The Effect of Azotobacter On Growth Indices, Yield and Essence Content Of Two Cultivars of Cumin (Cuminum cyminum L.) Under Salinity

Authors

1 Department of Agricultural Sciences, Payame Noor University (PNU), Iran.

2 Department of Biotechnology, Faculty of Chemistry, University of Kashan, Kashan, Iran.

Abstract

Henghameh Vosoughi Tabar1, Hamid Dehghanzadeh1*,Seid Ali Hosseini Tafreshi2
1- Department of Agricultural Sciences, Payame Noor University (PNU), Iran.
2-Department of Biotechnology, Faculty of Chemistry, University of Kashan, Kashan, Iran.
 
*Corresponding author Email: Dehghanzadeh@pnu.ac.ir
 
               Received: 6 April 2017                                                                                  Accepted: 12 May 20017
 
ABSTRACT
In order to investigate the effect of azotobacter on growth indices,grain yield and essence content of two cultivars of cumin (Cuminum cyminum L.) under saline conditions, an experiment was carried out in a factorial design arranged as randomized complete block with three replications in 2015 at Kashan University, Iran. Two cumin local varieties including Ardestān and Mashhad Ardehal were used. So, in a rudimentary experiment, the tolerance threshold was at 150 and 250 mM of Sodium Chloride for both varieties. The result showed that salinity and inoculation with bacteria had a significant effect on the percentage and germination rate, root length, plant height, and seed vigor. Increased concentrations up to 250 milli molar sodium chloride resulted in a significant reduction in germination percentage and germination rate, root length, stem height, seed vigor, grain yield and essence yield. Also, the results presented that in both varieties and at all levels of salinity,inoculation with bacteria compared to the control, lead to a significant increase in chlorophyll (a, b), carotenoids, catalase, ascorbate peroxidase, plant height, grain yield and essence yield. Overall, Ardestan variety showed higher value for plant height, Chlorophyll (a, b), the carotenoids, catalase, ascorbate peroxidase and essence content compared to Mashhad Ardehal variety. In general, the result of this experiment can be used to decrease salinity effects by azotobacter inoculation and increase grain yield and essence content of cumin.
 Keywords:Azotobacter,Cumin, Essence, Grain yield,Salinity

Keywords


       
 

Original Research

 
   

                 Research on Crop Ecophysiology                                  Vol.12/2, Issue 2 (2017), Pages: 70 - 81

 

 

 
 

 

 

 

 


The Effect of Azotobacter On Growth Indices, Yield and Essence Content Of Two Cultivars of Cumin (Cuminum cyminum L.) Under Salinity

 

Henghameh Vosoughi Tabar1, Hamid Dehghanzadeh1*,Seid Ali Hosseini Tafreshi2

1- Department of Agricultural Sciences, Payame Noor University (PNU), Iran.

2-Department of Biotechnology, Faculty of Chemistry, University of Kashan, Kashan, Iran.

 

*Corresponding author Email: Dehghanzadeh@pnu.ac.ir

 

               Received: 6 April 2017                                                                                  Accepted: 12 May 20017

 

ABSTRACT

In order to investigate the effect of azotobacter on growth indices,grain yield and essence content of two cultivars of cumin (Cuminum cyminum L.) under saline conditions, an experiment was carried out in a factorial design arranged as randomized complete block with three replications in 2015 at Kashan University, Iran. Two cumin local varieties including Ardestān and Mashhad Ardehal were used. So, in a rudimentary experiment, the tolerance threshold was at 150 and 250 mM of Sodium Chloride for both varieties. The result showed that salinity and inoculation with bacteria had a significant effect on the percentage and germination rate, root length, plant height, and seed vigor. Increased concentrations up to 250 milli molar sodium chloride resulted in a significant reduction in germination percentage and germination rate, root length, stem height, seed vigor, grain yield and essence yield. Also, the results presented that in both varieties and at all levels of salinity,inoculation with bacteria compared to the control, lead to a significant increase in chlorophyll (a, b), carotenoids, catalase, ascorbate peroxidase, plant height, grain yield and essence yield. Overall, Ardestan variety showed higher value for plant height, Chlorophyll (a, b), the carotenoids, catalase, ascorbate peroxidase and essence content compared to Mashhad Ardehal variety. In general, the result of this experiment can be used to decrease salinity effects by azotobacter inoculation and increase grain yield and essence content of cumin.

 Keywords:Azotobacter,Cumin, Essence, Grain yield,Salinity

 

Introduction

 

The cumin which is cultivated in arid and semiarid area; rain fed and irrigated, is considered as the second exporting economical medical plant in Iran (Alizadegan, et al., 2011; Haghirsasat, et al., 2011). There are many significant properties for cumin such as diuretic, diaphoretic, appetizer stomach strengthener, mucus enhancer, carminative, anti-cancer, anti-diarrhea controlling female discharge, regulating menstruation in young women, treats diabetes, and increasing lactation (Saeed najad and Rezvanimoghaddam, 2011; Haghirsadat, et al., 2011; Zargari, 1994).

A lot of the cumin planting zones are exposed to salinity. Recently, researchers were able to partially decrease the negative effects of salinity in most types of soil by means of transferring salinity resistance genes to plants or inoculating them in combination with growth stimulant bacteria (Arzanesh, et al., 2012; Rojas-Topias, et al., 2012; Kreps, et al., 2002). The prime example of salinity tolerance mechanisms is mainly due to the result of an increase in antioxidant enzymes such as catalase, ascorbate peroxidase, carotenoids, and chlorophyll protection (Mittova et al., 2002).  Azotobacter not only consolidate nitrogen, but also synthesis the stimulants growth and antibiotics. Azotobacter holds the most active cytochrom oxidase and dismotase systems. According to these properties, these bacteria can perform some utmost roles in plant stimulating growth and optimizing nutrition (Akhter et al., 2012; Koocheki et al., 2008). Also, they can be beneficial to the plants through multiple actions. In addition, it leads to germination strength and seedling vigor. Overall, it causes the base plant growth. It is proven that plants seem more sensitive to salinity in germination phase. Generally, a significant decrease of germination and growth is presented. Therefore, where, the salt density increases, a fall in growth value is observed, which presents proper indices for the tolerance determination rate to salinity with respect to different plants (Hasheminia et al., 2009). In fact, cumin is defined as a modest plant regarding its need for fertilized soil in order to obtain relative resistance to salinity (Kafi, 2002; Tatari, 2004).

Nabizadeh  Marvdasht  et al., (2003) reported that as the salinity increased, the cumin seed performance decreased.

Additionally, other research results showed that applying salinity to cumin created significant negative effects on root length, plant length, and seed vigor (Ekhtiari et al., 2010). Ghorbanli et al., (2012) reported that by increasing sodium chloride density on cumin, proline, catalaise, ascorbate proxidate, and malondialdehyde would  increase. Plants effected synchronously by sodium chloride and ascorbate, represented a high increase rate of proline, catalase, ascorbate proxidate in the sodium chloride density.  Rezaei et al., (2004) reported that applying azotobacter to cumin, caused higher germination rate in saline conditions. Accordingly, the positive effects raised by stimulating the growth of bacteria on the negative effects of salinity decreased upon plant growth. In accordance with the importance of cumin and the effect of soil or water salinity on its growth, yield and essence content; the impact of azetobacter on reducing the negative effects was studied considered essential.

 

Materials and methods

 

In order to investigate the effect of azotobacter on growth indices, grain yield and essence content of two cultivars of cumin (Cuminum cyminum L.) under saline conditions, an experiment was carried out in a factorial design arranged as randomized complete block with three replications in 2015 at Kashan University, Iran. Two cumin local varieties including Ardestān and Mashhad Ardehal were used. First, in a rudimentary randomized completed blocks design experiment with three ,under the condition of in vitro and pot planting, the levels of the variety tolerance to salinity was determined using six levels (0, 100, 150, 200, 250 and 300 (mM) of sodium chloride. The results showed that 150 and 250 mM the tolerance threshold for the tow varieties. Also, (7H
2
O) MgSO
4
solution was used to keep the azotobacter (Pierson, 1955). Based on the rudimentary experiment, salinity at 150 and 250 mM sodium chloride levels was used. Sterilized seeds were soaked for 24 hours in a solution of magnesium sulfate azotobacter. The irrigation and Hoagland fertigation consisting of 150 and 250mM density every 20 days was performed within 5 days regularly per day and based on field capacity. Germinated seeds were counted after planting time and every 5 days during 30 days. Harvesting and other measurements were done within 49 days after bacterial treatment.

 All the characters such as  percentage and rate of germination, root length , plant length, and seed vigor, ascorbate proxidate and catalase enzymes , chlorophyll (a, b), carotenoid, grain yield, essence  percentage and yield essence, were measured. The percentage of germination was calculated as equation (1), (Ellis et al., 1981):

(1) Germination percentage (GP) =    

Where:
 n= the number of germinated seeds, N= total seeds. 
On the other hand, by adding the total numbers of plumul and radical lengths seedling length was calculated and the induced vigor was obtained as equation 2 (, (Abdul-baki and Anderson, 1973) :
(2) Seed vigor indices (VI) = (plant and root) mean seedling length ×GP
          100

The rate of catalase enzyme action was calculated as equation (3), (Aebi, 1984):

(3)  
EA= represents the Enzymes action rate based on unit weight grams (based on revival of one mm H
2
O
2
/min), =based on presented amount by spectrophotometer, A=the spilled amount into Cuvette. B= added amount of phosphate to the sample in order to pulverize, EC= 0.03mM/cm (Catalase enzyme activity coefficient), C= to pulverize tissue weight in grams. 
The ascorbate proxidate activity rates was calculated as follow equation (4), (Nakano and Asada, 1981):
(4)  
EA= represents the enzymes action rate based on unit weight grams (based on ascorbate proxidate absorbtion /MIN), EC= 0.028 mM/cm (ascorbate proxidate enzyme activity coefficient). (Arnon, 1949) and (Lichtenthaler, 1987) method for chlorophyll measurement was performed. Then, Chlorophylls a, b and carotenoid in mg /g were calculated.

 

 

Analysis of variance was done by SPSS software. Means were compared Duncan test at 5% probability level. Figures were drown by Excel were used.
 
Results and discussion
 
   The results of variance analysis showed that salinity and inoculation with bacteria had significant effects on the germination rate and percentage (Table 1). Salinity of 250 mM led to the most decrease in germination (84% compared to the control) (Table 2). Germination percentage in salinity of 150 mM and the control treatments were not significantly differente. The increase of salinity to 250 mM, the significantly decreased germination rate (Table 2). So, inoculation with Azotobacter led to effective germination rate increases compared to control (Table 3). Germination rate increase with bacteria inoculation ( 37% compared to the control) (Table 2). Ekhtiari et al., (2010) also reported that cumin germination rate decreased in salinity conditions. The salinity effect can be adjusted according to plant genotype and varieties (Salami et al, 2006). The results showed that when salinity increased to higher than threshold, the nutrition absorption  disorder and also nitrogen absorption would decrease;in another study  the cumin germination rate and percentage decreased with salinity (Salami et al, 2006). Inoculation with azoobacter leads to significantly germination percentage. Although germination percentage decreased by salinity, inoculating by active bacteria would decreased the effect of salinity. It is potentially proven that bacteria are able to produce and change some plant hormones such as giberellin which plays an important role in germination (Hilhorst and Toorop, 1997). 
    Salinity, inoculating with bacteria and varieties had significant effects on plant and root length (Table 1). 150 mM sodium chloride had the highest root length and the increase was 15.6 percent over the control (Table 2). Increasing sodium chloride concentration to 250 mM led to a significant decrease in root length (Table 2). The presence of bacteria increased the root length up to 26.5% compared to no- inoculation treatment. Plant length variation among treatments was similar to root length (Table 2). Generally under hard salinity stress, plant length will reduce. Also, inoculation with bacteria compared to the control treatment led increase of the plant length by 22.8 percent (Table 2). Plant length and root length are the most effective and important characters in germination process under salinity stress. Root in direct contact with soil; therefore, salinity is considered as an obstacle to the root and plant length growth, because of reduction in water absorption. As the osmosis pressure gets higher, the osmosis potential lessens and therefore, less water feed the seeds (Jamil et al., 2005). Gilck et al., (1998) stated that rhyzobacteria have the power of production of ACC di-aminase cause an in increase in the root length through a reduction of ethylene levels.
   Seed vigor was affected by salinity, variety ,bacteria, salinity × variety, , salinity × bacteria. (Table 1). sodium chloride increased of to 250 mM, led to significantly decrease of seed vigor (Table 2). The control and 150 mM salinity treatment with inoculation by bacteria had significantly more seed vigor (Table 2).
 

 

Table 1. Analysis of variance of physiological and biochemical traits, growth characteristics and seed yield.

S.O.V

df

Germination Percent
 

Germination

rate

Root length

shoot length

Vigor

 

Carotenoid

Ascorbate peroxidase

Catalase

Chlorophyll a

Chlorophyll b

Essential oil content

Grain Yeild

Essence Yeild

 

Replication

2

17.44

0.03

0.49

0.56

0.541

0.04

0.004

1.63

0.003

0. 003

0./004

48.47**

0.03**

 

Salinity

2

** 20447.86

1971.10**

** 22.83

** 63.91

217.55**

** 7.63

** 11.59

** 428.93

** 2.43

** 0.166

ns 0.004

1243.71**

3.09**

 

Cultivar

1

ns 30.25

ns 1.99

2.15*

29.16**

23.51**

** 1.47

** 0.72

** 20.56

** 0.21

** 0.045

** 1.013

8.84ns

** 0.06

 

Salinity× Cultivar

2

ns 1.08

79.70**

ns 0.22

5.56**

5.07**

ns 0.04

ns 0.002

** 4.64

ns 0.003

ns 0.006

ns  0.021

13. 91**

** 0.026

 

Bacteria

1

** 1406.25

205.92**

41.39**

39.69**

54.1**

** 3.05

3.38**

124.58**

** 0.65

** 0.114

** 0.364

674.96**

1.08 **

 

Salinity× Bacteria

2

ns 7.58

61.02**

ns 1.15

ns 1.44

6.57**

ns 0.01

ns 0.01

* 3.37

ns 0.002

ns 0.002

ns  0.00

252.757**

0.32**

 

Cultivar× Bacteria

1

ns 2.25

ns 7.47

ns 0.25

0.001ns

ns 0.18

ns 0.00

ns 0.03

ns 1.17

ns 0.001

ns 0.007

ns 0.006

ns 11.07

0.002ns

 

Salinity× Cultivar× Bacteria

2

ns 5.25

ns 12.84

ns 0.66

ns 0.01

ns 0.002

ns 0.01

ns 0.05

ns 0.35

ns 0.008

ns 0.002

ns  0.001

27.77**

** 0.012

 

 

Error

22

29.14

4.78

0.44

0.49

0.517

0.02

0.03

0.81

0.009

0.004

0.017

3.11

0.0012

 

C.V.(%)

-

8.83

14.56

9.23

8.65

12.66

4.37

5.97

5.42

5.93

8.07

7.43

17.2

18.8

 

ns, * and **: Non-significant and significant at 5% and 1% levels of probability, respectively.  

 

 

 

 

 

 

                         

Table 2. Mean comparisons of physiological and biochemical traits, growth characteristics and cultivar grain yield under saline conditions and the use of bacteria

Salt treatment

Germination per (%)

Germination rate(no. /day)

Root Height (mm)

Shoot Length (mm)

Seed Vigor (mm)

Carotenoid (mg/gFW)

Ascorbate (unit mg/FW)

catalase (unit mg/FW)

Chlorophyll a (mg/gFW)

Chlorophyll b (mg/gFW)

Essential Oil (%)

grain Yield gr. per plant-1

Essentce Yield per plant

 

 

Control

86.58 a

26.27a

7.21 b

9.78 a

8.49 a

2.56 c

1.63 c

10.86 c

2.04 a

0.90 a

1.71 a

21.64 a

1.10 a

150 mM

83.3 a

17.72 b

8.54 a

9.17 a

7.76 a

3.49 b

3.07 b

16.23 b

1.63 b

0.78 b

1.75 a

7.07 b

0.39 b

150 mM

13.50 b

1.07 c

5.78 c

5.49 b

0.78 b

4.15 a

3.51 a

22.80 a

1.14 c

0.67 c

1.79 a

2.03 c

0.11 c

           

 

 

 

 

 

 

 

 

With Bacteria

68.19 a

17.41a

8.3 a

9.2 a

6.9 a

3.69 a

3.04 a

18.49 a

1.74 a

0.84 a

1.85 a

14.58 a

0.71 a

 

No Bacteria

 

54.09 b

12.63 b

6.1 b

7.1 b

4.45 b

3.11 b

2.43 b

14.00 b

1.47 b

0.73 b

1.65 b

5.92 b

0.36 b

Means followed by the same letters in each column are not significantly different at 5% level, according to Duncan's Multiple Range Test. 

 

The, reduce seed vigor in saline conditions have been reported in many studies (Fazeli-Kakhki et al., 2015; Aflaki et al., 2017). 
  Variance analysis showed that salinity, variety and bacteria significantly effected chlorophyll a, b and Carotenoid, (Table1). Increased salinity caused significant of increase carotenoid, where the most and the least amount belonged to 250 mM and control, treatments respectively. Also, inoculating with bacteria significantly increased carotenoid content (Table 2). Chlorophyll a contents decrease of significantly by increasing of salinity (Table 2). Chlorophylls a and b contents in Ardestan variety compared to Mashad Ardehal variety was higher (Figure 1). Moreover, inoculating with bacteria compared to control increased the Chlorophylls a and b contents (Table2). Javadi Poor et al., (2013) stated that increased salinity in some variety of safflower; the carotenoid content had a decreasing process. The studies (Rezaei et al., 2004) reported that the amount of chlorophylls a and b in cotton under the stress with different salinity levels, significantly decreased.
 
 
 

Figure1.Interaction effects of salinity, cultivar and bacterial inoculation                       Figure 2.Interaction effects of salinity, cultivar and bacterial  on chlorophyll a                                                                                                  inoculation on catalase

 
 
 
 
 
 
 
 
   
 
 
 
 
 
 
 
 

 

 

Figure3.Interaction effects of salinity, cultivar and bacterial inoculation on Essence percentage

 

   Catalase and ascorbate proxidase was affected by salinity, variety, and bacteria (Table 1). Salinity compared to control treatment led to increase of catalase enzyme’s activity. The most catalase enzyme activity was related to 250 mM sodium chloride treatment (Table 2). Applying bacteria significantly increaed catalase enzyme intense activity (Table 2). Increasing of sodium chloride concentration in the presence of bacteria extended catalase enzyme activity (Figure 2). Increasing salinity, increased ascorbate proxidase enzyme activity, so that the 250 mM sodium chloride and control treatments had the highest and lowest ascorbate proxidase contents, respectively. Ascorbate proxidase enzyme activity as affected by varieties (Table 1). Ascorbate proxidase enzyme content was 10% more in Ardestan varities compared to Mashhad Ardehal.  Also, inoculating with bacteria compared to control treatments significantly increased ascorbate proxidase activity (Table 2). Kohler et al., (2009) reported that in lettuce seedling (Lactuca sativa L.) inoculation with pseudomonas bacteria at the normal condition, decreases the rate of catalase enzyme activity but in stress condition, enzyme activity rate increased significantly. Ghorbanli et al., (2012) showed that increase of salinity in cumin increased the rate of ascorbate proxidase activity. 
  Grain yield was affected by salinity and inoculating with bacteria (Table 1). Varietiy had no significant effect on grain yield (Table 1). Inoculating with bacteria compared to control increased grain yield by 59.39 percent (Table 2). Zabihi et al., (2009) observed that by increasing salinity the yield and yield components decreased; and in pre-treatment with bacteria these trait will increase. In salvia (Salvia officinalis L.), inoculation with azotobacter and azospirillum, increased plant height (Saeed najad and Rezvanimoghaddam, 2010). Stimulating growth bacteria by producing indole acetic acid, giberellin, led to root length increased, root absorption level, and number of root hairs, and nutrition absorption and finally causes plant health improvement under stress condition (Egamberdieva and Kucharova, 2009).
    The essence yield and essence percentage were significantly affected by varieties and inoculating with bacteria (Table 1). However, salinity had no effect on exposed the essence percentage. The variety of Ardestan in comparison to Mashad Ardehal had more essence percentage (Figure 3). Inoculatiion with bacteria increased essence content by10.8 percent (Table 2). Also inoculating with bacteria increased essence yield (Table 2). The observed results showed that bio fertilizers application increased biological function, grail yield, and essence yield in cumin (Saeed najad and Rezvanimoghaddam, 2010). Bacteria application helps to increase nutrition absorption rate and growth rate, resulting in cumin’s increased yield (Saeed najad and Rezvani moghaddam, 2010). 
 
Conclusion
 
  The results of this study showed that increasing of salinity decreases the physiological characteristic, contents of chlorophylls a and b, grain yield and essence yield. However, the essence percentage did not affected by salinity. By increasing salinity the amount of carotenoid, catalase, and ascorbate proxidase increased significantly. However, increasing of salinity leads to a reduction in seed germination. It is observed that Ardestan variety  tolerates salinity condition more than Mashad Ardehal. In salinity condition inoculating seeds with azotobacter, decreade the negative effects on physiological characteristic. Generally, it is suggested to use azotobacter as bio-fertilizers to decrease the salinity effects on germination, grain yield, and essence yield of cumin.
 

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