Regulation of growth, nutritive, phytochemical and antioxidant potential of cultivated Drimiopsis maculata in response to biostimulant (vermicompost leachate, VCL) application

Abstract

The effect of vermicompost leachate (VCL, low-cost biostimulant) on the growth, elemental (macro and micro-nutrients) and phytochemical content as well as the antioxidant potential of Drimiopsis maculata was evaluated. Three dilutions (1:5; 1:10 and 1:20) of VCL were tested and the cultivation lasted for 3 months. In addition to the recorded growth parameters, dried and ground plant materials (leaves and bulbs) were evaluated for nutrients, phenolic acids and antioxidant capacity. Vermicompost leachate application enhanced the growth of D. maculata, particularly, the leaves (VCL 1:10) and bulbs (VCL 1:20) which were significantly bigger than the controls. Apart from the concentration of phosphorus which was significantly lower in the leaves of VCL (1:20)-treated plants, the quantity of all four macro-nutrients analysed were similar with and without VCL. Similar observations were also demonstrated in the majority of quantified micro-nutrients in D. maculata. Relative to the control, VCL-treated plants had higher concentrations of the 10 phenolic acids quantified in the leaves. However, the majority of the quantified phenolic acids were not significantly enhanced in bulbs. Antioxidant activity of D. maculata extracts was generally higher in leaves than in the bulbs. The leaf extract from VCL (1:10 and 1:20)-treated plants exhibited lower oxygen radical absorbance capacity (ORAC) when compared to the control. However, bulbs from VCL (1:5) treatment had significantly higher ORAC than the control. From a conservational perspective, the current findings provided insight on viable approaches useful for mitigating challenges associated with over-harvesting of highly utilized but slow-growing plant species.

Full text

1 23
Plant Growth Regulation
An International Journal on Plant
Growth and Development
ISSN 0167-6903
Plant Growth Regul
DOI 10.1007/s10725-018-0441-1
Regulation of growth, nutritive,
phytochemical and antioxidant potential of
cultivated Drimiopsis maculata in response
to biostimulant (vermicompost leachate,
VCL) application
Lister Dube, Kuben K.Naidoo, Georgina
D.Arthur, Adeyemi O.Aremu, Jiri
Gruz, Michaela Šubrtová, Monika
Jarošová, Petr Tarkowski, et al.

1 23
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Vol.:(0123456789)
1 3
Plant Growth Regulation
https://doi.org/10.1007/s10725-018-0441-1
ORIGINAL PAPER
Regulation ofgrowth, nutritive, phytochemical andantioxidant
potential ofcultivated Drimiopsis maculata inresponse
tobiostimulant (vermicompost leachate, VCL) application
ListerDube1· KubenK.Naidoo1· GeorginaD.Arthur1· AdeyemiO.Aremu2,3 · JiriGruz4· MichaelaŠubrtová4·
MonikaJarošová5· PetrTarkowski5,6· KarelDoležal4
Received: 17 January 2018 / Accepted: 12 September 2018
© Springer Nature B.V. 2018
Abstract
The effect of vermicompost leachate (VCL, low-cost biostimulant) on the growth, elemental (macro and micro-nutrients)
and phytochemical content as well as the antioxidant potential of Drimiopsis maculata was evaluated. Three dilutions (1:5;
1:10 and 1:20) of VCL were tested and the cultivation lasted for 3months. In addition to the recorded growth parameters,
dried and ground plant materials (leaves and bulbs) were evaluated for nutrients, phenolic acids and antioxidant capacity.
Vermicompost leachate application enhanced the growth of D. maculata, particularly, the leaves (VCL 1:10) and bulbs
(VCL 1:20) which were significantly bigger than the controls. Apart from the concentration of phosphorus which was sig-
nificantly lower in the leaves of VCL (1:20)-treated plants, the quantity of all four macro-nutrients analysed were similar
with and without VCL. Similar observations were also demonstrated in the majority of quantified micro-nutrients in D.
maculata. Relative to the control, VCL-treated plants had higher concentrations of the 10 phenolic acids quantified in the
leaves. However, the majority of the quantified phenolic acids were not significantly enhanced in bulbs. Antioxidant activity
of D. maculata extracts was generally higher in leaves than in the bulbs. The leaf extract from VCL (1:10 and 1:20)-treated
plants exhibited lower oxygen radical absorbance capacity (ORAC) when compared to the control. However, bulbs from
VCL (1:5) treatment had significantly higher ORAC than the control. From a conservational perspective, the current findings
provided insight on viable approaches useful for mitigating challenges associated with over-harvesting of highly utilized but
slow-growing plant species.
Keywords Asparagaceae· Conservation· Geophytes· Phenolic acids· Plant nutrients· Medicinal plants
* Adeyemi O. Aremu
aredeyemi@yahoo.com
1 Department ofNature Conservation, Faculty ofNatural
Sciences, Mangosuthu University ofTechnology, Jacobs, P.
O. Box12363, Durban4026, SouthAfrica
2 Indigenous Knowledge Systems (IKS) Centre, Faculty
ofNatural andAgricultural Sciences, North-West University,
Private Mail Bag X2046, Mmabatho2735, SouthAfrica
3 Food Security andSafety Niche Area, Faculty ofNatural
andAgricultural Sciences, North West University, Private
Mail Bag X2046, Mmabatho2790, NorthWestProvince,
SouthAfrica
4 Laboratory ofGrowth Regulators & Department ofChemical
Biology andGenetics, Centre oftheRegion Haná
forBiotechnological andAgricultural Research, Faculty
ofScience, Palacký University & Institute ofExperimental
Botany ASCR, Šlechtitelů 11, 78371Olomouc,
CzechRepublic
5 Centre oftheRegion Haná forBiotechnological
andAgricultural Research, Central Laboratories
andResearch Support, Faculty ofScience, Palacký
University, Šlechtitelů 27, 78371Olomouc, CzechRepublic
6 Centre oftheRegion Haná forBiotechnological
andAgricultural Research, Department ofGenetic Resources
forVegetables, Medicinal andSpecial Plants, Crop Research
Institute, Šlechtitelů 29, 78371Olomouc, CzechRepublic
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Abbreviations
AAPH 2,2-Azobis(2-methylpropionamidine)
dihydrochloride
CRD Completely randomised design
DW Dry weight
ICP-MS Inductively coupled plasma mass spectrometry
ORAC Oxygen radical absorbance capacity
ORS Octapole reaction system
TE Trolox equivalents
UHPLC Ultra-high performance liquid chromatography
VCL Vermicompost leachate
Introduction
Globally, there is a need for effective utilization of the lim-
ited arable land to enhance plant productivity. Although the
application of inorganic fertilizers and associated synthetic
chemicals have been extensively utilized to increase pro-
ductivity of plants, their resultant detrimental effects on the
environment and residual negative effects on human health
are a major concern (Lyson 2002; Tilman etal. 2002). As
a result, there is an increasing call for a shift in paradigm
which leans toward ‘green-based’ agricultural practices
including the use of biostimulants (Calvo etal. 2014; Lyson
2002; Sharma etal. 2014). Biostimulants are generally
sourced from diverse (natural) sources and hold tremendous
economic potential with an estimated $2billion worth of
sales in the global market by 2018 (Brown and Saa 2015;
Calvo etal. 2014). The application of biostimulants offers
a potential novel approach to enhanced growth and devel-
opment as well as mitigate biotic and abiotic stresses dur-
ing the cultivation of plants with diverse attributes such as
nutritional, ornamental and medicinal values (Craigie 2011;
Halpern etal. 2015; Sharma etal. 2014; Yakhin etal. 2017).
Interest in the cultivation of medicinal plants have gained
considerable attention as a result of the need to meet the
increasing demand from both local and international markets
(Affolter and Pengelly 2007; Canter etal. 2005; Lubbe and
Verpoorte 2011; Moyo etal. 2015). Particularly, the intense
harvesting of perennial herbs and geophytes for commercial
purposes has resulted in significant depletion in the wild
population of many species in sub-Saharan Africa (Dold
and Cocks 2002; Moyo etal. 2015; Williams etal. 2007). As
an alternative to harvesting medicinal plants from the wild
population, their cultivation has the potential to overcome
the challenges which entail quality control and ensure sus-
tainability as well as availability (Canter etal. 2005; Lubbe
and Verpoorte 2011; Wiersum etal. 2006). In addition, it
will be valuable to devise novel approaches that have the
potential to help reduce the long regeneration cycle of bulbs
which are often utilized in traditional medicine (Moyo etal.
2015). Recently, researchers have demonstrated the potential
of using biostimulants such as seaweed extracts, vermicom-
post and associated products for growth and cultivation
of plants with medicinal and nutritive value (Aremu etal.
2014, 2015a, 2016; Masondo etal. 2016; Pant etal. 2012;
Wang etal. 2014). Apart from enhancing the morphological
appearance of cultivated plants, the phytochemical integ-
rity of the medicinal plant need to be demonstrated in order
to guarantee their acceptability by local and international
consumers (Lubbe and Verpoorte 2011). Currently, there is
paucity of knowledge on the phytochemical integrity of cul-
tivated medicinal plants subjected to different biostimulants.
Drimiopsis maculata Lindl. & Paxton (syn Ledebouria
petiolata J.C.Manning & Goldblatt; family: Asparagaceae,
formerly: Hyacinthaceae) is a geophyte which is distributed
from Tanzania to South Africa (Manning etal. 2004). In
South Africa, the species is widespread in the eastern part of
the country particularly, in provinces such as Mpumalanga,
Gauteng, KwaZulu-Natal and Eastern Cape. Besides the
ornamental potential of D. maculata (Reinten etal. 2011),
it is well-known for its medicinal value among communities
in South Africa. For instance, infusions prepared from the
pound bulbs are applied as an enema for stomach-related
ailments in children (Hulme 1954). Bulb infusions are also
administered as purge for newly born suffering from ‘ipleyti’,
related to a marasmic condition among the Zulus (Hutch-
ings etal. 1996). In addition, traditional healers adminis-
ter the water extracts from the bulbs as enema to children
with stomach ailments. Given that such ailments are often
accompanied by fever, the ethno-medicinal rationale for D.
maculata may be related to its anti-inflammatory potential.
As a result, du Toit etal. (2005) isolated five (5) homoiso-
flavanones and structurally related compounds from D.
maculata which were screened for anti-inflammatory activ-
ity. While compound six exhibited high anti-inflammatory
activity, compounds 2, 5, 20 and 21 were moderately active
in term of inhibition in microsomal cells (%). The diver-
sity of phytochemicals in the bulb of D. maculata has been
well-demonstrated including norlignans, scillascillin-type
homoisoflavanone and xanthones (Koorbanally etal. 2001,
2006; Mulholland etal. 2004).
Given the rich chemical pool and susceptibility of D.
maculata to over-harvesting resulting from the use of the
bulbs in traditional medicine, the current study investigated
how the application of a low-cost and environment-friendly
biostimulant (vermicompost leachate, VCL) influences the
growth, nutrient (macro and micro) composition and phyto-
chemical content. Furthermore, the antioxidant activity of
the VCL-treated plants was evaluated to provide an indica-
tion of their biological efficacy.
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Plant Growth Regulation
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Materials andmethods
Source ofplant
Approximately 60 bulbs of D. maculata were collected from
Silverglen Nature Reserve (Chatsworth, KwaZulu-Natal,
South Africa). The identity of the plant was confirmed by
Mr Brian Abrahams (Nursery Manager, Silverglen Nature
Reserve) and a voucher specimen (Voucher number MUT/
KKN 25) of the plant including field data was prepared and
deposited at the Departmental Herbarium of Mangosuthu
University of Technology, Durban, South Africa.
Preparation andanalysis ofvermicompost leachate
Each worm composter consisted of a 3 tier rectangular bin
(145l) made of black polyethylene plastic (Global Worm-
ing CC). The middle bin housed the bedding the worms and
the feed, placed in layers as follows; first layer consisted of
moistened (by soaking overnight) shredded paper forming
a worm blanket to insulate the worms and prevent upward
migration. The second layer consisted of dried cow dung.
Then, 500 red wriggler worms (Eisenia foetida) measur-
ing approximately 2.5 cm each in length were added to the
composter. The worms were fed weekly with 1kg of lettuce
(Lactuca sativa, crisphead variety). The bottom bin served
as a collection compartment for the worm leachate. The bins
were kept away from direct sunlight. Approximately 1l of
water (kept in open air to remove chlorine) was added to
each composter monthly in order to keep the bedding moist.
Worms’ leachate (VCL) was collected after four months by
opening a tap at the bottom of the collection bin. The VCL
was stored in a polyethylene 25l drum in a cold room at 6°C
until application.
The pH and electrical conductivity of the VCL was meas-
ured using pH-200 pH meter and COM-80 EC meter from
HM, respectively. Primary nutrients in the leachate were
determined using a spectrophotometer (Cary 50 UV–Vis-
ible Spectrophotometer, Varian, Australia) following a modi-
fied method outlined by Kulkarni etal. (2014). The pH and
electrical conductivity of the liquid was 8.0 and 2.5dS/m,
respectively. The dark brown leachate contained 654mg/l
of potassium (K+), 220mg/l of nitrate (NO3−) and 186mg/l
of phosphate (PO43−).
Preparation, establishment ofplants
andexperimental design
Healthy bulbs were planted in plastic pots (diameter
of 0.30m at the bottom and 0.47m at the top and depth
of 0.115m) and watered daily for 2weeks. These bulbs
were left to sprout on the roof-top garden of Mangosuthu
University of Technology, Durban, South Africa (S 2958.142
E 3054.768) under natural field conditions with an average
temperature of 24°C and humidity of 79% (http://www.
timea nddat e.com). A 40m2 area was prepared consisting of
ten beds. Each bed had a length of 1.8m and width of 0.8m
and accommodated ten plants. The intra-row spacing and
inter-row spacing was 45 and 40cm, respectively which was
sufficient to accommodate the growth habit of the plants.
Plantlet were transplanted to each bed containing potting
mix (Grovida 30dm3) mulched into the soil. The pH of the
soil ranged from 7.6 to 8.0.
Plants were arranged in a completely randomised design
(CRD) consisting of ten plants (in triplicates) per treat-
ment. Four treatments namely: control (deionised water)
and 1:5, 1:10, 1:20 dilutions (i.e. 1ml VCL + 5ml water;
1ml VCL + 10ml water; 1ml VCL + 20ml water) of VCL
obtained from red wriggler worms (Eisenia foetida) were
applied once a week for 4weeks to the base of each seedling.
Fifty millilitre of the treatments were applied (via
soil drenching) once a week for a month (during the first
month). At the end of the 3months, morphological growth
parameters (for e.g. numbers of leaves, roots and bulbs).
Whole plants were thoroughly washed with distilled water
to remove soil and weighed to obtain fresh weights. Dry
weights were obtained after air drying for 2weeks at room
temperature.
Inductively coupled plasma mass spectrometry
(ICP‑MS)‑based macro andmicro‑nutrients analysis
In triplicates, the ground plant material (about 15–25mg)
from the leaves and bulbs of D. maculata were weighed into
polytetrafluoroethylene vessels. Thereafter, 2ml of HNO3
(67%, analpure) and 1ml of H2O2 (30%, analytical grade)
(Analytika Ltd., Prague, Czech Republic) were added and
digested in diffused microwave system (MLS 1200 Mega;
Milestone S.r.L., Sorisole, Italy).
The resultant solutions were diluted to 15ml in test tubes
with deionised water and analysed by ICP-MS (Agilent
7700x; Agilent Technologies, Tokyo, Japan) based on quad-
rupole mass analyser and octapole reaction system (ORS 3).
Collision cell in He-mode was used for elimination of pos-
sible polyatomic interferences and instrument was set-up by
using Tuning solution (Agilent Technologies, Santa Clara,
USA). Isotopes 23Na and 24Mg were measured in a gas mode
while isotopes 31P, 39K, 44Ca, 55Mn, 56Fe, 63Cu and 66Zn were
measured in He-mode. The internal standards included 6Li,
45Sc and 74Ge. The calibration solutions were prepared by
the appropriate dilution of the single element certified refer-
ence materials with 1.000 ± 0.002g/l for each element (Ana-
lytika Ltd., Prague, Czech Republic) with deionised water
(18.2MΩcm, Direct-Q; Millipore, Molsheim, France).
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Plant Growth Regulation
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Certified reference materials of strawberry leaves and
green algae (METRANAL® 3 and METRANAL® 8, Ana-
lytika Ltd., Prague, Czech Republic) were used for control-
ling the decomposition process and for method validation.
Measurement accuracy was verified by using certified refer-
ence material of water TM-15.2 (National Water Research
Institute, Ontario, Canada).
Ultra‑high performance liquid chromatography
(UHPLC)‑based phenolic acid quantification
Based on the methods described by Gruz etal. (2008), the
phenolic acids in the leaves and bulbs of D. maculata were
quantified using UHPLC (Waters, Milford, MA, USA)
linked to a Micromass Quattro micro™ API benchtop triple
quadrupole mass spectrometer (Waters MS Technologies,
Manchester, UK). Briefly, in triplicates, the ground plant
materials were homogenized with 80% methanol (40mg/
ml) in a 1.5ml Eppendorf tube, using an oscillation ball
mill (MM 301, Retsch, Haan, Germany) at a frequency of
25Hz for 3min. Deuterium-labelled internal standards
were added to the extraction solvent prior to plant material
homogenization. The extracts were centrifuged for 10min
at 26,000g and the supernatant was filtered through 0.45µm
nylon micro-filters (Alltech, Breda, Netherlands).
Oxygen radical absorbance capacity (ORAC) assay
Following 80% methanol extraction of ground plant materi-
als from the leaves and bulbs of D. maculata, ORAC of the
resultant extracts was measured using the protocol devel-
oped by Ou etal. (2001). In triplicates, fluorescein (100µl,
500mM) and plant extracts (25µl) were added into each
working well in a 96-well microplate and shaken. The reac-
tion was initiated by the addition of 2,2-Azobis(2-methyl-
propionamidine) dihydrochloride (AAPH, 25µl, 250mM)
pre-incubated at 37°C. The fluorescence (Ex. 485nm, Em.
510nm) was read every 3min over 90min in a microplate
reader Infinite M200 Pro (Tecan, Switzerland) incubated at
40°C. The net area under the curve was used to calculate
antioxidant capacity in trolox equivalents (µmol TE/g).
Data analysis
Growth response, elemental and phytochemical content as
well as antioxidant activity data were subjected to analysis
of variance (ANOVA) using SPSS software version 22.0.
For each of the evaluated parameters, the mean values were
further separated using Duncan’s multiple range test to check
for statistical significance (P ≤ 0.05).
Results
Growth data
Application of different dilutions of VCL improved the
morphological traits of D. maculata (Fig.1). For instance,
VCL-treated plants had more leaves which also translated
to higher fresh and dry biomass (Fig.1a–c). Similarly, VCL
treatment stimulated the production of higher numbers of
shoots in D. maculata (Fig.1d). However, the bulb biomass
was only significantly higher in VCL (1:20) treatments
(Fig.1e, f).
Macro‑ andmicro‑nutrient composition
The concentrations of the four macro-nutrients analysed
in D. maculata were generally higher in the leaves than in
the bulbs (Fig.2). The degree of abundance were in order
of K > Ca > P > Mg in both organs of D. maculata. In most
cases, no significant increase in the concentrations of macro-
nutrients was observed in VCL-treated plants (Fig.2a–d,
f–h). However, the P content was remarkably lower in leaves
of VCL (1:20) treatment than in the control (Fig.2e).
As depicted in Fig.3a–j, the leaves accumulated higher
concentrations of micro-nutrients than the bulbs regardless
of the treatment regime. While Na was the most abundant
(730–3366µg/g DW) micro-nutrient, Mn occurred in the
least concentration (10–20µg/g DW). An increase in Cu
content was observed in leaves of VCL (1:5)-treated D.
maculata (Fig.3a). However, the same treatment (VCL 1:5)
resulted in lower Fe content in leaves (Fig.3c). In the bulbs,
1:10 and 1:20 (dilutions) VCL treatments increased the Mn
and Zn contents, respectively (Fig.3f, j).
Phenolic acid content
The ten phenolic acids quantified in the extracts from the
two organs of D. maculata were classified as either hydroxy-
benzoic (Fig.4) or hydroxycinnamic (Fig.5) based on their
chemical structures. In most cases, both classes of phe-
nolic acid (with the exception of ferulic acid) accumulated
in higher concentrations in the leaves than in the bulbs. In
terms of quantity, p-hydroxybenzoic (11–19µg/g) and vanillic
(13–22µg/g) acids were the major hydroxybenzoic derivatives
while p-coumaric (28–41µg/g) and ferulic (5–8µg/g) acids
were the most abundant hydroxycinnamic derivative. The
remaining phenolic acids were generally in low concentra-
tions (< 5µg/g) in both the leaves and bulbs of D. maculata.
Among the six hydroxybenzoic quantified in the leaf
extracts of D. maculata, VCL application significantly
enhanced the phenolic acid content relative to the control
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Plant Growth Regulation
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(Fig.4a, c, e, g, i, k). Similarly, gallic acid content in the bulb
extracts was higher in VCL (1:5)-treated plants (Fig.4b).
Conversely, VCL application reduced the concentration of
p-hydroxybenzoic, protocatechuic, salicylic, syringic and
vanillic acids in the bulb extracts (Fig.4d, f, h, j, i).
In the leaf extracts, VCL application resulted into higher
concentration of the four hydroxycinnamic acids relative to
the control (Fig.5a, c, e, g). Although VCL (1:20) enhanced
the caffeic acid in bulbs (Fig.5b), no significant increase was
observed in the bulb extracts for the other three hydroxycin-
namic acids (Fig.5d, f, h).
Antioxidant activity
In terms of the ORAC, the extracts from the leaves were more
potent when compared to the bulbs (Table1). Application of
VCL had no significant stimulatory effect on the ORAC of the
extracts from the leaves of D. maculata. In fact, lower dilu-
tions (1:10 and 1:20) of VCL reduced the ORAC of the leaf
extracts. On the other hand, bulb extracts prepared from VCL
(1:5) treatment had higher ORAC activity than the control.
Discussion
Effect ofvermicompost leachate (VCL) application
ongrowth response
The beneficial effect of biostimulants including VCL
are often manifested over the life cycle of the plant. For
instance, effects such as improved seed germination, seed-
ling establishment, enhanced nutrient mobilization and
partitioning, improved rooting of cuttings, flowering, fruit
and crop yield have been demonstrated across a wide range
of plants (Craigie 2011; Halpern etal. 2015; Sharma etal.
2014). This is not surprising as these products are sourced
from diverse organisms and generally contain a wide range
of growth-stimulatory metabolites (Aremu etal. 2015b;
Brown and Saa 2015; Yakhin etal. 2017). In addition
to compounds such as humic acid as well as micro and
macro-nutrients, VCL contains cocktails of conventional
plant hormones which are possibly the active ingredients
and responsible for growth stimulatory effects (Aremu
etal. 2015b; Zhang etal. 2014).
In the current study, VCL-treated D. maculata generally
had bigger leaves and bulbs when compared to the control
(Fig.1). Given that biomass accumulation is a function of pho-
tosynthesis, it is possible to infer that the application of VCL
influenced this important process as demonstrated in many
species (Aremu etal. 2014; Arthur etal. 2012; Ievinsh 2011;
Masondo etal. 2016). For instance, the reduction of photo-
synthetic pigment content was significantly attenuated by sup-
plementing phosphorus-deficient Tulbaghia ludwigiana with
VCL (Aremu etal. 2014). Given the ability of VCL to enhance
morphological appearance of different plant organs especially
the underground part (e.g. bulbs), it affords a viable approach
useful for low-resource farmers to enhance the cultivation of
bulbous plants which are often in high demand by medicinal
plant vendors and users.
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Fig. 1 Effect of different dilutions of vermicompost leachate (VCL)
on morphological traits of D. maculata. a Number of leaves; b leaf
fresh weight; c leaf dry weight; d number of shoots; e bulb fresh
weight; f bulb dry weight. Data are presented as mean ± standard
error (n = 30). In each graph, bars with different letter(s) are signifi-
cantly different based on Duncan’s multiple range test (P ≤ 0.05)
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Plant Growth Regulation
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aaaa
Phosphorus (mg/g)
control1:5 1:10 1:20
0
2
4
6
a
aaa
VCL dilution (v/v)
Magnesium (mg/g)
control 1:5 1:10 1:20
0
2
4
6
aaaa
VCL dilution (v/v)
Magnesium (mg/g)
AB
CD
EF
GH
Fig. 2 Effect of different dilutions of vermicompost leachate (VCL)
on the concentration of macro-nutrients in leaves and bulbs of D.
maculata. a, b Calcium; c, d potassium; e, f phosphorus; g, h magne-
sium. Data are mean ± standard error (n = 3). In each graph, bars with
different letter(s) are significantly different based on Duncan’s multi-
ple range test (P ≤ 0.05)
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Plant Growth Regulation
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Fig. 3 Effect of different dilu-
tions of vermicompost leachate
(VCL) on the concentration of
micro-nutrients in leaves and
bulbs of D. maculata. a, b Cop-
per; c, d iron; e, f manganese;
g, h sodium; i, j zinc. Data are
mean ± standard error (n = 3). In
each graph, bars with different
letter(s) are significantly differ-
ent based on Duncan’s multiple
range test (P ≤ 0.05)
0
10
20
30
40
Leaves
b
a
bb
Copper (µg/g)
0
10
20
30
40
Bulbs
a
aa
a
Copper (µg/g)
0
200
400
600
800
a
b
ab
a
Iron (
µ
g/g)
0
200
400
600
800
a
a
a
a
Iron (µg/g)
0
5
10
15
20
25
aaaa
Manganese (µg/g)
0
5
10
15
20
25
a
bbb
Manganese (µg/g)
0
1000
2000
3000
4000
aaa
a
Sodium (µg/g)
0
1000
2000
3000
4000
aaaa
Sodium (µg/g)
control1:51:10 1:20
0
20
40
60
80
aa
a
a
VCL diluti on (v/v)
Zinc (µg/g)
control1:51:10 1:20
0
20
40
60
80
b
ab
b
a
VCL diluti on (v/v)
Zinc (µg/g)
AB
CD
EF
GH
IJ
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Plant Growth Regulation
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Fig. 4 Effect of different dilu-
tions of vermicompost leachate
(VCL) on the concentration of
hydroxybenzoic acid deriva-
tives in leaves and bulbs of
D. maculata. a, b Gallic acid;
c, d p-hydroxybenzoic acid;
e, f protocatechuic acid; g,
h salicylic acid; i, j syringic
acid; k, l vanillic acid. Data are
mean ± standard error (n = 3). In
each graph, bars with different
letter(s) are significantly differ-
ent based on Duncan’s multiple
range test (P ≤ 0.05)
0.00
0.05
0.10
0.15
0.20
0.25
Leaves
b
aaa
Gallic ac id (µg/g)
0.00
0.05
0.10
0.15
0.20
0.25
Bulbs
b
a
bb
Gallic ac id (µg/g)
0
5
10
15
20
25
c
b
a
b
p-Hydroxybenzoic acid ( µg/g)
0
5
10
15
20
25
abcc
p-Hydroxybenzoic acid ( µg/g)
0
1
2
3
4
5
b
a
a
a
Protocatechuic acid ( µg/g)
0
1
2
3
4
5
ab a
b
c
Protocatechuic acid ( µg/g)
0.0
0.2
0.4
0.6
0.8
1.0
b
aa
ab
Salicylic acid ( µg/g)
0.0
0.2
0.4
0.6
0.8
1.0
aab ab b
Salicylic acid ( µg/g)
0.0
0.2
0.4
0.6
0.8
1.0
c
b
a
ab
Syringic ac id ( µg/g)
0.0
0.2
0.4
0.6
0.8
1.0
a
bbb
Syringic ac id ( µg/g)
control1:5 1:10 1:20
0
10
20
30
c
b
a
b
VCL diluti on (v/v)
Vanillic acid (µg/g)
control1:5 1:10 1:20
0
10
20
30
acbc ab
VCL diluti on (v/v)
Vanillic acid (µg/g)
AB
CD
EF
GH
IJ
KL
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Plant Growth Regulation
1 3
0
1
2
3
4
5
Leaves
c
b
aa
Caffeic acid (µg/g)
0
1
2
3
4
5
Bulbs
bb
ab
a
Caffeic acid (µg/g)
0
10
20
30
40
50
c
a
a
b
p-Coumaric acid ( µg/g)
0
10
20
30
40
50
aa aa
p-Coumaric acid ( µg/g)
0.0
2.5
5.0
7.5
10.0
b
aaa
Ferulic acid ( µg/g)
0.0
2.5
5.0
7.5
10.0
a
aa
a
Ferulic acid ( µg/g)
control1:5 1:10 1:20
0.0
0.5
1.0
1.5
2.0
2.5
c
bbc
a
VCLdilution (v/v)
Sinapic acid (µg/g)
control1:5 1:10 1:20
0.0
0.5
1.0
1.5
2.0
2.5
aaaa
VCL diluti on (v/v)
Sinapic acid (µg/g)
AB
CD
EF
GH
Fig. 5 Effect of different dilutions of vermicompost leachate (VCL)
on the concentration of hydroxycinnamic acid derivatives in leaves
and bulbs of D. maculata. a, b Caffeic acid; c, d p-coumaric acid; e, f
ferulic acid; g, h sinapic acid. Data are mean ± standard error (n = 3).
In each graph, bars with different letter(s) are significantly different
based on Duncan’s multiple range test (P ≤ 0.05)
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Plant Growth Regulation
1 3
Effect ofvermicompost leachate (VCL) application
ontheconcentration ofmacro‑ andmicro‑nutrients
The influence of biostimulant application on plant nutri-
ent uptake and the underlying mechanisms are associated to
positive changes in soil structure or nutrient solubility, root
morphology and plant physiology (Halpern etal. 2015; Mar-
tínez-Ballesta etal. 2010). As emphasized by these authors,
the minerals in plants are related to the effect of agricultural
practices, especially the continuous debatable issue of organic
farming versus mineral fertilisation. In the current study, the
concentrations of macro-nutrients in D. maculata were similar
with and without VCL treatment. However, the macro-nutri-
ents were generally higher in the leaves than bulbs (Fig.2).
Similar trends were evident in the concentrations of micro-
nutrients with few exceptions. For instance, VCL application
increased the concentration of Cu in the leaves (VCL 1:5) and
Mn in bulbs (VCL 1:10) of D. maculata. In contrast, the leaves
of VCL (1:5)-treated plants had significantly lower Fe content
when compared to the control. As emphasized by Martínez-
Ballesta etal. (2010), this type of variable response has been
demonstrated in many plants. The effect of biostimulants on
nutrient (macro and micro) content are often variable depend-
ing on critical factors such as the crop, season cycle and year.
Thus, these aforementioned factors must be considered care-
fully prior to making conclusions and recommendations to
stakeholders.
Effect ofvermicompost leachate (VCL) application
onthephenolic acids (hydroxybenzoic
andhydroxycinnamic derivatives)
The therapeutic value of phytochemicals in D. maculata is
well recognized (Koorbanally etal. 2001). In addition, sev-
eral studies have also highlighted the diversity of chemicals
present in D. maculata (du Toit etal. 2005; Koorbanally
etal. 2001, 2006; Mulholland etal. 2004). In the current
study, ten phenolic acids comprising six hydroxybenzoic and
four hydroxycinnamic derivatives occurred in the leaves and
bulbs of D. maculata. Most of these phenolic acids are rec-
ognised as a potent bioactive compounds for treating differ-
ent diseases (Heleno etal. 2015). Ferulic acid was one of the
major phenolic acid in D. maculata and its diverse biological
effect such as antioxidant, anti-inflammatory, antimicrobial,
anti-allergic and hepato-protective effects have been demon-
strated (Kumar and Pruthi 2014).
As a result of the value associated with elevated level
of phytochemicals in plants, researchers often explored
different approaches especially the application of different
biostimulants during cultivation (Aremu etal. 2014, 2015a,
2016; Pant etal. 2012; Wang etal. 2014). Application of
VCL influenced the concentrations of phenolic acid accu-
mulated in the leaves and bulbs of D. maculata (Figs.4,
5). It has been established that the type and concentration
of phytochemicals in cultivated plants are often affected by
different factors. For instance, the phenolic compounds in
marionberry, strawberry and corn were significantly higher
when grown with organic supplements compared with non-
organic plants (Asami etal. 2003).
Effect ofvermicompost leachate (VCL) application
onantioxidant activity
Although biological screening efficacy of D. maculata is
limited, the anti-inflammatory potential have been demon-
strated (du Toit etal. 2005). Findings from the present study
provided an indication of the antioxidant potential. Antioxi-
dant activity is one of the most common biological activity
exhibited by many plants and this has increased the interest
in plant-derived antioxidants (Gülçin 2012). On the basis of
the effect of oxidative stress in the aetiology of many dis-
eases (Pham-Huy etal. 2008; Spector 2000), the presence of
potent antioxidants in medicinal plants is desired. The anti-
oxidant activity of cultivated plants are affected by different
factors including type of fertilizer applied, necessitating the
extensive focus in this area (Aremu etal. 2014; Masondo
etal. 2016; Pant etal. 2009; Rimmer 2006; Toor etal. 2006;
Wang etal. 2010). In the current study, application of VCL
(1:5 dilution) significantly enhanced the antioxidant activ-
ity of bulb extract of D. maculata. However, similar higher
antioxidant activity was absent in leaf extracts. In fact, the
antioxidant activity demonstrated was significantly lower in
leaves obtained from VCL (1:20)-treated plants. This find-
ing indicates that the positive influence of biostimulants on
antioxidant activity cannot be generalized as differences may
occur in the different organs of the same plant and/or among
different plants (plant specific). This plant specific-effect in
antioxidant response following treatment with biostimulants
have been documented in some studies (Aremu etal. 2014;
Asami etal. 2003; Masondo etal. 2016).
Table 1 Effect of different dilutions of vermicompost leachate (VCL)
on oxygen radical absorbance capacity (ORAC) of extracts from dif-
ferent organs of D. maculata
In each column, value (mean ± standard error, n = 3) with different
letter(s) are significantly different based on Duncan’s multiple range
test (P ≤ 0.05)
TE trolox equivalents
VCL dilutions (v/v) ORAC (µmol TE/g)
Leaves Bulbs
Control 151.0 ± 9.36a23.6 ± 1.28b
1:5 134.6 ± 7.23ab 32.6 ± 3.40a
1:10 121.7 ± 6.88b26.1 ± 0.92ab
1:20 88.0 ± 2.87c26.8 ± 1.55ab
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Plant Growth Regulation
1 3
Conclusions
Findings from the current study established the beneficial
effects from the application of low-cost biostimulants such
as VCL which enhanced the morphological appearance of
D. maculata. In addition, VCL application influenced the
quantity of macro and micro-nutrients as well as the accu-
mulated bioactive phytochemicals. This implies that the use
of VCL has the potential to ensure the desired quality and
quantity of cultivated medicinal plants. In terms of the dis-
tribution of chemical metabolites and ORAC potential, the
higher content and antioxidant demonstrated by the extracts
from the leaves relative to the bulbs provides a motivation
to support the call for plant part substitution (i.e. the use of
leaves rather than the bulbs) in the usage of D. maculata.
Overall, the present findings are valuable from a conserva-
tional perspective as useful insight for mitigating challenges
associated with over-harvesting of highly utilized but slow-
growing species especially geophytes.
Acknowledgements This work was financially supported by Mango-
suthu University of Technology under registered project NSci\05\2012
(LD, KKN, GDA), National Research Foundation (Incentive Funding
for Rated Researchers, UID: 109508) and Faculty Research Committee,
Faculty of Natural and Agricultural Sciences, North-West University,
Mmabatho, South Africa (AOA). MJ, JG, PT and KD were supported
by grant No. LO1204 (Sustainable development of research in Centre
of Region Haná) from the National Program of Sustainability I, MEYS,
Czech Republic. JG was also supported by the Czech Science Founda-
tion (No. 17-06613S). KD was also supported by MEYS of CR from
European Regional Development Fund-Project Centre for Experimen-
tal Plant Biology: No. CZ.02.1.01/0.0/0.0/16_019/0000738. We thank
the staff and management of Silverglen Nature Reserve for supplying
the bulbs used for the study.
Author contributions LD, KKN, GDA and AOA conceived and con-
ducted the field experiment. MJ and JG quantified the phenolic acids
and ORAC assay. MJ and PT conducted the elemental analysis. LD,
KKN, GDA and AOA analysed the data on growth parameters. KD
was also involved in conceptualization, design and provided technical
and editorial inputs. AOA wrote the manuscript with help from all the
other authors.
References
Affolter JM, Pengelly A (2007) Conserving medicinal plant biodiver-
sity. In: Wynn SG, Fougère BJ (eds) Veterinary herbal medicine.
Mosby, Saint Louis, pp257–263
Aremu AO, Masondo NA, Van Staden J (2014) Physiological and
phytochemical responses of three nutrient-stressed bulbous plants
subjected to vermicompost leachate treatment. Acta Physiol Plant
36:721–731
Aremu AO, Masondo NA, Rengasamy KRR, Amoo SO, Gruz J, Bíba
O, Šubrtová M, Pěnčík A, Novák O, Doležal K, Van Staden J
(2015a) Physiological role of phenolic biostimulants isolated from
brown seaweed Ecklonia maxima on plant growth and develop-
ment. Planta 241:1313–1324
Aremu AO, Stirk WA, Kulkarni MG, Tarkowská D, Turečková V,
Gruz J, Šubrtová M, Pěnčík A, Novák O, Doležal K, Strnad M,
Van Staden J (2015b) Evidence of phytohormones and phenolic
acids variability in garden-waste-derived vermicompost lea-
chate, a well-known plant growth stimulant. Plant Growth Regul
75:483–492
Aremu AO, Plačková L, Gruz J, Bíba O, Novák O, Stirk WA, Doležal
K, Van Staden J (2016) Seaweed-derived biostimulant (Kelpak®)
influences endogenous cytokinins and bioactive compounds in
hydroponically grown Eucomis autumnalis. J Plant Growth Regul
35:151–162
Arthur GD, Aremu AO, Kulkarni MG, Van Staden J (2012) Vermicom-
post leachate alleviates deficiency of phosphorus and potassium
in tomato seedlings. HortScience 47:1304–1307
Asami DK, Hong Y-J, Barrett DM, Mitchell AE (2003) Comparison
of the total phenolic and ascorbic acid content of freeze-dried
and air-dried marionberry, strawberry, and corn grown using con-
ventional, organic, and sustainable agricultural practices. J Agric
Food Chem 51:1237–1241
Brown P, Saa S (2015) Biostimulants in agriculture. Front Plant Sci.
https ://doi.org/10.3389/fpls.2015.00671
Calvo P, Nelson L, Kloepper JW (2014) Agricultural uses of plant
biostimulants. Plant Soil 383:3–41
Canter PH, Thomas H, Ernst E (2005) Bringing medicinal plants
into cultivation: opportunities and challenges for biotechnology.
Trends Biotechnol 23:180–185
Craigie JS (2011) Seaweed extract stimuli in plant science and agricul-
ture. J Appl Phycol 23:371–393
Dold AP, Cocks ML (2002) The trade in medicinal plants in the Eastern
Cape Province, South Africa. S Afr J Sci 98:589–597
du Toit K, Elgorashi EE, Malan SF, Drewes SE, van Staden J, Crouch
NR, Mulholland DA (2005) Anti-inflammatory activity and
QSAR studies of compounds isolated from Hyacinthaceae spe-
cies and Tachiadenus longiflorus Griseb. (Gentianaceae). Bioorg
Med Chem 13:2561–2568
Gruz J, Novák O, Strnad M (2008) Rapid analysis of phenolic acids in
beverages by UPLC–MS/MS. Food Chem 111:789–794
Gülçin İ (2012) Antioxidant activity of food constituents: an overview.
Arch Toxicol 86:345–391
Halpern M, Bar-Tal A, Ofek M, Minz D, Muller T, Yermiyahu U
(2015) The use of biostimulants for enhancing nutrient uptake.
In: Sparks DL (ed) Advances in agronomy, vol130. Academic
Press, Cambridge, pp141–174
Heleno SA, Martins A, Queiroz MJRP, Ferreira ICFR (2015) Bioac-
tivity of phenolic acids: metabolites versus parent compounds: a
review. Food Chem 173:501–513
Hulme M (1954) Wild flowers of Natal. Shuter & Shooter,
Pietermaritzburg
Hutchings A, Scott AH, Lewis G, Cunningham A (1996) Zulu
medicinal plants. An Inventory. University of Natal Press,
Pietermaritzburg
Ievinsh G (2011) Vermicompost treatment differentially affects seed
germination, seedling growth and physiological status of vegeta-
ble crop species. Plant Growth Regul 65:169–181
Koorbanally C, Crouch NR, Mulholland DA (2001) Scillascillin-type
homoisoflavanones from Drimiopsis maculata (Hyacinthaceae).
Biochem Syst Ecol 29:539–541
Koorbanally C, Mulholland DA, Crouch NR (2006) Norlignans and
homoisoflavanones from two South African Drimiopsis species
(Hyacinthaceae: Hyacinthoideae). Biochem Syst Ecol 34:588–592
Kulkarni Y, Warhade KK, Bahekar S (2014) Primary nutrients deter-
mination in the soil using UV spectroscopy. Int J Emerg Eng Res
Technol 2:198–204
Kumar N, Pruthi V (2014) Potential applications of ferulic acid from
natural sources. Biotechnol Rep 4:86–93
Author's personal copy

Plant Growth Regulation
1 3
Lubbe A, Verpoorte R (2011) Cultivation of medicinal and aro-
matic plants for specialty industrial materials. Ind Crops Prod
34:785–801
Lyson TA (2002) Advanced agricultural biotechnologies and sustain-
able agriculture. Trends Biotechnol 20:193–196
Manning JC, Goldblatt P, Fay MF (2004) A revised generic synop-
sis of Hyacinthaceae in sub-Saharan Africa, based on molecular
evidence, including new combinations and the new tribe Pseudo-
prospereae. Edinb J Bot 60:533–568
Martínez-Ballesta MC, Dominguez-Perles R, Moreno DA, Muries B,
Alcaraz-López C, Bastías E, García-Viguera C, Carvajal M (2010)
Minerals in plant food: effect of agricultural practices and role in
human health. A review. Agron Sustain Dev 30:295–309
Masondo NA, Kulkarni MG, Rengasamy KRR, Pendota SC, Finnie JF,
Van Staden J (2016) Effect of vermicompost leachate in Cerato-
theca triloba under nutrient deficiency. Acta Physiol Plant 38:236.
https ://doi.org/10.1007/s1173 8-016-2252-1
Moyo M, Aremu AO, Van Staden J (2015) Medicinal plants: an invalu-
able, dwindling resource in sub-Saharan Africa. J Ethnopharmacol
174:595–606
Mulholland DA, Koorbanally C, Crouch NR, Sandor P (2004) Xantho-
nes from Drimiopsis maculata. J Nat Prod 67:1726–1728
Ou B, Hampsch-Woodill M, Prior RL (2001) Development and vali-
dation of an improved oxygen radical absorbance capacity assay
using fluorescein as the fluorescent probe. J Agric Food Chem
49:4619–4626
Pant AP, Radovich TJK, Hue NV, Talcott ST, Krenek KA (2009) Ver-
micompost extracts influence growth, mineral nutrients, phyto-
nutrients and antioxidant activity in pak choi (Brassica rapa cv.
Bonsai, Chinensis group) grown under vermicompost and chemi-
cal fertiliser. J Sci Food Agric 89:2383–2392
Pant AP, Radovich TJK, Hue NV, Paull RE (2012) Biochemical proper-
ties of compost tea associated with compost quality and effects on
pak choi growth. Sci Hortic 148:138–146
Pham-Huy LA, He H, Pham-Huy C (2008) Free radicals, antioxidants
in disease and health. Int J Biomed Sci 4:89–96
Reinten EY, Coetzee JH, van Wyk B-E (2011) The potential of South
African indigenous plants for the international cut flower trade. S
Afr J Bot 77:934–946
Rimmer DL (2006) Free radicals, antioxidants, and soil organic matter
recalcitrance. Eur J Soil Sci 57:91–94
Sharma HSS, Fleming C, Selby C, Rao JR, Martin T (2014) Plant
biostimulants: 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
Spector A (2000) Oxidative stress and disease. J Ocul Pharmacol Ther
16:193–201
Tilman D, Cassman KG, Matson PA, Naylor R, Polasky S (2002) Agri-
cultural sustainability and intensive production practices. Nature
418:671–677
Toor RK, Savage GP, Heeb A (2006) Influence of different types of
fertilisers on the major antioxidant components of tomatoes. J
Food Compos Anal 19:20–27
Wang D, Shi Q, Wang X, Wei M, Hu J, Liu J, Yang F (2010) Influence
of cow manure vermicompost on the growth, metabolite contents,
and antioxidant activities of Chinese cabbage (Brassica campes-
tris ssp. chinensis). Biol Fertil Soils 46:689–696
Wang K-H, Radovich T, Pant A, Cheng Z (2014) Integration of cover
crops and vermicompost tea for soil and plant health manage-
ment in a short-term vegetable cropping system. Appl Soil Ecol
82:26–37
Wiersum KF, Dold AP, Husselman M, Cocks M (2006) Cultivation of
medicinal plants as a tool for biodiversity conservation and pov-
erty alleviation in the Amatola region, South Africa. In: Bogers
RJ, Craker LE, Lange D (eds) Medicinal and aromatic plants.
Springer, Dordrecht, pp43–57
Williams VL, Balkwill K, Witkowski ETF (2007) Size-class prevalence
of bulbous and perennial herbs sold in the Johannesburg medicinal
plant markets between 1995 and 2001. S Afr J Bot 73:144–155
Yakhin OI, Lubyanov AA, Yakhin IA, Brown PH (2017) Biostimulants
in plant science: a global perspective. Front Plant Sci 7:https ://doi.
org/10.3389/fpls.2016.02049
Zhang H, Tan SN, Wong WS, Ng CYL, Teo CH, Ge L, Chen X, Yong
JWH (2014) Mass spectrometric evidence for the occurrence of
plant growth promoting cytokinins in vermicompost tea. Biol Fert
Soils 50:401–403
Author's personal copy