Biostimulant Formulations and Moringa oleifera Extracts to Improve Yield, Quality, and Storability of Hydroponic Lettuce

The urgent need to increase the sustainability of crop production has pushed the agricultural sector towards the use of biostimulants based on natural products. The current work aimed to determine whether the preharvest application of two commercial formulations, based on a Fabaceae enzymatic hydrolysate or a blend of nitrogen sources including fulvic acids, and two lab-made aqueous extracts from Moringa oleifera leaves (MLEs), could improve yield, quality, and storability of lettuce grown in a hydroponic system, as compared to an untreated control. Lettuce plants treated with the MLEs showed significantly improved quality parameters (leaf number, area, and color), total phenolic content and antioxidant activity, and resistance against the fungal pathogen Botrytis cinerea, comparable to that obtained with commercial formulates, particularly those based on the protein hydrolysate. A difference between the M. oleifera extracts was observed, probably due to the different compositions. Although further large-scale trials are needed, the tested MLEs seem a promising safe and effective preharvest means to improve lettuce agronomic and quality parameters and decrease susceptibility to rots.


Introduction
Ready-to-eat products have shown the most rapid expansion in production among vegetable products, even in developing countries [1]. Among the most widely cultivated vegetables are lettuce (Lactuca sativa L.) and chicory, accounting for over 27 million tons harvested worldwide in 2020 [2]. The reasons for this are the quick and year-round production, as well as the beneficial phytochemical and bioactive contents (vitamins, carotenoid, polyunsaturated fatty acids, antioxidants, etc.) [3]. Lettuce is one of the most cultivated leafy green vegetables in Italy, comprising, together with chicory, a production of 963.280 tons in 2021 [2]. Moreover, due to the conversion of many farms to the production and sale of ready-to-eat and fresh-cut salad mixes, lettuce cultivation has gained substantial economic importance in recent years owing to constant varietal and technical progress [4].
However, vegetable production is under pressure to raise yields due to the predicted increase in world population from 8 to 9.6 billion around 2050 [5], of which 70% will live in urban centers. Another major challenge posing a significant risk to the stability of production is climate change [6], which will stress the food supply chain by increasing chemical inputs [7]. Consequently, the demand for raw or minimally processed vegetables is rising, and food industries have been urged to utilize new cultivation systems and  etables, mainly as the quercetin or kaempferol glycosides and less frequently as the luteolin or apigenin glycosides [33]. Their formation normally depends on light, so they are mainly concentrated in the outer tissues of the free-standing leaves. Quercetin's ability to induce resistance phenomena in plant tissues has been reported [34], and the induction of the phenylpropanoid pathway is considered to be involved in resistance mechanisms to stresses [35]. Glucomoringin and its acetyl derivative were reported to play an antioxidant and antimicrobial role [29].  The presence of neochlorogenic acid and quercetin 3-O-β-D-glucopyranoside were also confirmed by high-performance thin-layer chromatography (HPTLC) analysis using reference standards ( Figure 2). HPTLC analysis was also useful to confirm the exact nature of the chlorogenic acid isomer extracted. In fact, chlorogenic and neochlorogenic acids are classical diastereomers that differ in the stereochemistry at positions 1 and 4 of their sugar-like moieties, their corresponding overall stereochemistry notations being (1S,3R,4R,5R) and (1R,3R,4S,5R), respectively. They share the same molecular weight value and, although having quite different mass fragmentation patterns, an accurate and unquestionable identification is advisable. To the contrary, kaempferol acetylglucoside and quercetin acetylglucoside were identified only in MLEB (Table 1). Besides these compounds, MS analysis also revealed the presence in MLEB of the amino alcohol valinol; the organic acids quinic, malic, and gluconic acids; the phenylpropanoids caffeic, coumaroylquinic, and 3-feruloylquinic acids; the flavonoids vitexin, astragalin, quercetin malonylglucoside, kaempferol malonylglucoside, and quercetin 3-[6"-(3-hydroxy-3-methylglutaryl)glucoside]. Valinol, quinic, caffeic, and coumaroylquinic acids, and astragalin were also detected in MLES, with this latter containing additionally apigenin glucoside and kaempferol diglucoside (Table 2). Flavonol and flavone glycosides occur in common vegetables, mainly as the quercetin or kaempferol glycosides and less frequently as the luteolin or apigenin glycosides [33]. Their formation normally depends on light, so they are mainly concentrated in the outer tissues of the free-standing leaves. Quercetin's ability to induce resistance phenomena in plant tissues has been reported [34], and the induction of the phenylpropanoid pathway is considered to be involved in resistance mechanisms to stresses [35]. Glucomoringin and its acetyl derivative were reported to play an antioxidant and antimicrobial role [29].

Yield, Morphological Parameter Measurements and Quality Analysis Samplings
Results showed that the application of the commercial formulate based on Fabaceae enzymatic hydrolysate AgricostanD (AgriD) increased the marketable yield, head weight, and leaf area of lettuce plants by 38,37 and 22% (Table 3, Figure 3), respectively, as compared to the untreated control. Similar results were obtained using PHs on spinach [36], tomatoes [37], and wine grapes [38]. In addition, the tested MLEs even improved some agronomic parameters as compared to the control; in particular, the marketable lettuce yield and leaf area were increased by 30 and 14%, respectively, in MLEB-treated samples and the leaf number by 16% in the presence of MLES (Table 3). Other studies also reported that lettuce treated by

Yield, Morphological Parameter Measurements and Quality Analysis Samplings
Results showed that the application of the commercial formulate based on Fabaceae enzymatic hydrolysate AgricostanD (AgriD) increased the marketable yield, head weight, and leaf area of lettuce plants by 38, 37 and 22% (Table 3, Figure 3), respectively, as compared to the untreated control. Similar results were obtained using PHs on spinach [36], tomatoes [37], and wine grapes [38]. In addition, the tested MLEs even improved some agronomic parameters as compared to the control; in particular, the marketable lettuce yield and leaf area were increased by 30 and 14%, respectively, in MLEB-treated samples and the leaf number by 16% in the presence of MLES (Table 3). Other studies also reported that lettuce treated by MLEs showed increased plant growth parameters such as the number of leaves per plant, leaf area and yield [21,39]. Finally, Organor N12 (N12) increased leaf area by 13% as compared to the control, with an effect comparable to MLEB. Whereas, concerning the evaluation of the leaf water content (WC) and the dry matter (DM), no significant differences were observed in treated plants as compared to the control ( Table 3). The presence of bioactive peptides/compounds triggering signaling pathways involving phytohormone biosynthesis may explain the positive effects of the tested biostimulants on plant growth and yield [18]. Enhanced growth might also be ascribed to the presence of sugars in the biostimulants, which might represent a source of energy and stimulate nitrogen assimilation [21]. Furthermore, the addition of exogenous amino acids present in PHs might have an energy-saving role, so that the plant can better diverge this energy towards other biological functions [40]. In addition, flavonoids, among the main MLE constituents, have long been known to be responsible for many biological functions, including seedling growth and development [41]. As such, applications of MLEs could contribute to increased vegetative growth as well as nutrient uptake in different horticultural crops, ultimately increasing crop productivity and nutritional values and consequently attracting consumers' attention [25].

Yield, Morphological Parameter Measurements and Quality Analysis Samplings
Results showed that the application of the commercial formulate based on Fabacea enzymatic hydrolysate AgricostanD (AgriD) increased the marketable yield, head weight and leaf area of lettuce plants by 38,37 and 22% (Table 3, Figure 3), respectively, as com pared to the untreated control. Similar results were obtained using PHs on spinach [36], tomatoes [37], and win grapes [38]. In addition, the tested MLEs even improved some agronomic parameters a compared to the control; in particular, the marketable lettuce yield and leaf area were in creased by 30 and 14%, respectively, in MLEB-treated samples and the leaf number by 16% in the presence of MLES (Table 3). Other studies also reported that lettuce treated by

Leaf Color, SPAD Index, and Pigment Contents
Consumer preference is mainly determined by product appearance, which is linked particularly to color [42]. In the current study, all biostimulant treatments, except for MLEB, influenced the leaf hue angle (h • ), which corresponded to the common distinction of green among the colors ( Table 4). The highest h • value, as compared to the control, was obtained from samples treated with N12, followed by MLES and AgriD. The increase in hue angle gave lettuce leaves the appearance of a warm green color ( Figure 2) that might be attractive to consumers. Concerning the chroma value (C*), indicating the saturation of the color, only MLEB caused a 3% increase (Table 4). In contrast, none of the treatments increased the leaf lightness (L*). Finally, the Soil Plant Analysis Development (SPAD) index, a non-destructive indicator of N leaf content, showed a 10% increase in samples treated with N12 (Table 4), a finding which was not surprising given its high N content. Additionally, biostimulant application induced a significant increase in lettuce leaf pigments (Table 4), particularly total chlorophyll and carotenoid contents, reaching the highest increase with AgriD (15 and 16%, respectively), followed by MLES (10 and 11%, respectively). This ability to increase pigment content is in line with other reports on plant-based biostimulants, whose application may lead to the up-regulation of photosynthesis and improved nitrogen and carbon metabolism by enhancing N uptake efficiency and limiting chlorophyll degradation and leaf senescence [43]. Chlorophyll content and photosynthesis rate in plants are known to determine growth and yield. The application of PHs and MLEs are reported to enhance both these vital traits under normal and stress conditions. For instance, the application of MLEs on rocket was reported to increase photosynthetic pigments, photosynthetic rate, and stomatal conductance as compared to untreated plants [21]. In another study, snap bean (Phaseolus vulgaris) sprayed with MLE under normal growing conditions showed increased chlorophyll pigments compared to unsprayed plants [26]. The same findings were also reported in the foliar application of PHs in baby rocket leaves and lettuce [43,44], tomato [37], and corn [12].

Total Phenolic Compounds and Antioxidant Activities
The importance of antioxidant activity as a functional quality parameter of food is related to the beneficial effects of antioxidant molecules (hydrophilic and lipophilic) on human health due to their role in delaying or inhibiting oxidative damage, hence evading a broad range of diseases [25]. Leafy vegetables are considered important sources of antioxidant molecules, whose content might be conveniently enriched. In this study, biostimulant application significantly influenced the results from the antioxidant activity evaluation performed by DPPH and ABTS ( Figure 4); the highest values were registered in samples treated with MLES, producing a 72 and 53% increase in DPPH and ABTS values, respectively, followed by N12 (68 and 51%), and MLEB (43 and 26%). In contrast, AgriD elicited positive values only for ABTS antioxidant activity. Furthermore, total phenolic compounds (TPC) increased notably, by 34%, in samples treated with MLES as compared to untreated plants ( Figure 5).
In this regard, the application of plant biostimulants, including MLEs, can modify both primary and secondary metabolism, resulting in an increased concentration of antioxidant compounds. For instance, spinach plants supplemented with PHs showed an increase in the concentration of phenolic antioxidants, total soluble proteins, and other bioactive compounds [36]. Selected plant leaf extracts (mulberry, brassica, sorghum and moringa) significantly enhanced the radical scavenging capacity of radish as compared to the control [45]. Finally, MLE alone and in combination with K and Zn improved quality parameters, such as soluble solid contents, vitamin C, sugars, total antioxidant and phenolic contents, and the activities of superoxide dismutase and catalase enzymes in 'Kinnow' mandarin fruit [24]. It has been proposed that biostimulants might mimic a stressing event with the consequent induction of the plant responding system, including the antioxidant system, leading to an increase in antioxidant molecule production in plants [38,43]. Finally, the Soil Plant Analysis Development (SPAD) index, a non-destructive indicator of N leaf content, showed a 10% increase in samples treated with N12 (Table 4), a finding which was not surprising given its high N content. Additionally, biostimulant application induced a significant increase in lettuce leaf pigments (Table 4), particularly total chlorophyll and carotenoid contents, reaching the highest increase with AgriD (15 and 16%, respectively), followed by MLES (10 and 11%, respectively). This ability to increase pigment content is in line with other reports on plant-based biostimulants, whose application may lead to the up-regulation of photosynthesis and improved nitrogen and carbon metabolism by enhancing N uptake efficiency and limiting chlorophyll degradation and leaf senescence [43]. Chlorophyll content and photosynthesis rate in plants are known to determine growth and yield. The application of PHs and MLEs are reported to enhance both these vital traits under normal and stress conditions. For instance, the application of MLEs on rocket was reported to increase photosynthetic pigments, photosynthetic rate, and stomatal conductance as compared to untreated plants [21]. In another study, snap bean (Phaseolus vulgaris) sprayed with MLE under normal growing conditions showed increased chlorophyll pigments compared to unsprayed plants [26]. The same findings were also reported in the foliar application of PHs in baby rocket leaves and lettuce [43,44], tomato [37], and corn [12].

Total Phenolic Compounds and Antioxidant Activities
The importance of antioxidant activity as a functional quality parameter of food is related to the beneficial effects of antioxidant molecules (hydrophilic and lipophilic) on human health due to their role in delaying or inhibiting oxidative damage, hence evading a broad range of diseases [25]. Leafy vegetables are considered important sources of antioxidant molecules, whose content might be conveniently enriched. In this study, biostimulant application significantly influenced the results from the antioxidant activity evaluation performed by DPPH and ABTS ( Figure 4); the highest values were registered in samples treated with MLES, producing a 72 and 53% increase in DPPH and ABTS values, respectively, followed by N12 (68 and 51%), and MLEB (43 and 26%). In contrast, AgriD elicited positive values only for ABTS antioxidant activity. Furthermore, total phenolic compounds (TPC) increased notably, by 34%, in samples treated with MLES as compared to untreated plants ( Figure 5).  In this regard, the application of plant biostimulants, including MLEs, can modify both primary and secondary metabolism, resulting in an increased concentration of antioxidant compounds. For instance, spinach plants supplemented with PHs showed an increase in the concentration of phenolic antioxidants, total soluble proteins, and other the control [45]. Finally, MLE alone and in combination with K and Zn improved quality parameters, such as soluble solid contents, vitamin C, sugars, total antioxidant and phenolic contents, and the activities of superoxide dismutase and catalase enzymes in 'Kinnow' mandarin fruit [24]. It has been proposed that biostimulants might mimic a stressing event with the consequent induction of the plant responding system, including the antioxidant system, leading to an increase in antioxidant molecule production in plants [38,43].

Resistance to Postharvest Grey Mold
In our investigation, we also tested the resistance of treated plants to the economically dangerous fungal pathogen Botrytis cinerea, which can compromise yield and quality both during cultivation and after harvest. The lettuce leaves treated with MLEB and AgriD during the growth phase proved to be more resistant to the fungal infection, showing a reduced disease severity by 32 and 46%, respectively, as compared to control leaves (Figure 6). This finding is not surprising as soybean PHs proved to be effective against grey mold and downy mildew [40,46] on grapevines and green mold on citrus fruit [47]. The effect could be related to the induction of defense responses [46], including the closing of stomata [38]. A similar behavior might be supposed for MLEs, and particularly the MLEB, also given the inclusion of molecules such as quercetin and apigenin glucosides that might contribute to resistance to stresses [34,48,49].

Resistance to Postharvest Grey Mold
In our investigation, we also tested the resistance of treated plants to the economically dangerous fungal pathogen Botrytis cinerea, which can compromise yield and quality both during cultivation and after harvest. The lettuce leaves treated with MLEB and AgriD during the growth phase proved to be more resistant to the fungal infection, showing a reduced disease severity by 32 and 46%, respectively, as compared to control leaves ( Figure 6). This finding is not surprising as soybean PHs proved to be effective against grey mold and downy mildew [40,46] on grapevines and green mold on citrus fruit [47]. The effect could be related to the induction of defense responses [46], including the closing of stomata [38]. A similar behavior might be supposed for MLEs, and particularly the MLEB, also given the inclusion of molecules such as quercetin and apigenin glucosides that might contribute to resistance to stresses [34,48,49].
the control [45]. Finally, MLE alone and in combination with K and Zn improved quality parameters, such as soluble solid contents, vitamin C, sugars, total antioxidant and phenolic contents, and the activities of superoxide dismutase and catalase enzymes in 'Kinnow' mandarin fruit [24]. It has been proposed that biostimulants might mimic a stressing event with the consequent induction of the plant responding system, including the antioxidant system, leading to an increase in antioxidant molecule production in plants [38,43].

Resistance to Postharvest Grey Mold
In our investigation, we also tested the resistance of treated plants to the economically dangerous fungal pathogen Botrytis cinerea, which can compromise yield and quality both during cultivation and after harvest. The lettuce leaves treated with MLEB and AgriD during the growth phase proved to be more resistant to the fungal infection, showing a reduced disease severity by 32 and 46%, respectively, as compared to control leaves (Figure 6). This finding is not surprising as soybean PHs proved to be effective against grey mold and downy mildew [40,46] on grapevines and green mold on citrus fruit [47]. The effect could be related to the induction of defense responses [46], including the closing of stomata [38]. A similar behavior might be supposed for MLEs, and particularly the MLEB, also given the inclusion of molecules such as quercetin and apigenin glucosides that might contribute to resistance to stresses [34,48,49].

Lettuce Growth Conditions, Experimental Design, and Treatments
The study was carried out in a greenhouse at the CIHEAM Bari (Valenzano, Apulia, Italy) in the period March-May 2022. Seeds of lettuce [Lactuca sativa L. cv. ABAGO RZ , Rijk Zwaan, Italy] were sown into seed cells (0.0014 holes/m 2 ) filled with a commercial substrate (Brill®3 Special, Agrochimica, Bolzano, Italy). After 10 days, when lettuce seedlings had fully expanded cotyledons, they were transplanted to individual pots with perlite medium for hydroponic systems. The pots were randomly distributed in 5 rows; each row included 15 pots and was subjected to one treatment. Lettuce seedlings were fertigated using a drip system with a nutrient solution of FloraGrow/Micro and FloraBloom used according to the manufacturer's instructions (General Hydroponics, Portland, USA). The irrigation frequency was 5 times daily for a total of 1.25 L water/day/plant. Four treatments were applied: once at the transplanting by root immersion, and then three times at 10-day intervals during the growth period by foliar spray until dripping, using a steel spray bottle. The applied treatments were two commercial plant biostimulants based on blended nitrogen sources (Organor N12, ICAS International, Milan, Italy, 4 mL/L) and a Fabaceae enzymatic hydrolysate (AgricostanD, Costantino Srl, Favria, TO, Italy, 2.5 mL/L) and two aqueous extracts, namely MLES and MLEB, from leaves of M. oleifera plants cultivated in a greenhouse in Lecce province (South Apulia, Italy) and open field in Barletta area (North Apulia, Italy), respectively. Plants treated with water were used as a control.

Moringa Leaves Extract Preparation and Characterization
Based on our previous experience of microwave-assisted extraction of bioactive compounds from different plant matrices [50][51][52][53], the extraction was carried out under microwave irradiation. The fresh moringa leaves were dried before undergoing microwaveassisted extraction. Briefly, in a microwave tube, 200 mg of leaf powder was briefly suspended in 2 mL of bi-distilled water and extracted for 5 min at 80 • C. After filtration, the sample was centrifuged for 10 min at 8000× g, and the supernatant was lyophilized and resuspended in 500 mL of bi-distilled water. The extracts were qualitatively characterized using a High-Resolution Mass Spectrometry (HRMS) approach. Analyses were performed using a microTOF QII mass spectrometer (Bruker Daltonics, Bremen, Germany) equipped with ESI operating in both positive and negative ion modes. Bioactive compounds were tentatively identified through their MS spectra in comparison to literature data.
For HPTLC analysis, a mobile phase consisting of ethyl acetate:formic acid:acetic acid:water (100:11:11:26) was used. The application of standards and samples was performed using a semimicro applicator (Cellogel Electrophoresis Company, Milan, Italy). Approximately 1.5 µL of extract sample and 1.5 µL of 1 mg/mL solution of chlorogenic acid (CA), neochlorogenic acid (NCA), and quercetin 3-O-β-D-glucopyranoside (QGP) standards were separately applied in the form of bands (1.5 µL × 8 mm) at 1 cm from the bottom using TLC Silica gel 60 F254 pre-coated plate (Merck). The plate was developed up to the distance of 8 cm from the bottom, air dried, heated at 100 • C for 5 min, sprayed with 1% (w/v) diphenylboryloxyethylamine in methanol (NP), then sprayed with 5% (w/v) polyethylene glycol 4000 (PEG4000) in ethanol, air dried and visualized by viewing in UV-cabinet under long wavelength (366 nm).

Yield and Agronomic Parameters
The lettuce plants were harvested at commercial maturity. The external leaves were removed, and each head was weighted to determine marketable yield (g) per treatment including the control. Five homogenous plants, located at the center of the row, were selected per treatment for measuring leaf number and area. The leaf area of each plant was estimated using the open access software ImageJ 1.53 version (U.S. National Institutes of Health, Bethesda, MD, USA) and quantified in cm 2 . Samples from the abovementioned plants were put in a forced air-drying oven at 60 • C until a constant weight was reached for the successive determination of WC and DM. The WC was calculated according to the formula (1) whereas DM was calculated following Cristofano et al. [54] according to the formula (2) DM = (leaf dry weight/leaf fresh weight) ×100 For qualitative analysis, per treatment, fresh leaf sub-samples were frozen with liquid N, powdered, and stored at −80 • C.

Leaf Colour, SPAD Index, and Pigment Content
Colour CIELAB parameters (L*, C*, and a*, b*) were measured by a Chromameter (CR-300, Minolta Co. Ltd., Tokyo, Japan) between the midrib and the leaf margin in 10 undamaged lettuce leaves per replicate. The L* coordinate expressed the degree of lightness of the measured color (100 = white; 0 = black). The a* and b* values were used to calculate the hue angle (h • ) according to the formula (3) The h • expressed the color tones in degrees: 0 • (red color), 90 • (yellow), 180 • (green), and 270 • (blue). The C* described saturation of the color: the higher the C* value, the more saturated the color.
Moreover, the SPAD index, indicating N leaf content, was measured in 10 fully expanded leaves per replicate by SPAD-502Plus (Minolta) following manufacturer specifications.
Finally, pigment content of the leaves was spectrophotometrically determined [49].
The total chlorophyll was calculated as the sum of Chl a and Chl b (6).

Total Phenolic Compounds and Antioxidant Activities
One gram of fresh powdered lettuce leaves was homogenized with 5 mL ethanol 70% and then subjected to ultrasound for 15 min, centrifuged at 9000× g and filtered with 0.45 µm pore size nylon syringe filters. The determination of the total phenol content (TPC) was performed using the Folin-Ciocalteu method as described by Tarantino et al. [55] The antioxidant activity was determined using a free DPPH radical (1,1-diphenyl-2-picrylhydrazyl) and ABTS radical (2,20-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid) [56]. To measure the reduction in absorbance of the solutions a spectrophotometric assay was carried out at 517 and 734 nm wavelengths, respectively. The antioxidative activity of lettuce was expressed in mM Trolox Equivalents (TE) per g of fresh lettuce.

Resistance to Postharvest Grey Mold
A strain (FV52) of B. cinerea from the Culture Collection of the University of Bari Aldo Moro (Italy) was grown on PDA at 24 • C with a 14 h/10 h photoperiod for 7 days. Ten leaves per treatment (2 leaves per plant, 5 plants per treatment) were used. They were placed in plastic boxes containing a moistened filter paper with sterile distilled water at the bottom, this latter was topped by a plastic net to separate the leaves from the paper. The leaves were inoculated by depositing a mycelial plug, 5 mm in diameter, from the active growing margins of the B. cinerea colony [57]. The boxes were sealed in plastic bags and incubated at 18 • C for 4 days. Finally, leaves were assessed for the infected area as percentage (%) of leaf surface.

Statistical Analysis
All data were subjected to a one-way analysis of variance (One-way ANOVA) using Minitab 19 (Minitab Inc., State College, PA, USA). Means were separated utilizing Fisher, as a post-hoc test, performed at p ≤ 0.05 significance level.

Conclusions
The present study provides some evidence of the positive effect of biostimulant application on the yield, quality, and storability of greenhouse-grown lettuce. Effective commercial products exist, but a new promising biostimulant could be introduced. M. oleifera seems a good candidate as it is already used for human consumption, was recently proposed as a possible candidate for replacing animal proteins and is to be included among basic substances (i.e., active substances, not predominantly used as plant protection products but which may be of value for plant protection and for which the economic interest in applying for approval may be limited, EC Regulation 1107/2009). If included in this list, MLEs could be legally used in the EU. Although further studies on a larger scale and the mode of action are needed, the proposed data seem promising.