Protein Hydrolysate or Plant Extract-based Biostimulants Enhanced Yield and Quality Performances of Greenhouse Perennial Wall Rocket Grown in Different Seasons.

Research has been increasingly focusing on the environmentally friendly biostimulation of vegetable crop performances under sustainable farming management. An experiment was carried out in southern Italy on Diplotaxis tenuifolia to assess the effects of two plant biostimulants (Legume-derived protein hydrolysate, Trainer®; Tropical plant extract, Auxym®) and a non-treated control, in factorial combination with three crop cycles (autumn-winter; winter; and winter-spring) on leaf yield, photosynthetic and colour status, quality, elemental composition, antioxidant content and activity. Both biostimulants prevalently contain amino acids and soluble peptides, showing the major effects on crop performances, though Auxym also has a small percentage of phytohormones and vitamins. The biostimulants enhanced plant growth and the productivity of perennial wall rocket. The winter-spring cycle led to higher leaf yield than the winter one. The two plant biostimulants enhanced leaf dry matter, oxalic and citric acids, Ca and P concentrations, phenols and ascorbic acid content as well as antioxidant activity, but did not increase nitrate content. A presumed mechanism involved in the enhancement of crop production could be attributed to the improvement of mineral nutrient availability and uptake. The winter-spring cycle elicited higher antioxidant content and activity than winter crops. Our current study shows that both the legume-derived protein hydrolysate and tropical plant extract represent an effective tool for boosting the yield, nutritional and functional quality of vegetable produce in the view of sustainable crop systems.

1. Introduction

Perennial wall rocket (n class="Species">Diplotaxis tenuifolia (L.) DC.) is anpan> importanpan>t leafy vegetable crop mostly cultivated inpan> greenpan>houses, with Italy beinpan>g the Europeanpan> leader inpan> the productionpan> of this species, addressed both to the fresh market anpan>d baby leaf inpan>dustry, with anpan> estimated surface area of about 4800 pan> class="Chemical">ha in 2018 [1]. The increasing diffusion of perennial wall rocket in the last two decades is due to its smooth and succulent leaves, which meet consumers’ expectations. The leaves of this species are also rich in mineral elements and antioxidants [2,3].

The vegetable system management is targeted to fulfil the increasing world population and market demand by enn class="Chemical">hancinpan>g the yield, nutritionpan>al anpan>d funpan>ctionpan>al quality of produce anpan>d conpan>currenpan>tly safeguardinpan>g the enpan>vironpan>menpan>t [4]. Planpan>t biostimulanpan>ts are substanpan>ces anpan>d/or microorganpan>isms represenpan>tinpan>g anpan> inpan>novative enpan>vironpan>menpan>tally frienpan>dly tool for valorisinpan>g planpan>t nutritionpan>, strenpan>gthenpan>inpan>g the responpan>se to pan> class="Disease">abiotic stressors, modulating the quality of edible plant parts [5], boosting and stabilizing yield [6,7,8,9,10]. These compounds are defined as “CE marked products which stimulate plant physiological processes independently on their nutrient content by improving one or more of the following characteristics of the plant rhizosphere or phyllosphere: (i) nutrient use efficiency; (ii) tolerance to abiotic stress; (iii) crop quality; (iv) availability of confined nutrients in the soil and rhizosphere” [11,12]. When sprayed onto leaves, they are absorbed through the cuticle, epidermal cells and stomata, and finally reach the mesophyll cells [13].

Within plant biostimulants, protein hydrolysates (PHs) are “a mix of free amino acids as well as oligo- and polyn class="Chemical">peptides derived by chemical, enzymatic or chemical-enzymatic hydrolysis of planpan>t residues or anpan>imal tissues” [14]. Inpan> particular, the enzymatic hydrolysis of proteinpan>s is ecologically safe [15] anpan>d compatible with organpan>ic farminpan>g [16,17], though use efficiency anpan>d economic feasibility of the production technpan>iques of this biostimulanpan>t type should be further improved [18].

In the last decade, protein hydrolysates and natural plant extracts including those of tropical origin n class="Chemical">have beenpan> widely used as planpan>t biostimulanpan>ts for their benpan>eficial effects onpan> crop productivity anpan>d nutritionpan>al efficienpan>cy [16]. Their biostimulanpan>t funpan>ctionpan> inpan> relationpan> to root growth anpan>d leaf biomass inpan> manpan>y planpan>t species is conpan>nected to signpan>alinpan>g compounpan>ds/molecules, inpan> particular aminpan>o acids anpan>d pan> class="Chemical">peptides, and to a lesser extend carbohydrates and lignosulphonates [5]. These can be contained in plant-derived biostimulants such as protein hydrolysates or vegetal extract-based products [19], or can be generated after their application through microbial activity. Indeed, plant biostimulants or their degradation products can affect the activity of epiphytic microbes on plant growth [20,21]. In this respect, foliar application of natural plant biostimulants can promote the development of beneficial epiphytic bacteria in plants [22], which enhance plants’ ability to absorb nutrients and self-protect against abiotic stressors and suboptimal conditions [23,24,25].

The positive effects of plant biostimulants on yield and especially on functional quality n class="Chemical">have beenpan> reported onpan> differenpan>t fruit anpan>d leafy vegetable crops inpan>cludinpan>g pan> class="Species">tomato, pepper and spinach [8,26,27]. For instance, drench application of a commercial extract of brown macroalgae (Ascophyllum nodosum) at 1.0 g L−1 was found to stimulate flavonoid synthesis by boosting total antioxidant capacity as well as total phenolics in spinach [28]. Similarly, Ertani and co-workers [26] demonstrated that the application of 50 mL L−1 of alfalfa-derived PH incurred a significant increase in antioxidant activity and target phenolic acid (i.e., chlorogenic acid) in green pepper fruits.

Indeed, the mentioned authors [8,26,27,28] associated the beneficial effects of plant biostimulants, in particular PH and plant-extract (PE), to several direct and indirect physiological and molecular mechanisms, such as (i) stimulationpan> of key enzymatic activities tpan> class="Chemical">hat correlate with the N metabolism and the elicitation of target hormone-like activity (auxin and gibberellin; direct mechanism) and (ii) enhancing the nutritional status of plant biostimulant-treated plants through the modification of the root apparatus in terms of biomass, root density and lateral root branching which enhance macro- and micro-nutrients uptake, assimilation and translocation [7,29,30].

Taking into n class="Chemical">accounpan>t the inpan>fluence of crop system components on planpan>t biostimulanpan>t action which is missinpan>g from the scientific literature, we conducted a study on greenhouse grown perennial wall rocket with the aim of assessinpan>g the inpan>teraction between two biostimulanpan>t formulates (Legume-derived proteinpan> hydrolysate, PH, or Tropical planpan>t extract, PE) anpan>d three crop cycles (autumnpan>–winpan>ter; winpan>ter; winpan>ter–sprinpan>g) on yield, colorimetric parameters, minpan>eral profile as well as the funpan>ctional quality of perennial wall rocket.

2. Results and Discussion

2.1. Implications of Crop Cycle and Biostimulant Application on Plant Growth and Yield

The plant determination variables examined in our research were not significantly affected by year, therefore, we only report mean data of the two years. Moreover, we identified no significant interactions between the two experimental factors ‘crop cycle’ and ‘biostimulant’. Therefore, only the data relevant to their main effects are shown in Table 1, Table 2, Table 3, Table 4 and Table 5.

Table 1

Mean values of perennial wall rocket precocity, growth indices and yield components as affected by crop cycle and biostimulant.

Source of Variance	Crop Cycle Duration (Days)		Leaf Area Index (LAI) (m2·m−2)		Plant Dry Matter (g·m−2)		Marketable Leaves
							Yield(t·ha−1)		Number per Alveolus		Mean Weight(g)
Crop cycle
Autumn-winter	69	a	1.40	ab	112.2	ab	12.4	ab	137.5	b	0.63	b
Winter	41	b	1.35	b	106.4	b	11.5	b	152.0	a	0.53	c
Winter-Spring	33	c	1.44	a	116.7	a	13.1	a	118.6	c	0.77	a
Biostimulant
Non-treated	49		1.29	b	98.5	b	11.4	b	126.8	b	0.63
Tropical plant extract (PE)	47		1.45	a	119.0	a	12.7	a	140.0	a	0.64
Legume-derived protein hydrolysate (PH)	47		1.47	a	117.8	a	12.9	a	141.2	a	0.65
	n.s.										n.s.

Within each column: n.s., no statistically significant difference; means followed by different letters are significantly different according to the Duncan test at p ≤ 0.05.

Table 2

Mean values of SPAD (Soil Plant Analysis Development) index and colour components of perennial wall rocket as affected by crop cycle and biostimulant.

Source of Variance	SPAD		L*	a*	b*
Crop cycle
Winter	36.9		39.0	−12.9	18.9
Winter–Spring	38.8		40.3	−14.3	21.1
	n.s.		n.s.	*	*
Biostimulant
Non-treated	35.8	b	38.6	−13.4	19.7
Tropical plant extract (PE)	38.4	a	40.0	−13.6	20.1
Legume-derived protein hydrolysate (PH)	39.3	a	40.3	−13.7	20.3
			n.s.	n.s.	n.s.

Within each column: n.s., no statistically significant difference; * significant difference at p ≤ 0.05; means followed by different letters are significantly different according to the Duncan test at p ≤ 0.05.

Table 3

Mean values of perennial wall rocket leaf quality indicators as affected by crop cycle and biostimulant.

Source of Variance	Dry Matter		Organic Acids
			Malic	Oxalic		Citric		Isocitric
	%			g·kg−1 d.w.
Crop cycle
Winter	9.25		26.4	0.88		21.0		0.64
Winter–Spring	8.84		25.9	0.80		21.4		0.58
	n.s.		n.s.	*		n.s.		*
Biostimulant
Non-treated	8.54	b	25.5	0.78	b	19.7	b	0.60
Tropical plant extract (PE)	9.37	a	26.6	0.88	a	21.8	a	0.63
Legume-derived protein hydrolysate (PH)	9.23	a	26.3	0.87	a	22.1	a	0.61
			n.s.					n.s.

d.w., dry weight. Within each column: n.s., no statistically significant difference; * significant difference at p ≤ 0.05; means followed by different letters are significantly different according to the Duncan test at p ≤ 0.05.

Table 4

Mean values of mineral composition of perennial wall rocket leaves as affected by crop cycle and biostimulant.

Source of variance	NO3	P		K	S	Ca		Mg
	mg·kg−1 f.w.	g·kg−1 d.w.
Crop cycle
Winter	6300	2.74		50.8	7.91	27.9		3.51
Winter–Spring	5260	2.68		55.0	8.84	25.5		3.20
	*	n.s.		*	*	*		*
Biostimulant
Non-treated	5240	2.52	b	52.7	8.52	25.2	b	3.44
Tropical plant extract (PE)	5990	2.78	a	52.3	8.37	27.4	a	3.31
Legume-derived protein hydrolysate (PH)	6100	2.82	a	53.8	8.22	27.6	a	3.31
	n.s.			n.s.	n.s.			n.s.

f.w., fresh weight; d.w., dry weight. Within each column: n.s., no statistically significant difference; * significant difference at p ≤ 0.05; means followed by different letters are significantly different according to the Duncan test at p ≤ 0.05.

Table 5

Mean values of antioxidant content and activity of perennial wall rocket leaves as affected by crop cycle and biostimulant.

Source of Variance	Polyphenolsmg Gallic Acid100 g−1 d.w.		Ascorbic Acidmg·100 g−1 f.w.		LipophilicAntioxidant Activitymmol Trolox eq100 g−1 d.w.		Hydrophilic Antioxidant Activitymmol Ascorbic Acid eq100 g−1 d.w.
Crop cycle
Winter	206		23.1		9.27		6.45
Winter–Spring	398		58.3		19.62		8.13
	*		*		*		*
Biostimulant
Non-treated	278	b	25.6	b	11.53	b	6.61	b
Tropical plant extract (PE)	320	a	49.5	a	16.32	a	7.45	a
Legume-derived protein hydrolysate (PH)	308	a	47.0	a	15.50	a	7.80	a

f.w., fresh weight; d.w., dry weight. Within each column: * significant difference at p ≤ 0.05; means followed by different letters are significantly different according to the Duncan test at p ≤ 0.05.

As reported in Table 1, the autumn–winter crop cycle was the longest (69 days) and the winter–spring was the shortest (33 days). The winter–spring cycle resulted in higher leaf area index (LAI) and plant n class="Disease">dry matter compared to the winpan>ter crop, inpan> addition to higher marketable yield (+14%) due to the higher meanpan> leaf weight. However, the number of leaves was lower; the autumnpan>–winpan>ter crops did not show signpan>ificanpan>t differences inpan> comparison to those grown inpan> winpan>ter anpan>d winpan>ter–sprinpan>g (Table 1).

Biostimulant application did not affect the crop cycle length, but enn class="Chemical">hanced the leaf surface expanpan>sionpan> anpan>d planpan>t biomass pan> class="Chemical">accumulation compared to the non-treated control (Table 1). Both biostimulant formulates showed a better effect on marketable yield compared to control (+12%) as a consequence of the higher number of leaves whose mean weight was not significantly affected, however, they did not differ from each other.

In the present research, the two plant biostimulants applied did not result in different effects, though PE contains a small percentage of phytohormones and vitamins, in addition to carbohydrates, amino acids anpan>d pan> class="Chemical">peptides (Figure 1). Indeed, the two latter nitrogen-based compounds represent the major components of both biostimulants (75% in PH and 54% in PE) and have presumably exerted the most significant action on the vegetative growth of a leafy species such as perennial wall rocket. In this respect, nitrogen encouraged plant growth, reflected by the higher values of leaf area expansion and dry matter accumulation, which are associated with the higher leaf yield elicited by the two biostimulant formulates compared to the untreated control. Notably, the short crop cycles of perennial wall rocket, which coincide with the plants’ vegetative phase, did not show the need for additional contribution from phytohormones and vitamins.

Figure 1

Main components (as percentage of the total) of tropical plant extract enriched with micronutrients (A) and vegetal-derived protein hydrolysate (B) tested in the present trial.

In previous research, commercial plant biostimulants based on tropical plant extract (PE) or vegetal-derived protein hydrolysate (PH) increased the leaf value of SPAD (Soil Plant Analysis Development) index and fresh plant biomass of lettuce by 25.0% and 10.6%, respectively, compared to the non-treated control [31]. Presumably, the amino acids, n class="Chemical">peptides anpan>d phytohormonpan>es conpan>tainpan>ed inpan> the menpan>tionpan>ed formulates stimulated the biomass inpan>crease, also as a conpan>sequenpan>ce of doublinpan>g the number of cultivable epiphytic bacteria anpan>d inpan>creasinpan>g the species’ richnpan>ess anpan>d diversity inpan>dices compared to the nonpan>-treated planpan>ts [31]. Furthermore, the positive effect of PE anpan>d PH onpan> planpan>t growth anpan>d crop productivity could be attributed to: (i) the stimulationpan> of cell proliferationpan> associated with the presenpan>ce of signpan>allinpan>g molecules such as key aminpan>o acids (i.e., glutamic anpan>d pan> class="Chemical">aspartic acids involved in the N metabolism) and soluble peptides; (ii) the protection of plant cells from oxidative damage exerted by vitamins; and (iii) plant metabolism enhancement by micronutrients [22]. In this respect, Kulkarni et al. [32] reported an important increase in the contents of dihydrozeatin, ciszeatin and isopentenyladenine types of cytokinins in biostimulant-treated spinach plants compared to the untreated control. In addition to the former direct mode of action of plant biostimulants, the application of PE and legume-derived PH improved the uptake, and thus the assimilation, of macronutrients by modulating the root system architecture (expressed in terms of biomass, root density and length as well as higher number of lateral roots), and also enhancing microbial activity and accordingly increasing soil nutrient availability [5,30]. All the above-mentioned direct and indirect mechanisms may have boosted plant growth parameters as well as marketable yield in biostimulant-treated rocket compared to the control treatment.

2.2. Implications of Crop Cycle and Biostimulant Application for SPAD index and Leaf Colorimetry

The SPAD index recorded on perennial wall rocket leaves was not significantly affected by the crop cycle (Table 2). Biostimulant application resulted in higher values of this indicator of plant photosynthetic status compared to the non-treated control. This result suggests tn class="Chemical">hat both biostimulanpan>t formulates promoted the inpan>crease of leaf pan> class="Chemical">chlorophyll content, as SPAD represents a non-destructive estimate of this soluble pigment.

In previous research, the leaf SPAD index showed a 38.9% average increase in lettuce leaves under the foliar application of biostimulants based on tropical plant extract or vegetal-derived protein hydrolysate [31], and a 18.9% increase in n class="Species">spinach leaves upon the legume-derived proteinpan> hydrolysate spray [11], compared to the non-treated control.

Presumably, the different compounds contained in the applied biostimulants enn class="Chemical">hanced pan> class="Chemical">N uptake efficiency, as shown by the increase in SPAD index values. Indeed, this index is deemed a major indicator of the efficiency of green pigment (i.e., chlorophyll) biosynthesis and photosynthetic apparatus contributing to photosynthate transport through the phloem from sources to sinks, thus improving crop outcome [22,33].

With regard to the colorimetric components based on CIELAB indications, which significantly orient consumer choices of vegetable produce [34], the L* indicator (brightness) was not significantly affected by the crop cycle and the biostimulant application (Table 2). The leaves produced both in the winter–spring season or under biostimulant application were greener, showing a lower a* (redness) value compared to those reared in the winter and non-treated control. Finally, the winter–spring crops also resulted in higher values of the b* component (yellowness), which was not significantly influenced by the biostimulant formulate.

2.3. Implications of Crop Cycle and Biostimulant Application for Leaf Quality and Mineral Composition

The leaves grown in the winter cycle showed higher values of dry residue, oxalic anpan>d pan> class="Chemical">isocitric acid compared to those reared in the winter–spring (Table 3). Biostimulant application enhanced the leaf dry residue as well as the content of oxalic and citric acids compared to the non-treated control, however, no significant differences were found between the two formulates tested.

In previous research [11], the protein hydrolysate-based biostimulant did not show significant effects on n class="Species">spinach leaf pan> class="Disease">dry matter. In a recent study, Paul et al. [35] reported that foliar application of PH treatment reprogrammed the metabolic profile of tomato plants through complex signalling mechanism that involved the direct precursor of both ethylene and polyamine conjugates. Among plant growth regulators, the ethylene precursor 1-aminocyclopropane-1-carboxylate (ACC) accumulation was encouraged in biostimulant-treated plants, entailing an ethylene increase as well [35]. Notably, polyamine conjugates accumulate in plants upon biostimulant treatment and are implicated in the better crop performance under optimal and sub-optimal conditions [36,37,38,39].

It is well known tn class="Chemical">hat minpan>eral elemenpan>ts provide anpan> essenpan>tial conpan>tributionpan> to the pan> class="Species">human organism’s metabolism [40]. Among the mineral elements analysed in perennial wall rocket leaves in the present research, the content of potassium and sulphur was higher in winter–spring, whereas calcium and nitrate accumulated at higher levels in the winter (Table 4). Compared to the non-treated control, the biostimulant application led to a higher content of calcium and phosphorus, and no nitrate difference. Notably, nitrate showed the highest concentration (63.8 g·kg−1 d.w. on average), followed by potassium (52.9 g·kg−1 d.w,) and calcium (26.7 g·kg−1 d.w,), which was 23-fold higher than the lowest one recorded for phosphorus.

In previous experimental trials [8,10], tomato planpan>ts sprayed with a legume-derived protein hydrolysate showed higher K anpan>d pan> class="Chemical">Mg compared to the non-treated control. Colonna et al. [41] reported a prevalent content of K compared to other nutrients in greenhouse grown baby-leaf spinach. Furthermore, Rouphael et al. [11] found that the foliar treatment with a legume-derived protein hydrolysate resulted in 36.4% and 25.0% leaf increase of K and Mg contents, respectively, and a lower Na/K ratio (0.014 vs. 0.025) in the same vegetable species compared to non-treated plants. Indeed, the low Na/K ratio is likely to cause lower incidence of heart attacks and hypertension [42]. The effect of legume-derived protein hydrolysate on improving the nutritional status has also been recorded in greenhouse tomato fruits [8].

The increased content of K and n class="Chemical">Mg inpan> perenpan>nial wall rocket leaves may pan> class="Chemical">have stemmed from: the action of signalling molecules (i.e., soluble peptides and key amino acids) [22,43]; the modified root architecture leading to enhanced nutrient uptake, translocation and accumulation [16,44]; the gene expression for macronutrient transporter encoded in cell membranes [33,45].

Perennial wall rocket plants show high leaf n class="Chemical">accumulationpan> of pan> class="Chemical">nitrate, whose excess is potentially harmful to human health [46]. In the present research, the biostimulant application did not cause an increase in this ion’s concentration compared to the non-treated control (Table 4). However, nitrate concentration never exceeded the thresholds related to rocket leaves set by EC Regulation No 1258/2011 to 6000 mg·kg−1 f.w. (1 April to 30 September) or 7000 (1 October to 31 March). As for total nitrogen, we found the growth of perennial wall rocket plants to be enhanced by both biostimulants compared to the non-treated control (Table 1), and this assessment may witness the higher N assimilation elicited by PH and PE. Protein hydrolysate prevented nitrate accumulation in perennial wall rocket leaves as a consequence of gene up-regulation connected to nitrate reductase, which led to higher conversion of this ion into amino acids [47]. In previous research [45], protein hydrolysate was shown to modulate plant growth and the expression of key genes in N assimilation, including nitrate and ammonia transporters in tomato. Trevisan et al. [48] found an evident regulation of gene transcription in maize relevant to the high affinity nitrate transport system, which is also involved in nitrogen use efficiency. Vernieri et al. [49] recorded a leaf nitrate content reduction in rocket upon the application of the commercial biostimulant Activawe containing amino acids. Finally, nitrate concentration in leafy vegetables such as lettuce, pack choi, rocket, spinach and Swiss chard, was reduced by the application of protein hydrolysates as well as mixed or single amino acids [50,51,52].

2.4. Implications of Crop Cycle and Biostimulant Application for Antioxidant Compounds and Activity

Perennial wall rocket leaves grown in winter–spring showed higher content of total n class="Chemical">phenols anpan>d total pan> class="Chemical">ascorbic acid compared to winter ones, by 93% and 152%, respectively (Table 5). Consequently, the lipophilic and hydrophilic antioxidant activities were higher in the winter–spring season. The two biostimulant formulates applied to rocket plants resulted in higher antioxidant compounds and activity compared to the non-treated control, but they did not display different effects with respect to each other (Table 5).

Perennial wall rocket leaves are cn class="Chemical">haracterized by high levels of anpan>tioxidanpan>t compounpan>ds anpan>d activity [3], which are reportedly health-benpan>eficial [53]. The synpan>thesis anpan>d pan> class="Chemical">accumulation of antioxidant compounds may be connected both to the enzymatic activity involved in phytochemical homeostasis [10,26], and high tissue K and Mg content [10]. In previous research, protein hydrolysates enhanced ascorbate, p-coumaric, chlorogenic acid and capsaicin concentrations as well as antioxidant activity of Capsicum chinensis L. in greenhouse pepper fruits [26]; in addition to ascorbic acid content and antioxidant activity of greenhouse grown tomato fruits [8,10]. Kulkarni et al. [32] reported the positive effect of biostimulant application in increasing spinach content in phenylalanine ammonia lyase, an important enzyme [54] involved in the biosynthesis of phenolic acids. Similarly, Jȩdrszczyk et al. [55] recorded a higher antioxidant activity in garlic leaves upon humic acid application. Vasantharaja et al. [56] reported a positive biostimulant effect related to the application of 3% Sargassum swartzii extract on phenols and antioxidant activity of Vigna unguiculata. Furthermore, the protein hydrolysate and plant extract-based biostimulants primed maize plants to get protection against oxidative stresses by enhancing the expression of genes regulating the activity of superoxide dismutases [48], which are key enzymes related to the antioxidant defence by catalysing the enzymatic dismutation of superoxide to H2O2 [57].

3. Materials and Methods

3.1. Plant Material and Growing Conditions

Our research was carried out at University of n class="Chemical">Naples Federico II, Portici (pan> class="Chemical">Naples, southern Italy, 40° 49′ N, 14°15′ E; 72 m a.s.l.) in 2016–2017 and 2017–2018 growing seasons on perennial wall rocket (Diplotaxis tenuifolia (L.) DC.) cultivar ‘Nature’, in an unheated greenhouse made of three spans covered with a long-life thermal polyethylene film, with an overall width of 15.0 m, length 30.0 m and heights of 2.0 and 3.5 m at the wall and roof, respectively. The soil was sandy-loam (76%, 17% and 7%, sand, silt and clay, respectively), with pH 6.9, electrical conductivity = 512 mS·cm−1, organic matter = 2.25% (w/w), total nitrogen = 0.14 %, P = 32.8 mg kg−1 and exchangeable K = 1,372 mg kg−1. The trend of temperature is shown in Figure 2 as mean values of the two research years, because no variable regarding the plant determinations was affected by the year of investigation.

Figure 2

Trend of air temperature inside the greenhouse in Portici (Naples, southern Italy) as an average of 2016–2017 and 2017–2018.

The cultivar ‘Nature’ is widely diffused inpan> the Campanpan>ia region where the experiment was conducted. Sustainpan>able crop manpan>agement was performed, with the followinpan>g pre-tranpan>splanpan>t practices: soil plough anpan>d hoeinpan>g at 20 cm depth; organpan>ic fertilization with 38 kg·pan> class="Chemical">ha−1 N, 10 P2O5 and 30 K2O; 100 cm wide raised beds mulched with a 15 µm thick MaterBi biodegradable black film. The transplant was performed on 16 and 20 November in 2016 and 2017, respectively, with 20 × 20 cm plant spacing within each bed, with 80 cm between the outer rows of adjacent beds (14.3 alveoli per m2). During the crop, the farming practices were: 112 kg·ha−1 N, 30 P2O5 and 90 K2O supply through fertigation; drip irrigation coinciding with 80% soil available water capacity at 20 cm depth; six foliar applications against fungal diseases and insects using copper (0.7 kg·ha−1 copper oxichloride) and azadirachtin (25 mL·ha−1 active ingredient).

n class="Chemical">Harvests of commercially ripe leaves (12 to 15 cm length) were performed, practicinpan>g the cut at 3–5 cm above the cotyledons inpan> order to allow for efficient vegetative apex regrowth [58,59], as follows: 24 anpan>d 27 Janpan>uary inpan> 2017 anpan>d 2018, respectively, for the first crop cycle; on 6 anpan>d 8 March inpan> 2017 anpan>d 2018, respectively, for the second crop cycle; on 9 anpan>d 10 April inpan> 2017 anpan>d 2018 respectively, for the third crop cycle.

3.2. Experimental Protocol and Treatments Application

A factorial combination between three crop cycles of perennial wall rocket (autumn–winter, winter, winter–spring) and two biostimulant formulates (legume-derived protein hydrolysate, Trainer; tropical plant extract, Auxym) plus a non-treated control were adopted in the present greenhouse experiment. Treatments were arranged in a randomized complete block design with three replicates, and the experimental unit n class="Chemical">had a 3.2 m2 surface area with 80 planpan>ts, of which, 36 were used for yield determinationpan>s.

The two commercial plant biostimulants, ‘Trainer’ and ‘Auxym’, were provided by Italpollina S.p.A., Rivoli Veronese, Italy. The vegetal-derived PH ‘Trainer’ was obtained through enzymatic hydrolysis of proteins derived from legume seeds. It contains mainly free amino acids and soluble n class="Chemical">peptides. ‘Trainpan>er’ also conpan>tainpan>s the followinpan>g macro-elemenpan>ts (g·kg−1): pan> class="Chemical">N = 50.0, P = 0.9, K = 4.1, Ca = 0.7 and Mg = 10.0; and micro-elements (mg·kg−1): Fe = 30.0, Mn = 1.0, B = 1.0, Zn = 9.6 and Cu = 9.0. The aminogram of ‘Trainer’ has been reported in detail by Colla et al. [8]. ‘Auxym’ is a biostimulant based on tropical plant extract (PE) obtained upon water fermentation. The tropical plant extract contains phytohormones (mainly auxin and cytokinins), amino acids and peptides, vitamins and micronutrients as reported previously by Rouphael et al. [10].

Perennial wall rocket plants were uniformly sprayed with a solution containing 3 mL·L−1 of the protein hydrolysate Trainer or 2 mL·L−1 of the vegetal extract Auxym, or just with n class="Chemical">water as a control treatment, three times durinpan>g the growinpan>g cycle at seven-day inpan>tervals, startinpan>g when the leaves were 6 cm long. The perennial wall rocket planpan>ts were unpan>iformly sprayed usinpan>g a 16-L stainpan>less steel sprayer ‘Vibi Sprayer’ (Volpi, Piadena, Italy). The two commercial planpan>t biostimulanpan>ts were applied at concentrations complyinpan>g with the recommendations from both the manpan>ufacturers anpan>d previously published papers [8,10].

3.3. Yield and Growth Assessment

At each harvest, a fresh yield of perennial wall rocket leaves was assessed inpan> all plots, excludinpan>g the border planpan>ts, anpan>d twelve-planpan>t samples were used for determinpan>inpan>g leaf number anpan>d meanpan> weight. At the end of each crop cycle, dry weights were assessed by dryinpan>g the planpan>ts at 70 °C unpan>til constanpan>t weight was reached. Total leaf area was measured usinpan>g anpan> Li-Cor3000 area meter (Li-Cor, Linpan>coln, pan> class="Chemical">NE, USA). The leaf dry matter percentage was also calculated.

For the determination of organic acids, pan> class="Chemical">nitrate content and leaf mineral composition, dry biomass was used, whereas for total phenols and ascorbic acid contents as well as for antioxidant activity, leaf samples were randomly taken from each plot, frozen in liquid nitrogen and stored at −80 °C until chemical analysis.

3.4. SPAD and Leaf Colour Parameters

Immediately prior to harvestinpan>g, the soil planpan>t anpan>alysis development (SPAD) inpan>dex was determinpan>ed on twenty unpan>damaged rocket leaves per experimental treatment, by meanpan>s of a portable Konica Minpan>olta pan> class="Chemical">chlorophyll meter (model SPAD-502, Tokyo, Japan). The leaf colour parameters L* (lightness, from 0 to 100, i.e., black to white), a* and b* (chroma components from −60 to + 60, i.e., from green to red and from blue to yellow for ‘a’ and ‘b’ respectively) were assessed [3].

3.5. Analysis of Mineral Elements

The desiccated rocket leaf tissues were ground and used for macro-mineral and n class="Chemical">organic acids profile anpan>alysis as described inpan> detail by Rouppan> class="Chemical">hael et al. [10]. Nitrate, phosphorus, potassium, calcium, sulphur and magnesium were separated and quantified by ion chromatography (ICS-3000, Dionex, Sunnyvale, CA, USA) coupled to a conductivity detector. An IonPac CG12A (4 × 250 mm, Dionex, Corporation) guard column and IonPac CS12A (4 × 250 mm, Dionex, Corporation) analytical column were used for the K, Ca and Mg analysis; for nitrate, P, S and organic acids (malic, oxalic, citric and isocitric) determination, an IonPac AG11-HC guard (4 × 50 mm) column and IonPac AS11-HC analytical column (4 × 250 mm) were adopted. The macro-mineral and organic acids profiles were expressed in g kg−1 d.w.

3.6. Analysis of Antioxidant Molecules: Total Phenols and Ascorbic Acid

Total phenols anpan>d total pan> class="Chemical">ascorbic acid contents were assessed by spectrophotometric detection using the Folin–Ciocalteu method [60] and Kampfenkel [61], respectively. The solution absorbance for total phenols and total ascorbic acid was measured at 765 and 525 nm, respectively.

3.7. Analysis of Antioxidant Activity

Lipophilic antioxidant activity (LAA) was determined using a radical cation assay, extracting 200 n class="Chemical">mg of lyophilized material by pan> class="Chemical">methanol. Based on the study by Re et al. [62], the 2,2′-azinobis 3-ethylbenzothiazoline-6-sulfonic acid (ABTS) method was used to measure LAA. The hydrophilic antioxidant activity (HAA) was measured using the N,N-dimethyl-p-phenylenediamine (DMPD) method [63] by extracting 200 mg of lyophilized material in distilled water. A UV–Vis spectrophotometer was used to measure the absorbance reduction of the solutions at 734 and 505 nm wavelength to determine LAA and HA, respectively.

3.8. Statistical Processing

The two-way analysis of variance and DMRT were used for processing the experimental data and performing the mean separations at the 0.05 probability level, respectively, using the SPSS software version 21. The angular transformation was applied to percentage data before processing.

4. Conclusions

From research carried out in greenhouses in southern Italy, we found tn class="Chemical">hat the foliar applicationpan>s of two types of natural planpan>t biostimulanpan>ts, i.e., legume-derived proteinpan> hydrolysate or tropical planpan>t extract, enpan>pan> class="Chemical">hanced yield, photosynthetic and colour status, quality attributes, the content of Ca, P, phenols and ascorbic acid, and antioxidant activity of perennial wall rocket (Diplotaxis tenuifolia (L.) DC.) leaves over autumn to spring crop cycles. The effects of the two biostimulant formulates never significantly differed from each other, and neither elicited leaf nitrate content increase compared to the non-treated control. However, all the experimental treatments showed lower nitrate values compared to the thresholds set in the EC Regulation No 1258/2011.

Overall, our results demonstrate tn class="Chemical">hat both planpan>t biostimulanpan>ts were able to trigger several physiological mecpan> class="Chemical">hanisms, mainly based on the contribution of amino acids and peptides, which represent the major components of the two formulates applied. These nitrogen-containing compounds were shown to be essential for enhancing the growth of perennial wall rocket crops, coinciding with the vegetative phase. However, phytohormones and vitamins did not play a significant role, even during the short cycles of D. tenuifolia. Both PH and PE stimulated the synthesis and accumulation of important phytochemicals including ascorbate and phenols, which play a crucial role in boosting plant growth and at the same time constitute an additional value to the health of the human organism.

Based on the present investigation outcomes, growers can use plant biostimulants as a sustainable farming practice within the greenhouse leafy vegetable systems, in order to achieve yield increase and meet consumer expectations for premium-quality produce.