Scientia Horticulturae 183 (2015) 39–47

Contents lists available at ScienceDirect

Scientia Horticulturae
journal homepage: www.elsevier.com/locate/scihorti

Effects of A. nodosum seaweed extracts on spinach growth, physiology

and nutrition value under drought stress
Chenping Xu ∗ , Daniel I. Leskovar
Texas A&M AgriLife Research, Vegetable and Fruit Improvement Center, Department of Horticultural Sciences, Texas A&M University, Uvalde, TX 78801, USA

a r t i c l e i n f o a b s t r a c t

Article history: Seaweed extracts (SWE) are biodegradable and non-polluting to the environment, and have become
Received 27 June 2014 popular as biostimulants. Their effects on spinach growth, physiology and nutrition value under drought
Received in revised form stress were evaluated in a growth chamber study. Differential irrigations (100 and 50% evapotranspiration
12 November 2014
for full irrigation and drought stress, respectively) and Ascophyllum nodosum SWE application methods
Accepted 1 December 2014
(water foliar, SWE foliar, SWE drench, SWE foliar and drench) were applied every 4 d. After 35 d, leaf
Available online 26 December 2014
growth, chlorophyll content and fluorescence, gas exchange, and nutrition value were analyzed. Under
drought stress, leaf area, fresh and dry weight (FW and DW) were only 42, 42 and 60% of these under
Keywords:
Antioxidant capacity
full irrigation, respectively. Drought stress also decreased leaf relative water content (RWC) and specific
Drought leaf area (SLA) from 89 to 76% and 179 to 139 cm2 mg−1 , respectively. Gas exchange was reduced by
Nutrition value drought stress although chlorophyll content and fluorescence were not altered. Drought stress had no
Photosynthesis direct effects on the leaf nutrition quality except for reduced ferrous ion chelating ability (FICA). Under
Seaweed full irrigation SWE had no effects on leaf growth, physiology and nutrition value but under drought
Spinach stress SWE improved plant growth. Under drought stress A. nodosum SWE application increased leaf
RWC from 76 to approximately 82% and leaf area by 16, 21 and 38% with SWE foliar spray, drench or
both, respectively. Also, SWE increased leaf FW, DW and SLA by 20 to 32%, 11 to 22%, and 5 to 19%,
respectively. The inhibition of gas exchange and increases of stomatal limitation induced by drought
stress were reduced by SWE, irrespective of application methods. SWE application had no effects on
phenolic, flavonoid, carotenoid, and antioxidant capacity, but reduced FICA. These results indicated that
under mild drought stress A. nodosum SWE enhanced spinach growth by improving leaf water relations,
maintaining cell turgor pressure and reducing stomatal limitation, which in turn led to large leaf area
and high photosynthetic rate. A. nodosum SWE application negatively impacted nutritional quality by
reduced FICA under drought stress.
Published by Elsevier B.V.

1. Introduction nutrients, such as p-coumaric acid derivatives that exhibit strong

Spinach (Spinacia oleracea L.) is an important leafy green veg-
etable that contains large quantities of bioactive compounds and
Abbreviations: ABTS, 2,2-azino-bis(3-ethylbenzthiazoline-6-sulphonic acid);
Cond , stomatal conductance; DPPH, 2,2-diphenyl-1-picrylhydrazyl radical; DW, dry
weight; ET, evapotranspiration; ETR, electron transport rate; FICA, ferrous ion
chelating ability; FRAP, ferric reducing ability of plasma; FW, fresh weight; Ls , sto-
matal limitation; Pn , net photosynthetic rate; PS, photosystem; RWC, relative water
content; SLA, specific leaf area; SWC, soil water content; SWE, seaweed extracts;
TPTZ, 2,4,6-tris-2,4,6-tripyridyl-2-triazine; Tr , transpiration.
∗ Corresponding author. Current address: Agricultural Research Service, U.S. Dept.
of Agriculture, 1636 E Alisal Street, Salinas, CA 93905, USA. Tel.: +01 831 7552860;
fax: +01 831 7552814.
E-mail address: chenping.xu@ars.usda.gov (C. Xu).
antioxidant activity, and glucuronic acid derivatives of flavonoids
(e.g. patuletin and spinacetin) that are not common to most
other vegetables (Bergman et al., 2001; Edenharder et al., 2001;
Pandjaitan et al., 2005). Like most vegetables, spinach production
in semi-arid regions requires irrigation but water deficit is a sig-
nificant limitation. In addition, extensive applications of synthetic
chemical fertilizers as well as other chemicals, such as herbicides
and pesticides used to increase crop productivity, have caused con-
siderable damage to the ecology of agricultural systems and have
reduced the nutritional quality of crops (Kramer et al., 2006). The
use of biofertilizers and biostimulants to enhance crop growth and
yield has gained considerable momentum for ecological sustain-
ability and consumer health (Craigie, 2011).
Seaweed extracts (SWE) are biodegradable, non-toxic, non-
polluting and non-hazardous to humans, animals and ecology

http://dx.doi.org/10.1016/j.scienta.2014.12.004
0304-4238/Published by Elsevier B.V.
40 C. Xu, D.I. Leskovar / Scientia Horticulturae 183 (2015) 39–47
(Craigie, 2011; Khan et al., 2009). They contain major and minor
nutrients, amino acids, vitamins, and growth promoting substances
(Khan et al., 2009; Sharma et al., 2013). In recent years, the use of
natural seaweed as fertilizer has allowed for partial substitution
of conventional synthetic fertilizer (Dhargalkar and Pereira, 2005;
Hong et al., 2007; Khan et al., 2009; Zodape et al., 2010). Commercial
SWE products can be used as liquid extracts applied as foliar spray,
soil drench, or in granular/powder form as soil conditioners and
manure (Lingakumar et al., 2004; Thirumaran et al., 2009). Many
researchers have reported that SWE promoted growth, increased
yield and quality of horticultural crops such as citrus (Citrus sinen-
sis L.) (Fornes et al., 1995, 2002; Koo and Mayo, 1994), grapes
(Vitis vinifera L.) (Norrie et al., 2002), cucumber (Cucumis sativus
L.) (Sarhan et al., 2011), strawberry (Fragaria ananassa L.) (Alam
et al., 2013), tomato (Solanum lycopersicum L.) (Hernández-Herrera
et al., 2014; Kumari et al., 2011), broccoli (Brassica oleracea var. ital-
ica) (Mattner et al., 2013), olive (Olea europaea L.) (Chouliaras et al.,
2009), spinach (Fan et al., 2011), and bean (Phaseolus acutifolius
L.) (Beckett et al., 1994). While positive effects on plant growth
have been observed in many studies, others have found that SWE
did not provide improvements (Basher et al., 2012; Tourte et al.,
2000). Overall, these studies involved a wide range of SWE derived
from many different species, including Ascophyllum nodosum (A.
nodosum), Ecklonia maxima, Sargassum, Laminaria, Durvillaea pota-
torum, Ulva lactuca, Caulerpa sertularioides, Padina gymnospora,
Sargassum liebmannii, Sargassum johnstonii, and extracts produced
by a range of different groups. Hence, any variations between stud-
ies may be attributed to the different seaweed species used. Also
studies have shown that foliar and drench applications of SWE may
have different effects on crops and thus, application methods may
be an additional source of variation between studies (Crouch and
van Staden, 1992; Hernández-Herrera et al., 2014; Kumari et al.,
2011; Spann and Little, 2011).
In addition to growth enhancing effects of SWE, there is grow-
ing evidence for a role of SWE in increasing plant tolerance to
biotic stress such as diseases and insects, and abiotic stress such as
nutrition deficiency, salinity, drought, flooding and thermal stress
(Guinan et al., 2013; Jayaraman et al., 2011; Khan et al., 2009;
Sangha et al., 2010; Sharma et al., 2013). A study by Guinan et al.
(2013) found that while some commercial A. nodosum extracts were
effective in enhancing plant growth in abiotic stress conditions,
they may be less effective under biotic or stress-free conditions and
vice versa. The study also suggests that such specificity in stress-
tolerance induction by SWE may be associated with the type of
extract involved, their specific biochemical characteristics and the
underlying stress affecting the plant.
The current study aims to assess the efficacy of A. nodosum
SWE under drought stress using spinach as a model plant. Fur-
thermore, the study examines the potential differences in efficacy
between foliar and soil application. A commercially available alka-
line SWE extract of A. nodosum (Stimplex® Crop Biostimulant;
Acadian Seaplants, Nova Scotia, Canada) was used to investigate
its effects on leaf growth, chlorophyll content and fluorescence,
gas exchange, phytochemical contents, antioxidant capacity and
ferrous ion chelating ability (FICA, indirect antioxidant activity) in
spinach when exposed to long and mild drought stress.
2. Materials and methods
2.1. Plant materials and treatments
Five seeds of spinach (cv. Bloomsdale) were sown in each plastic
pot (15 cm diameter and 20-cm deep) filled with a mixture of sand
and topsoil (fine, montmorillonitic, mesic, aquic arqui-dolls) (1:1,
v/v) in a growth chamber with a temperature regime of 23/18 ◦ C
(day/night), a 14-h photoperiod, 65% relative humidity and a pho-
tosynthetically active radiation of 700 ␮mol m−2 s−1 at the canopy
level. Pots were watered to field capacity (allowing for free drainage
to occur) every 4 d before treatments were initiated. Plants were
thinned to one plant per pot two weeks after seeding.
Irrigation and A. nodosum SWE treatments started at 3 weeks
after seeding (3WAS). There were two irrigation treatments (full
irrigation and mild drought stress without visible wilting) and four
A. nodosum SWE treatments (water foliar, SWE foliar, SWE drench,
SWE foliar and drench). For the irrigation treatments, all pots were
watered to field capacity then differential irrigations were applied.
Irrigation volumes were determined by weighing each pot at field
capacity (after an irrigation and after allowing for free drainage
to occur) and again just before irrigation. The weight loss per pot
was assumed to equal total evapotranspiration (ET). The equiv-
alent amount of 100 and 50% ET was applied for full irrigation
and drought stress, respectively. A. nodosum SWE were applied
after irrigation according to the manufacturer’s guidelines. For SWE
foliar treatment, 0.5% SWE solutions were sprayed evenly over the
seedlings to the dripping point, for drench treatment plants were
drenched with 50 mL 0.5% SWE, and for SWE foliar and drench treat-
ment plants were sprayed with 0.5% SWE solution and drenched
with 50 mL 0.5% SWE. The A. nodosum SWE and irrigation treat-
ments were applied every 4 d. For plants with A. nodosum SWE
drench treatment, 50 mL less than the desired volume of water
was applied. Plants were fertilized with 50 mL of N 20–P 20–K 20
(8 g L−1 ) every 8 d.
The level of soil volumetric water content (SWC) in a 4–10 cm
soil layer of each treatment was monitored with ECH2 O® soil mois-
ture probes (EC-5, Decagon Devices Inc., WA). Leaf gas exchange
and fluorescence parameters were measured on the two largest
leaves from each plant at 35 days after treatment was applied, then
plants were harvested to measure leaf growth, relative water con-
tent (RWC), and nutrition quality. For RWC, specific leaf area (SLA),
chlorophyll content and nutrition quality analysis, two sets of leaf
discs (technical replicates) were collected using a cork borer from
five largest leaves of each plant.
2.2. Leaf growth and RWC
Leaf number, area, fresh and dry weight (FW and DW),
RWC, and specific leaf area (SLA) were measured. SLA was cal-
culated as: SLA = area/DW, where DW is leaf dry weight after
drying at 65 ◦ C for 3 d. Leaf RWC was calculated as: RWC
(%) = 100 × [(FW − DW)/(TW − DW)], where TW is turgid weight
after being soaked in water for 4 h at 4 ◦ C.
2.3. Leaf pigment and gas exchange
Leaf pigment was extracted with 80% (v/v) acetone and
absorbance of the extraction was measured at 663, 647 and 470 nm
with a spectrophotometer (Spectronic Genesys, Spectronic Instru-
ments, Rochester, NY). Chlorophyll and carotenoid content was
calculated using the formula described by Lichtenthaler (1987).
Leaf maximum photochemical efficiency (Fv /Fm ), quantum yield
(Y(II)) and electron transport rate (ETR) of photosystem II (PS
II) was measured on two largest leaves from each plant with a
chlorophyll fluorometer (OS5p-FL, OPTI-SCIENCES, Hudson, NH).
Measurements for Fv /Fm were made after leaves were adapted in
darkness for 30 min.
Net photosynthetic rate (Pn , ␮mol CO2 m−2 s−1 ), transpiration
(Tr , mmol H2 O m−2 s−1 ), stomatal conductance (Cond , mol m−2 s−1 ),
and stomatal limitation (Ls ) were determined for the two largest
leaves of each plant using a portable infrared gas analyzer (Li-6400,
LICOR, Inc, Lincoln, NB). The analyzer was set at 500 ␮mol s−1 flow
rate (leaf temperature of 25 ± 0.4 ◦ C, 60 ± 5% relative humidity) and
 C. Xu, D.I. Leskovar / Scientia Horticulturae 183 (2015) 39–47
Fig. 1. Soil volumetric water content (SWC) and irrigation dynamics for spinach plants, subjected to 100 (full irrigation) or 50% evapotranspiration (ET) irrigation (drought
stress).
a light emitting diode external light source providing a photosyn-
thetic photon flux density of 800 ␮mol m−2 s−1 . Ls was calculated
according the following equation: Ls = (1 − Ci /Ca ) × 100, where Ci
and Ca are intercellular and ambient CO2 concentration.
2.4. Nutrition value
About 1.2 g fresh leaf sample was harvested, soaked in liquid
nitrogen immediately, then stored at −80 ◦ C until analysis. Phy-
tochemicals were extracted with 12 mL 80% (v/v) acetone using a
Polytron homogenizer (Kinematica AG, Schweiz, Switzerland), then
incubated in darkness at −20 ◦ C overnight. After centrifuging at
18,000 rpm for 15 min, the supertanant was collected for nutrition
value analysis.
For total phenolics content, 0.1 mL extract was added to a
mixture of 0.15 mL H2 O, 0.75 mL of 1/10 dilution Folin–Ciocalteu
reagent. After 6 min, 0.60 mL of 7.5% (w/v) Na2 CO3 was added and
vortexed, then the mixture was incubated at 45 ◦ C in a water bath
for 10 min. Samples were allowed to cool to room temperature
before reading the absorbance at 765 nm (Slinkard and Singleton,
1997). A standard curve was prepared from a freshly made gallic
acid solution.
For total flavonoid content, 0.20 mL extract was mixed with
0.85 mL distilled water and 50 ␮L of 5% NaNO2 . After 6 min, 100 ␮L
of 10% AlCl3 ·6H2 O was added, and after another 5 min 0.35 mL of
1 M NaOH and 0.20 mL distilled water were added. The absorbance
was measured immediately at 510 nm (Dewanto et al., 2002). A (+)-
catechin hydrate equivalents standard curve was prepared from a
freshly made solution.
The antioxidant capacity was measured by the methods of 2,2-
azino-bis(3-ethylbenzthiazoline-6-sulphonic acid) (ABTS) (Miller
and Rice-Evans, 1996), ferric reducing ability of plasma (FRAP)
(Benzie and Strain, 1996) and 2,2-diphenyl-1-picrylhydrazyl rad-
ical (DPPH) (Brand-Williams et al., 1995). ABTS 2.5 mM stock
solution was prepared and 0.4 g MnO2 was added to the stock solu-
tion to generate ABTS radical cation (ABTS*), stirring the mixture
occasionally for 30 min at room temperature. Excess MnO2 was
removed by first filtering the solution through a Buchner funnel
and then with a 0.2 mM syringe-end filter. The ABTS* solution was
incubated at 30 ◦ C in a water bath and was diluted to an absorbance
of 0.7 (±0.02) at 730 nm using 5 mM phosphate buffer (pH 7.4).
Extract (20 ␮L) was added to 1.60 mL ABTS* solution and vortexed
41
for 10 s. The absorbance of the mixture was measured at 730 nm
after 1 min reaction.
For FRAP method, 10 mM 2,4,6-tris-2,4,6-tripyridyl-2-triazine
(TPTZ) and 20 mM ferric chloride was diluted in 300 mM sodium
acetate buffer (pH 3.6) at a ratio of 1:1:10. Extracts (50 ␮L) were
added to 2 mL TPTZ solution, and the absorbance at 593 nm
was determined after 4.5 min reaction. For DPPH method, DPPH
(20 mg L−1 ) was dissolved in 100% methanol. Extracts (20 ␮L) were
added to 2 mL DPPH solution, and the absorbance was determined
at 515 nm before and after 6 min reaction. Trolox (6-hydroxy-
2,5,7,8-tetramethylchroman-2-carboxyl acid) standard curve was
prepared.
For FICA measurement, the reaction mixture contained 40 ␮L
extracts, 1.48 mL methanol and 30 ␮L of 2 mM ferrous chloride.
After vortexing, 40 ␮L of 5 mM ferrozine was added followed by vig-
orous mixing. After 4 min the absorbance of the ferrozine–ferrous
ions complex was measured at 562 nm (Dinis et al., 1994). FICA
was calculated as the absorbance difference between control and
sample.
2.5. Statistical analysis
The experiment used a complete randomized design. Each bio-
logical replicate contained one pot and each treatment contained
six replicate pots. All data were subjected to analysis of vari-
ance using SAS (SAS Institute Inc., 1993). Mean differences among
irrigation and SWE were determined according to the Tukey’s Stu-
dentized range test at 0.05 significant level.
3. Results
3.1. Soil water content
Immediately after irrigation, the SWC was more than 30% for
full irrigation and around 20% for the drought stress treatment
(Fig. 1). Four days after irrigation and just before the next irriga-
tion event, the SWC difference between full irrigation and drought
stress was greatly reduced, but SWC was still higher in full irriga-
tion than in the drought stress (approximately 16% vs 13%). The
goal of the differential irrigation in this experiment was to main-
tain a relatively constant plant water status (either with or without
drought stress) and a long-term mild drought stress without wilting
symptoms.
42 C. Xu, D.I. Leskovar / Scientia Horticulturae 183 (2015) 39–47
Fig. 2. Effect of A. nodosum SWE foliar (F), drench (D) and F + D on leaf number, area, fresh and dry weight (FW and DW) of spinach under drought stress. The values are
means of six replicates ± standard error. Different letters on top of bars indicate significant different at P ≤ 0.05 according to the Tukey’s Studentized range test.
3.2. Leaf growth and RWC
As expected, drought stress greatly inhibited leaf growth (leaf
number, area, FW and DW) compared with full irrigation (Fig. 2).
The reduction in leaf area resulted from the combined decreases in
leaf number and leaf size. Also leaf RWC and SLA greatly decreased
under drought stress (Fig. 3).
Fig. 3. Effect of A. nodosum SWE foliar (F), drench (D) and F + D on leaf relative
water content (RWC) and specific leaf area (SLA) of spinach under drought stress.
The values are means of six replicates ± standard error. Different letters on top of
bars indicate significant different at P ≤ 0.05 according to the Tukey’s Studentized
range test.
Under full irrigation, A. nodosum SWE had no effect on leaf num-
ber, area, weight, RWC and SLA, regardless of application methods
(Figs. 2 and 3). Under drought stress, leaf growth was improved by
all A. nodosum SWE treatments and application with both foliar and
drench had the most pronounced effects (Figs. 2). All A. nodosum
SWE applications had no effect on leaf number but increased leaf
FW and DW. Also leaf area increased by all A. nodosum SWE treat-
ments but the combined foliar and drench application had most
pronounced effects as compared to single foliar or drench. Leaf RWC
and SLA increased similarly with all A. nodosum SWE applications
(Fig. 3).
3.3. Leaf chlorophyll and gas exchange
Leaf chlorophyll content increased under drought stress based
on FW (Fig. 4) but not on DW (data not shown). Leaf fluorescence
(photochemical efficiency, Y(II), and ETR) were not reduced after
35 d mild drought stress in this experiment (Fig. 4). Still, drought
stress greatly reduced Pn , Cond and Tr , which were only 63, 29 and
45% of these under full irrigation, respectively (Fig. 5). The Ls greatly
increased under drought stress from 19 to 35% (Fig. 5)
Under full irrigation, all A. nodosum SWE applications had no
effects on chlorophyll content and fluorescence (Fig. 4), and gas
exchange (Fig. 5). Under drought stress, A. nodosum SWE did not
affect chlorophyll content and fluorescence parameters (Fig. 4), but
it increased gas exchange similarly for all A. nodosum SWE applica-
tion methods (Fig. 5). Pn , Cond and Tr increased by approximately 25,
71 and 42%, respectively, with A. nodosum SWE application under
drought stress, while Ls decreased from 35% to approximately 27%
(Fig. 5).
3.4. Leaf nutrition value
Compared with full irrigation, drought stress increased leaf con-
tents of carotenoids, phenolics and flavonoids based on FW (Fig. 6),
but not on DW (data not shown). Similar leaf total antioxidant
capacity, irrespective of assay methods, increased under drought
stress based on FW (Fig. 7) but not on DW (data not shown). Leaf
FICA decreased under drought stress based on either FW or DW
(Fig. 8).
 C. Xu, D.I. Leskovar / Scientia Horticulturae 183 (2015) 39–47 43

Fig. 4. Effect of A. nodosum SWE foliar (F), drench (D) and F + D on leaf chlorophyll content and fluorescence of spinach under drought stress. The values are means of six
replicates ± standard error. Different letters on top of bars indicate significant different at P ≤ 0.05 according to the Tukey’s Studentized range test.

Under full irrigation, A. nodosum SWE had no effect on the con-
tent of carotenoids, phenolics and flavonoids, except that foliar
alone or combined with drench application reduced phenolics
content based on FW by approximately 15% (Fig. 6). The total
antioxidant capacity determined by FRAP method decreased by
foliar A. nodosum SWE application based on FW (Fig. 7). Other
than that, the total antioxidant capacity was not affected by any
A. nodosum SWE application. Leaf FICA was not impacted by A.
nodosum SWE applications (Fig. 8).
Under drought stress, A. nodosum SWE had no effects on phy-
tochemical content based on DW (data not shown) and only
decreased phenolics content based on FW (Fig. 6). Total antiox-
idant capacity based on FW might decrease depending on A.
nodosum SWE application methods (Fig. 7), but based on DW
it was not affected by A. nodosum SWE (data not shown). Leaf
FICA, based on either FW or DW, was reduced by A. nodosum
SWE regardless of application methods under drought stress
(Fig. 8).

Fig. 5. Effect of A. nodosum SWE foliar (F), drench (D) and F + D on leaf gas exchange of spinach under drought stress. The values are means of six replicates ± standard error.
Different letters on top of bars indicate significant different at P ≤ 0.05 according to the Tukey’s Studentized range test.
44 C. Xu, D.I. Leskovar / Scientia Horticulturae 183 (2015) 39–47
Fig. 6. Effect of A. nodosum SWE foliar (F), drench (D) and F + D on leaf total contents
of carotenoids, flavonoids and phenolics of spinach under drought stress. The values
are means of six replicates ± standard error. Different letters on top of bars indicate
significant different at P ≤ 0.05 according to the Tukey’s Studentized range test.
4. Discussion
4.1. Leaf growth and RWC
Loss of turgor pressure under drought usually results in reduc-
tion of cell expansion which leads to decreased leaf area (Taiz and
Zeiger, 2012a). As an indicator of leaf thickness, SLA has often been
reported to decrease under drought stress (Marcelis et al., 1998; Liu
and Stützel, 2004). A decrease in SLA under drought may be due
to the different sensitivity of photosynthesis and leaf expansion,
since drought stress reduces leaf expansion earlier than photo-
synthesis (Jensen et al., 1996; Tardieu et al., 1999). Reduction of
SLA is assumed to improve water use efficiency, because thicker
leaves usually have a higher density of chlorophyll and proteins
and, hence, have a greater photosynthetic capacity than thinner
leaves (Craufurd et al., 1999; Liu and Stützel, 2004; Wright et al.,
1994).
Under full irrigation, A. nodosum SWE had no effect on spinach
growth and leaf water content in the present study. However, sev-
eral researchers found that without abiotic stress, SWE enhanced
growth of horticultural crops. In a sand culture study, Beckett et al.
(1994) reported that SWE spray increased bean leaf area but did
not alter shoot and root biomass. In a greenhouse study, marigold
shoot weight and length increased with SWE applied either foliar
or to roots, but leaf area and root growth were unchanged (van
Fig. 7. Effect of A. nodosum SWE foliar (F), drench (D) and F + D on leaf total
antioxidant capacity of spinach under drought stress. The values are means of six
replicates ± standard error. Different letters on top of bars indicate significant dif-
ferent at P ≤ 0.05 according to the Tukey’s Studentized range test.
Staden et al., 1994). Kumari et al. (2011) found that tomato growth
was greatly enhanced by SWE, irrespective of application methods,
during a long term experiment. However, Crouch and van Staden
(1992) reported that plant FW and leaf area of young tomato plants
was improved by SWE drench, but not as foliar spray. In broccoli
grown in controlled conditions in a greenhouse, drench applica-
tion of SWE increased leaf area, stem diameter, and both shoot and
root biomass (Mattner et al., 2013). In the field, drench application
of SWE to clay-loam soil increased broccoli leaf number and area,
and stem diameter, but in sandy soil the effect was much less pro-
nounced (Mattner et al., 2013). Other researchers found that while
the efficacy of SWE were more pronounced in abiotic stress condi-
tions, SWE had more limited influence on growth of lettuce (Guinan
et al., 2013), orange (Spann and Little, 2011) and okra (Papenfus
et al., 2013) in stress-free conditions. E. maxima SWE were used
for okra under nutrient deficiency while A. nodosum extracts were
used for orange under drought stress and lettuce under salinity
and biotic stress. The current study on spinach is consistent with
these findings regardless of different seaweed species and stress
conditions.
Under drought stress, spinach leaf growth was improved by all
A. nodosum SWE treatments. Increased leaf RWC and area by A.
nodosum SWE under drought stress indicates that A. nodosum SWE
application could improve spinach leaf water relations and help
maintaining cell turgor pressure and expansion, which results in
 C. Xu, D.I. Leskovar / Scientia Horticulturae 183 (2015) 39–47
Fig. 8. Effect of A. nodosum SWE foliar (F), drench (D) and F + D on leaf ferrous ion
chelating ability (FICA) of spinach under drought stress. The values are means of
six replicates ± standard error. Different letters on top of bars indicate significant
different at P ≤ 0.05 according to the Tukey’s Studentized range test.
a large leaf area. Similarly, Spann and Little (2011) suggested that
the maintenance of orange plant growth by SWE under drought
stress might be related to improved water relations. Also Neily et al.
(2010) found that treatment of vegetable plants (lettuce, melon,
tomato, pepper and celery) with SWE greatly increased leaf water
content and improved the recovery of wilted plants. Increased
leaf area could result in more photosynthetic products, leading to
enhanced growth. In this study, leaf FW and DW increased by 20 to
32% and 11 to 22%, respectively, with A. nodosum SWE application.
4.2. Leaf chlorophyll and gas exchange
Mild drought stress simulated in this experiment just reduced
leaf water content while had no direct effects on chlorophyll con-
tent. Similarly, leaf fluorescence was not altered by mild drought
stress. The large increase in Ls under drought stress suggests that
reductions in Pn , Cond and Tr mainly resulted from stomatal closure.
Stomatal closure is a quick response to drought stress and an effi-
cient way to reduce water loss under these conditions (Taiz and
Zeiger, 2012b). However, low CO2 availability caused by stomatal
closure results in reduction in gas exchange. Under mild drought
stress in this experiment, the reduction in Pn , together with reduced
leaf areas as discussed above, led to decreased FW, DW and there-
fore plant growth.
Under full irrigation, A. nodosum SWE had no effects on spinach
leaf chlorophyll content and fluorescence, and gas exchange in
the present study. However, Kumari et al. (2011) found that SWE,
irrespective of application methods, could increase photosynthetic
pigments (chlorophyll and carotenoids) content in tomato leaves.
SWE was also found effective in increasing chlorophyll synthesis
in Zea mays and Phaseolus mungo (Lingakumar et al., 2004) and
Salvia officinalis (Kaoaua et al., 2013). There are a limited num-
ber of reports about the SWE effects on the function of PS II and
leaf gas exchange. Similarly to our results, no significant effects of
SWE under full irrigation were found in leaf photosynthetic rate of
orange trees (Spann and Little, 2011).
45
In the present study under drought stress, although A. nodosum
SWE did not affect chlorophyll content and fluorescence param-
eters, it increased gas exchange and decreased Ls from 35% to
approximately 27%. This result suggests that under drought stress
A. nodosum SWE could enhance leaf gas exchange mainly due to
reduced stomatal closure resulting from improved water relations.
Similarly, Cassan et al. (1992) found that spinach plants treated
with SWE did not exhibit significant increase in the O2 evolu-
tion rate of chloroplasts. However, under drought stress SWE were
found to increase chlorophyll content in S. officinalis (Kaoaua et al.,
2013), but had no effects on photosynthesis in orange leaves (Spann
and Little, 2011). This inconsistency might be due to differences
in drought severity and period, as well as growth condition and
plant species. Overall, under mild drought stress in the present
study A. nodosum SWE application improved leaf water relations
and helped maintaining cell turgor pressure and reducing stomatal
closure, which in turn resulted in a large leaf area and high photo-
synthetic rate, and consequently enhanced growth. The chlorophyll
content and function of PS II were not affected by the simulated
mild drought stress and the A. nodosum SWE application.
4.3. Leaf nutrition value
Polyphenols are a class of secondary metabolites which play
a key role as antioxidants. Among polyphenols, flavonoids have
attracted considerable interest due to their broad spectrum of
biological effects such as antioxidant, anti-inflammatory, vasore-
laxant, antimicrobial, antiviral, anticarcinogenic and antimutagenic
(Guo et al., 2011; Maimoona et al., 2011). Carotenoids have long
been recognized as essential nutrients and important health ben-
eficial compounds (Fraser and Bramley, 2004). Therefore, total
phenolics, flavonoids and carotenoid were quantified in this study.
Compared with full irrigation, drought stress increased leaf con-
tents of carotenoids, phenolics and flavonoids based on FW, but
not on DW, indicating that drought stress had no direct effects on
the phytochemical content.
The total antioxidant capacity of spinach leaf was measured by
three methods (ABTS, FRAP, and DPPH) because they are commonly
used to evaluate vegetable or fruit nutrition value (Ozgen et al.,
2006; Rekika et al., 2005). Similar to the response of phytochemical
content to drought stress, leaf total antioxidant capacity, irrespec-
tive of assay methods, increased under drought stress based on
FW but not on DW. This also indicates that drought stress in this
experiment did not directly affect leaf total antioxidant capacity.
Interestingly, leaf FICA decreased under drought stress based on
either FW or DW. Ferrous ion chelation is another indirect mecha-
nism of antioxidant activity. Although iron is an essential element,
excess iron may lead to high levels of active oxygen species. In
aqueous media ferrous ions generate superoxide anion radical and
hydrogen peroxide by autoxidation. Also they are involved in con-
version of superoxide anion radical and hydrogen peroxide into
more reactive hydroxyl radical (Huang, 2003). Ferrous ion chela-
tion can afford protection against oxidative damage (Huang, 2003).
In the present study, FICA response to drought stress was not con-
sistent with the responses of phenolics, carotenoids or flavonoids
content, which suggests that they are not mainly responsible for
chelating metal ions. A previous study also found that the fer-
rous ion chelating effects in pine extract did not correlate with the
phenolics content (Apetrei et al., 2011). Polysaccharides, peptides,
proteins, oleoresins and saponins have been reported to chelate
ferrous ions (Gülçin et al., 2006; Wang et al., 2009). Our results sug-
gest that spinach nutrition quality could be negatively influenced
by reduced FICA under drought stress.
Under full irrigation, A. nodosum SWE had little effect on the
content of carotenoids, phenolics and flavonoids, total antioxi-
dant capacity and FICA. However, previous research found that
46 C. Xu, D.I. Leskovar / Scientia Horticulturae 183 (2015) 39–47
drench application of SWE could enhance nutrition quality of
spinach and onion by increasing phenolics and flavanoids con-
tents (Fan et al., 2011; Lola-Luz et al., 2014), and that tomato leaf
phenolics and fruit lycopene and ascorbate content increased by
SWE, regardless of application methods (Kumari et al., 2011). The
inconsistency might be due to different growth medium or soil
texture since in sandy soil the effect of SWE drench application
was much less pronounced than in clay-loam soil (Mattner et al.,
2013).
In the present study under drought stress, A. nodosum SWE
had no effects on phytochemical content based on DW and only
decreased phenolics and flavonoids (SWE foliar) content based on
FW. These results indicate that phytochemicals content were not
directly altered by either drought or A. nodosum SWE, and the
content differences based on FW were caused by different water
content. Under drought stress, total antioxidant capacity based
on FW might decrease depending on A. nodosum SWE application
methods, but based on DW it was not affected by A. nodosum SWE
(data not shown). Consistent with the response of phytochemicals
content, leaf total antioxidant capacity was not directly altered by
either mild drought stress or A. nodosum SWE application. Con-
versely, leaf FICA, based on either FW or DW, was reduced by A.
nodosum SWE regardless of application methods under drought
stress. Our results suggest that spinach nutrition quality could be
negatively influenced by reduced FICA under drought stress, espe-
cially with A. nodosum SWE application.
In summary, the mild drought stress simulated in this study
deceased leaf water content, then lead to stomatal closure and
lose of cell turgor pressure and expansion, resulting in reduced
photosynthetic rate and leaf area. Mild drought stress had no
influence on leaf chlorophyll content, PS II function and nutrition
value, except for reduced FICA. With full irrigation A. nodosum
SWE had no effects on spinach growth, pigment content, PS II,
gas exchange and nutrition value. Under mild drought stress, A.
nodosum SWE enhanced spinach growth by improving leaf water
relations, which resulted in maintenance of cell turgor pressure and
reduction in stomatal limitation. A. nodosum SWE had no direct
influence on chlorophyll contents, PS II function and nutrition
quality, except for reduced FICA. A. nodosum SWE application, in
conjunction with deficit irrigation for spinach production in the
field, might be an efficient strategy for both water savings and
organic farming. Further research should be conducted to better
define and optimize application methods, rates and timing in the
field.
Acknowledgments
The authors thank Acadian Seaplants for providing seaweed
extracts.
References
Alam, M.Z., Braun, G., Norrie, J., Hodges, D.M., 2013. Effect of Ascophyllum extract
application on plant growth, fruit yield and soil microbial communities of straw-
berry. Can. J. Plant Sci. 93, 23–36.
Apetrei, C.L., Tuchilus, C., Aprotosoaie, A.C., Oprea, A., Malterud, K.E., Miron, A., 2011.
Chemical, antioxidant and antimicrobial investigations of Pinus cembra L. bark
and needles. Moleculars 16, 7773–7788.
Basher, A.A., Mohammed, A.J., Teeb, A.I.H., 2012. Effect of seaweed and drainage
water on germination and seedling growth of tomato (Lycopersicon spp.).
Euphrates J. Agric. Sci. 4, 24–39.
Beckett, R.P., Mathegka, A.D.M., Van Staden, J., 1994. Effect of seaweed concentrate
on yield of nutrient-stressed tepary bean (Phaseolus acutifolius Gray). J. Appl.
Phycol. 6, 429–430.
Benzie, I.F.F., Strain, J.J., 1996. The ferric reducing ability of plasma (FRAP) as a
measure of “antioxidant power”: the FRAP assay. Anal. Biochem. 239, 70–76.
Bergman, M., Varshavsky, L., Gottlieb, H.E., Grossman, S., 2001. The antioxidant
activity of aqueous spinach extract: chemical identification of active fractions.
Phytochemisty 58, 143–152.
Brand-Williams, W., Cuvelier, M.E., Berset, C., 1995. Use of a free radical method to
evaluate antioxidant activity. Lebensm. Wiss. Technol. 28, 25–30.
Cassan, L., Jeannin, I., Lamaze, T., Morot-Gaudry, J.F., 1992. The effect of the Asco-
phyllum nodosum extract Goëmar GA 14 on the growth of spinach. Bot. Marina
35, 437–439.
Chouliaras, V., Tasioula, M., Chatzissavvidis, C., Theriosa, I., Tsabolatidou, E., 2009.
The effects of a seaweed extract in addition to nitrogen and boron fertilization
on productivity, fruit maturation, leaf nutritional status and oil quality of the
olive (Olea europaea L.) cultivar Koroneiki. J. Sci. Food Agric. 89, 984–988.
Craigie, J.S., 2011. Seaweed extract stimuli in plant science and agriculture. J. Appl.
Phycol. 23, 371–393.
Craufurd, P.Q., Wheeler, T.R., Ellis, R.H., Summerfield, R.J., Williams, J.H., 1999. Effect
of temperature and water deficit on water-use efficiency, carbon isotope dis-
crimination, and specific leaf area in peanut. Crop Sci. 39, 136–142.
Crouch, I.J., Van Staden, J., 1992. Effect of seaweed concentrate on the establishment
and yield of greenhouse tomato plants. J. Appl. Phycol. 4, 291–296.
Dewanto, V., Wu, X., Adom, K.K., Liu, R.H., 2002. Thermal processing enhances the
nutritional value of tomatoes by increasing total antioxidant activity. J. Agric.
Food Chem. 50, 3010–3014.
Dhargalkar, V.K., Pereira, N., 2005. Seaweed: promising plant of the millennium. Sci.
Cult. 71, 60–66.
Dinis, T.C.P., Madeira, V.M.C., Almeida, M.L.M., 1994. Action of phenolic derivates
(acetoaminophen, salycilate and 5-aminosalycilate) as inhibitors of membrane
lipid peroxidation and as peroxyl radical scavengers. Arch. Biochem. Biophys.
315, 161–169.
Edenharder, R., Keller, G., Platt, K.L., Unger, K.K., 2001. Isolation and characterization
of structurally novel antimutagenic flavonoids from spinach (Spinacia oleracea).
J. Agric. Food Chem. 49, 2767–2773.
Fan, D., Hodges, M., Zhang, J., Kirby, C.W., Ji, X., Locke, S.J., Critchley, A.T., Prithiviraj, B.,
2011. Commercial extract of the brown seaweed Ascophyllum nodosum enhances
phenolic antioxidant content of spinach (Spinacia oleracea L.) which protects
Caenorhabditis elegans against oxidative and thermal stress. Food Chem. 124,
195–202.
Fornes, F., Sanchez-Perales, M., Guardiola, J.L., 1995. Effect of a seaweed extract on
citrus fruit maturation. Acta Hortic. 379, 75–82.
Fornes, F., Sanchez-Perales, M., Guardiola, J.L., 2002. Effect of a seaweed extract on
the productivity of ‘de Nules’ clementine mandarin and navelina orange. Bot.
Mar. 45, 486–489.
Fraser, P.D., Bramley, P.M., 2004. The biosynthesis and nutritional uses of
carotenoids. Prog. Lipid Res. 43, 228–265.
Guinan, K.J., Sujeeth, N., Copeland, R.B., Jones, P.W., O’Brien, N.M., Sharma, H.S.S.,
Prouteau, P.J.F., O’Sullivan, J.T., 2013. Discrete roles for extracts of Ascophyllum
nodosum in enhancing plant growth and tolerance to abiotic and biotic stress.
Acta Hortic. 1009, 127–135.
Gülçin, I., Mshvildadze, V., Gepdiremen, A., Elias, R., 2006. The antioxidant activity of
a triterpenoid glycoside isolated from the berries of Hedera colchica: 3-O-(beta-
d-glucopyranosyl)-hederagenin. Phytother. Res. 20, 130–134.
Guo, T., Wei, L., Sun, J., Hou, C., Fan, L., 2011. Antioxidant activities of
extract and fractions from Tuber indicum Cooke & Massee. Food Chem. 127,
1634–1640.
Hernández-Herrera, R.M., Santacruz-Ruvalcaba, F., Ruiz-López, M.A., Norrie, J.,
Hernández-Carmona, G., 2014. Effect of liquid seaweed extracts on growth of
tomato seedlings (Solanum lycopersicum L.). J. Appl. Phycol. 26, 619–628.
Hong, D.D., Hien, H.M., Son, P.N., 2007. Seaweeds from Vietnam used for functional
food, medicine and biofertilizer. J. Appl. Phycol. 19, 817–826.
Huang, X., 2003. Iron overload and its association with cancer risk in humans: evi-
dence for iron as a carcinogenic metal. Mutat. Res. 533, 153–171.
Jayaraman, J., Norrie, J., Punja, Z.K., 2011. Commercial extract from the brown sea-
weed Ascophyllum nodosum reduces fungal diseases in greenhouse cucumber. J.
Appl. Phycol. 23, 353–361.
Jensen, C.R., Mogensen, V.O., Mortensen, G., Andersen, M.N., Schjoerring, J.K., Thage,
J.H., Koribidis, J., 1996. Leaf photosynthesis and drought adaptation in field-
grown oilseed rape (Brassica napus L.). Aust. J. Plant Physiol. 23, 631–644.
Kaoaua, M.E., Chernane, H., Benaliat, A., Neamallah, L., 2013. Seaweed liquid extracts
effect on Salvia officinalis growth, biochemical compounds and water deficit
tolerance. Afr. J. Biotechnol. 12, 4481–4589.
Khan, W., Rayirath, U.P., Subramanian, S., Jithesh, M.N., Rayorath, P., Hodges, D.M.,
Critchley, A.T., Craigie, J.S., Norrie, J., Prithiviraj, B., 2009. Seaweed extracts as
biostimulants of plant growth and development. J. Plant Growth Regul. 28,
386–399.
Koo, R.C.J., Mayo, S., 1994. Effects of seaweed sprays on citrus fruit production. Proc.
Fla. State Hortic. Soc. 107, 82–85.
Kramer, S.B., Reganold, J.P., Glover, J.D., Bohannan, B.J.M., Mooney, H.A., 2006.
Reduced nitrate leaching and enhanced denitrifier activity and efficiency in
organically fertilized soils. PNAS 103, 4522–4527.
Kumari, R., Kaur, I., Bhatnagar, A.K., 2011. Effect of aqueous extract of Sargassum john-
stonii Setchell & Gardner on growth, yield and quality of Lycopersicon esculentum
Mill. J. Appl. Phycol. 23, 623–633.
Lichtenthaler, H.K., 1987. Chlorophylls and carotenoids-pigments of photosynthetic
biomembranes. Methods Enzymol. 148, 350–382.
Lingakumar, K., Jeyaprakash, R., Manimuthu, C., Haribaskar, A., 2004. Influence of
Sargassum sp. crude extract on vegetative growth and biochemical characteris-
tics in Zea mays and Phaseolus mungo. Seaweed Res. Utiln. 26, 155–160.
Liu, F., Stützel, H., 2004. Biomass partitioning, specific leaf area, and water use effi-
ciency of vegetable amaranth (Amaranthus spp.) in response to drought stress.
Sci. Hortic. 102, 15–27.
 C. Xu, D.I. Leskovar / Scientia Horticulturae 183 (2015) 39–47
Lola-Luz, T., Hennequart, F., Gaffney, M., 2014. Effect on health promot-
ing phytochemicals following seaweed application, in potato and onion
crops grown under a low input agricultural system. Sci. Hortic. 170,
224–227.
Maimoona, A., Naeem, I., Saddiqe, Z., Jameel, K., 2011. A review on biological,
nutraceutical and clinical aspects of French maritime pine bark extract. J.
Ethnopharmacol. 133, 261–277.
Marcelis, L.F.M., Heuvelink, E., Goudriaan, J., 1998. Modelling biomass production
and yield of horticultural crops: a review. Sci. Hortic. 74, 83–111.
Mattner, S.W., Wite, D., Riches, D.A., Porter, I.J., Arioli, T., 2013. The effect of kelp
extract on seedling establishment of broccoli on contrasting soil types in south-
ern Victoria, Australia. Biol. Agric. Hortic. 29, 258–270.
Miller, N.J., Rice-Evans, C.A., 1996. Spectrophotometric determination of antioxidant
activity. Redox Rep. 2, 161–171.
Neily, W., Shishkov, L., Nickerson, S., Titus, D., Norrie, J., 2010. Commercial extracts
from the brown seaweed Ascophyllum nodosum (Acadian) improves early estab-
lishment and helps resist water stress in vegetable and flower seedlings.
HortScience 45, S234.
Norrie, J., Branson, T., Keathley, P.E., 2002. Marine plant extracts impact on grape
yield and quality. Acta Hortic. 594, 315–319.
Ozgen, M., Reese, R.N., Tulio Jr., A.Z., Scheerens, J.C., Miller, A.R., 2006. Modified 2,2-
azino-bis-3-ethylbenzothiazoline-6-sulfonic acid (ABTS) method to measure
antioxidant capacity of selected small fruits and comparison to ferric reducing
antioxidant power (FRAP) and 2,2¢-diphenyl-1-picrylhydrazyl (DPPH) methods.
J. Agric. Food Chem. 54, 1151–1157.
Pandjaitan, N., Howard, L.R., Morelock, T., Gil, M.I., 2005. Antioxidant capacity and
phenolic content of spinach as affected by genetics and maturation. J. Agric. Food
Chem. 53, 8618–8623.
Papenfus, H.B., Kulkarni, M.G., Stirk, W.A., Finnie, J.F., Van Staden, J., 2013. Effect of
a commercial seaweed extract (Kelpak® ) and polyamines on nutrient-deprived
(N, P and K) okra seedlings. Sci. Hortic. 151, 142–146.
Rekika, D., Khanizadeh, S., Deschenes, M., Levasseur, A., Charles, M., Tsao, R., Yang, R.,
2005. Antioxidant capacity and phenolic content of selected strawberry geno-
types. HortScience 40, 1777–1781.
Sangha, J.S., Hobson, D., Hiltz, D., Critchley, A.T., Prithiviraj, B., 2010. The use of com-
mercial seaweed extracts as a means to alleviate abiotic stress in land plants: a
review. Algal Resour. 3, 153–168.
SAS Institute, 1993. SAS user’s guide: Statistics. SAS Institute, Cary, NC.
47
Sarhan, T.Z., Ali, S.T., Rasheed, S.M.S., 2011. Effect of bread yeast application and
seaweed extract on cucumber (Cucums sativus L.) plant growth, yield and fruit
quality. Mesopotamia J. Agric. 39, 26–34.
Sharma, H.S.S., Fleming, C.C., Selby, C., Rao, J.R., Martin, T.J.G., 2013. Plant bios-
timulants: a review on the processing of macroalgae and use of extracts for
crop management to reduce abiotic and biotic stresses. J. Appl. Phycol. 26,
465–490.
Slinkard, K., Singleton, V.L., 1997. Total phenol analysis: automation and comparison
with manual methods. Am. J. Enol. Viticult. 28, 49–55.
Spann, T.M., Little, H.A., 2011. Applications of a commercial extract of the brown
seaweed Ascophyllum nodosum increases drought tolerance in container-grown
‘Hamlin’ sweet orange nursery trees. HortScience 46, 577–582.
Taiz, L., Zeiger, E., 2012a. Plant Physiology. Sinauer Associates Inc., Publishers, Sun-
derland, MA, pp. 759.
Taiz, L., Zeiger, E., 2012b. Plant Physiology. Sinauer Associates Inc., Publishers, Sun-
derland, MA, pp. 769.
Tardieu, F., Granier, C., Muller, B., 1999. Modelling leaf expansion in a fluctuating
environment: are changes in specific leaf area a consequence of changes in
expansion rate? New Phytol. 143, 33–43.
Thirumaran, G., Arumugam, M., Arumugam, R., Anantharaman, P., 2009. Effect of
seaweed liquid fertilizer on growth and pigment concentration of Abelmoschus
esculentus (I) medikus. Am. Eurasian J. Agron. 2, 57–66.
Tourte, L., Bugg, R.L., Shennan, C., 2000. Foliar-applied seaweed and fish powder do
not improve yield and fruit quality of organically grown processing tomatoes.
Biol. Agric. Hortic. 18, 15–27.
van Staden, J., Upfold, S.J., Drewes, E.E., 1994. Effect of seaweed concentrate on
growth and development of the marigold Tagetes patula. J. Appl. Phycol. 6,
427–428.
Wang, T., Jónsdóttir, R., Ólafsdóttir, G., 2009. Total phenolic compounds, radical scav-
enging and metal chelation of extracts from Icelandic seaweeds. Food Chem. 116,
240–248.
Wright, G.C., Rao, R.C.N., Farquhar, G.D., 1994. Water-use efficiency and carbon iso-
tope discrimination in peanuts under water deficit conditions. Crop Sci. 34,
92–97.
Zodape, S.T., Mukhopadhyay, S., Eswaran, K., Reddy, M.P., Chikara, J., 2010.
Enhanced yield and nutritional quality in green gram (Phaseolus radiata L.)
treated with seaweed (Kappaphycus alvarezii) extract. J. Sci. Ind. Res. 69,
468–471.