Nitrate pollution reduction using biological fertilizers in paddy fields, the South Caspian Sea basin, Guilan Province, Iran

Document Type: Research Paper

Author

Department of Agriculture, Islamic Azad University of Lahijan branch, Lahijan, Iran

Abstract

Application of chemical fertilizers, especially urea, causes groundwater pollution. Therefore, to decrease environmental pollution, biological fertilizers may be employed. In order to investigate the effect of irrigation levels, Azospirillum and nitrogen levels on rice yield and its components, an experiment was conducted in the north of Iran during the crop season in 2013. The experiment was arranged in a split-split plot based on a completely randomized block design with 3 replications in which irrigation level was the main factor. The levels of factors used were: I1= continuous submergence and I2 = 11 days’ irrigation interval; A1 = application and A2= no application of Azospirillum as the sub factor; and nitrogen fertilizer levels as a sub-sub factor includingN1 = 0, N2 = 30, N3 = 60 and N4 = 90 kg ha-1. Continuous submergence and 11 days’ irrigation interval produced 4247 and 2720 kg ha-1, while the application of Azospirillum and the treatment without it produced 4064 and 2903 kg ha-1, respectively. Moreover, N4, N3, N2 and N1 produced 3950, 3800, 3225 and 2700 kg ha-1, while I1A1N4 and I1A1N3 had maximum and I2A2N1 had minimum mean values with 5381, 5330 and 1882 kg ha-1, respectively. Therefore, it is concluded that the application of a balanced nitrogen fertilizer with Azospirillum could prevent the indiscriminate use of chemical fertilizers and reduce nitrate pollution, leading towards more sustainable agriculture in the north of Iran.

Keywords


Nitrate pollution reduction using biological fertilizers in paddy fields, the South Caspian Sea basin, Guilan Province, Iran

 

Majid Ashouri

 

Department of Agriculture, Lahijan branch, Islamic Azad University, Lahijan, Iran

E-mail:mashouri48@yahoo.com

ABSTRACT

Application of chemical fertilizers, especially urea, causes groundwater pollution. Therefore, to decrease environmental pollution, biological fertilizers may be employed. In order to investigate the effect of irrigation levels, Azospirillum and nitrogen levels on rice yield and its components, an experiment was conducted in the north of Iran during the crop season in 2013. The experiment was arranged in a split-split plot based on a completely randomized block design with 3 replications in which irrigation level was the main factor. The levels of factors used were: I1= continuous submergence and I2 = 11 days’ irrigation interval; A1 = application and A2= no application of Azospirillum as the sub factor; and nitrogen fertilizer levels as a sub-sub factor includingN1 = 0, N2 = 30, N3 = 60 and N4 = 90 kg ha-1. Continuous submergence and 11 days’ irrigation interval produced 4247 and 2720 kg ha-1, while the application of Azospirillum and the treatment without it produced 4064 and 2903 kg ha-1, respectively. Moreover, N4, N3, N2 and N1 produced 3950, 3800, 3225 and 2700 kg ha-1, while I1A1N4 and I1A1N3 had maximum and I2A2N1 had minimum mean values with 5381, 5330 and 1882 kg ha-1, respectively. Therefore, it is concluded that the application of a balanced nitrogen fertilizer with Azospirillum could prevent the indiscriminate use of chemical fertilizers and reduce nitrate pollution, leading towards more sustainable agriculture in the north of Iran.

Key words: biological fertilizer, nitrate pollution, paddy fields, rice.

INTRODUCTION

Water and nutrients may interact with each other to produce a coupling effect. Some studies indicated that there was a significant interaction between nitrogen application and water management on nitrogen absorption and utilization, and grain yield in rice (Sun et al. 2010, 2014). Water and nitrogen are important inputs for rice production. The scarcity of fresh water resources now threatens rice production in China (Yao et al.  2014).

Chemical fertilizers have been used for over 50 years in Iran. Over 61% of the chemical fertilizers used are nitrogen fertilizers, of which urea constitutes 90.9%. The average application rate of urea is 33.3 kg ha-1 in Iran, which are 2.55, 3.58 and 2.64 times of those in Spain, Australia, and Canada, respectively. Despite the very heavy application of urea, the average grain yield of cereal is much lower than in other countries. Furthermore, there is not a positive and significant relationship between urea application and grain yield in Iran. Nitrogen fertilizer application has increased so the possibility of nitrate contamination of groundwater is high in paddy fields in the north of Iran (Mashayekhi & Lashkari 2010). In one study, the content of nitrate in a well near the paddy fields was measured and it was found that there was a positive correlation between nitrogen fertilizer application and groundwater pollution. Some studies have also been carried out in the north of Iran to determine the nitrate concentration in the soil, as well as surface and shallow ground waters (Mashayekhi & Lashkari 2010). In 1996, sampling from surface and groundwater, including water in rice fields, rivers, drains, domestic wells and semi deep wells, was performed in Guilan and Mazandaran provinces in Iran. The results showed that the most nitrate fluctuations were related to domestic wells. In the wet season, 13% of samples and, in a dry season, 3% of samples had concentrations which exceeded the standard level of 10 mg L-1 (Malakutim 1996).

Agricultural activities have a serious impact on nitrate contamination of groundwater (Macquarie et al. 2001; Buda et al. 2014).

Using nitrogen as a fertilizer and leakage of nitrate from livestock have decreased the ground water quality (Sweeten et al. 1995). Other studies have indicated that there was a close relation between nitrate contamination in groundwater and agricultural management (Hinkle & Tesoriero 2014). The utilization of biological nitrogen fixation technology can decrease the use of urea; prevent the depletion of soil organic matter and reduce environmental pollution. Nitrogen fixation by cyanobacteria helps to minimize the over-dependence on chemicals, particularly urea, in rice farming and also enhance the efficiency of nitrogen by releasing ammonia constantly to the rice crop (Geetha Lakshmi et al. 2012). Bio-fertilizers are becoming increasingly popular in many countries and for many crops, but very few studies on their effect on grain yield have been conducted in rice (Meynard Banayo et al. 2012).

The Azospirillums are soil bacteria capable of producing associative symbiosis in the roots of various plants including grain crops such as rice. The genus Azospirillum has several N-fixing species, which are rhizobacteria associated with monocots and dicots such as grasses, wheat, maize and Brassica chinensis L (Sing et al. 2011). The Azospirillums are the most famous microorganisms that can produce colonies in the rhizospher around cereals roots and lead to nitrogen fixation. Azospirillum strains have been isolated from rice repeatedly, and recently the strain Azospirillum sp. B510 has been sequenced (Kaneko et al. 2010).

Inoculation of plants with Azospirillum has been found to cause significant increases in growth and yield which is equivalent to that attainable by application of 15-20 kg N ha-1 (Rodrigues et al. 2008).

Phytohormone production and a beneficial effect on plant growth were also shown for a range of other microorganisms (Fernando et al. 2010).

Although in recent years the application rate of fertilizers in Iran has increased sharply and a large amount of fertilizers, in addition to that produced domestically, have been imported. Nevertheless, during this period, not only has the yield of crops not increased in accordance with the increased application rate of fertilizers, but also the yield of crops has declined. Reasons for this include water shortage, different irrigation methods, lack of scientific knowledge by farmers and methods of fertilizer application. Noteworthy, employment of nitrogen fertilizer is a very significant factor in the growth of rice. Since obtaining a higher grain yield is an important experimental goal, it has been suggested that 120 kg N ha-1 should be the basic fertilizer application model for further experiments of rice hybrids (Ashouri et al. 2013). A yield-increasing effect on rice by inoculation with Azospirillum sp. strain B510 has been shown but the experiment was conducted in pots only (Isawa et al. 2010). Results indicated that the influences of nitrogen on grain yield, straw yield, plant height, fertile tiller number/m2 and panicle length were significant. Maximum yield and yield components were achieved by applying 90 kg ha-1 and maximum mean of grain yield equal to 4343 kg ha-1 was related to this treatment (Hatamifar et al. 2013). The objectives of the present study were to investigate the effect of irrigation, nitrogen fertilizer management and the application of nitrogen fixation bacteria on grain yield of rice. The effect of Azospirillum employment was assessed on reducing the application of nitrogen fertilizer in rice fields and reducing the pollution of groundwater.

 

MATERIALS AND METHODS

In order to investigate the effect of irrigation levels, Azospirillum and nitrogen fertilizer levels on yield and yield components of rice, an experiment was conducted in the north of Iran during crop season 2013. The experiment was arranged in split-split plot based on completely randomized block design with 3 replications in which water levels were the main factor including I1 = continuous submergence and I2 = 11 days’ irrigation interval; A1= application and A2= no application of Azospirillum as was the sub factor; and nitrogen fertilizer levels were the sub-sub factor comprising N1= 0, N2 = 30, N3 = 60 and N4 = 90 kg ha-1. Moreover, Nitrogen fertilizer was split into three important growth stages, transplanting (50%), tillering (25%) and booting (25%). For all treatments, drainage basins were mounted from which waste water belonging to each replication treatment was excluded. Each experimental plot had 15 lines with five meters in length and the planting scheme was 25 ´ 25 cm. For bacteria inoculation, roots of seedlings were inoculated with bacteria for at least 12 hours. The nursery construction was performed in April and transplanted to the field in early May. Fertilizer application was based on the soil test and instructions of the technicians of the Rice Research Organization in Iran and the amount of P and K was calculated and applied to every plot.

The soil texture of the study area was clay-loam with a pH of 5.9., total organic matter, 3.08%, electrical conductivity, 0.68 ds m-1, total nitrogen, 0.43%, available phosphorus, 19.2 ppm and available potassium, 95 ppm. Grain yield was measured with 6 mharvesting of every plot. The yield and yield components were analyzed using MSTATC software. The Duncan’s multiple range test was used to compare the means at 1% significance.

 

RESULTS AND DISCUSSION

Grain yield

The results showed that the irrigation levels, application of Azospirillum and nitrogen levels, had significant relationships with grain yield (Table 1). Continuous submergence and 11 days’ irrigation interval produced 4247 and 2720 kg ha-1, respectively (Table 2).

The Application of Azospirillum compared to treatment without it yielded 4064 and 2903 kg ha-1, respectively (Table 2). N4, N3, N2 and N1 created 3950, 3800, 3225 and 2700 kg ha-1, respectively (Table 2). Noteworthy, I1A1N4 and I1A1N3 had maximum while I2A2N1 had minimum mean values with 5381, 5330 and 1882 kg ha-1, correspondingly (Fig. 1).

 Furthermore, N2 fixing efficiency of Azospirillum isolates were positively correlated with the grain yield which were showed by increasing the plant growth parameters such as number of roots, length of roots, the number, length and  width of leaves, length of stem, number of tiller and grains weight (Kanimozhi & Panneerselvam 2010). Fallah Amoli et al. (2014) showed that the treatments of Azospirillum and pseudomonas as well as 100% recommended N showed the importance of the role of PGPRs in appropriate transferring of nutrient and low pollution of environment, as well as the quality of rice and effective factors on quality based on genetic and breeding factors. In this study, local Tarom (rice) as a qualified cultivar was affected by N + PGPRs positively.

 

Number of tiller/m2

The effect of irrigation levels, application of Azospirillum and nitrogen fertilizer as well as interaction of irrigation levels, application of Azospirillum and nitrogen fertilizer on Number of tiller/m2 were significant (Table 1). Continuous submergence and 11 days’ irrigation interval produced 407 and 288 tiller/ m2 respectively (Table 2).

The application of Azospirillum compared to treatment without it produced 406 and 290 tiller m-2 respectively (Table 2). N4, N3, N2 and N1 produced 427, 370, 324 and 268 tiller/m2 respectively (Table 2). I1A1N4 had maximum while I2A2N1 had minimum mean values with 510 and 142 tiller/m2 respectively (Table 3).

 

Number of panicle/m2

The effect of irrigation levels, application of Azospirillum and nitrogen fertilizer as well as interaction of irrigation levels, application of Azospirillum and nitrogen fertilizer on Number of panicle/m2 were significant (Table 1). Continuous submergence and 11 days’ irrigation interval produced 347 and 245 panicle/m2 respectively (Table 2). The applic- ation of Azospirillum compared to treatment without it produced 354 and 237 panicle/m2 respectively (Table 2). N4, N3, N2 and N1 produced 373, 313, 280 and 225 panicle/m2, respectively (Table 2).

I1A1N4 had maximum, while I2A2N1 had minimum mean values with 420 and 170 panicle/m2 respectively (Table 3). Results indicated that the influence of nitrogen on grain yield, straw yield, plant height, fertile tiller number/m2 and panicle length was significant. Maximum yield and yield components achieved by applying 90 kg ha-1 and maximum mean of grain yield equal to 4343 kg ha-1 were related to this treatment (Hatamifar et al. 2013).

 

Number of grains in panicle

The effect of irrigation levels, application of Azospirillum and nitrogen fertilizer along with interaction of irrigation levels, the application of Azospirillum and nitrogen fertilizer on the number of grains in panicle were recognized significant (Table 1). Continuous submergence and 11 days’ irrigation interval produced 107 and 80 grains in the panicle, respectively (Table 2).

The application of Azospirillum and the treatment without it yielded 102 and 84 grains in the panicle, respectively (Table 2). N4, N3, N2 and N1 produced 100, 97, 90 and 84 grains in the panicle, respectively (Table 2). I1A1N4 had maximum, whereas I2A2N1 had minimum mean values of 116 and 60 grains in the panicle, respectively (Table 3). Consequently, the significant increase in the grain yield by Azospirillum application at 75 and 100% of the recommended dose of nitrogen (RDN) is mainly due to the higher number of panicles per m2, the increase in the mean panicle weight and also 1000-grain weights (Nayak et al. 2003).

 

Fig. 1. Interaction of irrigation levels, application of Azospirillum and nitrogen fertilizer on grain yield.

 

Unfilled grain percentage

The effects of irrigation levels, application of Azospirillum and nitrogen fertilizer, as well as interaction of irrigation levels, application of Azospirillum and nitrogen fertilizer on unfilled grain percentage were significant (Table 1). Continuous submergence and 11 days’ irrigation interval produced 7 and 11% unfilled grain, respectively (Table 2). The application of Azospirillum and the treatment without it produced 2 and 5% unfilled grain, respectively (Table 2). N4, N3, N2 and N1 produced 5, 6, 7 and 11% unfilled grain, respectively (Table 2). I1A1N4 had maximum whereas I2A2N1 had minimum mean values of 5.5 and 16.7% unfilled grain, respectively (Table 3).

 

 

1000-grains weight

The effects of irrigation levels, application of Azospirillum and nitrogen fertilizer along with interaction of irrigation levels, application of Azospirillum and nitrogen fertilizer on 1000-grains weight were significant (Table 1). Continuous submergence and 11 days’ irrigation interval produced 28.5 and 22.3 g, respectively (Table 2). The application of Azospirillum and the treatment without it produced 26.5 and 24.4 g, respectively (Table 2). N4, N3, N2 and N1 produced 26.2, 25.8, 24.5 and 23.5 g, respectively (Table 2). I1A1N4 had maximum while I2A2N1 had minimum mean values of 29.5 and 20.5 g, respectively (Table3). It has been reported that application of N fertilizer increased 1000-grain weight of rain-fed lowland rice even when the rice crop was encountered to water deficit (Castillo et al. 1992).

 

 

Biomass

The effects of irrigation levels, application of Azospirillum and nitrogen fertilizer as well as interaction of irrigation levels, application of Azospirillum and nitrogen fertilizer on biomass were significant (Table 1).

Continuous submergence and 11 days’ irrigation interval produced 9103 and 6442 kg ha-1, respectively (Table 2). The application of Azospirillum and treatment without it produced 8883 and 6602 kg ha-1 respectively (Table 2). N4, N3, N2 and N1 produced 8813, 8516, 7681 and 6860 kg ha-1 respectively (Table 2). I1A1N4 had maximum whereas I2A2N1 had minimum mean values of 12080 and 3931 kg ha-1 respectively (Table 3). Grain yield and biomass have been shown to increase as the applied N rate was increased (Ashouri 2015).

 

Harvest index

The effects of irrigation levels, the application of Azospirillum and nitrogen fertilizer along with interaction of irrigation levels, application of Azospirillum and nitrogen fertilizer on harvest index were significant (Table 1). Continuous submergence and 11 days’ irrigation interval produced 47 and 42% respectively (Table 2).

 

Table 1. Analysis of variance on yield and yield components of rice.

S.O.V

df

Grain yield

Number of tiller/m2

Number of panicles/m2

Number of grains in panicle

Unfilled grain (%)

1000-grains weight(g)

Biomass

Harvest index

R

2

289996ns

995ns

1028ns

2ns

0.008ns

0.019ns

20381ns

1.573

Irrigation(I)

1

28014352**

168625**

125052**

8721**

254**

470**

122985622**

8**

Ei

2

44044

141

91

2

2

0.198

26015

3

Azospirillum(A)

1

16154160**

207375**

163333**

3870**

67**

53**

9954**

0.738**

I×A

1

14770ns

792ns

1302ns

1645ns

118ns

5ns

72774ns

4ns

Ei

4

89131

240

169

0.332

0.213

0.029

17637

11

Nitrogen(N)

3

576230**

54417**

47048**

551**

2**

S

11596693**

1**

I×N

3

58209ns

590ns

920ns

11ns

3ns

s

160939ns

0.264ns

A×N

3

892136ns

4451ns

3993ns

35 ns

5ns

1ns

361483ns

0.005ns

I×A×N

3

659577**

813ns

364**

61**

1**

10**

514336**

205**

En

24

659577

227s

243

1

0.555

0.175

8062

0.740

CV (%)

-

6.24

4.33

5.27

6.19

8.37

5.16

9.12

8.12

**: Significant difference at 1% level

*: Significant difference at 5% level

 ns: non-significant difference

 

Table 2. The effect of irrigation levels, application of Azospirillum and nitrogen fertilizer on yield and yield components of rice.

Treatment

Grain yield

(kg ha-1)

Number of tiller/m2

Number of panicle/m2

Number of grains in panicle

Unfilled grain (%)

1000-grains weight(g)

Biomass

(kg ha-1)

Harvest index (%)

Irrigation

 

 

 

 

 

 

 

 

I1

4247a

407a

347a

107a

7b

28.5a

9103a

47a

I2

2720b

288b

245b

80b

11a

22.3b

6442b

42b

Azospirillum

 

 

 

 

 

 

 

 

A1

4064a

413a

354a

102a

2b

26.5a

8883a

46a

A2

2903b

282b

237b

84b

5a

24.4b

6602b

44b

Nitrogen

 

 

 

 

 

 

 

 

N1

2700c

268d

225d

84d

11a

23.5c

6860d

39c

N2

3225b

324c

280c

90c

7b

24.5b

7681c

42b

N3

3800ab

370b

313b

97b

6bc

25.8a

8516b

45a

N4

3950a

427a

373a

100a

5c

26.2a

8813a

45a

 

 

Table 3. Interactions of irrigation levels, application of Azospirillum and nitrogen fertilizer on yield and yield components of rice.

Treatment

Grain yield

(kg ha-1)

Number of tiller/m2

Number of panicle/m2

Number of grain in panicle

Unfilled grain (%)

1000-grains weight(g)

Biomass

(kg ha-1)

Harvest index(%)

I1A1N1

3877b-d

366e

317d

103d

6.4gh

28.8bc

9281c

42c

I1A1N2

4652b

442cd

367c

107c

6.5gh

28.7bc

10830b

43bc

I1A1N3

5330a

483ab

408ab

112b

6.2h

29.1b

11800a

45b

I1A1N4

5381a

510a

420a

116a

5.5i

29.9a

12080a

45b

I1A2N1

3943bc

292gh

242fg

95e

6.6gh

27.2d

7416g

43bc

I1A2N2

3345c-g

315fg

258ef

101d

6.9f

27.5d

7595g

44b

I1A2N3

3612b-e

367e

317d

107c

7.1f

28.1cd

8508e

42c

I1A2N4

3840b-d

408d

358c

110b

7.4e

28.5bc

9137f

42c

I2A1N1

3280c-g

275h

225fg

79g

7.9de

21.7g

6811d

48a

I2A1N2

3125c-g

333eh

283de

92f

8.8de

23.2f

7539h

41d

I2A1N3

3387c-f

367e

317d

102d

9.5d

24.7e

8392g

40de

I2A1N4

3331c-g

458bc

408b

104d

9.7d

25.2e

8629f

39e

I2A2N1

1882g

190j

170i

60j

16.7a

20.5h

3931l

48a

I2A2N2

2049fg

208i

187h

63i

14.5b

20.5h

4763k

43bc

I2A2N3

2187e-g

267h

208gh

67h

13.6bc

21.1gh

5366j

41d

I2A2N4

2369d-g

258h

216gh

70h

12.5c

21.3gh

6102i

39e

 

 

 

 

 

 

 

 

 

 

 

The application of Azospirillum and the treatment without it produced 46 and 44% respectively (Table 2). N4, N3, N2 and N1 produced 45, 45, 42 and 39%, respectively (Table 2). I2A1N1 and I2A2N1 had maximum, while I2A1N4 had minimum mean values with 48, 48 and 39%, respectively (Table 3). Drought stress had significant effects on harvest index, seed performance, straw performance, the number of fertile tiller/m2, the number of full seeds, and seed emptiness rate (Sabetfar et al. 2013).

 

CONCLUSION

Irrigation interval of 11days compared with continuous submergence decreased grain yield to 56% because the number of tiller and panicle per square meter, number of grains in the panicle and the weight of 1000 grains decreased and unfilled grain percentage increased in the 11-days’ irrigation interval. The use of biological fertilizer resulted in increased grain yield and it decreased the use of chemical fertilizers so that a reduced impact on the environment was achieved.

The application of different amounts of nitrogen showed positive effects on increasing grain yield and yield components. Also, the combination of applications of bacteria and different amounts of nitrogen improved these characteristics.

Therefore, it is concluded that the application of a balanced nitrogen fertilizer with Azospirillum could prevent the indiscriminate use of chemical fertilizers and reduce nitrate pollution, resulting in more sustainable agriculture in the north of Iran.

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