Effect of zinc and ascorbic acid on the agronomic characteristics of medicinal plant of purple conef by Journal of Research in Ecology

Journal of Research in Ecology

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An International Scientific Research Journal

Original Research

Journal of Research in Ecology

Effect of zinc and ascorbic acid on the agronomic characteristics of medicinal plant of purple coneflower under drought stress conditions in Khuzestan Authors: Ali Hasani1 and Maryam Hasani2

ABSTRACT: An experiment was conducted as factorial split plot based on completely 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-ethe 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:

Web Address: http://ecologyresearch.info/ documents/EC0351.pdf

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 Dates: Received: 09 Apr 2017

Accepted: 29 Apr 2017

Published: 25 May 2017

This article is governed by the Creative Commons Attribution License (http://creativecommons.org/ licenses/by/4.0), which gives permission for unrestricted use, non-commercial, distribution and reproduction in all medium, provided the original work is properly cited.

Journal of Research in Ecology An International Scientific Research Journal

700-714| JRE | 2017 | Vol 5 | No 1

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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

mental factors can have a negative effect on its growth

chemical fertilizers and supplying the

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): 700â&#x20AC;&#x201C;714

Hasani and Hasani, 2017 Table 1. The physical and chemical properties of soil in experimental site Soil texture

Sandy Loam

7.93

Electrical conductivity (dS/m) 1.91

Organic matter (%)

Nitrogen (mg kg-1)

Phosphorous (mg kg-1)

Potassium (mg kg-1)

Zinc (mg kg-1)

0.92

0.09

490

0.96

evaporation from class A pan (control), a2: irrigation af-

samples were turned into powder and the amount of zinc

ter 120 mm evaporation from class A pan and a3: irriga-

was estimated from the plant extracts by digestion meth-

tion after 170 mm evaporation from class A pan, and the

od. To determine the number of stomata, along with the

foliar application of zinc sulfate at two levels: b1: no

pollination stage, the fifth upper leaf per plant and in all

application (control) and b2: zinc sulfate with the concen-

treatments (three leaves from each treatment) were se-

tration of five per thousand and the foliar application of

lected and transferred to the Laboratory after separation

ascorbic acid at four levels; c1: no application, c2: 50

from the mother plant. The leaves were first washed by

ppm, c3: 100 ppm and c4: 150 ppm. The ground of design

the clean water and after dewatering, a solution was pre-

consisted of 72 plots with dimensions of 2×1.5 m that in

pared by liquid paste with white alcohol solution and

each plot three planting rows were considered as furrow

apply a thin layer of solution on the leaves by brush and

with the distance of 45 cm and one planting line in side

after drying the sample

of each other and also 1.5 m distance for the main plots

using the adhesive tape and then transferred on the lam,

and 2 m distance for experimental repetitions were con-

in each lam the number of stomata of five microscopic

sidered. The distribution of basic fertilizers needed was

view areas were counted and its mean were recorded.

conducted based on the results of soil analysis (Table 1). The foliar application of zinc sulfate element was conducted in the early morning before sunrise at 7-6 leaf stage of purple coneflower with a concentration of five

studied, it was wrapped by

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:

per thousand and the irrigation was conducted after foliar

CGR = W2-W1 / t2-t1 (g.m-2. Day-1)

application. The foliar application of ascorbic acid was

RGR = lnW2- lnW1 / t2-t1 (g.g-1.day-1)

conducted in the early morning before sunrise and before

NAR = LAI / CGR

the advent of Echinacea flowers (end of the growth peri-

where ‘W2 and W1’ were the dry weight of plants in the

od and before appearance of flowers) and for the control

last sampling and the sampling before it and ‘t2 and t1’

it was carried out by distilled water. The time of applying

were the time of these samplings. To determine the dry

the stress was after transferring the seedling to the main

weight in each sampling, the plant was dried for 48 hours

ground. In order to eliminate the marginal effects, two

in an electric oven at a temperature of 75°C and then

marginal rows were considered and also two plants from

samples were weighed with the help of a 0.001 digital

the beginning and end of each of remaining rows were

scale. The relative moisture content in the third leaf from

removed. In this study, the characteristics such as stem

the end of plant was taken in the middle of applying the

diameter, number of flowering branches, fresh plant

stress and the fresh weight of leaf was immediately deter-

yield, leaf area index that to measure it, at first the sam-

mined. Then, the leaves were saturated in the test tubes

pling was conducted from each experimental unit and the

for five hours by using the distilled water and re-

relationship between leaf area and weight was used. To

weighted. Then, the leaves were placed in the oven at 75°

determine the zinc

C for 24 hours and after drying, the dry weight of leaves

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

was determined and RWC was calculated. 702

Hasani and Hasani, 2017 RWC = (wf-wd) / (wt-wd) × 100

in the complete irrigation condition with 10.65 and 10.19

In this equation, ‘wf’ is the fresh weight of plant sample,

mm, respectively. The lowest diameter was related to the

‘wd’ is the dry weight and ‘wt’ is the saturated weight.

treatment with no foliar application of zinc sulfate and

The experiment was conducted in Sardasht at

ascorbic acid in the irrigation condition after 170 mm

Khuzestan after preparing the ground of farm and green-

evaporation from pan with 6.44 mm (Table 4). Decreas-

house. Purple coneflower seeds were planted in the rows

ing the cell division and enlargement result from the

with a distance of 10 cm and depth of 2 to 3 cm (5 March

drought stress (Cabuslay et al., 2002) that also decreases

2016) in the plant nursery. The seeds were soaked for 10

the stem diameter. Increasing the stem diameter in the

days before planting. The seeds were gradually germinat-

presence of zinc is due to the more production of trypto-

ed after 15 days with the regular and timely irrigation and

phan and growth hormones of auxin (IAA). By increas-

weed control of nursery surface. Due to the initial slow

ing IAA, more

growth of this plant, the weeds of nursery surface were

photosynthesis increases. Zinc also affects the metabo-

weeded by hands several times during the growth. On 20

lism of DNA and RNA and is involved in the production

June, the seedlings (3-4 leaf stage) in the rows with the

of nucleic acids. The presence of nucleic acids is neces-

distance of 45 cm and two-plant distance per row of 25

sary for cell growth and proliferation, so the zinc affects

cm and in the same conditions in terms of soil properties

the expansion of cells and their division and elongation

chlorophyll is made and the amount of

were transferred to the main land with an area of 685 m .

in meristematic tissues and ultimately the plant growth

The foliar application of zinc sulfate was conducted at 6-

(Malakooti et al., 1999). Smirnoff (2000) believed that

7 leaf stage of the plant. The sample size was 10% of the

ascorbic acid as a small molecule but with high physio-

plants of each experimental unit.

logical potential can induce the material making process-

Analysis of variance and mean comparison were

es, especially making the sugars in a way that ultimately

carried out and Duncan and LSD tests were also deter-

supply the growth (Barkosky and Einhelling, 2003).

mined at the appropriate probability level. The statistical

The number of flowering branches

software of SAS version 1.9 was used to analyze the data and Excel was used to draw the charts and figures.

The mean comparison for the interaction of drought stress and foliar application of zinc sulfate showed that the highest number of flowering branch was

RESULTS AND DISCUSSION

related to zinc sulfate foliar application treatment under

The results of analysis of variance of the meas-

complete irrigation condition with 8.1 flowering branch-

ured characteristics and calculated components are

es. The lowest number was related to the treatment of no

shown in Table 2.

zinc sulfate foliar application under the condition of irri-

Stem diameter

gation after 170 mm evaporation from pan with 2.23

Mean comparisons of tripartite interaction of

flowering branch (Table 3). In the ascorbic acid foliar

drought stress and foliar application of zinc sulfate and

application treatment, the highest flowering branch was

foliar application of ascorbic acid showed that the highest

related to 100 and 150 ppm ascorbic acid with 5.8 and

stem diameter was obtained by foliar application of

5.83 flowering branch, respectively. The lowest number

ascorbic acid along with foliar application of zinc sulfate

with 4.3 flowering branches was related to the treatment

in complete irrigation condition with 10.79 mm that had

of no ascorbic acid foliar application that had no signifi-

no significant difference with 100, 150 and 50 ppm of

cant difference with foliar application of 50 ppm ascorbic

ascorbic acid along with foliar application of zinc sulfate

acid, with 4.87 branches (Figure 1). The stress decreases

703

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 S.O.V.

2 2 4 1 3 2 6 3

Relative water content 0.3505 ns 2833.709** 2.331 516.2755** 49.8558** 35.4709** 1.1744 ns 1.375 ns

1.494 ns

0.03818 ns

0.80179 9.13

1.44146 6.26

Replication Water stress Error Zinc sulfate Ascorbic acid Stress× zinc sulfate Stress× ascorbic acid zinc sulfate× ascorbic acid Stress× zinc sulfate× ascorbic acid Error cv%

Zinc concentration

diameter of stem

0.9669 ns 1936.2969** 48.5878 5605.993** 0.5426 ns 146.7884** 0.01587 ns 0.0278 ns

0.9824 ns 16.97** 0.1085 30.7982** 3.4161** 1.4654** 0.0701 ns 0.0128 ns

Number of flowering branch 0.0762ns 48.0937** 0.6762 168.667** 5.646** 4.7801** 0.0387 ns 0.2572 ns

0.1312**

Leaf area index

Stomata density

0.0521ns 14.8301** 0.0202 1.7087** 1.013** 0.1252 ns 0.0063 ns 0.0631 ns

0.6709** 10.6834** 3.2288 37.845** 4.9581** 0.5487** 0.0116 ns 0.0246 ns

0.0173 ns

0.0149 ns

0.0606 ns

0.08487 6.64

0.10609 4.55

CGR4 1.63793* * 58.88183** 0.13199 980.72823** 9.84586* * 1.38855* * 0.191427ns 0.01524ns

CGR5 0.70625* * 55.0139** 0.19324 73.66754** 7.92851* * 9.50842n s 0.107318ns 0.071261ns

S.O.V.

Replication Water stress Error Zinc sulfate Ascorbic acid Stress× zinc sulfate Stress× ascorbic acid zinc sulfate× ascorbic acid Stress× zinc sulfate× ascorbic acid Error cv%

2 2 4 1 3 2 6 3

Proline 0.25413** 386.8166** 0.7682 176.635** 1.0551** 35.197** 0.0868ns 0.125ns

CGR1 0.28625* 18.93792** 0.45729 101.29389** 4.96037** 2.13597* * 039398ns 0.053518ns

0.03345 0.30466 4.59 6.74 Ms CGR2 CGR3 0.36430* 0.96555* ** 20.5668 56.79337** 0.33951 1.27082 97.76680** 798.46740** 4.71902** 9.35822** 2.61263n s 2.26885ns ns 0.08180 0.02538n s ns 0.02754 0.10424 ns

0.0321ns

0.061157ns

0.03837ns

0.089595 ns

0.05437ns

0.073116ns

0.04595 4.55

0.07647 6.64

0.10412 6.74

0.20454 4.59

0.30143 6.26

0.061466 9.13

RGR3 0.00108** 0.011881** 0.000739 0.05578** 0.00879** 0.002566**

RGR5 0.00066 ns 0.00075* 0.00054 0.00082* 0.00036 ns 0.00048 ns

NAR1 1.84993 ns 3.22438* 2.27651 59.11468** 0.86096 ns 0.88112 ns

S.O.V. Replication Water stress Error Zinc sulfate Ascorbic acid Stress× zinc sulfate S.O.V. Replication Water stress Error Zinc sulfate Ascorbic acid Stress× zinc sulfate Stress× ascorbic acid zinc sulfate× ascorbic acid Stress× zinc sulfate× ascorbic acid Error cv%

df 2 2 4 1 3 2

RGR1 0.010508 ns 0.035752* 0.015334 0.170528** 0.029678* 0.00155 ns

RGR2 0.00782** 0.03378** 0.008911 0.03046** 0.021640** 0.000726 ns

df 2 2 4 1 3 2 6

NAR2 0.10782* 0.44121** 0.19963 81.97547** 1.61762** 2.41715 ns 0.01376 ns

NAR3 0.12281 ns 10.69456** 0.17141 72.85049** 2.68033** 0.71001** 0.010327 ns

Ms RGR4 0.00122** 0.00392** 0.00047 0.01022** 0.00831** 0.000253 ns Ms NAR4 NAR5 0.30226** 0.78333** ** 8.94149 3.30595** 0.08619 0.30040 37.06318** 29.41573** 1.71915** 1.55535** ns 1.32498 0.20962 ns 0.11715** 0.026238 ns

0.03378 ns

0.00791 ns

0.02723 ns

0.07035 ns

123.569 ns

0.01430 ns

0.02668 ns

0.024661 ns

0.02723 ns

1760.168 ns

0.03249 8.79

0.11379 22.68

0.026271 7.99

0.13269 10.25

8739.22 4.91

Fresh yield of plant 32482.096 ns 1381458.776** 50666.114 7238293.373** 217134.325** 43297.623* 2200.22 ns

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 Stress 70 mm 120 mm 170 mm

Microelement Control With ZnSO4 Control With ZnSO4 Control With ZnSO4

Fresh weight of bush )Kg/ha( 827.93 1504.42 545.16 1167.32 432.15 1032.83

Proline concentration (mg/g fresh weight) 0.08 0.279 3.137 1.36 5.33 10.16

Relative water content of leaf 71.58 81.65 71.25 75.94 55.47 58.95

Number of flowering branch

Stomata density

4.3 8.1 3.23 7 2.23 4.57

15.87 17.33 15.33 17 14.67 16

Zinc concentration (mg/g dry weight) 23.51 45.76 16.56 34.96 10.59 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 the different levels of drought stress in ascorbic acid in microelement on the characteristics of the studied micronutrients according to LSD test Diameter Ascorbic Microelement of stem Stress acid (cm)

ar application of zinc sulfate increased LAI so that the

Control 50 ppm 70 mm

100 ppm 150 ppm Control

120 mm

50 ppm 100 ppm 150 ppm Control

170 mm

50 ppm 100 ppm 150 ppm

705

Control With ZnSO4 Control With ZnSO4 Control With ZnSO4 Control With ZnSO4 Control With ZnSO4 Control With ZnSO4 Control With ZnSO4 Control With ZnSO4 Control With ZnSO4 Control With ZnSO4 Control With ZnSO4 Control With ZnSO4

8.13 9.54 8.29 10.19 8.54 10.79 8.68 10.65 7.46 8.55 7.95 9.05 8.27 9.12 8.26 9.09 6.44 7.48 7.18 8 7.63 8.6 7.62 8.49

highest LAI was related to the foliar application of zinc sulfate with 3.32 and the lowest LAI with 2.85 was related to no foliar application (Figure 3). In ascorbic acid foliar application treatment, the highest LAI was related to the application of 100 and 150 ppm ascorbic acid with 3.50 and 3.47, respectively. The lowest LAI with 2.85 was related to no foliar application of ascorbic acid (Figure 4). Decreasing the leaf cell pressure potential as a result of stopping the growth of leaves, decreasing the cell division due to increased amount of abscisic acid and lack of providing photosynthetic products necessary for the growth of leaf have been stated as the most important possible causes for decreasing LAI due to drought stress (Shao et al., 2008). Water deficit decreases the number of leaves per plant and the leaf size and length. Extending the leaf area depends on the leaf turgor, temperature and amounts of Journal of Research in Ecology (2017) 5(1): 700-714

Hasani and Hasani, 2017 a

a b

6 Number of flowering branch of flowering branch Number

bc 5

2.5 b

4 2

LAI LAI

3 2

1.5

1 0.5

0 Control Control

p.p.m 5050ppm

p.p.m 100100 ppm

150 p.p.m 150ppm

0 70 mm 70mm

Ascorbic Acid Ascorbic acidAmount

120 mm 120mm

170 mm 170mm

Evaporationfrom frompan pan Evaporation

Figure 1. Effect of ascorbic acid spray on flowering

Figure 2. Effect of drought stress on leaf area index

branch numbers 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

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

3.3

3.5

3.2

LAI

2.5

3.1 3 2.9

2 1.5 1

0.5

2.8

2.7

‫شاهد‬

Control

2.6 wi thout ZnSO4 Without ZnSO4

Wi th ZnSO4

With ZnSO4

Figure 3. Effect of zinc sulfate on leaf area index Journal of Research in Ecology (2017) 5(1): 700-714

50 p.p.m

50 ppm

100 p.p.m

100 ppm

150 p.p.m

150ppm

Ascorbic acid Ascorbic Acid Amount

Figure 4. Effect of ascorbic acid spray on LAI 706

Hasani and Hasani, 2017 a 17

Fresh Yield (kg/ha) Fresh yield (kg\ha)

Stomata number Stomata number

16.6 16.4 b

16.2 16 15.8 15.6 15.4

Control

50 p.p.m

50ppm

100 p.p.m

100ppm

800 600 400 200 0

150 p.p.m

Control

150ppm

Ascorbic Acid Amount

1000

15.2

Control

1200

16.8

Control

50 p.p.m

50ppm

100 p.p.m

100ppm

150 p.p.m

150ppm

Ascorbic Acid Amount

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

quired for optimum plant growth is provided, even when

cells and the leaf area.

the existing zinc in the plant is more diluted due to high

The zinc concentration in the aerial parts

plant growth and the effect of phosphorus application

The mean comparisons for interaction of

(Malakooti et al., 1999). By using the zinc in corn fields,

drought stress and foliar application of zinc sulfate

Yasrebi et al. (1994) found that the use of zinc on most

showed that the highest amount of zinc in the aerial parts

soil leads to increase the zinc concentration and total

was related to the treatment of foliar application of zinc

uptake of zinc by the corn. In the experiment conducted

sulfate in complete irrigation condition with 45.76 mg/g

by Dehnavi and Sheshbahre (2017) in safflower with

dry weight. The lowest amount of zinc in the aerial parts

foliar application of zinc sulfate increased the zinc con-

were related to the treatment of foliar application of zinc

centration in its aerial parts.

sulfate in the irrigation after 170 mm evaporation from

Fresh weight of aerial parts

pan with 10.59 mg g dry weight (Figure 3). As a result of

The mean comparisons for the interaction of

the water deficit, due to reduced soil water content and

drought stress and foliar application of zinc sulfate

thus reduced food distribution in the soil texture, the nu-

showed that the highest plant yield was related to the zinc

trient absorption by the root decreases. In addition, the

sulfate foliar application treatment in the complete irriga-

transfer of nutrients from root to branches decreases.

tion with 1504.42 kg/ha. The lowest plant yield was re-

Increasing drought stress also causes the difficulties in

lated to no foliar application of zinc sulfate in the irriga-

the transfer of elements into the plant (Cakmak, 2008).

tion after 170 mm evaporation from pan with 432.15 kg/

By adequate intake of zinc in the soil, the amount re-

ha (Table 3). In the ascorbic acid foliar application treata

0.2 b 0.15

(%) RWC(%) RWC

(mg/g FW) Prolin(mg\g FW) Prolin

0.25

0.1 0.05 0 Control

Control

50 p.p.m

50ppm

100 p.p.m

100ppm

150 p.p.m

150ppm

Ascorbic Acid Amount

Ascorbic acid

Figure 7. Effect of ascorbic acid spray on proline

707

78 77 76 75 74 73 72 71 70 69 68

b c

Control

50 p.p.m

50ppm

100 p.p.m

100ppm

Ascorbic Acid Amount

150 p.p.m

150ppm

Ascorbic acid

Figure 8. Effect of ascorbic acid spray on RWC Journal of Research in Ecology (2017) 5(1): 700-714

Hasani and Hasani, 2017 8

70mm

170mm

y1 = -0.9963x 2 + 5.8655x - 2.782 R2 = 0.7758

CGR (g\m2.day) (g/m2.day) CGR

y2 = -0.83x 2 + 4.814x - 2.312 R2 = 0.8057

5 4 3 2

y3 = -0.7366x 2 + 4.1462x - 2.0446 R2 = 0.8903

‫شاهد‬

150 p.p.m

CGR CGR(g\m2.day) (g/m2.day)

120mm

50 p.p.m

y = -0.0041x2 + 0.8575x - 38.507

R2 = 0.731

R2 = 0.7078

6 5 4

y = -0.0041x2 + 0.8636x - 38.286

R2 = 0.692

y = -0.0044x2 + 0.9147x - 40.23

R2 = 0.7078

0 75

105

120

135

Figure 9. Effect of drought stress on CGR 16

Control

110

125

DAP

Figure 10. Effect of non-application ascorbic acid on CGR

With ZnSO4

70mm

120mm

170mm

0.25

14 y2 = -1.830x2 + 11.41x - 5.425 R² = 0.655

y 2= -0.041x + 0.198 R² = 0.924

0.2

(g\g.day) RGR RGR (g/g.day)

CGR(g\m2.day) (g/m2.day) (g\m2.day) CGR CGR

y = -0.0044x + 0.9147x - 40.23

8 6 4 y1 = -0.995x2 + 5.862x - 2.777 R² = 0.775

105

120

0.1 0.05 0

50 p.p.m

135

Figure 12. Effect of drought stress on RGR

100 p.p.m

150 p.p.m

‫شاهد‬

y 3= -0.064x + 0.3278 R2 = 0.9128

Zn ‫با‬

y4 = -0.0643x + 0.3295 R2 = 0.905

0.15 y1 = -0.0453x + 0.2229 R2 = 0.9325 y2 = -0.0531x + 0.2653 R2 = 0.8955

0.25 (g\g.day) RGR RGR (g\g.day) RGR(g/g.day)

0.2

0.05

120

0.3

0.25

0.1

105 DAP

Figure 11. Effect of zinc sulfate spray on CGR Control

-0.05

DAP

0.3

y3 = -0.035x + 0.160 R² = 0.922 75

135

y1 = -0.048x + 0.235 R² = 0.940

0.15

(g\m2\day) RGR RGR (g/m2/day)

100 p.p.m 2

y2 = -0.058x + 0.2984

0.2

R2 = 0.9689

0.15 0.1

y1 = -0.045x + 0.222 R² = 0.932

0.05

0 75

105

120

135

-0.05 DAP

Figure 13. Effect of non-application ascorbic acid on

0 -0.05

105

120

135

DAP

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

Hasani and Hasani, 2017 (Figure 6). The sharp decrease leaf length and width and

The specific role of proline is the osmotic pres-

subsequent decrease in leaf area, reduced height, all are

sure adjustment and protects the intracellular oxidative

in order to reduce the plant evaporation level and thus the

under stress and therefore, the proline accumulates in

plant fresh weight yield and photosynthesis decrease. It

plants that are under hard stresses. This result is con-

seems that decreasing the fresh weight in the plants under

sistent with the results obtained by Bayer (2007). Micro-

drought stress conditions due to prevention of the cell

nutrients, especially zinc, play role in osmotic adjustment

development and growth results from decreasing the Tur-

(because of increasing the proline content and soluble

gor pressure (Rane et al., 2001). Turanposhti et al.

sugars). Generally, proline is made of two ways: The

(2006) concluded that the effects of drought stress on

way of glutamate that its enzymes are placed in the cyto-

total fresh and dry weight of saffron was negative and

plasm and the way of ornithine that its enzymes are

significant. Micro-nutrients, particularly zinc, can in-

placed in the mitochondria. Glutamate way is more im-

crease the fresh yield of plants by affecting the

in-

portant in higher plants and it seems that the key en-

crease in the plant assimilates. Increasing the shoot fresh

zymes of the way have shown the positive reactions to

weight as a result of using zinc is due to an increase in

zinc, manganese, iron and copper foliar application

auxin biosynthesis (Khaladbarin and Eslamzadeh, 2001).

(Delauney et al., 1993). Gadallah (2000) showed that

Proline concentration

zinc treatment plays a critical role in improving the soy-

The mean comparisons for the interaction of

bean growth status under osmotic stress. Therefore, in-

drought stress and foliar application of zinc sulfate

creasing the proline and soluble sugars in response to the

showed that the highest proline concentration was related

foliar application of micro-nutrients in water stress con-

to the zinc sulfate foliar application treatment in the irri-

ditions can be attributed to the role of these elements and

gation after 170 mm evaporation from pan with 10.16

their effect in osmotic adjustment.

mg/g fresh weight. The lowest proline concentration was

Relative Water Content (RWC)

related to no foliar application of zinc sulfate in the com-

The mean comparisons for the interaction of

plete irrigation condition with 0.08 mg/g fresh weight

drought stress and foliar application of zinc sulfate

(Table 3). In the ascorbic acid foliar application treat-

showed that the highest RWC was related to the zinc

ment, the highest proline concentration was related to the

sulfate foliar application treatment in the complete irriga-

application of 100 and 150 ppm of ascorbic acid with

tion with 81.65. The lowest RWC was related to no foliar

0.212 and 0.206 mg/g fresh weight, respectively. The

application of zinc sulfate in the irrigation after 170 mm

lowest amount of proline with 0.08 mg/g fresh weight

evaporation from pan with 55.47% (Table 3). In the

was related to no foliar application of ascorbic acid

ascorbic acid foliar application treatment, the highest

(Figure 7). Water stress had a significant effect on

RWC was related to the application of 100 and 150 ppm

growth parameters, the yield of vegetative body, water

ascorbic acid with 76.26 and 77.89 and the lowest RWC

relations, chlorophyll, proline concentration, soluble sug-

with 71.58 was related to no foliar application of ascorbic

ars, amount and yield of basil essential oil (Hasani,

acid (Figure 8). Drought stress reduced the RWC. By

2002). Under drought stress, the plants stop their growth

reducing the water in the soil, the plant minimizes the

and increases the accumulation of solutes in the cells in

amount of water of its body by increasing the osmotic

order to have more access to water. This is called the

material in the tissues to enter the water into the plant

osmotic adjustment (Patakas et al., 2002).

with more power. This resulted in water loss in the tis-

709

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

Hasani and Hasani, 2017 70mm

120mm

Control

170mm

50 p.p.m

100 p.p.m

150 p.p.m

3.5

y 1= -0.190x2 + 0.586x + 2.037 R² = 0.967

2.5

2.5 NAR(g/m2/day) (g/m2/day) NAR

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

2 1.5 1 0.5

y4 = -0.2519x 2 + 0.9869x + 2.3316 R2 = 0.983

y 2= -0.189x2 + 0.620x + 1.643 R² = 0.985

1.5

y3 = -0.133x2 + 0.343x + 1.603 R² = 0.983

0.5

0 75

105

120

135

y1 = -0.19x 2 + 0.584x + 2.039 R2 = 0.967 y2 = -0.2014x 2 + 0.6726x + 2.344 R2 = 0.9942 y3 = -0.2259x 2 + 0.8259x + 2.4576 R2 = 0.9893

0 75

-0.5

105 DAP

DAP

120

135

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

Control

had a lower growth rate. With the increase in leaf area

w ith Zn

and thus better utilization of solar radiation, the amount NAR (g/m2.day) (g/m2.day) NAR

of dry matter production per unit area and growth rate of plant increased. In later stages, CGR decreased rapidly

because of shading the upper parts on the leaves, decreas-

ing the plant photosynthetic power and aging the leaves 2 1

(Gholipoor et al., 2004). In complete irrigation condition,

y1 = -0.1904x 2 + 0.5866x + 2.0372 R2 = 0.9671

CGR is high due to high LAI and dry matter production because some researchers believe that the CGR has a

0 75

105

120

135

DAP

direct

relationship

with

photosynthetic

surface

(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

Hasani and Hasani, 2017 ment of foliar application of 100 and 150 ppm and the 2

Net Assimilation Rate (NAR)

lowest growth rate with 6.77 g/m per day was related to

The highest NAR with 2.643 g/m2 per day was

the treatment of no foliar application (Figure 10). The

related to the control and the lowest with 1.912 g/m2 per

high growth rate in condition of ascorbic acid application

day was related to 170 mm stress treatment (Figure 15).

could be due to high LAI in purple coneflower. The high-

In the early stages of growth, the high NAR was due to

est growth rate with 13.833 g /m2 per day was related to

lower LAI. The leaves were fully exposed to light, so

the zinc sulfate application treatment. The lowest growth

their NAR was at maximum. About 105 days after germi-

rate with 6.73 g/m per day was related to control (Figure

nation, NAR decreased that it more results from an in-

11). The high growth rate in the condition of foliar appli-

crease in LAI and the number of leaves per plant of pur-

cation of micronutrients is due to the role of these ele-

ple coneflower and thus increase in shading among

ments, in particular zinc, in increasing LAI.

them. Balak (1993) stated that NAR decreases by in-

Relative Growth Rate (RGR)

creasing LAI and NAR has a decreasing trend from the

At the beginning of the growing season, RGR was in the

beginning to the end of the growing season. The highest

highest rate in all the cultivars and then, during the

NAR with 3.144 and 3.027 g/m2 per day was related to

growth period, in about 75 days after germination, it

the treatment of application of 100 and 150 ppm and the

gradually began to decrease due to the increased shading.

lowest NAR with 2.12 g/m2 per day was related to the

The highest growth rate with 0.19 g/m2 per day was relat-

treatment of no foliar application that had no significant

ed to control and the lowest with 0.14 g/m2 per day was

difference with the application of 50 ppm ascorbic acid

related to 170 mm stress treatment. Generally, RGR de-

with 2.12 g/m2 per day (Figure 16). The highest NAR

creased over time (Figure 12). At the beginning of the

with 5.3 g/m2 per day was related to the zinc sulfate ap-

growing season, RGR was in the highest rate in all treat-

plication treatment. The lowest NAR with 2.643 g/m 2 per

ments and then, after passing the growth period, in about

day was related to the control (Figure 17).

75 days after planting, it gradually began to decrease. The highest growth rate with 0.098 and 0.095 g/ 2

CONCLUSION

m per day was related to the treatment of application of

According to the results of this study, it can be

100 and 150 ppm and the lowest growth rate with 0.057

stated that the use of ascorbic acid as an antioxidant in-

g/m per day was related to the treatment of no foliar

creases the harmful effects of drought stress on some

application that had no significant difference with the

characteristics studied in purple coneflower and increases

application of 50 mg/l with 0.07 g/m (Figure 13). The

the biological yield of purple coneflower. The zinc sul-

high growth rate in condition of ascorbic acid application

fate foliar application in 6-7 leaf stage of purple cone-

was due to high LAI in purple Coneflower. The highest

flower and applying the moderate irrigation stress after

growth rate with 0.209 g/m per day was related to the

120 mm evaporation from pan can be suitable for pro-

zinc sulfate application treatment. The lowest growth rate

ducing this plant.

with 0.155 g/m2 per day was related to the control (Figure 14). The high growth rate in the condition of foliar application of micronutrients is due to the role of these elements, in particular zinc, in increasing LAI.

ACKNOWLEDGMENT It should be noted that the costs of the projects have been funded and allocated by the Young Researchers Club of Islamic Azad University, Yadegar -e- Imam

711

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

Hasani and Hasani, 2017 Khomeini (RAH) Branch, Shahr-e-Ray, Tehran, Iran. We

FranĂ§a MGC, Thi Pimentel AC, Rossiello ROP, Zuily

appreciate and thank.

-Fodil Y and Laffray D. (2000). Differences in growth and water relations among Phaseolus vulgaris cultivars

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