Journal of Research in Ecology ISSN No: Print: 2319 –1546; Online: 2319– 1554

An International Scientific Research Journal

Original Research

Effect of zinc and ascorbic acid on the agronomic characteristics of

medicinal plant of purple coneflower under drought stress conditions in
Khuzestan
Authors: ABSTRACT:
Journal of Research in Ecology

Ali Hasani1 and An experiment was conducted as factorial split plot based on completely
Maryam Hasani2 randomized block design with three replications at Sardasht in Khuzestan to evaluate the
effect of zinc sulfate and ascorbic acid on some morpho-physiological characteristics of
purple coneflower (Echinacea purpurea) in response to water deficit stress. The
experimental treatments included the water deficit stress as the main factor at three
levels (a1: irrigation after 70 mm evaporation, a2: irrigation after 120 mm evaporation, and
a3: irrigation after 170 mm evaporation from class A pan), and the treatment combination
Institution: included the zinc sulfate at two levels (b1: no application and b2: foliar application of zinc
1. Young Researchers and sulfate with the concentration of five (5) per thousand) and the foliar application of
Elite Club, Yadegar-e-Imam ascorbic acid at four levels (c1: no application, c2: 50 mg/l, c3: 100 mg/l and c4: 150 mg/l) as
Khomeini(RAH) Shahr-e- the secondary factor. The results showed that the application of water deficit stress on
Rey Branch, Islamic Azad purple coneflower caused significant differences in the stem diameter, number of
University, flowering branches, stomata density, zinc concentration of aerial parts, fresh weight yield,
Tehran, Iran RWC, LAI, proline concentration, CGR, RGR and NAR. The mean comparisons for the effect
of drought stress showed that the highest LAI was related to the complete irrigation
2. Department of treatment with 2.85. The mean comparisons for the treatment combination of drought
Microbiology, Science and stress and foliar application of zinc sulfate showed that the highest proline concentration
Research, Islamic Azad belonged to foliar application of zinc sulfate in the condition of irrigation after 170 mm
University, Tehran, Iran evaporation from class A pan with 10.16 mg/g fresh weight and the lowest proline
concentration was related to the treatment of no foliar application of zinc sulfate in
complete irrigation condition with 0.08 mg/g fresh weight. The highest CGR with 6.77 g/
m2/day was obtained in control and the lowest CGR with 4.16 g/m2/day was obtained in
the treatment of 170 mm stress. The highest growth rate with 0.19 g/m 2/day was
obtained in control and the lowest growth rate with 0.14 g/m2/day was obtained in the
treatment of 170 mm stress. The highest NAR with 2.64 g/m2/day belonged to control and
the lowest NAR with 1.91 g/m2/day belonged to the treatment of 170 mm stress.
Corresponding author: Considering the results of this study, the application of ascorbic acid as an antioxidant may
Ali Hasani decrease the harmful effects of drought stress on some characteristics of purple
coneflower including NAR, CGR, RGR, and RWC.

Keywords:
Ascorbic acid, water deficit stress, agronomic characteristics

Email ID:
Article Citation:
Ali Hasani and Maryam Hasani
Effect of zinc and ascorbic acid on the agronomic characteristics of medicinal plant of
purple coneflower under drought stress conditions in Khuzestan
Journal of Research in Ecology (2017) 5(1): 700-714
Web Address:
http://ecologyresearch.info/ Dates:
documents/EC0351.pdf Received: 09 Apr 2017 Accepted: 29 Apr 2017 Published: 25 May 2017

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Journal of Research 700-714| JRE | 2017 | Vol 5 | No 1

in Ecology
An International www.ecologyresearch.info
Scientific Research Journal
Hasani and Hasani, 2017
INTRODUCTION of ascorbic acid can increase the resistance to drought

Purple coneflower with the scientific name of and salinity stress and reduce the effects of oxidative

Monch Echinaceae purpurea (L.) is a perennial and stress (Shatala and Neumann, 2001). Also, ascorbic acid

herbaceous plant. This plant belongs to the Asteraceae plays a role in plant growth processes such as the pro-

family, and North America is reported as its origin cess of cell division and the cell wall expansion based

(Chevallier, 1996). One of the most important properties on the acid assumption (Smirnoff, 2000). Zinc foliar

of this plant is increasing the immunity against application increases the plant height, number of

pathogens, which has led this plant to be used as an branches per plant and shoot dry weight of mung bean

effective drug for the prevention and treatment of many and also the foliar application of zinc, manganese and

diseases such as colds, flu and infections (Li, 1998). All iron significantly increase the parameters of growth,

body of purple coneflower including the root and the yield and components of the plant (Thalooth et al.,

vegetative body contain the valuable active ingredients 2006). In a study, Hasani and Shrifabad (2003) observed

such as alkyl amide compounds, polysaccharide that in peppermint the foliar application with the micro-

compounds, as well as essential oils (Gruenwald et al., nutrients produced the highest dry yield and essential oil

1998). Providing the appropriate fertile soil by the yield per unit area. Meanwhile, the limiting environ-

balanced use of chemical fertilizers and supplying the mental factors can have a negative effect on its growth

nutrients needed by plants is one of the important as- and production.

pects of crop management for obtaining the maximum Drought is considered as one of the most im-

yield and the optimized quality of crops and decreasing portant inhibitors of plant production in many arid and

their harmful effects on the environment (Chaudhry and semi-arid parts of the world (Reddy et al., 2004). In

Sarwar, 1999). Zinc is absorbed by the plant roots as their study on Marigold, Shubhra et al. (2004) found

divalent ion. Zinc forms and activates a number of en- that the height and number of flowers per plant de-

zyme systems (Malakouti, 2005). One of the major tasks creased under drought stress. Increasing the stress in-

of zinc in the plant is the protein synthesis so that its creases the Proline and decreases leaf water potential

lack decreases the protein and increases the free amino and relative water content in Savory (Satureja hortensis

acids and amides. By increasing the zinc, the production L.) (Bahernik, 2004). Ghorbanli et al. (2010) studied the

of tryptophan and growth hormone of auxin (IAA) in- effects of drought and ascorbic acid on two rapeseed

creases because the auxin plays a role in photosynthesis, varieties. The research aimed to study the effect of zinc

growth and development of leaves and stems of the sulfate and ascorbic acid on some morpho-physiological

plant (Nasiri et al., 2010). characteristics of purple coneflower in response to water

Ascorbic acid is a small water-soluble antioxi- deficit stress.

dant that plays a role in the detoxification of active

oxygen species, especially hydrogen peroxide. In MATERIALS AND METHODS

addition, it directly plays a role in neutralizing the su- The study was conducted as factorial split plot

peroxide radicals, singlet oxygen, as a secondary antiox- based on the completely randomized block design with

idant in the reproduction of alpha-tocopherol and other three replications that the drought stress was used as the

lipophilic antioxidants (Noctor and Foyer, 1998). In main factor and the combinations of zinc and ascorbic

chloroplast, the ascorbic acid acts as a cofactor for the acid were the secondary factors. The experimental factors

violaxanthin cycle (Smirnoff, 2000). Foliar application included: stress at three levels; a1: irrigation after 70 mm
701 Journal of Research in Ecology (2017) 5(1): 700–714

Table 1. The physical and chemical properties of soil in experimental site
Electrical
Organic
Soil texture pH conductivity
matter (%)
(dS/m)
Sandy Loam 7.93 1.91 0.92
evaporation from class A pan (control), a2: irrigation af-
ter 120 mm evaporation from class A pan and a3: irriga-
tion after 170 mm evaporation from class A pan, and the
foliar application of zinc sulfate at two levels: b1: no
application (control) and b2: zinc sulfate with the concen-
tration of five per thousand and the foliar application of
ascorbic acid at four levels; c1: no application, c2: 50
ppm, c3: 100 ppm and c4: 150 ppm. The ground of design
2
consisted of 72 plots with dimensions of 2×1.5 m that in
each plot three planting rows were considered as furrow
with the distance of 45 cm and one planting line in side
of each other and also 1.5 m distance for the main plots
and 2 m distance for experimental repetitions were con-
sidered. The distribution of basic fertilizers needed was
conducted based on the results of soil analysis (Table 1).
The foliar application of zinc sulfate element was con-
ducted in the early morning before sunrise at 7-6 leaf
stage of purple coneflower with a concentration of five
per thousand and the irrigation was conducted after foliar
application. The foliar application of ascorbic acid was
conducted in the early morning before sunrise and before
the advent of Echinacea flowers (end of the growth peri-
od and before appearance of flowers) and for the control
it was carried out by distilled water. The time of applying
the stress was after transferring the seedling to the main
ground. In order to eliminate the marginal effects, two
marginal rows were considered and also two plants from
the beginning and end of each of remaining rows were
removed. In this study, the characteristics such as stem
diameter, number of flowering branches, fresh plant
yield, leaf area index that to measure it, at first the sam-
pling was conducted from each experimental unit and the
relationship between leaf area and weight was used. To
determine the zinc concentration in the aerial part, after
drying the aerial parts of plant (leaf, stem and flower) the
Journal of Research in Ecology (2017) 5(1): 700-714
Hasani and Hasani, 2017
Nitrogen Phosphorous Potassium Zinc
(mg kg-1) (mg kg-1) (mg kg-1) (mg kg-1)
0.09 15 490 0.96
samples were turned into powder and the amount of zinc
was estimated from the plant extracts by digestion meth-
od. To determine the number of stomata, along with the
pollination stage, the fifth upper leaf per plant and in all
treatments (three leaves from each treatment) were se-
lected and transferred to the Laboratory after separation
from the mother plant. The leaves were first washed by
the clean water and after dewatering, a solution was pre-
pared by liquid paste with white alcohol solution and
apply a thin layer of solution on the leaves by brush and
after drying the sample studied, it was wrapped by
using the adhesive tape and then transferred on the lam,
in each lam the number of stomata of five microscopic
view areas were counted and its mean were recorded.
The amount of proline in the underground parts
were measured by using Bates method (Bates, 1973).
The following equations were used to calculate
the components of growth analysis:
CGR = W2-W1 / t2-t1 (g.m-2. Day-1)
RGR = lnW2- lnW1 / t2-t1 (g.g-1.day-1)
NAR = LAI / CGR
where ‘W2 and W1’ were the dry weight of plants in the
last sampling and the sampling before it and ‘t2 and t1’
were the time of these samplings. To determine the dry
weight in each sampling, the plant was dried for 48 hours
in an electric oven at a temperature of 75°C and then
samples were weighed with the help of a 0.001 digital
scale. The relative moisture content in the third leaf from
the end of plant was taken in the middle of applying the
stress and the fresh weight of leaf was immediately deter-
mined. Then, the leaves were saturated in the test tubes
for five hours by using the distilled water and re-
weighted. Then, the leaves were placed in the oven at 75°
C for 24 hours and after drying, the dry weight of leaves
was determined and RWC was calculated.
702
Hasani and Hasani, 2017
RWC = (wf-wd) / (wt-wd) × 100
In this equation, ‘wf’ is the fresh weight of plant sample,
‘wd’ is the dry weight and ‘wt’ is the saturated weight.
The experiment was conducted in Sardasht at
Khuzestan after preparing the ground of farm and green-
house. Purple coneflower seeds were planted in the rows
with a distance of 10 cm and depth of 2 to 3 cm (5 March
2016) in the plant nursery. The seeds were soaked for 10
days before planting. The seeds were gradually germinat-
ed after 15 days with the regular and timely irrigation and
weed control of nursery surface. Due to the initial slow
growth of this plant, the weeds of nursery surface were
weeded by hands several times during the growth. On 20
June, the seedlings (3-4 leaf stage) in the rows with the
distance of 45 cm and two-plant distance per row of 25
cm and in the same conditions in terms of soil properties
2
were transferred to the main land with an area of 685 m .
The foliar application of zinc sulfate was conducted at 6-
7 leaf stage of the plant. The sample size was 10% of the
plants of each experimental unit.
Analysis of variance and mean comparison were
carried out and Duncan and LSD tests were also deter-
mined at the appropriate probability level. The statistical
software of SAS version 1.9 was used to analyze the data
and Excel was used to draw the charts and figures.
RESULTS AND DISCUSSION
The results of analysis of variance of the meas-
ured characteristics and calculated components are
shown in Table 2.
Stem diameter
Mean comparisons of tripartite interaction of
drought stress and foliar application of zinc sulfate and
foliar application of ascorbic acid showed that the highest
stem diameter was obtained by foliar application of
ascorbic acid along with foliar application of zinc sulfate
in complete irrigation condition with 10.79 mm that had
no significant difference with 100, 150 and 50 ppm of
ascorbic acid along with foliar application of zinc sulfate
703
in the complete irrigation condition with 10.65 and 10.19
mm, respectively. The lowest diameter was related to the
treatment with no foliar application of zinc sulfate and
ascorbic acid in the irrigation condition after 170 mm
evaporation from pan with 6.44 mm (Table 4). Decreas-
ing the cell division and enlargement result from the
drought stress (Cabuslay et al., 2002) that also decreases
the stem diameter. Increasing the stem diameter in the
presence of zinc is due to the more production of trypto-
phan and growth hormones of auxin (IAA). By increas-
ing IAA, more chlorophyll is made and the amount of
photosynthesis increases. Zinc also affects the metabo-
lism of DNA and RNA and is involved in the production
of nucleic acids. The presence of nucleic acids is neces-
sary for cell growth and proliferation, so the zinc affects
the expansion of cells and their division and elongation
in meristematic tissues and ultimately the plant growth
(Malakooti et al., 1999). Smirnoff (2000) believed that
ascorbic acid as a small molecule but with high physio-
logical potential can induce the material making process-
es, especially making the sugars in a way that ultimately
supply the growth (Barkosky and Einhelling, 2003).
The number of flowering branches
The mean comparison for the interaction of
drought stress and foliar application of zinc sulfate
showed that the highest number of flowering branch was
related to zinc sulfate foliar application treatment under
complete irrigation condition with 8.1 flowering branch-
es. The lowest number was related to the treatment of no
zinc sulfate foliar application under the condition of irri-
gation after 170 mm evaporation from pan with 2.23
flowering branch (Table 3). In the ascorbic acid foliar
application treatment, the highest flowering branch was
related to 100 and 150 ppm ascorbic acid with 5.8 and
5.83 flowering branch, respectively. The lowest number
with 4.3 flowering branches was related to the treatment
of no ascorbic acid foliar application that had no signifi-
cant difference with foliar application of 50 ppm ascorbic
acid, with 4.87 branches (Figure 1). The stress decreases
Journal of Research in Ecology (2017) 5(1): 700-714
 Hasani and Hasani, 2017
Table 2. Analysis of variance measured traits on the Echinacea purpurea
Ms
Relative Number of
S.O.V. Zinc con- diameter Leaf area Stomata
df water flowering
centration of stem index density
content branch
Replication 2 0.3505 ns 0.9669 ns 0.9824 ns 0.0762ns 0.0521ns 0.6709**
Water stress 2 2833.709** 1936.2969** 16.97** 48.0937** 14.8301** 10.6834**
Error 4 2.331 48.5878 0.1085 0.6762 0.0202 3.2288
Zinc sulfate 1 516.2755** 5605.993** 30.7982** 168.667** 1.7087** 37.845**
Ascorbic acid 3 49.8558** 0.5426 ns 3.4161** 5.646** 1.013** 4.9581**
Stress× zinc sulfate 2 35.4709** 146.7884** 1.4654** 4.7801** 0.1252 ns 0.5487**
Stress× ascorbic acid 6 1.1744 ns 0.01587 ns 0.0701 ns 0.0387 ns 0.0063 ns 0.0116 ns
zinc sulfate× ascorbic acid 3 1.375 ns 0.0278 ns 0.0128 ns 0.2572 ns 0.0631 ns 0.0246 ns
Stress× zinc sulfate× ascor-
6 1.494 ns 0.03818 ns 0.1312** 0.0173 ns 0.0149 ns 0.0606 ns
bic acid
Error 42 0.80179 1.44146 0.03345 0.30466 0.08487 0.10609
cv% 9.13 6.26 4.59 6.74 6.64 4.55
Ms
S.O.V. df Proline CGR1 CGR2 CGR3 CGR4 CGR5
Replication 2 0.25413** 0.28625* 0.36430* 0.96555* 1.63793* * 0.70625* *
Water stress 2 386.8166** 18.93792** 20.5668 **
56.79337** 58.88183** 55.0139**
Error 4 0.7682 0.45729 0.33951 1.27082 0.13199 0.19324
Zinc sulfate 1 176.635** 101.29389** 97.76680** 798.46740** 980.72823** 73.66754**
Ascorbic acid 3 1.0551** 4.96037** 4.71902** 9.35822** 9.84586* * 7.92851* *
Stress× zinc sulfate 2 35.197** 2.13597* * 2.61263n s 2.26885ns 1.38855* * 9.50842n s
Stress× ascorbic acid 6 0.0868ns 039398ns 0.08180 ns
0.02538n s 0.191427ns 0.107318ns
zinc sulfate× ascorbic acid 3 0.125ns 0.053518ns 0.02754 ns
0.10424 ns 0.01524ns 0.071261ns
Stress× zinc sulfate×
6 0.0321ns 0.061157ns 0.03837ns 0.089595 ns 0.05437ns 0.073116ns
ascorbic acid
Error 42 0.04595 0.07647 0.10412 0.20454 0.30143 0.061466
cv% 4.55 6.64 6.74 4.59 6.26 9.13
Ms
S.O.V.
df RGR1 RGR2 RGR3 RGR4 RGR5 NAR1
Replication 2 0.010508 ns 0.00782** 0.00108** 0.00122** 0.00066 ns 1.84993 ns
Water stress 2 0.035752* 0.03378** 0.011881** 0.00392** 0.00075* 3.22438*
Error 4 0.015334 0.008911 0.000739 0.00047 0.00054 2.27651
Zinc sulfate 1 0.170528** 0.03046** 0.05578** 0.01022** 0.00082* 59.11468**
Ascorbic acid 3 0.029678* 0.021640** 0.00879** 0.00831** 0.00036 ns 0.86096 ns
Stress× zinc sulfate 2 0.00155 ns 0.000726 ns 0.002566** 0.000253 ns 0.00048 ns 0.88112 ns
Ms
S.O.V.
df NAR2 NAR3 NAR4 NAR5 Fresh yield of plant
Replication 2 0.10782* 0.12281 ns 0.30226** 0.78333** 32482.096 ns
Water stress 2 0.44121** 10.69456** 8.94149 **
3.30595** 1381458.776**
Error 4 0.19963 0.17141 0.08619 0.30040 50666.114
Zinc sulfate 1 81.97547** 72.85049** 37.06318** 29.41573** 7238293.373**
Ascorbic acid 3 1.61762** 2.68033** 1.71915** 1.55535** 217134.325**
Stress× zinc sulfate 2 2.41715 ns 0.71001** 1.32498 ns
0.20962 ns 43297.623*
Stress× ascorbic acid 6 0.01376 ns 0.010327 ns 0.11715** 0.026238 ns 2200.22 ns
zinc sulfate×
3 0.03378 ns 0.00791 ns 0.02723 ns 0.07035 ns 123.569 ns
ascorbic acid
Stress× zinc sulfate×
6 0.01430 ns 0.02668 ns 0.024661 ns 0.02723 ns 1760.168 ns
ascorbic acid
Error 42 0.03249 0.11379 0.026271 0.13269 8739.22
cv% 8.79 22.68 7.99 10.25 4.91
ns, *, **: non significant, significant at the 5 and 1% probability, respectively

Journal of Research in Ecology (2017) 5(1): 700-714 704

 Hasani and Hasani, 2017
Table 3. Comparison between the different levels of drought stress on the characteristics of the studied
micronutrients according to LSD test
Fresh Proline Relative Zinc
Number of
weight of concentration water concentration
Stress Microelement flowering Stomata
bush (mg/g fresh content of (mg/g dry
branch density
)Kg/ha( weight) leaf weight)
Control 827.93 0.08 71.58 4.3 15.87 23.51
70 mm
With ZnSO4 1504.42 0.279 81.65 8.1 17.33 45.76
Control 545.16 3.137 71.25 3.23 15.33 16.56
120 mm
With ZnSO4 1167.32 1.36 75.94 7 17 34.96
Control 432.15 5.33 55.47 2.23 14.67 10.59
170 mm
With ZnSO4 1032.83 10.16 58.95 4.57 16 22.88
the photosynthetic production and vegetative develop- the number of flowering branches increase by increasing
ment in the plant. One of the symptoms of vegetative the photosynthetic production. The ascorbic acid can
development is decreasing the number of lateral branches induce the material-making processes, especially the pro-
per plant similar to Beigzadeh and Fatahi (2014) and duction of sugars, in a way that finally the growth is sup-
Valadabadi and Farahani (2011). Decreasing the number plied (Smirnoff, 2000).
of lateral branches due to drought stress has been report- Leaf Area Index (LAI)
ed by Koocheki et al. (2008) in basil and by Hasani The mean comparisons for the effect of drought
(2006) in Badrashbu (Dracocephalum moldavica). The showed that the highest LAI was related to the complete
foliar application with all four zinc sulfate, ferrous sul- irrigation with 2.85 that had a significant difference with
fate, manganese sulfate and copper sulfate fertilizers the irrigation after 120 mm evaporation from pan with a
leads to increase the carbohydrate metabolism and acti- 2.03. The lowest LAI was related to the irrigation after
vate the enzymes (Mezer et al., 2010), so it seems that 170 mm evaporation from pan with 1.28 (Figure 2). Foli-
Table 4. Comparison treatment combination between ar application of zinc sulfate increased LAI so that the
the different levels of drought stress in ascorbic acid highest LAI was related to the foliar application of zinc
in microelement on the characteristics of the studied
micronutrients according to LSD test sulfate with 3.32 and the lowest LAI with 2.85 was relat-

Ascorbic Diameter ed to no foliar application (Figure 3). In ascorbic acid

Stress acid Microelement of stem
(cm) foliar application treatment, the highest LAI was related
Control Control 8.13
With ZnSO4 9.54 to the application of 100 and 150 ppm ascorbic acid with
50 ppm Control 8.29 3.50 and 3.47, respectively. The lowest LAI with 2.85
With ZnSO4 10.19
100 ppm Control 8.54 was related to no foliar application of ascorbic acid
70 mm With ZnSO4 10.79
150 ppm Control 8.68 (Figure 4). Decreasing the leaf cell pressure potential as a
With ZnSO4 10.65
Control Control 7.46 result of stopping the growth of leaves, decreasing the
With ZnSO4 8.55
Control 7.95 cell division due to increased amount of abscisic acid
50 ppm With ZnSO4 9.05
120 mm Control 8.27 and lack of providing photosynthetic products necessary
100 ppm With ZnSO4 9.12
Control 8.26 for the growth of leaf have been stated as the most im-
150 ppm With ZnSO4 9.09 portant possible causes for decreasing LAI due to
Control Control 6.44
With ZnSO4 7.48 drought stress (Shao et al., 2008).
50 ppm Control 7.18
With ZnSO4 8 Water deficit decreases the number of leaves per
170 mm Control 7.63
100 ppm With ZnSO4 8.6 plant and the leaf size and length. Extending the leaf area
Control 7.62
150 ppm 8.49 depends on the leaf turgor, temperature and amounts of
With ZnSO4
705 Journal of Research in Ecology (2017) 5(1): 700-714
Hasani and Hasani, 2017
a a
6 b
3 a
bc
of flowering branch
Number of flowering branch

5
2.5
4 b
2
3

LAI
LAI
1.5 c
2
Number

1
1
0.5
0
Control
Control 5050ppm
p.p.m 100100
ppmp.p.m 150 p.p.m
150ppm 0
Ascorbic Acid 70 mm
70mm 120 mm
120mm 170 mm
170mm
Ascorbic acidAmount
Evaporation
Evaporationfrom
frompan
pan
Figure 1. Effect of ascorbic acid spray on flowering
branch numbers Figure 2. Effect of drought stress on leaf area index
assimilates for growth that all of them are affected by the zinc sulfate foliar application treatment in the com-
drought stress (Reddy et al., 2004). The nutrients develop plete irrigation with 17.33 stomata that had no significant
the leaf area by improving the number and size of leaf difference with the zinc sulfate foliar application treat-
cells. LAI is considered as a quantitative aspect of plant ment in the irrigation after 120 mm evaporation from pan
development and implies an increase in the number and with a 17 stomata. The lowest number was related to no
size of cells that the use of mineral elements, especially foliar application of zinc sulfate in the irrigation after 170
micronutrients, is positively correlated with the increas- mm evaporation from pan with 14.67 stomata (Table 3).
ing trend (Keshmiri et al., 2004). Fathi and In the ascorbic acid foliar application treatment,
Enayatgholizade (2009) reported the increased leaf area the highest number of stomata was related to the applica-
of barley due to foliar application of iron, zinc and cop- tion of 50, 100 and 150 ppm of ascorbic acid with 16.4,
per. Smirnoff (2000) believed that the ascorbic acid can 16.8 and 17 numbers of stomata, respectively. The lowest
induce the material-making processes, especially the pro- number of stomata with 15.87 numbers was related to the
duction of sugars, in a way that finally the growth is sup- treatment of no foliar application of ascorbic acid (Figure
plied. 5). It seems that decreasing the leaf area under drought
Stomatal density in the upper epidermis stress decreased the number of leaf stomata.
The mean comparisons for the interaction of Foliar application of micronutrients and ascor-
drought stress and foliar application of zinc sulfate bic acid increase the number of stomata in the leaves
showed that the highest stomatal density was related to

3.4 a a a
4 ab
3.3 c
3.5
3.2 3
2.5
3.1
2
LAI
LAI

3
1.5
2.9 b 1

2.8 0.5
0
2.7 ‫شاهد‬ 50 p.p.m 100 p.p.m 150 p.p.m
Control 50 ppm 100 ppm 150ppm
2.6
wi thout ZnSO4 Wi th ZnSO4
Ascorbic
Ascorbic acid
Acid Amount
Without ZnSO4 With ZnSO4

Figure 3. Effect of zinc sulfate on leaf area index Figure 4. Effect of ascorbic acid spray on LAI

Journal of Research in Ecology (2017) 5(1): 700-714 706

 Hasani and Hasani, 2017
a
17 a ab a
1200
16.8 c
a

Fresh yield (kg\ha)

16.6 1000 d
Stomata number
Stomata number

Fresh Yield (kg/ha)

16.4 800
16.2 b
16 600
15.8 400
15.6
15.4 200
15.2 0
Control 50 p.p.m 100 p.p.m 150 p.p.m Control 50 p.p.m 100 p.p.m 150 p.p.m
Control 50ppm 100ppm 150ppm Control 50ppm 100ppm 150ppm
Ascorbic Acid Amount
Ascorbic Acid Amount
Ascorbic acid Ascorbic acid
Figure 5. Effect of ascorbic acid spray on numbers of Figure 6. Effect of ascorbic acid spray on fresh grain
stomata yield

considering their role in the growth and development of
cells and the leaf area.
The zinc concentration in the aerial parts
The mean comparisons for interaction of
drought stress and foliar application of zinc sulfate
showed that the highest amount of zinc in the aerial parts
was related to the treatment of foliar application of zinc
sulfate in complete irrigation condition with 45.76 mg/g
dry weight. The lowest amount of zinc in the aerial parts
were related to the treatment of foliar application of zinc
sulfate in the irrigation after 170 mm evaporation from
pan with 10.59 mg g dry weight (Figure 3). As a result of
the water deficit, due to reduced soil water content and
thus reduced food distribution in the soil texture, the nu-
trient absorption by the root decreases. In addition, the
transfer of nutrients from root to branches decreases.
Increasing drought stress also causes the difficulties in
the transfer of elements into the plant (Cakmak, 2008).
By adequate intake of zinc in the soil, the amount re-
quired for optimum plant growth is provided, even when
the existing zinc in the plant is more diluted due to high
plant growth and the effect of phosphorus application
(Malakooti et al., 1999). By using the zinc in corn fields,
Yasrebi et al. (1994) found that the use of zinc on most
soil leads to increase the zinc concentration and total
uptake of zinc by the corn. In the experiment conducted
by Dehnavi and Sheshbahre (2017) in safflower with
foliar application of zinc sulfate increased the zinc con-
centration in its aerial parts.
Fresh weight of aerial parts
The mean comparisons for the interaction of
drought stress and foliar application of zinc sulfate
showed that the highest plant yield was related to the zinc
sulfate foliar application treatment in the complete irriga-
tion with 1504.42 kg/ha. The lowest plant yield was re-
lated to no foliar application of zinc sulfate in the irriga-
tion after 170 mm evaporation from pan with 432.15 kg/
ha (Table 3). In the ascorbic acid foliar application treat-

a
a a 78 b
0.25
77 c
76
FW)

0.2
75
(mg/g FW)

b
RWC(%)
(%)

0.15 74
Prolin(mg\g

c 73 d
RWC

0.1 72
71
Prolin

0.05 70
69
0 68
Control 50 p.p.m 100 p.p.m 150 p.p.m
Control 50ppm 100ppm 150ppm Control
Control
50 p.p.m
50ppm
100 p.p.m
100ppm
150 p.p.m
150ppm
Ascorbic Acid Amount
Ascorbic Acid Amount
Ascorbic acid Ascorbic acid

Figure 7. Effect of ascorbic acid spray on proline Figure 8. Effect of ascorbic acid spray on RWC

707 Journal of Research in Ecology (2017) 5(1): 700-714

Hasani and Hasani, 2017
8 70mm 120mm 170mm
150 p.p.m ‫شاهد‬ 50 p.p.m 100 p.p.m
10
7 2
y = -0.0044x + 0.9147x - 40.23
y1 = -0.9963x 2 + 5.8655x - 2.782 y2 = -0.83x 2 + 4.814x - 2.312 9 y = -0.0041x2 + 0.8575x - 38.507
R2 = 0.7758 R2 = 0.8057 R2 = 0.731 R2 = 0.7078
6 8
(g/m2.day)
CGR(g\m2.day)

CGR (g\m2.day)
7

(g/m2.day)
5
6
4
5
CGR

y = -0.0041x2 + 0.8636x - 38.286

CGR
3 4
3 R2 = 0.692
2 y3 = -0.7366x 2 + 4.1462x - 2.0446 2
R2 = 0.8903 y = -0.0044x2 + 0.9147x - 40.23 y = -0.0044x2 + 0.9147x - 40.23
1 1
R2 = 0.7078 R2 = 0.7078
0
0
75 90 105 120 135
65 80 95 110 125
DAP
DAP

Figure 10. Effect of non-application ascorbic acid on

Figure 9. Effect of drought stress on CGR CGR
16 Control With ZnSO4 70mm 120mm 170mm
14 0.25
y2 = -1.830x2 + 11.41x - 5.425 y 2= -0.041x + 0.198
12 0.2 y1 = -0.048x + 0.235
R² = 0.655
(g/m2.day)
(g\m2.day)

R² = 0.924
CGR(g\m2.day)

R² = 0.940
10
0.15
(g\g.day)
RGR (g/g.day)

8
CGR
CGR

6 0.1
RGR

4
0.05
y1 = -0.995x2 + 5.862x - 2.777 y3 = -0.035x + 0.160
2 R² = 0.922
R² = 0.775
0
0
75 90 105 120 135
75 90 105 120 135
-0.05
DAP DAP

Figure 11. Effect of zinc sulfate spray on CGR Figure 12. Effect of drought stress on RGR

0.3 Control 50 p.p.m 100 p.p.m 150 p.p.m

‫شاهد‬ Zn ‫با‬
0.25 0.3
y 3= -0.064x + 0.3278
R2 = 0.9128 0.25
0.2
y2 = -0.058x + 0.2984
(g/m2/day)
(g\m2\day)

y4 = -0.0643x + 0.3295 0.2

0.15 R2 = 0.9689
(g\g.day)
RGR(g/g.day)

R2 = 0.905
(g\g.day)

0.15
0.1 y1 = -0.0453x + 0.2229
RGR

R2 = 0.9325
RGR

0.1 y1 = -0.045x + 0.222

RGR
RGR

0.05 y2 = -0.0531x + 0.2653 R² = 0.932

R2 = 0.8955 0.05
0
75 90 105 120 135 0
-0.05 75 90 105 120 135
DAP -0.05
DAP

Figure 13. Effect of non-application ascorbic acid on

Figure 14. Effect of zinc sulfate spray on RGR
RGR

ment, the highest plant yield was related to the application and 1047.17 kg/ ha, respectively. The lowest fresh weight
of 100 and 150 mg of ascorbic acid per liter with 1046.27 yield was related to no foliar application of ascorbic acid

Journal of Research in Ecology (2017) 5(1): 700-714 708


(Figure 6). The sharp decrease leaf length and width and
subsequent decrease in leaf area, reduced height, all are
in order to reduce the plant evaporation level and thus the
plant fresh weight yield and photosynthesis decrease. It
seems that decreasing the fresh weight in the plants under
drought stress conditions due to prevention of the cell
development and growth results from decreasing the Tur-
gor pressure (Rane et al., 2001). Turanposhti et al.
(2006) concluded that the effects of drought stress on
total fresh and dry weight of saffron was negative and
significant. Micro-nutrients, particularly zinc, can in-
crease the fresh yield of plants by affecting the in-
crease in the plant assimilates. Increasing the shoot fresh
weight as a result of using zinc is due to an increase in
auxin biosynthesis (Khaladbarin and Eslamzadeh, 2001).
Proline concentration
The mean comparisons for the interaction of
drought stress and foliar application of zinc sulfate
showed that the highest proline concentration was related
to the zinc sulfate foliar application treatment in the irri-
gation after 170 mm evaporation from pan with 10.16
mg/g fresh weight. The lowest proline concentration was
related to no foliar application of zinc sulfate in the com-
plete irrigation condition with 0.08 mg/g fresh weight
(Table 3). In the ascorbic acid foliar application treat-
ment, the highest proline concentration was related to the
application of 100 and 150 ppm of ascorbic acid with
0.212 and 0.206 mg/g fresh weight, respectively. The
lowest amount of proline with 0.08 mg/g fresh weight
was related to no foliar application of ascorbic acid
(Figure 7). Water stress had a significant effect on
growth parameters, the yield of vegetative body, water
relations, chlorophyll, proline concentration, soluble sug-
ars, amount and yield of basil essential oil (Hasani,
2002). Under drought stress, the plants stop their growth
and increases the accumulation of solutes in the cells in
order to have more access to water. This is called the
osmotic adjustment (Patakas et al., 2002).
709
Hasani and Hasani, 2017
The specific role of proline is the osmotic pres-
sure adjustment and protects the intracellular oxidative
under stress and therefore, the proline accumulates in
plants that are under hard stresses. This result is con-
sistent with the results obtained by Bayer (2007). Micro-
nutrients, especially zinc, play role in osmotic adjustment
(because of increasing the proline content and soluble
sugars). Generally, proline is made of two ways: The
way of glutamate that its enzymes are placed in the cyto-
plasm and the way of ornithine that its enzymes are
placed in the mitochondria. Glutamate way is more im-
portant in higher plants and it seems that the key en-
zymes of the way have shown the positive reactions to
zinc, manganese, iron and copper foliar application
(Delauney et al., 1993). Gadallah (2000) showed that
zinc treatment plays a critical role in improving the soy-
bean growth status under osmotic stress. Therefore, in-
creasing the proline and soluble sugars in response to the
foliar application of micro-nutrients in water stress con-
ditions can be attributed to the role of these elements and
their effect in osmotic adjustment.
Relative Water Content (RWC)
The mean comparisons for the interaction of
drought stress and foliar application of zinc sulfate
showed that the highest RWC was related to the zinc
sulfate foliar application treatment in the complete irriga-
tion with 81.65. The lowest RWC was related to no foliar
application of zinc sulfate in the irrigation after 170 mm
evaporation from pan with 55.47% (Table 3). In the
ascorbic acid foliar application treatment, the highest
RWC was related to the application of 100 and 150 ppm
ascorbic acid with 76.26 and 77.89 and the lowest RWC
with 71.58 was related to no foliar application of ascorbic
acid (Figure 8). Drought stress reduced the RWC. By
reducing the water in the soil, the plant minimizes the
amount of water of its body by increasing the osmotic
material in the tissues to enter the water into the plant
with more power. This resulted in water loss in the tis-
Journal of Research in Ecology (2017) 5(1): 700-714
Hasani and Hasani, 2017
70mm 120mm 170mm Control 50 p.p.m 100 p.p.m 150 p.p.m
3
3.5

2.5 y 1= -0.190x2 + 0.586x + 2.037 y4 = -0.2519x 2 + 0.9869x + 2.3316

R² = 0.967 3 R2 = 0.983

2 2.5

NAR(g/m2/day)
(g/m2/day)
(g/m2.day)
(g/m2.day)

1.5 2
y 2= -0.189x2 + 0.620x + 1.643 y1 = -0.19x 2 + 0.584x + 2.039
R² = 0.985 1.5 R2 = 0.967
1
NAR

NAR
NAR

y2 = -0.2014x 2 + 0.6726x + 2.344

y3 = -0.133x2 + 0.343x + 1.603 1
R2 = 0.9942
0.5
R² = 0.983
0.5 y3 = -0.2259x 2 + 0.8259x + 2.4576
0 R2 = 0.9893
75 90 105 120 135 0
-0.5 75 90 105 120 135
DAP DAP

Figure 15. Effect of drought stress on NAR Figure 16. Effect of ascorbic acid on NAR
sues under drought conditions compared to non-stress ing RWC due to the effect of micro-nutrients has been
conditions. reported. Most likely, the role of ascorbic acid in plant
This result is consistent with the results obtained root development and increasing the soil moisture ab-
by Soriano et al. (2001). Decreasing RWC due to sorption capacity increase the RWC.
drought stress, had a high and positive correlation with Crop Growth Rate (CGR)
soil moisture content (Nautiyal et al., 2002). Decreasing CGR was somewhat similar in all treatments.
the growth and root activity and increasing the rate of Thus, at the beginning of the season CGR was slowly
evapotranspiration of the plant community are known as increased and then faster reached to its maximum, and
the factor involved in reducing RWC (Tarumingkeng and then decreased. The highest CGR with 6.773 g m2 per
Cotto, 2003). (França et al., 2000) reported that the RWC day was related to control and the amount CGR with 4.16
in bean is reduced by drought. The role of micronutrients g m2 per day was related to the treatment of 170 mm
in the osmotic adjustment and the preservation of plant stress (Figure 9). The high growth rate in complete irriga-
water potential has the highest effect in keeping the leaf tion condition was due to high LAI and subsequently
moisture and showed the highest RWC. In the research high dry weight production. In the early growth, due to
conducted by Hamrahi et al. (2008) on rapeseed, increas- lack of vegetation and low radiation absorption, the plant

6 Control w ith Zn
had a lower growth rate. With the increase in leaf area
and thus better utilization of solar radiation, the amount
5
of dry matter production per unit area and growth rate of
plant increased. In later stages, CGR decreased rapidly
NAR (g/m2.day)

4
(g/m2.day)

3
because of shading the upper parts on the leaves, decreas-
ing the plant photosynthetic power and aging the leaves
NAR

2
(Gholipoor et al., 2004). In complete irrigation condition,
y1 = -0.1904x 2 + 0.5866x + 2.0372
1 R2 = 0.9671 CGR is high due to high LAI and dry matter production

0
because some researchers believe that the CGR has a
75 90 105 120 135 direct relationship with photosynthetic surface
DAP
(Sidlauskas and Bernotas, 2003). The highest growth rate
Figure 17. Effect of zinc sulfate spray on NAR with 8.63 and 8.6 g/m2 per day was related to the treat-

Journal of Research in Ecology (2017) 5(1): 700-714 710


ment of foliar application of 100 and 150 ppm and the
2
lowest growth rate with 6.77 g/m per day was related to
the treatment of no foliar application (Figure 10). The
high growth rate in condition of ascorbic acid application
could be due to high LAI in purple coneflower. The high-
est growth rate with 13.833 g /m2 per day was related to
the zinc sulfate application treatment. The lowest growth
2
rate with 6.73 g/m per day was related to control (Figure
11). The high growth rate in the condition of foliar appli-
cation of micronutrients is due to the role of these ele-
ments, in particular zinc, in increasing LAI.
Relative Growth Rate (RGR)
At the beginning of the growing season, RGR was in the
highest rate in all the cultivars and then, during the
growth period, in about 75 days after germination, it
gradually began to decrease due to the increased shading.
The highest growth rate with 0.19 g/m2 per day was relat-
ed to control and the lowest with 0.14 g/m2 per day was
related to 170 mm stress treatment. Generally, RGR de-
creased over time (Figure 12). At the beginning of the
growing season, RGR was in the highest rate in all treat-
ments and then, after passing the growth period, in about
75 days after planting, it gradually began to decrease.
The highest growth rate with 0.098 and 0.095 g/
2
m per day was related to the treatment of application of
100 and 150 ppm and the lowest growth rate with 0.057
2
g/m per day was related to the treatment of no foliar
application that had no significant difference with the
2
application of 50 mg/l with 0.07 g/m (Figure 13). The
high growth rate in condition of ascorbic acid application
was due to high LAI in purple Coneflower. The highest
2
growth rate with 0.209 g/m per day was related to the
zinc sulfate application treatment. The lowest growth rate
with 0.155 g/m2 per day was related to the control
(Figure 14). The high growth rate in the condition of foli-
ar application of micronutrients is due to the role of these
elements, in particular zinc, in increasing LAI.
711
Hasani and Hasani, 2017
Net Assimilation Rate (NAR)
The highest NAR with 2.643 g/m2 per day was
related to the control and the lowest with 1.912 g/m2 per
day was related to 170 mm stress treatment (Figure 15).
In the early stages of growth, the high NAR was due to
lower LAI. The leaves were fully exposed to light, so
their NAR was at maximum. About 105 days after germi-
nation, NAR decreased that it more results from an in-
crease in LAI and the number of leaves per plant of pur-
ple coneflower and thus increase in shading among
them. Balak (1993) stated that NAR decreases by in-
creasing LAI and NAR has a decreasing trend from the
beginning to the end of the growing season. The highest
NAR with 3.144 and 3.027 g/m2 per day was related to
the treatment of application of 100 and 150 ppm and the
lowest NAR with 2.12 g/m2 per day was related to the
treatment of no foliar application that had no significant
difference with the application of 50 ppm ascorbic acid
with 2.12 g/m2 per day (Figure 16). The highest NAR
with 5.3 g/m2 per day was related to the zinc sulfate ap-
plication treatment. The lowest NAR with 2.643 g/m 2 per
day was related to the control (Figure 17).
CONCLUSION
According to the results of this study, it can be
stated that the use of ascorbic acid as an antioxidant in-
creases the harmful effects of drought stress on some
characteristics studied in purple coneflower and increases
the biological yield of purple coneflower. The zinc sul-
fate foliar application in 6-7 leaf stage of purple cone-
flower and applying the moderate irrigation stress after
120 mm evaporation from pan can be suitable for pro-
ducing this plant.
ACKNOWLEDGMENT
It should be noted that the costs of the projects
have been funded and allocated by the Young Research-
ers Club of Islamic Azad University, Yadegar -e- Imam
Journal of Research in Ecology (2017) 5(1): 700-714
Hasani and Hasani, 2017
Khomeini (RAH) Branch, Shahr-e-Ray, Tehran, Iran. We
appreciate and thank.
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