Introduction
Zinc (Zn) is an essential
micronutrient for plant growth and metabolism. It functions as a cofactor to
many enzymes; and therefore plays a key role in various biochemical processes
in plants, such as the metabolism of carbohydrates, lipids, auxins, and nucleic
acids (Wood et al. 2004; Zhang et al. 2016). It performs important functions in many
physiological reactions, such as enzyme activation, protein synthesis, gene
expression and regulation, and reproductive development (Ojeda-Barrios et al. 2014; Mattiello et al. 2015). Zn deficiency severely affects the productivity
and quality of plants (Hafeez et al. 2013). It causes chlorotic stripes and purple shading
on the edges and sheath of maize (Zea
mays L.) seedling leaves (Mattiello et al. 2015), decreases the leaf area of apple (Malus domestica L.) (Fu et al. 2015) and causes biomass reduction in red cabbage (Brassica oleracea L. var. capitata f. rubra) and chickpea (Cicer
arietinum L.) (Hajiboland and Amirazad 2010; Ullah et al. 2019,
2020). In pecan trees, Zn-deficiency-related phenotypic traits are observed as
follows: dwarfing of tree organs, curling of leaves, yellowing of leaves
between veins, shortening of shoot sections, loss of apical dominance, failure
to germinate, delayed bud breaking, resetting and poor development of the root
system (Wood et al. 2004). Further studies show that Zn deficiency could lead
to nutrient element imbalance, decreased leaf chlorophyll content, reduced
palisade parenchyma thickness, and increased stomatal density and pore size in
the foliage of pecan trees (Ojeda-Barrios et al. 2012). In summary, Zn deficiency is a typical
nutritional disorder in pecan trees (Peng et al. 2012; Smith et al. 2012).
Zn fertilization could significantly improve the agronomic traits and
yield of food crops, such as mungbean (Vigna
radiata L.), pea (Pisum sativum
L.), olive (Olea europea L.), apples,
and wheat (Triticum aestivum L.) (Saadati et al. 2013; Rafique et al. 2015; Gomez-Coronado
et al. 2016; Zhang et al. 2016; Haider et al. 2019, 2020). The symptoms of Zn deficiency in young or mature
pecan trees could be alleviated by applying Zn fertilizers within a short
period of time (Heerema et al. 2017; Haider et al. 2018). In pecan trees, Zn fertilization methods include
drip irrigation, soil banding in autumn, and foliar spraying in spring. Foliar
spraying is considered to be superior to the other two strategies (Walworth
2013). The compounds in Zn fertilizers include Zn
sulphate (ZnSO4), Zn oxide (ZnO), Zn nitrate (ZnNO3),
chelated Zn ethylenediaminetetraacetic acid (Zn-EDTA), and Zn trisodium
diethylenetriaminepentaacetate (Zn-DTPA) (Smith et al. 2012). Among these, Zn sulphate is most effective for
young pecan trees when considering economic and practical factors (Núńez-Moreno et al. 2015).
Despite its importance as an essential element, excess Zn can have toxic
effect on plants by disturbing the balance of nutrients and inducing oxidative
stress in plants (Wang et al. 2009). Excess Zn decreases the net photosynthetic rate
transpiration rate, stomatal conductance, and levels of chlorophylls a and b in
tea (Camellia sinensis (L.) O. Kuntze)
plants and young bean (Phaseolus vulgaris
L. cv. Lodi) plants (Vassilev et al. 2011; Mukhopadhyay et al. 2012). In addition, Zn toxicity led to decreased
catalase activity and reduced antioxidative substance activity in young bean
plants and pigeon pea (Cajanus cajan
(L.) Millspaugh) (Rao
and Sresty 2000). Therefore, it is important to apply an
appropriate concentration of Zn fertilizer to prevent Zn toxicity in plants.
A number of previous studies have mainly focused on Zn deficiency or Zn
toxicity in plants and on methods to improve the production of food crops using
Zn fertilizers during the reproductive period. Moreover, the types of Zn
fertilizers and methods of application have received much attention (Ashraf et al. 2013). However, there is limited information on Zn
application for pecan seedlings during the vegetative growth. To explore the
optimum Zn levels, this pot study was conducted to evaluate the effects of
different levels of Zn application on germination, growth, mineral elements
contents, and antioxidant enzyme activity of pecan seedlings with the
hypothesis that Zn application can enhance pecan growth.
Materials and Methods
Seed collection and storage
Healthy pecan seeds, which were
collected from ‘Pawnee’ trees in early October 2017 from Nanjing Green Universe
Pecan Science & Technology Co., Ltd., in Shanbei Village, Luhe District,
Nanjing, Jiangsu Province, China (lat. 31ş52'45"N, long. 119ş9'6"E,
altitude 170 m asl.). All seeds were peeled, dried in a cool and ventilated
room and then stored in refrigerator at 4℃ for cold stratification and
simulating natural dormancy.
Zn treatment and seed germination
The germination study began on
November 18, 2017. A total of 360 pecan seeds were selected and divided into
four treatments with three replicates. Each treatment consisted of a particular
concentration of Zn in the form of ZnSO4·7H2O (Zn
content, 22.57%). The seeds were soaked in solutions containing 10 L of
purified water (Biosafer-30TBA; Soffice (China) Co. Ltd.), 30 mg/L gibberellin
(GA, BR, Q/CYDZ 1315-2008, Sinopharm Chemical Reagent Co., Ltd.), and different
concentrations of ZnSO4 solutions (CK: 0.0 mg/L Zn; Z1:
0.1 mg/L Zn; Z2: 1.0 mg/L Zn; Z3: 10.0 mg/L Zn). The
soaking solutions were placed in plastic buckets for 10 days. While soaking,
the seeds were pressed under the solution’s surface constantly using a sealed
heavy water bag. The soaking solutions were replaced every five days.
The soaked seeds were removed, air dried, and stored in wet sand at 0℃ in
darkness in an artificial climate incubator for 30 days to disrupt seed
dormancy. The seeds and wet sand were placed in hollowed-out plastic crates
(length × width × height = 45.5×31×24 cm) with one layer of seeds covered with
one layer of wet sand. Each layer of wet sand was 10 cm thick. Each seed layer
contained 30 tiled seeds. During the storage period, the moisture of the wet
sand remained at 60~80% and was checked every day. Following this standard,
purified water was used to spray the seeds in a timely manner.
After the storage period, all the seeds were removed and rinsed with tap
water followed by purified water. After air drying, the seeds were transferred
into square plastic flowerpots with round holes at the bottom (length × width ×
height = 49×20×14 cm). Each pot contained a 10 cm thick vermiculite layer to
maintain humectation, and then, 30 seeds were buried in the pot, which was then
sealed with plastic wrap to prevent water loss. Each treatment was carried out
with three replicates. All the pots were placed in an artificial climate
incubator for 30 days. The culture conditions were as follows: light 16 h/d,
light level 400~500 μmol·m-2 s-1, temperature
25/22oC, relative humidity 65/40%.
Measured parameters
After culturing for 30 days, the germination
number of the pecan seeds was determined. Ten young seedlings were randomly
selected from each treatment for analysis of biomass, growth, mineral elements,
and antioxidant enzyme activity. The seedlings were then rinsed with tap water
followed by purified water, and dried in air. After removing the endosperm,
growth parameters were measured for pecan seedlings, which were then stored at -80℃ until further use.
Germination rate: Seed germination rate (%) = (germinated seeds /
total seeds sown) × 100%; total seeds sown = 30; three replicates each
treatment.
Biomass weight: Fresh weight per seedling (g) = total fresh
weight/total number of seedlings per treatment; dry weight per seedling (g) =
total dry weight/total number of seedlings per treatment. Total number of
seedlings per treatment = 10, with three replicates per treatment.
Growth parameters: Growth parameters (shoot and root length and
diameter) were measured with steel tape (5 × 16 mm, Deli, Deli Stationery Co.,
Ltd.) and Vernier calipers (150 ± 0.01 mm, Master Proof, Germany),
respectively. Each growth was determined as the mean value per treatment, and
each treatment contained 10 pecan seedlings, with three replicates each treatment.
Mineral
elements: To measure the seedling mineral
concentrations, five dry seedlings per treatment were sampled and mixed,
immediately blotted and oven-dried at 105℃ for 20 min and then kept at 80℃ for 72
h, with three replicates per treatment. The dried material was ground. To
measure the nitrogen (N), 0.5 g of powdered sample was digested with 10 mL of a
mixed acid solution (v/v, 10:1) consisting of concentrated sulfuric acid
(density 1.84 g/mL, AR) and perchloric acid (70%, AR), and heated at 100~200℃ until boiling to yield a colorless
solution. The boiled solution was dissolved in 5 mL of 5% sulfuric acid and
ultrapure water was added up to a final volume of 25 mL. The total N content
was determined by the indophenol blue colorimetric method in the boiled
solution (Piper 1945). To measure the phosphorus (P),
potassium (K) and zinc (Zn) content, 0.5 g of powdered sample was digested with
10 mL of a mixed acid solution (v/v, 5:1) consisting of concentrated nitric
acid (68%, AR) and perchloric acid (70%, AR), and heated at 100~200℃
until near dryness. The boiled solution was dissolved in 5 mL of 5% dilute
nitric acid, and distilled water was added up to a final volume of 25 mL. The
total P content was measured with ultraviolet absorption spectrophotometry
(UV-mini1240, Island Ferry, Japan) by the molybdenum-antimony-scandium
colorimetric method. The total K and Zn content was measured by a flame atomic
absorption spectrometer (PE900T, USA) (Wallace 1951).
Antioxidant
enzyme activity: For
estimation of the superoxide dismutase (SOD), peroxidase (POD), catalase (CAT)
activities and malondialdehyde (MDA) content, 0.5 g of frozen sample was ground
in liquid N2 and homogenized with 1 mL of 50 mM potassium
phosphate buffer (PBS, pH 7.8). The homogenate in 10 mL of ice-cold PBS was
centrifuged at 4000 rpm for 20 min at 4°C (Wang et al.
2009). The supernatant obtained was used
as the enzyme extract and stored at 4°C. All operations were carried out at 4°C
and completed in one week.
To initiate SOD activity, an
aliquot of 0.01 mL of enzyme extract was mixed with 3 mL of reaction solution
consisting of 50 mM phosphate buffer (pH 7.8), 13 mM L-methionine,
75 μM nitroblue
tebrazolium (NBT) for photochemical reduction, 10 μM EDTA-Na2, 2 μM riboflavin, and distilled water
(15:3:3:3:3:2.5, v/v). The reaction solution was placed in a test tube without
enzyme extract as the control group. The tubes were cultured for 30 min at 4000
Lux light in an illuminated incubator, and the reaction was stopped by covering
with a black cloth. The absorbance of the reaction mixture tube was measured at
560 nm (Liu et al. 2018). SOD activity was calculated as U·g-1
FW (unit of SOD activity per gram of fresh weight).
POD activity was determined following the method described by (Heath and Packer 1968). The mixture for the POD activity assay comprised
50 mL of 0.1 M phosphate buffer (pH
6.0), 28 μL of guaiacol and 19 μL of 30% H2O2.
An aliquot of 20 μL of enzyme extract was reacted with the assay
mixture for 3 min and then, the absorbance was measured at 470 nm. The recorded
activity was calculated as U·g-1·FW min-1 (unit of POD
activity per gram of fresh weight per minute).
CAT activity was estimated by monitoring the decrease in absorbance due to
decomposition of H2O2 at 240 nm. For this assay, 0.1 mL
of enzyme extract was mixed with 3 mL of reaction mixture containing 0.6 mL of
0.1 M phosphate buffer (pH 7.0). A
blank was run simultaneously as described above, substituting the 0.1 mL of
enzyme extract with 0.1 mL of phosphate buffer. CAT activity was expressed as
the change in absorbance per min and calculated as U·g-1·FW min-1
(unit of CAT activity per gram of fresh weight per minute).
%201417-1422_files/image002.jpg)
Fig. 1: Effect of
zinc application on growth of pecan seedling
The MDA content was determined as the lipid peroxidation product levels in
the fresh frozen samples via the method described by (Chen et al. 2003) using a spectrophotometer (UVmini-1240
ultraviolet spectrophotometer, Shimadzu Corporation, Suzhou, China). For this
assay, 0.5 g of frozen sample was ground in 5 mL of 10% trichloroacetic acid
(TCA, w/v) using a mortar and pestle, and then centrifuged at 4000 rpm for 20
min. The supernatant (1 mL) was mixed with 3 mL of 6% thiobarbituric acid (TBA,
w/v). After boiling at 100℃ for 15 min, the mixture was cooled rapidly in
an ice bath. The absorbance values (OD values) were recorded at 450 nm, 532 nm
and 600 nm. The MDA content was calculated using the following formula:
%201417-1422_files/image004.png){kind=small}
%201417-1422_files/image006.png){kind=small}
Vt is the total volume
of the supernatant, Vi is the volume of supernatant used for
analysis, and W is the weight of the sample.
Data analysis
The data were processed using Excel 2016
(Microsoft, Redmond, WA) and analysed using one-way analysis of variance
(ANOVA) technique. In case of significant effect, means were separated using
Tukey’s test at P ≤ 0.05. All the figures were plotted
by Origin Pro, version 9.1, and all the tables were prepared in Microsoft
Office Excel 2016.
Results
Effects
of Zn on pecan seedling growth parameters
Analysed data indicated significant improvement in
biomass and shoot and root length and diameter at 0.1 mg/L Zn (P ≤ 0.05). At 0.1 mg/L Zn, the maximum seedling fresh weight (3.98 g), dry
weight (2.54 g), shoot length (146.07 mm), shoot diameter (2.53 mm), root
length (228.44 mm), and root diameter (4.06 mm) were recorded, despite the
minimum germination rate (87.33%) being observed at this concentration. At high
Zn concentrations (≥1 mg/L Zn), slight inhibition was observed. High
concentrations significantly improved the germination rate (P ≤ 0.05). The maximum germination rate was 93.33%, obtained with 1.0 mg/L Zn
(Table 1 and Fig. 1).
Table
1:
Effect of zinc application on growth traits of pecan seedlings
|
Zinc level (mg/L) |
Biomass weight |
Germination (%) |
Shoot length (mm) |
Shoot diameter (mm) |
Root length (mm) |
Root diameter (mm) |
|
|
Fresh weight (g) |
Dry weight (g) |
||||||
|
0.0 |
3.56 ± 0.16b |
2.25 ± 0.03b |
90.33 ± 1.53ab |
119.95 ± 33.74b |
2.49 ± 0.40a |
148.77 ± 38.79bc |
3.73 ± 0.74ab |
|
0.1 |
3.98 ± 0.20a |
2.54 ± 0.23a |
87.33 ± 2.08b |
146.07 ± 28.17a |
2.53 ± 0.53a |
228.44 ± 179.35a |
4.06 ± 0.73a |
|
1.0 |
3.79 ± 0.22ab |
2.29 ± 0.05b |
93.33 ± 1.53a |
51.89 ± 10.49c |
2.51 ± 0.42a |
138.61 ± 50.64c |
3.93 ± 0.60a |
|
10.0 |
3.69 ± 0.11ab |
2.22 ± 0.04b |
90.33 ± 1.53ab |
35.06 ± 8.15c |
2.13 ± 0.38b |
172.44 ± 44.81b |
3.49 ± 0.63b |
Means ± SD in the same column
with different letters are statistically different from
each other at P ≤ 0.05
according to Tukey’s test
Effects
of Zn on pecan seedling mineral elements
Upon Zn application, Zn, N and K accumulation in
pecan seedlings significantly increased, while the P content decreased (Fig. 2,
P ≤ 0.05). At 10 mg/L Zn, the highest Zn and K levels were 112.57 mg/kg and
1560.89 mg/kg, respectively (Fig. 2A, D), while the highest N content was
9835.14 mg/kg at 0.1 mg/L Zn (Fig. 2B). Application of Zn decreased the
seedling P content. The lowest P content was 6201.73 mg/kg, observed upon
application of 1.0 mg/L Zn (Fig. 2C).
Effects
of Zn on pecan seedling antioxidant enzyme activity
Compared to the controls, the SOD, POD, and CAT
activities and MDA content of pecan seedlings exhibited an increase with
increasing Zn concentration. However, these increasing trends were not
significant (Fig. 3). At 10.0 mg/L Zn, the maximum POD activity and MDA content
were 3235.83 µmol g-1 min-1 and
8.91 µmol g-1, respectively; and
were significantly higher than those at 0.1 and 1.0 mg/L Zn (Fig. 3A, D; P ≤ 0.05). At a Zn concentration of 1.0 mg/L, the SOD and CAT activities
gradually reached their highest values of 563.49 µmol g-1 min-1
and 1550 µmol g-1 respectively (Fig. 3B, C).
%201417-1422_files/image008.png)
Fig. 2: Effect of
zinc application on Zn (A), N (B), P (C) and K (D)
contents in pecan seedlings
Means ± S.E. with different
letters are significantly different from each other at P ≤ 0.05 according to Tukey’s test
Here Zn= Zinc; N= Nitrogen;
P= Phosphorus; K= Potassium
%201417-1422_files/image010.png)
Fig. 3: Effect of
Zn application on activities POD (A), SOD (B), CAT (C) and
MDA (D) contents of pecan seedlings
Means ± S.E. with different
letters are significantly different from each other at P ≤ 0.05 according to Tukey’s test
Here
SOD= Superoxide dismutase;
POD= Peroxidase; CAT= Catalase; MDA= Malondialdehyde
Discussion
In this study, the seedling biomass
increased with 0.1 mg/L Zn application. At this concentration, the shoot and
root length and diameter also improved. No toxicity symptoms were observed.
Studies on woody as well as herbaceous plants have demonstrated that
application of Zn promotes plant growth (Hafeez et al. 2013). In a pot study conducted by (Prom-u-thai et al. 2012) on seedling vigour and
viability using rice (Oryza sativa
L.) as the test crop, Zn priming significantly enhanced the germination rate,
root number and dry weight at up to 5 mM ZnSO4. Based on a
2-year field study, accumulation of Zn has been reported to increase seed yield
significantly for pea (Rafique et al. 2015). The Zn concentration range for 95% maximum pea
yield was 42~53 mg kg-1 in leaves and 45~60 mg kg-1 in
seeds. Our findings were consistent with these results. There were obvious
symptoms of Zn application in pecan indicating that Zn may have a function in
pecan seedlings.
As a response to Zn application, nutrient absorption may enhance or reduce
in plant growth. The pecan seedlings exhibited significantly increased Zn, N
and K levels under a high Zn nutrient level. The results were similar to those
of (Ojeda-Barrios et al. 2014). Ojeda-Barrios reported that the accumulation of
Zn was related to other mineral nutrients. Based on a 3-year study, it was
demonstrated that foliar Zn compounds (ZnNO3, Zn-EDTA, Zn-DTPA)
significantly increased the N, K, Ca, Mg, Fe, Cu, Mn levels in the leaves of
eight-year-old ‘Western Schley’ pecan trees. Usually, Zn fertilizers promote N
absorption in plants (Lošák
2007). For the same reason, during the rapid growth
period of pecan, increased amounts of N and Zn fertilizers are required (Smith et al. 2012). During the growing season, combining zinc
sulphate with urea in foliar applications increased the concentration of Zn
from 0.7 to 1.5 mg per kg of apple tissue (Amiri et al. 2008). There was a slight increase in the N and K
levels in apple leaves between June and July in all the Zn fertilizer
treatments.
As the Zn levels improved, the P content in pecan seedlings decreased. It
was suggested that there was P-Zn antagonism regulation in pecan seedlings.
Wang reported that trees are considered healthy when the P/Zn value is ≤ 100 in the leaves of apple trees (Wang et al. 2010). This regulation may result from the interaction
between P and Zn termed ‘P-induced Zn deficiency’ (Yan et al. 2010). The nutritional disorder could be alleviated by
reducing the P content and increasing the Zn content (Fu et al. 2015). This regulation also occurs in pecan trees. In
orchard nutrient management, commercial pecan growers routinely apply
supplemental nitrogen and zinc. Only 40% routinely apply phosphorus, and fewer
use boron, iron, and copper (Walworth
2013). Eight-year-old ‘Western Schley’ pecan trees with
the best appearance were treated with ZnNO3 (100 mg/L Zn) and
Zn-DTPA (100 mg/L Zn), which led to increases of 73 and 69%, respectively, in
leaf Zn concentration (Ojeda-Barrios et al. 2014). Therefore, it was indicated that ZnNO3
and Zn-DTPA are good options for foliar Zn fertilization in pecan trees.
The results showed that the SOD, POD, CAT activities and MDA content in
the seedlings increased concomitantly with increasing Zn concentrations up to
10 mg/L, with all of these parameters showing a slight increase. This increase
could eliminate the production of reactive oxygen species (ROS). ROS induce
lipid peroxidation and are harmful to plant growth (Candan et al. 2018). Heavy metals usually cause lipid peroxidation in
a concentration-dependent manner (Wójcik et al. 2006). Madhava reported that 6-day-old seedlings of two
pigeon pea (Cajanus cajan (L.)
Millspaugh) cultivars, namely, LRG30 and ICPL87, were studied under excess Zn
and Ni, and the activities of antioxidative enzymes such as SOD, POD and
glutathione reductase (GSHR) registered higher values than the activity of CAT
and antioxidative substances such as ascorbic acid (Vc) and total glutathione (Rao
and Sresty 2000). Under external Zn stress with 0.07~1.12 mM
Zn, the NADH oxidase and POD activity increased in the leaves and roots of
repeseed (Brassica napus) seedlings,
but the SOD, CAT and POD activities decreased (Wang et al. 2009). Tavallali reported that salt stress
significantly increased the activity of antioxidant enzymes, but Zn
supplementation efficiently reduced the adverse effects of salt stress, such as
inducing high oxidative stress and increasing lipid peroxidation, electrolyte
leakage, and lipoxygenase activity to high levels in pistachio (Pistacia vera L. ‘Badami’) seedlings (Tavallali et al. 2010).
Conclusion
The growth of pecan seedlings
increased with low Zn levels (0.1 mg/L Zn), and was inhibited under high Zn
levels. In this study, pecan seedlings exhibited significantly higher Zn, N and
K levels under increased Zn nutrition than control, while P content decreased.
The SOD, POD, and CAT activities and MDA content in the seedlings showed a
slight increase with increasing Zn levels up to 10 mg/L. We inferred that the
increase in antioxidant enzymes activities alleviated the effects of Zn
application. Hence, it could be concluded that Zn supplementation was
beneficial to pecan seedlings.
Acknowledgements
This study was supported by the
Major Integration Projects of Forestry Technological Innovation and Promotion
of Jiangsu Province (LYKJ [2018]05-1) and the Priority Academic Program
Development of Jiangsu Higher Education Institutions (PAPD).
Author Contributions
This work was carried out in
collaboration between all authors. YS collected the literature along with references
pertaining to zinc and wrote the first draft of the manuscript. PT, XZ, ZL and
FC conducted the experiment, collected and analyzed the data while FP and YL
gave the suggestions of the experiment process and improved the English. All
authors read and approved the final manuscript.
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