International Journal of Microbiology & Parasitologymanuscript.advancejournals.org/uploads/8543823da6ea42759f2b711f9f... · International Journal of Microbiology & Parasitology

International Journal of Microbiology & Parasitology

Research Article

Nitrosoguanidine Derived Mutants of Azospirillum spp. and Their Relationship

With in vitro Nitrogen Fixation and Plant Growth Promoting Substances

Production 1Dayamani, K.J., 2Savalgi, V.P., 3 Srinivasa Murthy, R. and 4Krishnaraj, P.U.,

1Asssistant Professor, COH, UHS Campus, GKVK, Bangalore-65 2 Professor, Dept. of Agril. Microbiology, UAS, Dharwad -5

3 Jr. Scientific Officer, NCOF, Ghaziabad, New Delhi

4Professor and Head, Dept. of Agril. Microbiology, COH, Bijapur, UAS, Dharwad -5

Correspondence should be addressed to Dayamani, K.J.

Received 31 October 2014; Accepted 14 November 2014; Published 03 December 2014

Copyright: © 2014 Dayamani, K.J. et al. This is an open access article distributed under the Creative Commons Attribution

License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Keywords: acetylene reduction, Azospirillum, azide resistance, in vitro nitrogen fixation, , PGPR,

Introduction

The azospirilla are potentially useful nitrogen fixing

bacterium in agriculture, because of their close

association with the roots of economically

important crops. Mutation works on Azospirillum

have been done to induce higher nitrogen fixation

by using ethylene diamine resistant mutants.

Sodium azide (NaN3), a potent inhibitor of the

terminal segment of electron transport chain can be

reduced to ammonia and dinitrogen by nitrogenase.

The process requires ATP and a strong reducing

agent during nitrogen fixation. Nitrogenase, besides

reducing N2, also reduces some of the toxic

metabolites like cyanide and azide to harmless

compounds and thus helps in the detoxification of

these compounds. Resistance to azide has been

used to isolate mutants of Rhizobium with enhanced

nitrogen fixing ability [1]. However, such reports on

free living diazotrophs like Azospirillum are not

many. Therefore, the ability to make a change in the

electron transport chain along with the acquired

resistance might show higher nitrogenase or

Year: 2014; Volume: 1; Issue: 2

Article ID: IJMP14 17; Pages: 1-11

Abstract

Twenty-two strains of Azospirillum spp. were obtained from the 56 maize endorhizosphere samples. Two

isolates ASD-7 and ASD-8 were selected on the basis of in vitro N2 fixation and nitrogenase activity (ARA).

The inherent sodium azide resistance was recorded and were subjected to NTG mutagenesis. Sixteen

mutants were obtained and were further tested for their resistance to higher concentration of sodium

azide. Six azide resistant mutants examined for their N2 fixing ability, higher nitrogenase activity and

production of plant growth promoting substances. Among the mutants ASD-802 and ASD-801 fixed higher

amount of nitrogen (63.01 and 47.53 mg/g of malate respectively) and showed higher acetylene reduction

activity (624 and 586 n moles per mg of protein/hr).


2 www.advancejournals.org

nitrogen fixing activity. Having this in view, a study

was undertaken to determine azide resistance in

Azospirillum and to examine its relationship with

the rate of nitrogen fixation and nitrogenase

activity.

Materials and Methods

Azospirillum strains were isolated by following the

enrichment culture technique as described by Day

and Doboreiner [2]. The tentatively identified

isolates were subjected to morphological

characterization using different media such as Nfb

malate agar, BMS agar and cangored medium. In the

physiological characterization was done as enlisted

for identification of Azospirillum in Bergey’s manual. Then the isolates were screened for

beneficial traits like in vitro nitrogen fixation,

nitrogenase activity and the production of plant

growth promoting substances. In vitro nitrogen

fixation by each Azospirillum isolates was studied

according to the method described by Humphries

[3]. The reduction of acetylene to ethylene by

nitrogenase was measured by using NUAON gas

chromatogrophy fitted with flame ionization

detector (FIB) with poropak-T column. Nitrogen

carrier gas flow rate was maintained at 30 ml/min

oven temperature was 28°C iso thermal. The

temperature of injector port was 140°C. The FID

detector temperature was 150°C. The nitrogenase

activity (ARA) was expressed as n moles of ethylene

produced per mg of protein hr-1 [4]. The cell protein

of the Azospirillum isolates used for ARA was estimated by Lowry’s method [5] by using Folin

cicalteau reagent. The ARA was calculated by using

the formula.

Concentration of the

sample =

Area of the sample x

Concentration of the standard

Area of the standard

Inherent resistance of the Azospirillum isolates was

to sodium azide tested at different concentrations

of 5 to 40 ppm on N free malate solid media. Two

strains of Azospirillum (ASD-7 and ASD-8) were

selected and subjected to NTG mutagenesis and the

mutants obtained were again characterised for

sodium azide resistance, intrinsic antibiotic

resistance (IAR), in vitro nitrogen fixed per gram of

malate and nitrogenase activity as per the methods

detailed for wild types.

Results

Totally twenty-two isolates were obtained from 53

endorhizosphere soils from different locations.

Characteristically all the isolates were Gram –

negative, vibriod and exhibited spiral (cork-screw)

movement when observed using the hanging drop

technique.

Nitrogen fixation and nitrogenase activity

In the present investigation the total nitrogen fixed

by the isolates ranged from 2.03 mg of N/g of

carbon utilised to 18.88 mg of N/g of carbon (Table

1). The highest nitrogen fixation was observed by

ASD-8 followed by ASD-7. The selected isolates

could reduce acetylene between 65-464 n moles

per mg protein per hr. Among the isolates, ASD-8,

ASD-7, ASD-39 and ASD-97 have shown higher ARA

activity i.e. 464, 422, 409 and 388 n moles of

ethylene produced per mg protein per hr

respectively (TABLE 1). The least ARA activity was

seen in the strain ASD-53 i.e. 65 n moles per mg

protein per hr. Wherein this studies, it was ranged

from 1.01 to 63.01 mg. The nitrogenase activity of

the mutants recorded considerable variations from

178 to 684 n moles which is high compared to their

wild types. The variation in the nitrogenase activity

was observed [8] and reported that nitrogenase

activity ranged from 65.0 to 464 n moles of

C2H4/mg of protein/hr in wheat.



Table 1. In vitro nitrogen fixation and acetylene reduction activity of Azospirillum isolates

Strain No. N2 fixed mg/g of malate n mole/mg of protein/h

ASD-5 11.34 172

ASD-7 18.41 422

ASD-8 18.83 464

ASD-9 9.80 278

ASD-12 12.25 188

ASD-16 13.37 246

ASD-18 7.63 302

ASD-19 9.10 189

ASD-22 11.83 223

ASD-25 14.84 248

ASD-26 8.82 176

ASD-27 10.85 268

ASD-33 15.61 294

ASD-36 15.05 247

ASD-37 17.99 388

ASD-39 15.61 409

ASD-44 2.03 218

ASD-45 13.23 184

ASD-49 11.90 148

ASD-50 13.23 276

ASD-51 16.31 178

ASD-53 5.67 65

ACD-15 18.13 401

ACD-20 17.29 412

Among the 16 mutants, four mutants derived from

ASD-7 and the two mutants derived from ASD-8

have showed higher resistance to sodium azide (35

ppm). Two mutants ASD-802 and ASD-801 derived

from ASD-8 showed highest in vitro N fixed per g of

malate (63.01 and 47.53 mg of nitrogen fixed per g

of malate respectively) and nitrogenase activity

(684 and 526 n moles of ethylene per mg of protein

per hr) and (TABLE 2). The above results showed

that sodium azide resistance in Azospirillum can be

used as a genetic tool for isolation of mutants with

enhanced N-fixing ability. All the isolates could

produce IAA, ranging from 2.56 g /100 ml to 29.91

g /100 ml. Among all the Azospirillum isolates

examined for IAA production, strain ASD-37

produced maximum amount of IAA. Azospirillum

isolates ASD-7 and ASD-8 also produced higher

amount of IAA. The detailed results are presented in

the TABLE 3.



Table 2. Acetylene reduction activity of NTG derived azide resistant mutants of Azospirillum

Strain No. Strain N2 fixed mg/g of malate n moles/mg of protein/h

ASD-7 Wild type 18.40 422

ASD-701 Mutant 38.84 472

ASD-702 Mutant 08.10 490

ASD-703 Mutant 13.62 329

ASD-704 Mutant 21.14 294

ASD-705 Mutant 28.77 458

ASD-706 Mutant 25.20 178

ASD-707 Mutant 17.15 294

ASD-708 Mutant 15.33 352

ASD-709 Mutant 25.83 430

ASD-710 Mutant 09.73 310

ASD-8 Wild type 18.83 464

ASD-801 Mutant 47.53 586

ASD-802 Mutant 63.01 624

ASD-803 Mutant 21.14 366

ASD-804 Mutant 02.94 410

ASD-805 Mutant 07.35 379

ASD-806 Mutant 01.61 285



Table 3. Production of plant growth promoting substances by Azospirillum isolates

Strain No. IAA (μg/100ml) (GA μg/25ml) ASD-5 19.12 1.92

ASD-7 29.80 2.84

ASD-8 28.70 3.02

ASD-9 24.23 2.23

ASD-12 19.85 1.84

ASD-16 17.26 1.09

ASD-18 11.84 0.86

ASD-19 22.23 2.08

ASD-22 26.87 2.09

ASD-25 24.33 1.98

ASD-26 19.23 1.93

ASD-27 16.78 0.98

ASD-33 15.83 1.21

ASD-36 18.92 1.01

ASD-37 29.91 3.04

ASD-39 16.12 1.86

ASD-44 22.35 2.06

ASD-45 09.85 0.25

ASD-49 13.84 1.62

ASD-50 02.56 0.22

ASD-51 05.82 0.51

ASD-53 13.12 1.23

ACD-15 07.36 2.92

ACD-20 04.77 2.10

Production of plant growth promoting

substances

In vitro synthesis of IAA by the 22 Azospirillum

isolates and two standard strains of Azospirillum

was examined on a medium. Isolate ASD-37 (3.04

g /25 ml) produced highest amount of GA

followed by ASD-8 (3.02 g /25 ml). Gibberellic acid

production of Azospirillum isolates ranged from

0.22 to 3.04 g /25 ml. Out of 16 mutants, 12

mutants had highest IAA and GA production than

the wild type. ASD-802 found to produce the

highest IAA of 42.34 g /100 ml and GA of 4.16 g

/25 ml followed by ASD-801 with IAA production of

39.53 g /100 ml and GA production of 3.92 g /25

ml (TABLE 4)



Table 4. Production of plant growth promoting substances by NTG derived azide resistant mutants of

Azospirillum

Strain Strain IAA (g/100 ml) GA (g/25 ml)

ASD-7 Wild type 29.80 2.84

ASD-701 Mutant 31.96 3.04

ASD-702 Mutant 37.82 2.88

ASD-703 Mutant 32.18 1.96

ASD-704 Mutant 27.86 0.88

ASD-705 Mutant 36.79 2.94

ASD-706 Mutant 39.62 3.00

ASD-707 Mutant 32.98 2.19

ASD-708 Mutant 30.88 2.91

ASD-709 Mutant 37.65 3.28

ASD-710 Mutant 29.89 2.78

ASD-8 Wild type 28.70 3.02

ASD-801 Mutant 39.53 3.92

ASD-802 Mutant 42.34 4.16

ASD-803 Mutant 29.53 3.67

ASD-804 Mutant 26.82 2.97

ASD-805 Mutant 18.93 1.86

ASD-806 Mutant 20.74 1.98

Discussion

The morphological characteristics of the isolates in

comparison with the reference culture of

Azospirillum ACD-15 and ACD-20 showed similarity

and were in accordance with the description of

Azospirillum spp. given by Krieg and Doberenier [6].

Reports on nitrogen fixing efficiency of Azospirillum

strain isolated from grasses ranged from as low as

3.4 mg to as high as 83.3 mg of nitrogen fixed per

gram carbon source consumed [7]. The

mutagenised survivors were subjected to direct

screening for mutants having higher sodium azide

resistance than their respective wild type. These

mutational results are in accordance with the

reports [1] and [9]. Studies shows that [11]

relationship between azide resistance and N-fixing



ability in case of R. loti may be attributed to

enhanced respiration and high level of cytochrome

O and aa2 under microaerophilic culture condition

has improved N-fixation activity. Results from this

investigation suggest that at least one of the

mechanisms may act for enhanced N-fixation by

azide resistant mutants. Production of growth

promoting substances was also in accordance with

the studies by using with tryptophan as precursor

[10].

Conclusion

Increasing costs of chemical fertilizers, the

environmental pollution caused by them and also

the depletion of fossil fuel resources, since the

production of chemical fertilizer is based on the

non-renewable and consistently depleting

petroleum feed stocks, have called for more

attention to the use of bioinoculants to supplement

chemical fertilizers. Taking into considering the

above factors, Azospirillum strains were isolated

and indirect selection for improved N fixation was

attempted through screening for sodium azide

resistance. From the present investigation, it can be

concluded that the AziR mutants obtained through

mutagenesis, were found to be more effective in

enhancing the nitrogenase activity and nitrogen

fixed per gram of malate. Mechanism involved in

this has to be harnessed for further strain

improvement.

Conflict of interest

Further, the molecular mechanism involved in

higher nitrogen fixing ability of AZiR mutants of

Azospirillum has to be investigated and the mutants

has to be tested for their efficiency under field

condition.

Acknowledgements

I thank Karnataka State Department of Agriculture

for providing the financial assistance for carrying

out this research

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International Journal of Microbiology & Parasitologymanuscript.advancejournals.org/uploads/8543823da6ea42759f2b711f9f... · International Journal of Microbiology & Parasitology