Biostimulants as a Means to Alleviate the Transplanting Shock in Lettuce

Abstract

When transplanting vegetable plants in the field, the transplants undergo a small or large setback in growth, known as transplanting shock. Various practices are commonly applied to reduce the transplanting shock. In the past two decades, several studies have shown that the application of microbial and non-microbial biostimulants can enhance plant tolerance against abiotic stresses. However, there is no information on the effect of applying biostimulants at the time of transplanting in mitigating the transplanting shock of lettuce transplants in the field. Lettuce seedlings transplanted into the soil of an unheated plastic greenhouse were treated with two biostimulants, one microbial (Bactiva^®) and one non-microbial (Isabion^®), on the day of transplanting and after 14 and 21 days. During production, plant growth, and development, chlorophyll fluorescence and compositional parameters were determined. According to the results, the application of the non-microbial biostimulant had a significant effect on some measured parameters, with the leaves of the treated plants having a higher chlorophyll index (CCI) by 8%, as well as higher fluorescence parameters Fm/Fo, Fv/Fo, and Fv/Fm and area by 7, 10, 3, and 27%, respectively, but fewer total soluble phenols and lower fluorescence parameter ABS/RC by 7 and 26%, respectively, compared to the control untreated ones. The above may constitute some effects on the transplanting shock, without, however, being accompanied by significant effects on the number of leaves/plant, the leaf color parameters (L*, a*, b*, C*, and ho), and the chlorophyll (a, b, a + b), total carotenoid, dry matter, and nitrate content, along with the antioxidant capacity and plant fresh weight at harvest. However, a notable effect was that a greater percentage of plants at harvest had a fresh weight in the 351–400 class, while the greatest percentage of the control plants had a fresh weight in the 301–350 g class. In contrast, the application of the microbial biostimulant had no significant effect on any of the parameters determined compared to the control. Therefore, under the conditions of the present study, the effectiveness of biostimulant application at the time of transplanting on lettuce transplants is questionable.

1. Introduction

Transplants are commonly used for crop establishment in most vegetable species and maintaining their continuous growth in the field is a challenge, given that they undergo a minor or major setback in growth, known as transplanting shock [1].

The recently planted seedlings have a limited and pinched root system that had been developed in tray cells of small volume, together with an imperfect root–soil contact, which limits water uptake capacity upon transplanting on the field. On the other hand, when the environmental conditions favor increased evapotranspiration while the leaf transpiration demand exceeds the water uptake capacity of the seedling root system, a detrimental transplanting shock may occur [2,3,4], limiting the uninterrupted continuation of seedling growth that results in non-uniform establishment, and subsequently, yield loss and even the collapse of the transplants. Limited water uptake by the seedling root system results in a reduction in leaf water potential (Ψw) and partial or complete stomatal closure within hours after transplanting [2,3]. Under such stressful conditions, the photosynthesis is partially or completely inhibited even without full recovery during the following days even when the leaf water potential returns to the pre-planting levels upon well watering [3].

Pre- or post-transplanting treatments that retard seedling moisture loss alleviate one of the main causes of transplanting shock. Hardening of seedlings, optimal transplant age, application of anti-transpiration compounds, thorough soil preparation, selection of suitable planting period to coincide with favorable weather conditions, and immediate irrigation after transplanting with starter fertilizer solution are recommended practices and commonly applied in practice in order to reduce the transplanting shock [1,5,6].

In the past two decades, several studies have indicated that the application of microbial and non-microbial plant biostimulants induces a series of morpho-anatomical, biochemical, physiological, and molecular plant responses, and is able to modify plant primary and secondary metabolism, resulting, among others, in their enhanced tolerance against abiotic stresses [7].

Among biostimulants, plant growth-promoting rhizobacteria (PGPR) may benefit plants by increasing tolerance to abiotic stresses. They have been applied to different vegetable crops with various application methods even as seed treatments or for transplant production [8,9]. The addition of PGPR to the growing mixtures for seedling production has enhanced the overall growth of tomato and pepper [8,10], muskmelon and watermelon [11], and cauliflower [12] transplants. Moreover, it has been hypothesized that PGPR could be used to produce more vigorous transplants for at least a few weeks after transplanting in the field [13]. In this direction, an improved establishment of the crop, increased transplant survival, enhanced growth during the first weeks upon transplanting, and higher crop yield have been reported [10,11].

The efficacy of a commercial bacterial biostimulant (1.3 × 10⁸ CFU/g of Bacillus spp.) under variable fertigation rates has been studied on lettuce seedling growth and quality in addition to plant performance after transplanting in the field. It has been concluded that the inoculation of the growing substrate with Bacillus spp. promoted transplant growth during nursery production; moreover, its positive effect was further extended in the field by increasing crop yield and reducing tissue nitrate content [14].

However, several factors related to the characteristics of the microbial strains, the crop, and the soil should be taken into consideration when using microbial biostimulants, in order to maximize their effectiveness, given that not every microbe is suitable for all plant hosts and all ecosystems [15,16,17]. In addition, the effectiveness of the application of microbial biostimulants is also influenced by the competition for nutrients and space with the native microflora [9], also by the method of application that influences the dispersion of microbes and mortality under extreme environmental conditions, such as temperature and UV light [18].

On the other hand, protein hydrolysates have been used under foliar and root applications and found to have both positive and negative effects [19], incorporated as a component of broccoli seed coating material [20], in gelatin capsules that can be placed adjacent to the seed of cucumber [21], and applied through roots or leaves in greenhouse tomato [22,23], open-field-grown okra [24], spinach [25], endive [26], and carrot [27].

Three molecular fractions of a Graminaceae-derived protein hydrolysate were applied on lettuce grown under an unheated greenhouse in plastic pots filled with a 90:10 (v/v) mixture of quartz sand and perlite and irrigated with two levels (0 and 30 mM) of NaCl. While no significant effect on plant fresh weight was observed under 0 mM NaCl conditions, the 1–10 kDa fraction induced an increase in fresh weight only under mild salinity conditions [28].

The addition of humic substances (1% v/v) to a peat-based growth medium has been studied on pepper, tomato, watermelon, and lettuce transplant production systems, as well on their post-transplant yield performance under drought (water deficit vs. well-watered) and heat (hot vs. cool season) stress conditions. It has been concluded that the application of humic substances in containerized transplant production systems as a media amendment differentially modulated root and shoot growth based on crop species, but improved stress tolerance by mitigating the yield loss [29].

Pre-transplanting application of tablets containing Glomus intraradices and Trichoderma atroviride spores by placing one tablet per 1.5 L pot filled with fluvial sand and irrigated with modified Hoagland and Arnon nutrient solution significantly improved plant height, number of leaves per plant, and shoot and root dry weight, but not the SPAD index of lettuce transplants at early growth stages (35 days) due to the greater absorption of the main and trace elements (N, P, Mg, Fe, Zn, and B) via an increase in root surface. When these tablets were applied to the soil by placing one tablet per hole under the transplant roots, the leaf number, leaf area, shoot dry weight, SPAD index, and yield of PSII in dark-adapted leaves (Fv/Fm) were increased at 13 days after transplanting, while at 35 days, shoot fresh and dry weight, shoot dry weight root, and the content of N, P, Fe, Zn, Mn, and B were increased, without affecting the nitrate content [30].

Little work has been conducted on biostimulants as a means to mitigate the effects of transplanting shock in lettuce, and there is still no information on the effect of biostimulant application at the time of transplanting on lettuce transplants performance in the field.

2. Materials and Methods

2.1. Plant Material, Cultivation, and Treatments

Leaf lettuce seedlings (cv. Starfighter RZ) grown in 220-cell expanded polystyrene trays were transplanted at the 5-true-leaf stage in the soil of an unheated plastic greenhouse located in Eleousa, Thessaloniki province, North Greece (lat. 40°44′42.1″ N, long. 22°37′40.0″ E; altitude 14 m), on 12 November 2022 at a density of 6.06 plants/m². The soil was a sandy loam one (textural analysis: 64.3% sand, 29.0% silt, and 6.7% clay) with pH 8.2 and organic matter 1.6%. Prior to planting, 30-10-10 fertilizer was broadcast at the rate of 25 kg/ha and incorporated into the soil. Additional fertilizer (25 kg Ν/ha, 20 kg P₂O₅/ha, and 20 kg K₂O/ha) was applied during the cultivation period using the drip irrigation system. The total amount of water applied during cultivation was 67 m³/ha, and the application of pesticides was not required.

On the day of transplanting and after 14 and 21 days, the commercial biostimulants Bactiva^® and Isabion^® were applied. Bactiva^® (0.2 g/L) was applied as a solution in roots at a rate of 100 mL/plant, while Isabion^® was applied both in roots (0.4 mL/L) with 100 mL/plant and through foliar spray (2 mL/L) until runoff. Untreated plants, without any biostimulant supply, were used as control.

Bactiva^® (Bactiva GmbH, Heronger str. 2, D-47638 Straelen, Germany, agent-distributor Agripro OE, Ιonia, Thessaloniki, Greece) is a rooting agent which restores damaged roots and stimulates strong root growth through the plant hormones (gibberellins, cytokinins) produced upon the containing bacteria and fungi activity. In addition, bacteria fix nitrogen and solubilize insoluble phosphorus, thus providing minerals that the plants would not otherwise be able to take up. It contains beneficial bacteria (Bacillus subtilis, B. polymyxa, B. megaterium, Pseudomonas fluorescens 10⁸ CFU/g), fungi (Trichoderma harzianum, T. reesei, T. viride, Gliocladium virens), vitamins (B1-thiamin, B2-riboflavin, B3-niacin, B6-pyridoxine, B7-biotin, Β9-folic acid, B12-cyanocobalamin, C-ascorbic acid, and K-phylloquinone), plant protein amino acids, and soluble extracts of Yucca schidigera and Ascophyllum nodosum [31].

Isabion^® (manufacturer: Sicit Group SPA, via Arzignano 80, 36072 Chiampo (Vicenza), Italy; distributor: Syngenta Hellas Monoprosopi AEVE, L. Anthousas, Anthousa Attica) is a natural biostimulant. It contains a mixture of free amino acids and peptides (62.5%) of low molecular weight, which are derived from the hydrolysis of protein of animal origin (collagen). Free amino acids represent 11% w/w, the average molecular weight of the hydrolyzed protein is <2000 Da, and the standard density is 1.27 g/mL at 20 °C. It favors the development of the root system, the quick recovery from transplanting stress, the improvement of absorbability of nutrients, and the avoidance of frost damage [32].

2.2. Environmental Conditions

The air temperature and relative humidity in the greenhouse environment were recorded hourly with a sensor placed 15 cm above the soil surface and connected to an Elitech RC-5+ (Elitech Technology, Inc., London, UK). The solar radiation intensity data were recorded every 10 min from the nearest (20 km) meteorological station owned by the National Observatory of Athens and installed in Sindos, Thessaloniki province.

2.3. Determinations

During the plant growth on a weekly basis, the number of leaves per plant (at 14, 21, 28, and 35 days after transplanting) and the chlorophyll content index (CCI) (at 14, 21, 28, 35, 42, 49, 56, and 63 days after transplanting) were recorded. Moreover, at 21 and 35 days after transplanting, chlorophyll fluorescence was recorded.

At 35 and 63 days after transplanting, the plants were harvested and their quality, tip burn incidence, plant weight, and leaf color were determined. Then, they were frozen at −20 °C, and after being partially thawed and macerated in a blender, they were analyzed to determine their content in chlorophylls, total carotenoids, dry matter, nitrates, and total soluble phenols, along with their antioxidant capacity.

2.3.1. Chlorophyll Content Index (CCI)

Leaf chlorophyll content index was determined with a CCM-200 chlorophyll content meter (Opti-Sciences Inc., Hudson, NH, USA), and the measurements were taken on the fully expanded leaf.

2.3.2. Chlorophyll Fluorescence

Chlorophyll fluorescence was recorded with a Fluorpen FP100-MAX, PAM fluorometer (Photon Systems Instruments, Drásov, Czech Republic), and the measurements were taken on the fully expanded leaf. Based on Fo, Fm, and Fv parameters, Fv/Fm, Fv/Fo, and Fm/Fo were further calculated, as well as area and ABS/RC parameters.

2.3.3. Quality and Severity of Tip Burn

The quality and severity of tip burn were scored on a scale of 1 to 9, according to the system previously described [33,34,35]. For visual quality, 9 = excellent, virtually no defects; 7 = good, minor defects; 5 = average, satisfactory; slight to moderately acceptable defects; lower acceptable marketability limit; 3 = poor, excessive defects, with marketability; 1 = extremely poor. For the severity of tip burn, 1 = none; 3 = slight, acceptable for marketability; 5 = moderate, marginally acceptable for marketability; 7 = severe, not acceptable for marketability; 9 = extreme degree of severity.

2.3.4. Leaf Color

Color was determined with a CR-200 portable chroma meter (Konica Minolta, Osaka, Japan) equipped with an 8 mm measuring head and illumination medium C (6774 K), and the measurements were taken on the fully expanded leaf. The instrument was calibrated with the factory standard white color (Y = 93.9, X = 0.313, and y = 0.3209). From the readings of the instrument, the parameter L* (brightness, with values from 0 = black to 100 = white) was used, while from the parameters a* and b*, the chroma (C*) and hue (h°) were determined. C* = (a*² + b*²)^0.5 (color saturation, with low values showing a dull color and high values bright) and h° = tan⁻¹(b*/a*), when a* > 0 and b* > 0 or h° = 180° + tan⁻¹(b*/a*), when a* < 0 and b* > 0) (red-purple at 0°, yellow at 90°, blue-green at 180°, and blue at 270°) [36].

2.3.5. Dry Matter

Dry matter was determined after drying 50 g of macerated sample in an oven at 70 °C until a stable weight was attained.

2.3.6. Nitrates

For the extraction of nitrates, 5 g of macerated sample was mixed with 50 mL of distilled water and filtered through a Whatman No. 1 filter. The nitrate content was determined as described by Cataldo et al. [37], using a spectrophotometer Spectronic 30D (Thermo Spectronic, Cambridge, UK). Briefly, 0.2 mL of aqueous extract was thoroughly mixed with 0.8 mL of 5% (w/v) salicylic acid in concentrated sulfuric acid. After 20 min at room temperature, 19 mL of 2 M NaOH is slowly added to raise the pH to >12, and after cooling to room temperature, the absorbance at 410 nm was determined. A standard curve was developed with KNO₃. Nitrate concentration is presented on a fresh weight basis (as it is customary in commercial practice) and on a dry weight basis (to permit physiological comparisons).

2.3.7. Total Soluble Phenols

An amount of 5 g of macerated sample was homogenized with 25 mL of 95% methanol in a Polytron homogenizer (Kinematica GmbH, Malters, Switzerland), centrifuged at 12,857× g for 10 min at 20 °C, and the supernatant was filtered through a Whatman No. 1 filter. The determination of total soluble phenol content was carried out according to the method of Scalbert et al. [38], using a spectrophotometer Thermospectronic Helios Alpha (Thermo Fisher Scientific, Waltham, MA, USA). Briefly, a quantity of 0.5 mL of methanolic extract was mixed with 2.5 mL of Folin–Ciocalteu reagent and 2 mL of sodium carbonate (75 g/L). The samples were placed in a water bath at 50 °C for 5 min, and after cooling to room temperature, the absorption of the reaction mixture was measured at 760 nm. Data were expressed as mg gallic acid equivalent (GAE)/g fw. The standard reference curve was made by measuring the absorbance of solutions of known concentration of gallic acid (8–80 µg/mL) resulting from dilutions of a concentrated solution (80 mg/100 mL).

2.3.8. Chlorophylls and Total Carotenoids

For the extraction of chlorophylls and total carotenoids, 1 g of macerated sample was mixed with 10 mL of 100% acetone in capped plastic tubes and kept at −20 °C. After thawing, the samples were vortexed and centrifuged at 12,857× g for 10 min at 20 °C, and the supernatant was filtered through a Whatman No. 1 filter into 25 mL volumetric flasks. An additional 10 mL of 100% acetone was added to the pellet, and the samples were vortexed at 150× rpm for 10 min. The samples were refiltrated and the filtrates were added to the previous ones. The flasks were filled with 100% acetone and the absorbance of the filtrates was measured at wavelengths of 470, 645, and 662 nm, using a Thermospectronic Helios Alpha spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA) [39,40].

For the individual determination of chlorophylls and total carotenoids, the following equations were used:

Chlorophyll a (μg/g fw) = [(11.75 × Abs₆₆₂) − (2.35 × Abs₆₄₅)] × V/W

Chlorophyll b (μg/g fw) = [(18.61 × Abs₆₄₅) − (3.96 × Abs₆₆₂)] × V/W

Chlorophyll a + b (μg/g fw) = Chlorophyll a + Chlorophyll b

Total carotenoids (μg/g fw)= [(1000 × Abs₄₇₀ × V)/W] − [(2.27 × Chla) − (81.4 × Chlb)] × 227

where Abs = absorbance, W = tissue weight (g), and V = volume of extract (mL).

2.3.9. Total Antioxidant Capacity

Total antioxidant capacity was determined in the above supernatant following the Brand-Williams et al. method [41], using a Thermospectronic Helios Alpha spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA). Specifically, 200 μL of the above supernatant was added to 2800 μL of a 100 μM 2,2-diphenyl-1-picrylhydrazyl (DPPH) solution in 95% methanol in a test tube, vortexed, and kept in the dark for 1 h at room temperature, and the absorbance at 517 nm was measured. The ascorbic acid was used for the standard curve, and the total antioxidant capacity was expressed as mg ascorbic acid equivalents (AAE)/100 g fw.

2.3.10. Statistical Analysis

The analysis of variance (ANOVA) of the data was performed with SPSS v.22 software based on the experimental design of completely randomized groups, with 3 replications (Figure S1). When F-values were significant, means were compared with Duncan’s new multiple-rank test (p < 0.05).

3. Results

3.1. Environmental Conditions

The air temperature and relative humidity in the plant growth environment exhibited strong variation throughout the cultivation period (Figure 1). In particular, the average temperature was 12.3 °C, with a minimum of 0.0 and a maximum of 34.9 °C, and the average relative humidity was 87.0%, with a minimum of 21 and a maximum of 100%. In the first 35 days after transplanting, the average air temperature was 13.7 °C, with a minimum of 2.9 and a maximum of 34.9 °C, and the average relative humidity was 88.2%, with a minimum of 21 and a maximum of 100%. The total solar radiation was 514,054 W/m² (average daily value 8160 W/m²), while in the first 35 days after transplanting, the corresponding values were 246,435 W/m² (average daily value 7041 W/m²). These conditions are typical for an unheated greenhouse in the area during the winter season of the year.

3.2. Leaf Number/Plant

The sampling time but not the treatments significantly affected the number of leaves per plant in the first 35 days after transplanting (

Table 1. Effect of biostimulants for leaf number per lettuce plant during the first 35 days after transplanting. The plants were treated with the commercial biostimulants Bactiva^® and Isabion^®. Non-treated or control treatment was also included. At each sampling time, leaf number was determined in 20 plants per replicate.

Source of Variance		DF	p	%TV	η2
Sampling time (A)		4	***	97	39.2
Treatment (B)		2	ns	2	1.4
A × B		8	ns	0	0.4
Error		30
Sampling time (Days)
	0			5.01	d
	14			5.99	cd
	21			7.24	c
	28			13.51	b
	35			15.30	a
Treatment
	Control			8.86	a
	Bactiva			9.65	a
	Isabion			9.72	a
Sampling time × Treatment
0 days	Control Bactiva Isabion			5.03 5.03 4.97	c c c
14 days	Control Bactiva Isabion			5.97 6.10 5.90	c c c
21 days	Control Bactiva Isabion			6.90 7.23 7.60	c c c
28 days	Control Bactiva Isabion			12.03 14.13 14.37	b ab ab
35 days	Control Bactiva Isabion			14.37 15.77 15.77	ab a a

DF, degrees of freedom; p, probability; %TV, % of total variance; η², eta squared; *** significant effect at the 0.001 level; ns, non-significant effect. Different letters following values within each column indicate significantly different values at 0.05 level according to Duncan’s multiple range test.

). As an average of the three treatments, the number of leaves/plant increased significantly in the first 21 days (from 5.0 to 7.2 leaves/plant), and further increased 28 and 35 days after transplanting (13.5 and 15.3 leaves/plant, respectively) (Table 1), and this corresponds to increases of 45, 87, and 110%, at 21, 28, and 35 days after transplantation, respectively.

As an average of the three treatments, the daily rate of leaf production was the greatest (0.89 ± 0.082 leaves/day) 28 days after transplanting (Figure 2). The daily rate of leaf production 21 days after transplanting was greater in plants treated with Isabion^® (0.24 leaves/day) than in untreated ones (control) and those treated with Bactiva^® (0.13 and 0.16 leaves/day, respectively), while 28 days after transplanting, it was greater in plants treated with Bactiva^® and Isabion^® (0.99 and 0.96 leaves/day, respectively) compared with untreated plants (0.73 leaves/day).

3.3. Plant Fresh Weight

The sampling time but not the treatments significantly affected plant fresh weight during cultivation, while the interaction of sampling time × treatment was also not significant (

Table 2. Effect of biostimulants for lettuce plant fresh weight at 35 and 63 days after transplanting. The plants were treated with the commercial biostimulants Bactiva^® and Isabion^®. Non-treated or control treatment was also included. At each sampling time, plant fresh weight was determined in 10 plants per replicate.

Source of Variance		DF	p	%TV	η2
Sampling time (A)		1	***	100	95.5
Treatment (B)		2	ns	0	0
A × B		2	ns	0	0
Error		12
Sampling time (Days)
	35			55.6	b
	63			348.5	a
Treatment
	Control			199.8	a
	Bactiva			199.9	a
	Isabion			206.4	a
Sampling time × Treatment
35 days	Control Bactiva Isabion			53.6 56.6 56.7	b b b
63 days	Control Bactiva Isabion			346.0 343.2 356.2	a a a

DF, degrees of freedom; p, probability; %TV, % of total variance; η², eta squared; *** significant effect at the 0.001 level; ns, non-significant effect. Different letters following values within each column indicate significantly different values at 0.05 level according to Duncan’s multiple range test.

). As an average of the three treatments, the plants had the highest fresh weight at harvest at 63 days (348.5 g) compared to the plants at 35 days (56.6 g). At 63 days after transplanting, plants were 6.2 times heavier than at 35 days.

At harvest, 63 days after transplanting, the plant fresh weight did not differ significantly between treatments and ranged from 343 to 356 g (Table 2). However, significant variation between treatments was shown in the distribution of plant fresh weight in the different classes (Figure 3). In particular, in the untreated plants and those treated with Bactiva^®, the largest percentage (40.4 and 40.1%, respectively) had a fresh weight in the class of 301–350 g, while in the plants treated with Isabion^®, the largest percentage (43.0%) had a fresh weight in the 351–400 g class.

3.4. Quality and Tip Burn

At both sampling times (35 and 63 days after transplanting), the plant quality was excellent, without any defects, and there was also absolutely no development of tip burn.

3.5. Chlorophyll Content Index (CCI)

Sampling time and treatments significantly affected the chlorophyll content index, without a significant interaction among these factors. However, the proportion of total variance and η² due to the sampling time was much higher than that due to the treatments (

Table 3. Effect of biostimulants for chlorophyll content index (CCI) of lettuce plants during the cultivation period of 63 days. The plants were treated with the commercial biostimulants Bactiva^® and Isabion^®. Non-treated or control treatment was also included. At each sampling time, chlorophyll content index was determined in 10 plants per replicate.

Source of Variance		DF	p	%TV	η2
Sampling time (A)		7	***	92	40.1
Treatment (B)		2	**	7	5.8
A × B		14	ns	1	0.7
Error		48
Sampling time (Days)
	14			6.48	f
	21			8.41	d
	28			8.70	cd
	35			9.21	c
	42			7.43	e
	49			12.98	a
	56			11.25	ab
	63			10.87	ab
Treatment
	Control			9.07	b
	Bactiva			9.41	ab
	Isabion			9.77	a
Sampling time × Treatment
14 days	Control Bactiva Isabion			6.31 6.53 6.59	j j j
21 days	Control Bactiva Isabion			7.90 8.41 8.92	ghj fgh efg
28 days	Control Bactiva Isabion			7.99 8.83 9.27	fghi efg ef
35 days	Control Bactiva Isabion			8.72 9.07 9.85	efg efg de
42 days	Control Bactiva Isabion			6.85 7.42 8.01	ij hij fghi
49 days	Control Bactiva Isabion			12.67 13.30 12.99	ab a a
56 days	Control Bactiva Isabion			10.87 11.09 11.79	cd c bc
63 days	Control Bactiva Isabion			11.22 10.62 10.77	c cd cd

DF, degrees of freedom; p, probability; %TV, % of total variance; η², eta squared; ** and *** significant effect at the 0.001 and 0.001 level, respectively; ns, non-significant effect. Different letters following values within each column indicate significantly different values at 0.05 level according to Duncan’s multiple range test.

).

As an average of the three treatments, the chlorophyll content index increased up to 35 days by 42% (from 6.48 to 9.21), decreased at 42 days by 19% (from 9.21 to 7.43), and, after a sharp increase at 49 days by 75% (from 7.43 to 12.98), remained unchanged up to 63 days (10.87). As an average of all sampling times, the chlorophyll content index was higher by 8% in plants treated with Isabion^® (9.77) than in control plants (9.07), not significantly different compared to the plants treated with Bactiva^® (9.41).

3.6. Fluorescence Parameters

The sampling time significantly affected all eight fluorescence parameters determined, while the treatments significantly affected five out of eight parameters (except Fo, Fm, and Fv), without a significant interaction among these factors (

Table 4. Effect of biostimulants for lettuce leaf fluorescence parameters Fo, Fm, Fv, Fv/Fm, Fv/Fo, and Fm/Fo at 35 and 63 days after transplanting. The plants were treated with the commercial biostimulants Bactiva^® and Isabion^®. Non-treated or control treatment was also included. At each sampling time, fluorescence parameters were determined in 15 plants per replicate.

				Fo			Fm			Fv			Fv/Fm			Fv/Fo			Fm/Fo
Source of Variance		DF		p	%TV	η2	p	%TV	η2	p	%TV	η2	p	%TV	η2	p	%TV	η2	p	%TV	η2
Sampling time (A)		1		***	82	8.3	***	100	76.8	***	100	80.1	***	97	43.0	***	98	60.1	***	97	43.0
Treatment (B)		2		ns	10	2.1	ns	0	0.2	ns	0	0.3	*	2	1.7	**	2	1.9	*	2	1.7
A × B		2		ns	4	0.9	ns	0	0.1	ns	0	0.1	ns	0	0.3	ns	0	0.3	ns	0	0.3
Error		12
Sampling time (Days)
	35				20,492	b		57,637	b		37,145	b		0.64	b		1.87	b		2.87	b
	63				22,824	a		86,994	a		64,170	a		0.74	a		2.86	a		3.86	a
Treatment
	Control				22,378	a		72,842	a		50,464	a		0.68	b		2.27	b		3.27	b
	Bactiva				21,649	ab		71,353	a		49,704	a		0.69	ab		2.34	b		3.34	b
	Isabion				20,947	b		72,752	a		51,805	a		0.70	a		2.49	a		3.49	a
Sampling time × Treatment
35 days	Control Bactiva Isabion				21,735 20,119 19,623	ab bc c		58,923 56,720 57,269	b b b		37,188 36,601 37,646	c c c		0.63 0.64 0.66	c bc b		1.75 1.89 1.97	d bc b		2.75 2.89 2.97	d cd b
63 days	Control Bactiva Isabion				23,021 23,180 22,271	a a a		86,762 85,987 88,235	a a a		63,741 62,806 65,963	a a a		0.73 0.73 0.75	a a a		2.79 2.79 3.00	a a a		3.79 3.79 4.00	a a a

DF, degrees of freedom; p, probability; %TV, % of total variance; η², eta squared; *, **, and *** significant effect at the 0.05, 0.01, and 0.001 levels, respectively; ns, non-significant effect. Different letters following values within each column indicate significantly different values at 0.05 level according to Duncan’s multiple range test.

and

Table 5. Effect of biostimulants for lettuce leaf fluorescence parameters area and ABS/RC at 35 and 63 days after transplanting. The plants were treated with the commercial biostimulants Bactiva^® and Isabion^®. Non-treated or control treatment was also included. At each sampling time, fluorescence parameters were determined in 15 plants per replicate.

			Area			ABS/RC
Source of Variance		DF	p	%TV	η2	p	%TV	η2
Sampling time (A)		1	***	98	53.2	***	79	7.7
Treatment (B)		2	*	1	1.2	*	12	2.3
A × B		2	ns	1	0.9	ns	5	0.9
Error		12
Sampling time (Days)
	35		13,392,392		b		4.29	a
	63		116,939,172		a		3.87	b
Treatment
	Control		59,914,438		b		4.21	a
	Bactiva		59,317,866		b		4.10	ab
	Isabion		76,265,041		a		3.93	b
Sampling time × Treatment
35 days	Control Bactiva Isabion	15,118,185 9,575,242 15,483,749			c c c		4.52 4.25 4.09	a ab bc
63 days	Control Bactiva Isabion	104,710,692 109,060,490 137,046,333			b b a		4.52 4.25 4.09	c bc c

DF, degrees of freedom; p, probability; %TV, % of total variance; η², eta squared; *, and *** significant effect at the 0.05, and 0.001 level; ns, non-significant effect. Different letters following values within each column indicate significantly different values at 0.05 level according to Duncan’s multiple range test.

). In any case, both the proportion of total variance and η² due to the sampling time were much higher than those due to the treatments.

The highest values in all fluorescence parameters, apart from ABS/RC, were found at 63 days after transplanting (Table 4 and Table 5).

As an average of the two sampling times, in six out of eight fluorescence parameters, apart from Fm and Fv, significant differences were found between Isabion^® treated and the control plants. In contrast, no significant difference was found between the Bactiva^®-treated plants and the control ones in all of the parameters (Table 4 and Table 5). Finally, between Bactiva^® and Isabion^®, significant differences were found in three (Fm/Fo, Fv/Fo, area) out of eight fluorescence parameters (Table 4 and Table 5).

It is remarkable that the value of the Fv/Fm parameter increased from 0.64 at 35 days to 0.74 at 63 days (15% increase as an average of the three treatments), and in addition, the application of Isabion^® resulted in an increase in the value of the Fv/Fm parameter (0.70) compared to the control (0.68) as an average of the two sampling times as well as at 35 days after transplanting (0.66 vs. 0.63) (Table 4). Similarly, the value of the Fv/Fo parameter increased from 1.87 at 35 days to 2.86 at 63 days (53% increase as an average of the three treatments) (Table 4). The application of Isabion^® resulted in the increase in the value of the Fv/Fo parameter (2.49) compared to the control (2.27) as an average of the two sampling times as well as at 35 (1.97 vs. 1.75) and 63 (3.00 vs. 2.79) days after transplantation (Table 4). Accordingly, the value of the Fm/Fo parameter increased from 2.87 at 35 days to 3.86 at 63 days (34% increase as an average of the three treatments) (Table 4). The application of Isabion^® resulted in an increase in the value of the Fm/Fo parameter (3.49) compared to the control (3.27) as an average of the two sampling times as well as at 35 (2.97 vs. 2.75) and 63 (4.00 vs. 3.79) days after transplantation (Table 4).

3.7. Leaf Color

The sampling time significantly affected four of five leaf color parameters (L*, a*, b*, C*, and not h°), while the treatments significantly affected only the a* parameter, in which significant interaction of sampling time × treatment was also observed (

Table 6. Effect of biostimulants for lettuce leaf color parameters (L*, a*, b*, C*, and h°) at 35 and 63 days after transplanting. The plants were treated with the commercial biostimulants Bactiva^® and Isabion^®. Non-treated or control treatment was also included. At each sampling time, leaf color parameters were determined in 10 plants per replicate.

			L*			a*			b*			C*			h°
Source of Variance		DF	p	%TV	η2	p	%TV	η2	p	%TV	η2	p	%TV	η2	p	%TV	η2
Sampling time (A)		1	***	87	24.7	***	97	73.2	***	95	40.69	***	96	51.6	ns	65	2.3
Treatment (B)		2	ns	4	2.3	*	1	1.7	ns	2	1.8	ns	2	1.8	ns	6	0.5
A × B		2	ns	7	4.0	*	1	1.8	ns	2	1.8	ns	2	1.9	ns	6	0.4
Error		12
Sampling time (Days)
	35			48.09	a		−18.74	a		24.63	a		30.95	a		127.29	b
	63			44.47	b		−16.08	b		21.43	b		26.79	b		126.89	a
Treatment
	Control			45.71	a		−17.23	a		22.70	a		28.50	a		127.20	a
	Bactiva			47.02	a		−17.69	b		23.49	a		29.41	a		126.98	a
	Isabion			46.11	a		−17.30	a		22.90	a		28.70	a		127.08	a
Sampling time × Treatment
35 days	Control Bactiva Isabion			48.06 47.81 48.39	a a a		−18.62 −18.75 −18.86	b b b		−15.84 −16.64 −15.74	c c c		24.33 24.66 24.90	a a a		127.43 127.27 127.17	a a a
63 days	Control Bactiva Isabion			43.35 46.24 43.82	b a b		−15.84 −16.64 −15.74	a b a		21.07 22.32 20.89	bc b c		26.36 27.84 26.16	c b c		126.97 126.70 127.00	a a a

DF, degrees of freedom; p, probability; %TV, % of total variance; η², eta squared; *, and *** significant effect at the 0.05, and 0.001 levels, respectively; ns, non-significant effect. Different letters following values within each column indicate significantly different values at 0.05 level according to Duncan’s multiple range test.

). In any case, both the proportion of total variance and η² due to the sampling time were much higher than those due to the treatments.

As an average of the three treatments, the plants had the highest values for color parameters at 35 days (L* = 48.09, a* = −18.74, b* = 24.63, and C* = 30.95) compared to 63 days (L* = 44.47, a* = −16.08, b* = 21.43 and C* = 26.79) (Table 6).

3.8. Chlorophylls

Leaf chlorophyll content was not significantly affected by either sampling time or treatments, and the interaction of sampling time × treatment was also not significant (

Table 7. Effect of biostimulants for lettuce leaf chlorophyll content (a, b, and a/b) at 35 and 63 days after transplanting. The plants were treated with the commercial biostimulants Bactiva^® and Isabion^®. Non-treated or control treatment was also included. At 35 and 63 days, leaf chlorophyll content was determined in 3 plants and 1 plant per replicate, respectively.

			Chl a (μg/g fw)			Chl a (mg/g dw)			Chl b (μg/g fw)			Chl b (mg/g dw)			Chl a/b
Source of Variance		DF	p	%TV	η2	p	%TV	η2	p	%TV	η2	p	%TV	η2	p	%TV	η2
Sampling time (A)		1	ns	5	0.2	ns	55	3.4	ns	51	3.1	ns	4	0.1	***	98	50.7
Treatment (B)		2	ns	8	0.6	ns	2	0.2	ns	4	0.5	ns	5	0.3	ns	1	0.9
A × B		2	ns	64	4.5	ns	31	3.8	ns	32	4.0	ns	62	3.5	ns	1	0.9
Error		12
Sampling time (Days)
	35			226	a		3.34	a		64.18	a		0.95	a		3.52	a
	63			219	a		2.94	a		72.34	a		0.97	a		3.03	b
Treatment
	Control			231	a		3.20	a		70.22	a		0.97	a		3.28	a
	Bactiva			220	a		3.14	a		68.32	a		0.97	a		3.23	a
	Isabion			218	a		3.08	a		66.25	a		0.94	a		3.31	a
Sampling time × Treatment
35 days	Control Bactiva Isabion			252 228 198	a a a		3.62 3.41 2.99	a ab ab		71.53 64.67 56.33	ab ab b		1.03 0.97 0.85	a a a		3.52 3.52 3.52	a a a
63 days	Control Bactiva Isabion			210 212 237	a a a		2.78 2.86 3.16	b ab ab		68.90 71.97 71.97	ab ab a		0.91 0.97 1.02	a a a		3.05 2.94 3.10	b b b

DF, degrees of freedom; p, probability; %TV, % of total variance; η², eta squared; *** significant effect at the 0.001 level; ns, non-significant effect. Different letters following values within each column indicate significantly different values at 0.05 level according to Duncan’s multiple range test.

and

Table 8. Effect of biostimulants for lettuce leaf chlorophyll (a+ b) and total carotenoid content at 35 and 63 days after transplanting. The plants were treated with the commercial biostimulants Bactiva^® and Isabion^®. Non-treated or control treatment was also included. At 35 and 63 days, leaf chlorophyll (a + b) and total carotenoid content were determined in 3 plants and 1 plant per replicate, respectively.

			Chl a + b (μg/g fw)			Chl a + b (mg/g dw)			Total Carotenoids (μg/g fw)			Total Carotenoids (mg/g dw)
Source of Variance		DF	p	%TV	η2	p	%TV	η2	p	%TV	η2	p	%TV	η2
Sampling time (A)		1	ns	0	0.0	ns	38	1.8	ns	0	0.0	ns	44	2.5
Treatment (B)		2	ns	8	0.5	ns	3	0.3	ns	12	0.9	ns	4	0.5
A × B		2	ns	67	4.4	ns	42	3.8	ns	67	5.1	ns	39	4.4
Error		12
Sampling time (Days)
	35			289	a		4.27	a		61.26	a		0.90	a
	63			292	a		3.91	a		60.86	a		0.81	a
Treatment
	Control			301	a		4.17	a		63.73	a		0.88	a
	Bactiva			288	a		4.10	a		60.25	a		0.86	a
	Isabion			288	a		4.00	a		59.18	a		0.84	a
Sampling time × Treatment
35 days	Control Bactiva Isabion			322 292 254	a a a		4.64 4.36 3.82	a a a		68.97 61.50 53.30	a ab b		0.99 0.92 0.80	a ab ab
63 days	Control Bactiva Isabion			279 284 312	b b b		3.70 3.84 4.18	a a a		58.50 59.00 65.07	ab ab ab		0.78 0.80 0.87	b ab ab

DF, degrees of freedom; p, probability; %TV, % of total variance; η², eta squared; ns, non-significant effect. Different letters following values within each column indicate significantly different values at 0.05 level according to Duncan’s multiple range test.

). Leaf content was 198–252 µg/g fw and 2.78–3.62 mg/g dw for chlorophyll a, 56.3–76.2 μg/g fw and 0.85–1.03 mg/g dw for chlorophyll b, and 254–322 μg/g fw and 3.70–4.64 mg/g dw for chlorophyll a + b.

In contrast, the chlorophyll a/b ratio was significantly affected by the sampling time. As an average of the three treatments, the plants had the highest ratio at harvest at 35 days (3.52) compared to 63 days (3.03) (Table 7), and this corresponds to a difference of 16%.

3.9. Total Carotenoids

The content of total carotenoids in the leaves was not significantly affected by either sampling time or treatments. Moreover, the interaction of sampling time × treatment was also not significant (Table 8). Leaf content for total carotenoids was 53.3–69.0 µg/g fw and 0.78–0.99 mg/g dw.

3.10. Dry Matter

The sampling time but not treatments significantly affected dry matter content, while the interaction of sampling time × treatment was not significant (

Table 9. Effect of biostimulants for lettuce dry matter, nitrate, and total soluble phenol content at 35 and 63 days after transplanting. The plants were treated with the commercial biostimulants Bactiva^® and Isabion^®. Non-treated or control treatment was also included. At 35 and 63 days, dry matter, nitrate, and soluble content were determined in 3 and 1 plants per replicate, respectively.

			Dry Matter (%)			Nitrates (mg/kg fw)			Nitrates (% dw)			Total Soluble Phenols (μg/kg fw)			Total Soluble Phenols (μg/g dw)
Source of Variance		DF	p	%TV	η2	p	%TV	η2	p	%TV	η2	p	%TV	η2	p	%TV	η2
Sampling time (A)		1	***	94	32.7	***	95	27.2	***	96	51.6	ns	0	0.0	ns	22	1.8
Treatment (B)		2	ns	4	2.6	ns	2	1.0	ns	2	1.8	**	88	14.0	**	68	11.6
A × B		2	ns	1	0.5	ns	2	0.9	ns	2	1.9	ns	3	0.5	ns	2	0.4
Error		12
Sampling time (Days)
	35			6.75	b		369	a		0.55	a		247	a		3.65	a
	63			7.47	a		266	b		0.36	b		248	a		3.33	a
Treatment
	Control			7.25	a		317	a		0.44	a		276	a		3.81	a
	Bactiva			7.02	a		330	a		0.48	a		261	a		3.73	a
	Isabion			7.06	a		307	a		0.44	a		205	b		2.93	b
Sampling time × Treatment
35 days	Control Bactiva Isabion			6.94 6.68 6.64	b b b		366 394 348	a a a		0.53 0.59 0.52	a a a		279 252 208	a ab b		4.02 3.78 3.14	a ab bc
63 days	Control Bactiva Isabion			7.57 7.37 7.48	a a a		268 266 265	b b b		0.35 0.36 0.35	b b b		273 270 202	a a b		3.60 3.68 2.71	ab ab c

DF, degrees of freedom; p, probability; %TV, % of total variance; η², eta squared; ** and *** significant effect at the 0.01 and 0.001 level, respectively; ns, non-significant effect. Different letters following values within each column indicate significantly different values at 0.05 level according to Duncan’s multiple range test.

). As an average of the three treatments, the plants had the highest dry matter content at harvest after 63 days (7.47%) compared to 35 days after harvest (6.75%), and this corresponds to a difference of 11%.

3.11. Nitrates

The sampling time but not the treatments significantly affected the nitrate content during cultivation, while the interaction of sampling time × treatment was also not significant (Table 9). As an average of the three treatments, the plants had the highest nitrate content at 35 days (369 mg/kg fw and 0.55% dw) relative to harvest at 63 days (266 mg/kg fw and 0.36% dw), and this corresponds to a difference of 39% on a fresh weight basis.

3.12. Total Soluble Phenols

The treatments but not the sampling time significantly affected the content of total soluble phenols, while the interaction of sampling time × treatment was also not significant (Table 9). As an average of the two sampling times, the plants treated with Isabion^® had the lowest content of total soluble phenols (205 μg/kg fw and 2.93 μg/g dw) compared to the control plants and those treated with Bactiva^® (276 and 261 μg/kg fw and 3.81 and 3.73 μg/g dw, respectively), both without significant differences between them, and this corresponds to a difference of 26 and 21%, respectively, on a fresh weight basis.

3.13. Antioxidant Capacity

The sampling time but not the treatments significantly affected the antioxidant capacity expressed on the basis of dry weight during cultivation, while the interaction of sampling time × treatment was also not significant (

Table 10. Effect of biostimulants for lettuce antioxidant capacity at 35 and 63 days after transplanting. The plants were treated with the commercial biostimulants Bactiva^® and Isabion^®. Non-treated or control treatment was also included. At 35 and 63 days, antioxidant capacity was determined in 3 plants and 1 plant per replicate, respectively.

			Antioxidant Capacity (mg AAE/100 g fw)			Antioxidant Capacity (mg AAE/g dw)
Source of Variance		DF	p	%TV	η2	p	%TV	η2
Sampling time (A)		1	ns	15	0.8	*	55	4.3
Treatment (B)		2	ns	18	1.9	ns	5	0.8
A × B		2	ns	52	5.4	ns	31	4.9
Error		12
Sampling time (Days)
	35		11.26		a		1.66	a
	63		10.56		a		1.42	b
Treatment
	Control		11.67		a		1.62	a
	Bactiva		10.38		a		1.49	a
	Isabion		10.67		a		1.52	a
Sampling time × Treatment
35 days	Control Bactiva Isabion	11.03 11.97 10.77			ab a ab		1.59 1.79 1.62	ab a a
63 days	Control Bactiva Isabion	12.30 8.80 10.57			a a ab		1.64 1.19 1.41	a b ab

DF, degrees of freedom; p, probability; %TV, % of total variance; η², eta squared; * significant effect at the 0.05 level; ns, non-significant effect. Different letters following values within each column indicate significantly different values at 0.05 level according to Duncan’s multiple range test.

). As an average for the three treatments, the plants had the highest antioxidant capacity at 35 days (1.66 mg AAE/g dw) in relation to harvest at 63 days (1.42 mg AAE/g dw), and this corresponds to a difference of 17%.

4. Discussion

In the literature, it is generally reported that biostimulants can affect the primary or even secondary metabolism by increasing the photosynthetic activity and stimulating specific biosynthetic pathways [42,43,44,45]. Indicatively, it has been reported that biostimulants can increase photosynthetic efficiency, light use efficiency, and excitation energy dissipation in PSII antennae, together with photosynthetic pigments [43]. On the other hand, it has also been reported that the application of biostimulants resulted in an increase in plant biomass without causing an improvement in photochemistry parameters, suggesting a beneficial effect on stomatal conductance and not directly on PSII [46]. Therefore, the exact activated mechanisms are difficult to identify and are still under investigation.

The chlorophyll fluorescence technique is considered highly important in plant ecophysiology research and in particular for plant photosynthetic performance under field conditions, as its measurements are widely used as an indicator of functional changes in the photosynthetic mechanism [47,48]. The chlorophyll fluorescence measurements provide information related to the state and function of photosystem II (PSII) reaction centers and antennas on the electron donor (P680) and acceptor (pheophytin) sites [49]. Although this information may seem highly specialized, it has been extensively used to measure and in some cases categorize a range of effects on photosynthetic processes [50] and to identify plant responses under stressful conditions [51]. In fact, certain chlorophyll fluorescence parameters have been proposed for specific plant stresses [50].

Chlorophyll fluorescence assay equipment is cheaper in cost than photosynthesis assay equipment and has a faster measurement time, making it an important tool for determining plant stress in the field before other effects become apparent [47,48]. However, despite the simplicity of the measurements, a large number of different coefficients have been proposed to quantify photochemical and non-photochemical quenching, and the same parameter can often be reported in different ways, so that data interpretation remains complex and even controversial [47,49,52,53].

Fo is the fluorescence emission level when all major quinone acceptors (Q_A) are in the oxidized or open state [54]. An increase in Fo has been attributed to the physical separation of PSII reaction centers from their corresponding pigment antenna, thereby preventing energy transfer to PSII traps [55]. Fm is the maximum fluorescence level, where all reaction centers are closed, and its comparison with Fo gives information about the efficiency of photochemical quenching and, by extension, the efficiency of PSII. Fj and Fi are the fluorescence levels at the j = 2 ms and I = 30 ms stages, while an additional parameter often included due to its diffuse nature is the fluorescence variable or Fv = Fm − Fo [56].

The Fv/Fm parameter is the most widely used measurement parameter of chlorophyll fluorescence and tests whether or not plant stress affects PSII [57,58]. An Fv/Fm value in the range of 0.74–0.85 is considered optimal for many plant species, while lower values indicate a decrease in the quantum efficiency of PSII photochemistry and a disturbance or damage to the photosynthetic mechanism, implying plant stress [47,58,59,60], which is greater as the Fv/Fm ratio further decreases and fewer open reaction centers are available [47,49,52].

A much more sensitive parameter than Fv/Fm (in which stress-induced changes are detected rather slowly) is Fv/Fo, which contains the same basic information but exhibits higher values and a higher dynamic range than Fv/Fm. The Fv/Fo ratio shows a greater range in stress conditions, since all changes in Fv or Fo are immediately reflected in it [60].

The parameter Fm/Fo is equivalent to Fv/Fo + 1, and in partially photoinhibited leaves it shows a significant decrease, as does the parameter Fv/Fo, in contrast to the parameter Fv/Fm, which changes very little [60].

The results of the present work show that the value of the Fv/Fm parameter increased from 35 to 63 days (Table 5), suggesting that stress was diminished over time after transplanting. In addition, the application of Isabion^® resulted in an increase in the value of the Fv/Fm parameter compared to the control (Table 5), suggesting that plants treated with Isabion^® suffered less stress than control plants. Similarly, the value of the Fv/Fo and Fm/Fo parameters increased from 35 to 63 days, and the application of Isabion^® resulted in the increase in the value of both parameters compared to the control as an average of the two sampling times, as well as at 35 and 63 days after transplantation (Table 5).

The parameter ABS/RC (absorption (ABS) per active reaction center (RC)) refers to the flux of photons absorbed by the antenna pigments. Antenna pigments are the pigment molecules (chlorophyll b, carotenoids, xanthophylls, etc.) that surround the RC of chlorophyll a (known as the core pigment) and absorb and transfer solar energy to it. Part of this excitation energy is dissipated mainly as heat and less as fluorescence emission (F), while another is trapped (TR) in RC and converted to redox energy, reducing the primary quinone electron acceptor Q_A in PSII complexes to Q_A⁻, which is then reoxidized to Q_A, thus creating an electron transfer (ET) that ultimately leads to CO₂ fixation. The more electrons are transferred from Q_A^– to the electron transport (ET) chain, the longer the fluorescence signals remain below Fm and the larger Sm becomes, which is a measure of the energy required to close all reaction centers. The smaller Sm corresponds to the case where each Q_A is reduced only once and can then be denoted as S_S, denoting a single turnover [56].

The results of the present work showed that the value of the ABS/RC parameter decreased from 35 to 63 days (Table 5) and the Sm parameter increased from 387 at 35 days to 1.833 at 63 days, indicating a lower photon flux and thus a higher energy requirement to close all reaction centers. In addition, the application of Isabion^® resulted in a reduction in the value of the ABS/RC parameter compared to the control as an average of the two sampling times as well as at 35 days after transplanting (Table 5), with a parallel increase in the value of the Sm parameter at 63 days after transplanting (2083 vs. 1641).

The area parameter expresses the area between the fluorescence curve and the maximum fluorescence intensity (Fm) and is proportional to the size of the pool of Q_A electron acceptors on the reducing side of PSII [51].

The results of the present work show that the value of the area parameter increased from 13.4 × 10⁶ at 35 days to 116.9 × 10⁶ at 63 days (Table 6), indicating a larger pool size of Q_A electron acceptors on the reducing side of PSII. In addition, the application of Isabion^® resulted in an increase in the value of the area parameter (76.3 × 10⁶) compared to the control (59.9 × 10⁶) as an average of the two sampling times as well as in 63 days after transplanting (137.0 × 10⁶ vs. 104.7 × 10⁶) (Table 5), with a parallel increase in the Sm parameter value at 63 days after transplanting.

In addition to the changes occurring in the photochemical efficiency, changes are also observed in the efficiency of heat dissipation (i.e., non-photochemical damping), which are reflected as changes in the values of the Fm parameter [47].

The chlorophyll content index is derived from instruments that use light transmission through a leaf at two wavelengths (red: 650–660 nm; infrared region: 930–940 nm) to determine the intensity of the green color and the thickness of the leaves, respectively. The ratio of the transmission of the two wavelengths provides an index of chlorophyll content referred to as the CCI or alternatively the SPAD index. CCI is a linear scale and SPAD is a log scale [61,62]. The chlorophyll content index is commonly used to determine plant nutrient stress, particularly nitrogen and sulfur, and is considered reliable for crop fertilization management [63].

The results of the present work show that the chlorophyll content index (CCI) showed strong fluctuations over time, indicating a possible stress on the plants in nutrients and particularly nitrogen in certain stages of the growth and development of the plants until their harvest. In addition, the application of Isabion^® resulted in an increase in the value of the chlorophyll content index on average for all sampling times (9.77) compared to the control plants (9.07).

Chlorophylls a and b have a characteristic green color due to strong absorption mainly in red-orange and blue-violet, respectively. The chlorophyll a/b ratio ranges from 2.0–2.8 for shade-adapted plants to 3.5–4.9 for plants growing in full sunlight. Variation in chlorophyll a/b ratios is due to differences in the ratio of PSI to PSII and the size and composition of the chlorophyll–protein sunlight–harvesting complexes (LHCs) associated with each photosystem. Photosystems contain chlorophyll a, but no chlorophyll b, while LHCs contain significant amounts of chlorophyll b. Shade-adapted plants tend to have more LHCs associated with their photosystems than plants growing in full sunlight and therefore have a lower chlorophyll a/b ratio [64].

Plants modify their chlorophyll content (a, b, a + b, and a/b) to adapt to a given environment and optimize photosynthesis. According to the theory of optimal nitrogen allocation within a leaf, the chlorophyll a/b ratio is expected to increase when nitrogen availability decreases, especially under high light conditions [65]. Thus, the chlorophyll a/b ratio may be a useful indicator of nitrogen partitioning within a leaf because this ratio should be positively correlated with the ratio of PSII cores to the chlorophyll–protein complex that binds sunlight [66].

The results of the present work showed that the chlorophyll a/b ratio was significantly affected by the sampling time, and on average, for the three treatments, it decreased at 63 (3.03) compared to 35 days (3.52) (Table 8) without being significantly affected by the treatments, indicating an adaptation of the plants due to the increased number of leaves and the distribution of nitrogen in them, but not due to the application of the biostimulants.

Phenolic compounds are secondary natural metabolites produced by plants mainly for protection against stresses, participating in a wide range of physiological activities [67]. The reduced content of total soluble phenols found in the present work as a result of the application of Isabion^® probably indicates that plants treated with Isabion^® suffered less stress than control plants. However, various effects of the application of biostimulants on the content of phenolic compounds in horticultural crops have been reported [68]. The fact that the antioxidant capacity found in the present study was not significantly correlated with the content of total soluble phenolics suggests that the antioxidant capacity can be attributed, to a large extent, to components other than total soluble phenolics. Similarly, although significant differences among different treatments in lettuce phenolic content have been reported, antioxidant capacity was similar [69].

Nitrate accumulation in green leafy vegetables (such as lettuce, rocket, spinach) is an undesirable quality characteristic [70], and the results from the application of biostimulants on this physiological phenomenon are conflicting [71]. The results of the present work showed that the biostimulants had no significant effect on the nitrate content and that the nitrate content of the lettuce at harvest was much lower than the maximum permissible limits established by the European Union. This is in line with the results of previous research under Greek climatic conditions [72].

On the other hand, the failure of the microbial biostimulant to exhibit any effect reinforces the common belief [15,16,17] that not all microbial biostimulants are suitable for all plant species and for all ecosystems, and also suggests that the effectiveness of their application is influenced by the native microflora [9], the method of application, and the prevailing environmental conditions [18].

It has been reported that the application of biostimulants allows plants to face various environmental challenges, resulting in beneficial effects on crops [7,10,11]. However, there is great variation in the criteria for evaluating efficacy, there is no clear and consistent classification, and limited efficacy has been reported for non-microbial due to competitive interactions with soil bacteria and fungi that may affect the function of symbiotic microorganisms [9,15,16,17,18]. These interactions between mycorrhizal fungi and other soil organisms are complex and not fully elucidated [15,73,74].

5. Conclusions

In conclusion, the application of a non-microbial biostimulant had some effects on mechanisms involved in the transplanting shock, without, however, being accompanied by significant effects on plant growth parameters and composition at harvest. However, a notable effect was that a greater percentage of plants at harvest had a fresh weight in the 351–400 class, while the greatest percentage of the control plants had a fresh weight in the 301–350 g class. In contrast, the application of the microbial biostimulant had no significant effect on any of the parameters determined compared to the control. Therefore, under the conditions of the present study, the effectiveness of biostimulant application at the time of transplanting on lettuce transplants is questionable.