Ecotoxicology and Environmental Safety 216 (2021) 112195

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Ecotoxicology and Environmental Safety

journal homepage: www.elsevier.com/locate/ecoenv

Improving growth and photosynthetic performance of drought stressed

tomato by application of nano-organic fertilizer involves up-regulation of
nitrogen, antioxidant and osmolyte metabolism
Mohammad Abass Ahanger a, Maodong Qi a, Ziguang Huang a, Xuedong Xu a, Naheeda Begum a,
Cheng Qin a, Chenxi Zhang b, Nadeem Ahmad a, c, Nabil S. Mustafa d, Muhammad Ashraf e,
Lixin Zhang a, *
a
College of Life Sciences, Northwest A&F University, Yaangling, Shaanxi, China
b
Institute of Molecular Biology and Biotechnology, Zoology, The University of Lahore, Lahore, Pakistan
c
Islamabad Model College for Boys, Federal Directorate of Education, H-9, Islamabad, Pakistan
d
Department of Pomology, National Research Centre, Cairo, Egypt
e
University of Agriculture, Faisalabad, Pakistan

A R T I C L E I N F O A B S T R A C T

Edited by Professor Bing Yan Organic fertilizer usage is been introduced into agricultural practices for preventing the damaging effects of
chemical fertilizers. Present study investigated the beneficial role of organic fertilizer (nano-vermicompost) on
Keywords: the growth, oxidative stress parameters, antioxidant and nitrogen metabolism, osmolyte accumulation and
Drought mineral elements in tomato under drought stress. Drought stress resulted in reduced growth and biomass
Antioxidant system
accumulation by triggering oxidative stress due to excess accumulation of reactive oxygen species (ROS) and
Osmolytes
reduced mineral uptake. Application of nano-vermicompost proved significantly beneficial in improving growth
Nano-vermicompost
Tomato and mitigating the drought induced growth decline. Nano-vermicompost increased growth and dry matter
content and ameliorated the decline in chlorophyll contents, photosynthesis and PSII activity more significantly
at higher concentration (100 mg kg− 1 soil). ROS accumulation was significantly reduced by nano-vermicompost
application thereby enhancing the membrane stability under normal as well as drought conditions. Furthermore,
lipid peroxidation and activities of protease and lypoxygenase were significantly reduced. Drought up-regulated
antioxidant system and application of nano-vermicompost further enhanced the activities of antioxidant enzymes
and the contents of non-enzymatic antioxidant components. Accumulation of osmolytes including proline,
glycine betaine and sugars increased significantly due to nano-vermicompost application. Besides, decline in the
activity of nitrate reductase and content of essential mineral elements like nitrogen, potassium and phosphorous
was also ameliorated by nano-vermicompost application.

1. Introduction efficiency of agricultural crops. Drought stress reduces germination,

Drought stress is a global problem and rapid climate change has
further aggravated the situation. Restricted availability of water results
in significant reduction in the yield potential of major crops (Jatav et al.,
2014; Ahanger et al., 2014). Drought stress is experienced by plants
when availability of water to roots is low and the rate of transpiration
exceeds absorption, and such conditions often exist in arid and semi arid
regions of the globe. Under drought stress conditions water use effi­
ciency (WUE) is one of the important traits employed to evaluate the
seedling growth and photosynthesis, membrane functioning, enzyme
activity, mineral and water uptake hence significantly declines the yield
(Anjum et al., 2015; Jatav et al., 2014; Ahanger and Agarwal, 2017).
With respect to the plant phenological stage water deficit affects the
yield by depressing sink as well as source depending on the timing and
the severity of stress (Blum, 2005). The generative phase and the
beginning of flowering are the most sensitive phases of development.
Plants respond differently to drought in different periods of their
growth, and different strategies are triggered to prevent the damaging

* Corresponding author.
E-mail address: zhanglixin@nwafu.edu.cn (L. Zhang).

https://doi.org/10.1016/j.ecoenv.2021.112195
Received 14 September 2020; Received in revised form 19 March 2021; Accepted 24 March 2021
Available online 3 April 2021
0147-6513/© 2021 The Author(s). Published by Elsevier Inc. This is an open access article under the CC BY-NC-ND license
(http://creativecommons.org/licenses/by-nc-nd/4.0/).
M.A. Ahanger et al.
effects of drought (Touchette et al., 2007).
Drought stress results in the excess generation of ROS imparting
oxidative damage to plants and hence the functioning of key metabolic
pathways is altered (Begum et al., 2019, 2020). Excess ROS accumula­
tion reduces membrane stability and cellular functioning by hampering
pathways like photosynthesis, mineral uptake and assimilation (Ahan­
ger et al., 2017, 2019a). Plants up-regulate antioxidant system and
accumulation of osmolytes for neutralizing the excess ROS so that
metabolism is protected (Ahanger and Agarwal, 2017; Begum et al.,
2020). Antioxidants include both enzymatic and non-enzymatic com­
ponents which exist almost in every cellular compartment and bring
stabilization of cells by eliminating the excess ROS (Ahmad et al., 2019).
Osmolytes including proline, glycine betaine and soluble sugars have
key roles in maintaining the water potential, protecting enzyme activity
and stress signaling (Hayat et al., 2012; Ahanger et al., 2014, 2018).
Introduction of modern agricultural techniques like excessive
chemical usage for improving agricultural productivity has rendered
most of agricultural fields unproductive (Salehi et al., 2016). However,
organic fertilizer (vermicompost) is prepared from different raw mate­
rials after following thorough decomposition by the earthworm. Organic
fertilizer is rich in growth promoting components like mineral elements
and other related biochemical components (Zhang et al., 2020).
Therefore can be an ideal choice for improving crop growth and yield in
areas with low soil fertility and frequent stress outbreaks. Vermicompost
reduces the soil acidity and makes key macro- and micro-elements
available in suffice quantities (Mahmud et al., 2020). Recent advance­
ments in manufacturing techniques have led to fabrication of nano­
materials ranging in size and shape, making base for the further
advancement for specific application. Several fields including medicine,
food processing and environmental science have successfully and safely
employed the nanomaterials (Khot et al., 2012), however agricultural
and plant science particularly for plant protection and production is less
explored area. Nanomaterials enter plant either through apoplast or
symplast and can impart either positive or negative effect (Per­
ez-de-Luque, 2017).
Tomato (Solanum lycopersicon) is a vegetable crop within family
solanaceae and is grown worldwide. It is consumed either raw or as
ingredient with other foods. Being rich in lycopene, carotene and an­
thocyanins it is considered as natural antioxidants. It is believed that
lycopene is affective against prostate cancer and cares skin from the UV
radiations. Drought being a global problem influences the growth and
yield productivity of tomato considerably. Therefore present investiga­
tion envisages that application of nano-organic fertilizer (nano-vermi­
compost) can significantly reduce the damaging effects of drought by
up-regulating the defence mechanisms.
Plate 1. Transmission electron microscopy images showing 1 µm and 200 nm particle size of nano-compost.
2
Ecotoxicology and Environmental Safety 216 (2021) 112195
2. Material and methods
2.1. Production and characterization of nano-vermicompost
Raw material used for production of organic fertilizer (vermicom­
post) was fresh cow dung provided by the improved breed dairy cattle
breeding center of Yangling demonstration zone in Shaanxi Province.
Vermicomposting was done using earthworm variety Daping 2. Vermi­
compost was air dried and subjected to nano-mill for three hours at 200
rpm to obtain nano-vermicompost. The particle size of obtained compost
ranged from 1 µm to 200 nm as confirmed by transmission electron
microscope (Plate 1). Certain key properties of nano-compost include:
pH – 7.8, N, P and K – 124, 33.27 and 72.93 mg kg− 1 nano-compost
respectively.
2.2. Experimental design and growth conditions
Tomato (Solanum lycopersicon L. cultivar Huang 108) were sterilized
with 0.01% HgCl2 followed by thorough washing with distilled water.
Sterilized seeds were sown in trays filled with peat based substrate
(pindstrup) which was wetted with distilled water. Trays were regularly
monitored and were supplied with half strength Hoagland solution to
maintain the nutrient status. Twenty days after germination seedlings
were transplanted into pots filled with soil and nano-vermicompost.
Organic fertilizer (nano-vermicompost) was supplemented in two
doses i.e., 10 and 100 mg kg− 1 soil. Soil used was having pH of 7.5 and
available N, P and K were 65.98, 18.78 and 80.67 mg kg− 1 soil respec­
tively. Ten days after seedling establishment pots were divided into two
groups and one group was exposed to drought stress for another fifteen
days. Therefore the different groups of treatments included: (a) control
(5 kg garden soil), (b) NV1 (nano-vermicompost 10 mg kg− 1 soil), (c)
NV2 (nano-vermicompost, 100 mg kg− 1 soil), (d) drought (D), (e) D +
NV1, and (f) D + NV2. Pots were arranged in complete randomized
block design in growth chamber at College of Life Sciences, Northwest
A&F University, Yangling, Shaanxi, China with four replicates for each
treatment. The day/night temperature in the growth chamber was set as
32 ◦ C/27 ◦ C with a light density of 12,000-lux and relative humidity of
70%. Uppermost fully expanded leaves were collected from fourty five
days (fifteen days after drought and twenty five days after trans­
plantation) old seedlings and were put to analysis. Photosynthetic and
oxidative stress parameters, antioxidant system, secondary metabolites
and osmoregulatory components were analysed.
2.3. Plant height and dry weight estimation
Plant height was measured manually using scale and dry weight was
taken by oven drying the plant tissue at 70 ◦ C for 48 h.
M.A. Ahanger et al.
2.4. Estimation of δ-Amino levulinic acid (ALA), total chlorophylls and
carotenoid content, photosynthetic parameters and PSII activity
Method described by Harel and Klein (1972) was used for estimation
of δ-ALA content. Briefly two sets of leaf samples (200 mg each) were
taken in which one set was incubated in 60 mM levulinic acid (LA) for
4 h under light while another set was processed immediately for ALA
estimation. Tissue was extracted in 1 M sodium acetate buffer (pH 4.6)
using chilled mortar and pestle. Homogenate was centrifuged at 15,
000 g for 10 min. Thereafter, 1 mL supernatant was made 5 mL using
distilled water followed by addition of acetyl-acetone and mixture was
heated in a boiling water bath for 10 min and subsequently cooled at
room temperature. Thereafter, Ehrlich’s reagent was added and after
10 min of incubation absorbance was recorded at 555 nm. ALA syn­
thesized during 4 h incubation period was measured by subtracting the
0 h ALA from 4 h ALA content.
For determination of total chlorophyll and carotenoids, 100 mg fresh
leaf tissues was extracted in acetone as per method described by Arnon
(1949) and absorbance of supernatant was recorded at 480, 645 and
663 nm. For measurement of photosynthesis, intercellular CO2 concen­
tration and transpiration rate portable photosynthetic apparatus Li-6400
(LI-COR Inc., USA) was used and measurements were recorded using
fully expanded leaf. Chlorophyll fluorescence parameters including PSII
activity (Fv/Fm), photochemical quenching (qP), non- photochemical
quenching (NPQ) and electron transport rate (ETR) were measured
using Modulated Chlorophyll Fluorometer (PAM 2500; Walz, Germany)
after dark adapting the leaves for 25 min.
2.5. Determination of leaf water content, soluble sugars, free proline, free
amino acids and glycine betaine content
For measurement of relative water content (RWC) method of Smart
and Bihgham (1974) was followed. Content of sugars (Fong et al., 1953;
Jain and Guruprasad, 1989), free proline (Bates et al., 1973), free amino
acids (described by Ahanger et al., 2015) and glycine betaine (Grieve
and Grattan, 1983) were estimated in dry powdered samples.
2.6. Hydrogen peroxide, superoxide, lipid peroxidation and electrolyte
leakage
For estimation of H2O2, 100 mg fresh leaf tissue was homogenized in
5 mL of 0.1% trichloro acetic acid (TCA). After centrifuging the ho­
mogenate at 10,000 g for 10 min, 0.5 mL of supernatant was mixed with
equal amount of potassium phosphate buffer (pH 7.0) followed by
addition of 1 mL potassium iodide. Absorbance was recorded at 390 nm
(Velikova et al., 2000). Determination of superoxide was done following
Yang et al. (2011). Briefly, fresh 100 mg tissue was extracted in 65 mM
potassium phosphate buffer (pH 7.8) and homogenate was centrifuged
at 5000 g for 10 min. Supernatant was reacted with hydroxylamine
hydrochloride (10 mM) and left for 20 min followed by the addition of
sulfanilamide and naphthylamine. After incubation for 20 min at 25 ◦ C,
absorbance was taken at 530 nm and standard curve of NaNO2 was used
for calculation.
Lipid peroxidation was measured interms of content of malonalde­
hyde (MDA) formation and was determined in accordance of Heath and
Packer’s (1968) method. Briefly, fresh tissue was extracted in 1% TCA
and extract was centrifuged at 10,000 g for 5 min. Supernatant was
reacted with 0.5% thiobarbituric acid at 95 ◦ C for 30 min. After cooling
samples were centrifuged again at 5000 g and optical density of super­
natant was measured at 532 and 600 nm. For measurement of electro­
lyte leakage (EL) method of Dionisio-Sese and Tobita (1998) was
followed and calculations were done by following formulae:
ECb − ECa
EL(%) = X100
ECc
3
Ecotoxicology and Environmental Safety 216 (2021) 112195
2.7. Assay of lipoxygenase and protease activity
Activity of LOX (EC 1.13.11.12) was measured by following method
of Doderer et al. (1992) and linoleic acid was used as substrate. Change
in optical density was recorded at 234 nm and extinction coefficient of
25 mM− 1 cm− 1 was used for calculation. Protease (EC 3.4.21.40) ac­
tivity was assayed by method of Green and Neurath (1954). Briefly,
fresh plant tissue was macerated in cold 50 mM sodium potassium buffer
(pH 7.4) containing 1% PVP. After centrifuging the homogenate at
5000 g for 5 min, supernatant was incubated with casein at 40 ◦ C and
amount of tyrosine released was determined by reacting the mixture
with Folin Ciocalteu’s reagent in alkaline medium. Optical density was
recorded at 660 nm and activity was expressed as µg tyrosine released
mg− 1 protein (Green and Neurath, 1954).
2.8. Estimation of nitric oxide
Nitric oxide (NO) was measured following the method described by
Zhou et al. (2005). Fresh 500 mg tissue was extracted in cold 50 mM
acetic acid buffer (pH 3.6), containing 4% zinc diacetate. After centri­
fuging the homogenate at 11,500 g for 15 min, pellet was resuspended
in 1 mL extraction buffer and supernatants were pooled together.
Charcoal was added to neutralize the supernatant followed by vortexing
and filtration. Thereafter, 1 mL filtrate was mixed with 1 mL Greiss re­
agent and incubated for 30 min at room temperature. Absorbance was
taken at 540 nm and concentration of NO was calculated from standard
curve of sodium nitrite (NaNO2).
2.9. Determination of nitrate reductase
For measuring the activity of nitrate reductase (NR) fresh 300 mg
leaf tissue was incubated in 100 mM potassium phosphate buffer (pH
7.5) containing KNO3 (200 mM) and 0.5% n-propanol for 3 h in dark at
30 ◦ C. Thereafter, 1 mL aliquot was taken and sulphanilamide (1%) and
1-naphthylethylene diamine dihydrochloride (0.2%) were added.
Absorbance was recorded at 540 nm (Srivastava, 1974).
2.10. Activity of antioxidant enzymes and contents of ascorbate and
reduced glutathione
For extraction of antioxidant enzymes fresh 1.0 gm leaf tissue was
macerated in cold 100 mM phosphate buffer (pH 7.8) containing poly­
vinyl pyrolidine (1%), EDTA (1 mM) and phenylmethylsulfonyl fluoride
(0.1 mM) using prechilled pestle and mortar. Homogenate was centri­
fuged at 12,000 g for 15 min at 4 ◦ C and supernatant was used for
enzyme assay.
Assay of superoxide dismutase (SOD, EC 1.15.1.1) activity was car­
ried according to Bayer and Fridovich (1987) and optical density was
recorded at 560 nm. Catalase (EC 1.11.1.6) activity was measured using
method of Aebi (1984) and change in absorbance was recorded for 2 min
at 240 nm. Ascorbate peroxidase (APX, EC 1.11.1.11) was assayed ac­
cording to Nakano and Asada (1981). Activity of glutathione reductase
(GR; EC 1.6.4.2) was assayed according to Foyer and Halliwell (1976)
and change in absorbance at 340 nm was monitored for 2 min. Activity
of DHAR (EC: 1.8.5.1) was assayed in accordance of Nakano and Asada
(1981) and absorbance was taken at 265 nm for 2 min. MDHAR (EC:
1.6.5.4) was assayed following Hossain et al. (1984) and absorbance was
recorded at 340 nm for 2 min. Activities of antioxidant enzymes were
expressed as EU mg− 1 protein.
Methods described by Mukherjee and Choudhuri (1983) and Ellman
(1959) were employed for estimation of ascorbate (AsA) and reduced
glutathione (GSH) content respectively. Standard curve of AsA and GSH
were used for calculation.
M.A. Ahanger et al. Ecotoxicology and Environmental Safety 216 (2021) 112195

2.11. Determination of total phenols, flavonoids and activity
phenylalanine ammonia lyase
Content of total phenols was estimated according to Malick and
Singh (1980). Briefly, dry powdered sample was extracted in 80%
ethanol and extract was subjected to centrifugation for 10 min at 10,
000 g. 100 µL supernatant was made to 2 mL followed by addition of 1 N
Folin–Ciocalteu reagent and 1 mL Na2CO3. Optical density was taken at
650 nm and standard curve of catechol was used for calculation. Fla­
vonoids were extracted in methanol and homogenate was centrifuged at
10,000 g for 10 min 1 mL supernatant was reacted with 5% NaNO2 and
10% AlCl3. After 5 min, NaOH (2 mL) was added and optical density was
recorded at 510 nm (Zhishen et al., 1999). Calculation was done using
standard curve of catechin. For assaying the activity of PAL, method of
Zucker (1965) was followed and formation of trans-cinnamic acid was
measured at 290 nm.
2.12. Estimation of N, Na, K and P
Nitrogen was determined by modified micro- Kjeldahl method
(Jackson, 1973; Iswaran and Marwaha, 1980). Potassium and sodium
were estimated using flame photometer connected with continuous flow
systems (microflow automated continuous- flow analyzer III, Italy). The
Phosphorus was estimated using vanado-molybdophosphoric colori­
metric method.
amelioration of 21.1% and 94.9% for N, 12.0% and 87.5% for P, 15.2%
and 74.6% for K and 16.2% and 90.0% for Na was observed due to NV1
and NV2 respectively. However under normal conditions application of
nano compost increased N, P, K and Na maximally by 60.5%, 36.1%,
64.3% and 6.6% at 100 mg kg− 1 soil (Table 1).
3.3. Content of pigments and photosynthesis in drought stressed and
compost treated tomato
Drought stress resulted in significant reduction in the synthesis of
total chlorophylls and carotenoids, photosynthesis and PSII functioning,
however application of nanocompost ameliorated decline in pigments
and photosynthesis considerably (Figs. 1 and 2). Relative to control,
δ-ALA (27.4%), total chlorophylls (69.0%), carotenoids (36.5%),
photosynthesis (Pn, 42.0%), intercellular CO2 concentrations (Ci,
42.00%) and transpiration rate (E, 54.1%) increased significantly due to
NV2. Drought resulted in 27.4% decline in δ-ALA, 38.1% in total chlo­
rophylls, 31.2% in carotenoids, 37.77% in Pn, 37.8% in Ci and 41.61% in
E over control, however D + NV2 resulted in amelioration of the decline
maximally (Fig. 2). Drought significantly declined PSII activity (28.8%)
and qP (20.7%) while as increased NPQ (50.1%) over control. Maximal
amelioration of the decline in PSII activity, qP and NPQ was observed in
D + NV2 (Fig. 2).
3.4. Nano fertilizer reduced the oxidative stress

2.13. Statistical analysis
Data presented is mean (±SE) of four replicates. Duncan’s Multiple
Range Test was performed to test the significance of data and least
significant difference (LSD) was calculated at p < 0.05.
3. Results
3.1. Height and dry weight increased due to organic fertilizer
Relative to control, height and dry weight increased due to organic
fertilizer treatment attaining maximal increment of 44.7% and 61.6%
due to 100 mg kg− 1 soil (NV2). Drought reduced height (34.2%) and dry
weight (62.0%) considerably, however seedlings grown with D + NV2
exhibited maximal amelioration of 73.1% and 109.5% over the drought
stressed plants (Table 1).
3.2. Nano organic fertilizer mitigated the decline in uptake of mineral
elements
Drought resulted in increased accumulation of free radicals like H2O2
and O-2 as compared to control and compost supplemented counterparts.
Increase in H2O2 and O-2 due to drought was 130.7% and 112.5%
respectively resulting in 119.4% and 222.9% increase in lipid peroxi­
dation and electrolyte leakage over the control. However, NV2 supple­
mentation maximally declined the generation of H2O2 and O-2 by 1.5 and
1.7 fold respectively imparting a decline of 1.76 and 1.45 fold in lipid
peroxidation and electrolyte leakage. In D + NV2 treated plants, decline
in the accumulation of H2O2 and O-2 was maximal of 60.9% and 49.8%
resulting in amelioration of 56.8% in lipid peroxidation and 64.7% in
electrolyte leakage over the drought stressed plants (Table 1).
Activities of protease and lipoxygenase increased by 55.3% and
55.4% due to drought over the control plants. Decline in protease and
lypoxygenase was 1.3 and 1.5 folds in NV2 treated plants and this effect
was maintained even under drought conditions. In D + NV2 treated
plants, protease and lypoxygenase decreased by 37.7% and 60.7%
respectively over the drought stress plants (Fig. 3).
3.5. Content of osmolytes increased due to nano-compost treatment

Drought induced effect on the content of mineral elements is shown Content of proline, sugars, free amino acids and glycine betaine
in Table 1. Drought reduced content of N, P, K and Na by 34.5%, 24.2%, exhibited an increased in drought stressed tomato seedlings, however
27.6% and 47.3% respectively over the control, however an the increase was more conspicuous in nano organic fertilizer treated

Table 1
Shoot length, dry weight, nitrogen, phosphorous, potassium and sodium content in Solanum lycopersicum L. cultivar Huange 108 subjected to drought stress and organic
fertilizer supplementation.
Control NV1 NV2 Drought Drought + NV1 Drought + NV2

Shoot length (cm) 30.67 ± 2.4c 33.78 ± 2.6b 44.38 ± 3.7a 20.17 ± 2.1e 25.39 ± 2.5d 34.92 ± 2.9b
Shoot dry weight (g plant− 1) 4.732 ± 0.34c 4.809 ± 0.38b 7.649 ± 0.67a 1.796 ± 0.16e 2.339 ± 0.22d 4.901 ± 0.31b
Nitrogen (mg g− 1 DW) 17.15 ± 1.1c 19.20 ± 1.4b 27.53 ± 2.3a 11.22 ± 0.67e 13.59 ± 0.55d 21.87 ± 2.01b
Phosphorous (mg g− 1 DW) 8.25 ± 0.45c 8.33 ± 0.33b 11.23 ± 0.66a 6.25 ± 0.44d 8.33 ± 0.46b 11.23 ± 0.55a
Potassium (mg g− 1 DW) 18.14 ± 1.09c 21.39 ± 1.2b 29.82 ± 2.8a 13.12 ± 0.87e 15.12 ± 0.98d 22.92 ± 2.02b
Sodium (mg g− 1 DW) 2.11 ± 0.093b 2.16 ± 0.088b 2.25 ± 0.078a 1.11 ± 0.067c 1.29 ± 0.032c 2.11 ± 0.063b
Superoxide (nmol g− 1 FW) 12.28 ± 0.47c 10.02 ± 0.44d 6.85 ± 0.24e 26.1 ± 1.92a 21.2 ± 1.88b 13.08 ± 0.55c
Hydrogen peroxide (nmol g− 1 FW) 21.30 ± 1.1c 17.19 ± 1.09d 13.46 ± 0.66e 49.14 ± 3.7a 35.31 ± 2.8b 19.20 ± 1.7c
Lipid peroxidation (nmol MDA g− 1 FW) 17.00 ± 1.2c 15.61 ± 1.0d 9.65 ± 0.78e 37.31 ± 2.7a 29.38 ± 2.1b 16.11 ± 1.01c
Electrolyte leakage (percent) 14.11 ± 0.98c 13.05 ± 0.78d 9.69 ± 0.67e 45.57 ± 3.1a 37.43 ± 2.6b 16.06 ± 1.3c

Note: Mean (±SE) values of four replicates have been presented and mean values followed by different letters denote significant difference at P < 0.05 according to
Duncun’s multiple range test.

4
M.A. Ahanger et al.
Fig. 1. Content of (A) δ-amino levulinic acid, (B) total chlorophylls and (C)
carotenoids in Solanum lycopersicum L. cultivar Huange 108 subjected to
drought stress and organic fertilizer supplementation. Mean (±SE) values of
four replicates have been presented and different letters denote significant
difference at P < 0.05 according to Duncan’s multiple range test.
seedlings. Relative to control, proline, sugars, free amino acids and
glycine betaine increased by 66.6%, 69.5%, 37.0% and 84.0% due to
NV2 and by 146.3%, 115.2%, 98.7% and 140.8% due to drought stress.
Maximum percent increase of 229.16%, 165.0%, 142.4% and 219.7%
respectively for proline, sugars, free amino acids and glycine betaine
was observed in D + NV2 over the control (Table 2).
3.6. Activity of NR and NO concentration in drought and nano fertilizer
treated plants
Supplementation of nano compost significantly increased the activity
of NR with effect being much obvious due to higher (100 mg kg− 1 soil)
dose. Relative to control, increase of 52.7% was observed due to NV2
and a decline of 43.9% due to drought. Decline in activity of NR was
ameliorated by 21.3% and 49.4% due to NV1 and NV2 over the drought
stressed plants (Table 2). Drought stress resulted in increased (274.1%)
accumulation of NO over control however, supplementation of nano-
organic fertilizer resulted in significant decline as compared to
drought stressed counterparts. Relative to control, under normal con­
ditions NO increased by 30.1% and 54.7% due to NV1 and NV2
respectively (Table 2).
3.7. Nanocompost supplementation up-regulates the antioxidant system
Application of nano-vermicompost resulted in significant enhance­
ment in the functioning of antioxidant system by up-regulating the ac­
tivities of SOD, CAT, APX, MDHAR, DHAR, GR and the content of non-
5
Ecotoxicology and Environmental Safety 216 (2021) 112195
enzymatic components including AsA and GSH (Fig. 4). Relative to
control, NV2 application increased SOD by 73.1%, CAT by 16.3%, APX
by 81.8%, GR by 68.4%, MDHAR by 8.4%, DHAR by 75.7%, AsA by
17.1%, and GSH by 11.6%. Drought significantly increased the activities
of antioxidant enzymes assayed and the content of AsA and GSH with
maximal increase of 4.0 fold for SOD, 1.9 fold for CAT, 2.0 fold for APX,
2.2 fold for GR, 1.8 fold for MDHAR, 2.1 fold for DHAR, 1.3 fold for AsA
and 1.3 fold for GSH exhibited in D + NV2 treated seedlings (Fig. 4).
3.8. Effect of nano-compost on the content of total phenols and
flavonoids, and activity of PAL
Supplementation of nano-compost increased the content of total
phenols, flavonoids and PAL activity under normal as well as drought
conditions. Drought triggered the synthesis of total phenols (17.2%),
flavonoids (47.0%) and PAL activity (21.3%) over the control. Maximal
increase of 66.9% for total phenols, 112.9% for flavonoids and 132.4%
for PAL was observed in D + NV2 treated plants over control. Under
normal conditions, NV2 application increased total phenol, flavonoids
and PAL activity by 48.8%, 52.5% and 58.9% respectively (Fig. 5).
4. Discussion
Drought has proved to be one of most damaging environmental
factor restricting the growth and productivity of most crop plants.
Several management strategies have been introduced for preventing the
damage but could not succeed. In this backdrop the application of
organic fertilizer (nano-vermicompost) was tested in preventing the
drought induced growth reduction in tomato. Drought mediated growth
decline interms of height and dry weight was significantly mitigated due
to the application of vermicompost more conspicuously at 100 mg kg− 1
soil. Drought stress reduces cellular division, histone kinase activity and
restricts cell cycle transition (Schuppler et al., 1998) thereby leading to
considerable reduction in cell production. Earlier decline in plant height
and dry weight due to drought has been reported by Jatav et al. (2014)
and Ahanger and Agarwal (2017). Nano-vermicompost treated seedlings
exhibited significant enhancement in growth and biomass production
which can be attributed to maintenance of greater tissue water for
maintenance of enzyme functioning, photosynthesis and mineral up­
take. Increased growth interms of length and biomass accumulation due
to compost treatment may be ascribed to increased water use efficiency
and photosynthesis as reported by Xu and Mou (2016). Recently, Zhang
et al. (2020) have demonstrated increased growth and biomass pro­
duction in cotton due to organic fertilizer through modification of root
attributes like length, volume and surface area. Increased growth due to
vermicompost has been attributed to its several beneficial attributes like
(a) presence of high mineral content, (b) high water content, and (c)
presence of beneficial microbes (Usmani et al., 2019). Vermicompost
supplementation favors plant growth by reducing soil acidity (Mahmud
et al., 2020). Akhzari and Pessarakli (2017) have also demonstrated
increased growth, biomass accumulation and chlorophyll content in
Vetiveria Zizanioides due to vermicompost under water stress. Beneficial
impact of nano-materials like nano-biochar in enhancing the growth,
chlorophyll and root functioning has been reported in rice (Yue et al.,
2019) under heavy metal stress, however the use of nano-vermicompost
has not been explored. In drought stressed Cicer arietinum, Hosseinzadeh
et al. (2016) has demonstrated significant amelioration of the decline in
total chlorophylls, Pn, Ci, E and Fv/Fm due to vermicompost application
reflecting in significant modulation of both stomatal and non-stomatal
photosynthetic parameters. In present study it was also demonstrated
that nano-compost (organic fertilizer) proved potentially effective in
enhancing the photosynthetic capacity of tomato seedlings under
drought. Increased photosynthesis exhibited as higher Fv/Fm, qP and
ETR in nano-compost treated seedlings can be attributed to direct in­
fluence on the production of ROS like superoxide which are accumulated
to damaging levels under stress (Mamnabi et al., 2020). Nanomaterials
M.A. Ahanger et al.
Fig. 2. (A) Photosynthesis, (B) intercellular CO2 concentration, (C) transpiration rate, (D) Fv/Fm, (E) photochemical quenching (qP) and (F) non-photochemical
quenching (NPQ) in Solanum lycopersicum L. cultivar Huange 108 subjected to drought stress and organic fertilizer supplementation. Mean (±SE) values of four
replicates have been presented and different letters denote significant difference at P < 0.05 according to Duncan’s multiple range test.
having key functional properties can improve solar energy harvest
thereby facilitating chloroplast carbon capture, solar energy harnessing
and electron transport, moreover can reduce the ROS within chloroplast
and influence the sensing process (Khatri and Rathore, 2018). In present
study as well nano-vermicompost may have enhanced the capacity of
tomato to interact and optimize the light usage thereby contributing to
increased productivity and also improving the tolerance potential which
has been postulated by Swift et al. (2018) as well. Baghbani-Arani et al.
(2017) has also demonstrated increased chlorophyll content and PSII
functioning in water stressed fenugreek grown on vermicompost
amended soil. Drought mediated reduction in Ci due to stomatal closure
results in declined Pn, and together with this reduced pigment content,
Rubisco synthesis and activity and reduced transport electrons to PSII
result in photoinhibition (Pagter et al., 2005). Drought declines the
synthesis of chlorophyll intermediates including δ-ALA (Dalal and Tri­
pathy, 2012), however application of nano-compost was affective in
improving content of δ-ALA ultimately chlorophyll and photosynthetic
functioning in tomato and also proved beneficial in averting the drought
mediated decline. However the actual mechanisms are largely unknown
making further studies a need.
Increased growth and chlorophyll synthesis in compost treated
seedlings can be due to presence of key minerals like N, P, K, Mg and Ca
(Mathivanan et al., 2013) regulating key enzyme functions and the
synthesis of molecules like chlorophyll. Improved N and K contents
observed in compost treated seedlings under normal and drought con­
ditions may contribute significantly in strengthening of tolerance
mechanisms by improving enzyme activity, up-regulating ROS
6
Ecotoxicology and Environmental Safety 216 (2021) 112195
scavenging system and enhance tissue water content through enhanced
osmolyte synthesis (Ahanger and Agarwal, 2017; Ahanger et al., 2019a).
Azarmi et al. (2008) has also demonstrated improved growth and yield
in tomato treated with vermicompost through increased uptake of P, K,
Fe, Zn, Mn and Cu. Vermicompost improves soil properties like nutrient
level, organic matter and cation exchange capacity as well as water
holding capacity (Xu and Mou, 2016) which could have contributed
significantly to tomato growth to counteract the drought mediated
damage.
Drought resulted in considerable oxidative damage through
enhancement in the ROS accumulation, lipid peroxidation and electro­
lyte leakage. Similar to our results increased oxidative damage due to
drought has been reported by several workers (Ahmed et al., 2013;
Ahanger and Agarwal, 2017; Mamnabi et al., 2020) however influence
of applied nano-vermicompost on the mitigation of oxidative damage
has not been reported. Recently addition of vermicompost has been
reported to reduce the oxidative damage by declining lipid peroxidation
and increasing membrane stability in spring rapeseed (Mamnabi et al.,
2020). Enhanced ROS accumulation lead lipid peroxidation is accom­
panied by increased lypoxygenase and protease activity which results in
greater damage to lipids and proteins therefore structural and functional
alteration of major macromolecules including proteins and fatty acids is
triggered (Nahar et al., 2016). Nano-materials mediated decline in
oxidative effects of ROS is the result of their cumulative effect on anti­
oxidant metabolites like phenols, flavonoids, osmolytes like proline etc,
and antioxidant enzymes (Sadak and Bakry, 2020; Hussain et al., 2020).
Drought stress results in significant alteration in the lipid and fatty acid
M.A. Ahanger et al.
Fig. 3. Activity of (A) protease and (B) lipoxygenase in Solanum lycopersicum L.
cultivar Huange 108 subjected to drought stress and organic fertilizer supple­
mentation. Mean (±SE) values of four replicates have been presented and
different letters denote significant difference at P < 0.05 according to Duncan’s
multiple range test.
profile of plants (Sánchez-Martín et al., 2018). In addition drought stress
reduces the activity of enzymes involved in lipid synthesis concomitant
with increase in the ones involved in degradation (Gigon et al., 2004). In
present study also we observed increased protease and lipoxygenase
activity due to drought which was significantly reduced due to
nano-compost supplementation depicting the beneficial involvement in
protecting the proteins and lipids in tomato under such conditions.
Reduced oxidative damage observed in compost supplemented
seedlings was accompanied by significant up-regulation of the antioxi­
dant system. Stresses result in up-regulation of antioxidant system to
counter the ROS triggered damage (Ahmad et al., 2010; Ahanger et al.,
2017). Increased activity of antioxidant system due to drought stress has
been reported by other workers as well (Ahmed et al., 2013; Yang et al.,
2014; Ahanger and Agarwal, 2017; Begum et al., 2019, 2020). Increased
activity of antioxidant enzymes has been reported to prevent drought
mediated ROS triggered decline in photosynthesis by improving mem­
brane stability through quick elimination of ROS (Yang et al., 2014).
SOD forms first line of defense against toxic radicals eliminating su­
peroxide from cells thereby reducing the damage to metabolic pathways
like photosynthesis (Choudhary et al., 2017; Ahanger et al., 2017,
2019b). Increased SOD and CAT activity due to vermicompost supple­
mentation under water stress has been reported by Mamnabi et al.
Table 2
Content of proline, sugars, free amino acids and glycine betaine, relative water content (RWC), nitric oxide and activity of nitrate reductase in Solanum lycopersicum L.
cultivar Huange 108 subjected to drought stress and organic fertilizer supplementation.
Control
Proline (µmol g− 1 DW) 21.12 ± 1.7e
Sugars (mg g− 1 DW) 2.23 ± 0.21e
Free amino acids (mg g− 1 DW) 3.89 ± 0.42e
Glycine betaine µg g− 1 DW) 2.13 ± 0.17e
RWC (percent) 83.89 ± 5.5b
Nitric oxide content (µmol g− 1 FW) 0.232 ± 0.011 f
Nitrate reductase activity (µmol NO-2 released g− 1
FW) 1.46 ± 0.12c
Note: Mean (±SE) values of four replicates have been presented and mean values followed by different letters denote significant difference at P < 0.05 according to
Duncun’s multiple range test.
Ecotoxicology and Environmental Safety 216 (2021) 112195
(2020). Besides this CAT, APX, DHAR, MDHAR and GR form key anti­
oxidant enzymes involved in neutralizing the H2O2. However reports
discussing the effect of nano-vermicompost on antioxidant functioning
are not available. Nano-materials significantly improve antioxidant
enzyme activities reflecting in reduced accumulation of ROS and hence
greater protection to growth, photosynthesis and key metabolic path­
ways is attained (Sharma et al., 2019), however the impact may vary
considerably with concentration of nano-material used. Neutralization
of O-2 and H2O2 prevents the formation of more toxic radicals like OH
thereby stabilizing the membrane and cellular functioning (Ahmad
et al., 2010). CAT neutralises H2O2 in cytosol while as APX, DHAR,
MDHAR, GR, AsA and GSH work co-ordinately AsA-GSH cycle to elim­
inate H2O2 from mitochondria and chloroplast (Ahanger et al., 2017).
Increased AsA-GSH cycle prevents photoinhibition by maintaining the
NADP+/NADPH ratio so that ETR gets least affected (Ahmad et al.,
2018). Recently Ahmad et al. (2020) have also demonstrated significant
up-regulation of the antioxidant functioning in due to application of ZnO
nano-particles resulting in growth and photosynthetic regulation under
arsenic stress. Reports discussing the role of nano-organic fertilizer
application on the antioxidant system functioning are rare. Greater
availability of ions like Fe, Zn, Cu and Mn in vermicompost (Najafi-Ghiri
et al., 2019) may be the possible reason for increased antioxidant ac­
tivity as they act as co-factors for several enzymes like SOD. Mahmud
et al. (2019) have reported increased radical scavenging potential in
pine apple grown with organic fertilizer supplementation which has
been attributed to greater contents of AsA and polyphenols.
Up-regulation of the antioxidant system due to nano-fertilizer supple­
mentation resulted in amelioration of the drought mediated decline in
growth and photosynthesis significantly, further studies are required to
unravel the exact mechanism.
Antioxidant system in vermicompost supplemented seedlings was
further strengthened by increased synthesis of phenols and flavonoids in
them. Both phenols and flavonoids have the tendency to prevent ROS
mediated lipid peroxidation and maintain fluidity and functioning of
membranes by protruding into the lipid bilayer (Oteiza et al., 2005).
Besides they also assist in ROS scavenging by donating electrons to
guaiacol peroxidases and polyphenol oxidases (Ahanger et al., 2018). It
is believed that significant accumulation of phenolics, phytoalexins and
the activation enzymes like peroxidases as well as the enzymes regu­
lating phenylpropanoid and isoflavonoid biosynthesis crucially regulate
stress tolerance (Cherif et al., 2007). Recently vermicompost mediated
up-regulation of antioxidant activity has been reported in terms of
DPPH, ABTS and FRAP assays in Ananas comosus (Mahmud et al., 2020).
Increased PAL activity results in enhanced synthesis of polyphenols and
lignin precursors (Lee et al., 2007). Up-regulation of antioxidant system
and increased contents of secondary metabolites in nano-vermicompost
treated seedlings protects photosynthesis by optimizing the redox buffer
components for efficient electron transport. Further increased synthesis
of osmolytes including proline, sugars, free amino acids and glycine
betaine due to organic fertilizer was conspicuous which may have
contributed significantly to maintenance of tissue water content. Similar
to our results increased osmolyte accumulation due to drought has been
NV1 NV2 Drought Drought + NV1 Drought + NV2
23.02 ± 1.9e 35.19 ± 2.6d 52.02 ± 4.1c 57.45 ± 4.5b 69.52 ± 4.8a
2.29 ± 0.18e 3.78 ± 0.18d 4.80 ± 0.22c 5.29 ± 0.28b 5.91 ± 0.29a
4.01 ± 0.39e 5.33 ± 0.41d 7.63 ± 0.58c 7.98 ± 0.56b 9.43 ± 0.67a
2.19 ± 0.19e 3.92 ± 0.21d 5.13 ± 0.37c 5.43 ± 0.34b 6.81 ± 0.55a
84.5 ± 5.4b 89.62 ± 5.7a 55.56 ± 3.4e 59.89 ± 4.1d 69.83 ± 4.5c
0.302 ± 0.020e 0.359 ± 0.021d 0.868 ± 0.034a 0.419 ± 0.035c 0.459 ± 0.033b
1.54 ± 0.19b 2.23 ± 0.20a 0.819 ± 0.43e 1.041 ± 0.16d 1.619 ± 0.18b
7
M.A. Ahanger et al. Ecotoxicology and Environmental Safety 216 (2021) 112195

Fig. 4. Activity of (A) superoxide dismutase and (B) catalase (C) ascorbate peroxidase, (D) dehydroascorbate reductase, (E) monodehydro-ascorbate reductase, (F)
glutathione reductase and content of (E) ascorbate and (F) reduced glutathione activity in Solanum lycopersicum L. cultivar Huange 108 subjected to drought stress
and organic fertilizer supplementation. Mean (±SE) values of four replicates have been presented and different letters denote significant difference at P < 0.05
according to Duncan’s multiple range test.

reported by Pirzad et al. (2011), Jatav et al. (2014), Ahanger and
Agarwal (2017) in different crops. Protective role of osmolytes under
drought results mostly due to their ability to maintain water potential
gradient for continuous water uptake (Ahanger et al., 2014). Sufficient
accumulation of osmolytes protect major cellular structures like proteins
including Rubisco (Sivakumar et al., 2000), membranes etc (Ashraf and
Foolad, 2007). Greater accumulation of proline and sugar content under
drought due to supplementation of organic fertilizer has been demon­
strated by Salehi et al. (2016). Enhanced accumulation of compatible
osmolytes significantly influence the growth and yield performance of
crop plants therefore in present study organic fertilizer mediated in­
crease in osmolytes may contribute to better growth and yield produc­
tion of tomato. Recently application of nano-ZnO and compost to Linum
usitatissimum has been demonstrated to increase the accumulation of

8
M.A. Ahanger et al.
Fig. 5. Content of (A) total phenols and (B) flavonoids, and the activity of (C)
phenylalanine ammonia lyase in Solanum lycopersicum L. cultivar Huange 108
subjected to drought stress and organic fertilizer supplementation. Mean (±SE)
values of four replicates have been presented and different letters denote sig­
nificant difference at P < 0.05 according to Duncan’s multiple range test.
compatible osmolytes including proline, free amino acids and carbo­
hydrate thereby enhancing the growth and yield (Sadak and Bakry,
2020).
5. Conclusion
Conclusively, drought declined growth by reducing photosynthesis
through increased generation of toxic ROS. Drought induced oxidative
stress was ameliorated by the supplementation of nano-organic fertilizer
(nano-vermicompost) which was obvious as reduced ROS accumulation,
lipid peroxidation and enhanced membrane functioning. Up-regulation
of the antioxidant system, osmolytes and secondary metabolites due to
nano-vermicompost supplementation justifies its beneficial role in pre­
venting drought mediated damage to tomato.
CRediT authorship contribution statement
Mohammad Abass Ahanger, Lixin Zhang: Conceived and
designed. Mohammad Abass Ahanger, Maodong Qi, Ziguang Huang,
Xuedong Xu: Experimentation. Mohammad Abass Ahanger, Naheeda
Begum, Cheng Qin, Chenxi Zhang: Literature compilation. Moham­
mad Abass Ahanger, Maodong Qi, Naheeda Begum, Nadeem
Ahmad: Writing. Nabil S. Mustafa, Muhammad Ashraf, Lixin Zhang:
Critical reading. Mohammad Abass Ahanger, Lixin Zhang: Revision.
Lixin Zhang: Supervision, Funding acquisition.
9
Ecotoxicology and Environmental Safety 216 (2021) 112195
Declaration of Competing Interest
The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to influence
the work reported in this paper.
Acknowledgment
This study was funded by the National Key Research and Develop­
ment Program of 21 China [grant number 2017YFE0114000]. First
author is thankful to Northwest A&F University for Post Doctoral
Fellowship.
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