in nitrate content (−7%) in plant tissues compared to the control condition, confirming what was previously described by Aktsoglou et al. [80]. On the contrary, the use of the double dose of V-PH (0.30 mL L^–1) led to a significant increase in nitrate concentration (+9%), probably due to better transpirative activity (+12.4%) of the biostimulated plants. The dose-dependent effect of V-PH treatments was also observed by Carillo et al. [82] on greenhouse-grown lettuce. Although the authors recorded an increase in fresh yield, leaf area, and root dry weight regardless of the applied dose of 8, 9, and 47%, respectively, the activated mechanisms were ambiguous. Compared to the maximum dose of V-PH (5.0 mL L^–1), the dose of 2.5 mL L^–1 was more efficient in increasing the root-to-shoot ratio. A lower dose of V-PH induced a more pronounced remodelling of the root system, increasing nitrate uptake, translocation, and utilization efficiency [82]. On the contrary, the use of the maximum dose of V-PH resulted in an earlier improvement in photosynthetic performance, immediately providing a greater amount of assimilates to support higher production compared to in control conditions.

Table 1. Effects of vegetal-PH application on yield, quality, N uptake, and efficiency of leafy greens under optimal and sub-optimal N conditions.

Crops	V-PH Application Mode	Experimental Conditions	Nitrogen Fertilisation Management	Effects	Reference
Diplotaxis erucoides L.	Foliar	Soil culture in greenhouse	Four different nitrogen levels: 0, 60, 80, and 100 kg ha−1	The application of V-PH increased, under N-deficient conditions (0 and 60 kg ha−1), the production of fresh rocket (+35% on average). The biostimulant treatment increased, compared to the control plants, chlorophyll and carotenoid contents, but also nitrate content.	[75]
Diplotaxis tenuifolia (L.)	Foliar	Soil culture in greenhouse	Optimal	V-PH improved marketable yield (+15%), colorimetric parameters, mineral composition, and antioxidant activity of the plants.	[71]
Lactuca sativa L.	Foliar	Soil culture in greenhouse	Optimal	A significant increase in yield (26%), nutritional and functional traits, and nitrogen indices (N use efficiency and N physiological use efficiency).	[68]
Lactuca sativa L.	Foliar and root	Floating raft system	Optimal	The application of V-PH directly into the nutrient solution or by foliar application increased the fresh production and mineral status of lettuce plants.	[77]
Lactuca sativa L.	Foliar and root	Pot culture in greenhouse	Four different levels of nitrogen in the nutrient solution: 2, 5, 10, and 15 mM	Compared to foliar application, root application of V-PH significantly increased yield, chlorophyll concentrations, and antioxidant activities, associated with increased utilization efficiency and nitrogen uptake.	[78]
Lactuca sativa L.	Root	Soil-less culture in growth chamber	Three different nitrogen levels: 2.4, 4.8, and 8 mmol L−1 NaNO3	Improved N utilization and uptake efficiency as well as production and physiological performance of V-PH-treated plants.	[74]
Lactuca sativa L.	Foliar	Pot culture in greenhouse	Optimal	Regardless of the dose used, V-PH increased root biomass, leaf area, polyphenol content, and NUE compared to control plants.	[82]
Mentha × piperita	Root	Floating raft system	Optimal	Improved nutritional status and bioactive compounds (total phenols and carotenoids) with a significant reduction in nitrate content.	[80]
Ocimum basilicum L.	Foliar	Soil culture in greenhouse	Four different nitrogen levels: 0, 50, 100, and 150 kg ha−1	The application of V-PH increased fresh production and NUE under N-deficient conditions without increasing the nitrate concentration. Independent of the nitrogen regime, the application of V-PH increased polyphenol content.	[73]
Ocimum basilicum L.	Foliar	Soil culture in greenhouse	Optimal	Compared to control conditions, the application of V-PH increased fresh production, net CO2 assimilation, and the nutritional status of basil plants (higher contents of K and N).	[72]
Ocimum basilicum L.	Root	Floating raft system	Two different nitrogen levels in nutrient solution: 7.5 and 14 mM	Significant increase in marketable yield due to improved photosynthetic performance of V-PH-treated plants.	[81]
Spinacia oleracea L.	Foliar	Soil culture in greenhouse	Four different nitrogen levels: 0, 15, 30, and 45 kg ha−1	The application of V-PH increased the fresh and dry yield under N-deficient conditions without altering the nitrate concentration in the leaves. Independent of the nitrogen regime, V-PH increased Ca and Mg contents while reducing polyphenol content.	[69]
Spinacia oleracea L.	Foliar	Soil culture in greenhouse	Three different nitrogen levels: 0, 2.25, and 4.5 g m−2	Improvement of both utilization efficiency and nitrogen uptake efficiency. For each nitrogen level, V-PH significantly increased fresh production, with the greatest effect observed under optimal nitrogen conditions.	[79]
Spinacia oleracea L.	Foliar	Ebb and flow soil-less cultivation system	Three different nitrogen levels in nutrient solution: 2, 8, and 14 mM	V-PH increased fresh yield under optimal nitrogen conditions (14 mM) inducing an improvement in N uptake, foliar expansion, and photosynthetic activities.	[76]
Valerianella locusta L.	Foliar	Soil culture in greenhouse	Three different nitrogen levels: 0, 2.25, and 4.5 g m−2	The use of V-PH increased fresh production at sub-optimal nitrogen levels. The biostimulant increased not only the NUE but also nitrate content.	[79]

Table 1. Effects of vegetal-PH application on yield, quality, N uptake, and efficiency of leafy greens under optimal and sub-optimal N conditions.

Crops	V-PH Application Mode	Experimental Conditions	Nitrogen Fertilisation Management	Effects	Reference
Diplotaxis erucoides L.	Foliar	Soil culture in greenhouse	Four different nitrogen levels: 0, 60, 80, and 100 kg ha−1	The application of V-PH increased, under N-deficient conditions (0 and 60 kg ha−1), the production of fresh rocket (+35% on average). The biostimulant treatment increased, compared to the control plants, chlorophyll and carotenoid contents, but also nitrate content.	[75]
Diplotaxis tenuifolia (L.)	Foliar	Soil culture in greenhouse	Optimal	V-PH improved marketable yield (+15%), colorimetric parameters, mineral composition, and antioxidant activity of the plants.	[71]
Lactuca sativa L.	Foliar	Soil culture in greenhouse	Optimal	A significant increase in yield (26%), nutritional and functional traits, and nitrogen indices (N use efficiency and N physiological use efficiency).	[68]
Lactuca sativa L.	Foliar and root	Floating raft system	Optimal	The application of V-PH directly into the nutrient solution or by foliar application increased the fresh production and mineral status of lettuce plants.	[77]
Lactuca sativa L.	Foliar and root	Pot culture in greenhouse	Four different levels of nitrogen in the nutrient solution: 2, 5, 10, and 15 mM	Compared to foliar application, root application of V-PH significantly increased yield, chlorophyll concentrations, and antioxidant activities, associated with increased utilization efficiency and nitrogen uptake.	[78]
Lactuca sativa L.	Root	Soil-less culture in growth chamber	Three different nitrogen levels: 2.4, 4.8, and 8 mmol L−1 NaNO3	Improved N utilization and uptake efficiency as well as production and physiological performance of V-PH-treated plants.	[74]
Lactuca sativa L.	Foliar	Pot culture in greenhouse	Optimal	Regardless of the dose used, V-PH increased root biomass, leaf area, polyphenol content, and NUE compared to control plants.	[82]
Mentha × piperita	Root	Floating raft system	Optimal	Improved nutritional status and bioactive compounds (total phenols and carotenoids) with a significant reduction in nitrate content.	[80]
Ocimum basilicum L.	Foliar	Soil culture in greenhouse	Four different nitrogen levels: 0, 50, 100, and 150 kg ha−1	The application of V-PH increased fresh production and NUE under N-deficient conditions without increasing the nitrate concentration. Independent of the nitrogen regime, the application of V-PH increased polyphenol content.	[73]
Ocimum basilicum L.	Foliar	Soil culture in greenhouse	Optimal	Compared to control conditions, the application of V-PH increased fresh production, net CO2 assimilation, and the nutritional status of basil plants (higher contents of K and N).	[72]
Ocimum basilicum L.	Root	Floating raft system	Two different nitrogen levels in nutrient solution: 7.5 and 14 mM	Significant increase in marketable yield due to improved photosynthetic performance of V-PH-treated plants.	[81]
Spinacia oleracea L.	Foliar	Soil culture in greenhouse	Four different nitrogen levels: 0, 15, 30, and 45 kg ha−1	The application of V-PH increased the fresh and dry yield under N-deficient conditions without altering the nitrate concentration in the leaves. Independent of the nitrogen regime, V-PH increased Ca and Mg contents while reducing polyphenol content.	[69]
Spinacia oleracea L.	Foliar	Soil culture in greenhouse	Three different nitrogen levels: 0, 2.25, and 4.5 g m−2	Improvement of both utilization efficiency and nitrogen uptake efficiency. For each nitrogen level, V-PH significantly increased fresh production, with the greatest effect observed under optimal nitrogen conditions.	[79]
Spinacia oleracea L.	Foliar	Ebb and flow soil-less cultivation system	Three different nitrogen levels in nutrient solution: 2, 8, and 14 mM	V-PH increased fresh yield under optimal nitrogen conditions (14 mM) inducing an improvement in N uptake, foliar expansion, and photosynthetic activities.	[76]
Valerianella locusta L.	Foliar	Soil culture in greenhouse	Three different nitrogen levels: 0, 2.25, and 4.5 g m−2	The use of V-PH increased fresh production at sub-optimal nitrogen levels. The biostimulant increased not only the NUE but also nitrate content.	[79]

5. Conclusions and Challenges Ahead

The balanced supply of nutrients, especially nitrogen, is a fundamental prerequisite for achieving food security [83]. Farmers are currently tasked with producing more while fertilizer prices are continually rising. Therefore, it is crucial that strategies aimed at improving NUE do not compromise economic sustainability [84]. It is essential to develop and adopt production-efficient and environmentally sustainable practices. V-PH biostimulants have been identified as one of the most promising tools available in the horticultural sector [19]. These plant biostimulants not only stimulate growth under optimal and/or environmental stress conditions but also positively impact resource use efficiency. These biostimulants, as well as being a useful tool to reduce the use of chemical fertilizers, would be the best strategy to promote organic farming. The aim of this review was to discuss in detail the effects of V-PH application on the nitrogen metabolism of major leafy vegetables. Although many of the mechanisms enacted by various V-PH constituents are still not well understood, significant progress has been made in recent years. The results reported in this review underscore the positive role of these substances, especially under nitrogen-deficient conditions, paving the way for interesting scenarios that could involve the partial replacement of chemical inputs with natural products. Numerous studies have demonstrated the ability of V-PH to actively regulate nitrogen metabolism in a non-uniform and nitrogen-dependent manner. However, it is important to consider the current challenges that research must address:

Standardizing V-PH production processes will be necessary, taking into account that the final product composition depends on the raw material used;

Elucidating the mode of action in relation to the specific composition of the V-PH used.

Once these challenges are overcome, future research should focus on identifying the best V-PH combinations (application method and dose) × plant species × nitrogen fertilization, both in terms of productivity and environmental sustainability.