Industrial Crops and Products 94 (2016) 574–585

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Industrial Crops and Products

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

Vermicompost improves the physiological and biochemical responses

of blessed thistle (Silybum marianum Gaertn.) and peppermint
(Mentha haplocalyx Briq) to salinity stress
Li Xu a , Dong Yan a,b , Xuyang Ren a,b , Yiyang Wei a,b , Jing Zhou a , Haiyan Zhao a ,
Mingxiang Liang a,b,∗
a
College of Resources and Environmental Sciences, Nanjing Agricultural University, Jiangsu Province, People’s Republic of China
b
Jiangsu Key Lab of Marine Biology, Nanjing, People’s Republic of China

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

Article history: In this study, we examined whether vermicompost enhances a plant’s tolerance to salinity. We analyzed
Received 21 June 2016 the physiological responses of the aerial parts and roots of the herbal stress-resistant plants blessed
Received in revised form 7 September 2016 thistle and peppermint with NaCl and cow manure vermicompost and inorganic fertilizer. Salinity greatly
Accepted 9 September 2016
enhanced the accumulation of malondialdehyde (MDA) and proline in the aerial parts and roots of the
Available online 21 September 2016
two species, but did not affect chlorophyll content. The K+ /Na+ and Ca2+ /Na+ ratios and the total soluble
protein content were decreased in the aerial parts and roots under salinity conditions in both species.
Keywords:
Under normal conditions, vermicompost enhanced plant growth and increased the K+ /Na+ and Ca2+ /Na+
Blessed thistle
Peppermint
ratios and total soluble protein content while inorganic fertilizer treatment increased the total soluble
Vermicompost protein content and proline content in both species. Proline content in both species was greatly decreased
Salinity under vermicompost treatment than inorganic treatment under normal conditions. Furthermore, under
high salinity conditions, vermicompost treatment significantly reduced the MDA content and increased
total soluble protein content and the K+ /Na+ and Ca2+ /Na+ ratios than stress-treated plants. Vermicompost
had complex effects on the antioxidant enzyme activities of plants grown under high salinity treatment.
Our results show vermicompost mitigates the effects of salinity stress.
© 2016 Elsevier B.V. All rights reserved.

1. Introduction
Global warming and the widespread application of inorganic
fertilizers have greatly increased soil salinity. Approximately 20% of
the world’s agricultural fields are affected by salinity (Chinnusamy
et al., 2005). Salinity negatively impacts plant growth and delays
development, thus decreasing crop production. Whereas engineer-
ing practices to ameliorate saline soil are being developed, there
is also an interest in identifying and cultivating stress-resistant
plants, which have increased resistance to soil salinity. Stress-
resistant plants can be used as medicine, forage, or wood. For
instance, the flowering tops, leaves, and upper stems of the stress-
resistant plant blessed thistle (Silybum marianum Gaertn.) are used
in traditional medicines to stimulate appetite and relieve indi-
gestion and have antibacterial and antifungal activity. Peppermint
∗ Corresponding author at: College of Resources and Environmental Sciences,
Nanjing Agricultural University, Jiangsu Province, People’s Republic of China.
E-mail address: liangmx@njau.edu.cn (M. Liang).
(Mentha haplocalyx Briq), which has a high menthol content and is
used in the food and toothpaste industries, also tolerates high levels
of salt. Peppermint was shown to be a safe and effective treatment
for irritable bowel syndrome and has traditionally been used in folk
medicine and aromatherapy.
Malondialdehyde is considered to be an index of stress, includ-
ing salinity stress. Salinity induces plant oxidative stress by
increasing the production of reactive oxygen species (ROS), such
as superoxide radicals and hydrogen peroxide (H2 O2 ). To mitigate
ROS-mediated damage, plants have developed a complex defense
system that regulates the activity of antioxidative enzymes, scav-
enges ROS, and adjusts ionic and osmotic levels (Ashraf and Harris,
2004). Antioxidative enzymes such as superoxide dismutase (SOD),
peroxidase (POD), and catalase (CAT) alleviate the negative effects
of oxidative stress in plants (Alscher et al., 2002). Plants can greatly
minimize ionic toxicity by establishing ionic compartments within
cells or excreting ions. Furthermore, plants increase cellular levels
of somatically compatible solutes, such as proline, to maintain ionic
homeostasis and stabilize proteins in response to saline conditions.
Plants differ greatly in their ability to withstand salt stress and their

http://dx.doi.org/10.1016/j.indcrop.2016.09.023
0926-6690/© 2016 Elsevier B.V. All rights reserved.
 L. Xu et al. / Industrial Crops and Products 94 (2016) 574–585 575

internal mechanisms have evolved to adapt to the external envi- Table 1

Experimental design.
ronment. Although blessed thistle and peppermint are known to be
tolerant to salinity stress, the underlying mechanism that regulates Treatment NaCl Vermicompost NPK
the salinity response in these species has not been elucidated. CK / / /
Interestingly, blessed thistle and peppermint are only salt toler- T1 1‰ / /
ant when grown in soil that contains sufficient levels of nutrients T2 1‰ 5% /
and microorganisms. Earthworms consume animal waste and crop T3 1‰ 15% /
T4 4‰ / /
residues to produce vermicompost, which improves the nutrient
T5 4‰ 5% /
content and physicochemical and microbiological properties of the T6 4‰ 15% /
soil. Much evidence shows that vermicompost positively regulates T7 / 5% /
the growth and productivity of plants (Ibrahim et al., 2015; Lim T8 / 15% /
T9 / / 5%
et al., 2015; Oo et al., 2015), partly by increasing the electrical con-
T10 / / 15%
ductivity, overall microbial activity, and nitrate concentrations of
the soil. Few reports have addressed whether vermicompost pro-
motes the growth of halophytes under saline conditions and, if iments (i.e., young seedlings were first grown for two months in
so, how vermicompost enhances the plant’s tolerance to salinity. sand and watered with Hoagland’s solution and then transferred to
Furthermore, vermicompost has multiple effects on plant growth, pots), with the exception of the final salinity or vermicompost treat-
and it is unclear how a single factor from vermicompost, such as ment. The final treatments were as follows: plant potting media
nutrient value, affects plant growth. (0.5 kg vermiculite and 0.5 kg peat, control (CK)), plant potting
In this study, we probed the salt tolerance mechanism in blessed media plus 1‰ NaCl (treatment 1 (T1), 1 g NaCl in one pot), plant
thistle and peppermint by examining the growth of these plants potting media plus 5% vermicompost and 1‰ NaCl (T2, 1 g NaCl plus
in containers of horticultural media supplemented with differ- 50 g vermicompost in one pot), plant potting media plus 15% vermi-
ent amounts of vermicompost, NaCl, and inorganic fertilizer. In compost and 1‰ NaCl (T3), plant potting media plus 4‰ NaCl (T4),
addition to evaluating the plant’s physiological and biochemical plant potting media plus 5% vermicompost and 4‰ NaCl (T5), plant
responses to the different media, we examined whether vermi- potting media plus 15% vermicompost and 4‰ NaCl (T6), plant pot-
compost alleviated the effects of salinity stress in blessed thistle ting media plus 5% vermicompost (T7), plant potting media plus
and peppermint. 15% vermicompost (T8), plant potting media plus 5% inorganic fer-
tilizer (T9), and plant potting media plus 15% inorganic fertilizer
(T10, Table 1). Inorganic fertilizer (mixture of urea (N, 46.4%) and
2. Materials and methods calcium superphosphate (P2 O5 , 12%) and potassic fertilizer (K2 O,
56%)) was chemical fertilizer that had the same amount of available
2.1. Plant materials and growth conditions N, P, and K as did the vermicompost. Thus, 5% inorganic fertilizer
had the same amount of available N, P, and K as 5% vermicom-
Blessed thistle (Silybum marianum Gaertn.) and peppermint post. Five plants were grown per pot and four pots (replication)
(Mentha haplocalyx Briq) seeds were purchased from Wangtong were used. Plants were treated in different pots for 2 weeks under
Horticultural Company (Nanjing, China). Cow manure vermicom- a daily cycle of 16 h of light at 25 ◦ C and 8 h of darkness at 18 ◦ C in
post was kindly provided by the Nanjing Institute of Vegetable the greenhouse and watered with 500 ml every week (no leachate).
Science. The vermicompost had the following characteristics:
organic matter, 29.9%; total N, 2.47%; available N, 1.96 g kg−1 ;
2.2. Malondialdehyde content measurement
total phosphates 2.5%; available phosphates (extracted with 0.5 M
NaHCO3 (Page et al., 1982)), 8.7 g kg−1 ; total potassium 0.48%; avail-
Malondialdehyde (MDA) content was measured according to
able potassium (extracted with 1.0 M NH4 OAc (Rowell, 1994)),
(Lv et al., 2011). Approximately 0.1 g of tissue (aerial parts
2.0 g kg−1 ; pH (CaCl2 ), 6.6; and salt, 4.1 g kg−1 . The peat had
or roots) was used and absorbance values at 450, 532, and
the following characteristics: organic matter, 19.0%; total N,
600 nm were determined with a spectrophotometer. The concen-
0.45%; available N, 0.012 g kg−1 ; total phosphates, 0.016%; avail-
tration of MDA was calculated using the following formula: C
able phosphates (extracted with 0.5 M NaHCO3 (Page et al., 1982)),
(␮mol/L) = 6.45 × (OD532 − OD600 ) − 0.56 × OD450 .
0.55 g kg−1 ; pH (CaCl2 ), 5.6; and salt, 1.0 g kg−1 .
For cow manure vermicompost dosage experiments, seeds were
surface-sterilized with 10% H2 O2 solution for 30 min to soften the 2.3. Chlorophyll content measurement
coat and then rinsed with sterilized water. Seeds were transferred
to moist gauze and germinated for 5 days under a daily cycle of The chlorophyll content was measured according to a previ-
16 h of light at 25 ◦ C and 8 h of darkness at 18 ◦ C in the green- ously described method (Lichtenthaler, 1987). Briefly, leaf tissue
house. Young seedlings were placed in sand and watered with was frozen and ground in liquid nitrogen, and 1 ml of 100%
Hoagland’s solution every 5 days in the greenhouse. After two acetone was added to extract the chlorophyll. The concentra-
months, seedlings of uniform length were transferred to pots in the tions of chlorophyll a (Chl a), chlorophyll b (Chl b), and total
same greenhouse and subjected to various treatments. Plant pot- chlorophyll (T-Chl) were calculated using the following equations:
ting media consisted of a mixture of 0.5 kg vermiculite and 0.5 kg Chl a = 11.24 × OD661.6-2.04 × OD644.8; Chl b = 21.13 × OD644.8-
peat with 5% (50 g), 10% (100 g), 15% (150 g), or 20% (200 g) vermi- 4.19 × OD661.6; and tChl = 18.09 × OD644.8+ 7.05 × OD661.6.
compost (by dry weight). Five plants were grown per pot and four
pots (replication) were used. Plants were watered every week with 2.4. Ion content measurement
500 ml deionized water for 40 days (no leachate) and harvested to
measure the fresh weight. To determine the dry weight, fresh plants Ion content was measured according to a previously described
were dried for an initial 10 min at 100 ◦ C and then kept at 65 ◦ C until method (Huang and Schulte, 1985). Plant tissue samples (aerial
the weight of the plants was constant. parts or roots) were dried at 65 ◦ C for 6 d, weighed, and ground in
For salinity and vermicompost treatments, seeds and seedlings a grinder to pass through a 40 mesh screen. Samples were digested
were prepared as indicated for the vermicompost dosage exper- in hydrogen peroxide (H2 O2 )/nitric acid (HNO3 ) and analyzed by
576 L. Xu et al. / Industrial Crops and Products 94 (2016) 574–585

12 2.5
A a B
a
Fresh Weight of Aerial Parts (g) 10 a a
2.0

Fresh Weight of Roots (g)

ab ab ab
ab abcd abcd
bc
8 bc cd bcd abc
1.5 abcd
cd
6 c c c d
d
1.0

.5
2

0 0.0

5 .8 a
C a a D
bc ab
Fresh Weight of Aerial Parts (g)

ab ab a
4 ab ab

Fresh Weight of Roots (g)

cd cd bcd .6 bc abc
bc
cd de cd d
3
e e
.4

.2
1

0 0.0
CK T1 T2 T3 T4 T5 T6 T7 T8 T9 T10 CK T1 T2 T3 T4 T5 T6 T7 T8 T9 T10

Fig. 1. The effects of various treatments on the fresh weight of the aerial and root parts of blessed thistle and peppermint (A-B: blessed thistle; C-D: peppermint).

inductively coupled emission spectroscopy (ICP-OES) at the Nan- among treatments. Figures were created using SigmaPlot 10.0 (Sys-
jing Agricultural University Life Science Analysis Laboratory. tat Software, Inc., Germany).

2.5. Soluble protein and proline content measurement 3. Results

The soluble protein and proline content of the aerial parts or 3.1. The effects of salinity and vermicompost on blessed thistle
roots were measured as described (Lowry Oh and Farr, 1951). Total and peppermint growth
soluble protein content was determined using bovine serum albu-
min as a standard. The proline content was measured according to Our preliminary dosage experiments demonstrated that vermi-
a previously described method (Bates, 1973). compost increased the fresh and dry weight of both blessed thistle
To determine antioxidant enzyme activities, 0.5 g of aerial or and peppermint over a 40-day period, with the increase being pro-
root tissue was homogenized in ice-cold 50 mM KPi (pH 7.0) con- portional to the amount of vermicompost added (up to 20%). A 5%
taining 0.1 mM EDTA, 1% (w/v) polyvinylpyrrolidone (PVP), and or 10% vermicompost treatment increased the fresh weight ∼4 fold
0.5% (v/v) Triton X-100 at 4 ◦ C. The homogenate was filtered and dry weight ∼2 fold of the aerial parts of blessed thistle and pep-
through four layers of gauze and centrifuged at 14,000g for 15 min permint when compared to the untreated control (Supplementary
at 4 ◦ C. The supernatant was collected to determine the antioxidant Fig. 1A, B). The fresh and dry weights of the aerial parts of seedlings
enzyme activities and stored at −80 ◦ C for additional analyses(Hu, subjected to a 15% or 20% vermicompost treatment were ∼5 fold
1970). (fresh weight) and ∼4 fold (dry weight) greater than the untreated
control, respectively. Vermicompost application increased the root
2.6. CAT activity measurement biomass of both species; a 5% and 10% vermicompost treatment
resulted in a ∼2 fold increase, while a 15% and 20% vermicompost
The CAT activity was assayed by measuring the initial H2 O2 - treatment resulted in a ∼4 fold increase (Supplementary Fig. 1C,
scavenging rate. The extinction coefficient for H2 O2 at 240 nm was D). Considering the cost associated with applying vermicompost to
40 mM−1 cm−1 . The SOD activity was assayed according to the fields, we next evaluated the effects of supplementation with low
method described by Kwak et al. (1995) using pyrogallol (Sigma) concentrations (i.e., 5% and 15%) of vermicompost.
as a substrate (Kwak et al., 1995). The molar extinction coefficient Both species were exposed to a range of concentrations of vermi-
of purpurogallin is 2.47 mM−1 cm−1 . compost, NaCl, and inorganic fertilizer for two weeks. The inorganic
fertilizer (NPK) had the same concentration of available nitrogen,
2.7. Statistical analysis phosphorus, and potassium as the vermicompost. All plants grown
in soil supplemented with vermicompost or NPK exhibited signif-
Two-way ANOVA using Dunnett’s t-test was performed using icant increases in the biomass of the aerial parts when compared
SPSS 19.0 (SPSS Corp., Chicago, IL, USA) to evaluate differences to the untreated controls (Fig. 1). Whereas treatment with 1‰ or
 L. Xu et al. / Industrial Crops and Products 94 (2016) 574–585 577

4‰ NaCl inhibited the growth of the aerial tissues of peppermint under high NaCl stress. The water content of the aerial plant parts
(Fig. 1C), it did not affect blessed thistle growth. The fresh weight and roots of blessed thistle was increased by the addition of vermi-
of the aerial and root parts of blessed thistle was unaffected by 1‰ compost, NPK, or 1‰ NaCl and vermicompost (Supplementary Fig.
or 4‰ NaCl, while vermicompost treatments increased the fresh 2A).
weight of the aerial and root parts of blessed thistle under 1‰ NaCl For peppermint, vermicompost and NPK treatments generally
stress, but did not affect the fresh weight of blessed thistle roots increased the fresh weight of the aerial plant parts, while NaCl treat-

3.0
MDA Content of Aerial Parts (umol/g)

1.4
A B a
a

MDA Content of Roots (umol/g)

2.5 1.2

ab b
1.0
2.0
bc c
c .8
1.5 cd
cde de de c c
c
def def .6 c
d c
1.0 ef e d
.4
d
.5
.2

0.0 0.0

1.0 1.2
C D
MDA Content of Aerial Parts (umol/g)

a a
MDA Content of Roots (umol/g)

a
b 1.0
.8
bc
bc .8
cd c
.6
cd cd b b b
cd
.6 c c
c cd
.4
d
cd cd
.4

.2 .2
e

0.0 0.0

CK T1 T2 T3 T4 T5 T6 T7 T8 T9 T10 CK T1 T2 T3 T4 T5 T6 T7 T8 T9 T10

Fig. 2. The effects of various treatments on the malondialdehyde (MDA) content of the aerial and root parts of blessed thistle and peppermint (A-B: blessed thistle; C-D:
peppermint).

600 700
B ab a
A
a
Total Chlorophyll content (μg/g)

600 abc abc abc ab bc

500 abc ab abc
abc abc
abc ab 500
bc
400 abc bc c
bc
400
300 c bc
300

200
200

100
100

0 0

CK T1 T2 T3 T4 T5 T6 T7 T8 T9 T10 CK T1 T2 T3 T4 T5 T6 T7 T8 T9 T10

Fig. 3. The effects of various treatments on the total chlorophyll content of the above-ground parts and roots of blessed thistle and peppermint (A-B: blessed thistle; C-D:
peppermint).
578 L. Xu et al. / Industrial Crops and Products 94 (2016) 574–585

Table 2
The effects of various treatments on the ionic homeostasis of the aerial and root parts of blessed thistle and peppermint.

A. Ion content of the aerial parts of blessed thistle

Treatment Ca2+ (mg/g) K+ (mg/g) Na+ (mg/g) K+ /Na+ Ca2+ /Na+

CK 17.23 ± 1.03 cd 5.24 ± 0.55 ab 1.59 ± 0.49 d 3.29 10.82

T1 17.18 ± 1.97 d 3.75 ± 0.69 c 6.84 ± 0.51 b 0.55 2.51
T2 19.33 ± 5.66 a 4.26 ± 1.79 bc 4.30 ± 0.28 c 0.99 4.5
T3 18.4 ± 2.1 abcd 4.87 ± 0.42 ab 5.12 ± 1.11 c 0.95 3.6
T4 10.33 ± 1.67 e 1.97 ± 0.27 d 8.29 ± 0.87 ab 0.24 1.25
T5 17.04 ± 1.38 d 4.66 ± 0.53 ab 7.46 ± 0.33 ab 0.62 2.29
T6 17.77 ± 1.52bcd 3.68 ± 0.46 c 8.75 ± 1.53 a 0.42 2.03
T7 20.63 ± 0.59 ab 5.06 ± 0.18 ab 1.11 ± 0.12 d 4.55 18.55
T8 20.55 ± 1.49 abc 5.52 ± 0.72 a 1.08 ± 0.29 d 5.1 18.99
T9 18.6 ± 1.09 abcd 5.18 ± 0.55 ab 2.22 ± 0.64 d 2.34 8.38
T10 17.4 ± 1.96 abcd 4.92 ± 0.48 ab 1.73 ± 0.55 d 2.85 1.11

B. Ion content of blessed thistle roots

Treatment Ca2+ (mg/g) K+ (mg/g) Na+ (mg/g) K+ /Na+ Ca2+ /Na+

CK 10.84 ± 0.66 b 11.47 ± 3.07 cd 8.11 ± 1.04 ab 1.41 1.34

T1 9.78 ± 0.65 b 7.45 ± 0.87 e 9.87 ± 0.87 a 0.76 0.99
T2 10.80 ± 1.14 b 15.45 ± 3.45 a 6.10 ± 2.30 bc 2.53 1.77
T3 10.52 ± 0.54 b 11.59 ± 1.18bcd 5.19 ± 0.75 c 2.23 2.03
T4 8.68 ± 1.14 b 6.74 ± 2.55 e 10.93 ± 2.55 a 0.62 0.79
T5 9.5 ± 0.65 b 14.86 ± 2.20 ab 8.90 ± 1.67 ab 1.67 1.07
T6 10.93 ± 0.97 b 11.07 ± 0.70 cd 7.80 ± 1.13 ab 1.42 1.40
T7 8.77 ± 0.98 b 11.25 ± 0.99 cd 4.91 ± 0.48 c 2.29 1.79
T8 10.20 ± 0.29 b 14.13 ± 2.53abc 4.26 ± 0.62 cd 3.31 2.39
T9 29.35 ± 5.71 a 11.69 ± 2.03bcd 9.18 ± 1.98 a 1.27 3.2
T10 31.75 ± 7.5 a 10.09 ± 0.72 de 2.26 ± 0.65 d 4.46 14.03

C. Ion content of the aerial parts of peppermint

Treatment Ca2+ (mg/g) K+ (mg/g) Na+ (mg/g) K+ /Na+ Ca2+ /Na+

CK 15.75 ± 1.02 b 19.29 ± 3.29bcd 0.37 ± 0.08 de 42.58 52.13

T1 14.32 ± 2.12 cd 17.24 ± 2.50 cd 1.94 ± 0.33 a 7.39 8.89
T2 10.41 ± 0.53 c 16.74 ± 1.90 d 1.05 ± 0.34 b 9.91 15.94
T3 17.90 ± 1.39 ab 18.07 ± 1.80 cd 0.92 ± 0.28 bc 19.48 19.66
T4 6.68 ± 1.94 d 17.76 ± 2.98 cd 2.16 ± 0.58 a 3.09 8.20
T5 19.21 ± 1.53 a 23.15 ± 2.64 ab 1.23 ± 0.19 b 15.60 18.80
T6 19.48 ± 0.77 a 23.48 ± 3.91 ab 0.77 ± 0.10bcd 25.14 30.30
T7 14.1 ± 2.50 b 19.03 ± 3.48bcd 0.14 ± 0.05 e 104.09 140.12
T8 16.36 ± 2.36 b 18.34 ± 2.74 cd 0.34 ± 0.14 de 48.50 54.36
T9 19.14 ± 2.57 a 21.71 ± 3.62abc 0.44 ± 0.05 cde 43.49 49.33
T10 17.71 ± 1.79 ab 24.64 ± 3.07 a 0.41 ± 0.09 cde 43.26 60.17

D. Ion content of peppermint roots

Treatment Ca2+ (mg/g) K+ (mg/g) Na+ (mg/g) K+ /Na+ Ca2+ /Na+

CK 6.78 ± 0.25 c 10.60 ± 2.4 abc 1.35 ± 0.38 c 7.88 5.04

T1 6.70 ± 0.88 c 9.14 ± 0.43 bc 12.45 ± 2.49 a 0.73 0.54
T2 8.43 ± 1.29 c 12.57 ± 2.77abc 3.59 ± 0.67 c 3.5 2.35
T3 15.68 ± 2.89 d 15.74 ± 7.64 a 9.39 ± 1.52 b 1.68 1.67
T4 6.60 ± 1.90 c 6.14 ± 1.71 c 12.83 ± 2.23ab 0.48 0.51
T5 12.84 ± 3.71 b 9.43 ± 1.24 bc 8.48 ± 1.93 b 1.11 1.52
T6 21.10 ± 2.15 a 11.90 ± 2.76abc 8.68 ± 1.15 b 1.37 2.43
T7 12.11 ± 1.23 b 12.97 ± 2.13 ab 1.26 ± 0.21 c 10.26 9.58
T8 8.08 ± 0.92 c 13.54 ± 2.27 ab 1.05 ± 0.34 c 12.88 7.69
T9 13.96 ± 1.25 b 12.6 ± 2.40 abc 1.87 ± 0.29 c 6.74 7.46
T10 14.25 ± 4.19 b 13.19 ± 2.30 ab 2.16 ± 0.50 c 6.12 6.61

ment decreased the fresh weight of aerial parts. The fresh weight of
roots treated with NaCl, vermicompost, or NPK did not differ from
that of the control (Fig. 1C, D). The change in water content of the
aerial parts of peppermint was similar to that of blessed thistle.
However, the water content of the roots of plants subjected to all
treatments remained the same (Supplementary Fig. 2C, D).
3.2. The effects of salinity and vermicompost on malondialdehyde
(MDA) content
MDA is often used as an indicator to assess abiotic stress dam-
age. We found that treatment with 4‰ NaCl greatly increased MDA
levels in both blessed thistle and peppermint (Fig. 2). For blessed
thistle, the lowest MDA concentrations were measured in the aerial
parts of plants subjected to a 15% vermicompost treatment and
in the roots of plants treated with 15% NPK. Vermicompost treat-
ment significantly decreased the MDA content in blessed thistle
plants grown under salinity stress (1‰ and 4‰; Fig. 2A, B). For pep-
permint, similar results were observed: treatment with both NaCl
and vermicompost reduced the MDA content when compared to
treatment with NaCl alone (Fig. 2C, D). The lowest MDA content in
the aerial parts and roots was observed after a 15% vermicompost
treatment.
 L. Xu et al. / Industrial Crops and Products 94 (2016) 574–585 579

7 10

Soluble Protein Content of Aerial Parts (mg/g)

A B

Soluble Protein Content of Roots (mg/g)

a
a
6
ab 8 ab
5 bc b
bcd ab
6 bc
4
cde de
3
de de de 4 cd cd de

2
e e 2 e
e
1 e

0 0

Root Soluble Protein Content of Roots (mg/g)

35 12
Soluble Protein Content of Aerial Parts (mg/g)

C a
D
30 a a
10
b
25
8
c
b
20 b
6
d
b de
15 ef
c f
c c 4
10 cd f
de
e 2 g
5 g
e
0 0
CK T1 T2 T3 T4 T5 T6 T7 T8 T9 T10 CK T1 T2 T3 T4 T5 T6 T7 T8 T9 T10

Fig. 4. The effects of various treatments on the protein content of the aerial and root parts of blessed thistle and peppermint (A-B: blessed thistle; C-D: peppermint).

3.3. The effects of salinity and vermicompost on photosynthetic approximately 2 ∼ 3 fold more than the control. The lowest K+ /Na+
pigments in blessed thistle and peppermint leaves and Ca2+ /Na+ ratios were found in plants exposed to 4‰ NaCl.
In the roots, a 15% vermicompost treatment caused the highest
Long-term salinity treatments hinder photosynthesis. The pig- K+ /Na+ ratio and a 5% vermicompost treatment caused the high-
ment chlorophyll is the main player in photosynthesis, with est Ca2+ /Na+ ratio (Table 2D). In plants grown under salinity stress,
chlorophyll a and chlorophyll b capturing light and activated vermicompost supplementation always increased the K+ /Na+ and
chlorophyll a transforming the light into chemical energy. Under Ca2+ /Na+ ratios.
all conditions, the total chlorophyll content was not affected when
compared to the control in blessed thistle or peppermint (Fig. 3A, 3.5. The effects of salinity and vermicompost treatment on
B). The levels of chlorophyll a and b in plants subjected to salinity protein content of blessed thistle and peppermint
stress, vermicompost, NPK, or a combination of salinity stress and
vermicompost were similar to the levels in the untreated control Salinity stress did not affect the protein content of the aerial
(Supplemental Fig. 3). plant parts of blessed thistle supplemented or not with vermicom-
post, but vermicompost or NPK treatment in the absence of salinity
3.4. The effects of salinity and vermicompost on ionic stress significantly increased the protein content, with the excep-
homeostasis in blessed thistle and peppermint tion of a 5% NPK treatment (Fig. 4). For roots of blessed thistle, only
a 15% vermicompost treatment resulted in an increase of the pro-
Excess Na+ causes an ionic imbalance in plants. Under high salin- tein content (Fig. 4B). At 1‰ salinity, all vermicompost treatments
ity stress, plants maintain ionic homeostasis by regulating K+ and increased the protein content of the roots, while vermicompost did
Ca2+ levels. We determined the Na+ , K+ , and Ca2+ concentrations not increase the protein content at 4‰ salinity.
and calculated the K+ /Na+ and Ca2+ /Na+ ratios. The highest K+ /Na+ For peppermint, all vermicompost and NPK treatments
ratio was observed in the aerial parts of blessed thistle plants increased the protein levels in the aerial and root parts (Fig. 4C,
treated with 15% vermicompost, followed by those treated with D). Vermicompost treatment increased protein levels in the aerial
5% vermicompost. Similar results were obtained for the Ca2+ /Na+ and root parts under low salinity conditions, but not under high
ratios (Table 2A). The lowest K+ /Na+ and Ca2+ /Na+ ratios were salinity conditions in comparison to salinity treatment alone.
observed at 4‰ NaCl treatment. Treatment with 15% fertilizer
increased the K+ /Na+ and Ca2+ /Na+ ratios of roots to a greater extent 3.6. The effects of salinity and vermicompost treatment on the
than did treatment with 15% vermicompost (Table 2B). proline content of blessed thistle and peppermint
For peppermint, the highest K+ /Na+ and Ca2+ /Na+ ratios were
observed in the aerial parts of plants treated with 5% vermicompost, Proline has important roles in plant abiotic stress responses.
namely 104 and 140, respectively (Table 2C). Those values were Interestingly, vermicompost treatment did not affect the proline
580 L. Xu et al. / Industrial Crops and Products 94 (2016) 574–585

100 20
A B
Proline Content of Aerial Parts (ug/g)
a 18 a

Proline Content of Roots (ug/g)

a b
b 16
ab
80 bc
b
cd 14
de c
cde bcd
60 12
cd
e 10
f de
40 f 8 e
e
6 e e

20 4

0 0
Proline Content of Aerial Parts (ug/g)

100 14
D a
C

Proline Content of Roots (ug/g)

a 12
a a
80 ab
10 b
ab bc
60 8
bc
bcd cd cd cd cd cd bc
d 6 cd
40 cd
d cd
4

20
d
2

0 0

CK T1 T2 T3 T4 T5 T6 T7 T8 T9 T10 CK T1 T2 T3 T4 T5 T6 T7 T8 T9 T10

Fig. 5. The effects of various treatments on the proline content of the aerial and root parts of blessed thistle and peppermint (A-B: blessed thistle; C-D: peppermint).

content in the aerial parts of blessed thistle seedlings compared to or without vermicompost supplementation (Fig. 7B). CAT activity
the control, whereas single fertilizer treatment did (Fig. 5A;). Ver- was up-regulated only in plants subjected to vermicompost or NPK
micompost treatment decreased proline levels at 1‰ NaCl, but not treatment; CAT activity in the aerial parts was the same as the con-
at 4‰. Similar to the aerial plant parts, roots accumulated more pro- trol for all other treatments (Fig. 8A). CAT activity was the same in
line than the control when treated with NPK alone, but not when roots under all treatments (Fig. 8B).
treated with 15% vermicompost alone (Fig. 5B). In roots, vermi- For peppermint, most aerial tissues showed increased SOD
compost does not change proline levels under low or high salinity activities in response to vermicompost or NPK treatment (Fig. 6C).
treatment. Only treatment with 15% NPK up-regulated SOD activity in the
In peppermint, treatment with 15% NPK resulted in an increase roots compared to the control (Fig. 6d). All high salinity treatments
of the proline content compared to the control in all plant parts decreased the SOD activity in roots. Single vermicompost or NPK
(Fig. 5C, D). Both the aerial and root parts of plants grown under treatment induced POD activity in the aerial tissues, but treatment
salinity stress in soil supplemented with vermicompost had lower with 4‰ NaCl greatly inhibited POD activity (Fig. 7C). By contrast,
proline levels. vermicompost or NPK treatment did not affect POD activity in the
roots (Fig. 7D). Salinity treatment did not increase POD activity in
the presence or absence of 5% vermicompost. Only the 4‰ NaCl
3.7. The effects of salinity and vermicompost on the antioxidant treatment up-regulated CAT enzyme activity compared to the con-
enzyme activities of blessed thistle and peppermint trol, while all other treatments down-regulated CAT activity in the
aerial parts of the seedlings (Fig. 8C). Roots treated with both 4‰
SODs, PODs, and CATs are major antioxidant enzymes that func- NaCl and 15% vermicompost or with 5% vermicompost alone exhib-
tion in the plant stress response. SODs convert O2− into H2 O2 , ited increased CAT activity, whereas the other treatments either did
thus minimizing the toxic effect of free radicals. In blessed this- not affect CAT activity or decreased it (Fig. 8D).
tle, treatment with vermicompost alone increased the SOD enzyme
activities in the aerial plant parts, whereas treatment with NPK
did not (Fig. 6A). Only treatments with 15% vermicompost or with 4. Discussion
1‰ NaCl and 5% vermicompost increased SOD activity in the roots
(Fig. 6B). Peroxide and H2 O2 can be further reduced to H2 O by the 4.1. Blessed thistle and peppermint respond differently to high
POD and CAT enzymes. In the aerial parts of blessed thistle seedlings salinity conditions
grown in 4‰ NaCl, POD enzyme activity was only induced when the
plants were supplemented with 15% vermicompost (Fig. 7A). Inter- Salinity affects many aspects of plant metabolism and inhibits
estingly, POD activity was down-regulated in roots in response to nitrogen uptake (Hawkins and Lewis, 1993), mineral assimilation
most treatments, with the exception of the high salt treatment with (Keutgen and Pawelzik, 2009), ion distribution (Rogers and Noble,
 L. Xu et al. / Industrial Crops and Products 94 (2016) 574–585 581

50 60
A B a

SOD Activity of Aerial Parts (U/g)

a 50 ab
40 ab

SOD Activity of Roots (U/g)

bc bcd
bcd bcd abc bcd
40
cde cde bcd
30

cd cd cd
30

20
d
de de de 20
de e
10
10

0 0

50 50
C a
D
SOD Activity of Aerial Parts (U/g)

a
a

SOD Activity of Roots (U/g)

40 a 40 a
b
ab bc
bcd bc bc bcd abc
30 bcd bcd 30

cd cd
d de cd
20 20
e e

10 10

0 0

CK T1 T2 T3 T4 T5 T6 T7 T8 T9 T10 CK T1 T2 T3 T4 T5 T6 T7 T8 T9 T10

Fig. 6. The effects of various treatments on the superoxide dismutase (SOD) enzyme activities of the aerial and root parts of blessed thistle and peppermint (A-B: blessed
thistle; C-D: peppermint).

1992), water uptake (Koyro, 2006), secondary metabolite produc- (Chen et al., 2014). Long-term exposure to high salinity conditions
tion (Chen et al., 2014), stomatal conductance, photosynthesis usually results in dramatic changes in chlorophyll content.
(Turan et al., 2009, 2010), and plant growth and yield (Muhling K+ /Na+ or Ca2+ /Na+ ratios are also good indexes for assessing
and Lauchli, 2002). These negative effects are species-dependent damage due to salinity, and these two ratios were strongly reduced
and some plant species are more resistant to salinity stress than compared to the control in both species (Table 2C, D). Our find-
are others. The observation that the shoots and leaves of pepper- ing that treatment with high concentrations of NaCl caused lower
mint are more sensitive to salinity than are the roots is consistent K+ /Na+ and Ca2+ /Na+ ratios is in accordance with previous findings
with previous findings (Hawkins and Lewis, 1993). Several studies (Zhao et al., 2014). Interestingly, the K+ /Na+ and Ca2+ /Na+ ratios of
found that low concentrations of salinity increase the number of the aerial parts of peppermint at two salt concentrations were dra-
leaves or the fresh weight of shoots (Chen et al., 2014; Koyro, 2006). matically higher than those of the roots or aerial and root parts of
High NaCl concentrations (4‰) have complex effects on the water blessed thistle. The soluble protein content of both blessed thistle
content of blessed thistle, reducing the water content in the aerial and peppermint decreased when plants were subjected to increas-
plant parts and increasing the water content in the roots (Supple- ing levels of salinity stress, implying that protein synthesis was
mentary Fig. 2A, B). All concentrations of NaCl decreased the water inhibited in a dose-dependent manner (Fig. 4). This phenomenon
content of the aerial parts of peppermint, but not of the roots. Pre- seems reasonable, as plants may limit energy consumption under
vious reports demonstrated that the leaves of plants grown under salinity stress in order to survive. One soluble amino acid, pro-
saline conditions contain less water than do those of the untreated line, accumulated to high levels in salt-stressed plants, partly due
control, while the water content of roots was slightly increased or to increased proline synthesis (Huang et al., 2013). In this study,
maintained at the same level (Keutgen and Pawelzik, 2009). proline mainly accumulated under 4‰ salt stress, especially in the
MDA levels are used to assess the extent of damage in response roots (Fig. 5). Our finding is in agreement with the observation that
to abiotic stress ( Lim et al., 2015). In this study, we used MDA levels high salinity generally causes an increase in proline content (Gorai
as an indicator of plant stress in response to high saline conditions. et al., 2010).
The MDA content was significantly increased under two different Scavenging of ROS can improve salt tolerance by inducing
salt conditions for both species (Fig. 2). Interestingly, salinity is gen- antioxidant enzymes such as SOD, POD, and CAT (Ibrahim et al.,
erally thought to reduce chlorophyll content (Turan et al., 2009), but 2015). However, we found that SOD activities in the aerial and root
in our experiments, neither blessed thistle nor peppermint showed parts were strongly inhibited under 4‰ NaCl treatments in both
a reduction in chlorophyll content. In fact, the content of chloro- species (Fig. 6). Similar results have been reported before and seem
phyll a and b did not differ between salt treatment and the control to be species-dependent (Nusrat et al., 2014; Zhao et al., 2014).
(Fig. 3). This may be partly due to the short duration of our analysis POD activity was also inhibited in aerial tissues, but increased in
(2 weeks). A previous study showed that the change in chlorophyll the roots of peppermint (Fig. 7). POD activity under salinity stress
content was also dependent on the concentration of salt in the soil may be dosage-dependent in some species (Chen et al., 2014). CAT
582 L. Xu et al. / Industrial Crops and Products 94 (2016) 574–585

25 50
A a B
bcd
POD Activity of Aerial Parts (U/g)

b bc a
20 40

POD Activity of Roots (U/g)

a ab
a ab

15
bcd bcd 30
bcd cd
c bc
cd cd
d c
10 20 c c
c

5 10

0 0

70
C a 25
D
POD Activity of Aerial Parts (U/g)

a
60 bc b ab bc

POD Activity of Roots (U/g)

20
bc cd cd
50 c
e
de e e de
40 15 de
e ef
ef ef
30 f
f 10

20

5
10

0 0
CK T1 T2 T3 T4 T5 T6 T7 T8 T9 T10 CK T1 T2 T3 T4 T5 T6 T7 T8 T9 T10

Fig. 7. The effects of various treatments on the peroxidase (POD) enzyme activities of the aerial and root parts of blessed thistle and peppermint (A-B: blessed thistle; C-D:
peppermint).

activity was generally lower under salinity stress, except in the with 15% vermicompost mildly reduced the MDA content in roots.
aerial parts of peppermint (Fig. 8). CAT activity sometimes lev- Vermicompost and NPK treatment did not alter the total chloro-
eled and then gradually decreased at different NaCl concentrations phyll content in either species. These findings are different from
compared to the control in Ginkgo biloba (Chen et al., 2014). results in ginger, where chlorophyll content was slightly increased
in plants subjected to vermicompost treatments (Ahmad et al.,
2009). A five-month vermicompost treatment also increased the
4.2. Inorganic fertilizer not vermicompost induced the total chlorophyll content in onion (Allium cepa L) (Hawkins and
accumulation of proline in both species under normal conditions Lewis, 1993).
Vermicompost treatments dramatically enhanced the accumu-
Due to different fermentation processes, the nutrient compo- lation of Ca2+ and reduced the accumulation of Na+ in the aerial
sition of vermicompost may vary, but it is generally enriched in tissues of blessed thistle, thus increasing the Ca2+ /Na+ and K+ /Na+
nitrogen (N), phosphorus (P), potassium (K), and organic mat- ratios. However, NPK treatment lowered the Ca2+ /Na+ and K+ /Na+
ter. We found that vermicompost or NPK treatments increased ratios in all tissues, except in plants subjected to the 15% NPK
the fresh weight of the aerial and root parts of blessed thistle treatment. Our findings were similar in peppermint and blessed
and peppermint, with the exception of peppermint roots under thistle: ionic ratios increased under all vermicompost treatments,
the 15% vermicompost treatment. The fresh weight of the roots but decreased or remained similar under the NPK treatment
of peppermint plants grown in 15% vermicompost was slightly (Table 2). Those findings that vermicompost increased the Ca2+ /Na+
lower than that of the untreated control. Vermicompost and NPK and K+ /Na+ ratios are consistent with previous findings (Ahmad
increased the water content of both blessed thistle and pepper- et al., 2009; Belda et al., 2013; Mahmoud and Ibrahim, 2012;
mint compared to the untreated control, with the exception of Mendoza-Hernandez et al., 2014). Both vermicompost and NPK
peppermint roots under the 15% NPK treatment (Supplementary treatment significantly increased the total soluble protein levels
Fig. 2). These results are consistent with experiments in banana in both species compared to the control, but a 15% vermicom-
(Musa acuminate)(Aremu et al., 2012). The MDA content of the post treatment resulted in the greatest increase. Similar results
aerial tissues of blessed thistle treated with vermicompost or NPK were obtained in ginger (Zingiber officinale) (Ahmad et al., 2009;
was lower than that of the control but, in roots, the MDA content Srivastava et al., 2012). One striking finding is that two concentra-
was only decreased after NPK treatment (Fig. 2). Interestingly, the tions of NPK treatment caused proline to accumulate in both the
MDA content of the aerial parts of peppermint grown in the pres- aerial and root parts of both species, whereas vermicompost treat-
ence of vermicompost or NPK remained the same, while treatment
 L. Xu et al. / Industrial Crops and Products 94 (2016) 574–585 583

3.0 1.6
CAT Activity of Aerial Parts (U/g) A B
a 1.4
a a

CAT Activity of Roots (U/g)

2.5 a
ab
1.2 abcd abc ab
abcd
2.0
b 1.0 bcd abcd
bc bc cd
1.5 .8
bc bc d
bc
.6
1.0 bc
bc c
.4
.5
.2

0.0 0.0

8 5 a
C a D ab
CAT Activity of Aerial Parts (U/g)

bc

CAT Activity of Roots (U/g)

4
6
cde
b
def
c cde cd 3 cd
cde cde
4 e de defg
e efgh fgh
e 2
gh
g
2
1

0 0
CK T1 T2 T3 T4 T5 T6 T7 T8 T9 T10 CK T1 T2 T3 T4 T5 T6 T7 T8 T9 T10

Fig. 8. The effects of various treatments on the catalase (CAT) enzyme activities of the aerial and root parts of blessed thistle and peppermint (A-B: blessed thistle; C-D:
peppermint).

ment did not (Fig. 5). Previous studies showed that vermicompost micompost treatment was most effective when plants were grown
prompted an increase in proline content (Chinsamy et al., 2013; under low salinity stress (Figs. 1and 2). Vermicompost treatment
Patel and Saraf, 2013). For unknown reasons, our findings differ increased the shoot height and fresh/dry weight of plants subjected
from these previous reports (Fig. 5). SOD and CAT activities were to salinity stress (Ahmad et al., 2009; Ashraf and Harris, 2004;
dramatically up-regulated in the aerial tissues of blessed thistle Chinsamy et al., 2013; Keutgen and Pawelzik, 2009; Patel and Saraf,
after supplementation with vermicompost as compared with NPK 2013). Vermicompost dramatically decreased the MDA content in
(Figs. 6 A, 8 A). In peppermint, SOD activity was up-regulated in the the aerial and root parts of both species when grown under salinity
aerial parts and CAT activity was induced in the roots under ver- stress (Fig. 2). This effect seems to be dose-dependent. However,
micompost treatment in comparison with NPK treatment (Fig. 6C, in the case of total chlorophyll content, vermicompost treatment
8D). NPK did not influence antioxidant enzyme activities, except displayed complex effects. In our study, the total chlorophyll a and
that a 15% NPK treatment caused an increase of POD activity in b content did not differ in plants grown under salinity stress with
peppermint (Fig. 7C). CAT and ascorbate peroxidase activities were or without vermicompost treatment. These findings are in agree-
enhanced in Jatropha curcas by the application of vermicompost ment with those of several other groups (Ahmad et al., 2009; Patel
(Patel and Saraf, 2013) and the application of vermicompost also and Saraf, 2013). However, garden-waste-vermicompost leachate
induced antioxidant enzyme activity in the aerial parts of Eucomis increased the content of both chlorophyll a and b in Solanum lycop-
autumnalis and Tulbaghia ludwigiana, but not of Tulbaghia violacea ersicum (tomato) (Chinsamy et al., 2013). This phenomenon may
(Aremu et al., 2014). be related to the vermicompost type or treatment conditions.
Vermicompost treatment increased the K+ /Na+ and Ca2+ /Na+
4.3. Vermicompost greatly improves salinity tolerance ratios of plants grown under salinity stress (Table 2). This effect
also seems to be dose-dependent. The change in K+ /Na+ ratio has
Vermicompost has a higher density, electrical conductivity, been described before (Ahmad et al., 2009). The mechanisms under-
microbial activity, and nitrate concentration than does manure lying these changes are not clear. Vermicompost also increased
(Atiyeh et al., 2001; Chaoui et al., 2003). Vermicompost also the total soluble protein content (of the roots in blessed thistle or
improves soil respiration, biomass, and soil organism abundance of the aerial parts in peppermint under 1‰ NaCl treatment) and
to a greater extent than does manure (Chaoui et al., 2003; Wu et al., decreased the proline content of the aerial parts under 1‰ saline
2013). Vermicompost treatment increased the fresh weight and conditions in both blessed thistle and peppermint (Figs. 4 and 5).
water content of both species grown under salinity stress and ver- These findings were also observed previously (Ahmad et al., 2009).
584 L. Xu et al. / Industrial Crops and Products 94 (2016) 574–585
By contrast, some reports found that vermicompost increased the
proline levels, for example in tomato treated with vermicompost
leachate (Chinsamy et al., 2013).
Vermicompost treatment did not alter the antioxidant enzyme
activities in plants grown under saline conditions, with some
exceptions (Figs. 6–8). For instance, a 5% vermicompost treat-
ment increased the SOD activities in the aerial or root parts of
blessed thistle grown under salinity stress. Vermicompost treat-
ment decreased the POD activities in the aerial and root parts of
peppermint plants grown under saline conditions. No previous
reports discuss the role of antioxidant enzymes in plants grown
in saline soil supplemented with vermicompost. Our results sug-
gest that the effect of vermicompost treatment on the activities of
antioxidant enzymes in plants subjected to salinity stress is species-
dependent. The two species examined in our study had different
responses to vermicompost treatment when grown under saline
conditions.
5. Conclusion
In this study, we examined whether supplementing soil with
vermicompost alleviated the negative effects of salinity on plant
growth and general plant physiology. Low amounts of vermicom-
post improved plant growth in saline conditions. The beneficial
effect of vermicompost may not be attributed to a single factor,
such as a nutrient in the soil. Therefore, vermicompost may con-
tain plant growth regulators or microorganisms or change the soil
structure that contributes to its positive effect on plant growth.
Author contributions statement
LX and MXL conceived and designed the study. DY, XYR, and
YYW acquired the data. LX, JZ, and HYZ analyzed and interpreted
the data. LX and MXL wrote the managed the whole project.
Competing financial interests
The authors declare no competing financial interests.
Acknowledgements
We thank Aline Valster and Kathleen Farquharson for valuable
comments regarding the manuscript text. This research was sup-
ported by grants from the Social Development Project in Jiangsu
Province (BE2015681), the National Natural Science Foundation
of China (31571702), the Priority Academic Program Develop-
ment of Jiangsu Higher Education Institutions (PAPD program,
809001), and Fundamental Research Funds for the Central Univer-
sities (KYZ201516).
Appendix A. Supplementary data
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.indcrop.2016.09.
023.
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