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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
Advance Journals
The Open Access Publisher
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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).
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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.
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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.
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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
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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)
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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
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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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