Introduction
Salt stress reduces plant growth and development due to
water deficit, ionic imbalance and ionic toxicity (Munns and Tester 2008; Lin et
al. 2017; Zahra et al. 2018), and nutrient deficiencies of various
macro- and micro-nutrients (Niste et al.
2014). These nutrients are a prerequisite for essential physiological processes
like the synthesis of macromolecules, enzymes activation, stomatal regulation
and osmotic homeostasis (Fageria and Moreira 2011; Shahid et al. 2020). Reduction in relative
water content (RWC), photosynthesis and dry matter yield is a common effect of
salinity stress (Polash et al. 2018; Saddiq et al. 2019). Ionic
toxicity due to Na+ and Cl- reduces the uptake of other
nutrients and causes metabolic disturbances (Munns 2002). It is notable that nutrient
uptake varied widely in the milk thistle ecotypes collected from broad range of
geographical areas in the western USA for
selenium uptake and soil characteristics (Feist and Parker 2001). Other
studies showed the existence of genetic diversity for nutrient uptake in
alfalfa under salt stress (Benabderrahim et
al. 2020; Bhattarai et al. 2020).
Plant growth
promoters (PGPs) modulate plant responses to biotic and abiotic stresses and
regulate their growth and developmental cascades (Zahra et al. 2020). Different approaches have been beneficial in
ameliorating salt affected soils including physical, biological, and amending
soil properties with organic and inorganic chemicals (Ansari et al. 2019;
Niamat et al. 2019). There are some
important chemical growth promoters that have been used in different ways
including, seed pretreatment, foliar spray and medium supplementation. For
instance, thiourea (TU) is a synthetic compound, which contains sulfur (as –SH)
and nitrogen (as –NH2) functional groups in its structure
(https://byjus.com/chemistry/thiourea/). It is an important PGP, which
influences several plant growth-related processes under different abiotic
stress factors (Wahid et al. 2017).
Exogenous application of TU significantly improved the achene yield and
nutrient status (N, P, and K) and maintained higher nitrate reductase
activities in sunflower crop (Akladious et
al. 2014). Ascorbic acid (AsA), a PGP and an antioxidant, was implicated in
the promotion of plant growth and development, and in improving anti-oxidative
defense by participating in phytohormone-mediated signaling networks under
different abiotic stresses (Akram et al. 2017). AsA also alleviated
salinity stress effects by reducing the uptake of toxic ions Na+ and
Cl- ions whilst regulating the plant metabolism by increasing the
availability of water and enhancing nutrients assimilation under salt stress
(Barakat 2003; Aliniaeifard et al. 2016).
Moringa is a valuable miracle tree, whose leaves are enriched with vitamins,
amino acids, antioxidants, and mineral nutrients. The leaf extract of moringa
leaves (MLE) was used as bio-stimulant of growth. The exogenous application of
MLE efficiently increased the uptake of different macronutrients (N, P, K, and
S) and increased plant growth and development (Hoque et al. 2020) and mitigates salt-induced adversities (Merwad 2018).
Milk thistle [Silybum marianum (L.) Gaertn.] is a
herbaceous plant, belonging to family Asteraceae (Omidbaigi and Nobakht 2001).
The seed of milk thistle is enriched with flavonolignans such as silymarin,
silybin, sliychristin, silydianin, and isosilybin (Bhattacharya 2011). Its
silymarin constituent manifests excellent anti-oxidative properties and is used
to cure different diseases (Lucini et al.
2016). The seeds of milk thistle are extracted for pharmaceutical industries as
a common drug to protect many types of liver disorders including cirrhosis,
fatty liver, viral and toxic hepatitis, and damage induced by toxic agents (Lucini
et al. 2016; Bhattacharya 2011).
Therefore, the cultivation demand for milk thistle is increasing worldwide
(Karkanis et al. 2011; Bhattacharya 2011).
But to dismay, literature shows that no coherent and systematic research has
been conducted to domesticate milk thistle and produce cultivars for efficient
nutrient uptake and silymarin biosynthesis under abiotic stresses. We
hypothesize that the phenotypic plasticity in milk thistle may be closely
linked with inherent ability of ecotypes to adapt and survive in saline areas.
Moreover, soil supplementation of PGPs may limit salt stress effects and
improve growth, water and tissue nutrient status. The objective of this study
was to explore phenotypic plasticity for changes in growth, water and nutrient
contents in different milk thistle ecotypes with soil supplementation of
different selected PGPs under salinity stress.
Materials
and Methods
Source
of milk thistle ecotypes and field simulation
Milk thistle ecotypes were collected from Quetta (QTA;
30°N latitude, 66°E longitude and 1679 m above sea level); Faisalabad (FSD;
31°N latitude, 73°E longitude and 184 m above sea level); Gujranwala (GUJ; 58°N latitude, 45°E
longitude and 231 m above sea level) and Kallar Kahar (KK; 46°N latitude; 72°E
longitude; 554 m above sea level). These ecozones were commonly dissected into
three ecological zones cold semi-arid (QTA), semi-arid (FSD-and KK) and hot
semi-arid (GUJ). After collecting the plants from their natural habitats, the
ecotypes were assessed for their growth and multiplication in Faisalabad
conditions.
Simulated field-plot experiments were conducted to
determine the innate behavior of different milk thistle ecotypes in Faisalabad
conditions for nutrient uptake, and data were evaluated for salinity tolerance
in terms of different morphological and physiological responses. The plot size
was 2 m × 4 m. Before sowing seeds, the soil was dug out to the depth of 60 cm;
the trenches lined with polythene sheets and soil refilled. The seeds of milk
thistle ecotypes were sown on November 17th in 2017 and on November
20th in 2018 under open field conditions Table 1: The physico-chemical properties of soil collected from the
native areas of each ecotype
|
Sample |
AB-DTPA Extractable |
pH |
ECe (dS/m) |
mmol/L |
SAR |
|||||
|
(mg/kg) |
||||||||||
|
P |
K |
Organic carbon (%) |
Saturation (%) |
Na+ |
Cl- |
Ca+Mg |
||||
|
Faisalabad |
2.24 |
169 |
1.15 |
38.29 |
7.97 |
0.56 |
2.39 |
2 |
5.10 |
1.49 |
|
Faisalabad |
2.15 |
164 |
1.06 |
38.16 |
8.05 |
0.58 |
2.65 |
2 |
5.50 |
1.60 |
|
Gujranwala |
1.25 |
159 |
1.13 |
35.21 |
8.20 |
0.65 |
3.68 |
3 |
5.70 |
1.50 |
|
Gujranwala |
1.04 |
167 |
0.85 |
36.24 |
8.10 |
0.75 |
4.03 |
3 |
5.90 |
1.39 |
|
Quetta |
0.58 |
244 |
0.49 |
41.21 |
7.86 |
2.23 |
8.30 |
6 |
18.40 |
2.73 |
|
Quetta |
0.44 |
212 |
0.34 |
40.63 |
7.81 |
2.29 |
8.17 |
6 |
18.60 |
2.68 |
|
Kallar Kahar |
2.2 |
157 |
0.81 |
34.20 |
8.30 |
4.20 |
6.28 |
4 |
10.12 |
1.85 |
|
Kallar Kahar |
2.1 |
195 |
0.85 |
36.21 |
8.40 |
3.90 |
6.38 |
5 |
11.24 |
1.67 |
at 1–2 cm
depth and 60 cm row spacing with a seeding rate of 100 seeds m-2.
Two times weed hoeing was done manually 25 and 55 days after sowing. The
salinity level (to achieve 12 dS/m) was developed at seedling stage by using
NaCl salt (99.2% pure) with three irrigation intervals. Moringa leaf extract
was prepared according to Khan et al. (2017). The seedlings were
assigned to 4 sub-plots with three replicates each. At BBCH principal growth
stage 3 (Martinelli et al. 2015), salt stress (12 dS/m) and PGPs viz., TU (500 µM), AsA (500 µM), and MLE (3%) solutions
were soil supplements were applied to maintain their final concentrations at
soil field capacity. The experiment was performed in randomized complete block
design with three replicates. The soils from these four locations were analyzed
for physicochemical properties. Faisalabad soil was more fertile due to having
higher organic matter and P contents. The K was higher in QTA soils while NO3--N
were similar in the soil from both locations. The decreasing order of
electrical conductivity of soil extract (ECe) was obtained as FSD < GUJ <
QTA and KK. Availabale sodium in QTA and KK soil was higher than from GUJ and
FSD soil. In Faisalabad soil, the Cl- concentration was higher than
the QTA soil. Besides, sodium adsorption ratio (SAR), and Ca and Mg were also
higher in QTA and KK soil, respectively (Table 1).
Plant
dry matter yield
Plants were harvested at BBCH principal growth stage 5
on May 17 of 2017 and May 13 in 2018. Shoot and root fresh and dry weights were
measured after harvesting the plants (nine plants per treatment). The intact
plants were carefully dug out from the soil to ensure maximum recovery of root
mass; excess of soil removed, washed and blotted dry. Fresh weight of shoot and
root was taken after cutting the shoot from root with a portable balance. To
measure dry weight, these parts were put in paper bags, dried in an oven at 65oC
for seven days and dry weight recorded for each treatment in triplicate.
Relative
leaf water content (RLWC)
Leaves from the
mature plants were collected to determine RLWC. A 0.5 g of fresh discs cut from
fully expanded leaves (FW) were floated in petri dishes containing distilled
water under light for 4 h. To get turgid weight (TW), the excess water was
blotted away. These discs were dried for 72 h at 80°C to get dry weight (DW).
The RLWC was calculated with the equation:
RLWC (%) = (FW-DW) × 100 / (TW-DW)
Measurement
of tissue nutrients
To measure nitrate-N with the methods of Kowalenko and
Lowe (1973), 0.5 g of dried grinded material was extracted in 5 mL of distilled
water by boiling for 1 h, filtered and made the volume up to 50 mL. To 3 mL of
the extract, 7 mL of working chromotropic acid solution was pipetted with a
thrust and briefly vortexed. After 20 min, the intensity of yellow colored
complex was read at 430 nm with UV-VIS spectrophotometer (U-2001, Tokyo, Japan)
using distilled water as blank. The nitrate-N content in the unknown samples
was ascertained by preparing a standard curve (10‒100 mg/L NO3-).
To measure
phosphate-P, K and Ca contents, the plant samples were digested in 2 mL of acid
mixture (HNO3 and HClO4 in 3:1 ratio) with the methods of
Yoshida et al. (1976). To estimate
phosphate-P, 1 mL of the extract from the above was added to 2 mL of the 2N HNO3
and diluted to 8 mL. After adding 1 mL of molybdatevandate reagent, the final
volume was made up to 10 mL, briefly vortexed and let stand for 20 min at room
temperature. The color intensity was measured at 420 nm using UV-VIS
spectrophotometer (U-2001, Tokyo, Japan) using distilled water as blank. The amount
of phosphate-P was determined from the unknown samples by preparing a standard
curve (2.5 to 15.0 mg/L PO43-). The amounts of K and Ca
in the samples were determined using flame photometer (Sherwood 410, UK), while
standard curves were constructed by preparing graded series (0‒40 mg/mL)
separately for both the ions.
Tendon et al. (1993) method was used for
determination of sulfur-S. Ten mL of extract was taken in a 50 mL volumetric
flask and added with 1 mL of 6N HCl and 1 mL of 0.5% gum acacia solutions.
Swirled and added 0.5 g barium chloride crystals and waited for 1 min. Flasks
were swirled again until the crystals were dissolved. Transmittance of the solution
was taken on a UV-VIS spectrophotometer (U-2001, Tokyo, Japan) at 440 nm. The
sulfate-S in the unknown samples was estimated by preparing graded sulfate
standards (0, 4, 8, 12, 16, and 20 mg/L) from 100 ppm stock solution prepared
from K2SO4.
Statistical
analysis
The data were subjected to statistical analysis using
online software Statisix8.1 to find out significance of variance sources while
Least Significant Difference (LSD) test was applied to compare the treatment
means at 5% level of probability. The Principal Component Analysis (PCA) was made
keeping the ecotypes and nutrients as the components grown under salinity and
soil supplementation. Furthermore, Pearson’s correlations were drawn between
dry weight and their nutrient contents of shoot and root.
Results
Plant
dry matter yield
Statistical data revealed significant (P<0.01)
differences in root and shoot dry weight of salinity, ecotypes, and PGPs, and
the interaction among them was also significant (P<0.01) in both years.
Importantly, irrespective of the soil supplementation, the shoot dry weight
reduced to higher extent under salinity stress in all ecotypes during both
experimental years; however, during 2017 the shoot dry weight of MLE treated
plants in KK ecotype was higher under salinity stress as compared to control
plants. Data further revealed that AsA followed by MLE, and TU was effective in
increasing shoot dry weight during 2017 and 2018 under control and salt stress
conditions. Conversely, highest shoot dry weight was recorded in FSD by
following QTA, KK, and GUJ under saline and non-saline conditions during 2017,
while during 2018 the order of change in this attribute was: FSD > QTA >
GUJ and KK. As for QTA ecotype, the shoot dry weight during 2017 was much
higher as compared to 2018 (Fig. 1). Data recorded for root dry weight of FSD
ecotype followed by QTA, KK, and GUJ was higher under saline and non-saline
conditions during 2017 and 2018. Overall, root dry weight increased with the
soil supplementation of all the treatments in different ecotypes, and salinity
stress caused antagonistic effects in both year studies (Fig. 1).
%20692-700_files/image002.png)
Fig.
1: Plant dry weight (shoot and root) under control and salinity stress
conditions. The plants were soil supplemented with different PGPs during 2017
and 2018
%20692-700_files/image004.png)
Fig. 2: RWC of milk thistle under control and salinity stress conditions. The plants were soil
supplemented with different PGPs during 2017 and 2018. In this and subsequent
figures, the data points labeled with alphabets show significant (P<0.05)
overall interaction of all the factors
Relative
water content (RWC)
Results showed that RWC revealed significant (P<0.01)
differences in all ecotypes and different soil supplementations under salt stress,
and the interactions of different factors were also significant in both years.
Under control conditions during 2017 the order of improvement in RWC of FSD and
KK ecotypes was at maximum with the supplementation of TU, while in QTA and GUJ
ecotypes the application of TU and MLE showed remarkable differences as
compared to other PGPs. Under stress condition, MLE supplementation showed
maximum water content in FSD ecotype, whereas AsA supplemented plants had
retained higher RWC in other three ecotypes. Considering the ecotypes, QTA
ecotype was efficient in retaining higher water contents, while GUJ ecotype was
the least efficient under control and salinity stress (Fig. 2). Data recorded during 2018 revealed
that under control conditions during 2018 the order of improvement in FSD
ecotypes was at its maximum with the supplementation of TU, while in other
three ecotypes the application of AsA was more effective as compared to other
PGPs. However, under salt stress condition the order of improvement of FSD and
GUJ ecotypes was the highest with the supplementation of MLE, while in QTA and
KK ecotypes the application of MLE showed remarkable differences. The
decreasing order in respect to ecotypes was observed as: FSD < GUJ < QTA
< KK (Fig. 2).
Nutrient
status
Nitrate-N: Shoot NO3--N
content manifested significant (P<0.01) differences in ecotypes and salt
stress under different soil supplementations and the interaction among them was
also highly significant in year 2017, while it was non-significant (P>0.05) during
2018. Furthermore, shoot and root NO3--N content was
non-significant (P>0.05) during 2017, while the interaction between these
factors was significant (P<0.01) during 2018. During 2017 maximum shoot NO3--N
content was noted in QTA and followed by FSD > KK > GUJ ecotypes (Fig.
3). Soil supplementations with AsA was effective for FSD and QTA ecotypes,
while in others the MLE was more effective as compared to other PGPs under
control conditions during 2017 and 2018. Under salt stress, AsA supplementation
was effective for all ecotypes during 2017, while MLE improved the NO3--N
content during 2018. Root NO3--N content in control
plants of all ecotypes improved maximally with MLE supplementation during 2017.
However, during 2018, soil supplementation of AsA was the most effective in
increasing NO3--N content in FSD followed by GUJ, KK and
QTA ecotypes. Under salt stress, MLE treatment was the most effective in FSD
and GUJ ecotypes, and AsA soil supplementation proved better in QTA and KK
ecotype as compared to other PGPs. Ecotype from QTA in 2017 and FSD in 2018
displayed higher NO3--N content irrespective of the
salinity treatment in roots. Nonetheless, salinity stress during both the years
tended to decrease the NO3--N content in all ecotypes in
comparison to their control plants. Furthermore, greater NO3--N
content was noted in shoot as compared to root, and that also was higher in the
year 2017 as compared to 2018 (Fig. 4).
%20692-700_files/image006.png)
Fig. 4: Changes in the root
nutrient contents of milk thistle ecotypes under control and salinity stress conditions. The plants
were soil supplemented with different PGPs during 2017 and 2018
%20692-700_files/image008.png)
Fig. 3: Changes in the shoot
nutrient contents of milk thistle ecotypes under control and salinity stress conditions. The plants were
soil supplemented with different PGPs during 2017 and 2018
Phosphate-P: Results for
shoot and root phosphate-P contents displayed
non-significant (P>0.05) differences in ecotypes and salinity under
different soil supplementations, and the interaction among these three factors
was also non-significant (P>0.05) in 2017 and 2018. In 2017 under control
conditions, MLE was more effective in improving this attribute in the shoot of
FSD and GUJ ecotypes while in other ecotypes, the effect of AsA was more
evident. Under salt stress, the soil supplementation with AsA was more
effective for GUJ, while for other ecotypes, MLE was more effective. During 2018
under control conditions, AsA was more effective in improving shoot phosphate-P
in FSD and QTA ecotype, while MLE and TU, respectively were more effective in
GUJ and KK ecotypes (Fig. 3). Data recorded for root PO4--P contents revealed that in 2017, AsA was
effective for FSD, QTA and KK ecotypes, while TU was more effective for GUJ as
compared to other PGPs under control and stress conditions. Data recorded
during 2018, revealed that under control conditions, AsA was effective for FSD
ecotype, while MLE and TU showed more effectiveness in GUJ and QTA ecotypes,
respectively; while for KK ecotype, TU was more effective. Under salt stress,
MLE supplementation was effective for FSD and GUJ ecotypes, while for QTA and
KK the effect of TU was greater. Overall, result revealed that salinity during
both experiment years tend to decrease the shoot and root PO4--P contents in all
ecotypes in comparison to control plants. Nonetheless, QTA ecotype exhibited
highest improvement in this attribute with
soil supplementation during 2017–2018 under control than under salinity stress
(Fig. 4).
Sulfate-S: Data for shoot SO4--S contents in both shoot and root showed
significant (P<0.01) differences in ecotypes and salinity under different
soil supplementations and the interaction of these factors was non-sugnificant
(P>0.05) during 2017 but significant (P<0.01) during 2018. In 2017,
considering shoot SO4--S
contents of control plants, AsA was
more effective for FSD ecotype, and MLE was more supportive for GUJ and KK
ecotypes, while in QTA the effect AsA was more evident. A similar trend of
increase was found under stress in all ecotypes for improving this attribute.
Data recorded during 2018 exhibited that MLE supplementation was effective for
FSD and GUJ ecotypes, while TU supplied plants from QTA and KK ecotypes showed
higher content of SO4--S as compared to other PGPs.
Importantly, AsA soil supplementation performed better results in saline
subjected plants (Fig. 3). Considering root SO4--S in
2017, the AsA was highly effective for FSD and QTA ecotypes, while in GUJ and
KK the effect of control and TU supplementation was at its maximum However,
under salt stress, AsA increased sulfate-S content more in FSD, while in GUJ no
difference was seen with soil supplementations, while in QTA and KK the effect
of TU was greater. Data recorded during 2018 exposed that MLE was effective for
FSD and GUJ ecotypes, while TU and AsA showed more effectiveness in QTA and KK
ecotypes, respectively. However, under stress the effect of AsA was at maximum
in all ecotypes. Data recorded for root SO4--S contents revealed that all soil treatments were
effective in enhancing root SO4--S
contents irrespective of stress and
ecotypes differences. On the other hand, highest root SO4--S
contents was recorded in FSD by following KK, QTA and GUJ under non-saline
conditions but under saline was noted as KK > FSD > QTA > GUJ in 2017.
However, in 2018 maximum root SO4--S contents were
recorded in QTA by following FSD, KK and GUJ under saline and non-saline
conditions (Fig. 4).
K: Results for
both shoot and root K contents showed significant (P<0.01) differences in
ecotypes and salinity under different soil supplementations, and the
interaction among them was non-significant (P>0.05) during 2017 but highly
significant (P<0.01) during 2018. In 2017, the MLE supplementation indicated
maximum K content in the shoot of all ecotypes under control conditions.
However, under salt stress the AsA was more effective in all ecotypes except
for KK, in which the MLE supply was more effective in improving this attribute.
During 2018, AsA supplementation was better in increasing shoot K content in
all ecotypes except for FSD, in which MLE treatment showed maximum increase as
compared to other PGPs under control conditions. Centrality, under stress the
effect of MLE was more profound for GUJ, QTA and KK ecotypes, while in FSD the
AsA treatment was more effective for attaining more K. Considering root part
during 2017, the MLE supplied had more K content as compared to other soil
supplements under control conditions. Contrarily under salt stress AsA
supplementation was effective for FSD, GUJ, and QTA ecotypes, while MLE
increase K content in KK ecotype. Similar results were confirmed during 2018
under control conditions, while under stress AsA treatment was effective for
all ecotypes except for QTA ecotype, in which the TU treatment attained
superiority over all other treatments. Graphical data showed that during both
experimental years (i.e., 2017 and 2018) ecotype QTA had higher K content
followed by KK > FSD > GUJ under control and stressed plants (Fig. 3).
Data recorded for root K content revealed that all soil treatments were rather
ineffective in enhancing root K content irrespective of stress and ecotypes
differences during 2017. On the other hand, highest root K content was recorded
in QTA by following FSD, KK, and GUJ under saline and non-saline conditions
during 2017. In 2018 maximum root K was noted in QTA followed by KK, FSD, and
GUJ. Overall, result revealed that salinity during both experiment years tend
to decrease the root K content in all ecotypes in comparison to their control
plants (Fig. 4).
Ca: Results
obtained for shoot Ca content displayed non-significant (P>0.05) differences
in ecotypes and salinity under different soil supplementations, and the
interaction among these three factors was also non-significant (P>0.05) in
both the years (2017–2018). Furthermore, root Ca content was found
statistically significant (P<0.01) during 2017, while the interaction
between the factors observed non-significant (P>0.05) during 2018.
Considering shoot part during 2017 maximum Ca content was noted in QTA followed
by FSD, KK and GUJ ecotypes, while all soil supplements were effective in all
ecotypes in improving shoot Ca. Likewise, AsA supplementation was effective for
FSD, while MLE improved Ca in all other ecotypes. While under salt stress AsA
was effective for FSD and QTA ecotype, while MLE was more effective for GUJ and
KK ecotypes. During 2018 AsA supplementation was effective in all ecotypes as
compared to other PGPs under control or salt stress conditions. During 2018,
higher Ca content was observed in QTA followed by KK, FSD and GUJ under control
and saline condition (Fig. 3). As regards root Ca contents of control plants
during 2017, MLE and AsA were equally effective for FSD and QTA ecotype, while
AsA did so for KK ecotype. However, under salt stress the MLE was more
effective for all ecotypes except for GUJ, in which AsA was more effective.
During 2018, under control conditions, MLE was effective for FSD and QTA
ecotype, while TU showed more effectiveness in GUJ and KK ecotypes. Ca content in roots was maximally increased during 2017
as compared to 2018. Furthermore, it was observed that salinity during both
experiment years tend to decrease the Ca content in all ecotypes in comparison
to their control plants. Furthermore, maximum Ca content was found in shoot as
compared to root (Fig. 4).
Na: Results
showed that Na content in both shoot and root had significant (P<0.01)
differences in ecotypes and salinity treatment under different soil
supplementations and the interaction among them was also highly significant
during 2017, while non-significant (P>0.05) during 2018. Considering shoot
Na content of control plants, during 2017 the order of increase in shoot Na
was: control > TU=MLE > AsA in FSD ecotype, and in GUJ ecotype the
effectiveness was: MLE > control > TU > AsA >, while in QTA this
order was: control > TU=MLE=AsA, and in KK the order was: control > TU
> MLE=AsA. Under salinity stress, this order in FSD was: MLE > control
> TU > AsA, and in GUJ ecotype the effectiveness was: TU=AsA=MLE >
Control, while in QTA this order was: Control > TU > AsA > MLE, and in
KK the trend was found as: Control > TU > AsA=MLE. During 2018 the effect
of AsA supplementation was more effective in lowering the Na content in all
collected ecotypes irrespective of salinity and control conditions. It was
observed that during both experimental years (i.e., 2017 and 2018)
ecotype GUJ had higher Na content followed by KK > QTA > FSD under
control and stressed plants (Fig. 3). Data recorded for root Na content
revealed that all soil treatments were highly effective in lowering root Na
content irrespective of stress and ecotypes differences during 2017. On the other
hand, highest root Na content was recorded in GUJ by following KK, FSD and QTA
under saline and non-saline conditions during 2017. Under control conditions,
the effect of AsA supplementation was more in all ecotypes except for GUJ, in
which MLE supplementation was more effective in reducing Na uptake. Under salt
stress, the effect of MLE was more profound in lowering Na content in all
ecotypes than the other PGPs. Furthermore, data recorded during 2018 revealed
that AsA supplementation was more effective for reducing Na uptake irrespective
of salinity treatments. Overall, result revealed that GUJ and KK ecotypes
exhibited highest increase in Na content with salinity stress during 2017–2018
while soil supplementation proved quite effective in reducing both shoot and
root Na content. Overall, higher Na was present in root part as compared to
shoot, and year 2017 was more effective in lowering the Na uptake (Fig. 4).
Principal
component analysis (PCA)
According to the PCA, the components showed 98.24% variance.
Of the components, PC1 (F1) had 70.94% variance and PC2 (F2) exhibited 27.30%
variance. First link was more positively associated with Ca and phosphate-P.
The phosphate-P was closely linked with ecotype from QTA, while K was weekly
associated with FSD ecotype. Na contents showed strong association with KK
ecotype; while all ions showed no or weak association with GUJ area (Fig. 5).
Correlation
Among the essential nutrients, shoot nitrate-N,
sulfate-S, phosphate-P, K and Ca showed
positive correlation with SDW during both experimental years under
control and stressed conditions. However, the K and Ca showed more
statistically significant correlation (P<0.01) as compared to other
nutrients. Contrarily, Na content
exhibited a negative correlation with SDW. Besides, root NO3--N
indicated significant (P<0.01) positive correlation with RDW during both
experimental years. For RDW, PO43--P
showed positive correlation during 2017 irrespective to salinity treatment,
while non-significant relationship was noted during 2018. The SO42--S
indicated non-significant relationship with RDW during 2017, whereas positive
association was seen during 2018. Conversely, K and Ca showed non-significant
correlation with RDW. Na contents indicated negative correlation with RDW during
2017 and 2018 (Table 2).
Table
2: Correlation coefficient of shoot and root dry weight
with changing nutrient status of milk thistle under salinity during 2017 and 2018
|
Independent variables |
Dependent variables |
2017 |
2018 |
||
|
Control |
Salinity |
Control |
Salinity |
||
|
Shoot dry weight |
Shoot nitrate-N |
0.77** |
0.92** |
0.44ns |
0.23ns |
|
Shoot phosphate-P |
0.49ns |
0.48ns |
0.79** |
0.77** |
|
|
Shoot sulfate-S |
0.60* |
0.48 ns |
0.72** |
0.65** |
|
|
Shoot K |
0.60* |
0.73** |
0.82** |
0.96** |
|
|
Shoot Ca |
0.65** |
0.66** |
0.82** |
0.90** |
|
|
Shoot Na |
-0.55* |
-0.31ns |
-0.80** |
-0.07ns |
|
|
Root dry weight |
Root nitrate-N |
0.62** |
0.66** |
0.76** |
0.77** |
|
Root phosphate-P |
0.60* |
0.65** |
0.38ns |
0.25ns |
|
|
Root sulfate-S |
0.33ns |
0.04ns |
0.75** |
0.72** |
|
|
Root K |
0.38ns |
0.76** |
0.41ns |
0.34ns |
|
|
Root Ca |
0.15ns |
0.67** |
0.23ns |
-0.12ns |
|
|
Root Na |
-0.61* |
-0.47ns |
-0.65** |
-0.64** |
|
Significant at: * P<0.05; ** P<0.01, ns
non-significant
%20692-700_files/image010.png)
Fig.
5: Principal component analysis of Na, K, phosphate-P and
Ca in different milk thistle ecotypes
Discussion
There exist substantial phenotypic plasticity in milk
thistle for tolerance to salinity and responsiveness of milk thistle of
different PGPs both under control and salinity conditions. From these findings,
the milk thistle can be ranked as moderately tolerant to salinity based on
changes in plant dry weight, but this aspect needs further probe by using a
broader range of salinity levels. It has been reported that milk thistle shows
the variable response in salinity from different ecological zones of the world
(Ghavami and Ramin 2007; Egamberdieva et al. 2013; Hammami et al. 2020),
and salinity response ranges from 9–30 dS/m. Salinity is an adverse
environmental abiotic factor for plant growth and development as it is known
for arresting the cell elongation and expansion (Munns and Tester 2008; Zahra et
al. 2018). In this study, in addition to reduction in dry weight (Fig. 1),
there was a substantial decline in the RLWC (Fig. 2), which is attributed to
ion-toxicity and induced water deficit effects of salinity stress (Munns and
Tester 2008).
Like other
plants, milk thistly acquires and assimilates nutrients in requisite quantities
for better growth and yield (Geneva et
al. 2007; Školníková et al.
2019). Some nutrients are both structurally and functionally important (e.g.,
nitrogen, phosphorous, calcium and sulfur), while others (e.g., K and
Ca) are not part of plant structure but are required for physiological
processes in soluble form (Taiz et al.
2015). The salinity has an adverse effect on the uptake and assimilation of
these essential nutrients by having an ion-specificity effect, which results in
reduced tissue nutrient content (Volkov and Beilby 2017). It has been reported
that soil supplementation with PGPs has beneficial effects on the root uptake
and shoot assimilation of essential nutrients both under saline and non-saline
conditions (Abdallah et al. 2020). In
this study, the analysis of root and shoot tissues of milk thistle ecotypes for
nitrate-N, sulfate-S, phosphate-P, Na, K and Ca
supplemented with of PGPs revealed that salinity reduced the tissue
concentration of all the essential nutrients, except Na concentration, which
was increased many folds under salt condition (Fig. 3–4). It is important to
indicate that nitrate-N is among the most vital nutrients absorbed by the roots
and assimilates in the number of macromolecules such as proteins, alkaloids and
many others nitrogen containing metabolites (Taiz et al. 2015).
Worthily, the
ecotypes from QTA and FSD displayed a lesser decline in the shoot and root Na
content under salt stress as compared to those from and GUJ and KK (Fig. 3–4).
This appears to be due to innate ability of these ecotypes to physiologically
regulate this toxic ion. As regards changes in the tissue concentration of
essential nutrient measured in this study, we found a great deal of variability
in the ecotypes to acquire and assimilate different nutrients in shoot and root
tissues. Differences in the correlation coefficients of control and salinity
stressed plants (Table 2) combined with PCA results (Fig. 5) supported the view
that great phenotypic flexibility exists in milk thistle for maintenance of
tissue nutrients and eventually salinity tolerance in milk thistle.
Conclusion
Differences in the four milk thistle ecotypes for
growth, leaf water status and contents of analyzed nutrient indicated
phenotypic plasticity, in the two years of study under salinity as revealed
from correlation coefficient and PCA data. AsA and MLE were more effective PGPs
in reducing salt stress effect on the ecotypes. The benefit of soil
supplementation with PGPs was not only seen in terms of improved plant biomass
but also in the form of higher tissue content of the studied nutrients in
curtailing the effect of salinity on milk thistle ecotypes.
Acknowledgments
This paper is part of PhD thesis of first author. Thanks
to Department of Botany for provision of chemicals and other facilities for
performing this work, and ORIC, UAF for providing funds for lab facilities for
the current research.
Author
Contributions
NZ and AW conceived the idea; SMA and MA contributed in
planning the experiments; NZ analyzed data and prepared initial draft; AW, SMA
and MA finalized the paper
References
Abdallah MMS, TN El Sebai, AAE-M Ramadan, HMS
El-Bassiouny (2020). Physiological and biochemical role of proline, trehalose,
and compost onenhancing salinity tolerance of quinoa plant. Bull Natl Res Ctr 44; Article 96
Akladious SA (2014). Influence of thiourea application
on some physiological and molecular criteria of sunflower (Helianthus annuus
L.) plants under conditions of heat stress. Protoplasma 251:625‒638
Akram NA, F Shafique, M Ashraf (2017). Ascorbic acid–a
potential oxidant scavenger and its role in plant development and abiotic
stress tolerance. Front Plant Sci 8;
Article 613
Aliniaeifard S, J Hajilou, SJ Tabatabaei, M Sifi-Kalhor (2016).
Effects of ascorbic acid and reduced glutathione on the alleviation of salinity
stress in olive plants. Intl J Fruit Sci 16:395‒409
Ansari FA, I Ahmad, J Pichtel (2019). Growth stimulation
and alleviation of salinity stress to wheat by the biofilm forming Bacillus
pumilus strain FAB10. Appl Soil Ecol 143:45‒54
Barakat H (2003). Interactive effects of salinity and
certain vitamin on gene expression and cell division. Intl J Agric Biol
5:219‒225
Benabderrahim MA, M Guiza, M Haddad (2020). Genetic
diversity of salt tolerance in tetraploid alfalfa (Medicago sativa L.). Act
Physiol Plantarum 42; Article 5
Bhattacharya S (2011). Milk thistle (Silybum marianum
L. Gaert.) seeds in health. In: Nuts and Seeds in Health and Disease
Prevention, pp:759‒766. Academic Press, London
Bhattarai S, D Biswas, YB Fu, B Biligetu (2020).
Morphological, physiological, and genetic responses to salt stress in alfalfa:
A review. Agronomy 10; Article 5
Egamberdieva D, D Jabborova, N Mamadalieva (2013). Salt
tolerant Pseudomonas extremorientalis
able to stimulate growth of Silybum
marianum under salt stress. Med
Aromat Plant Sci Biotechnol 7:7‒10
Fageria NK, A Moreira (2011). The role of mineral
nutrition on root growth of crop plants. Adv
Agron 110:251‒331
Feist LJ, DR Parker (2001). Ecotypic variation in
selenium accumulation among populations of Stanleya pinnata. New
Phytol 149:61‒69
Geneva M, G Zehirov, I Stancheva, L Iliev, G Georgiev
(2007). Effect of soil fertilizer, foliar fertilizer, and growth regulator
application on milk thistle development, seed yield, and silymarin content. Commun Soil Sci Plant Anal 39:17‒24
Ghavami N, AA Ramin (2007). Salinity and temperature
effects on seed germination of milk thistle. Commun Soil Sci Plant 38:2681‒2691
Hammami H, B Saadatian, SAH Hosseini (2020).
Geographical variation in seed germination and biochemical response of milk
thistle (Silybum marianum) ecotypes
exposed to osmotic and salinity stresses. Indust Crops Prod 152;
Article 112507
Hoque TS, MS Rana, SA Zahan, I Jahan, MA Abedin (2020).
Moringa leaf extract as a bio-stimulant on growth, yield and nutritional
improvement in cabbage. Asian J Med Biol Res 6:196‒203
Karkanis A, D Bilalis, A Efthimiadou (2011). Cultivation
of milk thistle (Silybum marianum L. Gaertn.), a medicinal weed. Indust
Crops Prod 34:825‒830
Khan S, SMA Basra, I Afzal, A Wahid (2017). Screening of
moringa landraces for leaf extract as biostimulant in wheat. Intl J Agric Biol 19:999‒1006
Kowalenko CG, LE Lowe (1973). Determination of nitrates
in soil extracts. Soil Sci Soc Amer Proc 37:660
Lin J, Y Wang, S Sun, C Mu, X. Yan (2017). Effects of
arbuscular mycorrhizal fungi on the growth, photosynthesis and photosynthetic
pigments of Leymus chinensis seedlings under salt-alkali
stress and nitrogen deposition. Sci Total Environ 576:234‒241
Lucini L, D Kane, M Pellizzoni, A Ferrari, E Trevisi, G
Ruzickova, D Arslan (2016). Phenolic profile and in vitro antioxidant power of
different milk thistle [Silybum marianum
(L.) Gaertn.] cultivars. Indust Crops
Prod 83:11–16
Martinelli T, J Andrzejewska, M Salis, L Sulas (2015).
Phenological growth stages of Silybum
marianum according to the extended BBCH scale. Ann Appl Biol 166:53‒66
Merwad ARM (2018). Using humic substances and foliar
spray with Moringa leaf extract to alleviate salinity stress on wheat. In:
Sustainability of Agricultural Environment in Egypt: Part II, pp:265‒286.
Springer, Cham, Switzerland
Munns R (2002). Comparative physiology of salt and water
stress. Plant Cell Environ 25:239‒250
Munns R, M Tester (2008). Mechanisms of salinity
tolerance. Annu Rev Plant Biol 59:651‒681
Niamat B, M Naveed, Z Ahmad, M Yaseen, A Ditta, A
Mustafa, M Rafique, R Bibi, N Sun, M Xu (2019). Calcium-enriched animal manure
alleviates the adverse effects of salt stress on growth, physiology and
nutrients homeostasis of Zea mays L. Plants 8; Article 480
Niste M, R Vidican, I Rotar, V Stoian, R Pop, R Miclea
(2014). Plant nutrition affected by soil salinity and response of Rhizobium
regarding the nutrients accumulation. ProEnvironment 7:71‒75
Omidbaigi R, A Nobakht (2001). Nitrogen fertilizer
affecting growth, seed yield and active substances of milk thistle (Silybum
marianum). Pak J Biol Sci 4:1345‒1349
Polash MAS, MA Sakil, M Tahjib-Ul-Arif, MA Hossain
(2018). Effect of salinity on osmolytes and RWC of selected rice genotypes. Trop
Plant Res 5:227‒232
Saddiq MS, S Iqbal, I Afzal,
AM Ibrahim, MA Bakhtavar, MB Hafeez, Jahanzaib, MM Maqbool (2019). Mitigation
of salinity stress in wheat (Triticum aestivum L.) seedlings through
physiological seed enhancements. J Plant Nutr 42:1192‒1204
Shahid MA, A Sarkhosh, N Khan, RM Balal, S Ali, L Rossi,
C Gómez, N Mattson, W Nasim, F Garcia-Sanchez (2020). Insights into the
physiological and biochemical impacts of salt stress on plant growth and
development. Agronomy 10;
Article 938
Školníková M, P Škarpa, P Ryant (2019). Effect of
nitrogen fertilization on yield and quality of milk thistle [Silybum marianum L. (Gaertn.)] achenes. J Elelmentol 24:701‒710
Taiz L, E Zeiger, IM Mřller, A Murphy (2015). Plant Physiology and Development, 6th
edn. Sinauer Associates Inc. Press, Sunderland, Massachusetts, USA
Tendon HLS (1993). Methods of Analysis of Soil,
Plants, Water and Fertilizers. Fertilization Development and Consultation
Organization, New Delhi, India
Volkov V, MJ Beilby (2017). Editorial: Salinity
tolerance in plants: Mechanisms and regulation of ion transport. Front Plant Sci 8; Article 1795
Wahid A, SMA Basra, M Farooq (2017). Thiourea: A
molecule with immense biological significance for plants. Intl J Agric Biol 19:911‒920
Yoshida S, DA Forno, JH Cock, KA Gomez (1976). Laboratory
Manual for Physiological Studies of Rice. International Rice Research
Institute (IRRI), Los Banos, The Philippines
Zahra N, A Wahid, K Shaukat, T Rasheed (2020). Role of
seed priming and foliar spray of calcium in improving flag leaf growth, grain
filling and yield characteristics in wheat (Triticum aestivum) – a field
appraisal. Intl J Agric Biol 24:1591‒1600
Zahra N, S Mahmood, ZA Raza (2018). Salinity stress on
various physiological and biochemical attributes of two distinct maize (Zea
mays L.) genotypes. J Plant Nutr 41:1368‒1380