See

discussions, stats, and author profiles for this publication at: https://www.researchgate.net/publication/324728330

Growth and physiological response of

lemongrass (Cymbopogon citratus (D.C.)
Stapf.) under different levels of fly ash....

Article in International Journal of Phytoremediation · April 2018

DOI: 10.1080/15226514.2017.1393394

CITATIONS READS

0 41

4 authors, including:

Debabrata Panda Bandana Padhan

Central University of Orissa, Koraput, India Central University of Orissa
55 PUBLICATIONS 426 CITATIONS 7 PUBLICATIONS 4 CITATIONS

SEE PROFILE SEE PROFILE

Some of the authors of this publication are also working on these related projects:

Ethnomedicinal Knowledge of Tribes of Koraput, Odisha, India View project

Inter species genetic diversity study of wild Dioscorea View project

All content following this page was uploaded by Bandana Padhan on 24 April 2018.

The user has requested enhancement of the downloaded file.

 International Journal of Phytoremediation

ISSN: 1522-6514 (Print) 1549-7879 (Online) Journal homepage: http://www.tandfonline.com/loi/bijp20

Growth and physiological response of lemongrass

(Cymbopogon citratus (D.C.) Stapf.) under different
levels of fly ash-amended soil

Debabrata Panda, Dibyajyoti Panda, Bandana Padhan & Meghali Biswas

To cite this article: Debabrata Panda, Dibyajyoti Panda, Bandana Padhan & Meghali Biswas
(2018) Growth and physiological response of lemongrass (Cymbopogon citratus (D.C.) Stapf.)
under different levels of fly ash-amended soil, International Journal of Phytoremediation, 20:6,
538-544, DOI: 10.1080/15226514.2017.1393394

To link to this article: https://doi.org/10.1080/15226514.2017.1393394

View supplementary material

Published online: 24 Apr 2018.

Submit your article to this journal

View related articles

View Crossmark data

Full Terms & Conditions of access and use can be found at

http://www.tandfonline.com/action/journalInformation?journalCode=bijp20
INTERNATIONAL JOURNAL OF PHYTOREMEDIATION
2018, VOL. 20, NO. 6, 538–544
https://doi.org/10.1080/15226514.2017.1393394

Growth and physiological response of lemongrass (Cymbopogon citratus (D.C.) Stapf.)

under different levels of fly ash-amended soil
Debabrata Panda, Dibyajyoti Panda, Bandana Padhan, and Meghali Biswas
Department of Biodiversity and Conservation of Natural Resources, Central University of Orissa, Koraput, Odisha, India

ABSTRACT KEYWORDS
Revegetation with metal tolerant plants for management of fly ash deposits is an important environmental Antioxidants; Chlorophyll
perspective nowadays. Growth performance, photosynthesis, and antioxidant defense of lemongrass fluorescence; Fly ash;
(Cymbopogon citratus (D.C.) Stapf.) were evaluated under various combination of fly ash amended with Lemongrass; Photosynthesis;
garden soil in order to assess its fly ash tolerance potential. Under low level of fly ash (25%) amended soil, Phytoremediation
the plant growth parameters such as shoot, root, and total plant biomass as well as metal tolerance index
were increased compared to the control plants grown on garden soil, followed by decline under higher
concentration of fly ash (50%, 75% and 100%). In addition, leaf photosynthetic rate, stomatal conductance,
and photosystem (PS) II activity were not significantly changed under low level of fly ash (25%) amended
soil compared to the garden soil but these parameters were significantly decreased further with increase
of fly ash concentrations. Furthermore, increase of activities of some antioxidant enzymes such as
superoxide dismutase, ascorbate peroxidase, and guaiacol peroxidase over control were noticed in
lemongrass under all fly ash treatments. Taken together, the study suggests that lemongrass can be used
for phytoremediation of fly ash at 25% amended soil.

Introduction
Fly ash deposits have been recognized as a major environmental
threat, which are the results of coal combustion of thermal power
plants worldwide (Pandey et al. 2016). Continuous increase in the
number and area of fly ash dumpsites is a worldwide concern due
to their potential toxic effects and heavy metals released in the
environment (Pandey 2015). In India, fly ash generation is
expected to be 300–400 million tons per year by 2016–2017 (Maiti
and Prasad 2016). Despite its utilization in cement, sanitary, and
brick industries is in practice, huge quantities remain unused and
dumped (Verma et al. 2014). Therefore, a holistic approach is
urgently required for management of fly ash deposits. It has been
reported that revegetation and phytoremediation are the environ-
mental-friendly technologies, which utilizes plants to reduce,
remove, and degrade environmental pollutants, with the aim to
eliminate the hazardous effects of fly ash (Qu et al. 2012; Srivastva
et al. 2014; Pandey et al. 2016). However, phytorestoration of fly
ash deposit is a challenging task due to its high alkalinity, elevated
levels of potentially toxic metals, and poor nitrogen and organic
carbon that limit establishment of plants on fly ash (Gupta et al.
2007; Pandey and Singh 2010; Verma et al. 2014). Therefore, fly
ash with soil amendment may offer correction of these deficien-
cies and suitable combination to support plant growth with
reduced risk of metal toxicity (Verma et al. 2014). There are sev-
eral recent studies pertaining to reclamation, revegetation, and
phytoremediation of fly ash deposits (Srivastva et al. 2014; Pandey
2015; Maiti and Prasad 2016; Bisoi et al. 2017; Ghosh et al. 2017).
To date several metal hyperaccumulator plant species such
as, Azadirachta indica, Pongamia pinnata, Tectona grandis,
Ricinus communis, Cynodon dactylon, Cymbopogan flexuosus,
Cymbopogon winterianus, Saccharum munja, Oryza nivara, Oryza
rufipogan etc. have been identified (Stoffella et al. 2008; Pandey
and Singh 2012; Srivastva et al. 2014; Verma et al. 2014; Maiti
and Prasad 2016) for better in situ management of fly ash dump-
ing sites. However, their remediation potential of many of these
plants was limited because of their slow growth and low biomass
in the metal contaminated site. The characteristics that make any
species useful for phytoremediation include fast-growth with
capability to accumulate larger biomass, high metal accumulation
capacity, and unpalatable by livestock (Pandey and Singh 2012;
Bisoi et al. 2017). Recently several researchers suggested the use of
aromatic grasses for better in situ management of fly ash dumping
sites because these are high value crops due to their essential oil
production and societal benefits (Pandey et al. 2014; Verma et al.
2014; Maiti and Prasad 2016; Ghosh et al. 2017).
Cymbopogon citratus (D.C.) Stapf., commonly known as
lemongrass, is a metal tolerant aromatic grass which withstands
harsh environmental conditions (Das and Maiti 2009; Gupta
et al. 2013). Lemongrass cultivation is also widely practiced for
stabilization of slopes and for valuable aromatic chemical con-
stituents, which are used in perfumery, pharmaceuticals, cos-
metics and aromatherapy as well as toiletry products (Bienes
et al. 2010; Verma et al. 2014). Recently researchers have iden-
tified the lemongrass as a potential metal accumulator and

CONTACT Debabrata Panda dpanda80@gmail.com Department of Biodiversity and Conservation of Natural Resources, Central University of Orissa, Koraput,
764 021, Odisha, India.
Supplemental data for this article can be accessed on the publisher’s website.
© 2018 Taylor & Francis Group, LLC

suitability for phytoremediation of metal contaminated sites
(Israila et al. 2015; Gautam et al. 2017). The study was con-
ducted by Srivastva et al. (2014) pertaining to physiology of
lemongrass grown under fly ash treatment. Referring to this
study, the growth performance and antioxidant response of
lemongrass under varying levels of fly ash has not yet been
studied.
Fly ash has potential benefits for use in agronomy due to the
presence of macro- and micro-nutrients, which are conducive
for plant growth (Verma et al. 2014). However, the augmented
levels of non-essential and potentially toxic metals may pose a
problem for plant growth (Prajapati 2012). Several studies have
been carried out using fly ash in combination with other
organic amendments and in soil promotes the plant growth
and the yield of plants (Pandey and Singh 2010; Verma et al.
2014). But the exposure of plants to toxic levels of metals in fly
ash triggers a wide range of physiological and metabolic altera-
tions along with effect on leaf gas exchange (Dubey 2011; Raja
et al. 2014; Bisoi et al. 2017). The toxic elements from ash can
inhibit photosynthesis at several physiological levels: pigments,
gas exchange, structure, and function of chloroplasts (Reid
et al. 2004; Guidi et al. 2011; Gaji
c et al. 2013). Chlorophyll
fluorescence has been used frequently in the past as a conve-
nient informative tool for studying the effects of various envi-
ronmental stresses on the process of photosynthesis,
particularly on the function of photosystem (PS) II (Maxwell
and Johnson, 2000; Sayed 2003; Baker and Rosenqvist 2004).
However, the photosynthetic response of lemongrass to differ-
ent levels of fly ash amendments is not yet been studied.
The metals in contaminated soil trigger oxidative damage in
plants because the balance between the production of reactive
oxygen species (ROS) and their detoxification by the antioxida-
tive system is altered (Bhaduri and Fulekar 2012). Tolerance of
plants to metal stresses is correlated with an increased capacity
to scavenge or detoxify ROS (Bhaduri and Fulekar 2012; Bisoi
et al. 2017). In comparison to other heavy metal stress, rela-
tively little information is available on fly ash-induced oxidative
stress in grasses (Nadg
orska-Socha et al. 2013). The relation-
ship of antioxidant defense with photosynthesis and PSII activ-
ity in lemongrass under fly ash-amended soil is not yet been
studied. Keeping in view of the above, the present study aims to
evaluate the growth performance, photosynthesis, and antioxi-
dant response in lemongrass under different proportions of fly
ash-amended soil. The present study would be pertinent in
phytorestoration of fly ash dumps.
Material and methods
Collection and characterization of fly ash and garden soil
The fly ash used in the study was collected from the fly ash
deposits of National Aluminum Corporation Limited (NALCO),
Koraput, Odisha, India (18
460 2200 N to 82
530 2300 E) and the
garden soil was collected from the campus of Central University
of Orissa, Koraput (82
440 5400 E to 18
460 4700 N). After collec-
tion, the fly ash and garden soil samples were air dried for
7 days. Five different amendments of fly ash and garden soil
were prepared by mixing these two in different ratio in dry
weight, and were placed in 5 kg plastic pots, (45 cm in diameter
INTERNATIONAL JOURNAL OF PHYTOREMEDIATION 539
and 30 cm in height) i.e., garden soil (100%), fly ash (100%), fly
ash:garden soil (25:75%), fly ash:garden soil (50:50%), fly ash:gar-
den soil (75:25%). Pots were left at experimental site for 14 days
prior to plantation for physico-chemical stabilization and proper
conditioning of treated soil. Analysis for different chemical prop-
erties such as pH, electrical conductivity (EC), total N, organic C,
available P and available K of fly ash and garden soil amend-
ments were carried out as recently described in our laboratory
(Bisoi et al. 2017). For metal analysis, the fly ash and garden soil
samples (1 g each) were digested with a mixture of nitric, sulphu-
ric, and perchloric acid (6:1:2) at 100
C. Digested material was
diluted with double distilled water and Al, Fe, Cr, Cu, Mn, Zn,
Cd, and B contents were analyzed using an Atomic Absorption
Spectrophotometer (Parkin Elmer atomic absorption spectropho-
tometer, Analyst AA-200) (APHA 1989). All chemicals and
reagents used were of analytical grade and supplied by Sigma-
Aldrich. To manage accuracy and precision, all the experiments
were carried out in triplicates and the mean of triplicate analysis
was used in the result. The standard stock solutions were further
diluted to multilevel standards for all the metals. To minimize
the error, blanks were run simultaneously for the metal determi-
nation. Analytical quality was checked by the analysis of certified
reference materials.
Plant material and growth condition
The study was conducted by taking lemongrass (Cymbopogon
citratus (D.C.) Stapf.) collected from MS Swaminathan Research
Foundation (MSSRF), Jeypore, India. The healthy shoot stump
of lemongrass of 10 cm each were directly planted in the plastic
pots and the experiments were carried out in 3 replications in a
complete randomize block design. Plants were grown in the
campus of Central University of Orissa, India, irrigated every
alternate day with tap water and maintained for 90 days after
plantation. The plants were subjected to natural solar radiation,
with daily maximum photosynthetic photon flux density
(PPFD), air temperature, and relative humidity being about 1460
§ 20 m mol/m2/s, 33.6 § 2
C and 70–75%, respectively.
Plant growth parameters and metal tolerance index
Plant growth in terms of different growth parameters such as
fresh and dry weight of shoot and root were measured after
90 days of different treatments in each replication. Total plant
biomass was obtained after drying at 80
C until a constant
weight was recorded. Metal tolerance ability of the plant was
determined through metal tolerance index (MTI) using the for-
mula by Qihang et al. (2011).
MTI ð%Þ D ðTotal plant biomass under treatment
6 Total plant biomass under controlÞ £ 100
Leaf CO2 photosynthetic rate (PN) and stomatal
conductance (gs)
The measurements of PN and gs of 90-day-old lemongrass
plants were carried out on fully expanded leaves of 2 different
plants in each replication using an open system photosynthetic
540 D. PANDA ET AL.
gas analyzer (CI-304, CID, USA) under normal ambient envi-
ronmental condition at 11 § 1 hours. The second leaf from the
top was selected and kept inside the chamber under natural
irradiance until stable reading was recorded. The measure-
ments were carried out at 32 § 2
C, 70% relative humidity,
1024 § 33 mmol/m2/s photosynthetic active radiation,
370 mmol CO2/m2/s and 21% O2.
Measurement of chlorophyll fluorescence and SPAD index
Chlorophyll fluorescence parameters were measured on the
same leaves used for gas exchange measurements of 90-day-old
plants under different treatment using a portable Chl fluorome-
ter (JUNIOR-PAM, WALZ, Germany). Different parameters
like minimal fluorescence (Fo), maximal fluorescence (Fm), var-
iable fluorescence (Fv D Fm ¡ Fo) and Maximum photochemi-
cal efficiency of PSII (Fv/Fm) was measured in 20 minutes dark-
adapted leaves (Maxwell and Johnson 2000). In light adapted
leaves at a PPFD of 400 mmol/m2/s (for 15 minutes) steady
state fluorescence yield (Fs), maximal fluorescence (Fm0 ) after
0.8 seconds saturating white light pulse and minimal fluores-
cence (Fo0 ) were measured when actinic light was turned off.
Further, yield of PSII photochemistry (Y II), Quenching value
due to non-photochemical dissipation of absorbed light energy
(NPQ) and the coefficient for photochemical quenching (qP)
was also calculated (Maxwell and Johnson 2000).
Chlorophyll index of 90-day-old plants were made on the
fully expanded leaf of 2 different plants in each replication
using an SPAD 502 chlorophyll meter (KONIKA MINOTA
SENSING, JAPAN) by Shrestha et al. (2012).
Measurement of antioxidant enzyme activity and protein
content
After measurement of photosynthesis and Chl fluorescence,
same leaf tissue of lemongrass (0.5 g) was homogenized in 10 ml
of 50 mM potassium phosphate buffer (pH 7.8) as recently
described in our laboratory (Bisoi et al. 2017) for measurement
of antioxidative enzyme activity. An aliquot of the extract was
used to determine protein content following Lowry et al. (1951).
In brief, superoxide dismutase (SOD) activity was measured by
the photochemical method according to Gianopolitis and Ries
(1977) with modification suggested by Choudhury and Choud-
hury (1985). Ascorbate peroxidase (APX) activity was measured
according to Nakano and Asada (1981) by monitoring the rate
of ascorbate oxidation at 290 nm (E D 2.8 mmol/cm).The activ-
ity of guaiacol peroxidase (GPX) was assayed according to Rao
et al. (1995) and Catalase (CAT) activity was measured accord-
ing to Cakmak and Marschner (1992).
Determination of malondialdehyde (MDA) content
Lipid peroxidation product in terms of malondialdehyde
(MDA) content was measured by thiobarbituric acid (TBA)
reactions followed by Heath and Packer (1968). Leaf tissue
(0.1 g) was homogenized in 5 mL of 0.1% (w/v) trichloroacetic
acid (TCA) in a pre-chilled mortar and pestle. The homogenate
was centrifuged at 10,000 g for 20 minutes. In total, 1 ml of
20% TCA containing 0.5% TBA and 0.01 ml of butylated
hydroxy toluene (BHT) were added to 1 ml supernatant extract.
The assay mixture was kept at 95
C for 30 minutes. The con-
tent was cooled and centrifuged at 10,000 g for 15 minutes and
the absorbance was recorded at 532 nm and corrected for
600 nm. The content of MDA was expressed as nmol MDA/g
fwt by using E D 155 mmol/cm.
Statistical analysis
Differences between various growth, physiological, and bio-
chemical parameters were compared by analysis of variance
(ANOVA) using CROPSTAT (International Rice Research
Institute, Philippines). Mean values were compared by the least
significant difference (LSD, P < 0.05), wherever the F-test was
significant. Correlation analysis and Duncan’s multiple range
tests were done by the CROPSTAT software.
Results and discussion
Physico-chemical properties of garden soil and fly ash
The utilization of fly ash for vegetation is restricted because of
the presence of non-essential and potentially toxic metals,
which may pose a problem for plant growth. Therefore, fly ash
with soil amendment may offer suitable combination to sup-
port plant growth with reduced risk of metal toxicity (Pandey
and Singh 2010). In the present study, the nature of the fly ash
was alkaline and content of nitrogen (N), potassium (K), and
organic carbon (OC) was lower in comparison to garden soil
(Table 1). The electrical conductivity and the available phos-
phorus content were higher in the fly ash compared to garden
soil. In addition, fly ash contains the larger amount of Al, Fe,
Cr, Mn, Cu, Cd, and B in comparison to garden soil, except for
Zn (Table 1). Results indicate that in garden soil the concentra-
tion of Zn and Cu were within the range of average value for
soil whereas the concentrations of B and Mn were deficient
(Kabata Pendias 2011). However, in the fly ash, Cr, Cu, and Cd
were toxic and concentration of Mn was deficient, whereas con-
centrations of Zn and B were within the range of average values
for soils (Kabata Pendias 2011). Application of low level of fly
ash (25%) in soil amendments significantly (P < 0.05)
improved the pH, EC, and N compared to other treatments.
The phosphorus concentration in fly ash is quite high com-
pared to garden soil. The gradual increase of available P of the
soil was observed with an increased application rate of fly ash
(Sarangi et al. 2001). Thus the application of fly ash to soil sig-
nificantly improves the permeability of soil, moisture holding
capacity, reduces acidity, and heavy metal availability that
might be useful as soil amendment (Pandey and Singh 2010;
Ram and Masto 2014). Fly ash application is therefore poten-
tially valuable particularly in conditions where trace metals are
deficient (Gupta et al. 2006).
Plant growth and metal tolerance index
The growth parameters such as root, shoot, and total plant bio-
mass of 90-day-old lemongrass plants were significantly
increased after exposure to low level of fly ash (25%) amended
soil compared to the plants grown in garden soil (Table 2), but
 INTERNATIONAL JOURNAL OF PHYTOREMEDIATION 541

Table 1. Chemical properties of garden soils, fly ash, and different percentage of fly ash-amended soil used for the experiment.

Parameters Garden soil FA 25% FA 50% FA 75% Fly ash LSD (P < 0.05)

pH 6.64 § 0.11 6.78 § 0.10 7.20 § 0.04 7.50 § 0.02 8.70 § 0.10 0.14
EC (mS/cm) 68.5 § 11.0 98.0 § 8.5 102.5 § 3.5 110.1 § 11.0 157 § 12.0 13.2
Nitrogen (mg/kg) 0.25 § 0.02 0.29 § 0.03 0.16 § 0.04 0.12 § 0.02 0.10 § 0.02 0.04
Phosphorus (mg/kg) 0.12 § 0.00 0.13 § 0.01 0.16 § 0.02 0.16 § 0.05 0.22 § 0.06 0.07
Potassium (mg/kg) 0.86 § 0.05 0.81 § 0.03 0.59 § 0.07 0.39 § 0.04 0.42 § 0.03 0.06
Organic Carbon (%) 0.16 § 0.01 0.17 § 0.02 0.16 § 0.06 0.12 § 0.03 0.08 § 0.02 0.06
Metals (mg/g dwt)
Al 112 § 20 234 § 23 645 § 45 2520 § 98 4650 § 165 151
Fe 108 § 18 118 § 21 1345 § 42 1612 § 56 3150 § 125 121
Cr 12.2 § 1.3 18.4 § 3.5 26.3 § 5.2 33.5 § 3.4 41.2 § 2.5 10.1
Zn 83.5 § 2.4 78.4 § 2.8 71.3 § 7.3 66.4 § 6.5 62.0 § 4.3 11.3
Mn 83.4 § 5.6 89.3 § 4.4 102.2 § 7.3 106.5 § 8.9 115.6 § 12.2 7.4
Cu 12.3 § 1.2 21.4 § 2.3 32.3 § 4.2 45.7 § 4.5 56.3 § 2.5 10.3
Cd 21.2 § 1.8 26.5 § 2.4 34.3 § 3.5 39.7 § 3.8 46.3 § 3.1 11.2
B 12.0 § 1.1 14.5 § 2.0 21.3 § 2.7 25.6 § 1.6 28.6 § 2.3 3.5

Data are the mean of 3 replications with standard deviation. FA: fly ash; LSD: least significance difference.

these parameters were further decreased with increasing con-
centration of fly ash (75% and 100%). The plant biomass of
lemongrass was increased by 11% under low level of fly ash
(25%) amendments whereas the decrease of shoot and root and
total plant biomass was 70%, 73% and 71% respectively, under
100% of fly ash compared to the control. The study showed
that fly ash (25%) in soil amendments has the promoting effect
on growth of lemongrass. However, some studies showed that
high concentrations of metals in fly ash can inhibit plant
growth (Parveen et al. 2006; Bisoi et al. 2017). Fly ash has some
physical and chemical properties that might be useful in low
level of soil amendment, which are conducive for plant growth
(Verma et al. 2014; Ram and Masto 2014). In metal-polluted
soils, decrease in biomass of plants is closely related to growth
and development of roots (Gautam et al. 2017). In the present
study, the roots of lemongrass were more affected than shoots
since they exposed to elevated concentrations of metals in fly
ash. Due to metal toxicity in fly ash, poorly-developed roots
may lead to decrease in nutrient transport and water uptake by
plant, thereby affecting shoot and total plant biomass (Boonya-
pookana et al. 2005).
To determine metal tolerant capacity of the plant, one of
the most common parameters used is MTI (K€ ohl and L€osch
1999). Assessment of MTI of plants that can grow on fly ash
deposits is important for phytorestoration and phytoreme-
diation of fly ash (Zacchini et al. 2009). Based on total plant
biomass of lemongrass under different fly ash amendments,
the MTI was significantly increased in 25% of fly ash-
amended soil and further declined with increase of fly ash
concentration (Table 2). The lemongrass showed high toler-
ance to low level of fly ash-amended soil and high capability
to grow on metal-polluted soils as has been reported in red
mud (Gautam et al. 2017).
CO2 photosynthetic rate, stomatal conductance,
transpiration, and SPAD index
The CO2 photosynthetic rate (PN) was not significantly
changed (p < 0.05) under low level of fly ash (25%)
amended soil compare to control plants but further
decreased with increasing concentration of fly ash in soil
amendments (Table 2). The percentage of reduction of PN
was 68% under 100% of fly ash compared to the control
plants. Similarly, the gs and E were also remarkably
decreased under 100% of fly ash as compared to the control
plant. The percentage of reduction of gs and E was 62%
and 51%, respectively, under 100% fly ash compared to the
control plant grown under garden soil (Table 2). Further,
the SPAD Chl index of lemongrass was not significantly
changed in 25% and 50% of fly ash-amended soil compared
to the control plants but under higher concentration (75%
and 100%) of fly ash remarkably reduced the SPAD value
(Table 2). Leaf photosynthesis is known to be sensitive to
heavy metal in fly ash (Gupta et al. 2002; Raja et al. 2014;
Srivastva et al. 2014). The present study indicated that 25%
of fly ash-amended soil maintained the leaf PN in lemon-
grass along with the maintenance of gs and E. Furthermore,
the rapid drop in PN under high concentration of fly ash

Table 2. Plant biomass and metal tolerance Index (MTI) of 90-day-old lemongrass grown under different concentration of fly ash-amended soil.

Treatments SFM (g/plant) SDM (g/plant) RFM (g/plant) RDM (g/plant) PBM (g/plant) MTI (%)

GS 8.84 § 0.8 b
2.13 § 0.04 b
4.02 § 0.01 b
1.49 § 0.02 b
3.62 § 0.11b
100 § 0b
FA 25% 9.59 § 0.2a 2.28 § 0.04a 4.16 § 0.02a 1.76 § 0.10a 4.04 § 0.08a 111 § 1a
FA 50% 9.15 § 0.3b 2.12 § 0.01b 4.11 § 0.10a 1.07 § 0.10c 3.18 § 0.03c 87 § 3c
FA 75% 7.29 § 0.2c 1.85 § 0.10c 3.15 § 0.03c 0.70 § 0.10d 2.55 § 0.02d 70 § 2d
FA 100% 2.80 § 0.4d 0.66 § 0.20d 1.64 § 0.10d 0.41 § 0.02e 1.07 § 0.02e 29 § 2e
LSDP < 0.05 0.39 0.13 0.14 0.27 0.35 9.0
F value 29.80** 68.40** 272.03** 63.74** 96.6** 28.5**

Data are the mean of three replications (n D 3). Means followed by a common letter a, b, c, d and e are not significantly different at P < 0.05 by Duncans multiple range test.
GS: garden soil, FA: fly ash, SFM: shoot fresh mass, SDM: shoot dry mass, RFM: root fresh mass, RDM: root dry mass, PBM: total plant biomass; significance at P < 0.01.
542 D. PANDA ET AL.

was probably due to the structural damage of the photosyn-

thetic apparatus which can be evident in the fall in the val-
ues of SPAD index and/or may be due to functional
alteration of stomata that has been reported for Phaseolus
vulgaris and Oryza sativa (Miteva and Merakchiyska 2002;
Raja et al. 2014). The significant reduction in SPAD index
in lemongrass under high fly ash may be due to the pres-
ence of elevated level of metal ions or degradation of Chl
by free radicals generated by metals present in fly ash
(Mathur et al. 2016; Bisoi et al. 2017).

Photosystem II activity
To clarify the alterations of PSII activity in lemongrass
under different levels of fly ash-amended soil, we measured
different Chl fluorescence parameters such as Fo, Fm, Fv/Fm,
Y (II), qP, and NPQ. In the present study, Fo, Fm, Fv/Fm, Y
(II), and qP were not significantly changed under low level
of fly ash-amended soil (25%) in compared to the control
plants (Figure 1; Table S1). However, the higher concentra-
tion of fly ash (50%, 75% and 100%) amended soil inhibits
the PSII activity that was reflected in the significant decline
in the values of Fm, Fv/Fm, Y (II), and qP (Figure 1;
Table S1). In this study, the increase of Fo under high fly
ash concentration can be due to the disorganization of
antenna pigment level in lemongrass. This result is in
accordance with Calatayud and Barreno (2001) who
reported that abiotic stress, such as soil salinity and drought
can increase the Fo value. The Fm, Fv/Fm, qP, and Y (II) can
be used to estimate the functional efficiency of PSII (Cala-
tayud et al. 2006; Murchie and Lawson 2013). These param-
eters were significantly decreased in higher concentration of
fly ash indicates the sensitivity of PSII due to metal toxicity
in fly ash. In addition, increase of NPQ in lemongrass
under high fly ash concentration suggested that there was
decrease in the rate of primary charge separation or by
increase of heat dissipation (Maxwell and Johnson 2000; Fu
et al. 2012).
Figure 2. Spider type visual plot showing quantitative extend of changes in differ-
ent antioxidant enzymes activities and protein content in lemongrass exposed to
different concentration of fly ash-amended soil. The black circle with radius 1 rep-
resents garden soil (GS) and 25, 50, 75 and 100 are different percentage of fly ash-
amended soil. Data are the mean of 3 replications. SOD: superoxide dismutase;
APX: ascorbate peroxidase; GPX: guaiacol peroxidase and CAT: catalase.
Level of lipid peroxidation, protein content, and
antioxidant enzyme activity
High concentrations of fly ash in soil amendments can induce
oxidative stress, causing an increased production of ROS
(Arora et al. 2002; Bisoi et al. 2017). Lipid peroxidation is con-
sidered an indicator of oxidative stress (Panda 2007) and MDA
is a decomposition product of polyunsaturated fatty acids that
has been used as a biomarker for lipid peroxidation (Panda
2007). In the present study, MDA levels were not changed in
low level of fly ash (25% and 50%) but prominently increased
under high fly ash concentrations (75% and 100%) in soil
amendments (Figure 2; Table S2). Results in this study suggest
that low level of fly ash in soil amendment did not cause oxida-
tive stress whereas elevated levels of metals in fly ash can dam-
age the cell membrane, disturb metabolic processes, and inhibit
the plant growth that has been reported for other crops such as
Salix integra, Triticum aestivum and Oryza nivara (Zhang et al.
2010; Wang et al. 2014; Bisoi et al. 2017). This effect was also
noted in the present study under high concentration of fly ash
in the form of decrease in photosynthesis activity (Table 3) and
proteins level (Figure 2). The protein content was significantly
declined under different fly ash treatments probably due to the
toxic effects of ROS (Verma and Dubey 2003) or to the
increased protease activity affecting the total protein and
growth of plants (Sharma and Dubey 2007; Xu et al. 2010).

Table 3. Changes of CO2 photosynthetic rate (PN), transpiration rate (E), stomatal
conductance (gs) and SPAD index in lemongrass leaves grown under different con-
centration of fly ash.
PN E Gs SPAD
Treatments (ɥM CO2 m2/s) (mM H2O/m2/s) (mM H2O/m2/s) (rel.)

GS 11.8 § 1.0a 1.45 § 0.4b 56.2 § 3.40a 52.3 § 2.6a

FA 25% 11.9 § 0.2a 2.49 § 0.1a 55.4 § 0.10a 52.5 § 3.1a
Figure 1. Spider type visual plot showing quantitative extend of changes in vari- FA 50% 6.80 § 0.3b 0.93 § 0.1c 26.8 § 20.5b 52.0 § 2.2a
ous chlorophyll fluorescence parameter in lemongrass exposed to different con- FA 75% 4.90 § 1.8b 0.91 § 0.1c 22.8 § 10.6b 47.5 § 1.9b
centration of fly ash. The black circle with radius 1 represents garden soil (GS) and FA 100% 3.80 § 2.3b 0.72 § 0.1c 21.9 § 13.0b 40.5 § 1.3c
25, 50, 75 and 100 are different percentages of fly ash amendments. Data are the LSDP < 0.05 3.9 0.4 12.5 3.4
mean of 3 replications. Fo: minimum fluorescence yield obtained with dark- F value 14.2** 45.2** 9.4** 6.0**
adapted leaf; Fm: maximum Chl fluorescence yield obtained with dark-adapted
leaf; Fv: variable fluorescence (Fm – Fo); Fv/Fm: maximal photochemical efficiency of Data are the mean of 3 replications with standard deviation. Means followed by a
PSII; NPQ, non-photochemical quenching; qP: photochemical quenching; Y(II): yield common letter a, b, c, d and e are not significantly different at P < 0.05 by Dun-
of PSII photochemistry. cans multiple range test. GS: garden soil; FA: fly ash; significance at P < 0.01.

Table 4. Correlation co-efficient (r value) between photosynthetic parameters with antioxidative enzymes as well as protein and MDA level in lemongrass grown in differ-
ent levels of fly ash-amended soil.
Parameters Fv/Fm qP NPQ
** ** **
SOD ¡0.75 ¡0.64 0.72 ¡0.66
APX ¡0.93** ¡0.88** 0.81** ¡0.88**
GPX ¡0.85** ¡0.70** 0.71** ¡0.71**
CAT 0.87** 0.77** ¡0.72**
Protein 0.42** ¡0.06ns 0.10ns
MDA ¡0.88** ¡0.85** 0.71** ¡0.82**
Total degrees of freedom D 15; significance at P < 0.05; significance at P < 0.01; nsnon-significant.
Tolerance capacity of plants to metal stresses is correlated
with an increase of antioxidants to scavenge or detoxify ROS
(Bhaduri and Fulekar 2012; Bisoi et al. 2017). In the present
study, the activities of some antioxidative enzymes such as
SOD, APX, and GPX in the lemongrass plants were not
significantly changed under low level of fly ash (25%)
amendments compared to the control plants. However, the
activities of these enzymes were remarkably increased under
50%, 75% and 100% fly ash (Figure 2; Table S2). This rise of
enzyme activity in lemongrass can be considered as plant
response to active oxygen species caused by metal ion pre-
sented in fly ash. According to Bhaduri and Fulekar (2012),
increased levels of active oxygen may stimulate the cellular
protective mechanism to mitigate the cells’ damage. Results
obtained in this study were in agreement with findings of
Arora et al. (2002) for Zea mays and Oryza nivara (Bisoi
et al. 2017). However, remarkable inhibition of CAT activity
in lemongrass under different treatments of fly ash was
observed (Figure 2) suggesting that CAT was more sensitive
than other antioxidative enzymes in lemongrass under high
fly ash concentration.
Relationship between antioxidant enzymes with growth
and photosynthesis
Our experiment showed a negative correlation between activi-
ties of antioxidant enzymes such as SOD, APX, and GPX with
growth and photosynthetic parameters in lemongrass (Table 4).
These results suggested that the antioxidant enzyme systems of
lemongrass are unable to effectively scavenge the ROS caused
by metal ions in the high fly ash concentrations which nega-
tively affects growth and photosynthesis.
Conclusion
The study showed that garden soil amended with 25% of fly ash
could be beneficial for lemongrass improving the growth and
leaf photosynthesis and PSII activity. Therefore, lemongrass
has the capability to tolerate 25% of fly ash due to high MTI
which was more than 100%. In addition, the decline in photo-
synthesis in lemongrass under high concentrations of fly ash
can be due to chlorophyll damage, structural and functional
alteration of PSII. The induction of some antioxidative enzymes
in lemongrass under elevated fly ash concentration indicates
metal tolerance ability of this plant. These findings indicate
that lemongrass can be identified as plant species that can grow
on fly ash deposits and can be used for phytorestoration of fly
ash dumps.
INTERNATIONAL JOURNAL OF PHYTOREMEDIATION 543
Y(II) PN E gs SPAD PBM
** ** ** ** **
¡0.87 ¡0.69 ¡0.74 ¡0.45 ¡0.81**
¡0.94** ¡0.70** ¡0.87** ¡0.68** ¡0.62**
¡0.80** ¡0.68** ¡0.59** ¡0.60** ¡0.86**
0.80** 0.86** 0.68** 0.91** 0.60** 0.51**
0.003ns 0.31* 0.07ns 0.41** ¡0.20ns 0.29*
¡0.91** ¡0.57** ¡0.76** ¡0.68** ¡0.66**
Acknowledgments
The authors are grateful to the Head, Department of Biodiversity and Conser-
vation of Natural Resources for providing necessary facilities for the work.
The Regional Director, MS Swaminathan Research Foundation (MSSRF), Jey-
pore is highly acknowledged for providing the lemongrass for the experiment.
References
American Public Health Association (APHA). 1989. Standard methods for
examination of water and wastewaters. 14th ed. Washington (DC).
Arora A, Sairam RK, Srivastava GC. 2002. Oxidative stress and antioxida-
tive system in plants. Curr Sci. 82:1227–1238.
Baker NR, Rosenqvist E. 2004. Applications of chlorophyll fluorescence
can improve crop production strategies: an examination of future pos-
sibilities. J Exp Bot. 55:1607–1621.
Bhaduri MA, Fulekar MH. 2012. Antioxidant enzyme responses of plants
to heavy metal stress. Rev Environ Sci Biotechnol. 11:55–69.
Bienes R, Jimenez-Ballesta R, Ruiz-Colmenero M, Arevalo D, Alvarez A,
Marques MJ. 2010. Incidence of the growing of aromatics and medicinal
plants on the erosion of agricultural soils. Span J Rural Develop. 1:19–28.
Bisoi SS, Mishra SS, Barik J, Panda D. 2017. Effects of different treatments
of fly ash and mining soil on growth and antioxidant protection of
Indian wild rice. Int J Phytorem. 19(5):446–452.
Boonyapookana B, Parkplan P, Techapinyawat S, DeLaune RD, Jugsujinda
A. 2005. Phytoaccumulation of lead by sunflower (Helianthus annuus),
tobacco (Nicotiana tabacum), and vetiver (Vetiveria zizanioides). J Envi-
ron Sci Health Part A-Toxic/Hazard Subst Environ Engin. 40:117–137.
Cakmak I, Marschner H. 1992. Magnesium deficiency and highlight inten-
sity enhance activities of superoxide dismutase, ascorbate peroxidase
and glutathione reductase in bean leaves. Plant Physiol. 98:1222–1227.
Calatayud A, Barreno E. 2001. Chlorophyll a fluorescence, antioxidant
enzymes and lipid peroxidation in tomato in response to ozone and
benomyl. Environ Pollut. 115:283–289.
Calatayud A, Roca D, Mart
ınez PF. 2006. Spatial-temporal variations in
rose leaves under water stress conditions studied by chlorophyll fluo-
rescence imaging. Plant Physiol Biochem. 44:564–573.
Choudhury SR, Choudhury MA. 1985. Hydrogen peroxide metabolism as
an index of water stress tolerance in jute. Physiol Planta. 65:503–507.
Das M, Maiti SK. 2009. Growth of Cymbopogon citratus and Vetiveria ziza-
nioides on Cu mine tailings amended with chicken manure and
manure-soil mixtures: a pot scale study. Int J Phytorem. 11:651–663.
Dubey RS. 2011. Metal toxicity, oxidative stress and antioxidative defense
system in plants. In: Gupta SD, editor. Reactive oxygen species and
antioxidants in higher plants. Boca Raton (FL): CRC Press, pp. 177–
203.
Fu WG, Li PP, Wu YY. 2012. Effects of different light intensities on chloro-
phyll fluorescence characteristics and yield in lettuce. Sci Hortic-
Amesterdam. 135:45–51.
Gaji
c G, Pavlovi
c P, Kosti
c O, Jari
c S, ĐurĐevi
c L, Pavlovi
c D, Mitrovi
c M.
2013. Ecophysiological and biochemical traits of three herbaceous
plants growing on the disposed coal combustion fly ash of different
weathering stage. Arch Biol Sci. 65(4):1651–1667.
Gautam M, Pandey D, Agrawal M. 2017. Phytoremediation of metals using
lemongrass (Cymbopogon citratus (D.C.) Stapf.) grown under different
levels of red mud in soil amended with biowastes. Int J Phytorem. 12
(6):555–562.
544 D. PANDA ET AL.
Ghosh I, Ghosh M, Mukherjee A. 2017. Remediation of mine tailings and
fly ash dumpsites: role of Poaceae Family members and aromatic
grasses. In: Anjum AN, Gill SS, Tuteja N, editors. Enhancing cleanup
of environmental pollutants. Berlin Heidelberg: Springer. p. 117–167.
Giannopolitis CN, Ries SK. 1977. Superoxide dismutases: occurrence in
higher plants. Plant Physiol. 115:159–169.
Guidi L, Degl’Innocenti E, Carmassi G, Massa D, Pardossi A. 2011. Effects
of boron on leaf chlorophyll fluorescence of greenhouse tomato grown
with saline water. Environ Exp Bot. 73:57–63.
Gupta AK, Verma SK, Khan K, Verma RK. 2013. Phytoremediation using
aromatic plants: a sustainable approach for remediation of heavy met-
als polluted sites. Environ Sci Technol. 47:10115–10116.
Gupta DK, Raj U N, Tripathi RD, Inouhe M. 2002. Impacts of fly-ash on
soil and plant responses. J Plant Res. 115:401–409.
Gupta DK, Tripathi RD, Rai UN, Dwivedi S, Mishra S, Srivastava S, Inouhe
M. 2006. Changes in amino acid profile and amino acid content in
seeds of Cicer arietinum L. (chick pea) grown under various fly ash
amendments. Chemosphere 65: 939–945.
Gupta DK, Tripathi RD, Rai UN, Mishra S, Srivastava S, Dwivedi S, Maa-
thuis FJM. 2007. Growth and biochemical parameters of Cicer arietinum
L. grown on amended fly ash. Environ Monit Assess. 134:479–487.
Heath RL, Packer L. 1968. Photoperoxidation in isolated chloroplast I.
Kinetics and stoichiometry of fatty acid peroxidation. Arch Biochem
Biophys. 125:189–198.
Israila YZ, Bola AE, Emmanuel GC, Ola IS. 2015. The effect of application
of EDTA on the phytoextraction of heavy metals by Vetivera ziza-
nioides, Cymbopogon citrates and Helianthus annus. Int J Environ
Monit Anal. 3:38–43.
Kabata Pendias A. 2011. Trace element in soils and plants. Boca Raton
(FL): CRC press.
K€ohl L€osch R. 1999. Experimental characterization of heavy metal toler-
ance in plants. In: Prasad MNV, Hagemeyer J, editors. Heavy metal
stress in plants. Berlin Heidelberg: Springer. p. 371–389.
Lowry OH, Rosebrough NL, Farr AL, Randall RI. 1951. Protein measure-
ment with Folin phenol reagent. J Biol Chem. 193:265–275.
Maiti D, Prasad B. 2016. Revegetation of fly ash - a review with emphasis
on grass-legume plantation and bioaccumulation of metals. Appl Ecol
Env Res. 14(2):185–212.
Mathur S, Kalaji HM, Jajoo A. 2016. Investigation of deleterious effects of
chromium phytotoxicity and photosynthesis in wheat plant. Photosyn-
thetica. 54:185–192.
Maxwell K, Johnson GN. 2000. Chlorophyll fluorescence practical guide. J
Exp Bot. 51:659–668.
Miteva E, Merakchiyska M. 2002. Response of chloroplasts and photosyn-
thetic mechanism of bean plants to excess arsenic in soil. Bulg J Agric
Sci. 8:151–156.
Murchie EH, Lawson T. 2013. Chlorophyll fluorescence analysis: a guide to
good practice and understanding some new applications. J Exp Bot.
64:3983–3998.
Nadg
orska-Socha A, Kafel A, Kandziora-Ciupa A, Gospodarek J, Zawisza-
Raszka A. 2013. Accumulation of heavy metals and antioxidant
responses in Vicia faba plants grown on monometallic contaminated
soil. Environ Sci Pollut Res 20: 1124–1134.
Nakano Y, Asada K. 1981. Hydrogen peroxide is scavenged by ascorbate specific
peroxidase in spinach chloroplasts. Plant Cell Physiol. 22:867–880.
Panda SK. 2007. Chromium-mediated oxidative stress and ultrastructural
changes in root cells of developing rice seedlings. J Plant Physiol.
164:1419–1428.
Pandey SK, Bhattacharya T, Chakraborty S. 2016. Metal phytoremediation
potential of naturally growing plants on fly ash dumpsite of Patratu ther-
mal power station, Jharkhand, India. Intl J of Phytorem. 18(1):87–93.
Pandey VC, Prakash P, Bajpai O, Kumar A, Singh N. 2014. Phytodiversity
on fly ash deposits: evaluation of naturally colonized species for sus-
tainable phytorestoration. J Env Sci Pollu Res. 22(4):2776–2787.
View publication stats
Pandey VC, Singh B. 2012. Rehabilitation of coal fly ash basins: current
need to use ecological engineering. Ecol Eng. 49:190–192.
Pandey VC, Singh N. 2010. Impact of fly ash incorporation in soil systems.
Agri Eco Environ. 136:16–27.
Pandey VC. 2015. Assisted phytoremediation of fly ash dumps through
naturally colonized plants. Ecol Eng. 82:1–5.
Parveen R, Hisamuddin Azam T, Niyaz T, Singh S. 2006. Effect of fly ash
amended soil on the plant growth, yield, chlorophyll and oil content of
Mentha citrate. National Symposium on Issue and Challenges for Envi-
ronmental Management, Vision 2025, p55.
Prajapati SK. 2012. Biomonitoring and speciation of road dust for heavy
metals using Calotropis procera and Delbergia sissoo. Environ Skep
Crit. 1:61–64.
Qihang W, Wang S, Thangavel P, Qingfei L, Zheng H, Jun B, Qui R. 2011.
Phytostabilization of Jatropha curcas L. in polymetalic acid mine tail-
ings. Int J Phytorem. 13:788–804.
Qu J, Luo C, Cong Q, Yuan X. 2012. Carbon nanotubes and Cu–Zn nano-
particles synthesis using hyper accumulator plants. Environ Chem Lett.
10:153–158.
Raja R, Nayak AK, Rao KS, Puree C, Shahid M, Panda BB, Kumar A, Tri-
pathi R, Bhattacharyya P, Baig MJ, et al. 2014. Effect of fly ash deposi-
tion on photosynthesis, growth and yield of rice. Bull. Environ Contam
Toxicol. 93(1):106–112.
Ram LC, Masto RE. 2014. Fly ash for soil amelioration: a review on the
influence of ash blending with inorganic and organic amendments.
Earth Sci Rev. 128:52–74.
Rao MV, Hale BA, Ormrod DP. 1995. Amelioration of ozone-induced oxi-
dative damage in wheat plants grown under high carbon dioxide: role
of antioxidant enzymes. Plant Physiol. 109:421–432.
Reid RJ, Hayes JE, Post A, Stangoulis JCR, Graham RD. 2004. A critical
analysis of the causes of boron toxicity in plants. Plant Cell Environ.
27:1405–1414.
Sarangi PK, Mahakur D, Mishra PC. 2001. Soil biochemical activity and
growth response of rice Oriza sativa in fly ash amended soil. Bioresour
Technol. 76:199–205.
Sayed OH. 2003. Chlorophyll fluorescence as a tool in cereal crop research.
Photosynthetica. 41:321–330.
Sharma P, Dubey RS. 2007. Involvement of oxidative stress and role of
antioxidative defense system in growing rice seedlings exposed to toxic
concentrations of aluminum. Plant Cell Rep. 26(11):2027–2038.
Shrestha S, Bruect H, Asch H. 2012. Chlorophyll index, photochemical
reflectance index and chlorophyll fluorescence measurements of rice
leaves supplied with different N levels. Photochem Photobiol. 113:7–13.
Stoffella PJ, Lone MI, Zhen-Ii HE, Yang X. 2008. Phytoremediation of
heavy metal polluted soils and water: progress and perspectives. J of
Zhejiang Uni Sci B. 9(3):210–220.
Verma S, Dubey RS. 2003. Lead toxicity induces lipid peroxidation and
alters the activities of antioxidant enzymes in growing rice plants. Plant
Sci. 164:645–655.
Verma SK, Singh K, Gupta AK, Pandey VC, Trivedi P, Verma RJ, Patra
DD. 2014. Aromatic grasses for phytomanagement of coal fly ash haz-
ards. Eco Eng. 73:425–428.
Wang S, Shi X, Sun H, Chen Y, Pan H, Yang X, Rafiq T. 2014.Variations in
metal tolerance and accumulation in three hydroponically cultivated
varieties of Salix integra treated with lead. PloS one. 9:108–568.
Xu QS, Hu JZ, Xie KB, Yang HY, Du KH, Shi GX. 2010. Accumulation and
acute toxicity of silver in Potamogeton crispus (L.). J Hazard Mater.
173:186–193.
Zacchini M, Pietrini F, Mugnozza GS, Iori V, Pietrosanti L, Massacci A.
2009. Metal tolerance, accumulation and translocation in poplar and
willow clones treated with cadmium in hydroponics. Water Air Soil
Pollut. 197:23–34.
Zhang H, Hu LY, Li P, Hu KD, Jiang CX, Luo JP. 2010. Hydrogen sulfide
alleviated chromium toxicity in wheat. Biologia Plantarum. 54:743–747.