Comparative Effects of Root and Foliar Leonardite-Suspension Concentrate Application on Plant Growth and Photosynthetic Efficiency of Lettuce Plants (Lactuca sativa L.)

Abstract

Humic substances (HS) have been defined as a potential plant biostimulant to improve crop yield in a sustainable and environmentally friendly way. Leonardite-suspension concentrate (SC) is a type of HS extracted from lignite that is currently employed to enhance various physiological aspects of plants. However, the different effects between both modes of SC application (root and foliar) are poorly understood, especially on photosynthesis performance. Therefore, this study aimed to investigate the influence of a leonardite-SC-based product (BLACKJAK®), on lettuce growth and photosynthesis efficiency, while comparing both methods of application. For this purpose, four root (R): R1 (0.20 mL/L), R2 (0.40 mL/L), R3 (0.60 mL/L), and R4 (0.80 mL/L), and four foliar: F1 (5.00 mL/L), F2 (7.50 mL/L), F3 (10.00 mL/L), and F4 (12.50 mL/L) BLACKJAK® doses were applied to lettuce plants. Related shoot and root growth parameters, photosynthetic efficiency, and sugar and starch content were assessed in lettuce plants. The results showed that BLACKJAK® improved shoot and root biomass, foliar area, and root length, especially at intermediate doses (R2, R3, F2, and F3), with R3 demonstrating the greatest growth increases. Similarly, the main photosynthetic parameters analyzed (net photosynthetic rate and Rubisco carboxylation efficiency), and the soluble sugars and starch content were improved by the same doses, with R3 showing the best photosynthetic performance. Hence, our study suggests that BLACKJAK® improves lettuce yield and photosynthetic efficiency, particularly with radicular application at R3.

Full text

Vol.:(0123456789)
Journal of Plant Growth Regulation (2024) 43:4667–4679
https://doi.org/10.1007/s00344-024-11424-6
Comparative Effects ofRoot andFoliar Leonardite‑Suspension
Concentrate Application onPlant Growth andPhotosynthetic
Efficiency ofLettuce Plants (Lactuca sativa L.)
SantiagoAtero‑Calvo1 · FrancescoMagro2· GiacomoMasetti2· EloyNavarro‑León1· JuanJoseRios1·
BegoñaBlasco1· JuanManuelRuiz1
Received: 25 October 2023 / Accepted: 27 June 2024 / Published online: 20 July 2024
© The Author(s) 2024
Abstract
Humic substances (HS) have been defined as a potential plant biostimulant to improve crop yield in a sustainable and envi-
ronmentally friendly way. Leonardite-suspension concentrate (SC) is a type of HS extracted from lignite that is currently
employed to enhance various physiological aspects of plants. However, the different effects between both modes of SC
application (root and foliar) are poorly understood, especially on photosynthesis performance. Therefore, this study aimed
to investigate the influence of a leonardite-SC-based product (BLACKJAK®), on lettuce growth and photosynthesis effi-
ciency, while comparing both methods of application. For this purpose, four root (R): R1 (0.20mL/L), R2 (0.40mL/L), R3
(0.60mL/L), and R4 (0.80mL/L), and four foliar: F1 (5.00mL/L), F2 (7.50mL/L), F3 (10.00mL/L), and F4 (12.50mL/L)
BLACKJAK® doses were applied to lettuce plants. Related shoot and root growth parameters, photosynthetic efficiency,
and sugar and starch content were assessed in lettuce plants. The results showed that BLACKJAK® improved shoot and
root biomass, foliar area, and root length, especially at intermediate doses (R2, R3, F2, and F3), with R3 demonstrating
the greatest growth increases. Similarly, the main photosynthetic parameters analyzed (net photosynthetic rate and Rubisco
carboxylation efficiency), and the soluble sugars and starch content were improved by the same doses, with R3 showing the
best photosynthetic performance. Hence, our study suggests that BLACKJAK® improves lettuce yield and photosynthetic
efficiency, particularly with radicular application at R3.
Keywords Biostimulant· Humic substances· Lettuce· Plant growth· Photosynthesis
Abbreviations
A Net photosynthetic rate
CE Carboxylation efficiency
Ci Intercellular CO2
E Transpiration rate
FA Fulvic acids
gs Stomatal conductance
Jmax Maximum rate of electron transport
Ls Stomatal limitation
HA Humic acids
HS Humic substances
PS Photosystem
RGR Relative growth rate
Handling Editor: Soumya Mukherjee.
* Santiago Atero-Calvo
satero@ugr.es
Francesco Magro
fmagro@sipcam.com
Giacomo Masetti
gmasetti@sofbey.com
Eloy Navarro-León
enleon@ugr.es
Juan Jose Rios
jjrios@ugr.es
Begoña Blasco
bblasco@ugr.es
Juan Manuel Ruiz
jmrs@ugr.es
1 Department ofPlant Physiology, Faculty ofSciences,
University ofGranada, 18071Granada, Spain
2 Sofbey S.A., Via San Martino 16/A, 6850Mendrisio,
Switzerland
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4668 Journal of Plant Growth Regulation (2024) 43:4667–4679
Vcmax Rubisco maximum carboxylation rate
WUE Water use efficiency
Introduction
Feeding the world’s growing population has been and
continues to be the main challenge for most agricultural
researchers. It is estimated that the global population will
increase by 2 billion people by 2050, reaching a total of
9.7 billion people. As a result, food production will have to
increase by 60% over current production to feed the grow-
ing population (Muhie 2022; Simkin etal. 2019). Besides,
climate change and the abiotic stress conditions (salinity,
drought, high temperatures, etc.) are expected to consider-
ably reduce crop yield. Furthermore, a quarter of greenhouse
gas emissions come from agricultural practices which con-
tributes significantly to climate change (Muhie 2022). In
this way, chemical fertilizers used in an uncontrolled manner
greatly promote the release of carbon dioxide (CO2) into the
atmosphere, by altering physical and chemical soil proper-
ties such as pH, aeration, C/N ratio, affecting soil carbon
dynamics (Chi etal. 2020; Cao etal. 2023). Therefore, it is
imperative to discover an equilibrium between augmenting
food production for feeding the population while reducing
the overuse of chemical fertilizers.
Plant biostimulants have been described as a potential
tool to increase crop yield in a sustainable and environmen-
tally friendly way, while reducing the uncontrolled use of
chemical fertilizers. A brief definition of plant biostimulant
could be “any substance (organic or inorganic compound)
or microorganism that when it is applied to plant, improve
crop yield and quality, nutrient uptake and utilization, and
abiotic stress tolerance.” According to this definition, the
main categories of biostimulants are plant and seaweed
extracts, humic substances, protein hydrolysates, inorganic
compounds such as silica (Si), and beneficial bacteria, and
fungi (du Jardin 2015).
Humification is the process of biodegradation of the
soil organic matter (plants, animals, and microorganisms
waste) that results in humic substances (HS) generation.
Based on their physicochemical properties, HS are clas-
sified into three categories: (i) humin (insoluble in all pH
conditions); (ii) humic acids (HA) (soluble in alkaline
pH); and (iii) fulvic acids (FA) (soluble in alkaline and
acidic conditions) (Yang etal. 2021). Different natural
sources are used for HS extraction: composts and ver-
micomposts, volcanic soils, peat, and oxidation products
of lignite as leonardite (du Jardin 2015; Wei etal. 2023).
Leonardite is widely used by companies for commercial
HS generation as suspension concentrate (SC), which are
frequently employed by farmers and researchers (Consel-
van etal. 2017). HS may be applied to plants by foliar
spraying or to the soil solution (radicular or root applica-
tion), and the most studies published that can be found in
literature employ a single method of application.
Concerning to the beneficial effects of humic substances
on plant growth, they can exert their influence at various lev-
els, spanning from the soil to the leaves. Thus, HS improve
the bioavailability of essential nutrients such as nitrogen,
phosphorous, potassium, and others, as well as their uptake
by roots and the nutrient use efficiency (Yuan etal. 2022;
Zanin etal. 2019). Besides, different research studies have
shown the positive influence of HS in primary and second-
ary metabolism, improving photosynthetic efficiency (Fan
etal. 2014), tricarboxylic acid cycle (Nardi etal. 2007),
nitrogen and sulfur assimilation (Jannin etal. 2012; Zanin
etal. 2018), and phenolic metabolism (Savarese etal. 2022).
In addition, HS may change phytohormonal profile and act
as physiological signal emulating hormone functions (Chen
etal. 2022). As a result, plant growth can be enhanced by
HS application, increasing fresh and dry matter of shoots
and roots. Therefore, HS play an important role in modern
agriculture to increase crop production in a sustainable way
with a reduced environmental impact compared to chemical
fertilizers (Tiwari etal. 2023).
With respect to photosynthesis, improving the process of
light conversion into biomass is synonymous with increased
plant growth and productivity (Simkin etal. 2019). Some
studies have shown that biomass production under glass-
house and field conditions may be increased by enhancing
the photosynthesis process (Ding etal. 2016; Simkin etal.
2017). There are three main avenues for increase photosyn-
thetic efficiency: (i) improving the stomatal conductance (g)
and consequently the mesophyll intercellular CO2 (Ci) (ii)
enhancing the carboxylation efficiency of Rubisco, and (iii)
improving electron transport flux efficiency at photosyn-
thetic complex level (Araus etal. 2021). To achieve these
goals, most studies use techniques for the genetic manipula-
tion of enzymes in the photosynthetic process. Alternatively,
plant biostimulants such as HS have significance potential
to enhance photosynthesis by reducing fluorescence dissipa-
tion. This reduction results in more light energy utilization
for photosynthesis process (Canellas etal. 2013; Fan etal.
2014). Furthermore, HS may enhance Rubisco activity, lead-
ing to increased CO2 fixation and, consequently, a higher net
photosynthetic rate (Chen etal. 2022). However, research
comparing the effect on photosynthesis process between
root and foliar application of HS extracted from different
sources, such as leonardite, is scarce. Therefore, this study
aims to evaluate the growth and photosynthesis performance
of lettuce plants subjected to both root and foliar applica-
tions of HS. As the HS source, we used a leonardite-SC
named BLACKJAK®, which has yet to be examined for its
effects on photosynthesis performance of horticultural crops
through different application strategies.
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4669Journal of Plant Growth Regulation (2024) 43:4667–4679
Materials andMethods
Plant Material andGrowing Conditions
Lettuce plants (Lactuca sativa cv. Capitata) were used
as plant material for this work. Seeds of these plants
were germinated and grown in a tray with cells (size
3 × 3 × 10cm) for 45days. Afterward, lettuce seedlings
were transferred to individual pots (13cm upper diameter,
10cm lower diameter, 12.5cm high, and a volume of 2 L)
filled with 3:1 mixture of vermiculite:perlite, and distrib-
uted in a culture chamber under controlled environmental
conditions with temperature 25/15°C (day/night), rela-
tive humidity 60–80%, and 16/8h ofphotoperiod with a
photosynthetic photon-flux density of 350μmol m−1 s−1
(measured with a sensor SB quantum 190, LI-COR Inc.,
Lincoln, NE, USA). Throughout the experiment, lettuce
plants were irrigated with a complete Hoagland nutritive
solution (Hoagland and Arnon 1950), with small modi-
fications for the correct growth of lettuce, composed of
4mM KNO3, 1 mM KH2PO4, 1mM NaH2PO4·2H2O,
3mM Ca(NO3)2·4H2O, 2mM MgSO4·7H2O, 5µM Fe-
chelate (Sequestrene; 138FeG100), 2µM MnCl2·4H2O,
0.25mM CuSO4·5H2O, 1µM ZnSO4·7H2O, 10µM HBO3,
and 0.1µM Na2MoO4·2H2O (pH 5.5–6). The nutrient solu-
tion was renewed every 3 days.
Humic Substances Application andExperimental
Design
Treatments started 7days after transplantation and main-
tained for 30days. These treatments consisted of the appli-
cation of HS using BLACKJAK®, a leonardite-SC-based
product provided by the company Sofbey, S.A. (Mendrisio,
Switzerland), composed of 30% of organic matter, with
an acidic pH (4, 5). BLACKJAK® was applied to lettuce
plants 3 times with 10-days intervals by both radicular
and foliar application. Radicular (‘R’) HS were added
to Hoagland nutritive solution at four different concen-
trations: 0.20mL/L (R1), 0.40mL/L (R2), 0.60mL/L
(R3), and 0.80mL/L (R4). On the other hand, foliar (‘F’)
application was carried out by spraying lettuce leaves
with HS at four doses: 5.00mL/L (F1), 7.50mL/L (F2),
10.00mL/L (F3), and 12.50mL/L (F4). Those concen-
trations were selected based on commercial ranges and
according to a previous screening using radicular and
foliar doses on lettuce, where lower doses than those
employed had no physiological effect, whereas higher
doses resulted in toxicity. The foliar treatments were made
2h after switching on the light of the growth chamber.
Furthermore, a control treatment was conducted applying
Hoagland nutritive solution without HS. The experimen-
tal design consisted of a complete randomized block with
nine treatments, eight plants per treatment arranged in
individual pots with the treatments randomly distributed
in the culture chamber.
Sampling andPlant Growth Measurements
Plants of each treatment were sampled 30days after the first
HS application. The leaves and roots of all lettuce plants
were sampled, washed using distilled water, dried on filter
paper, and weighed for the fresh weight (FW) determina-
tion. These lettuce leaves of each treatment were frozen at
−45°C for subsequent biochemical analyses. To deter-
mine the relative growth rate (RGR), leaves and roots were
sampled before starting the HS application (initial time,
Ti = 0days) and weighed (initial fresh weight, FWi). At the
end of the experiment (final time, Tf = 30days), the FW of
leaves and roots (final fresh weight, FWf) from each treat-
ment was used to estimate the RGR, using the equation
RGR = (ln FWf − ln FWi)/(Tf−Ti) (Navarro-León etal.
2019). On the other hand, a LI-COR optical reader, model
LI-3000A (LI-COR Inc. Nebraska, USA), was employed to
determine the leaf area and root surface area, while the root
length was measured using a ruler.
Quantification ofPhotosynthetic Pigments
Chlorophylls (Chl a and Chl b) as well as carotenoids were
extracted from 0.1g of frozen lettuce leaves by adding 1mL
of methanol and then centrifugated at 5000×g for 5min.
Absorbance from the supernatant was measured at 3 differ-
ent wavelengths: 653nm, 666nm, and 470nm. From the
absorbance values, photosynthetic pigments concentration
was calculated using the equations described by Wellburn
(1994).
Estimation ofChl a Fluorescence Parameters
Six plants from each treatment were adapted to dark for
30min before Chl a fluorescence measure using a special
leaf clip holder allocated in fully expanded leaves at mid-
stem position. Chl a fluorescence kinetics was measured
using the Handy PEA Chlorophyll Fluorimeter (Hansatech
Ltd., King’s Lynn, Norfolk, UK) by the induction of red light
(650nm) with 3000μmol photons m−2 s−1 light intensity.
Chla=15.65 ×A666−7.34 ×A653
Chlb=27.05 ×A653−11.21 ×A666
Carotenoids
=
(
1000 ×A470−2.86 ×Chla−129.2 ×Chlb
)
∕
221
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4670 Journal of Plant Growth Regulation (2024) 43:4667–4679
Before taking measurements in the experiment, the Handy
PEA was calibrated by measuring a lettuce leaf. The JIP-test
was used to analyze the Chl a fluorescence transient. The
parameters employed in the present study to determine the
effect of HS on photosynthetic activities were as follows:
maximum quantum yield for primary (Fv/Fm, where Fv is
variable fluorescence calculated as Fv = Fm − Fo, being Fm
the maximum fluorescence and Fo the initial fluorescence),
proportion of active reaction centers (RCs) (RC/ABS),
performance index for energy conservation from photons
absorbed by photosystem II (PSII) antenna to the reduc-
tion of QB (PIABS), the efficiency/probability with which a
PSII trapped electron is transferred from QAto QB(ΨEo),
maximum quantum yield of electron transport (ΦEo), and
the number of times that QAis reduced from time 0 to time
that Fm is reached (N) (Roháček 2002; Strasser etal. 2004).
Determination ofLeaf Gas Exchange Measurements
Gas exchange parameters were recorded between 10.00 a.m
and 02.00 p.m in fully expanded leaves at midstem posi-
tion in six plants per treatment using a LI-6800 Portable
Photosynthesis System infrared gas analyzer (IRGA: LI-
COR Inc. Nebraska, USA), equipped with 6800-01A leaf
chamber (6 cm2 aperture). Version 1.3.17 of the LI-COR
software was used for data collection in the present study,
and system warmup tests were run before measurements
according to manufacturer’s recommendations. After suc-
cessfully passing warmup tests, environmental conditions
of leaf chamber were adapted to physiological demands of
lettuce as described by some authors (Hidalgo-Santiago
etal. 2021). In this way, all gas exchange parameters were
measured at 350μmol m−2 s−1of photosynthetically active
radiation (PAR), 400µmol mol−1 CO2 concentration, leaf
temperature at 30°C, relative humidity at 70%, and chamber
fan mixing speed at 10,000rpm. For each plant, 9 meas-
urements were taken, and the mean was expressed for the
parameters analyzed: net photosynthetic rate (A), transpira-
tion rate (E), intercellular CO2 (Ci), and stomatal conduct-
ance (gs) (Márquez etal. 2021; Saathoff and Welles 2021).
The water use efficiency (WUE) was calculated as A/E, the
carboxylation efficiency (CE) was estimated as A/Ci, and the
stomatal limitation (Ls) was assayed as 1−Ci/Ca (where Ca
represents the ambient CO2 concentration) (Ma etal. 2019;
do Rosário Rosa etal. 2021).
On the other hand, after each measurement of these
parameters, a rapid A-Ci response curve (RACiR) was
used to estimate the Rubisco maximum carboxylation rate
(Vcmax) and the maximum rate of electron transport (Jmax).
For the RACiR curve, the reference CO2 was adjusted so that
increasing concentrations of CO2 from 10 to 510µmol mol−1
were applied to the leaf through the chamber. As the applied
CO2 concentration increased, the LI-6800 measured the net
photosynthetic rate and the intercellular CO2 every 2s for
7min. To correct the RACiR curve data recorded in LI-6800
software, a RACiR curve was done with the chamber closed
and without plant leaf before the first measure. To determine
Vcmax and Jmax, the ‘plantecophys’ package in R described
by Duursma (2015) was used by fitting the data with the
traditional Farquhar etal. (1980) model.
Quantification ofSoluble Sugars andStarch
Concentration
The extraction and quantification of soluble sugars (sucrose,
glucose, and fructose) and starch were assayed according to
Dien etal. (2019) with small modifications. 0.1g of frozen
lettuce leaves were homogenized in 1mL of ethanol 83%.
After centrifugation (2800×g for 10min), supernatant was
used for soluble sugars determination, while the pellet was
oven-dried at 40°C for 48h and the resulting dry residue
was employed to quantify the starch content. 100 µL of
supernatant was added into glass tubes of 50mL capacity
with 3mL anthrone 0.1%. This mixture was incubated at
100°C for 10min. Afterward, soluble sugars were deter-
mined at 650nm against a standard curve of glucose. On
the other hand, 250 µL of distilled water, 250 µL of 4M
sodium acetate buffer (pH 4.5), and 250µL of glucoamylase
0.5% were added to the dry residue for starch determination.
After incubation at 40°C for 48h, the samples were filtered
and 100 µL of supernatant were added into glass tubes of
50mL capacity with 3mL anthrone 0.1%. After 10min of
incubation at 100°C, starch was quantified at 650nm using
a standard curve of glucose.
Statistical Procedures
The data were subjected to a simple ANOVA at 95% confi-
dence, using the Statgraphics Centurion 16.1.03 software.
Means were compared by Fisher’s least significant differ-
ences (LSD) and the significance levels were expressed as
*p < 0.05, **p < 0.01, ***p < 0.001, or NS (not significant).
For growth parameters, a total of eight replicates were
employed, whereas six replicates were used for photosyn-
thetic parameters and nine for biochemical analysis.
Results
Effect ofHS onLettuce Plants Growth
Leonardite-based product significantly increased biomass
production in terms of shoot fresh weight with respect to the
control at doses: R1 (16%), R2 (17%), R3 (23%), F2 (13%),
and F3 (13%). In this way, the leaf area and leaf RGR were
also increased by the same HS doses. The application of
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4671Journal of Plant Growth Regulation (2024) 43:4667–4679
root-HS offered better results in terms of shoot growth than
foliar HS, showing plants treated with R3 dose the highest
values (Table1; Fig.1A, B). On the other hand, radicular
HS applied at doses R2, R3, F2, F3, and F4 enhanced root
fresh weight with an increase of 32%, 33%, 15%, 16%, and
21%, respectively, compared to control. Besides, root RGR
was also improved by the same treatments. Therefore, as
for shoot growth, radicular applications resulted in greatest
increase in root biomass production and root RGR, with R3
presenting the highest values. Furthermore, R2, R4, and F3
Table 1 Effect of root and foliar application of humic substances on leaf and root biomass, leaf, and root RGR, foliar area, root length, and root
surface area
Values are means ± standard deviation (n = 8). The levels of significance were represented as NS (p>0.05), *(p < 0.05), ***(p < 0.001). Values
with different letters indicate significant differences
Shoot FW
(g−1 plant)
Leaf RGR
(g g−1 day−1)
Leaf area
(cm2)
Root FW
(g−1 plant)
Root RGR
(g g−1 day−1)
Root length (cm) Root surface area (cm2)
Control 49.65 ± 1.30c0.098 ± 0.001de 479.38 ± 13.67b2.67 ± 0.09d0.082 ± 0.001d16.68 ± 0.66bc 27.66 ± 0.97ab
R1 57.35 ± 2.76a0.104 ± 0.002ab 529.96 ± 15.81a2.73 ± 0.16cd 0.083 ± 0.002cd 19.28 ± 1.45abc 32.09 ± 2.85ab
R2 58.14 ± 2.07a0.105 ± 0.001ab 526.89 ± 13.41a3.52 ± 0.14a0.091 ± 0.001a21.27 ± 1.82a35.12 ± 2.36ab
R3 61.17 ± 1.90a0.106 ± 0.001a532.05 ± 12.38a3.56 ± 0.10a0.092 ± 0.001a19.99 ± 1.68ab 37.43 ± 4.77a
R4 45.90 ± 2.19c0.097 ± 0.0016e477.38 ± 12.15b2.72 ± 0.08cd 0.083 ± 0.001cd 20.59 ± 0.79a35.51 ± 4.32ab
F1 50.83 ± 1.80bc 0.099 ± 0.001cd 483.72 ± 9.49b2.79 ± 0.08cd 0.084 ± 0.001cd 15.55 ± 0.97c26.80 ± 2.09b
F2 56.07 ± 0.66ab 0.103 ± 0.001bc 528.17 ± 13.08a3.08 ± 0.21bc 0.087 ± 0.002bc 15.68 ± 1.02c34.40 ± 5.59ab
F3 55.91 ± 2.39ab 0.104 ± 0.001ab 528.90 ± 14.17a3.09 ± 0.19bc 0.087 ± 0.002bc 20.59 ± 1.56a31.87 ± 3.12ab
F4 48.36 ± 1.35c0.098 ± 0.001de 498.98 ± 9.72ab 3.22 ± 0.12ab 0.087 ± 0.001ab 20.35 ± 1.86ab 36.61 ± 3.26ab
p-value *** *** * *** *** * NS
LSD0.05 5.43 0.003 37.65 0.39 0.004 3.90 9.97
Fig. 1 Photography showing frontal (A), and zenithal (B) shoot growth of lettuce plants subjected to root and foliar application of humic sub-
stances. Scale represents 10cm
Fig. 2 Photography showing
root growth of lettuce plants
subjected to root and foliar
application of humic sub-
stances. Scale represents 3cm
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4672 Journal of Plant Growth Regulation (2024) 43:4667–4679
significantly enhanced root length with respect to control
plants, whereas no significant difference was observed for
root surface area at any HS dose and form (Table1; Fig.2).
Photosynthetic Efficiency ofLettuce Plants
Subjected toRadicular andFoliar HS
Foliar HS at dose F2 increased Chl a concentration, while
R1 and F1 showed a significant reduction of Chl a compared
to control plants. R4 enhanced Chl b, whereas F2 increased
total chlorophylls concentration. Furthermore, all radicu-
lar doses decreased carotenoids concentration, whereas F3
enhanced it (Table2).
With respect to leaf gas exchange parameters, A was gen-
erally increased by radicular and foliar humic substances
compared to control plants, except for R1 and F4 doses.
Plants subjected to radicular R3 HS presented the high-
est A. The foliar application of HS at doses F1, F2, and F3
enhanced E, Ci, and gs, while F4 significantly decreased E.
Furthermore, plants treated with radicular HS R2 showed
an increased in E and gs, while Ci was enhanced by R2, and
R3. Concerning Ls, a significant decrease was observed in
R2, F1, F2, and F3 treatments. The WUE was enhanced by
R2, R3, R4, F3, and F4, showing plants of R3 treatment the
highest value (Table3). Respect to Vcmax and CE, all doses
and forms of HS increased both parameters except R1 and
F4, with the highest values presented in R3 dose (Fig.3A,
B). Besides, R3, R4, F1, and F2 significantly enhanced Jmax
(Fig.3C).
Regarding fluorescence parameters, no significant differ-
ences were recorded for Fv/Fm (Fig.2A). Lettuce plants
treated with R2, R3, and F4 showed higher values of RC/
Table 2 Effect of root and foliar
application of humic substances
on photosynthetic pigments
concentration
Values are means ± standard deviation (n = 9). The level of significance was represented as ***(p < 0.001).
Values with different letters indicate significant differences
Chl a
(mg g−1 FW)
Chl b
(mg g−1 FW)
Total Chlorophylls
(mg g−1 FW)
Carotenoids
(µg g−1 FW)
Control 0.174 ± 0.006bc 0.084 ± 0.003b0.256 ± 0.009bcd 21.893 ± 0.630bc
R1 0.159 ± 0.006d0.087 ± 0.005ab 0.245 ± 0.011de 18.816 ± 1.938de
R2 0.170 ± 0.003cd 0.084 ± 0.002b0.254 ± 0.004cd 18.990 ± 0.348de
R3 0.163 ± 0.003cd 0.085 ± 0.002b0.248 ± 0.004de 17.576 ± 0.430e
R4 0.181 ± 0.002ab 0.092 ± 0.001a0.273 ± 0.003ab 19.388 ± 0.417de
F1 0.159 ± 0.005d0.076 ± 0.002c0.236 ± 0.006e19.776 ± 0.684cde
F2 0.188 ± 0.004a0.087 ± 0.001ab 0.275 ± 0.005a24.113 ± 0.487ab
F3 0.183 ± 0.002ab 0.086 ± 0.001ab 0.268 ± 0.003abc 26.087 ± 0.934a
F4 0.167 ± 0.003cd 0.081 ± 0.001bc 0.248 ± 0.004de 20.647 ± 0.774cd
p-value *** *** *** ***
LSD0.05 0.011 0.006 0.017 2.453
Table 3 Effect of root and foliar application of humic substances on leaf gas exchange parameters
Values are means ± standard deviation (n = 6). The levels of significance were represented as NS (p > 0.05), *(p < 0.05), **(p < 0.01), and
***(p < 0.001). Values with different letters indicate significant differences
A
(µmol m−2 s−1)E
(mmol m−2 s−1)Ci
(µmol mol−1)gs
(mol m−2 s−1)Ls WUE
Control 3.87 ± 0.49c1.17 ± 0.04de 258.30 ± 2.83c0.100 ± 0.003de 0.32 ± 0.02ab 3.28 ± 0.36d
R1 4.21 ± 0.22c1.21 ± 0.02de 263.41 ± 5.24c0.078 ± 0.013e0.33 ± 0.01a3.49 ± 0.19d
R2 6.99 ± 0.45ab 1.51 ± 0.10b285.54 ± 7.68ab 0.134 ± 0.009bc 0.25 ± 0.04c4.65 ± 0.34bc
R3 7.52 ± 0.55a1.29 ± 0.01cd 284.02 ± 2.20ab 0.108 ± 0.001cd 0.26 ± 0.03bc 5.37 ± 0.12a
R4 5.81 ± 0.28b1.08 ± 0.07e274.43 ± 4.70bc 0.108 ± 0.018cd 0.28 ± 0.02abc 5.08 ± 0.37abc
F1 6.51 ± 0.21ab 1.80 ± 0.07a284.02 ± 7.44ab 0.168 ± 0.010a0.25 ± 0.03c3.62 ± 0.13d
F2 6.12 ± 0.32b1.78 ± 0.05a295.60 ± 2.67a0.156 ± 0.005ab 0.24 ± 0.01c3.45 ± 0.26d
F3 6.92 ± 0.69ab 1.38 ± 0.06bc 295.53 ± 4.03a0.151 ± 0.011ab 0.25 ± 0.01c4.43 ± 0.17c
F4 4.38 ± 0.12c0.77 ± 0.06f270.28 ± 13.20bc 0.073 ± 0.011e0.29 ± 0.02abc 5.30 ± 0.02ab
p-value *** *** ** *** * ***
LSD0.05 1.19 0.17 18.72 0.030 0.06 0.71
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4673Journal of Plant Growth Regulation (2024) 43:4667–4679
ABS (Fig.2B). Concerning PIABS, it was also enhanced by
HS application at R2, R3, F2, F3, and F4, with the high-
est value recorded by R3 dose (Fig.4C). Besides, ΨEo and
ΦEo were increased by F3 and F4 application (Fig.4D, E),
while a significant reduction of N was found in lettuce plants
treated with R2 and R4 humic substances doses (Fig.4F).
Effect ofHS onSoluble Sugars andStarch
Concentration
The soluble sugars and starch concentration followed the
same trend. Thus, radicular application of HS at doses R1,
R2, and R4 as well as foliar doses F1, F2, and F3 signifi-
cantly enhanced soluble sugars and starch concentrations,
showing radicular doses the largest increases. However, R3
and F4 decreased soluble sugars, with the lowest value pre-
sented by plants treated with R3, whereas both doses did not
affect starch concentration (Fig.5A, B).
Discussion
Plant biostimulants have been defined as a potential tool
to reduce the uncontrolled use of chemical fertilizers due
to their low impact on the environment and their positive
effects on plant growth and agricultural production (Li etal.
2022). A biostimulant product that has been studied for dec-
ades is humic substances, which constitute more than 60%
of soil organic matter (Canellas etal. 2015). The irrigation
or spraying with humic and/or fulvic acids increase shoot
growth, as has been previously demonstrated by different
authors. In this way, Maji etal. (2017) observed a signifi-
cant increase in the fresh weight of shoots in Pisum sativum
L. plants treated with humic acid-rich vermicompost mixed
with soil. Likewise, the pretreatment of Oryza sativa L.
with HA as well as the foliar application of HA to Physalis
alkekengi L. plants also enhanced shoot growth (Huertas
Tavares etal. 2019; Kazemi etal. 2023). For leafy vegeta-
bles that are consumed fresh, such as lettuce, the shoot fresh
weight, the RGR, and the leaf area are reliable parameters
of crop productivity (Tan etal. 2020). In the present study,
we used BLACKJAK®, a product previously demonstrated
to enhance crop yield under field conditions (Černý etal.
2018). However, under environmental controlled growth
conditions,it has not been analyzed. Additionally, the com-
parative efficacy of its application methods (root versus
foliar) remains unstudied. Therefore, the present study evalu-
ated which application method is more effective in enhance
lettuce growth. Thus, BLACKJAK® application increased
all growth parameters at doses R1, R2, R3, F2, and F3 com-
pared to control plants. As previously reported, the physi-
ological effects of HS depend on different factors such as the
mode and rate of application (Canellas etal. 2015). Thereby,
our results suggest that intermediate radicular doses of the
HS-based product used for this study could provide a greater
shoot growth compared to foliar applications, being R3 the
dose that offered better results.
Fig. 3 Effect of root and foliar application of humic substances
on Vcmax (A), CE (B), and Jmax (C). Values are expressed as
means ± standard error (n = 6). Columns marked with the same letters
were not significantly different based on the LSD test (p < 0.05)
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4674 Journal of Plant Growth Regulation (2024) 43:4667–4679
Similarly, changes in root growth stimulated by HS are
one of the most reported beneficial effects of this type of
plant biostimulant (Canellas etal. 2015; Olaetxea etal.
2017; Rathor etal. 2023). Nunes etal. (2019) observed an
enhanced in root biomass production in maize plants by add-
ing HA to the culture. Similar results were obtained in wheat
plants by radicular FA (Yao etal. 2019), in maize seedlings
roots treated with HA (Zandonadi etal. 2019), in cucumber
Fig. 4 Effect of root and foliar
application of humic substances
on fluorescence parameters Fv/
Fm (A), RC/ABS (B), PIABS
(C), ΨEo (D), ΦEo. (E), and N
(F). Values are expressed as
means ± standard error (n = 6).
Columns marked with the same
letters were not significantly
different based on the LSD test
(p < 0.05)
Fig. 5 Effect of root and foliar
application of humic substances
on soluble sugars (A) and starch
concentration (B). Values are
expressed as means ± standard
error (n = 9). Columns marked
with the same letters were not
significantly different based on
the LSD test (p < 0.05)
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4675Journal of Plant Growth Regulation (2024) 43:4667–4679
plants after foliar spraying with sedimentary HA (De Hita
etal. 2020), as well as in cucumber and Arabidopsis plants
after radicular HA application (Aranaz etal. 2023). In this
experiment, root biomass and root RGR were improved by
HS application at doses R2, R3, F2, F3, and F4 with the
highest values presented by plants treated with radicular HS,
especially at R3 dose. In addition, the positive effects of HA
and FA on crop productivity are mainly due to the modifica-
tions in root architecture with the subsequent stimulation
of nutrients and water uptake (Nunes etal. 2019). Qin and
Leskovar (2020) observed that the application of a com-
mercial lignite HS-based product, composed by 32% HA
and 3% FA, increased root length and root surface area in
four different species (pepper, tomato, watermelon, and let-
tuce). In the present work, only R2, R4, and F3 increased
root length of lettuce plants, although HS application did not
affect root surface area. As it is well known, the source from
which humic substances are obtained, the type of humic sub-
stance applied, the proportion of HA and FA, the chemi-
cal composition, among other factors, may cause different
physiological results between different HS (Canellas etal.
2015). This could explain that our HS-based product did
not affect the root surface area in lettuce plants grown under
control conditions.
Fahramand etal. (2014) suggested that the increase of
root growth induced by HS is more pronounced than that
in the shoot which may be verified with the results obtained
in our experiment. The improvement of root growth by HS
has been defined as “auxin-like effect” through the induction
of root plasma membrane ATPase activity, with the sub-
sequent apoplast acidification and increased root cell wall
plasticity (de Azevedo etal. 2019; Monda etal. 2021; Rathor
etal. 2023). This could explain the increased root growth
observed in our study, both in terms of root biomass and
length. Comparing both modes of application, radicular HS
doses offered better results than foliar HS, which could be
attributed to the direct contact of HS with the root during
radicular applications, subsequently leading to the activa-
tion of the root plasma membrane ATPase (Olaetxea etal.
2017). In addition, previous studies have reported that HS
increase nutrient bioavailability through the formation of
HS-nutrient complexes and also enhance the activity of root
nutrient transporters (García-Mina etal. 2004; Jindo etal.
2016; Tomasi etal. 2013). All of these factors contribute to
improved plant growth. Consequently, direct contact of HS
applied at root level may enhance water and nutrient uptake,
resulting in increased shoot growth compared to foliar appli-
cations. Therefore, the root application of HS used in this
experiment, particularly at R3, could be a better option than
foliar spraying for lettuce growth.
The mechanisms of HS-induced growth enhancement
have been studied with a focus on different aspects of plant
physiology, particularly nutrient uptake and use efficiency
(Canellas etal. 2015; Jindo etal. 2016) as well as induced
changes in phytohormonal profile and the emulation of
hormone functions (Chen etal. 2022; De Hita etal. 2020).
In the present experiment, we focused on photosynthesis
performance as a mechanism of action, aiming to compare
between both modes of application effects on this essential
process. The hypothesis that an increase in photosynthe-
sis efficiency results in an increase in crop yield has been
extensively studied and confirmed (Milenković etal. 2021).
During the photosynthesis process, photosynthetic pigments
such as Chl a and b, and carotenoids are responsible for light
harvesting as well as photoprotection (Simkin etal. 2022).
Although the enhancement in photosynthetic pigments con-
centration induced by humic substances has been extensively
studied by different research authors (Bayat etal. 2021; Fan
etal. 2014), no effect on pigments has also been observed
by others under control conditions (Ali etal. 2015; Bijanza-
deh etal. 2021; Hernandez etal. 2015). In our experiment,
only F2 and F3 enhanced total chlorophylls and carotenoids
concentration, respectively, while radicular HS decreased
carotenoids. Hence, the results obtained suggest that only
foliar spraying with HS positively affected pigments con-
centration at doses that enhanced plant biomass. Moreover,
photosynthetic pigments concentration is not necessarily
linked with plant growth (Trevisan etal. 2010), as can be
observed in radicular-treated plants in our study.
The leaf gas exchange parameters such as A, gs, Ci,
Vcmax, Jmax, and PSII activity offer us an approximation
about leaf photosynthesis efficiency (Coursolle etal. 2019;
Fan etal. 2014; Mumtaz etal. 2020). In our study, R2, R3,
R4, F1, F2, and F3 enhanced A and Jmax by R3, R4, F1,
and F2 treatments. Hence, the results obtained in our experi-
ment suggest that the leonardite-SC-based product generally
enhanced the photosynthesis performance of lettuce plants.
If we compare both application methods, intermediate root
doses, especially R3, offered better results in terms of pho-
tosynthesis efficiency compared to foliar applications. Fur-
thermore, an increase in WUE was observed in plants treated
with R2, R3, R4, F3, and F4, which suggest that these doses
of leonardite-HS could be appropriate for future experi-
ments to increase drought stress tolerance (Rabbani and
Kazemi 2022). The positive influence of HS on photosyn-
thesis has been reported by different authors. Thus, Azcona
etal. (2011) observed an increase in net photosynthesis
in pepper plants subjected to radicular HS derived from
composted sludge, although they did not observe changes
in chlorophylls content. Similar results were obtained by
Haghighi etal. (2012), where HA application to nutrient
solution significantly enhanced photosynthesis efficiency in
lettuce plants. Likewise, foliar HA derived from sediments
improved photosynthesis rate in chrysanthemum (Fan etal.
2014), while similar results were shown in canola plants
treated with foliar HA extracted from vermicompost (Hemati
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4676 Journal of Plant Growth Regulation (2024) 43:4667–4679
etal. 2022), and in maize after foliar HA application (Wang
etal. 2023).
One of the main mechanisms by which HS enhance pho-
tosynthesis performance is the improvement of Rubisco
activity and its CE. In this way, Ertani etal. (2011) found
that a commercial lignosulfonate-humate enhanced Rubisco
activity and consequently the photosynthesis performance,
as has been observed by recent studies in maize plants (Chen
etal. 2022; Ertani etal. 2019). These results are in line with
those obtained in our experiment, where HS doses that
enhanced photosynthesis efficiency also improved Vcmax,
which is synonymous with Rubisco activity (Coursolle
etal. 2019) and CE. Therefore, an increase in Rubisco CE
and activity that results in better photosynthesis perfor-
mance is correlated with an enhance in biomass production
(Milenković etal. 2021; Simkin etal. 2019). Likewise, in
the present experiment, the doses that most enhanced the
photosynthetic efficiency of lettuce plants also increased
shoot biomass, except for F1 dose which did not affect shoot
growth. In addition, consistent with biomass production
results, applying HS to the nutrient solution, particularly
at the R3 dose, could be more effective in enhancing pho-
tosynthesis efficiency compared to foliar HS applications.
As previously described, an increase in plasma membrane
ATPase activity is associated with improved nutrient uptake,
which directly enhances photosynthesis performance (Zhang
etal. 2021). Furthermore, as discussed earlier, direct contact
of radicular HS with roots could lead to a greater increase
in water and nutrient bioavailability, uptake, and utilization
compared to foliar HS, as previously observed by Atero-
Calvo etal. (2023). These positive effects could result in
better stomatal gas exchange, thereby enhancing Rubisco
activity, and WUE, which as described by Guo etal. (2019),
Liu etal. (2013), and Wang etal. (2022), enhances net pho-
tosynthesis and plant growth.
Chlorophyll a fluorescence measurement is used to study
the photochemical reactions of photosynthesis through dif-
ferent parameters (Navarro-León etal. 2023). Thus, RC/
ABS represents how much energy is usable for photosyn-
thesis, PIABS determines the plant vitality, while ΨEo and
ΦEo indicate the electron transport efficiency (Navarro-León
etal. 2018, 2023). Overall, the application of HS improved
the PSII capability to capture light energy according to the
results of fluorescence parameters, especially at doses R2,
R3, F2, F3, and F4, which contributed to photosynthesis
performance of lettuce plants. Fan etal. (2014) found that
the foliar fertilization with HA enhanced the capability of
PSII to use light energy with the consequent increase in A,
which agrees with the results obtained in our study.
The CO2 fixation by Rubisco enzyme results in the gen-
eration of soluble sugars such as sucrose, glucose, and fruc-
tose, and starch, through the named Calvin-Benson cycle
(Simkin etal. 2019). These carbohydrates are the final
products of the photosynthesis process, and its generation
and concentration is correlated with crop yield (Simkin etal.
2019). The increase in sucrose and starch in tobacco plants
with highest photosynthetic CO2 assimilation rates, resulted
in a 30% increase in biomass (Lefebvre etal. 2005), as was
later found in tomato plants with increases in biomass,
sucrose, and starch (Ding etal. 2016). Similarly, our results
showed that the increase in photosynthesis efficiency in
leonardite-HS treatments at doses R2, F2, and F3 enhanced
soluble sugars and starch concentration, which resulted in
enhanced shoot biomass production. Radicular-HS applied
at dose R2 showed higher soluble sugars and starch concen-
tration increment compared to foliar applications. Similar
results were found by Ertani etal. (2011) in maize plants
treated with two different lignosulfonate-humate. Neverthe-
less, the R3 dose reduced soluble sugars and had no effect on
starch concentration. This result might be attributed to the
soluble sugars being converted into complex organic mol-
ecules that facilitate plant growth, similar to the mechanism
proposed by Rosa etal. (2009). Thus, Canellas etal. (2013)
showed that total carbohydrate content and soluble sugars
decreased (−60%) in maize plants after HS application,
which was accompanied by a 17% increase in grain produc-
tion. These results indicated that the soluble sugars may be
redirected toward supporting growth.
In conclusion, the leonardite-SC-based product (BLACK-
JAK®) used in the present study clearly enhanced plant
growth and photosynthesis efficiency at most of the doses
applied, especially at intermediate doses (R2, R3, F2, and
F3). Therefore, our findings show the potential implications
of BLACKJAK® in enhancing crop yields by improving
photosynthesis performance, which is of great importance
for practical applications in agriculture given the need to
feed the world’s growing population. Furthermore, the most
suitable application method for farmers to achieve higher
yields would be through irrigation (radicular application).
Particularly, R3 was the dose that produced the largest
increases in shoot and root growth, as well as photosyn-
thetic activity.
Acknowledgements This research was supported by the PAI program
(Plan Andaluz de Investigación, Grupo de Investigación AGR282) and
by a Grant from the FPU of the Ministerio de Educación y Ciencia
awarded to S.A.C. grant number [FPU20/05049].
Author Contributions FM, GM, JJR, BB, and JMR conceived and
designed research. SAC and ENL conducted experiments. FM and GM
provided biostimulant. SAC and ENL analyzed data. SAC wrote the
manuscript. FM, GM, JJR, BB, and JMR did a critical revision of the
article. All authors read and approved the manuscript.
Funding Funding for open access publishing: Universidad de Granada/
CBUA.
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4677Journal of Plant Growth Regulation (2024) 43:4667–4679
Declarations
Conflict of interest The authors declare that they have no known com-
peting financial interests or personal relationships that could have ap-
peared to influence the work reported in this paper.
Ethical Approval The authors declare that manuscript reporting studies
do not involve any human participants, human data, or human tissue.
Open Access This article is licensed under a Creative Commons Attri-
bution 4.0 International License, which permits use, sharing, adapta-
tion, distribution and reproduction in any medium or format, as long
as you give appropriate credit to the original author(s) and the source,
provide a link to the Creative Commons licence, and indicate if changes
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otherwise in a credit line to the material. If material is not included in
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need to obtain permission directly from the copyright holder. To view a
copy of this licence, visit http://creativecommons.org/licenses/by/4.0/.
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