agronomy

Article
Comparing Efficacy of Different Biostimulants for
Hydroponically Grown Lettuce (Lactuca sativa L.)
Zheng Wang 1 , Rui Yang 1, * , Ying Liang 1,2 , Shulan Zhang 3 , Zejin Zhang 2 , Chaohua Sun 4 , Jing Li 5 ,
Zhiyong Qi 1 and Qichang Yang 1, *

1 Institute of Urban Agriculture, Chinese Academy of Agricultural Sciences (IUA-CAAS), Chengdu National
Agricultural Science and Technology Center (NASC), Chengdu 610213, China; [email protected] (Z.W.);
[email protected] (Y.L.); [email protected] (Z.Q.)
2 Vegetable Germplasm Innovation and Variety Improvement Key Laboratory of Sichuan Province, Horticulture
Research Institute, Sichuan Academy of Agricultural Sciences, Chengdu 610066, China; [email protected]
3 Department of Resources and Environmental Sciences, College of Natural Resources and Environment,
Northwest A & F University, Xi’an 712100, China; [email protected]
4 Boe Houji Technology (Beijing) Co., Beijing 100144, China; [email protected]
5 Leading Bio-Agricultural Co., Ltd., Qinhuangdao 066004, China; [email protected]
* Correspondence: [email protected] (R.Y.); [email protected] (Q.Y.)

Abstract: Biostimulants can enhance horticultural crop production. However, their application in
hydroponically grown lettuce is still limited, and information regarding their relative efficacy is
lacking. A greenhouse trial was conducted to address this issue. Five nutrient solution treatments
were evaluated on two lettuce cultivars: butterhead and red oak-leaf. The treatments included
a half-strength modified Hoagland solution (Hs-H); a full-strength modified Hoagland solution



(Fs-H); and Hs-H supplemented with 50 mg L−1 fulvic acid (FA), 334 mg L−1 seaweed extract (SE),


or 5 mL L−1 gamma polyglutamic acid (PGA). The results indicated that the shoot biomass observed
Citation: Wang, Z.; Yang, R.; Liang,
after biostimulant supplementation was significantly greater than or comparable to that observed
Y.; Zhang, S.; Zhang, Z.; Sun, C.; Li, J.;
with Fs-H. Nutrient solutions supplemented with SE and PGA led to a greater increase in the root
Qi, Z.; Yang, Q. Comparing Efficacy
biomass than that realized with Hs-H and Fs-H treatments. The Hs-H + FA treatment resulted in the
of Different Biostimulants for
lowest root-to-shoot ratio on a fresh weight basis among all treatments. The nitrate concentration
Hydroponically Grown Lettuce
(Lactuca sativa L.). Agronomy 2022, 12,
in the shoot was significantly reduced following biostimulant supplementation compared to that
786. https://doi.org/10.3390/ realized with Fs-H and Hs-H treatments. Nutrient solutions supplemented with SE and PGA also
agronomy12040786 decreased soluble sugar concentrations compared to that achieved using Hs-H and Fs-H treatments.
FA and SE improved nutrient uptake for both cultivars, but PGA had a minimal effect on nutrient
Academic Editors: Andrea Ertani and
uptake. The two cultivars varied in their responses to biostimulant supplementation with regard
Michela Schiavon
to biomass, quality traits, and nutrient uptake. This study supports using fulvic acid and seaweed
Received: 22 February 2022 extract, rather than γ-PGA, in hydroponic lettuce production systems.
Accepted: 21 March 2022
Published: 24 March 2022 Keywords: biostimulants; seaweed extract; fulvic acid; γ-PGA; biomass; quality; nutrient uptake
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with regard to jurisdictional claims in
published maps and institutional affil-
iations. 1. Introduction
The global population is expected to grow in the next three decades and reach 9.7 bil-
lion by 2050 [1]. It will be challenging to manage resources, reduce environmental pollution,
and satisfy the growing demand for food under ever-changing climate conditions [2,3].
Copyright: © 2022 by the authors.
Increasing productivity through controlled environment agriculture (CEA) is widely con-
Licensee MDPI, Basel, Switzerland.
sidered a sustainable approach to feed the entire world by 2050. Hydroponics is a common
This article is an open access article
cultivation practice for CEA, offering greater yield and higher-quality products while
distributed under the terms and
conditions of the Creative Commons
maintaining a lower environmental impact. Generally, hydroponic systems use water and
Attribution (CC BY) license (https://
nutrients more efficiently than traditional open-field production systems [4,5].
creativecommons.org/licenses/by/ Among the factors determining crop yield and quality in hydroponic systems, a proper
4.0/). understanding of nutrient solution management is critical. To date, numerous nutrient

Agronomy 2022, 12, 786. https://doi.org/10.3390/agronomy12040786 https://www.mdpi.com/journal/agronomy

Agronomy 2022, 12, 786 2 of 17

solution formulas for hydroponic production systems have been designed, and most
of them contain only inorganic ions from soluble salts of elements essential for higher
plants [6]. However, it has been reported that the concentration of currently used nutrient
solutions can be reduced by up to 50% without adverse effects on biomass and quality
of floral crops, such as gerbera [7] and anthurium [8], as well as horticultural crops, such
as tomato [9] and zucchini squash [10]. Such results indicate inefficient nutrient use in
hydroponic production, possibly because the nutrient composition for the best plant growth
and nutrient uptake by plants differs [11,12]. Balancing supply and demand may decrease
production costs and reduce the risk of water pollution; however, the best plant growth may
not be achieved if a nutrient solution based on the nutrient uptake is used [13]. One way to
reduce nutrient use without damaging crop productivity in hydroponic production is to
enhance crop uptake of nutrients by using biostimulants [14,15].
Plant biostimulants are “substance(s) and/or microorganisms whose function when
applied to plants or the rhizosphere is to stimulate natural processes to enhance/benefit
nutrient uptake, nutrient efficiency, tolerance to abiotic stress, and crop quality” [16]; the
most researched plant biostimulants are humic-like substances, seaweed extracts, and
protein hydrolysates [15,17–19]. In terms of application, most plant biostimulants are foliar
sprays or applied directly to the soil [20,21]. In contrast, Vernieri et al. [22] considered
the combination of hydroponics and biostimulants as a promising environment-friendly
production strategy for vegetables grown in greenhouses. However, studies regarding the
direct addition of biostimulants to hydroponic solutions are still limited, and their effects
on crop production still need verification.
Different biostimulants play different roles in plant growth [23–26]. For example, fulvic
acid (FA), the main constituent of humic-like substances with small molecular size [27], can
improve root growth by increasing the number and length of lateral roots [28]; shoot weight;
and uptake of N, P, K, Ca, Mg, Cu, Fe, and Zn [15]. Seaweed extract (SE) is highly nutritious
in terms of essential macro and micronutrients for higher plants and growth-promoting
hormones [18]. Gamma poly glutamic acid (γ-PGA) was reported to enhance nutrient
uptake, promote nitrogen utilization and metabolism, and alleviate abiotic stress [29–31].
As the most important leafy vegetable and the most consumed salad vegetable world-
wide, lettuce is rich in health-promoting compounds [32,33]. The total growing area of
lettuce (Lactuca sativa L.) is 186 million hectares globally [34]. China has the largest lettuce
production worldwide and annual consumption of 15.96 million tons [34]. Hydroponic
cultivation is widely used for lettuce production, especially at agricultural facilities. Typical
hydroponic nutrient solutions have been modified from the classic Hoagland nutrient
solution, which has a high electrical conductivity (EC) [35]. Several studies have modified
the composition of the Hoagland nutrient solution for leafy vegetables [36–38], but there
is potential for improved lettuce yield and quality [39]. Previous studies have reported
positive effects on leafy vegetable growth, nutrient absorption, and qualities following the
application of fulvic acid, seaweed extract, and γ-PGA [40]. Despite this, studies directly
comparing the effectiveness of various types of biostimulants on hydroponically grown
lettuce are limited and warrant further investigation. To address this issue, this study
assesses the effect of a traditional hydroponic nutrient solution combined with supplemen-
tary biostimulants on the yield, quality, leaf pigment, and nutrient uptake in butterhead
and red oak-leaf lettuce.

2. Materials and Methods

2.1. Experimental Design and Growth Conditions
A greenhouse trial was conducted at the Chengdu Academy of Agricultural and
Forestry Sciences in Chengdu, Sichuan, China (103.9◦ N, 30.7◦ E). The experiment had a
split-plot design with three replicates, and both the main plots and subplots were randomly
arranged. Two lettuce (Lactuca sativa L.) cultivars, butterhead and red oak-leaf, were evalu-
ated as the main-plot factors due to their popularity among growers. The sub-plot factors
included five treatments of nutrient solutions: a half-strength modified Hoagland solu-
Agronomy 2022, 12, 786 3 of 17

tion (Hs-H), which also served as the negative control treatment; a full-strength modified
Hoagland solution (Fs-H), which also served as the positive control treatment; Hs-H with
50 mg L−1 fulvic acid (FA); Hs-H with 334 mg L−1 seaweed extract (SE); and Hs-H with
5 mL L−1 γ-PGA (PGA). The Fs-H treatment was prepared following the formula presented
by Hoagland and Arnon [35]. The nutrient composition of the Hs-H treatment was 4 mM
KNO3 , 0.8 mM KH2 PO4 , 0.3 mM K2 HPO4 , 1.5 mM MgSO4 , 3 mM Ca(NO3 )2 , 0.08 mM
Fe-Na EDTA, 60 µM H3 BO3 , 3 µM ZnSO4 , 20 µM MnSO4 , 0.4 µM CuSO4 , and 0.03 µM
(NH4 )6 Mo7 O24 . All biostimulants evaluated in this study were commercially available. The
fulvic acid was extracted from leonardite (Zhongnongrunze Biotech Co., Ltd., Chengdu,
China), the seaweed extract was derived from Sargassum spp. using alkaline extraction,
and the γ-PGA was produced by fermentation using proprietary microbial strains and
substrates (Leading Bio-agricultural Co., Ltd., Qinhuangdao, China). The nutrient con-
centrations of the evaluated biostimulants are listed in Table 1. The description, EC, and
original pH of all nutrient solution treatments are presented in Table 2. The evaluated
biostimulants were added directly to the nutrient solutions. The application rate was ex-
trapolated from the manufacturer’s instructions for foliar spray and soil application, as no
recommended rate for a hydroponic production system has been defined. To eliminate the
effect of different pH values among treatments, the pH of each nutrient solution treatment
was adjusted weekly and maintained at pH 5.8 during the entire growing period.

Table 1. Nutrient concentration in evaluated biostimulants.

C N P K S Ca Mg Fe Mn Cu Zn
Biostimulant (%) (%) (mg kg−1 ) (mg kg−1 ) (mg kg−1 ) (mg kg−1 ) (mg kg−1 ) (mg kg−1 ) (mg kg−1 ) (mg kg−1 ) (mg kg−1 )
FA 29.21 0.71 200.34 60,198.85 31,793.12 1054.90 1111.34 10,174.43 83.08 17.78 23.19
SE 27.26 0.86 64.50 83,309.58 35,666.93 242.19 425.13 1556.07 22.99 1.39 6.34
PGA 3.58 1.28 616.10 6137.83 1.88 7.88 105.56 7.60 0.34 0.23 0.49

Table 2. Description, electrical conductivity (EC), and original pH of nutrient solution treatments.

EC
Treatment Description Original pH
(mS cm−1 )
Hs-H Half-strength Hoagland solution 1.28 6.46
Fs-H Full-strength Hoagland solution 2.74 5.94
FA Hs-H + 50 mg/L fluvic acid 1.79 6.02
SE Hs-H + 334 mg/L seaweed extract 1.88 6.92
PGA Hs-H + 5 ml/L gamma polyglutamic acid 1.96 6.74
Note: To eliminate the effect of the original pH, the pH of each treatment was adjusted weekly and maintained at
pH 5.8 throughout the entire growing period.

Seeds of the two lettuce cultivars were germinated on a sponge in deionized (DI) water
in a growth chamber with day/night temperatures of 24/22 ◦ C and relative humidity of
60%. Seeds were germinated in darkness for three days before subsequent cultivation under
LED lights with a photosynthetic photon flux density of 150 µmol m−2 s−1 and a photope-
riod of 16 h (06:00–22:00). Upon unfolding of the first pair of true leaves (i.e., 10 day after
sowing), uniform seedlings were transferred to a deep-floating hydroponic system in the
greenhouse and grown for another 45 day under natural sunlight and temperature. The
individual plot size was 5 m2 , and the spacing between plants was 0.2 m. The daily maxi-
mum and minimum temperatures and daily light integral in the greenhouse are illustrated
in Figure S1.

2.2. Harvest and Sample Analysis

Upon harvest, the chlorophyll, flavonoid, and anthocyanin in the second pair of fully
expanded leaves were measured in 24 uniform plants within each subplot using a Dualex 4
Scientific optical leaf-clip meter (FORCE-A, Orsay, France). Three plants from each sub-plot
were randomly collected, thoroughly mixed to represent a composite sample, and stored in
a −80 ◦ C freezer to determine crop quality. Nitrate, soluble sugar, and soluble protein were
Agronomy 2022, 12, 786 4 of 17

analyzed using commercial testing kits that utilized classic and widely used colorimetric
methods (Suzhou Grace Biotechnology Co., Ltd., Suzhou, China). For nitrate determination,
0.1 g of fresh leaves was homogenized in 1 mL deionized water, extracted in a water bath
at 100 ◦ C for 30 min, cooled to room temperature, and centrifuged at 12,000× g for 15 min.
Then, 10 µL of the supernatant was thoroughly mixed with 40 µL of 5% salicylic acid
solution in concentrated sulfuric acid and 950 µL of 8% sodium hydroxide solution. The
absorbance was measured at 410 nm (http://www.geruisi-bio.com/pro/proshow/id/268,
(accessed on 20 March 2022) [41]. For soluble sugar determination, fresh leaves (0.1 g) were
homogenized in 1.5 mL of 80% ethanol in an ice bath, extracted in a 50 ◦ C water bath for
20 min, and centrifuged at 12,000× g for 15 min. Then, 100 µL of the supernatant was
thoroughly mixed with 300 µL DI water, 120 µL anthrone reagent, and 1 mL concentrated
sulfuric acid and incubated in a 95 ◦ C water bath for 10 min. After cooling to room
temperature, the absorbance was measured at 620 nm (http://www.geruisi-bio.com/pro/
proshow/id/431, (accessed on 20 March 2022) [42]. For soluble protein determination, 0.1 g
of fresh leaves were homogenized in 1 mL 0.1 M phosphatic buffer solution (pH = 6) in
an ice bath and centrifuged at 12,000× g for 15 min at 4 ◦ C. The supernatant (160 µL) was
thoroughly mixed with 800 µL Coomassie Brilliant Blue G250 solution, and the absorbance
was measured at 600 nm (http://www.geruisi-bio.com/pro/proshow/id/247, (accessed
on 20 March 2022) [43].
Thirty plants from each sub-plot were harvested to determine the fresh weight of the
shoots and roots. The shoot and root samples were dried in an air-forced oven at 75 ◦ C
for 48 h and weighed. Dried shoot samples were ground into a fine powder using a ball
mill and digested with concentrated sulfuric acid and 30% hydrogen peroxide. Nitrogen
was determined using the Kjeldahl method, and phosphorus was determined using the
molybdenum blue colorimetric method [44]. Sulfur, calcium, magnesium, potassium, copper,
manganese, iron, and zinc were analyzed using an Agilent 7700e inductively coupled plasma
mass spectrometer (ICP-MS; Agilent Technologies, Inc., Santa Clara, CA, USA). Nutrient
content was calculated by multiplying nutrient concentration with dry biomass.

2.3. Data Analysis

A mixed-model methodology was used to analyze the data. Fixed effects included
nutrient solution treatment and lettuce cultivar, while random effects included the replicate
and replicate × cultivar. Statistical analysis was performed using SAS software (version 9.4;
SAS Institute, Cary, NC, USA). To test for differences among fixed effects and their inter-
actions, p-values were generated using Tukey’s honestly significant difference method as
implemented by the PROC GLIMMIX procedure. Principal component analysis (PCA) and
heatmap analysis were performed using GraphPad Prism 9 software (GraphPad Software,
San Diego, CA, USA).

3. Results
Significant treatment × cultivar interactions (p < 0.05) were observed for most of the
biomass, quality, and nutrient uptake traits and were thus demonstrated separately by
using nutrient solution treatment and cultivar in the following analyses.

3.1. Biomass
Shoot biomass of both cultivars responded to the nutrient solution treatment
(Figure 1A,B). The positive control treatment (i.e., Fs-H) significantly increased shoot
biomass relative to the negative control treatment (i.e., Hs-H); however, the shoot dry
weight of butterhead decreased relatively (Figure 1C,D). The Hs-H and Fs-H treatments
did not lead to significantly different root fresh weights (Figure 1E), but the Fs-H treatment
led to a significantly higher root dry weight (Figure 1F). The Hs-H and Fs-H treatments
afforded similar root-to-shoot (R/S) ratios based on the fresh weight (Figure 1G). However,
on a dry weight basis, the Fs-H treatment notably improved the R/S ratio for butterhead
but decreased it for red oak-leaf (Figure 1H).
 Agronomy 2022, 12, x FOR PEER REVIEW 6 of 17
Agronomy 2022, 12, 786 5 of 17

Figure
Figure 1. (A,B)
1. (A,B) Growth
Growth status,
status, (C,D)(C,D) fresh weight
fresh weight (FW) and(FW)
dryand dry(DW)
weight weight (DW)(E,F)
of shoot, of shoot,
FW and (E,F) FW and
DWDW of of
root, andand
root, (G,H) root-to-shoot
(G,H) (R/S) ratio
root-to-shoot (R/S)byratio
nutrient solution treatments
by nutrient and lettuceand
solution treatments cultivars.
lettuce cultivars.
Data
Datarepresent mean
represent mean± standard deviation
± standard (SD) (n = (SD)
deviation 30). Different
(n = 30).upper- and lowercase
Different upper- letters indi-
and lowercase letters
cate significant differences among nutrient solution treatments for butterhead and red oak-leaf, re-
indicate significant differences among nutrient solution treatments for butterhead and red oak-leaf,
spectively (α = 0.05). T—main effect of treatment; C—main effect of cultivar; T × C—treatment by
respectively
cultivar (α = *,0.05).
interaction. T—main
** and effect
*** indicate of treatment;
significant C—main
difference at α =effect of cultivar;
0.05, 0.01, T ×respec-
and 0.001, C—treatment by
tively; n.s. indicates
cultivar not significant.
interaction. * and *** indicate significant difference at α = 0.05 and 0.001, respectively; n.s.
indicates not significant.
3.2. Crop Quality
Allcrop
The three biostimulants
quality significantly
of lettuce leaves grown inimproved
Hs-H and shoot
Fs-Hbiomass
nutrient compared
solutions didto not
that achieved
with the Hs-H
significantly differ treatment (Figureexcept
for either cultivar, 1C,D).forBiostimulant supplementation
the fact that Fs-H notably increasedalsosolu-resulted in
ble proteingreater
notably content compared to that shoot
or comparable achieved with therelative
biomass Hs-H treatment for red oak-leaf
to that achieved with the Fs-H
(Figure 2). PGA
treatment; treatmentthe
however, for PGA
butterhead and FA
treatment treatment
led for red oak-leaf
to a significantly significantly
lower shoot dry weight
decreased
than thatnitrate concentrations
achieved using thecompared to that achieved
Fs-H treatment using Hs-H
(Figure 1C,D). The and Fs-HPGA
SE and treat-treatments
ments (Figure 2A). SE and PGA significantly reduced soluble sugar
produced significantly greater root biomass than that achieved using the concentrations rela-
Hs-H, Fs-H,
tive to that achieved using Hs-H and Fs-H treatments in both cultivars (Figure 2B). Soluble
and FA treatments. Only the SE treatment notably improved the root fresh weight when
protein was not significantly affected by biostimulant supplementation for butterhead,
compared to that achieved using Hs-H and FA treatments (Figure 1E,F). The R/S ratio on
whereas FA significantly increased soluble protein in red oak-leaf compared to that
a fresh weight
achieved basis(Figure
using Hs-H following PGA
2C). FA andtreatment was similar
PGA resulted to thatcontents
in chlorophyll realizedthat
with Hs-H and Fs-
were
H treatments, but it was significantly greater than that achieved using FA and SE treatments
(Figure 1G). An inconsistent trend was observed for the root-to-shoot ratio (R/S ratio) on
a dry-weight basis. For butterhead, all three biostimulants resulted in a significantly higher
R/S ratio relative to that achieved using the Hs-H treatment but a significantly lower R/S
ratio relative to that realized using the Fs-H treatment (Figure 1H). In contrast, the R/S
Agronomy 2022, 12, 786 6 of 17

ratio following PGA treatment was significantly greater than that observed after FA, SE,
and both negative and positive control treatments (Figure 1H).

3.2. Crop Quality

The crop quality of lettuce leaves grown in Hs-H and Fs-H nutrient solutions did
not significantly differ for either cultivar, except for the fact that Fs-H notably increased
soluble protein content compared to that achieved with the Hs-H treatment for red oak-leaf
(Figure 2). PGA treatment for butterhead and FA treatment for red oak-leaf significantly de-
creased nitrate concentrations compared to that achieved using Hs-H and Fs-H treatments
(Figure 2A). SE and PGA significantly reduced soluble sugar concentrations relative to that
achieved using Hs-H and Fs-H treatments in both cultivars (Figure 2B). Soluble protein
Agronomy 2022, 12, x FOR PEER REVIEW not significantly affected by biostimulant supplementation for butterhead,
was 7 of whereas
17
FA significantly increased soluble protein in red oak-leaf compared to that achieved using
Hs-H (Figure 2C). FA and PGA resulted in chlorophyll contents that were not significantly
different from those achieved using Hs-H and Fs-H treatments; however, SE induced
not significantly different from those achieved using Hs-H and Fs-H treatments; however,
aSE
notably
inducedlower chlorophyll
a notably content than
lower chlorophyll thatthan
content realized using the
that realized Fs-H,
using FA, and
the Fs-H, FA, PGA
and treat-
ments
PGA treatments (Figure 2D). FA did not significantly change the flavonoid content com- to that
(Figure 2D). FA did not significantly change the flavonoid content compared
achieved using
pared to that the Hs-H
achieved usingand
theFs-H
Hs-Htreatments in either in
and Fs-H treatments cultivar (Figure(Figure
either cultivar 2E). SE2E).
and PGA
significantly
SE and PGA significantly reduced flavonoids content in butterhead compared to that re- using
reduced flavonoids content in butterhead compared to that realized
Fs-H,
alizedwhereas PGA
using Fs-H, significantly
whereas increasedincreased
PGA significantly flavonoids contentcontent
flavonoids in red oak-leaf (Figure 2E).
in red oak-leaf
The nutrient
(Figure solution
2E). The treatments
nutrient did not significantly
solution treatments affect theaffect
did not significantly anthocyanin content in red
the anthocyanin
oak-leaf (Figure
content in 2F). (Figure 2F).
red oak-leaf

Figure 2.
Figure 2. Concentration
Concentration of (A) nitrate,
of (A) (B) soluble
nitrate, sugar,sugar,
(B) soluble and (C)and
soluble
(C) protein,
soluble and content
protein, andof (D)
content of
chlorophyll, (E) flavonoids, and (F) anthocyanins in fresh leaves by nutrient solution treatments and
(D) chlorophyll, (E) flavonoids, and (F) anthocyanins in fresh leaves by nutrient solution treatments
lettuce cultivars. Anthocyanin content was below the detection limit for butterhead and is thus not
and
shownlettuce
here. cultivars. Anthocyanin
Data represent mean ± SD (n content was nbelow
= 3, (A–C); the detection
= 24 (D–F)). Different limit
upper-for
and butterhead
lowercase and is
thus not
letters shownsignificant
indicate here. Data representamong
differences ± SD (nsolution
mean nutrient = 3, (A–C); n = 24 for
treatments (D–F)). Different
butterhead andupper-
red and
oak−leaf, respectively
lowercase (α = significant
letters indicate 0.05). T—main effect of among
differences treatment; C—main
nutrient effect treatments
solution of cultivar; Tfor
× butterhead
C—
treatment by cultivar interaction. ** and *** indicate significant difference at α = 0.05, 0.01, and 0.001,
and red oak-leaf, respectively (α = 0.05). T—main effect of treatment; C—main effect of cultivar;
respectively; n.s. indicates not significant.
T × C—treatment by cultivar interaction. ** and *** indicate significant difference at α = 0.05, 0.01,
and
3.3. 0.001,
Nutrientrespectively; n.s. and
Concentration indicates
Content not significant.
The heatmap associated with cluster analysis in the current study depicted a clear
hierarchy of nutrient uptake (Figures 3 and 4). Butterhead and red oak-leaf responded
differently to the nutrient solution treatments. Within those responses, the different bi-
ostimulants had inconsistent impacts on the concentration and uptake of different mineral
Agronomy 2022, 12, 786 7 of 17

3.3. Nutrient Concentration and Content

The heatmap associated with cluster analysis in the current study depicted a clear
hierarchy of nutrient uptake (Figures 3 and 4). Butterhead and red oak-leaf responded
differently to the nutrient solution treatments. Within those responses, the different bios-
timulants had inconsistent impacts on the concentration and uptake of different mineral
nutrients in the shoot.
A more thorough examination of the data indicated that increasing the nutrient con-
centration from half-strength (Hs-H) to full-strength (Fs-H) did not increase the nutrient
concentration in the shoot proportionately and significantly (Table 3). Fs-H notably reduced
Ca concentration in both cultivars (Table 3). FA, SE, and PGA significantly reduced the
N concentration in butterhead compared to that achieved with Hs-H and Fs-H treatments.
In contrast, for red oak-leaf, the evaluated biostimulants either significantly increased
(i.e., FA and SE) or maintained a consistent N concentration (i.e., PGA) relative to that
realized with Hs-H and Fs-H treatments (Table 3). All three biostimulants significantly
reduced S concentrations in both cultivars compared to that achieved using Fs-H. SE and
PGA significantly increased the Mn concentration in both cultivars relative to that achieved
using Hs-H and Fs-H treatments, whereas FA did not. FA and PGA significantly increased
Agronomy 2022, 12, x FOR PEER REVIEW 8 of 17
and decreased Zn concentrations in both cultivars, respectively, compared to that realized
using Hs-H and Fs-H treatments (Table 3).

Figure3.3.Macro
Figure Macro and
and micronutrient
micronutrient concentration
concentration in theinshoots
the shoots
of (A) of (A) butterhead
butterhead and (B) and (B) red oak-
red oak-leaf
leaf under different nutrient solution treatments. Nutrient concentration was normalized
under different nutrient solution treatments. Nutrient concentration was normalized by the z-score; by the
z−score; the red and blue colors indicate upregulation and downregulation,
the red and blue colors indicate upregulation and downregulation, respectively. respectively.
 Figure 3. Macro and micronutrient concentration in the shoots of (A) butterhead and (B) red oak
Agronomy 2022, 12, 786 8 of 17
leaf under different nutrient solution treatments. Nutrient concentration was normalized by th
z−score; the red and blue colors indicate upregulation and downregulation, respectively.

Figure 4. Macro and micronutrient content in the shoot of (A) butterhead and (B) red oak-leaf under
different nutrient solution treatments. Nutrient concentration was normalized by the z-score; the red
and blue colors indicate upregulation and downregulation, respectively.

The effect of nutrient solution treatment on nutrient content in the shoot was inconsis-
tent with that of the nutrient concentration. For example, significant differences in N content
among treatments did not occur for butterhead (Table 4), despite significantly lower N
concentrations following biostimulant supplementation (Table 3). For red oak-leaf, Fs-H
resulted in a significantly higher N content relative to that achieved with Hs-H (Table 4),
although the N concentration was similar after Hs-H and Fs-H treatments (Table 3). In
butterhead, FA enhanced the Mn and Zn contents compared to those achieved using the
Hs-H treatment; however, in red oak-leaf, all but S, Ca, Mg, and Cu contents were enhanced.
Only the Zn content was significantly elevated by FA when compared to that achieved
using the Fs-H treatment (Table 4). For both cultivars, SE significantly improved or tended
to improve the content of most nutrients compared to that realized using Hs-H; however,
PGA had a minimal effect on nutrient content (Table 4).
Agronomy 2022, 12, 786 9 of 17

Table 3. Macro and micronutrient concentration in the shoot of butterhead and red oak-leaf lettuce. Data represent mean ± SD (n = 3). Means within each column
followed by different uppercase and lowercase letters are significantly different for butterhead and red oak-leaf (α = 0.05).

N P K S Ca Mg Fe Mn Cu Zn
Treatment (g kg−1 ) (g kg−1 ) (g kg−1 ) (g kg−1 ) (g kg−1 ) (mg kg−1 ) (mg kg−1 ) (mg kg−1 ) (mg kg−1 )
(g kg−1 )
Butterhead
Hs-H 33.1 ± 1.21 A 9.9 ± 0.54 A 79.2 ± 1.55 B 2.45 ± 0.06 AB 1.55 ± 0.04 A 3.55 ± 0.03 A 81.8 ± 4.75 A 48.6 ± 0.29 C 7.48 ± 0.32 AB 54.1 ± 2.58 B
Fs-H 31.7 ± 1.22 A 10.1 ± 0.09 A 80.3 ± 2.21 B 3.09 ± 0.16 A 1.14 ± 0.06 B 3.20 ± 0.07 AB 81.9 ± 6.93 A 64.6 ± 2.42 B 7.16 ± 0.50 AB 51.3 ± 0.81 B
FA 27.3 ± 3.46 B 10.8 ± 0.19 A 78.4 ± 3.91 B 2.15 ± 0.21 B 1.12 ± 0.01 B 3.69 ± 0.17 A 75.2 ± 4.94 A 60.6 ± 2.29 B 5.33 ± 0.43 B 74.4 ± 1.19 A
SE 24.7 ± 0.41 B 10.1 ± 0.05 A 107 ± 2.91 A 1.64 ± 0.04 B 1.37 ± 0.05 AB 3.64 ± 0.12 A 86.8 ± 0.15 A 101 ± 2.72 A 7.86 ± 0.19 A 71.9 ± 3.37 A
PGA 27.2 ± 0.74 B 9.7 ± 0.40 A 81.4 ± 1.65 B 2.25 ± 0.01 B 1.19 ± 0.02 B 2.58 ± 0.31 B 54.1 ± 2.61 B 96.0 ± 0.68 A 6.23 ± 0.28 B 41.9 ± 2.37 C
Red oak-leaf
Hs-H 24.5 ± 0.44 b 12.6 ± 0.92 ab 81.7 ± 2.79 c 3.88 ± 0.43 a 2.46 ± 0.14 a 4.22 ± 0.11 a 58.4 ± 2.93 b 53.5 ± 0.29 c 8.55 ± 0.74 a 68.8 ± 1.68 b
Fs-H 27.9 ± 1.11 b 12.0 ± 0.41 b 89.8 ± 0.89 b 3.76 ± 0.21 a 1.65 ± 0.06 c 3.02 ± 0.11 bc 68.7 ± 1.20 b 55.2 ± 1.26 c 6.69 ± 0.84 b 70.9 ± 4.00 b
FA 39.5 ± 1.14 a 12.4 ± 0.52 ab 84.5 ± 4.27 b 1.96 ± 0.06 b 1.57 ± 0.11 c 3.33 ± 0.26 b 67.0 ± 2.60 b 55.0 ± 5.35 c 4.21 ±0.10 c 86.3 ± 5.89 a
SE 40.3 ± 0.49 a 13.6 ± 0.21 a 118 ± 1.94 a 2.09 ± 0.00 b 2.14 ± 0.06 b 3.53 ± 0.09 b 96.4 ± 1.00 a 95.6 ± 1.55 a 5.30 ± 0.36 bc 71.1 ± 3.09 b
PGA 25.9 ± 1.72 b 10.8 ± 0.33 b 82.0 ± 2.29 b 2.42 ± 0.13 b 1.69 ± 0.10 c 2.60 ± 0.27 c 36.7 ± 3.47 c 85.3 ± 1.06 b 4.57 ± 0.48 c 35.3 ± 3.69 c

Table 4. Macro and micronutrient content in the shoot of butterhead and red oak-leaf lettuce. Data represent mean ± SD (n = 3). Means within each column followed
by different uppercase and lowercase letters are significantly different for butterhead and red oak-leaf (α = 0.05).

N P K S Ca Mg Fe Mn Cu Zn
Treatment (mg plant−1 ) (mg plant−1 ) (mg plant−1 ) (mg plant−1 ) (mg plant−1 ) (µg plant−1 ) (µg plant−1 ) (µg plant−1 ) (µg plant−1 )
(mg plant−1 )
Butterhead
Hs-H 107 ± 15.8 A 31.9 ± 4.20 B 255 ± 25.2 B 7.89 ± 0.92 B 4.99 ± 0.59 A 11.5 ± 1.36 B 265 ± 43.0 B 157 ± 17.4 C 24.0 ± 2.22 B 174 ± 12.6 B
Fs-H 116 ± 7.10 A 36.7 ± 2.76 AB 293 ± 27.2 B 11.3 ± 1.37 A 4.15 ± 0.23 A 11.7 ± 0.71 B 298 ± 27.2 B 236 ± 17.2 BC 26.2 ± 3.30 B 187 ± 16.0 B
FA 120 ± 3.76 A 47.9 ± 4.39 AB 348 ± 26.9 B 9.53 ± 1.06 AB 4.97 ± 0.50 A 16.4 ± 1.43 AB 336 ± 47.3 AB 271 ± 38.9 B 24.0 ± 4.48 B 332 ± 36.2 A
SE 128 ± 10.9 A 52.4 ± 3.56 A 554 ± 50.2 A 8.54 ± 0.78 AB 7.11 ± 0.60 A 18.9 ± 1.50 A 451 ± 32.4 A 525 ± 25.9 A 40.8 ± 3.26 A 375 ± 42.2 A
PGA 119 ± 6.28 A 42.3 ± 4.36 AB 356 ± 29.2 B 9.84 ± 0.61 AB 5.21 ± 0.40 A 11.4 ± 2.05 B 236 ± 7.36 B 420 ± 28.6 A 27.3 ± 2.98 B 184 ± 22.1 B
Hs-H 81.7 ± 10.7 c 42.3 ± 7.26 b 273 ± 39.2 b 13.1 ± 2.89 b 8.27 ± 1.47 ab 14.1 ± 1.62 ab 195 ± 28.7 b 179 ± 22.6 c 28.5 ± 4.57 ab 230 ± 31.6 bc
Fs-H 139 ± 12.4 b 59.8±7.18 ab 450±51.7 a 18.9±2.78 a 8.32±1.32 ab 15.2±2.28 ab 344±39.2 a 277±40.1 bc 33.2±4.04 a 353±24.9 b
FA 22 6± 18.9 a 71.1 ± 6.18 a 485 ± 51.6 a 11.2 ± 0.80 b 9.02 ± 1.13 ab 19.2 ± 2.53 a 384 ± 29.6 a 315 ± 18.7 b 24.1 ± 1.70 ab 496 ± 61.9 a
SE 186 ± 22.8 a 63.0 ± 7.81 a 545 ± 64.2 a 9.64 ± 1.06 b 9.87 ± 0.84 a 16.3 ± 1.52 a 446 ± 52.9 a 442 ± 51.4 a 24.7 ± 4.48 ab 330 ± 50.8 b
PGA 93.6 ± 2.81 c 39.2 ± 2.78 b 297 ± 19.5 b 8.75 ± 0.59 b 6.14 ± 0.63 b 9.44 ± 1.36 b 133 ± 16.7 b 309 ± 13.9 b 16.6 ± 2.27 b 128 ± 18.2 c
Agronomy 2022, 12, 786 10 of 17

3.4. Principal Component Analysis

PCA was performed to explore relationships among growth traits, quality traits, pig-
ment contents, and macro and micronutrients and their responses to nutrient solution and
lettuce cultivar treatments. The PCA results are presented as biplots, including variables
leading to each selected trait component being combined with treatment component scores.
The association between the traits represented by the first and second principal components
is shown in Figure 5. These two primary components accounted for approximately 57%
of the total variation. The first principal component (x-axis) mainly includes biomass,
soluble protein, and concentrations of Fe and Mn on the positive side of the scale, and
crop quality traits (including chlorophyll, anthocyanins, flavonoids, soluble sugar, nitrate,
and concentrations of Ca, S, and P) on the negative side of the scale. The second principal
component (y-axis) mainly includes the Cu concentration and the R/S ratio (on a dry
weight basis) on the positive side and the N, K, and Zn concentrations on the negative side.
These results indicate that the Ca, S, and P concentrations and most crop qualities were
negatively correlated with shoot biomass. In contrast, the Fe, Mn, and K concentrations
were positively correlated with biomass. The vector for the concentration of Mg was shorter
Agronomy 2022, 12, x FOR PEER REVIEW 11 of 17
than that for other traits, indicating that only a small proportion of variability was explained
by principal components 1 and 2.

Figure 5.
Figure 5. Principal
Principal component
component pattern
pattern for
for correlations
correlations of
of yield,
yield, quality,
quality, and
and nutrient
nutrient concentration
concentration
traits in butterhead and red oak-leaf. SFW—shoot fresh weight; SDW—shoot dry
traits in butterhead and red oak-leaf. SFW—shoot fresh weight; SDW—shoot dry weight; RFW—rootweight; RFW—
root fresh weight; RDW—root dry weight; TFW—total fresh weight; TDW—total dry weight;
fresh weight; RDW—root dry weight; TFW—total fresh weight; TDW—total dry weight; R/SFW
R/SFW—ratio of root to shoot in fresh weight; R/SDW—ratio of root to shoot in dry weight; SN—
—ratio of root to shoot in fresh weight; R/SDW—ratio of root to shoot in dry weight; SN—shoot nitrate
shoot nitrate concentration; SSS—shoot soluble sugar concentration; SP—soluble protein; Chl—leaf
concentration; SSS—shoot
chlorophyll content; soluble
Flav—leaf sugar concentration;
flavonoids SP—soluble
content; Anth—leaf protein; content;
anthocyanins Chl—leaf N,chlorophyll
P, K, S, Ca,
content;
Mg, Fe, Mn,Flav—leaf
Cu, andflavonoids
Zn—theircontent; Anth—leaf
respective anthocyanins
concentrations content; N, P, K, S, Ca, Mg, Fe, Mn,
in the shoot.
Cu, and Zn—their respective concentrations in the shoot.
4. Discussion
4. Discussion
4.1. Biomass
4.1. Biomass
Hs-H, which
Hs-H, which served
served as
as the
the negative
negative control
control treatment
treatment in
in this
this study,
study, is
is widely
widely used
used inin
lettuceproduction.
lettuce production.However,
However, our
our study
study found
found thatthat
Fs-HFs-H produced
produced ~23%~23%
greatergreater
shoot shoot
fresh
biomass than that realized using Hs-H, indicating that a higher EC of nutrient solutionsolu-
fresh biomass than that realized using Hs-H, indicating that a higher EC of nutrient (i.e.,
tion (i.e.,
higher higherconcentration)
nutrient nutrient concentration) may
may increase theincrease
yield of the yield of hydroponically
hydroponically pro-
produced lettuce.
duced lettuce. Previous studies have reached the same conclusion, but an interaction be-
tween the EC of the nutrient solution and air temperature may exist and needs to be con-
sidered when determining the optimal EC for lettuce production in a hydroponic system
[45,46].
In the current study, Hs-H combined with each biostimulant (i.e., FA, SE, and PGA)
elevated lettuce yield to a level comparable to or higher than that of the Fs-H treatment,
Agronomy 2022, 12, 786 11 of 17

Previous studies have reached the same conclusion, but an interaction between the EC
of the nutrient solution and air temperature may exist and needs to be considered when
determining the optimal EC for lettuce production in a hydroponic system [45,46].
In the current study, Hs-H combined with each biostimulant (i.e., FA, SE, and PGA)
elevated lettuce yield to a level comparable to or higher than that of the Fs-H treatment,
which is in agreement with previous studies [15,47,48]. As fertilizer prices have been
increasing and may continue to increase in the future, biostimulant supplementation
in the currently adopted hydroponic solutions may be a more sustainable approach to
increase crop production. Additionally, we compared the efficacy of different categories of
biostimulants on plant growth. SE and FA improved shoot biomass more efficiently than
PGA did in both cultivars. SE and FA were equally effective in butterhead with regard to the
shoot biomass, which is consistent with a previous study reporting no significant difference
in shoot FW between Ascophyllum nodosum extract and humic acid treatments [48]. In
contrast, SE was more effective than FA in improving shoot FW of red oak-leaf, indicating
that different cultivars differed in their response to exogenous biostimulants.
Root biomass responded differently to biostimulant treatments compared to shoot
biomass. FA exhibited a lower performance relative to that of SE and PGA with regard
to enhancing root FW; consequently, the lowest and highest R/S was observed for FA
and PGA, respectively. A similar result was reported in a study evaluating the impact of
different types of leonardite-derived humic acid on 60-day old corn seedlings [49]; this
study indicated that fulvic acid (i.e., low molecular weight humic acid) enhanced shoot
and root biomass, but a decrease in the R/S ratio from 1.3 to 0.84 was observed. A meta-
analysis also revealed that a brown coal-derived humic substance had no significant effect
on root growth [50]. Moreover, Wang et al. [51] found that the addition of PGA significantly
improved the shoot and root biomass concentrations and R/S ratio of cucumber seedlings.
Taken together, these results indicate that the effect of FA on lettuce growth is more evident
in shoots than in roots, and this trend is reversed for PGA.

4.2. Crop Quality

Biostimulants affect biomass production and improve crop quality by altering plant
physiology and metabolism [24,40]. Excessive accumulation of nitrates in vegetables is
a common issue that poses a potential threat to human health [52]. Our studies demon-
strated a decreasing trend in nitrate concentration following the addition of biostimulants
relative to that achieved using Hs-H (Figure 2A). This is especially true for the PGA treat-
ment, which decreased the nitrate concentration in the shoot by ~50% in butterhead lettuce.
A similar conclusion was reached in a study evaluating hydroponically cultivated maize
seedlings [53]. Their results indicated that adding protein hydrolysates could increase plant
shoot and root growth while decreasing nitrate concentration by >85%. In addition, Di
Mola et al. [54] found that the foliar application of legume-derived protein hydrolysates
and seaweed extract can increase lettuce yield and quality. However, PGA resulted in
significantly lower leaf nitrate content than SE content under various mineral N rates
(i.e., 0–30 kg N kg−1 ). Protein hydrolysates offer abundant organic nitrogen, including
polypeptides and amino acids [55]. Compared to mineral fertilizers that directly provide
nitrate, organic fertilizers gradually release nitrogen, limiting the excessive accumulation
of nitrate in vegetables [52]. The beneficial effects of seaweed extract on the quality of
horticultural crops such as tomato, mint, basil, grape, and olive have been previously
reported [21]. However, studies on lettuce are limited. Soluble sugar, soluble protein,
flavonoids, anthocyanins, and chlorophyll content were the common traits used to evalu-
ate the quality of lettuce. These components modulate the plant photosynthesis process
and nitrate assimilation and play important roles in human health. Flavonoids also play
an important role in flavor (i.e., bitterness) [56–58]. The current study found that the SE
treatment significantly decreased leaf soluble sugar concentration compared to that of
the negative control treatment in both butterhead and red oak-leaf cultivars (Figure 2B,E).
PGA slightly reduced flavonoid content in butterhead but significantly increased it in red
Agronomy 2022, 12, 786 12 of 17

oak-leaf. Similar inconsistent effects on quality traits (i.e., soluble sugars and carotenoids)
following the application of protein hydrolysates and amino acids have been reported in
two carrot cultivars [59]. Such changes in physiochemical quality traits are possibly due to
SE and PGA being able to alter carbon metabolism [47,60].

4.3. Nutrient Uptake

Although extensive studies indicate that different categories of biostimulants could pro-
mote the uptake of plant macro and micronutrients by stimulating root growth [23,24,40,50],
inconsistencies in their effectiveness may exist. For example, the effect of applied biostim-
ulants on shoot nitrogen concentration and content showed an opposite trend between
a butterhead and red oak-leaf, indicating an apparent genotypic effect on nitrogen accu-
mulation. Typical leonardite-derived humic substances contain only 1–2% nitrogen, which
may not be an adequate contributor to the nitrogen pool and may not be readily available
for plant uptake [61]. In the current study, root biomass did not notably improve following
the FA treatment. However, FA treatment still significantly enhanced N uptake in red
oak-leaf, suggesting that other mechanisms may be involved in FA-mediated enhancement
in N assimilation in certain cultivars. In addition, Xu et al. [31] reported that γ-PGA was
involved in nitrogen metabolism, improving nitrogen reduction and assimilation. However,
this study showed that the application of PGA did not significantly improve N uptake, even
though the N content in PGA was greater than that in FA and SE (Table 2). A relatively low
utilization rate (6–25%) of amino acids that are added to the cultivation medium by plants,
mainly due to microorganism competition and hydrolysis of amino acids, may explain this
phenomenon [62]. Taken together, the impact of biostimulants on nutrient uptake varies
depending on plant variety, substrate conditions, and application rates [63]. Our study
evaluated only one application rate for each biostimulant according to the recommended
foliar spray or soil application rate. Thus, the optimal rate for hydroponic cultivation needs
to be determined in the future.
Higher Zn and Mn uptake was detected in the FA and SE treatments in this study.
One possible reason is that the FA and SE treatments contained a high concentration of
the respective micronutrients, possibly in a form readily available for plant absorption
(Table 2). Meanwhile, an increase in root growth, particularly the lateral root, could
enhance the capability of plants to acquire nutrients [24,40,47,60]. In contrast, the Fe
concentrations in FA and SE were also abundant. However, an increase in Fe uptake was
not observed, possibly because Fe exists in forms that are not readily available to plants
or for the competitive uptake of other cations. In this study, the two evaluated cultivars
responded differently to added biostimulants with respect to nutrient concentration and
uptake, indicating that the role of genotype should not be ignored when comparing the
efficacy of different biostimulants.

4.4. Mode of Action

Currently, determining the mode of action of biostimulants is difficult because of the
complex nature of the components. Standardization is challenging because the source
material may vary in composition and quality [20]. The underlying mechanism affecting
lettuce growth may differ among seaweed extracts, fulvic acid, and γ-PGA. For exam-
ple, extensive studies have reported that Ascophyllum nodosum-based commercial sea-
weed extract could enhance the fresh matter production of different crops. Craigie [64]
suggested that the higher content of plant hormones in seaweed extract (including ab-
scisic acid, gibberellic acid, auxins, brassinosteroids, and cytokinins) is responsible for
improved plant growth [60], especially root growth [65]. However, experiments conducted
by Mondal et al. [66] demonstrated that K. alvarezii extracts, from which gibberellins and
auxins were removed by solvent extraction, maintained their activity (such as increasing
maize (Zea mays L.) biomass production and grain yield). This suggests that gibberellins
and auxins may not be the active ingredients in the seaweed extract. Fulvic acid is the major
constituent of humic substances with low molecular weight and has been reported to have
Agronomy 2022, 12, 786 13 of 17

the greatest stimulatory effect on plant growth, physiological metabolism, and assimilate
accumulation. Unlike seaweed extract, phytohormones in fulvic acid were detected in
many studies [67–69]; however, it has been well established that humic substances are
positively correlated with plant shoot and root growth, physiological performance, and
nitrogen metabolism in higher plants [23,24,40,50], as other molecules in fulvic acid may
exhibit auxin-like effects.
Biostimulants may play a more critical role when plants are subjected to abiotic
stress [21]. In the current study, the shoot FW after the SE treatment was enhanced two-fold
when compared to that observed after the Hs-H treatment in both butterhead and red oak-
leaf varieties. This may be explained by enhanced tolerance to cold environments irradiated
with weak light after SE application. The present study was performed in Chengdu, China,
during winter, which is characterized by low temperatures and lack of sunlight irradiation,
as illustrated in Figure S1 [70]. During the study period, the daily mean temperature
(approximately 10 ◦ C) was significantly lower than the optimum temperature for lettuce
growth (20–24 ◦ C) [71,72]. Studies on Arabidopsis thaliana have indicated that the lipophilic
components extracted from the brown seaweed Ascophyllum nodosum could alleviate cold
stress by elevating synthesis and accumulation of osmoprotectants and modifying cellular
fatty acid composition [73].
It is generally accepted that biostimulants can enhance crop production. However,
a lack of a yield increase or a negative impact on yield has also been reported in many crops
such as lettuce [74], tomato [75], and wheat [76,77]. Therefore, one should proceed with
caution when deciding whether to use biostimulant directly or the type and application
rate of a particular biostimulant [54,78]. For example, the effectiveness of humic substances
is coordinated by the application rate, properties, environmental conditions, and plant
type [50]. Humic substances could be divided into different categories based on the
extracted sources, and brown-coal-extracted humic substances are common commercial
products [50]. However, humic substances extracted from brown coal did not notably
improve root biomass, whereas those extracted from peat and manure promoted root
growth by 12–40% [50].

5. Conclusions
Compared to the half-strength nutrient solution treatment, the full-strength nutrient
solution further improved biomass; however, it did not increase crop quality traits or
nutrient uptake. The supplemental fulvic acid, seaweed extract, and γ-PGA solutions
improved shoot and root biomass by different magnitudes when compared to that achieved
using the half-strength nutrient solution. FA resulted in the lowest R/S ratio, while
SE resulted in the lowest soluble sugar and chlorophyll contents among all treatments.
The addition of biostimulants tended to reduce nitrate accumulation in the leaves. Crop
responses to biostimulants with regard to nutrient concentration varied according to
cultivar and mineral nutrient contents.

Supplementary Materials: The following supporting information can be downloaded at: https://
www.mdpi.com/article/10.3390/agronomy12040786/s1, Figure S1: Daily maximum and minimum
temperatures and daily light integral during the period of lettuce growth.
Author Contributions: Conceptualization, Z.W. and R.Y.; data curation, Z.W., R.Y., Z.Z. and Y.L.;
formal analysis, Y.L. and Z.Z.; investigation and methodology, S.Z. and C.S.; resources, J.L.; funding
acquisition and validation, Z.Q.; supervision, Q.Y.; writing—original draft, Z.W., R.Y. and Y.L.;
writing—review and editing, Z.W., R.Y. and Q.Y. All authors have read and agreed to the published
version of the manuscript.
Funding: This research was funded by the National Key Research and Development Program, Min-
istry of Science and Technology of China (No. 2020YFE0203600), Sichuan Science and Technology
Program (No. 2021YJ0500 and 2021JDRC0120), Central Public-Interest Scientific Institution Basal
Research Fund (No. Y2021XK04 and Y2021XK02), the Agricultural Science and Technology Inno-
vation Program of Chinese Academy of Agricultural Sciences (No. ASTIP-CAAS, 34-IUA-03 and
Agronomy 2022, 12, 786 14 of 17

34-IUA-04), the Local Financial Project of the National Agricultural Science and Technology Center
(No. NASC2021ST02 and NASC2020AR09), and the Key Research and Development Program of
Chengdu (No. 2021-YF09-00108-SN).
Institutional Review Board Statement: Not applicable.
Informed Consent Statement: Not applicable.
Data Availability Statement: Not applicable.
Acknowledgments: We acknowledge Tao Huang, Hui Jiang and Jiamin Wang for their assistance
with the experimental setup, sample collection, and analysis.
Conflicts of Interest: The authors declare no conflict of interest.

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