Eckols and seaweed-biostimulant (Kelpak) improve adaptative responses for phosphorus acquisition in white lupin and cucumber seedlings under phosphorus deficiency

Abstract

Bioactivities of eckol-type phlorotannins, i.e., eckol and EA6 (a fraction that contains 2-phloroeckol and dibenzodioxin-fucodiphloroethol), extracted from Ecklonia maxima, and a commercial seaweed biostimulant (Kelpak®) were investigated in white lupin and cucumber seedlings under different P-supplies. Lupin seedlings were grown with P-supplemented, P-free or P-free media with either eckol or Kelpak®. The cucumber seedlings were raised in hydroponic media with an insoluble salt of P as the sole P source and either supplemented with eckol, EA6, Kelpak® or no biostimulant. In the lupin seedlings, P starvation led to a reduction in root dry matter accumulation which was reversed when eckol or Kelpak® were applied. In cucumber seedlings, all the biostimulants significantly increased root dry matter but only eckol and Kelpak® induced total dry matter accumulation with Kelpak® also promoting shoot dry matter accumulation. In both lupin and cucumber seedlings, phlorotannins and Kelpak® elicited an increase in dry matter allocation to the roots. Eckol and Kelpak® elicited similar effects on cluster root development in lupin seedlings. All the biostimulants increased phosphatase activities in root exudates of lupin and cucumber seedlings. While tissue P contents were only augmented in Kelpak®-treated lupins, eckol, EA6 and Kelpak® treatments led to P-accumulation in the cucumber seedling leaves. The results showed that eckols influenced physiological traits linked to P-acquisition while Kelpak® altered both morphological and physiological traits for coping with P-limitation in both plants. The data suggest that eckol and seaweed biostimulants like Kelpak® may serve as sustainable tools for managing crops under P deprivation.

Full text

Vol.:(0123456789)
Journal of Applied Phycology
https://doi.org/10.1007/s10811-025-03485-1
RESEARCH
Eckols andseaweed‑biostimulant (Kelpak®) improve adaptative
responses forphosphorus acquisition inwhite lupin andcucumber
seedlings underphosphorus deficiency
LukeO.Omoarelojie1 · WendyA.Stirk1 · ManojG.Kulkarni1 · JohannesvanStaden1
Received: 21 January 2025 / Revised: 24 February 2025 / Accepted: 25 February 2025
© The Author(s) 2025
Abstract
Bioactivities of eckol-type phlorotannins, i.e., eckol and EA6 (a fraction that contains 2-phloroeckol and dibenzodioxin-
fucodiphloroethol), extracted from Ecklonia maxima, and a commercial seaweed biostimulant (Kelpak®) were investigated
in white lupin and cucumber seedlings under different P-supplies. Lupin seedlings were grown with P-supplemented, P-free
or P-free media with either eckol or Kelpak®. The cucumber seedlings were raised in hydroponic media with an insoluble
salt of P as the sole P source and either supplemented with eckol, EA6, Kelpak® or no biostimulant. In the lupin seedlings,
P starvation led to a reduction in root dry matter accumulation which was reversed when eckol or Kelpak® were applied.
In cucumber seedlings, all the biostimulants significantly increased root dry matter but only eckol and Kelpak® induced
total dry matter accumulation with Kelpak® also promoting shoot dry matter accumulation. In both lupin and cucumber
seedlings, phlorotannins and Kelpak® elicited an increase in dry matter allocation to the roots. Eckol and Kelpak® elicited
similar effects on cluster root development in lupin seedlings. All the biostimulants increased phosphatase activities in
root exudates of lupin and cucumber seedlings. While tissue P contents were only augmented in Kelpak®-treated lupins,
eckol, EA6 and Kelpak® treatments led to P-accumulation in the cucumber seedling leaves. The results showed that eckols
influenced physiological traits linked to P-acquisition while Kelpak® altered both morphological and physiological traits
for coping with P-limitation in both plants. The data suggest that eckol and seaweed biostimulants like Kelpak® may serve
as sustainable tools for managing crops under P deprivation.
Keywords Eckol· 2-Phloroeckol· Dibenzodioxin-fucodiphloroethol· Kelpak®· Phosphorus deficiency· Seaweed
biostimulant
Introduction
Phosphorus (P) is a critical, yet limiting, mineral element
with immense significance on soil fertility and productivity
in crop production systems. Inorganic phosphate (Pi:
PO2−
4
),
the chemical form of P that plant roots can absorb, consti-
tutes between 20 – 60% of the total soil P content (Wang
etal. 2022). However, most of the soil Pi are largely immo-
bile and unavailable for plant uptake (Tian etal. 2021). This
is due to the strong affinity of Pi to form insoluble salts with
cations like Ca2+, Fe2+ and Mg2+ with a very small fraction
of Pi (usually < 1%) in soil solution (Yuan etal. 2022).
While soil organic P is not readily available to plant roots
for uptake, they can be transformed into soluble Pi via biotic
mineralisation processes by root exudates and microbial con-
version (Tian etal. 2021; Vengavasi etal. 2021).
Plants have evolved several distinct adaptive morpho-
logical and physiological traits to acquire phosphorus since
it is an indispensable element in metabolic processes, cell
and tissue development and overall plant growth. Several
vital cellular processes (e.g., cell signalling and signal
transduction; synthesis of adenosine phosphates, nucleic
acids and phospholipids; enzyme activation and deacti-
vation; and cytoskeleton) require Pi or metabolites with
phosphate group(s) (Ham etal. 2018; Goldy and Caillaud
2023). Under P-limiting conditions, plants generally alter
their root architecture by inhibiting primary root growth and
* Johannes van Staden
rcpgd@ukzn.ac.za
1 Research Centre forPlant Growth andDevelopment, School
ofLife Sciences, University ofKwaZulu-Natal, Private Bag
X01, Pietermaritzburg3209, Scottsville, SouthAfrica
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Journal of Applied Phycology
increasing lateral root formation (Lambers etal. 2006; Ven-
gavasi etal. 2021). In some species, such as members of the
Proteaceae and Fabaceae families, distinct root structures
called “cluster” or “proteoid” roots are formed (Dinkelaker
etal. 1995). Morphologically, these are lateral roots with
numerous and closely spaced short rootlets that develop
from the pericycle of lateral roots to form one to several
dense clusters (Watt and Evans 1999; Lambers etal. 2015;
Vengavasi etal. 2021) which increase the overall root sur-
face area and enhance rhizosphere exploration for improved
Pi-acquisition. Another common physiological response in
plants under Pi-deprivation is an increase in the release of
phosphatases into the rhizosphere via root exudates (Lam-
bers etal. 2006; Han etal. 2022). Phosphatases cleave
phosphoricester bonds in the soil organic P pool thereby
releasing Pi for uptake by plant roots (Han etal. 2022). In
addition to organic acid and proton exudation, cluster roots
also exude phosphatases to facilitate Pi-mobilisation from
the soil (Skene 2001; Egle etal. 2003; Lambers etal. 2006;
Lambers etal. 2015).
Increased demand for food and agricultural products
and the need for sustainable crop production technologies,
both occasioned by a fast-growing global human popula-
tion, have driven the development of a myriad of natural and
environmentally safe agricultural inputs and alternatives to
chemical fertilisers for addressing the challenges of min-
eral nutrient deficiencies in soils and the attendant low crop
productivity. Among these technologies, seaweed-derived
plant biostimulants have gained significant scientific and
industrial interests due to their broad range of bioactivities.
These algal (mainly macroalgae) extracts stimulate defence
and growth responses when applied to plants (Nanda etal.
2022), including in plants under nutrient deprivation (Chrys-
argyris etal. 2018; Shukla and Prithiviraj 2021; Shukla etal.
2024). A diverse class of bioactive compounds including
phytohormones, polyamines, saccharides (Stirk etal. 2020;
Ali etal. 2021) and polyphenols (Rengasamy etal. 2015a, b;
Rengasamy etal. 2016) have been identified as the mediators
of the bioactivities of seaweed-derived biostimulants. Con-
siderable research efforts have been invested in elucidating
the bioactivities of whole or fractions of seaweed extracts
in enhancing nutrient acquisition in plants under nutrient
deprivation, such as potassium (Chrysargyris etal. 2018),
nitrogen (Shukla etal. 2024) and P (Shukla and Prithiviraj
2021) limitations.
Two eckol-type phlorotannins, namely eckol and diben-
zodioxin-fucodiphloroeckol, have been isolated and iden-
tified in the commercial seaweed biostimulant Kelpak®
which is produced from the kelp, Ecklonia maxima (Osbeck)
Papenfuss (Kannan etal. 2013; Omoarelojie etal. 2024).
The concentrations of the phlorotannins in Kelpak® varies
between batches, with eckol ranging from 589.1 to 822.54
μg L−1 and dibenzodioxin-fucodiphloroethol ranging from
85 to 895 μg L−1 (Omoarelojie etal. 2024). Phlorotannins
enhance plant growth and development by altering plant
metabolism. Eckol improved α-amylase activity and elicited
auxin-like bioactivities in rooting assays (Rengasamy etal.
2015b). Bioactive phytochemicals, vegetative growth, and
tissue auxin content were significantly increased in Eucomis
autumnalis that were treated with eckol and phloroglucinol
(Aremu etal. 2015). Foliar application of eckol on cabbage
plants increased vegetative growth, iridoid glycoside con-
tent, and myrosinase activity which contributes to resistance
to cabbage aphid (Rengasamy etal. 2016). Foliar applica-
tion of eckol promoted the accumulation of cis-zeatin, dihy-
drozeatin, and isopentenyladenine types of cytokinins and
free sinapic acid in treated Spinacia oleracea (Kulkarni etal.
2019). In addition to phlorotannins, other bioactive com-
pounds such as polyamines (Papenfus etal. 2012) and phyto-
hormones (Stirk etal. 2014) have been identified in Kelpak®.
Despite the well documented bioactivities of seaweed
derived-biostimulants in plant growth and development, the
role(s) and bioactivities of phlorotannins in plant P acqui-
sition remain largely unexplored. In this study, the effects
of eckol class of phlorotannins (eckol and EA6) that were
extracted from E. maxima were evaluated on the adaptive
responses and P acquisition in white lupin and cucum-
ber seedlings grown in P-deficient media. The efficacy of
Kelpak® on the adaptive responses and P acquisition in
white lupin and cucumber seedlings was also evaluated for
comparison.
Materials andmethods
Seeds of white lupin (Lupinus albuscultivar Amiga) and
cucumber (Cucumis sativus cultivar Ashley) were used
in this study. Phlorotannins (Fig.1) isolated fromEcklo-
niamaxima were used for the treatments. Eckol isolated
from the ethyl acetate fraction of a methanolic extract of
E. maxima following the method described in Kannan
etal. (2013) was used for the treatment of lupin seedlings.
For the cucumber seedlings, phlorotannins, i.e., eckol and
EA6 (a fraction containing 2-phloroeckol and dibenzodi-
oxin-fucodiphloroeckol in a ratio 1:1) were obtained from
ethyl acetate fractions of an ethanolic extract of E. maxima
(Omoarelojie etal. 2024). Kelpak®samples were supplied
by Kelp Products International (Pty) Ltd, Simon’s Town,
South Africa.
White lupin seedling experiment
Lupin seeds were washed and imbibed overnight in deion-
ized water at 24 °C. They were then rinsed several times
with deionized water before being transferred into Petri-
dishes lined with filter paper, irrigated with deionized
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Journal of Applied Phycology
water and incubated for 3 days in the dark. The germi-
nated seeds were transferred into vermiculite in 1-L plas-
tic garden bags (one seed per bag). Growth medium (200
mL) lacking in Pi, slightly modified from Le Thanh etal.
(2021) with the following mineral nutrient components
was supplied to the seedlings: MgSO4.7H2O 54 μM;
Ca(NO3)2.4H2O 400 μM; K2SO4200 μM; Na-Fe-EDTA 10
μM; H3BO3 2.4 μM; MnSO4.4H2O 0.24 μM; ZnSO4.7H2O
0.1 μM; CuSO4.5H2O 0.04 μM; Na2MoO4.2H2O 0.042
μM. The seedlings were raised in a growth chamber under
controlled conditions with 16 h light:8 h dark, 24 ± 1 °C
ambient temperature and PAR intensity 180 μmol photons
m–2 s–1 for 12 days. The lupin seedlings were assigned into
four treatment groups and supplied with 200 mL treatment
solutions (Table1). The seedlings were raised for a fur-
ther 10 days under the same growth conditions as above.
Thereafter, the seedlings were harvested for root exudate
and vegetative data collection.
Cucumber seedling experiment
Cucumber seeds were rinsed and imbibed in deionised
water for 6 h. The seeds were germinated in a 2-L plas-
tic tub lined with paper towel and moistened with 30 mL
deionised water. The seeds were incubated for 8 days in
the dark at 26 ± 1 °C. Thereafter, one etiolated seedling
was transferred to a labelled 25-mL flat-bottom conical
flask and supplied with 10 mL treatment solution. The
treatment groups and their respective treatment solutions
were prepared in a modified ½-strength Hoagland solu-
tion (HS) (Hoagland and Arnon 1950) in which the only
source of Pi was supplied as immobile Pi [i.e., insoluble
25 µM Ca3(PO4)2] (Table1). To prevent the drying out
of the flasks as the seedlings developed, 10 mL treatment
solution was added to each flask every two days. The tem-
perature was 24 ± 1 °C, 15 h light:9 h dark photoperiod
and PAR intensity of 180 photons μmol m–2 s–1. Twenty
days after treatment started, the cucumber seedlings were
prepared for exudate collection.
Root exudate collection
Lupin seedlings were carefully cleaned to remove adhering
vermiculite and transferred to glass jars containing 150 mL
exudate collection medium [50 µM CaCl2 (pH 7.04) (Egle
etal. 2003)]. The lupin seedlings were incubated in the exu-
date collection medium for 3 days at 24 ± 1 °C, 16 h light:8
h dark and PAR intensity 180 μmol photons m–2 s–1. The
roots of the cucumber seedlings and their respective flasks
were rinsed with deionized water and 15 mL exudate col-
lection medium added to each flask. After a 24 h incubation
period, 1 mL exudate medium was removed from each jar or
flask and transferred into Eppendorf tubes for phosphatase
activity assay. After root exudate collection, all the seedlings
were harvested for vegetative data collection and Pi content
determination.
Fig. 1 Structural formula of eckol (1) and the phlorotannin constitu-
ents of EA6, i.e., 2-phloroeckol (2) and dibenzodioxin-fucodiphloroe-
ckol (3)
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Journal of Applied Phycology
Vegetative data collection
Harvested lupin seedlings were separated into shoot and root
tissues and their fresh weight measured. The root tissues of
the lupin seedlings were further separated into cluster root
sections and non-cluster root sections. The shoot tissues,
roots and cluster root sections were dried at 95 °C for 7
days. The cucumber seedlings were separated into root and
shoot tissues and their fresh weights measured. Thereafter,
the shoots and roots were dried at 95 °C for 5 days. The dry
biomasses were measured after drying.
Phosphate content determination
The soluble cellular P in lupin seedlings was quantified as
described by Wang etal. (2011). Briefly, pre-weighed shoot
or root tissues (5 samples per treatment) were homogenised
and extracted with 1000 µL 1% (v/v) glacial acetic acid in
a bead beater. The tubes were centrifuged at 5000×g for 10
min at room temperature. To quantify the extracted soluble
P, 100 or 150 µL supernatant from roots or shoots, respec-
tively, were mixed with 700 µL phosphomolybdate reagent
(a mixture of 0.48% NH4MoO4 in 2.85% (v/v) H2SO4, and
10% (w/v) ascorbic acid in a ratio of 6:1) and the volume
made up to 1000 µL with deionised water. The absorbance
of this mixture was measured at 820 nm using a UV-Vis
spectrophotometer. The soluble P content was determined
from a standard curve prepared with known concentrations
of Pi (KH2PO4).
Total phosphate contentswere determined in lupin and
cucumber seedlings (5 samples per treatment) follow-
ing the method described by Ames (1966). Briefly, 30 mg
dried leaf biomass was soaked in 300 µL 10% (w/v) of
Mg(NO3)2.6H2O (prepared in 95% v/v alcohol) and ashed
at 550 °C for 6 h in a muffle furnace. The ashes were col-
lected into Eppendorf tubes, dissolved in 900 µL 0.5 M HCl,
sealed and heated for 15 min. After cooling, 300 µL of each
sample was mixed with 700 µL ammonium molybdate mix
[1 part 10% (w/v) ascorbic acid mixed with 6 parts 0.42%
(w/v) ammonium heptamolybdate tetrahydrate in 1N H2SO4]
and incubated at 37 °C for 1 h. The absorbance of the result-
ing samples was read at 820 nm and the phosphate content
determined from a standard curve prepared using potassium
phosphate.
Estimation ofphosphatase activity
Phosphatase activity was determined in the lupin and
cucumber seedlings in a spectrophotometric assay follow-
ing the method of Tagad and Sabharwal (2018) from 5 sam-
pled plants per treatment. This is based on the ability of
phosphatases to catalyse the hydrolysis of para-nitrophenyl
phosphate (p-NPP) to para-nitrophenol (p-NP), a chromo-
genic product with absorbance at 405 nm. Briefly, 200 µL
p-NPP in 200 mM acetate buffer (pH 5.0) mixed with 200
µL root exudate solution was incubated for 1 h. Then, 1 mL
0.5 M NaCl solution was added to terminate the reaction
and develop the yellow colour of the p-NP formed. The
absorbance was read at 405 nm in a spectrophotometer.
The corresponding amount of p-NP was determined from
a standard curve prepared with p-NP (3 – 100 µg mL−1).
The phosphatase activities of the exudates were expressed
in nanogram of p-NP formed per min per gram of total root
fresh weight (ng p-NP min−1 g−1 fwt).
Data analysis
Data collation and statistical analysis were carried out
using dplyr and ggplot packages in R software (v4.4.1).
All the data were subjected to one-way analysis of variance
(ANOVA) with the treatments as the grouping factor and
post hoc analysis was carried out using the Fisher’s least sig-
nificant differences (LSD) test to compare the means of all
the variables. Principal component analysis (PCA) was con-
ducted using prcomp function with data scaling. The cumu-
lative variability, Eigen values and principal component
Table 1 Description of
biostimulants treatments applied
to white lupin and cucumber
seedlings
Treatments Descriptions
P status Components
White lupin seedlings
+P P-supplied 32µM Pi (K2HPO4) in P-free medium
-P P-free P-free medium only
-P+Eck P-free 1 μM eckol in P-free medium
-P+KPK P-free 1% (v/v) Kelpak® solution in P-free medium
Cucumber seedlings
Ctl P-limited: insoluble P ½-strength Hoagland solution (HS) with 25 µM Ca3(PO4)2
Eck 10 µg L−1 eckol in ½-strength HS with 25 µM Ca3(PO4)2
EA6 10 µg L−1 EA6 in ½-strength HS with 25 µM Ca3(PO4)2
KPK 1% (v/v) Kelpak® in ½-strength HS with 25 µM Ca3(PO4)2
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Journal of Applied Phycology
scores were determined. The PCA outcome were visualised
in biplots using fviz_pca function (factoextra package) to
provide a graphical overview of the relationships among the
measured adaptive traits and biostimulant treatments and to
provide insight into the adaptive traits that contributed to
the observed delineation of P starvation responses under the
different treatments.
Results
Biomass accumulation
The P-free medium and treatment with eckol or Kelpak®
altered vegetative growth in white lupin seedlings especially
with respect to root development (Fig.2a-b). There was a
marginally significant difference in the means of fresh shoot
biomass among the treatments (ANOVA: F3,16 = 3.091, p
= 0.057). Fresh shoot matter in seedlings grown in P-free
medium (-P: m = 3.318, sd = 0.243) and eckol supplemented
P-free medium (-P+Eck: m = 3.3668, sd = 0.563) were
significantly lower (LSD = 1.147, α = 0.05) compared to
seedlings grown in P-supplied medium (+P: m = 4.606, sd
= 0.643) (Fig.2a). In contrast, lupin seedlings grown in
P-free medium and supplemented with Kelpak® (-P+KPK)
had fresh shoot biomass (m = 4.387, sd = 1.461) that were
not significantly different (LSD = 1.147, α = 0.05) from +P
seedlings (Fig.2a). There was no significant difference in
the means of fresh root biomass of all the treatment groups
(ANOVA: F3,16 = 1.576, p = 0.234) (Fig.2a). Total fresh
biomass in -P seedlings (m = 5.402, sd = 0.441) was sig-
nificantly lower than that of the +P seedlings (m = 7.367,
sd = 0.993), LSD = 1.865, α = 0.05. In contrast, total fresh
biomass in -P+Eck seedlings (m = 5.585, sd = 0.918) and
-P+KPK seedlings (m = 6.572, sd = 2.390) were not statisti-
cally different from +P seedlings, LSD = 1.865, α = 0.05.
P-deprivation and biostimulant treatments did not signifi-
cantly alter dry shoot biomass although the ANOVA (F3,16 =
2.689, p = 0.081) alludes to a trend towards some differences
in shoot dry weight between the treatments (Fig.2b). The
pairwise comparisons showed that dry shoot biomass of +P
seedlings (m = 0.497, sd = 0.069) was significantly different
from those in -P (m = 0.357, sd = 0.0507) and -P+Eck (m =
0.382, sd = 0.023) seedlings, LSD = 0.112, α = 0.05. Sup-
plementing P-free medium with Kelpak® increased dry shoot
matter (m = 0.418, sd = 0.142) to similar levels as in +P
seedlings, this was however not significantly different from
the dry shoot weights of -P seedlings, LSD = 0.112, α =
0.05. Dry root biomass was significantly altered in the treat-
ment groups (ANOVA: F3,16 = 9.512, p = 0.0008). Seedlings
grown in P-free medium had the lowest dry root matter (m
= 0.116, sd = 0.0185) while supplementing the medium
with eckol or Kelpak® increased seedling dry root matter
to similar levels as in P-supplied seedlings (m = 0.180, sd
= 0.028), LSD = 0.040, α = 0.05. Total dry biomass was
significantly different between treatments (ANOVA: F3,16
= 3.49, p = 0.040). While +P (m = 0.678, sd = 0.095) and
-P+KPK (m = 0.626, sd = 0.17) seedlings had the highest
dry weights, followed by -P+Eck (m = 0.581, sd = 0.034),
and the -P (m = 0.473, sd = 0.068) seedlings had the low-
est total dry weight. The difference between +P and -P
seedlings, as well as between +P and -P+Eck, was statisti-
cally significant, LSD = 0.14, α = 0.05. However, total dry
weights in +P and -P+KPK seedlings were not significantly
different.
In cucumber seedlings grown in ½-strength HS with
insoluble P [Ca3(PO4)2] as the sole source of P supply, sup-
plementing the nutrient media with phlorotannins (eckol or
EA6) or Kelpak® altered biomass accumulation (Fig.3a-c).
Fig. 2 Effects of eckol and Kelpak® supplementation on (a) fresh
and (b) dry biomass of white lupin seedlings under P-starvation and
biostimulant treatments. Treatments include seedlings grown in: +P,
P-supplied medium; -P, P-free medium; -P+Eck, P-free medium sup-
plemented with 1 μM eckol; and -P+KPK, P-free medium supple-
mented with 1% (v/v) Kelpak®. Data are the means ± SE (n = 5) and
different letters above the error bars indicate significantly different
means (Fisher’s LSD, α = 0.05)
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Journal of Applied Phycology
Fresh shoot biomass was significantly altered by Kelpak®
treatment (m = 0.883, sd = 0.225) compared to the control
(m = 0.611, sd = 0.093), LSD = 0.239, α = 0.05 (Fig.3b).
In P-limited cucumber seedlings, the addition of phlorotan-
nin or Kelpak® to the nutrient medium led to changes in
fresh root biomass (ANOVA: F3,16 = 21.51, p = 7.35e−06).
There was significantly higher fresh root weight in P-limited
cucumber seedlings that were treated with Kelpak® (m =
2.279, sd = 0.283) and EA6 (m = 1.696, sd = 0.130) com-
pared to the untreated control (m = 1.428, sd = 0.144), LSD
= 0.254, α = 0.05. As with fresh shoot matter, fresh root
matter was not significantly different in P-limited cucum-
ber seedlings that received eckol compared to EA6. There
was a similar trend with total fresh biomass (Fig.3b) where
Kelpak® treatment elicited a significantly higher total fresh
biomass (m = 3.162, sd = 0.490), a similar fresh matter
accumulation in Eck- (m = 2.222, sd = 0.401) and EA6-
treated seedlings (m = 2.502, sd = 0.124) while the P-limited
control seedlings had the lowest fresh weight (m = 2.039, sd
= 0.139), LSD = 0.442, α = 0.05.
The supplementation of growth media with phlorotan-
nins or Kelpak® led to changes in dry matter accumulation
(Fig.3c). Dry shoot weight in Kelpak®-treated P-limited
cucumber seedlings (m = 0.179, sd = 0.020) was signifi-
cantly higher than the controls (m = 0.142, sd = 0.011), LSD
= 0.026, α = 0.05. There was no significant difference in the
dry shoot weights of P-limited seedlings that were treated
with eckol (m = 0.155, sd = 0.024) or EA6 (m = 0.160, sd
= 0.020) in comparison to the control. There was a clear and
significant delineation of dry root matter among the treat-
ment groups. Kelpak®-treated seedlings had the highest (m
= 0.087, sd = 0.0174), both eckol (m = 0.072, sd = 0.006)
and EA6 (m = 0.061, sd = 0.006) had similar root weights
which were significantly lower than Kelpak®-treated seed-
lings but higher than the control seedlings which had the
least dry root matter (m = 0.037, sd = 0.006), LSD = 0.014,
α = 0.05. Total dry weights followed a similar trend, with
P-limited cucumber seedlings having the least dry matter,
the phlorotannin-treated (Eck and EA6) seedlings had simi-
lar dry weights that were significantly higher than the con-
trols, and Kelpak®-treated seedlings had the highest which
was significantly different from the control and phlorotannin
treated seedlings.
Biomass partitioning
A defining symptom of P-deficiency in plants is an enhanced
root-to-shoot ratio which arises from the prioritisation of
root development either via a reduction in shoot growth or
an increase in root production, or both. Root-to-shoot ratios
(computed on a dry weight basis) in +P lupin seedlings was
not significantly different from -P seedlings (Fig.4a). In con-
trast, root-to-shoot ratios in -P+Eck (m = 0.52, sd = 0.054)
Fig. 3 Biomass accumulation in cucumber seedlings grown in P-limited
Hoagland solution (HS) with insoluble P. (a) Cucumber seedlings grown
in Ctl (HS with insoluble P [25 µM Ca3(PO4)2]); Eck (HS with insoluble
P supplemented with 10 µg L−1 eckol); EA6 (HS with insoluble P sup-
plemented with 10 µg L−1 EA6); and KPK (HS with insoluble P supple-
mented with 1% v/v Kelpak® solution). (b) Fresh and (c) dry biomasses of
cucumber seedlings under P-limitation and biostimulant treatments. Data
represent means ± SE (n = 5) and different letters above the error bars
indicate significantly different means (Fisher’s LSD, α = 0.05)
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Journal of Applied Phycology
and -P+KPK (m = 0.531, sd = 0.184) seedlings showed
a significantly increased biomass allocation to the roots
compared to the +P seedlings (m = 0.363, sd = 0.027) and
untreated -P lupin seedlings (m = 0.326, sd = 0.02), LSD
= 0.130, α = 0.05. Root-to-shoot ratios in -P+Eck seed-
lings were not significantly different from that of -P+KPK
seedlings.
Cucumber seedlings that were grown with an insolu-
ble form of Pi [Ca3(PO4)2] as the sole source of P supply
showed a significant increase in root-to-shoot ratios under
eckol (m = 0.472, sd = 0.066), EA6 (m = 0.389, sd =
0.063), and Kelpak® (m = 0.487, sd = 0.065) treatments
compared to the control (m = 0.258, sd = 0.04), LSD =
0.079, α = 0.05 (Fig.4b). Root-to-shoot ratios in eckol-
treated cucumber seedlings were not statistically different
from Kelpak®-treated seedlings.
Cluster root development inwhite lupin
The development of cluster roots is central to the adaptive
strategies for coping with P-limiting conditions in white
lupin. There was a clear delineation of cluster root forma-
tion in white lupin seedlings grown in P-supplied medium
and P-free medium (Figs.5and6a-c). The elimination of
P from the growth medium led to a significant increase in
the number of cluster roots in -P seedlings (m = 18.0, sd =
3.535) compared to +P seedlings (m = 12.2, sd = 1.924),
LSD = 4.208, α = 0.05 (Fig.6a). Cluster root formation
was further increased in eckol (m = 27.0, sd = 2.915) and
Kelpak®-treated (m = 25.8, sd = 3.834) P-deprived seed-
lings. The dry weights of cluster roots from seedlings under
biostimulant treatments, -P+Eck (m = 0.047, sd = 0.013)
and -P+KPK (m = 0.046, sd = 0.012) were significantly
higher compared to the untreated -P (m = 0.03, sd = 0.013)
and +P (m = 0.024, sd = 0.007) seedlings, LSD = 0.016, α
= 0.05 (Fig.6b).
The allocation of biomass between cluster root and non-
cluster root was significantly influenced by P-supply but not
biostimulant treatments (Fig.6c) (ANOVA: F3,16 = 4.802,
p = 0.014). There was no significant difference between
the untreated -P seedlings when compared to eckol- and
Kelpak®-treated -P seedlings. However, cluster root-to-total
root ratio was lower in the +P seedlings (m = 0.131, sd =
0.027) compared to -P (m = 0.246, sd = 0.072), -P+Eck
(m = 0.238, sd = 0.0588) and -P+KPK (m = 0.222, sd =
0.05) seedlings, LSD = 0.073, α = 0.05, thus indicating a
prioritisation of biomass allocation for cluster root formation
in the P-deprived seedlings. More cluster roots per unit of
shoot were formed as indicated by cluster root-to-total shoot
ratios, in -P+Eck (m = 0.123, sd = 0.030) and -P+KPK (m
= 0.115, sd = 0.036) seedlings than in -P (m = 0.08, sd =
0.026) and +P (m = 0.047, sd = 0.011) seedlings, LSD =
0.037, α = 0.05.
Phosphate content
The concentrations of soluble cellular P were significantly
altered by P-supply and biostimulant treatments in the leaves
(ANOVA: F3,16 = 24.14, p = 3.51e−06) and roots (ANOVA:
F3,16 = 28.6, p = 1.15e−06) of lupin seedlings (Fig.7a). There
was no significant difference in the foliar soluble P of -P
seedlings (m =1.555, sd = 0.809) and -P+Eck seedlings
(m = 1.607, sd = 0.406), LSD = 0.763, α = 0.05. However,
foliar soluble P of +P seedlings (m = 4.229, sd =0.644)
was significantly higher than that of -P+KPK seedlings (m
= 2.574, sd = 0.244) and these were both higher than in -P
and -P+Eck seedlings. In the roots, soluble cellular P was
the highest in +P seedlings (m = 3.334, sd = 0.871) and
significantly different from that of -P+KPK (m = 1.547, sd
= 0.287) and -P+Eck (m = 0.966, sd = 0.447), these were
not significantly different, LSD = 0.689, α = 0.05. The least
soluble P in root tissues was in -P seedlings (m = 0.544, sd
= 0.12) and this was significantly different from that of +P,
-P+KPK, and -P+Eck seedlings.
Total P was also significantly different among the
treatment groups with both P-supply and biostimulants
inducing changes in the shoots (ANOVA: F3,16 = 13.29, p
= 1.3e−04) and roots (ANOVA: F3,16 = 28.2, p = 1.27e−06)
of lupin seedlings (Fig.7b). While the total P in the shoot
of -P+Eck seedlings (m = 23.18, sd = 4.259) was not
significantly different from that of -P+KPK seedlings (m
= 30.933, sd = 9.279) and -P seedlings (m = 19.615, sd
= 3.134), +P seedlings (m = 45.589, sd = 9.255) had a
significantly higher shoot total P than untreated -P and
biostimulant-treated -P seedlings. A similar trend was
Fig. 4 Phlorotannins and Kelpak® treatments reprogram biomass
allocation in (a) white lupin and (b) cucumber seedlings grown under
P-limiting conditions. Data represent means ± SE (n = 5) and differ-
ent letters above the error bars indicate significantly different means
(Fisher’s LSD, α = 0.05)
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Journal of Applied Phycology
observed with total P in the roots of the treatment groups
with -P+Eck seedlings having similar root total P as that
of -P+KPK and -P seedlings while root total P in all three
-P groups (-P, -P+ECK, and -P+KPK) were significantly
lower than that of +P seedlings.
In the P-limited cucumber seedlings, supplementing
the nutrient medium with eckol, EA6 or Kelpak® led to
significant increase (ANOVA: F3,16 = 24.09, p = 3.56e−06)
in the foliar P contents of the treated seedlings compared
to the untreated control (Fig.8). Eckol induced a 1.9-fold
increase in foliar P contents (m = 54.635, sd = 9.064) of
cucumber seedlings compared to the untreated control (m
= 28.504, sd = 5.184). Foliar total P in Kelpak®-treated
seedlings (m = 52.927, sd = 5.456) and EA6-treated seed-
lings (m = 61.438, sd = 5.761) was 2.1- and 1.8-fold
higher, respectively, compared to the untreated control
seedlings. Total P contents were not statistically different
in cucumber seedlings that received either phlorotannins
(eckol or EA6) and Kelpak®, LSD = 8.792, α = 0.05.
Phosphatase activity
The elimination of P from the nutrient medium and treat-
ments with either eckol or Kelpak® led to significant changes
(ANOVA: F3,16 = 26.35, p = 1.99e−06) in the phosphatase
activities in the root exudates of white lupin seedlings
(Fig.9a). Phosphatase activity in the root exudates from +P
lupin seedlings (m = 2.734, sd = 0.496) was 1.6-fold and
significantly (LSD = 2.17, α = 0.05) lower, compared to
exudates from -P seedlings (m = 5.126, sd = 0.756). When
-P lupin seedlings were treated with eckol or Kelpak®,
the phosphatase activities of root exudates were 2.2-fold
(m = 11.445, sd = 2.1066) and 1.5-fold (m = 7.521, sd =
2.285), respectively, and significantly higher compared to
the untreated -P seedlings. Phosphatase activities in the root
exudates of -P+Eck seedlings was significantly higher than
that -P+KPK seedlings.
In cucumber seedlings grown with insoluble Ca3(PO4)2
as the sole source of P, the phosphatase activities of root
Fig. 5 Effects of eckol and
Kelpak® supplementation on
cluster root development in
white lupin seedlings grown in
(a) P-supplied (+P), (b) P-free
medium (-P), (c) P-free medium
supplemented with 1 μM eckol
(-P+Eck) and (d) 1% (v/v)
Kelpak® (-P+KPK). Scale bar =
70 mm. Arrows indicate cluster
root sections
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Journal of Applied Phycology
exudates were significantly increased (ANOVA: F3,16 =
24.57, p = 3.13e−06) when the nutrient medium were sup-
plemented with phlorotannins (Eck or EA6) or Kelpak®
(Fig.9b). Phosphatase activities in the root exudates
from cucumber seedlings that received eckol, Kelpak® or
EA6, were 1.6-fold (m = 50.622, sd = 3.786), 2.3-fold
(m = 72.836, sd = 7.244) and 2.4-fold (m = 76.663, sd
= 14.741) higher, respectively, and significantly higher
compared to untreated control seedlings (m = 31.405,
sd = 8.759), LSD = 12.734, α = 0.05. While EA6 elic-
ited similar phosphatase activities as in root exudates of
Kelpak®-treated seedling, eckol-treated cucumber seed-
lings had significantly lower phosphate activities in their
root exudates.
Principal component analyses
The PCA of the adaptive traits in lupin seedlings in response
to P-deprivation and biostimulant (eckol and Kelpak®) treat-
ments showed that PC1, PC2 and PC3 with Eigen values
greater than 1 and accounted for 89.135% of the observed
variance (Online Resource 1). The variable loadings (Online
Resource 2) showed that fresh matter accumulation (FSht,
FRt, and FWt), dry matter accumulation (DSht and DWt),
soluble cellular P contents (ShtSolP and RtSolP) and total
P contents (ShtP and RtP) were positively associated with
PC1. Cluster root development (NCR, CRRtR, CRShtR) and
phosphatase activity (Phtase) showed strong but negative
associations with PC1. Both fresh and dry matter accumula-
tion, cluster root development parameters and phosphatase
activity were positively associated with PC2. Tissue P con-
tents, i.e., soluble P and total P, in theroots and shoots were
Fig. 6 Cluster root formation in white lupin seedlings grown in
P-supplied medium (+P), P-free medium (-P), and P-free medium
supplemented with 1 μM eckol (Eck) or 1% (v/v) Kelpak® (KPK).
(a) Average number of cluster roots formed per plant, (b) dry cluster
root biomass, (c) ratios of cluster roots to total root or shoot biomass.
The data represent means ± SE (n = 5) and different letters above the
error bars indicate significantly different means (Fisher’s LSD, α =
0.05)
Fig. 7 (a) Soluble cellular P and (b) total P contents in white lupin
seedlings raised in P-supplied medium (+P), P-free medium (-P),
and P-free medium supplemented with 1 μM eckol (Eck) or 1% (v/v)
Kelpak® (KPK). Data represent means ± SE (n = 5) and different let-
ters above the error bars indicate significantly different means (Fish-
er’s LSD, α = 0.05)
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Journal of Applied Phycology
negatively associated with PC2. The biplot of PC1 vs PC2
defined four clusters with conspicuous spatial separation of
+P seedlings from -P seedlings (Fig.10a). Fresh (FSht, FRt,
and FWt) biomass, dry shoot biomass and P contents (i.e.,
soluble P and total P in the roots and shoots) defined the +P
cluster and the eckol-cluster was defined by biomass alloca-
tion (RtShtR and CRRtR, CRShtR), cluster root develop-
ment (NCR and DCRt) and phosphatase activity (Phtase).
The -P cluster was only defined by biomass partitioning
between above- and below-ground tissues (RtShtR) while all
the adaptive traits contributed to defining the KPKcluster.
The PCA performed on the responses of P-limited
cucumber seedlings showed PC1 and PC2 with Eigen values
greater than 1 and explained 66.508% and 13.294% of total
variance, respectively (Online Resource 3). All the meas-
ured traits contributed to determining PC1. Fresh biomass
(FSht, FRt, FWt) and dry shoot (DSht) with positive asso-
ciation and biomass partitioning (RtShtR), foliar P content
and phosphatase activity (Phtase) with negative association
explained PC2 (Online Resource 4). Four clusters delineated
by treatments are presented in the biplot of PC1 vs PC2.
There was a clear spatial delineation of Ctl cluster away
from, and opposite, the phlorotannins (eckol and EA6) and
Kelpak® clusters (Fig.10b). While all morphological and
physiological traits were influenced and closely associated
with EA6 treatment, all response traits except shoot P and
biomass partitioning were influenced by eckol and Kelpak®
treatments.
Discussion
Supplying white lupin seedlings with P-free nutrient
medium, or cucumber seedlings with media containing
only insoluble P and supplementing these with phlorotan-
nins (eckol or EA6) or Kelpak® led to significant changes
in the adaptive responses of both white lupin and cucumber
seedlings to P-limitation. Eckol and Kelpak® treatments
mediated an increased allocation of resources for root
development as evident in the increase in root-to-shoot
ratios in the treated P-starved lupin seedlings compared
to the untreated P-starved seedlings. Kelpak®-mediated
prioritisation of root development was not detrimental
to shoot growth since shoot dry biomass in the P-starved
lupin seedlings treated with Kelpak® did not decrease
when compared to the untreated P-starved seedlings.
For P-limited cucumber seedlings treated with Kelpak®,
despite increased root accumulation, shoot growth was
still significantly higher than that of the controls. From a
Fig. 8 Foliar phosphate contents of cucumber seedlings grown in
nutrient medium with immobile P (Ctl) and media with immobile P
that were supplemented with 10 µg L−1 eckol (Eck), 10 µg L−1 EA6
(EA6), or 1% (v/v) Kelpak® (KPK). Data represent means ± SE (n =
5) and different letters above the error bars indicate significantly dif-
ferent means (Fisher’s LSD, α = 0.05)
Fig. 9 Phlorotannins and Kelpak® treatments alter phosphatase activ-
ities in root exudates of (a) white lupin and (b) cucumber seedlings
under P-limiting conditions. Data represent means ± SE (n = 5) and
different letters above the error bars indicate significantly different
means (Fisher’s LSD, α = 0.05)
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Journal of Applied Phycology
rhizoeconomic perspective, increased allocation of pho-
tosynthates to roots compromises overall growth due to
the greater respiratory burden of root tissues (Lynch and
Ho 2005; Honvault etal. 2021). The results of the present
study raise questions about the biochemical or physiologi-
cal processes that are influenced by these biostimulants to
achieve this balanced resource allocation, i.e., driving root
proliferation without compromising shoot growth.
The similarities in shoot P contents of untreated P-starved
lupin seedlings and the eckol-treated P-starved seedlings
may be attributed to the complete absence of P in the nutri-
ent medium. Thus, despite the eckol-mediated improvements
in the morphological and physiological adaptive traits for
coping with P-starvation, these did not translate into higher
P-contents in the shoot tissues since the treated seedlings
were grown in P-free nutrient media. The significantly
higher P-contents (soluble and total) in the Kelpak®-treated
P-starved lupin seedlings may be linked to the scavenging of
trace amounts of P that may be present in Kelpak® products.
An elemental analysis of Kelpak® product that was used for
this study showed the presence of trace amounts of total P
in the range of 1.6484 µmoles. In the cucumber seedlings
grown in nutrient medium with insoluble P, phlorotannins
(eckol and EA6) and Kelpak® treatments led to higher foliar
P contents, indicating that the biostimulants enhanced the
mobilisation of P from the immobile P-form. Similarly, sup-
plementation of P-limited Zea mays with an Ascophyllum
nodosum-derived biostimulant resulted in a higher P content
(Shukla and Prithiviraj 2021).
In both lupin and cucumber seedlings, phlorotannins and
Kelpak® treatments enhanced phosphatase activities in the
root exudates of P-deprived seedlings. This may be linked to
biostimulant-induced increase in the concentration of phos-
phatases in the root exudates or the ability of phlorotannins
or components of Kelpak® to act as cofactors or prosthetic
groups that enhance the activities of phosphatases. A num-
ber of studies have demonstrated the abilities of phlorotan-
nins to influence enzyme activities (Kumar etal. 2022) in a
dose-dependent manner (Kannan etal. 2013). Further inves-
tigations will be required to unravel the precise mechanism
that underpin phlorotannin and Kelpak®-mediated enhance-
ment of phosphatase activity.
Treating P-starved lupin seedlings with eckol or Kelpak®
and P-limited cucumber seedlings with eckol, EA6 or
Kelpak® led to increased phosphatase activities in root
exudates. These enhanced phosphatase activities could not
have contributed to the increase in P contents in the treated
P-starved lupin and cucumber seedlings as phosphatases
mobilise P from phosphorylated metabolites and phospho-
proteins (Widlanski etal. 1999) which were not supplied in
the media used in the present study. However, the relevance
of phosphatases in P acquisition in natural systems and as
an adaptive trait that is highly responsive to P-starvation
necessitated the investigation of this trait.
The treatment clusters in the PCA analysis clearly deline-
ate how P-supply, P-starvation and the biostimulant treat-
ments shaped P-responsive traits in the lupin seedlings. The
strong association of root-to-shoot ratios with untreated
P-starved lupin seedlings is consistent with biomass alloca-
tion to below-ground tissues under P-deficiency to enhance
root foraging for P (Vengavasi etal. 2021). The spatial
Fig. 10 Principal component analysis biplot of morphophysiological
adaptive responses in P-starved white lupin (a) and P-limited cucum-
ber (b) seedlings under phlorotannins or Kelpak® treatments. Fresh
shoot (FSht), root (FRt), total (FWt) biomass; dry shoot (DSht), root
(DRt), total (DWt) biomass; RTShtR, root-to-shoot ratio; CRRtR,
cluster root-to-total root ratio; CRShtR, cluster root-to-shoot ratio;
NCR, number of cluster roots; DCRt, dry cluster root biomass; total P
in shoot (ShtP) and root (RtP); soluble P in shoot (ShtSolP) and root
(RtSolP); Phtase, phosphatase activities
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Journal of Applied Phycology
aggregation of biomass and P-contents (i.e., traits associ-
ated with P-sufficiency) and their separation (in opposite
position) to cluster root development and phosphatase activ-
ity (i.e., adaptive traits associated with P-deficiency) along
PC1 is indicative of the negative correlations between these
parameters. The association of adaptive morphophysiologi-
cal traits (i.e., cluster root development, biomass partition-
ing, and phosphatase activity) with P-starved lupin seedlings
that received eckol treatment suggests that eckol augment
these processes in the P-starved seedlings in a bid to improve
P-mobilisation from the medium. The PCA showed that
treating the P-starved lupin seedlings with Kelpak® influ-
enced all the adaptive parameters, i.e., morphological, and
physiological traits that are associated with both P-suffi-
ciency and P-limitation.
The observations with cucumber seedlings follow a simi-
lar trend as in white lupin seedlings. Phlorotannins (eckol
and EA6) and Kelpak® treatments influenced both mor-
phological and physiological responses in P-limited lupin
seedlings with eckol and Kelpak® being less effective than
EA6 at enhancing foliar P content and biomass allocation.
The more potent actions of EA6 compared to eckol may be
attributed to the presence of two phlorotannins in EA6, i.e.,
2-phloroeckol and dibenzodioxin-fucodiphloroeckol.
Conclusions
Root application of eckol-type phlorotannins (eckol or
EA6) or seaweed-derived biostimulant, Kelpak® enhanced
the adaptive responses of white lupin and cucumber seed-
lings to suboptimal P conditions. The phlorotannins induced
similar bioactivities to those of Kelpak®, specifically eckol
and EA6 in P-limited cucumber seedlings and eckol in
P-starved white lupin seedlings. These findings suggest that
phlorotannins contribute to the bioactivities of Kelpak® in
improving seedling responses to P-limitation. Other bioac-
tive compounds present in Kelpak® may also act together
with phlorotannins to enhance plant adaptive responses to
P-deprivation.
These results suggest that seaweed-derived plant biostim-
ulants, like Kelpak® and its bioactive eckol-type phlorotan-
nins, provide viable and sustainable alternatives to chemical
fertilizers for managing crops in P-limited conditions. How-
ever, trials under natural field conditions are needed to assess
the potential of phlorotannins as standalone biostimulants
for large-scale agricultural use.
Acknowledgments The authors are grateful to Kelp Products Interna-
tional (Pty) Ltd, Simon’s Town, South Africa for providing the Kelpak®
samples used in this study.
Author contribution L.O.O. and J.vS. conceived the idea; L.O.O.
designed and conducted the experiments with support from W.A.S.
and M.G.K.; L.O.O. processed and analysed the data. L.O.O. wrote
the manuscript. All authors contributed to the final version of the
manuscript.
Funding Open access funding provided by University of KwaZulu-
Natal. Financial support was received from Kelp Products International
(Pty) Ltd, Simon’s Town, South Africa for this research.
Data availability The data supporting the conclusions of this article
will be made available on request, and without undue reservation.
Declarations
Competing interests The authors declare no competing interests.
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
were made. The images or other third party material in this article are
included in the article’s Creative Commons licence, unless indicated
otherwise in a credit line to the material. If material is not included in
the article’s Creative Commons licence and your intended use is not
permitted by statutory regulation or exceeds the permitted use, you will
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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